WO2022054060A1

WO2022054060A1 - Use of uniconazole for potentiating abscisic acid effects in plants

Info

Publication number: WO2022054060A1
Application number: PCT/IL2021/051104
Authority: WO
Inventors: Alon SAMEACH; Patricia FRESCNILLO; Omer CRANE; Michal ACKERMAN-LAVERT
Original assignee: Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd.; Migal Galilee Research Institute Ltd.
Priority date: 2020-09-13
Filing date: 2021-09-12
Publication date: 2022-03-17

Abstract

The present invention provides methods of potentiating an abscisic acid effect in a plant by a pretreatment with uniconazole. In particular, the present invention provides the use of a combination of uniconazole and ABA for fruitlet thinning and fruit coloring.

Description

USE OF UNICONAZOLE FOR POTENTIATING ABSCISIC ACID EFFECTS IN PLANTS

FIELD OF THE INVENTION

The present invention is in the agriculture field and relates to methods of potentiating Abscisic acid (ABA) effects in plants. In particular, the present invention relates to methods of fruitlet thinning by sequential treatment with uniconazole and ABA.

BACKGROUND OF THE INVENTION

Abscission of young fruitlets is a widespread phenomenon in fruit trees. For some fruit crops, the rate of natural abscission is agriculturally sufficient, and in many cases too intense. In apple (Malus domestica Borkh), flowering occurs at the beginning of spring when the terminal bud is in the form of a cyme-like inflorescence of four to six flowers. In commercially grown apples natural abscission is insufficient since without additional fruitlet thinning, fruits will not reach commercial size. The final size of fruits when more than two fruits remain per inflorescence is small and non-commercial (Byers & Carbaugh, 2002).

Flower induction in apples occurs -60-80 days after full bloom. In other words, the flowers blooming next spring are microscopically formed in the beginning of summer of the previous year. Transition of the meristem into inflorescence meristem (typically around July) is affected by the amount of fruit load on the tree at the time of transition. Large number of fruits in a current year will lead to no flowering and fruit at the next year, entering a cycle termed 'alternate' or biennial bearing (Haberman et al., 2016). Thus, farmers want to lose unnecessary fruitlets at an early stage to increase final fruit size of remaining fruit and to avoid entering a cycle of alternate bearing.

Within the apple inflorescence the terminal 'king' flower is the first to initiate, the first to reach anthesis, and is considered the fruitlet with the lowest chance to go through abscission. The consecutive fruit is called lateral 3 or 'L3' followed by two L2 fruits, and the last flower to initiate and later reach anthesis is termed LI (Botton et al., 2011). LI has the highest probability to enter abscission within a few weeks after full bloom (Botton et al., 2011). The number of fruitlets surviving the abscission period per inflorescence is highly dependent on genotype. In cultivar ‘Golden Delicious’, after natural abscission, the final number of fruits per inflorescence is between zero to four. On the other hand, cultivar 'Ariane' trees tend to retain almost all fruitlets thus this cultivar needs to be thinned intensively in order for the fruit to reach commercial size (Laurens et al., 2005; Celton et al., 2014).

The decision of farmers whether to thin fruitlets or not varies between cultivars, regions and years. Many scientists and farmers use a phenological stage that describes the majority of inflorescences on the trees: Full Bloom or Petal Fall which occurs 7-12 days after full bloom (DAFB). Others also use the diameter of the developing King fruitlet as a phenological parameter. The timing of thinning is crucial as at a certain point most varieties are no longer responsive to most chemical thinners. Once the window of opportunity to use chemical agents has closed, the only option is manual hand thinning, which is very time consuming and thus costly. A late fruitlet thinning is not effective, since final fruit size may no longer be improved. In addition, the ability to improve next year flowering is also time limited, up to around 30-40 DAFB (Haberman et al., 2016).

The agronomical use of fruitlet chemical thinners started in the 1940s and naphthalene acetic acid (NAA), an auxin, was the first growth regulator used (Davidson et al., 1945). It’s been proposed that NAA reduces the amount of sugar translocated from leaves (Schneider & Lasheen, 1973) and also CO2 assimilation in leaves (Stopar et al., 1997).

Another growth regulator widely used is benzylaminopurine (BA), a cytokinin that induces abscission and increases size of retained fruit either directly or as a result of reduced competition from neighboring fruit (Costa et al., 2018).

The commercial product ProTone SG (Valent Biosciences, IL, USA) contains 20% of s-abscisic acid (S-ABA). ProTone SG application has the ability to thin fruitlets in peach and apple trees. The use of ProTone SG in combination with gibberellic acid (GA) (in grapes) or cytokinin (in apples) for thinning is known. However, the concentrations of ABA (0.25% commercial ProTone SG, e.g. 0.5 gr/L ABA, 1.89 mM) that were efficient as other chemicals for apple fruitlet abscission also caused unacceptable leaf abscission in the specific farm conditions (McArtney et al., 2014).

In the recent years, Metamitron, a photosynthesis inhibitor, has been released to the market as 'Brevis' (Stopar, 2018). It acts through the inhibition of photosystem II. High concentrations and/or inadequate application conditions may cause an undesired overthinning (Greene, 2002). The consensus among researchers is that variability in response to chemical thinners such as the photosynthesis inhibitor Metamitron associates with the internal levels of carbohydrates in the trees, wherein higher levels in the beginning of the season would lead to less fruitlet abscission. Differences in reserve carbohydrates would be affected by environmental conditions in the orchard, like temperature and irradiance before the treatment and after the treatment (Duane et al., 2013, Greene, 2002, Greene & Costa, 2013). Some developed models such as the "The Cornell Apple Carbohydrate Thinning Model" that instruct the farmers regarding what concentration of a thinning agent to use in order to get correct thinning based on present and forecasted environmental conditions in the orchard (Lakso, 2017, Lakso & Robinson, 2014). Recommendations based on the model include when and what concentration should be used.

There are no exact means of predicting the extent of the natural fruitlet abscission in apple trees. Therefore, out of caution, most farmers refrain from full chemical thinning, which normally includes a recommended protocol of two spraying dates. In addition, there are some important cultivars with no successful and approved chemical thinner, for example cultivar ‘Starking’ (‘Top Red’) in Israel. One treatment that appeared to be efficient in ‘Starking’ thinning was spraying with a product called "Sevin", which is Carbaryl (1-naphthyl methylcarbamate), commercially used as an insecticide. Surprisingly, it also causes fruit thinning in apple and was used for ‘Top Red’ (Stern, 2008). However, because of the neurotoxic nature of carbaryl it has been banned in Europe and is continually undergoing risk assessment in other countries due to consumer awareness of its toxicity, particularly to bees. Other studies with ‘Starking’, mainly for the purpose of inhibiting vegetative growth, have found that spraying uniconazole P (Sold commercially in Israel as 'Magic' which contains 50 gr/L of uniconazole P by Adama- Agan, Airport city, Israel) at ‘Petal fall’ can cause increased flowering the following year (Stern, 2008). The major effect of uniconazole is to inhibit the biosynthesis of the hormone gibberellin, and in many fruit trees gibberellin inhibits flower induction, so it is speculated that uniconazole is increasing next year's flowering by reducing internal gibberellin levels. Several studies have attempted to reveal the molecular mechanisms of abscission. Many have relied on the transcriptomic analysis of fruitlets with or without thinning treatment at different stages. One study induced abscission by applying BA to L3 fruitlets and compared gene expression in these fruitlets compared to king fruitlets without treatment (Botton et al., 2011). The expression of genes involved in ethylene, ABA signaling, reactive oxygen species (ROS) production and senescence was first induced in cortex and later on in seeds of abscising fruitlets. Later on, the accumulation of sucrose and ROS was detected on those abscising fruitlets. The study proposed a model in which BA increases the competition for assimilates between fruitlets by stimulating shoot growth and nutritional shortage. Isoprene was also detected early on in abscising fruitlets and it was correlated with ABA in the cortex (Eccher et al., 2013). Additionally, it was found that in King and L3 fruitlets the proportion between ethylene biosynthesis and ethylene receptor genes favors survival (Eccher et al., 2015). Overall, there is a lack of data on abscission and how to manipulate it.

Uniconazole is an isomeric mixture of (aS,pE)-P-[(4-chlorophenyl)methylene]-a- (1, 1 -dimethylethyl)- IH-1, 2,4-triazole-l -ethanol. The geometrical isomers were first disclosed in JP Patent No. 56108773 as intermediates for the corresponding ethers, and uniconazole was subsequently disclosed in US Patent No. 4,554,007. Uniconazole is currently approved for use as a fungicide or plant growth regulator, in particular for ornamental plants, for example for reducing plant height and increasing flowering in some plants

International (PCT) Application Publication No. WO 2009/016628 discloses methods for thinning fruit in a fruit-carrying plant comprising applying uniconazole to the plant during anthesis.

US Patent Application Publication No. 2016/0338351 discloses the treatment of grapes with 3'-methyl-(S)-abscisic acid, 3'-propargyl-(S)-abscisic acid, and/or salts thereof in order to enhance the color of the grapes.

US Patent Application Publication No. 2016/0338352 discloses the treatment of apple trees, peach trees, and grape vines with 3'-methyl-(S)-abscisic acid, 3'-propargyl- (S)-abscisic acid, and/or salts thereof in order to reduce the number of fruits on the trees or vines that grow to maturity. US Patent No. 9,808,004 describes the use of S-abscisic acid (S-ABA) and ethylene producing-agents such as ethephon to synergistically improve red color in grapes and to alter the sensory characteristics of wine.

There remains a need for methods of enhancing abscisic acid activity in different agricultural applications such as in fruit thinning and fruit coloring.

SUMMARY OF THE INVENTION

The present invention provides methods of potentiating ABA effects associated with different plant phenomena which are of significance agricultural practices. The present invention provides in some embodiments methods for fruitlet thinning using uniconazole treatment followed by ABA treatment. The present invention further provides in some embodiments methods for fruit coloring using uniconazole treatment followed by ABA treatment. The present invention further provides in some embodiments, methods of enhancing ABA levels by applying a combination of ABA and uniconazole.

Unexpectedly, the present invention discloses that pretreatment of a plant with uniconazole enhances the effect of exogenously applied ABA on plant endogenous processes depended on ABA, such that the amounts of exogenous ABA required for achieving a desired activity are significantly reduced. The findings of the present invention thus enable cost-effective use of ABA, hitherto not broadly applied in agriculture practices due to its high cost. The present invention is based in part on the unexpected finding that pretreatment of apple tress with uniconazole at the fruitlet level significantly enhances the efficiency of exogenously applied ABA. The use of uniconazole as a pretreatment to ABA application enables a more accurate and controlled fruitlet thinning. Furthermore, the methods disclosed herein, including the use of reduced amounts of ABA, reduce undesired activities of ABA such as abscission of leaves.

According to an aspect of the present invention there is provided a method of potentiating an abscisic acid (ABA) effect in a plant, the method comprising applying to the plant: (1) uniconazole, a derivative or an analog thereof; and (2) ABA, a derivative or an analog thereof.

According to some embodiments, the ABA effect is selected from the group consisting of fruit thinning, fruit coloring and fall defoliation.

According to some embodiments, the ABA effect is fruitlet thinning. According to some embodiments, the method enhances fruitlet thinning.

According to some embodiments, the ABA effect is coloring induction of the plant fruit. According to some embodiments, the ABA effect is anthocyanin production. According to these embodiments, the method results in enhanced anthocyanin production. According to some embodiments, the enhanced anthocyanin production results in enhancing a purple color of the plant fruit.

According to some embodiments, the method enhances the effect of the exogenously applied ABA in the plant.

According to some embodiments, the uniconazole and ABA are applied sequentially. According to some embodiments, the uniconazole is applied at least 1, 2, 3, 4, or 5 hours before ABA application. According to some embodiments, the uniconazole is applied at least 1, 2, 3, 4, or 5 days before ABA application. According to some embodiments, the uniconazole is applied between 1 and 5 hours before ABA application. According to certain embodiments, the uniconazole is applied between 1 and 5 days, between 2 and 5 days, or between 2 and 4 days before ABA application. According to certain embodiments, the uniconazole is applied between 2 and 3 days before ABA application.

According to certain embodiments, the uniconazole and the ABA are applied concomitantly. According to certain embodiments, the uniconazole and the ABA each is applied separately. According to other embodiments, the uniconazole and the ABA are applied together.

According to some embodiments, the ABA is being applied in an amount of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% lower than the amount needed to cause the same level of the at least one ABA effect in the absence of uniconazole. Each possibility represents a separate embodiment of the invention.

According to some embodiments, the plant is a deciduous fruit tree or vine.

According to certain embodiments, the plant is a fruit tree selected from the group consisting of apples, grapes, pears, peaches, plums, nectarines, cherries, apricots, avocados and kiwis. According to specific embodiments, the fruit tree is an apple tree. According to certain embodiments, the vine is a grape vine (Vids viniferd).

According to some embodiments, the plant is a fruit tree wherein fruit thinning is required to obtain commercially acceptable fruit number and return bloom the following year.

According to some embodiments, the uniconazole is applied after full bloom of the fruit tree.

According to some embodiments, the uniconazole is applied at the fruitlet stage. According to some embodiments, the uniconazole is applied at least one day, two days, three days, four days or five days after full bloom. According to some embodiments, the uniconazole is applied at least one week after full bloom.

According to some embodiments, the uniconazole is applied at a late fruitlet stage. According to some embodiments, the plant is an apple tree and the uniconazole is applied at a fruitlet stage of from 5 to 25 mm king fruit diameter.

According to certain embodiment, the uniconazole is uniconazole P. According to some embodiments, the uniconazole derivative is abscinazole-E3M.

According to some embodiments, the uniconazole is applied at a concentration ranging between 0.3 mg/L and 20 g/L.

