Yaping Yuan

Analysis of the DNA methylation of maize (Zea mays L.) in response to cold stress based on methylation-sensitive amplified polymorphisms

DNA methylation plays a vital role in tuning gene expression in response to environmental stimuli. Here, methylation-sensitive amplified polymorphisms (MSAP) were used to assess the effect of cold stress on the extent and patterns of DNA methylation in maize seedlings. Overall, cold-induced genome-wide DNA methylation polymorphisms accounted for 32.6 to 34.8% of the total bands at the different treatment time-points. It was demonstrated that the extent and pattern of DNA methylation was induced by cold stress through the cold treatment process and that the demethylation of fully methylated fragments was the main contributor of the DNA methylation alterations. The sequences of 28 differentially amplified fragments relevant to stress were successfully obtained. Under the cold stress, demethylation was detected in most fragments. BLAST results indicate that the homologues of these fragments are involved in many processes, including hormone regulation, cold response, photosynthesis, and transposon activation. The expression analysis demonstrated an increase in the transcription of five demethylated genes. Despite the fact that DNA methylation changes and cold acclimation are not directly associated, our results may indicate that the specific demethylation of genes is an active and rapid epigenetic response to cold in maize during the seedling stage, further elucidating the mechanism of maize adaptation to cold stress.

iTRAQ-based quantitative proteomic analysis reveals new metabolic pathways responding to chilling stress in maize seedlings

UNLABELLED To date, transcriptome profile analysis of maize seedlings in response to cold stress have been well documented; however, changes in protein species abundance of maize seedlings in response to cold stress are still unknown. Herein, leaves from the maize inbred line W9816 (a cold-resistance genotype) were harvested at three-leaf stage, and were used to identify the differential abundance protein species (DAPS) between chilling stress (4°C) and control conditions (25°C). iTRAQ-based quantitative proteomic were used in this study. As a result, 173 DAPS were identified after chilling stress. Bioinformatic analysis showed that 159 DAPS were annotated in 38 Gene Ontology functional groups, 108 DAPS were classified into 20 clusters of orthologous groups of protein categories, 99 DAPS were enrichment in KEGG pathways. Antioxidants assays validated that the iTRAQ results were reliable. Based on functional analysis, we concluded that the adaptive response of maize seedlings to chilling stress might be related to alleviation of photodamage caused by the over-energized state of thylakoid membrane, more energy produced through glycolysis, increased abundance of stress-responsive protein species, and improvement in the overall ability to scavenge ROS. Posttranscriptional regulation and posttranslational modifications also play important roles for maize to adapt to chilling stress. BIOLOGICAL SIGNIFICANCE The major challenge for maize breeders is the complexity of the response to chilling stress. Although extensive researches have been focus on maize chilling stress using segregating populations, epigenetics, transcriptomics, molecular biology, however, the molecular mechanism of chilling stress in maize remains to be further elucidated. In the present paper, a differential proteomic analysis was performed and the results revealed the adaptive response of maize seedlings to chilling stress might be related to alleviation of photodamage caused by the over-energized state of thylakoid membrane, more energy produced through glycolysis, increased abundance of stress-responsive protein species, improvement in the overall ability to scavenge ROS, including detoxifying enzymes and antioxidants. Posttranscriptional regulation and posttranslational modifications also play important roles for maize to adapt to chilling stress. This approach identified new protein species involved in posttranslational modifications, signal transduction, lipid metabolism, inorganic ion transport and metabolism and other biological processes that were not previously known to be associated with chilling stress response.

Development of EST-PCR markers for Thinopyrum intermedium chromosome 2Ai#2 and their application in characterization of novel wheat-grass recombinants.

A series of expressed sequence tags-derived polymerase chain reaction (EST-PCR) markers specific to chromosome 2Ai#2 from Thinopyrum intermedium were developed in this study using a new integrative approach. The target alien chromosome confers high resistance to barley yellow dwarf virus (BYDV), which is a severe virus disease in wheat. To generate markers evenly distributed on 2Ai#2, a total of 105 primer pairs were designed based on mapped ESTs from 8 bins of wheat chromosome 2B with intron-prediction by aligning ESTs with genomic sequences of the new model plant Brachypodium distachyon. Eight and seven polymorphic markers on the short arm and the long arm of chromosome 2Ai#2, respectively, were obtained with a polymorphism rate of 14.3%. These chromosome 2Ai#2-specific EST-PCR markers were then used in tracing and exploring the structural variation of the alien chromosome in the population derived from the immature embryo culture of the cross between N452, a 2Ai#2(2D) substitution line, and common wheat CB037. Two centric fusion of translocations involving 2Ai#2 short or long arm with wheat chromosome 2D and some new genetic stocks including telosomes with the alien chromosome short or long arm were identified in the SC3 generations, which provided basic materials to further study the mechanism of the BYDV resistance. BYDV tests in two field seasons suggest that the BYDV resistance was mainly conferred by the short arm, gene interaction on both arms of the alien chromosome was discussed.

