Differential Transcription Profiling Reveals the MicroRNAs Involved in Alleviating Damage to Photosynthesis under Drought Stress during the Grain Filling Stage in Wheat.

To explore the possible novel microRNA (miRNA) regulatory pathways in Zhengmai 1860, a newly cultivated drought-tolerant wheat (Triticum aestivum L.) cultivar, miRNA transcriptome sequencing of the flag leaves of Zhengmai 1860, drought-sensitive variety Zhoumai 18, and drought-resistant variety Bainong 207 was performed during the grain filling stage. We also observed changes in the chloroplast ultrastructure, phytohormone levels, and antioxidant- and photosynthesis-related physiological indicators in three wheat varieties. The results showed that the flag leaves of the drought-tolerant variety Zhengmai 1860 had higher chlorophyll contents and net photosynthetic rates than those of Zhoumai 18 under drought stress during the grain filling stage; in addition, the chloroplast structure was more complete. However, there was no significant difference between Zhengmai 1860 and Bainong 207. MiRNA transcriptome analysis revealed that the differential expression of the miRNAs and mRNAs exhibited variable specificity. The KEGG pathway enrichment results indicated that most of the genes were enriched in the MAPK signaling pathway, plant hormone signal transduction, photosynthetic antennae protein, and amino acid and carbohydrate metabolism. In the drought-tolerant cultivar Zhengmai 1860, tae-miR408 was targeted to regulate the allene oxide synthase (AOS) gene, inhibit its expression, reduce the AOS content, and decrease the synthesis of jasmonic acid (JA) and abscisic acid (ABA). The results of this study suggest that Zhengmai 1860 could improve the photosynthetic performance of flag leaves by inhibiting the expression of genes involved in the JA pathway through miRNAs under drought conditions. Moreover, multiple miRNAs may target chlorophyll, antioxidant enzymes, phytohormone signal transduction, and other related pathways; thus, it is possible to provide a more theoretical basis for wheat molecular breeding.

Genome-wide association study of agronomic traits related to nitrogen use efficiency in Henan wheat.

BACKGROUND: Nitrogen use efficiency (NUE) is closely related to crop yield and nitrogen fertilizer application rate. Although NUE is susceptible to environments, quantitative trait nucleotides (QTNs) for NUE in wheat germplasm populations have been rarely reported in genome-wide associated study. RESULTS: In this study, 244 wheat accessions were phenotyped by three NUE-related traits in three environments and genotyped by 203,224 SNPs. All the phenotypes for each trait were used to associate with all the genotypes of these SNP markers for identifying QTNs and QTN-by-environment interactions via 3VmrMLM. Among 279 QTNs and one QTN-by-environment interaction for low nitrogen tolerance, 33 were stably identified, especially, one large QTN (r2 > 10%), qPHR3A.2, was newly identified for plant height ratio in one environment and multi-environment joint analysis. Among 52 genes around qPHR3A.2, four genes (TraesCS3A01G101900, TraesCS3A01G102200, TraesCS3A01G104100, and TraesCS3A01G105400) were found to be differentially expressed in low-nitrogen-tolerant wheat genotypes, while TaCLH2 (TraesCS3A01G101900) was putatively involved in porphyrin metabolism in KEGG enrichment analyses. CONCLUSIONS: This study identified valuable candidate gene for low-N-tolerant wheat breeding and provides new insights into the genetic basis of low N tolerance in wheat.

Overexpression of the Wheat TaPsb28 Gene Enhances Drought Tolerance in Transgenic Arabidopsis.

