Jianming Zeng

Molecular cloning and expression analysis of tea plant aquaporin (AQP) gene family.

The role of aquaporin proteins (AQPs) has been extensively studied in plants. However, the information of AQPs in the tea plant (Camellia sinensis) is unclear. In this manuscript, we isolated 20 full-length AQP cDNAs from the tea plant, and these sequences were classified into five subfamilies. The genes in these subfamilies displayed differential expression profiles in the studied tissues. The CsAQP expression patterns correlated with flower development and opening (FDO) and bud endodormancy (BED). To better understand the short-term expression patterns of CsAQPs in response to abiotic stress, tea plants were treated with abscisic acid (ABA), cold, salt or drought. ABA treatment down-regulated the expression of various CsAQPs. Salt up-regulated the transcription of most CsAQP genes. Cold treatment resulted in a complicated transcriptional regulation pattern for various CsAQPs. The expression of CsAQPs, especially plasma membrane intrinsic proteins (CsPIPs) and tonoplast intrinsic proteins (CsTIPs), was induced by drought and remained relatively high after rehydration in leaves, whereas almost all the CsAQPs were repressed in roots. Our results highlighted the diversity of CsAQPs in the tea plant and demonstrated that the CsPIP and CsTIP genes play a vital role in the stress response as well as in FDO and BED. Furthermore, certain CsSIPs (small basic intrinsic proteins), CsNIPs (NOD26-like intrinsic proteins) and CsXIPs (X intrinsic proteins) may regulate BED and FDO.

Biochemical and transcriptome analyses of a novel chlorophyll-deficient chlorina tea plant cultivar

BackgroundThe tea plant (Camellia sinensis (L.) O. Kuntze) is one of the most economically important woody crops. Recently, many leaf color genotypes have been developed during tea plant breeding and have become valuable materials in the processing of green tea. Although the physiological characteristics of some leaf color mutants of tea plants have been partially revealed, little is known about the molecular mechanisms leading to the chlorina phenotype in tea plants.ResultsThe yellow-leaf tea cultivar Zhonghuang 2 (ZH2) was selected during tea plant breeding. In comparison with Longjing 43 (LJ43), a widely planted green tea cultivar, ZH2 exhibited the chlorina phenotype and displayed significantly decreased chlorophyll contents. Transmission electron microscopy analysis revealed that the ultrastructure of the chloroplasts was disrupted, and the grana were poorly stacked in ZH2. Moreover, the contents of theanine and free amino acids were significantly higher, whereas the contents of carotenoids, catechins and anthocyanin were lower in ZH2 than in LJ43. Microarray analysis showed that the expression of 259 genes related to amino acid metabolism, photosynthesis and pigment metabolism was significantly altered in ZH2 shoots compared with those of LJ43 plants. Pathway analysis of 4,902 differentially expressed genes identified 24 pathways as being significantly regulated, including ‘cysteine and methionine metabolism’, ‘glycine, serine and threonine metabolism’, ‘flavonoid biosynthesis’, ‘porphyrin and chlorophyll metabolism’ and ‘carotenoid biosynthesis’. Furthermore, a number of differentially expressed genes could be mapped to the ‘theanine biosynthesis’, ‘chlorophyll biosynthesis’ and ‘flavonoid biosynthesis’ pathways. Changes in the expression of genes involved in these pathways might be responsible for the different phenotype of ZH2.ConclusionA novel chlorophyll-deficient chlorina tea plant cultivar was identified. Biochemical characteristics were analyzed and gene expression profiling was performed using a custom oligonucleotide-based microarray. This study provides further insights into the molecular mechanisms underlying the phenotype of the chlorina cultivar of Camellia sinensis.

Transcriptome Analysis of an Anthracnose-Resistant Tea Plant Cultivar Reveals Genes Associated with Resistance to Colletotrichum camelliae.

