Genome-wide identification of PYL/PYR-PP2C (A)-SnRK2 genes in Eutrema and their co-expression analysis in response to ABA and abiotic stresses.

ABA signaling core components PYR/PYL, group A PP2C and SnRK2 play important roles in various environmental stress responses of plants. This study identified 14 PYR/PYL, 9 PP2C (A), and 10 SnRK2 genes from halophytic Eutrema. Phylogenetic analysis showed 4 EsPYR/PYL, 4 EsPP2C (A) and 3 EsSnRK2 subfamilies characterized, which was supported by their gene structures and protein motifs. Large-scale segmental duplication event was demonstrated to be a major contributor to expansion of the EsPYL-PP2C (A)-SnRK2 gene families. Synteny relationship analysis revealed more orthologous PYL-PP2C (A)-SnRK2 gene pairs located in collinear blocks between Eutrema and Brassica than that between Eutrema and Arabidopsis. RNA-seq and qRT-PCR revealed EsABI1, EsABI2 and EsHAL2 showed a significantly up-regulated expression in leaves and roots in response to ABA, NaCl or cold stress. Three markedly co-expression modules of ABA/R-brown, NaCl/L-lightsteelblue1 and Cold/R-lightgreen were uncovered to contain EsPYL-PP2C (A)-SnRK2 genes by WGCNA analysis. GO and KEGG analysis indicated that the genes of ABA/R-brown module containing EsHAB1, EsHAI2 and EsSnRK2.6 were enriched in proteasome pathway. Further, EsHAI2-OE transgenic Arabidopsis lines showed significantly enhanced seeds germination and seedlings growth. This work provides a new insight for elucidating potential molecular functions of PYL-PP2C (A)-SnRK2 responding to ABA and abiotic stresses.

Histone H2A monoubiquitination marks are targeted to specific sites by cohesin subunits in Arabidopsis.

Histone H2A monoubiquitination (H2Aub1) functions as a conserved posttranslational modification in eukaryotes to maintain gene expression and guarantee cellular identity. Arabidopsis H2Aub1 is catalyzed by the core components AtRING1s and AtBMI1s of polycomb repressive complex 1 (PRC1). Because PRC1 components lack known DNA binding domains, it is unclear how H2Aub1 is established at specific genomic locations. Here, we show that the Arabidopsis cohesin subunits AtSYN4 and AtSCC3 interact with each other, and AtSCC3 binds to AtBMI1s. H2Aub1 levels are reduced in atsyn4 mutant or AtSCC3 artificial microRNA knockdown plants. ChIP-seq assays indicate that most binding events of AtSYN4 and AtSCC3 are associated with H2Aub1 along the genome where transcription is activated independently of H3K27me3. Finally, we show that AtSYN4 binds directly to the G-box motif and directs H2Aub1 to these sites. Our study thus reveals a mechanism for cohesin-mediated recruitment of AtBMI1s to specific genomic loci to mediate H2Aub1.

Limited conservation in cross-species comparison of GLK transcription factor binding suggested wide-spread cistrome divergence.

Non-coding cis-regulatory variants in animal genomes are an important driving force in the evolution of transcription regulation and phenotype diversity. However, cistrome dynamics in plants remain largely underexplored. Here, we compare the binding of GOLDEN2-LIKE (GLK) transcription factors in tomato, tobacco, Arabidopsis, maize and rice. Although the function of GLKs is conserved, most of their binding sites are species-specific. Conserved binding sites are often found near photosynthetic genes dependent on GLK for expression, but sites near non-differentially expressed genes in the glk mutant are nevertheless under purifying selection. The binding sites' regulatory potential can be predicted by machine learning model using quantitative genome features and TF co-binding information. Our study show that genome cis-variation caused wide-spread TF binding divergence, and most of the TF binding sites are genetically redundant. This poses a major challenge for interpreting the effect of individual sites and highlights the importance of quantitatively measuring TF occupancy.

Adaptative Mechanisms of Halophytic Eutrema salsugineum Encountering Saline Environment.