Psb28 is a soluble protein in the photosystem II (PSII) complex, but its role in the drought stress response of wheat remains unclear. Here, we functionally characterized the TaPsb28 gene, which positively regulates drought tolerance in wheat. When the full-length 546-bp TaPsb28 cDNA was transferred into Arabidopsis thaliana, it was located in the guard cell chloroplast around the stroma. Overexpression of TaPsb28 conferred drought tolerance, as exhibited by the increases in the survival rate. Transgenic plants maintained lower MDA content and higher chlorophyll content by inducing chlorophyll synthase (ChlG) gene transcription. The content of abscisic acid (ABA) and zeatin increased significantly in wild-type (WT) plants under drought stress, and the transcriptional expression levels of RD22, dihydroflavonol 4-reductase (DFR) and anthocyanin reductase (ANR) genes were induced, thus enhancing the contents of endogenous cyanidin, delphinidin, and proanthocyanidins. However, in transgenic plants, although anthocyanins were further aggregated, the ABA increase was inhibited, zeatin was restored to the control level under drought stress, and stomatal closure was promoted. These findings indicate ABA and zeatin have opposite synergistic effects in the process of drought tolerance caused by TaPsb28 because only after the effect of zeatin is alleviated can ABA better play its role in promoting anthocyanin accumulation and stomatal closure, thus enhancing the drought tolerance of transgenic plants. The results suggest that overexpression of TaPsb28 exerts a positive role in the drought response by influencing the functional metabolism of endogenous hormones. The understanding acquired through the research laid a foundation for further in-depth investigation of the function of TaPsb28 in drought resistance in wheat, especially its relationship with anthocyanidin accumulation.

GmTDN1 improves wheat yields by inducing dual tolerance to both drought and low-N stress.

Genetically enhancing drought tolerance and nutrient use efficacy enables sustainable and stable wheat production in drought-prone areas exposed to water shortages and low soil fertility, due to global warming and declining natural resources. In this study, wheat plants, exhibiting improved drought tolerance and N-use efficacy, were developed by introducing GmTDN1, a gene encoding a DREB-like transcription factor, into two modern winter wheat varieties, cv Shi4185 and Jimai22. Overexpressing GmTDN1 in wheat resulted in significantly improved drought and low-N tolerance under drought and N-deficient conditions in the greenhouse. Field trials conducted at three different locations over a period of 2-3 consecutive years showed that both Shi4185 and Jimai22 GmTDN1 transgenic lines were agronomically superior to wild-type plants, and produced significantly higher yields under both drought and N-deficient conditions. No yield penalties were observed in these transgenic lines under normal well irrigation conditions. Overexpressing GmTDN1 enhanced photosynthetic and osmotic adjustment capacity, antioxidant metabolism, and root mass of wheat plants, compared to those of wild-type plants, by orchestrating the expression of a set of drought stress-related genes as well as the nitrate transporter, NRT2.5. Furthermore, transgenic wheat with overexpressed NRT2.5 can improve drought tolerance and nitrogen (N) absorption, suggesting that improving N absorption in GmTDN1 transgenic wheat may contribute to drought tolerance. These findings may lead to the development of new methodologies with the capacity to simultaneously improve drought tolerance and N-use efficacy in cereal crops to ensure sustainable agriculture and global food security.

Transgenic expression of rice OsPHR2 increases phosphorus uptake and yield in wheat.

Oryza sativa PHOSPHATE RESPONSE2 (OsPHR2) can promote the uptake and use of phosphorus (P) in rice. We introduced OsPHR2 into the winter wheat (Triticum aestivum L.) variety "Zhengmai0856." OsPHR2 was integrated into the wheat genome with two copy numbers and could be correctly transcribed and expressed. OsPHR2 was mainly expressed in the leaves at the seedling stage. From the jointing to filling stage, OsPHR2 was mainly expressed in the roots, followed by the leaves, with a low expression level in detected the tassels and stems. The transgenic lines exhibited higher P accumulation at each growth stage and increased P uptake intensity in comparison to the wild type under low P and high P conditions. Analysis of the root characteristics showed that the transgenic expression of OsPHR2 increased the maximum root length, total root length, root-to-shoot ratio, and root volume under the conditions of P deficiency or low P. A field experiment showed that the transgenic lines had a higher grain yield than the wild type under low P and high P conditions. The yield of the transgenic lines increased by 6.29% and 3.73% on average compared with the wild type under low P and high P conditions, respectively. Thus, the transgenic expression of OsPHR2 could increase P uptake and yield in wheat, but the effect was more prominent under low P conditions.

Assessment of chronic disease management mode (CDMM) on participants with primary hypertension.

