The high-affinity transporter BnPHT1;4 is involved in phosphorus acquisition and mobilization for facilitating seed germination and early seedling growth of Brassica napus.

BACKGROUND: Seed germination and seedling establishment are two of the most critical phases in plant development. However, the molecular mechanisms underlying the effect of phosphorus on seed germination and post-germinated growth of oilseed rape are unclear so far. Here, we report the role of BnPHT1;4 in seed germination and early seedling development of Brassica napus. RESULTS: Our results show that BnPHT1;4 is preferentially expressed in cotyledons of early developing seedlings. Overexpression of BnPHT1;4 in oilseed rape promoted seed germination and seedling growth. Expression levels of the genes related to ABA and GA biosynthesis and signaling were significantly altered in BnPHT1;4 transgenic seedlings. Consequently, active GA level was up-regulated, whereas ABA content was down-regulated in BnPHT1;4 transgenic seedlings. Furthermore, exogenous GA could promote seed germination of wild type, while exogenous ABA could partially recover the advanced-germination phenotype of BnPHT1;4 transgenic seeds. Total phosphorus content in cotyledons of the transgenic seedlings was decreased more rapidly than that in wild type when Pi was supplied or deficient, and Pi contents in shoots and roots of the BnPHT1;4 transgenic plants were higher than those in wild type under high and low Pi conditions. CONCLUSIONS: Our data suggest that the high-affinity transporter BnPHT1;4 is involved in phosphorus acquisition and mobilization for facilitating seed germination and seedling growth of Brassica napus by modulating ABA and GA biosynthesis.

Characterization of squalene-induced PgCYP736B involved in salt tolerance by modulating key genes of abscisic acid biosynthesis.

Cytochrome P450 genes as the one of the largest superfamily genes mediate a wide range of plant biochemical pathways. In this study, a full-length cytochrome P450 monooxygenase (CYP736B) cDNA was isolated and characterized from Panax ginseng. It was revealed that the deduced amino acid of PgCYP736B shares a high degree of sequence homology with CYP736A12 encoded by P. ginseng. Expression of PgCYP736B was differentially induced not only during a Pseudomonas syringae infection (7.7-fold) and wounding (47.3-fold) but also after exposure to salt (7.4-fold), cold (8.3-fold), and drought stress (3.24-fold). The gene transcription was highly affected by methyl jasmonate (476-fold) in the ginseng, suggesting that PgCYP736B was elicitor-responsive. Furthermore, we overexpressed the PgCYP736B gene in Arabidopsis and found that PgCYP736B is a transmembrane protein. Overexpression of PgCYP736B in Arabidopsis conferred enhanced resistance to salt stress via decreased H2O2 accumulation, increased carotenoid levels, and through abscisic acid biosynthesis gene expression. Our results suggest that the induction of ginsenoside biosynthetic pathway genes along with PgCYP736B by an exogenous supply of 10-100 µM of squalene most likely affects the metabolite profile of ginsenoside triterpenoid. Overall, our findings indicate that PgCYP736B protects ginseng from salt stress and may contribute to triterpenoid biosynthesis.

Overexpression of annexin gene AnnSp2, enhances drought and salt tolerance through modulation of ABA synthesis and scavenging ROS in tomato.

Drought and high salinity are two major abiotic stresses that significantly affect agricultural crop productivity worldwide. Annexins are a multigene family that plays an essential role in plant stress responses and various cellular processes. Here, the AnnSp2 gene was cloned from drought-resistant wild tomato (Solanum pennellii) and functionally characterized in cultivated tomato. AnnSp2 protein was localized in the nucleus and had higher expression in leave, flower and fruit. It was induced by several phytohormones and some abiotic stresses. Tomato plants overexpressing AnnSp2 had increased tolerance to drought and salt stress, as determined by analysis of various physiological parameters. AnnSp2-transgenic plants were less sensitive to ABA during the seed germination and seedling stages. However, under drought stress, the ABA content significantly increased in the AnnSp2-overexpressing plants, inducing stomatal closure and reducing water loss, which underlay the plants' enhanced stress tolerance. Furthermore, scavenging reactive oxygen species (ROS), higher total chlorophyll content, lower lipid peroxidation levels, increased peroxidase activities (including APX, CAT and SOD) and higher levels of proline were observed in AnnSp2-overexpressing plants. These results indicate that overexpression of AnnSp2 in transgenic tomato improves salt and drought tolerance through ABA synthesis and the elimination of ROS.