According to some embodiments, the uniconazole is applied at a concentration ranging between 5 mg/L and 10 g/L. According to some embodiments, the uniconazole is applied at a concentration ranging between 0.1 g/L and 5 g/L. According to some embodiments, the uniconazole is applied at a concentration ranging between 0.5 g/L and 3 g/L. According to some embodiments, the uniconazole is applied at a concentration of about 1 g/L.

According to some embodiments, the ABA is S-ABA. According to additional embodiments, the ABA is 3'-methyl-(S)-abscisic acid or 3'-propargyl-(S)-abscisic acid.

Any formulation of ABA and uniconazole as is known for agricultural application can be used according to the teachings of the present invention.

According to some embodiments, the ABA is applied at a concentration of less than 10 g/L. According to some embodiments, the ABA is applied at a concentration of less than 8 g/L. According to some embodiments, the ABA is applied at a concentration of between 0.01 g/L and 5 g/L. According to some embodiments, the ABA is applied at a concentration of between 0.01 g/L and 1 g/L. According to some embodiments, the ABA is applied at a concentration of about 2 g/L. According to some embodiments, the ABA is applied at a concentration of about 1 g/L. According to some embodiments, the ABA is applied at a concentration of about 0.5 g/L. According to some embodiments, the ABA is applied at a concentration of 0.6 g/L.

According to some embodiments, the uniconazole is applied by aerosol spray, pressure spray, direct watering, and/or dipping.

According to some embodiments, the uniconazole is applied with an air blast sprayer.

According to some embodiments, the ABA is applied by aerosol spray, pressure spray, direct watering, and/or dipping.

According to some embodiments, the ABA is applied with an air blast sprayer.

According to some embodiments, the method further comprising applying an additional chemical agent that increase abscission.

Each of the active ingredients, uniconazole, ABA, or a combination thereof is preferably present in an agricultural composition. According to certain embodiments, the agricultural composition may further comprise agriculturally suitable auxiliaries, like solvents, carriers, surfactants or extenders.

According to an aspect, the present invention provides an effective amount of uniconazole, a derivative or analog thereof, for use in a method comprising administering ABA, a derivative or analog thereof to a plant, wherein the effective amount of uniconazole is selected from the group consisting of (i) an amount potentiating at least one effect associated with the administered ABA in the plant, (ii) an amount reducing the amount of exogenously administered ABA required to produce the effect, and (iii) an amount according to any one of (i) and (ii) wherein said amount is not associated with an undesired side effect of ABA.

According to some embodiments, the uniconazole is in a ready to use form. The uniconazole concentration is as described hereinabove. According to some embodiments, the uniconazole is at a concentration ranging between 0.1 mg/L and 20 g/L.

According to some embodiments, the effect of ABA is fruitlet thinning.

According to some embodiments, the effect of ABA is fruit coloring.

According to an additional aspect, the present invention provides an effective amount of ABA for use in a method of potentiating of at least one ABA effect in a plant, wherein the method comprising administering uniconazole to the plant, said effective amount of ABA being at least 10% lower than the amount needed to cause the same level of the at least one ABA effect in the absence of uniconazole.

According to some embodiments, the effective amount of ABA being at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% lower than the amount needed to cause the same level of the at least one ABA effect in the absence of uniconazole. Each possibility represents a separate embodiment of the invention.

According to some embodiments, the ABA is in a ready to use form.

The ABA concentration is as described hereinabove.

According to some embodiments, the ABA is at a concentration of less than 10 g/L.

According to some embodiments, the ABA effect is fruitlet thinning.

According to some embodiments, the ABA effect is fruit coloring.

According to an aspect, the present invention provides a combination of uniconazole, a derivative or an analog thereof and ABA, a derivative or an analog thereof.

The uniconazole and/or the ABA may be in a form of a powder, a granule or a ready-to-use solution.

According to a certain aspect, the present invention provides an agricultural composition comprising uniconazole, a derivative or an analog thereof and ABA, a derivative or an analog thereof.

According to some embodiments, the agricultural composition comprising more than 80%, 85%, 90%, or 95% water. According to some embodiments, the agricultural composition further comprises an agricultural acceptable carrier.

According to some embodiments, the agricultural composition further comprises additives, other thinning agents, growth regulators, and/or other agricultural active substances.

According to some embodiments, the ABA is formulated in a slow-release form. According to some embodiments, the ABA is encapsulated in a slow-release medium.

According to another aspect, the present invention provides a kit comprising (i) ABA, a derivative or analog thereof, and (ii) uniconazole, a derivative, or an analog thereof, the kit further comprising instruction material directing the use of the ABA and the uniconazole.

According to some embodiments, the ABA and the uniconazole are present in separate reservoirs or containers, optionally each with a carrier suitable for its administration.

According to some embodiments, the ABA and/or the uniconazole are in a ready to use form.

The ABA and the uniconazole are present in amounts as described hereinabove.

According to some embodiments, the uniconazole is at a concentration ranging between 0.3 mg/L and 20 g/L. According to some embodiments, the uniconazole concentration is about 1 g/L.

According to some embodiments, the instruction material directs the use of ABA and uniconazole for potentiating at least one effect associated with ABA activity in a plant.

According to some embodiments, the instructions material directs administering the uniconazole prior to administering the ABA.

It is to be understood that any combination of each of the aspects and the embodiments disclosed herein is explicitly encompassed within the disclosure of the present invention. Other objects, features and advantages of the present invention will become clear from the following description and drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIGs. 1A-1C. ‘Golden Delicious’ fruitlet survival under local conditions during two seasons (2014-2015). Fruitlets in an inflorescence are divided into categories based on their position in the inflorescence- king, big lateral (L3), medium laterals (L2) and small lateral (LI) (in 2014, L3 and L2 groups were followed as one group). Figures 1A-1B - Average percent fruitlet survival in 2014 and 2015. Figure 1C - Distribution of inflorescences based on number of persisting fruitlets (independent of position) in 2014 and 2015. Numbers are mean values of independent biological repeats (trees; n=5, 10 inflorescences per tree) ± standard error of the mean (bars). In Figures 1A and IB, different letters represent a significant difference in survival rate of the different fruitlet categories according to Tukey-Kramer HSD (P<0.05). In Figure 1C, asterisks represent a significant difference between the different years, according to student’s t test (P<0.05).

FIGs. 2A-2C. Fruitlet survival in two cultivars, ‘Ariane’ and ‘Golden Delicious’ in the same orchard in different years. Survival rate was assessed at the end of natural abscission. Figure 2A - Average percent fruitlet survival per fruitlet in 2015. Figure 2B - Distribution of inflorescences based on number of persisting fruitlets in 2015. Figure 2C - comparing ‘Golden Delicious’ (GD) to ‘Ariane’ in 2020. Average percent survival of LI fruitlets, with (L1WN) or without (LI A) neighbors, and of all fruitlets in the inflorescence. In Figures 2A-2B, Numbers are mean values of independent biological repeats (trees; ‘Golden delicious' n=5, 10 inflorescences per tree; ‘Ariane’ n=3, 12 inflorescences per tree) ± standard error of the mean (bars). In Figure 2C, means are values of 4 independent biological repeats. Asterisks represent a significant difference between the two cultivars, according to student’s t test (P<0.05). In Figure 2A different letters represent a significant difference is survival rate of the different fruitlet categories of 'Ariane' according to Tukey-Kramer HSD statistical analysis performed on ranked data. A similar analysis for 'Golden Delicious’ is shown in Figure IB.

FIGs. 3A-3B. Identifying the time point when the fate of the ‘Golden Delicious’ LI fruitlet to abscise is irreversible. The effect of removing all other fruitlets on LI survival (measured at the end of natural abscission). All other flowers/fruitlets were removed at different dates. Figure 3A, experiment in 2014. Figure 3B, experiment in 2015. For each time point, 5 inflorescences from each tree (5 trees) were randomly chosen, all in the same phenological stage. Numbers are mean values of independent biological repeats (trees) ± standard error of the mean (bars). Different letters represent a significant difference in survival rate between the different removal dates to the control (LI in an intact inflorescence; LI with neighbors; L1WN), according to Dunnett’s method (P<0.05).

FIGs. 4A-4C. Molecular markers for LI fruitlets abscission / survival potential ‘Golden Delicious’ (2014). Expression level of molecular markers that distinguishing early on between LI with neighboring fruit (L1WN) and LI alone (L1A). Samples were taken at 4,7,11 DAFB. Figure 4A - MdACOl, Figure 4B - MdCDKl-2, Figure 4C - MdCDK2-2. Relative gene expression was measured using quantitative real-time RT-PCR, using as housekeeping a histone H3-encoding gene (see materials and methods). Numbers are mean values of independent biological repeats (3 trees) ± standard error of the mean (bars). Asterisks represent a significant difference between the different fruitlets in a specific time point, according to student’s t test (P<0.05).

FIGs. 5A-5B. Venn diagrams comparing up regulated genes, in abscising fruitlets identified in different experiments. Figure 5A - Venn diagram comparing up regulated genes with an expression ratio of at least 1.5-fold change between different kinds of ‘towards abscising’ and ‘surviving’ apple fruitlets, identified in four different experiments. (I) king versus L2 fruitlets from the same inflorescence, at 20 DAFB (Ferrero et al., 2015), samples were analyzed using RNAseq; (II and III) king versus LI fruitlets from the same inflorescence at 15 DAFB (Botton et al., 2011), separated into two tissues- (II cortex and (III) seed, samples were analyzed using microarray; (IV) current RNAseq data, L1WN versus LI A fruitlets, at 7 DAFB. 171 genes were commonly up regulated in abscising fruitlets in all four datasets. In Figure 5B these 171 genes were compared to 1982 genes significantly induced at 11 DAFB by uniconazole treatment to L1A fruitlets, compared to mock treated L1A fruitlets. 66.6% of the commonly upregulated genes were also induced by uniconazole. Analysis was preformed using ‘Venny- An interactive tool for comparing lists with Venn's diagrams’ (bioinfogp . cnb . c sic . es/tools/venny/) .

FIG. 6. BAR analysis of Arabidopsis genes encoding proteins similar to Apple genes that appear to be involved in fruitlet abscission. The identified 3414 Apple differently expressed genes (DEGs; FPKM ratio of at least 1.5-fold change and statistically significant at 7 DAFB) were divided into 2 groups: significantly higher in El with neighbors at 7 DAFB (Genes induced in Apple fruitlet abscission) and Genes significantly lower in LI with neighbors (Genes repressed in Apple Fruitlet Abscission). Their homologs were identified in Arabidopsis and the response of the Arabidopsis genes to different stimuli was studied using the ‘BAR expression angler’ (http://bar.utoronto.ca/affydb/cgi-bin/affy_db_exprss_browser_in.cgi). Here it is shown that the response of these Arabidopsis genes to uniconazole (GA biosynthesis inhibitor) after 3 or 12 hours. Some genes were induced by uniconazole (Green, bottom part of the column), some repressed (Red; upper part of the column) and some were unaffected by uniconazole (White; middle). Two groups of Arabidopsis genes were also added (groups A and B) that encode proteins homologous to Apple genes for which expression was not altered in fruitlet abscission. After 12 hours of Arabidopsis seedling exposure to uniconazole, -30% of these genes were induced by uniconazole and 23% were repressed by uniconazole. Within the group of genes for which expression is affected in apple by abscission, the ratio is altered compared to controls (asterisks) suggesting a link between uniconazole treatment in Arabidopsis and a tendency to abscise in Apple fruitlets.

FIGs. 7A-7C. Uniconazole treatments causing abscission of fruitlets in apple ‘Golden Delicious’ fruitlets at Matityahu in 2017. 3 DAFB flowers were treated with 1 gr/L uniconazole P (2% Magic), 0.025% Triton X-100 surfactant (Sigma-Aldrich, St. Louis, MO. USA) or with mock (0.025% Triton X-100). Figure 7A - Treatment of King and L3 flowers. Figure 7B - Treatment of whole inflorescences. Figure 7C - Treatment of LI alone flowers. Asterisks represent significant difference between Control and Treatment in a specific fruitlet according to student’s t test (* P<0.05, ** P<0.01).

FIGs. 8A-8E. GAI, GA3 and ABA internal levels in apple ‘Golden Delicious’ fruitlets of King, LI With Neighbors (L1WN) and LI Alone (L1A) in years 2016 and 2018. 11 DAFB fruitlet samples were examined by High Performance Liquid Chromatography and tandem mass spectrometry (HPLC-MS), comparing King, LI of intact inflorescences (L1WN) and LI of inflorescences where only one flower was left (L1A). Compounds measured are GAI (Gibberellin 1) in Figure 8 A, GA3 (Gibberellin 3) in Figure 8B and cis-Abscisic acid (ABA) in Figure 8C. Data, based on of 3 biological repeats (trees), is represented by a ‘box and whiskers’ plot in which the lower and upper boundaries of the boxes denotate the first and third quartiles, respectively. The second quartile is also the median flowering percentage and is shown as a line in the center of the box. The mean value is marked with an “x”. Maximum and minimum values are denoted by the lines above and below the box with outliers shown as individual dots extending beyond the lines. Asterisks represent a statistical difference between L1WN fruitlets and other fruitlet types of the same year according to a student's t test (* P<0.05, ** P<0.01). Figure 8D - LI With Neighbors (L1WN) and LI Alone (L1A) fruitlet survival in ‘Golden Delicious’ and ‘Ariane’ fruitlets in 2020 in Matityahu. Figure 8E - ABA internal levels in apple ‘Golden Delicious’ and ‘Ariane’ fruitlets of LI With Neighbors (L1WN) and LI Alone (L1A) in 2020. 12 DAFB fruitlet samples from the fruit described in Figure 8D were examined by High Performance Liquid Chromatography and tandem mass spectrometry (HPLC-MS), for cis-Abscisic acid (ABA) levels. Averages of 4 biological repeats in Figures 8D and 8E.

