HIGH PLOIDY2-mediated SUMOylation of transcription factor ARR1 controls two-component signaling in Arabidopsis.

Cytokinins regulate plant growth, development, and responses to environmental stresses such as cold via phosphorelay from cytokinin receptors to the ARABIDOPSIS RESPONSE REGULATORs (ARRs). However, the molecular mechanisms underlying the activation of type-B ARR transcriptional activity in Arabidopsis (Arabidopsis thaliana) remain unclear. Here, we show that the E3 SUMO ligase HIGH PLOIDY2 SUMOylates ARR1, a type-B ARR, at K236, triggering its activation. Cold- or cytokinin-induced phosphorylation of ARR1 at D89 is crucial for its interaction with HPY2. Lysine 236 is critical for ARR1's transactivation without compromising its DNA-binding ability, while D89 is crucial for ARR1's binding to target gene promoters. Cytokinin enhances ARR1's chromatin binding, but cold does not. ARR1 K236 plays a critical role in promoting histone H3 acetylation in response to both cytokinin and cold without affecting chromatin binding. The K236R mutation in ARR1 reduces target gene expression and alters cytokinin and cold response phenotypes. This study unveils a mechanism of ARR1 activation wherein phosphorylated ARR1 interacts with HPY2 and binds to chromatin in response to cytokinin. Cold triggers a phosphorelay targeting chromatin-bound ARR1. HPY2 then catalyzes ARR1 SUMOylation at K236, enhancing histone H3 acetylation and leading to transcriptional activation of ARR1 in response to both cold and cytokinin.

Transcription factors WRKY2 and WRKY34 control LATERAL ORGAN BOUNDARIES DOMAIN10 expression in pollen vegetative cell nuclei.

The intricate regulation of gene expression determining cell fate during male gametogenesis involves a complex interplay of multiple transcriptional regulators. In Arabidopsis (Arabidopsis thaliana), the LATERAL ORGAN BOUNDARIES DOMAIN 10 (LBD10) transcription factor is prominent in early microspores and both the germ and vegetative cells of bicellular pollen, playing an important role in pollen development. However, in mature pollen, LBD10 exclusively localizes in the vegetative cell nucleus. Here, we identify cis-acting elements and trans-acting factors responsible for the specific expression of LBD10 in the vegetative cell nucleus during pollen maturation. Using a series of LBD10 promoter deletion constructs fused with GUS or GFP reporters, we pinpoint two crucial core promoter sequences. These sequences are situated within two 200 bp regions upstream of the start codon and independently govern LBD10 expression in the vegetative cell nucleus. We demonstrate that a W-box motif (AGTCAC) at -770 bp is essential for activating the expression of LBD10 in vegetative cells during pollen maturation. Our transient gene expression assays using Arabidopsis protoplasts and chromatin immunoprecipitation assays show that the transcription factors WRKY2 and WRKY34 recognize the LBD10 promoter region containing W-box motifs. Collectively, our findings suggest that WRKY2 and WRKY34 binding to the W-box motifs plays a role in the vegetative cell nucleus-specific expression of LBD10 in pollen. This interaction may contribute to male gametophyte development, shedding light on the intricate regulatory network governing this critical biological process.

The transcription factor LBD10 sustains pollen tube growth and integrity by modulating reactive oxygen species homeostasis via the regulation of flavonol biosynthesis in Arabidopsis.

