Takuji Wada

Role of a positive regulator of root hair development, CAPRICE ,in Arabidopsis root epidermal cell differentiation

In Arabidopsis, root hairs are formed only from a set of epidermal cells named trichoblasts or hair-forming cells. Previous studies showed CAPRICE (CPC) promotes differentiation of hair-forming cells by controlling a negative regulator, GLABRA2 (GL2), which is preferentially expressed in hairless cells. Here, we show that CPC is also predominantly expressed in the hairless cells, but not in the neighboring hair-forming cells, and that CPC protein moves to the hair-forming cells and represses the GL2 expression. We also show that the N terminus of bHLH protein interacts with CPC and is responsible for the GL2 expression. We propose a model in which CPC plays a key role in the fate-determination of hair-forming cells.

Cell-to-cell movement of the CAPRICE protein in Arabidopsis root epidermal cell differentiation.

CAPRICE (CPC), a small, R3-type Myb-like protein, is a positive regulator of root hair development in Arabidopsis. Cell-to-cell movement of CPC is important for the differentiation of epidermal cells into trichoblasts (root hair cells). CPC is transported from atrichoblasts (hairless cells), where it is expressed, to trichoblasts, and generally accumulates in their nuclei. Using truncated versions of CPC fused to GFP, we identified a signal domain that is necessary and sufficient for CPC cell-to-cell movement. This domain includes the N-terminal region and a part of the Myb domain. Amino acid substitution experiments indicated that W76 and M78 in the Myb domain are critical for targeted transport, and that W76 is crucial for the nuclear accumulation of CPC:GFP. To evaluate the tissue-specificity of CPC movement, CPC:GFP was expressed in the stele using the SHR promoter and in trichoblasts using the EGL3 promoter. CPC:GFP was able to move from trichoblasts to atrichoblasts but could not exit from the stele, suggesting the involvement of tissue-specific regulatory factors in the intercellular movement of CPC. Analyses with a secretion inhibitor, Brefeldin A, and with an rhd3 mutant defective in the secretion process in root epidermis suggested that intercellular CPC movement is mediated through plasmodesmata. Furthermore, the fusion of CPC to tandem-GFPs defined the capability of CPC to increase the size exclusion limit of plasmodesmata.

Arabidopsis TRANSPARENT TESTA GLABRA2 Is Directly Regulated by R2R3 MYB Transcription Factors and Is Involved in Regulation of GLABRA2 Transcription in Epidermal Differentiation

Arabidopsis thaliana TRANSPARENT TESTA GLABRA2 (TTG2) encodes a WRKY transcription factor and is expressed in young leaves, trichomes, seed coats, and root hairless cells. An examination of several trichome and root hair mutants indicates that MYB and bHLH genes regulate TTG2 expression. Two MYB binding sites in the TTG2 5′ regulatory region act as cis regulatory elements and as direct targets of R2R3 MYB transcription factors such as WEREWOLF, GLABRA1, and TRANSPARENT TESTA2. Mutations in TTG2 cause phenotypic defects in trichome development and seed color pigmentation. Transgenic plants expressing a chimeric repressor version of the TTG2 protein (TTG2:SRDX) showed defects in trichome formation, anthocyanin accumulation, seed color pigmentation, and differentiation of root hairless cells. GLABRA2 (GL2) expression was markedly reduced in roots of ProTTG2:TTG2:SRDX transgenic plants, suggesting that TTG2 is involved in the regulation of GL2 expression, although GL2 expression in the ttg2 mutant was similar to that in the wild type. Our analysis suggests a new step in a regulatory cascade of epidermal differentiation, in which complexes containing R2R3 MYB and bHLH transcription factors regulate the expression of TTG2, which then regulates GL2 expression with complexes containing R2R3 MYB and bHLH in the differentiation of trichomes and root hairless cells.

