CPR5-mediated nucleo-cytoplasmic localization of IAA12 and IAA19 controls lateral root development during abiotic stress.

Plasticity of the root system architecture (RSA) is essential in enabling plants to cope with various environmental stresses and is mainly controlled by the phytohormone auxin. Lateral root development is a major determinant of RSA. Abiotic stresses reduce auxin signaling output, inhibiting lateral root development; however, how abiotic stress translates into a lower auxin signaling output is not fully understood. Here, we show that the nucleo-cytoplasmic distribution of the negative regulators of auxin signaling AUXIN/INDOLE-3-ACETIC ACID INDUCIBLE 12 (AUX/IAA12 or IAA12) and IAA19 determines lateral root development under various abiotic stress conditions. The cytoplasmic localization of IAA12 and IAA19 in the root elongation zone enforces auxin signaling output, allowing lateral root development. Among components of the nuclear pore complex, we show that CONSTITUTIVE EXPRESSOR OF PATHOGENESIS-RELATED GENES 5 (CPR5) selectively mediates the cytoplasmic translocation of IAA12/19. Under abiotic stress conditions, CPR5 expression is strongly decreased, resulting in the accumulation of nucleus-localized IAA12/19 in the root elongation zone and the suppression of lateral root development, which is reiterated in the cpr5 mutant. This study reveals a regulatory mechanism for auxin signaling whereby the spatial distribution of AUX/IAA regulators is critical for lateral root development, especially in fluctuating environmental conditions.

The Arabidopsis thaliana NGATHA transcription factors negatively regulate cell proliferation of lateral organs.

The cell proliferation process of aerial lateral organs, such as leaves and flowers, is coordinated by complex genetic networks that, in general, converge on the cell cycle. The Arabidopsis thaliana NGATHA (AtNGA) family comprises four members that belong to the B3-type transcription factor superfamily, and has been suggested to be involved in growth and development of aerial lateral organs, although its role in the cell proliferation and expansion processes remains to be resolved in more detail. In order to clarify the role of AtNGAs in lateral organ growth, we took a systematic approach using both the loss- and gain-of-functional mutants of all four members. Our results showed that overexpressors of AtNGA1 to AtNGA4 developed small, narrow lateral organs, whereas the nga1 nga2 nga3 nga4 quadruple mutant produced large, wide lateral organs. We found that cell numbers of the lateral organs were significantly affected: a decrease in overexpressors and, inversely, an increase in the quadruple mutant. Kinematic analyses on leaf growth revealed that, compared with the wild type, the overexpressors displayed a lower activity of cell proliferation and yet the mutant a higher activity. Changes in expression of cell cycle-regulating genes were well in accordance with the cell proliferation activities, establishing that the AtNGA transcription factors act as bona fide negative regulators of the cell proliferation of aerial lateral organs.

ANGUSTIFOLIA mediates one of the multiple SCRAMBLED signaling pathways regulating cell growth pattern in Arabidopsis thaliana.

In Arabidopsis thaliana, an atypical leucine-rich repeat receptor-like kinase, SCRAMBLED (SCM), is required for multiple developmental processes including root epidermal cell fate determination, silique dehiscence, inflorescence growth, ovule morphogenesis, and tissue morphology. Previous work suggested that SCM regulates these multiple pathways using distinct mechanisms via interactions with specific downstream factors. ANGUSTIFOLIA (AN) is known to regulate cell and tissue morphogenesis by influencing cortical microtubule arrangement, and recently, the AN protein was reported to interact with the SCM protein. Therefore, we examined whether AN might be responsible for mediating some of the SCM-dependent phenotypes. We discovered that both scm and an mutant lines cause an abnormal spiral or twisting growth of roots, but only the scm mutant affected root epidermal patterning. The siliques of the an and scm mutants also exhibited spiral growth, as previously reported, but only the scm mutant altered silique dehiscence. Interestingly, we discovered that the spiral growth of roots and siliques of the scm mutant is rescued by a truncated SCM protein that lacks its kinase domain, and that a juxtamembrane domain of SCM was sufficient for AN binding in the yeast two-hybrid analysis. These results suggest that the AN protein is one of the critical downstream factors of SCM pathways specifically responsible for mediating its effects on cell/tissue morphogenesis through cortical microtubule arrangement.

