CN113557914B - Application of PIFs dark stabilizer in promoting dicotyledon top end hook development - Google Patents

Abstract

The invention relates to an application of a dark stabilizer of PIFs in promoting the development of a top hook of a dicotyledonous plant. In this application, the dark stabilizer comprises an active ingredient comprising a cytokinin and/or a precursor of said cytokinin. Cytokinin can be horizontally stabilized after transcription by stabilizing PIFs in a dark place, so that the maintenance of a top hook in the dark place is promoted, seedling unearthing is assisted, and the degradation process of the PIFs cannot be obviously influenced by the cytokinin under light, so that the top hook cannot be opened after the light is emitted, and subsequent photosynthesis cannot be inhibited.

Description

Application of PIFs dark stabilizer in promoting dicotyledon top end hook development

Technical Field

The invention relates to the technical field of plant cultivation, in particular to application of a dark stabilizer of PIFs in promoting the development of a top hook of a dicotyledonous plant.

Background

The top end hook of the dicotyledon is one of the key structures of early dark form establishment of the plant, is formed by bending the upper end of the hypocotyl of a etiolated seedling growing in a dark place, and can protect young cotyledons from being damaged by mechanical pressure of soil in the unearthing stage of plant buds, so that seeds buried in the soil can be unearthed smoothly. The indexes for measuring the development degree of the top end hook are mainly the bending angle of the upper end of the hypocotyl, and the mutant plants with defects in the development of the top end hook, including ethylene-related mutants and hls1-1 mutants which can not form the top end hook at all, have obvious soil emergence obstacles.

The development of the apical hook is generally divided into three stages: formation (bending from 0 to 180 degrees early in development), maintenance (angle is maintained at 180 degrees during which hypocotyls rapidly elongate to help the plant break out of the ground), and opening after exposure to light (process in which angle rapidly decreases to 0 degrees after exposure to light). Since the nineties of the last century, aggravation of tip hooks has been found as a phenotype of ethylene processing markers, and researchers have conducted intensive studies on the hormone regulation network of tip hooks, and found that plant hormones such as ethylene and gibberellin positively promote tip hooks, whereas hormones such as jasmonate and salicylic acid antagonize the development of tip hooks. In addition, the regulation and control of the development stage of the top end hook are very important, if the angle of the top end hook is intensified too obviously in the early formation stage, the cotyledon is exposed in soil, the plant is not favorable for protecting young and tender cotyledon, and simultaneously the rapid opening of the top end hook after the light is emitted, and the subsequent processes of cotyledon opening, photosynthesis and the like are also not favorable.

Therefore, the chemical regulating agent is used for dynamically regulating and controlling different development stages of the top end hook, so that the emergence of the plant seedling can be helped, the subsequent photosynthesis and early life activities can be carried out, and the emergence rate of the seed and the survival rate of the seedling are improved, so that the application value is important.

Disclosure of Invention

The application discovers that cytokinin can promote the accumulation of an ethylene downstream transcription factor EIN3/EIL1 by promoting ethylene synthesis to enhance the early formation of tip hooks; and the cytokinin can also be horizontally stabilized after transcription by stabilizing transcription Factors PIFs (Phytochrome Interacting Factors, PIFs for short) in the dark, so that the maintenance of a top hook in the dark (such as soil or culture medium) is promoted, the seedling emergence is assisted, the degradation process of the PIFs cannot be obviously influenced by the cytokinin under light, the opening of the top hook after the light exposure cannot be delayed, and the subsequent photosynthesis cannot be inhibited. Therefore, cytokinin or its precursor is a good active ingredient of a dark stabilizer of PIFs and can promote the development of the top hook of dicotyledons.

In this regard, there is a need to provide the use of dark stabilizers of PIFs comprising active ingredients including cytokinins and/or precursors of said cytokinins in the promotion of the development of the apical hook of dicotyledons.

In addition, a preparation method of the dicotyledonous plant seedling is also provided. The seeds prepared by the method have high unearthing rate and high survival rate.

A preparation method of dicotyledonous plant seedlings comprises the following steps:

after placing seeds of dicotyledonous plants in a substrate containing a dark stabilizer of PIFs, preparing seedlings, wherein the dark stabilizer comprises an active ingredient, and the active ingredient comprises cytokinin and/or a precursor of the cytokinin.

In one embodiment, the active ingredient comprises a cytokinin selected from at least one of kinetin, 6-benzylaminopurine, and trans-zeatin.

In one embodiment, the step of preparing the seedling comprises:

placing seeds of said dicot plant in a substrate comprising a dark stabilizer of PIFs to imbibe said seeds; and

and culturing the imbibed seeds in dark, and then culturing the seeds in light to prepare seedlings.

