G-box Element Research Articles

A viroid-derived small interfering RNA targets bHLH transcription factor MdPIF1 to regulate anthocyanin biosynthesis in Malus domestica.

Fruit colour is a critical determinant for the appearance quality and commercial value of apple fruits. Viroid-induced dapple symptom severely affects the fruit coloration, however, the underlying mechanism remains unknown. In this study, we identified an apple dimple fruit viroid (ADFVd)-derived small interfering RNA, named vsiR693, which targeted the mRNA coding for a bHLH transcription factor MdPIF1 (PHYTOCHROME-INTERACTING FACTOR 1) to regulate anthocyanin biosynthesis in apple. 5' RLM-RACE and artificial microRNA transient expression system proved that vsiR693 directly targeted the mRNA of MdPIF1 for cleavage. MdPIF1 positively regulated anthocyanin biosynthesis in both apple calli and fruits, and it directly bound to G-box element in the promoter of MdPAL and MdF3H, two anthocyanin biosynthetic genes, to promote their transcription. Expression of vsiR693 negatively regulated anthocyanin biosynthesis in both apple calli and fruits. Furthermore, co-expression of vsiR693 and MdPIF1 suppressed MdPIF1-promoted anthocyanin biosynthesis in apple fruits. Infiltration of ADFVd infectious clone suppressed coloration surrounding the injection sites in apple fruits, while a mutated version of ADFVd, in which the vsiR693 producing region was mutated, failed to repress fruit coloration around the injection sites. These data provide evidence that a viroid-derived small interfering RNA targets host transcription factor to regulate anthocyanin biosynthesis in apple.

PagbHLH35 Enhances Salt Tolerance through Improving ROS Scavenging in Transgenic Poplar.

The bHLH transcription factor family plays crucial roles in plant growth and development and their responses to adversity. In this study, a highly salt-induced bHLH gene, PagbHLH35 (Potri.018G141600), was identified from Populus alba × P. glandullosa (84K poplar). PagbHLH35 contains a highly conserved bHLH domain within the region of 52-114 amino acids. A subcellular localization result confirmed its nuclear localization. A yeast two-hybrid assay indicated PagbHLH35 lacks transcriptional activation activity, while a yeast one-hybrid assay indicated it could specifically bind to G-box and E-box elements. The expression of PagbHLH35 reached its peak at 12 h and 36 h time points under salt stress in the leaves and roots, respectively. A total of three positive transgenic poplar lines overexpressing PagbHLH35 were generated via Agrobacterium-mediated leaf disk transformation. Under NaCl stress, the transgenic poplars exhibited significantly enhanced morphological and physiological advantages such as higher POD activity, SOD activity, chlorophyll content, and proline content, and lower dehydration rate, MDA content and hydrogen peroxide (H2O2) content, compared to wild-type (WT) plants. In addition, histological staining showed that there was lower ROS accumulation in the transgenic poplars under salt stress. Moreover, the relative expression levels of several antioxidant genes in the transgenic poplars were significantly higher than those in the WT. All the results indicate that PagbHLH35 can improve salt tolerance by enhancing ROS scavenging in transgenic poplars.

Regulatory Module FtMYB5/6-FtGBF1-FtUFGT163 Promotes Rutin Biosynthesis in Tartary Buckwheat.

Tartary buckwheat is highly valued for its abundant rutin (quercetin 3-O-rutinoside). As a flavonoid glycoside, rutin is synthesized with the crucial involvement of UDP-dependent glycosyltransferases (UGTs). However, the functions and transcriptional regulation of the UGT-encoded genes remain poorly understood. This study identified a key gene, FtUFGT163, potentially encoding flavonol 3-O-glucoside (1 → 6) rhamnosyltransferase in Tartary buckwheat through omics analysis and molecular docking methods. The recombinant FtUFGT163 expressed in Escherichia coli demonstrated the capacity to glycosylate isoquercetin into rutin. Overexpression of FtUFGT163 significantly enhanced the rutin content in Tartary buckwheat. Further investigation identified a novel bZIP transcription factor, FtGBF1, that enhances FtUFGT163 expression by binding to the G-box element within its promoter, thereby augmenting rutin biosynthesis. Additional molecular biology experiments indicated that the specific positive regulator of rutin, FtMYB5/6, could directly activate the FtGBF1 promoter. Collectively, this study elucidates a novel regulatory module, termed "FtMYB5/6-FtGBF1-FtUFGT163", which effectively coordinates the biosynthesis of rutin in Tartary buckwheat, offering insights into the genetic enhancement of nutraceutical components in crops.

