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.

Integrated metabolome and transcriptome analysis of the anthocyanin biosynthetic pathway in relation to color mutation in miniature roses

Background Roses are famous ornamental plants worldwide. Floral coloration is one of the most prominent traits in roses and is mainly regulated through the anthocyanin biosynthetic pathway. In this study, we investigated the key genes and metabolites of the anthocyanin biosynthetic pathway involved in color mutation in miniature roses. A comparative metabolome and transcriptome analysis was carried out on the Neptune King rose and its color mutant, Queen rose, at the blooming stage. Neptune King rose has light pink colored petals while Queen rose has deep pink colored petals. Result A total of 190 flavonoid-related metabolites and 38,551 unique genes were identified. The contents of 45 flavonoid-related metabolites, and the expression of 15 genes participating in the flavonoid pathway, varied significantly between the two cultivars. Seven anthocyanins (cyanidin 3-O-glucosyl-malonylglucoside, cyanidin O-syringic acid, cyanidin 3-O-rutinoside, cyanidin 3-O-galactoside, cyanidin 3-O-glucoside, peonidin 3-O-glucoside chloride, and pelargonidin 3-O-glucoside) were found to be the major metabolites, with higher abundance in the Queen rose. Thirteen anthocyanin biosynthetic related genes showed an upregulation trend in the mutant flower, which may favor the higher levels of anthocyanins in the mutant. Besides, eight TRANSPARENT TESTA 12 genes were found upregulated in Queen rose, probably contributing to a high vacuolar sequestration of anthocyanins. Thirty transcription factors, including two MYB and one bHLH, were differentially expressed between the two cultivars. Conclusions This study provides important insights into major genes and metabolites of the anthocyanin biosynthetic pathway modulating flower coloration in miniature rose. The results will be conducive for manipulating the anthocyanin pathways in order to engineer novel miniature rose cultivars with specific colors.

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.

A distant pathway-cross regulation designed with transcription factors and JAZ repressors downregulates the biosynthesis of tobacco alkaloids and specific nitrosamines

Decrease of nicotine, other tobacco alkaloids (OTAs), and carcinogenic tobacco specific nitrosamines (TSNAs) is important for health. To reduce these compounds, we selected MRE and G-box elements of promoters of four Nicotiana tabacum Jasmonate ZIM-domain (NtJAZ) repressor genes as targets to screen transcription factors (TFs) and then identified Arabidopsis Production of Anthocyanin Pigment 1 (PAP1) and Transparent Testa 8 (TT8) for a novel regulation design. Electrophoretic mobility shift, cross-linked chromatin immunoprecipitation, and dual luciferase analyses demonstrated that PAP1, TT8, PAP1/TT8, and the PAP1/TT8/Transparent Testa Glabra 1 (TTG1) complex bound to and activated NtJAZ promoters. Stacked PAP1 (P) and TT8 (T), which were introduced to commercial tobacco cultivars, Narrow Leaf Madole (NL) and KY171 (KY), upregulated four NtJAZs, downregulated seven/eight nicotine biosynthetic genes, and reduced nicotine and nornicotine in transgenic red P+T-NL and P+L-KY plants. Field trials of red P+T-NL, P+L-KY, and PAP1 tobacco genotypes and metabolic quantification showed significant reduction of nicotine and four OTAs in most or all cured leaves. Three TSNAs and the fourth one were diminished by 63-92% and 30-74% in contents in all leaves of P+T-NL and P+L-KY genotypes, respectively. The total content of each TSNA was significantly reduced by 25-60% in leaves of PAP1 plants. Taken together, this regulation designed crossing from Arabidopsis anthocyanin to tobacco alkaloid pathway is defined as a distant pathway-cross regulation (DPCR). The proof of concept of DPCR unearths new functions of two known TFs, new activators of NtJAZs, and a negative regulation pathway of nicotine biosynthesis in red tobacco.

TOP1α, UPF1, and TTG2 regulate seed size in a parental dosage–dependent manner

Cues of maternal and paternal origins interact to control seed development, and the underlying molecular mechanisms are still far from clear. Here, we show that TOPOISOMERASE Iα (TOP1α), UP-FRAMESHIFT SUPPRESSOR 1 (UPF1), and TRANSPARENT TESTA GLABRA2 (TTG2) gametophytically, biparentally regulate seed size in Arabidopsis. TOP1α and UPF1 are mainly expressed in antipodal cells, and loss of their function leads to ectopic TTG2 expression in these female gametophytic cells. We further demonstrate that TOP1α and UPF1 directly repress TTG2 expression through affecting its chromatin status and determine its relative expression in antipodal cells versus sperm cells, which controls seed size in a dosage-dependent and parent-of-origin-dependent manner. The molecular interplay among these three genes explains their biparental gametophytic effect during diploidy and interploidy reciprocal crosses. Taken together, our findings reveal a molecular framework of parental interaction for seed size control.