FIG. 9. MdNCED3a (Md05g 1207300) gene expression analysis in apple ‘Golden Delicious’ and ‘Ariane fruitlets’, in years 2014, 2015, 2016 and 2018. Gene expression level comparing LI with neighbors (L1WN) and LI alone (L1A) from ‘Golden Delicious’ (2014, 2015, 2016 and 2018) and ‘Ariane’ (2015 and 2016) at indicated days after full bloom. Relative gene expression was measured by quantitative real-time PCR (RT- qPCR), using as housekeeping a histone H3-encoding gene. Bars are mean values of independent biological repeats (3-5 trees), error bars are Standard Error. Asterisks represent significant difference between L1WN and L1A at the specific timepoint in the specific cultivar, according to student’s t test (* P<0.05, *** P<0.001).

FIG. 10. Chemical induction of abscission in apple ‘Anna’ fruitlets at Pethahia, Israel, in 2019. 21-30 DAFB fruitlets were treated with the indicated chemical or with mock (Triton 0.025% and DMSO 0.8%).

FIG. 11. Chemical induction of abscission in K (King) and L3 (Lateral 3) in apple ‘Cripps Pink’ fruitlets in Merom Golan in 2019. 9 DAFB fruitlets were treated with the indicated chemical or with mock (Triton 0.025% and DMSO 0.8%). Average of biological repeats (trees; n=3, 16 inflorescences treated per treatment and tree) is presented and error bars represent Standard Error. Letters mean statistical difference between control and treatments of the indicated type of fruitlet according to Tukey-Kramer HSD (Red for K and L3, Blue for L2s and LI) (P<0.001 in K and L3 fruitlets and P<0.05 in L2s and LI fruitlets). Data was analyzed by one-way analysis of variance (ANOVA) using JMP Pro version 14 software (SAS Institute, Cary, NC, USA).

FIGs. 12A-12D. Fruitlet survival percentage (Figure 12 A) and Percentage of persisting fruitlets (Figure 12B), average fruit weight at harvest (Figure 12C) and distribution of fruit diameter (Figure 12D) in apple ‘Top Red’ at Matityahu in 2020. Average of biological repeats (trees; n=5) is presented, survival is based on 30 inflorescences per tree. Final fruit weight and size distribution is based on all fruits on the tree at harvest. Different letters in Figures 12A and 12B indicate a significant difference between treatments according to Tukey-Kramer HSD test (P<0.05). If the data were not normal and/or hetero scedastic, the statistical tests were conducted on transformation to ranks of the values. Data were analyzed by one-way analysis of variance (ANOVA) using JMP Pro version 14 software (SAS Institute, Cary, NC, USA).

FIGs. 13A-13C. Fruitlet survival percentage (Figure 13A), percentage of persisting fruitlets (Figure 13B) and average fruit weight at harvest (Figure 13C) in apple ‘Top Red’ at El-Rom in 2020. Average of biological repeats (trees; n=5) is presented, 30 inflorescences per tree were measured in Figures 13A-13B. All fruits on the tree were measured and shown in Figure 13C. For multiple comparisons, different letters indicate a significant difference between treatments according to Tukey-Kramer HSD test (P<0.05). If the data were not normal and/or hetero scedastic, the statistical tests were conducted on transformation to ranks of the values. For one comparison, asterisks represent significant difference between control and treatment according to t Test **(P<0.01). Data were analyzed by one-way analysis of variance (ANOVA) using JMP Pro version 14 software (SAS Institute, Cary, NC, USA).

FIGs. 14A-14B. Fruitlet survival percentage (Figure 14 A) and percentage of fruitlets with additional abscission (Figure 14B) after late treatment in apple ‘Golden Delicious’ and ‘Ariane’ at Matityahu in 2020 after very late treatments. One repeat per treatment was treated, except for treatment with 1 gr/L uniconazole P (2% Magic) and 0.6 gr/L S- ABA (0.3% ProTone SG), that included 2 repeats.

FIG. 15. Anthocyanin levels in grapes as calculated based on light emissions measured using a portable fluorometer (Multiplex III). Crimson Seedless grape clusters were treated at the ‘veraison’ stage with different combinations of chemicals, starting with Ethephon on the 18th of August 2020, followed by Uniconazole-P on August 24th, and ProTone on August 26th. Clusters were harvested on September 17th 2020 measured for color accumulation using a portable fluorometer (Multiplex III, Force A, France; Bahar et al., 2012). The Fluorometer measures Far-Red light emission after excitation with Red or Green light and the Fog of the Florescence excitation ratio (FER_RG) was used to calculate quantity of Anthocyanins (ANTH). Measurements were conducted for 10 berries in each cluster and the average ANTH was calculated, 3-6 clusters were measured per treatment.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods of potentiating effects of abscisic acid (ABA) by pretreatment with uniconazole. The ABA may be applied hours or days after the uniconazole.

The inventors of the present invention identified early changes in gene expression that will later lead to fruitlet abscission. The gathered information helped to better understand the initial processes occurring in fruitlets that are destined to abscise. The data was used to develop molecular markers that help predict the level of natural abscission. The information obtained allowed to develop a novel combination of chemicals, uniconazole and ABA, that together achieve robust and inexpensive fruitlet thinning.

According to an aspect of the present invention there is provided a method of enhancing abscisic acid (ABA) levels in a plant, the method comprises the step of applying to the plant: (1) uniconazole, a derivative or an analog thereof; and (2) ABA, derivative, or an analog thereof.

According to an aspect of the present invention there is provided a method of potentiating abscisic acid (ABA) effect in a plant, the method comprising applying to the plant: (1) uniconazole, a derivative or an analog thereof; and (2) ABA, a derivative or an analog thereof.

Definitions

The term "Abscisic acid" as used herein also includes salts and derivatives (collectively referred to as "ABA"). The term includes a racemic mixture of both isomers S-ABA and R-ABA as well as S-ABA isomer only. The ABA structure is as follows:

“Uniconazole” is a triazole chemical used as a plant growth retardant. It is an inhibitor of specific P450 enzymes. It is active on a wide range of plants and its most known activity is inhibiting the production of gibberellins by inhibiting P450 ent-kaurene oxidase (CYP701), an enzyme in gibberellin biosynthesis. The term uniconazole also includes salts and analogs thereof. Uniconazole has the structure (Formula I):

The term “potentiating”, when refers to ABA effect, means increasing or strengthening at least one ABA effect in a plant compared to the ABA effect in a corresponding plant that has not received treatment with uniconazole. According to certain exemplary embodiments, potentiation of ABA effect is assessed by quantitative measure of an ABA-related process or phenomenon, e.g., fruitlets abscission.

The term "derivative" or "analog" in the context of ABA or uniconazole refers broadly to a substance that contains the same basic chemical “skeleton” and functionality as the parent compound, but further has one or more modifications. The derivative or analog share part or all of the biological activity of the parent compound. Uniconazole derivatives are known in the art. For example, uniconazole derivatives are described in Takeuchi, J., et al. (2016. Scientific Reports 6: 37060).

According to some embodiments, the uniconazole derivative is abscinazole-E3M which has the structure (Formula II):

According to some embodiments, the uniconazole derivative is represented by the following structure (Formula III):

AbrfZB (2)

According to some embodiments, the uniconazole derivative is represented by the following structure (Formula IV):

The term “fruitlet thinning” as used herein refers to the action of removing individual fruits or flowers to achieve a preferred quantity of fruits. The desired fruit quantity may be several fruits but also zero or one fruit in order to increase yield in the following season(s). The action of removing individual fruits or flowers can be a direct action (e.g., hand-removal by men labor) or indirect (i.e., by applying chemical thinners).

Full bloom is defined herein as the time when all flower buds on a flowering plant have developed into mature flowers. A full bloom in apples is defined herein as a day within an apple orchard in which the majority of inflorescences within a cultivar have already open (reaching anthesis) king, L3 and L2 flowers while most LI flowers are at a pre-anthesis ‘balloon’ stage.

The terms 'king fruitlet' or “king fruit” are used herein interchangeably and refer to the main, centrally positioned fruitlet in the apple flower cluster.

All plants and plant parts can be treated in accordance with the invention. Plants are understood here to mean all plants and plant populations, such as desired wild plants or crop plants (including naturally occurring crop plants). Crop plants may be plants which can be obtained by conventional breeding and optimization methods or by biotechnological and genetic engineering methods or combinations of these methods, including the transgenic plants and including the plant cultivars which are protectable and non-protectable by plant breeders' rights. Plant parts are understood to mean all parts and organs of plants above and below the ground, such as shoot, leaf, flower and root, examples of which include leaves, needles, stalks, stems, flowers, fruit bodies, fruits and seeds, and also roots, tubers and rhizomes.

According to certain embodiments, the uniconazole and the ABA are applied separately. According to other embodiments, the uniconazole and the ABA are applied together within a single agricultural composition.

According to some embodiments, the ABA is applied at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours after treating with uniconazole. According to some embodiments, the ABA is applied at least 1, 2, 3, 4, or 5 days after treating with uniconazole.

According to some embodiments, the uniconazole is applied after full bloom of the plant.

According to an aspect, the present invention provides a method of potentiating abscisic acid (ABA) effect in a plant, the method comprising applying to the plant: (1) an agricultural composition comprising uniconazole, a derivative or an analog thereof; and (2) an agricultural composition comprising ABA, a derivative or an analog thereof.

According to some embodiments, the present invention provides an agricultural composition comprising: (1) uniconazole, a derivative or an analog thereof; and (2) ABA, a derivative or an analog thereof.

The active ingredient according to the inventions, i.e. uniconazole and ABA or analogs or derivatives thereof, may be present in an agricultural composition.

According to some embodiments, the uniconazole and the ABA are present in separate agricultural compositions.

According to specific embodiments, the uniconazole and the ABA are present in the same agricultural composition.

According to some embodiments, the agricultural composition comprising more than 80%, 85%, 90%, or 95% water.

According to some embodiments, the agricultural composition comprises agriculturally suitable auxiliaries, like solvents, carriers, surfactants or extenders.

According to some embodiments, the agricultural composition comprises surfactant. According to specific embodiments, the agricultural composition comprises TRITON™ X-100 Surfactant.

According to some embodiments, the agricultural composition comprises additives, other thinning agents, growth regulators, foliar fertilizers and other agricultural active substances.

According to some embodiments, the carrier is a natural or synthetic, organic or inorganic substance with which the active ingredients are mixed or combined for better applicability, in particular for application to plants or plant parts. The carrier is generally inert and should be suitable for use in agriculture.

The active ingredients can be applied as such or in the form of formulations, such as ready-to-use solutions, emulsions, water- or oil-based suspensions, wettable powders, pastes, soluble powders, soluble granules, etc. Application is accomplished in a customary manner, for example by spraying, watering, atomizing, dusting, spreading-on and the like.

The active agents according to the invention may be present as such or in their (commercial) formulations and in the use forms prepared from these formulations as a mixture with other (known) active ingredients, such as insecticides, attractants, sterilants, bactericides, acaricides, nematicides, growth regulators, herbicides, and/or fertilizers.

According to some embodiments, the uniconazole is applied at a concentration ranging between 0.3 mg/L and 20 g/L. According to some embodiments, the uniconazole is applied at a concentration ranging between 0.5 mg/L and 15 g/L. According to some embodiments, the uniconazole is applied at a concentration ranging between 100 mg/L and 10 g/L. According to some embodiments, the uniconazole is applied at a concentration ranging between 200 mg/L and 8 g/L. According to some embodiments, the uniconazole is applied at a concentration ranging between 0.5 g/L and 20 g/L. According to some embodiments, the uniconazole is applied at a concentration ranging between 0.5 g/L and 15 g/L. According to some embodiments, the uniconazole is applied at a concentration ranging between 0.5 g/L and 10 g/L. According to some embodiments, the uniconazole is applied at a concentration of about 1 g/L.

According to some embodiments, the uniconazole is applied at a concentration ranging between 0.5 g/L and 5 g/L.

According to some embodiments, the uniconazole is applied at a concentration ranging between 0.005% and 5%. According to some embodiments, the uniconazole is applied at a concentration ranging between 0.02% and 4%. According to some embodiments, the uniconazole is applied at a concentration ranging between 0.05% and 3%. According to some embodiments, the uniconazole is applied at a concentration ranging between 0.1% and 3%. According to some embodiments, the uniconazole is applied at a concentration ranging between 0.5% and 3%. According to some embodiments, the uniconazole is applied at a concentration ranging between 1% and 3%. According to some embodiments, the uniconazole is applied at a concentration ranging between 1.5% and 2.5%. According to some embodiments, the uniconazole is applied at a concentration ranging between 0.05% and 1%.

According to additional embodiments, the amount of ABA being 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10% of the amount required to cause the same level of the at least one ABA effect in the absence of uniconazole.

According to some embodiments, the ABA is S-ABA.

According to some embodiments, the ABA is applied at a concentration of less than 10 g/L. According to some embodiments, the ABA is applied at a concentration of less than 9 g/L. According to some embodiments, the ABA is applied at a concentration of less than 8 g/L. According to some embodiments, the ABA is applied at a concentration of less than 7 g/L. According to some embodiments, the ABA is applied at a concentration of less than 6 g/L. According to some embodiments, the ABA is applied at a concentration of less than 5 g/L. According to some embodiments, the ABA is applied at a concentration of less than 4 g/L. According to some embodiments, the ABA is applied at a concentration of between 0.1 g/L and 4 g/L. According to some embodiments, the ABA is applied at a concentration of between 0.2 g/L and 3 g/L. According to some embodiments, the ABA is applied at a concentration of between 0.2 g/L and 1.5 g/L. According to some embodiments, the ABA is applied at a concentration of between 0.3 g/L and 1 g/L. According to some embodiments, the ABA is applied at a concentration of about 1 g/L. According to some embodiments, the ABA is applied at a concentration of about 0.5 g/L. According to some embodiments, the ABA is applied at a concentration of about 0.6 g/L.

