Transcriptome analysis and identification of the low potassium stress-responsive gene SiSnRK2.6 in foxtail millet (Setaria italica L.).

KEY MESSAGE: The transcriptome is beneficial for dissecting the mechanism of millet in response to low potassium stress and SiSnRK2.6 was identified as a potential target for improving low potassium stress tolerance. Foxtail millet (Setaria italica L.), which originated in China, has high nutrient utilization character. Nevertheless, the molecular mechanism of its tolerance to low potassium stress is largely unclear. In this research, the low potassium tolerant variety "Yugu28" was screened out by low potassium stress treatment, and the transcriptome of "Yugu28" under low potassium stress was comprehensively analyzed. A total of 4254 differentially expressed genes (DEGs) were identified, including 1618 up-regulated and 2636 down-regulated genes, respectively. In addition, there were 302 transcription factor (TF) genes in the DEGs and MYB TFs accounted for the highest proportion, which was 14.9%. After functional analysis of all DEGs, a total of 7 genes involved in potassium transport and potassium ion channels and 50 genes corresponding to hormones were screened. The expression levels of randomly selected 17 DEGs were verified by qRT-PCR and the results coincided well with the RNA-seq analysis, indicating the reliability of our transcriptome data. Moreover, one of the ABA signaling pathway genes, SiSnRK2.6, was identified and selected for further functional verification. Compared with the wild type, transgenic rice with ecotopic expression of SiSnRK2.6 showed remarkably increased root length and root number, indicating that overexpression of SiSnRK2.6 can enhance the resistance of transgenic plants to low potassium stress.

Genetic Diversity, Population Structure and Mating Type Distribution of Setosphaeria turcica on Corn in Midwestern China.

Setosphaeria turcica is the causal agent of northern corn leaf blight (NCLB), which is a destructive foliar disease of corn around the world. To date, limited information is available on the genetic diversity, population structure, and mating type distribution of the pathogen in the mid-west of China. In this study, based on single nucleotide polymorphism (SNP) markers and mating type-specific primers, we characterized 117 S. turcica isolates collected from Henan, Hebei, Shanxi, and Shaanxi provinces in China. Based on the developed 33 SNP markers, all isolates can be categorized into two genetic groups. Each group consisted of isolates from all four provinces. The Nei's gene diversity of four populations ranged from 0.328 to 0.419 with a mean of 0.391. The analysis of fixation index (Fst) and gene flow (Nm) suggested that low genetic differentiation and high gene flow existed among four geographic populations. The analysis of molecular variance (AMOVA) demonstrated that the principal molecular variance existed within populations (98%) rather than among populations (2%). The analysis of mating type loci revealed that two mating types (MAT1-1 and MAT1-2) were basically in equilibrium in all four populations. These findings advance our understanding of the genetic diversity, population structure and mating type distribution of S. turcica on corn in the mid-west of China and will aid in developing efficient strategies to control NCLB.

Identification of miRNAs and their target genes associated with improved maize seed vigor induced by gibberellin.

High seed vigor is crucial for agricultural production owing to its potential in high quality and yield of crops and a better understanding of the molecular mechanism associated with maize seed vigor is highly necessary. To better understand the involvement and regulatory mechanism of miRNAs correlated with maize seed vigor, small RNAs and degradome sequencing of two inbred lines Yu537A and Yu82 were performed. A total of 791 mature miRNAs were obtained with different expressions, among of which 505 miRNAs were newly identified and the rest miRNAs have been reported before by comparing the miRNAs with the sequences in miRbase database. Analysis of miRNA families showed maize seeds contain fewer miRNA families and larger miRNA families compared with animals, indicating that functions of miRNAs in maize seeds were more synergistic than animals. Degradome sequencing was used to identify the targets of miRNAs and the results showed a total of 6,196 targets were obtained. Function analysis of differentially expressed miRNAs and targets showed Glycan degradation and galactose metabolism were closely correlated with improved maize seed vigor. These findings provide valuable information to understand the involvement of miRNAs with maize seed vigor and these putative genes will be valuable resources for improving the seed vigor in future maize breeding.

