Genetic variation in the aquaporin TONOPLAST INTRINSIC PROTEIN 4;3 modulates maize cold tolerance.

Cold stress is a major abiotic stress that threatens maize (Zea mays L.) production worldwide. Understanding the molecular mechanisms underlying cold tolerance is crucial for breeding resilient maize varieties. Tonoplast intrinsic proteins (TIPs) are a subfamily of aquaporins in plants. Here, we report that TIP family proteins are involved in maize cold tolerance. The expression of most TIP genes was responsive to cold stress. Overexpressing TIP2;1, TIP3;2 or TIP4;3 reduced the cold tolerance of maize seedlings, while loss-of-function mutants of TIP4;3 exhibited enhanced cold tolerance. Candidate gene-based association analysis revealed that a 328-bp transposon insertion in the promoter region of TIP4;3 was strongly associated with maize cold tolerance. This transposon insertion conferred cold tolerance by repressing TIP4;3 expression through increased methylation of its promoter region. Moreover, TIP4;3 was found to suppress stomatal closure and facilitate reactive oxygen species (ROS) accumulation under cold stress, thereby inhibiting the expression of cold-responsive genes, including DEHYDRATION-RESPONSIVE ELEMENT BINDING FACTOR 1 (DREB1) genes and a subset of peroxidase genes, ultimately attenuating maize cold tolerance. This study thus elucidates the mechanism underlying TIP-mediated cold tolerance and identifies a favourable TIP4;3 allele as a potential genetic resource for breeding cold-tolerant maize varieties.

Generation and characterization of caprine chymosin in corn seed.

Chymosin is widely used in the dairy industry, and much is produced through recombinant DNA in organisms such as bacteria and tobacco. In this study, we used a new transgenic method to express caprine chymosin in corn seeds with lower cost and better storage capability. The recombinant chymosin protein was successfully expressed at an average level of 0.37 mg/g dry weight, which is 0.27% of the total soluble protein in the corn seed. Prochymosin can be activated to produce a chymosin protein with the ability to induce clotting in milk, similar to the commercial protein. The activity of the purified recombinant chymosin was as high as 178.5 U/mg. These results indicate that we have successfully established a technology for generating corn seed-derived caprine chymosin for potential use in the dairy industry.

Overexpression of a GmGBP1 ortholog of soybean enhances the responses to flowering, stem elongation and heat tolerance in transgenic tobaccos.

Soybean is a typical short-day crop, and its photoperiodic and gibberellin (GA) responses for the control of flowering are critical to seed yield. The GmGBP1 mRNA abundance in leaves was dramatically increased in short-days (SDs) compared to that in long-days in which it was consistently low at all time points from 0 to 6 days (days after transfer to SDs). GmGBP1 was highly expressed in leaves and exhibited a circadian rhythm in SDs. Ectopic overexpression of GmGBP1 in tobaccos caused photoperiod-insensitive early flowering by increasing NtCO mRNA levels. GmGBP1 mRNA abundance was also increased by GAs. Transgenic GmGBP1 overexpressing (-ox) tobacco plants exhibited increased GA signaling-related phenotypes including flowering and plant height promotion. Furthermore, the hypocotyl elongation, early-flowering and longer internode phenotypes were largely accelerated by GA3 application in the GmGBP1-ox tobacco seedlings. Being consistent, overexpression of GmGBP1 resulted in significantly enhanced GA signaling (evidenced suppressed expression of NtGA20ox) both with and without GA treatments. GmGBP1 was a positive regulator of both photoperiod and GA-mediated flowering responses. In addition, GmGBP1-ox tobaccos were hypersensitive to ABA, salt and osmotic stresses during seed germination. Heat-inducible GmGBP1 also enhanced thermotolerance in transgenic GmGBP1-ox tobaccos during seed germination and growth. GmGBP1 protein was localized in the nucleus. Analyses of a series of 5'-deletions of the GmGBP1 promoter suggested that several cis-acting elements, including P-BOX, TCA-motif and three HSE elements necessary to induce gene expression by GA, salicic acid and heat stress, were specifically localized in the GmGBP1 promoter region.

Genomic and Transcriptomic Analyses Reveal Pathways and Genes Associated With Brittle Stalk Phenotype in Maize.

