bHLH TF Research Articles

Transcriptomic analysis reveals the mechanism underlying the anthocyanin changes in Fragaria nilgerrensis Schlecht. and its interspecific hybrids

BackgroundFragaria nilgerrensis (FN) provides a rich source of genetic variations for strawberry germplasm innovation. The color of strawberry fruits is a key factor affecting consumer preferences. However, the genetic basis of the fruit color formation in F. nilgerrensis and its interspecific hybrids has rarely been researched.ResultsIn this study, the fruit transcriptomes and flavonoid contents of FN (white skin; control) and its interspecific hybrids BF1 and BF2 (pale red skin) were compared. A total of 31 flavonoids were identified. Notably, two pelargonidin derivatives (pelargonidin-3-O-glucoside and pelargonidin-3-O-rutinoside) were revealed as potential key pigments for the coloration of BF1 and BF2 fruits. Additionally, dihydroflavonol 4-reductase (DFR) (LOC101293459 and LOC101293749) and anthocyanidin 3-O-glucosyltransferase (BZ1) (LOC101300000), which are crucial structural genes in the anthocyanidin biosynthetic pathway, had significantly up-regulated expression levels in the two FN interspecific hybrids. Moreover, most of the genes encoding transcription factors (e.g., MYB, WRKY, TCP, bHLH, AP2, and WD40) related to anthocyanin accumulation were differentially expressed. We also identified two DFR genes (LOC101293749 and LOC101293459) that were significantly correlated with members in bHLH, MYB, WD40, AP2, and bZIP families. Two chalcone synthase (CHS) (LOC101298162 and LOC101298456) and a BZ1 gene (LOC101300000) were highly correlated with members in bHLH, WD40 and AP2 families.ConclusionsPelargonidin-3-O-glucoside and pelargonidin-3-O-rutinoside may be the key pigments contributing to the formation of pale red fruit skin. DFR and BZ1 structural genes and some bHLH, MYB, WD40, AP2, and bZIP TF family members enhance the accumulation of two pelargonidin derivatives. This study provides important insights into the regulation of anthocyanidin biosynthesis in FN and its interspecific hybrids. The presented data may be relevant for improving strawberry fruit coloration via genetic engineering.

De Novo Transcriptome Profiling for the Generation and Validation of Microsatellite Markers, Transcription Factors, and Database Development for Andrographis paniculata.

Andrographis paniculata belongs to the family Acanthaceae and is known for its medicinal properties owing to the presence of unique constituents belonging to the lactones, diterpenoids, diterpene glycosides, flavonoids, and flavonoid glycosides groups of chemicals. Andrographolide, a major therapeutic constituent of A. paniculata, is extracted primarily from the leaves of this plant and exhibits antimicrobial and anti-inflammatory activities. Using 454 GS-FLX pyrosequencing, we have generated a whole transcriptome profile of entire leaves of A. paniculata. A total of 22,402 high-quality transcripts were generated, with an average transcript length and N50 of 884 bp and 1007 bp, respectively. Functional annotation revealed that 19,264 (86%) of the total transcripts showed significant similarity with the NCBI-Nr database and were successfully annotated. Out of the 19,264 BLAST hits, 17,623 transcripts were assigned GO terms and distributed into three major functional categories: molecular function (44.62%), biological processes (29.19%), and cellular component (26.18%) based on BLAST2GO. Transcription factor analysis showed 6669 transcripts, belonging to 57 different transcription factor families. Fifteen TF genes that belong to the NAC, MYB, and bHLH TF categories were validated by RT PCR amplification. In silico analysis of gene families involved in the synthesis of biochemical compounds having medicinal values, such as cytochrome p450, protein kinases, heat shock proteins, and transporters, was completed and a total of 102 different transcripts encoding enzymes involved in the biosynthesis of terpenoids were predicted. Out of these, 33 transcripts belonged to terpenoid backbone biosynthesis. This study also identified 4254 EST-SSRs from 3661 transcripts, representing 16.34% of the total transcripts. Fifty-three novel EST-SSR markers generated from our EST dataset were used to assess the genetic diversity among eighteen A. paniculata accessions. The genetic diversity analysis revealed two distinct sub-clusters and all accessions based on the genetic similarity index were distinct from each other. A database based on EST transcripts, EST-SSR markers, and transcription factors has been developed using data generated from the present study combined with available transcriptomic resources from a public database using Meta transcriptome analysis to make genomic resources available in one place to the researchers working on this medicinal plant.

