Identification of six CPC-like genes and their differential expression in leaves of tea plant, Camellia sinensis.

Tea is one of the most consumed beverages worldwide, and trichome formation in tea plant leaves impairs their commercial value. In Arabidopsis thaliana leaves, trichome formation is negatively regulated by the CPC family genes, which encode R3-type MYB transcription factors. Here, we identified six CPC-like genes in a tea plant (Camellia sinensis var. sinensis) for the first time. Simulated three-dimensional structure of the MYB domains of all the six CPC-like proteins exhibited negative charge on the surface, as observed on that of the Arabidopsis CPC protein that does not bind to DNA, indicating their similarity with regard to molecular interaction. We further found that the six CPC-like genes were differentially expressed in different developmental stages of tea leaves, and four out of the six genes were upregulated in the youngest 1st leaves, which formed more trichomes than other older leaves. Although it does not establish a causal link, the correlation between differential expression of CPC-like genes and variable trichome formation suggests that the R3-type MYB transcription factors are potential precipitating factors in affecting the value of tea leaf.

Identification of genes involved in Meloidogyne incognita-induced gall formation processes in Arabidopsis thaliana.

Root-knot nematodes (RKN; Meloidogyne incognita) are phytoparasitic nematodes that cause significant damage to crop plants worldwide. Recent studies have revealed that RKNs disrupt various physiological processes in host plant cells to induce gall formation. However, little is known about the molecular mechanisms of gall formation induced by nematodes. We have previously found that RNA expression levels of some of genes related to micro-RNA, cell division, membrane traffic, vascular formation, and meristem maintenance system were modified by nematode infection. Here we evaluated these genes importance during nematode infection by using Arabidopsis mutants and/or ß-glucronidase (GUS) marker genes, particularly after inoculation with nematodes, to identify the genes involved in successful nematode infection. Our results provide new insights not only for the basic biology of plant-nematode interactions but also to improve nematode control in an agricultural setting.

Effect of the CLE14 polypeptide on GLABRA2 homolog gene expression in rice and tomato roots.

The CLAVATA3/ESR (CLE) plant polypeptides act as peptide hormones in various physiological and developmental aspects in a diverse array of land plants. One of the CLE family of genes, CLE14, is reported to induce root hair formation in Arabidopsis thaliana roots. Previously, we demonstrated that the application of synthetic CLE14 polypeptide treatment induced excess root hairs, and reduced the expression level of the non-hair cell fate determinant gene, GLABRA2 (GL2) in Arabidopsis roots. In this study, we investigated the function of synthetic CLE14 polypeptide in rice (Oryza sativa) and tomato (Solanum lycopersicum) roots. We measured the expression levels of the OsGL2 and SlGL2 genes, i.e., homologs of the Arabidopsis GL2 gene, in rice and tomato seedlings, respectively. Although CLE14 polypeptide treatment induced excess root hair formation in rice roots, substantial root hair induction was not observed in tomato roots. However, the CLE14 polypeptide treatment significantly inhibited the expression of the GL2 homolog genes of rice (OsGL2) and tomato (SlGL2). Our findings thus indicated that CLE14 can inhibit the GL2 gene expression in both rice and tomato plants, similar to the effect seen in Arabidopsis.

Contribution of Root Hair Development to Sulfate Uptake in Arabidopsis.

Root hairs often contribute to nutrient uptake from environments, but the contribution varies among nutrients. In Arabidopsis, two high-affinity sulfate transporters, SULTR1;1 and SULTR1;2, are responsible for sulfate uptake by roots. Their increased expression under sulfur deficiency (-S) stimulates sulfate uptake. Inspired by the higher and lower expression, respectively, of SULTR1;1 in mutants with more (werwolf [wer]) and fewer (caprice [cpc]) root hairs, we examined the contribution of root hairs to sulfate uptake. Sulfate uptake rates were similar among plant lines under both sulfur sufficiency (+S) and -S. Under -S, the expression of SULTR1;1 and SULTR1;2 was negatively correlated with the number of root hairs. These results suggest that both -S-induced SULTR expression and sulfate uptake rates were independent of the number of root hairs. In addition, we observed (1) a negative correlation between primary root lengths and number of root hairs and (2) a greater number of root hairs under -S than under +S. These observations suggested that under both +S and -S, sulfate uptake was influenced by the root biomass rather than the number of root hairs.

