TRANSPARENT TESTA GLABRA1 Research Articles

Negative feedback regulation of GLABRA1 contributes to epidermal cell patterning in the Arabidopsis root

GLABRA1 (GL1), which encodes an R2R3 MYB transcription factor, is a key regulator of trichome patterning in the aerial organs of Arabidopsis thaliana. Although it has been generally assumed that GL1 functions exclusively in shoots and is not expressed in roots, reverse transcription polymerase chain reaction (RT-PCR) analysis has revealed that GL1 is indeed expressed in roots. To investigate whether GL1 plays a role in root epidermal patterning, we analyzed the effects of gl1 mutations in sensitized genetic backgrounds. Our findings show that gl1 mutants enhance the root epidermal phenotype of a weak allele of the werewolf (wer) mutant and suppress the phenotype of the caprice (cpc) mutant. We also demonstrate that the GL1 promoter is active in N-position epidermal cells, and that the GFP-GL1 fusion protein is predominantly localized in the nucleus of N-position cells. Furthermore, we provide evidence that GL1 expression is positively regulated by WER, GLABRA3, ENHANCER OF GLABRA3, and TRANSPARENT TESTA GLABRA1, while negatively regulated by CPC, TRIPTYCHON, and GLABRA2 (GL2). Notably, GL2, which is positively regulated by GL1, moderately represses GL1 expression, and both GL1 and GL2 are positively regulated by WER in N-position cells. These findings suggest that a negative feedback regulation of GL1 expression via GL2 contributes to the fine-tuning of non-hair cell fate determination in Arabidopsis root epidermis.

The R2R3-MYB transcription factor PgTT2 from Panax ginseng interacts with the WD40-repeat protein PgTTG1 during the regulation of proanthocyanidin biosynthesis and the response to salt stress

Proanthocyanidins (PAs) are flavonoid compounds with important defensive roles in plants. The application of PAs in industries such as the pharmaceutical industry has led to increased interest in enhancing their biosynthesis. In Arabidopsis thaliana, PAs are biosynthesized under the regulation of an R2R3-MYB transcription factor TRANSPARENT TESTA 2 (TT2), which can interact with other proteins, including TRANSPARENT TESTA GLABRA 1 (TTG1), while also regulating a plant's response to abiotic stressors. However, the regulation of PA biosynthesis in the high-value medicinal plant Panax ginseng (ginseng) has not yet been studied. Understanding the mechanism of PAs biosynthesis regulation in ginseng may be helpful in increasing the plant's range of pharmacological applications. This study found that the overexpression of PgTT2 increased PA biosynthesis by an average of 67.3% in ginseng adventitious roots and 50.5% in arabidopsis seeds. Furthermore, transgenic arabidopsis plants overexpressing PgTT2 produced increased reactive oxygen species (ROS) scavenging ability by influencing abscisic acid synthesis and signaling. However, under high salinity stress, seed germination and growth rate of seedlings were decreased. An expression analysis of plants facing salt stress revealed increased transcripts of an ABA biosynthetic gene, NCED3, and ABA signaling genes ABI5 and ABI3. Moreover, the PgTT2 protein showed a direct interaction with PgTTG1 in yeast two-hybrid assays. This study therefore reveals novel information on the transcriptional regulation of PA production in ginseng and shows how PgTT2 influences the ABA response pathway to regulate responses to ROS and salt stress.

TRANSPARENT TESTA GLABRA1 Regulates High-Intensity Blue Light-Induced Phototropism by Reducing CRYPTOCHROME1 Levels.

