Chengjin Guo

Overexpression of VP, a vacuolar H+-pyrophosphatase gene in wheat (Triticum aestivum L.), improves tobacco plant growth under Pi and N deprivation, high salinity, and drought

Establishing crop cultivars with strong tolerance to P and N deprivation, high salinity, and drought is an effective way to improve crop yield and promote sustainable agriculture worldwide. A vacuolar H+-pyrophosphatase (V-H+-PPase) gene in wheat (TaVP) was functionally characterized in this study. TaVP cDNA is 2586-bp long and encodes a 775-amino-acid polypeptide that contains 10 conserved membrane-spanning domains. Transcription of TaVP was upregulated by inorganic phosphate (Pi) and N deprivation, high salinity, and drought. Transgene analysis revealed that TaVP overexpression improved plant growth under normal conditions and specifically under Pi and N deprivation stresses, high salinity, and drought. The improvement of growth of the transgenic plants was found to be closely related to elevated V-H+-PPase activities in their tonoplasts and enlarged root systems, which possibly resulted from elevated expression of auxin transport-associated genes. TaVP-overexpressing plants showed high dry mass, photosynthetic efficiencies, antioxidant enzyme activities, and P, N, and soluble carbohydrate concentrations under various growth conditions, particularly under the stress conditions. The transcription of phosphate and nitrate transporter genes was not altered in TaVP-overexpressing plants compared with the wild type, suggesting that high P and N concentrations regulated by TaVP were caused by increased root absorption area instead of alteration of Pi and NO3 − acquisition kinetics. TaVP is important in the tolerance of multiple stresses and can serve as a useful genetic resource to improve plant P- and N-use efficiencies and to increase tolerance to high salinity and drought.

Identification and characterization of microRNAs from wheat (Triticum aestivum L.) under phosphorus deprivation

Plant microRNAs (miRNAs) are non-coding RNAs (19–24 nucleotides long) that play a critical role in the sequence-specific regulation of target gene transcripts. In this study, 32 miRNAs from wheat (Triticum aestivum L.) (TaMIRs) currently released in the miRBase database were subjected to expression pattern analysis under conditions of normal inorganic phosphate (Pi) supply and Pi deprivation stress. Semi-quantitative and quantitative reverse transcriptase polymerase chain reaction analysis revealed that 9 TaMIRs responded to Pi starvation: TaMIR159b, TaMIR167, TaMIR399, TaMIR408, TaMIR1122, TaMIR1125, TaMIR1135, TaMIR1136, and TaMIR1136 were up-regulated, whereas TaMIR408 was down-regulated. Small RNA blot analysis confirmed these results. Target prediction analysis indicated that the low Pi-responsive TaMIRs possessed variable target genes, ranging from none in TaMIR399 and TaMIR1122 to more than 20 in TaMIR1136. The target genes randomly selected from each low Pi-responsive TaMIR (except TaMIR399 and TaMIR1122) demonstrated an opposite expression pattern to the TaMIR, suggesting that the target genes were transcriptionally regulated by miRNA-mediated pathways. The target genes that interacted with the low Pi-responsive TaMIRs could be classified into diverse gene families, such as those involving transcription regulation, cell cycling, chromosome establishment, signal transduction, primary metabolism, phytohormone response, trafficking, defense response, and protein degradation. This study helps elucidate the plant regulatory mechanisms in response to low Pi signaling via the miRNA-mediated pathways in wheat.

Overexpression of TaNHX3, a vacuolar Na+/H+ antiporter gene in wheat, enhances salt stress tolerance in tobacco by improving related physiological processes

