FERONIA and microtubules independently contribute to mechanical integrity in the Arabidopsis shoot.

To survive, cells must constantly resist mechanical stress. In plants, this involves the reinforcement of cell walls, notably through microtubule-dependent cellulose deposition. How wall sensing might contribute to this response is unknown. Here, we tested whether the microtubule response to stress acts downstream of known wall sensors. Using a multistep screen with 11 mutant lines, we identify FERONIA (FER) as the primary candidate for the cell's response to stress in the shoot. However, this does not imply that FER acts upstream of the microtubule response to stress. In fact, when performing mechanical perturbations, we instead show that the expected microtubule response to stress does not require FER. We reveal that the feronia phenotype can be partially rescued by reducing tensile stress levels. Conversely, in the absence of both microtubules and FER, cells appear to swell and burst. Altogether, this shows that the microtubule response to stress acts as an independent pathway to resist stress, in parallel to FER. We propose that both pathways are required to maintain the mechanical integrity of plant cells.

Karrikins control seedling photomorphogenesis and anthocyanin biosynthesis through a HY5-BBX transcriptional module.

The butenolide molecule, karrikin (KAR), emerging in smoke of burned plant material, enhances light responses such as germination, inhibition of hypocotyl elongation, and anthocyanin accumulation in Arabidopsis. The KAR signaling pathway consists of KARRIKIN INSENSITIVE 2 (KAI2) and MORE AXILLARY GROWTH 2 (MAX2), which, upon activation, act in an SCF E3 ubiquitin ligase complex to target the downstream signaling components SUPPRESSOR OF MAX2 1 (SMAX1) and SMAX1-LIKE 2 (SMXL2) for degradation. How degradation of SMAX1 and SMXL2 is translated into growth responses remains unknown. Although light clearly influences the activity of KAR, the molecular connection between the two pathways is still poorly understood. Here, we demonstrate that the KAR signaling pathway promotes the activity of a transcriptional module consisting of ELONGATED HYPOCOTYL 5 (HY5), B-BOX DOMAIN PROTEIN 20 (BBX20), and BBX21. The bbx20 bbx21 mutant is largely insensitive to treatment with KAR2 , similar to a hy5 mutant, with regards to inhibition of hypocotyl elongation and anthocyanin accumulation. Detailed analysis of higher order mutants in combination with RNA-sequencing analysis revealed that anthocyanin accumulation downstream of SMAX1 and SMXL2 is fully dependent on the HY5-BBX module. However, the promotion of hypocotyl elongation by SMAX1 and SMXL2 is, in contrast to KAR2 treatment, only partially dependent on BBX20, BBX21, and HY5. Taken together, these results suggest that light- and KAR-dependent signaling intersect at the HY5-BBX transcriptional module.

Sugar inhibits brassinosteroid signaling by enhancing BIN2 phosphorylation of BZR1.

Sugar, light, and hormones are major signals regulating plant growth and development, however, the interactions among these signals are not fully understood at the molecular level. Recent studies showed that sugar promotes hypocotyl elongation by activating the brassinosteroid (BR) signaling pathway after shifting Arabidopsis seedlings from light to extended darkness. Here, we show that sugar inhibits BR signaling in Arabidopsis seedlings grown under light. BR induction of hypocotyl elongation in seedlings grown under light is inhibited by increasing concentration of sucrose. The sugar inhibition of BR response is correlated with decreased effect of BR on the dephosphorylation of BZR1, the master transcription factor of the BR signaling pathway. This sugar effect is independent of the sugar sensors Hexokinase 1 (HXK1) and Target of Rapamycin (TOR), but requires the GSK3-like kinase Brassinosteroid-Insensitive 2 (BIN2), which is stabilized by sugar. Our study uncovers an inhibitory effect of sugar on BR signaling in plants grown under light, in contrast to its promotive effect in the dark. Such light-dependent sugar-BR crosstalk apparently contributes to optimal growth responses to photosynthate availability according to light-dark conditions.

Strigolactone mimic 2-nitrodebranone is highly active in Arabidopsis growth and development.