In one embodiment, the concentration of the active ingredient in the matrix is 0.1 μ M to 30 μ M.

In one embodiment, before culturing the seed after imbibition in dark, the method further comprises a step of pregermination.

In one embodiment, the step of pregerminating comprises: and placing the seed after imbibition under illumination for 4-8 hours.

In one embodiment, the dark culture time is 72-108 hours;

and/or the temperature of the dark culture is 18-25 ℃;

and/or the temperature of the illumination culture is 18-25 ℃;

and/or, the dicotyledonous plant is arabidopsis, soybean or tomato.

In one embodiment, the method further comprises the step of sterilizing the seed of the dicotyledonous plant before the step of post-culturing the seed in the substrate containing the dark stabilizers of the PIFs.

Drawings

FIG. 1 is a plant of Arabidopsis thaliana ethylene insensitive mutant ein3eil1 seed and Col-0 Arabidopsis thaliana seed of example 1, which were treated with ACC and KR, respectively, and cultured in the dark for 3.5 days;

FIG. 2 shows the results of phenotype analysis of plants treated with different concentrations of KR and KT from the Arabidopsis thaliana ethylene insensitive mutant ein3eil1 seed and Col-0 Arabidopsis thaliana seed in example 2;

FIGS. 3 to 4 show the hook angle at the top and the plant after the seeds of Col-0 Arabidopsis and the seeds of different mutants of Arabidopsis in example 3 are treated with ACC, KR and KT, respectively;

FIG. 5 shows the expression of TCS gene in the tip hook in example 3;

FIGS. 6 to 7 show the tip hook angle and the plant of the Col-0 Arabidopsis seeds and the Arabidopsis ethylene insensitive mutant ein3eil1 seeds treated with ACC, KR, KT, 6-BA and trans-Zeatin, respectively, in example 3;

FIG. 8 is a phenotypic plot of the apical hook formation period of DMSO and KT treated seeds of the Arabidopsis ethylene insensitive mutant ein3eil1 of example 4;

FIG. 9 is the apical hook angle for 3 days, 3.5 days, 4 days, 4.5 days and 5 days of growth of Arabidopsis thaliana ethylene insensitive mutant ein3eil1 seeds in example 4 treated with DMSO, ACC and KT;

FIG. 10 shows the expression of HLSI/PP2A in the whole plant seedlings of the Arabidopsis ethylene insensitive mutant ein3eil1 seeds treated with DMSO, ACC and KT in example 4;

FIG. 11 shows the distribution of auxin in plants treated with ACC and KT respectively from seeds of the Arabidopsis thaliana ethylene insensitive mutant ein3eil1 of example 4;

FIG. 12 is a graph showing the change with time of the angle of top hook of seeds of Col-0 type Arabidopsis thaliana, seeds of Arabidopsis thaliana ethylene insensitive mutant ein3eil1 and seeds of Arabidopsis thaliana pifq mutation after treatment with ACC and KT, respectively, in example 4;

FIGS. 13 to 14 show the results of the change of the plant and the angle of the top hook with the illumination time after KT and ACC treatment of the seed of the transgenic plant PIF4ox/pifq in example 5;

FIGS. 15 to 16 show the results of the change of the plant and the angle of the top hook with the illumination time after KT and ACC treatment of the seed of the transgenic plant PIF5ox/pifq in example 5;

FIG. 17 shows the change of the content of GFP protein and HSP90 protein in the transgenic plant of example 5, in plants treated with KT, in seeds of the plant PIF4ox/pifq with respect to illumination time;

FIG. 18 shows the change of the content of GFP protein and HSP90 protein in the plants of transgenic plant PIF5ox/pifq of example 5 after seed treatment with KT as a function of the time of light.

Detailed Description

The present invention will now be described more fully hereinafter for purposes of facilitating an understanding thereof, and may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete. The terms "first," "second," and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Herein, DMSO refers to the abbreviation for dimethyl sulfoxide; ACC is an abbreviation for 1-aminocyclopropane-1-carboxylic acid, an ethylene synthesis precursor; KT is an abbreviation of Kinetin and refers to Kinetin, and the chemical structural formula of KT is as follows:

KR is an abbreviation for Kinetin Riboside, a nuclear glycated Kinetin, and has the chemical structural formula:

in general, the term "plant" relates to any of the various photosynthetic multicellular organisms of the kingdom Plantae that characteristically grow by cell division, contain chloroplasts, and have cell walls composed of cellulose. In particular, plants are intended to include, but are not limited to, angiosperms, which may be dicotyledonous plants.

