Ryosuke Sano

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.

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.

Contribution of NAC Transcription Factors to Plant Adaptation to Land

From Drips to Tubes In the evolutionary transition from aquatic to terrestrial habitats, plants acquired internal systems to transport water and provide structural support. Xu et al. (p. 1505, published online 20 March) studied a family of genes and the cells they control to better understand the innovations required to adapt to dry land. In Arabidopsis, specific transcription factors regulate development of xylem—the plant tissue that transports water. The moss Physcomitrella patens has similar genes, which regulate development of hydroids and stereids, cells specialized in water transport and structural support. The similarity in the genes and their functions suggests the evolutionary origins of land-plant vascular systems. Similarities are revealed in the generation of internal water transport systems in moss and Arabidopsis. The development of cells specialized for water conduction or support is a striking innovation of plants that has enabled them to colonize land. The NAC transcription factors regulate the differentiation of these cells in vascular plants. However, the path by which plants with these cells have evolved from their nonvascular ancestors is unclear. We investigated genes of the moss Physcomitrella patens that encode NAC proteins. Loss-of-function mutants formed abnormal water-conducting and supporting cells, as well as malformed sporophyte cells, and overexpression induced ectopic differentiation of water-conducting–like cells. Our results show conservation of transcriptional regulation and cellular function between moss and Arabidopsis thaliana water-conducting cells. The conserved genetic basis suggests roles for NAC proteins in the adaptation of plants to land.

PHYLOGENETIC RELATIONSHIPS WITHIN THE COLONIAL VOLVOCALES (CHLOROPHYTA) INFERRED FROM rbcL GENE SEQUENCE DATA

The chloroplast‐encoded large subunit of the ribulose‐1, 5‐bisphosphate carboxylase / oxygenase (rbcL) gene was sequenced from 20 species of the colonial Volvocales (the Volvacaceae, Goniaceae, and Tetrabaenaceae) in order to elucidate phylogenetic relationships within the colonial Volvocales. Eleven hundred twenty‐eight base pairs in the coding regions of the (rbcL) gene were analyzed by the neighbor‐joining (NJ) method using three kinds of distance estimations, as well as by the maximum parsimony (MP) method. A large group comprising all the anisogamous and oogamous volvocacean species was resolved in the MP tree as well as in the NJ trees based on overall and synonymous substitutions. In all the trees constructed, Basichlamys and Tetrabaena (Tetrabaenaceae) constituted a very robust phylogenetic group. Although not supported by high bootstrap values, the MP tree and the NJ tree based on nonsynonymous substitutions indicated that the Tetrabaenaceae is the sister group to the large group comprising the Volvocaceae and the Goniaceae. In addition, the present analysis strongly suggested that Pandorina and Astrephomene are monophyletic genera whereas Eudorina is nonmonophyletic. These results are essentially consistent with the results of the recent cladistic analyses of morphological data. However, the monophyly of the Volvocaceae previously supported by four morphological synapomorphies is found only in the NJ tree based on nonsynonymous substitutions (with very low bootstrap values). The genus Volvox was clearly resolved as a polyphyletic group with V. rousseletii Pocock separated from other species of Volvox in the rbcL gene comparisons, although this genus represents a monophyletic group in the previous morphological analyses. Furthermore, none of the rbcL gene trees supported the monophyly of the Goniaceae; Astrephomene was placed in various phylogenetic positions.

KNOX homeobox genes potentially have similar function in both diploid unicellular and multicellular meristems, but not in haploid meristems.

Summary Members of the class 1 knotted‐like homeobox (KNOX) gene family are important regulators of shoot apical meristem development in angiosperms. To determine whether they function similarly in seedless plants, three KNOX genes (two class 1 genes and one class 2 gene) from the fern Ceratopteris richardii were characterized. Expression of both class 1 genes was detected in the shoot apical cell, leaf primordia, marginal part of the leaves, and vascular bundles by in situ hybridization, a pattern that closely resembles that of class 1 KNOX genes in angiosperms with compound leaves. The fern class 2 gene was expressed in all sporophyte tissues examined, which is characteristic of class 2 gene expression in angiosperms. All three CRKNOX genes were not detected in gametophyte tissues by RNA gel blot analysis. Arabidopsis plants overexpressing the fern class 1 genes resembled plants that overexpress seed plant class 1 KNOX genes in leaf morphology. Ectopic expression of the class 2 gene in Arabidopsis did not result in any unusual phenotypes. Taken together with phylogenetic analysis, our results suggest that (a) the class 1 and 2 KNOX genes diverged prior to the divergence of fern and seed plant lineages, (b) the class 1 KNOX genes function similarly in seed plant and fern sporophyte meristem development despite their differences in structure, (c) KNOX gene expression is not required for the development of the fern gametophyte, and (d) the sporophyte and gametophyte meristems of ferns are not regulated by the same developmental mechanisms at the molecular level.