According to some embodiments, the ABA is applied at a concentration of less than 1%. According to some embodiments, the ABA is applied at a concentration of less than 0.9%. According to some embodiments, the ABA is applied at a concentration of less than 0.8%. According to some embodiments, the ABA is applied at a concentration of less than 0.7%. According to some embodiments, the ABA is applied at a concentration of less than 0.6%. According to some embodiments, the ABA is applied at a concentration of less than 0.5%. According to some embodiments, the ABA is applied at a concentration of between 0.01% and 2%. According to some embodiments, the ABA is applied at a concentration of between 0.02% and 1%. According to some embodiments, the ABA is applied at a concentration of between 0.03% and 0.5%. According to some embodiments, the ABA is applied at a concentration of between 0.04% and 0.06%. According to some embodiments, the ABA is applied at a concentration of about 0.05%.

The percentage (%) of uniconazole and ABA when refers to their concentration can be by volume (e.g., gr/L) or by weight (e.g., gr/gr). The concentration may be calculated as amount in volume, for example, when the compound is in solution or by weight, for example, when the compound is administered as powder.

The active agents and composition as described herein can be applied by any methods typically used in the agricultural industry for the application of chemicals. According to some embodiments, the agricultural composition is applied by aerosol spray, pressure spray, direct watering, and/or dipping.

According to some embodiments, the composition is applied by common spraying techniques used in the agricultural industry.

According to some embodiments, the amount of the agricultural composition comprising the uniconazole used for application of the fruitlet thinning according to the invention is from 1,000 to 2,000 liter per hectare of the orchard.

EXAMPLES

Materials and Methods

Plant material

The experiments were conducted over 7 years (2014, 2015, 2016, 2017, 2018, 2019 and 2020) with ‘Golden Delicious’, ‘Ariane’, ‘Cripps Pink’ ‘Anna’ and 'Top red' apple (Malus x domestica Borkh.) cultivars, as well as ‘Crimson Seedless’ table grape (Vitis I’inifera). ‘Golden Delicious’, ‘Ariane’ and some ‘Top Red’ trees were in a commercial apple orchard at the ‘Matityahu’ research station of the Agricultural Research Organization (ARO) in Northern Israel (33°04'04"N, 35°27'04"E, altitude 667 m). Other ‘Top Red’ trees were in a commercial apple orchard at El-Rom (33°10'18.4"N, 35°48'09.7"E). ‘Cripps Pink’ trees were in a commercial orchard in the Golan Heights (33°07'57.1"N 35°48'16.0"E, altitude 940 m). ‘Anna’ trees were in a commercial orchard in Petahia (31°50'52.5"N 34°54'08.8"E, altitude 166 m). ‘Crimson Seedless’ vines were in a commercial orchard in Pedaya (31°50'59.6"N 34°53'12.6"E, altitude 99 m). ‘Golden Delicious’ trees are grafted on ‘M9’ rootstock, planted in 1997. ‘Ariane’ trees are grafted on two types of rootstocks, ‘Mains’ and ‘MM106’, planted in a young introduction plot in 2012. ‘Cripps Pink’ trees are grafted on ‘106’ rootstock, planted in 2013. ‘Top Red’ trees at Matityahu are grafted on ‘M9 rootstock, planted in 2000. ‘Top Red’ trees at El- Rom are grafted on ‘ 106’ rootstock, planted in 2012. ‘Crimson Seedless’ vines are grafted on ‘Richter 110’ rootstock. All trees were grown and pruned according to commercial practice.

Trees were selected in the spring based on their level of flowering. For most experiments, selected trees were with relatively high and uniform flowering. The date of Full Bloom (DAFB) is the day within an apple orchard in which the majority of inflorescences within a specific cultivar have already open (reaching anthesis) king, L3 and L2 flowers, while most LI flowers are at a preanthesis ‘balloon’ stage’. DAFB for ’Golden Delicious’ was 6, 9, 19, 3 and 9 April in 2014, 2015, 2017, 2018 and 2020 respectively. In 2016, DAFB was 31 March. The date of full bloom in ‘Anna’ was 21 February 2019. The date of full bloom in ‘Cripps Pink’ was 14 April 2019. The date of full bloom in ‘Top Red’ full bloom was 14 and 20 April 2020 at Matityahu and El-Rom orchards, respectively. The date of full bloom in ‘Ariane’ was 09 April 2020.

LI with neighboring fruit, LI alone and King experiments for gene expression and hormone analysis

For LI with neighboring fruit, LI alone and King experiment, inflorescences at phenological stage II at full bloom were chosen randomly at both sides of the tree. King flowers were marked with a ring and LI flower was either marked with a ring (LI with neighboring fruit) or all the rest of the flowers of the inflorescence were removed (LI Alone). Samples (fruitlet without crown and pedicel) were collected at the same time of the day at different dates, removed with secateurs, placed immediately in liquid nitrogen and stored at -80 °C until further analysis. Each sample consisted of 5 flowers/fruitlets in 2014, 2015 and 2016 and 10 fruitlets in 2018.

‘Golden Delicious’ at 2014: 3 trees, strongly flowering, 40 inflorescences per tree, samples were collected at 11 DAFB. ‘Golden Delicious’ at 2015, 4 trees, strongly flowering, 60 inflorescences per tree, samples were collected at 9 and 11 DAFB. ‘Ariane’ at 2015: 3 trees, medium flowering, 30 inflorescences per tree, samples were collected at 11 DAFB. ‘Golden Delicious’ at 2016: 3 trees, strongly flowering, 40 inflorescences per tree, samples were collected at 9 and 11 DAFB. ‘Ariane’ at 2016: 3 trees, medium flowering, 20 inflorescences per tree at 11 DAFB. ‘Golden Delicious’ at 2018: 5 trees, strongly flowering, 20 inflorescences per tree, samples were collected at 12 DAFB.

Fruitlet samples of El ‘Golden Delicious’ with neighboring fruit (L1WN) and LI alone (LI A) from 4,7,11 DAFB of the year 2014 were used for the analysis. L1WN and L1A samples from 7 DAFB were sequenced in 3 biological repeats from 4, 11 DAFB, 1 biological repeat.

Bioinformatic analysis of published data

Previous studies have attempt at characterizing molecular events leading to apple fruitlet abscission, using RNAseq and microarray (Botton et al., 2011, Eccher et al., 2013, Ferrero et al., 2015). Each of these experiments compared different kinds of surviving vs. towards-ab seising apple fruitlets. Here, the raw data of these experiments were analyzed, and filtered differentially expressed genes (DEGs) that have an expression ratio of at least 1.5 FC between ‘towards abscising’ and ‘surviving’ fruitlets.

One group compared king to LI fruitlets from the same inflorescence at 15 DAFB (Botton et al., 2011, Eccher et al., 2013). They separated the fruitlet into two tissues- cortex and seed, samples were analyzed using microarray. Cortex comparisons identified 5,157 DEGs while seed comparisons identified 10,142 DEGs.

Another group compared seeds of king and L2 fruitlets from the same inflorescence, at 20 DAFB (Ferrero et al., 2015), samples were analyzed using RNAseq and from this data the present analysis identified 2,322 DEGs (based on three biological repeats).

Illumina

Transcriptome libraries were prepared using the Illumina TruSeq RNA library preparation kit Of Illumina (Illumina #RS- 122-2001), according to the manufactures’ recommended protocol, starting with around 3 pg of total RNA. The amplified indexed libraries were quantified using Invitrogen Qubit fluorometer and equally pooled according to pool design. Pooled libraries were run on a 4% agarose gel and DNA around 270 bp (the length of RNA inserts plus the 3' and 5' adaptors) was size selected and recovered in 15 pL elution buffer (QIAGEN). Size selected libraries were then quantified again using the Qubit Fluorometer. Size was verified using the High Sensitive DNA gels on Agilent 2200 TapeStation instrument.

Libraries were sequenced on NextSeq 500 sequencer (Illumina, San Diego, CA, USA) using the NextSeq 500 High Output V 1 sequencing Kit (FC-404-2005), in a paired- end configuration, reading 40 bases from each direction (40 bp paired-end reads) derived from fragments with an average size of 263 bp.

Transcriptome analysis

Analysis was performed by Prof. Hector Anton-Candela, Miguel Hernandez University of Elche, Spain. The quality of the reads was evaluated using FastQC, and the reads were processed using Trimmomatic v.0.32 (Bolger et al., 2014) for trimming lower quality ends and any remaining adaptor sequences that might be present. The reference genome sequence (in FASTA format) for the Malus x domestica genome vl.O was downloaded from the Phytozome portal (phytozome.jgi.doe.gov/pz/portal.html) (Goodstein et al., 2012). To determine gene expression levels, the Tophat/Cufflinks pipeline using a previously described protocol (Trapnell et al., 2012) was used as follows: The reads were first aligned to the genome sequence using the TopHat v.2.0.13 split aligner (Trapnell et al., 2009) running with Bowtie2 v.2.2.5 (Langmead & Salzberg, 2012) and specifying the exon boundaries with the -G option. For this, a file in gff3 format downloaded from Phytozome (Mdomestica_196_vL0.gene_exons.gff3) was used. Transcripts were then assembled using Cufflinks and Cuffmerge, and their differential expression was assessed with Cuffdiff (Trapnell et al., 2012). A total of 73,673 genes were identified and FPKM values (fragments per kilobase of transcript per million mapped reads) were calculated.

Differently expressed genes (DEGs) between LI fruitlets with or without neighbors at 7 DAFB were identified. Since there were 3 biological repeats for each condition, the criteria for a DEG was that the average FPKM levels for the three repeats were at least 1.5 fold higher/lower between treatments and that the FPKM levels between treatments were significantly different as determined by Cuffdiff using the ‘pooled’ dispersion estimation method. 3414 DEGs were identified, including 2815 previously annotated (MDPOOOOxxxx) and 599 new genes/transcripts defined by the Tophat/Cufflinks pipeline. Of the 2815 previously annotated genes, 1959 DEGs were higher in fruitlets with neighbors, and 856 DEGs were lower in fruitlets with neighbors.

The annotation of the genome was retrieved from the Phytozome portal as a text file (Mdomestica_196_vL0.annotation_info.txt). This file includes information on sequence motifs, Gene Ontology terms assigned to each gene, as well as on their best hits in the Arabidopsis genome.

Transcriptome analysis using published data

Previous studies compared different kinds of surviving vs. towards abscising apple fruitlets. One group used a 30K microarray technique, with 30,419 experimental probes in random triplicates (Botton et al., 2011, Eccher et al., 2013). In the present application the raw data of this experiment (http://www.ebi.ac.uk/arrayexpress/arrays/A-MEXP- 1852/) was analyzed, and filtered out probes that could not be verified by their presence in the Malus x domestica genome vl.0. Probes homologous to a gene in the database were renamed using the current ‘MDPOOOOxxxx’ nomenclature.

Four samples from their experiment collected from fruitlets 15 days after petal fall (DAPF) were compared. Two types of fruitlets: large king versus LI, and two types of tissue: seed versus cortex. Differently expressed genes (DEG) between the two types of fruitlets were looked for, based on at least 1.5-fold change in expression. The cortex samples identified 5157 DEGs, while the seed samples identified 10142 DEGs.

Another group (Ferrero et al., 2015) compared RNA from seeds of king and L2 fruitlets, at 20 DAFB in three biological repeats, using Illumina (HiSeq 2000) RNAseq (http://www. ncbi.nlm.nih.gov/geo/query/acc. cgi?acc=GSE62415). Their raw data was used here to find DEGs that have an expression ratio of at least 1.5-fold difference in expression between fruitlet types (based on three biological repeats), and 2322 DEGs were identified.

Vennv analysis

The publicly available software ‘Venny’ was used. It is an interactive tool for comparing lists with Venn's diagrams (http://bioinfogp.cnb.csic.es/tools/venny/). This software allows the sorting of DEGs which were unique or common to different experiments.

BAR analysis

The annotated DEGs were sorted for those that encode a protein homologous to an Arabidopsis protein: 1791 DEGs were higher in fruitlets with neighbors, and 793 DEGs were lower in fruitlets with neighbors. The question how the Arabidopsis genes, encoding these proteins, respond to different stimuli was asked. The ‘BAR expression angler’ (http://bar.utoronto.ca/affydb/cgi-bin/affy_db_exprss_browser_in.cgi) was used for this analysis (Toufighi et al., 2005). The software allows to scan a large set of Arabidopsis genes for data on their expression in response to many different treatments and conditions, such as stress, hormone, different tissues and more.

Hormone analysis

For experiments following different hormones, three samples were analyzed per repeat and year (LI Alone, LI with neighboring fruit and King) from 2016 and 2018. Tissue was grinded and lyophilized and hormone measurement analysis was performed by the Plant hormone profiling service at the National Research Council (Canada). In experiments comparing ABA levels in LI fruitlets of cultivars ‘Golden Delicious’ and ‘Ariane’ four repeats were used per treatment, ABA analysis was conducted at the Volcani Center Metabolomic Unit, Israel

Chemicals and Calibration Curves

ABA (A4906, CAS#21293-29-8) was purchased from Sigma- Aldrich (Sigma- Aldrich, St. Louis, MO. USA) and GAs 1 and 3 were purchased from OlChemim Ltd. (Olomouc, Czech Republic). Deuterated forms of the hormones were used as internal standards: d4-ABA was synthesized and prepared at NRCC SK as previously described (Abrams et al., 2003, Zaharia et al., 2005) and d2-GAs 1 and 3 were purchased from OlChemim Ltd. (Olomouc, Czech Republic). Calibration curves were created for all compounds of interest. Quality control samples (QCs) were run along with the tissue samples.

