Chaohai Li

Proteomic analysis of seed viability in maize

To identify specific proteins related to maize seed viability, seeds of Zhengdan 958 (one of the high-yield maize hybrids in China) were sorted based on viability evaluation with triphenyltetrazolium chloride (TTC) assay and used for comparative proteomic analysis. After TTC staining, embryos of high-viability seeds were deep red (R type), while embryos of dead seeds were white (W type). Proteomic analysis revealed that 28 protein spots identified were differently expressed significantly between R and W embryos, of which 20 were up-regulated and 8 down-regulated in R embryos. Among them were proteins involved in stress response, protein folding, and stabilization, as wells as proteins related to nutrient reservoir and metabolism. Prominently, small heat shock proteins, late embryogenesis abundant (LEA) proteins, and antioxidant enzymes were highly up-regulated, while two proteases were highly down-regulated in R embryos compared to W embryos. One of LEA proteins was EMB564, which declined in abundance during artificial aging of seeds. Our results suggested an association of EMB564 with maize seed viability. It would be of interest to use these small proteins to develop quick tests for seed quality.

Abscisic Acid Refines the Synthesis of Chloroplast Proteins in Maize (Zea mays) in Response to Drought and Light

To better understand abscisic acid (ABA) regulation of the synthesis of chloroplast proteins in maize (Zea mays L.) in response to drought and light, we compared leaf proteome differences between maize ABA-deficient mutant vp5 and corresponding wild-type Vp5 green and etiolated seedlings exposed to drought stress. Proteins extracted from the leaves of Vp5 and vp5 seedlings were used for two-dimensional electrophoresis (2-DE) and subsequent matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). After Coomassie brilliant blue staining, approximately 450 protein spots were reproducibly detected on 2-DE gels. A total of 36 differentially expressed protein spots in response to drought and light were identified using MALDI-TOF MS and their subcellular localization was determined based on the annotation of reviewed accession in UniProt Knowledgebase and the software prediction. As a result, corresponding 13 proteins of the 24 differentially expressed protein spots were definitely localized in chloroplasts and their expression was in an ABA-dependent way, including 6 up-regulated by both drought and light, 5 up-regulated by drought but down-regulated by light, 5 up-regulated by light but down-regulated by drought; 5 proteins down-regulated by drought were mainly those involved in photosynthesis and ATP synthesis. Thus, the results in the present study supported the vital role of ABA in regulating the synthesis of drought- and/or light-induced proteins in maize chloroplasts and would facilitate the functional characterization of ABA-induced chloroplast proteins in C4 plants.

The Difference of Physiological and Proteomic Changes in Maize Leaves Adaptation to Drought, Heat, and Combined Both Stresses

At the eight-leaf stage, maize is highly sensitive to stresses such as drought, heat, and their combination, which greatly affect its yield. At present, few studies have analyzed maize response to combined drought and heat stress at the eight-leaf stage. In this study, we measured certain physical parameters of maize at the eight-leaf stage when it was exposed to drought, heat, and their combination. The results showed an increase in the content of H2O2 and malondialdehyde (MDA), and in the enzyme activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), and glutathione reductase (GR), but a decrease in the quantum efficiency of photosystem II (ΦPSII). The most obvious increase or decrease in physical parameters was found under the combined stress condition. Moreover, to identify proteins differentially regulated by the three stress conditions at the eight-leaf stage, total proteins from the maize leaves were identified and quantified using multiplex iTRAQ-based quantitative proteomic and LC-MS/MS methods. In summary, the expression levels of 135, 65, and 201 proteins were significantly changed under the heat, drought and combined stress conditions, respectively. Of the 135, 65, and 201 differentially expressed proteins, 61, 28, and 16 responded exclusively to drought stress, heat stress, and combined stress, respectively. Bioinformatics analysis implied that chaperone proteins and proteases play important roles in the adaptive response of maize to heat stress and combined stress, and that the leaf senescence promoted by ethylene-responsive protein and ripening-related protein may play active roles in maize tolerance to combined drought and heat stress. The signaling pathways related to differentially expressed proteins were obviously different under all three stress conditions. Thus, the functional characterization of these differentially expressed proteins will be helpful for discovering new targets to enhance maize tolerance to stress.

Quantitative iTRAQ-based proteomic analysis of phosphoproteins and ABA-regulated phosphoproteins in maize leaves under osmotic stress.