Integrated analysis of miRNAome transcriptome and degradome reveals miRNA-target modules governing floral florescence development and senescence across early- and late-flowering genotypes in tree peony.

As a candidate national flower of China, tree peony has extremely high ornamental, medicinal and oil value. However, the short florescence and rarity of early-flowering and late-flowering varieties restrict further improvement of the economic value of tree peony. Specific miRNAs and their target genes engaged in tree peony floral florescence, development and senescence remain unknown. This report presents the integrated analysis of the miRNAome, transcriptome and degradome of tree peony petals collected from blooming, initial flowering, full blooming and decay stages in early-flowering variety Paeonia ostii 'Fengdan', an early-flowering mutant line of Paeonia ostii 'Fengdan' and late-flowering variety Paeonia suffruticosa 'Lianhe'. Transcriptome analysis revealed a transcript ('psu.G.00014095') which was annotated as a xyloglucan endotransglycosylase/hydrolase precursor XTH-25 and found to be differentially expressed across flower developmental stages in Paeonia ostii 'Fengdan' and Paeonia suffruticosa 'Lianhe'. The miRNA-mRNA modules were presented significant enrichment in various pathways such as plant hormone signal transduction, indole alkaloid biosynthesis, arachidonic acid metabolism, folate biosynthesis, fatty acid elongation, and the MAPK signaling pathway. Multiple miRNA-mRNA-TF modules demonstrated the potential functions of MYB-related, bHLH, Trihelix, NAC, GRAS and HD-ZIP TF families in floral florescence, development, and senescence of tree peony. Comparative spatio-temporal expression investigation of eight floral-favored miRNA-target modules suggested that transcript 'psu.T.00024044' and microRNA mtr-miR166g-5p are involved in the floral florescence, development and senescence associated agronomic traits of tree peony. The results might accelerate the understanding of the potential regulation mechanism in regards to floral florescence, development and abscission, and supply guidance for tree peony breeding of varieties with later and longer florescence characteristics.

Homodimeric and Heterodimeric Interactions among Vertebrate Basic Helix–Loop–Helix Transcription Factors

The basic helix–loop–helix transcription factor (bHLH TF) family is involved in tissue development, cell differentiation, and disease. These factors have transcriptionally positive, negative, and inactive functions by combining dimeric interactions among family members. The best known bHLH TFs are the E-protein homodimers and heterodimers with the tissue-specific TFs or ID proteins. These cooperative and dynamic interactions result in a complex transcriptional network that helps define the cell’s fate. Here, the reported dimeric interactions of 67 vertebrate bHLH TFs with other family members are summarized in tables, including specifications of the experimental techniques that defined the dimers. The compilation of these extensive data underscores homodimers of tissue-specific bHLH TFs as a central part of the bHLH regulatory network, with relevant positive and negative transcriptional regulatory roles. Furthermore, some sequence-specific TFs can also form transcriptionally inactive heterodimers with each other. The function, classification, and developmental role for all vertebrate bHLH TFs in four major classes are detailed.

An OsKala3, R2R3 MYB TF, Is a Common Key Player for Black Rice Pericarp as Main Partner of an OsKala4, bHLH TF.

