CAPRICE Research Articles

Complementation and protein localization analyses of R3 MYBs in an Arabidopsis caprice mutant.

Root hairs play vital roles in plant growth since they enable the efficient absorption of water and nutrients from the soil. Recent advances in Arabidopsis research have provided a deeper understanding of the molecular genetic mechanisms underlying root hair differentiation. CAPRICE (CPC) and its four homologs, which belong to the CPC gene family and encode R3 MYB transcription factors, play central roles in root hair differentiation. In this study, to better understand the functional specificity and contribution of these five CPC family genes, we conducted phenotypic and expression analyses of the CPC family proteins in a cpc mutant background. As a result, ENHANCER OF TRY AND CPC1 (ETC1) and ETC3 were found to complement the hairless root phenotype of the cpc mutant, as did CPC, whereas TRIPTYCHON (TRY) and ETC2 did not rescue the cpc phenotype. Protein expression analysis revealed that GFP fluorescence was nearly undetectable in pCPC::TRY:GFP/cpc and pCPC::ETC2:GFP/cpc plants, supporting the incapability of root hair formation in these plants. Interestingly, the fluorescence intensity of the CPC:GFP fusion protein was weaker than that of ETC1:GFP and ETC3:GFP fusion proteins. These results were inconsistent with the result of the phenotypic analysis, in which the three genes promoted root hair formation to almost the same degree in the cpc mutant background. We further discuss the discrepancy between the root hair phenotypes and the expression levels of CPC family proteins.

Amino acid substitution in CAPRICE (CPC) protein affects its cell-to-cell movement in the root epidermis of Arabidopsis thaliana

Amino acid substitution in CAPRICE (CPC) protein affects its cell-to-cell movement in the root epidermis of Arabidopsis thaliana

Regulation of Phytohormones on the Growth and Development of Plant Root Hair.

The tubular-shaped unicellular extensions of plant epidermal cells known as root hairs are important components of plant roots and play crucial roles in absorbing nutrients and water and in responding to stress. The growth and development of root hair include, mainly, fate determination of root hair cells, root hair initiation, and root hair elongation. Phytohormones play important regulatory roles as signal molecules in the growth and development of root hair. In this review, we describe the regulatory roles of auxin, ethylene (ETH), jasmonate (JA), abscisic acid (ABA), gibberellin (GA), strigolactone (SL), cytokinin (CK), and brassinosteroid (BR) in the growth and development of plant root hairs. Auxin, ETH, and CK play positive regulation while BR plays negative regulation in the fate determination of root hair cells; Auxin, ETH, JA, CK, and ABA play positive regulation while BR plays negative regulation in the root hair initiation; Auxin, ETH, CK, and JA play positive regulation while BR, GA, and ABA play negative regulation in the root hair elongation. Phytohormones regulate root hair growth and development mainly by regulating transcription of root hair associated genes, including WEREWOLF (WER), GLABRA2 (GL2), CAPRICE (CPC), and HAIR DEFECTIVE 6 (RHD6). Auxin and ETH play vital roles in this regulation, with JA, ABA, SL, and BR interacting with auxin and ETH to regulate further the growth and development of root hairs.

Ectopic Expression of JcCPL1, 2, and 4 Affects Epidermal Cell Differentiation, Anthocyanin Biosynthesis and Leaf Senescence in Arabidopsis thaliana.

The CAPRICE (CPC)-like (CPL) genes belong to a single-repeat R3 MYB family, whose roles in physic nut (Jatropha curcas L.), an important energy plant, remain unclear. In this study, we identified a total of six CPL genes (JcCPL1–6) in physic nut. The JcCPL3, 4, and 6 proteins were localized mainly in the nucleus, while proteins JcCPL1, 2, and 5 were localized in both the nucleus and the cytoplasm. Ectopic overexpression of JcCPL1, 2, and 4 in Arabidopsis thaliana resulted in an increase in root hair number and decrease in trichome number. Consistent with the phenotype of reduced anthocyanin in shoots, the expression levels of anthocyanin biosynthesis genes were down-regulated in the shoots of these three transgenic A. thaliana lines. Moreover, we observed that OeJcCPL1, 2, 4 plants attained earlier leaf senescence, especially at the late developmental stage. Consistent with this, the expression levels of several senescence-associated and photosynthesis-related genes were, respectively, up-regulated and down-regulated in leaves. Taken together, our results indicate functional divergence of the six CPL proteins in physic nut. These findings also provide insight into the underlying roles of CPL transcription factors in leaf senescence.