Salt cress (Eutrema salsugineum), an Arabidopsis-related halophyte, can naturally adapt to various harsh climates and soil conditions; thus, it is considered a desirable model plant for deciphering mechanisms of salt and other abiotic stresses. Accumulating evidence has revealed that compared with Arabidopsis, salt cress possesses stomata that close more tightly and more succulent leaves during extreme salt stress, a noticeably higher level of proline, inositols, sugars, and organic acids, as well as stress-associated transcripts in unstressed plants, and they are induced rapidly under stress. In this review, we systematically summarize the research on the morphology, physiology, genome, gene expression and regulation, and protein and metabolite profile of salt cress under salt stress. We emphasize the latest advances in research on the genome adaptive evolution encountering saline environments, and epigenetic regulation, and discuss the mechanisms underlying salt tolerance in salt cress. Finally, we discuss the existing questions and opportunities for future research in halophytic Eutrema. Together, the review fosters a better understanding of the mechanism of plant salt tolerance and provides a reference for the research and utilization of Eutrema as a model extremophile in the future. Furthermore, the prospects for salt cress applied to explore the mechanism of salt tolerance provide a theoretical basis to develop new strategies for agricultural biotechnology.

Eutrema EsMYB90 Gene Improves Growth and Antioxidant Capacity of Transgenic Wheat Under Salinity Stress.

The ectopic expression of the EsMYB90 transcription factor gene from halophytic Eutrema salsugineum has been reported to enhance the level of anthocyanin and other flavonoid metabolites in transgenic tobacco. In this study, the wheat JW1 overexpressing EsMYB90 showed longer roots and higher fresh weight than that in wild type (WT) under salt stress. In addition, the transgenic wheat plants displayed significantly higher peroxidase (POD) and glutathione S-transferase (GST) activity, as well as markedly lower malondialdehyde (MDA) content than that of the WT during salt stress conditions. The analysis of histochemical staining and H2O2 level indicated that the accumulation of reactive oxygen species (ROS) was significantly lower in the roots of transgenic wheat plants compared to the WT under salt stress. Transcriptome analysis revealed that the EsMYB90 gene affected the expression of considerable amounts of stress-related genes that were involved in phenylpropanoid biosynthesis and antioxidant activity in transgenic plants subjected to NaCl treatment. Importantly, the significantly upregulated expression genes in transgenic wheat under salt stress were mainly associated with the antioxidative enzymes POD and GST encoding genes compared with the WT. Furthermore, EsMYB90 is suggested to bind with the MYB-binding elements of pTaANS2 and pTaDFR1 by dual luciferase assay, to activate the transcription of TaANS2 and TaDFR1 genes that are encoding key enzymes of anthocyanin biosynthesis in transgenic wheat plants. All the results indicated that, under salt stress, the EsMYB90 gene plays a crucial role in preventing wheat seedlings from oxidative stress damage via enhancing the accumulation of non-enzymatic flavonoids and activities of antioxidative enzymes, which suggested that EsMYB90 is an ideal candidate gene for the genetic engineering of crops.

Transcriptome Profiling of the Salt Stress Response in the Leaves and Roots of Halophytic Eutrema salsugineum.

Eutrema salsugineum can grow in natural harsh environments; however, the underlying mechanisms for salt tolerance of Eutrema need to be further understood. Herein, the transcriptome profiling of Eutrema leaves and roots exposed to 300 mM NaCl is investigated, and the result emphasized the role of genes involved in lignin biosynthesis, autophagy, peroxisome, and sugar metabolism upon salt stress. Furthermore, the expression of the lignin biosynthesis and autophagy-related genes, as well as 16 random selected genes, was validated by qRT-PCR. Notably, the transcript abundance of a large number of lignin biosynthesis genes such as CCoAOMT, C4H, CCR, CAD, POD, and C3'H in leaves was markedly elevated by salt shock. And the examined lignin content in leaves and roots demonstrated salt stress led to lignin accumulation, which indicated the enhanced lignin level could be an important mechanism for Eutrema responding to salt stress. Additionally, the differentially expressed genes (DEGs) assigned in the autophagy pathway including Vac8, Atg8, and Atg4, as well as DEGs enriched in the peroxisome pathway such as EsPEX7, EsCAT, and EsSOD2, were markedly induced in leaves and/or roots. In sugar metabolism pathways, the transcript levels of most DEGs associated with the synthesis of sucrose, trehalose, raffinose, and xylose were significantly enhanced. Furthermore, the expression of various stress-related transcription factor genes including WRKY, AP2/ERF-ERF, NAC, bZIP, MYB, C2H2, and HSF was strikingly improved. Collectively, the increased expression of biosynthesis genes of lignin and soluble sugars, as well as the genes in the autophagy and peroxisome pathways, suggested that Eutrema encountering salt shock possibly possess a higher capacity to adjust osmotically and facilitate water transport and scavenge reactive oxidative species and oxidative proteins to cope with the salt environment. Thus, this study provides a new insight for exploring the salt tolerance mechanism of halophytic Eutrema and discovering new gene targets for the genetic improvement of crops.