Quantitative RUBY reporter assay for gene regulation analysis.

Various reporter genes have been developed to study gene expression pattern and gene regulation. The RUBY reporter gene was recently developed and widely used, because of its visible and noninvasive advantages. However, quantitative analysis of RUBY gene expression levels was lacking. In this study, we introduce a novel betalain quantification method in combination with the tobacco transient expression system. The betalain produced in tobacco leaves was extracted and purified, and its concentration was quantitatively measured. We successfully applied this approach in studying the transcriptional regulation of ARC5 gene by transcription factors CPD25 and CPD45. Furthermore, with this method, we showed that the gene expression of RCA and Rbcs1A gene were regulated by light, transcription factors HY5 and PIFs through G-box and I-box elements. The development of this betalain quantification approach with the tobacco transient expression system offers a cost-effective and intuitive strategy for studying the regulatory mechanism of gene expression.

A light responsive transcription factor CsbHLH89 positively regulates anthocyanidin synthesis in tea (Camellia sinensis)

Anthocyanins are a class of secondary metabolites with rich physiological functions and many benefits for the human body. Although there have been numerous reports on anthocyanin synthesis in tea plants, the mechanism by which bHLH transcription factors regulate anthocyanin synthesis in tea plants is still unclear. In this study, we identified CsbHLH89, a basic helix–loop–helix (bHLH) transcription factor that is located in the nucleus and cytoplasm and has transcriptional activation activity. CsbHLH89 directly binds to G-box elements and activates the CsCHS, CsFLS, and CsDFR promoters in vitro and in vivo. Further experiments demonstrated that CsbHLH89 could be involved in the synthesis of tea plant anthocyanins as homodimers or heterodimers. Our research results show that CsbHLH89 can positively regulate anthocyanin synthesis in tea. In addition, it was found that CsHY5, a light signal transduction factor, could bind to the promoter of CsbHLH89 to indirectly promote anthocyanin accumulation. Silencing of CsHY5 and CsbHLH89 by antisense oligonucleotide, respectively, revealed that the expression of structural genes and transcription factors were down-regulated. Shading treatment of tea plants revealed a significant reduction in anthocyanin content and down-regulation of the expression of genes involved in anthocyanin synthesis. Our research provides new insight into the regulatory network of anthocyanin synthesis in tea and a new strategy for breeding rich anthocyanin.

PbrbZIP15 promotes sugar accumulation in pear via activating the transcription of the glucose isomerase gene PbrXylA1.

The composition and abundance of soluble sugars in mature pear (Pyrus) fruit are important for its acceptance by consumers. However, our understanding of the genes responsible for soluble sugar accumulation remains limited. In this study, a S1-group member of bZIP gene family, PbrbZIP15, was characterized from pear genome through the combined analyses of metabolite and transcriptome data followed by experimental validation. PbrbZIP15, located in nucleus, was found to function in fructose, sucrose, and total soluble sugar accumulation in pear fruit and calli. After analyzing the expression profiles of sugar-metabolism-related genes and the distribution of cis-acting elements in their promoters, the glucose isomerase 1 gene (PbrXylA1), whose corresponding protein catalyzed the isomerization of glucose and fructose in vitro, was identified as a downstream target gene of PbrbZIP15. PbrbZIP15 could directly bind to the G-box element in PbrXylA1 promoter and activate its transcription, as evidenced by chromatin immunoprecipitation-quantitative PCR, yeast one-hybrid, electrophoretic mobility shift assay, and dual-luciferase assay. PbrXylA1, featuring a leucine-rich signal peptide in its N-terminal, was localized to the endoplasmic reticulum. It was validated to play a significant role in fructose, sucrose, and total soluble sugar accumulation in pear fruit and calli, which was associated with the upregulated fructose/glucose ratio. Further studies revealed a positive correlation between the sucrose content and the expression levels of several sucrose-biosynthesis-related genes (PbrFRK3/8, PbrSPS1/3/4/8, and PbrSPP1) in PbrbZIP15-/PbrXylA1-transgenic fruit/calli. In conclusion, our results suggest that PbrbZIP15-induced soluble sugar accumulation during pear development is at least partly attributed to the activation of PbrXylA1 transcription.

