Genome-wide analysis of fatty acid desaturase genes in chia (Salvia hispanica) reveals their crucial roles in cold response and seed oil formation.

Chia (Salvia hispanica) is a functional food crop with high α-linolenic acid (ALA), the omega-3 essential fatty acid, but its worldwide plantation is limited by cold-intolerance and strict short-photoperiod flowering feature. Fatty acid desaturases (FADs) are responsible for seed oil accumulation, and play important roles in cold stress tolerance of plants. To date, there is no report on systemically genome-wide analysis of FAD genes in chia (ShiFADs). In this study, 31 ShiFAD genes were identified, 3 of which contained 2 alternative splicing transcripts, and they were located in 6 chromosomes of chia. Phylogenetic analysis classified the ShiFAD proteins into 7 groups, with conserved gene structure and MEME motifs within each group. Tandem and segmental duplications coursed the expansion of ShiFAD genes. Numerous cis-regulatory elements, including hormone response elements, growth and development elements, biotic/abiotic stress response elements, and transcription factor binding sites, were predicted in ShiFAD promoters. 24 miRNAs targeting ShiFAD genes were identified at whole-genome level. In total, 15 SSR loci were predicted in ShiFAD genes/promoters. RNA-seq data showed that ShiFAD genes were expressed in various organs with different levels. qRT-PCR detection revealed the inducibility of ShiSAD2 and ShiSAD7 in response to cold stress, and validated the seed-specific expression of ShiSAD11a. Yeast expression of ShiSAD11a confirmed the catalytic activity of its encoded protein, and its heterologous expression in Arabidopsis thaliana significantly increased seed oleic acid content. This work lays a foundation for molecular dissection of chia high-ALA trait and functional study of ShiFAD genes in cold tolerance.

MYB43 in Oilseed Rape (Brassica napus) Positively Regulates Vascular Lignification, Plant Morphology and Yield Potential but Negatively Affects Resistance to Sclerotinia sclerotiorum.

Arabidopsis thaliana MYB43 (AtMYB43) is suggested to be involved in cell wall lignification. PtrMYB152, the Populus orthologue of AtMYB43, is a transcriptional activator of lignin biosynthesis and vessel wall deposition. In this research, MYB43 genes from Brassica napus (rapeseed) and its parental species B. rapa and B. oleracea were molecularly characterized, which were dominantly expressed in stem and other vascular organs and showed responsiveness to Sclerotinia sclerotiorum infection. The BnMYB43 family was silenced by RNAi, and the transgenic rapeseed lines showed retardation in growth and development with smaller organs, reduced lodging resistance, fewer silique number and lower yield potential. The thickness of the xylem layer decreased by 28%; the numbers of sclerenchymatous cells, vessels, interfascicular fibers, sieve tubes and pith cells in the whole cross section of the stem decreased by 28%, 59%, 48%, 34% and 21% in these lines, respectively. The contents of cellulose and lignin decreased by 17.49% and 16.21% respectively, while the pectin content increased by 71.92% in stems of RNAi lines. When inoculated with S. sclerotiorum, the lesion length was drastically decreased by 52.10% in the stems of transgenic plants compared with WT, implying great increase in disease resistance. Correspondingly, changes in the gene expression patterns of lignin biosynthesis, cellulose biosynthesis, pectin biosynthesis, cell cycle, SA- and JA-signals, and defensive pathways were in accordance with above phenotypic modifications. These results show that BnMYB43, being a growth-defense trade-off participant, positively regulates vascular lignification, plant morphology and yield potential, but negatively affects resistance to S. sclerotiorum. Moreover, this lignification activator influences cell biogenesis of both lignified and non-lignified tissues of the whole vascular organ.

Cloning and characterization of phosphorus starvation inducible Brassica napus PURPLE ACID PHOSPHATASE 12 gene family, and imprinting of a recently evolved MITE-minisatellite twin structure.

