GLABRA2 Research Articles

SABRE is required for stabilization of root hair patterning in Arabidopsis thaliana.

Patterned differentiation of distinct cell types is essential for the development of multicellular organisms. The root epidermis of Arabidopsis thaliana is composed of alternating files of root hair and non-hair cells and represents a model system for studying the control of cell-fate acquisition. Epidermal cell fate is regulated by a network of genes that translate positional information from the underlying cortical cell layer into a specific pattern of differentiated cells. While much is known about the genes of this network, new players continue to be discovered. Here we show that the SABRE (SAB) gene, known to mediate microtubule organization, anisotropic cell growth and planar polarity, has an effect on root epidermal hair cell patterning. Loss of SAB function results in ectopic root hair formation and destabilizes the expression of cell fate and differentiation markers in the root epidermis, including expression of the WEREWOLF (WER) and GLABRA2 (GL2) genes. Double mutant analysis reveal that wer and caprice (cpc) mutants, defective in core components of the epidermal patterning pathway, genetically interact with sab. This suggests that SAB may act on epidermal patterning upstream of WER and CPC. Hence, we provide evidence for a role of SAB in root epidermal patterning by affecting cell-fate stabilization. Our work opens the door for future studies addressing SAB-dependent functions of the cytoskeleton during root epidermal patterning.

Shared functions of plant and mammalian StAR-related lipid transfer (START) domains in modulating transcription factor activity.

BackgroundSteroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domains were first identified from mammalian proteins that bind lipid/sterol ligands via a hydrophobic pocket. In plants, predicted START domains are predominantly found in homeodomain leucine zipper (HD-Zip) transcription factors that are master regulators of cell-type differentiation in development. Here we utilized studies of Arabidopsis in parallel with heterologous expression of START domains in yeast to investigate the hypothesis that START domains are versatile ligand-binding motifs that can modulate transcription factor activity.ResultsOur results show that deletion of the START domain from Arabidopsis Glabra2 (GL2), a representative HD-Zip transcription factor involved in differentiation of the epidermis, results in a complete loss-of-function phenotype, although the protein is correctly localized to the nucleus. Despite low sequence similarly, the mammalian START domain from StAR can functionally replace the HD-Zip-derived START domain. Embedding the START domain within a synthetic transcription factor in yeast, we found that several mammalian START domains from StAR, MLN64 and PCTP stimulated transcription factor activity, as did START domains from two Arabidopsis HD-Zip transcription factors. Mutation of ligand-binding residues within StAR START reduced this activity, consistent with the yeast assay monitoring ligand-binding. The D182L missense mutation in StAR START was shown to affect GL2 transcription factor activity in maintenance of the leaf trichome cell fate. Analysis of in vivo protein–metabolite interactions by mass spectrometry provided direct evidence for analogous lipid-binding activity in mammalian and plant START domains in the yeast system. Structural modeling predicted similar sized ligand-binding cavities of a subset of plant START domains in comparison to mammalian counterparts.ConclusionsThe START domain is required for transcription factor activity in HD-Zip proteins from plants, although it is not strictly necessary for the protein’s nuclear localization. START domains from both mammals and plants are modular in that they can bind lipid ligands to regulate transcription factor function in a yeast system. The data provide evidence for an evolutionarily conserved mechanism by which lipid metabolites can orchestrate transcription. We propose a model in which the START domain is used by both plants and mammals to regulate transcription factor activity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-014-0070-8) contains supplementary material, which is available to authorized users.

Investigation of the contribution of oil biosynthetic enzymes to seed oil content in Brassica napus and Arabidopsis thaliana