Tea plant breeding is a topic of great economic importance. However, disease remains a major cause of yield and quality losses. In this study, an anthracnose-resistant cultivar, ZC108, was developed. An infection assay revealed different responses to Colletotrichum sp. infection between ZC108 and its parent cultivar LJ43. ZC108 had greater resistance than LJ43 to Colletotrichum camelliae. Additionally, ZC108 exhibited earlier sprouting in the spring, as well as different leaf shape and plant architecture. Microarray data revealed that the genes that are differentially expressed between LJ43 and ZC108 mapped to secondary metabolism-related pathways, including phenylpropanoid biosynthesis, phenylalanine metabolism, and flavonoid biosynthesis pathways. In addition, genes involved in plant hormone biosynthesis and signaling as well as plant-pathogen interaction pathways were also changed. Quantitative real-time PCR was used to examine the expression of 27 selected genes in infected and uninfected tea plant leaves. Genes encoding a MADS-box transcription factor, NBS-LRR disease-resistance protein, and phenylpropanoid metabolism pathway components (CAD, CCR, POD, beta-glucosidase, ALDH and PAL) were among those differentially expressed in ZC108.

Identification of differential gene expression profiles between winter dormant and sprouting axillary buds in tea plant (Camellia sinensis) by suppression subtractive hybridization

Tea plant (Camellia sinensis (L.) O. Kuntze) is an important cash crop. In temperate regions, bud dormancy in winter and budbreak in spring are important biological phenomena for the tea plant life cycle. To understand the molecular mechanism of dormancy maintenance and release in tea plant, the differentially expressed genes in dormant and sprouting axillary buds were investigated in two cultivars (special early-sprouting tea cultivar ‘Camellia sinensis cv. Longjing 43’ (‘LJ’) and late-sprouting tea cultivar ‘C. sinensis cv. Zhenghe Dabaicha’ (‘ZD’)), using suppression subtractive hybridization (SSH) approach. Four high performance complementary DNA (cDNA)-SSH libraries (‘LJ’ sprouting bud library, ‘LJ’ dormant bud library, ‘ZD’ sprouting bud library and ‘ZD’ dormant bud library) were constructed, and 1,736 valid ESTs were obtained, in which 1,242 ESTs were unique to the sprouting bud libraries and 494 ESTs were unique to the dormant bud libraries. Based on sequence matching and gene ontology analysis, 1,287 unigenes consisting of 208 contigs and 1,079 singletons were identified, in which 995 had Blast hits. The putative functions of differentially regulated sequences were involved in most aspects of plant biological processes. The quality and expression patterns of partial ESTs from these four libraries were validated by qRT-PCR. We identified numerous differentially expressed genes mainly involved in stress response, water metabolism, cell division regulation, energy metabolism and hormone regulation from dormant and sprouting bud libraries. This study provides general information on tea plant axillary bud dormancy and release at the transcriptional level and provides some hypotheses for further exploration on the mechanism of bud dormancy and budbreak in tea plant.

Transcriptomic analysis of the effects of three different light treatments on the biosynthesis of characteristic compounds in the tea plant by RNA-Seq

The tea plant (Camellia sinensis) is considered an environment-sensitive plant, because the biosynthesis of tea’s characteristic compounds is easily influenced by light quality, ambient temperature, and humidity. The characteristic compounds in tea leaves, especially polyphenols, theanine, and caffeine, are important components that contribute to tea’s flavor. To investigate the effects of light quality on secondary metabolism in young shoots of tea plants at the messenger RNA (mRNA) transcript level, RNA-Seq was performed using one leaf and a bud from tea cultivar 108, which was reared in three different colored light conditions. Plastic film (blue, purple, and yellow) was used to create different monochromatic light conditions, which were compared to natural (white) light treatment as a control. Our results showed that, compared to the control, the 2667, 1938, and 2326 genes were differentially expressed and the 1518, 1032, and 1316 genes were upregulated in blue, purple, and yellow light conditions, respectively. Gene Ontology (GO) analysis indicated that oxidation-reduction, regulation of transcription, and protein phosphorylation were the main enriched biological process terms in all three treatments. Conversely, the top enriched terms in the cellular component and molecular function categories were plasma membrane and ATP binding. Furthermore, KEGG analysis showed that the genes which were downregulated in blue light were enriched for genes in the amino acid biosynthesis, flavonoid biosynthesis, and phenylpropanoid biosynthesis pathways. Similarly, phenylpropanoid biosynthesis and phenylalanine metabolism pathway genes were enriched among the transcripts that were downregulated in purple light and yellow light. Particularly, a few genes encoding enzymes involved in the biosynthesis of catechins, theanine, and caffeine exhibited significantly different expression levels in the different monochromatic light conditions. Compared to purple and yellow light treatments, more genes were differentially expressed in blue light relative to white light. These results demonstrated that light quality affects secondary metabolism in young shoots of the tea plant, and that light color can influence the biosynthesis of characteristic compounds in tea leaves.

