Presence Of Exogenous Abscisic Acid Research Articles

Phytohormones enhance heavy metal responses in Euglena gracilis: Evidence from uptake of Ni, Pb and Cd and linkages to hormonomic and metabolomic dynamics

Over the last decade, significant effort has been made to understand phytohormonal functions (e.g., cytokinins (CKs) and abscisic acid (ABA)) in metal stress responses of higher plants and algae. Despite the potential for these phytohormones to improve industrial remediation by Euglena gracilis (Euglenophyceae), no such roles have been elucidated for this highly adaptive species and its response to heavy metals. This study demonstrates that toxic metals (nickel, lead, cadmium) modify hormonal activity profiles (i.e., CK forms and their concentrations) in E. gracilis. Furthermore, exogenous ABA or CK (tZ) enabled higher metal uptake efficiency (i.e., 9.35% in lead and 9.2% in cadmium uptake with CK) and alleviated metal toxicity through the regulation of endogenous CKs (i.e., total CK, isoprenoid CK) and gibberellin (GAs, GA1 and GA3) levels. These responses suggest that E. gracilis regulates multiple phytohormone signals during metal stress acclimation. A deeper approach, using untargeted metabolomic analyses, gave more detailed insight into phytohormone-controlled pathways and associated modified metabolites, which were frequently related to metal accumulation and the physiological acclimation to metal presence. Significant changes in the levels of cellular metabolites, especially those involved in acclimation to metal stress, were under the influence of phytohormones in algal cells. When grown under metal stress conditions, the presence of exogenous ABA or CKs, caused changes in cellular metabolites which included those from: lipid pathways, riboflavin metabolism, the biosynthesis of cofactors/vitamins, and carbohydrate metabolism. Also, bioactive secondary metabolites (e.g., terpenoids, alkaloids, flavonoids, carotenoids) were modified in algal cells treated with phytohormones. Thus, the study gives a detailed view on the regulatory functions of ABA and CKs in algal metal bioremediation strategies, which are attributed to enhanced metal uptake and in the fine-tuning of plant hormone levels during metal stress response. The results can guide efforts to develop efficient, low-cost and environmentally friendly methods for bioremediation.

Synthesis of Abscisic Acid in Neopyropia yezoensis and Its Regulation of Antioxidase Genes Expressions Under Hypersaline Stress.

Abscisic acid (ABA) is regarded as crucial for plant adaptation to water-limited conditions and it functions evolutionarily conserved. Thus, insights into the synthesis of ABA and its regulation on downstream stress-responsive genes in Neopyropia yezoensis, a typical Archaeplastida distributed in intertidal zone, will improve the knowledge about how ABA signaling evolved in plants. Here, the variations in ABA contents, antioxidant enzyme activities and expression of the target genes were determined under the presence of exogenous ABA and two specific inhibitors of the ABA precursor synthesis. ABA content was down-regulated under the treatments of each or the combination of the two inhibitors. Antioxidant enzyme activities like SOD, CAT and APX were decreased slightly with inhibitors, but up-regulated when the addition of exogenous ABA. The quantitative assays using real-time PCR (qRT-PCR) results were consistent with the enzyme activities. All the results suggested that ABA can also alleviate oxidative stress in N. yezoensis as it in terrestrial plant. Combined with the transcriptome assay, it was hypothesized that ABA is synthesized in N. yezoensis via a pathway that is similar to the carotenoid pathway in higher plants, and both the MVA and that the MEP pathways for isoprenyl pyrophosphate (IPP) synthesis likely exist simultaneously. The ABA signaling pathway in N. yezoensis was also analyzed from an evolutionary standpoint and it was illustrated that the emergence of the ABA signaling pathway in this alga is an ancestral one. In addition, the presence of the ABRE motif in the promoter region of antioxidase genes suggested that the antioxidase system is regulated by the ABA signaling pathway.

