Identification and Analysis of Genes Involved in Auxin, Abscisic Acid, Gibberellin, and Brassinosteroid Metabolisms Under Drought Stress in Tender Shoots of Tea Plants.

Endogenous phytohormones auxin (indole-3-acetic acid [IAA]), abscisic acid (ABA), gibberellin (GA3), and brassinosteroid (BR) play a role in responses to drought stress in higher plants. Tea plant is one of the major economic corps worldwide. The tender shoots of tea plants are the main source for tea production. The effects of drought stress on endogenous IAA, ABA, GA3, and BR metabolisms in tender shoots of tea plants need to be illustrated. In this study, a total of 17 IAA-related genes, 17 ABA-related genes, 18 GA3-related genes, and 8 BR-related genes were identified under drought stress in tender shoots of tea plants, respectively. By using a combination of phytohormone determination, phylogenetic tree construction and sequence analysis, gene expression profiles, functional classification, Kyoto encyclopedia of genes and genomes enrichment, and distribution of genes analysis, we have demonstrated that IAA, ABA, GA3, and BR metabolisms might participate in the regulation of the response to drought stress in tender shoots of tea plants. The expression level of CsLYCE negatively correlated with ABA accumulation under drought stress. Our findings could shed new light on the effects of drought stress on the IAA, ABA, GA3, and BR metabolisms in tender shoots of tea plants.

Preharvest UV-C treatment affected postharvest senescence and phytochemicals alternation of strawberry fruit with the possible involvement of abscisic acid regulation.

As an environmentally friendly approach for fruit quality improvement, the effect of preharvest UV-C on the physiology of strawberry fruit during postharvest storage remains to be assessed. Strawberry fruit developed with supplementary UV-C were stored at room temperature for 2 weeks. Preharvest UV-C attenuated fruit postharvest senescence and altered phytochemicals composition. Higher ester titer was found in the treated fruit at harvest, whereas higher terpene and furanone contents were detected after 72 h of storage. At harvest, polyphenolics accumulated to a higher level in UV-C group, but the difference disappeared after 24 h of storage. Meanwhile, the intrinsic level of abscisic acid and the expressions of FaPYR1, SnRK2, and FaASR in the UV-C-treated fruit was enhanced at harvest but returned to a lower level as storage proceeded. This study highlights the time-dependent effect of preharvest UV-C on strawberry fruit postharvest biochemical indexes and the possible involvement of abscisic acid signaling factors.

The regulatory mechanism of chilling-induced dormancy transition from endo-dormancy to non-dormancy in Polygonatum kingianum Coll.et Hemsl rhizome bud.

KEY MESSAGE: We identified three dormant stages of Polygonatum kingianum and changes that occurred during dormancy transition in the following aspects including cell wall and hormones, as well as interaction among them. Polygonatum kingianum Coll.et Hemsl (P. kingianum) is an important traditional Chinese medicine, but the mechanism of its rhizome bud dormancy has not yet been studied systematically. In this study, three dormancy phases were induced under controlled conditions, and changes occurring during the transition were examined, focusing on phytohormones and the cell wall. As revealed by HPLC-MS (High Performance Liquid Chromatography-Mass Spectrometry) analysis, the endo- to non-dormancy transition was association with a reduced abscisic acid (ABA)/gibberellin (GA3) ratio, a decreased level of auxin (IAA) and an increased level of trans-zeatin (tZR). Transmission electron microscopy showed that plasmodesmata (PDs) and the cell wall of the bud underwent significant changes between endo- and eco-dormancy. A total of 95,462 differentially expressed genes (DEGs) were identified based on transcriptomics, and clustering and principal component analysis confirmed the different physiological statuses of the three types of bud samples. Changes in the abundance of transcripts associated with IAA, cytokinins (CTKs), GA, ABA, brassinolide (BR), jasmonic acid (JA), ethylene, salicylic acid (SA), PDs and cell wall-loosening factors were analysed during the bud dormancy transition in P. kingianum. Furthermore, nitrilase 4 (NIT4) and tryptophan synthase alpha chain (TSA1), which are related to IAA synthesis, were identified as hub genes of the co-expression network, and strong interactions between hormones and cell wall-related factors were observed. This research will provide a good model for chilling-treated rhizome bud dormancy in P. kingianum and cultivation of this plant.

Genome-Wide Identification and Characterization of the AREB/ABF/ABI5 Subfamily Members from Solanum tuberosum.

