Chenxi Xu

Drought-Responsive Mechanisms in Plant Leaves Revealed by Proteomics

Plant drought tolerance is a complex trait that requires a global view to understand its underlying mechanism. The proteomic aspects of plant drought response have been extensively investigated in model plants, crops and wood plants. In this review, we summarize recent proteomic studies on drought response in leaves to reveal the common and specialized drought-responsive mechanisms in different plants. Although drought-responsive proteins exhibit various patterns depending on plant species, genotypes and stress intensity, proteomic analyses show that dominant changes occurred in sensing and signal transduction, reactive oxygen species scavenging, osmotic regulation, gene expression, protein synthesis/turnover, cell structure modulation, as well as carbohydrate and energy metabolism. In combination with physiological and molecular results, proteomic studies in leaves have helped to discover some potential proteins and/or metabolic pathways for drought tolerance. These findings provide new clues for understanding the molecular basis of plant drought tolerance.

De novo and comparative transcriptome analysis of cultivated and wild spinach

Spinach (Spinacia oleracea L.) is an economically important green leafy vegetable crop. In this study, we performed deep transcriptome sequencing for nine spinach accessions: three from cultivated S. oleracea, three from wild S. turkestanica and three from wild S. tetrandra. A total of approximately 100 million high-quality reads were generated, which were de novo assembled into 72,151 unigenes with a total length of 46.5 Mb. By comparing sequences of these unigenes against different protein databases, nearly 60% of them were annotated and 50% could be assigned with Gene Ontology terms. A total of 387 metabolic pathways were predicted from the assembled spinach unigenes. From the transcriptome sequencing data, we were able to identify a total of ~320,000 high-quality single nucleotide polymorphisms (SNPs). Phylogenetic analyses using SNPs as well as gene expression profiles indicated that S. turkestanica was more closely related to the cultivated S. oleracea than S. tetrandra. A large number of genes involved in responses to biotic and abiotic stresses were found to be differentially expressed between the cultivated and wild spinach. Finally, an interactive online database (http://www.spinachbase.org) was developed to allow the research community to efficiently retrieve, query, mine and analyze our transcriptome dataset.

Draft genome of spinach and transcriptome diversity of 120 Spinacia accessions

Spinach is an important leafy vegetable enriched with multiple necessary nutrients. Here we report the draft genome sequence of spinach (Spinacia oleracea, 2n=12), which contains 25,495 protein-coding genes. The spinach genome is highly repetitive with 74.4% of its content in the form of transposable elements. No recent whole genome duplication events are observed in spinach. Genome syntenic analysis between spinach and sugar beet suggests substantial inter- and intra-chromosome rearrangements during the Caryophyllales genome evolution. Transcriptome sequencing of 120 cultivated and wild spinach accessions reveals more than 420 K variants. Our data suggests that S. turkestanica is likely the direct progenitor of cultivated spinach and spinach domestication has a weak bottleneck. We identify 93 domestication sweeps in the spinach genome, some of which are associated with important agronomic traits including bolting, flowering and leaf numbers. This study offers insights into spinach evolution and domestication and provides resources for spinach research and improvement.

Heat-Responsive Photosynthetic and Signaling Pathways in Plants: Insight from Proteomics

Heat stress is a major abiotic stress posing a serious threat to plants. Heat-responsive mechanisms in plants are complicated and fine-tuned. Heat signaling transduction and photosynthesis are highly sensitive. Therefore, a thorough understanding of the molecular mechanism in heat stressed-signaling transduction and photosynthesis is necessary to protect crop yield. Current high-throughput proteomics investigations provide more useful information for underlying heat-responsive signaling pathways and photosynthesis modulation in plants. Several signaling components, such as guanosine triphosphate (GTP)-binding protein, nucleoside diphosphate kinase, annexin, and brassinosteroid-insensitive I-kinase domain interacting protein 114, were proposed to be important in heat signaling transduction. Moreover, diverse protein patterns of photosynthetic proteins imply that the modulations of stomatal CO2 exchange, photosystem II, Calvin cycle, ATP synthesis, and chlorophyll biosynthesis are crucial for plant heat tolerance.

Expression Analysis of Oxalate Metabolic Pathway Genes Reveals Oxalate Regulation Patterns in Spinach

Spinach (Spinacia oleracea L.) is one of most important leafy vegetables because of its high nutritional value and high oxalate content, which can be toxic with negative effects on human nutrition. Ammonium and nitrate can effectively regulate oxalate accumulation, although the mechanisms underlying the oxalate biosynthesis and regulation are still undetermined in plants. In the present study, we identified 25 putative genes that are involved in the oxalate biosynthetic and degradation pathway, before analyzing the oxalate content and the expression levels of the corresponding proteins under normal growth conditions, with or without ammonium and nitrate treatments, using high and low oxalate-accumulated spinach genotypes. The two cultivars exhibited different profiles of total oxalate and soluble oxalate accumulation. The high oxalate concentrations in spinach were as a result of the high transcription levels of the genes that are involved in oxalate biosynthesis under normal growth conditions, such as SoGLO2, SoGLO3, three SoOXACs, SoMLS, SoMDH1, SoMDH2, and SoMDH4. The results revealed that the ammonium and nitrate were able to control the oxalate content in leaves, possibly because of the different transcription levels of the genes. The oxalate content is regulated by complex regulatory mechanisms and is varied in the different varieties of spinach. The results from this research may be used to assist the investigation of the mechanism of oxalate regulation and breeding for reduced oxalate content in spinach.

