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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.

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. miR408-5p typically regulates target IAA30 via translation repression, but switches to cleaving IAA30 mRNA under high auxin conditions. miR393, miR156, miR408-5p and their targets could hierarchically act in auxin pathway and regulate leaf inclination.

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.

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.

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 H 2 O 2 , and overexpressing miR529a can increase plant tolerance to high level of H 2 O 2 , 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 H 2 O 2 related response genes could improve oxidative stress tolerance in rice.

Free amino acids play a key role as flavor components and metabolic precursors in fresh corn, with umami taste largely attributing to their concentration, particularly the umami amino acid glutamate and aspartate. While several genes involved in the biosynthesis of these amino acids have been identified, their regulatory pathways remain poorly understood. Sweet waxy corn ‘Qianjiangnuo No.3’ (Q3) and waxy corn ‘Huayunhuanuo 402’ (H402), with contrasting umami taste, were used in this study. Transcriptomic, metabolomic, and targeted metabolite profiling were conducted at kernel-filling stage. Our analysis showed that Q3 ears possessed significantly higher levels of free amino acids than H402, and its umami amino acids were highly enriched. Diverse enrichment of amino acids was observed across the isolated kernels distribution in ears. The O2 (bZIP1), Naked endosperm1 (NKD1), and bZIP genes were found significantly downregulated in Q3 compared to H402. Conversely, genes involved in glutamate and aspartate biosynthesis showed higher expression in Q3 than H402 while translation-associated genes snoRNA U3-2 and rRNA28S exhibited lower expression in Q3 than H402, correlating with a notable enrichment of free umami amino acids. Genes promoter analysis revealed an abundance of bZIPs binding motifs. These findings suggest that bZIP members may act as central regulators modulating free amino acid synthesis and accumulation in fresh kernels.

Mitigating heavy metals (HMs) contamination and ensuring the safe production of crops is of paramount importance for sustainable agriculture development. The purpose of the current field plot study was to select maize varieties with low HMs and metalloids in their edible parts but high accumulation in other parts. The cadmium (Cd), arsenic (As), lead (Pb), and chromium (Cr) contents of 11 maize varieties were measured by atomic absorption spectrometry, and the plant growth and bioconcentration factors (BFs) were examined. Furthermore, the average daily intake (ADDi) of HMs in maize grains was calculated to assess the associated health risks. The results revealed that the growth of variety TZ23 was minimally impacted HMs and metalloids. The grains of all of the tested maize varieties contained Cr, As, and Pb contents in accordance with National Food Safety Standards (NFSSs, GB2762-2017, ≤0.1 mg·kg−1), while the Cd concentration in grains of varieties QJN1, LSCR, and JN20 were 0.084 mg·kg−1, 0.094 mg·kg−1, and 0.077 mg·kg−1, respectively, in accordance with NFSSs. The translocation factor (TF) of As, Pb and Cr in the grains of 11 maize varieties were found to be less than 1. However, the TF of grain Cd in varieties LYN9, JYN9, and QJN3 exceeded 1. For varieties HNY21, TZ23, and LYN9, the TF of Cd, As, Pb, and Cr in the stems/leaves was less than 1. Cluster analysis revealed that the grains of variety HNY21 had the lowest accumulation capacity of all four HMs. Importantly, the variety JN20 exhibited a high accumulation capacity for Pb and a low capacity for As, while both varieties SKN11 and QJN3 had high accumulation capacities for Cd and low capacities for As. Health risk (HR) indices of the different age groups displayed an overall trend of children > elderly > young adult. Among the HMs and metalloids, Cd and Cr pose the greatest health risks of maize intake. Variety QJN3 posed a significant HR due to chronic toxicity. This study provides a scientific basis for multi-element pollution control and screening of maize varieties suitable for cultivation in mining areas and the remediation of HMs-contaminated soils.

In fresh maize breeding, developing robust and accurate near‐infrared (NIR) calibration models traditionally requires significant time, cost, and labor. To address these challenges, a novel machine learning approach is proposed using a Prediction‐Correction Neural Network (PCNN) that enables effective modeling from small sample sets augmented with synthetic data based on NIR spectroscopy. For key quality traits such as amylopectin, protein, crude fiber, and total sugar, the PCNN achieved residual predictive deviation (RPD) values between 2.821 and 4.862, and coefficients of determination (RV2 $R_V^2$ ) ranging from 0.869 to 0.951, using an average of only 32 calibration samples. For sugars including fructose, glucose, and sucrose, the model yielded RPD >2 and RV2≥0.747 $R_V^2 \\ge 0.747$with just 62 samples. The PCNN method has also been successfully applied to NIR model development for small sample sets in intact kernel of fresh maize and other crops, including forage maize, rice, wheat, and barley. Compared to Partial Least Squares (PLS) and traditional Artificial Neural Networks (ANN), PCNN delivered RPD improvements of 38.99%−63.20% over PLS and 7.07%−25.82% over ANN. These results highlight the PCNN's high efficiency and accuracy, offering a scalable and cost‐effective solution for rapid quality evaluation in fresh maize and other cereals. A cost‐effective and scalable PCNN approach is developed for NIR model construction that has compatibility with multiple instruments and multiple crops. It is time and cost saving, rapid and nondestructive, small sample set based, various quality properties and extensible for breeding application in fresh maize, forage maize, rice, wheat, and barley.

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 A t and D t 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 A t and D t 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.