ABI1 protein phosphatase 2C is a negative regulator of abscisic acid signaling.

The plant hormone abscisic acid (ABA) is a key regulator of seed maturation and germination and mediates adaptive responses to environmental stress. In Arabidopsis, the ABI1 gene encodes a member of the 2C class of protein serine/threonine phosphatases (PP2C), and the abi1-1 mutation markedly reduces ABA responsiveness in both seeds and vegetative tissues. However, this mutation is dominant and has been the only mutant allele available for the ABI1 gene. Hence, it remained unclear whether ABI1 contributes to ABA signaling, and in case ABI1 does regulate ABA responsiveness, whether it is a positive or negative regulator of ABA action. In this study, we isolated seven novel alleles of the ABI1 gene as intragenic revertants of the abi1-1 mutant. In contrast to the ABA-resistant abi1-1 mutant, these revertants were more sensitive than the wild type to the inhibition of seed germination and seedling root growth by applied ABA. They also displayed increases in seed dormancy and drought adaptive responses that are indicative of a higher responsiveness to endogenous ABA. The revertant alleles were recessive to the wild-type ABI1 allele in enhancing ABA sensitivity, indicating that this ABA-supersensitive phenotype results from a loss of function in ABI1. The seven suppressor mutations are missense mutations in conserved regions of the PP2C domain of ABI1, and each of the corresponding revertant alleles encodes an ABI1 protein that lacked any detectable PP2C activity in an in vitro enzymatic assay. These results indicate that a loss of ABI1 PP2C activity leads to an enhanced responsiveness to ABA. Thus, the wild-type ABI1 phosphatase is a negative regulator of ABA responses.     

ASG2 is a farnesylated DWD protein that acts as ABA negative regulator in Arabidopsis

The tagging‐via‐substrate approach designed for the capture of mammal prenylated proteins was adapted to Arabidopsis cell culture. In this way, proteins are in vivo tagged with an azide‐modified farnesyl moiety and captured thanks to biotin alkyne Click‐iT® chemistry with further streptavidin‐affinity chromatography. Mass spectrometry analyses identified four small GTPases and ASG2 (ALTERED SEED GERMINATION 2), a protein previously associated to the seed germination gene network. ASG2 is a conserved protein in plants and displays a unique feature that associates WD40 domains and tetratricopeptide repeats. Additionally, we show that ASG2 has a C‐terminal CaaX‐box that is farnesylated in vitro. Protoplast transfections using CaaX prenyltransferase mutants show that farnesylation provokes ASG2 nucleus exclusion. Moreover, ASG2 interacts with DDB1 (DAMAGE DNA BINDING protein 1), and the subcellular localization of this complex depends on ASG2 farnesylation status. Finally, germination and root elongation experiments reveal that asg2 and the farnesyltransferase mutant era1 (ENHANCED RESPONSE TO ABSCISIC ACID (ABA) 1) behave in similar manners when exposed to ABA or salt stress. To our knowledge, ASG2 is the first farnesylated DWD (DDB1 binding WD40) protein related to ABA response in Arabidopsis that may be linked to era1 phenotypes.     

ATHB17 is a positive regulator of abscisic acid response during early seedling growth

We performed activation tagging screen to isolate abscisic acid (ABA) response mutants. One of the mutants, designated ahs10 (ABA-hypersensitive 10), exhibited ABA-hypersensitive phenotypes. TAIL-PCR analysis of the mutant revealed that T-DNA was inserted in the promoter region of the Arabidopsis gene, At2g01430, which encodes a homeodomain-leucine zipper protein ATHB17. Subsequent expression analysis indicated that ATHB17 was activated in ahs10. To recapitulate the mutant phenotypes, we prepared ATHB17 OX lines and investigated their phenotypes. The results showed that ATHB17 confers ABA-hypersensitivity and drought tolerance. On the contrary, ATHB17 knockout lines were ABA-insensitive and drought-sensitive, further demonstrating that ATHB17 is involved in ABA and water-stress responses. Interestingly, the ATHB17 effect on seedling growth in the presence of ABA was observed only during the postgermination seedling establishment stage, suggesting that it functions during a narrow developmental window of early seedling growth.     

