The Arabidopsis spliceosomal protein SmEb modulates ABA responses by maintaining proper alternative splicing of HAB1

Abscisic acid (ABA) signaling is critical for seed germination and abiotic stress responses in terrestrial plants. Pre-mRNA splicing is known to regulate ABA signaling. However, the involvement of canonical spliceosomal components in regulating ABA signaling is poorly understood. Here, we show that the spliceosome component Sm core protein SmEb plays an important role in ABA signaling. SmEb expression is up-regulated by ABA treatment, and analysis of Arabidopsis smeb mutant plants suggest that SmEb modulates the alternative splicing of the ABA signaling component HAB1 by enhancing the HAB1.1 splicing variant while repressing HAB1.2. Overexpression of HAB1.1 but not HAB1.2 rescues the ABA-hypersensitive phenotype of smeb mutants. Mutations in the transcription factor ABI3, 4, or 5 also reduce the ABA hypersensitivity of smeb mutants during seed germination. Our results show that the spliceosomal component SmEb plays an important role in ABA regulation of seed germination and early seedling development.

The PHD finger of Arabidopsis SIZ1 recognizes trimethylated histone H3K4 mediating SIZ1 function and abiotic stress response

Arabidopsis SIZ1 encodes a SUMO E3 ligase to regulate abiotic and biotic stress responses. Among SIZ1 or mammalian PIAS orthologs, plant SIZ1 proteins contain the plant homeodomain (PHD) finger, a C4HC3 zinc finger. Here, we investigated the importance of PHD of Arabidopsis SIZ1. The ProSIZ1::SIZ1(ΔPHD):GFP was unable to complement growth retardation, ABA hypersensitivity, and the cold-sensitive phenotype of the siz1 mutant, but ProSIZ1::SIZ1:GFP could. Substitution of C162S in the PHD finger was unable to complement the siz1 mutation. Tri-methylated histone H3K4 (H3K4me3) was recognized by PHD, not by PHD(C162S). WRKY70 was up-regulated in the siz1-2 mutant and H3K4me3 accumulated at high levels in the WRKY70 promoter. PHD interacts with ATX, which mediates methylation of histone, probably leading to suppression of ATX’s function. These results suggest that the PHD finger of SIZ1 is important for recognition of the histone code and is required for SIZ1 function and transcriptional suppression.

A Stress-Responsive NAC Transcription Factor from Tiger Lily (LlNAC2) Interacts with LlDREB1 and LlZHFD4 and Enhances Various Abiotic Stress Tolerance in Arabidopsis

Our previous studies have indicated that a partial NAC domain protein gene is strongly up-regulated by cold stress (4 °C) in tiger lily (Lilium lancifolium). In this study, we cloned the full-length of this NAC gene, LlNAC2, to further investigate the function of LlNAC2 in response to various abiotic stresses and the possible involvement in stress tolerance of the tiger lily plant. LlNAC2 was noticeably induced by cold, drought, salt stresses, and abscisic acid (ABA) treatment. Promoter analysis showed that various stress-related cis-acting regulatory elements were located in the promoter of LlNAC2; and the promoter was sufficient to enhance activity of GUS protein under cold, salt stresses and ABA treatment. DREB1 (dehydration-responsive binding protein1) from tiger lily (LlDREB1) was proved to be able to bind to the promoter of LlNAC2 by yeast one-hybrid (Y1H) assay. LlNAC2 was shown to physically interact with LlDREB1 and zinc finger-homeodomain ZFHD4 from the tiger lily (LlZFHD4) by bimolecular fluorescence complementation (BiFC) assay. Overexpressing LlNAC2 in Arabidopsis thaliana showed ABA hypersensitivity and enhanced tolerance to cold, drought, and salt stresses. These findings indicated LlNAC2 is involved in both DREB/CBF-COR and ABA signaling pathways to regulate stress tolerance of the tiger lily.

The Arabidopsis thaliana NAC transcription factor family: structure–function relationships and determinants of ANAC019 stress signalling

TFs (transcription factors) are modular proteins minimally containing a DBD (DNA-binding domain) and a TRD (transcription regulatory domain). NAC [for NAM (no apical meristem), ATAF, CUC (cup-shaped cotyledon)] proteins comprise one of the largest plant TF families. They are key regulators of stress perception and developmental programmes, and most share an N-terminal NAC domain. On the basis of analyses of gene expression data and the phylogeny of Arabidopsis thaliana NAC TFs we systematically decipher structural and functional specificities of the conserved NAC domains and the divergent C-termini. Nine of the ten NAC domains analysed bind a previously identified conserved DNA target sequence with a CGT[GA] core, although with different affinities. Likewise, all but one of the NAC proteins analysed is dependent on the C-terminal region for transactivational activity. In silico analyses show that the NAC TRDs contain group-specific sequence motifs and are characterized by a high degree of intrinsic disorder. Furthermore, ANAC019 was identified as a new positive regulator of ABA (abscisic acid) signalling, conferring ABA hypersensitivity when ectopically expressed in plants. Interestingly, ectopic expression of the ANAC019 DBD or TRD alone also resulted in ABA hypersensitivity. Expression of stress-responsive marker genes [COR47 (cold-responsive 47), RD29b (responsive-to-desiccation 29b) and ERD11 (early-responsive-to-dehydration 11)] were also induced by full-length and truncated ANAC019. Domain-swapping experiments were used to analyse the specificity of this function. Chimaeric proteins, where the NAC domain of ANAC019 was replaced with the analogous regions from other NAC TFs, also have the ability to positively regulate ABA signalling. In contrast, replacing the ANAC019 TRD with other TRDs abolished ANAC019-mediated ABA hypersensitivity. In conclusion, our results demonstrate that the biochemical and functional specificity of NAC TFs is associated with both the DBDs and the TRDs.