Strigolactones play crucial roles in regulating plant architecture and development, as endogenous hormones, and orchestrating symbiotic interactions with fungi and parasitic plants, as components of root exudates. rac-GR24 is currently the most widely used strigolactone analog and serves as a reference compound in investigating the action of strigolactones. In this study, we evaluated a suite of debranones and found that 2-nitrodebranone (2NOD) exhibited higher biological activity than rac-GR24 in various aspects of plant growth and development in Arabidopsis, including hypocotyl elongation inhibition, root hair promotion and senescence acceleration. The enhanced activity of 2NOD in promoting AtD14-SMXL7 and AtD14-MAX2 interactions indicates that the molecular structure of 2NOD is a better match for the ligand perception site pocket of D14. Moreover, 2NOD showed lower activity than rac-GR24 in promoting Orobanche cumana seed germination, suggesting its higher ability to control plant architecture than parasitic interactions. In combination with the improved stability of 2NOD, these results demonstrate that 2NOD is a strigolactone analog that can specifically mimic the activity of strigolactones and that 2NOD exhibits strong potential as a tool for studying the strigolactone signaling pathway in plants.

The chemical compound 'Heatin' stimulates hypocotyl elongation and interferes with the Arabidopsis NIT1-subfamily of nitrilases.

Temperature passively affects biological processes involved in plant growth. Therefore, it is challenging to study the dedicated temperature signalling pathways that orchestrate thermomorphogenesis, a suite of elongation growth-based adaptations that enhance leaf-cooling capacity. We screened a chemical library for compounds that restored hypocotyl elongation in the pif4-2-deficient mutant background at warm temperature conditions in Arabidopsis thaliana to identify modulators of thermomorphogenesis. The small aromatic compound 'Heatin', containing 1-iminomethyl-2-naphthol as a pharmacophore, was selected as an enhancer of elongation growth. We show that ARABIDOPSIS ALDEHYDE OXIDASES redundantly contribute to Heatin-mediated hypocotyl elongation. Following a chemical proteomics approach, the members of the NITRILASE1-subfamily of auxin biosynthesis enzymes were identified among the molecular targets of Heatin. Our data reveal that nitrilases are involved in promotion of hypocotyl elongation in response to high temperature and Heatin-mediated hypocotyl elongation requires the NITRILASE1-subfamily members, NIT1 and NIT2. Heatin inhibits NIT1-subfamily enzymatic activity in vitro and the application of Heatin accordingly results in the accumulation of NIT1-subfamily substrate indole-3-acetonitrile in vivo. However, levels of the NIT1-subfamily product, bioactive auxin (indole-3-acetic acid), were also significantly increased. It is likely that the stimulation of hypocotyl elongation by Heatin might be independent of its observed interaction with NITRILASE1-subfamily members. However, nitrilases may contribute to the Heatin response by stimulating indole-3-acetic acid biosynthesis in an indirect way. Heatin and its functional analogues present novel chemical entities for studying auxin biology.

Experimental Procedures for Studying Skotomorphogenesis in Arabidopsis thaliana.

Seedlings grown in darkness exhibit distinct morphologies comparing with light-grown seedlings. Elongated hypocotyls, closed yellow cotyledons, and the formation of apical hooks are typical characteristics for etiolated seedlings, which are collectively named skotomorphogenesis. Various plant hormones and environmental factors are essential for maintaining skotomorphogenesis. Due to the diverse morphological outcomes in etiolated seedlings grown under different treatments, studies on skotomorphogenesis are of particular importance to reveal the molecular mechanisms underlying plant response to environmental cues. Here, we detailed experimental procedures to facilitate researchers who are investigating etiolation growth-related studies.

Method for Phenotypic Chemical Screening to Identify Cryptochrome Inhibitors.

After germination, plants determine their morphogenesis, such as hypocotyl elongation and cotyledon opening, by responding to various wavelengths of light (photomorphogenesis). Cryptochrome is a blue-light photoreceptor that controls de-etiolation, stomatal opening and closing, flowering time, and shade avoidance. Successful incorporation of these phenotypes as indicators into a chemical screening system results in faster selection of candidate compounds. Here, we describe phenotypic screening for the blue-light response of Arabidopsis thaliana seedling and the resulting process that clarifies that the compound obtained in the screening is an inhibitor of cryptochromes.

Plant growth promotion effect of plasma activated water on Lactuca sativa L. cultivated in two different volumes of substrate.