Arabidopsis Tóxicos en Levadura 78 (AtATL78) mediates ABA-dependent ROS signaling in response to drought stress

Plants have developed a variety of complicated responses to cope with drought, one of the most challenging environmental stresses. As a quick response, plants rapidly inhibit stomatal opening under the control of abscisic acid (ABA) signaling pathway, in order to preserve water. Here, we report that Arabidopsis Tóxicos en Levadura (ATL), a RING-type E3 ubiquitin ligase, mediates the ABA-dependent stomatal closure. In contrast to wild-type plants, the stomatal closure was fully impaired in atatl78 mutant plants even in the presence of exogenous ABA and reactive oxygen species (ROS). Besides, under high concentrations of Ca2+, a down-stream signaling molecule of ABA signaling pathway, atatl78 mutant plants successfully closed the pores. Furthermore, AtATL78 protein indirectly associated with catalases and the deficiency of AtATL78 led the reduction of catalase activity and H2O2, implying the function of AtATL78 in the modulation of ROS activity. Based on these results, we suggest that AtATL78 possibly plays a role in promoting ROS-mediated ABA signaling pathway during drought stress.

Functional characterization of Arabidopsis HsfA6a as a heat‐shock transcription factor under high salinity and dehydration conditions

Although heat-shock transcription factors are well characterized in the heat stress-related pathway, they are poorly understood in other stress responses. Here, we functionally characterized AtHsfA6a in the presence of exogenous abscisic acid (ABA) and under high salinity and dehydration conditions. AtHsfA6a expression under normal conditions is very low, but was highly induced by exogenous ABA, NaCl and drought. Unexpectedly, the levels of AtHsfA6a transcript were not significantly altered under heat and cold stresses. Electrophoretic mobility shift assays and transient transactivation assays indicated that AtHsfA6a is transcriptionally regulated by ABA-responsive element binding factor/ABA-responsive element binding protein, which are key regulators of the ABA signalling pathway. Additionally, fractionation and protoplast transient assays showed that AtHsfA6a was in cytoplasm and nucleus simultaneously; however, under conditions of high salinity the majority of AtHsfA6A was in the nucleus. Furthermore, at both seed germination and seedlings stage, plants overexpressing AtHsfA6a were hypersensitive to ABA and exhibited enhanced tolerance against salt and drought stresses. Finally, the microarray and qRT-PCR analyses revealed that many stress-responsive genes were up-regulated in the plants overexpressing AtHsfA6a. Taken together, the data strongly suggest that AtHsfA6a acts as a transcriptional activator of stress-responsive genes via the ABA-dependent signalling pathway.

Functional analysis of TaABF1 during abscisic acid and gibberellin signalling in aleurone cells of cereal grains

The wheat transcription factor TaABF1 physically interacts with the protein kinase PKABA1 and mediates both abscisic acid (ABA)-induced and ABA-suppressed gene expression. In bombarded aleurone cells of imbibing grains, the effect of TaABF1 in down-regulating the gibberellin (GA)-induced Amy32b promoter was stronger in the presence of exogenous ABA. As these grains contained low levels of endogenous ABA, the effect of TaABF1 may also be mediated by ABA-induced activation even in the absence of exogenous ABA. Levels of TaABF1 protein decreased slightly during imbibition of afterripened grains. However, TaABF1 levels (especially in aleurone layers) were not substantially affected by exogenous ABA or GA, indicating that changes in TaABF1 protein level are not an important part of regulating its role in hormone signalling. We found that TaABF1 was phosphorylated in vivo in aleurone cells, suggesting a role for post-translational modification in regulating TaABF1 activity. Induction of Amy32b by overexpression of the transcription factor GAMyb could not be prevented by TaABF1, indicating that TaABF1 acts upstream of GAMyb transcription in the signalling pathway. Supporting this view, knockdown of TaABF1 by RNA interference resulted in increased expression from the GAMyb promoter. These results are consistent with a model in which TaABF1 is constitutively present in aleurone cells, while its ability to down-regulate GAMyb is regulated in response to ABA.