Reproduction in angiosperms relies on the precise growth of pollen tubes, facilitating the delivery of sperm cells to the ovule for double fertilization. LATERAL ORGAN BOUNDARIES DOMAIN10 (LBD10), a plant-specific transcription factor, plays a pivotal role in Arabidopsis pollen development. Here, we uncovered LBD10's function in sustaining pollen tube growth and integrity. The lbd10 mutant exhibited elevated levels of reactive oxygen species (ROS) and hydrogen peroxide (H2O2) in both pollen grains and tubes, leading to compromised pollen tube growth. The inhibition of ROS synthesis and scavenging of excess ROS with an antioxidant treatment each alleviated these defects in lbd10. The lbd10 mutant displayed reduced flavonol accumulation in both pollen grains and tubes. All the altered phenotypes of lbd10 were complemented by expressing LBD10 under its native promoter. Exogenous application of flavonoids recused the defects in pollen tube growth and integrity in lbd10, along with reducing the excess levels of ROS and H2O2. LBD10 directly binds the promoters of key flavonol biosynthesis genes in chromatin and promotes reporter gene expression in Arabidopsis mesophyll protoplasts. Our findings indicate that LBD10 modulates ROS homeostasis by transcriptionally activating genes crucial for flavonol biosynthesis, thereby maintaining pollen tube growth and integrity.

Histone modification-dependent production of peptide hormones facilitates acquisition of pluripotency during leaf-to-callus transition in Arabidopsis.

Chromatin configuration is critical for establishing tissue identity and changes substantially during tissue identity transitions. The crucial scientific and agricultural technology of in vitro tissue culture exploits callus formation from diverse tissue explants and tissue regeneration via de novo organogenesis. We investigated the dynamic changes in H3ac and H3K4me3 histone modifications during leaf-to-callus transition in Arabidopsis thaliana. We analyzed changes in the global distribution of H3ac and H3K4me3 during the leaf-to-callus transition, focusing on transcriptionally active regions in calli relative to leaf explants, defined by increased accumulation of both H3ac and H3K4me3. Peptide signaling was particularly activated during callus formation; the peptide hormones RGF3, RGF8, PIP1 and PIPL3 were upregulated, promoting callus proliferation and conferring competence for de novo shoot organogenesis. The corresponding peptide receptors were also implicated in peptide-regulated callus proliferation and regeneration capacity. The effect of peptide hormones in plant regeneration is likely at least partly conserved in crop plants. Our results indicate that chromatin-dependent regulation of peptide hormone production not only stimulates callus proliferation but also establishes pluripotency, improving the overall efficiency of two-step regeneration in plant systems.

Control of lateral root formation by rapamycin-induced dimerization of engineered RGI/BAK1 and by BIR3 chimera.

Leucine-rich repeat receptor kinases (LRR-RKs) play a pivotal role in diverse aspects of growth, development, and immunity in plants by sensing extracellular signals. Typically, LRR-RKs are activated through the ligand-induced interaction with a SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) coreceptor, triggering downstream signaling. ROOT MERISTEM GROWTH FACTOR1 (RGF1) INSENSITIVEs (RGIs) LRR-RLK receptors promote primary root meristem activity while inhibiting lateral root (LR) development in response to RGF peptide. In this study, we employed rapamycin-induced dimerization (RiD) and BAK1-INTERACTING RECEPTOR-LIKE KINASE3 (BIR3) chimera approaches to explore the gain-of-function of RGI1, RGI4, and RGI5. Rapamycin induced the association of cytosolic kinase domains (CKDs) of RGI1 and the BAK1 coreceptor, activating both mitogen-activated protein kinase 3 (MPK3) and MPK6. Rapamycin significantly inhibited LR formation in RiD-RGI1/RGI4/RGI5-BAK1 plants. Using transgenic Arabidopsis expressing RGI1CKD fused to the BIR3-LRR chimera under estradiol control, we observed a substantial reduction in LR density upon ß-estradiol treatment. Additionally, we identified a decrease in root gravitropism in BIR3 chimera plants. In contrast, RiD-RGI/BAK1 plants did not exhibit defects in root gravitropism, implying the importance of combinatorial interactions between RGIs and SERK coreceptors in the inhibition of root gravitropism. Constitutive activation of RGIs with BAK1 in RiD-RGI/BAK1 plants by rapamycin treatment resulted in the inhibition of primary root growth, resembling the inhibitory effects observed with high concentrations of phytohormones on primary root elongation. Our findings highlight that the interactions between CKDs of RGIs and BAK1, constitutively induced by rapamycin or BIR3 chimera, efficiently control LR development.