Katavic, V., Shi, L., Yu, Y., Zhao, L., Haughn, G. W. and Kunst, L. 2014. Investigation of the contribution of oil biosynthetic enzymes to seed oil content in Brassica napus and Arabidopsis thaliana. Can. J. Plant Sci. 94: 1109–1112. One of the critical reactions in triacylglycerol (TAG) biosynthesis is activation of fatty acyl chains to fatty acyl CoAs, catalyzed by long-chain acyl CoA synthetases (LACS). In Arabidopsis thaliana there is a family of nine genes that encode LACSs. Studies to determine whether the products of two of these genes, LACS8 and LACS9, function together to contribute acyl-CoAs for storage oil biosynthesis in A. thaliana resulted in discovery that it is not LACS8 but LACS1 that functionally overlaps with LACS9 in TAG biosynthesis (published in Plant Journal). To elucidate regulatory mechanisms of seed oil synthesis, the potential roles of phospholipase D zeta (PLDZ) and rhamnose synthase 2 (RHM2/MUM4) in transcription factor GLABRA2 (GL2)-mediated regulation of seed oil biosynthesis and deposition were investigated. Results demonstrated that PLDZ genes are not involved in GL2-mediated seed oil accumulation and that GL2 regulates seed oil production, at least in part, through its influence on expression of the gene RHM2/MUM4 required for the seed coat mucilage biosynthesis (published in Plant Journal). A novel Arabidopsis mutant with speckled seed coat and reduced seed oil phenotypes resulting from a mutation in a single unknown gene was identified, but attempts to isolate the gene by positional cloning have not been successful to date (unpublished results). Finally, seed oil content in near-isogenic double haploid Brassica napus lines was analyzed, “low oil” and “high oil” lines were identified, and developing seeds for expression profiling of target seed oil biosynthesis/bioassembly genes in selected double haploid lines were collected (unpublished results).

Control of trichome formation in Arabidopsis by poplar single-repeat R3 MYB transcription factors.

In Arabidopsis, trichome formation is regulated by the interplay of R3 MYBs and several others transcription factors including the WD40-repeat protein TRANSPARENT TESTA GLABRA1 (TTG1), the R2R3 MYB transcription factor GLABRA1 (GL1), the bHLH transcription factor GLABRA3 (GL3) or ENHANCER OF GLABRA3 (EGL3), and the homeodomain protein GLABRA2 (GL2). R3 MYBs including TRICHOMELESS1 (TCL1), TCL2, TRYPTICHON (TRY), CAPRICE (CPC), ENHANCER OF TRY AND CPC1 (ETC1), ETC2 and ETC3 negatively regulate trichome formation by competing with GL1 for binding GL3 or EGL3, thus blocking the formation of TTG1–GL3/EGL3–GL1, an activator complex required for the activation of the trichome positive regulator gene GL2. However, it is largely unknown if R3 MYBs in other plant species especially woody plants have similar functions. By BLASTing the Populus trichocarpa protein database using the entire amino acid sequence of TCL1, an Arabidopsis R3 MYB transcription factor, we identified a total of eight R3 MYB transcription factor genes in poplar, namely P. trichocarpa TRICHOMELESS1 through 8 (PtrTCL1–PtrTCL8). The amino acid signature required for interacting with bHLH transcription factors and the amino acids required for cell-to-cell movement of R3 MYBs are not fully conserved in all PtrTCLs. When tested in Arabidopsis protoplasts, however, all PtrTCLs interacted with GL3. Expressing each of the eight PtrTCL genes in Arabidopsis resulted in either glabrous phenotypes or plants with reduced trichome numbers, and expression levels of GL2 in all transgenic plants tested were greatly reduced. Expression of PtrTCL1 under the control of TCL1 native promoter almost completely complemented the mutant phenotype of tcl. In contrast, expression of PtrTCL1 under the control of TRY native promoter in the try mutant, or under the control of CPC native promoter in the cpc mutant resulted in glabrous phenotypes, suggesting that PtrTCL1 functions similarly to TCL1, but not TRY and CPC.

HD-Zip Proteins GL2 and HDG11 Have Redundant Functions in Arabidopsis Trichomes, and GL2 Activates a Positive Feedback Loop via MYB23.

