Cotton GhERF38 gene is involved in plant response to salt/drought and ABA.

ERF (ethylene-responsive factor) transcription factors play important roles in plant stress signaling transduction pathways. However, their specific roles during diverse abiotic stresses tolerance in Gossypium hirsutum are largely unknown. Here, a novel ERF transcription factor, designated GhERF38, homologous to AtERF38 in Arabidopsis, was isolated from cotton (Gossypium hirsutum L). GhERF38 expression was up-regulated by salt, drought and ABA treatments. Subcellular localization results indicated that GhERF38 was localized in the cell nucleus. Over-expression of GhERF38 in Arabidopsis reduced plant tolerance to salt and drought stress as indicated by a decline of seed germination, plant greenness frequency, primary roots length and the survival rate in transgenic plants compared to those of wild type plants under salt or drought treatment. Besides, stress tolerance related physiological parameters such as proline content, relative water content, soluble sugar and chlorophyll content were all significantly lower in transgenic plants than those of wild type plants under salt or drought treatment. Furthermore, over-expression of GhERF38 in Arabidopsis resulted in ABA sensitivity in transgenic plants during both seed germination and seedling growth. Interestingly, the stomatal aperture of guard cells in the transgenic plants was larger than that in transgenic plant after ABA treatment, suggesting that GhERF38-overexpressing plants were insensitive to ABA in terms of stomatal closure. Furthermore, expressions of the stress-related genes were altered in the GhERF38 transgenic plants under high salinity, drought or ABA treatment. Together, our results revealed that GhERF38 functions as a novel regulator that is involved in response to salt/drought stress and ABA signaling during plant development.

Isolation and functional characterization of a R2R3-MYB regulator of the anthocyanin biosynthetic pathway from Epimedium sagittatum.

KEY MESSAGE: A R2R3-MYB transcription factor EsAN2 was isolated from Epimedium sagittatum and functionally characterized to regulate the anthocyanin biosynthetic pathway. Epimedium plants are used widely both as traditional Chinese medicinal herbs and ornamental perennials. Anthocyanins, acting as major contributors to plant color diversity, their biosynthesis are regulated by a series of transcription factors, including MYB, bHLH and WD40 protein. Previously, a MYB transcription factor involved in regulation of the anthocyanin pathway from Epimedium sagittatum, EsMYBA1 has been isolated, but was found to be expressed mostly in leaves. In this research, another MYB transcription factor, designated as EsAN2, was isolated from flowers by the screening of E. sagittatum EST database. Preferential expression of EsAN2 in flowers and flower buds was found. Ectopic expression of EsAN2 in tobacco significantly enhanced the anthocyanin biosynthesis and accumulation, both in leaves and flowers. Most structural genes of the anthocyanin biosynthetic pathway were strongly upregulated, as well as two bHLH regulators (NtAn1a and NtAn1b) in old leaves of tobacco overexpressing EsAN2, compared to the control plants. While only three structural genes, chalcone synthase (CHS), chalcone isomerase (CHI) and anthocyanidin synthase (ANS), were upregulated by EsAN2 ectopic expression in tobacco flowers. Yeast two-hybrid assay showed that EsAN2 was capable of interacting with four bHLH regulators of the anthocyanin biosynthetic pathway. These results suggest that EsAN2 is involved in regulation of the anthocyanin biosynthesis in Epimedium flowers. Identification and characterization of EsAN2 provide insight into the coloration of Epimedium flowers and a potential candidate gene for metabolic engineering of flavonoids in the future.

Transcriptomic profiling revealed the role of 24-epibrassinolide in alleviating salt stress damage in tall fescue (Festuca arundinacea).