Purple acid phosphatase (PAP) is important for phosphorus assimilation and in planta redistribution. In this study, seven Brassica napus PAP12 (BnPAP12) genes orthologous to Arabidopsis thaliana PAP12 (AtPAP12) are isolated and characterized. NCBI BLASTs, multi-alignments, conserved domain prediction, and featured motif/residue characterization indicate that all BnPAP12 members encode dimeric high molecular weight plant PAPs. BnPAP12-1, BnPAP12-2, BnPAP12-3 and BnPAP12-7 (Group I) have six introns and encode 469-aa polypeptides structurally comparable to AtPAP12. BnPAP12-4 and BnPAP12-6 (Group II) have seven introns and encode 526-aa PAP12s. Encoding a 475-aa polypeptide, BnPAP12-5 (Group III) is evolved from a chimera of 5' part of Group I and 3' part of Group II. Sequence characterization and Southern detection suggest that there are about five BnPAP12 alleles. Homoeologous non-allelic fragment exchanges exist among BnPAP12 genes. BnPAP12-4 and BnPAP12-6 are imprinted with a Tourist-like miniature inverted-repeat transposable element (MITE) which is tightly associated with a novel minisatellite composed of four 36-bp tandem repeats. Existing solely in B. rapa/oleracea lineage, this recently evolved MITE-minisatellite twin structure does not impair transcription and coding capacity of the imprinted genes, and could be used to identify close relatives of B. rapa/oleracea lineage within Brassica. It is also useful for studying MITE activities especially possible involvement in minisatellite formation and gene structure evolution. BnPAP12-6 is silent in transcription. All other BnPAP12 genes basically imitate AtPAP12 in tissue specificity and Pi-starvation induced expression pattern, but divergence and complementation are distinct among them. Alternative polyadenylation and intron retention also exist in BnPAP12 mRNAs.

Molecular cloning and characterization of a novel stem-specific gene from Camptotheca acuminata.

In higher plants, P450s participate in the biosynthesis of many important secondary metabolites. Here we reported for the first time the isolation of a new cytochrome P450 cDNA that expressed in a stem-specific manner from Camptotheca acuminata (designated as CaSS), a native medicinal plant species in China, using RACE-PCR. The full-length cDNA of CaSS was 1735 bp long containing a 1530 bp open reading frame (ORF) encoding a polypeptide of 509 amino acids. Bioinformatic analysis revealed that CASS contained a heme-binding domain PFGXGRRXCX and showed homology to other plant cytochrome P450 monooxygenases and hydroxylases. Southern blotting analysis revealed that there was only one copy of the CaSS present in the genome of Camptotheca acuminata. Northern blotting analysis revealed that CaSS expressed, in a tissuespecific manner, highly in stem and lowly in root, leaf and flower. Our study suggests that CaSS is likely to be involved in the phenylpropanoid pathway.

CDNA cloning and characterization of the Ve homologue gene StVe from Solanum torvum Swartz.

Verticillium wilt is a disastrous disease causing significant yield losses of many crops. Isolation of verticillium wilt resistance gene is a fundamental work for controlling this disease through genetic engineering. In this report, we describe the cloning and characterization of a Ve like gene (StVe) from Solanum torvum Swartz. The nucleotide sequence of StVe is 3640 bp long with an open reading frame of 3414 bp encoding a protein precursor of 1138 aa. Sharing high homologies to tomato verticillium wilt disease resistance genes Ve1 and Ve2, the leucine rich (15.89%) protein StVe has a calculated molecular weight of 126.48kDa with an isoelectric point of 5.62. It possesses a hydrophobic N-terminal signal peptide of 20 aa and 38 predicted leucine-rich repeats containing 32 potential N-glycosylation sites (28 being significant). Fifty-seven predicted phosphorylation sites (36 for S, 8 for T and 13 for Y) distribute in StVe protein. A PEST-like sequence and a mammalian endocytosis signals YCVF are found within the C-terminal region. The C terminus of StVe concludes with the residues KKF similar to the KKX motif that confers endoplasmic reticulum localization in plants as well as mammals and yeast. The sequence analysis of the StVe gene implies that the StVe is a potential verticillium wilt disease resistance gene encoding a cell surface-like receptor protein.

Engineering tropane biosynthetic pathway in Hyoscyamus niger hairy root cultures.

