KR20210034514A - A Method for De novo Root Regeneration of Plant based on an Ultradian Rhythm - Google Patents

Abstract

The present invention relates to a method for promoting new root regeneration in ultradian rhythm-based plants. The method comprises a step of transforming a plant with a gene for encoding an enhancer of ABA co-receptor 1 (EAR1). The plant is Arabidopsis. According to the present invention, new root regeneration can be promoted.

Description

A Method for De novo Root Regeneration of Plant based on an Ultradian Rhythm}

The present invention is a plant transformed by a gene encoding EAR1 (Enhancer of ABA co-Receptor 1), a method for promoting new root regeneration of a plant comprising the step of transforming the plant with a gene encoding EAR1, and encoding EAR1 It relates to a method for inhibiting aging of a plant comprising the step of transforming the plant with the gene.

Biological rhythms exist in several species and play an important role in the health and well-being of organisms (Laje et al., Front Integr Neurosci. 2018 Mar 13; 12:10). In addition to circadian rhythms (CR) with a period of ~24 hours, short rhythms, called ultradian rhythms (UR), represent periods ranging from seconds to hours. UR is a bacterium (Cerveny et al., Proc Natl Acad Sci USA. 2013 Aug 6;110(32):13210-5), plants (Iijima and Matsushita, J Plant Physiol. 2011 Nov 15;168(17):2072- 80) and animals (den Boon et al., Endocrinology. 2019 Apr 1;160(4):791-802). UR tends to be considered a fragmentary event because of its intermittent, transient, and aperiodic pattern, but control of hibernation (Diatroptov et al., Bull Exp Biol Med. 2019 Dec;168(2):291-294), hormone secretion pattern. (Steyn and Ngo, Best Pract Res Clin Endocrinol Metab. 2017 Dec;31(6):521-533), rapid eye movements during sleep in mammals (Weber et al., Nat Commun. 2018 Jan 24;9(1): 354), metabolic cycle (Brodsky, Biochemistry (Mosc). 2014 Jun;79(6):483-95) and gene expression (van der Veen and Gerkema, FASEB J. 2017 Feb;31(2):743-750) It is widespread in a variety of biological processes, including. Destructive UR has a detrimental effect on the development of brain, spine and ribs in animals (Santorelli et al., ACS Synth Biol. 2018 May 18;7(5):1447-1455), damage to the root structure of plants (Moreno-Risueno et al., Science. 2010 Sep 10;329(5997):1306-11), induces metabolic syndrome in humans, Cushing's disease, Alzheimer's disease and cancer (den Boon and Sarabdjitsingh, Best Pract Res Clin Endocrinol Metab. 2017 Octocrinol Metab. ;31(5):445-457; Flynn et al., Ann Endocrinol (Paris). 2018 Jun;79(3):112-114). Despite the broad presence and essential role in physiological regulation, UR regulation remains unclear. Little is known about the regulatory mechanisms driving UR or the mechanisms underlying their functional significance. UR in plants is understood phenomenologically (Solheim et al., New Phytol. 2009;183(4):1043-52).

Many plant organs, such as new branches and flowers, are produced in the later stages. Plant cells are highly reprogrammable and have amazing plasticity and regenerative abilities. Because of this regenerative ability, plants can replace dead tissues and organs, and each individual plant cell has the ability to regenerate the whole plant (Ikeuchi et al., Development. 2016 May 1;143(9):1442-51 ). This unique ability allows plants to withstand adverse conditions such as wound stress, insect and herbivore attacks, and adverse climates despite being immobile (Sugimoto et al., Curr Opin Plant Biol. 2019 Feb;47:138- 150). De novo organogenesis allows plants to form new shoots or roots from explants, and is a powerful regenerative mechanism that is evident both in nature and in vitro. It has been used for propagation of rare genotype plants in agriculture, horticulture and forestry through cutting or grafting techniques (Sang et al., New Phytol. 2018 Jun;218(4):1334-1339). Resected Arabidopsis thaliana leaves are capable of regenerating adventitious roots (ARs) in medium without B5 hormone (Chen et al., Front Plant Sci. 2014 May 15; 5:208). The root production capacity of Arabidopsis leaf explants has been used to study the molecular mechanisms underlying de novo root regeneration (DNRR) as well as general plant regeneration. Leaf explants trigger the biosynthesis of compounds required for adventitious root initiation by detecting early signals including wound stress, environmental signals, and developmental stages (Pan et al., J Genet Genomics. 2019 Mar 20;46(3):133- 140). After activation of the initial signal, the level of endogenous auxin, which is a key element of DNRR, increases in the leaf blade, and excessive auxin causes the regenerative ability such as pericycle or procambium cells in the vascular tube. Move to the petiole where the cell is located (Bustillo-Avendano et al., Plant Physiol. 2018 Feb;176(2):1709-1727). When auxin accumulates in cells with regenerative capacity, cell fate changes occur, forming root-generating cells, followed by root primordium, root apical meristem, and finally adventitious roots (Bustillo). -Avendano et al., Plant Physiol. 2018 Feb;176(2):1709-1727). Although recent studies have revealed to some extent the molecular mechanisms underlying DNRR, much of this process, which is critical to survival, remains unclear.

Accordingly, the present inventors confirmed that the superweek rhythm (UR) caused by leaf excision in Arabidopsis induces new root regeneration (DNRR) through various biological processes, and completed the present invention. I did.

The information described in the background section is only for improving an understanding of the background of the present invention, and thus does not include information forming the prior art known to those of ordinary skill in the art to which the present invention pertains. May not.

It is an object of the present invention to provide a plant in which new root regeneration is promoted based on the rhythm of Choju-il.

Another object of the present invention is to provide a method for promoting new root regeneration of plants and a method for inhibiting aging of plants.

In order to achieve the above object, the present invention provides a plant transformed by a gene encoding EAR1 (Enhancer of ABA co-Receptor 1).

The present invention also provides a method for promoting de novo root regeneration of a plant comprising the step of transforming the plant with a gene encoding Enhancer of ABA co-Receptor 1 (EAR1).

The present invention also provides a method for inhibiting aging of a plant comprising transforming the plant with a gene encoding EAR1 (Enhancer of ABA co-Receptor 1).

According to the present invention, since it is possible to promote new root regeneration through various biological processes based on the rhythm of the second week in cut Arabidopsis leaves, it can be usefully used in various fields such as agriculture, horticulture and forestry, as well as general plant regeneration have.