WEREWOLF and ENHANCER of GLABRA3 are interdependent regulators of the spatial expression pattern of GLABRA2 in Arabidopsis.

In multicellular organisms, cell fates are specified through differential regulation of transcription. Epidermal cell fates in the Arabidopsis thaliana root are precisely specified by several transcription factors, with the GLABRA2 (GL2) homeodomain protein acting at the farthest downstream in this process. To better understand the regulation of GL2 expression, we ectopically expressed WEREWOLF (WER) and ENHANCER OF GLABRA3 (EGL3) in various tissues and examined GL2 expression. Here we show that WER expressed ubiquitously in the root induced GL2 expression only in the root epidermis, whereas co-expression of WER and EGL3 induced GL2 expression in the corresponding tissues. We also found that GL3 accumulated in the nucleus at the early meristematic region and EGL3 accumulated later in the nucleus of epidermal cells. We further found that ectopic expression of WER and EGL3 in ground tissues inhibited GL2 expression in the epidermis. Our results suggest that the co-expression of WER and EGL3 is sufficient for driving GL2 and CPC expression.

Distinct signaling mechanisms in multiple developmental pathways by the SCRAMBLED receptor of Arabidopsis.

SCRAMBLED (SCM), a leucine-rich repeat receptor-like kinase in Arabidopsis (Arabidopsis thaliana), is required for positional signaling in the root epidermis and for tissue/organ development in the shoot. To further understand SCM action, we generated a series of kinase domain variants and analyzed their ability to complement scm mutant defects. We found that the SCM kinase domain, but not kinase activity, is required for its role in root epidermal patterning, supporting the view that SCM is an atypical receptor kinase. We also describe a previously uncharacterized role for SCM in fruit dehiscence, because mature siliques from scm mutants fail to open properly. Interestingly, the kinase domain of SCM appears to be dispensable for this developmental process. Furthermore, we found that most of the SCM kinase domain mutations dramatically inhibit inflorescence development. Because this process is not affected in scm null mutants, it is likely that SCM acts redundantly to regulate inflorescence size. The importance of distinct kinase residues for these three developmental processes provides an explanation for the maintenance of the conserved kinase domain in the SCM protein, and it may generally explain its conservation in other atypical kinases. Furthermore, these results indicate that individual leucine-rich repeat receptor-like kinases may participate in multiple pathways using distinct signaling mechanisms to mediate diverse cellular communication events.

A gene regulatory network for root epidermis cell differentiation in Arabidopsis.

The root epidermis of Arabidopsis provides an exceptional model for studying the molecular basis of cell fate and differentiation. To obtain a systems-level view of root epidermal cell differentiation, we used a genome-wide transcriptome approach to define and organize a large set of genes into a transcriptional regulatory network. Using cell fate mutants that produce only one of the two epidermal cell types, together with fluorescence-activated cell-sorting to preferentially analyze the root epidermis transcriptome, we identified 1,582 genes differentially expressed in the root-hair or non-hair cell types, including a set of 208 "core" root epidermal genes. The organization of the core genes into a network was accomplished by using 17 distinct root epidermis mutants and 2 hormone treatments to perturb the system and assess the effects on each gene's transcript accumulation. In addition, temporal gene expression information from a developmental time series dataset and predicted gene associations derived from a Bayesian modeling approach were used to aid the positioning of genes within the network. Further, a detailed functional analysis of likely bHLH regulatory genes within the network, including MYC1, bHLH54, bHLH66, and bHLH82, showed that three distinct subfamilies of bHLH proteins participate in root epidermis development in a stage-specific manner. The integration of genetic, genomic, and computational analyses provides a new view of the composition, architecture, and logic of the root epidermal transcriptional network, and it demonstrates the utility of a comprehensive systems approach for dissecting a complex regulatory network.