LBD18 and IAA14 antagonistically interact with ARF7 via the invariant Lys and acidic residues of the OPCA motif in the PB1 domain.

MAIN CONCLUSION: LBD18 and IAA14 antagonistically interact with ARF7 through the electrostatic faces in the ARF7PB1 domain, modulating ARF7 transcriptional activity. Auxin Response Factor 7 (ARF7)/ARF19 control lateral root development by directly activating Lateral Organ Boundaries Domain 16 (LBD16)/LBD18 genes in Arabidopsis. LBD18 upregulates ARF19 expression by binding to the ARF19 promoter. It also interacts with ARF7 through the Phox and Bem1 (PB1) domain to enhance the ARF7 transcriptional activity, forming a dual mode of positive feedback loop. LBD18 competes with the repressor indole-3-acetic acid 14 (IAA14) for ARF7 binding through the PB1 domain. In this study, we examined the molecular determinant of the ARF7 PB1 domain for interacting with LBD18 and showed that the electronic faces in the ARF7 PB1 domain are critical for interacting with LBD18 and IAA14/17. We used a luminescence complementation imaging assay to determine protein-protein interactions. The results showed that mutation of the invariant lysine residue and the OPCA motif in the PB1 domain in ARF7 significantly reduces the protein interaction between ARF7 and LBD18. Transient gene expression assays with Arabidopsis protoplasts showed that IAA14 suppressed transcription-enhancing activity of LBD18 on the LUC reporter gene fused to the ARF19 promoter harboring an auxin response element, but mutation of the invariant lysine residue and OPCA motif in the PB1 domain of IAA14 reduced the repression capability of IAA14 for transcription-enhancing activity of LBD18. We further showed that the same mutation in the PB1 domain of IAA14 reduces its repression capability, thereby increasing the LUC activity induced by both ARF7 and LBD18 compared with IAA14. These results suggest that LBD18 competes with IAA14 for ARF7 binding via the electrostatic faces of the ARF7 PB1 domain to modulate ARF7 transcriptional activity.

ROOT MERISTEM GROWTH FACTOR1 (RGF1)-RGF1 INSENSITIVE 1 peptide-receptor pair inhibits lateral root development via the MPK6-PUCHI module in Arabidopsis.

ROOT MERISTEM GROWTH FACTOR1 (RGF1) and its receptors RGF1 INSENSITIVEs (RGIs) regulate primary root meristem activity via a mitogen-activated protein kinase (MPK) signaling cascade in Arabidopsis. However, it is unknown how RGF1 regulates lateral root (LR) development. Here, we show that the RGF1-RGI1 peptide-receptor pair negatively regulates LR development via activation of PUCHI encoding AP2/EREBP. Exogenous RGF1 peptides inhibited LR development of the wild type. However, the rgi1 mutants were partially or fully insensitive to RGF1 during LR development, whereas four other rgi single mutants, namely rgi2, rgi3, rgi4, and rgi5, were sensitive to RGF1 in inhibiting LR formation. Consistent with this, the red fluorescent protein (RFP) signals driven by the RGF1 promoter were detected at stage I and the following stages, overlapping with RGI1 expression. PUCHI expression was significantly up-regulated by RGF1 but completely inhibited in rgi1. LR development of puchi1-1 was insensitive to RGF1. PUCHI expression driven by the RGI1 promoter reduced LR density in both the wild type and rgi1,2,3. Further, mpk6, but not mpk3, displayed significantly down-regulated PUCHI expression and insensitive LR development in response to RGF1. Collectively, these results suggest that the RGF1-RGI1 module negatively regulates LR development by activating PUCHI expression via MPK6.

Recent advances in peptide signaling during Arabidopsis root development.