Nitrate Accumulation and Expression Patterns of Genes Involved in Nitrate Transport and Assimilation in Spinach

Excessive accumulation of nitrate in spinach is not only harmful to human beings, but also limits the efficiency of nitrogen usage. However, the underlying mechanism of nitrate accumulation in plants remains unclear. This study analyzed the physiological and molecular characteristics of nitrate uptake and assimilation in the spinach varieties with high or low nitrate accumulation. Our results showed that the variety of spinach with a high nitrate content (So18) had higher nitrate uptake compared to the variety with a low nitrate content (So10). However, the nitrate reductase activities of both varieties were similar, which suggests that the differential capacity to uptake and transport nitrate may account for the differences in nitrate accumulation. The quantitative PCR analysis showed that there was a higher level of expression of spinach nitrate transporter (SoNRT) genes in So18 compared to those in So10. Based on the function of Arabidopsis homologs AtNRTs, the role of spinach SoNRTs in nitrate accumulation is discussed. It is concluded that further work focusing on the expression of SoNRTs (especially for SoNRT1.4, SoNRT1.5 and SoNRT1.3) may help us to elucidate the molecular mechanism of nitrate accumulation in spinach.

Higher anthocyanin accumulation was associated with higher transcription levels of anthocyanin biosynthesis genes in spinach

Spinach (Spinacia oleracea L.) is widely cultivated as an economically important green leafy vegetable crop for fresh and processing consumption. The red-purple spinach shows abundant anthocyanin accumulation in the leaf and leaf petiole. However, the molecular mechanisms of anthocyanin synthesis in this species are still undetermined. In the present study, we investigated pigment formation and identified anthocyanin biosynthetic genes in spinach. We also analyzed the expression of these genes in purple and green cultivars by quantitative PCR. The accumulation of anthocyanin showed that it was the dominant pigment resulting in the red coloration in spinach. In total, 22 biosynthesis genes and 25 regulatory genes were identified in spinach, based on the spinach genomic and transcriptomic database. Furthermore, the expression patterns of genes encoding enzymes indicated that SoPAL, SoUFGT3, and SoUFGT4 were possible candidate genes for anthocyanin biosynthesis in red-purple spinach. The expression patterns of transcription factors indicated that two SoMYB genes, three SobHLH genes, and one SoWD40 gene were drastically up-regulated and co-expression in red-purple spinach, suggesting an essential role of regulatory genes in the anthocyanin biosynthesis of spinach. These results will enhance our understanding of the molecular mechanisms of anthocyanin biosynthesis in purple spinach.

Proteomics and Phosphoproteomics of Heat Stress-Responsive Mechanisms in Spinach

Elevated temperatures limit plant growth and reproduction and pose a growing threat to agriculture. Plant heat stress response is highly conserved and fine-tuned in multiple pathways. Spinach (Spinacia oleracea L.) is a cold tolerant but heat sensitive green leafy vegetable. In this study, heat adaptation mechanisms in a spinach sibling inbred heat-tolerant line Sp75 were investigated using physiological, proteomic, and phosphoproteomic approaches. The abundance patterns of 911 heat stress-responsive proteins, and phosphorylation level changes of 45 phosphoproteins indicated heat-induced calcium-mediated signaling, ROS homeostasis, endomembrane trafficking, and cross-membrane transport pathways, as well as more than 15 transcription regulation factors. Although photosynthesis was inhibited, diverse primary and secondary metabolic pathways were employed for defense against heat stress, such as glycolysis, pentose phosphate pathway, amino acid metabolism, fatty acid metabolism, nucleotide metabolism, vitamin metabolism, and isoprenoid biosynthesis. These data constitute a heat stress-responsive metabolic atlas in spinach, which will springboard further investigations into the sophisticated molecular mechanisms of plant heat adaptation and inform spinach molecular breeding initiatives.

Functional analysis of spinach CONSTITUTIVE EXPRESSOR OF PATHOGENESIS-RELATED GENES5 (SoCPR5) in plant immunity

Spinach is one of the most nutritious green-leaf vegetables. In the spinach production, diseases cause a significant loss in both yield and quality. Improving disease resistance is one of the major challenges in spinach breeding. Arabidopsis nucleoporin CONSTITUTIVE EXPRESSER OF PATHOGENESIS-RELATED GENES 5 (CPR5) functions as a negative regulator of plant cell death and immunity as cpr5 mutant exhibits spontaneous cell death and heightened immunity. In addition, CPR5 play a role in trichome development as the majority of cpr5 mutant trichomes are branchless whereas wild type trichomes are often three-branched. In the spinach genome, we identified a homolog of Arabidopsis CPR5, referred to as Spinacia oleracea CPR5 (SoCPR5). To investigate the function of SoCPR5, we introduced SoCPR5 into Arabidopsis cpr5 mutant. Our data showed that both spontaneous cell death and heightened immunity were suppressed in the SoCPR5-transgenic cpr5 mutants, verifying that SoCPR5 functions as its Arabidopsis counterpart in plant cell death and immunity. SoCPR5 also fully restored wild type trichome phenotype of the cpr5 mutant. Our study therefore indicates that the function of SoCPR5 is conserved between plant species and SoCPR5 can be applied for genetic manipulation of plant immunity in spinach.

The complete mitochondrial genome sequence of spinach, Spinacia oleracea L

Abstract Spinach (Spinacia oleracea L.) is an economically important vegetable crop. Here we describe the complete mitochondrial DNA sequence of spinach, which has a length of 329,613 bp and a GC content of 43.4%. It is separated by a pair of directly repeated elements of 7286 bp, to form a large single copy region of 229,375 bp and a small single copy region of 85,666 bp. The genome contains 29 protein-coding genes, 4 pseudogenes, 24 tRNA genes, and 3 rRNA genes. A phylogenetic analysis revealed that spinach was closely related to Beta vulgaris (sugar beet), both belonging to the Amaranthaceae family.