Histidine Regulates Seed Oil Deposition through Abscisic Acid Biosynthesis and ß-Oxidation.

The storage compounds are deposited into plant seeds during maturation. As the model oilseed species, Arabidopsis (Arabidopsis thaliana) has long been studied for seed oil deposition. However, the regulation of this process remains unclear. Through genetic screen with a seed oil body-specific reporter, we isolated low oil1 (loo1) mutant. LOO1 was mapped to HISTIDINE BIOSYNTHESIS NUMBER 1A (HISN1A). HISN1A catalyzes the first step of His biosynthesis. Oil significantly decreased, and conversely proteins markedly increased in hisn1a mutants, indicating that HISN1A regulates both oil accumulation and the oil-protein balance. HISN1A was predominantly expressed in embryos and root tips. Accordingly, the hisn1a mutants exhibited developmental phenotype especially of seeds and roots. Transcriptional profiling displayed that ß-oxidation was the major metabolic pathway downstream of HISN1A ß-Oxidation was induced in hisn1a mutants, whereas it was reduced in 35S:HISN1A-transgenic plants. In plants, seed storage oil is broken-down by ß-oxidation, which is controlled by abscisic acid (ABA). We found that His activated genes of ABA biosynthesis and correspondingly advanced ABA accumulation. Exogenous ABA rescued the defects of hisn1a mutants, whereas mutation of ABA DEFICIENT2, a key enzyme in ABA biosynthesis, blocked the effect of His on ß-oxidation, indicating that ABA mediates His regulation in ß-oxidation. Intriguingly, structural analysis showed that a potential His-binding domain was present in the general amino acid sensors GENERAL CONTROL NON-DEREPRESSIBLE2 and PII, suggesting that His may serve as a signal molecule. Taken together, our study reveals that His promotes plant seed oil deposition through ABA biosynthesis and ß-oxidation.

[SYNTHESIS OF PHYTOHORMONES BY NOCARDIA VACCINII IMV B-7405--PRODUCER OF SURFACTANTS].

AIM: To study the synthesis of phytohormones (auxins, cytokinins, abscisic acid) under cultivation of Nocardia vaccinii IMV B-7405 (surfactants producer) in media containing different carbon sources (glycerol, refined sunflower oil, as well as waste oil after frying potatoes and meat). METHODS: Phytohormones were extracted from supernatants of culture liquid (before or after surfactant separation) by ethylacetate (auxins, abscisic acid) and n-butanol (cytokinins), concentrated and purified by thin-layer chromatography, then quantitative determination was performed using a scanning Sorbfil spectrodensitometer. RESULTS: While growing in medium with refined oil IMV B-7405 strain synthesized 1.8 ± 0.09 g/l extracellular surfactant, also maximum amount of auxins (245-770 µ/l) and cytokinins (134-348 µl). Cultivation of N. vaccini LMV B-7405 on waste oil was accompanied by decreasing amount of phytohormones to 23-84 µ/l (auxins) and 16-90 µ/l (cytokinins) and increasing surfactant concentration to 2.3-2.6 g/l. The level of abscisic acid synthesis was practically not dependent on the nature of growth substrate, was substantially lower than that of auxins and cytokinins and ranged from 2 to 12 µ/l. CONCLUSIONS: Obtained data demonstrate the possibility of using oil-containing industrial waste for the simultaneous synthesis of both surfactants and phytohormones, and indicate the need for studies of the effect of producer cultivation conditions on the biological properties of the target products of microbial synthesis.

Plastidic isoprenoid biosynthesis in tomato: physiological and molecular analysis in genotypes resistant and sensitive to drought stress.

