Abscisic Acid Glucosyl Ester Research Articles

A New Abscisic Acid Conjugate, ABA-L-Glutamate, Determined in Different Plant Species by Combined Immunoaffinity Chromatography-Tandem Mass Spectrometry

Abscisic acid (ABA) is a phytohormone that occurs in plants at very low concentration (pmol/g fresh weight) and regulates multiple biological processes, including stomatal closure, seed germination, and responses to environmental stresses. In the present study, isolation of ABA, ABA glucosyl ester, and 11 ABA amino acid conjugates from minute quantities of plant tissue (less than 20 mg fresh weight) was achieved using a purification method based on the combination of an Oasis HLB column and an immunoaffinity sorbent. New monoclonal antibodies raised against (+)-cis,trans-ABA conjugated to BSA through its carboxyl group (C1) were characterised by enzyme-linked immunosorbent assay (ELISA) and used for immunoaffinity chromatography (IAC) gel preparation. The use of immunoaffinity purification significantly reduced matrix effects and increased the selectivity and sensitivity of subsequent UHPLC-MS/MS analysis. In addition to (+)-cis,trans-ABA and its glucosyl ester, a new abscisic acid conjugate, ABA-L-glutamate, was isolated by IAC and identified by tandem mass spectrometry in pea (Pisum sativum L.), Lepidium sativum L. and wheat (Triticum aestivum L.) seedlings. However, it was not found in 10-day-old seedlings of Arabidopsis thaliana or water-stressed tobacco (Nicotiana tabacum L.) leaves. Here, the identification of an ABA conjugate with glutamic acid in plants is described for the first time.

Physiological and Transcriptomic Analysis of Citrus Fruit Segment Drying under Facility-Forcing Cultivation

Facility-forcing cultivation could effectively improve the quality of ‘Beni Madonna’ citrus (Citrus nankao × C. amakusa) and advance the ripening period. However, segment drying (KS) before harvest caused fruit quality deterioration and commodity value loss. In this research, we investigated the physiological and molecular characteristics involved in citrus segment drying under facility-forcing cultivation. The juice yield, sugar, acid, vitamin C, and lignin contents in KS fruits were significantly decreased, and the contents of pectin and cellulose were significantly increased. The relative contents of abscisic acid and abscisic acid glucosyl ester in KS fruits were significantly decreased. A total of 1215 differentially expressed genes (DEGs) were screened by transcriptome sequencing. DEGs were significantly enriched in water metabolism, sugar metabolism, transportation, cell wall and phenylpropanoid biosynthesis, and plant hormone signal transduction. The decrease in water absorption and sugar synthesis, the increase of pectin and cellulose synthesis, and the decrease in ABA accumulation may be the main reasons that cause citrus fruit segments to dry under facility-forcing cultivation.

Abscisic acid root-to-shoot translocation by transporter AtABCG25 mediates stomatal movements in Arabidopsis.

The phytohormone abscisic acid (ABA) plays a central role in regulating stomatal movements under drought conditions. The root-derived peptide CLAVATA3/EMBRYO SURROUNDING REGION-RELATED 25 (CLE25) moves from the root to shoot for activating ABA biosynthesis under drought conditions. However, the root-to-shoot translocation of root-derived ABA and its regulation of stomatal movements in the shoot remain to be clarified. Here, we reveal that the ABA transporter ATP-binding cassette subfamily G member 25 (AtABCG25) mediates root-to-shoot translocation of ABA and ABA-glucosyl ester (ABA-GE) in Arabidopsis (Arabidopsis thaliana). Isotope-labeled ABA tracer experiments and hormone quantification in xylem sap showed that the root-to-shoot translocation of ABA and ABA-GE was substantially impaired in the atabcg25 mutant under nondrought and drought conditions. However, the contents of ABA and ABA-GE in the leaves were lower in the atabcg25 mutant than in the wild type (WT) under nondrought but similar under drought conditions. Consistently, the stomatal closure was suppressed in the atabcg25 mutant under nondrought but not under drought conditions. The transporter activity assays showed that AtABCG25 directly exported ABA and ABA-GE in planta and in yeast (Saccharomyces cerevisiae) cells. Thus, we proposed a working model in which root-derived ABA transported by AtABCG25 via xylem mediates stomatal movements in the shoot under nondrought conditions but might exhibit little effect on stomatal movements under drought conditions. These findings extend the functions of AtABCG25 and provide insights into the long-distance translocation of ABA and its role in stomatal movements.

