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Drought is a global threat that affects agricultural production. Plants have evolved several adaptive strategies to cope with drought. Stomata are essential structures for plants to control water status and photosynthesis rate. Stomatal closure is an efficient way for plants to reduce water loss and improve survivability under drought conditions. The opening and closure of stomata depend on the turgor pressure in guard cells. Three key signaling molecules, including abscisic acid (ABA), reactive oxygen species (ROS), and calcium ion (Ca

Cysteine proteases (CPs) are vital proteolytic enzymes that play critical roles in various plant processes. However, the particular functions of CPs in maize remain largely unknown. We recently identified a pollen-specific CP (named PCP), which highly accumulated on the surface of maize pollen. Here, we reported that PCP played an important role in pollen germination and drought response in maize. Overexpression of PCP inhibited pollen germination, while mutation of PCP promoted pollen germination to some extent. Furthermore, we observed that germinal apertures of pollen grains in the PCP-overexpression transgenic lines were excessively covered, whereas this phenomenon was not observed in the wild type (WT), suggesting that PCP regulated pollen germination by affecting the germinal aperture structure. In addition, overexpression of PCP enhanced drought tolerance in maize plants, along with the increased activities of the antioxidant enzymes and the decreased numbers of the root cortical cells. Conversely, mutation of PCP significantly impaired drought tolerance. These results may aid in clarifying the precise functions of CPs in maize and contribute to the development of drought-tolerant maize materials.

The GATA transcription factors are important transcriptional regulators of plant growth and responses to environmental stimuli. Here, a total of 25 CaGATA genes was identified in chickpea (Cicer arietinum), and their basic characteristics, including gene structure, duplication patterns, conserved domains and various physical and chemical parameters were subsequently determined. Of our interest, the enrichment of the hormone- and stress-responsive cis-regulatory elements in the promoters of CaGATA genes has been analyzed to predict the CaGATA members with potential hormone-mediated functions in stress tolerance. Furthermore, the tissue-specific expression patterns of the CaGATA genes were assessed using the available transcriptome data. More importantly, transcript levels of the identified CaGATA genes were quantified in roots and leaves of chickpea seedlings exposed to ABA (abscisic acid) or dehydration treatment using real-time quantitative PCR. Expression levels of a total of 12 CaGATA genes were significantly altered in roots and/or leaves by both ABA and dehydration treatments, suggesting that these genes might play roles in regulation of chickpea response to water stress in an ABA-dependent manner. Out of these genes, only CaGATA04 was induced in both roots and leaves by ABA and dehydration treatments. Furthermore, CaGATA05 and 21 were the most highly induced in roots (8.55-fold) and leaves (4.90-fold), respectively, by dehydration. Findings of this study have provided important insights into the CaGATA family of chickpea, as well as useful information for selection of CaGATA genes of interest for in-depth functional characterizations that might lead to development of chickpea cultivars with improved performance under water-deficit conditions.

Drought is a major environmental threat that limits crop growth, development, and productivity worldwide. Improving drought resistance with genetic engineering methods is necessary to tackle global climate change. It is well known that NAC (NAM, ATAF and CUC) transcription factors play a critical role in coping with drought stress in plants. In this study, we identified an NAC transcription factor ZmNAC20, which regulates drought stress response in maize. ZmNAC20 expression was rapidly upregulated by drought and abscisic acid (ABA). Under drought conditions, the ZmNAC20-overexpressing plants had higher relative water content and survival rate than the wild-type maize inbred B104, suggesting that overexpression of ZmNAC20 improved drought resistance in maize. The detached leaves of ZmNAC20-overexpressing plants lost less water than those of wild-type B104 after dehydration. Overexpression of ZmNAC20 promoted stomatal closure in response to ABA. ZmNAC20 was localized in the nucleus and regulated the expression of many genes involved in drought stress response using RNA-Seq analysis. The study indicated that ZmNAC20 improved drought resistance by promoting stomatal closure and activating the expression of stress-responsible genes in maize. Our findings provide a valuable gene and new clues on improving crop drought resistance.