Soil salinization is a major problem all over the world. The accumulation of salt in soil reduces the root water uptake and directly affects plant growth and metabolic activities. Brassinosteroid is a plant hormone that plays an important role in regulation of plant growth and physiological process, including promotion of cell expansion and elongation, signal transduction and stress response. Exogenous 24-epibrassinolide (EBL) has been proved to alleviate various environmental stress in plants. However, the role that EBL plays in salt stress response is still unknown in tall fescue (Festuca arundinacea). In this study, the physiology and molecular mechanisms regulated by exogenous EBL of salt stress response in tall fescue was investigated. Tall fescue plants were divided into four groups, including control (CK), NaCl solution (SALT), 24-epibrassinolide (EBL), NaCl solution + 24-epibrassinolide (SE). During the growth period of tall fescue, we found that electrolyte leakage (EL) and malondialdehyde (MDA) were decreased, chlorophyll (Chl) content and antioxidant enzyme activity were increased in leaves of tall fescue in SE group compared with SALT group, indicating that EBL improved the salt tolerance in grasses. Transcriptomic profiling analysis showed that after 12 h of treatments, 10,265, 13,830 and 10,537 differential genes were expressed in EBL, SALT, and SE groups compared with control, respectively. These differentially expressed genes (DEGs) mainly focused on binding, catalytic activity, cellular process, metabolic process, cellular anatomical entity. Moreover, most of the differential genes were expressed in the plant hormone signal transduction pathway. These results helped us to better understand the mechanism of exogenous 24-epibrassinolide to improve the salt tolerance of tall fescue.

A R2R3-MYB Transcription Factor Regulates the Flavonol Biosynthetic Pathway in a Traditional Chinese Medicinal Plant, Epimedium sagittatum.

Flavonols as plant secondary metabolites with vital roles in plant development and defense against UV light, have been demonstrated to be the main bioactive components (BCs) in the genus Epimedium plants, several species of which are used as materials for Herba Epimedii, an important traditional Chinese medicine. The flavonol biosynthetic pathway genes had been already isolated from Epimedium sagittatum, but a R2R3-MYB transcription factor regulating the flavonol synthesis has not been functionally characterized so far in Epimedium plants. In this study, we isolated and characterized the R2R3-MYB transcription factor EsMYBF1 involved in regulation of the flavonol biosynthetic pathway from E. sagittatum. Sequence analysis indicated that EsMYBF1 belongs to the subgroup 7 of R2R3-MYB family which contains the flavonol-specific MYB regulators identified to date. Transient reporter assay showed that EsMYBF1 strongly activated the promoters of EsF3H (flavanone 3-hydroxylase) and EsFLS (flavonol synthase), but not the promoters of EsDFRs (dihydroflavonol 4-reductase) and EsANS (anthocyanidin synthase) in transiently transformed Nicotiana benthamiana leaves. Both yeast two-hybrid assay and transient reporter assay validated EsMYBF1 to be independent of EsTT8, or AtTT8 bHLH regulators of the flavonoid pathway as cofactors. Ectopic expression of EsMYBF1 in transgenic tobacco resulted in the increased flavonol content and the decreased anthocyanin content in flowers. Correspondingly, the structural genes involved in flavonol synthesis were upregulated in the EsMYBF1 overexpression lines, including NtCHS (chalcone synthase), NtCHI (chalcone isomerase), NtF3H and NtFLS, whereas the late biosynthetic genes of the anthocyanin pathway (NtDFR and NtANS) were remarkably downregulated, compared to the controls. These results suggest that EsMYBF1 is a flavonol-specific R2R3-MYB regulator, and involved in regulation of the biosynthesis of the flavonol-derived BCs in E. sagittatum. Thus, identification and functional characterization of EsMYBF1 provide insight into understanding the biosynthesis and regulation of the flavonol-derived BCs in Epimedium plants, and also provide an effective tool gene for genetic manipulation to improve the flavonol synthesis.

Comparative Profiling of miRNAs and Target Gene Identification in Distant-Grafting between Tomato and Lycium (Goji Berry).