The mechanical strength of the stalk affects the lodging resistance and digestibility of the stalk in maize. The molecular mechanisms regulating the brittleness of stalks in maize remain undefined. In this study, we constructed the maize brittle stalk mutant (bk5) by crossing the W22:Mu line with the Zheng 58 line. The brittle phenotype of the mutant bk5 existed in all of the plant organs after the five-leaf stage. Compared to wild-type (WT) plants, the sclerenchyma cells of bk5 stalks had a looser cell arrangement and thinner cell wall. Determination of cell wall composition showed that obvious differences in cellulose content, lignin content, starch content, and total soluble sugar were found between bk5 and WT stalks. Furthermore, we identified 226 differentially expressed genes (DEGs), with 164 genes significantly upregulated and 62 genes significantly downregulated in RNA-seq analysis. Some pathways related to cellulose and lignin synthesis, such as endocytosis and glycosylphosphatidylinositol (GPI)-anchored biosynthesis, were identified by the Kyoto Encyclopedia of Gene and Genomes (KEGG) and gene ontology (GO) analysis. In bulked-segregant sequence analysis (BSA-seq), we detected 2,931,692 high-quality Single Nucleotide Polymorphisms (SNPs) and identified five overlapped regions (11.2 Mb) containing 17 candidate genes with missense mutations or premature termination codons using the SNP-index methods. Some genes were involved in the cellulose synthesis-related genes such as ENTH/ANTH/VHS superfamily protein gene (endocytosis-related gene) and the lignin synthesis-related genes such as the cytochrome p450 gene. Some of these candidate genes identified from BSA-seq also existed with differential expression in RNA-seq analysis. These findings increase our understanding of the molecular mechanisms regulating the brittle stalk phenotype in maize.

Dynamic Changes in Genome-Wide Histone3 Lysine27 Trimethylation and Gene Expression of Soybean Roots in Response to Salt Stress.

Soybean is an important economic crop for human diet, animal feeds and biodiesel due to high protein and oil content. Its productivity is significantly hampered by salt stress, which impairs plant growth and development by affecting gene expression, in part, through epigenetic modification of chromatin status. However, little is known about epigenetic regulation of stress response in soybean roots. Here, we used RNA-seq and ChIP-seq technologies to study the dynamics of genome-wide transcription and histone methylation patterns in soybean roots under salt stress. Eight thousand seven hundred ninety eight soybean genes changed their expression under salt stress treatment. Whole-genome ChIP-seq study of an epigenetic repressive mark, histone H3 lysine 27 trimethylation (H3K27me3), revealed the changes in H3K27me3 deposition during the response to salt stress. Unexpectedly, we found that most of the inactivation of genes under salt stress is strongly correlated with the de novo establishment of H3K27me3 in various parts of the promoter or coding regions where there is no H3K27me3 in control plants. In addition, the soybean histone modifiers were identified which may contribute to de novo histone methylation and gene silencing under salt stress. Thus, dynamic chromatin regulation, switch between active and inactive modes, occur at target loci in order to respond to salt stress in soybean. Our analysis demonstrates histone methylation modifications are correlated with the activation or inactivation of salt-inducible genes in soybean roots.

Identification of MicroRNAs in Response to Different Day Lengths in Soybean Using High-Throughput Sequencing and qRT-PCR.

MicroRNAs (miRNAs) are short, non-coding single-strand RNA molecules that play important roles in plant growth, development and stress responses. Flowering time affects the seed yield and quality of soybean. However, the miRNAs involved in the regulation of flowering time in soybean have not been reported until recently. Here, high-throughput sequencing and qRT-PCR were used to identify miRNAs involved in soybean photoperiodic pathways. The first trifoliate leaves of soybean that receive the signal of light treatment were used to construct six libraries (0, 8, and 16 h under short-day (SD) treatment and 0, 8, and 16 h under long-day (LD) treatment). The libraries were sequenced using Illumina Solexa. A total of 318 known plant miRNAs belonging to 163 miRNA families and 81 novel predicted miRNAs were identified. Among these, 23 miRNAs at 0 h, 65 miRNAs at 8 h and 83 miRNAs at 16 h, including six novel predicted miRNAs at 8 h and six novel predicted miRNAs at 16 h, showed differences in abundance between LD and SD treatments. Furthermore, the results of GO and KEGG analyses indicated that most of the miRNA targets were transcription factors. Seven miRNAs at 0 h, 23 miRNAs (including four novel predicted miRNAs) at 8 h, 16 miRNAs (including one novel predicted miRNA) at 16 h and miRNA targets were selected for qRT-PCR analysis to assess the accuracy of the sequencing and target prediction. The results indicated that the expression patterns of the selected miRNAs and miRNA targets showed no differences between the qRT-PCR and sequencing results. In addition, 23 miRNAs at 0 h, 65 miRNAs at 8 h and 83 miRNAs at 16 h responded to day length changes in soybean, including six novel predicted miRNAs at 8 h and six novel predicted miRNAs at 16 h. These results provided an important molecular basis to understand the regulation of flowering time through photoperiodic pathways in soybean.