Transcriptome Analysis Of Leaves Research Articles

CRISPR/Cas9-mediated mutagenesis of the mediator complex subunits MED5a and MED5b genes impaired secondary metabolite accumulation in hop (Humulus lupulus)

Hop (Humulus lupulus L.) is an important commercial crop known for the biosynthesis of valuable specialized secondary metabolites in glandular trichomes (lupulin glands), which are used for the brewing industry. To achieve burgeoning market demands is the essentiality of comprehensive understanding of the mechanisms of biosynthesis of secondary metabolites in hop. Over the past year, several studies using structural biology and functional genomics approaches have shown that Mediator (MED) serves as an integrative hub for RNAP II-mediated transcriptional regulation of various physiological and cellular processes, including involvement of MED5a and MED5b in hyperaccumulation of phenylpropanoid in A. thaliana. In the present work, an unprecedented attempt was made to generate Hlmed5a/med5b double loci mutant lines in hop using a CRISPR/Cas9-based genome editing system. The Hlmed5a/med5b double loci mutant lines showed reduced expression of structural genes of the flavonoid, humulone, and terpenoid biosynthetic pathways, which was more pronounced in the lupulin gland compared to leaf tissue and was consistent with their reduced accumulation. Phenotypic and anatomical observations revealed that Hlmed5a/med5b double loci mutant line exhibited robust growth, earlier flowering, earlier cone maturity, reduced cone size, variations in floral structure patterns, and distorted lupulin glands without any remarkable changes in leaf morphology, intensity of leaf color, and chlorophyll content. Comparative transcriptome analysis of leaf and lupulin gland tissues indicates that the expression of enzymatic genes related to secondary metabolite biosynthesis, phytohormone biosynthesis, floral organs, flowering time, and trichome development, including other genes related to starch and sucrose metabolism and defense mechanisms, were differentially modulated in the Hlmed5a/med5b lines. The combined results from functional and transcriptomic analyses illuminates the pivotal function of HlMED5a and HlMED5b in homeostasis of secondary meatbolites accumulation in hop.

Genome-wide identification and expression analysis of the ZF-HD gene family in pea (Pisum sativum L.).

Pea is a conventional grain-feed-grass crop in Tibet and the only high-protein legume in the region; therefore, it plays an important role in Tibetan food and grass security. Zinc finger-homeodomain (ZF-HD) belongs to a family of homozygous heterotypic cassette genes, which play an important role in plant growth, development, and response to adversity stress. Using a bioinformatics approach, 18 PsZF-HD family members were identified. These genes were distributed across seven chromosomes and two scaffold fragments, and evolutionary analysis classified them into two subgroups, MIF and ZHD. The MIF subgroup was subdivided into three subclasses (PsMIFⅠ-III), and the ZHD subgroup was subdivided into five subclasses (ZHDⅠ-V). The PsZF-HD members were named PsMIF1-PsMIF4 and PsZHD1-PsZHD14. Twelve conserved motifs and four conserved domains were identified from PsZF-HD family, of which MIF subgroup only contained one domain, while ZHD subgroup contained two types of domains. In addition, there were significant differences in the three-dimensional structures of the protein members of the two subgroups. Most PsZF-HD genes had no introns (13/18), and only five genes had one intron. Forty-five cis-acting elements were predicted and screened, involving four categories: light response, stress, hormone, and growth and development. Transcriptome analysis of different tissues during pea growth and development showed that PsZHD11, 8, 13, 14 and MIF4 were not expressed or were individually expressed in low amounts in the tissues, while the other 13 PsZF-HDs genes were differentially expressed and showed tissue preference, as seen in aboveground reproductive organs, where PsZHD6, 2, 10 and MIF1 (except immature seeds) were highly expressed. In the aerial vegetative organs, PsZHD6, 1, and 10 were significantly overexpressed, while in the underground root system, PsMIF3 was specifically overexpressed. The leaf transcriptome under a low-nitrogen environment showed that the expression levels of 17 PsZF-HDs members were upregulated in shoot organs. The leaf transcriptome analysis under a low-temperature environment showed stress-induced upregulation of PsZHD10 and one genes and down-regulation of PsZHD6 gene. These results laid the foundation for deeper exploration of the functions of the PsZF-HD genes and also improved the reference for molecular breeding for stress resistance in peas.