The class IV homeodomain leucine zipper transcription factor GLABRA2 (GL2) acts in a complex regulatory circuit that regulates the differentiation of trichomes in Arabidopsis thaliana. We describe a genetic interaction with HOMEODOMAIN GLABROUS11 (HDG11), previously identified as a negative regulator of trichome branching. gl2 hdg11 double mutants display enhanced trichome cell-type differentiation defects. Transgenic expression of HDG11 using the GL2 promoter partially suppresses gl2 trichome phenotypes. Vice versa, expression of GL2 under the control of its native promoter partially complements hdg11 ectopic branching. Since gl2 hdg11 and gl2 myb23 double mutants and the triple mutant display similar trichome differentiation defects, we investigated a connection to the R2R3-MYB transcription factor MYB23. We show that MYB23 transcript levels are significantly reduced in shoots from gl2 mutants and that GL2 can drive the expression of a MYB23-promoter fusion to green fluorescent protein. Yeast one-hybrid, chromatin immunoprecipitation, and in planta reporter gene experiments indicate that an L1-box in the MYB23 promoter acts as a GL2 binding site. Taken together, our findings reveal a functional redundancy between GL2 and HDG11, two homeodomain leucine zipper transcription factors previously thought to mediate opposing functions in trichome morphogenesis. A model is proposed in which GL2 transcript levels are maintained through a positive feedback loop involving GL2 activation of MYB23.

MYB82 functions in regulation of trichome development in Arabidopsis.

Trichome initiation and patterning are controlled by the TTG1–bHLH–MYB regulatory complex. Several MYB transcription factors have been determined to function in trichome development via incorporation into this complex. This study examined the role of MYB82, an R2R3-MYB transcription factor, in Arabidopsis trichome development. MYB82 was revealed to be a nuclear-localized transcription activator. Suppression of MYB82 function by fusion with a dominant repression domain (SRDX) resulted in glabrous leaves, as did overexpression of N-terminal-truncated MYB82. Overexpression of MYB82 genomic sequence, but not its cDNA sequence, led to reduced trichome numbers. Further investigation indicated that at least one of the two introns in MYB82 is essential to the protein’s trichome developmental function. An MYB-binding box was identified in the third exon of MYB82, which was inferred to be crucial for MYB82 function because the mutation of this box interfered with the ability of MYB82 to rescue the gl1 mutant. Protein interaction analysis revealed that MYB82 physically interacts with GLABRA3 (GL3). In addition, MYB82 and GL1 can form homodimers and heterodimers at R2R3-MYB domains, which may explain why their overexpression reduces trichome numbers. These results demonstrate the functional diversification of MYB82 and GL1 in trichome development.

MiR828 and miR858 regulate homoeologous MYB2 gene functions in Arabidopsis trichome and cotton fibre development

Although polyploidy is common in plants and some animals, mechanisms for functional divergence between homoeologous genes are poorly understood. MYB2 gene promotes cotton fibre development and is functionally homologous to Arabidopsis GLABROUS1 (GL1) in trichome formation. The most widely cultivated cotton is an allotetraploid (Gossypium hirsutum, AADD) that contains GhMYB2A and GhMYB2D homoeologs. Here we show that GhMYB2D mRNA accumulates more than GhMYB2A during fibre initiation and is targeted by miR828 and miR858, generating trans-acting siRNAs (ta-siRNAs) in the TAS4 family. Overexpressing GhMYB2A but not GhMYB2D complements the gl1 phenotype. Mutating the miR828-binding site or replacing its downstream sequence in GhMYB2D abolishes ta-siRNA production and restores trichome development in gl1 mutants. Moreover, disrupting Dicer-like protein 4 or RDR6, the biogenesis genes for ta-siRNAs, in the gl1 GhMYB2D overexpressors restores trichome development. These data support a unique role for microRNAs in functional divergence between target homoeologous genes that are important for evolution and selection of morphological traits.

Shared functions of plant and mammalian StAR-related lipid transfer (START) domains in modulating transcription factor activity

Steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domains were first identified from mammalian proteins that bind lipid/sterol ligands via a hydrophobic pocket. In plants, predicted START domains are predominantly found in homeodomain leucine zipper (HD-Zip) transcription factors that are master regulators of cell-type differentiation in development. Here we utilized studies of Arabidopsis in parallel with heterologous expression of START domains in yeast to investigate the hypothesis that START domains are versatile ligand-binding motifs that can modulate transcription factor activity. Our results show that deletion of the START domain from Arabidopsis Glabra2 (GL2), a representative HD-Zip transcription factor involved in differentiation of the epidermis, results in a complete loss-of-function phenotype, although the protein is correctly localized to the nucleus. Despite low sequence similarly, the mammalian START domain from StAR can functionally replace the HD-Zip-derived START domain. Embedding the START domain within a synthetic transcription factor in yeast, we found that several mammalian START domains from StAR, MLN64 and PCTP stimulated transcription factor activity, as did START domains from two Arabidopsis HD-Zip transcription factors. Mutation of ligand-binding residues within StAR START reduced this activity, consistent with the yeast assay monitoring ligand-binding. The D182L missense mutation in StAR START was shown to affect GL2 transcription factor activity in maintenance of the leaf trichome cell fate. Analysis of in vivo protein–metabolite interactions by mass spectrometry provided direct evidence for analogous lipid-binding activity in mammalian and plant START domains in the yeast system. Structural modeling predicted similar sized ligand-binding cavities of a subset of plant START domains in comparison to mammalian counterparts. The START domain is required for transcription factor activity in HD-Zip proteins from plants, although it is not strictly necessary for the protein’s nuclear localization. START domains from both mammals and plants are modular in that they can bind lipid ligands to regulate transcription factor function in a yeast system. The data provide evidence for an evolutionarily conserved mechanism by which lipid metabolites can orchestrate transcription. We propose a model in which the START domain is used by both plants and mammals to regulate transcription factor activity.

Identification and Characterization of Cotton Genes Involved in Fuzz‐Fiber Development

Cotton fuzz fibers, like Arabidopsis trichomes, are elongated unicells. It is postulated that a transcriptional complex of GLABRA1 (GL1), GLABRA3 (GL3), and TRANSPARENT TESTAGLABRA1 (TTG1) might be in existence in Arabidopsis as evidenced by their physical interaction in yeast, and the complex regulates expression of GLABRA2 (GL2) controlling trichome cell differentiation; it is also assumed that TRIPTYCHON (TRY) and CAPRICE (CPC) counteract the complex formation in neighboring cells. Here, the homologs GaMYB23 (a homolog of GL1), GaDEL65 (a homolog of GL3), GaTTG1, GaCPC and GaTRY were identified in Gossypium arboreum. We show that GaMYB23 can bind to and activate the promoters of GaCPC, GaGL2 and GaTRY, and that GaMYB23, GaTRY and GaTTG1 could interact with GaDEL65 in yeast and in planta. In situ analysis showed that GaMYB23, GaGL2, GaDEL65, and GaTRY were predominantly expressed in fuzz fiber, but GaTRY proteins were primarily found in undeveloped epidermal cells. A G. arboreum fuzzless mutant with consistently high level GaMYB23 transcript has lost the detectable GaMYB23-promoter of GaGL2 complex, corresponding to sharply reduced transcription of GaGL2. Our results support that cotton homologs to the genetic molecules regulating Arabidopsis trichome differentiation interacted in the epidermis of ovules and the redundant GaMYB23 serves as a negative regulator in fuzz-fiber patterning.

Theglabra1mutation affects the stomatal patterning ofArabidopsis thalianarosette leaves

Abstract. In Arabidopsis thaliana, the GLABRA 1 (GL1) gene encodes a R2R3-MYB transcription factor that regulates epidermal genes necessary to promote trichome initiation. To determine whether the GL1 gene is also involved in the stomatal patterning of rosette leaves, the epidermal cell pattern of a glabrous mutant (gl1-1) was compared to that of the wild type Columbia ecotype. The gl1-1 mutant was found to have lower stomatal densities and greater distances between stomata as compared to the Columbia ecotype. These findings suggest that stomatal patterning and trichome developmental processes share genetic components to specify cell fate and identity.

Nucleotide sequence variation of GLABRA1 contributing to phenotypic variation of leaf hairiness in Brassicaceae vegetables

GLABRA1 (GL1) belongs to the group of R2R3-MYB transcription factors and is known to be essential for trichome initiation in Arabidopsis. In our previous study, we identified a GL1 ortholog in Brassica rapa as a candidate for the gene controlling leaf hairiness by QTL analysis and suggested that a 5-bp deletion (B-allele) and a 2-bp deletion (D-allele) in the exon 3 of BrGL1 and a non-synonymous SNP (C-allele) in the second nucleotide of exon 3 possibly cause leaf hairlessness. In this study, we transformed a B. rapa line having the B-allele with the A-allele (wild type) or the C-allele of BrGL1 under the control of the CaMV 35S promoter. The transgenic plants with the A-allele showed dense coverage of seedling tissues including stems, young leaves and hypocotyls with trichomes, whereas the phenotypes of those with the C-allele were unchanged. In order to obtain more information about allelic variation of GL1 in different plant lineages and its correlation with leaf hairiness, two GL1 homologs, i.e., RsGL1a and RsGL1b, in Raphanus sativus were analyzed. Allelic variation of RsGL1a between a hairless line and a hairy line was completely associated with hairiness in their BC1F1 population. Comparison of the full-length of RsGL1a in the hairless and hairy lines showed great variation of nucleotides in the 3' end, which might be essential for its function and expression.