Roots provide the plant with water and nutrients and anchor it in a substrate. Root development is controlled by plant hormones and various sets of transcription factors. Recently, various small peptides and their cognate receptors have been identified as controlling root development. Small peptides bind to membrane-localized receptor-like kinases, inducing their dimerization with co-receptor proteins for signaling activation and giving rise to cellular signaling outputs. Small peptides function as local and long-distance signaling molecules involved in cell-to-cell communication networks, coordinating root development. In this review, we survey recent advances in the peptide ligand-mediated signaling pathways involved in the control of root development in Arabidopsis. We describe the interconnection between peptide signaling and conventional phytohormone signaling. Additionally, we discuss the diversity of identified peptide-receptor interactions during plant root development.

LBD18 uses a dual mode of a positive feedback loop to regulate ARF expression and transcriptional activity in Arabidopsis.

A hierarchy of transcriptional regulators controlling lateral root formation in Arabidopsis thaliana has been identified, including the AUXIN RESPONSE FACTOR 7 (ARF7)/ARF19-LATERAL ORGAN BOUNDARIES DOMAIN 16 (LBD16)/LBD18 transcriptional network; however, their feedback regulation mechanisms are not known. Here we show that LBD18 controls ARF activity using the dual mode of a positive feedback loop. We showed that ARF7 and ARF19 directly bind AuxRE in the LBD18 promoter. A variety of molecular and biochemical experiments demonstrated that LBD18 binds a specific DNA motif in the ARF19 promoter to regulate its expression in vivo as well as in vitro. LBD18 interacts with ARFs including ARF7 and ARF19 via the Phox and Bem1 domain of ARF to enhance the transcriptional activity of ARF7 on AuxRE, and competes with auxin/indole-3-acetic acid (IAA) repressors for ARF binding, overriding the negative feedback loop exerted by Aux/IAA repressors. Taken together, these results show that LBD18 and ARFs form a double positive feedback loop, and that LBD18 uses the dual mode of a positive feedback loop by binding directly to the ARF19 promoter and through the protein-protein interactions with ARF7 and ARF19. This novel mechanism of feedback loops may constitute a robust feedback mechanism that ensures continued lateral root growth in response to auxin in Arabidopsis.

LBD16 and LBD18 acting downstream of ARF7 and ARF19 are involved in adventitious root formation in Arabidopsis.

BACKGROUND: Adventitious root (AR) formation is a complex genetic trait, which is controlled by various endogenous and environmental cues. Auxin is known to play a central role in AR formation; however, the mechanisms underlying this role are not well understood. RESULTS: In this study, we showed that a previously identified auxin signaling module, AUXIN RESPONSE FACTOR(ARF)7/ARF19-LATERAL ORGAN BOUNDARIES DOMAIN(LBD)16/LBD18 via AUXIN1(AUX1)/LIKE-AUXIN3 (LAX3) auxin influx carriers, which plays important roles in lateral root formation, is involved in AR formation in Arabidopsis. In aux1, lax3, arf7, arf19, lbd16 and lbd18 single mutants, we observed reduced numbers of ARs than in the wild type. Double and triple mutants exhibited an additional decrease in AR numbers compared with the corresponding single or double mutants, respectively, and the aux1 lax3 lbd16 lbd18 quadruple mutant was devoid of ARs. Expression of LBD16 or LBD18 under their own promoters in lbd16 or lbd18 mutants rescued the reduced number of ARs to wild-type levels. LBD16 or LBD18 fused to a dominant SRDX repressor suppressed promoter activity of the cell cycle gene, Cyclin-Dependent Kinase(CDK)A1;1, to some extent. Expression of LBD16 or LBD18 was significantly reduced in arf7 and arf19 mutants during AR formation in a light-dependent manner, but not in arf6 and arf8. GUS expression analysis of promoter-GUS reporter transgenic lines revealed overlapping expression patterns for LBD16, LBD18, ARF7, ARF19 and LAX3 in AR primordia. CONCLUSION: These results suggest that the ARF7/ARF19-LBD16/LBD18 transcriptional module via the AUX1/LAX3 auxin influx carriers plays an important role in AR formation in Arabidopsis.

LBD13 positively regulates lateral root formation in Arabidopsis.