ZmCCA1a on Chromosome 10 of Maize Delays Flowering of Arabidopsis thaliana.

Maize (Zea mays) is a major cereal crop that originated at low latitudes, and thus photoperiod sensitivity is an important barrier to the use of tropical/subtropical germplasm in temperate regions. However, studies of the mechanisms underlying circadian regulation in maize are at an early stage. In this study we cloned ZmCCA1a on chromosome 10 of maize by map-based cloning. The gene is homologous to the Myb transcription factor genes AtCCA1/AtLHY in Arabidopsis thaliana; the deduced Myb domain of ZmCCA1a showed high similarity with that of AtCCA1/AtLHY and ZmCCA1b. Transiently or constitutively expressed ZmCCA1a-YFPs were localized to nuclei of Arabidopsis mesophyll protoplasts, agroinfiltrated tobacco leaves, and leaf and root cells of transgenic seedlings of Arabidopsis thaliana. Unlike AtCCA1/AtLHY, ZmCCA1a did not form homodimers nor interact with ZmCCA1b. Transcripts of ZmCCA1a showed circadian rhythm with peak expression around sunrise in maize inbred lines CML288 (photoperiod sensitive) and Huangzao 4 (HZ4; photoperiod insensitive). Under short days, transcription of ZmCCA1a in CML288 and HZ4 was repressed compared with that under long days, whereas the effect of photoperiod on ZmCCA1a expression was moderate in HZ4. In ZmCCA1a-overexpressing A. thaliana (ZmCCA1a-ox) lines, the circadian rhythm was disrupted under constant light and flowering was delayed under long days, but the hypocotyl length was not affected. In addition, expression of endogenous AtCCA1/AtLHY and the downstream genes AtGI, AtCO, and AtFt was repressed in ZmCCA1a-ox seedlings. The present results suggest that the function of ZmCCA1a is similar, at least in part, to that of AtCCA1/AtLHY and ZmCCA1b, implying that ZmCCA1a is likely to be an important component of the circadian clock pathway in maize.

Comprehensive dynamic transcriptome analysis at two seed germination stages in maize (Zea mays L.).

Seed germination, as an integral stage of crop production, directly affects Zea mays (maize) yield and grain quality. However, the molecular mechanisms of seed germination remain unclear in maize. We performed comparative transcriptome analysis of two maize inbred lines, Yu82 and Yu537A, at two stages of seed germination. Expression profile analysis during seed germination revealed that a total of 3381 and 4560 differentially expressed genes (DEGs) were identified in Yu82 and Yu537A at the two stages. Transcription factors were detected from several families, such as the bZIP, ERF, WRKY, MYB and bHLH families, which indicated that these transcription factor families might be involved in driving seed germination in maize. Prominent DEGs were submitted for KEGG enrichment analysis, which included plant hormones, amino acid mechanism, nutrient reservoir, metabolic pathways and ribosome. Of these pathways, genes associated with plant hormones, especially gibberellins, abscisic acid and auxin may be important for early germination in Yu82. In addition, DEGs involved in amino acid mechanism showed significantly higher expression levels in Yu82 than in Yu537A, which indicated that energy supply from soluble sugars and amino acid metabolism may contribute to early germination in Yu82. This results provide novel insights into transcriptional changes and gene interactions in maize during seed germination.

Alternative splicing of ZmCCA1 mediates drought response in tropical maize.

The circadian clock regulates numerous biological processes in plants, especially development and stress responses. CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) is one of the core components of the day-night rhythm response and is reportedly associated with ambient temperature in Arabidopsis thaliana. However, it remains unknown if alternative splicing of ZmCCA1 is modulated by external stress in maize, such as drought stress and photoperiod. Here, we identified three ZmCCA1 splice variants in the tropical maize line CML288, which are predicted to encode three different protein isoforms, i.e., ZmCCA1.1, ZmCCA1.2, and ZmCCA1.3, which all retain the MYB domain. In maize, the expression levels of ZmCCA1 splice variants were influenced by photoperiod, tissue type, and drought stress. In transgenic A. thaliana, ZmCCA1.1 may be more effective than ZmCCA1.3 in increasing drought tolerance while ZmCCA1.2 may have only a small effect on tolerance to drought stress. Additionally, although CCA1 genes have been found in many plant species, alternative CCA1 splicing events are known to occur in species-specific ways. Our study provides new sight to explore the function of ZmCCA1 splice variants' response to abiotic stress, and clarify the linkage between circadian clock and environmental stress in maize.