Salt stress is one of the major abiotic stresses affecting plant growth, development, and productivity. In this study, we functionally characterized a wheat vacuolar Na(+)/H(+) antiporter gene (TaNHX3). TaNHX3 is 78.9% identical with TaNHX2 in nucleic acid level, encoding a polypeptide of 522 amino acids (aa). TaNHX3 is targeted onto tonoplast after ER sorting and can complement the growth under salt stress in a yeast mutant with a defective vacuolar Na(+)/H(+) antiporter exchange. TaNHX3 transcripts were induced by applying salt stress in wheat cultivars. More TaNHX3 were detected in the salt-stress-resistant cultivar Ji 7369 compared with the salt-stress-sensitive cultivar Shimai 12 and Ji-Shi-3, an isogenic line derived from aforementioned cultivars with Shimai 12 genetic background. The ectopic TaNHX3 expression in tobacco significantly enhanced the plant tolerance to salt stress. Compared with control plants, the TaNHX3 overexpressing plants displayed no varied Na(+) contents and accumulated more Na(+) amount in plants. However, they exhibited higher fresh and dry weights, more accumulative nitrogen, phosphorus, and potassium, higher contents of chlorophyll, carotenoid, soluble protein, higher activities of the antioxidant enzymes including superoxide dismutase, catalase, and peroxidase, and lower malondialdehyde and H2O2 amount. Our results indicated that TaNHX3 plays an important role in regulating the cytosolic Na(+) transportation within vacuoles under high salinity, alleviating the Na(+) damage effects. The improved salt stress tolerance in TaNHX3 overexpressing tobacco plants is closely associated with the improvement of the aforementioned physiological processes. TaNHX3 can be used as a candidate gene for molecular breeding of salt-tolerant plants.

Overexpression of TaSRK2C1 , a Wheat SNF1-Related Protein Kinase 2 Gene, Increases Tolerance to Dehydration, Salt, and Low Temperature in Transgenic Tobacco

Protein phosphorylation–dephosphorylations are major signaling events induced by osmotic stress in plants. In this study, a wheat SNF1-related protein kinase 2 (SnRK2) gene, TaSRK2C1, was functionally characterized. The results from the sequence analysis showed that TaSRK2C1 contains conserved domains typified in SnRK2 protein kinases, including the ATP binding site, N-myristoylation site, protein kinase-activating signature, and transmembrane-spanning region. The transcripts of TaSRK2C1 in roots were induced by treatments of dehydration, high salinity, low temperature, and exogenous abscisic acid, which suggest its potential roles relative to osmotic stress signal transductions. The ectopic expression of TaSRK2C1 in tobacco significantly up-regulated the expression levels of three putative central regulators, namely, RD29a, DREB1A, and DREB2, which are involved in responding to osmotic stresses. Thus, higher levels of free proline and soluble carbohydrates in transgenic plants were detected, and conferred tolerance to high salinity, dehydration stress, and low temperature in plants. The overall results in this study indicate that TaSRK2C1 have important functions in plant response and adaptation to osmotic stresses via mediation of signal transductions initiated by distinct abiotic stresses. Manipulating TaSRK2C1 toward improving the osmotic-stress tolerance in crop plants is feasible.

Wheat microRNA Member TaMIR444a Is Nitrogen Deprivation-Responsive and Involves Plant Adaptation to the Nitrogen-Starvation Stress

AbstractmicroRNAs (miRNAs) are involved in regulating various plant developmental processes and mediating plant-adaptive responses to nutrient deprivation. In this study, the characterization of a wheat miRNA member TaMIR444a and the role of this miRNA in mediating plant tolerance to the N-starvation stress were investigated. Results indicated that the expression levels of TaMIR444a and NtMIR444a, the homologue of TaMIR444a in tobacco, were upregulated in roots and leaves under N deprivation, whereas the transcription of their target genes showed reverse expression patterns in above tissues. These results suggest that miR444a is conserved across plant species of dicots and monocots and can possibly establish the miRNA/target modules for mediating plant response to N deficiency. Overexpression of TaMIR444a in tobacco improved the plant growth feature, biomass, N content, photosynthetic parameters, and antioxidant enzymatic activities under N deprivation. Based on microarray analyses, a large number of genes were identified to be differentially expressed in the TaMIR444a-overexpressing plants; these differential genes are categorized into functional groups of signal perception and transduction, transcription regulation, primary and secondary metabolism, phytohormone response, cellular protection and defensive responsiveness, etc. qPCR analyses revealed that the nitrate transporter (NRT) genes NtNRT1.1-s, NtNET1.1-t, and NtNRT2.1 and the antioxidant enzyme genes (AEEs) NtCAT1;1, NtPOD1;3, and NtPOD4 were significantly upregulated by TaMIR444a, suggesting that the altered transcription of these NRT and AEE genes is associated with the improvement of the N acquisition and the cellular ROS detoxification in the N-deprived transgenic plants. Together, our findings demonstrate that miR444a acts as one critical regulator in mediating plant tolerance to the N-starvation stress through modulation of the regulatory networks associated with N acquisition, cellular ROS homeostasis, and carbon assimilation. Our findings have provided insights into the mechanisms of plant tolerance to N deficiency mediated by the distinct miRNA pathways.