Leaf transcriptome analysis of Medicago ruthenica revealed its response and adaptive strategy to drought and drought recovery

BackgroundDrought is one of the main causes of losses in forage crop yield and animal production. Medicago ruthenica (L.) cv. Zhilixing is a high-yielding alfalfa cultivar also known for its high tolerance to drought. We analyzed the transcriptome profile of this cultivar throughout drought stress and recovery and we were able to describe its phased response through the expression profiles of overlapping gene networks and drought-specific genes.ResultsThe ABA and auxin signal transduction pathways are overlapping pathways in response to drought and drought recovery in forage crops. Medicago ruthenica (L.) cv. Zhilixing adopts different strategies at different degrees of drought stress. On the 9th day of drought, transcriptional regulations related to osmoregulation are enhanced mainly through increased activities of carbohydrate and amino acid metabolism, while photosynthetic activities were reduced to slow down growth. With drought prolonging, on the 12th day of drought, the synthesis of proline and other stored organic substances was suppressed in general. After recovery, Medicago ruthenica synthesizes flavonoids through the flavonoid biosynthesis pathway to remove accumulated ROS and repair the oxidative damage from water stress. In addition, the regulation of circadian rhythm seems to accelerate the drought recovery process.ConclusionsMedicago ruthenica adapts to drought by regulating the osmoregulatory system and photosynthesis, which appears to involve the ABA and auxin signaling pathways as key regulators. Furthermore, the synthesis of flavonoids and the regulation of the circadian rhythm can accelerate the recovery process. These results enriched our knowledge of molecular responses to drought and drought recovery in Medicago ruthenica and provide useful information for the development of new legume forage grass varieties with improved adaptability to drought stress.

Comparative transcriptome analysis of leaf, stem, and root tissues of Semiliquidambar cathayensis reveals candidate genes involved in terpenoid biosynthesis.

Semiliquidambar cathayensis is a traditional medicinal plant and endemic species in China. Its roots, branches, leaves, bark, and nectar are known to have therapeutic effects against rheumatoid arthritis, lumbar muscle strain, and several other diseases. However, limited knowledge regarding the molecular properties of S. cathayensis highlights the need for further research in order to elucidate the underlying pathways governing the synthesis of its active ingredients and regulation of its accumulation processes. We conducted transcriptome sequencing of the leaf, stem and root epidermises, and stem and root xylems of S. cathayensis with three biological replicates. Moreover, candidate genes involved in terpenoid biosynthesis, such as IDI, FPPS, DXR, SQS, GPPS, and HMGR were selected for quantitative real-time PCR analysis. We identified 88,582 unigenes. Among which, 36,144 unigenes were annotated to the nr protein database, 21,981 to the Gene Ontology database, 11,565 to the Clusters of Orthologous Groups database, 24,209 to the Pfam database, 21,685 to the SWISS-PROT database, and 12,753 to the Kyoto Encyclopedia of Genes and Genomes (KEGG), with 5072 unigenes common to all six databases. Of those annotated using the KEGG database, 187 unigenes were related to the terpenoid metabolism pathway, and expression analysis of the related genes indicated that the mevalonate and methylerythritol 4-phosphate pathways play different roles in terpenoid biosynthesis in different tissues of S. cathayensis. These findings greatly expand gene resources of S. cathayensis and provide basic data for the study of the biosynthetic pathways and molecular mechanisms of terpenoids.

