Project description:Red clover (Trifolium pratense L.) is an important cool-season legume plant, which is used as forage. Leaf senescence is a critical developmental process that negatively affects plant quality and yield. The regulatory mechanism of leaf senescence has been studied, and genes involved in leaf senescence have been cloned and characterized in many plants. However, those works mainly focused on model plants. Information about regulatory pathways and the genes involved in leaf senescence in red clover is very sparse. In this study, to better understand leaf senescence in red clover, transcriptome analysis of mature and senescent leaves was investigated using RNA-Seq. A total of about 35,067 genes were identified, and 481 genes were differentially expressed in mature and senescent leaves. Some identified differentially expressed genes showed similar expression patterns as those involved in leaf senescence in other species, such as Arabidopsis, Medicago truncatula and rice. Differentially expressed genes were confirmed by quantitative real-time PCR (qRT-PCR). Genes involved in signal transduction, transportation and metabolism of plant hormones, transcription factors and plant senescence were upregulated, while the downregulated genes were primarily involved in nutrient cycling, lipid/carbohydrate metabolism, hormone response and other processes. There were 64 differentially expressed transcription factor genes identified by RNA-Seq, including ERF, WRKY, bHLH, MYB and NAC. A total of 90 genes involved in biosynthesis, metabolism and transduction of plant hormones, including abscisic acid, jasmonic acid, cyokinin, brassinosteroid, salicylic acid and ethylene, were identified. Furthermore, 207 genes with direct roles in leaf senescence were demonstrated, such as senescence-associated genes. These genes were associated with senescence in other plants. Transcriptome analysis of mature and senescent leaves in red clover provides a large number of differentially expressed genes. Further analysis and identification of senescence-associated genes can provide new insight into the regulatory mechanisms of leaf development and senescence in legume plant and red clover.
Project description:Red clover (Trifolium pratense L.) is a diploid, naturally cross-pollinated, cool-season species. As a perennial forage legume, red clover is mostly cultivated in temperate regions worldwide. Being a non-model crop species, genomic resources for red clover have been underdeveloped. Thus far, genomic analysis used in red clover has mainly relied on simple sequence repeat (SSR) markers. However, SSR markers are sparse in the genome and it is often difficult to unambiguously map them using short reads generated by next generation sequencing technology. Single nucleotide polymorphisms (SNPs) have been successfully applied in genomics assisted breeding in several agriculturally important species. Due to increasing importance of legumes in forage production, there is a clear need to develop SNP based markers for red clover that can be applied in breeding applications. In this study, we first developed an analytical pipeline that can confidently identify SNPs in a set of 72 different red clover genotypes using sequences generated by targeted amplicon sequencing. Then, with the same filtering stringency used in this pipeline, we used sequences from publicly available RNA-seq data to identify confident SNPs in different red clover varieties. Using this strategy, we have identified a total of 69,975 SNPs across red clover varieties. Among these, 28% (19,116) of them are missense mutations. Using Medicago truncatula as the reference, we annotated the regions affected by these missense mutations. We identified 2,909 protein coding regions with missense mutations. Pathway analysis of these coding regions indicated several biological processes impacted by these mutations. Specifically, three domains (homeobox domain, pentatricopeptide repeat containing plant-like, and regulator of Vps4 activity) were identified with five or more missense SNPs. These domain might also be a functional contributor in the molecular mechanisms of self-incompatibility in red clover. Future in-depth sequence diversity analysis of these three genes may yield valuable insights into the molecular mechanism involved in self-incompatibility in red clover.
Project description:Nutrient remobilization during leaf senescence nourishes the growing plant. Understanding the regulation of this process is essential for reducing our dependence on nitrogen fertilizers and increasing agricultural sustainability. Our laboratory is interested in chromatin changes that accompany the transition to leaf senescence. Previously, darker green leaves were reported for Arabidopsis thaliana hac1 mutants, defective in a gene encoding a histone acetyltransferase in the CREB-binding protein family. Here, we show that two Arabidopsis hac1 alleles display delayed age-related developmental senescence, but have normal dark-induced senescence. Using a combination of ChIP-seq for H3K9ac and RNA-seq for gene expression, we identified 43 potential HAC1 targets during age-related developmental senescence. Genetic analysis demonstrated that one of these potential targets, ERF022, is a positive regulator of leaf senescence. ERF022 is regulated additively by HAC1 and MED25, suggesting MED25 may recruit HAC1 to the ERF022 promoter to increase its expression in older leaves.
