Analysis of carotenogenic genes promoters and WRKY transcription factors in response to salt stress in Dunaliella bardawil

Abstract

The unicellular alga Dunaliella bardawil is a highly salt-tolerant organism, capable of accumulating glycerol, glycine betaine and β-carotene under salt stress, and has been considered as an excellent model organism to investigate the molecular mechanisms of salt stress responses. In this study, several carotenogenic genes (DbCRTISO, DbZISO, DbLycE and DbChyB), DbBADH genes involved in glycine betaine synthesis and genes encoding probable WRKY transcription factors from D. bardawil were isolated, and promoters of DbCRTISO and DbChyB were cloned. The promoters of DbPSY, DbLycB, DbGGPS, DbCRTISO and DbChyB contained the salt-regulated element (SRE), GT1GMSCAM4, while the DbGGPS promoter has another SRE, DRECRTCOREAT. All promoters of the carotenogenic genes had light-regulated elements and W-box cis-acting elements. Most WRKY transcription factors can bind to the W-box, and play roles in abiotic stress. qRT-PCR analysis showed that salt stress up-regulated both carotenogenic genes and WRKY transcription factors. In contrast, the transcription levels of DbBADH showed minor changes. In D. bardawil, it appears that carotenoid over-accumulation allows for the long-term adaptation to salt stress, while the rapid modulation of glycine betaine biosynthesis provides an initial response.

Figure 1. Conserved domain analysis and alignment of four probable WRKY transcription factors from D. bardawil.

(A) Conserved domain analysis of four probable WRKY transcription factors from D. bardawil. (B) Alignment of the four WRKY protein domains from D. bardawil by ClustalX2. The WRKY motif is highlighted in a red frame and the cysteines and histidines that form the zinc-finger structures are shown in red triangles.

Figure 2. Isolation of promoters of DbCRTISO and DbChyB in D. bardawil.

(A) Promoter isolation of DbCRTISO by genome walking. (B) Promoter isolation of DbChyB by genome walking. Lane M, 500 bp DNA ladder marker; lanes 1-3, products of 1st PCR, 2nd nested PCR, and 3rd nested PCR, respectively.

Figure 3. Distribution of exons and introns in the genomic DNA of CRTISOs and ChyBs.

(A) Distribution of exons and introns in the genomic DNA of CRTISOs. The sequences used for analysis are as follows: CRTISO from green algae: DbCRTISO (in this study); CrCRTISO, Chlamydomonas reinhardtii (NW_001843787.1); CsCRTISO, Coccomyxa subellipsoidea C-169 (NW_005178040.1); CRTISO from diatoms: TpCRTISO, Thalassiosira pseudonana (NC_012070.1); PtCRTISO, Phaeodactylum tricornutum (NC_011675.1); CRTISO from red algae: GsCRTISO, Galdieria sulphuraria (NW_005178468.1); CmCRTISO, Cyanidioschyzon merolae (NC_010140.1); CRTISO from higher plants: AtCRTISO, Arabidopsis thaliana (NC_003070.9); ZmCRTISO, Zea mays (NC_024462.1). (B) Distribution of exons and introns in the genomic DNA of ChyBs. The sequences used for analysis are as follows: ChyB from green algae: DbChyB (in this study); CrChyB, Chlamydomonas reinhardtii (NW_001843792.1); BpChyB, Bathycoccus prasinos (NC_023998.1); MpChyB, Micromonas pusilla CCMP1545 (NW_003315882.1); VcChyB, Volvox carteri f. nagariensis (NW_003307618.1); ChyB from higher plants: AtChyB, Arabidopsis thaliana (NW_003302549.1); CsChyB, Citrus sinensis cultivar Valencia (NC_023054.1); SlChyB, Solanum lycopersicum cultivar Heinz 1706 (NC_015440.2); ZmChyB, Zea mays (NC_024468.1).

Figure 4. qRT-PCR analysis of genes under salt stress in D. bardawil.

(A) qRT-PCR analysis of the transcription levels of genes involved in carotenoid biosynthesis. (B) qRT-PCR analysis of the transcription levels of genes involved in glycine betaine biosynthesis and encoding WRKY transcription factors.