Cloning and functional characterization of SAD genes in potato

Abstract

Stearoyl-acyl carrier protein desaturase (SAD), locating in the plastid stroma, is an important fatty acid biosynthetic enzyme in higher plants. SAD catalyzes desaturation of stearoyl-ACP to oleyl-ACP and plays a key role in determining the homeostasis between saturated fatty acids and unsaturated fatty acids, which is an important player in cold acclimation in plants. Here, four new full-length cDNA of SADs (ScoSAD, SaSAD, ScaSAD and StSAD) were cloned from four Solanum species, Solanum commersonii, S. acaule, S. cardiophyllum and S. tuberosum, respectively. The ORF of the four SADs were 1182 bp in length, encoding 393 amino acids. A sequence alignment indicated 13 amino acids varied among the SADs of three wild species. Further analysis showed that the freezing tolerance and cold acclimation capacity of S. commersonii are similar to S. acaule and their SAD amino acid sequences were identical but differed from that of S. cardiophyllum, which is sensitive to freezing. Furthermore, the sequence alignments between StSAD and ScoSAD indicated that only 7 different amino acids at residues were found in SAD of S. tuberosum (Zhongshu8) against the protein sequence of ScoSAD. A phylogenetic analysis showed the three wild potato species had the closest genetic relationship with the SAD of S. lycopersicum and Nicotiana tomentosiformis but not S. tuberosum. The SAD gene from S. commersonii (ScoSAD) was cloned into multiple sites of the pBI121 plant binary vector and transformed into the cultivated potato variety Zhongshu 8. A freeze tolerance analysis showed overexpression of the ScoSAD gene in transgenic plants significantly enhanced freeze tolerance in cv. Zhongshu 8 and increased their linoleic acid content, suggesting that linoleic acid likely plays a key role in improving freeze tolerance in potato plants. This study provided some new insights into how SAD regulates in the freezing tolerance and cold acclimation in potato.

Fig 1. Alignment amino acid sequences of stearoyl-acyl carrier protein desaturases (SAD) of different potato species, including S. acaule, S. cardiophyllum and S. commersonii (A), S. commersonii and S.tuberosum, cv Zhongshu8 (B).

Red arrow indicates the putative cleavage site of the chloroplast-transit peptide, and blue underlined sequence indicates the conserved iron-binding sites of the catalytic domain. The acyl-ACP desaturase and ferritin-like family domains are underlined in black and red, respectively.

Fig 2. Neighbor-joining tree of the stearoyl-acyl carrier protein desaturase (SAD) amino acid sequences of 10 species.

Fig 3. Process of buds induction from potato tuber dish A: Nov.26th, 2012; B: Dec.1th, 2012; C: Dec.5th, 2012; D: Dec.10th, 2012.

Fig 4. The pBI121 binary vector (A) and Southern blots of transgenic plants harboring the stearoyl-acyl carrier protein desaturase gene from S. commersonii (ScoSAD) (B).

PCK, pBI121:ScoSAD plasmid; NCK, wild-type Zhongshu8 plant; T13-T33, ScoSAD-overexpressing lines.

Fig 5. Expression of the stearoyl-acyl carrier protein desaturase (SAD) gene in four potato genotypes following cold acclimation.

Fig 6. Chromatogram of fatty acid methyl ester products in potato leaves.