Genome-wide identification of C2H2-ZFPs and functional analysis of BcZAT12 and BcZAT10 under cold stress in non-heading Chinese cabbage

Low temperature affects the quality and yield of non-heading Chinese cabbage.C2H2-type zinc finger proteins (C2H2-ZFPs) play an important role in various environmental stresses. To date, the functions of C2H2-ZFPs in non-heading Chinese cabbage are still uncharacterized. Here, 163 C2H2-ZFPs in non-heading Chinese cabbage were identified using the sequenced NHCC001 genome and classified into nine subfamilies. Then characterization of the C2H2-ZFPs was comprehensively investigated. Synteny analysis suggested that these genes these genes were highly conserved during evolution, which may be related to the triploid (WGT) event of NHCC001. Through a combination of genome-wide and transcriptome analysis, we identified two zinc finger protein factors BcZAT12 and BcZAT10 that are closely related to cold. Furthermore, it was found that both BcZAT12 and BcZAT10 were localized in the nucleus and highly expressed in leaves. VIGS and transient overexpression experiments indicated that BcZAT12 and BcZAT10 acted as transcriptional repressors to negatively regulate cold tolerance in non-heading Chinese cabbage during cold resistance. Surprisingly, we demonstrated that BcZAT12 can directly bind to the ACT core sequence of A (G/C) T-X3–4-A (G/C) T on the BcCBF1 promoter and inhibit its activity. Meanwhile, we discovered that BcNTL4 and BcNAC055 can bind to the G-BOX element on the promoter of BcZAT12 and negatively regulate the expression of BcZAT12. Interestingly, we found that BcABF2/4 can interact with BcZAT12, speculating that BcZAT12 may interact with BcABF2/4 to regulate the expression of cold-related genes. This study laid a foundation to further understand the functional role and regulatory mechanism of C2H2-ZFPs of non-heading Chinese cabbage under cold stress.

Overexpression of a pseudo-etiolated-in-light-like protein in Taraxacum koksaghyz leads to a pale green phenotype and enables transcriptome-based network analysis of photomorphogenesis and isoprenoid biosynthesis.

Plant growth and greening in response to light require the synthesis of photosynthetic pigments such as chlorophylls and carotenoids, which are derived from isoprenoid precursors. In Arabidopsis, the pseudo-etiolated-in-light phenotype is caused by the overexpression of repressor of photosynthetic genes 2 (RPGE2), which regulates chlorophyll synthesis and photosynthetic genes. We investigated a homologous protein in the Russian dandelion (Taraxacum koksaghyz) to determine its influence on the rich isoprenoid network in this species, using a combination of in silico analysis, gene overexpression, transcriptomics and metabolic profiling. Homology-based screening revealed a gene designated pseudo-etiolated-in-light-like (TkPEL-like), and in silico analysis identified a light-responsive G-box element in its promoter. TkPEL-like overexpression in dandelion plants and other systems reduced the levels of chlorophylls and carotenoids, but this was ameliorated by the mutation of one or both conserved cysteine residues. Comparative transcriptomics in dandelions overexpressing TkPEL-like showed that genes responsible for the synthesis of isoprenoid precursors and chlorophyll were downregulated, probably explaining the observed pale green leaf phenotype. In contrast, genes responsible for carotenoid synthesis were upregulated, possibly in response to feedback signaling. The evaluation of additional differentially expressed genes revealed interactions between pathways. We propose that TkPEL-like negatively regulates chlorophyll- and photosynthesis-related genes in a light-dependent manner, which appears to be conserved across species. Our data will inform future studies addressing the regulation of leaf isoprenoid biosynthesis and photomorphogenesis and could be used in future breeding strategies to optimize selected plant isoprenoid profiles and generate suitable plant-based production platforms.