A pair of DUF538 domain-containing proteins modulates plant growth and trichome development through the transcriptional regulation of GLABRA1 in Arabidopsis thaliana.

SMALLER TRICHOMES WITH VARIABLE BRANCHES (SVB) is an emerging plant growth regulator in trichome development, endoplasmic reticulum stress response, and phosphoinositide signaling, and belongs to the land plant-specific DUF538 domain-containing protein family. Despite its multifaceted roles, the functions of this protein family are poorly understood in plant growth and development. Here, we report that SVB-like (SVBL), the closest homolog of SVB, modulates plant growth and trichome development with SVB in Arabidopsis thaliana. Although none of the single mutants showed an obvious growth defect, the double mutants of svb svbl exhibited dwarfed plant growth. In trichome development, the defects in svb mutant were greatly enhanced by the additional mutation in SVBL, despite the single knockout of SVBL showing the mild defects. The double mutation reduced the transcript level of one of the central hub genes for trichome development, GLABRA1 (GL1), which in turn affects the other downstream genes, GLABRA2 (GL2), TRANSPARENT TESTA GLABRA2 (TTG2), TRIPTYCHON (TRY), CAPRICE (CPC), and ENHANCER OF TRY AND CPC1 (ETC1). In situ translational reporter assays showed that SVB and SVBL share highly similar localization patterns both at tissue and subcellular levels. The present study suggests that SVB and SVBL play a pivotal role in plant growth and trichome development by affecting a specific subset of known trichome developmental regulators, highlighting the importance of the DUF538 protein family in higher plants.

The Carboxyl-Terminus of TRANSPARENT TESTA GLABRA1 Is Critical for Its Functions in Arabidopsis.

The Arabidopsis WD40 repeat protein TRANSPARENT TESTA GLABRA1 (TTG1) regulates cell fate determination, including trichome initiation and root hair formation, as well as secondary metabolism such as flavonoid biosynthesis and seed coat mucilage production. TTG1 regulates different processes via regulating the expression of its downstream target genes by forming MYB-bHLH-WD40 (MBW) activator complexes with different R2R3 MYB and bHLH transcription factors. Here, we report the identification of the carboxyl (C)-terminus as a critical domain for TTG1′s functions in Arabidopsis. We found that the ttg1Δ15aa mutant shows pleiotropic phenotypes identical to a TTG1 loss-of-function mutant. Gene sequencing indicates that a single nucleotide substitution in TTG1 led to a premature stop at the W327 residue, leading to the production of a truncated TTG1 protein with a deletion of the last 15 C-terminal amino acids. The expression of TTG1 under the control of its native promoter fully restored the ttg1Δ15aa mutant phenotypes. Consistent with these observations, the expression levels of TTG1 downstream genes such as GLABRA2 (GL2) and CAPRICE (CPC) were reduced in the ttg1Δ15aa mutant. Assays in Arabidopsis protoplast show that TTG1Δ15aa failed to interact with the bHLH transcription factor GL3, and the deletion of the last 3 C-terminal amino acids or the 339L amino acid alone fully abolished the interaction of TTG1 with GL3. Furthermore, the expression of TTG1Δ3aa under the control of TTG1 native promoter failed to restore the ttg1Δ15aa mutant phenotypes. Taken together, our results suggest that the C-terminal domain of TTG1 is required for its proper function in Arabidopsis.

Effect of phosphate starvation on CAPRICE homolog gene expression in the root of Arabidopsis.