OBJECTIVE: Hypertension requires continuous and long-term care to prevent associated complications. Chronic disease management mode (CDMM) was developed to improve patients' self-management. We aimed to evaluate quality of care and clinical outcomes of CDMM versus routine care. METHODS: 300 patients aged >30 years with primary hypertension were randomly allocated to the CDMM intervention group (n = 162) or the usual care control group (n = 138). CDMM comprised nursing consultations, telephone contact, online WeChat link, health education, and appropriate referrals during hospitalisation and after discharge. QLICD-HY (V 2.0) scale was used to evaluate the quality of life. Care outcomes were biochemical parameters, body mass index, blood pressure levels, waist circumference, psychological indicators and quality of life assessed on admission (baseline) and one month post-care for both groups. Data were collected and analysed using SPSS 20.0. RESULTS: After one month, the intervention group had 6 mm Hg (95% CI: -5.12 to -9.08) lower SBP and 0.6 mM/L (95% CI: -0.52 to -0.68) lower LDL than the control group. In terms of improvements in BMI, UmAlb or waist circumference, there were no differences between both groups. The intervention group scored better on psychological indicators than controls (P < 0.05), and scores reflecting social and psychological function in the intervention group were significantly higher than scores at baseline, and higher than scores of controls after one month (P < 0.05). In the control group, there was no statistically significant difference between the scores at baseline and after one month. CONCLUSIONS: Under CDMM hypertension care, improvement of blood pressure and LDL was clinically significant. Intervention care further improves social and psychological function among participants with primary hypertension.

Response of transgenic Arabidopsis expressing maize C4 photosynthetic enzyme genes to high light.

This study assessed the responses of wild-type (WT) and transgenic Arabidopsis expressing seven combinations of maize (Zea mays) genes phosphoenolpyruvate carboxylase (pepc), pyruvate phosphate dikinase (ppdk), and NADP-malic enzyme (nadp-me) to high light. Our results showed that the net CO2 assimilation rate (Pn) and shoot dry weight of four of the transgenic Arabidopsis genotypes were significantly different from those of WT under high-light treatment, being in the order of Zmpepc+Zmppdk+Zmnadp-me (PC-K-M) > Zmpepc+Zmppdk (PC-K) > Zmpepc (PC), Zmpepc+Zmnadp-me (PC-M) > WT. The other genotypes did not differ from WT. This indicated that Zmpepc was essential for maintaining high photosynthetic performance under high light, Zmppdk had a positive synergistic effect on Zmpepc, and the combination of all three genes had the greatest synergistic effect. These four genotypes also maintained higher photosystem II (PSII) activity (K-phase, J-phase, RC/CSm), electron transfer capacity (J-phase), and photochemical efficiency (TRo/ABS), and accumulated less reactive oxygen species (O2·-, H2O2) and suffered less damage to the membrane system (MDA) than WT under high light. Collectively, PC, PC-K, PC-M, and PC-K-M used most of the absorbed energy for CO2 assimilation through a significantly higher Pn, which reduced the generation of excess electrons in the photosynthetic apparatus, thereby reducing damage to the membrane system and PSII. This ultimately resulted in improved high-light tolerance. Pn was the main reason for the significant difference in the high-light tolerance of the four genotypes. Joint expression of the three maize genes may be of great value in the genetic improvement of high-light tolerance in C3 crops.

Joint expression of Zmpepc, Zmppdk, and Zmnadp-me is more efficient than expression of one or two of those genes in improving the photosynthesis of Arabidopsis.

This study assessed the effects of seven combinations of maize (Zea mays) genes phosphoenolpyruvate carboxylase (pepc), pyruvate phosphate dikinase (ppdk), and NADP-malic enzyme (nadp-me), on the photosynthesis of Arabidopsis. The photosynthetic rate, carboxylation efficiency, and shoot-dry-weight of Zmpepc (PC), Zmpepc + Zmppdk (PCK), Zmpepc + Zmnadp-me (PCM), and Zmpepc + Zmppdk + Zmnadp-me (PCKM) were significantly higher than those of the control wild-type (WT), with a trends to be PCKM > PCK > PC and PCM > WT. This indicated that Zmpepc was a prerequisite for improved photosynthetic performance; Zmppdk had a positive effect on Zmpepc, and the triple gene combination had the most significant synergistic effects. PCKM significantly enhanced activity of photosystem (PS)II (K, J phase) and PSI, light energy absorption (ABS/CSm) and conversion (TRo/ABS), and electron transfer (ETo/TRo). PCKM up-regulated 18 photosynthesis-related proteins, among which, 11 were involved in light reaction resulting in improved light-energy absorption and conversion efficiency, electron transfer, activity and stability of PSII and PSI, and the ATP and NADPH production. The remaining seven proteins were involved in dark reaction. The up-regulation of these proteins in PCKM improved the coordinated operation of light and dark reaction, increasing the photosynthesis and dry weight ultimately. These results also provide a promising strategy for the genetic improvement of the photosynthetic performance of C3 crops by inserting major C4 photosynthetic genes.