Rice (Oryza sativa) pericarp exhibits various colors due to the accumulation of anthocyanins and/or proanthocyanidins. Previous work revealed that the two basic helix-loop-helix (bHLH) transcription factors OsKala4 and OsRc are key regulators for the black and red pericarp traits, respectively, and their inactivation results in rice with white pericarp. However, their pericarp-specific R2R3 MYB partner remained unknown. Here, we characterized the role of the R2R3 MYB gene OsKala3 in rice pericarp pigmentation through genetic and molecular approaches. A rice protoplast transfection assay showed that OsKala3 is a nuclear-localized protein. Furthermore, OsKala3 physically interacted with OsKala4 in a yeast two-hybrid analysis. Co-transfection assays in rice protoplasts revealed that OsKala3 and OsKala4 mediate the activation of anthocyanin biosynthetic genes. Notably, the OsKala3 promoter region exhibited an insertion polymorphism specifically in rice cultivars with black pericarp, creating two tandem repeats while red and white varieties harbor only one. The number of repeats within the OsKala3 promoter correlated with increased transactivation by OsKala3, thus providing a rationale for the black pericarp characteristic of cultivars with two repeats. These results thus provide evidence for the molecular basis of anthocyanin biosynthesis in rice pericarp and may facilitate the introduction of this beneficial trait to other rice cultivars through marker-assisted breeding.

Secretory lipid transfer protein OsLTPL94 acts as a target of EAT1 and is required for rice pollen wall development.

The plant pollen wall protects the male gametophyte from various biotic and abiotic stresses. The formation of a unique pollen wall structure and elaborate exine pattern is a well-organized process, which needs coordination between reproductive cells and the neighboring somatic cells. However, molecular mechanisms underlying this process remain largely unknown. Here, we report a rice male-sterile mutant (l94) that exhibits defective pollen exine patterning and abnormal tapetal cell development. MutMap and knockout analyses demonstrated that the causal gene encodes a type-G non-specific lipid transfer protein (OsLTPL94). Histological and cellular analyses established that OsLTPL94 is strongly expressed in the developing microspores and tapetal cells, and its protein is secreted to the plasma membrane. The l94 mutation impeded the secretory ability of OsLTPL94 protein. Further in vivo and in vitro investigations supported the hypothesis that ETERNAL TAPETUM 1 (EAT1), a basic helix-loop-helix transcription factor (bHLH TF), activated OsLTPL94 expression through direct binding to the E-box motif of the OsLTPL94 promoter, which was supported by the positive correlation between the expression of EAT1 and OsLTPL94 in two independent eat1 mutants. Our findings suggest that the secretory OsLTPL94 plays a key role in the coordinated development of tapetum and microspores with the regulation of EAT1.

Exploring the genes involved in biosynthesis of dihydroquercetin and dihydromyricetin in Ampelopsis grossedentata

Dihydroquercetin (DHQ), an extremely low content compound (less than 3%) in plants, is an important component of dietary supplements and used as functional food for its antioxidant activity. Moreover, as downstream metabolites of DHQ, an extremely high content of dihydromyricetin (DHM) is up to 38.5% in Ampelopsis grossedentata. However, the mechanisms involved in the biosynthesis and regulation from DHQ to DHM in A. grossedentata remain unclear. In this study, a comparative transcriptome analysis of A. grossedentata containing extreme amounts of DHM was performed on the Illumina HiSeq 2000 sequencing platform. A total of 167,415,597 high-quality clean reads were obtained and assembled into 100,584 unigenes having an N50 value of 1489. Among these contigs, 57,016 (56.68%) were successfully annotated in seven public protein databases. From the differentially expressed gene (DEG) analysis, 926 DEGs were identified between the B group (low DHM: 210.31 mg/g) and D group (high DHM: 359.12 mg/g) libraries, including 446 up-regulated genes and 480 down-regulated genes (B vs. D). Flavonoids (DHQ, DHM)-related DEGs of ten structural enzyme genes, three myeloblastosis transcription factors (MYB TFs), one basic helix–loop–helix (bHLH) TF, and one WD40 domain-containing protein were obtained. The enzyme genes comprised three PALs, two CLs, two CHSs, one F3’H, one F3’5’H (directly converts DHQ to DHM), and one ANS. The expression profiles of randomly selected genes were consistent with the RNA-seq results. Our findings thus provide comprehensive gene expression resources for revealing the molecular mechanism from DHQ to DHM in A. grossedentata. Importantly, this work will spur further genetic studies about A. grossedentata and may eventually lead to genetic improvements of the DHQ content in this plant.