The application finds that the cytokinin can promote the accumulation of ethylene downstream transcription factors EIN3/EIL1 by promoting ethylene synthesis to enhance the early formation of tip hooks; and cytokinin can also be subjected to horizontal stabilization after transcription by stabilizing the PIFs in a dark place, so that the maintenance of top hooks in the dark place (such as soil or culture medium) is promoted, the seedling emergence is assisted, and the degradation process of the PIFs cannot be obviously influenced by the cytokinin under light, so that the opening of the top hooks after the light exposure cannot be delayed, and the subsequent photosynthesis cannot be inhibited.

Based on the above, one embodiment of the present application provides the use of a dark stabilizer of PIFs, the active ingredient of which comprises a cytokinin and/or a precursor of the cytokinin, for promoting the development of the apical hook of dicotyledonous plants. It is to be noted that, in the present context, a cytokinin precursor refers to a substance that is converted into cytokinins during the development of the seed into seedlings. In some embodiments, the precursor of cytokinin is a nuclear glycated cytokinin. For example, the precursor of kinetin is nuclear glycated kinetin. Optionally, the PIFs are PIF4 and/or PIF5.

Optionally, the active ingredient of the dark stabilizer comprises at least one selected from the group consisting of Kinetin (KT), nuclear glycated Kinetin (KR), 6-benzylaminopurine (6-BA), and trans-Zeatin (trans-Zeatin). In some embodiments, the dark stabilizer is a cytokinin and/or a precursor of a cytokinin. In this case, the stabilizer in the dark has only the active ingredient and does not include other adjuvants. In other embodiments, the dark place stabilizer includes other adjuvants in addition to the active ingredient.

In addition, an embodiment of the present application further provides a method for preparing a dicotyledonous plant seedling, which includes steps S110 to S120:

and step S110, sterilizing the dicotyledonous plant seeds.

Alternatively, the dicot is arabidopsis, soybean or tomato. In this embodiment, the dicot is arabidopsis thaliana.

Optionally, the dicot seeds are sterilized with ethanol. Specifically, the step of sterilizing the dicotyledonous plant seed comprises: soaking the seeds in 75% ethanol for more than 10 min. Further, in the step of the sterilization treatment, the soaking time is 10 to 25 minutes. In one specific example, the time for soaking is 15 minutes. It is understood that in other embodiments, other disinfection reagents may be used to disinfect dicotyledonous seeds, and correspondingly, the disinfection steps may be adapted.

It is understood that in some embodiments, step S110 may be omitted. For example, the purchased seeds are seeds that have been sterilized.

Step S120, putting the dicotyledonous plant seeds into a matrix containing a dark stabilizer of PIFs to make the seeds imbibe.

In particular, dark stabilizers of PIFs, as described above, include active ingredients including cytokinins and/or precursors of the cytokinins. Further, the precursor of cytokinins is nuclear glycated cytokinins.

Optionally, the active ingredient of the dark stabilizer comprises at least one selected from the group consisting of Kinetin (KT), nuclear glycated Kinetin (KR), 6-benzylaminopurine (6-BA), and trans-Zeatin (trans-Zeatin).

Optionally, the concentration of the active ingredients of the dark stabilizers of the PIFs in the matrix is between 0.1. Mu.M and 30. Mu.M. Further, the concentration of the active ingredients of the dark stabilizers of PIFs in the matrix is 1. Mu.M to 10. Mu.M.

In an alternative embodiment, the dicot seed is a Col-0 Arabidopsis seed, and the concentration of the active ingredient of the dark stabilizers of the PIFs in the substrate is from 5 μ M to 15 μ M.

In another alternative embodiment, the dicotyledonous plant seed is an Arabidopsis thaliana ethylene insensitive mutant ein3eil1, and the concentration of the active ingredient of the dark stabilizer of the PIFs in the medium is 0.1. Mu.M to 5. Mu.M.

Optionally, the substrate is soil, culture medium or solvent. It is understood that the substrate is not limited to the above, but may be other substances that provide water for seed imbibition and that allow sufficient contact between the seeds and the dark stabilizers of the PIFs.

And S130, culturing the imbibed seeds in dark, then culturing the seeds in light, and culturing the seeds to seedlings.

Specifically, the seed after imbibition is buried in a matrix for dark culture and then is subjected to light culture. Optionally, the substrate here is soil or culture medium.

Dark culture refers to culturing the imbibed seeds in a dark environment. For example, the imbibed seeds are buried in soil. Alternatively, the dark culture time is 72 hours to 108 hours. Further, the dark culture time is 84 to 96 hours. Optionally, the temperature of the dark culture is 18 ℃ to 25 ℃. Further, the temperature of dark culture is 21 ℃ to 23 ℃. It is understood that in other embodiments, the time and temperature of the dark culture can be adjusted for a particular plant. It is understood that, in the dark culture stage, if a transparent container is used to hold the transparent substrate for culturing seeds, the substrate in the culture container should be protected from light.