Characterization and expression pattern analysis of microRNAs in wheat under drought stress

Plant microRNAs (miRNAs) play important roles in regulating plant growth, development, and responses to abiotic stresses. In this study, 38 miRNAs (TaMIRs) from wheat (Triticum aestivum L.), 36 from the miRBase database, and two from our previous work were characterized and subjected to an expression pattern analysis under normal conditions and a drought stress. A semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR), real-time quantitative PCR (qPCR), and small RNA blot analyses revealed that two TaMIRs (TaMIR1120 and TaMIR1123) were root-predominant and two TaMIRs (TaMIR1121 and TaMIR1134) were leaf-predominant. Seven TaMIR precursors showed altered expressions after the drought; of these, TaMIR1136 was upregulated, whereas TaMIR156, TaMIR408, TaMIR1119, TaMIR1129, TaMIR1133, and TaMIR1139 were downregulated. These seven drought-responsive TaMIRs showed dose-dependent and typical temporal expression patterns during drought induction, and they gradually returned back under the normal growth conditions. The drought-responsive and the tissue-predominant TaMIRs had varying numbers of target genes. Randomly selected target genes exhibited opposite expression patterns to their corresponding TaMIRs suggesting that they were regulated by distinct TaMIRs through a post-transcriptional cleavage. The target genes regulated by drought-responsive and tissue-predominant TaMIRs are involved in various cellular processes, such as signal transduction, transcriptional regulation, primary and secondary metabolisms, development, and defense responses. These results provide a novel insight into the miRNA-mediated responses of wheat to drought stress.

Characterization and expression analysis of mitogen-activated protein kinase cascade genes in wheat subjected to phosphorus and nitrogen deprivation, high salinity, and drought

Mitogen-activated protein kinase (MAPK) cascades are conserved signal-transducing modules that have important functions in plant growth and development as well as in diverse biotic and abiotic stress responses. In this study, six MAP kinase kinase kinase genes, two MAP kinase kinase genes, and 11 MAP kinase genes in wheat were characterized. The MAPK cascade members of wheat were named based on their homologs in Brachypodium distachyon and Arabidopsis. The polypeptides translated from these MAPK cascade genes share conserved domains similar to those reported in B. distachyon and Arabidopsis, and such domains are involved in protein interaction and phosphorylation. Expression analysis results of the MAPK cascade members revealed that some of the selected genes were involved in responses to phosphorus (P; ten genes) and nitrogen (N; five genes) deprivation, salinity (five genes), and drought (three genes). Such genes were either upregulated or downregulated. Temporal expression profile analysis of the stress-responsive MAPK members indicated that all of these genes were progressively regulated by stress and exhibited typical recovery responses when these genes were exposed again to normal growth conditions. Two MAPK members, TaMPK6 and TaMPK16, responded to four stress factors, including deprivations of P and N as well as salt and drought stress. TaMPK4, TaMPK12;1, TaMPK14, and TaMPK17 were responsive to two stress factors. These results suggested that various MAPK cascade members in wheat are involved in mediating signal transductions by transcriptionally regulating P and N deprivations, high salinity, and drought. TaMPK6 and TaMPK16, together with TaMPK4, TaMPK12;1, TaMPK14, and TaMPK17 have potential functions in mediating plant responses and tolerance to two or more stress factors by distinct MAPK cascade modules. Our data have provided information about MAPK modules and put further insight into their biological functions in abiotic stress response of wheat.