Maize accumulates large amounts of starch in seeds which have been used as food for human and animals. Maize starch is an importantly industrial raw material for bioethanol production. One critical step in bioethanol production is degrading starch to oligosaccharides and glucose by α-amylase and glucoamylase. This step usually requires high temperature and additional equipment, leading to an increased production cost. Currently, there remains a lack of specially designed maize cultivars with optimized starch (amylose and amylopectin) compositions for bioethanol production. We discussed the features of starch granules suitable for efficient enzymatic digestion. Thus far, great advances have been made in molecular characterization of the key proteins involved in starch metabolism in maize seeds. The review explores how these proteins affect starch metabolism pathway, especially in controlling the composition, size and features of starch. We highlight the roles of key enzymes in controlling amylose/amylopectin ratio and granules architecture. Based on current technological process of bioethanol production using maize starch, we propose that several key enzymes can be modified in abundance or activities via genetic engineering to synthesize easily degraded starch granules in maize seeds. The review provides a clue for developing special maize cultivars as raw material in the bioethanol industry.

Background Currently, mechanical maize kernel harvesting has not been fully utilized in developing countries including China, partly due to the absence of suitable cultivars capable of rapid desiccation during seed maturation. The initiation of rapid desiccation during seed maturation is regulated by abscisic acid (ABA). For further characterization of ABA-regulated key genes and cellular events, it is necessary to perform transcriptome analysis of maize developing embryos. The ABA synthesis-deficient mutant (vp5) and normal maize (Vp5) seeds are suitable materials for such purpose. Results In the present work, developing vp5 and Vp5 embryos were compared by ABA content and transcriptome analyses. Quantitative analysis revealed the significant difference in ABA synthesis between both genotypes. From 29 days after pollination (DAP), ABA content increased rapidly in Vp5 embryos, but decreased gradually in vp5 embryos. At 36 DAP, ABA level in vp5 decreased to 1/4 that of Vp5, suggesting that the differential ABA levels would affect seed maturation. Comparative transcriptomic analysis has found 1019 differentially expressed genes (DEGs) between both genotypes, with the most DEGs (818) at 36 DAP. Further, weighted correlation network analysis (WGCNA) revealed eight DEGs co-expression modules. Particularly, a module was negatively correlated with ABA content in vp5 embryos. The module was mainly involved in metabolic and cellular processes, and its hub genes encoded thiamine, NPF proteins, calmodulin, metallothionein etc. Moreover, the expression of a set of key genes regulated by ABA was further verified by RT-qPCR. The results of the present work suggested that because of ABA deficiency, the vp5 seeds maintained strong metabolic activities and lacked dormancy initiation during seed maturation. Conclusion Transcriptome and WGCNA analyses revealed significant ABA-related changes in metabolic pathways and DEGs between vp5 and Vp5 during seed maturation. The results would provide insights for elucidating the molecular mechanism of ABA signaling and developing high dehydration tolerance maize suitable for mechanical harvesting.

Starch is the major form of carbohydrate storage in plants and exists as discrete starch granules (SGs). Isolation of high-quality SGs in different plant tissues is a prerequisite for studying the roles of SGs during plant growth, development, and responses to abiotic stress. However, it is difficult to isolate transitory SGs from leaves and storage SGs from pollen grains due to their small sizes and low quantities. Herein, we develop a novel method for isolating SGs by using the aqueous two-phase system (ATS) of ethanol/NaH2 PO4 . The ATS method efficiently separated SGs from contaminants based on their differences in density, solubility, and polarity. Using this method, we first isolated and purified three kinds of SGs from maize seeds, pollen, and leaves. The biochemical, microscopic, and proteomic analyses demonstrated the high purity of the isolated SGs. Proteomic analysis revealed distinct differences in SG-bound proteins between seed SGs and pollen SGs. As a simple, rapid, and low-cost method, the ATS-based method exhibits highly universal and reproducible results for starch-containing tissues in various plant species.

Maize (Zea mays) starch is an important agricultural commodity that serves as food, feed, and a raw material for industrial purposes. It is organized into starch granules (SG) inside amyloplasts and is highly accumulated in endosperms. Maize hybrids, which exhibits heterosis over their parents, are globally grown due to higher vigor of the F1 plants. However, the parental effect on the size of SG in F1 hybrid seeds remains unclear. Here we compared the seed SG sizes among two parental inbred lines (Chang7-2 and Zheng58) as well as their reciprocal hybrids. SG was observed in situ and in vitro with SEM. The size of seed SG in hybrids was more like that of female parents, especially for large SG population. Thus, the control of SG size exhibits a maternal inheritance trend in the context of plastid (amyloplast) inheritance. Our results provide some insight on selecting parental inbred lines for breeding maize hybrids with different SG sizes.