Endomembrane-biased dimerization of ABCG16 and ABCG25 transporters determines their substrate selectivity in ABA-regulated plant growth and stress responses

ATP-binding cassette (ABC) transporters are integral membrane proteins that have evolved diverse functions fulfilled via the transport of various substrates. In Arabidopsis, the G subfamily of ABC proteins is particularly abundant and participates in multiple signaling pathways during plant development and stress responses. In this study, we revealed that two Arabidopsis ABCG transporters, ABCG16 and ABCG25, engage in ABA-mediated stress responses and early plant growth through endomembrane-specific dimerization-coupled transport of ABA and ABA-glucosyl ester (ABA-GE), respectively. We first revealed that ABCG16 contributes to osmotic stress tolerance via ABA signaling. More specifically, ABCG16 induces cellular ABA efflux in both yeast and plant cells. Using FRET analysis, we showed that ABCG16 forms obligatory homodimers for ABA export activity and that the plasma membrane-resident ABCG16 homodimers specifically respond to ABA, undergoing notable conformational changes. Furthermore, we demonstrated that ABCG16 heterodimerizes with ABCG25 at the endoplasmic reticulum (ER) membrane and facilitates the ER entry of ABA-GE in both Arabidopsis and tobacco cells. The specific responsiveness of the ABCG16-ABCG25 heterodimer to ABA-GE and the superior growth of their double mutant support an inhibitory role of these two ABCGs in early seedling establishment via regulation of ABA-GE translocation across the ER membrane. Our endomembrane-specific analysis of the FRET signals derived from the homo- or heterodimerized ABCG complexes allowed us to link endomembrane-biased dimerization to the translocation of distinct substrates by ABCG transporters, providing a prototypic framework for understanding the omnipotence of ABCG transporters in plant development and stress responses.

A comprehensive analysis of carotenoids metabolism in two red-fleshed mutants of Navel and Valencia sweet oranges (Citrus sinensis).

Kirkwood Navel and Ruby Valencia are two spontaneous bud mutations of the respective parental lines of sweet orange (Citrus sinensis) Palmer Navel and Olinda Valencia, showing an atypical red pigmentation of the pulp. These red-fleshed varieties are commercially available and highly attractive for consumers but their carotenoid metabolism and the basis of the mutation have not been investigated. The red colour of Kirkwood and Ruby pulp was observed from the very early stages of fruit development until full maturity and associated with an altered carotenoid profiling. The red-fleshed varieties accumulated from 6- up to 1000-times more total carotenoids compared to the standard oranges. Specifically, the pulp of Kirkwood and Ruby accumulated large amounts of phytoene and phytofluene, and moderate contents of lycopene. Moreover, the red-fleshed oranges contained other unusual carotenes as δ-carotene, and lower concentrations of downstream products such as β,β-xanthophylls, abscisic acid (ABA) and ABA-glucosyl ester. This peculiar profile was associated with chromoplasts with lycopene crystalloid structures and round vesicles likely containing colourless carotenes. The flavedo and leaves of Kirkwood and Ruby showed minor changes in carotenoids, mainly limited to higher levels of phytoene. The carotenoid composition in Kirkwood and Ruby fruits was not explained by differences in the transcriptional profile of 26 genes related to carotenoid metabolism, covering the main steps of biosynthesis, catabolism and other processes related to carotenoid accumulation. Moreover, sequence analysis of the lycopene cyclase genes revealed no alterations in those of the red-fleshed oranges compared to the genes of the standard varieties. A striking event observed in Kirkwood and Ruby trees was the reddish coloration of the inner side of the bark tissue, with larger amounts of phytoene, accumulation of lycopene and lower ABA content. These observation lead to the conclusion that the mutation is not only manifested in fruit, affecting other carotenogenic tissues of the mutant plants, but with different consequences in the carotenoid profile. Overall, the carotenoid composition in the red-fleshed mutants suggests a partial blockage of the lycopene β-cyclization in the carotenoid pathway, rendering a high accumulation of carotenes upstream lycopene and a reduced flow to downstream xanthophylls and ABA.

