Switching action modes of miR408-5p mediates auxin signaling in rice.

MicroRNAs (miRNAs) play fundamental roles in many developmental and physiological processes in eukaryotes. MiRNAs in plants generally regulate their targets via either mRNA cleavage or translation repression; however, which approach plays a major role and whether these two function modes can shift remains elusive. Here, we identify a miRNA, miR408-5p that regulates AUXIN/INDOLE ACETIC ACID 30 (IAA30), a critical repressor in the auxin pathway via switching action modes in rice. We find that miR408-5p usually inhibits IAA30 protein translation, but in a high auxin environment, it promotes the decay of IAA30 mRNA when it is overproduced. We further demonstrate that IDEAL PLANT ARCHITECTURE1 (IPA1), an SPL transcription factor regulated by miR156, mediates leaf inclination through association with miR408-5p precursor promoter. We finally show that the miR156-IPA1-miR408-5p-IAA30 module could be controlled by miR393, which silences auxin receptors. Together, our results define an alternative auxin transduction signaling pathway in rice that involves the switching of function modes by miR408-5p, which contributes to a better understanding of the action machinery as well as the cooperative network of miRNAs in plants.

Full-length transcriptome reveals the pivotal role of ABA and ethylene in the cold stress response of Tetrastigma hemsleyanum.

Tetrastigma hemsleyanum is a valuable herb widely used in Chinese traditional and modern medicine. Winter cold severely limits the artificial cultivation of this plant, but the physiological and molecular mechanisms upon exposure to cold stress in T. hemsleyanum are unclear. T. hemsleyanum plants with different geographical origins exhibit large differences in response to cold stress. In this research study, using T. hemsleyanum ecotypes that exhibit frost tolerance (FR) and frost sensitivity (FS), we analyzed the response of cottage seedlings to a simulated frost treatment; plant hormones were induced with both short (2 h) and long (9 h) frost treatments, which were used to construct the full-length transcriptome and obtained 76,750 transcripts with all transcripts mapped to 28,805 genes, and 27,215 genes, respectively, annotated to databases. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed enrichment in plant hormone signaling pathways. Further analysis shows that differently expressed genes (DEGs) concentrated on calcium signaling, ABA biosynthesis and signal transduction, and ethylene in response to cold stress. We also found that endogenous ABA and ethylene content were increased after cold treatment, and exogenous ABA and ethylene significantly improved cold tolerance in both ecotypes. Our results elucidated the pivotal role of ABA and ethylene in response to cold stress in T. hemsleyanum and identified key genes.

Facilitating Growth of Maize (Zea mays L.) by Biostimulants: A Perspective from the Interaction between Root Transcriptome and Rhizosphere Microbiome.

The plant growth-promoting effects of biostimulants have been widely documented, while little is known about the intrinsic mechanism. In our study, a pot experiment was conducted to investigate the effects of biostimulants on maize, and the maize root transcriptome and rhizosphere microbiome were assessed. The physicochemical properties of the soil were significantly altered with various trends, and the growth and yield of maize were promoted by biostimulants. Sampling time and maize strain were the strongest factors that altered the rhizosphere microorganisms. Rhizosphere microbiota with biostimulant application exhibited high community robustness. Root transcriptome analysis suggested an altered expression profile induced by biostimulants and maize strains. An integrated correlation analysis demonstrated that phosphate and nitrate metabolism genes are tightly associated with some rhizosphere microbiota. These results implied the plant growth-promoting effects of biostimulants might act in a rhizosphere microorganism-dependent manner and help to expand the use of biostimulants in sustainable agriculture.

Cadmium induced a non-coding RNA microRNA535 mediates Cd accumulation in rice.