The asymmetrical distribution of auxin supports high intensity blue light (HBL)-mediated phototropism. Flavonoids, secondary metabolites induced by blue light and TRANSPARENT TESTA GLABRA1 (TTG1), alter auxin transport. However, the role of TTG1 in HBL-induced phototropism in Arabidopsis (Arabidopsis thaliana) remains unclear. We found that TTG1 regulates HBL-mediated phototropism. HBL-induced degradation of CRYPTOCHROME 1 (CRY1) was repressed in ttg1-1, and depletion of CRY1 rescued the phototropic defects of the ttg1-1 mutant. Moreover, overexpression of CRY1 in a cry1 mutant background led to phototropic defects in response to HBL. These results indicated that CRY1 is involved in the regulation of TTG1-mediated phototropism in response to HBL. Further investigation showed that TTG1 physically interacts with CRY1 via its N-terminus and that the added TTG1 promotes the dimerization of CRY1. The interaction between TTG1 and CRY1 may promote HBL-mediated degradation of CRY1. TTG1 also physically interacted with blue light inhibitor of cryptochrome 1 (BIC1) and Light-Response Bric-a-Brack/Tramtrack/Broad 2 (LRB2), and these interactions either inhibited or promoted their interaction with CRY1. Exogenous gibberellins (GA) and auxins, two key plant hormones that crosstalk with CRY1, may confer the recovery of phototropic defects in the ttg1-1 mutant and CRY1-overexpressing plants. Our results revealed that TTG1 participates in the regulation of HBL-induced phototropism by modulating CRY1 levels, which are coordinated with GA or IAA signaling.

Transcription factors Pbr3RAV2 and PbrTTG1 regulate pear resistance to Botryosphaeria dothidea via the autophagy pathway.

Pear ring rot, caused by Botryosphaeria dothidea, is the most serious disease of pear (Pyrus spp.) trees. However, the molecular mechanisms underlying pear resistance to B. dothidea remain elusive. In this study, we demonstrated that the pear AuTophagy-related Gene 1a (PbrATG1a) plays a key role in autophagic activity and resistance to B. dothidea. Stable overexpression of PbrATG1a enhanced resistance to B. dothidea in pear calli. Autophagy activity was greater in PbrATG1a-overexpressing calli than in wild-type calli. We used yeast 1-hybrid screening to identify a transcription factor, related to ABI3 and VP1 (Pbr3RAV2), that binds the promoter of PbrATG1a and enhances pear resistance to B. dothidea by regulating autophagic activity. Specifically, the overexpression of Pbr3RAV2 enhanced resistance to B. dothidea in pear calli, while transient silencing of Pbr3RAV2 resulted in compromised resistance to B. dothidea in Pyrus betulifolia. In addition, we identified Transparent Testa Glabra 1 (PbrTTG1), which interacts with Pbr3RAV2. Pathogen infection enhanced the interaction between Pbr3RAV2 and PbrTTG1. The Pbr3RAV2-PbrTTG1 complex increased the binding capacity of Pbr3RAV2 and transcription of PbrATG1a. In addition to providing insights into the molecular mechanisms underlying pear disease resistance, these findings suggest potential genetic targets for enhancing disease resistance in pear.

BrTTG1 regulates seed coat proanthocyanidin formation through a direct interaction with structural gene promoters of flavonoid pathway and glutathione S-transferases in Brassica rapa L.

Seed coat color is a significant agronomic trait in horticultural crops such as Brassica rapa which is characterized by brown or yellow seed coat coloration. Previous Brassica rapa studies have shown that BrTTG1 is responsible for seed coat proanthocyanidin formation, which is dependent on the MYB-bHLH-WD40 complex, whereas some studies have reported that TRANSPARENT TESTA GLABRA 1 (TTG1) directly interacts with the structural gene promoters of the flavonoid pathway. Herein, the brown-seeded inbred B147 and ttg1 yellow-seeded inbred B80 mutants were used as plant materials for gene expression level analysis, gene promoter clone and transient overexpression. The analysis identified eleven structural genes involved in the flavonoid biosynthesis pathway, which are potentially responsible for BrTTG1- dependent seed coat proanthocyanidin formation. The promoters of these genes were cloned and cis-acting elements were identified. Yeast one-hybrid and dual-luciferase assays confirmed that BrTTG1 directly and independently interacted with proCHS-Bra008792, proDFR-Bra027457, proTT12-Bra003361, proTT19-Bra008570, proTT19-Bra023602 and proAHA10-Bra016610. A TTG1-binding motif (RTWWGTRGM) was also identified. Overexpression of TTG1 in the yellow-seed B. rapa inbred induced proanthocyanidin accumulation by increasing the expression levels of related genes. Our study unveiled, for the first time, the direct interaction between TTG1 and the promoters of the flavonoid biosynthesis pathway structural genes and glutathione S-transferases in Brassica rapa. Additionally, we have identified a novel TTG1-binding motif, providing a basis for further exploration into the function of TTG1 and the accumulation of proanthocyanidins in seed coats.