1 is a diagram showing that the generation of a second week rhythm (UR) in a three-hour period is independent of a circadian clock.
(A) A diagram showing the experimental setup used to measure the luciferase (LUC) signal in the excised leaves. The third and fourth rosette leaves were excised from 21-day-old transgenic Arabidopsis plants expressing the ORE1::LUC structure, and the LUC intensity was measured every 30 minutes at 22° C. under continuous white light (LL) conditions using a CCD camera. .
(B) In Arabidopsis leaves excised at the indicated time points. ORE1 It is a diagram showing promoter activity. Promoter activity was measured in 24 leaves (n = 24), and representative data of 3 leaf samples are shown. At least 3 different experiments were conducted showing similar results.
(C) Wavelet (wavelet) analysis result. Using the ORE1 promoter activity (top panel), signals were detected in both -24 hours circadian rhythm (CR; left) and -3 hours UR (right). The black line represents the LUC intensity, and the line reconstructed by wavelet analysis is displayed in red. The wavelet power plot (bottom panel) was plotted using the wavelet-derived period. Red and blue represent high and low wavelet powers, respectively, and the plot shows the approximate pattern of each rhythm.
(D and E) A diagram showing the wavelet power of CR(D) and UR(E) in the promoter activity of clock-related genes quantified by wavelet analysis. Wavelet power values (Y-axis) were plotted against periods (X-axis), and data represent mean ± standard error of mean (SEM) ( n = 24).
(F) A diagram showing the phase distribution of the ORE1 and CCA1 promoter activity patterns. The direction of the arrow represents the average of the phases, and the length (r) of the arrow represents the inverse proportion of the phase standard deviation. Each dot represents the phase measured on one individual leaf ( n = 24), and black and red represent CR and UR, respectively.
(G) It is the result of box-plot analysis of the phase distribution of the promoter activity of the clock-related gene ( n = 24).
(H) ORE1 promoter activity was shown in the circadian clock variants toc1 and elf4 , and three representative samples are shown ( n = 24), and at least three other experiments showing similar results were performed.
2 is a diagram showing that UR has a strong correlation with de novo root regeneration (DNRR).
(AC) In leaves excised from ORE1 ::LUC plants of various ages, ORE1 It is a diagram showing promoter activity (A), wavelet power (B) and rooting rate (C). The fourth leaf was excised from the indicated plants and placed in a sucrose-free half-strength B5 (½ B5) medium under LL conditions at 22°C. In (A) 24 leaves were measured ( n = 24), and representative data for 3 samples were shown. Root regeneration was measured based on root tip emergence at the indicated time point (DAC; days after culturing). Data represent the mean ± SEM of 3 independent biological replicates ( n = 10).
(DF) A diagram showing ORE1 promoter activity in cut leaves cultured at 22°C (D), 17°C (E) and 27°C (F). The top panel shows three representative samples ( n = 24) and the bottom panel shows the merged UR wavelet power plot of all samples with low transparency.
(G) A diagram showing the rooting rate of ORE1 ::LUC leaves at different temperatures. Fourth leaves excised from 21 day old plants were incubated at the indicated temperatures. Data represent the mean ± SEM of 3 independent biological replicates ( n = 10).
ORE1 in the treated as; (cytokinin BA) or methyl jasmonate (MeJA) leaf; (H) plant hormone (control) are not treated or 0.2μM indole-3-acetic acid ( IAA auxin), 6-benzyladenine It is a diagram showing promoter activity. The graph shows data from three representative samples ( n = 24).
(I) A diagram showing the UR wavelet power, and the data was measured by wavelet analysis of the sample indicated in (H).
(J) A diagram showing the rooting rate of ORE1 ::LUC leaves treated or untreated with plant hormones. Fourth leaves were excised from 21 day old plants, and data represent the mean ± SEM of 3 independent biological replicates ( n = 10).
(K) This is a photograph of a plant hormone-treated or untreated resected leaf. The third or fourth leaves were excised from 21-day-old ORE1 ::LUC plants and placed in ½ B5 medium containing or without (-) 0.2 μM auxin (IAA), cytokinin (CK) or methyl jasmonate (JA). . The small picture shown together in the CK picture is a picture showing an enlarged petiole treated with 1 μM CK. Root playback image was taken at 10 DAC, White bar = 5mm; red bar = 0.1mm.
3 is a diagram showing that a wide variety of Arabidopsis genes exhibit vibrations per week.
(A) Heatmap showing the expression levels of UR oscillating genes belonging to two anti-phasic groups (group 1 and group 2), yellow indicates high expression level, blue indicates low expression level.
(B) A graph showing the promoter activity of two vibrating genes selected based on RNA-seq data, and the graph shows data from three representative samples (n = 24).
(C) As a result of gene ontology (GO) enrichment analysis of the UR vibration gene, the number of group 1 and group 2 genes enriched in each GO term was displayed in different colors.
(D) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis result of UR vibration gene.
(E and F) A diagram showing the UR wavelet power (top panel) and rooting rate (bottom panel) of knockout mutants of genes involved in auxin (E) and cytokinin (F) signaling. UR wavelet power was quantified by wavelet analysis, and rooting rates of variant leaves were normalized for wild-type leaves. Data are expressed as mean ± standard deviation (SD, n = 4), with asterisks indicating statistically significant differences (** p <0.01, *** p <0.001; two-tailed Student's t -test).
4 is a diagram showing that impaired mutations in UR production also exhibit reduced DNRR.
(A) ORE1 ::LUC construct (Col/ ORE1 ::LUC ) or Col/ ORE1 ::LUC homozygous mutant lines derived from EMS mutagenesis of plants (M21, M23, M38 and M83 ) In leaves resected from wild-type (Col-0) plants expressing ORE1 As a diagram showing the promoter activity, the graph shows the data of three representative samples (n = 24).
(BD) A diagram showing UR wavelet power (B), rooting rate (C) and flowering time (D) of leaves excised from wild-type plants and mutant candidates. Data represent the mean ± SEM of 4 independent biological replicates ( n = 10). Wavelet power was measured through wavelet analysis, and the flowering time was recorded as the number of days it took for the plant to show 1 cm of inflorescence.
(E) A diagram showing the UR wavelet power of wild-type and mutant candidates. The third and fourth excised leaves were treated with 0.2 μM IAA (n = 24).
(F) A diagram showing the structure of the rooting rate phenotype by exogenous auxin treatment, and the fourth leaf was excised from a 24-day-old plant and cultured in ½ B5 medium containing 0.2 μM IAA. Data represent the mean ± SEM of 3 independent biological replicates ( n = 10).
(G) A diagram showing GUS staining analysis of whole leaves excised from 21-day-old DR5::GUS Arabidopsis plants, where WT was wild-type (unmutated DR5:: GUS GUS plants); M21 and M38 refer to EMS mutated DR5::GUS lines. Images were captured at 0 DAC, at least 3 different experiments with similar results were performed, Scale bar = 5mm.
(H) Images showing the petioles of the leaves indicated in (G) in 0, 1 and 2 DACs, at least 3 different experiments showing similar results were performed, Scale bar = 5 mm.
5 is a diagram showing that ROU1/EAR1 and Abscisic Acid (ABA) signaling control UR generation and DNRR.
(A) A schematic diagram showing mutations in mutant lines rou1 -1 (M21), rou1 -2 (M23) and rou1 -3 (M83).
(B and C) box plot showing the UR phenotypic structure in EAR1 complementary lines ( COM-9 and COM-24 ) (n = 24) (B), Rooting rates of complementary lines expressing EAR1 ( EAR1::EAR1-GFP ) in rou1 -1 mutant background (C). Fourth leaves were excised from 21-day-old plants and cultured in ½ B5 medium without sucrose. Data represent the mean ± SEM of 3 independent biological replicates ( n = 10).
(D and E) A plot showing UR wavelet power (D) and rooting rate (E) at 10 DACs of excised leaves treated with various concentrations of exogenous ABA.The data in (E) are from 3 independent biological replicates. Mean±SEM is represented ( n =22).
(F) A diagram showing the ABA content of the leaves excised from wild-type and aba2 mutant plants, and the ABA content was measured at the indicated time point. Data represent mean±SEM ( n =4), and at least 2 different experiments were performed showing similar results.
(G and H) A diagram showing promoter activity in wild type (ORE1 ::LUC ) (G) and aba2 mutant (H) leaves, the graph shows three representatives of the measured samples (n = 24). The upper graph shows the data of three representative samples ( n = 24), and the lower panel shows the merged UR wavelet power plot of all samples with low transparency.
(I) A plot showing the rooting rates of wild-type and aba2 mutant leaves, and the data represent the mean ± SEM of 3 independent biological replicates ( n = 10).
6 is a diagram showing that ROU1/EAR1 mediates hormone signaling, regulates UR production and promotes DNRR.
(A) is determined by qRT-PCR in the excised leaves standardized ACT2 expression as a diagram showing the level of expression of ROU1 / EAR1, the data represents the mean ± SEM of three independent biological replicates (n = 6).
Showing the level of expression of ROU1 / EAR1 determined by qRT-PCR in leaf - (B) 10μM auxin (IAA), abscisic acid (ABA), between talkie Nin (BA) or processed, or a non-hormone treated with () As a figure, expression levels were measured at 0h (black) and 24h (red) after resection. Values were first normalized based on ACT2 expression, and then normalized based on each value of the control (-) sample at 0h. Data represent the mean ± SEM of 3 independent biological replicates ( n = 6).
(C) A diagram showing the expression of EAR1 ::GUS in leaves excised from 21-day-old plants at 0 and 1 DAC.
(D) ROU1 :: ROU1 - GFP ROU1 - GFP in the petioles of leaves excised from plants and cultured in the absence of 10 μM auxin (IAA), ABA, cytokinin (BA) or hormones (-) This is a confocal micrograph showing the expression. The end of the white arrow indicates the nucleus, and Scale bar = 0.1mm.
(E) CSV :: GFP (control) and ROU1 :: ROU1 - GFP at 0h and 24h after resection As a figure showing the intensity of the GFP signal in leaves, it was treated with 10 μM auxin (IAA), ABA, cytokinin (BA), or no hormone. GFP intensity values were calculated from the confocal images shown in (C), and the data represent the mean ± SD ( n = 7).
(F) ROU1 :: ROU1 - GFP is a diagram showing the level of EAR1 protein in leaves that were excised from the transformation line and treated with 10 μM auxin (IAA), ABA, cytokinin (BA) or hormone-free (control) leaves. EAR1 protein levels were measured at 0, 8, 24 and 48 h after resection, and α-tubulin was used as a loading control.
(G) A diagram showing the gene expression pattern of cluster 2 containing the EAR1 gene. Clustering was performed on differentially expressed genes (DEGs) in the time series (0, 24, 48, 72 and 96h), and petiole regions of wild type and rou1 -1 mutant leaves were compared.
(H) Heatmap of DNRR-related genes selected from the set of identified DEGs, expression values obtained from RNA-seq data were normalized for comparison.
(I) A diagram showing a model explaining the regulation of UR and DNRR by ROU1/EAR1 and hormone signaling in resected leaves.
7 is a diagram showing the promoter activity of a circadian clock-related gene in resected Arabidopsis leaves.
(A) Arabidopsis excised at the indicated time points As a diagram showing the promoter activity of circadian clock-related genes in leaves, a total of 24 leaves of each genotype were measured ( n = 24), and representative data of 3 samples are displayed. At least 3 different experiments were conducted showing similar results.
(B) A wavelet analysis and phase distribution of the promoter activity of a clock-related gene. Wavelet analysis was performed using the luciferase (LUC) intensity of the clock gene. The top left panel shows the reconstructed signal; The lower left panel shows the wavelet spectrum according to the circadian rhythm (CR) parameter; The middle panel shows the results of Super Week Rhythm (UR). Rayleigh's plot showing the phase distribution is displayed on the right panel, and the arrow direction of the Rayleigh's plot represents the phase average, and the arrow length (r) represents the inverse proportion of the phase standard deviation. Each point represents the phase measured on an individual leaf ( n = 24), and black and red represent CR and UR, respectively.
8 is a diagram showing ORE1 promoter activity in wild-type and circadian clock mutant leaves.
(A) As a graph showing the correlation between CR and UR power, the average values of CR and UR quantified by wavelet analysis from the promoter activity of a clock-related gene were plotted and linear regression analysis was performed.
(B) wild type (Col-0), lhy And elf3 A diagram showing the promoter activity pattern of ORE1 in the mutant, the graph shows three representative samples. At least 3 independent experiments with similar results were performed.
(C) As a result of wavelet analysis of each mutation according to the UR parameter, the average wavelet power (Y-axis) was plotted against the period (X-axis). Data represent mean ± standard error of the mean (SEM) ( n = 24).
9 shows ORE1 in whole seedlings and attached or detached organs. It is a diagram showing promoter activity.
(AC) A diagram showing the activity of the ORE1 promoter in plant organs isolated from plants on days 7 (A), 21 (B) and 35 (C), various plant organs were indicated, and the'resected 'Leaf' means 4th rosette leaf.
10 is a diagram showing that the UR is synchronized under certain dark and low temperature conditions.
(A) A graph showing the ORE1 promoter activity pattern under various light and temperature conditions, LL is continuous light; DD stands for continuous dark condition.
(B) A diagram showing the reconstructed wavelet result of each iteration under various light and temperature conditions.
(C) A diagram showing the reconstructed wavelet result of the average value of the samples under various conditions.
(D) Expression of ORE1 was determined by RT-qPCR and was relatively normalized to ACT2 under synchronized UR conditions. Data represent the mean ± SEM of 3 independent biological replicates ( n = 6).
(E) A graph showing the promoter activity of the vibrating gene identified in the RNA-seq data, and shows the data of three representative samples (n = 24).
11 is a diagram showing that EAR1 is an important UR modulator.
(A) A diagram showing the structure of the UR phenotype after mutual crossing between the parent (Col / ORE1 ::LUC ) and mutation candidates (M21, M23, M38 and M83) or two mutation candidates. '+' is the restored UR; '-' for unstructured UR; 'x' means do not intersect.
(B) rou1 -1 (M21) determined by whole genome sequencing and in view of the rou1 -3 (M83) genome total distribution of mutations in the chromosome of each line, the position of the detected SNP in the mutant (X- axis) and the allele (allele) Frequency (Y- axis) being shown. Gene ID represents the amino acid substitutions could lead to GC-to-AT (EMS effects ) that a candidate gene for holding a mutation, a red asterisk rou1 rou1 -1 and - represents a common gene in all three lines.
(C) wild type, rou1 -1, rou1 -2 and In rou1 -3 genotypes is a view showing the amino acid sequence of the EAR1.
12 is a diagram showing clustering of gene expression data.
(A) a diagram showing the expression patterns of differentially expressed genes (DEG), clustering analysis, wild-type and rou1 - it was carried out using the temporal expression pattern of DEG in the first leaf.
(B) Heatmap of DNRR-related genes. Genes marked with superscript UR refer to genes whose expression follows UR, and expression values obtained from RNA-seq data were normalized for comparison.
(C) A Venn diagram showing the overlap between DNRR-related genes, DEG, UR vibration genes, and auxin-, cytokinin (CK)-, and abscisic acid (ABA)-related genes.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by an expert skilled in the art to which the present invention belongs. In general, the nomenclature used in this specification is well known and commonly used in the art.

