Oxylipins, including jasmonic acid (JA) and volatiles, are important for signaling in plants, and these are formed by the lipoxygenase (LOX) enzyme family. There is a large gap in understanding of the underlying molecular basis of their roles in tea plants. Here, we identified 11 CsLOX genes from the tea plant (Camellia sinensis), and characterized their phylogeny, gene structure and protein features into three subclasses. We then examined their enzymatic activities, LOX expression and alternative splicing of transcripts during development and in response to abiotic or biotic stresses in tea plants. In vitro expressed protein assays showed that the CsLOX2, 3 and 9 enzymatically function to produce 9/13-HPOT, 13-HPOT and 9-HPOT, respectively. CsLOX2 and CsLOX9 green fluorescent protein (GFP) fusion proteins localized to chloroplasts and the cytoplasm, respectively. RNA sequencing, quantitative reverse transcription–PCR and Northern blot analysis suggested that CsLOX5, 6 and 9 were predominantly expressed in seeds, flowers and roots, respectively. CsLOX2, 3, 4, 6 and 7 were up-regulated after attack by the insect Ectropis oblique, while CsLOX1 was induced after infection with the pathogen Glomerella cingulata. CsLOX3, 7 and 10 were up-regulated by JA but not ABA or salicylic acid. Long-term cold stress down-regulated CsLOX expression while a short duration of cold induced the expression of CsLOX1, 6 and 7. Alternatively spliced transcripts of six CsLOX genes were dynamically regulated through time and varied in relative abundances under the investigated stresses; we propose a mechanism of competing or compensating regulation between isoforms. This study improves our understanding of evolution of LOXs and regulation of their diverse functions in plants.
Tea,
made from leaves of Camellia sinensis, has
long been consumed worldwide for its unique taste and aroma. Terpenoids
play important roles not only in tea beverage aroma formation, but
also in the productivity and quality of tea plantation due to their
significant contribution to light harvesting pigments and phytohormones.
To date, however, the regulation of terpenoid synthase genes remains
unclear. Herein, the analyses of metabolomics, sRNAs, degradome, and
transcriptomics were performed and integrated for identifying key
regulatory miRNA-target circuits on terpenoid biosynthesis in leaf
tissues over five different months in which the amount of terpenoids
in tea leaves varies greatly. Four classes of miRNA-TF pairs that
might play a central role in the regulation of terpenoid biosynthesis
were also uncovered. Ultimately, a hypothetical model was proposed
that mature miRNAs maintained by light regulator at both the transcriptional
and posttranscriptional levels negatively regulate the targets to
control terpenoid biosynthesis.
Based
on the abundance of taste compounds in leaves at different
leaf positions on the same shoot, green tea made from one bud and
one leaf, or even just one bud, has the best quality. To elucidate
the mechanism underlying the regulation of the biosynthesis of these
compounds, we profiled the metabolome, transcriptome, sRNA, degradome,
and WGCNA using leaves from five leaf positions of shoots. Through
this analysis, we found 139 miRNA-target pairs related to taste compound
biosynthesis and 96 miRNA-target pairs involved in phytohormone synthesis
or signal transduction. Moreover, miR166-HD-ZIP,
miR169-NF-YA, IAA, ZA, ABA, and JA were positively
related to the accumulation of gallated catechin, caffeine, and theanine.
However, miR396-GRF, miR393-bHLH, miR156-SBP, and SA were negatively correlated
with these compounds. Among these important pairs, the miR396-GRF and miR156-SBP pairs were further validated
by using qRT-PCR, Northern blots, and cotransformation. This is the
first report describing that miRNA-TF pairs and phytohormones might
synergistically regulate the biosynthesis of taste compounds in the
leaves of tea plants.
Alcohol
dehydrogenase (ADH) is a vital enzyme in the biosynthesis
pathway of six-carbon volatiles in plants. However, little is known
about its functions in tea plants. Here, we identified two ADH genes (CsADH1 and CsADH2). An in vitro protein expression assay showed that
both CsADH1 and CsADH2 proteins can catalyze the reduction of (Z)-3-hexenal into (Z)-3-hexenol. Subcellular
localization revealed that both CsADH1 and CsADH2 proteins were predominantly
localized in the nucleus and cytosol. CsADH1 had
high transcripts in young stems in autumn, while CsADH2 showed extremely high expression levels in stems and roots. The
expression of CsADH2 was mainly downregulated under
ABA treatment, while CsADH1 and CsADH2 transcripts were significantly lower under MeJA treatment at 12
and 24 h. Under cold treatment, CsADH1 transcripts
first decreased and then increased, while CsADH2 demonstrated
an almost opposite expression pattern. Notably, CsADH2 was significantly upregulated under simulated Ectropis
obliqua invasion. Gene suppression by antisense oligonucleotides
(AsODNs) demonstrated that AsODN_ADH2 treatment significantly
reduced CsADH2 transcripts and the abundance of (Z)-3-hexenol products. The results indicate that the two CsADH genes may play an important role in response to (a)biotic
stresses and in the process of (Z)-3-hexenol biosynthesis.
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