Abscisic acid (ABA) plays crucial roles in plant development and adaption to environmental stresses. The ABA-responsive element binding protein/ABRE-binding factor and ABA INSENSITIVE 5 (AREB/ABF/ABI5) gene subfamily members, which belong to the basic domain/leucine zipper (bZIP) transcription factors family, participate in the ABA-mediated signaling pathway by regulating the expression of their target genes. However, information about potato (Solanum tuberosum) AREB/ABF/ABI5 subfamily members remains scarce. Here, seven putative AREB/ABF/ABI5 members were identified in the potato genome. Sequences alignment revealed that these members shared high protein sequence similarity, especially in the bZIP region, indicating that they might possess overlapping roles in regulating gene expression. Subcellular localization analysis illustrated that all seven AREB/ABF/ABI5 members were localized in the nucleus. Transactivation activity assays in yeast demonstrated that these AREB/ABF/ABI5 members possessed distinct transcriptional activity. Electrophoretic mobility shift assays (EMSA) confirmed that all of these AREB/ABF/ABI5 members could have an affinity to ABRE in vitro. The expression patterns of these AREB/ABF/ABI5 genes showed that they were in response to ABA or osmotic stresses in varying degrees. Moreover, most AREB/ABF/ABI5 genes were induced during stolon swelling. Overall, these results provide the first comprehensive identification of the potato AREB/ABF/ABI5 subfamily and would facilitate further functional characterization of these subfamily members in future work.

Abscisic acid prevents pollen abortion under high-temperature stress by mediating sugar metabolism in rice spikelets.

Heat stress at the pollen mother cell (PMC) meiotic stage leads to pollen sterility in rice, in which the reactive oxygen species (ROS) and sugar homeostasis are always adversely affected. This damage is reversed by abscisic acid (ABA), but the mechanisms underlying the interactions among the ABA, sugar metabolism, ROS and heat shock proteins in rice spikelets under heat stress are unclear. Two rice genotypes, Zhefu802 (a recurrent parent) and fgl (its near-isogenic line) were subjected to heat stress of 40°C after pre-foliage sprayed with ABA and its biosynthetic inhibitor fluridone at the meiotic stage of PMC. The results revealed that exogenous application of ABA reduced pollen sterility caused by heat stress. This was achieved through various means, including: increased levels of soluble sugars, starch and non-structural carbohydrates, markedly higher relative expression levels of heat shock proteins (HSP24.1 and HSP71.1) and genes related to sugar metabolism and transport, such as sucrose transporters (SUT) genes, sucrose synthase (SUS) genes and invertase (INV) genes as well as increased antioxidant activities and increased content of adenosine triphosphate and endogenous ABA in spikelets. In short, exogenous application of ABA prior to heat stress enhanced sucrose transport and accelerated sucrose metabolism to maintain the carbon balance and energy homeostasis, thus ABA contributed to heat tolerance in rice.

Identification and Expression Analyses of SBP-Box Genes Reveal Their Involvement in Abiotic Stress and Hormone Response in Tea Plant (Camellia sinensis).

The SQUAMOSA promoter binding protein (SBP)-box gene family is a plant-specific transcription factor family. This family plays a crucial role in plant growth and development. In this study, 20 SBP-box genes were identified in the tea plant genome and classified into six groups. The genes in each group shared similar exon-intron structures and motif positions. Expression pattern analyses in five different tissues demonstrated that expression in the buds and leaves was higher than that in other tissues. The cis-elements and expression patterns of the CsSBP genes suggested that the CsSBP genes play active roles in abiotic stress responses; these responses may depend on the abscisic acid (ABA), gibberellic acid (GA), and methyl jasmonate (MeJA) signaling pathways. Our work provides a comprehensive understanding of the CsSBP family and will aid in genetically improving tea plants.

Type 2C phosphatase 1 of Artemisia annua L. is a negative regulator of ABA signaling.

The phytohormone abscisic acid (ABA) plays an important role in plant development and environmental stress response. Additionally, ABA also regulates secondary metabolism such as artemisinin in the medicinal plant Artemisia annua L. Although an earlier study showed that ABA receptor, AaPYL9, plays a positive role in ABA-induced artemisinin content improvement, many components in the ABA signaling pathway remain to be elucidated in Artemisia annua L. To get insight of the function of AaPYL9, we isolated and characterized an AaPYL9-interacting partner, AaPP2C1. The coding sequence of AaPP2C1 encodes a deduced protein of 464 amino acids, with all the features of plant type clade A PP2C. Transcriptional analysis showed that the expression level of AaPP2C1 is increased after ABA, salt, and drought treatments. Yeast two-hybrid and bimolecular fluorescence complementation assays (BiFC) showed that AaPYL9 interacted with AaPP2C1. The P89S, H116A substitution in AaPYL9 as well as G199D substitution or deletion of the third phosphorylation site-like motif in AaPP2C1 abolished this interaction. Furthermore, constitutive expression of AaPP2C1 conferred ABA insensitivity compared with the wild type. In summary, our data reveals that AaPP2C1 is an AaPYL9-interacting partner and involved in the negative modulation of the ABA signaling pathway in A. annua L.

