An Arabidopsis homolog of importin β1 is required for ABA response and drought tolerance

The import of proteins into the nucleus in response to drought is critical for mediating the reprogramming of gene expression that leads to drought tolerance. However, regulatory mechanisms involved in nuclear protein import remain largely unknown. Here, we have identified an Arabidopsis gene (AtKPNB1) as a homolog of human KPNB1 (importin β1). AtKPNB1 was expressed in multiple organs, and the protein was localized in the cytoplasm and nucleus. AtKPNB1 was able to facilitate nuclear import of a model protein. Null mutation of AtKPNB1 delayed development under normal growth conditions and increased sensitivity to abscisic acid (ABA) during seed germination and cotyledon development. Inactivation of AtKPNB1 increased stomatal closure in response to ABA, reduced the rate of water loss, and substantially enhanced drought tolerance. AtKPNB1 interacted with several importin α proteins, nucleoporin AtNUP62, and the Arabidopsis Ran proteins. Inactivation of AtKPNB1 did not affect the ABA responsiveness or the expression level or subcellular localization of ABI1, ABI2 or ABI5, key regulators of the ABA signaling pathway. Moreover, phenotypic analysis of epistasis revealed that AtKPNB1 modulates the ABA response and drought tolerance through a pathway that is independent of ABI1 and ABI5. Collectively, our results show that AtKPNB1 is an Arabidopsis importin β that functions in ABA signaling.     

Interaction between sugar and abscisic acid signalling during early seedling development in Arabidopsis

Sugars regulate important processes and affect the expression of many genes in plants. Characterization of Arabidopsis (Arabidopsis thaliana) mutants with altered sugar sensitivity revealed the function of abscisic acid (ABA) signalling in sugar responses. However, the exact interaction between sugar signalling and ABA is obscure. Therefore ABA deficient plants with constitutive ABI4 expression (aba2-1/35S::ABI4) were generated. Enhanced ABI4 expression did not rescue the glucose insensitive (gin) phenotype of aba2 seedlings indicating that other ABA regulated factors are essential as well. Interestingly, both glucose and ABA treatment of Arabidopsis seeds trigger a post-germination seedling developmental arrest. The glucose-arrested seedlings had a drought tolerant phenotype and showed glucose-induced expression of ABSCISIC ACID INSENSITIVE3 (ABI3), ABI5 and LATE EMBRYOGENESIS ABUNDANT (LEA) genes reminiscent of ABA signalling during early seedling development. ABI3 is a key regulator of the ABA-induced arrest and it is shown here that ABI3 functions in glucose signalling as well. Multiple abi3 alleles have a glucose insensitive (gin) phenotype comparable to that of other known gin mutants. Importantly, glucose-regulated gene expression is disturbed in the abi3 background. Moreover, abi3 was insensitive to sugars during germination and showed sugar insensitive (sis) and sucrose uncoupled (sun) phenotypes. Mutant analysis further identified the ABA response pathway genes ENHANCED RESPONSE TO ABA1 (ERA1) and ABI2 as intermediates in glucose signalling. Hence, three previously unidentified sugar signalling genes have been identified, showing that ABA and glucose signalling overlap to a larger extend than originally thought. Bas J. W. Dekkers and Jolanda A. M. J. Schuurmans contributed equally to this paper.     

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.     

Comparative genomics and regulatory evolution: conservation and function of the Chs and Apetala3 promoters

DNA sequence variations of chalcone synthase (Chs) and Apetala3 gene promoters from 22 cruciferous plant species were analyzed to identify putative conserved regulatory elements. Our comparative approach confirmed the existence of numerous conserved sequences which may act as regulatory elements in both investigated promoters. To confirm the correct identification of a well-conserved UV-light-responsive promoter region, a subset of Chs promoter fragments were tested in Arabidopsis thaliana protoplasts. All promoters displayed similar light responsivenesses, indicating the general functional relevance of the conserved regulatory element. In addition to known regulatory elements, other highly conserved regions were detected which are likely to be of functional importance. Phylogenetic trees based on DNA sequences from both promoters (gene trees) were compared with the hypothesized phylogenetic relationships (species trees) of these taxa. The data derived from both promoter sequences were congruent with the phylogenies obtained from coding regions of other nuclear genes and from chloroplast DNA sequences. This indicates that promoter sequence evolution generally is reflective of species phylogeny. Our study also demonstrates the great value of comparative genomics and phylogenetics as a basis for functional analysis of promoter action and gene regulation.