New ABA-Hypersensitive Arabidopsis Mutants Are Affected in Loci Mediating Responses to Water Deficit and Dickeya dadantii Infection

Abstract

On water deficit, abscisic acid (ABA) induces stomata closure to reduce water loss by transpiration. To identify Arabidopsis thaliana mutants which transpire less on drought, infrared thermal imaging of leaf temperature has been used to screen for suppressors of an ABA-deficient mutant (aba3-1) cold-leaf phenotype. Three novel mutants, called hot ABA-deficiency suppressor (has), have been identified with hot-leaf phenotypes in the absence of the aba3 mutation. The defective genes imparted no apparent modification to ABA production on water deficit, were inherited recessively and enhanced ABA responses indicating that the proteins encoded are negative regulators of ABA signalling. All three mutants showed ABA-hypersensitive stomata closure and inhibition of root elongation with little modification of growth and development in non-stressed conditions. The has2 mutant also exhibited increased germination inhibition by ABA, while ABA-inducible gene expression was not modified on dehydration, indicating the mutated gene affects early ABA-signalling responses that do not modify transcript levels. In contrast, weak ABA-hypersensitivity relative to mutant developmental phenotypes suggests that HAS3 regulates drought responses by both ABA-dependent and independent pathways. has1 mutant phenotypes were only apparent on stress or ABA treatments, and included reduced water loss on rapid dehydration. The HAS1 locus thus has the required characteristics for a targeted approach to improving resistance to water deficit. In contrast to has2, has1 exhibited only minor changes in susceptibility to Dickeya dadantii despite similar ABA-hypersensitivity, indicating that crosstalk between ABA responses to this pathogen and drought stress can occur through more than one point in the signalling pathway.

Abscisic Acid Synthesis and Response

“…Negative regulators of ABA signaling identified in forward genetic screens include the enhanced response to ABA (ERA), ABA hypersensitive at germination (AHG and ABH1), supersensitive to ABA, drought and NaCl (SAD1), and hot ABA-deficiency suppressor (HAS) (Plessis et al, 2011) loci. In contrast to the PP2C-encoding AHG1 and AHG3, AHG2 (AT1G55870), AHG11 (AT2G44880), ABH1 (AT2G13540), and SAD1 (AT5G48870) all encode enzymes involved in RNA processing or degradation (Hugouvieux et al, 2001;Nishimura et al, 2005;Murayama et al, 2012) that could affect RNA accumulation at a post-transcriptional step.…”

PECTIN METHYLESTERASE INHIBITOR6 Promotes Arabidopsis Mucilage Release by Limiting Methylesterification of Homogalacturonan in Seed Coat Epidermal Cells

“…For amplification of PCR products from single-stranded cDNA from the wild type and pmei6-1 (Figures 1G and 1H), the following exon primers were used: PMEI6 CDS, forward primer 59-GTCCCAATTTCTTAAAAAGTTTGG-ATTCCTCCAATAAAGAACATGACTTC-39 and reverse primer 59-CATA-GCGTAGATTACAAGCCATCAAAAGCAAGCTTCTTGAGGAGAGCCAG-39; PMEI6 59, reverse primer 59-GCAAGAGCCTCAGCACCCGT-39; PMEI6 39, forward primer 59-GAGTAGAACACGGACGGTCA-39; EF1a4 primers were as described by North et al (2007). Quantitative real-time PCR reactions were performed as previously described using primers that had been tested for their efficiency rates and sensitivity on dilution series of cDNAs (Plessis et al, 2011). PMEI6 and seed reference gene At4g12590 (Dekkers et al, 2012) specific primers were as follows: PMEI6, forward primer, 59-GGCAGA-TAAGCGATCTCGCCAC-39; PMEI6, reverse primer 59-AGCCAGAGCATTG-CTGCATAGTC-39; At4g12590, forward primer 59-TGGCATTGACTTGAG-CACTGTCG-39; and At4g12590, reverse primer 59-TCGAGGTAGTGCC-CATTCGTGCT-39.…”

“…PMEI6 and seed reference gene At4g12590 (Dekkers et al, 2012) specific primers were as follows: PMEI6, forward primer, 59-GGCAGA-TAAGCGATCTCGCCAC-39; PMEI6, reverse primer 59-AGCCAGAGCATTG-CTGCATAGTC-39; At4g12590, forward primer 59-TGGCATTGACTTGAG-CACTGTCG-39; and At4g12590, reverse primer 59-TCGAGGTAGTGCC-CATTCGTGCT-39. EF1a4 primers were as described by Plessis et al (2011).…”

Inactivation of pecS restores the virulence of mutants devoid of osmoregulated periplasmic glucans in the phytopathogenic bacterium Dickeya dadantii

“…The essential resistance mechanisms required by D. dadantii to counteract the defence generated by plant hosts are resistance to acid, oxidative molecules, osmotic stress and antimicrobial peptides (Plessis et al, 2011). Resistance of the wild-type strain, the single opgG and pecS mutant strains, and the opgG pecS double mutant strain was compared under these various stress conditions.…”

“…Expression of these factors during pathogenesis is strictly controlled by a complex regulatory network required for adaptation, resulting in bacterial response to host injury after entry into the host. In particular, bacteria must respond to acid stress upon penetration of host cells, oxidative stress during invasion and osmotic stress during maceration (Plessis et al, 2011).…”