Red and Blue Light Affect the Formation of Adventitious Roots of Tea Cuttings (Camellia sinensis) by Regulating Hormone Synthesis and Signal Transduction Pathways of Mature Leaves.

Light is an important environmental factor which affects plant growth, through changes of intensity and quality. In this study, monochromatic white (control), red (660 nm), and blue (430 nm) light-emitting diodes (LEDs) were used to treat tea short cuttings. The results showed the most adventitious roots in blue light treated tea cuttings, but the lowest roots in that treated by red light. In order to explore the molecular mechanism of light quality affecting adventitious root formation, we performed full-length transcriptome and metabolome analyses of mature leaves under three light qualities, and then conducted weighted gene co-expression network analysis (WGCNA). Phytohormone analysis showed that Indole-3-carboxylic acid (ICA), Abscisic acid (ABA), ABA-glucosyl ester (ABA-GE), trans-Zeatin (tZ), and Jasmonic acid (JA) contents in mature leaves under blue light were significantly higher than those under white and red light. A crosstalk regulatory network comprising 23 co-expression modules was successfully constructed. Among them, the “MEblue” module which had a highly positive correlation with ICA (R = 0.92, P = 4e-04). KEGG analysis showed that related genes were significantly enriched in the “Plant hormone signal transduction (ko04075)” pathway. YUC (a flavin-containing monooxygenase), AUX1, AUX/IAA, and ARF were identified as hub genes, and gene expression analysis showed that the expression levels of these hub genes under blue light were higher than those under white and red light. In addition, we also identified 6 auxin transport-related genes, including PIN1, PIN3, PIN4, PILS5, PILS6, and PILS7. Except PILS5, all of these genes showed the highest expression level under blue light. In conclusion, this study elucidated the molecular mechanism of light quality regulating adventitious root formation of tea short cutting through WGCNA analysis, which provided an innovation for “rapid seedling” of tea plants.

Plant Hormone Response to Low-Temperature Stress in Cold-Tolerant and Cold-Sensitive Varieties of Zanthoxylum bungeanum Maxim.

Plant growth and survival in nature, its growth process, will be affected by various factors from the environment, among which temperature has a greater impact. In recent years, extreme weather has frequently appeared, and the growth of crops has been increasingly affected by the environment. As an important flavoring and Chinese herbal medicine crop, Zanthoxylum bungeanum is also facing the harm of low-temperature stress. Plant hormones play a vital role in the response of plants to low temperatures. In this study, ultra-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to determine the hormone components of cold-tolerant and cold-sensitive varieties of Z. bungeanum. Combined with chemometric analysis and weighted gene co-expression network analysis (WGCNA), the hormone component differences and hormone response strategies of Z. bungeanum under low-temperature stress were comprehensively studied. The results showed that 45 hormones were detected in Z. bungeanum. Among them, there were 7 kinds of components with high content and were detected in both two varieties. At the late stage of low-temperature stress, the contents of abscisic acid (ABA) and ABA-glucosyl ester (ABA-GE) in Fuguhuajiao (FG) were significantly increased, and the latter served as the storage of the former to supplement the active ABA. Orthogonal partial least squares discriminant analysis (OPLS-DA) found that indole-3-carboxylic acid (ICA), indole-3-carboxaldehyde (ICAld), meta-Topolin riboside (mTR), cis-Zeatin-O-glucoside riboside (cZROG), and N6-isopentenyladenosine (IPR) in FG were the upregulated important difference components, and IPR and 2-methylthio-cis-zeatin riboside (2MeScZR) in Fengxiandahongpao (FX) were the upregulated important difference components. There were common crossing points and independent response pathways in response to low temperature in two varieties. WGCNA analysis found that the main hormone components were associated with multiple metabolic pathways including carbon, fatty acid, amino acid, and sugar metabolism, indicating that hormone regulation plays an important role in the response of Z. bungeanum to low temperature. This study clarified the hormone response mechanism of Z. bungeanum under low-temperature stress and provided a reference and basis for further improving the cold resistance of Z. bungeanum and cultivating new varieties.