The illumination culture refers to culturing the substrate embedded with seeds under illumination. Optionally, the temperature of the light culture is 18 ℃ to 25 ℃. Furthermore, the temperature of the light culture is 21 ℃ to 23 ℃. Optionally, the time of the light culture is 1 hour to 24 hours. Further, the time for light culture is 1 to 10 hours. It is understood that in other embodiments, the time and temperature of the light culture may be adjusted according to the particular plant.

In some embodiments, the method further comprises the step of accelerating germination before the step of growing the imbibed seeds by burying the seeds in the matrix. Optionally, the step of pregerminating comprises: placing the seed after imbibition under the illumination for 4-8 hours. Optionally, the temperature during pregermination is 21 ℃ to 23 ℃.

It is understood that in some embodiments, steps S120 and S130 may be combined. At this time, the dicotyledonous plant seeds were buried in a medium culture medium containing a dark stabilizer of PIFs, and then grown to seedlings.

According to the preparation method of the dicotyledonous plant seedlings, the seeds are treated by the dark stabilizer containing the PIFs, so that the top end hooks can be maintained at about 180 degrees before unearthing, unearthing is facilitated, young and tender cotyledons are protected from damage caused by mechanical pressure of soil, the dark stabilizer does not affect degradation of the PIFs after light is emitted, the top end hooks can be rapidly reduced to about 0 degree, subsequent photosynthesis is not affected, and the unearthing rate of the seeds and the survival rate of the seedlings are improved.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The following detailed description is given with reference to specific examples. The following examples are not specifically described, and other components except for inevitable impurities are not included. Reagents and equipment used in the examples, unless otherwise specified, are all conventional in the art. The experimental procedures, in which specific conditions are not indicated in the examples, were carried out according to conventional conditions, such as those described in the literature, in books, or as recommended by the manufacturer. Hereinafter and in the drawings, "Apical hook angle" refers to the top hook angle; "hypocotyl" refers to hypocotyl length; "Root length" means Root length "degrees" is a unit of angle, °.

In the following examples, KT, KR or ACC added to 1/2MS (Murashige and Skoog) medium was added after dissolved in DMSO, and DMSO was added to the medium at a final concentration of 0.1% (v/v), unless otherwise specified. For example, 1/2MS medium containing 10. Mu.M ACC means that ACC is dissolved in DMSO and added to 1/2MS medium at a concentration of 10. Mu.M ACC and 0.1% (v/v) DMSO in the medium. Hereinafter, "0.1% DMSO" means DMSO at a volume percentage concentration of 0.1%.

Example 1

(1) Seeds of dried Arabidopsis ethylene-insensitive mutant ein3eil1 (abbreviated as ein3eil 1) were soaked in 75% ethanol for 15 minutes to sterilize, and then rinsed three times with sterile water and spotted on 1/2MS (Murashige and Skoog) medium containing 0.1% DMSO, 1/2MS medium containing 10. Mu.M ACC and medium containing 10. Mu.M KR. The seeds and medium were then placed in a four-degree freezer together and imbibed for three days. The plates were then removed, placed under light for 4 hours to promote germination and then incubated in the dark at 22 ℃ for 3.5 days (82 hours). The plants were then phenotyped. The results are shown in FIG. 1, 1mm on the scale of FIG. 1.

(2) Dried Col-0 type Arabidopsis seeds (i.e., columbia type 0 Arabidopsis seeds, wild type Arabidopsis seeds) were soaked in 75% ethanol for 15 minutes to sterilize, and then washed three times with sterile water before spotting on 1/2MS medium containing 0.1% DMSO, 1/2MS medium containing 10. Mu.M ACC and 0.1% DMSO, and medium containing 10. Mu.M KR and 0.1% DMSO. The seeds and medium were then placed in a four-degree freezer together and imbibed for three days. The plates were then removed, placed under light for 4 hours to promote germination and then incubated in the dark at 22 ℃ for 3.5 days (82 hours). As a result, as shown in FIG. 1, the length of the scale bar in FIG. 1 was 1mm.

As can be seen in fig. 1, KR increases the apical hook angle of the ethylene insensitive mutant ein3eil1 from 90 degrees to around 180 degrees, which indicates that KR does not rely on ethylene signaling to regulate apical hook development.