Comprehensive analysis of JAZ family members in Ginkgo biloba reveals the regulatory role of the GbCOI1/GbJAZs/GbMYC2 module in ginkgolide biosynthesis.

Ginkgo biloba L., an ancient relict plant known as a 'living fossil', has a high medicinal and nutritional value in its kernels and leaves. Ginkgolides are unique diterpene lactone compounds in G. biloba, with favorable therapeutic effects on cardiovascular and cerebrovascular diseases. Thus, it is essential to study the biosynthesis and regulatory mechanism of ginkgolide, which will contribute to quality improvement and medication requirements. In this study, the regulatory roles of the JAZ gene family and GbCOI1/GbJAZs/GbMYC2 module in ginkgolide biosynthesis were explored based on genome and methyl jasmonate-induced transcriptome. Firstly, 18 JAZ proteins were identified from G. biloba, and the gene characteristics and expansion patterns along with evolutionary relationships of these GbJAZs were analyzed systematically. Expression patterns analysis indicated that most GbJAZs expressed highly in the fibrous root and were induced significantly by methyl jasmonate. Mechanistically, yeast two-hybrid assays suggested that GbJAZ3/11 interacted with both GbMYC2 and GbCOI1, and several GbJAZ proteins could form homodimers or heterodimers between the GbJAZ family. Moreover, GbMYC2 is directly bound to the G-box element in the promoter of GbLPS, to regulate the biosynthesis of ginkgolide. Collectively, these results systematically characterized the JAZ gene family in G. biloba and demonstrated that the GbCOI1/GbJAZs/GbMYC2 module could regulate ginkgolides biosynthesis, which provides a novel insight for studying the mechanism of JA regulating ginkgolide biosynthesis.

Transcriptomic and Targeted Metabolomics Analysis of Detached Lycium ruthenicum Leaves Reveals Mechanisms of Anthocyanin Biosynthesis Induction through Light Quality and Sucrose Treatments.

Light quality and sucrose-induced osmotic stress are known to cause anthocyanin synthesis in detached Lycium ruthenicum leaves. To identify the mechanisms by which the kind of light quality and sucrose concentration are induced, here, we conducted transcriptome sequencing in detached L. ruthenicum leaves treated with different qualities of light and sucrose concentrations. Leaves treated with blue light or sucrose showed a significantly increased total anthocyanins content compared to those treated with white light. Delphinidin-3-O-rutinoside and delphinidin-3-O-glucoside production were differentially regulated by the BL(-S), BL(+S), and WL(+S) treatments. The structural genes CHS, CHI, F3'H, F3'5'H, ANS, and UFGT were significantly up-regulated in leaves treated with blue light or sucrose. Leaves treated with blue light additionally showed up-regulation of the light photoreceptors CRY1, PIF3, COP1, and HY5. The anthocyanin-related genes NCED1, PYR/PYL, PP2C, SnRK2, and ABI5 were significantly up-regulated in leaves treated with sucrose, promoting adaptability to sucrose osmotic stress. Co-expression and cis-regulatory analyses suggested that HY5 and ABI5 could regulate LrMYB44 and LrMYB48 through binding to the G-box element and ABRE element, respectively, inducing anthocyanin synthesis in response to blue light or sucrose treatment. Candidate genes responsive to blue light or sucrose osmotic stress in the anthocyanin biosynthesis pathway were validated through quantitative reverse transcription PCR. These findings deepen our understanding of the mechanisms by which blue light and sucrose-induced osmotic stress regulate anthocyanin synthesis, providing valuable target genes for the future improvement in anthocyanin production in L. ruthenicum.