Quantitative analysis of changes in the phosphoproteome of maize induced by the plant hormone salicylic acid.

Phytohormone salicylic acid (SA) plays an important role in regulating various physiological and biochemical processes. Our previous study identified several protein kinases responsive to SA, suggesting that phosphorylation events play an important role in the plant response to SA. In this study, we characterized the phosphoproteome of maize in response to SA using isotope tags for relative and absolute quantification (iTRAQ) technology and TiO2 enrichment method. Based on LC-MS/MS analysis, we found a total of 858 phosphoproteins among 1495 phosphopeptides. Among them, 291 phosphopeptides corresponding to 244 phosphoproteins were found to be significantly changed after SA treatment. The phosphoproteins identified are involved in a wide range of biological processes, which indicate that the response to SA encompasses a reformatting of major cellular processes. Furthermore, some of the phosphoproteins which were not previously known to be involved with SA were found to have significantly changed phosphorylation levels. Many of these changes are phosphorylation decreases, indicating that other currently unknown SA signaling pathways that result in decreased phosphorylation of downstream targets must be involved. Our study represents the first attempt at global phosphoproteome profiling in response to SA, and provides a better understanding of the molecular mechanisms regulated by SA.

Phosphoproteomic analysis of the resistant and susceptible genotypes of maize infected with sugarcane mosaic virus.

Protein phosphorylation plays a pivotal role in the regulation of many cellular events. No information is yet available, however, on protein phosphorylation in plants in response to virus infection. In this study, we characterized phosphoproteomes of resistant and susceptible genotypes of maize (Zea mays L.) in response to Sugarcane mosaic virus (SCMV) infection. Based on isotope tags for relative and absolute quantification technology, TiO2 enrichment method and LC-MS/MS analysis, we identified 65 and 59 phosphoproteins respectively, whose phosphorylation level regulated significantly in susceptible and resistant plants. Some identified phosphoproteins were shared by both genotypes, suggesting a partial overlapping of the responsive pathways to virus infection. While several phosphoproteins are well-known pathogen response phosphoproteins, virus infection differentially regulates most other phosphoproteins, which has not been reported in literature. Changes in protein phosphorylation status indicated that response to SCMV infection encompass a reformatting of major cellular processes. Our data provide new valuable insights into plant-virus interactions.

QTLs for seed vigor-related traits identified in maize seeds germinated under artificial aging conditions.

High seed vigor is important for agricultural production due to the associated potential for increased growth and productivity. However, a better understanding of the underlying molecular mechanisms is required because the genetic basis for seed vigor remains unknown. We used single-nucleotide polymorphism (SNP) markers to map quantitative trait loci (QTLs) for four seed vigor traits in two connected recombinant inbred line (RIL) maize populations under four treatment conditions during seed germination. Sixty-five QTLs distributed between the two populations were identified and a meta-analysis was used to integrate genetic maps. Sixty-one initially identified QTLs were integrated into 18 meta-QTLs (mQTLs). Initial QTLs with contribution to phenotypic variation values of R(2)>10% were integrated into mQTLs. Twenty-three candidate genes for association with seed vigor traits coincided with 13 mQTLs. The candidate genes had functions in the glycolytic pathway and in protein metabolism. QTLs with major effects (R(2)>10%) were identified under at least one treatment condition for mQTL2, mQTL3-2, and mQTL3-4. Candidate genes included a calcium-dependent protein kinase gene (302810918) involved in signal transduction that mapped in the mQTL3-2 interval associated with germination energy (GE) and germination percentage (GP), and an hsp20/alpha crystallin family protein gene (At5g51440) that mapped in the mQTL3-4 interval associated with GE and GP. Two initial QTLs with a major effect under at least two treatment conditions were identified for mQTL5-2. A cucumisin-like Ser protease gene (At5g67360) mapped in the mQTL5-2 interval associated with GP. The chromosome regions for mQTL2, mQTL3-2, mQTL3-4, and mQTL5-2 may be hot spots for QTLs related to seed vigor traits. The mQTLs and candidate genes identified in this study provide valuable information for the identification of additional quantitative trait genes.

