Abscisic Acid and Abiotic Stress Signaling

Introduction

Plants experience many different kinds of abiotic stresses including higher concentration of salt (salinity), extremes of temperature (low temperature, i.e., cold [chilling or freezing], higher temperature [heat]and water shortage (drought or dehydration). These stresses are the principal cause of reducing the yield of crops significantly.¹ Overall, stress tolerance is a complex phenomenon because plants may go through multiple stresses at the same time during their development. Among abiotic stresses, the high salinity stress is the most severe environmental stress, which impairs crop production on at least 20% of irrigated land worldwide. In addition, increased salinity of arable land is expected to have devastating global effects, resulting in up to 50% land loss by the middle of 21 ^century.1

Plants perceive and respond adaptively to abiotic stress imposed by salt, cold, drought and wounding and the adaptive process is controlled mainly by the phytohormone, abscisic acid (ABA), which acts as an endogenous messenger in the regulation of the plant's water status.² ABA is generated as a signal during a plant's life cycle to control seed germination and developmental processes. The action of ABA can target specifically guard cells for induction of stomatal closure but may also signal systemically for adjustment towards severe water shortage. Since various stresses induce ABA synthesis, it is now considered as a plant stress hormone.¹2 Various transcription factors are known to regulate the ABA-responsive gene expression.¹3 Stress responsive genes can be expressed either through an ABA-dependent or ABA-independent pathway.⁴ This review article first describes the general pathway for plant stress response followed by roles of ABA and transcription factors in stress tolerance. The regulation of ABA biosynthesis pathway is also described.

Generic Pathway for Plant Response to Stress

Plants can respond to stresses as individual cells and synergistically as a whole organism. The extracellular stress signal is first perceived at membrane level by the membrane receptors, ion channel, receptor-like kinase (RLK) or histidine kinase (HK) etc and then activate large and complex signaling cascade intracellularly including the generation of secondary signal molecules such as Ca^{, inositol phosphates (InsP), reactive oxygen species (ROS) and ABA. The stress signal then transduces inside the nucleus to induce multiple stress responsive genes, the products of which ultimately lead to plant adaptation to stress tolerance directly or indirectly.1} Overall, the stress response could be a coordinted action of many genes, which may cross-talk with each others. The stress-induced gene products are also involved in the generation of regulatory molecules like ABA, salicylic acid and ethylene, which can initiate the second round of signaling. Small molecules like ABA play important role in this process.¹4

ABA and Abiotic Stress Signaling

ABA is an important phytohormone and plays a critical role in response to various stress signals. The application of ABA to plant mimics the effect of a stress condition. As many abiotic stresses ultimately results in desiccation of the cell and osmotic imbalance, there is an overlap in the expression pattern of stress genes after cold, drought, high salt or ABA application. This suggests that various stress signals and ABA share common elements in the signaling pathway and these common elements cross-talk with each other, to maintain cellular homeostasis.⁵6 ABA also play important roles in many other physiological processes such as seed dormancy and delays its germination, development of seeds, promotion of stomatal closure, embryo morphogenesis, synthesis of storage proteins and lipids, leaf senescence and also defense against pathogens.² ABA levels are induced in response to various stress signals. ABA actually helps the seeds to surpass the stress conditions and geminate only when the conditions are conducive for seed germination and growth. ABA also prevents the precocious germination of premature embryos. Stomatal closure under drought conditions prevents the intracellular water loss and thus ABA is aptly called as a stress hormone.

Main function of ABA seems to be the regulation of plant water balance and osmotic stress tolerance. Several ABA deficient mutants namely aba1, aba2 and aba3 have been reported for Arabidopsis.⁷ ABA deficient mutants for tobacco, tomato and maize have also been reported.² Without any stress treatment the growth of these mutants is comparable to wild type plants. Under drought stress, ABA deficient mutants readily wilt and die if the stress persists. Under salt stress also ABA deficient mutants show poor growth.⁸ In addition, ABA is required for freezing tolerance, which also involves the induction of dehydration tolerance genes.⁸

Studies suggest that osmotic stress imposed by high salt or drought is transmitted through at least two pathways; one is ABA-dependent and the other ABA-independent. Cold exerts its effects on gene expression largely through an ABA-independent pathway.⁵6 ABA induced expression often relies on the presence of cis-acting element called ABRE element (ABA-responsive element).⁵69 Genetic analysis indicates that there is no clear line of demarcation between ABA-dependent and ABA-independent pathways and the components involved may often cross talk or even converge in the signaling pathway. Calcium, which serves as a second messenger for various stresses, represents a strong candidate, which can mediate such cross talks. Several studies have demonstrated that ABA, drought, cold and high salt result in rapid increase in calcium levels in plant cells.¹4