Plasma activated water (PAW) can represent an alternative to chemical fertilizers in agriculture. The effects of PAW treatment applied in two concentrations (1.5 or 3.0 mg L-1 NO3-) on some morphological, physiological, biochemical parameters and yield of Lactuca sativa L. grown in two different pot volumes (400 or 3200 cm3) were investigated in this study. The results showed that both PAW concentrations did not influence the germination, once the process was initiated. Positive effects of the treatments were registered on the length of radicle and hypocotyls of lettuce at a concentration of 1.5 mg L-1 NO3- (PAW I), the chlorophyll content was significantly increased at a concentration of 3.0 mg L-1 NO3- (PAW II) and bigger pot volume, also the foliar weight and area. No significant differences between the treated and untreated plants were recorded for the root weight, leaf length and width. The dry weight was significantly higher for the lettuce treated with PAW I and II grown in big volume pots at 57 days after transplanting (DAT) and small volume pots at 64 DAT. The nitrites content of the lettuce grown in big pots was lower than of the lettuce grown in small pots, regardless of the PAW treatment. Contrary, the nitrates content was higher in the lettuce grown in big pots (up to 36.4 mg KNO3/g DW), compared to small pots (under 0.3 mg KNO3/g DW).

Plant grafting relieves asymmetry of jasmonic acid response induced by wounding between scion and rootstock in tomato hypocotyl.

Plant grafting is a sequential wound healing process. However, whether wounding induces a different jasmonic acid (JA) response within half a day (12 h) after grafting or non-grafting remains unclear. Using the tomato hypocotyl grafting method, we show that grafting alleviates the asymmetrical accumulation of JA and jasmonic acid isoleucine conjugate (JA-Ile) in scion and rootstock caused by wounding, and from 2 h after tomato micrografting, grafting obviously restored the level of JA-Ile in the scion and rootstock. Meanwhile, five JA-related genes, SlLOX11, SlAOS, SlCOI1, SlLAPA and SlJA2L, are detected and show significant changes in transcriptional expression patterns within 12 h of grafting, from asymmetrical to symmetrical, when the expression of 30 JA- and defense-related genes were analyzed. The results indicated that grafting alleviates the asymmetrical JA and defense response between scion and rootstock of the tomato hypocotyl within 12 h as induced by wounding. Moreover, we demonstrate that in the very early hours after grafting, JA-related genes may be involved in a molecular mechanism that changes asymmetrical expression as induced by wounding between scion and rootstock, thereby promoting wound healing and grafting success.

The Arabidopsis RLCK VI_A2 Kinase Controls Seedling and Plant Growth in Parallel with Gibberellin.

The plant-specific receptor-like cytoplasmic kinases (RLCKs) form a large, poorly characterized family. Members of the RLCK VI_A class of dicots have a unique characteristic: their activity is regulated by Rho-of-plants (ROP) GTPases. The biological function of one of these kinases was investigated using a T-DNA insertion mutant and RNA interference. Loss of RLCK VI_A2 function resulted in restricted cell expansion and seedling growth. Although these phenotypes could be rescued by exogenous gibberellin, the mutant did not exhibit lower levels of active gibberellins nor decreased gibberellin sensitivity. Transcriptome analysis confirmed that gibberellin is not the direct target of the kinase; its absence rather affected the metabolism and signalling of other hormones such as auxin. It is hypothesized that gibberellins and the RLCK VI_A2 kinase act in parallel to regulate cell expansion and plant growth. Gene expression studies also indicated that the kinase might have an overlapping role with the transcription factor circuit (PIF4-BZR1-ARF6) controlling skotomorphogenesis-related hypocotyl/cotyledon elongation. Furthermore, the transcriptomic changes revealed that the loss of RLCK VI_A2 function alters cellular processes that are associated with cell membranes, take place at the cell periphery or in the apoplast, and are related to cellular transport and/or cell wall reorganisation.

Enhanced thermotolerance of Arabidopsis by chitooligosaccharides-induced CERK1n-ERc fusion gene.

Heat stress is a major growth-limiting factor for most crops over the world. Chitin elicitor receptor kinase 1 (CERK1) is a chitin/chitooligosaccharides receptor, and ERECTA (ER) plays a crucial role in plant resistance to heat stress. In the present study, a chitooligosaccharides-induced CERK1n-ERc fusion gene was designed and synthesized, in which the extracellular domain and transmembrane domain of CERK1 gene is connected with the response region of ER gene. We successfully constructed the CERK1n-ERc fusion gene by Overlap PCR and introduced it into Arabidopsis by Agrobacterium-medicated infection. Genetically modified (GM) plants had a greater germination rate and germination index, as well as a shorter mean germination time, indicating that they had a better thermotolerance compared with the wild-type (WT) lines under heat stress. Moreover, the GM lines showed a lower level of hydrogen peroxide (H2O2) and relative electrolyte leakage (REL), suggesting that they were in better state compared with the WT plants when exposed to high temperature. UPLC-MS/MS was employed to assess the phytohormone level, suggesting that the GM lines acquired a better thermotolerance via jasmonic acid (JA) signaling pathways. In general, we constructed a COS-induced fusion gene to enhance the thermotolerance of Arabidopsis during seed germination and postgermination growth.