Isoprenoid compounds synthesised in the plastids are involved in plant response to water deficit. The functionality of the biosynthetic pathway of these compounds under drought stress has been analysed at the physiological and molecular levels in two related species of tomato (Solanum chilense and Solanum lycopersicum) that differ in their tolerance to abiotic challenge. Expression analysis of the genes encoding enzymes of these pathways (DXS, IPI, GGPPS, PSY1, NCED and HPT1) in plants at different RWC values shows significant differences for only GGPPS and HPT1, with higher expression in the tolerant S. chilense. Chlorophyll, carotenoids, α-tocopherol and ABA content was also determined in both species under different drought conditions. In agreement with HPT1 transcriptional activity, higher α-tocopherol content was observed in S. chilense than in S. lycopersicum, which correlates with a lower degree of lipoperoxidation in the former species. These results suggest that, in addition to lower stomatal conductance, α-tocopherol biosynthesis is part of the adaptation mechanisms of S. chilense to adverse environmental conditions.

Suppression of 9-cis-epoxycarotenoid dioxygenase, which encodes a key enzyme in abscisic acid biosynthesis, alters fruit texture in transgenic tomato.

Cell wall catabolism during fruit ripening is under complex control and is key for fruit quality and shelf life. To examine the role of abscisic acid (ABA) in tomato (Solanum lycopersicum) fruit ripening, we suppressed SlNCED1, which encodes 9-cis-epoxycarotenoid dioxygenase (NCED), a key enzyme in the biosynthesis of ABA. To suppress SlNCED1 specifically in tomato fruits, and thus avoid the pleiotropic phenotypes associated with ABA deficiency, we used an RNA interference construct driven by the fruit-specific E8 promoter. ABA accumulation and SlNCED1 transcript levels in the transgenic fruit were down-regulated to between 20% and 50% of the levels measured in the control fruit. This significant reduction in NCED activity led to a down-regulation in the transcription of genes encoding major cell wall catabolic enzymes, specifically polygalacturonase (SlPG), pectin methyl esterase (SlPME), ß-galactosidase precursor mRNA (SlTBG), xyloglucan endotransglycosylase (SlXET), endo-1,4-ß-cellulose (SlCels), and expansin (SlExp). This resulted in an increased accumulation of pectin during ripening. In turn, this led to a significant extension of the shelf life to 15 to 29 d compared with a shelf life of only 7 d for the control fruit and an enhancement of fruit firmness at the mature stage by 30% to 45%. In conclusion, ABA affects cell wall catabolism during tomato fruit ripening via down-regulation of the expression of major catabolic genes (SlPG, SlPME, SlTBG, SlXET, SlCels, and SlExp).

Ethylene-dependent/ethylene-independent ABA regulation of tomato plants colonized by arbuscular mycorrhiza fungi.

We investigated the relationship between ABA and ethylene regulating the formation of the arbuscular mycorrhiza (AM) symbiosis in tomato (Solanum lycopersicum) plants and tried to define the specific roles played by each of these phytohormones in the mycorrhization process. We analysed the impact of ABA biosynthesis inhibition on mycorrhization by Glomus intraradices in transgenic tomato plants with an altered ethylene pathway. We also studied the effects on mycorrhization in sitiens plants treated with the aminoethoxyvinyl glycine hydrochloride (AVG) ethylene biosynthesis inhibitor and supplemented with ABA. In addition, the expression of plant and fungal genes involved in the mycorrhization process was studied. ABA biosynthesis inhibition qualitatively altered the parameters of mycorrhization in accordance with the plant's ethylene perception and ethylene biosynthesis abilities. Inhibition of ABA biosynthesis in wild-type plants negatively affected all the mycorrhization parameters studied, while tomato mutants impaired in ethylene synthesis only showed a reduced arbuscular abundance in mycorrhizal roots. Inhibition of ethylene synthesis in ABA-deficient sitiens plants increased the intensity of mycorrhiza development, while ABA application rescued arbuscule abundance in the root's mycorrhizal zones. The results of our study show an antagonistic interaction between ABA and ethylene, and different roles of each of the two hormones during AM formation. This suggests that a dual ethylene-dependent/ethylene-independent mechanism is involved in ABA regulation of AM formation.

Cloning and expression analysis of an aldehyde oxidase gene in Arachis hygogaea L.