Phosphate (Pi) starvation affects root hair formation to increase the absorptive surface area of the roots. CAPRICE (CPC) and its homolog genes, including TRIPTYCHON (TRY), ENHANCER OF TRY AND CPC1 (ETC1), ETC2, and ETC3, positively regulate root hair formation in a partially redundant manner. In particular, ETC1 responds to Pi deficiency. To clarify role sharing among the CPC homolog genes under Pi-deficient condition, we analyzed the expression of five CPC homolog genes under Pi-deficient condition, using the real-time polymerase chain reaction analysis. Pi starvation enhanced the expression of not only ETC1, but also ETC3. Furthermore, ETC3, which is rarely expressed in the roots, was induced by Pi deficiency. The expression levels of CPC, TRY, and ETC2 in response to Pi deficiency were not significantly different from those under the control conditions. These results suggest that CPC homologs can be divided into two groups, genes that respond to Pi deficiency (ETC1 and ETC3) and those that do not (CPC, TRY, and ETC2).

Heterologous Expression of GbTCP4, a Class II TCP Transcription Factor, Regulates Trichome Formation and Root Hair Development in Arabidopsis.

Two class I family teosinte branched1/cycloidea/proliferating cell factor1 (TCP) proteins from allotetraploid cotton are involved in cotton fiber cell differentiation and elongation and root hair development. However, the biological function of most class II TCP proteins is unclear. This study sought to reveal the characteristics and functions of the sea-island cotton class II TCP gene GbTCP4 by biochemical, genetic, and molecular biology methods. GbTCP4 protein localizes to nuclei, binding two types of TCP-binding cis-acting elements, including the one in its promoter. Expression pattern analysis revealed that GbTCP4 is widely expressed in tissues, with the highest level in flowers. GbTCP4 is expressed at different fiber development stages and has high transcription in fibers beginning at 5 days post anthesis (DPA). GbTCP4 overexpression increases primary root hair length and density and leaf and stem trichomes in transgenic Arabidopsis relative to wild-type plants (WT). GbTCP4 binds directly to the CAPRICE (CPC) promoter, increasing CPC transcript levels in roots and reducing them in leaves. Compared with WT plants, lignin content in the stems of transgenic Arabidopsis overexpressing GbTCP4 increased, and AtCAD5 gene transcript levels increased. These results suggest that GbTCP4 regulates trichome formation and root hair development in Arabidopsis and may be a candidate gene for regulating cotton fiber elongation.

Determination of Arsenic Content in Different Brands of Rice Sold in Port Harcourt, Nigeria

Arsenic, a naturally occurring element seen in the environment and cycled by water has been seen to be a pollutant in soil, water and air. The inorganic form of arsenic is associated with major health problems and cancer mainly arising due to its anthropogenic activities. The content of arsenic was assayed in six rice brands sold in Port Harcourt, Rivers State, Nigeria. The rice samples were ground to powdered forms and subjected to acid digestion before analysis. The Agilent Micro Plasma Atomic Emission Spectrophotometer (MP-AES) was used to analyze the digested samples. Results showed mean arsenic concentration in Vico Rice, the highest as 1.0073. This was followed by Local Rice 0.9420, Marori Benz 0.9140, Golden Stallion Rice 0.861 and the Cap Rice 0.8077 while the least mean concentration of arsenic was recorded in African Princess Rice 0.6417, all in mg/kg. The mean concentration was significantly different in the six brands of rice (P<0.05). The arsenic content in the different rice brand was within the tolerable concentration of 1.4 mg/kg as in cereals and vegetables. This concentration did not exceed the Maximum Permissible Limit of 1 mg/kg hence considered safe for consumption except Vico rice with a higher concentration above 1 mg/kg. This shows that rice brands sold in Port Harcourt pose no health risk for consumers with respect to arsenic content.

Single-repeat R3 MYB transcription factors from Platanus acerifolia negatively regulate trichome formation in Arabidopsis.