TRANSPARENT TESTA GLABRA2 defines trichome cell shape by modulating actin cytoskeleton in Arabidopsis thaliana.

The Arabidopsis (Arabidopsis thaliana) TRANSPARENT TESTA GLABRA2 (TTG2) gene encodes a WRKY transcription factor that regulates a range of development events like trichome, seed coat, and atrichoblast formation. Loss-of-function of TTG2 was previously shown to reduce or eliminate trichome specification and branching. Here, we report the identification of an allele of TTG2, ttg2-6. In contrast to the ttg2 mutants described before, ttg2-6 displayed unique trichome phenotypes. Some ttg2-6 mutant trichomes were hyper-branched, whereas others were hypo-branched, distorted, or clustered. Further, we found that in addition to specifically activating R3 MYB transcription factor TRIPTYCHON (TRY) to modulate trichome specification, TTG2 also integrated cytoskeletal signaling to regulate trichome morphogenesis. The ttg2-6 trichomes displayed aberrant cortical microtubules (cMTs) and actin filaments (F-actin) configurations. Moreover, genetic and biochemical analyses showed that TTG2 could directly bind to the promoter and regulate the expression of BRICK1 (BRK1), which encodes a subunit of the actin nucleation promoting complex suppressor of cyclic AMP repressor (SCAR)/Wiskott-Aldrich syndrome protein family verprolin homologous protein (WAVE). Collectively, taking advantage of ttg2-6, we uncovered a function for TTG2 in facilitating cMTs and F-actin cytoskeleton-dependent trichome development, providing insight into cellular signaling events downstream of the core transcriptional regulation during trichome development in Arabidopsis.

MYB-bHLH-TTG1 ina Multi-tiered Pathway Regulates Arabidopsis Seed Coat Mucilage Biosynthesis Genes Including PECTIN METHYLESTERASE INHIBITOR14 Required forHomogalacturonan Demethylesterification.

MYB-bHLH-TTG1 (MBW) transcription factor (TF) complexes regulate Arabidopsis seed coat biosynthesis pathways via a multi-tiered regulatory mechanism. The MYB genes include MYB5, MYB23 and TRANSPARENT TESTA2 (TT2), which regulate GLABRA2 (GL2), HOMEODOMAIN GLABROUS2 (HDG2) and TRANSPARENT TESTA GLABRA2 (TTG2). Here, we examine the role of PECTIN METHYLESTERASE INHIBITOR14 (PMEI14) in seed coat mucilage pectin methylesterification and provide evidence in support of multi-tiered regulation of seed coat mucilage biosynthesis genes including PMEI14. The PMEI14 promoter was active in the seed coat and developing embryo. A pmei14 mutant exhibited stronger attachment of the outer layer of seed coat mucilage, increased mucilage homogalacturonan demethylesterification and reduced seed coat radial cell wall thickness, results consistent with decreased PMEI activity giving rise to increased PME activity. Reduced mucilage release from the seeds of myb5, myb23, tt2 and gl2, hdg2, ttg2 triple mutants indicated that HDG2 and MYB23 play minor roles in seed coat mucilage deposition. Chromatin immunoprecipitation analysis found that MYB5, TT8 and seven mucilage pathway structural genes are directly regulated by MYB5. Expression levels of GL2, HDG2, TTG2 and nine mucilage biosynthesis genes including PMEI14 in the combinatorial mutant seeds indicated that these genes are positively regulated by at least two of those six TFs and that TTG1 and TTG2 are major regulators of PMEI14 expression. Our results show that MYB-bHLH-TTG1 complexes regulate mucilage biosynthesis genes, including PMEI14, both directly and indirectly via a three-tiered mechanism involving GL2, HDG2 and TTG2.

TARGET OF EAT3 (TOE3) specifically regulates fruit spine initiation in cucumber (Cucumis sativus L.).