Genome-Wide Identification of Laccase Gene Family from Punica granatum and Functional Analysis towards Potential Involvement in Lignin Biosynthesis

Laccase (LAC) is the key enzyme responsible for lignin biosynthesis. Here, 57 PgLACs from pomegranate were identified and distributed on eight chromosomes and one unplaced scaffold. They were divided into six groups containing three typical Cu-oxidase domains. Totally, 51 cis-acting elements in the promoter region of PgLACs are involved in response to ABA, GA, light, stress, etc., indicating diverse functions of PgLACs. The expression profiles of 13 PgLACs during the seed development stage showed that most PgLACs expressed at a higher level earlier than at the later seed development stage in two pomegranate cultivars except PgLAC4. Also, PgLAC1/6/7/16 expressed at a significantly higher level in soft-seed ‘Tunisia’; on the contrary, PgLAC37 and PgLAC50 with a significantly higher expression in hard-seed ‘Taishanhong’. Combined with their distinguishing cis-acting elements, it was concluded that PgLAC1/6/7 may respond to GA via TATC-box and GARE-motif, and PgLAC16 repressed the promotor activity of embryo mid-maturation genes via RY-element so as to contribute to softer seed formation, whereas PgLAC37/50 may participate in seed formation and accelerate seed maturity via ABRE and G-box elements. Collectively, the dramatic gene expressions of PgLAC1/6/7/16/37/50 will provide valuable information to explore the formation of soft- and hard-seed in pomegranate.

The B-box transcription factor PabBBX27 in the regulation of chlorophyll biosynthesis and photosynthesis in poplar (Populus alba × P. Berolinensis)

Chlorophyll is responsible for absorbing light which is required to convert solar energy into chemical energy during photosynthesis. Despite remarkable progresses achieved in chlorophyll biosynthesis, the relevant mechanisms in poplar remain unclear. Here, we identified a BBX gene, PabBBX27, which was from Populus alba × P. Berolinensis, may regulate the biosynthesis of chlorophyll in poplar. PabBBX27 transcripts were most abundant in young leaves of poplar. Transgenic lines overexpressing (OE) PabBBX27 were characterized by dark green leaf. The contents of chlorophyll and photosynthates in the OE lines were significantly increased. In addition, photosynthetic rate, stomatal guidance, intercellular CO2 concentration, transpiration rate, initial fluorescence F0, maximum light efficiency (Fv/Fm), actual photochemical quantum yield and photochemical quenching of the OE lines were all increased. Genes relate to chlorophyll metabolic pathway and photosynthesis were activated in the OE lines as well. Yeast one-hybrid results showed that PabBBX27 may enhance their activities by binding to the G-box elements in the promoter sequences of these genes. Taken together, our results provide a new insight for the molecular mechanism of chlorophyll metabolic pathway and the cultivation of new varieties with high photosynthetic utilization efficiency for poplar.

GATA zinc finger protein p66β promotes breast cancer cell migration by acting as a co-activator of Snail

The transcriptional repressor Snail induces EMT during embryonic development and tumor metastasis. Growing evidence indicates that Snail functions as a trans-activator to induce gene expression; however, the underlying mechanism remains elusive. Here, we report that Snail cooperates with GATA zinc finger protein p66β to transactivate genes in breast cancer cells. Biologically, depletion of p66β reduces cell migration and lung metastasis in BALB/c mice. Mechanistically, Snail interacts with p66β and cooperatively induces gene transcription. Notably, a group of genes induced by Snail harbor conserved G-rich cis-elements (5′-GGGAGG-3′, designated as G-box) in their proximal promoter regions. Snail directly binds to G-box via its zinc fingers and transactivates the G-box-containing promoters. p66β enhances Snail binding affinity to G-box, whereas depletion of p66β results in a decreased binding affinity of Snail to the endogenous promoters and concomitantly reduces the transcription of Snail-induced genes. Taken together, these data demonstrated that p66β is critical for Snail-mediated cell migration by acting as a co-activator of Snail to induce genes containing G-box elements in the promoters.