Retrograde Induction of phyB Orchestrates Ethylene-Auxin Hierarchy to Regulate Growth.

Exquisitely regulated plastid-to-nucleus communication by retrograde signaling pathways is essential for fine-tuning of responses to the prevailing environmental conditions. The plastidial retrograde signaling metabolite methylerythritol cyclodiphosphate (MEcPP) has emerged as a stress signal transduced into a diverse ensemble of response outputs. Here, we demonstrate enhanced phytochrome B protein abundance in red light-grown MEcPP-accumulating ceh1 mutant Arabidopsis (Arabidopsis thaliana) plants relative to wild-type seedlings. We further establish MEcPP-mediated coordination of phytochrome B with auxin and ethylene signaling pathways and uncover differential hypocotyl growth of red light-grown seedlings in response to these phytohormones. Genetic and pharmacological interference with ethylene and auxin pathways outlines the hierarchy of responses, placing ethylene epistatic to the auxin signaling pathway. Collectively, our findings establish a key role of a plastidial retrograde metabolite in orchestrating the transduction of a repertoire of signaling cascades. This work positions plastids at the zenith of relaying information coordinating external signals and internal regulatory circuitry to secure organismal integrity.

Strigolactone and Karrikin Signaling Pathways Elicit Ubiquitination and Proteolysis of SMXL2 to Regulate Hypocotyl Elongation in Arabidopsis.

Strigolactones (SLs) and karrikins (KARs) are related butenolide signaling molecules that control plant development. In Arabidopsis (Arabidopsis thaliana), they are recognized separately by two closely related receptors but use the same F-box protein MORE AXILLARY GROWTH2 (MAX2) for signal transduction, targeting different members of the SMAX1-LIKE (SMXL) family of transcriptional repressors for degradation. Both signals inhibit hypocotyl elongation in seedlings, raising the question of whether signaling is convergent or parallel. Here, we show that synthetic SL analog GR244DO enhanced the interaction between the SL receptor DWARF14 (D14) and SMXL2, while the KAR surrogate GR24 ent-5DS induced association of the KAR receptor KARRIKIN INSENSITIVE2 (KAI2) with SMAX1 and SMXL2. Both signals trigger polyubiquitination and degradation of SMXL2, with GR244DO dependent on D14 and GR24 ent-5DS dependent mainly on KAI2. SMXL2 is critical for hypocotyl responses to GR244DO and functions redundantly with SMAX1 in hypocotyl response to GR24 ent-5DS Furthermore, GR244DO induced response of D14-LIKE2 and KAR-UP F-BOX1 through SMXL2, whereas GR24 ent-5DS induced expression of these genes via both SMAX1 and SMXL2. These findings demonstrate that both SLs and KARs could trigger polyubiquitination and degradation of SMXL2, thus uncovering an unexpected but important convergent pathway in SL- and KAR-regulated gene expression and hypocotyl elongation.

IPyA glucosylation mediates light and temperature signaling to regulate auxin-dependent hypocotyl elongation in Arabidopsis.

Auxin is a class of plant hormone that plays a crucial role in the life cycle of plants, particularly in the growth response of plants to ever-changing environments. Since the auxin responses are concentration-dependent and higher auxin concentrations might often be inhibitory, the optimal endogenous auxin level must be closely controlled. However, the underlying mechanism governing auxin homeostasis remains largely unknown. In this study, a UDP-glycosyltransferase (UGT76F1) was identified from Arabidopsis thaliana, which participates in the regulation of auxin homeostasis by glucosylation of indole-3-pyruvic acid (IPyA), a major precursor of the auxin indole-3-acetic acid (IAA) biosynthesis, in the formation of IPyA glucose conjugates (IPyA-Glc). In addition, UGT76F1 was found to mediate hypocotyl growth by modulating active auxin levels in a light- and temperature-dependent manner. Moreover, the transcription of UGT76F1 was demonstrated to be directly and negatively regulated by PIF4, which is a key integrator of both light and temperature signaling pathways. This study sheds a light on the trade-off between IAA biosynthesis and IPyA-Glc formation in controlling auxin levels and reveals a regulatory mechanism for plant growth adaptation to environmental changes through glucosylation of IPyA.