Control of Plant Trichome and Root-Hair Development by a Tomato (Solanum lycopersicum) R3 MYB Transcription Factor

In Arabidopsis thaliana the CPC-like MYB transcription factors [CAPRICE (CPC), TRIPTYCHON (TRY), ENHANCER OF TRY AND CPC 1, 2, 3/CPC-LIKE MYB 3 (ETC1, ETC2, ETC3/CPL3), TRICHOMELESS 1, 2/CPC-LIKE MYB 4 (TCL1, TCL2/CPL4)] and the bHLH transcription factors [GLABRA3 (GL3) and ENHANCER OF GLABRA 3 (EGL3)] are central regulators of trichome and root-hair development. We identified TRY and GL3 homologous genes from the tomato genome and named them SlTRY and SlGL3, respectively. Phylogenic analyses revealed a close relationship between the tomato and Arabidopsis genes. Real-time reverse transcription PCR analyses showed that SlTRY and SlGL3 were predominantly expressed in aerial parts of developing tomato. After transformation into Arabidopsis, CPC::SlTRY inhibited trichome formation and enhanced root-hair differentiation by strongly repressing GL2 expression. On the other hand, GL3::SlGL3 transformation did not show any obvious effect on trichome or non-hair cell differentiation. These results suggest that tomato and Arabidopsis partially use similar transcription factors for epidermal cell differentiation, and that a CPC-like R3 MYB may be a key common regulator of plant trichome and root-hair development.

Natural variation in GL1 and its effects on trichome density in Arabidopsis thaliana

The ultimate understanding of how biological diversity arises, is maintained, and lost depends on identifying the genes responsible. Although a good deal has been discovered about gene function over the past few decades, far less is understood about gene effects, that is, how natural variation in a gene contributes to natural variation in phenotypes. Trichome density in Arabidopsis thaliana is an ideal trait for studies of natural molecular and phenotypic variation, as trichome initiation is genetically well-characterized and trichome density is highly variable in and among natural populations. Here, we show that variation at GLABRA1 (GL1), an R2R3 MYB transcription factor gene, which has a role in trichome initiation, has qualitative and likely quantitative effects on trichome density in natural accessions of A.thaliana. Specifically, we characterize four independent loss-of-function alleles for GL1, each of which yields a glabrous phenotype. Further, we find that a pattern of common polymorphisms confined to the GL1 locus is associated with quantitative variation for trichome density. While mutations resulting in a glabrous phenotype are primarily coding changes, the pattern resulting in quantitative variation spans both coding and regulatory regions. These data show that GL1 is an important source of trichome density variation within A.thaliana and, along with recent reports, suggest that the TTG1 epidermal cell fate pathway generally may be the key molecular genetic source of natural trichome density variation and an important model for the study of molecular evolution.

A Single Amino Acid Substitution in IIIf Subfamily of Basic Helix-Loop-Helix Transcription Factor AtMYC1 Leads to Trichome and Root Hair Patterning Defects by Abolishing Its Interaction with Partner Proteins in Arabidopsis