Tightly controlled expression of OsbHLH35 is critical for anther development in rice

Anther development is a complex process regulated by a myriad of transcription factors belonging to distinct protein families. In this study, we focus on the functional characterization of OsbHLH35, a basic Helix-Loop-Helix (bHLH) TF that regulates anther development in rice. Plants overexpressing OsbHLH35 presented small and curved anthers, leading to a reduction of 72 % on seed production. Rice transgenic plants expressing GUS reporter gene under the control of OsbHLH35 promoter (pOsbHLH35::GUS) showed that this TF specifically accumulates in anthers at the meiosis stage and in other spikelet tissues. Yeast one-hybrid screening identified three members of the Growth-Regulating Factor (GRF) family, OsGRF3, OsGRF4, and OsGRF11, as transcriptional regulators of OsbHLH35. Transactivation assay showed that OsGRF11 negatively regulates OsbHLH35 expression in Arabidopsis protoplasts. This regulation was also observed in planta through the analysis of transgenic plants overexpressing OsGRF11 (OsGRF11OE), confirming that OsGRF11 is a negative regulator of OsbHLH35 in rice. Our data suggest that OsbHLH35 plays an essential role in anther development in rice and the fine control of its expression is crucial to ensure proper seed production.

The apple bHLH transcription factor MdbHLH3 functions in determining the fruit carbohydrates and malate.

SummaryChanges in carbohydrates and organic acids largely determine the palatability of edible tissues of horticulture crops. Elucidating the potential molecular mechanisms involved in the change in carbohydrates and organic acids, and their temporal and spatial crosstalk are key steps in understanding fruit developmental processes. Here, we used apple (Malus domestica Borkh.) as research materials and found that MdbHLH3, a basic helix–loop–helix transcription factor (bHLH TF), modulates the accumulation of malate and carbohydrates. Biochemical analyses demonstrated that MdbHLH3 directly binds to the promoter of MdcyMDH that encodes an apple cytosolic NAD‐dependent malate dehydrogenase, activating its transcriptional expression, thereby promoting malate accumulation in apple fruits. Additionally, MdbHLH3 overexpression increased the photosynthetic capacity and carbohydrate levels in apple leaves and also enhanced the carbohydrate accumulation in fruits by adjusting carbohydrate allocation from sources to sinks. Overall, our findings provide new insights into the mechanism of how the bHLH TF MdbHLH3 modulates the fruit quality. It directly regulates the expression of cytosolic malate dehydrogenase MdcyMDH to coordinate carbohydrate allocation and malate accumulation in apple.

A basic/helix–loop–helix transcription factor controls leaf shape by regulating auxin signaling in apple

Climate-driven phenological change across local spatial gradients leads to leaf shape variation. At higher elevations, leaves of broadleaf species tend to become narrower, but the underlying molecular mechanism is largely unknown. In this study, a series of morphometric analyses and biochemical assays, combined with functional identification in apple, were performed. We show that the decrease in apple leaf width with increasing altitude is controlled by a basic/helix-loop-helix transcription factor (bHLH TF), MdbHLH3. The MdbHLH3-overexpressing lines have a lower transcript abundance of MdPIN1 encoding an auxin efflux carrier but a higher transcript abundance of MdGH3-2 encoding a putative auxin amido conjugate synthase, resulting in a lower free auxin concentration; feeding the transgenic leaves with exogenous auxin partially restores leaf width. MdbHLH3 transcriptionally suppresses and activates MdPIN1 and MdGH3-2, respectively, by specifically binding to their promoters. This alters auxin homeostasis and transport, consequently leading to changes in leaf shape. These findings suggest that the bHLH TF MdbHLH3 directly modulates auxin signaling in controlling leaf shape in response to local spatial gradients in apple.