Abscisic acid (ABA) regulates various developmental processes and stress responses in plants. Protein phosphorylation/dephosphorylation is a central post-translational modification (PTM) in ABA signaling. However, the phosphoproteins regulated by ABA under osmotic stress remain unknown in maize. In this study, maize mutant vp5 (deficient in ABA biosynthesis) and wild-type Vp5 were used to identify leaf phosphoproteins regulated by ABA under osmotic stress. Up to 4052 phosphopeptides, corresponding to 3017 phosphoproteins, were identified by Multiplex run iTRAQ-based quantitative proteomic and LC-MS/MS methods. The 4052 phosphopeptides contained 5723 non-redundant phosphosites; 512 phosphopeptides (379 in Vp5, 133 in vp5) displayed at least a 1.5-fold change of phosphorylation level under osmotic stress, of which 40 shared common in both genotypes and were differentially regulated by ABA. Comparing the signaling pathways involved in vp5 response to osmotic stress and those that in Vp5, indicated that ABA played a vital role in regulating these pathways related to mRNA synthesis, protein synthesis and photosynthesis. Our results provide a comprehensive dataset of phosphopeptides and phosphorylation sites regulated by ABA in maize adaptation to osmotic stress. This will be helpful to elucidate the ABA-mediate mechanism of maize endurance to drought by triggering phosphorylation or dephosphorylation cascades.

In Situ Nitrogen Mineralization, Nitrification, and Ammonia Volatilization in Maize Field Fertilized with Urea in Huanghuaihai Region of Northern China

Nitrogen (N) fertilization potentially affects soil N mineralization and leaching, and can enhance NH3 volatilization, thus impacting crop production. A fertilizer experiment with five levels of N addition (0, 79, 147, 215 and 375 kg N ha-1) was performed in 2009 and 2010 in a maize field in Huanghuaihai region, China, where > 300 kg N ha-1 has been routinely applied to soil during maize growth period of 120 days. Responses of net N mineralization, inorganic N flux (0–10cm), NH3 volatilization, and maize yield to N fertilization were measured. During the growth period, net N mineralization and nitrification varied seasonally, with higher rates occurring in August and coinciding with the R1 stage of maize growth. Soil NO3 −-N contributed to more than 60% of inorganic N flux during maize growth. Cumulative NH3 volatilization increased with N additions, with total NH3 volatilization during maize growth accounting for about 4% of added N. Relative to the control, mean maize yield in the fertilizer treatments increased by 17% and 20% in 2009 and 2010, respectively. However, grain yield, aboveground biomass, and plant N accumulation did not increase with added N at levels > 215 kg N ha-1. These results suggest that the current N rate of 300 kg N ha-1 is not only excessive, but also reduces fertilizer efficacy and may contribute to environmental problems such as global warming and eutrophication of ground water and streams.

QTL analysis of shading sensitive related traits in maize under two shading treatments.

During maize development and reproduction, shading stress is an important abiotic factor influencing grain yield. To elucidate the genetic basis of shading stress in maize, an F2:3 population derived from two inbred lines, Zhong72 and 502, was used to evaluate the performance of six traits under shading treatment and full-light treatment at two locations. The results showed that shading treatment significantly decreased plant height and ear height, reduced stem diameter, delayed day-to-tassel (DTT) and day-to-silk (DTS), and increased anthesis-silking interval (ASI). Forty-three different QTLs were identified for the six measured traits under shading and full light treatment at two locations, including seven QTL for plant height, nine QTL for ear height, six QTL for stem diameter, seven QTL for day-to-tassel, six QTL for day-to-silk, and eight QTL for ASI. Interestingly, three QTLs, qPH4, qEH4a, and qDTT1b were detected under full sunlight and shading treatment at two locations simultaneously, these QTL could be used for selecting elite hybrids with high tolerance to shading and high plant density. And the two QTL, qPH10 and qDTS1a, were only detected under shading treatment at two locations, should be quit for selecting insensitive inbred line in maize breeding procedure by using MAS method.

Differential morphology and transcriptome profile between the incompletely fused carpels ovary and its wild-type in maize.