Overexpression of V-type H+ pyrophosphatase gene EdVP1 from Elymus dahuricus increases yield and potassium uptake of transgenic wheat under low potassium conditions.

Lack of potassium in soil limits crop yield. Increasing yield and conserving potassium ore requires improving K use efficiency (KUE). Many genes influence KUE in plants, but it is not clear how these genes function in the field. We identified the V-type H+-pyrophosphatase gene EdVP1 from Elymus dahurica. Gene expression analysis showed that EdVP1 was induced by low potassium stress. Protein subcellular localization analysis demonstrated that EdVP1 localized on the plasma membrane. We overexpressed EdVP1 in two wheat varieties and conducted K tolerance experiments across years. Yield per plant, grain number per spike, plant height, and K uptake of four transgenic wheat lines increased significantly compared with WT; results from two consecutive years showed that EdVP1 significantly increased yield and KUE of transgenic wheat. Pot experiments showed that transgenic plants had significantly longer shoots and roots, and higher K accumulation in shoots and roots and H+-PPase activity in shoots than WT under low K. A fluidity assay of potassium ion in EdVP1 transgenic plant roots showed that potassium ion influx and H+ outflow in transgenic plants were higher than WT. Overexpressing EdVP1 significantly improved yield and KUE of transgenic wheat and was related to higher K uptake capacity in root.

Author Correction: Molecular marker assisted breeding and genome composition analysis of Zhengmai 7698, an elite winter wheat cultivar.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Molecular marker assisted breeding and genome composition analysis of Zhengmai 7698, an elite winter wheat cultivar.

Zhengmai 7698 is an elite winter wheat variety widely cultivated in the Southern regions of the Yellow-Huai River Valley of China. Here, we report the molecular markers used for breeding Zhengmai 7698 and the genome composition of this cultivar revealed using genome-wide SNPs. A total of 26 DNA markers derived from the genes controlling gluten protein quality, grain hardness, flour color, disease resistance, or pre-harvesting sprouting resistance were used during breeding. Consequently, Zhengmai 7698 had strong gluten, high grain hardness index, white flour color, and high levels of resistance to powdery mildew, stripe rust infections, and pre-harvesting sprouting. Using genome complexity reduction, 28,996 high-quality SNPs distributed on 21 wheat chromosomes were identified among Zhengmai 7698 and its three parental lines (4B269, Zhengmai 9405 and Zhoumai 16). Zhengmai 7698 shared 12,776, 14,411 and 16,085 SNPs with 4B269, Zhengmai 9405 and Zhoumai 16, respectively. Thus, the contributions of 4B269, Zhengmai 9405 and Zhoumai 16 to the genome of Zhengmai 7698 were comparable. Interestingly, Zhengmai 7698 had 307 unique SNPs that are absent in all three parents. We suggest that molecular markers facilitate selection of a wheat cultivar with multiple elite traits. Analysis of genome composition with SNPs may provide useful clues for further dissecting the genetic basis of improved wheat performance.

Physiological characteristics and metabolomics of transgenic wheat containing the maize C4 phosphoenolpyruvate carboxylase (PEPC) gene under high temperature stress.