Scopolamine is a pharmaceutically important tropane alkaloid extensively used as an anticholinergic agent. Here, we report the simultaneous introduction and overexpression of genes encoding the rate-limiting upstream enzyme putrescine N-methyltransferase (PMT) and the downstream enzyme hyoscyamine 6 beta-hydroxylase (H6H) of scopolamine biosynthesis in transgenic henbane (Hyoscyamus niger) hairy root cultures. Transgenic hairy root lines expressing both pmt and h6h produced significantly higher (P < 0.05) levels of scopolamine compared with the wild-type and transgenic lines harboring a single gene (pmt or h6h). The best line (T(3)) produced 411 mg/liter scopolamine, which was over nine times more than that in the wild type (43 mg/liter) and more than twice the amount in the highest scopolamine-producing h6h single-gene transgenic line H(11) (184 mg/liter). To our knowledge, this is the highest scopolamine content achieved through genetic engineering of a plant. We conclude that transgenic plants harboring both pmt and h6h possessed an increased flux in the tropane alkaloid biosynthetic pathway that enhanced scopolamine yield, which was more efficient than plants harboring only one of the two genes. It seems that the pulling force of the downstream enzyme (the faucet enzyme) H6H plays a more important role in stimulating scopolamine accumulation in H. niger whereas the functioning of the upstream enzyme PMT is increased proportionally. This study provides an effective approach for large-scale commercial production of scopolamine by using hairy root culture systems as bioreactors.

Molecular cloning and characterization of a mannose-binding lectin gene from Crinum asiaticum.

Full-length cDNA of a mannose-binding lectin or agglutinin gene was cloned from a traditional Chinese medicinal herb Crinum asiaticum var. sinicum through RACE-PCR cloning. The full-length cDNA of C. asiaticum agglutinin (caa) was 820 bp and contained a 528 bp open reading frame encoding a lectin precursor (preproprotein) of 175 amino acid residues with a 22 aa signal peptide. The coding region of the caa gene was high in G/C content. The first 20 bp of the 5' UTR had a dC content of 50%, which was a typical feature of the leader sequence. By cutting away the signal peptide, the CAA proprotein was 15.79 kDa with a pl of 9.27 and contained 3 mannose-binding sites (QDNY). Random coil and extended strand constituted interlaced domination of the main part of the secondary structure. B-lectin conserved domain existed within N24 to G130. Predicted three-dimensional structure of CAA proprotein was very similar to that of GNA (Galanthus nivalis agglutinin). It is significant that besides certain homologies to known monocot mannose-binding lectins from Amaryllidaceae, Orchidaceae, Alliaceae and Liliaceae, caa also showed high similarity to gastrodianin type antifungal proteins. No intron was detected within the region of genomic sequence corresponding to the caa full-length cDNA. Southern blot analysis indicated that the caa gene belonged to a low-copy gene family. Northern blot analysis demonstrated that caa mRNA was constitutively expressed in all the tested tissue types including the root, bulb, leaf, rachise, flower and fruit tissues.

Molecular cloning of a potential Verticillium dahliae resistance gene SlVe1 with multi-site polyadenylation from Solanum licopersicoides.

Caused by Verticillium spp. pathogens, verticillium wilt is a common detrimental disease damaging yield production of many important crops. Isolation of verticillium wilt resistance genes and their transgenic application is a fundamental way to control this disease. Here we report the cloning and sequence characterization of a potential Verticillium dahliae Kleb. resistance gene (Ve) from Solanum lycopersicoides Dun. (designated as SlVe1). The nucleotide sequence of SlVe1 is 3400 bp with an ORF of 3156 bp encoding a protein precursor of 1051 amino acids (aa). Unlike tomato Ve1, SlVe1 had a short leader sequence of 22 bp. Multiple polyadenylation sites were detected, which may result from alternative cleavages directed by the common polyadenylation signal AATAAA, and nucleotide sequences of the cleavage sites for polyadenylation conform to PyPyA. Sharing high homologies to tomato verticillium wilt disease resistance genes Ve1 and Ve2, SlVe1 encoded a cell-surface glycoprotein with receptor-mediated endocytosis-like signal. The leucine rich (16.51%) putative SlVe1 protein had a calculated molecular weight of 116.97 kDa with an isoelectric point of 5.22. It possessed a hydrophobic N-terminal signal peptide of 23 aa and 28 predicted significant leucine-rich repeats (LRRs) containing 29 potential N-glycosylation sites (18 being significant). A membrane-associated hydrophobic domain resided within the C-terminal, flanked by a neutral/acidic aa rich domain and a neutral/basic aa rich domain. Forty-four predicted phosphorylation sites (28 for S, 5 for T and 11 for Y) distributed in SlVe1, and an endocytosis signal EKWLLW resided in the neutral/basic aa rich C-terminal domain. As compared with Ve1, several clues of variations have been detected in SlVe1 and their possible implications are discussed.