Juntao Gu

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.

Wheat nuclear factor Y (NF-Y) B subfamily gene TaNF-YB3;l confers critical drought tolerance through modulation of the ABA-associated signaling pathway

Acting as an indispensible subunit of nuclear factor-Y (NF-Y), a heterotrimeric transcription factor in eukaryotes, NF-YB proteins play important roles in regulating plant responses to abiotic stresses. In this study, a wheat (Triticum aestivum) NY-YB gene referred to as NtNF-YB3;l was functionally characterized for mediation of the drought stress. NtNF-YB3;l shares a high similarity to its counterparts across various plant species. The transcript abundance of TaNF-YB3;l was significantly up-regulated in roots and leaves upon drought and exogenous abiscisic acid (ABA), suggesting its role in regulating drought response possibly through an ABA-associated pathway. The TaNF-YB3;l overexpression lines exhibited improved growth under drought, which is consistent with behaviors of the transgenic lines with promoted stomata closure rate, enhanced leaf water retention capacity, and increased antioxidant enzyme activities and osmolyte accumulation. The genes encoding peroxidase (POD) genes (i.e., NtPOD1;3, NtPOD1;5, and NtPOD2;1) showed up-regulated expression in TaNF-YB3;l -overexpressing plants treated by simulated drought, suggesting their contribution to the TaNF-YB3;l-mediated POD activities. Transcripts of the ABA receptor gene NtPYL4 and PIN-FORMed (PIN) gene NtPIN4 were up-regulated in TaNF-YB3;l overexpression lines; overexpression or knockdown of them validated their roles in regulating plant drought response and modifying root system architecture (RSA) establishment, respectively. Our investigation indicates that TaNF-YB3;l is crucial for plant drought tolerance largely through its function in modulating an ABA-associated signaling pathway, which impacts on the physiological processes associated with stomata movement, reactive oxygen species (ROS) metabolsim, osmolyte accumulation, and RSA establishment.

Transcriptional Regulation of the Rice Phytase Gene OsPHY1 by Several Phytohormones and Osmotic Stresses Using Promoter-GUS Analysis

During germination, phytate, a major phosphate compound in seeds, is degraded into inorganic phosphorus (Pi) for differentiation of new tissues via catalysis. OsPHY1, a member of phytase genes in rice, has previously been found to show high expression levels in the endosperm and hypocotyl during germination and is involved in the degradation of seed phytate. In this study, we investigated the transcriptional mechanisms of OsPHY1 via OsPHY1 promoter-GUS analysis. The results of GUS histochemical staining and activities in tobaccos harboring OsPHY1-GUS reveal that the reporter gene is strongly expressed in the hypocotyl and responds to diverse stimuli cues initiated by phytohormones abscisic acid (ABA), gibberellin (GA3), and indole-3-acetic acid (IAA), as well as osmotic stresses of salt, drought, and cold. The results indicate that the cis-regulatory element CANBNNAPA regulates gene hypocotyl-predominant expression. ABRE and GAREAT are involved in gene responses to ABA and GA3, respectively, and DRECRTCOREAT is involved in gene responses to stresses of salt, drought, and cold. In addition, the responses of OsPHY1 and part-osmotic stress-responsive genes to salt, drought, and cold mediated by DRECRTCOREAT are largely accomplished through the ABA-dependent pathway with the involvement of the ABA responsive cis-regulatory element ABRE. Our results reveal that the degradation of seed phytate during germination mediated by OsPHY1 is regulated in a fine-tune manner with the involvement of diverse cis-regulatory elements at the transcription level.