MAIN CONCLUSION: Lateral Organ Boundaries Domain 13 (LBD13), which is expressed in emerged lateral roots and encodes a transcriptional activator, plays an important role in lateral root formation in Arabidopsis. Lateral roots (LRs) are major determinants of root system architecture, contributing to the survival strategies of plants. Members of the LBD gene family encode plant-specific transcription factors that play key roles in plant organ development. Several LBD genes, such as LBD14, 16, 18, 29, and 33, have been shown to play important roles in regulating LR development in Arabidopsis. In the present study, we show that LBD13 is expressed in emerged LRs and LR meristems of elongated LRs and regulates LR formation in Arabidopsis. Transient gene expression assays with Arabidopsis protoplasts showed that LBD13 is localized to the nucleus and harbors transcription-activating potential. Knock-down of LBD13 expression by RNA interference resulted in reduced LR formation, whereas overexpression of LBD13 enhanced LR formation in transgenic Arabidopsis. Analysis of ß-glucuronidase (GUS) expression under the control of the LBD13 promoter showed that GUS staining was detected in LRs emerged from the primary root, but not in LR primordia. Moreover, both the distribution of LR primordium number and developmental kinetics of LR primordia were not affected either by knock-down or by overexpression of LBD13. Taken together, these results suggest that LBD13 is a nuclear-localized transcriptional activator and controls LR formation during or after LR emergence.

CYTOKININ RESPONSE FACTOR2 (CRF2) and CRF3 Regulate Lateral Root Development in Response to Cold Stress in Arabidopsis.

Lateral roots (LRs) are a major determinant of the root system architecture in plants, and developmental plasticity of LR formation is critical for the survival of plants in changing environmental conditions. In Arabidopsis thaliana, genetic pathways have been identified that regulate LR branching in response to numerous environmental cues, including some nutrients, salt, and gravity. However, it is not known how genetic components are involved in the LR adaptation response to cold. Here, we demonstrate that CYTOKININ RESPONSE FACTOR2 (CRF2) and CRF3, encoding APETALA2 transcription factors, play an important role in regulating Arabidopsis LR initiation under cold stress. Analysis of LR developmental kinetics demonstrated that both CRF2 and CRF3 regulate LR initiation. crf2 and crf3 single mutants exhibited decreased LR initiation under cold stress compared with the wild type, and the crf2 crf3 double mutants showed additively decreased LR densities compared with the single mutants. Conversely, CRF2 or CRF3 overexpression caused increased LR densities. CRF2 was induced by cold via a subset of the cytokinin two-component signaling (TCS) pathway, whereas CRF3 was upregulated by cold via TCS-independent pathways. Our results suggest that CRF2 and CRF3 respond to cold via TCS-dependent and TCS-independent pathways and control LR initiation and development, contributing to LR adaptation to cold stress.

Dimerization in LBD16 and LBD18 Transcription Factors Is Critical for Lateral Root Formation.

LATERAL ORGAN BOUNDARIES DOMAIN/ASYMMETRIC LEAVES2-LIKEs (hereafter referred to as LBD) are plant-specific transcription factors that play important roles in a plethora of plant growth and development. The leucine (Leu) zipper-like coiled-coil motif in the lateral organ boundaries domain of the class I LBD proteins has been proposed to mediate protein dimerization, but it has not been experimentally assessed yet. LBD16 and LBD18 have been well characterized to play important roles in lateral root development in Arabidopsis (Arabidopsis thaliana). Here, we investigated the role of the coiled-coil motif in the dimerization of LBD16 and LBD18 and in transcriptional regulation and biological function. We built the molecular models of the coiled coil of LBD16 and LBD18, providing the probable Leu zipper models of the helix dimer. Using a variety of molecular techniques, such as bimolecular fluorescence complementation, luciferase complementation imaging, GST pull down, and coimmunoprecipitation assays, we showed that the conserved Leu or valine residues in the coiled-coil motif are critical for the dimerization of LBD16 or LBD18. Using transgenic Arabidopsis plants that overexpress HA:LBD16 or HA:LBD16Q in lbd16 or HA:LBD18 or HA:LBD18Q in lbd18, we demonstrated that the homodimerization of LBD18 mediated by the coiled-coil motif is crucial for transcriptional regulation via promoter binding and for lateral root formation. In addition, we found that the carboxyl-terminal region beyond the coiled-coil motif in LBD18 acts as an additional dimerization domain. These results provide a molecular basis for homodimerization and heterodimerization among the 42 Arabidopsis LBD family members for displaying their biological functions.