Four R3 MYB genes were cloned and identified from Platanus acerifolia and analysed according to endogenous gene expression profiles, protein-protein interaction patterns, phenotypic effects and related gene expression profiles in transgenic Arabidopsis, suggesting that London plane R3 MYB genes inhibit trichome formation in Arabidopsis. The CPC-like MYB transcription factors including CAPRICE (CPC), TRIPTYCHON (TRY), ENHANCER OF TRY AND CPC 1, 2 and 3 (ETC1, ETC2 and ETC3), TRICHOMELESS1 (TCL1) and TRICHOMELESS2(TCL2) play important roles in controlling trichome patterning in Arabidopsis. In this study, four sequences homologous with the Arabidopsis CPC family were identified from London plane and named PaTRY, PaCPC-like1, PaCPC-like2 and PaCPC-like3. Over-expression of PaTRY, PaCPC-like1, PaCPC-like2 and PaCPC-like3 in Arabidopsis resulted in glabrous phenotypes. In addition, expression of endogenous GL2, GL1, MYB23, TTG2 and a set of R3 MYB-encoding genes was markedly reduced. Furthermore, the protein products of PaTRY, PaCPC-like1, PaCPC-like2 and PaCPC-like3 were shown to interact with PaGL3 in yeast two-hybrid assays. Together, these results likely suggest that the mechanisms of trichome regulation in London plane have similarities with those in Arabidopsis.

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.

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.

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 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.

Triptychon (TRY) Protein Accumulation in the Roots of Mutant Auxinresistant 1 (axr1) Arabidopsis thaliana

Plant root hairs play an essential role in water and nutrient uptake. CAPRICE (CPC) family transcription factors act to induce root hairs in Arabidopsis thaliana. Previously, using a proteasome inhibitor assay, we proposed that the CPC family protein TRIPTYCHON (TRY) was degraded through the ubiquitin/26S proteasome pathway. The Auxin resistant 1 (AXR1) gene encodes the ubiquitin-activating enzyme E1. In this study, to further investigate the mechanism of TRY degradation, we introduced a CPCp:TRY-GFP construct into axr1-3 A. thaliana mutants. The CPCp:TRY-GFP transgenic plant showed weak TRY-GFP fluorescence while the CPCp:TRY-GFP in axr1-3 transgenic plant showed strong TRY-GFP fluorescence. These results support the suggestion that TRY is degraded by the ubiquitin/26S proteasome mechanism.

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.

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.

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.

The paralogous R3 MYB proteins CAPRICE, TRIPTYCHON and ENHANCER OF TRY AND CPC1 play pleiotropic and partly non-redundant roles in the phosphate starvation response ofArabidopsisroots

Phosphate (Pi) deficiency alters root hair length and frequency as a means of increasing the absorptive surface area of roots. Three partly redundant single R3 MYB proteins, CAPRICE (CPC), ENHANCER OF TRY AND CPC1 (ETC1) and TRIPTYCHON (TRY), positively regulate the root hair cell fate by participating in a lateral inhibition mechanism. To identify putative targets and processes that are controlled by these three transcription factors (TFs), we conducted transcriptional profiling of roots from Arabidopsis thaliana wild-type plants, and cpc, etc1 and try mutants grown under Pi-replete and Pi-deficient conditions using RNA-seq. The data show that in an intricate interplay between the three MYBs regulate several developmental, physiological and metabolic processes that are putatively located in different tissues. When grown on media with a low Pi concentration, all three TFs acquire additional functions that are related to the Pi starvation response, including transition metal transport, membrane lipid remodelling, and the acquisition, uptake and storage of Pi. Control of gene activity is partly mediated through the regulation of potential antisense transcripts. The current dataset extends the known functions of R3 MYB proteins, provides a suite of novel candidates with critical function in root hair development under both control and Pi-deficient conditions, and challenges the definition of genetic redundancy by demonstrating that environmental perturbations may confer specific functions to orthologous proteins that could have similar roles under control conditions.

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.