Time-course transcriptome analysis of leaf cutting provides an insight into the mechanism of Zamioculcas zamiifolia vegetative regeneration

ISHS II International Symposium on Tropical and Subtropical Ornamentals Time-course transcriptome analysis of leaf cutting provides an insight into the mechanism of Zamioculcas zamiifolia vegetative regeneration

Transcriptional Analysis of Maize Leaf Tissue Treated With Seaweed Extract Under Drought Stress

Kappaphycus alvarezii seaweed extract (KSWE) has been known for its plant biostimulant and stress alleviation activities on various crops. However, very few reports are available depicting its impact at the molecular level, which is crucial in identifying the mechanism of action of KSWE on plants. Here, maize leaf tissue of control and KSWE-treated plants were analyzed for their transcriptional changes under drought stress. KSWE was applied foliarly at the V5 stage of maize crop under drought, and leaf transcriptome analysis was performed. It was found that a total of 380 and 631 genes were up- and downregulated, respectively, due to the application of KSWE. Genes involved in nitrate transportation, signal transmission, photosynthesis, transmembrane transport of various ions, glycogen, and starch biosynthetic processes were found upregulated in KSWE-treated plants, while genes involved in the catabolism of polysaccharide molecules such as starch as well as cell wall macromolecules like chitin and protein degradation were found downregulated. An overview of differentially expressed genes involved in metabolic as well as regulatory processes in KSWE-treated plants was also analyzed via Mapman tool. Phytohormone signaling genes such as cytokinin-independent 1 (involved in cytokine signal transduction), Ent-kaurene synthase and GA20 oxidase (involved in gibberellin synthesis), and gene of 2-oxoglutarate-dependent dioxygenase enzyme activity (involved in ethylene synthesis) were found upregulated while 9-cis-epoxycarotenoid dioxygenase (a gene involved in abscisic acid synthesis) was found downregulated due to the application of KSWE. Modulation of gene expression in maize leaf tissue in response to KSWE treatment elucidates mechanisms to ward off drought stress, which can be extended to understand similar phenomenon in other crops as well. This molecular knowledge can be utilized to make the use of KSWE more efficient and sustainable.

Drought Stress-Mediated Transcriptome Profile Reveals NCED as a Key Player Modulating Drought Tolerance in Populus davidiana.

Populus trichocarpa has been studied as a model poplar species through biomolecular approaches and was the first tree species to be genome sequenced. In this study, we employed a high throughput RNA-sequencing (RNA-seq) mediated leaf transcriptome analysis to investigate the response of four different Populus davidiana cultivars to drought stress. Following the RNA-seq, we compared the transcriptome profiles and identified two differentially expressed genes (DEGs) with contrasting expression patterns in the drought-sensitive and tolerant groups, i.e., upregulated in the drought-tolerant P. davidiana groups but downregulated in the sensitive group. Both these genes encode a 9-cis-epoxycarotenoid dioxygenase (NCED), a key enzyme required for abscisic acid (ABA) biosynthesis. The high-performance liquid chromatography (HPLC) measurements showed a significantly higher ABA accumulation in the cultivars of the drought-tolerant group following dehydration. The Arabidopsis nced3 loss-of-function mutants showed a significantly higher sensitivity to drought stress, ~90% of these plants died after 9 days of drought stress treatment. The real-time PCR analysis of several key genes indicated a strict regulation of drought stress at the transcriptional level in the P. davidiana drought-tolerant cultivars. The transgenic P. davidiana NCED3 overexpressing (OE) plants were significantly more tolerant to drought stress as compared with the NCED knock-down RNA interference (RNAi) lines. Further, the NCED OE plants accumulated a significantly higher quantity of ABA and exhibited strict regulation of drought stress at the transcriptional level. Furthermore, we identified several key differences in the amino acid sequence, predicted structure, and co-factor/ligand binding activity of NCED3 between drought-tolerant and susceptible P. davidiana cultivars. Here, we presented the first evidence of the significant role of NCED genes in regulating ABA-dependent drought stress responses in the forest tree P. davidiana and uncovered the molecular basis of NCED3 evolution associated with increased drought tolerance.