Stress-induced protein phosphatase 2C is a negative regulator of a mitogen-activated protein kinase

Protein phosphatases of type 2C (PP2Cs) play important roles in eukaryotic signal transduction. In contrast to other eukaryotes, plants such as Arabidopsis have an unusually large group of 69 different PP2C genes. At present, little is known about the functions and substrates of plant PP2Cs. We have previously shown that MP2C, a wound-induced alfalfa PP2C, is a negative regulator of mitogen-activated protein kinase (MAPK) pathways in yeast and plants. In this report, we provide evidence that alfalfa salt stress-inducible MAPK (SIMK) and stress-activated MAPK (SAMK) are activated by wounding and that MP2C is a MAPK phosphatase that directly inactivates SIMK but not the wound-activated MAPK, SAMK. SIMK is inactivated through threonine dephosphorylation of the pTEpY motif, which is essential for MAPK activity. Mutant analysis indicated that inactivation of SIMK depends on the catalytic activity of MP2C. A comparison of MP2C with two other PP2Cs, ABI2 and AtP2CHA, revealed that although all three phosphatases have similar activities toward casein as a substrate, only MP2C is able to dephosphorylate and inactivate SIMK. In agreement with the notion that MP2C interacts directly with SIMK, the MAPK was identified as an interacting partner of MP2C in a yeast two-hybrid screen. MP2C can be immunoprecipitated with SIMK in a complex in vivo and shows direct binding to SIMK in vitro in protein interaction assays. Wound-induced MP2C expression correlates with the time window when SIMK is inactivated, corroborating the notion that MP2C is involved in resetting the SIMK signaling pathway.     

Abscisic acid signalling when soil moisture is heterogeneous: decreased photoperiod sap flow from drying roots limits abscisic acid export to the shoots

To investigate the contribution of different parts of the root system to total sap flow and leaf xylem abscisic acid (ABA) concentration ([X-ABA]_leaf), individual sunflower ( Helianthus annuus L.) shoots were grafted onto the root systems of two plants grown in separate pots and sap flow through each hypocotyl measured below the graft union. During deficit irrigation (DI), both pots received the same irrigation volumes, while during partial root zone drying (PRD) one pot ('wet') was watered and another ('dry') was not. During PRD, once soil water content ( θ ) decreased below a threshold, the fraction of sap flow from drying roots declined. As θ declined, root xylem ABA concentration increased in both irrigation treatments, and [X-ABA]_leaf increased in DI plants, but [X-ABA]_leaf of PRD plants actually decreased within a certain θ range. A simple model that weighted ABA contributions of wet and dry root systems to [X-ABA]_leaf according to the sap flow from each, better predicted [X-ABA]_leaf of PRD plants than either [X-ABA]_dry, [X-ABA]_wet or their mean. Model simulations revealed that [X-ABA]_leaf during PRD exceeded that of DI with moderate soil drying, but continued soil drying (such that sap flow from roots in drying soil ceased) resulted in the opposite effect.     

The ABI1 and ABI2 protein phosphatases 2C act in a negative feedback regulatory loop of the abscisic acid signalling pathway

The Arabidopsis ABI1 and ABI2 genes encode two protein serine/threonine phosphatases 2C (PP2C). These genes have been originally identified by the dominant mutations abi1--1 and abi2--1, which reduce the plant's responsiveness to the hormone abscisic acid (ABA). However, recessive mutants of ABI1 were recently shown to be supersensitive to ABA, which demonstrated that the ABI1 phosphatase is a negative regulator of ABA signalling. We report here the isolation and characterisation of the first reduction-of-function allele of ABI2, abi2--1R1. The in vitro phosphatase activity of the abi2--1R1 protein is approximately 100-fold lower than that of the wild-type ABI2 protein. Abi2--1R1 plants displayed a wild-type ABA sensitivity. However, doubly mutant plants combining the abi2--1R1 allele and a loss-of-function allele at the ABI1 locus were more responsive to ABA than each of the parental single mutants. These data indicate that the wild-type ABI2 phosphatase is a negative regulator of ABA signalling, and that the ABI1 and ABI2 phosphatases have overlapping roles in controlling ABA action. Measurements of PP2C activity in plant extracts showed that the phosphatase activity of ABI1 and ABI2 increases in response to ABA. These results suggest that ABI1 and ABI2 act in a negative feedback regulatory loop of the ABA signalling pathway.     

Arabidopsis ROP‐interactive CRIB motif‐containing protein 1 (RIC1) positively regulates auxin signalling and negatively regulates abscisic acid (ABA) signalling during root development

Auxin and abscisic acid (ABA) modulate numerous aspects of plant development together, mostly in opposite directions, suggesting that extensive crosstalk occurs between the signalling pathways of the two hormones. However, little is known about the nature of this crosstalk. We demonstrate that ROP‐interactive CRIB motif‐containing protein 1 (RIC1) is involved in the interaction between auxin‐ and ABA‐regulated root growth and lateral root formation. RIC1 expression is highly induced by both hormones, and expressed in the roots of young seedlings. Whereas auxin‐responsive gene induction and the effect of auxin on root growth and lateral root formation were suppressed in the ric1 knockout, ABA‐responsive gene induction and the effect of ABA on seed germination, root growth and lateral root formation were potentiated. Thus, RIC1 positively regulates auxin responses, but negatively regulates ABA responses. Together, our results suggest that RIC1 is a component of the intricate signalling network that underlies auxin and ABA crosstalk.     

