Trehalose-6-phosphate phosphatases (TPPs), which are encoded by members of the TPP gene family, can improve the drought tolerance of plants. However, the molecular mechanisms underlying the dynamic regulation of TPP genes during drought stress remain unclear. In this study, we explored the function of an Arabidopsis TPP gene by conducting comparative analyses of a loss-of-function mutant and overexpression lines. The loss-of-function mutation of Arabidopsis thaliana TPPF, a member of the TPP gene family, resulted in a drought-sensitive phenotype, while a line overexpressing TPPF showed significantly increased drought tolerance and trehalose accumulation. Compared with wild-type plants, tppf1 mutants accumulated more H2O2 under drought, while AtTPPF-overexpressing plants accumulated less H2O2 under drought. Overexpression of AtTPPF led to increased contents of trehalose, sucrose, and total soluble sugars under drought conditions; these compounds may play a role in scavenging reactive oxygen species. Yeast one-hybrid and luciferase activity assays revealed that DREB1A could bind to the DRE/CRT element within the AtTPPF promoter and activate the expression of AtTPPF. A transcriptome analysis of the TPPF-overexpressing plants revealed that the expression levels of drought-repressed genes involved in electron transport activity and cell wall modification were upregulated, while those of stress-related transcription factors related to water deprivation were downregulated. These results indicate that, as well as its involvement in regulating trehalose and soluble sugars, AtTPPF is involved in regulating the transcription of stress-responsive genes. AtTPPF functions in regulating levels of trehalose, reactive oxygen species, and sucrose levels during drought stress, and the expression of AtTPPF is activated by DREB1A in Arabidopsis. These findings shed light on the molecular mechanism by which AtTPPF regulates the response to drought stress.

Deep sequencing of DNase-I treated chromatin (DNase-seq) can be used to identify DNase I-hypersensitive sites (DHSs) and facilitates genome-scale mining of de novo cis-regulatory DNA elements. Here, we adapted DNase-seq to generate genome-wide maps of DHSs using control and cold-treated leaf, stem, and root tissues of three widely studied grass species: Brachypodium distachyon, foxtail millet (Setaria italica), and sorghum (Sorghum bicolor). Functional validation demonstrated that 12 of 15 DHSs drove reporter gene expression in transiently transgenic B. distachyon protoplasts. DHSs under both normal and cold treatment substantially differed among tissues and species. Intriguingly, the putative DHS-derived transcription factors (TFs) are largely colocated among tissues and species and include 17 ubiquitous motifs covering all grass taxa and all tissues examined in this study. This feature allowed us to reconstruct a regulatory network that responds to cold stress. Ethylene-responsive TFs SHINE3, ERF2, and ERF9 occurred frequently in cold feedback loops in the tissues examined, pointing to their possible roles in the regulatory network. Overall, we provide experimental annotation of 322,713 DHSs and 93 derived cold-response TF binding motifs in multiple grasses, which could serve as a valuable resource for elucidating the transcriptional networks that function in the cold-stress response and other physiological processes.

Agrobacterium-mediated genetic transformation is well established in the model grass Brachypodium distachyon. However, most protocols employ immature embryos because of their better regenerative capacity. A major problem associated with the immature embryo system is that they are available only during a limited time window of growing plants. In this study, we have developed an optimized Agrobacterium-mediated genetic transformation protocol that utilizes mature embryos. We have adopted seed shearing and photoautotrophic rooting (PR) in callus induction and root regeneration, respectively, with evident significant improvement in these aspects. We have also revealed that the newly developed chemical inducer Fipexide (FPX) had the ability to induce callus, shoots, and roots. By comparison, we have demonstrated that FPX shows higher efficiency in shoot generation than other frequently used chemicals in our mature embryo-based system. In addition, we demonstrated that the age of embryogenetic callus severely affects the transformation efficiency (TE), with the seven-week-old embryogenetic callus having the highest TE reaching 52.6%, which is comparable with that in immature embryo transformation. The new methodologies reported here will advance the development and utilization of Brachypodium as a new model system for grass genomics.

Sugarcane (Saccharum spp.), a leading sugar and energy crop, is seriously impacted by drought stress. However, the molecular mechanisms underlying sugarcane drought resistance, especially the functions of epigenetic regulators, remains elusive. Here, we show that a S. spontaneum KDM4/JHDM3 group JmjC protein, SsJMJ4, negatively regulates drought-stress responses through its H3K27me3 demethylase activity. Ectopic overexpression of SsJMJ4 in Arabidopsis reduced drought resistance possibly by promoting expression of AtWRKY54 and AtWRKY70, two negative regulators of drought stress. SsJMJ4 directly bound to AtWRKY54 and AtWRKY70, and reduced H3K27me3 levels at these loci to ensure their proper transcription under normal conditions. Drought stress downregulated both transcription and protein abundances of SsJMJ4, which was correlated with the reduced occupancy of SsJMJ4 at AtWRKY54 and AtWRKY70 chromatin, the increased H3K27me3 levels at these loci, as well as the reduced transcription levels of these genes. In S. spontaneum, drought stress-repressed transcription of SsWRKY122, an ortholog of AtWRKY54 and AtWRKY70, was associated with the increased H3K27me3 levels at these loci. Transient overexpression of SsJMJ4 in S. spontaneum protoplasts raised transcription of SsWRKY122, paralleled with reduced H3K27me3 levels at its loci. These results suggest that the SsJMJ4-mediated dynamic deposition of H3K27me3 is required for proper response to drought stress.

Worldwide, cadmium (Cd) contamination severely threatens rice production and public health. Jasmonic acid (JA) is recognized to be involved in rice Cd stress responses, but the underlying mechanism remains unclear. In this study, we show that JA positively regulates Cd tolerance in rice by repressing Cd uptake and root-to-shoot translocation. Cd exposure rapidly elevated the endogenous JA in rice roots, which was associated with increased expression of JA synthesis and JA-responsive genes. Moreover, silencing the expression of either allene oxide synthase (OsAOS; active in JA biosynthesis) or CORONATINE INSENSITIVE1 (OsCOI1; active in JA perception) resulted in aggravated Cd toxicity and increased Cd accumulation in both the roots and shoots, as well as increased translocation from the root to the shoots. A short-term uptake experiment revealed that silencing of OsAOS and OsCOI1 enhanced root Cd uptake ability. Furthermore, the elevated transcript levels of genes for Cd uptake (OsNramp5, OsNramp1, and OsIRT1) and root-to-shoot translocation (OsHMA2) were observed in OsAOS and OsCOI1 RNAi plants in comparison with wild-type plants. Taken together, our findings suggest that JA enhances rice cadmium tolerance by suppressing Cd uptake and translocation.