Phenotype and mechanism analysis of plant dwarfing in pear regulated by abscisic acid

Close planting of dwarf varieties is currently the main cultivation direction for pear trees, and the screening of excellent dwarf varieties is an important goal for breeders. In this study, the dwarfing pear variety ‘601D’ and its vigorous mutant ‘601T’ were used to show their biological characteristics and further explore the dwarfing mechanism in ‘601D’. The biological characteristics showed that ‘601D’ had a shorter internode length, a shorter and more compact tree body, thicker and broader leaves, lower stomata density, larger stomata size (dimension), and higher photosynthetic capacity. The biological characteristics of ‘601T’ showed notable contrasts. The results of endogenous hormone tests indicated that the contents of abscisic acid (ABA), ABA-glucosyl ester, and GA4 were higher in ‘601D’, but the trans-zeatin content was lower. By transcriptomic analysis, significant differences were found in the biosynthetic and metabolic pathways of ABA. Related transcription factors such as bHLH, WRKY, and homeobox also participated in the regulation of plant dwarfing. We therefore examined three hormones with obvious differences with ‘601T’, and found that only ABA could induce ‘601T’ to return to a dwarfing plant phenotype. Therefore, we conclude that the dwarfing of ‘601D’ is caused by an excessive accumulation of ABA. This study provides a new theoretical basis for breeding dwarf varieties.

Changes in abscisic acid metabolism in relation to the maturation of grapevine (Vitis vinifera L., cv. Menc\xeda) somatic embryos

BackgroundSomatic embryogenesis in grapevines is a complex process that depends on many physiological and genetic factors. One of its main limitations is the process of precocious germination of the somatic embryos in differentiation medium. This process lowers plant conversion rates from the somatic embryos, and it is probably caused by a low endogenous abscisic acid (ABA) content.ResultsPrecocious germination of the somatic embryos was successfully avoided by culturing grapevine cv. Mencía embryogenic aggregates over a semipermeable membrane extended on top of the differentiation medium. The weekly analysis of the endogenous ABA and ABA-glucosyl ester (ABA-GE) contents in the aggregates showed their rapid accumulation. The expression profiles of 9-cis-epoxycarotenoid dioxygenase (VvNCED1), 8′-hydroxylase (VvHyd2), UDP-glucosyltransferase (VvUGT) and β-glucosidase (VvBG2) genes in grapevine revealed that the occurrence of a first accumulation peak of endogenous ABA in the second week of culture over the semipermeable membrane was mainly dependent on the expression of the VvNCED1 gene. A second increase in the endogenous ABA content was observed in the fourth week of culture. At this point in the culture, our results suggest that of those genes involved in ABA accumulation, one (VvNCED1) was repressed, while another (VvBG2) was activated. Similarly, of those genes related to a reduction in ABA levels, one (VvUGT) was repressed while another (VvHyd2) was activated. The relative expression level of the VvNCED1 gene in embryogenic aggregates cultured under the same conditions and treated with exogenous ABA revealed the significant downregulation of this gene.ConclusionsOur results demonstrated the involvement of ABA metabolism in the control of the maturation of grapevine somatic embryos cultured over a semipermeable membrane and two important control points for their endogenous ABA levels. Thus, subtle differences in the expression of the antagonistic genes that control ABA synthesis and degradation could be responsible for the final level of ABA during the maturation of grapevine somatic embryos in vitro. In addition, the treatment of somatic embryos with exogenous ABA suggested the feedback-based control of the expression of the VvNCED1 gene by ABA during the maturation of grapevine somatic embryos.

Fine tuning of hormonal signaling is linked to dormancy status in sweet cherry flower buds.