Aldehyde oxidase (AO) plays important role in plant hormone biosynthetic pathways, such as abscisic acid (ABA) and indole-3-acetic acid (IAA). The enzyme catalyzes the last step of the pathways. In this study a full-length cDNA encoding an aldyhyde oxidase was cloned and sequenced from leaves of peanut by RT-PCR, RACE-PCR and genomic DNA walking methods. The full-length cDNA, designated as Arachis hygogaea L. aldehyde oxidase 1 (AhAO1), consists of an open reading frame of 4131 bp, a 326 bp 5' untranslated region and a 128 bp 3' untranslated region including a poly (A) tail of 21 nucleotides. The gene encodes a polypeptide of 1377 amino acids with a calculated molecular weight of 150 kDa and an isoelectric point (pl) of 6.99. Analysis of amino acid sequence of AhAO1 shows that it had 61%, 59% and 55% identity with the AOs from tomato, Arabidopsis and maize, respectively The peanut AO polypeptide contains consensus sequences for iron-sulfur centers and a molybdenum cofactor (MoCo)-binding domain. Semi-quantitative RT-PCR analysis showed that AhAO1 expression was higher in leaves than in roots of peanut.

ABA regulates apoplastic sugar transport and is a potential signal for cold-induced pollen sterility in rice.

Cold temperatures cause pollen sterility and large reductions in grain yield in temperate rice growing regions of the world. Induction of pollen sterility by cold involves a disruption of sugar transport in anthers, caused by the cold-induced repression of the apoplastic sugar transport pathway in the tapetum. Here we demonstrate that the phytohormone ABA is a potential signal for cold-induced pollen sterility (CIPS). Cold treatment of the cold-sensitive cultivar Doongara resulted in increased anther ABA levels. Exogenous ABA treatment at the young microspore stage induced pollen sterility and affected cell wall invertase and monosaccharide transporter gene expression in a way similar to cold treatment. In the cold-tolerant cultivar R31, ABA levels were significantly lower under normal circumstances and remained low after cold treatment. The differences in endogenous ABA levels in Doongara and R31 correlated with differences in expression of the ABA biosynthetic genes encoding zeaxanthin epoxidase (OSZEP1) and 9-cis-epoxycarotenoid dioxygenase (OSNCED2, OSNCED3) in anthers. The expression of three ABA-8-hydroxylase genes (ABA8OX1, 2 and 3) in R31 anthers was higher under control conditions and was regulated differently by cold compared with Doongara. Our results indicate that the cold tolerance phenotype of R31 is correlated with lower endogenous ABA levels and a different regulation of ABA metabolism.

Effects of postharvest storage and dormancy status on ABA content, metabolism, and expression of genes involved in ABA biosynthesis and metabolism in potato tuber tissues.

At harvest, and for an indeterminate period thereafter, potato tubers will not sprout and are physiologically dormant. Abscisic acid (ABA) has been shown to play a critical role in tuber dormancy control but the mechanisms controlling ABA content during dormancy as well as the sites of ABA synthesis and catabolism are unknown. As a first step in defining the sites of synthesis and cognate processes regulating ABA turnover during storage and dormancy progression, gene sequences encoding the ABA biosynthetic enzymes zeaxanthin epoxidase (ZEP) and 9-cis-epoxycarotenoid dioxygenase (NCED) and three catabolism-related genes were used to quantify changes in their relative mRNA abundances in three specific tuber tissues (meristems, their surrounding periderm and underlying cortex) by qRT-PCR. During storage, StZEP expression was relatively constant in meristems, exhibited a biphasic pattern in periderm with transient increases during early and mid-to-late-storage, and peaked during mid-storage in cortex. Expression of two members of the potato NCED gene family was found to correlate with changes in ABA content in meristems (StNCED2) and cortex (StNCED1). Conversely, expression patterns of three putative ABA-8'-hydroxylase (CYP707A) genes during storage varied in a tissue-specific manner with expression of two of these genes rising in meristems and periderm and declining in cortex during storage. These results suggest that ABA synthesis and metabolism occur in all tuber tissues examined and that tuber ABA content during dormancy is the result of a balance of synthesis and metabolism that increasingly favors catabolism as dormancy ends and may be controlled at the level of StNCED and StCYP707A gene activities.

