The spatio-temporal specificity of PYR1/PYL/RCAR ABA receptors in response to developmental and environmental cues

ABSTRACT

From the different functions ABA exerted between the aboveground and belowground, seed and vegetative tissues, primary root and lateral root, stimulating stomatal closure and inhibiting stomatal opening, between young and senescence leaves in stomatal movement, among different cells in plasma membrane water permeability, we addressed the organ-, tissue-, cell-, physiological processes-, and development stage specificities of PYR1/PYL/RCAR ABA receptors. This specificity may reflect the spatio-temporal properties of water potentials as well as the endogenous ABA levels in detail context, which plus the various affinities among this receptor families, resulted in the specificity of the transcripts as well as genes functions. PYR1/PYL/RCAR ABA receptors may integrate the message of ABA resource (local signaling or long distance signaling) and concentration, thus fine-tuning ABA response to environmental- and developmental cues. It also evolutionally affording land plants sophisticated mechanism to survival adverse environments.

The identification of PYR1/PYL/RCAR (pyrabactin resistance 1/PYR1-like/regulatory components of ABA receptors) ABA receptors dramatically advances our understanding of the molecular mechanism of ABA perception and signaling transduction,^1-2 however, the distance closing to unveil the mystery marks of ABA sensing related with environmental- and developmental cues is still longer. Except the various combinations among the core signaling components, in which there are 14 PYR/PYL, 9 PP2C and 3 SnRK2 involved,^3-4 the spatiotenporal specificity of PYR1/PYL/RCAR ABA receptors themselves in response to endogenous and exgenous stimulus is a very complex regulation. Certainly, exploring this regulation is pivotal to uncover ABA-dependent sophisticated response mechanism.

Given the redundancy features, forward genetics cannot have identified the phenotype of single gene mutant within this receptor family. ¹ However, gene expression patterns, biochemical analysis of different PP2C-PYL receptor complexes, different affinity to ABA or ABA agonists,^5-8 and genetic analysis of different pyr/pyl mutants as well as over-expressing lines suggest that the function of PYR/PYL proteins is not completely redundant.^9-10 Moreover, this redundancy presents a clear organ-, developmental stage- and environmental conditions-specificity, and which is correspondence to ABA's distinct function among these organs.

The organ- or tissue- specificities of PYR1/PYL/RCAR ABA receptors

The distinction sensitivity to ABA between plants aboveground and belowground has been addressed several decades ago.¹¹ It is well known that osmotic stress-derived ABA can reduce leaf hydraulic conductivity and shoot growth, thus reducing the transpiration rate in Arabidopsis leaves,^12-14 whereas it can maintain root water uptake by enhancing root growth and root hydraulic conductivity.^8,11,15-17 Recently it is found that the single PYL8 ABA receptor play a key role in mediating ABA-controlled root growth and root response to moisture gradient, means hydrotropism,^17-18 while there is a obvious redundant characteristics of PYR1/PYL/RCAR ABA receptor family in stomatal aperture regulation.⁹ A naphthalene sulfonamide ABA agonist pyrabactin, which activates seed ABA responses but fails to trigger substantial responses in vegetative tissues in Arabidopsis thaliana. Moreover, pyrabactin-induced transcriptome is highly correlated with that of ABA in seeds than in vegetative tissues of Arabidopsis plants.¹ In crop plant, we recently found that activation of PYR1 by pyrabactin can significantly improve maize root hydraulic conductivity.⁸ Excluded species specificity,¹⁹ organ-specificity may be a common feature for this type receptors in response to ABA within plant kingdom.