Cysteine proteases, belonging to the C1-papain family, play a major role in plant growth and development, senescence, and immunity. There is evidence to suggest that pollen cysteine protease (CP) (ZmCP03) is involved in regulating the anther development and pollen formation in maize. However, there is no report on the genome-wide identification and comparison of CPs in the pollen coat and other tissues in maize. In this study, a total of 38 homologous genes of ZmCP03 in maize were identified. Subsequently, protein motifs, conserved domains, gene structures, and duplication patterns of 39 CPs are analyzed to explore their evolutionary relationship and potential functions. The cis-elements were identified in the upstream sequence of 39 CPs, especially those that are related to regulating growth and development and responding to environmental stresses and hormones. The expression patterns of these genes displayed remarked difference at a tissue or organ level in maize based on the available transcriptome data in the public database. Quantitative reverse transcription PCR (RT-qPCR) analysis showed that ZmCP03 was preferably expressed at a high level in maize pollen. Analyses by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot, immunofluorescence and immunogold electron microscopy all validated the cellular localization of ZmCP03 in both the pollen coat and pollen cytoplasm. In addition, 142 CP genes from Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa) and cotton (Gossypium hirsutum), together with 39 maize CPs, were retrieved to analyze their evolution by comparing with orthologous genes. The results suggested that ZmCP03 was relatively conservative and stable during evolution. This study may provide a referential evidence on the function of ZmCP03 in pollen development and germination in maize.

BackgroundThe mesocotyl connects the coleoptilar node and the basal part of the seminal root of maize (Zea mays) seedling. The mesocotyl pushes the shoot of the seedling out of the soil during seed germination; thus, its growth is highly related to deep-sowing tolerance. Although many studies on the maize mesocotyl have been carried out at physiological and molecular levels, the proteomic changes associated with cellular and physiological activities during mesocotyl growth are still unknown.ResultsIn the present study, the maize hybrid Zhengdan 958 was used to study mesocotyl growth and accompanying protein changes.The dark-grown etiolated mesocotyls exhibited a slow-fast-slow feature, with significant changes in the levels of indole-3-acetic acid (IAA) and cellulose and the activity of peroxidase (POD). In particular, POD activity increased with mesocotyl growth, showing higher activity at the mature (lower) end of the mesocotyl. For the proteomic analysis, soluble proteins were extracted from etiolated mesocotyls dark-grown for 48 h, 84 h, and 132 h, corresponding to the initial, rapid, and slow growth periods, respectively, and subjected to separation by two-dimensional gel electrophoresis (2-DE). As a result, 88 differentially abundant proteins (DAPs) were identified using MALDI-TOF-TOF analysis. At 48 h, most DAPs were stress proteins, heat shock proteins and storage proteins; at 84 h, oxidation/reduction proteins, carbohydrate biogenesis-related proteins and cytoskeleton-related proteins were highly accumulated; at 132 h, the most striking DAPs were those involved in the synthesis and modification of the cell wall and the biogenesis of carbohydrates. Gene ontology (GO) analysis showed that changes in the abundance and proportion of DAPs were consistent with cellular and physiological activities and biological processes during mesocotyl growth. The accumulation of nine DAPs of interest was verified by immunoblotting and RT-qPCR.ConclusionsThe present study revealed that the protein patterns in 2-D gels differed greatly with mesocotyl growth. At different growth periods, a specific set of DAPs participate in various biological processes and underlie the cellular and physiological activities of the mesocotyl. These results contributed to the understanding of mesocotyl growth and the cultivation of maize lines with deep-sowing tolerance.

Seeds store lipids in the form of lipid bodies (LBs) for germination and early seedling growth. LBs can be easily isolated by the established floating-extraction method from oleaginous seeds containing a large quantity of LBs. Compared to oleaginous seeds, maize and other cereal seeds contain a small quantity of LBs, so it is difficult to isolate a sufficient quantity of LBs from their embryos for 2DE-based proteomic analysis. At present, only a limited number of LBs-associated proteins in maize embryos have been identified. We here reported a modified floating-extraction method using polyvinylidene difluoride disc to collect floating LBs from maize embryo extracts. The LBs-associated proteins were resolved with two-dimensional electrophoresis and identified with mass spectrometry. As a result, several well-known LBs proteins were identified in the purified LBs fraction, such as oleosin, caleosin, and steroleosin. We also identified another two LBs proteins, corticosteroid 11-β-dehydrogenase 1 and 11-β-hydroxysteroid dehydrogenase-like 5. In particular, steroleosin, corticosteroid 11-β-dehydrogenase 1, 11-β-hydroxysteroid dehydrogenase-like, and hydroxyproline-rich glycoprotein were found as the most abundant protein components in maize LBs. The data set of maize LBs subproteome would provide insights into functional research of LBs-associated proteins during seed development and germination. Additionally, the protocol developed here is expected to be applicable for isolating LBs in other seeds or tissues containing a low quantity of LBs.