Differential gene expression during somatic embryogenesis in the maize ( Zea mays L.) inbred line H99

Somatic embryogenesis is a complex developmental process that offers great potential for plant propagation. Although many studies have shown that the generation of embryonic cells from somatic cells is accompanied by the synthesis of RNA and DNA and by elevated enzymatic activity, the mechanism of the onset of somatic embryogenesis is not well understood. cDNA-amplified fragment length polymorphism analysis was used to evaluate the gene expression pattern in embryogenic and non-embryogenic of the inbred maize line H99 during the process of embryogenesis. We identified a total of 101 candidate genes associated with the formation of maize embryonic calli. Based on the sequence analysis, these genes included 53 functionally-annotated TDFs involved in such processes as energy production and conversion, cell division and signal transduction, suggesting that somatic embryogenesis undergoes a complex process. Two full-length cDNA sequences, encoding KHCP (kinesin heavy chain like protein) and TypA (tyrosine phosphorylation protein A), and partial sequences, encoding ARF-GEP (guanine nucleotide-exchange protein of ADP ribosylation factor) homologs, were isolated from embryonic calli of maize and named ZmKHCP, ZmTypA and ZmARF-GEP, respectively. Finally, the real-time qRT-PCR results showed that the expression levels of the three genes were significantly higher in the embryonic calli than the non-embryonic calli. Thus, this study provides important clues to understanding the induction of somatic embryogenesis in maize. The candidate genes associated with the formation of embryonic calli may offer additional insights into the mechanism of somatic embryogenesis, and further research on the three candidate genes may determine their role in increasing the rate of induction of embryonic calli, which may aid in the development of cultivars through transgenic breeding.

Analysis of DNA cytosine methylation patterns in maize hybrids and their parents

In order to better understand the molecular basis of heterosis in maize, the methylation-sensitive amplification polymorphism method was used to estimate patterns of cytosine methylation in seedling roots and leaves and 15-d postfertilization embryo and endosperm tissues of hybrids and their parental lines Zheng58 and Chang7-2. In all tissues, total relative methylation levels in the hybrids were lower than corresponding mid-parent values, with a higher number of demethylation events inferred for the hybrids. The trend of reduced methylation and increased demethylation in the hybrids relative to their parents may allow de-repression and possibly the expression of various genes associated with a hybrid phenotypic variation. To further investigate observed methylation pattern changes, we sequenced 50 differentially displayed DNA fragments. The BLAST analysis revealed that 13 fragments shared similarity with known functional proteins in maize or other plant species including proteins related to metabolism, transposons/retrotransposons, development, stress response, and signal transduction. The genes associated with these proteins may thus contribute significantly to maize hybrid vigour.

Overexpression of maize chloride channel gene ZmCLC-d in Arabidopsis thaliana improved its stress resistance

In plant cells, anion channels and transporters are essential for key functions. Members of the chloride channel (CLC) family located in intracellular organelles are required for anion accumulation, pH adjustment, and salt tolerance. Here, we cloned a maize (Zea mays L.) CLC gene, named ZmCLC-d, and found that its transcription was up-regulated under cold, drought, salt, and heat stresses, and after hydrogen peroxide (H2O2) and abscisic acid (ABA) treatments. The overexpression of ZmCLC-d in Arabidopsis conferred tolerance to cold, drought, and salt stresses; this tolerance was primarily displayed by an increased germination rate, root length, plant survival rate, antioxidant enzyme (catalase, peroxidase, and superoxide dismutase) activities, and a reduced accumulation of Cl− in transgenic plants as compared with wild type (WT) plants. The accumulation of H2O2 and superoxide anion in leaves of the ZmCLC-d-overexpressing plants is much less than that of the WT plants. The expressions of some stress related genes, such as CBF1, CBF2, CBF3, DREB2A, and RCI2A, increased to a greater extent in the ZmCLC-d-overexpressing plants than in the WT. Our results strongly suggest that ZmCLC-d played an important role in stress tolerance.

Isolation and functional characterization of a cold responsive phosphatidylinositol transfer-associated protein, ZmSEC14p, from maize (Zea may L.)