The Arabidopsis STV1 Protein, Responsible for Translation Reinitiation, Is Required for Auxin-Mediated Gynoecium Patterning

Ribosomal protein L24 (RPL24) is implicated in translation reinitiation of polycistronic genes. A newly isolated Arabidopsis thaliana short valve1 (stv1) mutant, in which one of the RPL24-encoding genes, RPL24B, is deleted, shows specific defects in the apical-basal patterning of the gynoecium, in addition to phenotypes induced by ribosome deficiency. A similar gynoecium phenotype is caused by mutations in the auxin response factor (ARF) genes ETTIN (ETT) and MONOPTEROS (MP), which have upstream open reading frames (uORFs) in their 5′-transcript leader sequences. Gynoecia of a double mutant of stv1 and a weak ett mutant allele are similar to those of a strong ett allele, and transformation with a uORF-eliminated ETT construct partially suppressed the stv1 gynoecium phenotype, implying that STV1 could influence ETT translation through its uORFs. Analyses of 5′-leader-reporter gene fusions showed that the uORFs of ETT and MP negatively regulate the translation of the downstream major ORFs, indicating that translation reinitiation is an important step for the expression of these proteins. Taken together, we propose that perturbation of translation reinitiation of the ARF transcripts causes the defects in gynoecium patterning observed in the stv1 mutant.

Jasmonic acid control of GLABRA3 links inducible defense and trichome patterning in Arabidopsis

Once attacked by herbivores, plants regenerate new leaves with increased trichome density as an inducible defense. Trichomes are specified from neighboring epidermal cells through local cell-cell interactions in the leaf primordia. However, the molecular mechanism of how herbivore-induced damage at older leaves remodels the pattern of trichome fate specification at newly forming leaves is largely unknown. In this study, we show that mutations in either the biosynthetic or signaling pathway of jasmonates (JAs), long-distance wound signals, abolish the wound-induced formation of trichomes. To identify the factors linking JA signaling to trichome fate specification, we isolated a novel class of mutants, unarmed (urm), which lack trichome induction but show otherwise normal responses to JAs. URM9 encodes an Importin β family protein, and URM23 is identical to TRANSPARENT TESTA GLABRA1 (TTG1), the product of which interacts with the bHLH transcription factor GLABRA3 (GL3). Loss of either URM9 or URM23 disrupts the subnuclear localization of GL3, thus implicating GL3 in trichome induction. The expression of GL3 was enhanced by JA treatment prior to trichome initiation. Genetic analysis of multiple trichome mutants shows that GL3, in concert with the R2R3-Myb transcription factor GLABRA1 (GL1), promotes trichome fate in response to JA in a dosage-dependent manner. These results indicate that GL3 is a key transcription factor of wound-induced trichome formation acting downstream of JA signaling in Arabidopsis.

Arabidopsis CAPRICE-LIKE MYB 3 (CPL3) controls endoreduplication and flowering development in addition to trichome and root hair formation.

CAPRICE (CPC) encodes a small protein with an R3 MYB motif and promotes root hair cell differentiation in Arabidopsis thaliana. Three additional CPC-like MYB genes, TRY (TRIPTYCHON), ETC1 (ENHANCER OF TRY AND CPC 1) and ETC2 (ENHANCER OF TRY AND CPC 2) act in a redundant manner with CPC in trichome and root hair patterning. In this study, we identified an additional homolog, CPC-LIKE MYB 3 (CPL3), which has high sequence similarity to CPC, TRY, ETC1 and ETC2. Overexpression of CPL3 results in the suppression of trichomes and overproduction of root hairs, as has been observed for CPC, TRY, ETC1 and ETC2. Morphological studies with double, triple and quadruple homolog mutants indicate that the CPL3 gene cooperatively regulates epidermal cell differentiation with other CPC homologs. Promoter-GUS analyses indicate that CPL3 is specifically expressed in leaf epidermal cells, including stomate guard cells. Notably, the CPL3 gene has pleiotropic effects on flowering development, epidermal cell size and trichome branching through the regulation of endoreduplication.