Valid application of western blotting.

Western blotting is a powerful and commonly used tool to identify and quantify a specific protein in a complex mixture. However, the systematic errors in the application of western blotting analysis are frequently to be found, which may compromise the interpretation of results. To make a valid application of western blotting, it is essential to begin with three independent biological replicates. Subsequently, a more reliable normalization method is in urgent need for western blotting analysis and using reference proteins is the currently preferred method of normalization. Additionally, identification of valid reference proteins is crucial for western blotting analysis and it should be examined carefully in relation to the cell or tissue types when using housekeeping proteins as internal standards.

Comparative proteomic analysis of the plant-virus interaction in resistant and susceptible ecotypes of maize infected with sugarcane mosaic virus.

Sugarcane mosaic virus (SCMV) is an important viral pathogen and has caused serious losses in grain and forage yield. To identify candidate SCMV resistance proteins and to explore the molecular mechanisms involved in the plant-SCMV interaction, we conducted proteomic analyses of leaf samples from resistant and susceptible ecotypes of maize infected with SCMV. Proteins were analyzed by quantitative two-dimensional differential gel electrophoresis (2D-DIGE), and 93 protein spots showed statistically significant differences after virus inoculation. Functional categorization showed that SCMV-responsive proteins were mainly involved in energy and metabolism, stress and defense responses, photosynthesis, and carbon fixation. The majority of the identified proteins were located in chloroplast and cytoplasm based on bioinformatic analysis. Among these identified proteins, 17 have not been identified previously as virus-responsive proteins, and 7 were new and did not have assigned functions. Western blotting analyses confirmed the expression patterns of proteins of specific interest, and the genes encoding these proteins were further analyzed by real-time PCR. The results of this study showed overlapping and specific proteomic responses to SCMV infection between resistant and susceptible maize ecotypes. This study provides further insight into the molecular events during compatible and incompatible interactions between viruses and host plants. BIOLOGICAL SIGNIFICANCE: Sugarcane mosaic virus (SCMV) is an important viral pathogen and has caused serious losses in grain and forage yield. However, little is known about host-SCMV interactions from the proteome perspective. This study analyzed proteomic changes in resistant and susceptible plants that are infected with SCMV using DIGE based proteomics. We identified 17 proteins that have not been identified previously as virus-responsive proteins, and 7 new proteins without assigned functions. These proteins are interesting candidates for future research, as they may be associated with new biological functions and play important roles in plant-virus interactions. Real-time RT-PCR analysis of genes encoding several proteins of interest provided indication on whether the changes in protein abundance were regulated at the mRNA level. The results of this study showed overlapping and specific proteomic responses to SCMV infection between resistant and susceptible ecotypes. After inoculation, the proteins involved in energy and metabolism, stress and defense responses, photosynthesis and other four functional groups showed significant changes in both ecotypes, which suggested that SCMV infection influenced these physiological processes in both the resistant Siyi and the susceptible Mo17. However, the oxidative burst was more pronounced during incompatible plant-SCMV interactions, as compared to those defined as compatible. We also observed an increase of enzymes involved in glycolysis and gluconeogenesis pathways in the resistant maize ecotype Siyi, while decrease in the susceptible maize ecotype Mo17. In addition, there is a marked increase of guanine nucleotide-binding protein beta submit in the resistant Siyi, which suggests a possible involvement of G-protein associated pathways in the resistant responses of maize to SCMV. These observations may possibly reveal protein targets/markers that are useful in the design of future diagnosis or plant protection strategies and provide new insights into the molecular mechanism of plant-virus interactions.