Identifying key regulators related to cadmium (Cd) tolerance and accumulation is the main factor for genetic engineering to improve plants for bioremediation and ensure crop food safety. MicroRNAs (miRNAs), as fine-tuning regulators of genes, participate in various abiotic stress processes. MiR535 is an ancient conserved non-coding small RNA in land plants, positively responding to Cd stress. We investigated the effects of knocking out (mir535) and overexpressing miR535 (mir535 and OE535) under Cd stress in rice plants in this study. The mir535 plants showed better Cd tolerance than wild type (WT), whereas the OE535 showed the opposite effect. Cd accumulated approximately 71.9% and 127% in the roots of mir535 and OE535 plants, respectively, compared to WT, after exposure to 2 µmol/L Cd. In brown rice, the total Cd accumulation of OE535 and mir535 was about 78% greater and 35% lower than WT. When growing in 2 mg/kg Cd of soil, the Cd concentration was significantly lower in mir535 and higher in OE535 than in the WT; afterward, we further revealed the most possible target gene SQUAMOSA promoter binding-like transcription factor 7(SPL7) and it negatively regulates Nramp5 expression, which in turn regulates Cd metabolism. Therefore, the CRISPR/Cas9 technology may be a valuable strategy for creating new rice varieties to ensure food safety.

Role of plant metabolites in the formation of bacterial communities in the rhizosphere of Tetrastigma hemsleyanum.

Tetrastigma hemsleyanum Diels et Gilg, commonly known as Sanyeqing (SYQ), is an important traditional Chinese medicine. The content of bioactive constituents varies in different cultivars of SYQ. In the plant growth related researches, rhizosphere microbiome has gained significant attention. However, the role of bacterial communities in the accumulation of metabolites in plants have not been investigated. Herein, the composition of bacterial communities in the rhizosphere soils and the metabolites profile of different SYQ cultivars' roots were analyzed. It was found that the composition of microbial communities varied in the rhizosphere soils of different SYQ cultivars. The high abundance of Actinomadura, Streptomyces and other bacteria was found to be associated with the metabolites profile of SYQ roots. The findings suggest that the upregulation of rutin and hesperetin may contribute to the high bioactive constituent in SYQ roots. These results provide better understanding of the metabolite accumulation pattern in SYQ, and also provide a solution for enhancing the quality of SYQ by application of suitable microbial consortia.

Comparative Analysis of Proanthocyanidin Metabolism and Genes Regulatory Network in Fresh Leaves of Two Different Ecotypes of Tetrastigma hemsleyanum.

Tetrastigma hemsleyanum Diels et Gilg is a rare and wild medicinal resource. Metabolites, especially secondary metabolites, have an important influence on T. hemsleyanum adaptability and its medicinal quality. The metabolite proanthocyanidin (PA) is a polyphenol compound widely distributed in land plants, which can be used as antioxidants and anticancer agents. Here, we discovered that three types of PA accumulated in large amounts in purple leaves (PL), but not in green leaves (RG), based on widely non-targeted metabolomics. In addition, we further found that catechins and their derivatives, which are the structural units of PA, are also enriched in PL. Afterwards, we screened and obtained five key genes, DNR1/2, ANS, ANR and LAR closely related to PA biosynthesis through transcriptome analysis and found they were all highly expressed in PL compared to RG. Therefore, observed the regulatory relationship between the main compounds and genes network, and the PA metabolism regulatory pathway was complicated, which may be different to other species.

OsLHY is involved in regulating flowering through the Hd1- and Ehd1- mediated pathways in rice (Oryza sativa L.).

Flowering time (or heading date in crops) is a critical agronomic trait for rice reproduction and adaptation. The circadian clock is an endogenous oscillator that is involved in controlling photoperiodic flowering. The rice LATE ELONGATED HYPOCOTYL (OsLHY), the core oscillator component of circadian clock, is a homolog of the LHY/CCA1 in Arabidopsis. Here we showed that CRISPR/Cas9-engineered mutations in OsLHY caused late flowering in rice only under natural long-day (nLD) and short-day (nSD) conditions, but not artificial SD (10 h light/14 h dark) conditions. In the oslhy mutant, the diurnal expression of circadian clock-related genes was seriously affected under both LD and SD conditions. Furthermore, the expression of the flowering activators Ehd1, Hd3a and RFT1 was down-regulated and flowering repressors Hd1 and Ghd7 was up-regulated in the oslhy mutant under LD conditions. While the transcripts of flowering-related genes were not dramatically influenced under SD conditions. Dual-luciferase assays showed that OsLHY repressed the transcription of OsGI, Hd1, Ghd7, Hd3a, RFT1 and OsELF3, and activated the transcription of Ehd1. Moreover, the yeast one hybrid assay and electrophoretic mobility shift assay confirmed that OsLHY directly repressed OsGI, RFT1 and OsELF3 by binding to their promoters, which is consistent with that in Arabidopsis. These results suggested that the OsLHY can promote rice flowering mainly through regulating Hd1 and Ehd1.