Nuclear trapping controls the position-dependent localization of CAPRICE in the root epidermis of Arabidopsis.

Cell fate determination and differentiation are central processes in the development of multicellular organisms, and the Arabidopsis (Arabidopsis thaliana) root epidermis provides a model system to study the molecular basis of these processes. A lateral inhibition mechanism mediated by an R3 single-repeat MYB protein, CAPRICE (CPC), has been proposed to explain the specification of the two types of root epidermal cells (hair cells and nonhair cells). However, it is not clear how CPC acts preferentially in the H-position cells, rather than the N-position cells, where its gene is expressed. To explore this issue, we examined the effect of misexpressed CPC on cell fate specification and CPC localization in the root epidermis. We show that CPC is able to move readily within the root epidermis when its expression level is high and that CPC can induce the hair cell fate in a cell-autonomous manner. We provide evidence that CPC is capable of moving from the stele tissue in the center of the root to the outermost epidermal layer, where it can induce the hair cell fate. In addition, we show that CPC protein accumulates primarily in the nuclei of H-position cells in the early meristematic region, and this localization requires the H-cell-expressed ENHANCER OF GLABRA3 (EGL3) basic helix-loop-helix transcription factor. These results suggest that cell-cell movement of CPC occurs readily within the meristematic region of the root and that EGL3 preferentially traps the CPC protein in the H-position cells of the epidermis.

Funneling of gibberellin signaling by the GRAS transcription regulator scarecrow-like 3 in the Arabidopsis root.

During plant development, because no cell movement takes place, control of the timing and extent of cell division and coordination of the direction and extent of cell expansion are particularly important for growth and development. The plant hormone gibberellins (GAs) play key roles in the control of these developmental processes. However, little is known about the molecular components that integrate the generic GA signaling into a specific cell/tissue to coordinate cell division and cell expansion. Here we report that scarecrow-like 3 (SCL3), a GRAS protein, acts as a positive regulator to integrate and maintain a functional GA pathway by attenuating the DELLA repressors in the root endodermis. The tissue-specific maintenance of GA signaling in the root endodermis plays distinct roles along the longitudinal root axis. While in the elongation/differentiation zone (EDZ), the endodermis-confined GA pathway by SCL3 controls primarily coordination of root cell elongation; in the meristem zone (MZ) SCL3 in conjunction with the short-root/scarecrow (SHR/SCR) pathway controls GA-modulated ground tissue maturation. Our findings highlight the regulatory network of the GRAS transcription regulators (SCL3, DELLAs, and SHR/SCR) in the root endodermis, shedding light on how GA homeostasis is achieved and how the maintenance of GA signaling controls developmental processes in roots.

WEREWOLF, a regulator of root hair pattern formation, controls flowering time through the regulation of FT mRNA stability.

A key floral activator, FT, integrates stimuli from long-day, vernalization, and autonomous pathways and triggers flowering by directly regulating floral meristem identity genes in Arabidopsis (Arabidopsis thaliana). Since a small amount of FT transcript is sufficient for flowering, the FT level is strictly regulated by diverse genes. In this study, we show that WEREWOLF (WER), a MYB transcription factor regulating root hair pattern, is another regulator of FT. The mutant wer flowers late in long days but normal in short days and shows a weak sensitivity to vernalization, which indicates that WER controls flowering time through the photoperiod pathway. The expression and double mutant analyses showed that WER modulates FT transcript level independent of CONSTANS and FLOWERING LOCUS C. The histological analysis of WER shows that it is expressed in the epidermis of leaves, where FT is not expressed. Consistently, WER regulates not the transcription but the stability of FT mRNA. Our results reveal a novel regulatory mechanism of FT that is non cell autonomous.

Cell fate in the Arabidopsis root epidermis is determined by competition between WEREWOLF and CAPRICE.