Phenotypic and transcriptome analysis on red leaf and green leaf Eucommia ulmoides Oliver

Eucommia ulmoides Oliver. (E. ulmoides) is an important medicinal and economic tree species, and “Huazhong No.12” (HZ12) is the only existing red leaf genotype of E. ulmoides. In this study, phenotype/anatomic observation and photosynthetic pigment content detection were performed on “Huazhong No.11” (HZ11, green leaf E. ulmoides) and HZ12. Additionally, the transcriptome sequencing data of HZ11 and HZ12 leaves were also analyzed and compared. The results showed that the palisade tissue of HZ12 leaf was red, while the palisade tissue of HZ11 leaf was green. Chlorophyll a, b, a+b, and carotenoid contents of HZ12 leaf were significantly lower than that of HZ11 leaf. While the total flavonoids and anthocyanin contents in HZ12 leaf were significantly higher than that in HZ11 leaf. Transcriptome analysis indicated that, compared with HZ12, there were 26 MYB and 49 bHLH TF genes, and some structural genes were differentially expressed in HZ11, and among these differentially expressed genes (DEGs), 2 MYB, 1 bHLH, 1 F3′H, 1 F3′5′H, 5 DFR and 9 GT genes were involved with flavonoid biosynthesis and anthocyanin biosynthesis pathways. Taken together, the results that achieved in the present study not only provide valuable clues for the excavation on anthocyanin biosynthesis related genes and the corresponding gene regulation networks in E. ulmoides, but also provide reference for the leaf coloration mechanism research in other woody plant species.

Comparative transcriptome analysis reveals gene expression differences between two peach cultivars under saline-alkaline stress

BackgroundSaline-alkaline stress is a major abiotic stress that is harmful to plant growth worldwide. Two peach cultivars (GF677 and Maotao) display distinct phenotypes under saline-alkaline stress. The molecular mechanism explaining the differences between the two cultivars is still unclear.ResultsIn the present study, we systematically analysed the changes in GF677 and Maotao leaves upon saline-alkaline stress by using cytological and biochemical technologies as well as comparative transcriptome analysis. Transmission electron microscopy (TEM) observations showed that the structure of granum was dispersive in Maotao chloroplasts. The biochemical analysis revealed that POD activity and the contents of chlorophyll a and chlorophyll b, as well as iron, were notably decreased in Maotao. Comparative transcriptome analysis detected 881 genes with differential expression (including 294 upregulated and 587 downregulated) under the criteria of |log2 Ratio| ≥ 1 and FDR ≤0.01. Gene ontology (GO) analysis showed that all differentially expressed genes (DEGs) were grouped into 30 groups. MapMan annotation of DEGs showed that photosynthesis, antioxidation, ion metabolism, and WRKY TF were activated in GF677, while cell wall degradation, secondary metabolism, starch degradation, MYB TF, and bHLH TF were activated in Maotao. Several iron and stress-related TFs (ppa024966m, ppa010295m, ppa0271826m, ppa002645m, ppa010846m, ppa009439m, ppa008846m, and ppa007708m) were further discussed from a functional perspective based on the phylogenetic tree integration of other species homologues.ConclusionsAccording to the cytological and molecular differences between the two cultivars, we suggest that the integrity of chloroplast structure and the activation of photosynthesis as well as stress-related genes are crucial for saline-alkaline resistance in GF677. The results presented in this report provide a theoretical basis for cloning saline-alkaline tolerance genes and molecular breeding to improve saline-alkaline tolerance in peach.

Physiological and transcriptomic analyses reveal the mechanisms underlying the salt tolerance of Zoysia japonica Steud

BackgroundAreas with saline soils are sparsely populated and have fragile ecosystems, which severely restricts the sustainable development of local economies. Zoysia grasses are recognized as excellent warm-season turfgrasses worldwide, with high salt tolerance and superior growth in saline-alkali soils. However, the mechanism underlying the salt tolerance of Zoysia species remains unknown.ResultsThe phenotypic and physiological responses of two contrasting materials, Zoysia japonica Steud. Z004 (salt sensitive) and Z011 (salt tolerant) in response to salt stress were studied. The results show that Z011 was more salt tolerant than was Z004, with the former presenting greater K+/Na+ ratios in both its leaves and roots. To study the molecular mechanisms underlying salt tolerance further, we compared the transcriptomes of the two materials at different time points (0 h, 1 h, 24 h, and 72 h) and from different tissues (leaves and roots) under salt treatment. The 24-h time point and the roots might make significant contributions to the salt tolerance. Moreover, GO and KEGG analyses of different comparisons revealed that the key DEGs participating in the salt-stress response belonged to the hormone pathway, various TF families and the DUF family.ConclusionsZoysia salt treatment transcriptome shows the 24-h and roots may make significant contributions to the salt tolerance. The auxin signal transduction family, ABA signal transduction family, WRKY TF family and bHLH TF family may be the most important families in Zoysia salt-stress regulation.

