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Biochemical and transcriptomic analyses reveal different metabolite biosynthesis profiles among three color and developmental stages in ‘ Anji Baicha ’ ( Camellia sinensis )

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ABSTRACT

Background: The new shoots of the albino tea cultivar ‘Anji Baicha’ are yellow or white at low temperatures and turn green as the environmental temperatures increase during the early spring. ‘Anji Baicha’ metabolite profiles exhibit considerable variability over three color and developmental stages, especially regarding the carotenoid, chlorophyll, and theanine concentrations. Previous studies focused on physiological characteristics, gene expression differences, and variations in metabolite abundances in albino tea plant leaves at specific growth stages. However, the molecular mechanisms regulating metabolite biosynthesis in various color and developmental stages in albino tea leaves have not been fully characterized.

Results: We used RNA-sequencing to analyze ‘Anji Baicha’ leaves at the yellow-green, albescent, and re-greening stages. The leaf transcriptomes differed considerably among the three stages. Functional classifications based on Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes enrichment analyses revealed that differentially expressed unigenes were mainly related to metabolic pathways, biosynthesis of secondary metabolites, phenylpropanoid biosynthesis, and carbon fixation in photosynthetic organisms. Chemical analyses revealed higher β-carotene and theanine levels, but lower chlorophyll a levels, in the albescent stage than in the green stage. Furthermore, unigenes involved in carotenoid, chlorophyll, and theanine biosyntheses were identified, and the expression patterns of the differentially expressed unigenes in these biosynthesis pathways were characterized. Through co-expression analyses, we identified the key genes in these pathways. These genes may be responsible for the metabolite biosynthesis differences among the different leaf color and developmental stages of ‘Anji Baicha’ tea plants.

Conclusions: Our study presents the results of transcriptomic and biochemical analyses of ‘Anji Baicha’ tea plants at various stages. The distinct transcriptome profiles for each color and developmental stage enabled us to identify changes to biosynthesis pathways and revealed the contributions of such variations to the albino phenotype of tea plants. Furthermore, comparisons of the transcriptomes and related metabolites helped clarify the molecular regulatory mechanisms underlying the secondary metabolic pathways in different stages.

Electronic supplementary material: The online version of this article (doi:10.1186/s12870-016-0885-2) contains supplementary material, which is available to authorized users.

No MeSH data available.


Theanine biosynthesis pathway and the related unigenes. a The glutamine, glutamate, alanine, ethylamine, and theanine concentrations were determined at different stages. The double asterisks indicate a significant difference between the YG or W stages and the G stage (P < 0.01; Student’s t-test). b Theanine biosynthesis pathway. The bracketed numbers in red following each gene name indicate the number of corresponding unigenes identified in our database. c All differentially expressed genes involved in theanine biosynthesis were hierarchically clustered and mapped using the fragments per kilobase of exon per million mapped read values. Colors indicate the normalized signal intensity as defined in the bar
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Fig6: Theanine biosynthesis pathway and the related unigenes. a The glutamine, glutamate, alanine, ethylamine, and theanine concentrations were determined at different stages. The double asterisks indicate a significant difference between the YG or W stages and the G stage (P < 0.01; Student’s t-test). b Theanine biosynthesis pathway. The bracketed numbers in red following each gene name indicate the number of corresponding unigenes identified in our database. c All differentially expressed genes involved in theanine biosynthesis were hierarchically clustered and mapped using the fragments per kilobase of exon per million mapped read values. Colors indicate the normalized signal intensity as defined in the bar