In the present invention, it was confirmed the presence of ultradian rhythms (UR) of a 3-hour period that promotes de novo root regeneration (DNRR) in resected Arabidopsis leaves. As a result of monitoring the promoter activity of circadian rhythms (CR)-related genes in the excised leaves, UR whose oscillation was independent of CR was detected. Cells expressing the UR-cycling gene overlapped with the root area of the petiole region of the excised leaf. In addition, UR-related gene expression showed a strong correlation with DNRR in response to internal and external factors, suggesting that the two processes are linked. Transcriptome profiling identified 4,073 oscillating genes in a variety of biological processes, including the hormonal pathway linked to DNRR. The efficiency of DNRR was reduced in mutants of auxin- and cytokinin (CK)-related genes. Using a forward genetic screen, mutations in the Regulator Of the Ultradian rhythm ( ROU ) gene were isolated that indicated loss of UR and reduced DNRR efficiency. Abscisic acid (ABA) signaling component EAR1 (Enhancer of ABA co-Receptor 1) was identified as an important UR regulator and was named “ROU1”. Exogenous plant hormones (exogenous phytohormone) process ROU1 / EAR1 It affected the ROU1/EAR1 protein stability as well as the transcript level. In addition, DNRR- related gene expression was altered in the rou1 mutant, suggesting that ROU1/EAR1 acts as a mediator of UR-linked hormonal changes in resected leaves and promotes DNRR. Taken together, it is suggested that resected leaves produce UR, which promotes DNRR, as a survival strategy under adverse conditions.

In one aspect, the present invention relates to a plant transformed by a gene encoding EAR1 (Enhancer of ABA co-Receptor 1).

In the present invention,'EAR1 (Enhancer of ABA co-Receptor 1)' is a component that regulates abscisic acid (ABA) signaling, and according to an embodiment of the present invention, an important superweek rhythm ( Ultradian rhythm; UR) was identified as a regulator, and was named'regulator of ultradian rhythm 1 (ROU1)'.

In the present invention, the plant may be characterized in that Arabidopsis (Arabidopsis), but is not limited thereto.

In the present invention, the EAR1 may be characterized in that it generates an Ultradian rhythm from a petiole of a plant.