Comparative transcriptome analysis reveals that chlorophyll metabolism contributes to leaf color changes in wucai (Brassica campestris L.) in response to cold

BackgroundChlorophyll (Chl) is a vital photosynthetic pigment involved in capturing light energy and energy conversion. In this study, the color conversion of inner-leaves from green to yellow in the new wucai (Brassica campestris L.) cultivar W7–2 was detected under low temperature. The W7–2 displayed a normal green leaf phenotype at the seedling stage, but the inner leaves gradually turned yellow when the temperature was decreased to 10 °C/2 °C (day/night), This study facilitates us to understand the physiological and molecular mechanisms underlying leaf color changes in response to low temperature.ResultsA comparative leaf transcriptome analysis of W7–2 under low temperature treatment was performed on three stages (before, during and after leaf color change) with leaves that did not change color under normal temperature at the same period as a control. A total of 67,826 differentially expressed genes (DEGs) were identified. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) analysis revealed that the DEGs were mainly enriched in porphyrin and Chl metabolism, carotenoids metabolism, photosynthesis, and circadian rhythm. In the porphyrin and chlorophyll metabolic pathways, the expression of several genes was reduced [i.e. magnesium chelatase subunit H (CHLH)] under low temperature. Almost all genes [i.e. phytoene synthase (PSY)] in the carotenoids (Car) biosynthesis pathway were downregulated under low temperature. The genes associated with photosynthesis [i.e. photosystem II oxygen-evolving enhancer protein 1 (PsbO)] were also downregulated under LT. Our study also showed that elongated hypocotyl5 (HY5), which participates in circadian rhythm, and the metabolism of Chl and Car, is responsible for the regulation of leaf color change and cold tolerance in W7–2.ConclusionsThe color of inner-leaves was changed from green to yellow under low temperature in temperature-sensitive mutant W7–2. Physiological, biochemical and transcriptomic studies showed that HY5 transcription factor and the downstream genes such as CHLH and PSY, which regulate the accumulation of different pigments, are required for the modulation of leaf color change in wucai under low temperature.

Physiological and transcriptional response of carbohydrate and nitrogen metabolism in tomato plant leaves to nickel ion and nitrogen levels

This study investigated how nickel ion (Ni2+) improves carbohydrate and nitrogen (N) metabolism in tomato plant leaves under different N supply levels. We exposed the tomato plants to two levels of N (7.66 and 0.383 mmol•L−1) and two levels of Ni2+ (0 and 0.1 mg•L−1 NiSO4) under hydroponic conditions. After nine days of treatments, we harvested the leaves for physiological, biochemical, and transcriptome sequencing analysis. Low N (LN) levels reduced the concentration of total N and the activities of enzymes; however, Ni2+ can regulate these levels. Leaf transcriptome analysis identified 3277 differentially expressed genes (DEGs). The DEGs associated with the glycolytic pathway-tricarboxylic acid (EMP pathway-TCA) cycle, biosynthesis of amino acids, and N metabolism were downregulated after low N application, whereas those regulated by Ni2+ showed high transcript abundances. This study provides valuable insights into the carbohydrate and N metabolism mechanism of tomato plant leaves in response to Ni2+ and N levels.

Leaf transcriptome analysis of Lancaster versus other heterotic groups’ maize inbred lines revealed different regulation of cold‐responsive genes