The presence or absence of spines is an important economic trait of cucumber fruit. Spines are believed to be a type of specialized trichome on the fruit surface and all the identified cucumber trichome-less mutants lack fruit spines. However, genes that specifically regulate fruit spine initiation remain to be identified. Here, we found that knocking out cucumber TARGET OF EAT3 homolog (CsTOE3), belonging to the APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) family, affected flower development, and more interestingly, inhibited cucumber fruit spine initiation. On analyzing expression patterns by qRT-PCR and in situ hybridization assay, CsTOE3 was found to be highly expressed in male and female flowers, and its mRNA accumulated in the tips of sepal and petal primordia and in the cells of fruit spines and peels. Biochemical analyses indicated that CsTOE3 directly interacts with GLABRA1 (CsGL1) and TRANSPARENT TESTA GLABRA1 (CsTTG1), which are positive regulators of trichome formation. In addition, RNA-seq showed that the transcription levels of eight ERFs were significantly upregulated in CsTOE3 knockout lines. Phytohormone content analysis also revealed a significant increase in the amount of ethylene released by CsTOE3 knockout lines; treatment with the ethylene synthesis inhibitor aminoethoxyvinyl-glycine (AVG) partly restored the spineless phenotype. Our results suggest that CsTOE3 specifically regulates fruit spine initiation but does not affect the formation of trichomes on other organs in cucumber. Our findings may have a far-reaching significance for cucumber germplasm improvement and quality breeding using fruit spines as the target trait.

Tropaeolum majus R2R3 MYB Transcription Factor TmPAP2 Functions as a Positive Regulator of Anthocyanin Biosynthesis.

Anthocyanins are an important group of water-soluble and non-toxic natural pigments with antioxidant and anti-inflammatory properties that can be found in flowers, vegetables, and fruits. Anthocyanin biosynthesis is regulated by several different types of transcription factors, including the WD40-repeat protein Transparent Testa Glabra 1 (TTG1), the bHLH transcription factor Transparent Testa 8 (TT8), Glabra3 (GL3), Enhancer of GL3 (EGL3), and the R2R3 MYB transcription factor Production of Anthocyanin Pigment 1 (PAP1), PAP2, MYB113, and MYB114, which are able to form MYB-bHLH-WD40 (MBW) complexes to regulate the expression of late biosynthesis genes (LBGs) in the anthocyanin biosynthesis pathway. Nasturtium (Tropaeolum majus) is an edible flower plant that offers many health benefits, as it contains numerous medicinally important ingredients, including anthocyanins. By a comparative examination of the possible anthocyanin biosynthesis regulator genes in nasturtium varieties with different anthocyanin contents, we found that TmPAP2, an R2R3 MYB transcription factor gene, is highly expressed in “Empress of India”, a nasturtium variety with high anthocyanin content, while the expression of TmPAP2 in Arabidopsis led to the overproduction of anthocyanins. Protoplast transfection shows that TmPAP2 functions as a transcription activator; consistent with this finding, some of the biosynthesis genes in the general phenylpropanoid pathway and anthocyanin biosynthesis pathway were highly expressed in “Empress of India” and the 35S:TmPAP2 transgenic Arabidopsis plants. However, protoplast transfection indicates that TmPAP2 may not be able to form an MBW complex with TmGL3 and TmTTG1. These results suggest that TmPAP2 may function alone as a key regulator of anthocyanin biosynthesis in nasturtiums.

RsTTG1, a WD40 Protein, Interacts with the bHLH Transcription Factor RsTT8 to Regulate Anthocyanin and Proanthocyanidin Biosynthesis in Raphanus sativus.

MBW complexes, consisting of MYB, basic helix–loop–helix (bHLH), and WD40 proteins, regulate multiple traits in plants, including anthocyanin and proanthocyanidin (PA) biosynthesis and the determination of epidermal cell fate. Here, a WD40 gene from Raphanus sativus, designated TRANSPARENT TESTA GLABRA 1 (RsTTG1), was cloned and functionally characterized. Heterologous expression of RsTTG1 in the Arabidopsis thaliana mutant ttg1-22 background restored accumulation of anthocyanin and PA in the mutant and rescued trichome development. In radish, RsTTG1 was abundantly expressed in all root and leaf tissues, independently of anthocyanin accumulation, while its MBW partners RsMYB1 and TRANSPARENT TESTA 8 (RsTT8) were expressed at higher levels in pigment-accumulating tissues. In yeast two-hybrid analysis, the full-length RsTTG1 protein interacted with RsTT8. Moreover, transient protoplast co-expression assays demonstrated that RsTTG1, which localized to both the cytoplasm and nucleus, moves from the cytoplasm to the nucleus in the presence of RsTT8. When co-expressed with RsMYB1 and RsTT8, RsTTG1 stably activated the promoters of the anthocyanin biosynthesis genes CHALCONE SYNTHASE (RsCHS) and DIHYDROFLAVONOL 4-REDUCTASE (RsDFR). Transient expression of RsTTG1 in tobacco leaves exhibited an increase in anthocyanin accumulation due to activation of the expression of anthocyanin biosynthesis genes when simultaneously expressed with RsMYB1 and RsTT8. These results indicate that RsTTG1 is a vital regulator of pigmentation and trichome development as a functional homolog of AtTTG1.