Wounding of potato tubers induces increases in ABA biosynthesis and catabolism and alters expression of ABA metabolic genes.

The effects of physical wounding on ABA biosynthesis and catabolism and expression of genes encoding key ABA metabolic enzymes were determined in potato tubers. An increase in ABA and ABA metabolite content was observed 48h after wounding and remained elevated through 96h. Wounding induced dramatic increases in the expression of the ABA metabolic genes encoding zeaxanthin epoxidase (ZEP), 9-cis-epoxycarotenoid dioxygenase (NCED), and ABA-8'-hydroxylase. Although the patterns of wound-induced expression of individual genes varied, increased gene expression was observed within 3h of wounding and remained elevated through 96h. An apparent correlation between expression of the gene encoding ZEP and the increase in ABA content suggested that the wound-induced increase in ABA biosynthesis was regulated by both substrate availability and increased NCED activity. Suppression of wound-induced jasmonic acid accumulation by rinsing the wounded tissue with water did not inhibit the subsequent increase in ABA content. Exogenous ethylene completely suppressed the wound-induced increase in ABA content and dramatically reduced wound-induced up-regulation of ABA metabolic genes. This study is the first to identify the molecular bases for increased ABA accumulation following physical trauma in potato tubers and highlights the complex physiological interactions between various wound-induced hormones.

Molecular cloning and characterization of cDNAs encoding carotenoid cleavage dioxygenase in bitter melon (Momordica charantia).

Carotenoid cleavage dioxygenases (CCDs) are a family of enzymes that catalyze the oxidative cleavage of carotenoids at various chain positions to form a broad spectrum of apocarotenoids, including aromatic substances, pigments and phytohormones. Using the rapid amplification of cDNA ends (RACE) PCR method, we isolated three cDNA-encoding CCDs (McCCD1, McCCD4, and McNCED) from Momordica charantia. Amino acid sequence alignments showed that they share high sequence identity with other orthologous genes. Quantitative real-time RT PCR (reverse transcriptase PCR) analysis revealed that the expression of McCCD1 and McCCD4 was highest in flowers, and lowest in roots and old leaves (O-leaves). During fruit maturation, the two genes displayed differential expression, with McCCD1 peaking at mid-stage maturation while McCCD4 showed the lowest expression at that stage. The mRNA expression level of McNCED, a key enzyme involved in abscisic acid (ABA) biosynthesis, was high during fruit maturation and further increased at the beginning of seed germination. When first-leaf stage plants of M. charantia were exposed to dehydration stress, McNCED mRNA expression was induced primarily in the leaves and, to a lesser extend, in roots and stems. McNCED expression was also induced by high temperature and salinity, while treatment with exogenous ABA led to a decrease. These results should be helpful in determining the substrates and cleavage sites catalyzed by CCD genes in M. charantia, and also in defining the roles of CCDs in growth and development, and in the plant's response to environmental stress.

Cloning and expression analysis of cDNAs for ABA 8'-hydroxylase during sweet cherry fruit maturation and under stress conditions.

Abscisic acid (ABA) plays a key role in various aspects of plant growth and development, including adaptation to environmental stress and fruit maturation in sweet cherry fruit. In higher plants, the level of ABA is determined by synthesis and catabolism. In order to gain insight into ABA synthesis and catabolism in sweet cherry fruit during maturation and under stress conditions, four cDNAs of PacCYP707A1 -PacCYP707A4 for 8'-hydroxylase, a key enzyme in the oxidative catabolism of ABA, and one cDNA of PacNCED1 for 9-cis-epoxycarotenoid dioxygenase, a key enzyme in the ABA biosynthetic pathway, were isolated from sweet cherry fruit (Prunus avium L.). The timing and pattern of PacNCED1 expression was coincident with that of ABA accumulation, which was correlated to maturation of sweet cherry fruit. All four PacCYP707As were expressed at varying intensities throughout fruit development and appeared to play overlapping roles in ABA catabolism throughout sweet cherry fruit development. The application of ABA enhanced the expression of PacCYP707A1 -PacCYP707A3 as well as PacNCED1, but downregulated the PacCYP707A4 transcript level. Expressions of PacCYP707A1, PacCYP707A3 and PacNCED1 were strongly increased by water stress. No significant differences in PacCYP707A2 and PacCYP707A4 expression were observed between dehydrated and control fruits. The results suggest that endogenous ABA content is modulated by a dynamic balance between biosynthesis and catabolism, which are regulated by PacNCED1 and PacCYP707As transcripts, respectively, during fruit maturation and under stress conditions.

