TEAseq-based identification of 35,696 Dissociation insertional mutations facilitates functional genomic studies in maize.

In plants, transposable element (TE)-triggered mutants are important resources for functional genomic studies. However, conventional approaches for genome-wide identification of TE insertion sites are costly and laborious. This study developed a novel, rapid, and high-throughput TE insertion site identification workflow based on next-generation sequencing and named it Transposable Element Amplicon Sequencing (TEAseq). Using TEAseq, we systemically profiled the Dissociation (Ds) insertion sites in 1606 independent Ds insertional mutants in advanced backcross generation using K17 as background. The Ac-containing individuals were excluded for getting rid of the potential somatic insertions. We characterized 35,696 germinal Ds insertions tagging 10,323 genes, representing approximately 23.3% of the total genes in the maize genome. The insertion sites were presented in chromosomal hotspots around the ancestral Ds loci, and insertions occurred preferentially in gene body regions. Furthermore, we mapped a loss-of-function AGL2 gene using bulked segregant RNA-sequencing assay and proved that AGL2 is essential for seed development. We additionally established an open-access database named MEILAM for easy access to Ds insertional mutations. Overall, our results have provided an efficient workflow for TE insertion identification and rich sequence-indexed mutant resources for maize functional genomic studies.

The miR172/IDS1 signaling module confers salt tolerance through maintaining ROS homeostasis in cereal crops.

Salt stress triggers the overdose accumulation of reactive oxygen species (ROS) in crop plants, leading to severe oxidative damage to living tissues. MicroRNAs (miRNAs) act as master regulators orchestrating the stress responsive regulatory networks as well as salt tolerance. However, the fundamental roles of miRNAs in modulating salt tolerance in cereal crops, especially in salt-triggered ROS scavenging remain largely unknown. Through small RNA sequencing, a salt-responsive miRNA, miR172 was identified in rice. Further, by generating the miR172-overexpression or MIR172 gene loss-of-function mutant lines, the biological significance of miR172 and its downstream signaling pathways related to salt tolerance were defined. We demonstrated that miR172 is a positive regulator of salt tolerance in both rice and wheat. More interestingly, miR172a and miR172b, but not miR172c or miR172d are involved in salt stress response, emphasizing the functional differentiation within miR172 family members. Further evidence uncovers a novel miR172/IDS1 regulatory module that functions as a crucial molecular rheostat in maintaining ROS homeostasis during salt stress, mainly through balancing the expression of a group of ROS-scavenging genes. Our findings establish a direct molecular link between miRNAs and detoxification response in cereal crops for improving salt tolerance.

INDETERMINATE SPIKELET1 Recruits Histone Deacetylase and a Transcriptional Repression Complex to Regulate Rice Salt Tolerance.

Perception and transduction of salt stress signals are critical for plant survival, growth, and propagation. Thus, identification of components of the salt stress-signaling pathway is important for rice (Oryza sativa) molecular breeding of salt stress resistance. Here, we report the identification of an apetala2/ethylene response factor transcription factor INDETERMINATE SPIKELET1 (IDS1) and its roles in the regulation of rice salt tolerance. By genetic screening and phenotype analysis, we demonstrated that IDS1 conferred transcriptional repression activity and acted as a negative regulator of salt tolerance in rice. To identify potential downstream target genes regulated by IDS1, we conducted chromatin immunoprecipitation (ChIP) sequencing and ChIP-quantitative PCR assays and found that IDS1 may directly associate with the GCC-box-containing motifs in the promoter regions of abiotic stress-responsive genes, including LEA1 (LATE EMBRYOGENESIS ABUNDANT PROTEIN1) and SOS1 (SALT OVERLY SENSITIVE1), which are key genes regulating rice salt tolerance. IDS1 physically interacted with the transcriptional corepressor topless-related 1 and the histone deacetylase HDA1, contributing to the repression of LEA1 and SOS1 expression. Analyses of histone H3 acetylation status and RNA polymerase II occupation on the promoters of LEA1 and SOS1 further defined the molecular foundation of the transcriptional repression activity of IDS1. Our findings illustrate an epigenetic mechanism by which IDS1 modulates salt stress signaling as well as salt tolerance in rice.

miR156-Targeted SBP-Box Transcription Factors Interact with DWARF53 to Regulate TEOSINTE BRANCHED1 and BARREN STALK1 Expression in Bread Wheat.