Project description:Warm winters and hot springs may promote panicle leaf growing and repress floral development. To identify genes potentially involved in litchi panicle leaf senescence, eight RNA-sequencing (RNA-Seq) libraries of the senescing panicle leaves under low temperature (LT) conditions and the developing panicle leaves under high temperature (HT) conditions were constructed. For each library, 4.78?8.99 × 10? clean reads were generated. Digital expression of the genes was compared between the senescing and developing panicle leaves. A total of 6477 upregulated differentially expressed genes (DEGs) (from developing leaves to senescing leaves), and 6318 downregulated DEGs were identified, 158 abscisic acid (ABA)-, 68 ethylene-, 107 indole-3-acetic acid (IAA)-, 27 gibberellic acid (GA)-, 68 cytokinin (CTK)-, 37 salicylic acid (SA)-, and 23 brassinolide (BR)-related DEGs. Confirmation of the RNA-Seq data by quantitative real-time PCR (qRT-PCR) analysis suggested that expression trends of the 10 candidate genes using qRT-PCR were similar to those revealed by RNA-Seq, and a significantly positive correlation between the obtained data from qRT-PCR and RNA-Seq were found, indicating the reliability of our RNA-Seq data. The present studies provided potential genes for the future molecular breeding of new cultivars that can induce panicle leaf senescence and reduce floral abortion under warm climates.
Project description:Leaf growth and senescence are controlled by tight genetic factors involved regulation at multiple levels. Circular RNAs (circRNAs) have recently been reported as the microRNA sponge to accomplish corresponding regulatory roles. This study aims to explore the expression profile and functional role of circRNAs in Arabidopsis leaf growth and senescence. We used publically available RNA-seq data of Arabidopsis leaves to identify the circular RNA expression profile and used quantitative real-time PCR to validate our identified circRNAs. The functions of circRNAs were explored using distinct bioinformatics methods including analysis of network, gene ontology and KEGG pathway. We identified 168 circRNAs, including 40 novel circRNAs, in Arabidopsis thaliana leaves, with 158 (94.1%) circRNAs arising from the exons of genes. Real-time PCRs were used to verify 4 highly expressed circRNAs and they all showed consistent expression patterns with the RNA-seq results. Interestingly, 6 and 35 circRNAs were differentially expressed at G- to -M stage and M- to -S stage, respectively. The circRNAs display an upregulation trend during the lifespan of Arabidopsis leaves. Moreover, the expression of circRNAs during senescence is independent of host gene expression to a certain degree. The gene ontology (GO) and KEGG pathway analysis of the targeted mRNA of circRNA-miRNA-mRNA network showed that the circRNAs may be involved in plant hormone signal transduction, Porphyrin and chlorophyll metabolism during leaves senescence. Our comprehensive analysis of the expression profile of circRNAs and their potential functions during leaf growth and senescence suggest that circRNAs may function as new post-transcriptional regulators in the senescence of Arabidopsis leaves.
Project description:BACKGROUND:Auxin may have a positive effect on plants under drought stress. White clover is widely cultivated and often prone to water shortages. In the present study, we investigated the effects of exogenous indole -?3-acetic acid (IAA) on growth and physiological changes of white clover under drought stress condition. The contents of endogenous IAA and other hormones including ABA, CTK, JA, GA, IAA, and SA were assayed. Moreover, expressions of auxin-responsive genes, drought-responsive genes and leaf senescence-associated genes were detected in response to exogenous IAA. RESULTS:Compared to control, drought stress alone significantly diminished stem dry weigh, relative water content (RWC) and total chlorophyll content (Chl). Exogenous IAA treatment significantly increased RWC and Chl, whereas L-AOPP treatment drastically decreased stem dry weight, RWC and Chl under drought stress condition. Additionally, exogenous IAA treatment significantly increased ABA content and JA content, up-regulated expression of auxin responsive genes (GH3.1, GH3.9, IAA8), drought stress responsive genes (bZIP11, DREB2, MYB14, MYB48, WRKY2, WRKY56, WRKY108715 and RD22), and down-regulated expressions of auxin-responding genes (GH3.3, GH3.6, IAA27) and leaf senescence genes (SAG101 and SAG102) in the presence of PEG. Contrarily, L-AOPP treatment significantly reduced contents of ABA, GA3 and JA, down-regulated expressions of GH3.1, GH3.9, IAA8, bZIP11, DREB2, MYB14, MYB48, WRKY2, WRKY56, WRKY108715, ERD and RD22, and up-regulated SAG101 and SAG102. CONCLUSIONS:Exogenous IAA improved drought tolerance of white clover possibly due to endogenous plant hormone concentration changes and modulation of genes involving in drought stress response and leaf senescence. These results provided useful information to understand mechanisms of IAA improved drought tolerance in white clover.
Project description:Early leaf senescence is an important agronomic trait that affects crop yield and quality. To understand the molecular mechanism of early leaf senescence, Oryza sativa premature leaf senescence 1 (ospls1) mutant rice with a deletion of OsVHA-A and its wild type were employed in this study. The genotype-dependent differences in photosynthetic indexes, senescence-related physiological parameters, and yield characters were investigated during the grain-filling stage. Moreover, RNA sequencing (RNA-seq) was performed to determine the genotype differences in transcriptome during the grain-filling stage. Results showed that the ospls1 mutant underwent significant decreases in the maximal quantum yield of photosystem II (PSII) photochemistry (Fv/Fm), net photosynthesis rate (Pn), and soluble sugar and protein, followed by the decreases in OsVHA-A transcript and vacuolar H?-ATPase activity. Finally, yield traits were severely suppressed in the ospls1 mutant. RNA-seq results showed that 4827 differentially expressed genes (DEGs) were identified in ospls1 mutant between 0 day and 14 days, and the pathways of biosynthesis of secondary metabolites, carbon fixation in photosynthetic organisms, and photosynthesis were downregulated in the senescing leaves of ospls1 mutant during the grain-filling stage. In addition, 81 differentially expressed TFs were identified to be involved in leaf senescence. Eleven DEGs related to hormone signaling pathways were significantly enriched in auxin, cytokinins, brassinosteroids, and abscisic acid pathways, indicating that hormone signaling pathways participated in leaf senescence. Some antioxidative and carbohydrate metabolism-related genes were detected to be differentially expressed in the senescing leaves of ospls1 mutant, suggesting that these genes probably play response and regulatory roles in leaf senescence.