The root hair and nonhair cells in the Arabidopsis (Arabidopsis thaliana) root epidermis are specified by a suite of transcriptional regulators. Two of these are WEREWOLF (WER) and CAPRICE (CPC), which encode MYB transcription factors that are required for promoting the nonhair cell fate and the hair cell fate, respectively. However, the precise function and relationship between these transcriptional regulators have not been fully defined experimentally. Here, we examine these issues by misexpressing the WER gene using the GAL4-upstream activation sequence transactivation system. We find that WER overexpression in the Arabidopsis root tip is sufficient to cause epidermal cells to adopt the nonhair cell fate through direct induction of GLABRA2 (GL2) gene expression. We also show that GLABRA3 (GL3) and ENHANCER OF GLABRA3 (EGL3), two closely related bHLH proteins, are required for the action of the overexpressed WER and that WER interacts with these bHLHs in plant cells. Furthermore, we find that CPC suppresses the WER overexpression phenotype quantitatively. These results show that WER acts together with GL3/EGL3 to induce GL2 expression and that WER and CPC compete with one another to define cell fates in the Arabidopsis root epidermis.

Brassinosteroid-Insensitive 1-Associated Receptor Kinase 1 Modulates Abscisic Acid Signaling by Inducing PYR1 Monomerization and Association With ABI1 in Arabidopsis.

Brassinosteroid-Insensitive 1-Associated Receptor Kinase 1 (BAK1) is a versatile kinase involved in many different plant developmental responses. Previously, we showed that BAK1 interacts with open stomata 1 (OST1), a cytoplasmic kinase, to promote abscisic acid (ABA)-induced stomatal closure. ABA is a plant hormone that primarily regulates stress responses and is recognized by the PYRABACTIN RESISTANCE1 (PYR1)/PYR1-LIKE (PYL)/REGULATORY COMPONENT OF ABA RECEPTORS (RCAR), which activates ABA signaling. Here, we demonstrated that BAK1 interacts with PYR1 and phosphorylates PYR1 in response to ABA in plants. We identified T137 and S142 of PYR1 as the phosphosites targeted by BAK1. Using phosphomimetic (PYR1DD) and phospho-dead (PYR1AA) PYR1 compared with wild-type PYR1, we showed that transgenic plants overexpressing a phosphomimetic PYR1 exhibited hypersensitivity to the inhibition of ABA-induced root growth and seed germination and increased ABA-induced stomatal closure and ABA-inducible gene expression. As underlying reasons for these phenomena, we further demonstrated that phosphorylated PYR1 existed in a monomeric form, in which ABA binding was increased, and the degree of complex formation with ABI1 was also increased. These results suggest that BAK1 positively modulates ABA signaling through interaction with PYR1, in addition to OST1.

Characterization of cp3 reveals a new bri1 allele, bri1-120, and the importance of the LRR domain of BRI1 mediating BR signaling.

BACKGROUND: Since the identification of BRI1 (BRASSINOSTEROID-INSENSITIVE1), a brassinosteroids (BRs) receptor, most of the critical roles of BR in plant development have been assessed using various bri1 mutant alleles. The characterization of individual bri1 mutants has shown that both the extracellular and cytoplasmic domains of BRI1 are important to its proper functioning. Particularly, in the extracellular domain, regions near the 70-amino acid island are known to be critical to BR binding. In comparison, the exact function of the leucine rich-repeats (LRR) region located before the 70-amino acid island domain in the extracellular cellular portion of BRI1 has not yet been described, due to a lack of specific mutant alleles. RESULTS: Among the mutants showing altered growth patterns compared to wild type, we further characterized cp3, which displayed defective growth and reduced BR sensitivity. We sequenced the genomic DNA spanning BRI1 in the cp3 and found that cp3 has a point mutation in the region encoding the 13th LRR of BRI1, resulting in a change from serine to phenylalanine (S399F). We renamed it bri1-120. We also showed that overexpression of the wild type BRI1 protein rescued the phenotype of bri1-120. Using a GFP-tagged bri1-120 construct, we detected the bri1-120 protein in the plasma membrane, and showed that the phenotypic defects in the rosette leaves of bri1-301, a kinase-inactive weak allele of BRI1, can be restored by the overexpression of the bri1-120 proteins in bri1-301. We also produced bri1-301 mutants that were wild type in appearance by performing a genetic cross between bri1-301 and bri1-120 plants. CONCLUSIONS: We identified a new bri1 allele, bri1-120, whose mutation site has not yet been found or characterized. Our results indicated that the extracellular LRR regions before the 70-amino acid island domain of BRI1 are important for the appropriate cellular functioning of BRI1. Also, we confirmed that a successful interallelic complementation occurs between the extracellular domain mutant allele and the cytoplasmic kinase-inactive mutant allele of BRI1 in vivo.