A bHLH transcription factor, MYC2, imparts salt intolerance by regulating proline biosynthesis in Arabidopsis.

MYC2, a bHLH TF, acts as regulatory hub within several signaling pathways by integration of various endogenous and exogenous signals which shape plant growth and development. However, its involvement in salt stress regulation is still elusive. This study has deciphered a novel role of MYC2 in imparting salt stress intolerance by regulating delta1 -pyrroline-5-carboxylate synthase1 (P5CS1) gene and hence proline synthesis. P5CS1 is a rate-limiting enzyme in the biosynthesis of proline. Y-1-H and EMSA studies confirmed the binding of MYC2 with the 5'UTR region of P5CS1. Transcript and biochemical studies have revealed MYC2 as a negative regulator of proline biosynthesis. Proline is necessary for imparting tolerance toward abiotic stress; however, its overaccumulation is toxic for the plants. Hence, studying the regulation of proline biosynthesis is requisite to understand the mechanism of stress tolerance. We have also studied that MYC2 is regulated by mitogen-activated protein kinase (MAPK) cascade mitogen-activated protein kinase kinase 3-MPK6 and vice versa. Altogether, this study demonstrates salt stress-mediated activation of MYC2 by MAPK cascade, regulating proline biosynthesis and thus salt stress.

Transcriptome analysis and identification of genes associated with fruiting branch internode elongation in upland cotton

BackgroundAppropriate plant architecture can improve the amount of cotton boll opening and allow increased planting density, thus increasing the level of cotton mechanical harvesting and cotton yields. The internodes of cotton fruiting branches are an important part of cotton plant architecture. Thus, studying the molecular mechanism of internode elongation in cotton fruiting branches is highly important.ResultsIn this study, we selected internodes of cotton fruiting branches at three different stages from two cultivars whose internode lengths differed significantly. A total of 76,331 genes were detected by transcriptome sequencing. By KEGG pathway analysis, we found that DEGs were significantly enriched in the plant hormone signal transduction pathway. The transcriptional data and qRT-PCR results showed that members of the GH3 gene family, which are involved in auxin signal transduction, and CKX enzymes, which can reduce the level of CKs, were highly expressed in the cultivar XLZ77, which has relatively short internodes. Genes related to ethylene synthase (ACS), EIN2/3 and ERF in the ethylene signal transduction pathway and genes related to JAR1, COI1 and MYC2 in the JA signal transduction pathway were also highly expressed in XLZ77. Plant hormone determination results showed that the IAA and CK contents significantly decreased in cultivar XLZ77 compared with those in cultivar L28, while the ACC (the precursor of ethylene) and JA contents significantly increased. GO enrichment analysis revealed that the GO categories associated with promoting cell elongation, such as cell division, the cell cycle process and cell wall organization, were significantly enriched, and related genes were highly expressed in L28. However, genes related to the sphingolipid metabolic process and lignin biosynthetic process, whose expression can affect cell elongation, were highly expressed in XLZ77. In addition, 2067 TFs were differentially expressed. The WRKY, ERF and bHLH TF families were the top three largest families whose members were active in the two varieties, and the expression levels of most of the genes encoding these TFs were upregulated in XLZ77.ConclusionsAuxin and CK are positive regulators of internode elongation in cotton branches. In contrast, ethylene and JA may act as negative regulators of internode elongation in cotton branches. Furthermore, the WRKY, ERF and bHLH TFs were identified as important inhibitors of internode elongation in cotton. In XLZ77(a short-internode variety), the mass synthesis of ethylene and amino acid conjugation of auxin led to the inhibition of plant cell elongation, while an increase in JA content and degradation of CKs led to a slow rate of cell division, which eventually resulted in a phenotype that presented relatively short internodes on the fruiting branches. The results of this study not only provide gene resources for the genetic improvement of cotton plant architecture but also lay a foundation for improved understanding of the molecular mechanism of the internode elongation of cotton branches.

