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

Abstract

SummaryHAB1 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 Pro HAB1 : green¯uorescent 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 cauli¯ower mosaic virus (CaMV) 35S promoter led to reduced ABA sensitivity both in seeds and vegetative tissues, compared to wild-type plants. Thus, in the ®eld 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.

ABA signalling is fine-tuned by antagonistic HAB1 variants

“…When germinated on a medium containing various concentrations of ABA, hab1-1 and hab1-1HAB1.1 plants were more sensitive or more tolerant to ABA than wild type during both the germination and greening stages, respectively (Fig. 2d,e), in accordance with previous findings 16 . In sharp contrast, the hab1-1HAB1.2pm plants exhibited greater ABA sensitivity than hab1-1 (Fig.…”

“…It appears that a high HAB1.2/HAB1.1 ratio is correlated with delayed seed germination and a post-germination developmental arrest; therefore, the HAB1.2 splice variant may facilitate the ABA-induced germination delay and post-germinative growth arrest. Thus, the role of HAB1.2 is clearly opposite to that of HAB1.1, which positively modulates early development in response to ABA 16 . Our genetic evidence that the overexpression of HAB1.1 and HAB1.2 in hab1-1 reduced or increased ABA sensitivity during seed germination and postgerminative development, respectively (Fig.…”

“…1c). It has been shown that HAB1 plays a key role in the post-germination developmental arrest induced by ABA 16 . To understand the role of HAB1 AS in early plant development, we examined the expression of the two variants and the effects of ABA on HAB1 AS.…”

“…Because HAB1.1 is a negative regulator of ABA signalling, and given that a loss of function or overexpression of HAB1.1 results in ABA-hypersensitive or -insensitive phenotypes during germination and post germination, respectively 16 , we investigated the function of HAB1.2 in the ABA response during early development. We generated transgenic plants overexpressing c-Myc-tagged recombinant HAB1.2 protein in a HAB1 loss-offunction mutant, hab1-1.…”

“…HAB1 is among the best-characterized PP2Cs in ABA signalling 15 . HAB1 is broadly expressed in various tissues and organs and is induced by ABA treatment 16 . HAB1 binds to and dephosphorylates SnRK2s, especially SnRK2.6 (OST1), and negatively regulates ABA signalling 17,18 .…”