Expression Pattern Analysis of Zinc Finger Protein Genes in Wheat (Triticum aestivum L.) Under Phosphorus Deprivation

Abstract Zinc finger protein (ZFP) genes comprise a large and diverse gene family, and are involved in biotic and abiotic stress responses in plants. In this study, a total of 126 ZFP genes classified into various types in wheat were characterized and subjected to expression pattern analysis under inorganic phosphate (Pi) deprivation. The wheat ZFP genes and their corresponding GenBank numbers were obtained from the information of a 4×44K wheat gene expression microarray chip. They were confirmed by sequence similarity analysis and named based on their homologs in Brachypodium distachyon or Oriza sativa. Expression analysis based on the microarray chip revealed that these ZFP genes are categorized into 11 classes according to their gene expression patterns in a 24-h of Pi deprivation regime. Among them, ten genes were differentially up-regulated, ten genes differentially down-regulated, and two genes both differentially up- and down-regulated by Pi deprivation. The differentially up- or down-regulated genes exhibited significantly more or less transcripts at one, two, or all of the checking time points (1, 6, and 24 h) of Pi stress in comparison with those of normal growth, respectively. The both differentially up- and down-regulated genes exhibited contrasting expression patterns, of these, TaWRKY70;5 showed significantly up-regulated at 1 and 6 h and down-regulated at 24 h whereas TaAN1AN20-8;2 displayed significantly upregulated at 1 h and downregulated at 6 h under deprivation Pi condition. Real time PCR analysis confirmed the expression patterns of the differentially expressed genes obtained by the microarray chip. Our results indicate that numerous ZFP genes in wheat respond to Pi deprivation and have provided further insight into the molecular basis that plants respond to Pi deprivation mediated by the ZFP gene family.

Expression pattern analysis of microRNAs in root tissue of wheat (Triticum aestivum L.) under normal nitrogen and low nitrogen conditions

MicroRNAs (miRNAs) are highly conserved non-coding small RNAs involved in regulating plant growth and development, as well as plant responses, to diverse environmental signaling cues. In this study, the expression patterns of 38 wheat microRNAs (TaMIRs) were investigated under normal N and low N stress. Under normal N conditions, the TaMIRs exhibited four expression patterns: high, moderate, low, and undetectable expression. Seven TaMIRs (TaMIR156, TaMIR399, TaMIR444, TaMIR1118, TaMIR1129, TaMIR1133, and TaMIR1136) showed varied expression levels under N deprivation. TaMIR156, TaMIR444, TaMIR1118, TaMIR1129, and TaMIR1136 were upregulated, whereas TaMIR399 and TaMIR1133 were downregulated. The expression patterns of TaMIR444, TaMIR1118, and TaMIR1129 were further analyzed under various low N concentrations and then returned to normal N. The aforementioned TaMIRs exhibited ever higher expression at lower N concentrations and whose expressions returned to those before low N stress after they were restored to normal N conditions, which suggest that N concentration and low N-duration are inversely correlated with their expression. The putative target genes of the low N–responsive TaMIRs were identified and categorized into various functional groups. Expression analysis revealed that the target genes showed opposite pattern to that of their corresponding TaMIRs. Our results suggested that some low N–responsive TaMIRs are involved in regulating plant response to N deprivation by interacting with their target genes. Distinct TaMIRs are potentially involved in plant tolerance to low N stress through microRNA-mediated pathways.

Investigation of Differential Expressed Genes Responding to Deficient-Pi in Wheat as Revealed by cDNA-AFLP Analysis

Abstract The objective of this study was to disclose the possible mechanism of tolerance to Pi-deficiency in wheat (Triticum aestivum L.) at the gene-expression level. Seedlings of wheat cultivar Shixin 828, which has high phosphorus use efficiency, were treated with 20 µmol L−1 Pi for short (1–6 h), medium (12–48 h), and long terms (72–144 h). The differentially expressed sequence tags (ESTs) were identified using complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) technique. A total of 143 and 94 nonredundant differential ESTs with up- and down-regulated patterns were identified, of which 23, 54, and 66 ESTs enhanced expression after the short-, medium-, and long-term treatments, and 17, 39, and 38 ESTs declined expression after the 3 treatments, respectively. These ESTs were classified into several functional groups according to the BLAST analysis. The up-regulated ESTs, except for 44 with unknown function, confer functions of signal transduction, transcription regulation, metabolism, stress response, development, transport, and lipid metabolism. The down-regulated ESTs are involved in the above functions and protein synthesis and protein degradation. Under the Pi-deficient condition, some genes were specifically up-regulated, such as OsPTF1 and ZAT10 homologues and genes encoding mitogen activated protein kinase, calcium-dependent protein kinase and protein kinase, high-affinity phosphate transporter, peroxidase and glutathione S-transferase. These genes are conjectured to play important roles in promoting adaptation to Pi-deficient environment.