Reliable reference genes for normalization of gene expression data in tea plants (Camellia sinensis) exposed to metal stresses.

Tea plants [Camellia sinensis (L.) O. Kuntze] are an important leaf-type crop that are widely used for the production of non-alcoholic beverages in the world. Exposure to excessive amounts of heavy metals adversely affects the quality and yield of tea leaves. To analyze the molecular responses of tea plants to heavy metals, a reliable quantification of gene expression is important and of major importance herein is the normalization of the measured expression levels for the target genes. Ideally, stably expressed reference genes should be evaluated in all experimental systems. In this study, 12 candidate reference genes (i.e., 18S rRNA, Actin, CYP, EF-1α, eIF-4α, GAPDH, MON1, PP2AA3, TBP, TIP41, TUA, and UBC) were cloned from tea plants, and the stability of their expression was examined systematically in 60 samples exposed to diverse heavy metals (i.e., manganese, aluminum, copper, iron, and zinc). Three Excel-based algorithms (geNorm, NormFinder, and BestKeeper) were used to evaluate the expression stability of these genes. PP2AA3 and 18S rRNA were the most stably expressed genes, even though their expression profiles exhibited some variability. Moreover, commonly used reference genes (i.e., GAPDH and TBP) were the least appropriate reference genes for most samples. To further validate the suitability of the analyzed reference genes, the expression level of a phytochelatin synthase gene (i.e., CsPCS1) was determined using the putative reference genes for data normalizations. Our results may be beneficial for future studies involving the quantification of relative gene expression levels in tea plants.

Heterologous expression of three Camellia sinensis small heat shock protein genes confers temperature stress tolerance in yeast and Arabidopsis thaliana.

KEY MESSAGE: CsHSP17.7, CsHSP18.1, and CsHSP21.8 expressions are induced by heat and cold stresses, and CsHSP overexpression confers tolerance to heat and cold stresses in transgenic Pichia pastoris and Arabidopsis thaliana. Small heat shock proteins (sHSPs) are crucial for protecting plants against biotic and abiotic stresses, especially heat stress. However, knowledge concerning the functions of Camellia sinensis sHSP in heat and cold stresses remains poorly understood. In this study, three C. sinensis sHSP genes (i.e., CsHSP17.7, CsHSP18.1, and CsHSP21.8) were isolated and characterized using suppression subtractive hybridization (SSH) technology. The CsHSPs expression levels in C. sinensis leaves were significantly up-regulated by heat and cold stresses. Phylogenetic analyses revealed that CsHSP17.7, CsHSP18.1, and CsHSP21.8 belong to sHSP Classes I, II, and IV, respectively. Heterologous expression of the three CsHSP genes in Pichia pastoris cells enhanced heat and cold stress tolerance. When exposed to heat and cold treatments, transgenic Arabidopsis thaliana plants overexpressing CsHSP17.7, CsHSP18.1, and CsHSP21.8 had lower malondialdehyde contents, ion leakage, higher proline contents, and transcript levels of stress-related genes (e.g., AtPOD, AtAPX1, AtP5CS2, and AtProT1) compared with the control line. In addition, improved seed germination vigor was also observed in the CsHSP-overexpressing seeds under heat stress. Taken together, our results suggest that the three identified CsHSP genes play key roles in heat and cold tolerance.

Isolation and dynamic expression of four genes involving in shikimic acid pathway in Camellia sinensis 'Baicha 1' during periodic albinism.

Flavonoids are the main flavor components and functional ingredients in tea, and the shikimic acid pathway is considered as one of the most important pathways in flavonoid biosynthesis, but little was known about the function of regulatory genes in the metabolism phenolic compounds in tea plant (Camellia sinensis), especially related genes in shikimic acid pathway. The dynamic changes of catechin (predominant flavonoid) contents were analyzed in this study, and four genes (CsPPT, CsDAHPS, CsSDH and CsCS) involving in shikimic acid pathway in C. sinensis albino cultivar 'Baicha 1' were cloned and characterized. The full-length cDNA sequences of these genes were obtained using reverse transcription-PCR and rapid amplification of cDNA ends. At the albinistic stage, the amounts of all catechins decreased to the lowest levels, when epigallocatechin gallate was the highest, whereas gallocatechin-3-O-gallate the lowest. Gene expression patterns analyzed by qRT-PCR showed that CsPPT and CsDAHPS were highly expressed in flowers and buds, while CsSDH and CsCS showed high expression levels in buds and leaves. It was also found that the transcript abundance of shikimic acid biosynthetic genes followed a tightly regulated biphasic pattern, and was affected by albinism. The transcript levels of CsPPT and CsDAHPS were decreased at albinistic stage followed elevated expression, whereas CsSDH and CsCS were increased only at re-greening stage. Taken together, these findings suggested that these four genes in C. sinensis may play different roles in shikimic acid biosynthesis and these genes may have divergent functions.

Transcriptomic Analysis Reveals the Molecular Mechanisms of Drought-Stress-Induced Decreases in Camellia sinensis Leaf Quality.

The tea plant [Camellia sinensis (L.) O. Kuntze] is an important commercial crop rich in bioactive ingredients, especially catechins, caffeine, theanine and other free amino acids, which the quality of tea leaves depends on. Drought is the most important environmental stress affecting the yield and quality of this plant. In this study, the effects of drought stress on the phenotype, physiological characteristics and major bioactive ingredients accumulation of C. sinensis leaves were examined, and the results indicated that drought stress resulted in dehydration and wilt of the leaves, and significant decrease in the total polyphenols and free amino acids and increase in the total flavonoids. In addition, HPLC analysis showed that the catechins, caffeine, theanine and some free amino acids in C. sinensis leaves were significantly reduced in response to drought stress, implying that drought stress severely decreased the quality of C. sinensis leaves. Furthermore, differentially expressed genes (DEGs) related to amino acid metabolism and secondary metabolism were identified and quantified in C. sinensis leaves under drought stress using high-throughput Illumina RNA-Seq technology, especially the key regulatory genes of the catechins, caffeine, and theanine biosynthesis pathways. The expression levels of key regulatory genes were consistent with the results from the HPLC analysis, which indicate a potential molecular mechanism for the above results. Taken together, these data provide further insights into the mechanisms underlying the change in the quality of C. sinensis leaves under environmental stress, which involve changes in the accumulation of major bioactive ingredients, especially catechins, caffeine, theanine and other free amino acids.