Example 2

Soaking the dried Col-0 type Arabidopsis seeds and the seeds of the Arabidopsis ethylene insensitive mutant ein3eil1 in 75% ethanol for 15 minutes respectively for disinfection; then, the mixture is respectively washed by sterile water for three times and then is spotted in 1/2MS culture media containing KR with different concentrations and 1/2MS culture media containing KT with different concentrations. The seeds and medium were then placed in a four-degree freezer together and imbibed for three days. The plates were then removed, placed under light for 4 hours to promote germination, and then incubated in the dark at 22 ℃ for 3.5 days. The plants were then phenotyped (measuring tip hook angle, hypocotyl length and root length) and the results are shown in table 1 and figure 2. In FIG. 2, "Chemical concentrations" means the concentration of a compound, that is, KT or KR.

TABLE 1

As can be seen from FIG. 2, the action effect (tip hook bending) of KT and KR on Arabidopsis seeds shows high consistency, and the optimum concentration of KT and KR for promoting the tip hook of ein3eil1 is about 1 μ M, while the optimum concentration of KT and KR for promoting the tip hook of wild type (Col-0) does not obviously promote the tip hook of wild type plants, and both KT and KR can promote the tip hook of wild type plants at high concentration (10 μ M). Therefore, by controlling the applied concentrations of KR and KT, precise control of the top hook for different periods (formation and maintenance) can be achieved.

It can be seen from table 1 that the tip hook angle is more sensitive to KT and KR than to hypocotyl and root, and that the promotion of KT and KR to hypocotyl and root is mainly dependent on ethylene (as indicated by mutant ein3eil1 requiring higher concentration to achieve the same effect), whereas the tip hook is not. Also, as can be seen from fig. 2 and table 1, after application of KT and KR, the tip hooks of arabidopsis wild-type Col-0 and ein3eil1 both developed to different degrees, and the tip hook angle of ein3eil1 was more sensitive to KT or KR (less EC 50) than the wild-type. The tip hook angle increases with increasing KT and KR concentrations before the maximum concentration is reached, showing a concentration dependence. Thus, cytokinins exist to regulate apical hook in an ethylene-independent manner.

Example 3

Soaking dried Col-0 type Arabidopsis seeds, arabidopsis thaliana cytokinin receptor double mutant ahk2/3 (ahk 2/3) seeds, arabidopsis thaliana cytokinin receptor double mutant ahk2/4 (ahk 2/4) seeds, arabidopsis thaliana cytokinin receptor double mutant ahk3/4 (ahk 3/4) seeds, and Arabidopsis thaliana cytokinin downstream transcription factor triple mutant arr1/11/12 (arr 1/11/12) seeds in 75% ethanol for 15 minutes respectively to sterilize; then, they were each washed three times with sterile water and spotted on 1/2MS medium containing 0.1% DMSO (as a blank control), 1/2MS medium containing 10. Mu.M ACC, 1/2MS medium containing 10. Mu.M KR and 1/2MS medium containing 10. Mu.M KT, respectively. The seeds were then imbibed for three days with the medium in a four-degree freezer. The plates were then removed, placed under light for 4 hours to promote germination and then incubated in the dark at 22 ℃ for 3.5 days. The plants were then photographed and phenotyped (measuring the tip hook angle) and the results are shown in fig. 3 and 4, with the scale bar in fig. 4 representing 1mm.

In addition, dried transgenic Arabidopsis seeds (TCS:: GFP) were soaked in 75% ethanol for 15 minutes to be sterilized; then, they were each spotted on 1/2MS medium containing 0.1% DMSO (as a blank control), 1/2MS medium containing 10. Mu.M KR and 1/2MS medium containing 10. Mu.M KT, after three washes with sterile water, respectively. The seeds were then imbibed for three days with the medium in a four-degree freezer. The plates were then removed, placed under light for 4 hours to promote germination and then incubated in the dark at 22 ℃ for 3.5 days. The plants were then sectioned and observed under a laser confocal microscope ((Zeiss LSM-880) (excitation wavelength: 488nm, emission wavelength detected 510-525 nm) and the results are shown in FIG. 5, the length of the scale bar in FIG. 5 representing 50 μm.

As can be seen from FIGS. 3 and 4, the cytokinin-treated mutants had tip hook angles smaller than that of the KT-and KR-treated wild-types (Col-0). As can be seen from FIG. 5, both KT and KR can promote the expression of TCS (TCS is a cytokinin signal response element, i.e., where cytokinin signal is activated, green fluorescent protein is expressed), which also indicates that both KT and KR can activate cytokinin signal. As can be seen from fig. 3 to 5, the action effects of KT and KR are reduced to some extent in cytokinin signaling mutants, and both of them can activate cytokinin signaling reporter gene TCS: : expression of GFP.

From the above, both KT and KR regulate apical hook by cytokinin signaling, and it was confirmed by the present application that classical cytokinins such as 6-benzylaminopurine (6-BA) and trans-Zeatin (trans-Zeatin) have similar functions to KR except KT (FIG. 6 to FIG. 7, and the length of scale bar in FIG. 7 indicates 1 mm). Thus, cells have ethylene independent regulation of apical hook.