Extensively Current Activity of Transposable Elements in Natural Rice Accessions Revealed by Singleton Insertions.

Active transposable elements (TEs) have drawn more attention as they continue to create new insertions and contribute to genetic diversity of the genome. However, only a few have been discovered in rice up to now, and their activities are mostly induced by artificial treatments (e.g., tissue culture, hybridization etc.) rather than under normal growth conditions. To systematically survey the current activity of TEs in natural rice accessions and identify rice accessions carrying highly active TEs, the transposon insertion polymorphisms (TIPs) profile was used to identify singleton insertions, which were unique to a single accession and represented the new insertion of TEs in the genome. As a result, 10,924 high-confidence singletons from 251 TE families were obtained, covering all investigated TE types. The number of singletons varied substantially among different superfamilies/families, perhaps reflecting distinct current activity. Particularly, eight TE families maintained potentially higher activity in 3,000 natural rice accessions. Sixty percent of rice accessions were detected to contain singletons, indicating the extensive activity of TEs in natural rice accessions. Thirty-five TE families exhibited potentially high activity in at least one rice accession, and the majority of them showed variable activity among different rice groups/subgroups. These naturally active TEs would be ideal candidates for elucidating the molecular mechanisms underlying the transposition and activation of TEs, as well as investigating the interactions between TEs and the host genome.

Genome-wide analysis of microRNA156 and its targets, the genes encoding SQUAMOSA promoter-binding protein-like (SPL) transcription factors, in the grass family Poaceae.

MicroRNAs (miRNAs) are endogenous small non-coding RNAs that play an important role in post-transcriptional gene regulation in plants and animals by targeting messenger RNAs (mRNAs) for cleavage or repressing translation of specific mRNAs. The first miRNA identified in plants, miRNA156 (miR156), targets the SQUAMOSA promoter-binding protein-like (SPL) transcription factors, which play critical roles in plant phase transition, flower and plant architecture, and fruit development. We identified multiple copies of MIR156 and SPL in the rice, Brachypodium, sorghum, maize, and foxtail millet genomes. Sequence and chromosomal synteny analysis showed that both MIR156s and SPLs are conserved across species in the grass family. Analysis of expression data of the SPLs in eleven juvenile and adult rice tissues revealed that four non-miR156-targeted genes were highly expressed and three miR156-targeted genes were only slightly expressed in all tissues/developmental stages. The remaining SPLs were highly expressed in the juvenile stage, but their expression was lower in the adult stage. It has been proposed that under strong selective pressure, non-miR156-targeted mRNA may be able to re-structure to form a miRNA-responsive element. In our analysis, some non-miR156-targeted SPLs (SPL5/8/10) had gene structure and gene expression patterns similar to those of miR156-targeted genes, suggesting that they could diversify into miR156-targeted genes. DNA methylation profiles of SPLs and MIR156s in different rice tissues showed diverse methylation patterns, and hypomethylation of non-CG sites was observed in rice endosperm. Our findings suggested that MIR156s and SPLs had different origination and evolutionary mechanisms: the SPLs appear to have resulted from vertical evolution, whereas MIR156s appear to have resulted from strong evolutionary selection on mature sequences.

OsmiR535, a Potential Genetic Editing Target for Drought and Salinity Stress Tolerance in Oryza sativa.

OsmiR535 belongs to the miR156/miR529/miR535 superfamily, a highly conserved miRNA family in plants. OsmiR535 is involved in regulating the cold-stress response, modulating plant development, and determining panicle architecture and grain length. However, the role that OsmiR535 plays in plant responses to drought and salinity are elusive. In the current study, molecular and genetic engineering techniques were used to elucidate the possible role of OsmiR535 in response to NaCl, PEG(Poly ethylene glycol), ABA(Abscisic acid), and dehydration stresses. Our results showed that OsmiR535 is induced under stressed conditions as compared to control. With transgenic and CRISPR/Cas9 knockout system techniques, our results verified that either inhibition or knockout of OsmiR535 in rice could enhance the tolerance of plants to NaCl, ABA, dehydration and PEG stresses. In addition, the overexpression of OsmiR535 significantly reduced the survival rate of rice seedlings during PEG and dehydration post-stress recovery. Our results demonstrated that OsmiR535 negatively regulates the stress response in rice. Moreover, our practical application of CRISPR/Cas9 mediated genome editing created a homozygous 5 bp deletion in the coding sequence of OsmiR535, demonstrating that OsmiR535 could be a useful genetic editing target for drought and salinity tolerance and a new marker for molecular breeding of Oryza sativa.