Instrumentation

Analysis was performed on a UPLC/ESLMS/MS utilizing a Waters ACQUITY UPLC system, equipped with a binary solvent delivery manager and a sample manager coupled to a Waters Micromass Quattro Premier XE quadrupole tandem mass spectrometer via a Z-spray interface. MassLynx™ and QuanLynx™ (Micromass, Manchester, UK) were used for data acquisition and data analysis.

Hormone quantification bv HPLC-ESI-MS/MS

The procedure for quantification of ABA and gibberellins in plant tissue was performed using a modified procedure (Lulsdorf et al., 2013).

Briefly, the analyses utilize the Multiple Reaction Monitoring (MRM) function of the MassLynx v4.1 (Waters Inc) control software. The resulting chromatographic traces are quantified off-line by the QuanLynx v4.1 software (Waters Inc) wherein each trace is integrated and the resulting ratio of signals (non-deuterated/internal standard) is compared with a previously constructed calibration curve to yield the amount of analyte present (ng per sample). Calibration curves were generated from the MRM signals obtained from standard solutions based on the ratio of the chromatographic peak area for each analyte to that of the corresponding internal standard. The QC samples, internal standard blanks and solvent blanks were also prepared and analyzed along each batch of tissue samples.

RNA extraction and first-strand cDNA synthesis

Total RNA was extracted using the modified Cetyltrimethylammonium bromide method (White et al., 2008). RNA concentration was determined in a spectrophotometer (ND-1000, NanoDrop Technologies, Rockland, DE, USA). mRNA was Isolated from 7.5 pg of total RNA using magnetic oligo-dT beads (Dynabeads Oligo (dT) 25, (Thermo Fisher Scientific, Waltham, Massachusetts, USA)) according to manufacturer’s instructions. First-strand cDNA was synthesized using SuperScript II Reverse Transcriptase (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and Oligo (dT) 12-18 primers.

Expression analysis by quantitative real-time PCR

Analysis of cDNA samples was performed using the AB solute Blue QPCR ROX Mix (Thermo Fisher Scientific, Waltham, Massachusetts, USA) or qPCRBIO Fast qPCR SyGreen Blue Mix Lo-ROX (PCR Biosystems Ltd., London, UK). Reactions were run on a Rotor-Gene 6000 cycler (Corbett Life Science, Sydney, Australia) or qTOWER³ real-time thermal cycler (Analytik Jena AG, Jena, Germany), in two/three technical repeats. Quantification of each gene was performed using Corbett Research Rotor-Gene Software or qPCR soft software from Jena as previously described (Teper-Bamnolker & Samach, 2005).

Primers for quantitative real-time PCR

For each gene of interest, internal quantitative real-time PCR primers were designed. At least one of the real-time primers used spanned an exon-exon border, in order to assure cDNA amplification only. Each reaction was subjected to melting-point analysis to confirm that only a single product was amplified. Relative expression per sample was measured by comparing gene expression of NCED3 gene (MDP0000228070) to expression of the housekeeping Histone H3 encoding gene, (MDP0000263445), used by others (Kotoda et al., 2006, Mimida et al., 2009). Both Histone H3 and NCED3 cDNA fragments were cloned within a pGEM-T vector (Promega) and used a series of diluted DNA from these plasmids to form standard curves. For each gene examined in each time point, the average of three to five independent biological repeats was calculated. The mean (of a treatment at a specific time point) with the lowest relative expression was given a value of ‘1’ and relative expression of all other means was calculated as fold increase compared to this baseline treatment.

Relative expression of sample was calculated by manually determining the threshold point in the exponential phase, assuming that in this phase, DNA concentration is doubled in each cycle. Expression level was calculated using a Formula (2 (Ct max- Ct sample)). For each time point, the averages of three independent biological repeats were calculated.

Chemical treatments to fruitlets

In 2017, Application of 1 gr/L uniconazole P (2 % Magic of AD AMA Agan, Israel) on inflorescences of 'Golden Delicious’ trees, comparing fruitlet survival to natural conditions. Six strongly flowering 'Golden Delicious' trees were selected and divided randomly between the treatment and the control. 10 inflorescences per tree were marked using a colored ribbon. Flower rings of different colors were placed on K, fruitlets. Treatments were conducted at 3 DAFB. In three of the trees, the marked full inflorescences were each sprayed with 0.7 ml of 1 gr/L uniconazole P together with 0.025% Triton xlOO surfactant, 3 DAFB between 6-7 AM. In the three other trees the marked full inflorescences were sprayed with 0.7ml of the 0.025% Triton xlOO, as a control. Fruitlet survival in the marked inflorescences was compared in response to the different treatments.

In 2017, Application of 1 gr/L uniconazole P (2% Magic) on King and L3 flowers of 'Golden Delicious’ trees, and comparing survival of different fruitlets. Six strongly flowering 'Golden Delicious' trees were selected and divided randomly between the treatment and the control. 20 inflorescences per tree were marked using a colored ribbon. Flower rings of different colors were placed on K, L3 and LI fruitlets. In ten of the inflorescences the king and two L3 fruitlets received 0.1ml drops of 1 gr/L uniconazole P together with 0.025% Triton xlOO, 3 DAFB between 6-7 AM. In ten of the inflorescences the king and two L3 fruitlets received 0.1ml drops of 0.025% Triton x 100, 3 DAFB between 6-7 AM. Fruitlet survival was compared in the marked inflorescences in response to the different treatments.

In 2017, in young 'Golden Delicious’ trees with medium flowering we marked 90 inflorescences per repeat with a ribbon, each repeat consisting of 2-3 trees, 5 biological repeats. 30 inflorescences were marked with a blue ribbon, and the LI flower was marked with a ring. 30 inflorescences were marked with a red ribbon, and all neighbors accept the LI flower were removed at full bloom. 30 additional inflorescences were marked with a yellow ribbon, and all neighbors accept the LI flower were removed at full bloom. At 3 DAFB LI fruitlets were sprayed with 0.025% Triton x 100 (blue and Yellow ribbons), or with 1 gr/L uniconazole P (2% Magic) together with 0.025% Triton x 100 (red ribbons). Thus, blue ribbons marked inflorescences containing L1WN fruitlets, the yellow ribbons marked inflorescences containing L1A fruitlets, and the blue ribbons marked inflorescences containing LI A fruitlets treated with uniconazole P at 3 DAFB. At 12 DAFB 5-10 fruitlets were collected from each treatment and each repeat. These samples were used for RNA analysis. Fruitlet survival in all other marked inflorescences was measured at 60 DAFB.

In 2019, 30 fruitlets of ‘Anna’ trees were chosen per treatment and chemical was applied with a brush around 21-30 DAFB. Treatments were: Mock, 1 or 2 gr/L S-ABA (0.5 and 1% ProTone SG). All treatments included 0.025% Triton X100. Survival was assessed 16 days after treatment.

In 2019, 3 high flowering ‘Cripps Pink’ trees were chosen and treated 9 DAFB. K and L3 fruitlets were marked with a ring and treatment was applied only on those fruitlets with a brush. 16 inflorescences (32 fruitlets) per tree were treated with one of the following treatments: Mock, 2 gr/L S-ABA (1% ProTone SG) or 1 gr/L uniconazole P (2% Magic). All treatments included 0.025% of Triton xlOO. Survival of either K and L3 or L2s and LI was assessed 21 DAFB.

In 2020, on August 18^th in the evening, Crimson Seedless vines were sprayed with Ethrel (480 gram/ Liter Ethephon, Bayer Crop-Science, Monheim, Germany) at a concentration of 0.1% Ethrel which is 480 mg/Liter Ethephon, and an additional surfactant of 0.02% Triton xlOO. Further chemical treatments were performed to specific east and west facing clusters by immersion of the cluster in a bucket containing the chemical. The bucket was 16cm deep and 16cm diameter. On August 24th in the evening, some of the clusters were immersed for 10 seconds in 1 gr/L uniconazole P (2% Magic) together with 0.025% of Triton xlOO. On August 26th in the evening, some of the clusters were immersed for 10 seconds in 0.25 gr/L S-ABA (0.125% ProTone SG) together with 0.025% of Triton xlOO. Berrie clusters were harvested on September 17^th 2020 and brought in cold conditions to the nearby lab, where they were immediately measured for color accumulation using a portable fluorometer (Multiplex III, Force A, France; Bahar et al., 2012). The Fluorometer measures Far-Red light emission after excitation with Red or Green light and the Log of the Florescence excitation ratio (FER_RG) is used to calculate quantity of Anthocyanins (ANTH). Measurements were conducted for 10 berries in each cluster and the average ANTH was calculated, 3-6 clusters were measured per treatment.

Chemical treatments to the whole tree

Treatments applied in 2020 to ‘Top Red’ trees at Matityahu and El-Rom and to ‘Golden Delicious’ and ‘Ariane’ trees at Matityahu were 1 gr/L uniconazole P (2% Magic) and/ or 0.6g/L S-ABA (0.3% ProTone SG). All treatments included 0.025% surfactant Triton x 100. Treatments were performed at 8:00 AM with a 4-Gallon, Piston, 425 Backpack Sprayer (Solo Inc., Newport News, VA, USA).

‘Top Red’ trees fruitlet survival was assessed 37DAFB at Matityahu and 41 DAFB at El-Rom. At harvest the number of fruit and total fruit weight per tree were measured in the field. Fruit from control and double treatment was taken to the ‘Beresheet’ packing house where fruit for each of the trees were sorted and counted based on diameter. Survival was assessed 65 DAFB in ‘Golden Delicious’ trees and 67 DAFB in ‘Ariane’ trees at Matityahu.

Statistical Analysis

Data presented as average of biological repeats (trees), and analyzed by one-way analysis of variance (ANOVA) using JMP version 14 software (SAS Institute, Cary, NC, USA). Differences between treatment and control were determined by student's t tests. In multiple comparisons, Tukey-Kramer HSD or Dunnett’s method were implemented. If not stated differently, statistical significance was determined at P < 0.05. In cases the variance was not equal and/or the data did not show normal distribution, the statistical tests were conducted on transformation to ranks or Arcsine transformation of the values.

Example 1: Studying LI fruitlet abscission

Initially, fruitlet abscission in cv. ‘Golden Delicious’ under local natural conditions was tracked during two consecutive years, 2014-2015 (Fig. 1A-1B, respectively). In both years, abscission started at ~ 15-20 DAFB and lasted for ~ 4 weeks. In 2014, the L3 and L2 groups were surveyed as one group, and in 2015 they were surveyed separately. In 2014, the L2-3 fruitlets had the highest survival rate among the different fruitlet categories: 53.3%. In this year the king fruitlets had a lower survival rate of 22%, and LI fruitlets had the lowest survival rate (6%) (Fig. 1A). In 2015, king fruitlets had the highest survival rate 43%, followed by L3 (31%), L2 (15%) and LI (4%) (Fig. IB).

Average percent fruitlet survival in ‘Golden Delicious’ was higher (37.6%) in 2014 compared to 2015 (20%) (Fig. 1A-1B). In 2014 -86% of inflorescences retained 1-3 fruitlets, while in 2015 -85% retained only 0-2 fruitlets (Fig. 1C). This noticeable variation in abscission between the two years in the same cultivar- ‘Golden Delicious’ in the same location, emphasizes the dilemma faced by farmers regarding forecasting the level of natural thinning. Without such a forecast, the correct thinning practices remain obscure since a similar hormone treatment used during these two years will likely lead to different results. This variation is also an obstacle in understanding the molecular basis of abscission. Still, in one parameter there was less variation - the survival rate of LI fruitlets: 6% in 2014, 4% in 2015.

The variation among cultivars in LI survival was studied. Specifically, the literature was scanned for a cultivar with a much higher natural survival rate of LI fruitlets.

When comparing fruitlet abscission of different apple varieties, 'Ariane' trees was noted as a cultivar that tends to retain all its fruitlets thus needs to be thinned intensively in order for fruit to reach commercial size (Laurens et al., 2005, Celton et al., 2014). In this application fruitlet abscission of 'Ariane' was compared to that of ‘Golden Delicious’ grown in the same orchard in two years. In 2015 fruitlet survival in ‘Ariane’ (63%) was much higher than ‘Golden Delicious’ (20%) (Fig. 2A). A similar significant difference was noted in 2020 (59% vs. 22%; Fig 2C). In every fruitlet position, the survival rate in ‘Ariane’ was significantly higher compared to ‘Golden Delicious’ (Fig. 2A). As a result, the number of surviving fruitlets per inflorescence was significantly higher in ‘Ariane’ in 2015 (Fig 2B). While in ‘Golden Delicious’ most inflorescences (-85%) retained only 0- 2 fruitlets, in 'Ariane' most inflorescences (-80%) retained 3-5 fruitlets (Fig. 2B). LI fruitlets survival rate in 'Ariane' reached 46% (2015; Fig 2A) and 20% (2020; Fig.2C), while in ‘Golden Delicious’ LI survival was 4% (2015; Fig IB) and 0% (2020, Fig. 2C) in ‘Golden Delicious’ (Fig 2C).