Example 4

Soaking dry Col-0 type Arabidopsis seeds and Arabidopsis ethylene insensitive mutant ein3eil1 seeds in 75% ethanol for 15min respectively for disinfection; then, they were each washed three times with sterile water and spotted on 1/2MS medium containing 0.1% DMSO (as a blank) or 1/2MS medium containing 10. Mu.M KT, respectively. The seeds were then imbibed for three days with the medium in a four-degree freezer. The plate was then taken out, placed under light for 4 Hours to promote Germination, placed at 22 ℃ and, after culturing for 48 Hours in the dark on a dynamic plant phenotype platform (Dynaplant), one photograph was taken every one hour, with the results shown in fig. 8 ("HAI" in fig. 8 represents Hours After Germination, waters After Germination).

Soaking dry Col-0 type Arabidopsis seeds and Arabidopsis ethylene insensitive mutant ein3eil1 seeds in 75% ethanol for 15min respectively for disinfection; then, they were each washed three times with sterile water and spotted on 1/2MS medium containing 10. Mu.M ACC and 1/2MS medium containing 10. Mu.M KT, respectively, 1/2MS medium containing 0.1% DMSO as a blank. The seeds and medium were then placed in a four-degree freezer together and imbibed for three days. The plates were then removed, placed under light for 4 hours to promote germination, then placed at 22 ℃ and the phenotype (tip hook angle) was recorded every 12 hours, starting three days after imbibition, and ending five days after imbibition. The results are shown in FIG. 9 ("DAI" in FIG. 9 indicates the post-Imbibition hours, days After inhibition).

HLS1 gene expression level detection: (1) sample preparation: soaking dried Arabidopsis thaliana ethylene insensitive mutant ein3eil1 seeds in 75% ethanol for 15 minutes for disinfection; then, they were each washed three times with sterile water and spotted on 1/2MS medium containing 0.1% DMSO (as a blank control), 1/2MS medium containing 10. Mu.MACC, 1/2MS medium containing 10. Mu.M KT, respectively. The seeds were then imbibed for three days with the medium in a four-degree freezer. And then taking out the plate, irradiating for 4 hours under light to promote germination, placing at 22 ℃, culturing for 5 days in the dark, collecting plant samples in the dark, freezing and storing in liquid nitrogen for subsequent fluorescent quantitative PCR experiments. (2) fluorescent quantitative PCR experiment: after RNA extraction using Eastup Super total RNA extraction kit (Promega Shanghai) and reverse transcription using M-MLV reverse transcriptase, fluorescent quantitative PCR experiments were performed using Light Cycler 480system (Roche) instrument with SYBR Green Mix (Takara) enzyme to detect the RNA transcript levels, the results are shown in FIG. 10.

Asymmetric auxin signal distribution observation: dry transgenic Arabidopsis seeds (DR 5:: GFP/ein3eil 1) were soaked in 75% ethanol for 15 minutes to sterilize; then, they were each spotted on 1/2MS medium containing 0.1% DMSO (as a blank control), 1/2MS medium containing 10. Mu.M ACC and 1/2MS medium containing 10. Mu.M KT, after three washes with sterile water, respectively. The seeds and medium were then placed in a four-degree freezer together and imbibed for three days. The plates were then removed, placed under light for 4 hours to promote germination and then incubated in the dark at 22 ℃ for 3.5 days. The plants were then sectioned and observed under a laser confocal microscope ((Zeiss LSM-880) (excitation wavelength: 488nm, emission wavelength detected 510-525 nm) the results are shown in FIG. 11.

As can be seen from fig. 8, cytokinin can promote maintenance of the apical hook in the dark in an ethylene-independent manner; as can be seen from FIG. 9, the effect of cytokinin in promoting the maintenance of the tip hook of the ethylene-insensitive mutant ein3eil1 was at least five days after the culture; as can be seen from FIG. 10, cytokinins have an ethylene-independent pathway for promoting apical hook development, which is capable of promoting expression of the apical hook development-associated gene HLS1 in the ein3eil1 mutant; as can be seen from fig. 11, cytokinins have an ethylene-independent pathway that promotes the development of apical hooks, which promotes asymmetric distribution of auxin, resulting in maintenance of apical hooks. It can be excluded from FIG. 11 that cytokinin itself causes asymmetric growth at the tip hook by affecting cell division.

From FIGS. 8-11, it is clear that cytokinins strongly promote the maintenance of apical hooks, which enables ein3eil1 to remain closed (180 degrees) five days after culture, while also activating the expression of HOOKLESS1 and the asymmetric distribution of auxin (the most fundamental pathway for two apical hook development), compared to untreated plants in which apical hooks are now and almost completely open.