[This corrects the article DOI: 10.1371/journal.pone.0202238.].

Additional file 3: Table S3. Primers used in RT-qPCR.

Duckweed, a small floating aquatic plant in the family Lemnaceae, has considerable potential for agricultural and environmental applications. Proteomic analysis will facilitate the study of plant biology and plant-environment interactions. A reliable and reproducible protein extraction method is critical for successful proteomic analysis. To date, there have been no evaluation studies of extraction methods for duckweed proteomics. In this study, we developed a trichloroacetic acid (TCA)/acetone/TCA precipitation method (TAT) for Spirodela polyrhiza and compared it with three extraction methods, primarily by two-dimensional gel electrophoresis (2DE). These methods were based on TCA/acetone precipitation, phenol extraction or their combination to concentrate proteins and remove interfering substances. As a result, these methods produced significant differences in protein yield, and each revealed a unique set of proteins. In general, TAT produced better 2DE protein patterns with high reproducibility regarding the number, coverage and abundance of protein spots. Our results provide useful information for selecting suitable extraction methods for proteomic analysis of duckweed plants.

Comparative proteomics is widely used to detect protein changes, especially differential abundance proteins (DAPs) that are involved in plant responses to development, disease, or environment. Once DAPs are identified, it is essential to validate any change in their abundance, and their role in the biological process under study. In addition to common confirmation by quantitative RT-PCR, immunoblot, and multiple reaction monitoring analysis, it has been proposed that enzyme activity assay (EAA) can be complementary to the standard proteomics results, especially regarding the elucidation of protein (enzyme) function and the mechanism of enzyme-associated biochemical or metabolic pathways. The enzymes discussed here are the DAPs identified in comparative plant proteomics. Despite the small number of enzymes in a proteome, they often make up a substantial proportion of the DAPs identified in comparative studies. Currently, only a few studies have performed EAA to complement the interpretation of proteomic data, especially activity-based protein profiling. This viewpoint aims to arouse the attention of proteomic researchers on the promising role of EAA in plant proteomics and highlights the need for high-throughput assays of enzyme activities in comparative plant proteomics.

Maize starch is an important agricultural commodity that serves as food, feed, and a raw material for industrial purposes. Starch biosynthesis in maize endosperm is an essential process for maize productivity under changing environmental conditions. However, the molecular mechanism underlying starch synthesis during maize endosperm development remains largely unknown. Recently, proteomic analyses of starch synthesis in maize seeds have produced large amounts of data, which significantly add to our understanding of maize starch biosynthesis and facilitate knowledge‐based maize improvements. In this review, the current knowledge on starch biosynthesis in maize endosperm is introduced, key aspects of protein extraction for proteomic analysis of maize starch biosynthesis are discussed, and the recent proteomic findings are summarized. A model outlining the roles of specific enzymes is then proposed. The important role of proteomics in identifying the key proteins involved in starch biosynthesis is highlighted in order to further the understanding of the complex mechanisms underlying maize starch biosynthesis.

Protein extracts obtained from cells or tissues often require removal of interfering substances for the preparation of high-quality protein samples in proteomic analysis. A number of protein extraction methods have been applied to various biological samples. TCA/acetone precipitation and phenol extraction, a common method of protein extraction, is thought to minimize protein degradation and activity of proteases as well as reduce contaminants like salts and polyphenols. However, the TCA/acetone precipitation method relies on the complete pulverization and repeated rinsing of tissue powder to remove the interfering substances, which is laborious and time-consuming. In addition, by prolonged incubation in TCA/acetone, the precipitated proteins are more difficult to re-dissolve. We have described a modified method of TCA/acetone precipitation of plant proteins for proteomic analysis. Proteins of cells or tissues were extracted using SDS-containing buffer, precipitated with equal volume of 20% TCA/acetone, and washed with acetone. Compared to classical TCA/acetone precipitation and simple acetone precipitation, this protocol generates comparable yields, spot numbers, and proteome profiling, but takes less time (ca. 45 min), thus avoiding excess protein modification and degradation after extended-period incubation in TCA/acetone or acetone. The modified TCA/acetone precipitation method is simple, fast, and suitable for proteomic analysis of various plant tissues in proteomic analysis.