Basic helix-loop-helix proteins (bHLHs) comprise one of the largest families of transcription factors in plants. They have been shown to be involved in responses to various abiotic stresses, such as drought, salinity, chilling, heavy metal toxicity, iron deficiency, and osmotic damages. By specifically binding to cis-elements in the promoter region of stress related genes, bHLHs can regulate their transcriptional expression, thereby regulating the plant’s adaptive responses. This review focuses on the structural characteristics of bHLHs, the regulatory mechanism of how bHLHs are involved transcriptional activation, and the mechanism of how bHLHs regulate the transcription of target genes under various stresses. Finally, as increasing research demonstrates that flavonoids are usually induced under fluctuating environments, the latest research progress and future research prospects are described on the mechanisms of how flavonoid biosynthesis is regulated by bHLHs in the regulation of the plant’s responses to abiotic stresses.

Ethylene Responsive Factor (ERF) subfamily comprise the largest number of proteins in the plant AP2/ERF superfamily, and have been most extensively studied on the biological functions. Members of this subfamily have been proven to regulate plant resistances to various abiotic stresses, such as drought, salinity, chilling and some other adversities. Under these stresses, ERFs are usually activated by mitogen-activated protein kinase induced phosphorylation or escape from ubiquitin-ligase enzymes, and then form complex with nucleic proteins before binding to cis-element in promoter regions of stress responsive genes. In this review, we will discuss the phylogenetic relationships among the ERF subfamily proteins, summarize molecular mechanism how the transcriptional activity of ERFs been regulated and how ERFs of different subgroup regulate the transcription of stress responsive genes, such as high-affinity K⁺ transporter gene PalHKT1;2, reactive oxygen species related genes LcLTP, LcPrx, and LcRP, flavonoids synthesis related genes FtF3H and LhMYBSPLATTER, etc. Though increasing researches demonstrate that ERFs are involved in various abiotic stresses, very few interact proteins and target genes of them have been comprehensively annotated. Hence, future research prospects are described on the mechanisms of how stress signals been transited to ERFs and how ERFs regulate the transcriptional expression of stress responsive genes.

Salinity causes osmotic stress to crops and limits their productivity. To understand the mechanism underlying soybean salt tolerance, proteomics approach was used to identify phosphoproteins altered by NaCl treatment. Results revealed that 412 of the 4698 quantitatively analyzed phosphopeptides were significantly up-regulated on salt treatment, including a phosphopeptide covering the serine 59 in the transcription factor GmMYB173. Our data showed that GmMYB173 is one of the three MYB proteins differentially phosphorylated on salt treatment, and a substrate of the casein kinase-II. MYB recognition sites exist in the promoter of flavonoid synthase gene GmCHS5 and one was found to mediate its recognition by GmMYB173, an event facilitated by phosphorylation. Because GmCHS5 catalyzes the synthesis of chalcone, flavonoids derived from chalcone were monitored using metabolomics approach. Results revealed that 24 flavonoids of 6745 metabolites were significantly up-regulated after salt treatment. We further compared the salt tolerance and flavonoid accumulation in soybean transgenic roots expressing the ³⁵S promoter driven cds and RNAi constructs of GmMYB173 and GmCHS5, as well as phospho-mimic (GmMYB173_S59D) and phospho-ablative (GmMYB173_S59A) mutants of GmMYB173. Overexpression of GmMYB173_S59D and GmCHS5 resulted in the highest increase in salt tolerance and accumulation of cyaniding-3-arabinoside chloride, a dihydroxy B-ring flavonoid. The dihydroxy B-ring flavonoids are more effective as anti-oxidative agents when compared with monohydroxy B-ring flavonoids, such as formononetin. Hence the salt-triggered phosphorylation of GmMYB173, subsequent increase in its affinity to GmCHS5 promoter and the elevated transcription of GmCHS5 likely contribute to soybean salt tolerance by enhancing the accumulation of dihydroxy B-ring flavonoids.

Increasing biotic and abiotic stresses are seriously impeding the growth and yield of staple crops and threatening global food security. As one of the largest classes of regulators in vascular plants, WRKY transcription factors play critical roles governing flavonoid biosynthesis during stress responses. By binding major W-box cis-elements (TGACCA/T) in target promoters, WRKYs modulate diverse signaling pathways. In this review, we optimized existing WRKY phylogenetic trees by incorporating additional plant species with WRKY proteins implicated in stress tolerance and flavonoid regulation. Based on the improved frameworks and documented results, we aim to deduce unifying themes of distinct WRKY subfamilies governing specific stress responses and flavonoid metabolism. These analyses will generate experimentally testable hypotheses regarding the putative functions of uncharacterized WRKY homologs in tuning flavonoid accumulation to enhance stress resilience.

The bZIP family is one of the most widely distributed and conserved transcription factor categories in eukaryotes. They are widely involved in processes of plant abiotic stress responses, such as improving the ability of plants to adapt to drought, salinity and cold. To comprehensively understand how plant bZIPs regulating these tolerances, this article will have an overview of their distributions on different species, characteristics of protein architecture, regulation mechanisms for target genes’ transcriptional expression, and relevant research progresses in recent years. Specially, we will discuss the possible roles of flavonoids, whose synthesis could be regulated by bZIPs, in abovementioned plant tolerant responses.

Soybean (Glycine max (L.) Merrill) is an important component of the human diet and animal feed, but soybean production is limited by abiotic stresses especially salinity. We recently found that rhizobia inoculation enhances soybean tolerance to salt stress, but the underlying mechanisms are unaddressed. Here, we used quantitative phosphoproteomic and metabonomic approaches to identify changes in phosphoproteins and metabolites in soybean roots treated with rhizobia inoculation and salt. Results revealed differential regulation of 800 phosphopeptides, at least 32 of these phosphoproteins or their homologous were reported be involved in flavonoid synthesis or trafficking, and 27 out of 32 are transcription factors. We surveyed the functional impacts of all these 27 transcription factors by expressing their phospho-mimetic/ablative mutants in the roots of composite soybean plants and found that phosphorylation of GmMYB183 could affect the salt tolerance of the transgenic roots. Using data mining, ChIP and EMSA, we found that GmMYB183 binds to the promoter of the soybean GmCYP81E11 gene encoding for a Cytochrome P450 monooxygenase which contributes to the accumulation of ononin, a monohydroxy B-ring flavonoid that negatively regulates soybean tolerance to salinity. Phosphorylation of GmMYB183 was inhibited by rhizobia inoculation; overexpression of GmMYB183 enhanced the expression of GmCYP81E11 and rendered salt sensitivity to the transgenic roots; plants deficient in GmMYB183 function are more tolerant to salt stress as compared with wild-type soybean plants, these results correlate with the transcriptional induction of GmCYP81E11 by GmMYB183 and the subsequent accumulation of ononin. Our findings provide molecular insights into how rhizobia enhance salt tolerance of soybean plants. Graphical Abstract Highlights Rhizobia affects flavonoid metabolism via GmMYB183 phosphorylation. Flavonoids rebalance the cellular ROS under salt stress. Mechanistic insights into soybean responses to salinity revealed by proteomics.