Mentions: The chlorophyll metabolic pathway includes the following three phases: biosynthesis of chlorophyll a, interconversion between chlorophylls a and b, and degradation of chlorophyll a [41–44]. In our database, 71 unigenes were related to chlorophyll biosynthesis, including the genes for almost all of the key enzymes (Fig. 5b and Additional file 4). The expression levels of the DEGs were determined via a hierarchical cluster analysis (Fig. 5c). The expression levels of the gene encoding geranylgeranyl diphosphate reductase (CAO), which is an enzyme involved in chlorophyll b biosynthesis, are elevated in plants with insufficient amounts of chlorophyll b [45]. Two CAO unigenes (unigene 16722 and unigene 37586) were identified in our database, and both were significantly up-regulated in the G stage. This finding implies that high CAO expression levels might induce the efficient biosynthesis of chlorophyll b to increase its concentration during the G stage (Fig. 5a). NYC1 encodes chlorophyll b reductase, which catalyzes the degradation of chlorophyll b to 7-hydroxymethyl chlorophyll a [46, 47]. The degradation of chlorophyll b is suppressed in NYC1 mutant plants, which remain green until just before death due to natural senescence [48]. Three NYC1 unigenes were detected in our database, and the expression levels of two of these (unigene 37738 and unigene 26042) were significantly higher in the W stage than in the YG and G stages. High NYC1 unigene expression levels during the W stage might enhance the degradation of chlorophyll b (Fig. 4c). In plants, the NOL (NYC1-like) protein is closely related to NYC1, and NOL mutant plants also remain green, similar to wild-type plants [49]. Three NOL unigenes were identified in our database, but the expression levels of these unigenes were not significantly different between the YG and G stages. Hydroxymethyl chlorophyll a reductase (HCAR) converts 7-hydroxymethyl chlorophyll a to chlorophyll a [50]. The HCAR expression levels are strongly correlated with chlorophyll content in carnation flower petals [51]. Additionally, HCAR expression is strongly up-regulated during the stage in which etiolated A. thaliana seedlings turn green [52]. These results suggest that HCAR is essential for chlorophyll turnover during the greening stage. Three HCAR unigenes were identified in our database, and only one (unigene 168719) was significantly up-regulated during the G stage. High expression levels of this unigene in the G stage might contribute to the higher chlorophyll a concentrations during this stage compared with the other two stages (Fig. 5a). Pheophytinase (PPH) has a key function in chlorophyll degradation. The expression of PPH is induced in darkness, which accelerates chlorophyll degradation. In PPH mutant plants, chlorophyll degradation is inhibited, and the plants exhibit a sustained green phenotype during senescence [53]. In our study, the expression of PPH (unigene 5416) was lower in the YG stage than in the W and G stages (Fig. 5b), suggesting the lowest chlorophyll a degradation rate occurred during the YG stage. However, the chlorophyll a concentration in the YG stage was lower than that during the G stage. This may have been because the expression level for unigene 4795, which encodes chlorophyll synthase (CHLG), was significantly higher in the G stage than in the YG stage (Fig. 5c). The higher chlorophyll a concentration in the G stage than in the YG stage might be because chlorophyll a was synthesized faster than it was degraded. Chlorophyllase (CLH) catalyzes the conversion of chlorophyll a to chlorophyllide a. In citrus plants, CLH expression levels are negatively correlated with chlorophyll contents [54]. We identified six candidate CLH unigenes in our database (Fig. 5b), four of which were significantly differentially expressed between the YG and G stages. The expression levels of unigene 56286 and unigene 90639 were up-regulated during the YG and W stages, respectively, while two unigenes were up-regulated in the G stage (unigene 11687 and unigene 131928) (Fig. 6c). Unigene 56286 and unigene 90639 might contribute to chlorophyll a degradation to lower the chlorophyll concentration more during the YG and W stages than in the G stage. The expression levels of PaO, which encodes pheophorbide a oxygenase, are closely correlated with the rate of chlorophyll breakdown [55]. Three PaO unigenes were detected in our database, but only one (unigene 89999) was significantly up-regulated from the YG stage to the G stage. However, the chlorophyll content in the G stage was relatively high (Fig. 5a). Identical results were reported for carnations [51], potentially because the post-transcriptional regulation of PaO inhibits the activity of the encoded enzyme [56] or because chlorophyll a is synthesized faster than it is degraded.Fig. 6