Two B-box proteins, PavBBX6/9, positively regulate light-induced anthocyanin accumulation in sweet cherry.

Anthocyanin production in bicolored sweet cherry (Prunus avium cv. Rainier) fruit is induced by light exposure, leading to red coloration. The phytohormone abscisic acid (ABA) is essential for this process, but the regulatory relationships that link light and ABA with anthocyanin-associated coloration are currently unclear. In this study, we determined that light treatment of bicolored sweet cherry fruit increased anthocyanin accumulation and induced ABA production and that ABA participates in light-modulated anthocyanin accumulation in bicolored sweet cherry. Two B-box (BBX) genes, PavBBX6/9, were highly induced by light and ABA treatments, as was anthocyanin accumulation. The ectopic expression of PavBBX6 or PavBBX9 in Arabidopsis (Arabidopsis thaliana) increased anthocyanin biosynthesis and ABA accumulation. Overexpressing PavBBX6 or PavBBX9 in sweet cherry calli also enhanced light-induced anthocyanin biosynthesis and ABA accumulation. Additionally, transient overexpression of PavBBX6 or PavBBX9 in sweet cherry peel increased anthocyanin and ABA contents, whereas silencing either gene had the opposite effects. PavBBX6 and PavBBX9 directly bound to the G-box elements in the promoter of UDP glucose-flavonoid-3-O-glycosyltransferase (PavUFGT), a key gene for anthocyanin biosynthesis, and 9-cis-epoxycarotenoid dioxygenase 1 (PavNCED1), a key gene for ABA biosynthesis, and enhanced their activities. These results suggest that PavBBX6 and PavBBX9 positively regulate light-induced anthocyanin and ABA biosynthesis by promoting PavUFGT and PavNCED1 expression, respectively. Our study provides insights into the relationship between the light-induced ABA biosynthetic pathway and anthocyanin accumulation in bicolored sweet cherry fruit.

PhebZIP47, a bZIP transcription factor from moso bamboo (Phyllostachys edulis), positively regulates the drought tolerance of transgenic plants

The bZIP transcription factor (TF) family plays indispensable roles in plant response to environmental stresses. However, little is known about the functional roles of bZIP genes in moso bamboo (Phyllostachys edulis). Here, transcriptome data from public databases was used to analyse the expression patterns of 154 bZIP genes, and the results exhibited that the expression of many bZIP genes were strongly induced by drought. Combined with weighted gene co-expression network and qRT-PCR analysis, we screened a key candidate gene PhebZIP47 in response to drought and ABA. Next, the lines of transgenic PhebZIP47-overexpressing Arabidopsis and rice were generated. Transgenic plants showed enhanced resistance to drought at the seed germination and adult stage. Measurements of physiological indicators including chlorophyll content, relative water content, electrical conductivity, malondialdehyde and proline content were used to support this conclusion. Transcriptome analysis of transgenic rice showed that there were a large number of differentially expressed genes (DEGs) regulated by PhebZIP47, including genes of drought tolerance regulatory pathway and ABA signalling pathway. Meanwhile, a G-box element was significantly enriched in the promoters of the DEGs annotated as ‘response to stress’, and EMSA experiment suggested that this element could be bound by PhebZIP47. In addition, transgenic plants were less sensitive to ABA compared with wild-type plants under exogenous ABA treatment. Overall, our study provided a key candidate gene for the molecular breeding of moso bamboo and laid a foundation for the study of drought-resistance mechanism of bZIP genes.

Genome-wide identification of myeloblastosis gene family and its response to cadmium stress in Ipomoea aquatica.