Plant trichomes and root hairs are powerful models for the study of cell fate determination. In Arabidopsis thaliana, trichome and root hair initiation requires a combination of three groups of proteins, including the WD40 repeat protein transparent TESTA GLABRA1 (TTG1), R2R3 repeat MYB protein GLABRA1 (GL1), or werewolf (WER) and the IIIf subfamily of basic helix-loop-helix (bHLH) protein GLABRA3 (GL3) or enhancer of GLABRA3 (EGL3). The bHLH component acts as a docking site for TTG1 and MYB proteins. Here, we isolated a mutant showing defects in trichome and root hair patterning that carried a point mutation (R173H) in AtMYC1 that encodes the fourth member of IIIf bHLH family protein. Genetic analysis revealed partial redundant yet distinct function between AtMYC1 and GL3/EGL3. GLABRA2 (GL2), an important transcription factor involved in trichome and root hair control, was down-regulated in Atmyc1 plants, suggesting the requirement of AtMYC1 for appropriate GL2 transcription. Like its homologs, AtMYC1 formed a complex with TTG1 and MYB proteins but did not dimerized. In addition, the interaction of AtMYC1 with MYB proteins and TTG1 was abrogated by the R173H substitution in Atmyc1-1. We found that this amino acid (Arg) is conserved in the AtMYC1 homologs GL3/EGL3 and that it is essential for their interaction with MYB proteins and for their proper functions. Our findings indicate that AtMYC1 is an important regulator of trichome and root hair initiation, and they reveal a novel amino acid necessary for protein-protein interactions and gene function in IIIf subfamily bHLH transcription factors.

Functional characterization of TRICHOMELESS2, a new single-repeat R3 MYB transcription factor in the regulation of trichome patterning in Arabidopsis

BackgroundSingle-repeat R3 MYB transcription factors (single-repeat MYBs) play important roles in controlling trichome patterning in Arabidopsis. It was proposed that single-repeat MYBs negatively regulate trichome formation by competing with GLABRA1 (GL1) for binding GLABRA3/ENHANCER OF GLABRA3 (GL3/EGL3), thus inhibiting the formation of activator complex TTG1(TRANSPARENT TESTA GLABRA1)-GL3/EGL3-GL1 that is required for the activation of GLABRA2 (GL2), whose product is a positive regulator of trichome formation. Previously we identified a novel single-repeat MYB transcription factor, TRICHOMELESS1 (TCL1), which negatively regulates trichome formation on the inflorescence stems and pedicels by directly suppressing the expression of GL1.ResultsWe analyzed here the role of TRICHOMELESS2 (TCL2), a previously-uncharacterized single-repeat MYB transcription factor in trichome patterning in Arabidopsis. We showed that TCL2 is closely related to TCL1, and like TCL1 and other single-repeat MYBs, TCL2 interacts with GL3. Overexpression of TCL2 conferred glabrous phenotype while knockdown of TCL2 via RNAi induced ectopic trichome formation on the inflorescence stems and pedicels, a phenotype that was previously observed in tcl1 mutants. These results suggested that TCL2 may have overlapping function with TCL1 in controlling trichome formation on inflorescences. On the other hand, although the transcription of TCL2, like TCL1, is not controlled by the activator complex formed by GL1 and GL3, and TCL2 and TCL1 proteins are more than 80% identical at the amino acid level, the expression of TCL2 under the control of TCL1 promoter only partially recovered the mutant phenotype of tcl1, implying that TCL2 and TCL1 are not fully functional equivalent.ConclusionsTCL2 function redundantly with TCL1 in controlling trichome formation on inflorescences, but they are not fully functional equivalent. Transcription of TCL2 is not controlled by activator complex formed by GL1 and GL3, but MIR156 controlled SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) transcription factors. However, SPLs might require co-activators to regulate the expression of their target genes, including TCL1, TRY and possibly, TCL2.

Nuclear trapping by GL3 controls intercellular transport and redistribution of TTG1 protein inArabidopsis

Trichome patterning on Arabidopsis leaves is one of the best-studied model systems for two-dimensional de novo patterning. In addition to an activator-inhibitor-related mechanism, we previously proposed a depletion mechanism to operate during this process such that GLABRA3 (GL3) traps the trichome-promoting factor TRANSPARENT TESTA GLABRA1 (TTG1) in trichomes that, in turn, results in a depletion of TTG1 in trichome neighbouring cells. In this manuscript we analyze the molecular basis underlying this trapping mechanism. We demonstrate the ability of GL3 to regulate TTG1 mobility by expressing TTG1 and GL3 in different tissue layers in different combinations. We further show that TTG1 trapping by GL3 is based on direct interaction between both proteins and recruitment in the nucleus.