The distinct sensitivity to ABA is also existed between lateral root elongation and seed germination, in which the concentration that inhibit lateral root growth cannot inhibit seed germination.¹⁷ Even in the belowground, ABA exerted a different role in regulating primary root growth and lateral root growth under osmotic stress conditions.^20-22 It is known ABA has a much stronger effect on lateral root than on the primary root, suggesting that different signaling mechanisms underlying regulating these two types of roots growth.²¹ While PYL8 mediates the ability of ABA to inhibit primary root growth through the PP2C-SnRK2 pathway,¹⁸ it together with PYL9 promotes lateral root growth recovery from inhibition independently of the core signaling pathway by enhancing MYB77-dependent transcription of auxin-responsive genes.^17,23

The cell- specificities of PYR1/PYL/RCAR ABA receptors

It has been shown that application ABA directly enhance plasma membrane water permeability of roots cortical cells ¹⁵ and guard cells, ²⁴ while inhibit that of vascular bundle-sheath cells ¹² and has not effect to that of mesophyll cells.^12,24 The core signaling component SnRK2.6 has been documented implicated in plasma membrane intrinsic proteins PIP2;1 activation through protein phosphorylation at Ser-121, thus increase in guard cell permeability to water.²⁴ However, how this signaling model involved in PIP inhibition is still not clear, though 4 PIPs dephosphorylation has been documented by a phosphoproteome technology in Arabidopsis leaves following 5-30 min ABA treatment.²⁵ Other target proteins that dephosphorylated by ABA included PM H⁺-ATPase,²⁶ and several calcium-related proteins.²⁵ It is proposed that ABA make a dual effect on stomata in which ABA closes stomata in adverse hydraulic conditions via its biochemical effect on the guard cells, but also via an indirect hydraulic effect through a decrease in water permeability within the leaf vascular tissues.¹⁴ PYR1/PYL/RCAR ABA receptors-mediated core signaling pathway has been shown involved in the former effect, how this type receptors implicated in the latter is needed to be determined in the future.

Promoting stomatal closure and inhibiting it opening, two sides of ABA action on stomatal aperture, have been shown with different signaling mechanism and target ion channels.²⁷ While ABA-induced stomatal closure was impaired in the pyr1/pyl1/pyl2/pyl4 quadruple ABA receptor mutant, ABA inhibition of the opening of the mutant’s stomata remained intact.²⁷ Except an unidentified extracellular receptor remains functional in the quadruple mutant, some of the other 10 PYR/PYL/RCAR members might be involved in the opening inhibition. These results indicate that PYR1/PYL/RCAR ABA receptor displays a cell- and even physiological processes specific features.

The development stage- specificities of PYR1/PYL/RCAR ABA receptors

It is clear that the ABA level increased in senescencing or aging leaves, but their stomatal movement pattern is opposite from that of young mature leaves. ABA induced stomatal closure in young leaves, while inhibit stomatal closure in senescing leaves.²⁸ Zhao et al. ¹⁰ recently documented that the core signaling components were implicated in ABA-mediated leaf senescence. These authors further found that leaf senescence may benefit drought resistance by helping to generate an osmotic potential gradient, which causes water to preferentially flow to developing tissues. An ABA- and senescence up-regulated PP2C family PP named SENESCENCE ASSOCIATED GENE113 (SAG113), which is a negative regulator of ABA signaling, to specifically suppress the stomata from closing such that senescing leaves lose water rapidly, leading to senescence and ultimate desiccation.²⁹ It remains to be seen how PYR1/PYL/RCAR ABA receptors - AtNAP transcription factor - SAG were implicated in ABA-mediated stomata closure inhibition in senescing leaves. Leaf ABA sensitivity is reported closely related with its microclimate. In the rosette plant Arabidopsis, stomatal sensitivity to ABA is acquired during leaf development by exposure to an increasingly dryer atmosphere, while young immature leaves, which develop in the center of the rosette, do not close in response to ABA.³⁰ Taken together, during the process of leaf development, that means from immature to mature, from young to aging, its stomatal sensitivity to ABA initially increased, and then decreased again. The young mature leaves have the maximum stomatal sensitivity to ABA than both the young immature leaves and the senescing leaves. So far PYR1/ PYL/ RCAR ABA receptors has been confirmed implicated in mature leaves stomatal closure and Leaf senescence,^1,10 but whether they involved in the stomatal movement of young immature leaves is still not clear. These results also shown that leaf ABA sensitivity is closely related with its microclimate.