Integrated Metabolomic and Transcriptomic Analysis Reveals the Flavonoid Regulatory Network by Eutrema EsMYB90.

Flavonoids are representative secondary metabolites with different metabolic functions in plants. Previous study found that ectopic expression of EsMYB90 from Eutremasalsugineum could strongly increase anthocyanin content in transgenic tobacco via regulating the expression of anthocyanin biosynthesis genes. In the present research, metabolome analysis showed that there existed 130 significantly differential metabolites, of which 23 metabolites enhanced more than 1000 times in EsMYB90 transgenic tobacco leaves relative to the control, and the top 10 of the increased metabolites included caffeic acid, cyanidin O-syringic acid, myricetin and naringin. A total of 50 markedly differential flavonoids including flavones (14), flavonols (13), flavone C-glycosides (9), flavanones (7), catechin derivatives (5), anthocyanins (1) and isoflavone (1) were identified, of which 46 metabolites were at a significantly enhanced level. Integrated analysis of metabolome and transcriptome revealed that ectopic expression of EsMYB90 in transgenic tobacco leaves is highly associated with the prominent up-regulation of 16 flavonoid metabolites and the corresponding 42 flavonoid biosynthesis structure genes in phenylpropanoid/flavonoid pathways. Dual luciferase assay documented that EsMYB90 strongly activated the transcription of NtANS and NtDFR genes via improving their promoter activity in transiently expressed tobacco leaves, suggesting that EsMYB90 functions as a key regulator on anthocyanin and flavonoid biosynthesis. Taken together, the crucial regulatory role of EsMYB90 on enhancing many flavonoid metabolite levels is clearly demonstrated via modulating flavonoid biosynthesis gene expression in the leaves of transgenic tobacco, which extends our understanding of the regulating mechanism of MYB transcription factor in the phenylpropanoid/flavonoid pathways and provides a new clue and tool for further investigation and genetic engineering of flavonoid metabolism in plants.

Identification of the Eutrema salsugineum EsMYB90 gene important for anthocyanin biosynthesis.

BACKGROUND: Anthocyanins contribute to coloration and antioxidation effects in different plant tissues. MYB transcription factors have been demonstrated to be a key regulator for anthocyanin synthesis in many plants. However, little information was available about the MYB genes in the halophyte species Eutrema salsugineum. RESULT: Here we report the identification of an important anthocyanin biosynthesis regulator EsMYB90 from Eutrema salsugineum, which is a halophyte tolerant to multiple abiotic stresses. Our phylogenetic and localization analyses supported that EsMYB90 is an R2R3 type of MYB transcription factor. Ectopic expression of EsMYB90 in tobacco and Arabidopsis enhanced pigmentation and anthocyanin accumulation in various organs. The transcriptome analysis revealed that 42 genes upregulated by EsMYB90 in 35S:EsMYB90 tobacco transgenic plants are required for anthocyanin biosynthesis. Moreover, our qRT-PCR results showed that EsMYB90 promoted expression of early (PAL, CHS, and CHI) and late (DFR, ANS, and UFGT) anthocyanin biosynthesis genes in stems, leaves, and flowers of 35S:EsMYB90 tobacco transgenic plants. CONCLUSIONS: Our results indicated that EsMYB90 is a MYB transcription factor, which regulates anthocyanin biosynthesis genes to control anthocyanin biosynthesis. Our work provides a new tool to enhance anthocyanin production in various plants.