The transcript accumulation of RD29A gene is reported to be regulated in both ABA-dependent and ABA-independent manner.¹⁰ Proline accumulation in plants can be mediated by both ABA-dependent and ABA-independent signaling pathways.¹ The role of calcium in ABA-dependent induction of P5CS gene during salinity stress has been reported by.¹¹ It is known that the expression of RD29A, RD22, COR15A, COR47 and P5CS genes was reduced in the los5 mutant.⁸ The signaling mechanism behind the activation of these genes are not well known, but the transcriptional activation of few stress induced genes represented by RD29A is known to some extent.³ It is also suggested that phospholipase D (PLD) along with ABA and calcium act as a negative regulator of proline biosynthesis in Arabidopsis.¹² The salinity stress-induced upregulation of transcript of pea DNA helicase 45 (PDH45) followed ABA-dependent pathway¹³ while calcineurin B-like protein (CBL) and CBL-interacting protein kinase (CIPK) from pea followed the ABA-independent pathway.¹⁴15 Overall, the ABA-dependent pathways are involved essentially in osmotic stress gene expression. Recently, Lee et al. (2006)¹⁶ have proposed that the activation of inactive ABA pools by polymerized AtBG1 (a β-glucosidase, hydrolyzes glucose-conjugated) could be a mechanism by which plants rapidly adjust ABA levels and respond to changing environmental cues.

ABA Biosynthesis

ABA level is known to induce under stress condition, which is mainly due to the induction of genes for enzymes responsible for ABA biosynthesis. Several ABA biosynthesis genes have been cloned which includes Zeathanxin epoxidase (Known as ABA1 in Arabidopsis), 9-cis-epoxycarotenoid dioxygenase (NCED), ABA aldehyde oxidase and ABA3 also known as LOS5.³8 ABA is synthesized from β-carotene through several enzymatic steps (Fig. 1). The abiotic stress-induced activation of many ABA biosynthetic genes such as zeaxanthin oxidase (ZEP), 9-cis-epoxycarotenoid dioxygenase (NCED), ABA-aldehyde oxidase (AAO) and molybdenum cofactor sulphurase (MCSU) appeared to be regulated through calcium-dependent phosphorylation pathway.³417 The accumulation of ABA can also feedback stimulate the expression of ABA biosynthetic genes through calcium-signaling pathway and can also activate the ABA catabolic enzymes to degrade the ABA. The mechanisms by which the abiotic stress upregulate ABA biosynthesis genes are not well understood. Recently, Verslues et al. (2007)¹⁸ suggested that metabolic changes that alter hydrogen peroxide levels could also affect both ABA accumulation and ABA sensitivity.

Figure 1

ABA biosynthesis pathway and its regulation. ABA is synthesized from β- carotene via the oxidative cleavage of neoxanthin and conversion of xanthoxin to ABA via ABA-aldehyde. Abiotic stresses (Dehydration, cold, salinity) stimulate ABA biosynthesis and accumulation by activating genes involved in the ABA biosynthetic pathway, which itself could be mediated by calcium-dependent phosphorylation cascade. ABA could also upregulate expression of ABA biosynthetic genes via calcium signaling pathways (see Xiong et al., 2002; Zhu, 2002). AAO, ABA-aldehyde oxidase; MCSU, molybdenium cofactor sulfurase, NCED, 9-cis-epoxycarotenoid dioxygenase, ZEP, zeaxanthin epoxidase.