The Arabidopsis C3H2C3-Type RING E3 Ubiquitin Ligase AtAIRP1 Is a Positive Regulator of an Abscisic Acid-Dependent Response to Drought Stress

Ubiquitination is a eukaryotic posttranslational protein modification that is mediated by the cascade of E1, E2, and E3 ubiquitin (Ub) ligases and is involved in regulating numerous cellular functions. In this study, we obtained 100 different Arabidopsis (Arabidopsis thaliana) T-DNA insertion mutant plants in which RING E3 Ub ligase genes were suppressed and monitored their phenotypes in the presence of exogenous abscisic acid (ABA), a plant stress hormone. One of these loss-of-function mutants displayed ABA-insensitive phenotypes at the germination stage and was named atairp1 (for Arabidopsis ABA-insensitive RING protein 1). AtAIRP1 encodes a cytosolic protein containing a single C3H2C3-type RING motif with in vitro E3 Ub ligase activity. AtAIRP1 was significantly induced by ABA and drought stress. In contrast to atairp1 mutant plants, AtAIRP1-overexpressing transgenic plants (35S:AtAIRP1-sGFP) were hypersensitive to exogenous ABA in terms of radicle emergence, cotyledon development, root elongation, and stomatal closure. Ectopic expression of AtAIRP1-sGFP in atairp1 effectively rescued the loss-of-function ABA-insensitive phenotype. Both 35S:AtAIRP1-sGFP and atairp1/35S:AtAIRP1-sGFP plants accumulated higher amounts of hydrogen peroxide in response to exogenous ABA than did wild-type and atairp1 mutant plants. AtAIRP1 overexpressors were markedly tolerant to severe drought stress, as opposed to atairp1, which was highly susceptible. The levels of drought stress-related genes and basic leucine zipper transcription factor genes were up-regulated in the 35S:AtAIRP1-sGFP lines relative to wild-type and atairp1 mutant plants in response to ABA. Overall, these results suggest that AtAIRP1, a C3H2C3-type RING E3 Ub ligase, is a positive regulator in the Arabidopsis ABA-dependent drought response.

Involvement of Arabidopsis thaliana phospholipase Dζ2 in root hydrotropism through the suppression of root gravitropism

Root hydrotropism is the phenomenon of directional root growth toward moisture under water-deficient conditions. Although physiological and genetic studies have revealed the involvement of the root cap in the sensing of moisture gradients, and those of auxin and abscisic acid (ABA) in the signal transduction for asymmetric root elongation, the overall mechanism of root hydrotropism is still unclear. We found that the promoter activity of the Arabidopsis phospholipase Dzeta2 gene (PLDzeta2) was localized to epidermal cells in the distal root elongation zone and lateral root cap cells adjacent to them, and that exogenous ABA enhanced the activity and extended its area to the entire root cap. Although pldzeta2 mutant root caps did not exhibit a morphological phenotype in either the absence or presence of exogenous ABA, the inhibitory effect of ABA on gravitropism, which was significant in wild-type roots, was not observed in pldzeta2 mutant roots. In root hydrotropism experiments, pldzeta2 mutations significantly retarded or disturbed root hydrotropic responses. A drought condition similar to that used in a hydrotropism experiment enhanced the PLDzeta2 promoter activity in the root cap, as did exogenous ABA. These results suggest that PLDzeta2 responds to drought through ABA signaling in the root cap and accelerates root hydrotropism through the suppression of root gravitropism.

ABA- and ethylene-mediated responses in osmotically stressed tomato are regulated by the TSS2 and TOS1 loci

The study of mutants impaired in the sensitivity or synthesis of abscisic acid (ABA) has become a powerful tool to analyse the interactions occurring between the ABA and ethylene signalling pathways, with potential to change the traditional view of the role of ABA as just being involved in growth inhibition. The tss2 tomato mutant, which is hypersensitive to NaCl and osmotic stress, shows enhanced growth inhibition in the presence of exogenous ABA. The tos1 tomato mutant is also hypersensitive to osmotic stress, but in contrast to tss2, shows decreased sensitivity to ABA. Surprisingly, blocking ethylene signalling suppresses the growth defect of tss2 seedlings on ABA, NaCl, and osmotic stress, but not the osmotic hypersensitivity of tos1. The ethylene production of tss2 seedlings is increased compared with that of control seedlings under osmotic stress. In addition, the tss2 plants are hypersensitive to root growth inhibition by the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC). This suggests that, in addition to ABA regulation, TSS2 acts as a negative regulator of endogenous ethylene accumulation. As previously shown in Arabidopsis, it is shown here that extensive cross-talk occurs between the ABA and ethylene signalling pathways in tomato and that the TSS2 and TOS1 loci appear as regulators of this cross-talk.