Spatio-temporal specificities of PYR1/PYL/RCAR ABA receptors in transcriptional levels

It is well known that gene functions are closely related to the mechanism by which gene expression is regulated.⁷ Much of the core ABA signaling pathway is transcriptionally regulated by ABA, thus these core components provide a framework on which to expand the signaling network. So the transcriptional profiling of PYR1/PYL/RCAR ABA receptors in response to environmental- and developmental cues is contributed greatly to the specificities of this type receptor. Gene expression patterns obtained from public databases and GUS reporter gene analyses have revealed substantial differences among PYR1/ PYL/ RCAR ABA receptor family.⁹ In Arabidopsis, the most highly expressed receptors in most developmental stages are PYR1, PYL1, PYL4, PYL5, and PYL2. Among these, PYR1 is consistently highly expressed, but the others vary among stages and organs. At the tissue level, further variation is seen.⁹ GUS reporter analyses of PYR1, PYL1, PYL2, PYL4, PYL5, and PYL8 promoters has shown both overlapping and differential expression in different tissues.⁹ PYR1, PYL1, PYL2, and PYL4 appear to be predominantly expressed in guard cells, and their disruption resulted in insensitivity to ABA in the stomata.³² The relative nonredundant characteristic of PYL8 in root function of Arabidopsis is corresponded to its specific expression pattern in this organ.¹⁸ It is shown that PYL8 predominantly expressed in vascular tissue, columella cells, root epidermis and lateral root cap, which is correspondent to its role in regulating root growth and hydrotropism. Activation of PYR1 by the synthetic agonist pyrabactin is sufficient to activate ABA responses in seeds but yields a minimal response in vegetative tissues, which is consistent with the high level expression of PYR1 in seeds relative to other tissues.¹ Our recent results demonstrated that PYR1/PYL/RCAR ABA receptor genes in maize exhibit distinct expression patterns in response to abiotic stresses (or ABA) between roots and leaves, with isoforms that are upregulated in roots being down-regulated in leaves, and vice versa.³³

Except with the different downstream transcript factors, this receptor family themselves displays a clear development-specific features in transcription. PYL5 is the most highly expressed receptor at seed maturation, suggesting that it may play a major role in ABA response at this stage. It has been documented so far that PYR1/PYL/RCAR ABA receptors are implicated in seed maturation and germination, early seedling growth, root growth, fruit development and ripen, and leaf senescence etc.^10,34-37 As described in the above paragraph, this development-specific transcriptional response to ABA may reflect the correspondence different functions among this receptor family in varies development stages.

Spatio-temporal specificities of PYR1/PYL/RCAR ABA receptors reflect specificities of developmental and environmental cues-derived endogenous ABA levels status or water status

The various transcriptional responses integrate the different affinities of the receptor family members ultimately permit responses that vary over a wide range of ABA concentrations and cell types.^3-4 Given the dimeric receptors show a higher dissociation constant for ABA (greater than 50 μM; lower affinity) than monomeric ones (approximately 1μM), the endogenous ABA level determined the selection for this two types receptor sub-families.³⁸ According to the literatures, it is proposed that the dimeric receptors act as the key targets for chemical modulation of vegetative ABA responses as well as the objective receptors for protein engineering,^5-6,39 while the monomeric ones prefer implicated in genetic-modify plant traits for abiotic stress tolerance ² and also involved in root function regulation.^17-18,23 That the more sensitive of shoot growth than root growth in response to draft stress, is resulted from the insufficient ABA level in the former than the latter to restrict ethylene production.¹¹ It was shown that leaf ABA concentration ([ABA]) varied greatly as a result of plant growth under different levels of soil water deficit at moderate (60%) or high (90%) relative air humidity (RH). [ABA] of well-watered plants grown at moderate RH was sufficient to induce functional stomata. Lower [ABA] than this threshold level resulted in a proportional attenuation of stomatal sensitivity to desiccation, whereas higher [ABA] did not produce any vapour pressure deficit (VPD) plants due to the level of foliar ABA sharply decreased under this conditions, while spraying ABA during the exposure to low VPD prevented loss of stomatal closing response thereafter.⁴² Now it is confirmed that the endogenous ABA level was not only positively related to the stress tolerance, but also to the recovering ability of dehydrated tissues to rehydrate. The tissue- and even cell-specific ABA accumulation pattern may determine the spatiotemporal specificity of PYR1/PYL/RCAR ABA receptors.