Biochemical and Genetic Analysis Identify CSLD3 as a beta-1,4-Glucan Synthase That Functions during Plant Cell Wall Synthesis.

In plants, changes in cell size and shape during development fundamentally depend on the ability to synthesize and modify cell wall polysaccharides. The main classes of cell wall polysaccharides produced by terrestrial plants are cellulose, hemicelluloses, and pectins. Members of the cellulose synthase (CESA) and cellulose synthase-like (CSL) families encode glycosyltransferases that synthesize the ß-1,4-linked glycan backbones of cellulose and most hemicellulosic polysaccharides that comprise plant cell walls. Cellulose microfibrils are the major load-bearing component in plant cell walls and are assembled from individual ß-1,4-glucan polymers synthesized by CESA proteins that are organized into multimeric complexes called CESA complexes, in the plant plasma membrane. During distinct modes of polarized cell wall deposition, such as in the tip growth that occurs during the formation of root hairs and pollen tubes or de novo formation of cell plates during plant cytokinesis, newly synthesized cell wall polysaccharides are deposited in a restricted region of the cell. These processes require the activity of members of the CESA-like D subfamily. However, while these CSLD polysaccharide synthases are essential, the nature of the polysaccharides they synthesize has remained elusive. Here, we use a combination of genetic rescue experiments with CSLD-CESA chimeric proteins, in vitro biochemical reconstitution, and supporting computational modeling and simulation, to demonstrate that Arabidopsis (Arabidopsis thaliana) CSLD3 is a UDP-glucose-dependent ß-1,4-glucan synthase that forms protein complexes displaying similar ultrastructural features to those formed by CESA6.

High frequency regeneration of plants via callus-mediated organogenesis from cotyledon and hypocotyl cultures in a multipurpose tropical tree (Neolamarkia Cadamba).

In this works, a simple, efficient and repeatable protocol was developed for in vitro regeneration via callus-mediated organogenesis of Neolamarkia Cadamba using cotyledonary petioles and hypocotyls. Effects of basal medium, plant growth regulators, the types and age of explant on the formation of adventitious buds/shoots were studied. Meanwhile, histological analysis for early ontogenic stages and genetic stability assessment by flow cytometry were investigated. Our investigation demonstrated that, compared with 6-benzyladenine (BA), N6-(2-isopentenyl) adenine (2-ip), Thidiazuron (TDZ) was the optimal cytokinin for buds/shoots induction on cotyledon and hypocotyl explants. Douglas-fir and sugar pine medium (DCR) supplemented with 22.7 µM TDZ and 0.27 µM α-naphthalene acetic acid (NAA) was most effective on bud induction, with the highest bud-induction rate and numbers of buds on cotyledon and hypocotyl explants. The available shoot per explant hit 35.2 when the induced callus sub-cultured to a medium without TDZ. It was found that TDZ could promote induction of the callus and the buds, however, continuous exposure beyond 4 weeks of supplemented high concentration (exceed 11.35 µM), TDZ was harmful to the proliferation and growth of buds/shoots. DCR appeared more efficiency than Murashige and Skoog medium (MS), Woody Plant medium (WPM), anther culture of cereal crops medium (N6) on bud induction. Age of cotyledon and hypocotyl explants in 20-day to 25-day was most beneficial to adventitious buds/shoots formation. Histological investigation confirmed that the buds originated from the wounded incisions of cotyledonary petiole and hypocotyl fragments, with callus formation. The regeneration plantlets were successfully acclimatized in greenhouse, yielded above 95% survival rate in field, exhibited normal morphology and growth characteristics. The analysis of flow cytometry on N. cadamba indicated no variation in the ploidy levels between the regenerated plantlets and the donor trees. The developed procedure can be used for mass production, germplasm exchange and transgenic studies to improve the resistance of the species via Agrobacterium-mediated.

LPIAT, a lyso-Phosphatidylinositol Acyltransferase, Modulates Seed Germination in Arabidopsis thaliana through PIP Signalling Pathways and is Involved in Hyperosmotic Response.