Gain-of-function and loss-of-function phenotypes of the protein phosphatase 2C HAB1 reveal its role as a negative regulator of abscisic acid signalling.

HAB1 was originally cloned on the basis of sequence homology to ABI1 and ABI2, and indeed, a multiple sequence alignment of 32 Arabidopsis protein phosphatases type-2C (PP2Cs) reveals a cluster composed by the four closely related proteins, ABI1, ABI2, HAB1 and At1g17550 (here named HAB2). Characterisation of transgenic plants harbouring a transcriptional fusion ProHAB1: green fluorescent protein (GFP) indicates that HAB1 is broadly expressed within the plant, including key target sites of abscisic acid (ABA) action as guard cells or seeds. The expression of the HAB1 mRNA in vegetative tissues is strongly upregulated in response to exogenous ABA. In this work, we show that constitutive expression of HAB1 in Arabidopsis under a cauliflower mosaic virus (CaMV) 35S promoter led to reduced ABA sensitivity both in seeds and vegetative tissues, compared to wild-type plants. Thus, in the field of ABA signalling, this work represents an example of a stable phenotype in planta after sustained overexpression of a PP2C genes. Additionally, a recessive T-DNA insertion mutant of HAB1 was analysed in this work, whereas previous studies of recessive alleles of PP2C genes were carried out with intragenic revertants of the abi1-1 and abi2-1 mutants that carry missense mutations in conserved regions of the PP2C domain. In the presence of exogenous ABA, hab1-1 mutant shows ABA-hypersensitive inhibition of seed germination; however, its transpiration rate was similar to that of wild-type plants. The ABA-hypersensitive phenotype of hab1-1 seeds together with the reduced ABA sensitivity of 35S:HAB1 plants are consistent with a role of HAB1 as a negative regulator of ABA signalling. Finally, these results provide new genetic evidence on the function of a PP2C in ABA signalling.

The 5 UTR negatively regulates quantitative and spatial expression from the ABI3 promoter

The involvement of transcription factors Arabidopsis abscisic acid-insensitive3 (ABI3), maize viviparous1 (VP1) and Phaseolus vulgaris ABI3-like factor (PvALF) in the spatial control of storage protein gene expression is well established. However, little insight exists as to how they are themselves regulated. To address this, a 5.15 kb ABI3 upstream sequence including a 4.6 kb full-length promoter and 519 bp of 5'-untranslated region (UTR) was used to drive either beta-glucuronidase (GUS) or green fluorescent protein (GFP) expression in Arabidopsis. Expression from the full-length (- 4630/ + 519ABI3 ) and various 5'-truncated promoters was detected during embryogenesis in all lines, except those transgenic for promoter elements shorter than 364 bp. Two upstream activating regions, -3600 to -2033 and -2033 to -882, enhanced GUS expression in seeds. The -882 to -364 region was sufficient to confer seed-specific expression of GUS when fused to a - 64/ + 6CaMV 35S minimal promoter. Expression from the ABI3 promoter constructs was seed-specific, except in the presence of exogenous abscisic acid (ABA) (>0.3 microM), when GUS expression was detected in seedling roots. Excision of a 405 bp region containing three upstream open reading frames (uORFs) from the 5'-UTR dramatically increased GUS expression and debilitated constraint of reporter expression in roots. Negative regulation of ABI3 expression by the 5'-UTR may involve a post-transcriptional mechanism analogous to that of tumor suppressor genes which also bear long, uORF-containing, 5'-UTRs, or through interactions with RNA-binding proteins.

The ABSCISIC ACID INSENSITIVE 3 (ABI3) gene is modulated by farnesylation and is involved in auxin signaling and lateral root development in Arabidopsis.