Natural variation and evolutionary dynamics of transposable elements in Brassica oleracea based on next-generation sequencing data.

Brassica oleracea comprises various economically important vegetables and presents extremely diverse morphological variations. They provide a rich source of nutrition for human health and have been used as a model system for studying polyploidization. Transposable elements (TEs) account for nearly 40% of the B. oleracea genome and contribute greatly to genetic diversity and genome evolution. Although the proliferation of TEs has led to a large expansion of the B. oleracea genome, little is known about the population dynamics and evolutionary activity of TEs. A comprehensive mobilome profile of 45,737 TE loci was obtained from resequencing data from 121 diverse accessions across nine B. oleracea morphotypes. Approximately 70% (32,195) of the loci showed insertion polymorphisms between or within morphotypes. In particular, up to 1221 loci were differentially fixed among morphotypes. Further analysis revealed that the distribution of the population frequency of TE loci was highly variable across different TE superfamilies and families, implying a diverse expansion history during host genome evolution. These findings provide better insight into the evolutionary dynamics and genetic diversity of B. oleracea genomes and will potentially serve as a valuable resource for molecular markers and association studies between TE-based genomic variations and morphotype-specific phenotypic differentiation.

A guanine insert in OsBBS1 leads to early leaf senescence and salt stress sensitivity in rice (Oryza sativa L.).

KEY MESSAGE: A rice receptor-like kinase gene OSBBS1/OsRLCK109 was identified; this gene played vital roles in leaf senescence and the salt stress response. Early leaf senescence can cause negative effects on rice yield, but the underlying molecular regulation is not fully understood. bilateral blade senescence 1 (bbs1), an early leaf senescence mutant with a premature senescence phenotype that occurs mainly performing at the leaf margins, was isolated from a rice mutant population generated by ethylmethane sulfonate (EMS) treatment. The mutant showed premature leaf senescence beginning at the tillering stage and exhibited severe symptoms at the late grain-filling stage. bbs1 showed accelerated dark-induced leaf senescence. The OsBBS1 gene was cloned by a map-based cloning strategy, and a guanine (G) insertion was found in the first exon of LOC_Os03g24930. This gene encodes a receptor-like cytoplasmic kinase and was named OsRLCK109 in a previous study. Transgenic LOC_Os03g24930 knockout plants generated by a CRISPR/Cas9 strategy exhibited similar early leaf senescence phenotypes as did the bbs1 mutant, which confirmed that LOC_Os03g24930 was the OsBBS1 gene. OsBBS1/OsRLCK109 was expressed in all detected tissues and was predominantly expressed in the main vein region of mature leaves. The expression of OsBBS1 could be greatly induced by salt stress, and the bbs1 mutant exhibited hypersensitivity to salt stress. In conclusion, this is the first identification of OsRLCKs participating in leaf senescence and playing critical roles in the salt stress response in rice (Oryza sativa L.).

MiR529a modulates panicle architecture through regulating SQUAMOSA PROMOTER BINDING-LIKE genes in rice (Oryza sativa).

KEY MESSAGE: MiR529a affects rice panicle architecture by targeting OsSPL2,OsSPL14 and OsSPL17 genes that could regulate their downstream panicle related genes. The panicle architecture determines the grain yield and quality of rice, which could be regulated by many transcriptional factors. The SQUAMOSA PROMOTER BINDING-LIKE (SPL) transcription factors are involved in the regulation of panicle development, which are targeted by miR156 and miR529. The expression profile demonstrated that miR529a is preferentially expressed in the early panicle of rice and it might regulate panicle development in rice. However, the regulation mechanism of miR529-SPL is still not clear. In this study, we predicted five miR529a putative target genes, OsSPL2, OsSPL14, OsSPL16, OsSPL17 and OsSPL18, while only the expression of OsSPL2, OsSPL14, and OsSPL17 was regulated by miR529a in the rice panicle. Overexpression of miR529a dramatically affected panicle architecture, which was regulated by OsSPL2, OsSPL14, and OsSPL17. Furthermore, the 117, 35, and 25 pathway genes associated with OsSPL2, OsSPL14 and OsSPL17, respectively, were predicted, and they shared 20 putative pathway genes. Our results revealed that miR529a could play a vital role in the regulation of panicle architecture through regulating OsSPL2, OsSPL14, OsSPL17 and the complex networks formed by their pathway and downstream genes. These findings will provide new genetic resources for reshaping ideal plant architecture and breeding high yield rice varieties.