Amino acid substitutions in CPC-LIKE MYB reveal residues important for protein stability in Arabidopsis roots.

TRYPTICHON (TRY) and ENHANCER OF TRY AND CPC2 (ETC2) encode R3-type MYB transcription factors that are involved in epidermal cell differentiation in Arabidopsis thaliana. TRY and ETC2 belong to the CPC-like MYB gene family, which includes seven homolog genes. Previously, we showed that among the CPC family members, TRY and ETC2 are characterized by rapid proteolysis compared with that of other members, and we demonstrated that this proteolysis is mediated by the proteasome-dependent pathway. In this study, we compared the functions of the wild-type TRY and ETC2 proteins and their amino acid-substituted versions. Our results showed that the substitution of amino acids in the C-terminal of TRY and ETC2 conferred them the ability to induce root hair formation. Furthermore, we confirmed that these mutations enhanced the stability of the TRY and ETC2 proteins. These results revealed that the amino acids, which are important for the functions of TRY and ETC2, mediate morphological pattern formation and can be useful in understanding the pathway determining the fate of root hair cells.

Effect of amino acid substitution of CAPRICE on cell-to-cell movement ability in Arabidopsis root epidermis.

An R3-type MYB transcription factor, CAPRICE (CPC), is known to promote root hair cell differentiation in Arabidopsis root epidermis. The CPC protein moves from non-hair cells to the neighboring cells, and acts as an inducer of root hair formation. In contrast, we previously showed that the CPC homolog, ENHANCER OF TRY AND CPC1 (ETC1), does not move between the root epidermal cells. To clarify the critical difference in the cell-to-cell movement ability of CPC and ETC1 proteins, we generated five different chimeras of CPC and ETC1. As expected, four of the five chimeric proteins with substitution of CPC amino acids with those of ETC1 induced many root hair and no-trichome phenotype, like CPC. These chimeric proteins essentially maintained the cell-to-cell movement ability of CPC. However, one chimeric protein in which ETC1 was sandwiched between the CPC-specific movement motifs of S1 and S2 did not induce ectopic root hair formation. This chimeric protein did not move between the cells. These results indicate that the maintenance of not only the S1 and S2 motifs but also the precise structure of CPC protein might be necessary for the cell-to-cell movement of CPC. Our results should help in further unraveling of the roles of these MYB transcription factors in root hair formation.

CLE14 peptide signaling in Arabidopsis root hair cell fate determination.

Morphological adjustment is a critical strategy for the survival of plant species in various environments. The CLE (CLAVATA3/EMBRYO SURROUNDING REGION) family of plant polypeptides is known to play important roles in various physiological and developmental processes and the relevant signaling pathways are conserved in diverse land plants. Previously, it has been suggested that overexpression of CLE14 promotes root hair cell differentiation in Arabidopsis roots. To clarify this suggested function of CLE14 peptide on root hair induction, we examined the effect of synthetic CLE14 peptide on Arabidopsis root hair development. Consistent with the results of previous overexpression analyses of CLE14, we demonstrated that application of synthetic CLE14 peptide induced excess root hair formation on CLE14-treated Arabidopsis roots. In addition, CLE14 reduced the expression of the non-hair cell fate determinant gene, GLABRA2. Our results thus indicate that CLE14 can activate the transcriptional regulatory cascade of root hair formation.

Extended C termini of CPC-LIKE MYB proteins confer functional diversity in Arabidopsis epidermal cell differentiation.