Local translocation of small RNAs between cells is proved. Long distance translocation between rootstock and scion is also well documented in the homo-grafting system, but the process in distant-grafting is widely unexplored where rootstock and scion belonging to different genera. Micro RNAs are a class of small, endogenous, noncoding, gene silencing RNAs that regulate target genes of a wide range of important biological pathways in plants. In this study, tomato was grafted onto goji (Lycium chinense Mill.) to reveal the insight of miRNAs regulation and expression patterns within a distant-grafting system. Goji is an important traditional Chinese medicinal plant with enriched phytochemicals. Illumina sequencing technology has identified 68 evolutionary known miRNAs of 37 miRNA families. Moreover, 168 putative novel miRNAs were also identified. Compared with control tomato, 43 (11 known and 32 novels) and 163 (33 known and 130 novels) miRNAs were expressed significantly different in shoot and fruit of grafted tomato, respectively. The fruiting stage was identified as the most responsive in the distant-grafting approach and 123 miRNAs were found as up-regulating in the grafted fruit which is remarkably higher compare to the grafted shoot tip (28). Potential targets of differentially expressed miRNAs were found to be involved in diverse metabolic and regulatory pathways. ADP binding activities, molybdopterin synthase complex and RNA helicase activity were found as enriched terms in GO (Gene Ontology) analysis. Additionally, "metabolic pathways" was revealed as the most significant pathway in KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis. The information of the small RNA transcriptomes that are obtained from this study might be the first miRNAs elucidation for a distant-grafting system, particularly between goji and tomato. The results from this study will provide the insights into the molecular aspects of miRNA-mediated regulation in the medicinal plant goji, and in grafted tomato. Noteworthy, it would provide a basis how miRNA signals could exchange between rootstock and scion, and the relevance to diverse biological processes.

Comparative photosynthetic and metabolic analyses reveal mechanism of improved cold stress tolerance in bermudagrass by exogenous melatonin.

Melatonin (N-acetyl-5-methoxytryptamine) has been reported to participate in plant development and abiotic stress responses. The main objective of this study was to investigate the role of melatonin in the cold-sensitive (S) and the cold-tolerant (T) bermudagrass genotypes' response to cold stress. The genotypes were treated with 100 µM melatonin and exposed to 4 °C temperature for 3 days. In both genotypes, cold stress increased the endogenous melatonin levels, and more prominently in T than S. Physiological responses indicated that exogenous melatonin triggered antioxidant activities in both genotypes, while it alleviated cell damage in the T genotype response to cold stress. Melatonin treatment under cold stress increased fluorescence curve levels for both genotypes, and higher in T than S genotypes. In both genotypes, the alterations in photosynthetic fluorescence parameters after melatonin treatment highlighted the participation of melatonin in improving photosystem response to cold stress, particularly for the cold-tolerant genotype. The metabolic analyses revealed the alterations of 44 cold-responsive metabolites in the two genotypes, mainly including carbohydrates, organic acids and amino acids. After exogenous melatonin treatment under cold condition, there was high accumulation of metabolites in the cold-tolerant regimes than their cold-sensitive counterparts. Collectively, the present study revealed differential modulations of melatonin between the cold-sensitive and the cold-tolerant genotypes in response to cold stress. This was mainly by impacting antioxidant system, photosystem II, as well as metabolic homeostasis.

Identification of microRNAs and target genes in the fruit and shoot tip of Lycium chinense: a traditional Chinese medicinal plant.

Although Lycium chinense (goji berry) is an important traditional Chinese medicinal plant, little genome information is available for this plant, particularly at the small-RNA level. Recent findings indicate that the evolutionary role of miRNAs is very important for a better understanding of gene regulation in different plant species. To elucidate small RNAs and their potential target genes in fruit and shoot tissues, high-throughput RNA sequencing technology was used followed by qRT-PCR and RLM 5'-RACE experiments. A total of 60 conserved miRNAs belonging to 31 families and 30 putative novel miRNAs were identified. A total of 62 significantly differentially expressed miRNAs were identified, of which 15 (14 known and 1 novel) were shoot-specific, and 12 (7 known and 5 novel) were fruit-specific. Additionally, 28 differentially expressed miRNAs were recorded as up-regulated in fruit tissues. The predicted potential targets were involved in a wide range of metabolic and regulatory pathways. GO (Gene Ontology) enrichment analysis and the KEGG (Kyoto Encyclopedia of Genes and Genomes) database revealed that "metabolic pathways" is the most significant pathway with respect to the rich factor and gene numbers. Moreover, five miRNAs were related to fruit maturation, lycopene biosynthesis and signaling pathways, which might be important for the further study of fruit molecular biology. This study is the first, to detect known and novel miRNAs, and their potential targets, of L. chinense. The data and findings that are presented here might be a good source for the functional genomic study of medicinal plants and for understanding the links among diversified biological pathways.