TRANSPARENT TESTA GLABRA1, a Key Regulator in Plants with Multiple Roles and Multiple Function Mechanisms

TRANSPARENT TESTA GLABRA1 (TTG1) is a WD40 repeat protein. The phenotypes caused by loss-of-function of TTG1 were observed about half a century ago, but the TTG1 gene was identified only about twenty years ago. Since then, TTG1 has been found to be a plant-specific regulator with multiple roles and multiple functional mechanisms. TTG1 is involved in the regulation of cell fate determination, secondary metabolisms, accumulation of seed storage reserves, plant responses to biotic and abiotic stresses, and flowering time in plants. In some processes, TTG1 may directly or indirectly regulate the expression of downstream target genes via forming transcription activator complexes with R2R3 MYB and bHLH transcription factors. Whereas in other processes, TTG1 may function alone or interact with other proteins to regulate downstream target genes. On the other hand, the studies on the regulation of TTG1 are very limited. So far, only the B3-domain family transcription factor FUSCA3 (FUS3) has been found to regulate the expression of TTG1, phosphorylation of TTG1 affects its interaction with bHLH transcription factor TT2, and TTG1 proteins can be targeted for degradation by the 26S proteasome. Here, we provide an overview of TTG1, including the identification of TTG1, the functions of TTG1, the possible function mechanisms of TTG1, and the regulation of TTG1. We also proposed potential research directions that may shed new light on the regulation and functional mechanisms of TTG1 in plants.

MYB–bHLH–TTG1 Regulates Arabidopsis Seed Coat Biosynthesis Pathways Directly and Indirectly via Multiple Tiers of Transcription Factors

MYB–bHLH–WDR (MBW) transcription factor (TF) complexes regulate Arabidopsis seed coat development including mucilage and tannin biosynthesis. The R2R3 MYBs MYB5, MYB23 and TRANSPARENT TESTA2 (TT2) participate in the MBW complexes with the WD-repeat protein TRANSPARENT TESTA GLABRA1 (TTG1). These complexes regulate GLABRA2 (GL2) and TTG2 expression in developing seeds. Microarray transcriptome analysis of ttg1-1- and wild-type (Ler) developing seeds identified 246 TTG1-regulated genes, which include all known metabolic genes of the tannin biosynthetic pathway. The first detailed TTG1-dependent metabolic pathways could be proposed for the biosynthesis of mucilage, jasmonic acid (JA) and cuticle including wax ester in developing seeds. We also assigned many known and previously uncharacterized genes to the activation/inactivation of hormones, plant immunity and nutrient transport. The promoters of six cuticle pathway genes were active in developing seeds. Expression of 11 genes was determined in the developing seeds of the combinatorial mutants of MYB5, MYB23 and TT2, and in the combinatorial mutants of GL2, HOMEODOMAIN GLABROUS2 (HDG2) and TTG2. These six TFs positively co-regulated the expression of four repressor genes while three of the six TFs repressed the wax biosynthesis genes examined, suggesting that the three TFs upregulate the expression of these repressor genes, which, in turn, repress the wax biosynthesis genes. Chromatin immunoprecipitation analysis identified 21 genes directly regulated by MYB5 including GL2, HDG2, TTG2, four repressor genes and various metabolic genes. We propose a multi-tiered regulatory mechanism by which MBWs regulate tannin, mucilage, JA and cuticle biosynthetic pathways.

TRANSPARENT TESTA GLABRA 1 participates in flowering time regulation in Arabidopsis thaliana

Pleiotropic regulatory factors mediate concerted responses of the plant’s trait network to endogenous and exogenous cues. TRANSPARENT TESTA GLABRA 1 (TTG1) is such a factor that has been predominantly described as a regulator of early developmental traits. Although its closest homologs LIGHT-REGULATED WD1 (LWD1) and LWD2 affect photoperiodic flowering, a role of TTG1 in flowering time regulation has not been reported. Here we reveal that TTG1 is a regulator of flowering time in Arabidopsis thaliana and changes transcript levels of different targets within the flowering time regulatory pathway. TTG1 mutants flower early and TTG1 overexpression lines flower late at long-day conditions. Consistently, TTG1 can suppress the transcript levels of the floral integrators FLOWERING LOCUS T and SUPPRESSOR OF OVEREXPRESSION OF CO1 and can act as an activator of circadian clock components. Moreover, TTG1 might form feedback loops at the protein level. The TTG1 protein interacts with PSEUDO RESPONSE REGULATOR (PRR)s and basic HELIX-LOOP-HELIX 92 (bHLH92) in yeast. In planta, the respective pairs exhibit interesting patterns of localization including a recruitment of TTG1 by PRR5 to subnuclear foci. This mechanism proposes additional layers of regulation by TTG1 and might aid to specify the function of bHLH92. Within another branch of the pathway, TTG1 can elevate FLOWERING LOCUS C (FLC) transcript levels. FLC mediates signals from the vernalization, ambient temperature and autonomous pathway and the circadian clock is pivotal for the plant to synchronize with diurnal cycles of environmental stimuli like light and temperature. Our results suggest an unexpected positioning of TTG1 upstream of FLC and upstream of the circadian clock. In this light, this points to an adaptive value of the role of TTG1 in respect to flowering time regulation.