The CAPRICE (CPC) gene encodes a R3-type MYB transcription factor that promotes differentiation of root hair cells in Arabidopsis thaliana Here, we have compared the functions of five CPC-homologous genes for epidermal cell differentiation using CPC promoter-driven transgenic plants. Our results show that TRIPTYCHON (TRY) and ENHANCER OF TRY AND CPC2 (ETC2) were less effective in root hair cell differentiation and were unstable in root epidermal cells when compared with CPC, ETC1 or CPC LIKE MYB3 (CPL3). The deletion of the extended C-terminal domain of TRY and ETC2 enhanced protein stability and conferred the ability to induce root hair cell differentiation on them. Treatment with MG132, a proteasome inhibitor, also led to the accumulation of TRY, indicating that TRY proteolysis is mediated by the proteasome-dependent pathway. Our results indicate that the CPC family includes relatively stable (CPC, ETC1 and CPL3) and unstable (TRY and ETC2) proteins that might be degraded by the proteasome. Our findings provide new insights into the regulatory mechanism of CPC family proteins that mediate root hair cell differentiation and should be useful in understanding epidermal development.

Localization of the CAPRICE-ENHANCER OF TRY AND CPC1 chimera protein in Arabidopsis root epidermis.

The CAPRICE (CPC) encodes an R3-type MYB transcription factor, which promotes root-hair differentiation. Previously, we showed that the CPC protein moves from the non-hair cell to the neighboring cell and induces root-hair differentiation in Arabidopsis. In addition, we proposed two cell-to-cell movement signal sequences, S1 and S2, in CPC. However, an S1:2xGFP:S2 chimera protein did not move between root epidermal cells. Here, we show that the S1 and S2 sequences do not confer cell-to-cell movement or nuclear localization ability to a GFP protein. The ENHANCER OF TRY AND CPC1 (ETC1) gene encodes the CPC homolog R3 MYB; this protein does not possess cell-to-cell movement ability or the S1 sequence. To elucidate whether the S1 sequence can induce cell-to-cell movement ability in ETC1, CPCp:S1:ETC1:2xGFP was constructed and introduced into Arabidopsis. Our results indicate that the addition of the S1 sequence was not sufficient for ETC1 to acquire cell-to-cell movement ability.

Localization of ENHANCER OF TRY AND CPC1 protein in Arabidopsis root epidermis.

CAPRICE (CPC) is a R3-type MYB transcription factor, which induces root-hair cell differentiation in Arabidopsis thaliana. The CPC homologous gene ENHANCER TRY AND CPC1 (ETC1) has a similar function to CPC, and acts in concert with CPC. The CPC protein moves between root epidermal cells, from hairless cells to the neighboring cells, and promotes root-hair differentiation. Therefore, ETC1 is predicted to have movement ability similar to that of CPC. In this study, we generated ETC1:ETC1:GFP and CPC:ETC1:GFP transgenic plants to clarify whether ETC1 exhibits cell-to-cell movement. Transgenic plants showed many-root-haired and trichome-less phenotypes, similar to those observed in CPC:CPC:GFP plants, suggesting a similar function of ETC1 and CPC. However, the ETC1:GFP fusion protein located exclusively to the hairless cells in both ETC1:ETC1:GFP and CPC:ETC1:GFP transgenic plants. These results indicate that, unexpectedly, the ETC1 protein cannot move in the root epidermis from hairless cells to the neighboring cells.

Expression and protein localization analyses of Arabidopsis GLABRA3 (GL3) in tomato (Solanum lycopersicum) root epidermis.