The effect of abscisic acid on the growth and storage of germinating rape (Brassica napus L.) seed dried following selection on the basis of a newly-emerged radicle

Abscisic acid at 1 × 10^–4M concentration controlled the progress of the emerging radicle from germinating rape seeds and apparently restricted it to the dehydration tolerant phase. ABA treatment during germination followed by washing reduced the deleterious effects of drying the seeds following selection based on a newly-emerged radicle. Furthermore, the longevity of these low-moisture-content germinating seeds at a range of temperatures was improved by the ABA treatment. The viability of the treated seeds stored at –20°C was maintained for up to 100 days.     

INH, a negative regulator of MPF, is a form of protein phosphatase 2A.

MPF, a protein kinase complex consisting of cyclin and p34cdc2 subunits, promotes the G2 to M phase transition in eukaryotic cells. The pathway of activation and inactivation of MPF is not well understood, although there is strong evidence that removal of phosphate from a tyrosine residue on p34cdc2 is part of the activation process. INH was originally identified as an activity that could inhibit the posttranslational activation of a latent form of MPF, called pre-MPF, in immature (G2 phase-arrested) Xenopus oocytes. We have purified INH and demonstrated that it is a form of protein phosphatase 2A. Both INH and the catalytic subunit of protein phosphatase 2A can directly inactivate an isolated p34cdc2-cyclin complex. Both cyclin and p34cdc2 become dephosphorylated; the rate of inactivation closely parallels the removal of phosphate from a specific site on p34cdc2. We propose that INH opposes MPF activation by reversing this critical phosphorylation.     

Cell cycle-dependent regulation of telomerase activity by auxin, abscisic acid and protein phosphorylation in tobacco BY-2 suspension culture cells

Telomerase is a specialized RNA-directed DNA polymerase that adds telomeric repeats onto the ends of linear eukaryotic chromosomes. It was recently reported that the low, basal level of telomerase activity markedly increased at early S-phase of the cell cycle, and auxin further increased the S-phase-specific telomerase activity in tobacco BY-2 cells. In this study we show that abscisic acid (ABA), a phytohormone known to induce the cyclin-dependent protein kinase inhibitor, effectively abolished both the auxin- and S-phase-specific activation of telomerase in a concentration- and time-dependent fashion in synchronized tobacco BY-2 cells. These results suggest that there exists a hormonal cross-talk between auxin and ABA for the regulation of telomerase activity during the cell cycle of tobacco cells. Treatment of synchronized BY-2 cells with the protein kinase inhibitor staurosporine or H-7 effectively prevented the S-phase-specific activation of telomerase activity. By contrast, when okadaic acid or cantharidin, potent inhibitors of protein phosphatase 2A (PP2A), was applied to the cells, the S-phase-specific high level of telomerase activity was continuously maintained in the cell cycle for at least 14 h after release from M-phase arrest. Incubation of tobacco cell extracts with exogenous PP2A rapidly abrogated in vitro telomerase activity, while okadaic acid and cantharidin blocked the action of PP2A, effectively restoring in vitro telomerase activity. Taken together, these findings are discussed in the light of the suggestion that antagonistic functions of auxin and ABA, and reciprocal phosphorylation and dephosphorylation of telomerase complex, are necessarily involved in the cell cycle-dependent modulation of telomerase activity in tobacco cells.     

Phoslactomycin targets cysteine-269 of the protein phosphatase 2A catalytic subunit in cells

According to the chemical genetic approach, small molecules that bind directly to proteins are used to analyze protein function, thereby enabling the elucidation of complex mechanisms in mammal cells. Thus, it is very important to identify the molecular targets of compounds that induce a unique phenotype in a target cell. Phoslactomycin A (PLMA) is known to be a potent inhibitor of protein Ser/Thr phosphatase 2A (PP2A); however, the inhibitory mechanism of PP2A by PLMA has not yet been elucidated. Here, we demonstrated that PLMA directly binds to the PP2A catalytic subunit (PP2Ac) in cells by using biotinylated PLMA, and the PLMA-binding site was identified as the Cys-269 residue of PP2Ac. Moreover, we revealed that the Cys-269 contributes to the potent inhibition of PP2Ac activity by PLMA. These results suggest that PLMA is a PP2A-selective inhibitor and is therefore expected to be useful for future investigation of PP2A function in cells.     