Biochemical and transcriptomic analyses reveal different metabolite biosynthesis profiles among three color and developmental stages in ‘ Anji Baicha ’ ( Camellia sinensis )
Theanine biosynthesis pathway and the related unigenes. a The glutamine, glutamate, alanine, ethylamine, and theanine concentrations were determined at different stages. The double asterisks indicate a significant difference between the YG or W stages and the G stage (P < 0.01; Student’s t-test). b Theanine biosynthesis pathway. The bracketed numbers in red following each gene name indicate the number of corresponding unigenes identified in our database. c All differentially expressed genes involved in theanine biosynthesis were hierarchically clustered and mapped using the fragments per kilobase of exon per million mapped read values. Colors indicate the normalized signal intensity as defined in the bar
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Related In: Results  -  Collection

License 1 - License 2
Show All Figures
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Fig6: Theanine biosynthesis pathway and the related unigenes. a The glutamine, glutamate, alanine, ethylamine, and theanine concentrations were determined at different stages. The double asterisks indicate a significant difference between the YG or W stages and the G stage (P < 0.01; Student’s t-test). b Theanine biosynthesis pathway. The bracketed numbers in red following each gene name indicate the number of corresponding unigenes identified in our database. c All differentially expressed genes involved in theanine biosynthesis were hierarchically clustered and mapped using the fragments per kilobase of exon per million mapped read values. Colors indicate the normalized signal intensity as defined in the bar
Mentions: The chlorophyll metabolic pathway includes the following three phases: biosynthesis of chlorophyll a, interconversion between chlorophylls a and b, and degradation of chlorophyll a [41–44]. In our database, 71 unigenes were related to chlorophyll biosynthesis, including the genes for almost all of the key enzymes (Fig. 5b and Additional file 4). The expression levels of the DEGs were determined via a hierarchical cluster analysis (Fig. 5c). The expression levels of the gene encoding geranylgeranyl diphosphate reductase (CAO), which is an enzyme involved in chlorophyll b biosynthesis, are elevated in plants with insufficient amounts of chlorophyll b [45]. Two CAO unigenes (unigene 16722 and unigene 37586) were identified in our database, and both were significantly up-regulated in the G stage. This finding implies that high CAO expression levels might induce the efficient biosynthesis of chlorophyll b to increase its concentration during the G stage (Fig. 5a). NYC1 encodes chlorophyll b reductase, which catalyzes the degradation of chlorophyll b to 7-hydroxymethyl chlorophyll a [46, 47]. The degradation of chlorophyll b is suppressed in NYC1 mutant plants, which remain green until just before death due to natural senescence [48]. Three NYC1 unigenes were detected in our database, and the expression levels of two of these (unigene 37738 and unigene 26042) were significantly higher in the W stage than in the YG and G stages. High NYC1 unigene expression levels during the W stage might enhance the degradation of chlorophyll b (Fig. 4c). In plants, the NOL (NYC1-like) protein is closely related to NYC1, and NOL mutant plants also remain green, similar to wild-type plants [49]. Three NOL unigenes were identified in our database, but the expression levels of these unigenes were not significantly different between the YG and G stages. Hydroxymethyl chlorophyll a reductase (HCAR) converts 7-hydroxymethyl chlorophyll a to chlorophyll a [50]. The HCAR expression levels are strongly correlated with chlorophyll content in carnation flower petals [51]. Additionally, HCAR expression is strongly up-regulated during the stage in which etiolated A. thaliana seedlings turn green [52]. These results suggest that HCAR is essential for chlorophyll turnover during the greening stage. Three HCAR unigenes were identified in our database, and only one (unigene 168719) was significantly up-regulated during the G stage. High expression levels of this unigene in the G stage might contribute to the higher chlorophyll a concentrations during this stage compared with the other two stages (Fig. 5a). Pheophytinase (PPH) has a key function in chlorophyll degradation. The expression of PPH is induced in darkness, which accelerates chlorophyll degradation. In PPH mutant plants, chlorophyll degradation is inhibited, and the plants exhibit a sustained green phenotype during senescence [53]. In our study, the expression of PPH (unigene 5416) was lower in the YG stage than in the W and G stages (Fig. 5b), suggesting the lowest chlorophyll a degradation rate occurred during the YG stage. However, the chlorophyll a concentration in the YG stage was lower than that during the G stage. This may have been because the expression level for unigene 4795, which encodes chlorophyll synthase (CHLG), was significantly higher in the G stage than in the YG stage (Fig. 5c). The higher chlorophyll a concentration in the G stage than in the YG stage might be because chlorophyll a was synthesized faster than it was degraded. Chlorophyllase (CLH) catalyzes the conversion of chlorophyll a to chlorophyllide a. In citrus plants, CLH expression levels are negatively correlated with chlorophyll contents [54]. We identified six candidate CLH unigenes in our database (Fig. 5b), four of which were significantly differentially expressed between the YG and G stages. The expression levels of unigene 56286 and unigene 90639 were up-regulated during the YG and W stages, respectively, while two unigenes were up-regulated in the G stage (unigene 11687 and unigene 131928) (Fig. 6c). Unigene 56286 and unigene 90639 might contribute to chlorophyll a degradation to lower the chlorophyll concentration more during the YG and W stages than in the G stage. The expression levels of PaO, which encodes pheophorbide a oxygenase, are closely correlated with the rate of chlorophyll breakdown [55]. Three PaO unigenes were detected in our database, but only one (unigene 89999) was significantly up-regulated from the YG stage to the G stage. However, the chlorophyll content in the G stage was relatively high (Fig. 5a). Identical results were reported for carnations [51], potentially because the post-transcriptional regulation of PaO inhibits the activity of the encoded enzyme [56] or because chlorophyll a is synthesized faster than it is degraded.Fig. 6

