Heterologous Expression of the Transcription Factor EsNAC1 in Arabidopsis Enhances Abiotic Stress Resistance and Retards Growth by Regulating the Expression of Different Target Genes

Heterologous expression of a transcription factor (TF) gene in a related species is a useful method for crop breeding and the identification of gene function. The differences in phenotype and target gene expression between HE lines (with the heterologous expression of an ortholog) and OX lines (with an overexpressed native gene) must be understood. EsNAC1, encoding a NAC protein and the ortholog of RD26 in Arabidopsis, was cloned from Eutrema salsugineum and introduced into Arabidopsis. The heterologous expression of EsNAC1 retarded the vegetative growth of Arabidopsis, and the transgenic plants (HE lines) showed much greater resistance to salt and oxidative stress than the wild type, Col-0. The HE lines accumulated 2.8-fold (8-h light) of starch, 1.42-fold of Chlorophyll a and 1.31-fold of Chlorophyll b than Col-0 during the light period, with obvious differences compared to the RD26OX line. A genome-wide ChIP (chromatin immunoprecipitation analysis)-on-chip assay revealed that EsNAC1 targeted promoters of different genes compared to RD26. In HE lines, EsNAC1 could specifically upregulate the expression level of TF genes NAC DOMAIN CONTAINING PROTEIN 62 (ANAC062), INTEGRASE-TYPE DNA-BINDING PROTEIN (TINY2), and MYB HYPOCOTYL ELONGATION-RELATED (MYBH) to show more effective abiotic stress resistance than RD26OX lines. Moreover, DELTA1-PYRROLINE-5-CARBOXYLATE SYNTHASE 1 (P5CS1), TRYPTOPHAN BIOSYNTHESIS 2 (TRP2) or GALACTINOL SYNTHASE 2 (GOLS2), was also specifically regulated by EsNAC1 to retard the vegetative growth of HE lines, but not the brassinosteroid singling pathway in RD26OX lines. These differences in phenotypes and metabolism between the HE lines and the RD26OX line implied that the differential features could be produced from the diversity of target genes in the transgenic plants when the ortholog was introduced

Over-expression of glutamine synthetase genes Gln1-3/Gln1-4 improved nitrogen assimilation and maize yields

In agriculture, certain fertilizers that contain nitrogen generally tend to provide the most macronutrients for plant growth and development. The cDNAs of Gln1-3 and Gln1-4 genes, encoding glutamine synthetase isoenzymes (GS1), were fused to the rice actin1 promoter and over-expressed in the inbred maize line DH9632 by Agrobacte¬rium-mediated genetic transformation. PCR assays demonstrated the integration of these genes in six transgenic lines. Transcription of Gln1-3 or Gln1-4 in the transformants was also confirmed by semi-quantitative RT-PCR and qRT-PCR; the transgenic lines had significantly higher expression compared with wild type. Transgenic lines L2 and L7 expressed the most Gln1-3 and Gln1-4 mRNA, respectively, and had the most enzyme activity in leaves below the ear after pollination for 14 days. Over-expression of these two genes led to increased chlorophyll con¬tent and improved photosynthesis after 14 days. In addition, yield-related traits such as ear length, ear diameter, ear weight, grain weight per ear, and hundred-kernel weight were improved in the transgenic lines. The plot yield of transgenic L2 was increased by approximately 20%. These results suggest that overexpression of Gln1-3 and Gln1-4 in maize improves yields and enhances nitrogen using efficiency. Thus, transgenic lines overexpressing Gln1-3 or Gln1-4 in maize could potentially be used in maize breeding

Effects of Raised Ambient Temperature on the Local and Systemic Adaptions of Maize

Maize is a staple food, feed, and industrial crop. One of the major stresses on maize production is heat stress, which is usually accompanied by other stresses, such as drought or salinity. In this review, we compared the effects of high temperatures on maize production in China. Heat stress disturbs cellular homeostasis and impedes growth and development in plants. Plants have evolved a variety of responses to minimize the damage related to high temperatures. This review summarized the responses in different cell organelles at elevated temperatures, including transcriptional regulation control in the nuclei, unfolded protein response and endoplasmic reticulum-associated protein quality control in the endoplasmic reticulum (ER), photosynthesis in the chloroplast, and other cell activities. Cells coordinate their activities to mediate the collective stresses of unfavorable environments. Accordingly, we evaluated heat stress at the local and systemic levels in in maize. We discussed the physiological and morphological changes in sensing tissues in response to heat stress in maize and the existing knowledge on systemically acquired acclimation in plants. Finally, we discussed the challenges and prospects of promoting corn thermotolerance by breeding and genetic manipulation

ZmNF-YA1 Contributes to Maize Thermotolerance by Regulating Heat Shock Response

Zea mays (maize) is a staple food, feed, and industrial crop. Heat stress is one of the major stresses affecting maize production and is usually accompanied by other stresses, such as drought. Our previous study identified a heterotrimer complex, ZmNF-YA1-YB16-YC17, in maize. ZmNF-YA1 and ZmNF-YB16 were positive regulators of the drought stress response and were involved in maize root development. In this study, we investigated whether ZmNF-YA1 confers heat stress tolerance in maize. The nf-ya1 mutant and overexpression lines were used to test the role of ZmNF-YA1 in maize thermotolerance. The nf-ya1 mutant was more temperature-sensitive than the wild-type (WT), while the ZmNF-YA1 overexpression lines showed a thermotolerant phenotype. Higher malondialdehyde (MDA) content and reactive oxygen species (ROS) accumulation were observed in the mutant, followed by WT and overexpression lines after heat stress treatment, while an opposite trend was observed for chlorophyll content. RNA-seq was used to analyze transcriptome changes in nf-ya1 and its wild-type control W22 in response to heat stress. Based on their expression profiles, the heat stress response-related differentially expressed genes (DEGs) in nf-ya1 compared to WT were grouped into seven clusters via k-means clustering. Gene Ontology (GO) enrichment analysis of the DEGs in different clades was performed to elucidate the roles of ZmNF-YA1-mediated transcriptional regulation and their contribution to maize thermotolerance. The loss function of ZmNF-YA1 led to the failure induction of DEGs in GO terms of protein refolding, protein stabilization, and GO terms for various stress responses. Thus, the contribution of ZmNF-YA1 to protein stabilization, refolding, and regulation of abscisic acid (ABA), ROS, and heat/temperature signaling may be the major reason why ZmNF-YA1 overexpression enhanced heat tolerance, and the mutant showed a heat-sensitive phenotype