In temperate trees, optimal timing and quality of flowering directly depend on adequate winter dormancy progression, regulated by a combination of chilling and warm temperatures. Physiological, genetic and functional genomic studies have shown that hormones play a key role in bud dormancy establishment, maintenance and release. We combined physiological and transcriptional analyses, quantification of abscisic acid (ABA) and gibberellins (GAs), and modeling to further investigate how these signaling pathways are associated with dormancy progression in the flower buds of two sweet cherry cultivars. Our results demonstrated that GA-associated pathways have distinct functions and may be differentially related with dormancy. In addition, ABA levels rise at the onset of dormancy, associated with enhanced expression of ABA biosynthesis PavNCED genes, and decreased prior to dormancy release. Following the observations that ABA levels are correlated with dormancy depth, we identified PavUG71B6, a sweet cherry UDP-GLYCOSYLTRANSFERASE gene that up-regulates active catabolism of ABA to ABA glucosyl ester (ABA-GE) and may be associated with low ABA content in the early cultivar. Subsequently, we modeled ABA content and dormancy behavior in three cultivars based on the expression of a small set of genes regulating ABA levels. These results strongly suggest the central role of ABA pathway in the control of dormancy progression and open up new perspectives for the development of molecular-based phenological modeling.

Dormancy breaking in Fagus sylvatica seeds is linked to formation of abscisic acid-glucosyl ester

Seed dormancy is an adaptive mechanism that allows seed germination under suitable environmental conditions. Germination of stored dormant seeds proceeds after dormancy breaking induced by stratification. To improve understanding of dormancy breaking in beechnuts, we: investigated effects of moisture content and temperature during storage; analysed contents of abscisic acid, abscisic acid metabolites and indole-3-acetic acid in embryonic axes during storage and stratification; and histochemically localized storage proteins in embryogenic axes and cotyledons of beechnut embryos. The results show that storage conditions can affect the nuts’ abscisic acid contents, but not the stratification process. In addition, dormancy breaking is linked to a reduction in abscisic acid contents and depth of dormancy is not influenced by either of these storage factors. Detected changes that correlated most strongly with dormancy breaking were a big increase in abscisic acid-glucosyl ester levels and accompanying reduction in the abscisic acid-glucosyl ester to abscisic acid ratio. We detected extremely low concentrations of other abscisic acid metabolites-neophaseic, phaseic and dihydrophaseic acids—in non-dormant stored beechnuts, but somewhat higher concentrations during dormancy. No relationships between changes in indole-3-acetic acid levels with either storage conditions or dormancy breaking were detected. Changes in distributions of storage proteins were related to the seeds’ moisture content during storage and stratification rather than seed dormancy. We conclude that increases in endogenous abscisic acid-glucosyl ester levels and abscisic acid-glucosyl ester to abscisic acid ratios are good markers of depth of dormancy and/or effectiveness of stratification in beechnuts.

Comparative analysis of abscisic acid levels and expression of abscisic acid-related genes in Scots pine and Norway spruce seedlings under water deficit

Abscisic acid (ABA) is one of the main participants in the regulation of plant responses to water deficiency. Knowledge of the ABA signal transduction pathways in gymnosperms is rather limited, especially in comparison with those in angiosperms. Seedlings of Scots pine and Norway spruce are known for their contrasting behaviour strategies under water deficit. To characterize the possible role of ABA in these differences, ABA dynamics were investigated under conditions of water deficit in seedlings of these two species. The content of ABA and its catabolites was followed in the roots and needles of seedlings of Pinus sylvestris and Picea abies under conditions of polyethylene glycol (PEG)-induced water deficiency (−0.15 and −0.5 MPa) for 10 days. The expression of the main genes for ABA-biosynthetic enzymes was also analysed. ABA showed more pronounced stress-dependent dynamics in pine roots than in spruce roots, whereas in needles, the response was greater for spruce than pine. The ABA increase during drought was mainly due to de novo synthesis and the shift in the balance between ABA synthesis and catabolism towards synthesis. The ABA-glucosyl ester did not serve as a reserve for the release of free ABA under water deficiency. The expression levels of the main ABA biosynthetic genes showed a weak or no correlation with changes in ABA content under water stress, i.e., the ABA content in the seedlings of both species was not directly linked to the transcript levels of the main ABA biosynthetic genes. Less-pronounced stress-induced changes in ABA in pine needles than in spruce needles may be related to pine seedlings having a less conservative strategy of growth and maintenance of water balance under water deficit.