RrTTG1 promotes fruit prickle development through an MBW complex in Rosa roxburghii.

Fruit prickles are widely distributed on the pericarp and exhibit polymorphic traits at different developmental stages. Although they are multicellular appendages that are well-known for helping plants defend against biotic and abiotic stresses, their origination and molecular mechanism are still less known. Here, we studied the origination and molecular mechanism of fruit prickles in Rosa roxburghii. Using morphological and histological observations, we found that the fruit prickle primordium of R. roxburghii originated from the ground meristem that underwent cell division to form flagelliform prickles, continued to enlarge, and finally lignified to form mature fruit prickles. We amplified a homolog of candidate gene TRANSPARENT TESTA GLABRA1 (TTG1) from R. roxburghii, named RrTTG1. RrTTG1 harbored four conserved WD-repeat domains and was exclusively nuclear-localized. Using qRT-PCR and in situ hybridization, we found that RrTTG1 was constitutively expressed and highly expressed during the initiation and cell expansion phases of fruit prickles. Ectopic expression analysis in Arabidopsis proved that RrTTG1 substantially enhanced the number of trichome and pigmentation production and inhibited root hair formation. Besides, RrTTG1 complemented the phenotypes of the ttg1 mutant in Arabidopsis, thus indicating that RrTTG1 played pleiotropic roles akin to AtTTG1. We demonstrated that the RrTTG1 only interacted with RrEGL3, a homolog of ENHANCER OF GLABRA3 (EGL3), via yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays. Briefly, RrTTG1 might positively regulate the initiation of fruit prickle primordium and cell enlargement by forming the RrTTG1-RrEGL3-RrGL1 complex in R. roxburghii. Therefore, our results help characterize the RrTTG1 in R. roxburghii and also elucidate the establishment of the prickles regulatory system in the Rosaceae plants.

A molecular framework for signaling crosstalk between jasmonate and ethylene in anthocyanin biosynthesis, trichome development, and defenses against insect herbivores in Arabidopsis.

The phytohormones ethylene (ET) and jasmonate (JA) regulate plant development, growth, and defense responses; however, the molecular basis for their signaling crosstalk is unclear. Here, we show that JA-ZIM-domain (JAZ) proteins, which repress JA signaling, repress trichome initiation/branching and anthocyanin accumulation, and inhibit the transcriptional activity of the basic helix-loop-helix (bHLH)-MYB members (GLABRA3 (GL3)-GL1 and TRANSPARENT TESTA 8 (TT8)-MYB75) of WD-repeat/bHLH/MYB (WBM) complexes. The ET-stabilized transcription factors ETHYLENE-INSENSITIVE3 (EIN3) and EIN3-LIKE1 (EIL1) were found to bind to several members of WBM complexes, including GL3, ENHANCER OF GLABRA3 (EGL3), TT8, GL1, MYB75, and TRANSPARENT TESTA GLABRA1 (TTG1). This binding repressed the transcriptional activity of the bHLH-MYB proteins and inhibited anthocyanin accumulation, trichome formation, and defenses against insect herbivores while promoting root hair formation. Conversely, the JA-activated bHLH members GL3, EGL3, and TT8 of WBM complexes were able to interact with and attenuate the transcriptional activity of EIN3/EIL1 at the HOOKLESS1 promoter, and their overexpression inhibited apical hook formation. Thus, this study demonstrates a molecular framework for signaling crosstalk between JA and ET in plant development, secondary metabolism, and defense responses.