AbstractOne of the strategies for overcoming global climate change threatening to decrease maize yield is early sowing. To contribute to the development of cold‐tolerant hybrids this research focused on the genetic background's comparative analysis in maize inbreds with good combining ability. Leaf whole‐transcriptome sequencing of 46 maize genotypes revealed 77 differentially expressed genes (DEGs) between Lancaster and other heterotic groups (i.e. BSSS, Iowa dent, Ohio), referred to as non‐Lancaster group, under optimal growing conditions. Cold test of the subset of four Lancaster and four non‐Lancaster lines showed that the former were cold sensitive and the latter cold tolerant. Cold‐induced expression analysis of seven DEGs in eight lines revealed different expression regulation dependent on the duration of cold exposure and genetic background for six out of seven analysed genes—chloroplast ATP‐sulphurylase, photosystem II cytochrome b559 alpha subunit, CIPK serine‐threonine protein kinase 15, glutamyl‐tRNA reductase, photosystem II reaction centre protein I and Calvin cycle CP12‐chloroplastic‐like encoding genes. The results imply that differently regulated basic processes between Lancaster and non‐Lancaster maize group involve, at least, photosynthesis and sulphate assimilation, contributing to their different cold response and different adaptation to low temperatures.

Global Transcriptome and Weighted Gene Co-expression Network Analyses of Growth-Stage-Specific Drought Stress Responses in Maize.

Drought is the major abiotic stress threatening maize (Zea mays L.) production globally. Despite recent scientific headway in deciphering maize drought stress responses, the overall picture of key genes, pathways, and co-expression networks regulating maize drought tolerance is still fragmented. Therefore, deciphering the molecular basis of maize drought tolerance remains pertinent. Here, through a comprehensive comparative leaf transcriptome analysis of drought-tolerant hybrid ND476 plants subjected to water-sufficient and water-deficit treatment conditions at flared (V12), tasseling (VT), the prophase of grain filling (R2), and the anaphase of grain filling (R4) crop growth stages, we report growth-stage-specific molecular mechanisms regulating maize drought stress responses. Based on the transcriptome analysis, a total of 3,451 differentially expressed genes (DEGs) were identified from the four experimental comparisons, with 2,403, 650, 397, and 313 DEGs observed at the V12, VT, R1, and R4 stages, respectively. Subsequently, 3,451 DEGs were divided into 12 modules by weighted gene co-expression network analysis (WGCNA), comprising 277 hub genes. Interestingly, the co-expressed genes that clustered into similar modules exhibited diverse expression tendencies and got annotated to different GO terms at different stages. MapMan analysis revealed that DEGs related to stress signal transduction, detoxification, transcription factor regulation, hormone signaling, and secondary metabolites biosynthesis were universal across the four growth stages. However, DEGs associated with photosynthesis and amino acid metabolism; protein degradation; transport; and RNA transcriptional regulation were uniquely enriched at the V12, VT, R2, and R4 stages, respectively. Our results affirmed that maize drought stress adaptation is a growth-stage-specific response process, and aid in clarifying the fundamental growth-stage-specific mechanisms regulating drought stress responses in maize. Moreover, genes and metabolic pathways identified here can serve as valuable genetic resources or selection targets for further functional validation experiments.

The influence of soil drought stress onthe leaf transcriptome offaba bean (Vicia faba L.) intheQinghai-Tibet Plateau.

Water deficit hasasignificantimpactongrowth, development andyield of fava bean (Vicia fava L.) in arid and semi-arid climates. The aim of this study was to identify differentially expressed genes in the Qinghai 13 genotype under soil drought through leaf transcriptome analysis. A total of 256.95M clean reads were obtained and assembled into 176334 unigenes, with an average length of 766bp. A total of 9126 (4439 upregulated and 4687 downregulated) differentially expressed genes (DEGs) were identified in faba bean leaves under soil drought. In total, 324 putative transcriptionfactors were identified and classified as belonging to different transcription factor families. According to GO and KEGG analysis, the soil drought stress-inducible DEGs encoded proteins mainly involved in regulating photosynthesis, osmoticadjustment, detoxification, autophagy and other functions. In addition, alargeportion of DEGs appeared to be novel because they could not be annotated in any functional databases, therefore, suggesting a specific response to soil drought in faba bean. Finally, RNA-seq analysis was validated by quantitative reverse-transcription PCR analysis. This work provides comprehensiveand valuableinformation for understanding the molecular mechanisms which faba bean uses to respond to soil drought.