Differential expression of gibberellin- and abscisic acid-related genes implies their roles in the bud activity-dormancy transition of tea plants

Key messageThirty genes involved in GA and ABA metabolism and signalling were identified, and the expression profiles indicated that they play crucial roles in the bud activity-dormancy transition in tea plants.AbstractGibberellin (GA) and abscisic acid (ABA) are fundamental phytohormones that extensively regulate plant growth and development, especially bud dormancy and sprouting transition in perennial plants. However, there is little information on GA- and ABA-related genes and their expression profiles during the activity-dormancy transition in tea plants. In the present study, 30 genes involved in the metabolism and signalling pathways of GA and ABA were first identified, and their expression patterns in different tissues were assessed. Further evaluation of the expression patterns of selected genes in response to GA3 and ABA application showed that CsGA3ox, CsGA20ox, CsGA2ox, CsZEP and CsNCED transcripts were differentially expressed after exogenous treatment. The expression profiles of the studied genes during winter dormancy and spring sprouting were investigated, and somewhat diverse expression patterns were found for GA- and ABA-related genes. This diversity was associated with the bud activity-dormancy cycle of tea plants. These results indicate that the genes involved in the metabolism and signalling of GA and ABA are important for regulating the bud activity-dormancy transition in tea plants.

Integrative transcriptional and metabolic analyses provide insights into cold spell response mechanisms in young shoots of the tea plant

Green tea has attracted an increasing number of consumers worldwide due to its multiple health benefits. With the increase in global warming, more frequent cold spells in the spring often cause more serious damage to green tea production because of the young leaves used. We recorded the changes in climatic conditions during a typical cold spell and the damage symptoms caused by the cold spell in different tea cultivars and breeding lines. By simulating the low temperature of a cold spell under controlled conditions, comparative transcriptome and metabolic analyses were performed with sprouting shoots. Many pathways and genes were regulated differentially by the cold spell conditions. Taking into account the metabolic analysis, the results suggested that the mitogen-activated protein kinase (MAPK)-dependent ethylene and calcium signalling pathways were two major early cold-responsive mechanisms involved in sprouting shoots and were followed by the induction of the Inducer of CBF Expressions (ICE)-C-repeat binding factors (CBF)-cold-responsive (COR) signalling pathway to augment cold tolerance. During the cold shock, growth, photosynthesis and secondary metabolism-mainly involving flavonoid biosynthesis-were remarkably affected. Notably, the increased starch metabolism, which might be dependent on the high expression of β-amylase3 (BAM3) induced by CBF, played crucial roles in protecting young shoots against freezing cold. A schematic diagram of cold spell response mechanisms specifically involved in the sprouting shoots of the tea plant is ultimately proposed. Some essential transcriptional and metabolic changes were further confirmed in the plant materials under natural cold spell conditions. Our results provide a global view of the reprograming of transcription and metabolism in sprouting tea shoots during a cold spell and meaningful information for future practices.

Tea plant SWEET transporters: expression profiling, sugar transport, and the involvement of CsSWEET16 in modifying cold tolerance in Arabidopsis