Lyso-lipid acyltransferases are enzymes involved in various processes such as lipid synthesis and remodelling. Here, we characterized the activity of an acyltransferase from Arabidopsis thaliana (LPIAT). In vitro, this protein, expressed in Escherichia coli membrane, displayed a 2-lyso-phosphatidylinositol acyltransferase activity with a specificity towards saturated long chain acyl CoAs (C16:0- and C18:0-CoAs), allowing the remodelling of phosphatidylinositol. In planta, LPIAT gene was expressed in mature seeds and very transiently during seed imbibition, mostly in aleurone-like layer cells. Whereas the disruption of this gene did not alter the lipid composition of seed, its overexpression in leaves promoted a strong increase in the phosphatidylinositol phosphates (PIP) level without affecting the PIP2 content. The spatial and temporal narrow expression of this gene as well as the modification of PIP metabolism led us to investigate its role in the control of seed germination. Seeds from the lpiat mutant germinated faster and were less sensitive to abscisic acid (ABA) than wild-type or overexpressing lines. We also showed that the protective effect of ABA on young seedlings against dryness was reduced for lpiat line. In addition, germination of lpiat mutant seeds was more sensitive to hyperosmotic stress. All these results suggest a link between phosphoinositides and ABA signalling in the control of seed germination.

Gibberellins modulate shade-induced soybean hypocotyl elongation downstream of the mutual promotion of auxin and brassinosteroids.

Plants and crops are widely suffered by shade stress in the natural communities or in the agricultural fields. The three main phytohormones auxin, gibberellins (GAs) and brassinosteroids (BRs) were found essential in shade avoidance in Arabidopsis. However, their relationship have been seldom reported in plant shade avoidance control. Here, we report our investigation of the crosstalk of auxin, GAs and BRs in shade-induced hypocotyl elongation of soybean. Exogenous feeding of indol-3-acetic acid (IAA), GA3 or 24-epibrassinolide (EBL) distinctly promoted hypocotyl elongation in the white light, while the potent biosynthesis inhibitors of GA3, IAA, BRs severely diminished shade-induced hypocotyl elongation. Synergistic treatment of their biosynthesis inhibitors showed that GA3 fully, while EBL slightly, restored the hypocotyl elongation that was efficiently repressed by IAA biosynthesis inhibitor, GA3 and IAA dramatically suppressed the hypocotyl growth inhibition by BR biosynthesis inhibitor in the shade, whereas both IAA and EBL feeding cannot suppress the elongation inhibition by GA biosynthesis inhibitor. Further analyses revealed that shade remarkably upregulated expression of key genes of IAA, GA and BR biosynthesis in the soybean hypocotyls, and GA biosynthesis genes were effectively blocked by IAA, GA and BR biosynthesis inhibitors in the shade. Taken together, these results suggest that GAs modulate shade-induced hypocotyl elongation downstream of mutual promotion of auxin and BRs in soybean.

14-3-3 proteins contribute to leaf and root development via brassinosteroid insensitive 1 in Arabidopsis thaliana.

BACKGROUND: Brassinosteroids (BR) are essential growth hormone in plants. Various components involved in signal transduction pathway have been identified as targets of 14-3-3 phospho-binding proteins. Previously, we showed that 14-3-3 proteins directly interact with the Brassinosteroid Insensitive 1 (BRI1), the BR receptor kinase, and are also subject to phosphorylation in a BR-dependent manner. OBJECTIVE: In this study, we aimed to examine a potential interplay between 14-3-3 proteins and BRI1 in plant growth. METHODS: Morphological phenotypes of a T-DNA insertion mutant line, 14-3-3ψφε, defective in three 14-3-3 isoforms, psi, phi and epsilon, were characterized and compared with bri1-5 and two transgenic lines for BRI1, BRI1-Flag and BRI1-Flag (14-3-3ψφε). We also generated complementation lines carrying each of the three 14-3-3 genes and determined their differences in rosette growth. RESULTS: No significant differences between the wild-type and 14-3-3ψφε seedlings were observed regardless of BR applications. However, BRI1-Flag (14-3-3ψφε) showed a significantly reduced cold tolerance and BR sensitivity in hypocotyl and root development when compared to BRI1-Flag. In addition, narrower leaf shape and smaller rosette size were observed in BRI1-Flag (14-3-3ψφε), while the mutant phenotypes were partially restored in the complementation lines, two of which with 14-3-3φ and 14-3-3ε showed the rosette growth comparable to BRI1-Flag. CONCLUSION: Taken together, our results suggested that 14-3-3 proteins might positively regulate BRI1 activity and showed that 14-3-3 isoforms have different functional impacts in BR signaling.