Differential expression between drought-tolerant and drought-sensitive sugarcane under mild and moderate water stress as revealed by a comparative analysis of leaf transcriptome.

Sugarcane contributes 80% of global sugar production and to bioethanol generation for the bioenergy industry. Its productivity is threatened by drought that can cause up to 60% yield loss. This study used RNA-Seq to gain a better understanding of the underlying mechanism by which drought-tolerant sugarcane copes with water stress. We compared gene expression in KPS01-12 (drought-tolerant genotype) and UT12 (drought-sensitive genotype) that have significantly different yield loss rates under drought conditions. We treated KPS01-12 and UT12 with mild and moderate water stress and found differentially expressed genes in various biological processes. KPS01-12 had higher expression of genes that were involved in water retention, antioxidant secondary metabolite biosynthesis, and oxidative and osmotic stress response than UT12. In contrast, the sensitive genotype had more down-regulated genes that were involved in photosynthesis, carbon fixation and Calvin cycle than the tolerant genotype. Our obtained expression profiles suggest that the tolerant sugarcane has a more effective genetic response than the sensitive genotype at the initiation of drought stress. The knowledge gained from this study may be applied in breeding programs to improve sugarcane production in drought conditions.

SWEET Uniporter Gene Family Expression Profile in the Pitcher Development in the Carnivorous Plant Nepenthes sp.

Transcriptome analysis of leaf and pitcher at different developmental stages in the carnivorous plant Nepenthes sp. identified 20 cDNAs of the SWEET gene family encoding sugar uniporters of classes I–IV. The structure of NSWEET proteins generally corresponded to the 3-1-3 scheme typical of SWEET proteins in eukaryotes. The variability in the expression of NSWEET genes in the mature leaf and in the three stages of pitcher development indicates the possible functional diversity of these genes. It has been suggested that class I transporters (NSWEET2d, 2f, and 2h) may participate in export of sugars from the leaf to the site of pitcher meristem initiation, while proteins NSWEET1, 2a, 2k (class I), 4a, 4b, 4d (II), and 12c (III) may participate in the delivery of sugars for the primary development of the pitcher. In subsequent stages, they can be replaced by NSWEET2b, 2c, 2e, 2i, 2j (class I), 4c (II), and 12b (III), delivering hexoses and sucrose to the growing pitcher. In the fully formed pitcher, proteins NSWEET12a (III), 2g (I), and 16 (IV) can export sugars from the digestive fluid to the leaf.

Elucidating genes involved in sesquiterpenoid and flavonoid biosynthetic pathways in Saussurea lappa by de novo leaf transcriptome analysis

Saussurea lappa (family Asteraceae) possesses immense pharmacological potential mainly due to the presence of sesquiterpene lactones. In spite of its medicinal importance, S. lappa has been poorly explored at the molecular level. We initiated leaf transcriptome sequencing of S. lappa using the illumina highseq 2000 platform and generated 62,039,614 raw reads. Trinity assembler generated 122,434 contigs with an N50 value of 1053 bp. The assembled transcripts were compared against the non-redundant protein database at NCBI. The Blast2GO analysis assigned gene ontology (GO) terms, categorized into molecular functions (3132), biological processes (4477) and cellular components (1.927). Using KEGG, around 476 contigs were assigned to 39 pathways. For secondary metabolic pathways, we identified transcripts encoding genes involved in sesquiterpenoid and flavonoid biosynthesis. Relatively low number of transcripts were also found encoding for genes involved in the alkaloid pathway. Our data will contribute to functional genomics and metabolic engineering studies in this plant.