ABA and Transcription Factors in Stress Tolerance

Transcriptional regulatory network of cis-acting elements and transcription factors involved in ABA and abiotic stress responsive gene expression is depicted in Figure 2. The promoters of the stress-induced genes contain cis-regulatory elements such as DRE/CRT (A/GCCGAC), ABRE (PyACGTGGC), MYC recognition sequence (MYCRS; CANNTG) and MYB recognition sequence (MYBRS; C/TAACNA/G), which are regulated by various upstream transcriptional factors (Fig. 2).¹17 The ABA-dependent stress signaling activates basic leucine zipper transcription factors called AREB, which binds to ABRE element to induce the stress responsive gene (RD29B). In Arabidopsis, it is reported that two ABRE motofs are involved in the regulation of ABA-responsive expression of the RD29B gene, which encodes a LEA-like (late embryogenesis abundant) protein.⁹ Transcription factors like DREB2A and DREB2B trans-activate the DRE cis-element of osmotic stress genes and thereby are involved in maintaining the osmotic equilibrium of the cell.¹ Some genes like RD22 lack the typical CRT/DRE elements in their promoter suggesting their regulation by some other mechanism. RD22 gene encodes a protein having a homology to an unidentified seed protein. The drought-inducible expression of DREB1D is regulated by ABA-dependent pathways, indicating that DREB1D protein may function in the slow response to drought that depends upon the accumulation of ABA (Fig. 2). The MYC/MYB transcription factors, RD22BP1 and AtMYB2, could bind MYCRS and MYBRS elements, respectively and help in activation of RD22 gene (Fig. 2). These MYC and MYB proteins are known to synthesized only after endogenous levels of ABA accumulate, hence suggesting that their role is in a late stage of the stress responses.¹ The Clp protease regulatory subunit encoding gene, ERD1 (early responsive to dehydration 1), responds to dehydration and salinity stress before the accumulation of ABA, which suggested that an ABA-independent pathway also exists in the dehydration stress response of Arabidopsis.¹⁹ Overexpression of both ZF-HD and NAC proteins activate the expression of ERD1 gene in normal growth conditions (non-stressed) in the transgenic Arabidopsis plants. Overall, these transcription factors may also cross-talk with each other for their maximal response to stress tolerance. Kim et al. (2004)²⁰ reported that overexpression of a transcription factor regulating ABA-responsive gene expression confered multiple stress tolerance.

Figure 2

Transcriptional regulatory network of cis-acting elements and ABA-dependent transcription factors involved in drought, cold and salinity stress gene expression. Abiotic stress signaling seems to be mediated by transcription factors such as NAC, ZF-HD, DREB2A/DREB2B and DREB1/CBF, AREB1, RD22BP1 and MYC/MYB transcription activators, which interact with NACR, ZF-HDR, DRE/CRT, ABRE and MYCRS/MYBRS elements in the promoter of the stress genes, respectively. AtMYC2 and AtMYB2 act cooperatively to activate the expression of ABA-inducible genes such as RD22. Cis-acting elements that are involved in transcription of stress-responsive gene are shown in boxes. Transcription factor that regulate stress-inducible gene expression are shown in ovals. ABA, abscisic acid; ABRE, ABA-responsive element; AREB, ABRE-binding protein; CBF, C-repeat-binding factor; COR, cold regulated genes; CRT, C-repeat; DRE, dehydration-responsive element; DREB, DRE-binding protein; ERD, early responsive to dehydration, MYB, myeloblastosis; MYBRS, MYB-recognition sequence; MYC, myelocytomatosis; MYCRS, MYC-recognition sequence; NACR, NAC-recognition site; RD, genes responsive to dehydration; ZF-HD; zinc-finger homeodomain.

The ABA level goes down during seed imbibition, which allow embryos to germinate and develop into seedlings, while ABA level remains high during abiotic stress conditions, which can arrest the growth and development. Several transcription factors, including abscisic acid-insensitive, ABI3 and ABI5, are known to control this developmental checkpoint. Recently, Reyes and Chua (2007)²¹ have shown that in germinating Arabidopsis thaliana seeds, ABA induces the accumulation of microRNA 159 (miR159) in an ABI3-dependent fashion and miRNA159 mediates cleavage of MYB101 and MYB33 transcripts in vitro and in vivo. Here, they have shown that, the two MYB transcription factors function as positive regulators of ABA responses, as null mutants of myb33 and myb101 show hyposensitivity to the hormone. These results suggested that ABA-induced accumulation of miR159 controls transcript levels of two MYB factors during Arabidopsis seed germination. This is a homeostatic mechanism to direct MYB33 and MYB101 transcript degradation to desensitize hormone signaling during seedling stress responses.²¹

Conclusions and Perspectives

Overall, the abiotic stress signaling is an important area with respect to increase in crops yield under sub-optimal conditions. The involvement of the stress responsive genes in various metabolic processes to enhance the stress tolerance in plant might have general implications. The mechanism of stress tolerance is just beginning to be understood. The overall progress of research on ABA regulated stress responsive genes and their products reflect their central role in plant growth and development under stress conditions. A lot of effort is still required to uncover in detail of each products of gene induced by ABA and their interacting partners to understand the complexity of the high salinity stress signal transduction pathways. The role of endogenous siRNA in regulating the ABA induced stress tolerance will be further helpful in our better understanding mechanism of stress tolerance. Determination of the upstream receptors or sensors that monitor the stimuli as well as the downstream effectors that regulate the responses is essential, which will also expedite our understanding of ABA madiated stress signaling mechanisms in plants. The mechanisms by which the abiotic stress upregulate ABA biosynthesis genes are still need understand. Still we need to understand the functions of all the different kinds of ABA-responsive genes. Overall, it is becoming clear that ABA action enforces a sophisticated regulation at all levels.