Chemically forced dormancy termination mimics natural dormancy progression in potato tuber meristems by reducing ABA content and modifying expression of genes involved in regulating ABA synthesis and metabolism.

The length of potato tuber dormancy depends on both the genotype and the environmental conditions during growth and storage. Abscisic acid (ABA) has been shown to play a critical role in tuber dormancy control but the mechanisms regulating ABA content during dormancy, as well as the sites of ABA synthesis, and catabolism are unknown. Recently, a temporal correlation between changes in ABA content and certain ABA biosynthetic and catabolic genes has been reported in stored field tubers during physiological dormancy progression. However, the protracted length of natural dormancy progression complicated interpretation of these data. To address this issue, in this study the synthetic dormancy-terminating agent bromoethane (BE) was used to induce rapid and highly synchronous sprouting of dormant tubers. The endogenous ABA content of tuber meristems increased 2-fold 24 h after BE treatment and then declined dramatically. By 7 d post-treatment, meristem ABA content had declined by >80%. Exogenous [(3)H]ABA was readily metabolized by isolated meristems to phaseic and dihydrophaseic acids. BE treatment resulted in an almost 2-fold increase in the rate of ABA metabolism. A differential expression of both the StNCED and StCYP707A gene family members in meristems of BE-treated tubers is consistent with a regulatory role for StNCED2 and the StCYP707A1 and StCYP707A2 genes. The present results show that the changes in ABA content observed during tuber dormancy progression are the result of a dynamic equilibrium of ABA biosynthesis and degradation that increasingly favours catabolism as dormancy progresses.

Abscisic acid biosynthesis in tomato: regulation of zeaxanthin epoxidase and 9-cis-epoxycarotenoid dioxygenase mRNAs by light/dark cycles, water stress and abscisic acid.

Two genes encoding enzymes in the abscisic acid (ABA) biosynthesis pathway, zeaxanthin epoxidase (ZEP) and 9-cis-epoxycarotenoid dioxygenase (NCED), have previously been cloned by transposon tagging in Nicotiana plumbaginifolia and maize respectively. We demonstrate that antisense down-regulation of the tomato gene LeZEP1 causes accumulation of zeaxanthin in leaves, suggesting that this gene also encodes ZEP. LeNCED1 is known to encode NCED from characterization of a null mutation (notabilis) in tomato. We have used LeZEP1 and LeNCED1 as probes to study gene expression in leaves and roots of whole plants given drought treatments, during light/dark cycles, and during dehydration of detached leaves. During drought stress, NCED mRNA increased in both leaves and roots, whereas ZEP mRNA increased in roots but not leaves. When detached leaves were dehydrated, NCED mRNA responded rapidly to small reductions in water content. Using a detached leaf system with ABA-deficient mutants and ABA feeding, we investigated the possibility that NCED mRNA is regulated by the end product of the pathway, ABA, but found no evidence that this is the case. We also describe strong diurnal expression patterns for both ZEP and NCED, with the two genes displaying distinctly different patterns. ZEP mRNA oscillated with a phase very similar to light-harvesting complex II (LHCII) mRNA, and oscillations continued in a 48 h dark period. NCED mRNA oscillated with a different phase and remained low during a 48 h dark period. Implications for regulation of water stress-induced ABA biosynthesis are discussed.

A stress-inducible gene for 9-cis-epoxycarotenoid dioxygenase involved in abscisic acid biosynthesis under water stress in drought-tolerant cowpea.

Four cDNA clones named CPRD (cowpea responsive to dehydration) corresponding to genes that are responsive to dehydration were isolated using differential screening of a cDNA library prepared from 10-h dehydrated drought-tolerant cowpea (Vigna unguiculata) plants. One of the cDNA clones has a homology to 9-cis-epoxycarotenoid dioxygenase (named VuNCED1), which is supposed to be involved in abscisic acid (ABA) biosynthesis. The GST (glutathione S-transferase)-fused protein indicates a 9-cis-epoxycarotenoid dioxygenase activity, which catalyzes the cleavage of 9-cis-epoxycarotenoid. The N-terminal region of the VuNCED1 protein directed the fused sGFP (synthetic green-fluorescent protein) into the plastids of the protoplasts, indicating that the N-terminal sequence acts as a transit peptide. Both the accumulation of ABA and expression of VuNCED1 were strongly induced by drought stress in the 8-d-old cowpea plant, whereas drought stress did not trigger the expression of VuABA1 (accession no. AB030295) gene that encodes zeaxanthin epoxidase. These results indicate that the VuNCED1 cDNA encodes a 9-cis-epoxycarotenoid dioxygenase and that its product has a key role in the synthesis of ABA under drought stress.