Comparative transcriptome analysis reveals molecular response to salinity stress of salt-tolerant and sensitive genotypes of indica rice at seedling stage

Abiotic stresses, such as salinity, greatly threaten the growth and productivity of plants. Rice (Oryza sativa L.) is one of the most important food crops, as well as a monocot model for genomic research. To obtain a global view of the molecular response to salinity stress, we conducted a leaf transcriptome analysis on rice seedlings. Two cultivars of rice subspecies indica, including the salt-tolerant genotype Xian156 and the salt-sensitive genotype IR28, were used in the present study. Eighteen RNA libraries were obtained from these two genotypes at three timepoints (0 h, 48 h and 72 h) after applying salinity stress. We obtained the reference-guided assembly of the rice transcriptome, which resulted in 1,375 novel genes, including 1,371 annotated genes. A comparative analysis between genotypes and time points showed 5,273 differentially expressed genes (DEGs), of which 286 DEGs were only found in the tolerant genotype. The Disease resistance response protein 206 and TIFY 10 A were differentially expressed, which were validated by quantitative real-time PCR. The differentially expressed genes identified through the mRNA transcriptome, along with the structure, provide a revealing insight into rice molecular response to salinity stress and underlie the salinity tolerance mechanism between genotypes.

De novo transcriptome analyses reveals putative pathway genes involved in biosynthesis and regulation of camptothecin in Nothapodytes nimmoniana (Graham) Mabb.

Comprehensive transcriptome analysis of leaf and root tissues of Nothapodytes nimmoniana unravels several putative pathway genes, transcription factors and CYPs related to camptothecin (CPT) biosynthesis. Additionally, post-transcriptional suppression by artificial microRNA (aMIR) of NnCYP76B6 (geraniol 10-hydroxylase) suggests its role in CPT biosynthesis. Tissue-specific LC-MS/MS analysis revealed the presence of secologanin as the central intermediate of MIA pathway in N. nimmoniana. Nothapodytes nimmoniana is a rich source of potent anticancer drug camptothecin (CPT) whose biosynthetic pathway is unresolved due to the lack of genomic and transcriptomic information. Present investigation entails deep transcriptome analysis of N. nimmoniana which led to identification of putative pathway genes and regulatory components involved in CPT biosynthesis. Using Illumina HiSeq 2500 sequencing platform a total of 31,172,889 (6.23Gb) and 31,218,626 (6.24Gb) raw reads were generated from leaf and root wood, respectively. These were assembled de novo into 138,183 unique contigs. Additionally, 16 cytochrome P450 transcripts related to secondary metabolism were also identified. Further, transcriptome data pool presented 1683 putative transcription factors of which transcripts corresponding to WRKY TFs were the most abundant (14.14%). A total of 2741 transcripts were differentially expressed out of which 478 contigs showed downregulation in root wood and 2263 contigs were up-regulated. Further, comparative analyses of 17 genes involved in CPT biosynthetic pathway were validated by qRT-PCR. On basis of intermediates, two distinct seco-iridoid pathways are involved in the biosynthesis of monoterpene indole alkaloids either through multiple isomers of strictosidinic acid or strictosidine. Tissue-specific LC-MS/MS analysis revealed the presence of secologanin as the central intermediate of MIA pathway in N. nimmoniana. Geraniol-10 hydroxylase (NnCYP76B6) an important enzyme in CPT biosynthesis which specifically shunts geraniol into the secologanin pathway was also cloned from the trancriptome resource. In planta transient expression of NnCYP76B6 showed a significant enhancement in mRNA transcript levels coincident with enhanced CPT accumulation. Further, artificial microRNA (aMIR) mediated downregulation of NnCYP76B6 resulted in reduction of mRNA transcript levels as well as CPT content in comparison to control. These empirical results suggest a plausible regulatory role for NnCYP76B6 in CPT biosynthesis and also establish a valuable repository for deciphering various structural, rate limiting and regulatory genes of CPT biosynthetic pathway.