Overexpression of miR529a confers enhanced resistance to oxidative stress in rice (Oryza sativa L.).

KEY MESSAGE: Overexpressing miR529a can enhance oxidative stress resistance by targeting OsSPL2 and OsSPL14 genes that can regulate the expression of their downstream SOD and POD related genes. MicroRNAs are involved in the regulation of plant developmental and physiological processes, and their expression can be altered when plants suffered environment stresses, including salt, oxidative, drought and Cadmium. The expression of microRNA529 (miR529) can be induced under oxidative stress. However, its biological function under abiotic stress responses is still unclear. In this study, miR529a was overexpressed to investigate the function of miR529a under oxidative stress in rice. Our results demonstrated that the expression of miR529a can be induced by exogenous H2O2, and overexpressing miR529a can increase plant tolerance to high level of H2O2, resulting in increased seed germination rate, root tip cell viability, reduced leaf rolling rate and chlorophyll retention. The expression of oxidative stress responsive genes and the activities of superoxide dismutase (SOD) and peroxidase (POD) were increased in miR529a overexpression plant, which could help to reduce redundant reactive oxygen species (ROS). Furthermore, only OsSPL2 and OsSPL14 were targeted by miR529a in rice seedlings, repressing their expression in miR529aOE plants could lead to strengthen plant tolerance to oxidation stress. Our study provided the evidence that overexpression of miR529a could strengthen oxidation resistance, and its target genes OsSPL2 and OsSPL14 were responsible for oxidative tolerance, implied the manipulation of miR529a and its target genes regulation on H2O2 related response genes could improve oxidative stress tolerance in rice.

Genomic Identification and Comparative Expansion Analysis of the Non-Specific Lipid Transfer Protein Gene Family in Gossypium.

Plant non-specific lipid transfer proteins (nsLTPs) are involved in many biological processes. In this study, 51, 47 and 91 nsLTPs were identified in Gossypium arboreum, G. raimondii and their descendant allotetraploid G. hirsutum, respectively. All the nsLTPs were phylogenetically divided into 8 distinct subfamilies. Besides, the recent duplication, which is considered cotton-specific whole genome duplication, may have led to nsLTP expansion in Gossypium. Both tandem and segmental duplication contributed to nsLTP expansion in G. arboreum and G. hirsutum, while tandem duplication was the dominant pattern in G. raimondii. Additionally, the interspecific orthologous gene pairs in Gossypium were identified. Some GaLTPs and GrLTPs lost their orthologs in the At and Dt subgenomes, respectively, of G. hirsutum. The distribution of these GrLTPs and GaLTPs within each subfamily was complementary, suggesting that the loss and retention of nsLTPs in G. hirsutum might not be random. Moreover, the nsLTPs in the At and Dt subgenomes might have evolved symmetrically. Furthermore, both intraspecific and interspecific orthologous genes showed considerable expression variation, suggesting that their functions were strongly differentiated. Our results lay an important foundation for expansion and evolutionary analysis of the nsLTP family in Gossypium, and advance nsLTP studies in other plants, especially polyploid plants.

Flexible microRNA arm selection in rice.

MicroRNAs act at the post-transcriptional level and guide Argonaute proteins to cleave their corresponding target transcripts. However, little attention has been paid to arm selection in miRNA precursors. In this study, small RNA high-throughput sequencing data from 29 different rice libraries were pooled to investigate tissue- and abiotic stress-specific dynamic expression of miRNAs. We found that more than half of pre-miRNAs showed changes in arm selection in different tissues and/or under different abiotic stresses. Our findings suggest that miRNA selection is remarkably prevalent in plants, providing new insights into the role of miRNAs in plant growth and development.