Mutant phenotype dynamic observation experiment: soaking dry Col-0 type Arabidopsis seeds, an Arabidopsis ethylene insensitive mutant ein3eil1 and an Arabidopsis photoreceptor interaction factor quadruple mutant pifq in 75% ethanol for 15 minutes respectively to sterilize; then, after three washes with sterile water, respectively, were spotted on 1/2MS medium containing 0.1% DMSO (as a blank control), 1/2MS medium containing 10. Mu.M ACC and 1/2MS medium containing 10. Mu.M KT. The seeds and medium were then placed in a four-degree freezer together and imbibed for three days. The plates were then removed, placed under light for 4 hours to promote germination and incubated in the dark at 22 ℃. The phenotype (angle of tip hook) of the different mutants under different treatments was then counted every 12 hours starting 3 days after imbibition, and the results are shown in figure 12. In FIG. 12, pifq is a quadruple mutant of PIF (PIF 1 gene, PIF3 gene, PIF4 gene and PIF5 gene are deleted in addition to Col-0, abbreviated as pifq).

In fig. 12, in the wild type Col-0, the tip hook angles of the KT-treated group and the ACC-treated group showed similar degree of exacerbation (around 260 degrees), while the tip hook angle of the KT-treated group was maintained for a longer time. Furthermore, in ein3eil1, KT still has this function. In pifq, the tip hook angle of pifq cannot be maintained in the dark regardless of the KT process. From this, it is known that the maintenance of the apical hook by cytokinin (KT) is independent of ethylene signal, but dependent on the presence of PIF. That is, cytokinin exhibits a more permanent property in promoting tip hook maintenance, capable of significantly prolonging its maintenance time in the dark, compared to ethylene, another hormone capable of strongly promoting tip hook exacerbation, and this effect is dependent on the presence of PIFs proteins.

Example 5

To verify that cytokinin treatment did not prevent post-exposure opening of the top hook, thereby inhibiting subsequent development and photosynthesis, experiments were performed using transgenic plants overexpressing PIF4 and PIF5. The specific process comprises the following steps:

(1) Transgenic plant light treatment phenotype experiments: soaking seeds of the dried Arabidopsis PIF4 overexpression line (PIF 4 ox/pifq) and seeds of the dried Arabidopsis PIF5 overexpression line (PIF 5 ox/pifq) in 75% ethanol for 15 minutes respectively for sterilization; then, after three washes with sterile water, the cells were spotted on 1/2MS medium containing 0.1% DMSO (as a blank control), 1/2MS medium containing 10. Mu.M ACC, and 1/2MS medium containing 10. Mu.M KT. The seeds were then imbibed for three days with the medium in a four-degree freezer. The plates were then removed, placed under light for 4 hours to promote germination and then incubated in the dark at 22 ℃ for 3.5 days, after which the plants were placed under constant light for 0 hours, 6 hours, and 12 hours before recording the phenotype (top hook angle), respectively. The experimental results are shown in fig. 13 to 16, in which: FIG. 13 is a chart showing statistics of the phenotype data of PIF4ox/pifq ("HAE" in FIG. 13 indicates Hours After Exposure to light: hours After After Exposure, same applies hereinafter); FIG. 14 is a table of representative plants from the different treatment groups of FIG. 13; FIG. 15 is a graphical representation of the phenotypic data for PIF5 ox/pifq; FIG. 16 is a table of representative plants from the different treatment groups of FIG. 15.

(2) Western blot experiments: soaking seeds of a dried Arabidopsis thaliana PIF4 overexpression line (PIF 4 ox/pifq) and seeds of a dried Arabidopsis thaliana PIF5 overexpression line (PIF 5 ox/pifq) in 75% ethanol for 15 minutes respectively for sterilization; then, after three washes with sterile water, the cells were spotted on 1/2MS medium containing 0.1% DMSO (as a blank control), 1/2MS medium containing 10. Mu.M ACC, and 1/2MS medium containing 10. Mu.M KT. The seeds and medium were then placed in a four-degree freezer together and imbibed for three days. The plates were then removed, irradiated with light for 4 hours to promote germination and cultured in the dark at 22 ℃ for 3.5 days, after which the plants in the DMSO-treated group were cryopreserved in liquid nitrogen after 0min, 15min, 30 min and 45 min of light treatment, respectively, and the plants in the KT-treated group were cryopreserved in liquid nitrogen after 0min, 15min, 30 min, 45 min and 60 min of light treatment, respectively.