Project description:Survivor populations of red clover (Trifolium pratense L.) from plots in a field experiment in southern Norway were genetically characterized using genotyping by sequencing, and compared with the original population and each other. Genetic differentiation between populations was characterized on the basis of allele frequencies of single nucleotide polymorphisms (SNPs), using principal component analysis. SNPs that had been under selection, i.e., SNPs with significantly different allele frequencies in survivor populations relative to the original population, or between survivor populations that had received different treatments, were identified by analysis of F ST values, using BayeScan and a simple and stringent F ST-based test utilizing replicate populations from the field experiment. In addition, we tested the possibility of pooling DNA samples prior to sequencing, and pooling leaf samples prior to DNA extraction and sequencing, followed by allele frequency estimation on the basis of number of variant reads. Overall, survivor populations were more different from each other than from the original population, indicating random changes in allele frequency, selection in response to local variation in conditions between plots in the field experiment, or sampling error. However, some differentiation was observed between plots sown as pure stands or species mixtures, plots sown at different densities, and plots subjected to different harvesting regimes. Allele frequencies could be accurately estimated from pooled DNA, and SNPs under selection could be identified when leaf samples were pooled prior to DNA extraction. However, substantial sampling error required replicate populations and/or a high number of sampled individuals. We identified a number of chromosomal loci that had been under selection in pure stand plots relative to the original sown population, and loci that had been under differential selection in pure stands of red clover vs. red clover grown in species mixtures. These are all candidate loci for establishment success or persistence in red clover.
Project description:Leaf senescence is an important developmental programmed degeneration process that dramatically affects crop quality and yield. The regulation of senescence is highly complex. Although senescence regulatory genes have been well characterized in model species such as Arabidopsis and rice, there is little information on the control of this process in cotton. Here, the senescence process in cotton (Gossypium hirsutum L.) leaves was investigated over a time course including young leaf, mature leaf and leaf samples from different senescence stages using RNA-Seq.Of 24,846 genes detected by mapping the tags to Gossypium genomes, 3,624 genes were identified as differentially expressed during leaf senescence. There was some overlap between the genes identified here and senescence-associated genes previously identified in other species. Most of the genes related to photosynthesis, chlorophyll metabolism and carbon fixation were downregulated; whereas those for plant hormone signal transduction were upregulated. Quantitative real-time PCR was used to evaluate the results of RNA-Seq for gene expression profiles. Furthermore, 519 differentially expressed transcription factors were identified, notably WRKY, bHLH and C3H. In addition, 960 genes involved in the metabolism and regulation of eight hormones were identified, of which many genes involved in the abscisic acid, brassinosteroid, jasmonic acid, salicylic acid and ethylene pathways were upregulated, indicating that these hormone-related genes might play crucial roles in cotton leaf development and senescence. However, most auxin, cytokinin and gibberellin pathway-related genes were downregulated, suggesting that these three hormones may act as negative regulators of senescence.This is the first high-resolution, multiple time-course, genome-wide comprehensive analysis of gene expression in cotton. These data are the most comprehensive dataset currently available for cotton leaf senescence, and will serve as a useful resource for unraveling the functions of many specific genes involved in cotton leaf development and senescence.
Project description:Leaf senescence is the final stage of plant development. Many internal and external factors affect the senescence process in rice (Oryza sativa L.). In this study, we identified qCC2, a major quantitative trait locus (QTL) for chlorophyll content using a population derived from an interspecific cross between O. sativa (cv. Hwaseong) and Oryza grandiglumis. The O. grandiglumis allele at qCC2 increased chlorophyll content and delayed senescence. GW2 encoding E3 ubiquitin ligase in the qCC2 region was selected as a candidate for qCC2. To determine if GW2 is allelic to qCC2, a gw2-knockout mutant (gw2-ko) was examined using a dark-induced senescence assay. gw2-ko showed delayed leaf senescence in the dark with down-regulated expression of senescence-associated genes (SAGs) and chlorophyll degradation genes (CDGs). The association of the GW2 genotype with the delayed senescence phenotype was confirmed in an F2 population. RNA-seq analysis was conducted to investigate 30-day-old leaf transcriptome dynamics in Hwaseong and a backcross inbred line-CR2002-under dark treatment. This resulted in the identification of genes involved in phytohormone signaling and associated with senescence. These results suggested that transcriptional regulation was associated with delayed senescence in CR2002, and RING-type E3 ubiquitin ligase GW2 was a positive regulator of leaf senescence in rice.