Identification and characterization of Rubisco activase genes in Oryza punctata

AbstractRubisco activase (Rca), a specific chaperone, catalyzes the in vivo activation of Rubisco, and thus plays a major role in plant photosynthesis. Although the genes encoding Rubisco activase have been studied in many model or economic plants, few studies have analyzed their homologs in plants of closely related crop species. In this study, an Rca gene was identified and characterized in a wild relative of rice, Oryza punctata Kotschy ex Steud. The gene was 2747 bp long and possessed six exons and five introns with 47% GC content. Furthermore, cDNA sequencing produced two transcripts, RcaL and RcaS, that differed in the sequence by an inclusion of 99 bp at the carboxy terminus of RcaL. Sequence comparison between the two transcripts and the genomic DNA showed that there was a 20‐bp alternative splicing event that occurred at the fifth intron of the gene leading to the synthesis of a short polypeptide. The leaf transcriptome analysis showed that RcaS had a higher expression level than that of RcaL in a normal growth environment. In addition, the yeast two‐hybrid assays showed the small isoform of Rubisco activase in close contact with the large subunit of the Rubisco in this species, supporting the side‐on binding model of interaction between two proteins. This study broadens our understanding of the molecular characteristics of some essential genes in photosynthesis.

Transcriptome Analysis of Leaves, Flowers and Fruits Perisperm of Coffea arabica L. Reveals the Differential Expression of Genes Involved in Raffinose Biosynthesis.

Coffea arabica L. is an important crop in several developing countries. Despite its economic importance, minimal transcriptome data are available for fruit tissues, especially during fruit development where several compounds related to coffee quality are produced. To understand the molecular aspects related to coffee fruit and grain development, we report a large-scale transcriptome analysis of leaf, flower and perisperm fruit tissue development. Illumina sequencing yielded 41,881,572 high-quality filtered reads. De novo assembly generated 65,364 unigenes with an average length of 1,264 bp. A total of 24,548 unigenes were annotated as protein coding genes, including 12,560 full-length sequences. In the annotation process, we identified nine candidate genes related to the biosynthesis of raffinose family oligossacarides (RFOs). These sugars confer osmoprotection and are accumulated during initial fruit development. Four genes from this pathway had their transcriptional pattern validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Furthermore, we identified ~24,000 putative target sites for microRNAs (miRNAs) and 134 putative transcriptionally active transposable elements (TE) sequences in our dataset. This C. arabica transcriptomic atlas provides an important step for identifying candidate genes related to several coffee metabolic pathways, especially those related to fruit chemical composition and therefore beverage quality. Our results are the starting point for enhancing our knowledge about the coffee genes that are transcribed during the flowering and initial fruit development stages.

Whole transcriptome analysis of three leaf stages in two cultivars and one of their F1 hybrid of Camellia sinensis L. with differing EGCG content

Tea (Camellia sinensis (L.) O. Kuntze) is one of the most beneficial beverages due to its health attributes. Whole leaf transcriptome analysis of three leaf stages of C. sinensis has the potential to reveal candidate genes associated with these major beneficial compounds. Epigallocatechin-3-gallate (EGCG) is one of the most important bioactive catechin (antioxidant) in tea drink derived. EGCG has many important physiological functions and positive effects on human health, such as anticancer, antioxidation, controlling blood pressure, and blood sugar content. Although the major metabolic pathways of tea-specific compounds have been established, knowledge of the genes involved in the regulation and synthesis of EGCG in C. sinensis is limited. To gain insight into the molecular mechanisms that regulate EGCG levels, leaf transcriptomes were sequenced at three developmental stages (bud, second leaf, and fourth leaf) of two C. sinensis cultivars and one F1 hybrid line #1005 that possess varying EGCG levels. Approximately 470.9 M of quality controlled-filtered sequencing paired-end reads corresponding to 47.09 G nucleotides were obtained from which 130,762 unique sequences were identified. Genes whose expression correlated with changes in EGCG levels were identified, which may be associated with EGCG content. Prediction of the potential targets of microRNAs (miRNAs), the specific subset of long noncoding RNAs (LncRNAs), and mRNA-like noncoding RNAs (mlncRNAs) indicated that miRNAs may play a role in the expression regulation of the genes involved in EGCG levels.