RrMYB5- and RrMYB10-regulated flavonoid biosynthesis plays a pivotal role in feedback loop responding to wounding and oxidation in Rosa rugosa.

SummaryFlavonoids play critical roles in plant responses to various stresses. Few studies have been reported on what the mechanism of activating flavonoid biosynthesis in plant responses to wounding and oxidation is. In this study, flavonoid metabolites and many MYB transcript factors from Rosa rugosa were verified to be induced by wounding and oxidation. RrMYB5 and RrMYB10, which belong to PA1‐ and TT2‐type MYB TFs, respectively, showed extremely high induction. Overexpression of RrMYB5 and RrMYB10 resulted in an increased accumulation of proanthocyanidins in R. rugosa and tobacco by promoting the expression of flavonoid structural genes. Transcriptomic analysis of the transgenic plants showed that most genes, involved in wounding and oxidation response and ABA signalling modulation, were up‐regulated by the overexpression of RrMYB10, which was very much similar to that observed in RrANR and RrDFR overexpression transgenics. RrMYB5 and RrMYB10 physically interacted and mutually activated each other's expressions. They solely or synergistically activated the different sets of flavonoid pathway genes in a bHLH TF EGL3‐independent manner. Eventually, the accumulation of proanthocyanidins enhanced plant tolerance to wounding and oxidative stresses. Therefore, RrMYB5 and RrMYB10 regulated flavonoid synthesis in feedback loop responding to wounding and oxidation in R. rugosa. Our study provides new insights into the regulatory mechanisms of flavonoid biosynthesis by MYB TFs and their essential physiological functions in plant responses to wounding and oxidative stresses.

The regulatory module MdPUB29‐MdbHLH3 connects ethylene biosynthesis with fruit quality in apple

The plant hormone ethylene is critical for climacteric fruit ripening, while glucose and anthocyanins determine the fruit quality of climacteric fruits such as apple. Understanding the exact molecular mechanism for this process is important for elucidating the interconnection of ethylene and fruit quality. Overexpression of apple MdbHLH3 gene, an anthocyanin-related basic helix-loop-helix transcription factor (bHLH TF) gene, promotes ethylene production, and transgenic apple plantlets and trees exhibit ethylene-related root developmental abnormalities, premature leaf senescence, and fruit ripening. Biochemical analyses demonstrate that MdbHLH3 binds to the promoters of three genes that are involved in ethylene biosynthesis, including MdACO1, MdACS1, and MdACS5A, activating their transcriptional expression, thereby promoting ethylene biosynthesis. High glucose-inhibited U-box-type E3 ubiquitin ligase MdPUB29, the ortholog of Arabidopsis AtPUB29 in apple, influences the expression of ethylene biosynthetic genes and ethylene production by direct ubiquitination of the MdbHLH3 protein. Our findings provide new insights into the ubiquitination of MdbHLH3 by glucose-inhibited ubiquitin E3 ligase MdPUB29 in the regulation of ethylene biosynthesis as well as indicate that the regulatory module MdPUB29-MdbHLH3 connects ethylene biosynthesis with fruit quality in apple.

Overexpression of SmbHLH148 induced biosynthesis of tanshinones as well as phenolic acids in Salvia miltiorrhiza hairy roots