LBD14/ASL17 Positively Regulates Lateral Root Formation and is Involved in ABA Response for Root Architecture in Arabidopsis.

The LATERAL ORGAN BOUNDARIES (LOB) DOMAIN/ASYMMETRIC LEAVES2-LIKE (LBD/ASL) gene family members play key roles in diverse aspects of plant development. Previous studies have shown that LBD16, 18, 29 and 33 are critical for integrating the plant hormone auxin to control lateral root development in Arabidopsis thaliana. In the present study, we show that LBD14 is expressed exclusively in the root where it promotes lateral root (LR) emergence. Repression of LBD14 expression by ABA correlates with the inhibitory effects of ABA on LR emergence. Transient gene expression assays with Arabidopsis protoplasts demonstrated that LBD14 is a nuclear-localized transcriptional activator. The knock-down of LBD14 expression by RNA interference (RNAi) resulted in reduced LR formation by delaying both LR primordium development and LR emergence, whereas overexpression of LBD14 in Arabidopsis enhances LR formation. We show that ABA (but not other plant hormones such as auxin, brassinosteroids and cytokinin) specifically down-regulated ß-glucuronidase (GUS) expression under the control of the LBD14 promoter in transgenic Arabidopsis during LR development from initiation to emergence and endogenous LBD14 transcript levels in the root. Moreover, RNAi of LBD14 enhanced the LR suppression in response to ABA, whereas LBD14 overexpression did not alter the ABA-mediated suppression of LR formation. Taken together, these results suggest that LBD14 promoting LR formation is one of the critical factors regulated by ABA to inhibit LR growth, contributing to the regulation of the Arabidopsis root system architecture in response to ABA.

LATERAL ORGAN BOUNDARIES DOMAIN (LBD)10 interacts with SIDECAR POLLEN/LBD27 to control pollen development in Arabidopsis.

During male gametophyte development in Arabidopsis thaliana, the microspores undergo an asymmetric division to produce a vegetative cell and a generative cell, which undergoes a second division to give rise to two sperm cells. SIDECAR POLLEN/LATERAL ORGAN BOUNDARIES DOMAIN (LBD) 27 plays a key role in the asymmetric division of microspores. Here we provide molecular genetic evidence that a combinatorial role of LBD10 with LBD27 is crucial for male gametophyte development in Arabidopsis. Expression analysis, genetic transmission and pollen viability assays, and pollen development analysis demonstrated that LBD10 plays a role in the male gametophyte function primarily at germ cell mitosis. In the mature pollen of lbd10 and lbd10 expressing a dominant negative version of LBD10, LBD10:SRDX, aberrant microspores such as bicellular and smaller tricellular pollen appeared at a ratio of 10-15% with a correspondingly decreased ratio of normal tricellular pollen, whereas in lbd27 mutants, 70% of the pollen was aborted. All pollen in the lbd10 lbd27 double mutants was aborted and severely shrivelled compared with that of the single mutants, indicating that LBD10 and LBD27 are essential for pollen development. Gene expression and subcellular localization analyses of LBD10:GFP and LBD27:RFP during pollen development indicated that posttranscriptional and/or posttranslational controls are involved in differential accumulation and subcellular localization of LBD10 and LBD27 during pollen development, which may contribute in part to combinatorial and distinct roles of LBD10 with LBD27 in microspore development. In addition, we showed that LBD10 and LBD27 interact to form a heterodimer for nuclear localization.

Expression and Protein Interaction Analyses Reveal Combinatorial Interactions of LBD Transcription Factors During Arabidopsis Pollen Development.