Next, it was studied whether the survival rate of the LI fruitlet in ‘Golden Delicious’ depends on the presence of other fruitlets in the inflorescence. Removing all other fruit in the inflorescence one day after full bloom, increased the survival rate of the LI fruitlet (LI Alone; L1A) to 84% in 2014 (Fig. 3A) 85% in 2015 (Fig. 3B) and 78% in 2020 (Fig. 2C). This suggests that LI is not predestined to abscise, and its survival rate depends on the presence of other fruitlets in the inflorescence. In 'Ariane' in 2020, when neighbors were removed (LI Alone), LI survival rate also increased from 20% (L1WN) to 82% (L1A; Fig. 2C). Thus, without neighbors, the survival rate of L1A was similar in both cultivars (Fig. 2C). This might suggest that the difference between these cultivars is not within LI, but in how LI reacts to neighboring fruit.

Example 2: Identifying the time point of no return, when the fate of the LI fruitlet to abscise is irreversible in ‘Golden Delicious’

The LI fruitlet will normally abscise, starting at 15-20 DAFB. It was further asked until what time/stage the fate of this fruitlet can still be changed by removing all other fruitlets in the inflorescence. This will help to identify the time in which the initial events leading to LI abscission should be studied.

All flowers/fruitlets, except LI, were removed at either 1,4,7, 11,15 DAFB at 2014, and at 1,5,7,9,11,15,20 DAFB at 2015. On May 20, 2014 (44 DAFB) and June 6, 2015 (58 DAFB), at the end of natural fruitlet abscission, LI survival rate was assessed (see materials and methods).

Removal of all other fruitlets at 15-20 DAFB was too late to ‘rescue’ the LI fruitlets (4-16% survival, both years). On the other hand, if all other fruitlets were removed on 7 DAFB, 68-76% of LI fruitlets survived. Four days later (11 DAFB), removal of all other fruitlets could only slightly increase LI survival rate (28% survival in 2014) (Fig. 3A- 3B).

At 2015, all the fruitlets were removed at 9 DAFB as well, which also caused a slight increase in LI survival to 56%, while removal at 11 DAFB allowed 52% survival of LI fruitlets (Fig. 3B). Thus, in both years, 2014 and 2015, up to 7 DAFB, the LI fruitlet has not gone through any irreversible committing steps towards abscission. Something happens between 7-15 DAFB to make the fate of LI fruitlets irreversible.

Once the time in which the fate of the LI fruitlet to abscise is set (irreversible) was revealed, it was aimed to identify a molecular marker that distinguishes early on between LI fruitlets destined to abscise and those destined to survive. As previously shown, Li's chance of survival depends on the presence of other fruit in the inflorescence. LI with neighbors (L1WN) have a 4-6% chance of survival (Fig. 3), while LI fruitlets in inflorescences from which all other flowers were removed at full bloom (LI Alone; L1A) had an 84-85% chance of surviving the natural fruitlet abscission period. RNA expression of L1WN was compared with LI A fruitlets at 4,7,11 DAFB (2014). Relative gene expression was measured using quantitative real-time RT-PCR. For preliminary analysis previously characterized molecular markers for apple fruitlets abscission / survival potential were looked for.

MdACOl (1 -AMINOCYCLOPROPANE- 1 -CARBOXYLATE OXIDASE1) involved in ethylene biosynthesis, is a known marker for abscission induction in apple (Botton et al., 2011, Dal Cin et al., 2005). By 11 DAFB, expression of MdACOl was 36-fold change (FC) higher in LI with neighboring fruit (L1WN) compared to LI alone (L1A) fruitlets (Fig. 4A).

Development of apple fruit involves an early period of cell division, that lasts approximately 30 days after pollination (Janssen et al., 2008). Cyclin dependent kinases (CDKs) play a role in progression of the cell cycle. Expression levels of MdCDKl-2 and MdCDK2-2 decrease at the end of cell division period, thus, they appear to be useful as early markers of growth arrest in the developing fruitlet (Janssen et al., 2008). It is now shown that at 11 DAFB LI WN fruitlets had a 4-fold reduction in expression of MdCDK2- 2, and a ~7-fold reduction in expression of MdCDKl-2, compared to L1A fruitlets (Fig. 4B-4C). Reduced expression of both genes in fruitlets bound to abscise appears to be a useful early marker of growth arrest of the fruitlet.

After revealing the time in which LI fruitlet abscission becomes irreversible and showing that molecular markers can predict the fruitlet abscission / survival potential, this system was used to learn more about the initial molecular events that later lead to abscission. A broad and unbiased picture was achieved by comparing the transcriptome of LI fruitlets, using RNAseq, at early stages of development.

Example 3: Analysis of fruitlet RNAseu data

Fruitlet samples of L1WN and LI A from ‘Golden Delicious’ at 4,7,11 DAFB of 2014 were used for the analysis. Samples were sequenced in 3 biological repeats from 7 DAFB, 1 biological repeat from 4, 11 DAFB. This initial analysis was performed by Prof. Hector Anton-Candela, Miguel Hernandez University of Elche, Spain. A total of 73,673 genes were expressed in all samples. The initial criteria for identifying differently expressed genes (DEG) was to compare samples from 7 DAFB. Genes for which the FPKM (fragments per kilobase of transcript per million mapped reads) was at least 1.5- fold different between treatments (comparing L1WN to L1A) were selected (3 repeats). A total of 3,414 DEGs were found. Among them significantly higher in LI with neighbors at 7 DAFB (SHWN) - 2,368 genes. Significantly lower in LI with neighbors at 7 DAFB (SLWN) -1046 genes.

Out of the long and diverse list of SHWN DEGs identified, it was asked which are in common with the DEGS identified by other groups studying apple fruitlet abscission, using RNAseq and microarray (Botton et al., 2011, Eccher et al., 2013, Ferrero et al., 2015). As detailed in the materials and methods, the raw data of these experiments were analyzed, and DEGs that have an expression ratio of at least 1.5 FC between ‘towards abscising’ and ‘surviving’ fruitlets were filtered. An important thing to notice, is that in these other experiments, the comparison between surviving and ‘towards abscising’ fruitlets is from the same inflorescence- comparing different fruitlets, each reaching a different developmental stage at time of sampling. In the present experiments the same LI fruitlets are compared, at the same developmental stage, with or without neighboring fruitlets.

Using ‘Venny- An interactive tool for comparing lists with Venn's diagrams’ (bioinfogp.cnb.csic.es/tools/venny/) 171 genes that were commonly up regulated in abscising fruitlets- ‘common to all- upregulated in abscising fruitlets’ were identified (Fig. 5A). As expected, MdACOl mentioned above was among the 171 ‘common to all- upregulated in abscising fruitlets’ genes.

In the present experiment, two kinds of LI fruitlets with different abscission potential were compared. It is logical to assume that natural fruitlet abscission of the L1WN fruit is a phenomenon due to some kind of stress or signal caused or sent by neighbors, termed here "neighbor stress”.

It is assumed that "neighbor stress" has not been studied in model organisms using whole transcriptome data, so there are no public transcriptome data from other species it can be compared with. Still, neighbors can cause stress through mechanisms common to model species. For example, neighbors could send a hormone signal, or could limit the availability of some important factor (abiotic stress of some type, deficiency in sugars, minerals, hormones, water etc.). If so, perhaps the identified DEGs can be matched, at least partially, with those identified by others in Arabidopsis, studying some hormonal and chemical treatment.

It is hypothesized that by screening all available transcriptome data, specific treatments given in Arabidopsis that have the most similar “fingerprint” to the present data can be identified: sharing a group of genes induced by the treatment and a group of genes repressed by the treatment. In other words, perhaps at least part of the gene expression changes seen herein could be explained by a better defined and studied stress from Arabidopsis. Such a finding, if verified, might help to understand the essence of the initial events that occur in neighbor stress, causing these major changes in gene expression.

‘BAR expression angler’ (http://bar.utoronto.ca/affydb/cgi- bin/affy_db_exprss_browser_in.cgi) was used for this analysis (Toufighi et al., 2005). The software’s input is only Arabidopsis genes. Out of the 2,368 SHWN genes, 1,100 have a known and significant homolog in Arabidopsis present in the BAR expression angler (AtSHWN). Out of the 1046 SLWN genes, 594 genes have a known and significant homolog in Arabidopsis present in the BAR expression angler (AtSLWN). As a control, a list of two groups of "random genes" was also compiled. For this purpose, the list of Arabidopsis homologs of all the genes represented in the present RNAseq data was used, and 100 genes that are equally expressed in L1A compared to L1WN at 7 DAFB were chosen, then randomly divided to a list of two groups- group A and B. When the Arabidopsis data regarding a stress which is unlinked to "neighbor stress" is examined, it is expected that the AtSHWN, AtSEWN as well as A and B groups behave in a similar way. ~X % would be induced by the treatment, Y% would be repressed and Z% would be unaffected by the treatment. If, on the other hand, data regarding a treatment in Arabidopsis which partly mimics "neighbor stress" is analyzed, it is expected to see that some "linkage" so that the two groups (AtSHWN and AtSEWN) would be sorted differently by the treatment. The distribution of induced, repressed or unaffected genes would be expected to be quite different between the two groups. On the other hand, it is expected that groups A and B will have a similar distribution (no linkage). This criterion was used as a means to score the different treatments offered by the software (hormone treatment, tissue series, and stress series). Altogether, 169 datasets were scanned, and the treatment in which the two DEG groups- AtSHWN and AtSLWN, behaved most differently, was chosen.

This treatment was Gibberellin (GA) inhibitor (10 pM Uniconazole) for 12h, (Goda et al., 2008) (www.arabidopsis.org/servlets/ TairObject?type=expression_set&id =1008080692). (Fig 6). 46% of the AtSHWN genes were induced by this uniconazole treatment and 40% of the AtSLWN genes were repressed by this treatment (Fig 6). A statistic Chi-squared test showed that the AtSHWN and AtSLWN groups are significantly different in response to 12hr uniconazole treatment, while the two random groups A and B respond exactly the same to the uniconazole treatment (Fig. 6). Collection of tissues after a 3-hour treatment with uniconazole did not separate the two groups (Fig. 6). This might be because this chemical changes gene expression by first affecting internal hormone levels.

Example 5: Application of 1 sr/L Uniconazole P (2% Manic) onto Apple Inflorescences.

Since the analysis suggests that genes encoding similar proteins were upregulated by neighbors in apple and by uniconazole in Arabidopsis, it was then asked if treating particular apple flowers/fruitlets with uniconazole will cause abscission. Three DAFB inflorescences were treated with 1 gr/L uniconazole (2% Magic). When whole inflorescences were treated, this caused increased abscission of all fruitlets (Fig 7A). When only king and L3 flowers were treated, there was an increase in their abscission together with a decrease in L2 and LI abscission. (Fig 7B). Thus, by adding uniconazole to specific flowers, the interactions between fruitlets can be changed, causing some to abscise more and others to abscise less. Finally, L1A fruitlets were treated to examine if uniconazole treatment can increase abscission of fruitlets that are no longer under neighbor competition. Indeed, this treatment reduced LI A survival from 60% to 22% (Fig 7C).

The inventors of the present invention have identified 171 ‘common to all- upregulated in abscising fruitlets’ genes, based on their own gene expression analysis and those performed by others. In the present experiments the expression of these genes was significantly higher in L1WN compared to L1A fruitlets. Using Arabidopsis data, it was noticed that a similar fingerprint is caused by uniconazole (Arabidopsis) and neighbors (Apple). It was then showed that uniconazole can cause LI fruitlet abscission, having a similar affect as neighboring fruitlets. Although the responses to neighbors and uniconazole are similar with regard to abscission, the response in gene expression caused by these two treatments may be completely different. If the uniconazole treatment acts using similar changes in gene expression as neighbors, it would be assumed that many of the 'common to all- upregulated in abscising fruitlets’ genes would be also induced by Uniconazole. Using the above experiment, it can be now compared, using RNAseq, the changes in gene expression between L1A fruitlets (high survival), L1WN fruitlets (low survival) and LI A treated with uniconazole (low survival) AT 11 DAFB. It was found that 114 of the 171 (67%) ‘common to all- upregulated in abscising fruitlets’ genes were included in the 1,982 DEGs that had higher expression in LI A treated with uniconazole compared to untreated L1A (Fig. 5B). Thus, the majority of changes in gene expression occurring in fruitlets fated to abscise, can be mimicked by treating a fruitlet with uniconazole.

As mentioned above, uniconazole is considered a Gibberellin biosynthesis inhibitor, inhibiting the P450 ent-kaurene oxidase (CYP701), catalyzing the oxidation of entkaurene to ent-kaurenoic acid in gibberellin (GA) biosynthesis. It is therefore hypothesized that uniconazole treatment mimics neighbors because in growing fruitlets GA levels are lower in L1WN fruitlets compared to L1A or King fruitlets, and this reduction in GA is a trigger for abscission. Active GA levels (GAI and GA3) in 11 DAFB fruitlets were measured.

In order to further understand the involvement of GA in fruitlet abscission, hormone levels in different ‘Golden Delicious’ apple fruitlets, such as king (high survival rate) L1WN (low survival rate) and LI A (high survival rate), at 11 DAFB was measured. Surprisingly, no reduction in GAs in L1WN compared to other fruitlets was detected (Fig. 8A-8B).

Interestingly, uniconazole also inhibits ABA catabolism by inhibiting the ABA 80- hydroxylase enzyme (Saito et al., 2006), meaning that uniconazole simultaneously inhibits GA biosynthesis and ABA catabolism. Indeed, when ABA levels in these 11 DAFB fruitlets was measured, results showed that ABA levels were higher in fruitlets prone to abscise (L1WN) (Fig. 8C). Previous studies (Eccher et al., 2013) showed that ABA levels in LI fruitlets were significantly higher compared to King fruitlet at ~18 DAFB. The present finding suggests that this increase in ABA in LI fruitlets occurs at least a week sooner, close to the point in which the fate of the LI fruitlet is decided, and this increase is dependent on the presence of neighbors. In the ‘Ariane’ cultivar in which L1WN abscission is much reduced compared to ‘Golden Delicious’ (Fig. 8D), the internal levels of ABA within L1WN fruitlets at 12 DAFB were significantly lower (Fig. 8E). In LI A fruitlets with high survival in both ‘Golden Delicious’ and ‘Ariane’, the ABA levels are lower and similar (Fig. 8E). These results suggest that differences in internal ABA levels within fruitlets from different varieties may be the cause for different levels of natural abscission between cultivars.