In the present specification, the term'Ultradian rhythm (UR)' refers to a short rhythm from a few seconds to several hours among biological rhythms such as biochemical, physiological, or behavioral periodicity found in an organism, and the present invention According to an embodiment of, it means a periodicity of about 3 hours.

In one embodiment of the present invention, clock-related genes ORE1 (ORESARA 1), CAT3 (Catalase-3), CAB2 (chlorophyll A/B-binding protein 2), in the petiole site of an excised leaf of Arabidopsis, Gene promoters of TOC1 (Timing of CAB expression 1), LHY (Late Elongated Hypocotyl), GI (GIGANTEA), CCA1 (Circadian Clock Associated 1), PRR7 (Pseudo-response regulator7) and CCR2 (Cold and Circadian-Regulated 2) genes The activity of was confirmed, and it was confirmed that the circadian rhythm of ~24 hours showed a short periodicity of ~3 hours.

In the present invention, the "periodicity of gene promoter activity" refers to a period from when the activity is lowest to when the activity is highest, and then again until it is lowest again.

In another aspect, the present invention relates to a method for promoting de novo root regeneration of a plant comprising the step of transforming the plant with a gene encoding Enhancer of ABA co-Receptor 1 (EAR1).

In the present invention, the plant may be characterized in that Arabidopsis (Arabidopsis), but is not limited thereto.

In the present invention, the EAR1 may be characterized in that it generates an Ultradian rhythm from a petiole of a plant.

In one embodiment of the present invention, it was confirmed that the chosun rhythm in the excised leaves of Arabidopsis promotes new root regeneration by increasing the rooting rate and the like, and it was confirmed that the mutation of EAR1 breaks the chosun rhythm. In other words, it was confirmed that the EAR1 generates a 3-hour cycle of ultra-weekly rhythm, and as a result, de novo root regeneration (DNRR) is promoted.

In another aspect, the present invention relates to a method for inhibiting aging of a plant comprising transforming the plant with a gene encoding Enhancer of ABA co-Receptor 1 (EAR1).

In the present invention, the plant may be characterized in that Arabidopsis (Arabidopsis), but is not limited thereto.

In the present invention, the EAR1 may be characterized in that it generates an Ultradian rhythm from a petiole of a plant.

In one embodiment of the present invention, since it was confirmed that the EAR1-induced superweek rhythm promotes new root regeneration in the excised leaves of Arabidopsis, it is suggested that aging can be suppressed through the cell fate change of the plant.

Arabidopsis Ablation-triggered UR in the leaf

Unlike CR, which adapts to the Earth's 24-hour light/dark cycle, the UR found across species does not match known environmental cycles. The origin of UR has not yet been identified. Some studies suggest that UR originated from the interaction between circadian oscillators coupled to each other in separate phases (Hughes et al., PLoS Genet. 2009 Apr;5(4):e1000442 et al.). However, other studies show that UR is driven by a super-periodic pacemaker system independent of the circadian clock (Waite et al., Eur J Neurosci. 2012 Oct;36(8):3142-50, etc.). Indeed, UR with various rhythmic phenomena can be driven by various mechanisms (Iwasaki and Thomas, Trends Genet. 1997 Mar;13(3):111-5).

The present inventors confirmed UR in the resected Arabidopsis leaves, and confirmed that the characteristics of UR are different from those of CR. Since CR is well known that the biological cycle changes by a 24-hour light/dark cycle, the promoter activity of a circadian clock-related gene in individual leaves showed a synchronized CR phase between leaves under free-running conditions (Fig. 1G). -H). However, UR exhibited a scattered phase (Fig. 1G-H) and strong unsynchronization, suggesting that the biological cycle alteration mechanism of UR is distinct from CR.

UR also exhibits thermal reactivity, unlike CR. CR denotes temperature compensation (Beale et al., J Biol Rhythms. 2019 pr;34(2):144-153), while UR exhibited heat-sensitive properties (Fig. 2D-F). Importantly, the data according to the present invention showed strong retention of UR in irregular circadian clock mutants (Figs. 1J-K). Taken together, these results mean that the UR is driven by a vibrating mechanism referred to herein as an'ultradian clock', which is independent of the circadian clock system.

Because genes are often co-regulated by this rhythm, UR can be obscured by CR (van der Veen and Gerkema, FASEB J. 2017 Feb;31(2):743-750). The data in accordance with the present invention provides additional evidence to support this concept. The negative correlation between CR and UR wavelet power in the promoter activity of circadian clock-related genes (Fig. 8A) can be explained by co-regulation. Core circadian clock genes such as CCA1 , LHY , TOC1 and PRR7 , which tightly regulate themselves, showed relatively low UR wavelet power in promoter activity, and RNA-seq analysis did not reveal UR regulatory genes. The UR pattern of the promoter with high CR wavelet power (FIG. 7B) can be a clue as to how the CR regulator affects UR production. In addition, the increased UR wavelet power in the irregular circadian clock mutant (Fig. 1J-K) means that CR obscures UR. Further research into the interactions between different biological rhythms is important to advance an understanding of how various endogenous biological processes are organized and integrated in an organism so that they can adapt to the external environment.

The signal that triggers UR is an important issue. In the ORE1 promoter activity, ~3 hours UR was detected only in the resected leaves and not in the attached leaves or other resected organs, which means that resection is required, but the wound signal alone is not sufficient to induce UR. Although sucrose is reported to be required for DNRR in cancer conditions, the process of DNRR in leaf explants occurs in LL conditions without exogenous application of plant hormones (Chen et al., Front Plant Sci. 2014 May 15 ;5:208), which means DNRR requires energy. Likewise, the leaf-specific shape of UR (Figure 9) and re-establishment of UR by simultaneous treatment of cancer and low temperature (Figure 10) suggests that activation of photosynthetic machinery or energy sources can serve as important signals for UR production. do. The UR structure by exogenous auxin treatment in aged leaves (Fig. 4E) suggests that auxin induces UR.

In DNRR The role of UR

Although UR has been observed in many cases, its biological relevance has not yet been established due to 1) difficulty of detection and 2) aperiodic nature (Goh et al., Biology. 2019 Mar; 8(1): 15). However, some URs that show strong vibrational patterns with distinct spatial expression patterns are relatively well characterized. Segmentation and somitogenesis in Drosophila (Palmeirim et al., Cell. 1997 Nov 28;91(5):639-48) and root branching of Arabidopsis (Moreno-Risueno et al., Science. 2010 Sep 10;329(5997): 1306-11) is controlled by a developmental clock that requires vibrational transcriptional modulators. The present inventors confirmed a strong UR in the petiole region of the resected Arabidopsis leaves. In addition, RNA-seq analysis showed the regulation of transcriptional oscillations by UR and enrichment of GO term related to plant development (Fig. 3C), which shows that the regulation mechanism of UR identified in the petiole region of the resected leaf is similar to that of the existing UR regulation mechanism. It suggests that there is a possibility of similarity. In response to both internal and external factors, a consistent correlation between UR and DNRR, which is a developmental process, was confirmed (FIG. 2). In addition, a strong UR appearance was found in the petiole region cells overlapping the rooting site (FIG. 8), which suggests that UR participates in DNRR in the resected leaves. Indeed, the rou mutant defective in UR production showed delayed and reduced root regeneration (Figs. 4A-C), confirming the positive role of UR in promoting DNRR.

In the present invention, we also investigated the phase of DNRR in which UR plays a key role. Wounds are the first event to initiate DNRR through JA signaling and epigenetic regulation, triggering auxin production in resected leaves. Then, auxin accumulates in the AR forming region, followed by cell fate conversion from regenerative cells to AR (Bustillo-Avendano et al., Plant Physiol. 2018 Feb;176(2):1709-1727). During this phase, UR appears only in resected leaves during auxin accumulation and initial cell fate transition (~16-72 hours after resection), suggesting that UR is mainly involved in this process. Cell fate shifting requires complex regulatory networks including transcription factor interactions and chromatin restructuring (Ye et al., Science. 2019 Nov 15;366(6467):838-843). Auxin accumulation in the petiole region and subsequent expression of transcription factor genes such as WOX and LBD are involved in cell fate shifts during DNRR (Jing et al., Front Plant Sci. 2020; 11: 317). In addition, epigenetic regulation has been associated with JA-mediated wound signaling to promote DNRR (Zhang et al., Nat Rev Immunol. 2019 Jul;19(7):417-432). However, the molecular mechanisms underlying this process have not been fully elucidated. In the present invention, it is suggested that ablation-triggered UR controls large-scale changes in gene expression, creating a complex regulatory network for cell fate transformation. According to an embodiment of the present invention, more than 4,000 genes are regulated by UR, and it can be confirmed that various biological processes are involved in this regulation, which means that the DNRR process is more complex than expected.

