Genome-Wide Analysis of the bZIP Gene Family Identifies Two ABI5-Like bZIP Transcription Factors, BrABI5a and BrABI5b, as Positive Modulators of ABA Signalling in Chinese Cabbage

Abstract

bZIP (basic leucine zipper) transcription factors coordinate plant growth and development and control responses to environmental stimuli. The genome of Chinese cabbage (Brassica rapa) encodes 136 putative bZIP transcription factors. The bZIP transcription factors in Brassica rapa (BrbZIP) are classified into 10 subfamilies. Phylogenetic relationship analysis reveals that subfamily A consists of 23 BrbZIPs. Two BrbZIPs within subfamily A, Bra005287 and Bra017251, display high similarity to ABI5 (ABA Insensitive 5). Expression of subfamily A BrbZIPs, like BrABI5a (Bra005287/BrbZIP14) and BrABI5b (Bra017251/BrbZIP13), are significantly induced by the plant hormone ABA. Subcellular localization assay reveal that both BrABI5a and BrABI5b have a nuclear localization. BrABI5a and BrABI5b could directly stimulate ABA Responsive Element-driven HIS (a HIS3 reporter gene, which confers His prototrophy) or LUC (LUCIFERASE) expression in yeast and Arabidopsis protoplast. Deletion of the bZIP motif abolished BrABI5a and BrABI5b transcriptional activity. The ABA insensitive phenotype of Arabidopsis abi5-1 is completely suppressed in transgenic lines expressing BrABI5a or BrABI5b. Overall, these results suggest that ABI5 orthologs, BrABI5a and BrABI5b, have key roles in ABA signalling in Chinese cabbage.

Fig 1. The phylogenetic tree of bZIP transcription factors between Chinese cabbage (Brassica rapa) and Arabidopsis.

The 136 BrbZIPs and 75 AtbZIPs protein sequences were aligned by the MUSCLE tool; and the maximum likelihood tree was generated using MEGA 5.0. The 10 distinct subfamilies were designated as A~S and labeled with different colored branches respectively.

Fig 2. Gene structure of the subfamily A AtbZIP and BrbZIP genes in Arabidopsis and Chinese cabbage (Brassica rapa).

Exon/intron organization of subfamily A AtbZIP and BrbZIP genes was depicted with the online Gene Structure Display Server. The exons and introns are represented by green boxes and blue lines respectively. The purple box denotes the bZIP domain region.

Fig 3. Protein architecture of subfamily A AtbZIP and BrbZIP proteins in Arabidopsis and Chinese cabbage (Brassica rapa).

The distribution of conserved motifs identified from 23 BrbZIP and 13 AtbZIP proteins of the subfamily A are predicted by the MEME (Multiple Em for Motif Elicitation) tool. Each motif is represented by a number in colored box. See S4 Table for detailed motif information.

Fig 4. Expression profile of subfamily A BrbZIP genes in Chinese cabbage (Brassica rapa) after ABA treatment.

qRT-PCR analysis of the expression pattern of subfamily A BrbZIP genes in response to ABA treatment. Eleven-day-old seedlings were treated with 0.1 mM ABA followed by sampling at 0, 4, 8, 12, 16, 20 and 24 h. The relative expression of the subfamily A BrbZIP genes was normalized to the expression of the cabbage ACTIN2 gene (BrACTIN2) and expressed relative to the level in mock-treated seedlings. The expression of Bra003254 after ABA treatment was also determined, which is adjacent to Bra003253/BrbZIP10 (only 1630 bp far from it) and encodes a putative DHHC zinc binding domain like proteins.

Fig 5. Domain structure and expression pattern of BrABI5a and BrABI5b.

(A) Schematic diagram of domains in the BrABI5a and BrABI5b protein. Three N-terminal and one C-terminal conserved sequences (C1, C2, C3 and C4) are shown in the green box, the basic domain is shown in the blue box, the bipartite nuclear localization signal is shown in the black brown rectangle and the Leu residues defining the Leu zipper are shown in the red rectangle, #, the conserved sumoylation site is shown in brown. (B) Exon/intron organization of BrABI5a and BrABI5b genes. The exons and introns are represented by boxes and lines respectively. (C-D) qRT-PCR analysis of the expression patterns of BrABI5a and BrABI5b under various environmental stress conditions. The relative expression of BrABI5a or BrABI5b was normalized to the expression of cabbage ACTIN2 (BrACTIN2) and expressed relative to the level in mock-treated seedlings.

Fig 6. Subcellular localization of BrABI5a and BrABI5b.

(A–C) The GFP fluorescence signal of BrABI5a-GFP. (D–F) The GFP fluorescence signal of BrABI5b-GFP. (A, D) Green fluorescence under dark field. (B, E) Cell morphology of the lower epidermis of a tobacco leaf under bright field. (C, F) Overlay of bright-field and green fluorescence signals. (G) The carboxyl-terminal sequence of BrABI5a and BrABI5b are similar to the NLS of the ABF2. The NLS-like motif is underlined and shown in light blue.

Fig 7. Transactivation activities of BrABI5a and BrABI5b.

(A) Yeast one-hybrid analysis of BrABI5a and BrABI5b. Yeast lines yWAM2 expressing the indicated plasmids were grown on synthetic complete medium without Leu and Trp (SC-LW; left) and on synthetic complete medium without Leu, Trp, and His (SC-HLW; right). Yeast cells were incubated until the optical density at 600 nm reached 0.5 and then diluted 2-fold (×2), 10-fold (×10), 50-fold (×50), or 250-fold (×250) and used for assays. (B) Transactivation activity BrABI5a or BrABI5b in Arabidopsis leaf mesophyll protoplasts. Transactivation experiments were performed using protoplasts prepared from Col-0 leaves. Transfected cells were cultured for 16 h without or with 5μM ABA, and relative LUC activity was assayed according to the Dual-Luciferase Reporter Assay Protocol provided by Promega. The empty vector control was also included as a negative control. The values shown are average fLUC (firefly, Photinus pyralis, LUC) activities normalized to rLUC (sea pansy, Renilla reniformis, LUC) activities. BrABI5aΔbZIP and BrABI5bΔbZIP are forms of BrABI5a and BrABI5b that carries a deletion of the intact C-terminal bZIP region respectively.

Fig 8. Heterogeneous expression of BrABI5a and BrABI5b reverse the insensitivity of Arabidopsis abi5-1 to ABA during seed germination.

(A, B) Sensitivity of seed germination to ABA. The seeds of Ws-2, abi5-1, and transgenic abi5-1 lines carrying Myc-tagged BrABI5a or BrABI5b (abi5-1::Myc-BrABI5a or abi5-1::Myc-BrABI5b) were germinated on MS medium (A) and MS medium supplemented with 3μM ABA (B) for the indicated days. The emergence rate of green cotyledons (C) and radicle (D) from Ws-2, abi5-1 and abi5-1::Myc-BrABI5a or abi5-1::Myc-BrABI5b transgenic seeds plated on MS supplemented with ABA. Approximately 150 seeds were used in each experiment. Error bars represent SD (seed number > 100). (E) Immunoblots of Myc-BrABI5a or Myc-BrABI5b protein levels in the transgenic abi5-1 lines (abi5-1::Myc-BrABI5a or abi5-1::Myc-BrABI5b). N, transgenic abi5-1 lines carrying the empty Myc-tagged vector; CBB (Coomassie Brilliant Blue) R250-stained RLS (Rubisco large subunit) served as a loading control.