A putative novel transcription factor, AtSKIP, is involved in abscisic acid signalling and confers salt and osmotic tolerance in Arabidopsis.

We identified and functionally characterized the AtSKIP gene (At1g77180), an Arabidopsis homologue of SNW/SKIP, under abiotic stresses. Although the SNW/SKIP protein has been implicated as a critical transcription cofactor, its biological functions have yet to be reported in any plant. Recently, we have isolated Salt-tolerance genes (SATs) via the overexpression screening of yeast with a maize cDNA library. One of the selected genes (SAT2) appeared to confer elevated tolerance to salt. Maize SAT2 cDNA encodes a homologue of the human SNW/SKIP transcriptional coregulator. Treatment with salt, mannitol and abscisic acid induced AtSKIP expression. Ectopic expression of the AtSKIP gene modulated the induction of salt tolerance, dehydration resistance and insensitivity towards abscisic acid under stress conditions. By contrast, atskip antisense lines displayed reduced tolerance to abiotic stresses during germination. Moreover, a decrease in AtSKIP expression resulted in an abnormal phenotype. We further determined that the AtSKIP protein activated the transcription of a reporter gene in yeast. Green fluorescent protein-tagged AtSKIP was localized in the nuclei of both onion cells and transgenic Arabidopsis cells. Taken together, these results suggest that AtSKIP functions as both a positive regulator and putative potential transcription factor in the abiotic stress signalling pathway.

Two LTR retrotransposon elements within the abscisic acid gene cluster in Botrytis cinerea B05.10, but not in SAS56

The plant hormone abscisic acid has huge economic potential and can be applied in agriculture and forestry for it is considered to be involved in plant resistance to stresses such as cold, heat, salinity, drought, pathogens and wounding. Now overproducing strains of Botrytis cinerea are used for biotechnological production of abscisic acid. An LTR retrotransposon, Boty-aba, and a solo LTR were identified by in silico genomic sequence analysis, and both were detected within the abscisic acid gene cluster in B. cinerea B05.10, but not in B. cinerea SAS56. Boty-aba contains a pair of LTRs and two internal genes. The LTRs and the first gene have features characteristic of Ty3/gypsy LTR retrotransposons. The second gene is a novel gene, named brtn, which encodes for a protein (named BRTN) without putative conserved domains. The impressive divergence in structure of the abscisic acid gene clusters putatively gives new clues to investigate the divergence in the abscisic acid production yields of different B. cinerea strains.

Expression of an abscisic acid-binding single-chain antibody influences the subcellular distribution of abscisic acid and leads to developmental changes in transgenic potato plants.

Potato (Solanum tuberosum L. cv. Désirée) plants were transformed to express a single-chain variable-fragment antibody against abscisic acid (ABA), and present in the endoplasmic reticulum at to up to 0.24% of the soluble leaf protein. The resulting transgenic plants were only able to grow normally at 95% humidity and moderate light. Four-week-old plants accumulated ABA to high extent, were retarded in growth and their leaves were smaller than those of control plants. Leaf stomatal conductivity was increased due to larger stomates. The subcellular concentrations of ABA in the chloroplast, cytoplasm and vacuole, and the apoplastic space of leaves were determined. In the 4-week-old transgenic plants the concentration of ABA not bound to the antibody was identical to that of control plants and the stomates were able to close in response to lower humidity of the atmosphere. A detailed analysis of age-dependent changes in plant metabolism showed that leaves of young transformed plants developed in ABA deficiency and leaves of older plants in ABA excess. Phenotypic changes developed in ABA deficiency partly disappeared in older plants.

Characterization of the ABA-deficient tomato mutant notabilis and its relationship with maize Vp14.

The notabilis (not) mutant of tomato has a wilty phenotype due to a deficiency in the levels of the plant hormone abscisic acid (ABA). The mutant appears to have a defect in a key control step in ABA biosynthesis--the oxidative cleavage of a 9-cis xanthophyll precursor to form the C15 intermediate, xanthoxin. A maize mutant, viviparous 14 (vp14) was recently obtained by transposon mutagenesis. This maize genetic lesion also affects the oxidative cleavage step in ABA synthesis. Degenerate primers for PCR, based on the VP14 predicted amino acid sequence, have been used to provide probes for screening a wilt-related tomato cDNA library. A full-length cDNA clone was identified which is specific to the not gene locus. The ORFs of the tomato cDNA and maize Vp14 are very similar, apart from parts of their N-terminal sequences. The not mutation has been characterized at the DNA level. A specific A/T base pair deletion of the coding sequence has resulted in a frameshift mutation, indicating that not is a null mutant. This observation is discussed in connection with the relatively mild phenotype exhibited by not mutant homozygotes.