[Affection of exogenous gibberellic acid (GA3) on endogenus hormones of Panax quinquefolium seed during its morphological after ripening period].

According to the determination of endogenus hormones variation of Panax quinquefolium seed during its morphological afterripenging period, and the affection of exo-GA3 on the endogenus hormones dynamics, it suggested that exo-GA3 couldn't be used for accelerate the growth of Panax quinquefolium embryo, but was helpful in relieving seed dormancy during physiological afterripening period.

The 9-cis-epoxycarotenoid cleavage reaction is the key regulatory step of abscisic acid biosynthesis in water-stressed bean.

Abscisic acid (ABA), a cleavage product of carotenoids, is involved in stress responses in plants. A well known response of plants to water stress is accumulation of ABA, which is caused by de novo synthesis. The limiting step of ABA biosynthesis in plants is presumably the cleavage of 9-cis-epoxycarotenoids, the first committed step of ABA biosynthesis. This step generates the C(15) intermediate xanthoxin and C(25)-apocarotenoids. A cDNA, PvNCED1, was cloned from wilted bean (Phaseolus vulgaris L.) leaves. The 2, 398-bp full-length PvNCED1 has an ORF of 615 aa and encodes a 68-kDa protein. The PvNCED1 protein is imported into chloroplasts, where it is associated with the thylakoids. The recombinant protein PvNCED1 catalyzes the cleavage of 9-cis-violaxanthin and 9'-cis-neoxanthin, so that the enzyme is referred to as 9-cis-epoxycarotenoid dioxygenase. When detached bean leaves were water stressed, ABA accumulation was preceded by large increases in PvNCED1 mRNA and protein levels. Conversely, rehydration of stressed leaves caused a rapid decrease in PvNCED1 mRNA, protein, and ABA levels. In bean roots, a similar correlation among PvNCED1 mRNA, protein, and ABA levels was observed. However, the ABA content was much less than in leaves, presumably because of the much smaller carotenoid precursor pool in roots than in leaves. At 7 degrees C, PvNCED1 mRNA and ABA were slowly induced by water stress, but, at 2 degrees C, neither accumulated. The results provide evidence that drought-induced ABA biosynthesis is regulated by the 9-cis-epoxycarotenoid cleavage reaction and that this reaction takes place in the thylakoids, where the carotenoid substrate is located.

Molecular identification of zeaxanthin epoxidase of Nicotiana plumbaginifolia, a gene involved in abscisic acid biosynthesis and corresponding to the ABA locus of Arabidopsis thaliana.

Abscisic acid (ABA) is a plant hormone which plays an important role in seed development and dormancy and in plant response to environmental stresses. An ABA-deficient mutant of Nicotiana plumbaginifolia, aba2, was isolated by transposon tagging using the maize Activator transposon. The aba2 mutant exhibits precocious seed germination and a severe wilty phenotype. The mutant is impaired in the first step of the ABA biosynthesis pathway, the zeaxanthin epoxidation reaction. ABA2 cDNA is able to complement N.plumbaginifolia aba2 and Arabidopsis thaliana aba mutations indicating that these mutants are homologous. ABA2 cDNA encodes a chloroplast-imported protein of 72.5 kDa, sharing similarities with different mono-oxigenases and oxidases of bacterial origin and having an ADP-binding fold and an FAD-binding domain. ABA2 protein, produced in Escherichia coli, exhibits in vitro zeaxanthin epoxidase activity. This is the first report of the isolation of a gene of the ABA biosynthetic pathway. The molecular identification of ABA2 opens the possibility to study the regulation of ABA biosynthesis and its cellular location.