Genetic and environmental factors affect bread wheat (Triticum aestivum) plant architecture, which determines grain yield. In this study, we demonstrate that miR156 controls bread wheat plant architecture. We show that overexpression of tae-miR156 in bread wheat cultivar Kenong199 leads to increased tiller number and severe defects in spikelet formation, probably due to the tae-miR156-mediated repression of a group of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes. Furthermore, we found that the expression of two genes TEOSINTE BRANCHED1 (TaTB1) and BARREN STALK1 (TaBA1), whose orthologous genes in diverse plant species play conserved roles in regulating plant architecture, is markedly reduced in the tae-miR156-OE bread wheat plants. Significantly, we demonstrate that the strigolactone (SL) signaling repressor DWARF53 (TaD53), which physically associates with the transcriptional corepressor TOPLESS, can directly interact with the N-terminal domains of miR156-controlled TaSPL3/17. Most importantly, TaSPL3/17-mediated transcriptional activation of TaBA1 and TaTB1 can be largely repressed by TaD53 in the transient expression system. Our results reveal potential association between miR156-TaSPLs and SL signaling pathways during bread wheat tillering and spikelet development.

MicroRNA319-regulated TCPs interact with FBHs and PFT1 to activate CO transcription and control flowering time in Arabidopsis.

The transcription factor CONSTANS (CO) is a central component that promotes Arabidopsis flowering under long-day conditions (LDs). Here, we show that the microRNA319-regulated TEOSINTE BRANCHED/CYCLOIDEA/PCF (TCP) transcription factors promote photoperiodic flowering through binding to the CO promoter and activating its transcription. Meanwhile, these TCPs directly interact with the flowering activators FLOWERING BHLH (FBHs), but not the flowering repressors CYCLING DOF FACTORs (CDFs), to additively activate CO expression. Furthermore, both the TCPs and FBHs physically interact with the flowering time regulator PHYTOCHROME AND FLOWERING TIME 1 (PFT1) to facilitate CO transcription. Our findings provide evidence that a set of transcriptional activators act directly and additively at the CO promoter to promote CO transcription, and establish a molecular mechanism underlying the regulation of photoperiodic flowering time in Arabidopsis.

The miR9863 family regulates distinct Mla alleles in barley to attenuate NLR receptor-triggered disease resistance and cell-death signaling.

Barley (Hordeum vulgare L.) Mla alleles encode coiled-coil (CC), nucleotide binding, leucine-rich repeat (NB-LRR) receptors that trigger isolate-specific immune responses against the powdery mildew fungus, Blumeria graminis f. sp. hordei (Bgh). How Mla or NB-LRR genes in grass species are regulated at post-transcriptional level is not clear. The microRNA family, miR9863, comprises four members that differentially regulate distinct Mla alleles in barley. We show that miR9863 members guide the cleavage of Mla1 transcripts in barley, and block or reduce the accumulation of MLA1 protein in the heterologous Nicotiana benthamiana expression system. Regulation specificity is determined by variation in a unique single-nucleotide-polymorphism (SNP) in mature miR9863 family members and two SNPs in the Mla miR9863-binding site that separates these alleles into three groups. Further, we demonstrate that 22-nt miR9863s trigger the biogenesis of 21-nt phased siRNAs (phasiRNAs) and together these sRNAs form a feed-forward regulation network for repressing the expression of group I Mla alleles. Overexpression of miR9863 members specifically attenuates MLA1, but not MLA10-triggered disease resistance and cell-death signaling. We propose a key role of the miR9863 family in dampening immune response signaling triggered by a group of MLA immune receptors in barley.