The arrangement of root hair and non-hair cells in the root epidermis provides a useful model for understanding the cell fate determination system in plants. A network of related transcription factors, including GLABRA3 (GL3), influences the patterning of cell types in Arabidopsis. GL3 is expressed primarily in root hair cells and encodes a bHLH transcription factor, which inhibits root hair differentiation in Arabidopsis root epidermis. By transforming the GL3 promoter::GFP into tomato, we demonstrated that the Arabidopsis GL3 promoter can function in tomato root epidermis. GFP fluorescence was observed in almost all root epidermal cells in the GL3::GFP transgenic tomato plants, indicating that all root epidermal cells of tomato possess root hair cell identity similar to that of Arabidopsis root hair cells. This is consistent with the phenotype of the tomato root, in which all epidermal cells produce root hairs. Moreover, we observed the localization of a GL3:GFP fusion protein in GL3::GL3:GFP transgenic tomato; although GL3 is known to exclusively localize in non-hair cell nuclei in Arabidopsis root epidermis, GL3:GFP fluorescence was detected not in the nuclei but in the cytoplasm of transgenic tomato epidermal cells. These results suggest that the nuclear localization mechanism differs between tomato and Arabidopsis.

The ectopic localization of CAPRICE LIKE MYB3 protein in Arabidopsis root epidermis.

Cell fate determination is a critical step of plant morphogenesis. Root hair and trichome formation is a good model for studying cell fate determination. The gene CAPRICE (CPC) encodes an R3 type MYB transcription factor, promotes root hair formation, and inhibits trichome formation in Arabidopsis thaliana. The CPC homologous gene CPC LIKE MYB3 (CPL3) encoded 66% similar amino acid sequence to CPC, and it also possessed a cell-to-cell movement WxM motif. CPC protein moves from non-hair cells to neighboring root hair forming cells and induces root hair formation in Arabidopsis root epidermal cells. In this study, to investigate the function and cell-to-cell movement ability of CPL3, we generated CPC:CPL3:GFP transgenic plants to compare against CPL3:CPL3:GFP transgenic plants. CPC:CPL3:GFP transgenic plants showed no-trichome and many root-hair phenotypes, confirming similar function of CPL3 to CPC in root hair and trichome cell fate determination. However, CPL3:GFP fusion protein localized exclusively in non-hair cells in CPC:CPL3:GFP transgenic plants. Collectively, our results suggest that the CPL3 protein does not have cell-to-cell movement ability. Our findings indicate that the CPC family includes a movement protein and a protein that does not move. We believe our results provide new insight into the regulatory mechanism that mediates epidermal cell fate determination.

CAPRICE family genes control flowering time through both promoting and repressing CONSTANS and FLOWERING LOCUS T expression.

CAPRICE (CPC) and six additional CPC family genes encode R3-type MYB transcription factors involved in epidermal cell fate determination, including Arabidopsis root hair and trichome differentiation. Previously, we reported that the CPC and CPC family genes TRIPTYCHON (TRY) and CAPRICE LIKE MYB3 (CPL3) also affect flowering time. The cpl3 mutant plants flower earlier, with fewer but larger leaves, than do wild type plants, and mutations in CPC or TRY delay flowering in the cpl3 mutant. In this study, we examined flowering time, leaf number, and fresh weight for CPC family gene double and triple mutants. Mutation in ENHANCER OF TRY AND CPC1 (ETC1) shortened the flowering time of the cpl3 single mutant. Mutation in ETC2 significantly reduced fresh weight in the cpl3 mutant. Expression levels of the flowering-related genes CONSTANS (CO) and FLOWERING LOCUS T (FT) were higher in the cpl3 mutant than in wild type plants. The high expression levels of CO and FT in cpl3 were significantly reduced by mutations in CPC, TRY, ETC1, or ETC2. Our results suggest that CPC family genes antagonistically regulate flowering time through CO and FT expression.

Root hair formation at the root-hypocotyl junction in CPC-LIKE MYB double and triple mutants of Arabidopsis.