LATERAL ORGAN BOUNDARIES DOMAIN (LBD) transcription factor gene family members play key roles in diverse aspects of plant development. LBD10 and LBD27 have been shown to be essential for pollen development in Arabidopsis thaliana. From the previous RNA sequencing (RNA-Seq) data set of Arabidopsis pollen, we identified the mRNAs of LBD22, LBD25 and LBD36 in addition to LBD10 and LBD27 in Arabidopsis pollen. Here we conducted expression and cellular analysis using GFP:GUS (green fluorescent protein:ß-glucuronidase) reporter gene and subcellular localization assays using LBD:GFP fusion proteins expressed under the control of their own promoters in Arabidopsis. We found that these LBD proteins display spatially and temporally distinct and overlapping expression patterns during pollen development. Bimolecular fluorescence complementation and GST (glutathione S-transferase) pull-down assays demonstrated that protein-protein interactions occur among the LBDs exhibiting overlapping expression during pollen development. We further showed that LBD10, LBD22, LBD25, LBD27 and LBD36 interact with each other to form heterodimers, which are localized to the nucleus in Arabidopsis protoplasts. Taken together, these results suggest that combinatorial interactions among LBD proteins may be important for their function in pollen development in Arabidopsis.

Lateral Organ Boundaries Domain16 and 18 Act Downstream of the AUXIN1 and LIKE-AUXIN3 Auxin Influx Carriers to Control Lateral Root Development in Arabidopsis.

Several members of the Lateral Organ Boundaries Domain (LBD)/Asymmetric Leaves2-Like (ASL) gene family have been identified to play important roles in Arabidopsis (Arabidopsis thaliana) lateral root (LR) development during auxin response, but their functional relationship with auxin transporters has not been established yet. Here, we show that the AUXIN1 (AUX1) and LIKE-AUXIN3 (LAX3) auxin influx carriers are required for auxin signaling that activates LBD16/ASL18 and LBD18/ASL20 to control LR development. The lax3 mutant phenotype was not significantly enhanced when combined with lbd16 or lbd18. However, LBD18 overexpression could rescue the defects in LR emergence in lax3 with concomitant expression of the LBD18 target genes. Genetic and gene expression analyses indicated that LBD16 and LBD18 act with AUX1 to regulate LR initiation and LR primordium development, and that AUX1 and LAX3 are needed for auxin-responsive expression of LBD16 and LBD18. LBD18:SUPERMAN REPRESSIVE DOMAIN X in the lbd18 mutant inhibited LR initiation and LR primordium development in response to a gravitropic stimulus and suppressed promoter activities of the cell cycle genes Cyclin-Dependent Kinase A1;1 and CYCLINB1;1. Taken together, these results suggest that LBD16 and LBD18 are important regulators of LR initiation and development downstream of AUX1 and LAX3.

AtC3H14, a plant-specific tandem CCCH zinc-finger protein, binds to its target mRNAs in a sequence-specific manner and affects cell elongation in Arabidopsis thaliana.

AtC3H14 (At1 g66810) is a plant-specific tandem CCCH zinc-finger (TZF) protein that belongs to the 68-member CCCH family in Arabidopsis thaliana. In animals, TZFs have been shown to bind and recruit target mRNAs to the cytoplasmic foci where mRNA decay enzymes are active. However, it is not known whether plant TZF proteins such as AtC3H14 function. So far, no mRNA targets of plant TZFs have been identified. We have obtained several lines of experimental evidence in support of our hypothesis that AtC3H14 is involved in post-transcriptional regulation of its target genes. Nucleic acid binding assays using [(35) S]-labeled AtC3H14 protein showed that AtC3H14 could bind to ssDNA, dsDNA, and ribohomopolymers, suggesting its RNA-binding activity. RNA immunoprecipitation (RIP) assay identified several putative target RNAs of AtC3H14, including a polygalacturonase, a well-known cell wall modifying gene. RNA electrophoretic mobility shift assays (RNA-EMSA) were used to confirm the RIP results and demonstrate that the TZF domain of AtC3H14 is required for the target RNA binding. Microarray analysis of 35S::AtC3H14 plants revealed that many of the cell wall elongation and/or modification-associated genes were differentially expressed, which is consistent with the cell elongation defect phenotype and the changes in the cell wall monosaccharide composition. In addition, yeast activation assay showed that AtC3H14 also function as a transcriptional activator, which is consistent with the previous finding that AtC3H14 activate the secondary wall biosynthesis genes. Taken together, we conclude that AtC3H14 may play a key role in both transcriptional and post-transcriptional regulation.