It was further speculated that the common denominator in the responses to uniconazole or fruitlet neighbors is the accumulation of ABA. Indeed, expression levels of MdNCED3a, a key enzyme in ABA biosynthesis, were significantly higher at 11 DAFB in L1WN compared to L1A (Fig. 9). Much lower levels were measured in 'Ariane', in which abscission is very low for LI with or without neighbors (Fig. 9).

This led to hypothesize that the initial pattern of gene expression identified in apple fruitlets destined to abscise was similar to Arabidopsis after treatment with uniconazole because uniconazole causes ABA accumulation, and the gene expression response occurring in both future abscising L1WN apple fruitlets and Arabidopsis seedlings treated with uniconazole is likely due to ABA accumulation in both tissues (naturally in apple LI fruitlets, and uniconazole-induced in Arabidopsis).

Since applying uniconazole causes fruitlet abscission via accumulation of ABA, it was studied whether direct treatment of fruitlets with ABA will also cause fruitlet abscission.

Next, the effect of a direct application of ABA on apple fruitlets was tested.

A pilot test was initially performed by treating 'Anna' inflorescences with 1 or 2 gr/L of S-ABA (0.5 or 1% Pro tone SG). The 2 gr/L solution worked well while the Igr/L solution only had a marginal effect (Fig. 10).

Then a full experiment in the 'Cripps Pink' variety using the effective 2 gr/L of S- ABA was conducted. ABA and uniconazole treatments were compared given specifically to fruitlets that normally survive (King and L3 fruitlets). Both the treatments caused abscission of King and L3 treated fruitlets, leading to an increase in survival of LI and L2 fruitlets (Fig. 11).

Example 6: Pretreatment with uniconazole can cause lower, affordable concentrations of ProTone to have a significant fruitlet thinning effect in 'TOD Red'

ProTone (Valent BioSciences corporation, USA, a composition comprising S- ABA) is commercially used in Israel to increase skin color in grapes. It is given at a concentration of 0.15%-0.2% for ProTone-SG (150-200 grams to 100 liters water), costing farmers -600 NIS per dunam. A rough estimation is that applying an effective 1% Protone SG in apples would cost the farmer -0.9 NIS per kg of final fruit. This cost is far from economical for apple farmers and that is why agents selling ProTone in Israel are not interested in trying to convince apple farmers to use ProTone as a chemical thinner.

It is hypothesized that relatively high levels of ProTone are required for apple thinning since a large portion of the external ABA is efficiently catabolized within the plant on arrival, thus reducing the treatment efficiency. Additionally, the present results suggest that uniconazole is causing abscission in apples mostly by inhibiting ABA catabolism, so it is now hypothesized that in pre-treating apple trees with uniconazole followed by a treatment of ProTone, the ProTone would be more effective using much lower concentrations. An additional major advantage of pre-treating with uniconazole is its putative positive effect on return flowering (Haberman et al., 2016)

This hypothesis was examined in a cultivar that has difficulties in finding a successful treatment for fruit thinning: 'Top Red' (Starking/Red Delicious). Since 'Top Red' is not well thinned by chemical treatments, if not extensively hand thinned it usually enters steep cycles of alternate bearing causing growers to lose money and interest in this cultivar. A relatively late chemical application (14-16 DAFB) was tried since, as mentioned above, at this time point it was hoped to gather enough molecular information that will allow to estimate potential natural abscission in a specific orchard/cultivar/year. The experiments have started with 1 gr/L uniconazole P, waiting 2-3 days and then spraying 0.6 gr/L of S-ABA (0.3% ProTone SG). As a control, other trees in the same rows were sprayed with just one of the chemicals, or none of the chemicals. The experiment was performed in two locations, Matityahu in the Northern Galilee (Fig 12A- B), and El-Rom in the Golan Heights (Fig. 13A-B). In both locations only the double treatment caused a significant reduction in fruitlet survival (Figs. 12 and 13). The single treatments had no significant effect. No side effects of leaf abscission or leaf yellowing was observed. Thus, a concentration of 0.6 gr/L of S-ABA (0.3% ProTone SG) was sufficient to cause abscission after pre-treating with uniconazole. As can be seen (Figs. 12A, 13A), in untreated trees the percent of fruitlet survival in El-Rom this year (2020) was much lower than in Matityahu. Still, the double treatment increased abscission relative to the control in both locations. The double treatment also significantly increased the percent of inflorescences with 0-1 final fruitlets in Matityahu (Fig 12B), and 0 fruitlets in El-Rom (Fig. 13B). Although zero fruit does not seem desirable, it is actually very important in ‘Top Red’ since these are the only spurs that will contain flowers for the next year (Haberman et al., 2016). At harvest, in Matityahu, the average fruit weight in trees treated with the combination was significantly higher compared to untreated trees (Fig. 12C). Fruits from the control and double treatment were also analyzed for fruit diameter, and the double treatment increased the percent of fruit above a diameter of 65 mm (Fig. 12D). At harvest, in El-Rom only, average fruit weight was measured and the double treatment increased average fruit weight while each single treatment did not (Fig 13C).

In the preliminary experiment with 'Anna', relatively advanced fruitlets abscised in response to S-ABA treatment (Fig 10). It was examined whether in trees in which natural abscission was already advanced (40-42 DAFB), this double treatment would still increase abscission. This is a stage in which chemical thinners normally do not work anymore. This was tested in a preliminary experiment using two adjacent rows of young trees, one of 'Golden Delicious' and the other of 'Ariane'. As mentioned above, natural abscission in 'Ariane' is very low (Fig. 2C). Some trees were left untreated, others received 1 gr/L uniconazole P, and others received the 1 gr/L uniconazole P and an additional 0.6 gr/L of S-ABA treatment 4 days later. In this experiment the delay in the 2^nd spray was due to weather and workforce constraints. Unlike the previous experiments, there was no treatment just with S-ABA, and there were only one or two repeats per treatment so significance could be analyzed (Fig. 14A-14B). One clear unsurprising observation is that 'Ariane' survival is much higher, as expected (Fig. 14A). A second observation is that the double treatment did not seem to cause abscission in 'Ariane', and possibly slightly increased abscission in 'Golden Delicious'. When the first treatment was performed (40-42 DAFB), many fruitlets already abscised. Since fruitlet numbers in each inflorescence was recorded before the treatments, the number of additional fruitlets abscised in response to the treatment could also be measured (Fig. 14B). In over 60% of the inflorescences there was no additional abscission after 40-42 DAFB. It appears that the 2^nd repeat of the double treatment had more abscission in the two cultivars. The 2^nd repeat of both varieties was located next to each other. It is not known if the additional abscission over there was due to the double treatment or to the location within the row.

Example 7: Pretreatment with uniconazole can increase color in table grapes treated with ProTone.

Under warm summers as in Israel, several cultivars of table grapes often fail to accumulate sufficient anthocyanins to achieve good uniform berry color, and this has a commercial disadvantage. Recently, growers apply either one or a combination of Ethephon and S-ABA at the ‘veraison’ stage in order to increase grape color. Use of either chemical at a higher concentration may improve color even more yet this causes premature berry softening (Peppi et al., 2007). In a commercial orchard in which the commercial treatment is 480mg/L of Ethephon followed after a few weeks by 0.3 gr/L ProTone SG we asked whether adding a Uniconazole-P treatment before S-ABA applications will increase color, as measured by Anthocyanin accumulation. As shown in Fig. 15, when 1 gr/L Uniconazole P was added two days before a 0.25 gr/L S-ABA application, anthocyanin accumulation and therefore color appearance was higher. Conclusions:

The research leading to the present invention aimed at understanding the initial molecular and hormonal events that trigger fruitlet abscission in apples. Other researches aimed to understand this process mostly compared fruitlets in different positions within the inflorescence (Botton et al., 2011, Eccher et al., 2015, Eccher et al., 2013, Ferrero et al., 2015) or comparing fruitlets with or without a chemical thinning treatment (Botton et al., 2011). Fruitlets at different positions within an inflorescence are at different developmental stages and this can bias the findings when comparing between them (Ackerman & Samach, 2015). In the present invention, a different approach was used, studying LI fruitlets with or without neighbor fruitlets. This allowed to compare the same fruitlet under two opposite fates, with no chemical or hormonal intervention. Since the fate of LI fruitlets depends on the presence of its neighbors, the time in which the fate of LI fruitlets is set was revealed by removing neighbors at different time points. The present invention now shows that within a window of time, 7-11 DAFB, the fate of LI fruitlets is set. Thus, the initial changes in gene expression leading to LI abscission occur before or during this period. Changes in gene expression occurring only after this window of time are likely downstream events that are part of abscission or fruitlet death, yet not the cause of abscission. RNA samples from LI fruitlets with or without neighbors were collected at 4, 7, 11 DAFB and RNASeq analysis of these samples was performed. By 7 DAFB, 2368 genes that were significantly higher in LI with neighbors were identified. 171 of these genes came up in all screens performed by other groups. Without wishing to be bound by any theory or mechanism of action, neighbors may act on LI like a stress or a hormone, and thus a search was performed for Arabidopsis treated by stress or hormone for a common fingerprint of gene expression as neighbors. Uniconazole treatment to Arabidopsis seedlings was identified as a treatment that most resembled, in gene expression, the effect of neighbors on LI fruitlets. The present invention now shows that uniconazole treatment could mimic the effect of neighbors on apple fruitlet abscission. It also triggered expression of two thirds of the 171 genes common to all abscising fruitlets.

Since Uniconazole is known as an inhibitor of Gibberellin biosynthesis, it is assumed that LI fruitlets with neighbors would contain significantly lower amounts of active GAs. Surprisingly, measuring hormone levels showed that this was not the case. On the other hand, at 11 DAFB, LI fruitlets with neighbors had significantly higher levels of ABA compared to LI alone fruitlets or King fruitlets. Expression of an apple NCED3 encoding gene, a key step in ABA biosynthesis, is higher in L1WN compared to L1A by 11-12 DAFB and in some years this difference is significant at earlier dates. Others have already associated fruitlet abscission with ABA accumulation (Eccher et al., 2013), yet here this accumulation was identified one week earlier, suggesting it is part of the early trigger leading to abscission, and not a downstream event triggered by an earlier event. The application of the GA biosynthesis inhibitor uniconazole mimics internal events leading to abscission, yet no changes in internal GA together with accumulation of ABA occurred in fruitlets fated to abscise. This could be easily explained due to the ability of uniconazole to cause ABA accumulation by inhibiting the enzyme leading to its degradation (Saito et al., 2006). Treating apple fruitlets with a commercial product containing S-ABA, ProTone, can indeed cause abscission. This product cannot be used by apple farmers as a thinning agent due to its current high price, and the requirements of relatively high ProTone concentrations to cause fruitlet abscission. The research described herein aimed at identifying the initial events triggering abscission that identified ABA and uniconazole activity, raised the interest in finding a way to cause fruitlet abscission in apples using a much lower, affordable, concentration of ProTone. Accordingly, the present invention discloses that prespraying trees with uniconazole enables the use of lower concentration of ABA as a potent abscission inducer. Without wishing to be bound by specific theory or a mechanism of action, the reduction in the required ABA concentration is due to the inhibition of ABA catabolism by uniconazole.

References:

Abrams S.R., Nelson K. & Ambrose S.J. (2003) Deuterated abscisic acid analogs for mass spectrometry and metabolism studies. Journal of Labelled Compounds and Radiopharmaceuticals, 46, 273-283.

Ackerman M. & Samach A. (2015) Doubts regarding carbohydrate shortage as a trigger toward abscission of specific Apple (Malus domestica) fruitlets. New Negatives in Plant Science, doi: 10.1016/j. neps.2015.1006.1003.

Bahar A., Kaplunov T., Zutahy Y., Daus A., Lurie S. & Lichter A. (2012) Autofluorescence for analysis of ripening in Thompson Seedless and colour in Crimson Seedless table grapes. Australian Journal of Grape and Wine Research, 18, 353-359.

Botton A., Eccher G., Forcato C., Ferrarini A., Begheldo M., Zermiani M., Moscatello S., Battistelli A., Velasco R., Ruperti B. & Ramina A. (2011) Signaling Pathways Mediating the Induction of Apple Fruitlet Abscission. Plant Physiology, 155, 185-208.

Byers R. & Carbaugh D. (2002) Effects of Thinning Time on Yield, Fruit Size, and Return Bloom of ‘York’ and ‘Golden Delicious’ Apple Trees. Journal of Tree Fruit Production, 3, 55-62.

Celton J.M., Kelner J. J., Martinez S., Bechti A., Touhami A.K., James M.J., Durel C.E., Laurens F. & Costes E. (2014) Fruit Self-Thinning: A Trait to Consider for Genetic Improvement of Apple Tree. PLoS ONE, 9.

Dal Cin V., Danesin M., Boschetti A., Dorigoni A. & Ramina A. (2005) Ethylene biosynthesis and perception in apple fruitlet abscission (Malus domestica L. Borhk). J Exp Bot, 56, 2995 - 3005.

Davidson J.H., Hammer O.H., Reimer C.A. & Dutton W.C. (1945) Thinning apples with the sodium salt of naphthylacetic acid. Mich. Agr. Expt. Quart. Bui., 27, 352-356.