Abscisic Acid Related Metabolites in Sweet Cherry Buds (Prunus avium L.)

As our climate changes, plant mechanisms involved for dormancy release become increasingly important for commercial orchards. It is generally believed that abscisic acid (ABA) is a key hormone that responds to various environmental stresses which affects bud dormancy. For this reason, a multi-year study was initiated to obtain data on plant metabolites during winter rest and ontogenetic development in sweet cherry buds (Prunus avium L.). In this paper, we report on metabolites involved in ABA synthesis and catabolism and its effect on bud dormancy in the years 2014/15-2016/17. In previous work, the timings of the different phases of para-, endo-, ecodormancy and ontogenetic development for cherry flower buds of the cultivar ‘Summit’ were determined, based on classical climate chamber experiments and changes in the bud’s water content. Based on these time phases, we focused now on the different aspects of the ABA-metabolism. The results show that there is a continual synthesis of ABA about 5 weeks before leaf fall, and a degradation of ABA during ecodormancy and bud development until the phenological stage ‘open cluster’. This is confirmed by relating the ABA content to that of the total precursor carotenoids, neoxanthin and violaxanthin. The tentative monitoring of individual intermediate metabolites revealed that dihydroxyphaseic acid is the most abundant catabolite of ABA and ABA glucosyl ester is in terms of mass intensity, the most abundant ABA metabolite observed in this study. The results suggest that the direct route for ABA biosynthesis from farnesyl pyrophosphate may also be relevant in cherry flower buds.

Spectral quality of supplemental LED grow light permanently alters stomatal functioning and chilling tolerance in basil ( Ocimum basilicum L.)

Abstract The spectral qualities of light contain environmental information used by plants as cues to modify their biology in order to adapt and survive. LED based lamps, with their highly definable spectral properties, thus hold great perspectives for use in greenhouse production. The effects of the spectral qualities of light on plant physiology is still not fully understood, and a great fraction of the current knowledge further comes from studies looking at short term effects or using monochromatic light treatments in enclosed growth chambers, thus far from the light conditions of greenhouse production. The objective of this study was to determine whether spectral modifications to the supplemental LED lighting in greenhouse production could modify post-cultivation performance of basil, which is prone to chilling injuries (CI). We cultivated basil under greenhouse conditions using four different supplemental LED light treatments; 80%Red/20%Blue, 80%Red/20%Blue + UV-A, 40%Red/60%Blue and 80%Red/20%Green, followed by a post-cultivation shelf life simulation including a chilling treatment. Stomatal anatomical features and levels of hormonal regulators, abscisic acid (ABA) and ABA glucosylester (ABA-GE), along with soluble sugars, fatty acids and lipoxygenase activity (LOX) were determined at the end of the growing period. Without compromising grow rates, the light treatments showed marked effects on the chilling tolerance and shelf life performance with increasing ratios of blue light having negative effects, and green light having positive effects on chilling tolerance. Differences in shelf life performance seemed mediated solely by factors related to stomatal functioning. Stomatal density (number of stomata per leaf area, SD) increased with increasing ratios of blue light whereas green light showed indications to decrease SD. [ABA] and [ABA-GE] showed positive correlation to SD, whereas the ratio of [ABA-GE]/[ABA] conversely correlated negatively with increased SD. Our results thus suggest an important relationship between leaf water retention and post-cultivation chiling tolerance in basil.