Plant hormone -Regulated ROU1 / On EAR1 by Auxinaceae Cytokinin Through the balance of signaling DNRR Promotion

When detached, many physical, chemical and biological processes in the leaf change rapidly; This includes dynamic reconfiguration of the hormonal environment (Pan et al., J Genet Genomics. 2019 Mar 20;46(3):133-140). Many hormones are involved in DNRR regulation, which is consistent with the identification of the ABA signaling component, ROU1/EAR1, as a modulator of UR and DNRR. Mutation of ROU1 / EAR1 is the UR was completely void (Fig. 4A-B), which is ROU1 / EAR1 key component of 'second clock cycle ", suggesting that led to the generation UR. Furthermore, the dynamic changes in hormones are consistent with the UR pattern and ROU1/EAR1 levels (Figures 5G and 6A), which means that ROU1/EAR1 serves as a link between the dynamic hormonal environment and UR production. Compared to auxin, the role of ABA and CK in DNRR is not clear. According to the present invention, ABA inhibited UR production and DNRR by reducing ROU1/EAR1 levels. In addition, RNA-seq analysis showed that many ABA-related genes in the rou1 mutant were regulated by UR and DEG (Fig. 12C), suggesting a regulatory mechanism based on the role of ABA in UR production and DNRR. . However, the role of CK in regulating UR and DNRR appears to be more complex. Exogenous CK clearly inhibited UR and DNRR by reducing ROU1/EAR1 levels. However, mutation analysis suggested that CK signaling was required for adequate DNRR, consistent with previous studies (Bustillo-Avendano et al., Plant Physiol. 2018 Feb;176(2):1709-1727). According to previous studies, new root organogenesis and side root formation may share similar genetic pathways (Xu and Huang, Curr Top Dev Biol. 2014;108:1-33). During embryonic development and root tip regeneration, the overlapping and antagonistic hormonal domain between auxin and CK regulates root regeneration (Efroni et al., Cell. 2016 Jun 16;165(7):1721-1733). Likewise, DNRRs can share these regulatory developmental mechanisms to generate new roots. CK efficiently initiates DNRR by restricting the auxin domain in the petiole region, thereby inhibiting UR production. The increased number of de novo roots in CK-deficient (mutant) and auxin-treated resected leaves can be explained by the reduced CK domain; The auxin domain is expanded, consequently, the number of roots increases, and the results that exogenous auxin expand ROU1/EAR1 expression to the upper region of the petiole (Fig. 6D) support this hypothesis. This spatial limitation of the auxin domain by the CK domain can be the survival mechanism of resected leaves in natural conditions by optimizing the number of roots and the initiation site.

The importance of hormone regulation in DNRR was confirmed by DEG analysis of the rou1 mutant. Among the well-known DNRR regulators, many auxin- and CK-related genes were antagonistically regulated by ROU1/EAR1 in different temporal expression patterns (Figs. 6H and 12B), and regulatory mechanisms linked to auxin and CK signaling were combined. Suggests that you can. In addition, cell cycle regulators, transcription factors, and epigenetic factors are also involved in ROU1/EAR1 regulation, suggesting that ROU1/EAR1 is a major regulator of DNRR. Taken together, the present inventors propose a model in which the dynamic hormonal environment modulates ROU1/EAR1 to generate the UR required for efficient DNRR initiation (FIG. 6I). How EAR1 regulates UR production or how information of super-periodic vibration is transmitted as a control signal to promote DNRR has not been fully elucidated, but the present invention relates to the initiation of DNRR in resected leaves, wound and hormonal signals. In addition to the known role of Xu (Xu, Curr Opin Plant Biol. 2018 Feb;41:39-45), the role of hypercycle rhythm was first identified. Further analysis of EAR1 and its potential partners will help build a hypercycle clock model by improving our understanding of the underlying mechanisms of EAR1-mediated UR generation. The present invention improves the current understanding of the DNRR initiation mechanism. Additional knowledge of DNRR and general plant regeneration will contribute to an understanding of agriculture and plant development and crop integrity.

Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not construed as being limited by these examples.

Example One: CR -Resected leaf-specific UR identification driven by an independent vibration mechanism

High-resolution tracking of promoter activity at 30 minute intervals showed an increased activation cycle with a short periodicity in addition to the expected -24 hour CR. This shorter cycle rhythm was identified in many circadian clock-related genes of the third and fourth rosette leaves excised in 21-day-old Arabidopsis plants (Figs. 1A, 1B and 7A). Wavelet and time-frequency analysis were used to confirm that these short bursts reflected endogenous biological rhythms (Leise, J Circadian Rhythms. 2013; 11: 5).

First, the circadian signal was separated by subtracting the smooth circadian signal from the original vibration pattern (Fig. 1C). Wavelet spectra showed -24 hours CR in all tested promoters with varying wavelet powers (Figures 1D and 7B). In addition, the ORE1 and CAT3 promoters showed a ~3 hour UR with large and stable wavelet power (Figs. 1E and 7B). The promoters of some genes, including PRR7 and CCA1 , exhibited a cracked UR pattern detected at the peak time of the circadian cycle, thereby reducing the power of the super-cycle wavelet (FIG. 7B). For example, promoters showing lower circadian wavelet powers, including ORE1 and CAT3 , exhibited higher circadian wavelet powers (FIGS. 1D and 1E ). On the other hand, promoters with higher circadian wavelet power, such as PRR7 and CCA1 , exhibited lower super-cycle wavelet power (Figs. 1D and 1E). These results suggest that the UR pattern can be affected by the circadian wavelet power. Consistent with these data, each promoter exhibited a strong negative correlation between the initial and circadian wavelet powers (correlation coefficient [R] = -0.8217; P = 0.0067; Fig. 8A). Thus, promoter activity of circadian clock-related genes in resected Arabidopsis leaves shows UR, which can be negatively affected by CR.

To further characterize whether UR is a true rhythm, circadian and circadian phases were calculated in the promoter activity of circadian clock-related genes from wavelet analysis. While the circadian phases were very closely distributed, the super-periodic phases of all tested promoters were scattered (Figs. 1F-G and 7B), and thus, the UR Suggests that it is regulated independently of CR. To confirm this possibility, the UR pattern of the circadian clock mutation was measured. Since ORE1 promoter activity shows a dominant UR with high wavelet power compared to other gene promoters (Fig. 1E), ORE1 is selected as the representative promoter, and luciferase (LUC) gene under the control of the ORE1 promoter ( ORE1 ::LUC) for further experiments. A transgenic Arabidopsis line expressing a was used. As a result, similar to the wild type, UR persisted with an unchanged -3 hour period in all circadian clock mutants tested (Figs. 1H and 8B). Irregular circadian clock mutations, elf3 and elf4 , maintained an identifiable UR with much higher wavelet power (FIG. 8C ), supporting the notion that CR negatively influences the UR pattern.

UR was identified in the resected leaves, and UR was also present in the leaves attached to the stem and other resected organs. Therefore, LUC signal intensity was measured in 7-day-old ORE1 ::LUC seedlings and organs excised from these seedlings. UR was detected in the resected cotyledons, but not in whole seedlings or other resected organs such as shoots, hypocotyls and roots (FIG. 9A). In addition, ORE1 promoter activity was tested in attached leaves and organs excised from adult plants (Figs. 9B-C). As a result, only rosette leaves excised from 21-day-old plants and stem leaves excised from 35-day-old plants showed UR, and did not appear in attached leaves, excised stems, or excised flowers. These results indicate that UR was specifically triggered in the resected leaves. In addition, UR showed strong expression in the petiole region, and the development of UR only in the resected leaves along with the presence of the petiole region suggests that UR plays a spatially important role in the resected leaves. As a result, we identified UR driven by a circadian-independent oscillation mechanism that potentially plays a specific role in the petiole region of the resected leaf.