The genome of the recretohalophyte Limonium bicolor provides insights into salt gland development and salinity adaptation during terrestrial evolution

Halophytes have evolved specialized strategies to cope with high salinity. The extreme halophyte sea lavender (Limonium bicolor) lacks trichomes but possesses salt glands on its epidermis that can excrete harmful ions, such as sodium, to avoid salt damage. Here, we report a high-quality, 2.92-Gb, chromosome-scale L. bicolor genome assembly based on a combination of Illumina short reads, single-molecule, real-time long reads, chromosome conformation capture (Hi-C) data, and Bionano genome maps, greatly enriching the genomic information on recretohalophytes with multicellular salt glands. Although the L. bicolor genome contains genes that show similarity to trichome fate genes from Arabidopsis thaliana, it lacks homologs of the decision fate genes GLABRA3, ENHANCER OF GLABRA3, GLABRA2, TRANSPARENT TESTA GLABRA2, and SIAMESE, providing a molecular explanation for the absence of trichomes in this species. We identified key genes (LbHLH and LbTTG1) controlling salt gland development among classical trichome homologous genes and confirmed their roles by showing that their mutations markedly disrupted salt gland initiation, salt secretion, and salt tolerance, thus offering genetic support for the long-standing hypothesis that salt glands and trichomes may share a common origin. In addition, a whole-genome duplication event occurred in the L. bicolor genome after its divergence from Tartary buckwheat and may have contributed to its adaptation to high salinity. The L. bicolor genome resource and genetic evidence reported in this study provide profound insights into plant salt tolerance mechanisms that may facilitate the engineering of salt-tolerant crops.

A DE1 BINDING FACTOR 1-GLABRA2 module regulates rhamnogalacturonan I biosynthesis in Arabidopsis seed coat mucilage.

The mucilage surrounding hydrated Arabidopsis thaliana seeds is a specialized extracellular matrix composed mainly of the pectic polysaccharide rhamnogalacturonan I (RG-I). Although, several genes responsible for RG-I biosynthesis have been identified, the transcriptional regulatory mechanisms controlling RG-I production remain largely unknown. Here we report that the trihelix transcription factor DE1 BINDING FACTOR 1 (DF1) is a key regulator of mucilage RG-I biosynthesis. RG-I biosynthesis is significantly reduced in loss-of-function mutants of DF1. DF1 physically interacts with GLABRA2 (GL2) and both proteins transcriptionally regulate the expression of the RG-I biosynthesis genes MUCILAGE MODIFIED 4 (MUM4) and GALACTURONOSYLTRANSFERASE-LIKE5 (GATL5). Through chromatin immunoprecipitation-quantitative PCR and transcriptional activation assays, we uncover a cooperative mechanism of the DF1-GL2 module in activating MUM4 and GATL5 expression, in which DF1 binds to the promoters of MUM4 and GATL5 through interacting with GL2 and facilitates the transcriptional activity of GL2. The expression of DF1 and GL2 is directly regulated by TRANSPARENT TESTA GLABRA2 (TTG2) and, in turn, DF1 directly represses the expression of TTG2. Taken together, our data reveal that the transcriptional regulation of mucilage RG-I biosynthesis involves a regulatory module, comprising DF1, GL2, and TTG2.

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.

Conserved and non-conserved functions of the rice homologs of the Arabidopsis trichome initiation-regulating MBW complex proteins

BackgroundTrichome initiation in Arabidopsis is regulated by a MYB-bHLH-WD40 (MBW) transcriptional activator complex formed by the R2R3 MYB transcription factor GLABRA1 (GL1), MYB23 or MYB82, the bHLH transcription factor GLABRA3 (GL3), ENHANCER OF GLABRA3 (EGL3) or TRANSPARENT TESTA8 (TT8), and the WD40-repeat protein TRANSPARENT TESTA GLABRA1 (TTG1). However, the functions of the rice homologs of the MBW complex proteins remained uncharacterized.ResultsBased on amino acid sequence identity and similarity, and protein interaction prediction, we identified OsGL1s, OsGL3s and OsTTG1s as rice homologs of the MBW complex proteins. By using protoplast transfection, we show that OsGL1D, OsGL1E, OsGL3B and OsTTG1A were predominantly localized in the nucleus, OsGL3B functions as a transcriptional activator and is able to interact with GL1 and TTG1. By using yeast two-hybrid and protoplast transfection assays, we show that OsGL3B is able to interact with OsGL1E and OsTTG1A, and OsGL1E and OsTTG1A are also able to interact with GL3. On the other hand, we found that OsGL1D functions as a transcription activator, and it can interact with GL3 but not OsGL3B. Furthermore, our results show that expression of OsTTG1A in the ttg1 mutant restored the phenotypes including alternations in trichome and root hair formation, seed color, mucilage production and anthocyanin biosynthesis, indicating that OsTTG1A and TTG1 may have similar functions.ConclusionThese results suggest that the rice homologs of the Arabidopsis MBW complex proteins are able to form MBW complexes, but may have conserved and non-conserved functions.

