The NAC-like transcription factor CsNAC7 positively regulates the caffeine biosynthesis-related gene yhNMT1 in Camellia sinensis

Caffeine is an important functional substance and is abundant in tea plant, but little is known about how its biosynthesis is regulated by transcription factors. In this study, the NAC-like transcription factor-encoding gene CsNAC7, which is involved in caffeine synthesis, was isolated from a Yinghong 9 cDNA library using a yeast one-hybrid assay; this gene comprises 1371 bp nucleotides and is predicted to encode 456 amino acids. The expression of CsNAC7 at the transcriptional level in tea shoots shared a similar pattern with that of the caffeine synthase gene yhNMT1 in the spring and summer, and its expressed protein was localized in the nucleus. Assays of gene activity showed that CsNAC7 has self-activation activity in yeast, that the active region is at the N-terminus, and that the transient expression of CsNAC7 could significantly promote the expression of yhNMT1 in tobacco leaves. In addition, overexpression or silencing of CsNAC7 significantly increased or decreased the expression of yhNMT1 and the accumulation of caffeine in transgenic tea calli, respectively. Our data suggest that the isolated transcription factor CsNAC7 positively regulates the caffeine synthase gene yhNMT1 and promotes caffeine accumulation in tea plant.

The absence of the caffeine synthase gene is involved in the naturally decaffeinated status of Coffea humblotiana, a wild species from Comoro archipelago

Caffeine is the most consumed alkaloid stimulant in the world. It is synthesized through the activity of three known N-methyltransferase proteins. Here we are reporting on the 422-Mb chromosome-level assembly of the Coffea humblotiana genome, a wild and endangered, naturally caffeine-free, species from the Comoro archipelago. We predicted 32,874 genes and anchored 88.7% of the sequence onto the 11 chromosomes. Comparative analyses with the African Robusta coffee genome (C. canephora) revealed an extensive genome conservation, despite an estimated 11 million years of divergence and a broad diversity of genome sizes within the Coffea genus. In this genome, the absence of caffeine is likely due to the absence of the caffeine synthase gene which converts theobromine into caffeine through an illegitimate recombination mechanism. These findings pave the way for further characterization of caffeine-free species in the Coffea genus and will guide research towards naturally-decaffeinated coffee drinks for consumers.

Convergent Biochemical Pathways for Xanthine Alkaloid Production in Plants Evolved from Ancestral Enzymes with Different Catalytic Properties

Convergent evolution is widespread but the extent to which common ancestral conditions are necessary to facilitate the independent acquisition of similar traits remains unclear. In order to better understand how ancestral biosynthetic catalytic capabilities might lead to convergent evolution of similar modern-day biochemical pathways, we resurrected ancient enzymes of the caffeine synthase (CS) methyltransferases that are responsible for theobromine and caffeine production in flowering plants. Ancestral CS enzymes of Theobroma, Paullinia, and Camellia exhibited similar substrate preferences but these resulted in the formation of different sets of products. From these ancestral enzymes, descendants with similar substrate preference and product formation independently evolved after gene duplication events in Theobroma and Paullinia. Thus, it appears that the convergent modern-day pathways likely originated from ancestral pathways with different inferred flux. Subsequently, the modern-day enzymes originated independently via gene duplication and their convergent catalytic characteristics evolved to partition the multiple ancestral activities by different mutations that occurred in homologous regions of the ancestral proteins. These results show that even when modern-day pathways and recruited genes are similar, the antecedent conditions may be distinctive such that different evolutionary steps are required to generate convergence.

Inoculation with Clariodeoglomus etunicatum improves leaf food quality of tea exposed to P stress