We have isolated a new mutation in maize, incompletely fused carpels (ifc), which results in an open stylar canal on the ovary and an incomplete pericarp at the top of the kernel. The maize ovary derives from the fusion of three carpels; however, the molecular networks regulating maize carpel fusion remain largely unclear. In this study, RNA sequencing (RNA-seq) was performed on wild-type (WT) and ifc ovaries that were collected after carpel fusion defects could be morphologically distinguished. In total, 877 differentially expressed genes were identified. Functional analysis revealed overexpression of genes related to “DNA binding”, “transcription regulation”, “hormones”, and “stress responses”. Among the 88 differentially expressed transcription factor (TF) genes, five showed a high degree of conservation (77.7–88.0% amino acid identity) of their conserved domains with genes associated with carpel fusion deficiency in Arabidopsis thaliana, suggesting that these five genes might control carpel fusion in maize. In addition, 30 genes encoding components of hormone synthesis and signaling pathways were differentially expressed between ifc and WT ovaries, indicating complex hormonal regulation during carpel fusion. These results help elucidate the underlying mechanisms that regulate carpel fusion, supporting the functional analysis of genes involved in producing this phenotype.

Differential miRNA expression in maize ear subjected to shading tolerance

Maize is an important crop worldwide. Its grain yield is susceptible to decrease under conditions of abiotic stress, such as shade in subtropical and temperate zones. The genetic basis of shade tolerance has not been determined in maize. MicroRNAs (miRNAs) are known to play critical roles in plant stress responses, including responses to environmental stress; but shade-associated miRNAs have not previously been identified in maize. In this study, the shade-sensitive inbred line 502 was used to examine miRNA expression differences in maize ear, after a 10-day treatment of either shade or exposure to natural light. A total of 130 known miRNAs belonging to 21 families were identified, of which 45 miRNAs were differentially expressed between shaded and natural light treatments. Twelve novel miRNAs were also predicted. In total, 94 miRNAs were upregulated and 48 downregulated in plants exposed to shaded conditions, compared with those exposed to natural light. These differentially expressed miRNAs may participate in regulating hormone homeostasis, metabolism, development and flower timing. These results suggest that the decrease of maize yield under shaded conditions may partly be determined by the differential expression of shade-induced miRNAs.

Spatial variation in carbon and nitrogen in cultivated soils in Henan Province, China: potential effect on crop yield.

Improved management of soil carbon (C) and nitrogen (N) storage in agro-ecosystems represents an important strategy for ensuring food security and sustainable agricultural development in China. Accurate estimates of the distribution of soil C and N stores and their relationship to crop yield are crucial to developing appropriate cropland management policies. The current study examined the spatial variation of soil organic C (SOC), total soil N (TSN), and associated variables in the surface layer (0–40 cm) of soils from intensive agricultural systems in 19 counties within Henan Province, China, and compared these patterns with crop yield. Mean soil C and N concentrations were 14.9 g kg−1 and 1.37 g kg−1, respectively, whereas soil C and N stores were 4.1 kg m−2 and 0.4 kg m−2, respectively. Total crop production of each county was significantly, positively related to SOC, TSN, soil C and N store, and soil C and N stock. Soil C and N were positively correlated with soil bulk density but negatively correlated with soil porosity. These results indicate that variations in soil C could regulate crop yield in intensive agricultural systems, and that spatial patterns of C and N levels in soils may be regulated by both climatic factors and agro-ecosystem management. When developing suitable management programs, the importance of soil C and N stores and their effects on crop yield should be considered.

Development of Incompletely Fused Carpels in Maize Ovary Revealed by miRNA, Target Gene and Phytohormone Analysis

Although the molecular basis of carpel fusion in maize ovary development remains largely unknown, increasing evidence suggests a critical role of microRNAs (miRNAs). In this study, a combination of miRNA sequencing, degradome and physiological analyses was used to characterize carpel fusion development in maize ovaries showing incompletely (IFC) and completely fused carpels (CFC). A total of 162 known miRNAs distributed across 33 families were identified, of which 20 were differentially expressed. In addition, 53 miRNA candidates were identified, of which 10 were differentially expressed in the IFC and CFC ovaries. In degradome analysis, a total of 113 and 11 target genes were predicted for the known and novel miRNAs, respectively. Moreover, 24 (60%) target genes of the differentially expressed known miRNAs were found to code transcription factors, including auxin response factor (ARF), TB1-CYC-PCFs (TCP), APETALA2 (AP2), growth regulating factor (GRF), MYB, NAC, and NF-YA, all of which have been shown to play a role in carpel fusion development. Correlation analysis of these differentially expressed known miRNAs and their targets with phytohormone signals revealed significant correlations with at least one phytohormone signal, the main regulator of carpel fusion development. These results suggest that incomplete carpel fusion is partly the result of differential expression of certain miRNAs and their targets. Overall, these findings improve our knowledge of the effect of miRNA regulation on target expression, providing a useful resource for further analysis of the interactions between miRNAs, target genes and phytohormones during carpel fusion development in maize.