Functional Analysis of the Epidermal-Specific MYB Genes CAPRICE and WEREWOLF in Arabidopsis

Epidermis cell differentiation in Arabidopsis thaliana is a model system for understanding the developmental end state of plant cells. Two types of MYB transcription factors, R2R3-MYB and R3-MYB, are involved in cell fate determination. To examine the molecular basis of this process, we analyzed the functional relationship of the R2R3-type MYB gene WEREWOLF (WER) and the R3-type MYB gene CAPRICE (CPC). Chimeric constructs made from the R3 MYB regions of WER and CPC used in reciprocal complementation experiments showed that the CPC R3 region cannot functionally substitute for the WER R3 region in the differentiation of hairless cells. However, WER R3 can substantially substitute for CPC R3. There are no differences in yeast interaction assays of WER or WER chimera proteins with GLABRA3 (GL3) or ENHANCER OF GLABRA3 (EGL3). CPC and CPC chimera proteins also have similar activity in preventing GL3 WER and EGL3 WER interactions. Furthermore, we showed by gel mobility shift assays that WER chimera proteins do not bind to the GL2 promoter region. However, a CPC chimera protein, which harbors the WER R3 motif, still binds to the GL2 promoter region.

The HALTED ROOT gene encoding the 26S proteasome subunit RPT2a is essential for the maintenance of Arabidopsis meristems

In higher plants, post-embryonic development is dependent on the activity of the root and shoot apical meristem (RAM and SAM). The quiescent center (QC) in the RAM and the organizing center (OC) in the SAM are known to be essential for the maintenance of meristematic activity. To understand the mechanism that maintains post-embryonic meristems, we isolated an Arabidopsis mutant, halted root (hlr). In this mutant, the cellular organization was disrupted in post-embryonic meristems both in the root and in the shoot, and their meristematic activity was reduced or became abnormal. We showed that the mutant RAM lost its QC identity after germination, which was specified during embryogenesis, whereas the identity of differentiated tissues was maintained. In the post-embryonic SAM, the expression pattern of a typical OC marker gene, WUSCHEL, was disturbed in the mutant. These observations indicate that the HLR gene is essential to maintain the cellular organization and normal nature of the RAM and SAM. The HLR gene encodes RPT2a, which is a subunit of the 26S proteasome that degrades key proteins in diverse cellular processes. We showed that the HLR gene was expressed both in the RAM and in the SAM, including in the QC and the OC, respectively, and that the activity of proteasomes were reduced in the mutant. We propose that proteasome-dependent programmed proteolysis is required to maintain the meristem integrity both in the shoot and in the root.

New Insights into the Mechanism of Development of Arabidopsis Root Hairs and Trichomes

Epidermis cell differentiation in Arabidopsis thaliana is a model system for understanding the mechanisms leading to the developmental end state of plant cells. Both root hairs and trichomes differentiate from epidermal cells and molecular genetic analyses using Arabidopsis mutants have demonstrated that the differentiation of root hairs and trichomes is regulated by similar molecular mechanisms. Molecular-genetic approaches have led to the identification of many genes that are involved in epidermal cell differentiation, most of which encode transcription factors that induce the expression of genes active in both root hair and trichome development. Control of cell growth after fate determination has also been studied using Arabidopsis mutants.

The Trihelix Transcription Factor GTL1 Regulates Ploidy-Dependent Cell Growth in the Arabidopsis Trichome

Leaf trichomes in Arabidopsis thaliana develop through several distinct cellular processes, such as patterning, differentiation, and growth. Although recent studies have identified several key transcription factors as regulating early patterning and differentiation steps, it is still largely unknown how these regulatory proteins mediate subsequent trichome development, which is accompanied by rapid cell growth and branching. Here, we report a novel trichome mutation in Arabidopsis, which in contrast with previously identified mutants, increases trichome cell size without altering its overall patterning or branching. We show that the corresponding gene encodes a GT-2-LIKE1 (GTL1) protein, a member of the trihelix transcription factor family. GTL1 is present within the nucleus during the postbranching stages of trichome development, and its loss of function leads to an increase in the nuclear DNA content only in trichomes that have completed branching. Our data further demonstrate that the gtl1 mutation modifies the expression of several cell cycle genes and partially rescues the ploidy defects in the cyclin-dependent kinase inhibitor mutant siamese. Taken together, this study provides the genetic evidence for the requirement of transcriptional regulation in the repression of ploidy-dependent plant cell growth as well as for an involvement of GTL trihelix proteins in this regulation.