The present study aimed to evaluate the effect of an arbuscular mycorrhizal fungus (AMF), Clariodeoglomus etunicatum, on leaf food quality and relevant gene expression levels of tea (Camellia sinensis cv. ‘Fuding Dabaicha’) seedlings exposed to 0.5 μM P (P0.5) and 50 μM P (P50) levels. Twenty-four weeks later, the seedlings recorded higher root mycorrhizal fungal colonization in P50 than in P0.5. AMF-inoculated tea plants represented significantly higher leaf fructose and glucose contents and lower sucrose content than non-inoculated plants, irrespective of substate P levels. AMF treatment also increased total amino acids content in P0.5 and P50, accompanied with higher expression of glutamate dehydrogenase (CsGDH) and lower expression of glutamine synthetase (CsGS) and glutamine oxoglutarate aminotransferase (CsGOGAT). The total flavonoid content was higher in mycorrhizal versus non-mycorrhizal plants under P0.5 and P50, together with induced expression of phenylalanine ammonia-lyase (CsPAL) and cinnamic acid 4-hydroxylase (CsC4H). Mycorrhizal fungal inoculation improved catechins content, which is due to the up-regulated expression of flavanone 3-hydroxylase (CsF3H), flavonoid 3'-hydroxylase (CsF3'H), dihydroflavonol 4-reductase (CsDFR), leucoanthocyanidin reductase (CsLAR), anthocyanidin reductase (CsANR), and chalcone isomerase (CsCHI) under P0.5. However, under P50, the gene involved in catechins synthesis was not affected or down-regulated by mycorrhization, implying a complex mechanism (e.g. nutrient improvement). AMF also inhibited the tea caffeine synthase 1 (CsTCS1) expression regardless of P levels. Therefore, the results of this study concluded that inoculation with C. etunicatum improves leaf food quality of tea exposed to P stress, but the improved mechanisms were different between P0.5 and P50.

Convergent evolution of caffeine in plants by co-option of exapted ancestral enzymes

Convergent evolution is a process that has occurred throughout the tree of life, but the historical genetic and biochemical context promoting the repeated independent origins of a trait is rarely understood. The well-known stimulant caffeine, and its xanthine alkaloid precursors, has evolved multiple times in flowering plant history for various roles in plant defense and pollination. We have shown that convergent caffeine production, surprisingly, has evolved by two previously unknown biochemical pathways in chocolate, citrus, and guaraná plants using either caffeine synthase- or xanthine methyltransferase-like enzymes. However, the pathway and enzyme lineage used by any given plant species is not predictable from phylogenetic relatedness alone. Ancestral sequence resurrection reveals that this convergence was facilitated by co-option of genes maintained over 100 million y for alternative biochemical roles. The ancient enzymes of the Citrus lineage were exapted for reactions currently used for various steps of caffeine biosynthesis and required very few mutations to acquire modern-day enzymatic characteristics, allowing for the evolution of a complete pathway. Future studies aimed at manipulating caffeine content of plants will require the use of different approaches given the metabolic and genetic diversity revealed by this study.

Low Caffeine Content in Novel Grafted Tea with Camellia sinensis as Scions and Camellia oleifera as Stocks

Caffeine, a purine alkaloid, is a major secondary metabolite in tea leaves. The demand for low caffeine tea is increasing in recent years, especially for health reasons. We report a novel grafted tea material with low caffeine content. The grafted tea plant had Camellia sinensis as scions and C. oleifera as stocks. The content of purine alkaloids was determined in the leaves of one-year-old grafted tea plants by HPLC. We also characterized caffeine synthase (CS), a key enzyme involved in caffeine biosynthesis in tea plants, at the expression level. The expression patterns of CS were examined in grafted and control leaves by Western blot, using a self-prepared polyclonal antibody with high specificity and sensitivity. The expression of related genes ( TCS1, tea caffeine synthase gene, GenBank accession No. AB031280; sAMS, SAM synthetase gene, AJ277206; TIDH, IMP dehydrogenase gene, EU106658) in the caffeine biosynthetic pathway was investigated by qRT-PCR. HPLC showed that the caffeine content was only 38% as compared with the non-grafted tea leaves. Immunoblotting analysis showed that CS protein decreased by half in the leaves of grafted tea plants. qRT-PCR revealed no significant changes in the expression of two genes in the upstream pathway ( sAMS and TIDH), while the expression of TCS1 was greatly decreased (50%). Taken together, these data revealed that the low caffeine content in the grafted tea leaves is due to low TCS1 expression and CS protein accumulation.

Gene expression profile during coffee fruit development and identification of candidate markers for phenological stages

The objective of this work was to identify genes that could be used as suitable markers for molecular recognition of phenological stages during coffee (Coffea arabica) fruit development. Four cultivars were evaluated as to their differential expression of genes associated to fruit development and maturation processes. Gene expression was characterized by both semi-quantitative and quantitative RT-PCR, in fruit harvested at seven different developmental stages, during three different seasons. No size polymorphisms or differential expression were observed among the cultivars for the evaluated genes; however, distinct expression profiles along fruit development were determined for each gene. Four out of the 28 evaluated genes exhibited a regular expression profile in all cultivars and harvest seasons, and, therefore, they were validated as candidate phenological markers of coffee fruit. The gene α-galactosidase can be used as a marker of green stage, caffeine synthase as a marker of transition to green and yellowish-green stages, and isocitrate lyase and ethylene receptor 3 as markers of late maturation.