POLTERGEIST and POLTERGEIST-LIKE1 are essential for the maintenance of post-embryonic shoot and root apical meristems as revealed by a partial loss-of-function mutant allele of pll1 in Arabidopsis.

BACKGROUND: POLTERGEIST (POL) and POL-LIKE1 (PLL1) encoding related protein phosphatase 2Cs are essential for the establishment of both shoot and root meristems during embryogenesis. As the strong pol pll1 are seedling-lethal due to the lack of hypocotyl vasculature, the roles of POL/PLL1 for the post-embryonic development is difficult to be assessed. OBJECTIVE: To prepare a weak pol pll1 double mutant that are able to produce post-embryonic organs. METHODS: Several T-DNA insertion mutants of pll1 were crossed to pol-6 for the preparation of weak pol pll1. To understand the epistatic interactions between POL/PLL1 and CLAVATAs, the phenotypes of clvs pol pll1 were assessed and the expression patterns of stem cell markers were examined in pol pll1. POLpro:PLL1-GFP expression was examined during the embryogenesis with confocal microscopy. RESULTS: We isolated a pll1-3 (S544N) allele and prepared a weak pol-6 pll1-3. About 5% of pol-6 pll1-3 seedlings continued the post-embryonic development displaying short roots with reduced root meristem, wuschel-like adventitious phyllotaxis, and defective flowers lacking carpel. The clv1, clv2, and clv3 phenotypes led by enlarged shoot meristems were almost completely suppressed in the pol-6 pll1-3. POL/PLL1 were required for the indeterminate floral organ development displayed by agamous. PLL1-GFP asymmetrically localized in the shootward sides of columella cells and increased the size of distal root meristem region by enhancing the WUS-RELATED HOMEOBOX 5 expression suggesting that PLL1 might provide the stem cells and progenies with proper positional information for the asymmetric cell divisions. CONCLUSION: Together, POL/PLL1 are required for the maintenance of stem cell pools for the post-embryonic development in Arabidopsis.

Overexpression of three related root-cap outermost-cell-specific C2H2-type zinc-finger protein genes suppresses the growth of Arabidopsis in an EAR-motif-dependent manner.

The root meristem of Arabidopsis thaliana is protected by the root cap, the size of which is tightly regulated by the balance between the formative cell divisions and the dispersal of the outermost cells. We isolated an enhancer-tagged dominant mutant displaying the short and twisted root by the overexpression of ZINC-FINGER OF ARABIDOPSIS THALIANA1 (ZAT1) encoding an EAR motif-containing zinc-finger protein. The growth inhibition by ZAT1 was shared by ZAT4 and ZAT9, the ZAT1 homologues. The ZAT1 promoter was specifically active in the outermost cells of the root cap, in which ZAT1-GFP was localized when expressed by the ZAT1 promoter. The outermost cell-specific expression pattern of ZAT1 was not altered in the sombrero (smb) or smb bearskin1 (brn1) brn2 accumulating additional root-cap layers. In contrast, ZAT4-GFP and ZAT9- GFP fusion proteins were distributed to the inner root-cap cells in addition to the outermost cells where ZAT4 and ZAT9 promoters were active. Overexpression of ZAT1 induced the ectopic expression of PUTATIVE ASPARTIC PROTEASE3 involved in the programmed cell death. The EAR motif was essential for the growth inhibition by ZAT1. These results suggest that the three related ZATs might regulate the maturation of the outermost cells of the root cap. [BMB Reports 2020; 53(3): 160-165].

