ABSTRACT
ABF transcription factors are the key regulators of ABA signaling. Using RACE-PCR, we identified and sequenced the coding regions of four genes that encode ABF transcription factors in the extremophile plant Thellungiella salsuginea, a close relative of Arabidopsis thaliana that possesses high tolerance to abiotic stresses. An analysis of the deduced amino acid sequences revealed that the similarity between Thellungiella and Arabidopsis ABFs ranged from 71% to 88%. Similar to their Arabidopsis counterparts, Thellungiella ABFs share a bZIP domain and four conservative domains, including a highly conservative motif at the C-terminal tail, which was reported to be a canonical site for binding by 14-3-3 regulatory proteins. Gene expression analysis by real-time PCR revealed a rapid transcript induction of three of the ABF genes in response to salt stress. To check whether Thellungiella ABF transcription factors can interact with abundant 14-3-3 proteins, multiple constructs were designed, and yeast two-hybrid experiments were conducted. Six of the eight tested Ts14-3-3 proteins were able to bind the TsABFs in an isoform-specific manner. A serine-to-alanine substitution in the putative 14-3-3 binding motif resulted in the complete loss of interaction between the 14-3-3 proteins and the ABFs. The role of 14-3-3 interaction with ABFs in the salt and ABA signaling pathways is discussed in the context of Thellungiella survivability.
Subject(s)
14-3-3 Proteins/metabolism , Abscisic Acid/genetics , Arabidopsis Proteins/genetics , Basic-Leucine Zipper Transcription Factors/genetics , Brassicaceae/genetics , Gene Expression Regulation, Plant , Genes, Plant , Transcription Factors/genetics , Abscisic Acid/metabolism , Amino Acid Sequence , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/metabolism , Basic-Leucine Zipper Transcription Factors/metabolism , Brassicaceae/metabolism , Molecular Sequence Data , Molecular Structure , Protein Binding , Protein Isoforms , Salt Tolerance/genetics , Signal Transduction , Transcription Factors/metabolismABSTRACT
BACKGROUND: Thellungiella has been proposed as an extremophile alternative to Arabidopsis to investigate environmental stress tolerance. However, Arabidopsis accessions show large natural variation in their freezing tolerance and here the tolerance ranges of collections of accessions in the two species were compared. RESULTS: Leaf freezing tolerance of 16 Thellungiella accessions was assessed with an electrolyte leakage assay before and after 14 days of cold acclimation at 4°C. Soluble sugars (glucose, fructose, sucrose, raffinose) and free polyamines (putrescine, spermidine, spermine) were quantified by HPLC, proline photometrically. The ranges in nonacclimated freezing tolerance completely overlapped between Arabidopsis and Thellungiella. After cold acclimation, some Thellungiella accessions were more freezing tolerant than any Arabidopsis accessions. Acclimated freezing tolerance was correlated with sucrose levels in both species, but raffinose accumulation was lower in Thellungiella and only correlated with freezing tolerance in Arabidopsis. The reverse was true for leaf proline contents. Polyamine levels were generally similar between the species. Only spermine content was higher in nonacclimated Thellungiella plants, but decreased during acclimation and was negatively correlated with freezing tolerance. CONCLUSION: Thellungiella is not an extremophile with regard to freezing tolerance, but some accessions significantly expand the range present in Arabidopsis. The metabolite data indicate different metabolic adaptation strategies between the species.