Genetic screens have identified a number of genes that regulate abscisic acid (ABA) responsiveness in Arabidopsis. Using a combination of suppressor screens and double mutant analysis, we have determined a genetic relationship for a number of these ABA response loci. Based on germination in the presence of exogenous ABA, the ABI1 and ABI2 phosphatases act at or upstream of the ERA1 farnesyl transferase and the ABI3 and ABI5 transcription factors act at or downstream of ERA1. In contrast with ABI3 and ABI5, the ABI4 transcription factor appears to act at or upstream of ERA1. Based on reporter gene constructs, the upstream regulation of ABI3 by ERA1 occurs at least partially at the level of transcription, suggesting that this lipid modification is required to attenuate ABI3 expression. Similar experiments also indicate that ABI3 is auxin inducible in lateral root primordia. Related to this, loss-of-function abi3 alleles show reduced lateral root responsiveness in the presence of auxin and an auxin transport inhibitor, and era1 mutants have increased numbers of lateral roots. These results suggest the possibility that genes identified through ABA responsive germination screens such as ERA1 and ABI3 have functions in auxin action in Arabidopsis.

An abscisic acid-sensitive checkpoint in lateral root development of Arabidopsis.

Lateral root (LR) formation displays considerable plasticity in response to developmental and environmental signals. The mechanism whereby plants incorporate diverse regulatory signals into the developmental programme of LRs remains to be elucidated. Current concepts of lateral root regulation focus on the role of auxin. In this study, we show that another plant hormone, abscisic acid (ABA), also plays a critical role in the regulation of this post-embryonic developmental event. In the presence of exogenous ABA, LR development is inhibited. This occurs at a specific developmental stage, i.e. immediately after the emergence of the LR primordium (LRP) from the parent root and prior to the activation of the LR meristem, and is reversible. Interestingly, this inhibition requires 10-fold less ABA than the inhibition of seed germination and is only slightly reduced in characterised abi mutants, suggesting that it may involve novel ABA signalling mechanisms. We also present several lines of evidence to support the conclusion that the ABA-induced lateral root inhibition is mediated by an auxin-independent pathway. First, the inhibition could not be rescued by either exogenous auxin application or elevated auxin synthesis. Secondly, a mutation in the ALF3 gene, which is believed to encode an important component in the auxin-dependent regulatory pathway for the post-emergence LR development, does not affect the sensitivity of LRs to ABA. Thirdly, ABA and the alf3-1 mutation do not act at the same developmental point. To summarise, these results demonstrate a novel ABA-sensitive, auxin-independent checkpoint for lateral root development in Arabidopsis at the post-emergence stage. In addition, we also present data indicating that regulation of this developmental checkpoint may require novel ABA signalling mechanisms and that ABA suppresses auxin response in the LRPs.

The interaction of light and abscisic acid in the regulation of plant gene expression.

Extended dark treatments of light-grown plants of both Lemna gibba and Arabidopsis thaliana resulted in substantial increases in abscisic acid (ABA) concentrations. The concentration of ABA could be negatively regulated by phytochrome action in Lemna. As has been noted in other species, ABA treatment reduced Lemna rbcS and Lhcb RNA levels, which are positively regulated by phytochrome in many species. In view of these observations, the possibility that phytochrome effects on gene expression may be mediated primarily by changes in ABA was tested using a transient assay in intact plants. The phytochrome responsiveness of the Lemna Lhcb2*1 promoter was still apparent in the presence of exogenous ABA. Additionally, when 2-bp mutations were introduced into this promoter so that phytochrome responsiveness was lost, a response to exogenous ABA was still present. We conclude that phytochrome- and ABA-response elements are separable in the Lhcb2*1 promoter. We tested whether the effects of ABA on RNA abundance could be inhibited by treatment with gibberellin and found no evidence for such an inhibition. We have also found that the ABA-responsive Em promoter of wheat can be negatively regulated by phytochrome action. It is likely that this regulation is mediated at least in part by phytochrome-induced changes in ABA levels. Our results demonstrate that it is essential to take into account that dark treatments and the phytochrome system can affect ABA levels when interpreting studies of light-regulated genes.