Arabidopsis glabra2 mutant seeds deficient in mucilage biosynthesis produce more oil

Seed oil, one of the major seed storage compounds in plants, is of great economic importance for human consumption, as an industrial raw material and as a source of biofuels. Thus, improving the seed oil yield in crops is an important objective. The GLABRA2 (GL2) gene in Arabidopsis thaliana encodes a transcription factor that is required for the proper differentiation of several epidermal cell types. GL2 has also been shown to regulate seed oil levels, as a loss-of-function mutation in the GL2 gene results in plants with a higher seed oil content than wild-type. We have extended this observation by showing that loss-of-function mutations in several positive regulators of GL2 also result in a high seed oil phenotype. The GL2 gene is expressed in both the seed coat and embryo, but the embryo is the main site of seed oil accumulation. Surprisingly, our results indicate that it is loss of GL2 activity in the seed coat, not the embryo, that contributes to the high seed oil phenotype. One target of GL2 in the seed coat is the gene MUCILAGE MODIFIED 4 (MUM4), which encodes a rhamnose synthase that is required for seed mucilage biosynthesis. We found that mum4 mutant seeds, like those of gl2 mutants, have an increased seed oil content in comparison with wild-type. Therefore, GL2 regulates seed oil production at least partly through its influence on MUM4 expression in the seed coat. We propose that gl2 mutant seeds produce more oil due to increased carbon allocation to the embryo in the absence of seed coat mucilage biosynthesis.

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.

Zinc Finger Protein5Is Required for the Control of Trichome Initiation by Acting Upstream ofZinc Finger Protein8in Arabidopsis

Arabidopsis (Arabidopsis thaliana) trichome development is a model system for studying cell development, cell differentiation, and the cell cycle. Our previous studies have shown that the GLABROUS INFLORESCENCE STEMS (GIS) family genes, GIS, GIS2, and ZINC FINGER PROTEIN8 (ZFP8), control shoot maturation and epidermal cell fate by integrating gibberellins (GAs) and cytokinin signaling in Arabidopsis. Here, we show that a new C2H2 zinc finger protein, ZFP5, plays an important role in controlling trichome cell development through GA signaling. Overexpression of ZFP5 results in the formation of ectopic trichomes on carpels and other inflorescence organs. zfp5 loss-of-function mutants exhibit a reduced number of trichomes on sepals, cauline leaves, paraclades, and main inflorescence stems in comparison with wild-type plants. More importantly, it is found that ZFP5 mediates the regulation of trichome initiation by GAs. These results are consistent with ZFP5 expression patterns and the regional influence of GA on trichome initiation. The molecular analyses suggest that ZFP5 functions upstream of GIS, GIS2, ZFP8, and the key trichome initiation regulators GLABROUS1 (GL1) and GL3. Using a steroid-inducible activation of ZFP5 and chromatin immunoprecipitation experiments, we further demonstrate that ZFP8 is the direct target of ZFP5 in controlling epidermal cell differentiation.

Analysis of IIId, IIIe and IVa group basic-helix-loop-helix proteins expressed in Arabidopsis root epidermis

Differentiation of Arabidopsis epidermal cells into root hairs and trichomes is a functional model system for understanding plant cell development. Previous studies showed that one of the Arabidopsis basic-helix-loop-helix (AtbHLH) proteins, GLABRA3 (GL3), is involved in root-hair and trichome differentiation. We analyzed 11 additional AtbHLH genes with homology to GL3. Estimation of the phylogeny based on amino acid sequences of the bHLH region suggests that 11 AtbHLH genes used in this study evolved by duplications of a single common GL3 ancestor. Promoter-GUS analysis showed that AtbHLH006, AtbHLH013, AtbHLH017 and AtbHLH020 were expressed in roots. Among them, AtbHLH006 and AtbHLH020 were preferentially expressed in root epidermal non-hair cells. Consistent with the expression patterns from promoter -GUS analysis, GFP fluorescence was observed in the nuclei of root epidermal non-hair cells of AtbHLH006p::AtbHLH006:GFP and AtbHLH020p::AtbHLH020:GFP transgenic plants. However, AtbHLH006 and AtbHLH0020 proteins did not interact with epidermis-specific MYB proteins and TTG1. Taken together, AtbHLH006 and AtbHLH020 may function in root epidermal cells, but other GL3-like bHLH proteins may have evolved to regulate different processes.