Background: MicroRNAs are key regulators of plant growth and development with important roles in environmental adaptation. The microRNAs from the halophyte species Thellungiella salsuginea (salt cress), which exhibits extreme salt stress tolerance, remain to be investigated. The sequenced genome of T. salsuginea and the availability of high-throughput sequencing technology enabled us to discover the conserved and novel miRNAs in this plant species. It is interesting to identify the microRNAs from T. salsuginea genome wide and study their roles in salt stress response. Results: In this study, two T. salsuginea small RNA libraries were constructed and sequenced using Solexa technology. We identified 109 miRNAs that had previously been reported in other plant species. A total of 137 novel miRNA candidates were identified, among which the miR* sequence of 26 miRNAs was detected. In addition, 143 and 425 target mRNAs were predicted for the previously identified and Thellungiella-specific miRNAs, respectively. A quarter of these putative targets encode transcription factors. Furthermore, numerous signaling factor encoding genes, defense-related genes, and transporter encoding genes were amongst the identified targets, some of which were shown to be important for salt tolerance. Cleavage sites of seven target genes were validated by 5’ RACE, and some of the miRNAs were confirmed by qRT-PCR analysis. The expression levels of 26 known miRNAs in the roots and leaves of plants subjected to NaCl treatment were determined by Affymetrix microarray analysis. The expression of most tested miRNA families was up- or down-regulated upon NaCl treatment. Differential response patterns between the leaves and roots were observed for these miRNAs. Conclusions: Our results indicated that diverse set of miRNAs of T. salsuginea were responsive to salt stress and could play an important role in the salt stress response.

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.

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.

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.

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.