Example 2: UR indicating a strong positive correlation and DNRR Confirm

After leaf resection, the level of auxin in the transforming cells of the foliar increases and is transported to the cells with the regenerative capacity of the petiole basal, activating cell fate transformation and starting root regeneration (Xu, Curr Opin Plant Biol. 2018 Feb;41 :39-45). The specific localization of UR and DNRR to the petiole region suggests that UR and DNRR may be involved. To test this possibility, we checked the UR pattern and rooting rate of the resected leaves and measured the DNRR efficiency under various conditions.

Previous studies have shown that DNRR efficiency is highly dependent on the stage of leaf development (Pan et al., J Genet Genomics. 2019 Mar 20;46(3):133-140); Old leaves have significantly lower root regeneration ability than younger leaves. Therefore, the UR pattern of the leaves excised in plants of different ages was investigated. The wavelet power of UR was similar in the resected leaves in the 17- and 21-day-old plants, and gradually decreased with age (FIGS. 2A-B). The rooting rate also gradually decreased with the age of the leaves. Leaves excised in the youngest plants showed the highest rooting rate (FIG. 2C ), suggesting that other factors such as hormones may independently influence DNRR. These data show that UR and DNRR were continuously adjusted with leaf age.

Environmental conditions such as nutrition, light and temperature are also important for proper regeneration. Since many rhythmic biological processes have been temperature compensated, we tested whether UR is altered by temperature (Beale et al., J Biol Rhythms. 2019 pr;34(2):144-153). The excised leaves were cultured at different temperatures and the ORE1 promoter activity was measured. At 22° C., the optimum growth temperature of Arabidopsis, UR was stable for at least 36 hours (FIG. 2D ). However, below 17°C, the UR cycle continued to increase for 2 days (FIG. 2E), and at 27°C, UR rapidly weakened (FIG. 2F). This result implies that UR is temperature-dependent. Rooting rates were also changed at different temperatures. At temperatures lower or higher than 22° C., the DNRR efficiency decreased (FIG. 2G ). The appearance of AR at 17° C. was delayed by ~3 days compared to 22° C. consistent with the UR pattern (Fig. 2E, G). At 27°C, DNRR can be difficult to initiate due to the rapidly weakening UR cycle. This result suggests a close correlation between UR and DNRR.

Plant hormones such as auxin, CK and jasmonate (JA) are known to play an important role in plant regeneration during in vitro culture and in natural conditions (Zhang et al., Nat Rev Immunol. 2019 Jul;19(7):417) -432). Therefore, UR and DNRR were monitored in the presence of low concentrations (0.2 μM) of various plant hormones. Among the tested hormones, indole-3-acetic acid (IAA; auxin) and methyl jasmonate (MeJA), known to promote DNRR, did not affect UR or rooting rate compared to untreated leaves (Fig. 2H-K). However, 6-benzyladenine (BA), a synthetic CK, completely removed UR from leaves resected from 21-day-old plants, and reduced root regeneration (FIG. 2H-K). In addition, 1 μM BA initiated callus, but did not initiate root generation (FIG. 2K). These results imply that exogenous CK application inhibits UR production and prevents DNRR. Taken together, these data suggest a strong positive correlation between UR production and DNRR in resected leaves, meaning that UR may be the upstream signal for optimal DNRR initiation.

Example 3: Affect genes with various functions, DNRR Linkage-rich in genes Second cycle Vibration check

If UR acts as an upstream signal of DNRR, it was expected that DNRR-related genes would exhibit a super-periodic oscillation pattern. RNA-seq analysis was performed to investigate the expression pattern of DNRR-related genes. Since the super-periodic phase showed strong unsynchronization between the leaves, the environmental conditions were first optimized to ensure the sampling of the leaves in the synchronized super-periodic phase. Cyanothece 's hypercycle metabolic cycle occurs in continuous light (LL) after a light/dark biological cycle change (Cerveny et al., Proc Natl Acad Sci USA. 2013 Aug 6;110(32):13210-5). In addition, the delayed appearance of UR at 17° C. (Figure 2E) suggests that low temperatures reset UR. Therefore, after culturing in various dark and cold regions, ORE1 promoter activity was measured in the resected leaves under LL conditions at 22°C. As a result, it was confirmed that UR was greatly synchronized in the fourth rosette leaves excised from 21-day-old plants cultured in continuous dark (DD) conditions at 16° C. for 24 hours (FIGS. 10A-C). A total of 16 samples were collected at 30 minute intervals for further analysis. Synchronized UR was identified based on the ORE1 expression level determined by RT-qPCR (FIG. 10D ).

To identify genes representing UR based on RNA-seq data, MetaCycle analysis, an existing method used to evaluate the periodicity of temporal data, was performed by integrating various methods such as ARSER, JTK_CYCLE, and Lomb-Scargle (Wu et al., Bioinformatics. 2016 Nov 1;32(21):3351-3353). As a result, it was confirmed that 4,073 genes vibrating in a period of ~ 3 hours, these genes were divided into two antiphasic groups (Fig. 3A and Table 1).

Some of these putative vibrating genes were verified by constructing a transformation line expressing the LUC gene under the control of the corresponding promoter (Figs. 3B and 10E), supporting that these genes are regulated by UR at the transcription level.

Next, Gene Ontology (GO) analysis of the UR-cycling gene was performed. As a result, these genes were classified into biological process categories (Figure 3C), especially'root development' (GO: 0048364),'response to hormones' (GO: 0009725), and'hormone-mediated signaling pathways' (GO: 0009755). And'cell wall tissue' (GO: 0071555)', suggesting that UR regulates DNRR at the transcription level (Table 2).

To identify genes linked to different UR phases, the number of genes in different GO terms was examined. However, the two antiphase groups did not differ in the number of abundant genes in various GO terms (Fig. 3C), which means that the UR phase is not associated with a specific function. As a result, identification of multiple genes representing a ~3 hour period and enrichment in various biological processes validated UR in resected leaves and suggests an important role for UR.

In addition, in order to predict the major pathways linked to UR, the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was performed. As a result, it was confirmed that the hormone signal transduction gene was abundant (FIG. 3D). Vibration genes linked to different stages in hormonal function, from biosynthesis to receptors, as well as signaling-related and transcription factor-coding genes linked to various hormonal pathways were identified. Auxin, CK and JA are essential to initiate DNRR in resected leaves. Auxin and CK play important roles in promoting the proliferation and differentiation of regenerative cells in the petiole region, whereas JA promotes DNRR by transducing wound signals. Therefore, RNA-seq analysis-based identification of DNRR-related plant hormone-reactive oscillatory genes suggests that UR observed in resected Arabidopsis leaves plays an important role in DNRR and is linked to related hormones.

To investigate this possibility, the UR patterns and rooting rates of mutations in the oscillatory genes involved in auxin and CK signaling were identified. Loss of function mutations of five AUXIN RESPONSIVE FACTOR ( ARF ) genes ( ARF4 , 7 , 8 , 10 and 11 ), PIN-FORMED 3 ( PIN3 ), AUXIN SIGNALING F-BOX 2 ( AFB2 ) and TRANSPORT INHIBITOR RESPONSE 1 ( TIR1) Was isolated and transformed with ORE1::LUC transgene. Some mutations showed low UR wavelet power, but afb2 And tir1 Only the mutation was significantly impaired in DNRR efficiency (Figure 3E).

Since AFB2 and TIR1 belong to the TIR1/AFB F-box protein family, the role of ubiquitination in the auxin signaling pathway in the UR and DNRR process was investigated by examining the AUXIN RESISTANT 1 ( AXR1) mutation. UR wavelet power axr1 mutations, but similar to the wild-type, rooting rate of axr1 mutant was significantly reduced (Fig. 3E). These results imply that the auxin signaling components influence the UR pattern and DNRR process, but are independent of each other.