QUIRKY regulates root epidermal cell patterning through stabilizing SCRAMBLED to control CAPRICE movement in Arabidopsis.

SCM, a leucine-rich repeat receptor-like kinase, is required for root epidermal cells to appropriately interpret their location and generate the proper cell-type pattern during Arabidopsis root development. Here, via a screen for scm-like mutants we describe a new allele of the QKY gene. We find that QKY is required for the appropriate spatial expression of several epidermal cell fate regulators in a similar manner as SCM in roots, and that QKY and SCM are necessary for the efficient movement of CPC between epidermal cells. We also show that turnover of SCM is mediated by a vacuolar degradation pathway triggered by ubiquitination, and that QKY prevents this SCM ubiquitination through their physical interaction. These results suggest that QKY stabilizes SCM through interaction, and this complex facilitates CPC movement between the epidermal cells to help establish the cell-type pattern in the Arabidopsis root epidermis.

Distinct and overlapping roles of single-repeat MYB genes in root epidermal patterning.

Cell specification in the root epidermis of Arabidopsis generates a position-dependent pattern of root-hair cells and non-hair cells. Here we conduct a comprehensive analysis of the five members of a single-repeat R3 MYB gene family, including CAPRICE (CPC), TRIPTYCHON (TRY), ENHANCER of TRY and CPC 1, 2, and 3 (ETC1, ETC2, and ETC3), and study their role and functional relationship in root epidermal cell specification. Based on genetic and expression analyses, CPC, TRY and ETC1, but not ETC2 or ETC3, promote the hair cell fate by inhibiting non-hair specification. Further, we find that single-repeat MYB activity is required for epidermal patterning throughout root development, beginning during embryogenesis. We also identify a novel regulatory interaction whereby GLABRA2 (GL2) promotes TRY (but not CPC or ETC1) expression in the root epidermis, which generates a second lateral inhibition feedback loop. Gene fusion experiments combining CPC regulatory elements with protein-coding regions of each single-repeat MYB gene suggest that all five proteins are functionally similar, although TRY and ETC2 exhibit distinctions from CPC/ETC1/ETC3. These results provide new insight into the function of these single-repeat MYBs and suggest that divergence of their regulatory sequences is largely responsible for their distinct roles in epidermal cell patterning.

Involvement of Pyridoxine/Pyridoxamine 5'-Phosphate Oxidase (PDX3) in Ethylene-Induced Auxin Biosynthesis in the Arabidopsis Root.

As sessile organisms, plants have evolved to adjust their growth and development to environmental changes. It has been well documented that the crosstalk between different plant hormones plays important roles in the coordination of growth and development of the plant. Here, we describe a novel recessive mutant, mildly insensitive to ethylene (mine), which displayed insensitivity to the ethylene precursor, ACC (1-aminocyclopropane-1-carboxylic acid), in the root under the dark-grown conditions. By contrast, mine roots exhibited a normal growth response to exogenous IAA (indole-3-acetic acid). Thus, it appears that the growth responses of mine to ACC and IAA resemble those of weak ethylene insensitive (wei) mutants. To understand the molecular events underlying the crosstalk between ethylene and auxin in the root, we identified the MINE locus and found that the MINE gene encodes the pyridoxine 5'-phosphate (PNP)/pyridoxamine 5'-phosphate (PMP) oxidase, PDX3. Our results revealed that MINE/PDX3 likely plays a role in the conversion of the auxin precursor tryptophan to indole-3-pyruvic acid in the auxin biosynthesis pathway, in which TAA1 (TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1) and its related genes (TRYPTOPHAN AMINOTRANSFERASE RELATED 1 and 2; TAR1 and TAR2) are involved. Considering that TAA1 and TARs belong to a subgroup of PLP (pyridoxal-5'-phosphate)-dependent enzymes, we propose that PLP produced by MINE/PDX3 acts as a cofactor in TAA1/TAR-dependent auxin biosynthesis induced by ethylene, which in turn influences the crosstalk between ethylene and auxin in the Arabidopsis root.