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.

PHYLOGENETIC ANALYSIS OF YAMAGISHLELLA AND PLATYDORINA (VOLVOCACEAE, CHLOROPHYTA) BASED ON rbcL GENE SEQUENCES1

Yamagishiella, based on Pandorina unicocca Rayburn et Starr, is distinguished from Eudorina by its isogamous sexual reproduction, whereas Platydorina exhibits anisogamous sexual reproduction. In the present study, we sequenced the large subunit of ribulose‐1,5‐bisphosphate carboxylase/oxygenase (rbcL) genes from five Japanese and North American strains of Y. unicocca (Rayburn et Starr) Nozaki, two Platydorina caudata Kofoid strains, and two strains of Eudorina unicocca G. M. Smith, as well as eight related colonial and unicellular species. Phylogenetic trees were constructed based on these sequence data and on previously published rbcL gene sequences from 23 volvocalean species in order to deduce phylogenetic relationships within the colonial Volvocales, with particular regard to the phylogenetic positions and status of the genera Yamagishiella and Platydorina. Two robust monophyletic groups of the anisogamous/oogamous volvocacean species were resolved in the maximum‐parsimony tree as well as in the neighbor‐joining distance tree. One of the two groups comprises three species of Volvox section Volvox, whereas the other is composed of other sections of Volvox as well as of all the species of Eudorina and Pleodorina. Platydorina, however, was positioned outside these two monopliyletic groups. Therefore, derivation of the Platydorina lineage may be earlier than that of such anisogamous/oogamous groups, or orgin of “anisogamy with sperm packets” in Platydorina may be independent of sperm packet evolution in Eudorina, Pleodorina, and Volvox. It was also resolved with high bootstrap values that all of the Y. unicocca strains form a monophyletic group positioned outside the large monophyletic group including Eudorina and Pleodorina. These reject the possibility of the reverse evolution of isogamy from anisogamy to give rise to Yamagishiella within the lineage of Eudorina.

CHARACTERIZATION OF A FLORICAULA/LEAFY HOMOLOGUE OF GNETUM PARVIFOLIUM AND ITS IMPLICATIONS FOR THE EVOLUTION OF REPRODUCTIVE ORGANS IN SEED PLANTS

The morphological variation among reproductive organs of extant seed plants makes assessment of organ homology difficult. Comparisons of expression patterns of homeotic genes that control organ development will yield new information about the homology of organs to assess inferences deduced from previous morphological studies. In angiosperms, the FLORICAULA/LEAFY (FLO/LFY) genes convert a vegetative shoot meristem to a floral meristem by inducing floral homeotic genes, most of which belong to the MADS‐box gene family. To provide insights into the evolution of reproductive organs in seed plants, a FLO/LFY homologue (GpLFY) was cloned from Gnetum parvifolium. GpLFY mRNA was expressed in both the vegetative shoot apex and the female strobilus. The GpLFY mRNA signal was detected in early developmental stages of the collar and the ovule primordium, including the nucellus and three envelopes. A comparison of FLO/LFY gene expression in Gnetum and the conifer Pinus radiata indicates that the Gnetum collar and ovule are homologous with the conifer bract and ovule‐ovuliferous scale complex, respectively. Overexpression of GpLFY in transgenic Arabidopsis promoted a conversion of a shoot meristem to a floral primordium. The Arabidopsis LFY null mutant, lfy‐26, with a malformed flower, was complemented by overexpression of GpLFY. These results indicate that the inductive pathway from the FLO/LFY gene to the MADS‐box genes already existed in the common ancestor of angiosperms and gymnosperms.