To investigate CK- and UR-regulatory genes, ARABIDOPSIS HISTIDINE Loss of function mutations of KINASE 3 and 4 ( AHK3 , 4 ), CYTOKININ RESPONSE FACTOR 5 ( CRF5 ), KISS ME DEADLY 1 and 2 ( KMD1 , 2 ), PURINE PERMEASE 14 ( PUP14 ) and CYTOKININ-RESPONSIVE GATA FACTOR 1 ( CGA1 ) Was tested. Double mutant (ahk3 ahk4) with a single mutation (cga1) both showed an increased UR power with lower rooting rate, which indicates the CK signaling pathway is required for DNRR started (Fig. 3F). Inhibited extrinsic CK process all the processes of the UR generated and DNRR but (FIG. 2H-K), the constructive CK signaling activating factor, the enhancement mutation ore12 (oresara 12) of the AHK3 gene identified as the normal UR power and DNRR The process was shown (Fig. 3F). For UR generation and DNRR process, it may be necessary to control a specific CK in spatiotemporality. Overall, these results imply that the auxin and CK signaling pathways antagonistically regulate UR production, but are essential for proper DNRR through spatiotemporal regulation.

Example 4: due to impaired mutations in UR production DNRR Decrease confirmation

Previous studies have suggested that UR observed in resected leaves may be involved in AR production. To test this hypothesis and identify the factors that regulate UR, a forward genetic screen was performed. Transgenic Col-0 seeds expressing the ORE1::LUC construct (Col/ ORE1 ::LUC ) were treated with ethyl methane sulfonate (EMS) to generate random point mutations. Mutants showing loss of UR were isolated from the M2 population. After screening for ~16,000 M2 seeds, four homozygous lines (M21, M23, M38 and M83) that did not show UR in the excised leaves were identified (Figs. 4A-B). Next, the rooting rates of the third and fourth rosette leaves excised from 21-day-old plants of this mutant line were confirmed. It was confirmed that rooting initiation was delayed and rooting rate decreased in all four mutant lines compared to the wild type (FIG. 4C), and it was confirmed that UR has a positive role in DNRR regulation. In addition, M38, one of the mutant lines, showed very late flowering, and the other three lines showed a relatively delayed flowering compared to the wild type (Figure 4D), which indicates that these UR regulators are involved in other biological processes such as flowering. It suggests that you can do it. Genetic analysis showed that all four lines were recessive mutations (FIG. 11A ). M21, M23 and M83 were classified into the same complementation group (Figure 11A), suggesting that the mutant genes of these lines may be important for UR generation. Therefore this gene "second week rhythm regulators 1 (regulator of ultradian rhythm 1; ROU1) ' named, and focused on the rou1 mutants for further investigation.

In the case of DNRR, it has been reported that exogenous auxin treatment rescues regeneration defects in older leaves (Chen et al., Front Plant Sci. 2014 May 15;5:208). Therefore, we tested whether exogenous auxin treatment rescued the defects of UR and rooting rates in the rou1 -1 (M21) mutant leaves. Fourth rosette leaves excised from older (25 day old) wild type and rou1 -1 mutant plants were used to test the effect of auxin application on reduced UR and regenerative capacity. Exogenous auxin treatment of leaves excised from young (21 days old) plants did not affect UR wavelet power and rooting rate (Fig. 2H-J). However, as expected, the UR power and rooting rate of the resected leaves in aged wild-type plants were rescued after exogenous auxin treatment (Fig. 4E-F). In contrast, in rou1 mutant leaves, UR cycling was not rescued by exogenous auxin treatment, and the recovery of rooting rates was also lower than that observed in wild-type leaves (Fig. 4E-F). These results imply that auxin is important for UR production, and this process is mediated by ROU1.

ROU1-mediated UR is required to promote DNRR, but auxins may independently induce DNRR. However, spatiotemporal regulation of auxin can be altered in rou1 mutant leaves. To test this possibility, β- glucuronidase ( GUS ) under the control of a synthetic auxin-reactive DR5 promoter ( DR5::GUS ; Moreno-Risueno et al., Science. 2010 Sep 10;329(5997):1306-11). Transgenic plants expressing the gene were used, and rou1DR5 ::GUS and rou2 ( M38 ) DR5::GUS plants were made. Leaves excised from young wild-type or mutant plants had no auxin accumulation in the petiole region (Fig. 4G-H). In addition, the expression of DR5::GUS gradually increased in the petiole region of wild-type leaves, and showed a timing and pattern similar to that seen in the two rou mutants (Fig. 4H). These results suggest that the accumulation of auxin in the petiole region by resection is not regulated by rou -mediated UR, and that rooting rates are partially rescued through UR-independent pathways.

Example 5: ABA signaling component Of EAR1 Through UR adjustment DNRR Palpation confirmation

The effectiveness of complementary mutations made it possible to identify mutations by whole-genome sequencing (WGS). The F2 homozygous mutant progeny pool derived from the backcross of the Col/ ORE1::LUC parent and rou1 -1 (M21) or rou1 -3 (M83) was sequenced against the parent showing no UR pattern. Only one gene, EAR1, had a common intragene SNP, and the inheritance was consistent with the EMS mutated line (FIG. 11B ). WGS results rou1 -1, rou1-2 (M23) and rou1 - 3 line EAR1 Exons were verified by sequencing (Fig. 11C). rou1 rou1 -1 and - while the second line includes a nonsense mutation in the EAR1 and replacing tryptophan residues (W52 and W112 * *) at amino acid position 52 times and 112 times, respectively, as stop codons, rou1 - 3 is 157 amino acids It was confirmed that it contained a missense mutation (G157Q) that caused a substitution from glycine to glutamate at the position (FIG. 5A). To further confirm whether EAR1 is associated with the UR phenotype, complementation of expression of the green fluorescent protein ( GFP ) gene-fusion of EAR1 under the control of its cognate promoter ( EAR1 :: EAR1 - GFP ) in the background of the rou1 -1 mutation Lines (COM9 and COM24) were made. UR damaged and DNRR rou1 -1 phenotype of mutants EAR1 :: EAR1 - GFP It was complemented by the construct (Figs. 5B-C). These results confirm that EAR1 is a true UR regulator and promotes DNRR in resected Arabidopsis leaves.

EAR1 is known to function as a negative regulator of ABA signaling (Wang et al., Plant Cell. 2018 Apr;30(4):815-834). The EAR1 protein binds to the inhibitory domain of the type 2C protein phosphatase (PP2C) and enhances the activity of the enzyme. This in turn leads to a stronger inactivation of the downstream protein kinase, blocking the ABA signaling pathway. Based on this relationship, we tried to determine whether ABA signaling is involved in regulating UR and DNRR. When the excised leaves are treated with increasing ABA concentration, UR wavelet power and DNRR efficiency gradually decrease (Fig. 5D-E), suggesting that ABA negatively regulates UR generation and DNRR process. ABA2 is known to play a major function in ABA biosynthesis (Schwartz et al., Plant Physiol. 1997 May;114(1):161-6). Therefore, aba2 Because of the low endogenous ABA in mutant leaves, we hypothesized that aba2 mutant leaves exhibit improved UR and DNRR. To test this hypothesis, the ABA content of wild-type and aba2 mutant leaves before resection (0 hours) and 2-96 hours after resection was measured. In wild-type leaves, ABA levels decreased rapidly after resection (2 hours), and gradually increased with incubation time (FIG. 5F). However, as in previous reports (Leon-Kloosterziel et al., Plant J. 1996 Oct;10(4):655-61), the endogenous ABA content of aba2 leaves was lower than that of wild-type leaves at 0 hours, and increased significantly with further culture. Did not (Fig. 5F). Next, UR and DNRR were investigated in aba2 mutant and wild-type leaves. The UR pattern of wild-type leaves gradually decreased over time, but the UR pattern of the aba2 leaves expanded until after 3 days incubation (DAC) (Fig. 5G-H), consistent with the change in endogenous ABA content (Fig. 5F). . This stable UR pattern in aba2 leaves resulted in a higher rooting rate compared to that of wild-type leaves (Fig. 5I). These data suggest that high endogenous ABA content or enhanced ABA signaling inhibits UR production and DNRR.