Rhizosphere microbiome structure alters to enable wilt resistance in tomato.

Tomato variety Hawaii 7996 is resistant to the soil-borne pathogen Ralstonia solanacearum, whereas the Moneymaker variety is susceptible to the pathogen. To evaluate whether plant-associated microorganisms have a role in disease resistance, we analyzed the rhizosphere microbiomes of both varieties in a mesocosm experiment. Microbiome structures differed between the two cultivars. Transplantation of rhizosphere microbiota from resistant plants suppressed disease symptoms in susceptible plants. Comparative analyses of rhizosphere metagenomes from resistant and susceptible plants enabled the identification and assembly of a flavobacterial genome that was far more abundant in the resistant plant rhizosphere microbiome than in that of the susceptible plant. We cultivated this flavobacterium, named TRM1, and found that it could suppress R. solanacearum-disease development in a susceptible plant in pot experiments. Our findings reveal a role for native microbiota in protecting plants from microbial pathogens, and our approach charts a path toward the development of probiotics to ameliorate plant diseases.

BRI1-Associated Receptor Kinase 1 Regulates Guard Cell ABA Signaling Mediated by Open Stomata 1 in Arabidopsis.

Stomatal movements are critical in regulating gas exchange for photosynthesis and water balance between plant tissues and the atmosphere. The plant hormone abscisic acid (ABA) plays key roles in regulating stomatal closure under various abiotic stresses. In this study, we revealed a novel role of BAK1 in guard cell ABA signaling. We found that the brassinosteroid (BR) signaling mutant bak1 lost more water than wild-type plants and showed ABA insensitivity in stomatal closure. ABA-induced OST1 expression and reactive oxygen species (ROS) production were also impaired in bak1. Unlike direct treatment with H2O2, overexpression of OST1 did not completely rescue the insensitivity of bak1 to ABA. We demonstrated that BAK1 forms a complex with OST1 near the plasma membrane and that the BAK1/OST1 complex is increased in response to ABA in planta. Brassinolide, the most active BR, exerted a negative effect on ABA-induced formation of the BAK1/OST1 complex and OST1 expression. Moreover, we found that BAK1 and ABI1 oppositely regulate OST1 phosphorylation in vitro, and that ABI1 interacts with BAK1 and inhibits the interaction of BAK1 and OST1. Taken together, our results suggest that BAK1 regulates ABA-induced stomatal closure in guard cells.

Conservation and Diversification of the SHR-SCR-SCL23 Regulatory Network in the Development of the Functional Endodermis in Arabidopsis Shoots.

Development of the functional endodermis of Arabidopsis thaliana roots is controlled, in part, by GRAS transcription factors, namely SHORT-ROOT (SHR), SCARECROW (SCR), and SCARECROW-LIKE 23 (SCL23). Recently, it has been shown that the SHR-SCR-SCL23 regulatory module is also essential for specification of the endodermis (known as the bundle sheath) in leaves. Nevertheless, compared with what is known about the role of the SHR-SCR-SCL23 regulatory network in roots, the molecular interactions of SHR, SCR, and SCL23 are much less understood in shoots. Here, we show that SHR forms protein complexes with SCL23 to regulate transcription of SCL23 in shoots, similar to the regulation mode of SCR expression. Our results indicate that SHR acts as master regulator to directly activate the expression of SCR and SCL23. In the SHR-SCR-SCL23 network, we found a previously uncharacterized negative feedback loop whereby SCL23 modulates SHR levels. Through molecular, genetic, physiological, and morphological analyses, we also reveal that the SHR-SCR-SCL23 module plays a key role in the formation of the endodermis (known as the starch sheath) in hypocotyls. Taken together, our results provide new insights into the regulatory role of the SHR-SCR-SCL23 network in the endodermis development in both roots and shoots.