Key messageA Sec14-like protein,ZmSEC14p, from maize was structurally analyzed and functionally tested. Overexpression ofZmSEC14pin transgenicArabidopsisconferred tolerance to cold stress.AbstractSec14-like proteins are involved in essential biological processes, such as phospholipid metabolism, signal transduction, membrane trafficking, and stress response. Here, we reported a phosphatidylinositol transfer-associated protein, ZmSEC14p (accession no. KT932998), isolated from a cold-tolerant maize inbred line using the cDNA-AFLP approach and RACE-PCR method. Full-length cDNA that consisted of a single open reading frame (ORF) encoded a putative polypeptide of 295 amino acids. The ZmSEC14p protein was mainly localized in the nucleus, and its transcript was induced by cold, salt stresses, and abscisic acid (ABA) treatment in maize leaves and roots. Overexpression of ZmSEC14p in transgenic Arabidopsis conferred tolerance to cold stress. This tolerance was primarily displayed by the increased germination rate, root length, plant survival rate, accumulation of proline, activities of antioxidant enzymes, and the reduction of oxidative damage by reactive oxygen species (ROS). ZmSEC14p overexpression regulated the expression of phosphoinositide-specific phospholipase C, which cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) and generates second messengers (inositol 1,4,5-trisphosphate and 1,2-diacylglycerol) in the phosphoinositide signal transduction pathways. Moreover, up-regulation of some stress-responsive genes such as CBF3, COR6.6, and RD29B in transgenic plants under cold stress could be a possible mechanism for enhancing cold tolerance. Taken together, this study strongly suggests that ZmSEC14p plays an important role in plant tolerance to cold stress.

Histological and transcript analyses of intact somatic embryos in an elite maize (Zea mays L.) inbred line Y423

Intact somatic embryos were obtained from an elite maize inbred line Y423, bred in our laboratory. Using 13-day immature embryos after self-pollination as explants, and after 4-5 times subculture, a large number of somatic embryos were detected on the surface of the embryonic calli on the medium. The intact somatic embryos were transferred into the differential medium, where the plantlets regenerated with shoots and roots forming simultaneously. Histological analysis and scanning electron micrographs confirmed the different developmental stages of somatic embryogenesis, including globular-shaped embryo, pear-shaped embryo, scutiform embryo, and mature embryo. cDNA-amplified fragment length polymorphism (cDNA-AFLP) was used for comparative transcript profiling between embryogenic and non-embryogenic calli of a new elite maize inbred line Y423 during somatic embryogenesis. Differentially expressed genes were cloned and sequenced. Gene Ontology analysis of 117 candidate genes indicated their involvement in cellular component, biological process and molecular function. Nine of the candidate genes were selected. The changes in their expression levels during embryo induction and regeneration were analyzed in detail using quantitative real-time PCR. Two full-length cDNA sequences, encoding ZmSUF4 (suppressor of fir 4-like protein) and ZmDRP3A (dynamin-related protein), were cloned successfully from intact somatic embryos of the elite inbred maize line Y423. Here, a procedure for maize plant regeneration from somatic embryos is described. Additionally, the possible roles of some of these genes during the somatic embryogenesis has been discussed. This study is a systematic analysis of the cellular and molecular mechanism during the formation of intact somatic embryos in maize.

Identification of microRNAs involved in chilling response of maize by high-throughput sequencing

Chilling stress impedes growth, development, and productivity of maize (Zea mays L.). MicroRNAs (miRNAs) play critical roles in plant responses to biotic and abiotic stresses at the post-transcriptional level. Although some miRNAs have been identified in maize, little is known about the miRNAs that accumulate differently in the response to chilling stress. In this paper, we combined Illumina sequencing with Northern blot to identify chilling-responsive miRNAs in maize. Novel miRNAs (36) were predicted and some were validated. Twenty-eight known miRNAs and 24 novel miRNAs were found to be differentially expressed under various chilling (6 ºC) treatment times, and most of them were down-regulated after the chilling treatments. Northern blot and real time quantitative polymerase chain reaction proved that miR408b and miRn138 were up-regulated, miR168a, miR529, miRn120, miRn44, and miRn22 were down-regulated, miR166b, miR396c, and miRn59 undulated under 2, 6, and 12 h of the chilling stress. Analysis agriGO based on the target genes of differentially expressed miRNAs indicates that it might change hydrolase and phosphatase activities, nucleic acid metabolisms, and many cellular components to adapt to the chilling stress.