Differential hormonal and gene expression dynamics in two inbred sunflower lines with contrasting dormancy level

Seed germination and dormancy are tightly regulated by hormone metabolism and signaling pathway. We investigated the endogenous content of abscisic acid (ABA), its catabolites, and gibberellins (GAs), as well as the expression level of certain ABA and GAs metabolic and signaling genes in embryo of dry and imbibed cypselas of inbred sunflower (Helianthus annuus L., Asteraceae) lines: B123 (dormant) and B91 (non-dormant). Under our experimental conditions, the expression of RGL2 gene might be related to the ABA peak in B123 line at 3 h of imbibition. Indeed, RGL2 transcripts are absent in dry and early embedded cypselas of the non-dormant line B91. ABA increase was accompanied by a significant ABA-Glucosyl ester (ABA-GE) and phaseic acid (PA) (two ABA catabolites) decrease in B123 line (3 h) which indicates that ABA metabolism seems to be more active in this line, and that it would be involved in the imposition and maintenance of sunflower seed dormancy, as it has been reported for many species. Finally, an increase of bioactive GAs (GA1 and GA3) occurs at 12 h of imbibition in both lines after a decrease in ABA content. This study shows the first report about the RGL2 tissue-specific gene expression in sunflower inbred lines with contrasting dormancy level. Furthermore, our results provide evidence that ABA and GAs content and differential expression of metabolism and signaling genes would be interacting in seed dormancy regulation through a mechanism of action related to embryo itself.

Biochemical characterization of embryogenic calli of Vanilla planifolia in response to two years of thidiazuron treatment

Vanilla planifolia embryogenic calli were cultured for two years on a medium containing thidiazuron (TDZ). Due to the presence of TDZ, these calli were under permanent chemical treatment and the differentiation of adventitious shoots from protocorm-like-bodies (PLBs) was blocked. When embryogenic calli were transferred onto a medium without TDZ, shoot organogenesis and plantlet regeneration occurred. To gain better knowledge about the biochemical and molecular processes involved in the morphoregulatory role of TDZ, hormonal and metabolomic analyses were performed. Our results indicate that in the presence of TDZ, embryogenic calli contained a high amount of abscisic acid (ABA) essentially metabolized into abscisic acid glucosyl ester (ABAGE) and phaseic acid (PA), which was the most abundant. When transferred onto a medium without TDZ, shoot regeneration and development take place in four stages that include: embryogenic calli growth, differentiation of PLBs from meristmatic cells zones (MCZ), shoot organogenesis from PLBs and the elongation of well-formed shoots.From a hormonal perspective, the significant reduction in ABA metabolism and its readjustment in the ABAGE pathway triggered PLBs formation. However, this first morphogenesis was stimulated by a strong reduction in IAA metabolism. The organogenesis of PLBs into shoots is associated with an increase in ABA catabolism and a gradual shift in cellular metabolism towards shoot differentiation. Thus, the initiation of the elongation process in shoots is correlated with an alteration in metabolite composition, including an increase in energy reserves (sucrose/starch) and a rapid decrease in alanine content. Our data highlighted the relationship between endogenous hormone signalling, carbohydrate metabolism and shoot organogenesis in Orchid plants.

Signalling mechanisms involved in the response of two varieties of Humulus lupulus L. to soil drying: II. changes in the concentration of abscisic acid catabolites and stress-induced phytohormones

Abscisic acid (ABA) is one of the most common stress signals that appear in plant organs in response to soil drying. Equilibrium between ABA biosynthesis and catabolism regulates ABA accumulation in plants under water stress. The aim of our work was to explore the dynamics of changes in ABA metabolites as well as other stress-induced phytohormones such as jasmonic acid, indole-3-acetic acid, and their respective metabolites in hop [Humulus lupulus (L.)] plants during drying and to identify among them potential signals involved in drought signalling. We showed that the concentrations of all ABA metabolites (except the concentration of ABA glucosyl ester in leaves) increased in the same manner in leaves and xylem sap approximately at the same level of soil water content when the relative water content of leaves decreased. The predominant metabolites in leaves and xylem sap were phaseic acid and dihydroxyphaseic acid. ABA glucosyl ester was not a source of the increased concentration of ABA in leaves and xylem sap because of its considerably lower concentration compared to ABA. The concentration of jasmonates decreased in leaves of hop plants. Changes in auxin concentration suggest that this hormone is involved in the response of hop plants to soil drying.