In this paper, two transgenic wheat lines, PC27 and PC51, containing the maize PEPC gene and its wild-type (WT) were used as experimental material to study the effects of high temperature on their photosynthetic physiological characteristics and metabolome. The results showed that transgenic wheat lines had higher photosynthetic rate (P n) than WT under non-stress treatment (NT) and high temperature stress treatment (HT), and more significantly under HT. The change trends of F v/F m, Ф PSII, and q P were similar to P n, whereas that of non-photochemical quenching (NPQ) was the opposite. Compared with WT, no differences in chlorophyll content between the transgenic wheat and WT were observed under NT, but two transgenic lines had relatively higher contents than WT under HT. The change trends of Chlorophyll a/b radio, the decreased values of F m, Wk, and Vj, and the activity of the antioxidant enzyme were consistent with the chlorophyll content. Compared with WT, transgenic wheat lines exhibited lower rate of superoxide anion production, H2O2 and malondialdehyde content under HT, and no significant differences were observed under NT. The expression pattern of the ZmPEPC gene and wheat endogenous photosynthesis-related genes were in agreement with that of P n. Compared with WT, about 13 different metabolites including one organic acid, six amino acids, four sugars, and two polyols were identified under NT; 25 different metabolites including six organic acids, 12 amino acids, four sugars, and three polyols were identified under HT. Collectively, our results indicate that ZmPEPC gene can enhance photochemical and antioxidant enzyme activity, upregulate the expression of photosynthesis-related genes, delay degradation of chlorophyll, change contents of proline and other metabolites in wheat, and ultimately improves its heat tolerance.

Drought tolerance and proteomics studies of transgenic wheat containing the maize C4 phosphoenolpyruvate carboxylase (PEPC) gene.

Enhancing drought tolerance of crops has been a great challenge in crop improvement. Here, we report the maize phosphoenolpyruvate carboxylase (PEPC) gene was able to confer drought tolerance and increase grain yield in transgenic wheat (Triticum aestivum L.) plants. The improved of drought tolerance was associated with higher levels of proline, soluble sugar, soluble protein, and higher water use efficiency. The transgenic wheat plants had also a more extensive root system as well as increased photosynthetic capacity during stress treatments. The increased grain yield of the transgenic wheat was contributed by improved biomass, larger spike and grain numbers, and heavier 1000-grain weight under drought-stress conditions. Under non-stressed conditions, there were no significant increases in these of the measured traits except for photosynthetic rate when compared with parental wheat. Proteomic research showed that the expression levels of some proteins, including chlorophyll A-B binding protein and pyruvate, phosphate dikinase, which are related to photosynthesis, PAP fibrillin, which is involved in cytoskeleton synthesis, S-adenosylmethionine synthetase, which catalyzes methionine synthesis, were induced in the transgenic wheat under drought stress. Additionally, the expression of glutamine synthetase, which is involved in ammonia assimilation, was induced by drought stress in the wheat. Our study shows that PEPC can improve both stress tolerance and grain yield in wheat, demonstrating the efficacy of PEPC in crop improvement.

Pyramiding expression of maize genes encoding phosphoenolpyruvate carboxylase (PEPC) and pyruvate orthophosphate dikinase (PPDK) synergistically improve the photosynthetic characteristics of transgenic wheat.

Using particle bombardment transformation, we introduced maize pepc cDNA encoding phosphoenolpyruvate carboxylase (PEPC) and ppdk cDNA encoding pyruvate orthophosphate dikinase (PPDK) into the C3 crop wheat to generate transgenic wheat lines carrying cDNA of pepc (PC lines), ppdk (PK lines) or both (PKC lines). The integration, transcription, and expression of the foreign genes were confirmed by Southern blot, Real-time quantitative reverse transcription PCR (Q-RT-PCR), and Western blot analysis. Q-RT-PCR results indicated that the average relative expression levels of pepc and ppdk in the PKC lines reached 10 and 4.6, respectively, compared to their expressions in untransformed plants (set to 1). The enzyme activities of PEPC and PPDK in the PKC lines were 4.3- and 2.1-fold higher, respectively, than in the untransformed control. The maximum daily net photosynthetic rates of the PKC, PC, and PK lines were enhanced by 26.4, 13.3, and 4.5%, respectively, whereas the diurnal accumulations of photosynthesis were 21.3, 13.9, and 6.9%, respectively, higher than in the control. The Fv/Fm of the transgenic plants decreased less than in the control under high temperature and high light conditions (2 weeks after anthesis), suggesting that the transgenic wheat transports more absorbed light energy into a photochemical reaction. The exogenous maize C4-specific pepc gene was more effective than ppdk at improving the photosynthetic performance and yield characteristics of transgenic wheat, while the two genes showed a synergistic effect when they were transformed into the same genetic background, because the PKC lines exhibited improved photosynthetic and physiological traits.