Total proteins of plants were extracted with 2 XSDS buffer (60 mM Tris-HCl, pH 6.8,25%213glycerol,2% SDS, 14.4M. Beta. -merictoethanol, 0.1% bromophenol, and 1MDTT), respectively, followed by electrophoresis on 7.5% SDS polyacrylamide gel, followed by electrotransfer to PVDF membrane for hybridization and development with the corresponding Anti-GFP or Anti-Actin antibodies, as shown in FIGS. 17 and 18. In fig. 17 and 18, "L0m" represents a group of 0min of light exposure, "L15m" represents a group of 15min of light exposure, and the like.

As can be seen from fig. 13 to 16, the transgenic plants PIF4ox/pifq and PIF5ox/pifq treated with KT showed extremely strong tip hook aggravation in the early stage, but the tip hook had a significant opening tendency after 6 hours of the light treatment, and the angle was substantially halved and decreased to about 90 degrees after 12 hours. In contrast, the ethylene-treated transgenic plants PIF4ox/pifq and PIF5ox/pifq, although also capable of inducing apical hook exacerbation, responded to light more slowly than cytokinin, showed no significant opening trend for 6 hours of light exposure, and returned to an angle of around 180 degrees only for 12 hours of treatment.

It can be seen from FIGS. 17 and 18 that KT can promote the accumulation of PIFs in the dark (light treatment, 0HAE), and when plants are exposed to light, the treatment of KT does not significantly delay the degradation of PIFs, and after about one hour of light, the PIFs are degraded and become almost similar to the phenotypic conclusion.

From the above, the characteristic that cytokinin can promote the maintenance of top hooks in dark places and rapidly open under light provides a powerful guarantee for the emergence of young plants without affecting the subsequent photosynthesis and growth under light.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, so as to understand the technical solutions of the present invention specifically and in detail, but not to be understood as the limitation of the protection scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. It should be understood that the technical solutions provided by the present invention, which are obtained by logical analysis, reasoning or limited experiments by those skilled in the art, are within the scope of the present invention as set forth in the appended claims. Therefore, the protection scope of the present invention should be subject to the content of the appended claims, and the description and the drawings can be used for explaining the content of the claims.

Claims (8)

1. Use of a dark stabilizer of PIFs to maintain dicotyledonous top hooks in the dark and not to affect the opening of said top hooks after exposure to light, characterized in that said dark stabilizer comprises an active ingredient comprising kinetin and/or ribosylated kinetin.
2. 2. A method of maintaining dicot top hooks in the dark using dark stabilizers of PIFs without affecting the opening of the top hooks after exposure to light, comprising the steps of:

   after putting seeds of dicotyledonous plants in a substrate containing a dark stabilizer of PIFs, culturing, and preparing seedlings, wherein the dark stabilizer comprises an active ingredient, and the active ingredient comprises kinetin and/or ribosylated kinetin.
3. 3. The method according to claim 2, characterized in that the step of preparing the seedlings comprises:

   placing seeds of said dicot plant in a substrate comprising a dark stabilizer of PIFs to imbibe said seeds; and

   culturing the imbibed seeds in dark and then culturing the seeds in light to prepare seedlings.
4. 4. The method according to claim 3, wherein said active ingredient is present in said matrix at a concentration of 0.1 μ M to 30 μ M, using a dark stabilizer of PIFs to maintain dicotyledonous tip hooks in the dark and not to affect the opening of said tip hooks after exposure to light.
5. 5. The method according to claim 3, characterized in that it comprises a step of pregermination before culturing the imbibed seeds in dark, said method comprising the step of culturing the imbibed seeds in dark with a dark stabilizer of PIFs to maintain dicotyledonous plant tip hooks in dark and without affecting the opening of said tip hooks after exposure to light.
6. 6. The method according to claim 5, characterized in that said step of pregerminating comprises the steps of: and placing the seed after imbibition under illumination for 4-8 hours.
7. 7. The method of claim 4, wherein said dark cultivation is carried out for 72-108 hours, using a dark stabilizer of PIFs to maintain dicotyledonous plant top hooks in the dark and without affecting the opening of said top hooks after light exposure;

   and/or the temperature of the dark culture is 18-25 ℃;

   and/or the temperature of the illumination culture is 18-25 ℃;

   and/or, the dicotyledonous plant is arabidopsis, soybean or tomato.
8. 8. The method for maintaining the dicotyledonous plant top end hook in the dark by using the PIFs dark place stabilizer according to any one of claims 2 to 7, wherein the method does not influence the opening of the top end hook after the light is emitted, and is characterized by further comprising a step of disinfecting the dicotyledonous plant seeds before the step of putting the dicotyledonous plant seeds into a substrate containing the PIFs dark place stabilizer for post-culture.

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