Involvement of abscisic acid metabolites and the oxidative status of barley genotypes in response to drought

Thameur, A., Ferchichi, A. and López-Carbonell, M. 2014. Involvement of abscisic acid metabolites and the oxidative status of barley genotypes in response to drought. Can. J. Plant Sci. 94: 1481–1490. Endogenous concentrations of free abscisic acid (ABA), abscisic acid glucosyl ester (ABAGE), phaseic acid (PA), dihydrophaseic acid (DPA) and 7′-hydroxy ABA (7′-OH ABA) were analysed by means of a LC–MS/MS system in five genotypes of barley (Hordeum vulgare L.) grown under well-watered and drought stress conditions. For this purpose a drought treatment was conducted using genotypes: ‘Ardahoui’, ‘Manel’, ‘Pakistan’, ‘Rihane’ and ‘Roho’. Our results show that free and conjugated ABA levels increased in all genotypes grown under water stress, except in Manel genotype, in which ABAGE levels were the lowest. In contrast, genotypes Ardhaoui and Roho showed the highest ABA and ABAGE levels. Nevertheless, drought Rihane plants showed the maximum ability to increase the endogenous ABA concentrations. PA, DPA and 7′-OH ABA increased also in all drought genotypes, especially in the leaves of Manel and Roho genotypes, while the highest ability to increase the endogenous PA content corresponded to genotypes Ardahoui and Pakistan. To evaluate the plant oxidative status, some antioxidant compounds were analysed. Under drought conditions, small changes in some of them were seen. Among the genotypes studied, Manel was the only one which did not show increases in malondialdehyde (MDA) and, in parallel, showed a decrease in ABAGE content. These results provide us valuable information and contribute to the knowledge of the different responses of these genotypes to drought stress.

Involvement of abscisic acid metabolites and the oxidative status of barley genotypes in response to drought

Thameur, A., Ferchichi, A. and Lopez-Carbonell, M. 2014. Involvement of abscisic acid metabolites and the oxidative status of barley genotypes in response to drought. Can. J. Plant Sci. 94: 1481-1490. Endogenous concentrations of free abscisic acid (ABA), abscisic acid glucosyl ester (ABAGE), phaseic acid (PA), dihydrophaseic acid (DPA) and 7'-hydroxy ABA (7'-OH ABA) were analysed by means of a LC-MS/MS system in five genotypes of barley (Hordeum vulgare L.) grown under well-watered and drought stress conditions. For this purpose a drought treatment was conducted using genotypes: ‘Ardahoui’, ‘Manel’, ‘Pakistan’, ‘Rihane’ and ‘Roho’. Our results show that free and conjugated ABA levels increased in all genotypes grown under water stress, except in Manel genotype, in which ABAGE levels were the lowest. In contrast, genotypes Ardhaoui and Roho showed the highest ABA and ABAGE levels. Nevertheless, drought Rihane plants showed the maximum ability to increase the endogenous ABA concentrations. PA, DPA and 7'-O...

Transcription and enzymatic analysis of beta-glucosidase VvBG1 in grape berry ripening

Although beta-glucosidase was recently demonstrated to be involved at transcriptional level in fleshy fruit development, it remains unclear whether BG possesses biochemical activity. In the present study, real-time reverse transcription polymerase chain reaction analysis showed that the mRNA expression levels of β-glucosidase gene VvBG1 increased rapidly concomitant with berry red-colouring in Muscat Hamburg grapevine (Vitis vinifera), suggesting that the VvBG1 gene might be involved in the berry ripening. In order to explore VvBG1 biochemical activity, a 60-kD recombinant protein, VvBG1, was expressed in Escherichia coli BL21 (DE3) and purified. The results gained from a combination of VvBG1-pulp-homogeneity-incubation test and gas chromatography-mass spectroscopy analysis showed that VvBG1-treated berry samples contained more free ABA than the control, demonstrating that VvBG1 has high glycosyl hydrolase activity that can transform the ABA-glucosyl ester to free ABA. In conclusion, both the high expression and increased hydrolase activity of VvBG1 could suggest an important role of ABA in grape berry ripening.