Duane W.G., Alan N.L., Terence L.R. & Phillip S. (2013) Development of a Fruitlet Growth Model to Predict Thinner Response on Apples. HortScience horts, 48, 584-587.

Eccher G., Begheldo M., Boschetti A., Ruperti B. & Botton A. (2015) Roles of Ethylene Production and Ethylene Receptor Expression in Regulating Apple Fruitlet Abscission. Plant Physiol, 169, 125-137.

Eccher G., Botton A., Dimauro M., Boschetti A., Ruperti B. & Ramina A. (2013) Early Induction of Apple Fruitlet Abscission Is Characterized by an Increase of Both Isoprene Emission and Abscisic Acid Content. Plant Physiology, 161, 1952-1969.

Ferrero S., Carretero-Paulet L., Mendes M.A., Botton A., Eccher G., Masiero S. & Colombo L. (2015) Transcriptomic Signatures in Seeds of Apple (Malus domestica L. Borkh) during Fruitlet Abscission. PLOS ONE, 10.

Goda H., Sasaki E., Akiyama K., Maruyama-Nakashita A., Nakabayashi K., Li W.Q., Ogawa M., Yamauchi Y., Preston J., Aoki K., Kiba T., Takatsuto S., Fujioka S., Asami T., Nakano T., Kato H., Mizuno T., Sakakibara H., Yamaguchi S., Nambara E., Kamiya Y., Takahashi H., Hirai M.Y., Sakurai T., Shinozaki K., Saito K., Yoshida S. & Shimada Y. (2008) The AtGenExpress hormone and chemical treatment data set: experimental design, data evaluation, model data analysis and data access. Plant Journal, 55, 526-542.

Goodstein D.M., Shu S., Howson R., Neupane R., Hayes R.D., Fazo J., Mitros T., Dirks W., Hellsten U., Putnam N. & Rokhsar D.S. (2012) Phytozome: a comparative platform for green plant genomics. Nucleic Acids Res, 40, DI 178-1186.

Greene D. (2002) Chemicals, Timing, and Environmental Factors Involved in Thinner Efficacy on Apple. Hortscience, 37, 477-481.

Greene D. & Costa G. (2013) Fruit Thinning In Pome- and Stone-Fruit: State Of The Art.

Greene D.W., Schupp J.R. & Winzeler H.E. (2011) Effect of Abscisic Acid and Benzyladenine on Fruit Set and Fruit Quality of Apples. Hortscience, 46, 604-609.

Haberman A., Ackerman M., Crane O., Kelner J.J., Costes E. & Samach A. (2016) Different flowering response to various fruit loads in apple cultivars correlates with degree of transcript reaccumulation of a TFEl-encoding gene Plant Journal, 87, 161-173.

Janssen B., Thodey K., Schaffer R., Alba R., Balakrishnan L., Bishop R., Bowen J., Crowhurst R., Gleave A. & Ledger S. (2008) Global gene expression analysis of apple fruit development from the floral bud to ripe fruit. BMC Plant Biol, 8, 16.

Kotoda N., Iwanami H., Takahashi S. & Abe K. (2006) Antisense expression of MdTFLl, a TFLl-like gene, reduces the juvenile phase in apple. J. Amer. soc. Hort. Sci., 131, 74-81.

Lakso A.N. (2017) Perspectives on physiology in support of fruit production and future opportunities: lessons from 40 years of fruit research. Acta Hort, 1177.

Lakso A.N. & Robinson T.L. (2014) Integrating Physiological Models in Applied Fruit Crop Research Acta Hort, 1058, 285-290.

Langmead B. & Salzberg S.L. (2012) Fast gapped-read alignment with Bowtie 2. Nat Methods, 9, 357-359.

Laurens F., Lespinasse Y. & Fouillet A. (2005) A New Scab-resistant Apple: 'Ariane'. Hortscience, 40.

Lulsdorf M.M., Yuan H.Y., Slater S.M.H., Vandenberg A., Han X., Zaharia L.I. & Abrams S.R. (2013) Endogenous hormone profiles during early seed development of C. arietinum and C. anatolicum. Plant Growth Regulation, 71, 191-198.

McArtney S.J., Abrams S.R., Woolard D.D. & Petracek P.D. (2014) Effects of S- Abscisic Acid and (+)-8 '-Acetylene Abscisic Acid on Fruit Set and Stomatal Conductance in Apple. Hortscience, 49, 763-768.

Mimida N., Kotoda N., Ueda T., Igarashi M., Hatsuyama Y., Iwanami H., Moriya S. & Abe K. (2009) Four TFLl/CEN-Like Genes on Distinct Linkage Groups Show Different Expression Patterns to Regulate Vegetative and Reproductive Development in Apple (Malusxdomestica Borkh.). Plant and Cell Physiology, 50, 394-412.

Peppi M.C., Fidelibus M.W. & Dokoozlian N.K. (2008) Timing and Concentration of Abscisic Acid Applications Affect the Quality of ‘Crimson Seedless’ Grapes. International Journal of Fruit Science, 7, 71-83.

Saito S., Okamoto M., Shinoda S., Kushiro T., Koshiba T., Kamiya Y., Hirai N., Todoroki Y., Sakata K., Nambara E. & Mizutani M. (2006) A Plant Growth Retardant, Uniconazole, Is a Potent Inhibitor of ABA Catabolism in Arabidopsis. Bioscience, Biotechnology, and Biochemistry, 70, 1731-1739.

Schneider G.W. & Lasheen A.M. (1973) NAA and sevin on composition, development, and absission of apple fruit. Hortscience 8, 103-104.

Stern R.A. (2008) Chemical thinning in ‘Top Red’ Ortal 2008.

Stopar M., Black B. & Bukovac M. (1997) The effect of NAA and BA on carbon dioxide assimilation by shoot leaves of spur-type 'Delicious' and 'Empire' apple trees. J Amer Soc Hort Sci, 122, 837 - 840.

Teper-Bamnolker P. & Samach A. (2005) The flowering integrator FT regulates SEPALLATA3 and FRUITFULL accumulation in Arabidopsis leaves. Plant Cell, 17, 2661-2675.

Toufighi K., Brady S.M., Austin R., Ly E. & Provart N.J. (2005) The Botany Array Resource: e-Northerns, Expression Angling, and promoter analyses. Plant J, 43, 153-163.

Trapnell C., Pachter L. & Salzberg S.L. (2009) TopHat: discovering splice junctions with RNA-Seq. Bioinformatics, 25, 1105-1111.

Trapnell C., Roberts A., Goff L., Pertea G., Kim D., Kelley D.R., Pimentel H., Salzberg S.L., Rinn J.L. & Pachter L. (2012) Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc, 7, 562-578. White E.J., Venter M., Hiten N.F. & Burger J.T. (2008) Modified Cetyltrimethylammonium bromide method improves robustness and versatility: The benchmark for plant RNA extraction. Biotechnology Journal, 3, 1424-1428.

Woolard D.D.e.a. (2012) USE OF ABSCSIC ACID COMBINATIONS FOR THINNING US 2012/0220458 Al.

Zaharia E.I., Galka M.M., Ambrose S.J. & Abrams S.R. (2005) Preparation of deuterated abscisic acid metabolites for use in mass spectrometry and feeding studies. Journal of Eabelled Compounds and Radiopharmaceuticals, 48, 435-445. The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. The means, materials, and steps for carrying out various disclosed functions may take a variety of alternative forms without departing from the invention.

Claims

1. A method of potentiating an abscisic acid (ABA) effect in a plant, comprising a step of applying to the plant: (1) uniconazole, a derivative or an analog thereof; and (2) ABA, a derivative or an analog thereof.

2. The method of claim 1, wherein the ABA effect is fruitlet thinning.

3. The method of claim 1 , wherein the ABA effect is coloring induction of the plant fruit.

4. The method of claim 1, wherein the ABA effect is anthocyanin production.

5. The method according to any one of claims 1 to 4, wherein the uniconazole and ABA are applied sequentially.

6. The method of claim 5, wherein the uniconazole is applied at least 1, 2, 3, 4, or 5 hours before ABA application.

7. The method of claim 5, wherein the uniconazole is applied at least 1 or 2 days before ABA application.

8. The method according to any one of claims 1 to 4, wherein the uniconazole and the ABA are applied concomitantly.

9. The method of claim 8, wherein the uniconazole and the ABA are applied separately.

10. The method according to any one of claims 1 to 9, wherein the plant is a deciduous fruit tree or vine.

11. The method according to any one of claims 1 to 9, wherein the plant is a fruit tree selected from the group consisting of apples, grapes, pears, peaches, plums, nectarines, cherries, apricots, avocados and kiwis.

12. The method of claim 11, wherein the fruit tree is an apple tree.

13. The method of claim 12, wherein the plant is an apple tree and the uniconazole is applied at a fruitlet stage of from 5 to 25 mm king fruit diameter.

14. The method of claim 11, wherein the vine is a grape vine (Vitis viniferd).

15. The method according to any one of claims 1 to 14, wherein the uniconazole is applied after full bloom of the fruit tree.

16. The method of claim 15, wherein the uniconazole is applied at least one day, two days, three days, four days or five days after full bloom.

17. The method according to any one of claims 1 to 16, wherein the uniconazole is applied at a concentration ranging between 0.3 mg/L and 20 g/L.

18. The method according to any one of claims 1 to 16, wherein the uniconazole is applied at a concentration ranging between 0.1 g/L and 5 g/L.

19. The method according to any one of claims 1 to 18, wherein the ABA is S-ABA.

20. The method according to any one of claims 1 to 19, wherein the ABA is applied at a concentration of less than 10 g/L.

21. The method according to any one of claims 1 to 19, wherein the ABA is applied at a concentration of between 0.1 g/L and 2 g/L.

22. The method according to any one of claims 1 to 21, wherein the uniconazole and/or ABA is applied by aerosol spray, pressure spray, direct watering, and/or dipping.

23. The method according to any one of claims 1 to 21, wherein the uniconazole and/or ABA is applied by air blast sprayer.

24. The method according to any one of claims 1 to 23, wherein the method further comprising applying an additional chemical agent that increases abscission.

25. The method according to any one of claims 1 to 24, wherein the ABA is being applied in an amount of at least 10% lower than the amount needed to cause the same level of the at least one ABA effect in the absence of uniconazole.

26. An effective amount of uniconazole for use in a method comprising administering ABA to a plant, wherein the effective amount of uniconazole is selected from the group consisting of (i) an amount enhancing at least one effect associated with the administered ABA in the plant, (ii) an amount reducing the amount of exogenously administered ABA required to produce the effect, and (iii) an amount according to any one of (i) and (ii) wherein said amount is not associated with an undesired side effect of ABA.

27. The effective amount of uniconazole for use according to claim 26, wherein the uniconazole is in a ready to use form.

28. The effective amount of uniconazole for use according to any one of claims 26 to 27, wherein the uniconazole is at a concentration ranging between 0.3 mg/L and 20 g/L.

29. The effective amount of uniconazole for use according to any one of claims 26 to 28, wherein the effect of ABA is fruitlet thinning.

30. The effective amount of uniconazole for use according to any one of claims 26 to 28, wherein the effect of ABA is fruit coloring.

31. An effective amount of ABA for use in a method of enhancing the level of at least one ABA effect in a plant, wherein the method comprising administering uniconazole to the plant, said effective amount of ABA being at least 10% lower than the amount needed to cause the same level of the at least one effect in the absence of uniconazole.

32. The effective amount of ABA for use according to claim 31, wherein the ABA is in a ready to use form.

33. The effective amount of ABA for use according to any one of claims 31 or 32, wherein said amount is ABA concentration of less than 10 g/L.

34. The effective amount of ABA for use according to any one of claims 31 to 33, wherein the ABA effect is fruitlet thinning.

35. The effective amount of ABA for use of any one of claims 31 to 33, wherein the ABA effect is fruit coloring.

36. A combination of uniconazole, a derivative or an analog thereof and ABA, a derivative or an analog thereof.

37. An agricultural composition comprising uniconazole, a derivative or an analog thereof and ABA, derivative or an analog thereof.

38. The agricultural composition of claim 37, wherein the agricultural composition comprising more than 80%, 85%, 90%, or 95% water.

39. The agricultural composition according to any one of claims 37 or 38, wherein the agricultural composition further comprises an agricultural acceptable carrier.

40. The agricultural composition according to any one of claims 37 to 39, wherein the agricultural composition further comprises additives, other thinning agents, growth regulators, and/or other agricultural active substance.

41. The agricultural composition according to any one of claims 37 to 40, wherein the ABA is formulated in a slow-release form.

42. The agricultural composition according to any one of claims 37 to 40, wherein the ABA is encapsulated in a slow-release medium.

43. A kit comprising (i) ABA, a derivative or analog thereof and (ii) uniconazole, a derivative, or an analog thereof, the kit further comprising instruction material directing the use of the ABA and the uniconazole.

44. The kit of claim 43, wherein the ABA and the uniconazole are present in separate reservoirs or containers optionally each in a carrier suitable for its administration.

45. The kit according to any one of claims 43 or 44, wherein the ABA and/or the uniconazole are in a ready to use form.

46. The kit according to any one of claims 43 or 44, wherein the ABA is at a concentration of less than 10 g/L.

47. The kit according to any one of claims 43 or 44, wherein the ABA concentration is about 0.5 g/L.

48. The kit according to any one of claims 43 or 44, wherein the uniconazole is at a concentration ranging between 0.3 mg/L and 20 g/L.

49. The kit according to any one of claims 43 or 44, wherein the uniconazole concentration is about 1 g/L.

50. The kit according to any one of claims 43 to 49, wherein the instruction material directs the use of ABA and uniconazole for potentiating at least one effect associated with ABA activity in a plant.

51. The kit of claim 50, wherein the instructions material directs administering the uniconazole prior to administering the ABA.