Key messageThirteen SWEET transporters were identified in Camellia sinensis and the cold-suppression gene CsSWEET16 contributed to sugar compartmentation across the vacuole and function in modifying cold tolerance in Arabidopsis.AbstractThe sugars will eventually be exported transporters (SWEET) family of sugar transporters in plants is a recently identified protein family of sugar uniporters that contain seven transmembrane helices harbouring two MtN3 motifs. SWEETs play important roles in various biological processes, including plant responses to environmental stimuli. In this study, 13 SWEET transporters were identified in Camellia sinensis and were divided into four clades. Transcript abundances of CsSWEET genes were detected in various tissues. CsSWEET1a/1b/2a/2b/2c/3/9b/16/17 were expressed in all of the selected tissues, whereas the expression of CsSWEET5/7/9a/15 was not detected in some tissues, including those of mature leaves. Expression analysis of nine CsSWEET genes in leaves in response to abiotic stresses, natural cold acclimation and Colletotrichum camelliae infection revealed that eight CsSWEET genes responded to abiotic stress, while CsSWEET3 responded to C. camelliae infection. Functional analysis of 13 CsSWEET activities in yeast revealed that CsSWEET1a/1b/7/17 exhibit transport activity for glucose analogues and other types of hexose molecules. Further characterization of the cold-suppression gene CsSWEET16 revealed that this gene is localized in the vacuolar membrane. CsSWEET16 contributed to sugar compartmentation across the vacuole and function in modifying cold tolerance in Arabidopsis. Together, these findings demonstrate that CsSWEET genes play important roles in the response to abiotic and biotic stresses in tea plants and provide insights into the characteristics of SWEET genes in tea plants, which could serve as the basis for further functional identification of such genes.

Gene Characterization and Expression Analysis Reveal the Importance of Auxin Signaling in Bud Dormancy Regulation in Tea Plant

The tea plant is an economically important woody plant whose raw leaves are used for tea production. Winter bud dormancy is not only a useful biological strategy for tea plant survival but also a biological event that affects the economics of tea production. Based on our previous transcriptome analysis of axillary buds in different dormancy states, we reanalyzed a large number of differentially expressed auxin-related genes and determined the relative importance of the roles of auxin signaling in bud dormancy regulation in tea plant. Subsequently, we cloned the full-length cDNA sequence of several auxin-related genes in the AUX/LAX, PIN/PILS, AUX/IAA, GH3, and SAUR gene families, characterized these genes and performed a phylogenetic analysis, and conserved motif search using the sequences of their encoded proteins. Expression profile analyses, including tissue-specific expression and time-course expression during the active-dormant-active status transitions of overwinter buds, were carried out, combined with IAA content detection. Generally, the expression patterns of auxin-related genes were consistent with the IAA content changes in buds and their active-dormant status transition. In particular, we confirmed the crucial roles of the auxin transport gene CsLAX2 and the early auxin response genes CsGH3.6, CsGH3.9, CsGH3.10, CsIAA26, CsIAA33, CsSAUR50, and CsSAUR41 in bud dormancy regulation in tea plant. Our results validate the important role of auxin in tea plant dormancy regulation and provide useful information for further functional studies.

Cloning and Expression Analysis of Gibberellin Receptor Gene CsGID1a in Tea Plant (Camellia sinensis)

GID1(Gibberellin insensitive dwarf1),as the soluble gibberellin(GA) receptor in GA signaling pathway,plays the vital role in GA reactions.In this study,the homologous gene of GID1 was isolated with RT-PCR and RACE-PCR from tea plant(Camellia sinensis).The obtained cDNA sequence,named CsGID1a,had the full-length of 1411 bp containing a 1023 bp open reading frame(ORF),encoding 341 amino acid residues,and was submitted to GenBank with accession number JX235369.The bioinformatics characterization indicated that CsGID1a was a non-secretory protein without a signal peptide.The molecular weight and theoretic isoelectric point of CsGID1a are 38.53 kD and 5.62,respectively.CsGID1a was located in the nucleus,encoding a protein with one transmembrane domain.CsGID1a contained hormone sensitive lipsase family(HSL) conserved domains,HGG and GXSXG motif,and shared the plant carboxylesterase tertiary structure.Homologous alignment and phylogenetic tree showed that CsGID1a shared over 60% amino acid sequence similarity with that of other species,and had the highest similarity(87%) and the closest genetic relationship to Vitis vinifera.The real-time PCR analysis showed that the expression of CsGID1a was down-regulated by high concentration of GA 3(1.0×10-5 mol L-1) and reduced slowly during the treatment for five hours.The further experiments suggested that the expression of CsGID1a was also decreased in the process of bud sprouting.These results demonstrated that CsGID1a and GA could be associated with bud bursting in tea plant in spring.