In Arabidopsis thaliana, R3-type MYB genes, CAPRICE (CPC) and its family of genes including TRIPTYCHON (TRY), ENHANCER OF TRY AND CPC1 (ETC1), ETC2 and CPC-LIKE MYB3 cooperatively regulate epidermal cell differentiation. Root hair formation is greatly reduced by a mutation in CPC, and try and etc1 enhance this phenotype. In this study, we demonstrate that CPC, TRY and ETC1 are also involved in root hair formation at the root-hypocotyl junction. The cpc try and cpc etc1 double mutants showed a reduced number of root hairs in that area. Additionally, the expression of ETC1::GUS was higher near this area. These results suggest that CPC family of genes also cooperatively regulates root hair formation at the root-hypocotyl junction in unique ways.

Overexpressing CAPRICE and GLABRA3 did not change the anthocyanin content of tomato (Solanum lycopersicum) fruit peel.

In Arabidopsis thaliana, the R3-type MYB transcription factor CAPRICE (CPC) and bHLH transcription factor GLABRA3 (GL3) cooperatively regulate epidermal cell differentiation. CPC and GL3 are involved in root-hair differentiation, trichome initiation and anthocyanin biosynthesis in Arabidopsis epidermal cells. Previously, we showed that CPC and GL3 also influence anthocyanin accumulation in tomato. Introduction of 35S::CPC into tomato significantly inhibits anthocyanin accumulation in cotyledons, leaves and stems. In contrast, introduction of GL3::GL3 strongly enhances anthocyanin accumulation in cotyledons, leaves and stems of tomato. In this study, we investigated the effect of CPC and GL3 on anthocyanin accumulation in the epidermis of tomato fruit. Unlike the results with vegetative tissues, overexpression of CPC and GL3 did not influence anthocyanin biosynthesis in tomato fruit peel.

A plant U-box protein, PUB4, regulates asymmetric cell division and cell proliferation in the root meristem.

The root meristem (RM) is a fundamental structure that is responsible for postembryonic root growth. The RM contains the quiescent center (QC), stem cells and frequently dividing meristematic cells, in which the timing and the frequency of cell division are tightly regulated. In Arabidopsis thaliana, several gain-of-function analyses have demonstrated that peptide ligands of the Clavata3 (CLV3)/embryo surrounding region-related (CLE) family are important for maintaining RM size. Here, we demonstrate that a plant U-box E3 ubiquitin ligase, PUB4, is a novel downstream component of CLV3/CLE signaling in the RM. Mutations in PUB4 reduced the inhibitory effect of exogenous CLV3/CLE peptide on root cell proliferation and columella stem cell maintenance. Moreover, pub4 mutants grown without exogenous CLV3/CLE peptide exhibited characteristic phenotypes in the RM, such as enhanced root growth, increased number of cortex/endodermis stem cells and decreased number of columella layers. Our phenotypic and gene expression analyses indicated that PUB4 promotes expression of a cell cycle regulatory gene, CYCD6;1, and regulates formative periclinal asymmetric cell divisions in endodermis and cortex/endodermis initial daughters. These data suggest that PUB4 functions as a global regulator of cell proliferation and the timing of asymmetric cell division that are important for final root architecture.

Arabidopsis CAPRICE (MYB) and GLABRA3 (bHLH) control tomato (Solanum lycopersicum) anthocyanin biosynthesis.

In Arabidopsis thaliana the MYB transcription factor CAPRICE (CPC) and the bHLH transcription factor GLABRA3 (GL3) are central regulators of root-hair differentiation and trichome initiation. By transforming the orthologous tomato genes SlTRY (CPC) and SlGL3 (GL3) into Arabidopsis, we demonstrated that these genes influence epidermal cell differentiation in Arabidopsis, suggesting that tomato and Arabidopsis partially use similar transcription factors for epidermal cell differentiation. CPC and GL3 are also known to be involved in anthocyanin biosynthesis. After transformation into tomato, 35S::CPC inhibited anthocyanin accumulation, whereas GL3::GL3 enhanced anthocyanin accumulation. Real-time reverse transcription PCR analyses showed that the expression of anthocyanin biosynthetic genes including Phe-ammonia lyase (PAL), the flavonoid pathway genes chalcone synthase (CHS), dihydroflavonol reductase (DFR), and anthocyanidin synthase (ANS) were repressed in 35S::CPC tomato. In contrast, the expression levels of PAL, CHS, DFR, and ANS were significantly higher in GL3::GL3 tomato compared with control plants. These results suggest that CPC and GL3 also influence anthocyanin pigment synthesis in tomato.