Example 6: due to the hormonal environment EAR1 Control and DNRR Palpation confirmation

After ablation, we tested how ROU1/EAR1 modulates UR and DNRR. Since ROU1/EAR1 is a positive regulator of UR, the level of ROU1/EAR1 in leaves increased after resection, and this level was assumed to be higher in aba2 mutant leaves than in wild-type leaves. To test this possibility, ROU1 /EAR1 expression levels were measured after resection in wild-type and aba2 mutant leaves. For the wild-type leaves, ROU1 / EAR1 transcript levels are increasing but there early excision was reduced to 48 hours after the resection (Figure 6A); This pattern was opposite to the endogenous ABA content of the resected wild-type leaves (Fig. 5F). For aba2 mutant leaves, ROU1 / EAR1 The expression pattern was similar to that observed in wild-type leaves; However, ROU1 / EAR1 Transcript levels did not decrease at later time points, which was consistent with the results of stable UR wavelet power and improved DNRR efficiency in aba2 mutant leaves (Fig. 5H-I). These results suggest that the effect of ROU1/EAR1 on UR and DNRR is negatively regulated by ABA.

Since various hormones affect UR and DNRR, we hypothesized that the altered hormonal environment affects ROU1/EAR1 in the petiole. To test this hypothesis, ROU1/EAR1 transcript levels were measured under various hormonal conditions. Upregulation of ROU1 / EAR1 transcript levels at 24 hours after the resection is, IAA or did not change much by the ABA, was significantly suppressed by the BA (Fig. 6B), before the effect of these hormones on the UR and DNRR observations Did not match perfectly (Figs. 2H-J and 5D-E). In addition, the temporal expression pattern of ROU1/EAR1 in the excised leaves did not show a close correlation with the observed UR pattern. For example, ROU1/EAR1 transcript levels peaked at 12 hours after resection (Figure 6A), while UR remained robust until at least 72 hours after resection (Figure 5G). Previous studies have shown that ROU1/EAR1 is expressed in most tissues, including adult leaves (Wang et al., Plant Cell. 2018 Apr;30(4):815-834), but there may be additional spatiotemporal regulation. Check represents the specific localization - in order to test this possibility, ROU1 :: GUS transgenic plants were created, ROU1 / EAR1 This has been expressed in full leaf in moderation immediately (0 hour), strong petiole in the 24 hours after ablation (Fig. 6C).

Next, CsV :: GFP (control) and ROU1 :: ROU1 - using a GFP transgenic plants was detected temporal and spatial changes in ROU1 / EAR1 expressed in the leaf stalk region. CsV :: GFP as compared to the GFP expression from the cut leaf from a plant, ROU1 ROU1 :: - in the leaf cut from GFP plants ROU1 / EAR1 expression of the nucleus and cytoplasm of the, in particular petiole region in 24 hours of cutting cells ( cytoplasm) dramatically increased (Fig. 6D-E). Auxin treatment did not significantly affect ROU1/EAR1 protein levels, but ROU1/EAR1 protein levels were higher in the upper petiole region due to increased nuclear localization compared to the lower petiole region (Fig. 6D-E). In contrast, ABA and CK treatment inhibited ROU1/EAR1 accumulation in the petiole area (Fig. 6D-E). This result was supported by immunoblot analysis, and it was found that the ROU1/EAR1 protein accumulation pattern (detected by anti-GFP antibody) was similar to that of GFP fluorescence under various hormone treatments (FIG. 6F). In addition, the effect of ROU1/EAR1 on UR production and DNRR was supported by accumulation of ROU1/EAR1 up to 48 hours after resection. These results suggest that the efficacy of plant hormones on resected leaves controls ROU1/EAR1 levels, thereby regulating UR cycling and DNRR.

To determine the effect of ROU1/EAR1 on DNRR, RNA-seq analysis was performed on the petiole regions of wild-type and rou1-1 mutant leaves at 0, 24, 48, 72 and 96 hours after resection. As a result of RNA-seq analysis, 9,754 differentially expressed genes (DEGs) classified into 12 clusters based on the similarity of gene expression were revealed (Table 1). The first focuses on the Cluster 2 contains a ROU1 / EAR1. In wild-type leaves, ROU1 / EAR1 transcript levels were up-regulated in 24 hours, and then gradually decreased (Figure 6G). However, rou1 - The first leaves the transcriptional level of ROU1 / EAR1 did not increase from 24 to 48 hours (Fig. 6G).

For further understanding of the effect of ROU1/EAR1 on DNRR, GO and KEGG enrichment analyzes were performed for 325 genes belonging to cluster 2. These genes include'auxin-activated signaling pathway' (GO: 0009734),'cellular response to auxin stimulus' (GO: 0071365),'hormone-mediated signaling pathway' (GO: 0009755), and'cellular response to hormone stimulus' ( GO: 0032870),'response to auxin' (GO: 0009733) and'response to hormone' (KEGG: 04075) were very rich in GO terms related to the auxin and hormonal pathways, indicating that these genes may be related to DNRR. It means that you can (Table 2). In addition, some DNRR-related genes were identified in cluster 2 (Fig. 6H). Two YUCCA genes linked to auxin biosynthesis (YUC8 and YUC9 ) (Hentrich et al., Plant Signal Behav. 2013 Nov; 8(11): e26363) and WOX11 respond to wound stress-induced increased levels of auxin, and AR during DNRR It is involved in organogenesis and cell fate transformation (Haecker et al., Development. 2004 Feb;131(3):657-68 et al.). WOX11 also promotes callus formation and participates in entourage formation from multiple isolated plant tissues, the WOX5 /7 and LBD16 genes (Berckmans et al., Plant Cell. 2011 Oct;23(10):3671-83) Et al.) has been reported to directly bind to cis-elements in the promoter (Sheng et al., Development. 2017 Sep 1;144(17):3126-3133). However, the expression of WOX5 and LBD16 was not affected in the rou1 mutation.

DEG (8,684), a large number of belonging to the cluster of nine showed similar expression patterns, such as excessive up-regulated in rou1 -1 mutant leaves compared to wild type leaves and end at time series (FIG. 6H and 12). This group contains the CK genes associated with DNRR (Figure 6H). DNRR and AHP family genes known as entourage initiation inhibitors (AHP1 , 3, 4 ) and ARR1 (Lavenus et al., Trends Plant Sci. 2013 Aug;18(8):450-8) are upregulated at 48 hours after resection. (Figure 6H). Similar expression patterns include transcription factors such as SHR and SCR (Della Rovere et al., Ann Bot. 2015 Mar; 115(4): 617-628), which are involved in AR formation and xylogenesis, and GCN5 and GCN5, which affect growth and regeneration. Similar expression patterns were shown by genes encoding epigenetic regulatory factors such as SDG2 (Servet et al., Mol Plant. 2010 Jul;3(4):670-7). These results demonstrate that the absence of EAR1 alters the auxin and CK signals of the leaves at different time points after resection, and this plant hormone imbalance may lead to decreased DNRR. Taken together, it is suggested that ROU1/EAR1, an ABA signaling component that is affected by the hormonal environment, produces UR in resected leaves and promotes DNRR through regulation of a complex gene expression network. Thus, ROU1/EAR1 provides a mechanism for survival in adverse conditions.

As described above, specific parts of the present invention have been described in detail, and it will be apparent to those of ordinary skill in the art that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. will be. Accordingly, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims (5)

Plants transformed by the gene encoding EAR1 (Enhancer of ABA co-Receptor 1).
A method for promoting de novo root regeneration of a plant comprising the step of transforming a plant with a gene encoding EAR1 (Enhancer of ABA co-Receptor 1).
The method of claim 2, wherein the plant is Arabidopsis.
The method of claim 2, wherein the EAR1 generates an Ultradian rhythm in a petiole of a plant.
A method for inhibiting aging of a plant comprising transforming the plant with a gene encoding EAR1 (Enhancer of ABA co-Receptor 1).

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