CPK12: A Ca2+-dependent protein kinase balancer in abscisic acid signaling

Ca²⁺ is believed to be a critical second messenger in ABA signal transduction. Ca²⁺-dependent protein kinases (CDPKs) are the best characterized Ca²⁺ sensors in plants. Recently, we identified an Arabidopsis CDPK member CPK12 as a negative regulator of ABA signaling in seed germination and post-germination growth, which reveals that different members of the CDPK family may constitute a regulation loop by functioning positively and negatively in ABA signal transduction. We observed that both RNA interference and overexpression of CPK12 gene resulted in ABA-hypersensitive phenotypes in seed germination and post-germination growth, suggesting a high complexity of the CPK12-mediated ABA signaling pathway. CPK12 stimulates a negative ABA-signaling regulator (ABI2) and phosphorylates two positive ABA-signaling regulators (ABF1 and ABF4), which may partly explain the ABA hypersensitivity induced by both downregulation and upregulation of CPK12 expression. Our data indicate that CPK12 appears to function as a balancer in ABA signal transduction in Arabidopsis.     

Plasma membrane-associated proline-rich extensin-like receptor kinase 4, a novel regulator of Ca2+ signalling, is required for abscisic acid responses in Arabidopsis thaliana

Plant roots respond to environmental stresses or the exogenous plant hormone abscisic acid (ABA) by undergoing marked physiological and morphological changes. We show here that PERK4 , a gene that encodes a member of the Arabidopsis thaliana proline-rich extensin-like receptor kinase family, plays an important role in ABA responses. Mutation of PERK4 by T-DNA insertion decreased sensitivity to ABA with respect to seed germination, seedling growth and primary root tip growth. The effect on root growth was due to enhanced cell elongation rather than cell division. The cytosolic free calcium concentration and Ca²⁺ channel currents were lower in perk4 root cells than in wild-type cells in the presence of ABA. Root growth was similar in wild-type and perk4 plants after the application of a Ca²⁺ channel blocker. PERK4 localised to the plasma membrane, and was shown to be an ABA- and Ca²⁺-activated protein kinase. Our data suggest that the receptor-like kinase encoded by PERK4 functions at an early stage of ABA signalling to inhibit root cell elongation by perturbing Ca²⁺ homeostasis.     

Arabidopsis RNA-binding protein UBA2a relocalizes into nuclear speckles in response to abscisic acid

The Arabidopsis thaliana RNA binding protein UBA2a is the closest homologue of the Vicia faba AKIP1 (56% identity). Like AKIP1, UBA2a is a constitutively-expressed nuclear protein and in response to ABA it is also reorganized within the nucleus in "speckles" suggesting a possible role of this protein in the regulation of mRNA metabolism during ABA signaling. AKIP1 interacts with, and is phosphorylated by, the upstream ABA-activated protein kinase AAPK. We have investigated if a pathway similar to that described in Vicia faba also exists in Arabidopsis. Our results showed that despite the resemblance between the corresponding Vicia and Arabidopsis proteins, it appears that the function of UBA2a is independent of OST1 phosphorylation.     

Interaction between abscisic acid receptor PYL3 and protein phosphatase type 2C in response to ABA signaling in maize

Abscisic acid (ABA) is a ubiquitous hormone that regulates plant growth, development and responses to environmental stresses. In recent researches, pyrabactin resistance 1-like protein (PYL) and protein phosphatase type 2C (PP2C) were identified as the direct receptor and the second component of ABA signaling pathway, respectively. However, a lot of PYL and PP2C members were found in Arabidopsis and several other plants. Some of them were found not to be involved in ABA signaling. Because of the complex diversity of the genome, few documents have been available on the molecular details of the ABA signal perception system in maize. In the present study, we conducted bioinformatics analysis to find out the candidates (ZmPYL3 and ZmPP2C16) of the PYL and PP2C members most probably involved in ABA signaling in maize, cloned their encoding genes (ZmPYL3 and ZmPP2C16), verified the interaction between these two proteins in response to exogenous ABA induction by yeast two-hybrid assay and bimolecular fluorescence complementation, and investigated the expression patterns of these two genes under the induction of exogenous ABA by real-time fluorescence quantitative PCR. The results indicated that the ZmPYL3 and ZmPP2C16 proteins interacted in vitro and in vivo in response to the induction of exogenous ABA. The downregulated expression of the ZmPYL3 gene and the upregulated expression of the ZmPP2C16 gene are responsive to the induction of exogenous ABA. The ZmPYL3 and ZmPP2C16 proteins are the most probable members of the receptors and the second components of ABA signaling pathway, respectively.