Except the endogenous ABA levels ABA-resource also plays a key role in mediating ABA perception. When WT Arabidopsis leaves were xylem-supplied with 10μM ABA, leaf hydraulic conductance (K_leaf) decreased by c. 50%, yet foliar application of the same ABA concentration (while decreasing transpiration rate) had no statistically significant effect on detached leaf K_leaf.¹² While application ABA to root medium generally inhibit root growth, McAdam et al. recently shown that shoot-derived ABA promotes root growth.⁴³ It is clear that the different sourced ABA correspondence to its distinct action, even in the same tissues or cells.⁴⁴ Although several ABA transporters have been identified so far,⁴⁵ the selection for PYR1/PYL/RCAR ABA receptors between local ABA and long distance ABA may be different. Recently developed FÖrster resonance energy transfer (FRET) biosensors for direct ABA imaging will greatly advance our observation of ABA dynamics and transport in vivo,⁴⁶ which will also benefit our understanding of its correspondence perception by PYR1/PYL/RCAR ABA receptors. ABA perception is also related with water status both of plant tissues and the surrounding environment.^47-48 As discussed in the above paragraph, it is proposed that the water conditions in senescing leaves did not benefit its stomatal closure.^10,28 The different stomatal sensitivity to ABA between mature leaves and young leaves in Arabidopsis rosette is also due to their distinct surrounding microclimate.³⁰ It seems that exposure of a leaf for a long time (several days) to some environmental conditions generates a sort of memory in the guard cells that results in the loss of suitable responses of the stomata to closing stimuli, such as desiccation and ABA.⁴⁷ The involved secondary messengers in stomatal movement is also related with leaves water conditions or RH. It is indicated that nitric oxide (NO) mediated ABA-controlled stomatal closure in the turgid leaves but not the wilted leaves,⁴⁹ Although ABA can induce H₂O₂ production in guard cells, but does not close the stomata on Vicia faba leaves developed at high air humidity.⁵⁰ All these truths shown that the context water status exerts a pivotal role in ABA perception and signaling transduction.

Conclusions

In brief, PYR1/PYL/RCAR ABA receptors display an organ-, tissue-, cell-, and even physiological processes specificities in response to environmental- and developmental cues. This specificity may underlying the mechanism of different ABA sensitivities among varies cells, tissues or organs, thus different ABA functions. The spatio-temporal differences in water potentials (hydraulic signaling), ABA levels (chemical signaling), distribution of ABA between apoplastic and symplastic compartments, plus the various affinities among this receptors family, resulted in the specificity of the transcription as well as gene function. The diversity of PYLs therefore contributes to the versatility of ABA signaling and functions (Fig. 1). Further exploring the specificities in each layer in more plant species will benefit the understanding of the early ABA signaling events network in a detail context in the future.

Figure 1.

Proposed model of the spatio-temporal specificity of PYR1/PYL/RCAR ABA receptors in response to developmental and environmental cues.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Dr. Frantisek Baluska for kindly inviting us to prepare this article. This work has been partially funded by the Special Fund for Basic Scientific Research from Northwest A&F University (No. QN2012021).