SmbHLH148 activated the whole biosynthetic pathways of phenolic acids and tanshinones, thus upregulated the production of both the two groups of pharmaceutical ingredients in Salvia miltiorrhiza. Phenolic acids and tanshinones are the two important groups of pharmaceutical ingredients presented in Salvia miltiorrhiza Bunge. The bHLH transcription factors could regulate secondary metabolism efficiently in plants. However, there are only some MYCs have been studied on regulation of either phenolic acids or tanshinones biosynthesis. In this study, a bHLH TF named SmbHLH148, which is homologous to AtbHLH148, AtbHLH147 and CubHLH1, was isolated and functionally characterized from S. miltiorrhiza. Transcription of SmbHLH148 could be intensely induced by ABA and also be moderately induced by MeJA and GA. SmbHLH148 is present in all the six tissues and mostly expressed in fibrous root and flowers. Subcellular localization analysis found that SmbHLH148 was localized in the nucleus. Overexpression of SmbHLH148 significantly increased not only three phenolic acids components accumulation but also three tanshinones content. Content of caffeic acid, rosmarinic acid and salvianolic acid B were reached to 2.87-, 4.00- and 5.99-fold of the control in the ObHLH148-3, respectively. Content of dihydrotanshinone I, cryptotanshinone, and tanshinone I were also present highest in ObHLH148-3, reached 2.5-, 5.04- and 3.97-fold of the control, respectively. Expression analysis of pathway genes of phenolic acids and tanshinones in transgenic lines showed that most of them were obviously upregulated. Moreover, transcription of AREB and JAZs were also induced in SmbHLH148 overexpression lines. These results suggested that SmbHLH148 might be taken part in ABA and MeJA signaling and activated almost the whole biosynthetic pathways of phenolic acids and tanshinones, thus the production of phenolic acids and tanshinones were upregulated.

Characterization of the ZmbHLH122 transcription factor and its potential collaborators in maize male reproduction

OsEAT1 (DTD), a stamen-specific bHLH transcription factor, is a master regulator in rice male reproduction. To elucidate its functional mechanism in maize male reproduction, the maize orthologue ZmbHLH122 of OsEAT1 was isolated in this study. Sequence analysis revealed that the ZmbHLH122 coding sequence consists of 1422 bp which encode a protein of 473 amino acids. Bioinformatic analysis confirmed that ZmbHLH122 belongs to the bHLH TF family. The subcellular localization of ZmbHLH122-eGFP in rice protoplasts showed that ZmbHLH122 is a nuclear-localized protein. Yeast one-hybrid assays demonstrated that ZmbHLH122 exhibits weak transactivation ability. Genome-wide coexpression analysis identified 751 potential collaborators of ZmbHLH122, most of which are highly expressed in maize anthers and tassels, and sequence alignment analysis revealed that 43 coexpressed genes are homologous to Arabidopsis male fertility-related genes. Expression pattern analysis confirmed that ZmbHLH122 and its potential collaborators Ms23 (ZmbHLH16), ZmbHLH51, ZmbHLH66 (Ms32) and ZmMYB74 are preferentially expressed in the maize male reproductive organs, especially those at the uninucleate and early binucleate stages. Yeast two-hybrid assays revealed that ZmMYB74 directly interacts with two maize stamen development regulators, Ms23 (ZmbHLH16) and ZmbHLH51, and indirectly collaborates with ZmbHLH122. Our study lays the foundation for further understanding of the ZmbHLH122 molecular function in maize male reproduction.

The bHLH transcription factor CgbHLH001 is a potential interaction partner of CDPK in halophyte Chenopodium glaucum

Plants have evolved different abilities to adapt to the ever-fluctuating environments for sessility. Calcium-dependent protein kinase (CDPK) is believed to play a pivotal role in abiotic stress signaling. So far, study on the specific substrates that CDPK recognized in response to adversity is limited. In the present study, we revealed a potential interaction between CDPK and a bHLH transcription factor under salt stress in Chenopodium glaucum. First, we identified a CgCDPK, which was up-regulated under salt and drought stress; then by Y2H screening, CgCDPK was detected to be involved in interaction with a bHLH TF (named as CgbHLH001), which also positively respond to salt and drought stress. Further computational prediction and experiments including GST-pulldown and BiFC assays revealed that potential interaction existed between CgCDPK and CgbHLH001, and they might interact on the plasma membrane. In addition, CgCDPK-overexpressed transgenic tobacco line could significantly accumulate transcripts of NtbHLH (a homolog of CgbHLH001 in N. tabacum), which provided another evidence of correlation between CgCDPK and CgbHLH001. Our results suggest that CgbHLH001 can interact with CgCDPK in signal transduction pathway in response to abiotic stress, which should provide new evidence for further understanding of the substrate specificity of plant CDPK signaling pathway.