Regulation of root hair cell differentiation by R3 MYB transcription factors in tomato and Arabidopsis.

CAPRICE (CPC) encodes a small protein with an R3 MYB motif and regulates root hair and trichome cell differentiation in Arabidopsis thaliana. Six additional CPC-like MYB proteins including TRIPTYCHON (TRY), ENHANCER OF TRY AND CPC1 (ETC1), ENHANCER OF TRY AND CPC2 (ETC2), ENHANCER OF TRY AND CPC3/CPC-LIKE MYB3 (ETC3/CPL3), TRICHOMELESS1 (TCL1), and TRICHOMELESS2/CPC-LIKE MYB4 (TCL2/CPL4) also have the ability to regulate root hair and/or trichome cell differentiation in Arabidopsis. In this review, we describe our latest findings on how CPC-like MYB transcription factors regulate root hair cell differentiation. Recently, we identified the tomato SlTRY gene as an ortholog of the Arabidopsis TRY gene. Transgenic Arabidopsis plants harboring SlTRY produced more root hairs, a phenotype similar to that of 35S::CPC transgenic plants. CPC is also known to be involved in anthocyanin biosynthesis. Anthocyanin accumulation was repressed in the SlTRY transgenic plants, suggesting that SlTRY can also influence anthocyanin biosynthesis. We concluded that tomato and Arabidopsis partially use similar transcription factors for root hair cell differentiation, and that a CPC-like R3 MYB may be a key common regulator of plant root-hair development.

CLAVATA3-like genes are differentially expressed in grape vine (Vitis vinifera) tissues.

The CLAVATA3 (CLV3)/endosperm surrounding region [(ESR) CLE] peptides function as intercellular signaling molecules that regulate various physiological and developmental processes in diverse plant species. We identified five CLV3-like genes from grape vine (Vitis vinifera var. Pinot Noir): VvCLE 6, VvCLE 25-1, VvCLE 25-2, VvCLE 43 and VvCLE TDIF. These CLV3-like genes encode short proteins containing 43-128 amino acids. Except VvCLE TDIF, grape vine CLV3-like proteins possess a consensus amino acid sequence known as the CLE domain. Phylogenic analysis suggests that the VvCLE 6, VvCLE25-1, VvCLE25-2 and VvCLE43 genes have evolved from a single common ancestor to the Arabidopsis CLV3 gene. Expression analyses showed that the five grape CLV3-like genes are expressed in leaves, stems, roots and axillary buds with significant differences in their levels of expression. For example, while all of them were strongly expressed in axillary buds, VvCLE6 and VvCLE43 expression prevailed in roots, and VvCLE25-1, VvCLE25-2 and VvCLE TDIF expression in stems. The differential expression of the five grape CLV3-like peptides suggests that they play different roles in different organs and developmental stages.

Flowering is delayed by mutations in homologous genes CAPRICE and TRYPTICHON in the early flowering Arabidopsis cpl3 mutant.

CAPRICE (CPC) and CAPRICE-like (CPL) myeloblastosis (MYB) family members [including TRYPTICHON (TRY) and ENHANCER OF TRYPTICHON AND CAPRICE (ETC)] of Arabidopsis thaliana encode R3-type MYB transcription factors that promote root hair differentiation and inhibit trichome formation in a redundant manner. Previously, we reported that the CPL3 gene affects flowering. The cpl3 mutant plants flower earlier and with fewer leaves than the wild type. In this study, we show that mutations in CPC or TRY delay flowering of cpl3 plants. A mutation in ETC1 did not further delay flowering but reduced plant size. Our study provides insight into the regulation of flowering time by the CPC-like MYB gene family.