The Seed Development Factors TT2 and MYB5 Regulate Heat Stress Response in Arabidopsis.

HEAT SHOCK FACTOR A2 (HSFA2) is a regulator of multiple environmental stress responses required for stress acclimation. We analyzed HSFA2 co-regulated genes and identified 43 genes strongly co-regulated with HSFA2 during multiple stresses. Motif enrichment analysis revealed an over-representation of the site II element (SIIE) in the promoters of these genes. In a yeast 1-hybrid screen with the SIIE, we identified the closely related R2R3-MYB transcription factors TT2 and MYB5. We found overexpression of MYB5 or TT2 rendered plants heat stress tolerant. In contrast, tt2, myb5, and tt2/myb5 loss of function mutants showed heat stress hypersensitivity. Transient expression assays confirmed that MYB5 and TT2 can regulate the HSFA2 promoter together with the other members of the MBW complex, TT8 and TRANSPARENT TESTA GLABRA 1 (TTG1) and that the SIIE was involved in this regulation. Transcriptomic analysis revealed that TT2/MYB5 target promoters were enriched in SIIE. Overall, we report a new function of TT2 and MYB5 in stress resistance and a role in SIIE-mediated HSFA2 regulation.

Genomic analysis of WD40 protein family in the mango reveals a TTG1 protein enhances root growth and abiotic tolerance in Arabidopsis

WD40 domain-containing proteins constitute one of the most abundant protein families in all higher plants and play vital roles in the regulation of plant growth and developmental processes. To date, WD40 protein members have been identified in several plant species, but no report is available on the WD40 protein family in mango (Mangifera indica L.). In this study, a total of 315 WD40 protein members were identified in mango and further divided into 11 subgroups according to the phylogenetic tree. Here, we reported mango TRANSPARENT TESTA GLABRA 1 (MiTTG1) protein as a novel factor that functions in the regulation of Arabidopsis root growth and development. Bimolecular fluorescence complementation (BiFC) assay in tobacco leaves revealed that MiTTG1 protein physically interacts with MiMYB0, MiTT8 and MibHLH1, implying the formation of a new ternary regulatory complex (MYB-bHLH-WD40) in mango. Furthermore, the MiTTG1 transgenic lines were more adapted to abiotic stresses (mannitol, salt and drought stress) in terms of promoted root hairs and root lengths. Together, our findings indicated that MiTTG1 functions as a novel factor to modulate protein–protein interactions and enhance the plants abilities to adjust different abiotic stress responses.

The Arabidopsis MYB5 transcription factor interacts with CASEIN KINASE2 BETA3 subunit in a yeast two-hybrid system.

Arabidopsis thaliana MYB5 collaborates with TRANSPARENT TESTA GLABRA1 (TTG1) and basic-Helix-Loop-Helix (bHLH) transcription factors to regulate seed coat, trichome and root cell differentiation. Using a yeast two-hybrid system we show that the N-terminal region of MYB5 binds directly to the serine/threonine CASEIN KINASE2 BETA3 (CK2β3) subunit. Functions of the CASEIN KINASE2 (CK2) complex include facilitating phosphorylation of MYB transcription factors and cell cycle checkpoint regulatory proteins. Purified recombinant MYB5 protein was found to bind only weakly in vitro to the promoter of ALPHA/BETA ESTERASE/HYDROLASE4 (ABE4), a known MYB5 target gene. We propose that phosphorylation of MYB5 facilitated by the MYB5-CK2β3 interaction enhances MYB5 binding to its target gene promoters.