The myeloblastosis (MYB) proteins perform key functions in mediating cadmium (Cd) tolerance of plants. Ipomoea aquatica has strong adaptability to Cd Stress, while the roles of the I. aquatica MYB gene family with respect to Cd stress are still unclear. Here, we identified a total of 183 MYB genes in the I. aquatica genome (laMYB), which were classified into 66 1R-type IaMYB, 112 2R-type IaMYB, four 3R-type IaMYB, and one 4R-type IaMYB based on the number of the MYB repeat in each gene. The analysis of phylogenetic tree indicated that most of IaMYB genes are associated with the diverse biological processes including defense, development and metabolism. Analysis of sequence features showed that the IaMYB genes within identical subfamily have the similar patterns of the motif distributions and gene structures. Analysis of gene duplication events revealed that the dispersed duplication (DSD) and whole-genome duplication (WGD) modes play vital roles in the expansion of the IaMYB gene family. Expression profiling manifests that approximately 20% of IaMYB genes had significant role in the roots of I. aquatica under Cd stress. Promoter profiling implied that the differentially expressed genes might be induced by environmental factors or inherent hormones and thereby execute their function in Cd response. Remarkably, the 2R-type IaMYB157 with abundant light-responsive element G-box and ABA-responsive element ABRE in its promoter region exhibited very strong response to Cd stress. Taken together, our findings provide an important candidate IaMYB gene for further deciphering the molecular regulatory mechanism in plant with respect to Cd stress.

Genome-wide analysis and identification of the PEBP genes of Brassica juncea var. Tumida

BackgroundPhosphatidylethanolamine-binding protein (PEBP) is widely present in animals, plants, and microorganisms. Plant PEBP genes are mainly involved in flowering transition and nutritional growth. These genes have been studied in several plants; however, to the best of our knowledge, no studies have explored them in Brassica juncea var. tumida. This study identified and characterized the entire PEBP gene family of Brassica juncea var. tumida.ResultsA total of 21 PEBP genes were identified from Brassica juncea var. tumida. Through phylogenetic analysis, the 21 corresponding proteins were classified into the following four clusters: TERMINAL FLOWER 1 (TFL1)-like proteins (n = 8), MOTHER OF FT AND TFL1 (MFT)-like proteins (n = 5), FLOWERING LOCUS T (FT)-like proteins (n = 6), and ybhB-like proteins (n = 2). A total of 18 genes contained four exons and had similar gene structures in each subfamily except BjMFT1, BjPYBHB1, and Arabidopsis thaliana CENTRORADIALIS homolog of Brassica juncea var. tumida (BjATC1). In the analysis of conserved motif composition, the BjPEBP genes exhibited similar characteristics, except for BjFT3, BjMFT1, BjPYBHB1, BjPYBHB2, and BjATC1. The BjPEBP promoter includes multiple cis-acting elements such as the G-box and I-box elements that respond to light, ABRE and GARE-motif elements that respond to hormones, and MBSI and CAT-box elements that are associated with plant growth and development. Analysis of RNA-Seq data revealed that the expression of a few BjPEBP genes may be associated with the development of a tumorous stem. The results of qRT–PCR showed that BjTFL1 and BjPYBHB1 were highly expressed in the flower tissue, BjFT1 and BjATC1 were mainly expressed in the root, and BjMFT4 were highly detected in the stem. The results of yeast two-hybrid screening suggested that BjFT interacts with Bj14-3-3. These results indicate that BjFT is involved in flowering regulation.ConclusionsTo the best of our knowledge, this study is the first to perform a genome-wide analysis of PEBP genes family in Brassica juncea var. tumida. The findings of this study may help improve the yield and molecular breeding of Brassica juncea var. tumida.

PpybZIP43 contributes to sucrose synthesis in pear fruits by activating PpySPS3 expression and interacts with PpySTOP1.