Differential molecular responses to abscisic acid and osmotic stress in viviparous maize embryos

Substantial quantities of mRNA encoding the abundant “Em” polypeptide accumulate, in planta, in developing embryos of maize (Zea mays L.). By contrast, accumulation of “Em” mRNA is only barely detectable in embryos with the vp-5/vp-5 genotype [an abscisic acid (ABA)-deficient viviparous phenotype]. “Em” mRNA is not detectable within viviparous embryos of the vp-1/vp-1 genotype that are non-responsive to ABA. Culture of immature wild-type and vp-5/vp-5 embryos in the presence of exogenous ABA or of an osmotically active agent prevents precocious germination and results in expression of the “Em” genes. When vp-1/vp-1 embryos are cultured under similar conditions, only the application of osmotic stress prevents precocious germination. However, “Em” mRNA does not accumulate either in ABA-treated or stressed, arrested embryos, indicating a requirement for ABA perception through a VP-1-mediated mechanism for “Em” gene expression. Nevertheless, vp-1/vp-1 embryos do show both ABA and stress responses at the molecular level. Treatment with ABA causes the accumulation of mRNA encoding a polypeptide of approx. 30 kDa, whilst osmotic stress induces the accumulation both of a 30-kDa polypeptide and a set of approx. 20-kDa polypeptides. This indicates the existence of discrete, parallel ABA and stress response pathways in developing maize embryos.

Induction of Peroxidase Isozymes during Shoot Enhancement by ABA in Cotyledon Explants of Loblolly Pine ( Pinus taeda L.)

Summary Induction of peroxidase isozymes during shoot enhancement on cotyledon explants of loblolly pine by abscisic acid (ABA) was studied using native polyacrylamide gel electrophoresis (PAGE). Unique peroxidase bands were detected when cotyledon explants were cultured in the presence of exogenous ABA. The number of peroxidase bands increased with the length of exposure to ABA. Explants exposed to fluridone (FLU), an inhibitor of ABA biosynthesis, produced fewer bands showing peroxidase isozyme banding patterns that did not vary regardless of the length of treatment. Four weeks of FLU treatment produced the same peroxidase isozyme band pattern as the ABA-treated explants if exogenous ABA was present in the media for the first two weeks of treatment, but not after.

Expression of lipid body protein gene during maize seed development. Spatial, temporal, and hormonal regulation.

The storage lipids of seeds are packaged into lipid bodies, simple organelles containing a triacylglycerol core surrounded by a layer of phospholipids and proteins. A cDNA encoding the major protein from lipid bodies of maize (L3) was used as a hybridization probe to analyze the regulation of lipid body biosynthesis during seed development and germination. Immunofluorescent microscopy demonstrates that L3 protein accumulation is tissue-specific, confined to the embryo (scutellum and embryonic axis) and the aleurone layer of developing seed. Northern analyses show that L3 mRNA also accumulates to high levels in the embryo and is not found in any nonseed tissue. The steady-state level of L3 mRNA is developmentally regulated, increasing during early seed development and peaking at about the midpoint of seed development. It then decreases slowly, to 20% of the peak level, in the embryo of the mature seed and declines rapidly to undetectable levels as the seed germinates. The high level of L3 gene expression during seed development is not due to amplification of the L3 gene, since Southern analyses of maize genomic DNA indicate that the L3 gene has the same relatively low copy number throughout development (1-4 copies/haploid) genome). Rather, the increase in the steady-state level of L3 mRNA during seed development is transcriptionally regulated. The developmental changes in L3 mRNA levels are paralleled by changes in the transcription of the L3 gene, as measured by run-off transcription in isolated nuclei. The role of the plant growth regulator abscisic acid in regulating L3 gene expression was investigated. When the mature seed is allowed to imbibe in the presence of exogenous abscisic acid, germination is retarded, and the period of L3 gene expression is extended. The extended period of L3 gene expression in the presence of exogenous abscisic acid is due, at least in part, to a dramatic and specific increase in L3 gene transcription.