MYB46 directly regulates the gene expression of secondary wall-associated cellulose synthases in Arabidopsis.

Cellulose is the most abundant biopolymer on Earth. Three cellulose synthases (CESA4, CESA7 and CESA8) are necessary for cellulose production in the secondary cell walls of Arabidopsis. Little is known about how expression of these CESA genes is regulated. We recently identified a cis-regulatory element (M46RE) that is recognized by MYB46, which is a master switch for secondary wall formation in Arabidopsis. A genome-wide survey of promoter sequences for the presence of M46REs led to the hypothesis that MYB46 may function as a direct regulator of all three secondary wall-associated cellulose synthase genes: CESA4, CESA7 and CESA8. We tested this hypothesis using several lines of experimental evidence. All three CESA genes are highly up-regulated by both constitutive and inducible over-expression of MYB46 in planta. Using a steroid receptor-based inducible activation system, we show that MYB46 directly activates transcription of the three CESA genes. We then used an electrophoretic mobility shift assay and chromatin immunoprecipitation analysis to confirm that MYB46 protein directly binds to the promoters of the three CESA genes both in vitro and in vivo. Furthermore, ectopic up-regulation of MYB46 resulted in a significant increase of crystalline cellulose content in Arabidopsis. Taken together, we have identified MYB46 as a transcription factor that directly regulates all three secondary wall-associated CESA genes. Yeast one-hybrid screening identified additional transcription factors that regulate the CESA genes. However, none of the putative regulators appears to be regulated by MYB46, suggesting the multi-faceted nature of transcriptional regulation of secondary wall cellulose biosynthesis.

LBD18 acts as a transcriptional activator that directly binds to the EXPANSIN14 promoter in promoting lateral root emergence of Arabidopsis.

Lateral root formation, a developmental process under the control of the plant hormone auxin, is a major determinant of root architecture, and defines the ability of a plant to acquire nutrients and water. The LATERAL ORGAN BOUNDARIES DOMAIN/ASYMMETRIC LEAVES2-LIKE (LBD/ASL) proteins play an important role in the lateral organ development of plants, including lateral root formation. However, their downstream components and signalling mechanisms are largely unknown. Here, we show that auxin-responsive LBD18/ASL20 acts as a specific DNA-binding transcriptional activator that directly regulates EXPANSIN14 (EXP14), a gene encoding a cell wall-loosening factor that promotes lateral root emergence in Arabidopsis thaliana. We showed that LBD18 possesses transcription-activating function in both yeast and Arabidopsis protoplasts. We isolated putative LBD18 target genes by microarray analysis, and identified EXP14 as a direct target of LBD18. Dexamethasone-induced expression of LBD18 under the CaMV 35S promoter in transgenic Arabidopsis resulted in enhanced expression of GUS fused to the EXP14 promoter in primordium and overlaying tissues. In contrast, GUS expression under the EXP14 promoter in the lbd18 mutant background was significantly reduced in the same tissues. Experiments using a variety of molecular techniques demonstrated that LBD18 activates EXP14 by directly binding to a specific promoter element in vitro and in vivo. Overexpression of EXP14 in Arabidopsis resulted in the stimulation of emerged lateral roots, but not primordia, whereas EXP14 loss-of-function plants had reduced auxin-stimulated lateral root formation. This study revealed the molecular function of LBD18 as a specific DNA-binding transcription factor that activates EXP14 expression by directly binding to its promoter.