Sucrose is an important factor affecting sweetness and flavor in pear fruits, but the molecular mechanism of sucrose synthesis regulation is relatively unknown. Here, we characterized a transcription factor gene from pear (Pyrus pyrifolia Nakai cv. "Hosui") fruits, PpybZIP43, and found that the transient overexpression of PpybZIP43 in pear fruits significantly increased the sucrose content and the relative expression level of sucrose phosphate synthase genes (PpySPS3 and PpySPS8). Subcellular localization analysis in tobacco leaves showed that PpybZIP43 was localized in the nucleus. Yeast one-hybrid, electrophoretic mobility shift assay (EMSA), and dual-luciferase reporter assays indicated that PpybZIP43 was able to activate the expression of PpySPS3 by binding specifically to the G-box (CACGTG) element in the promoter. The protein-protein interaction assays using yeast two-hybrid, bimolecular fluorescence complementation (BiFC), firefly luciferase complementation imaging (LCI), and glutathione S-transferase (GST) pull-down demonstrated that PpybZIP43 could directly interact with PpySTOP1 to form a transcription complex. This study is helpful for understanding the molecular basis of sucrose synthesis and accumulation in pear fruits and provides candidate genes for breeding.

Jasmonate increases terpene synthase expression, leading to strawberry resistance to Botrytis cinerea infection.

Jasmonate induced FaTPS1 to produce terpene, and overexpression FaTPS1 led to fruit resistant against B. cinerea infection, FaMYC2 induced FaTPS1 by binding to its promoter that downstream of jasmonate. Jasmonic acid (JA) and its derivatives are associated with plant defence responses against pathogenic organisms. In the present study, a total of 10,631 differentially expressed genes, 239 differentially expressed proteins, and 229 differential metabolites were screened and found to be mainly involved in pathogen perception, hormone biosynthesis and signal transduction, photosynthesis, and secondary metabolism. In strawberry fruits, methyl jasmonate (MeJA) induced FaTPS1 expression and quickly increased the terpene content. Furthermore, FaTPS1 overexpression increased the emission of sesquiterpenes, especially germacrene D, and improved strawberry resistance against Botrytis cinerea infection, although the knockdown ofFaTPS1increased its susceptibility to the same pathogen. Using a yeast one-hybrid assay and transient expression analysis, we demonstrated that FaMYC2 can bind to the G-box element in the promoter region ofFaTPS1, thus inducing FaTPS1 expression. MeJA also stimulated FaMYC2 expression and regulated downstream signalling cascades. Moreover, we presented a possible model of the new signalling pathway of MeJA-mediated strawberry resistance to B. cinerea.

The methyl jasmonate-responsive transcription factor DobHLH4 promotes DoTPS10, which is involved in linalool biosynthesis in Dendrobium officinale during floral development

Linalool is an aromatic monoterpene produced in the Chinese medicinal plant Dendrobium officinale, but little information is available on the regulation of linalool biosynthesis. Here, a novel basic helix-loop-helix (bHLH) transcription factor, DobHLH4 from D. officinale, was identified and functionally characterized. The expression profile of DobHLH4 was positively correlated with that of DoTPS10 (R2 = 0.985, p < 0.01), which encodes linalool synthase that is responsible for linalool production, during floral development. DobHLH4 was highly expressed in petals, and was significantly induced by methyl jasmonate. Analysis of subcellular localization showed that DobHLH4 was located in the nucleus. Yeast one-hybrid and dual-luciferase assays indicated that DobHLH4 bound directly to the DoTPS10 promoter harboring the G-box element, and up-regulated DoTPS10 expression. A yeast two-hybrid screen confirmed that DobHLH4 physically interacted with DoJAZ1, suggesting that DobHLH4 might function in the jasmonic acid-mediated accumulation of linalool. Furthermore, transient overexpression of DobHLH4 in D. officinale petals significantly increased linalool production by triggering linalool biosynthetic pathway genes, especially DoTPS10. We suggest a hypothetical model that depicts how jasmonic acid signaling may regulate DoTPS10 by interacting with DobHLH4 and DoJAZ1. In doing so, the formation of linalool is controlled. Our results indicate that DobHLH4 is a positive regulator of linalool biosynthesis and may be a promising target for in vitro-based metabolic engineering to produce linalool.