View Article: PubMed Central - PubMed

ABSTRACT

Background: The new shoots of the albino tea cultivar &lsquo;Anji Baicha&rsquo; are yellow or white at low temperatures and turn green as the environmental temperatures increase during the early spring. &lsquo;Anji Baicha&rsquo; metabolite profiles exhibit considerable variability over three color and developmental stages, especially regarding the carotenoid, chlorophyll, and theanine concentrations. Previous studies focused on physiological characteristics, gene expression differences, and variations in metabolite abundances in albino tea plant leaves at specific growth stages. However, the molecular mechanisms regulating metabolite biosynthesis in various color and developmental stages in albino tea leaves have not been fully characterized.

Results: We used RNA-sequencing to analyze &lsquo;Anji Baicha&rsquo; leaves at the yellow-green, albescent, and re-greening stages. The leaf transcriptomes differed considerably among the three stages. Functional classifications based on Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes enrichment analyses revealed that differentially expressed unigenes were mainly related to metabolic pathways, biosynthesis of secondary metabolites, phenylpropanoid biosynthesis, and carbon fixation in photosynthetic organisms. Chemical analyses revealed higher &beta;-carotene and theanine levels, but lower chlorophyll a levels, in the albescent stage than in the green stage. Furthermore, unigenes involved in carotenoid, chlorophyll, and theanine biosyntheses were identified, and the expression patterns of the differentially expressed unigenes in these biosynthesis pathways were characterized. Through co-expression analyses, we identified the key genes in these pathways. These genes may be responsible for the metabolite biosynthesis differences among the different leaf color and developmental stages of &lsquo;Anji Baicha&rsquo; tea plants.

Conclusions: Our study presents the results of transcriptomic and biochemical analyses of &lsquo;Anji Baicha&rsquo; tea plants at various stages. The distinct transcriptome profiles for each color and developmental stage enabled us to identify changes to biosynthesis pathways and revealed the contributions of such variations to the albino phenotype of tea plants. Furthermore, comparisons of the transcriptomes and related metabolites helped clarify the molecular regulatory mechanisms underlying the secondary metabolic pathways in different stages.

Electronic supplementary material: The online version of this article (doi:10.1186/s12870-016-0885-2) contains supplementary material, which is available to authorized users.

No MeSH data available.