Structural, expression and evolutionary analysis of the non-specific phospholipase C gene family in Gossypium hirsutum

Abstract Background Nonspecific phospholipase C (NPC), which belongs to a phospholipase C subtype, is a class of phospholipases that hydrolyzes the primary membrane phospholipids, such as phosphatidylcholine, to yield sn-1, 2-diacylglycerol and a phosphorylated head-group. NPC plays multiple physiological roles in lipid metabolism and signaling in plants. To fully understand the putative roles of NPC genes in upland cotton, we cloned NPC genes from Gossypium hirsutum and carried out structural, expression and evolutionary analysis. Results Eleven NPC genes were cloned from G. hirsutum, which were found on chromosomes scaffold269.1, D03, A07, D07, A08, D11, and scaffold3511_A13. All GhNPCs had typical phosphoesterase domains and have hydrolase activity that acts on ester bonds. GhNPCs were annotated as phospholipase C, which was involved in glycerophospholipid metabolism, ether lipid metabolism, and biosynthesis of secondary metabolites. These GhNPCs showed differential expression patterns in distinct plant tissues and in response to various types of stress (low-phosphate, salt, drought, and abscisic acid). They also had different types and numbers of cis-element. GhNPCs could be classified into four subfamilies. Four pairs of GhNPCs were generated by whole-genome duplication and they underwent purifying selection. Conclusions Our results suggested that GhNPCs are involved in regulating key abiotic stress responses and ABA signaling transduction, and they may have various functional roles for different members under complex abiotic stress conditions. Functional divergence may be the evolutionary driving force for the retention of four pairs of duplicate NPCs. Our analysis provides a solid foundation for the further functional characterization of the GhNPC gene family, and leads to potential applications in the genetic improvement of cotton cultivars

Trihelix Transcription Factor ZmThx20 Is Required for Kernel Development in Maize

Maize kernels are the harvested portion of the plant and are related to the yield and quality of maize. The endosperm of maize is a large storage organ that constitutes 80–90% of the dry weight of mature kernels. Maize kernels have long been the study of cereal grain development to increase yield. In this study, a natural mutation that causes abnormal kernel development, and displays a shrunken kernel phenotype, was identified and named “shrunken 2008 (sh2008)”. The starch grains in sh2008 are loose and have a less proteinaceous matrix surrounding them. The total storage protein and the major storage protein zeins are ~70% of that in the wild-type control (WT); in particular, the 19 kDa and 22 kDa α-zeins. Map-based cloning revealed that sh2008 encodes a GT-2 trihelix transcription factor, ZmThx20. Using CRISPR/Cas9, two other alleles with mutated ZmThx20 were found to have the same abnormal kernel. Shrunken kernels can be rescued by overexpressing normal ZmThx20. Comparative transcriptome analysis of the kernels from sh2008 and WT showed that the GO terms of translation, ribosome, and nutrient reservoir activity were enriched in the down-regulated genes (sh2008/WT). In short, these changes can lead to defects in endosperm development and storage reserve filling in seeds

Identification of a new 130 bp <it>cis</it>-acting element in the <it>TsVP1 </it>promoter involved in the salt stress response from <it>Thellungiella halophila</it>

Abstract Background Salt stress is one of the major abiotic stresses affecting plant growth and productivity. Vacuolar H+-pyrophosphatase (H+-PPase) genes play an important role in salt stress tolerance in multiple species. Results In this study, the promoter from the vacuolar H+-pyrophosphatase from Thellungiella halophila (TsVP1) was cloned and compared with the AVP1 promoter from Arabidopsis thaliana. Sequence analysis indicated that these two promoters had seven similar motifs at similar positions. To determine which tissues the two promoters are active in, transgenic plants were produced with expression of the GUS reporter gene under the control of one of the promoters. In transgenic Arabidopsis with the TsVP1 promoter, the GUS reporter gene had strong activity in almost all tissues except the seeds and the activity was induced in both shoots and roots, especially in the root tips, when treated with salt stress. Such induction was not found in transgenic Arabidopsis with the AVP1 promoter. By analyzing different 5' deletion mutants of the TsVP1 promoter, an 856 bp region (-2200 to -1344) was found to contain enhancer elements that increased gene expression levels. Two AAATGA motifs, which may be the key elements for the anther specific expression profile, in the deleted TsVP1 promoters (PT2 to PT6) were also identified. A 130 bp region (-667 to -538) was finally identified as the key sequence for the salt stress response by analyzing the different mutants both with and without salt stress. GUS transient assay in tobacco leaves suggested the 130 bp region was sufficient for the salt stress response. Bioinformatic analysis also revealed that there may be novel motifs in this region that are the key elements for the salt stress responsive activity of the TsVP1 promoter. Conclusions The TsVP1 promoter had strong activity in almost all tissues except the seeds. In addition, its activity was induced by salt stress in leaves and roots, especially in root tips. A 130 bp region (-667 to -538) was identified as the key region for responding to salt stress.</p