Structure and expression of GSL1 and GSL2 genes encoding gibberellin stimulated-like proteins in diploid and highly heterozygous tetraploid potato reveals their highly conserved and essential status.

BACKGROUND: GSL1 and GSL2, Gibberellin Stimulated-Like proteins (also known as Snakin-1 and Snakin-2), are cysteine-rich peptides from potato (Solanum tuberosum L.) with antimicrobial properties. Similar peptides in other species have been implicated in diverse biological processes and are hypothesised to play a role in several aspects of plant development, plant responses to biotic or abiotic stress through their participation in hormone crosstalk, and redox homeostasis. To help resolve the biological roles of GSL1 and GSL2 peptides we have undertaken an in depth analysis of the structure and expression of these genes in potato. RESULTS: We have characterised the full length genes for both GSL1 (chromosome 4) and GSL2 (chromosome 1) from diploid and tetraploid potato using the reference genome sequence of potato, coupled with further next generation sequencing of four highly heterozygous tetraploid cultivars. The frequency of SNPs in GSL1 and GSL2 were very low with only one SNP every 67 and 53 nucleotides in exon regions of GSL1 and GSL2, respectively. Analysis of comprehensive RNA-seq data substantiated the role of specific promoter motifs in transcriptional control of gene expression. Expression analysis based on the frequency of next generation sequence reads established that GSL2 was expressed at a higher level than GSL1 in 30 out of 32 tissue and treatment libraries. Furthermore, both the GSL1 and GSL2 genes exhibited constitutive expression that was not up regulated in response to biotic or abiotic stresses, hormone treatments or wounding. Potato transformation with antisense knock-down expression cassettes failed to recover viable plants. CONCLUSIONS: The potato GSL1 and GSL2 genes are very highly conserved suggesting they contribute to an important biological function. The known antimicrobial activity of the GSL proteins, coupled with the FPKM analysis from RNA-seq data, implies that both genes contribute to the constitutive defence barriers in potatoes. The lethality of antisense knock-down expression of GSL1 and GSL2, coupled with the rare incidence of SNPs in these genes, suggests an essential role for this gene family. These features are consistent with the GSL protein family playing a role in several aspects of plant development in addition to plant defence against biotic stresses.

Inhibition of platelet activation by lachrymatory factor synthase (LFS)-silenced (tearless) onion juice.

Onion and garlic are renowned for their roles as functional foods. The health benefits of garlic are attributed to di-2-propenyl thiosulfinate (allicin), a sulfur compound found in disrupted garlic but not found in disrupted onion. Recently, onions have been grown with repressed lachrymatory factor synthase (LFS) activity, which causes these onions to produce increased amounts of di-1-propenyl thiosulfinate, an isomer of allicin. This investigation into the key health attributes of LFS-silenced (tearless) onions demonstrates that they have some attributes more similar to garlic and that this is likely due to the production of novel thiosulfinate or metabolites. The key finding was that collagen-induced in vitro platelet aggregation was significantly reduced by tearless onion extract over normal onion extract. Thiosulfinate or derived compounds were shown not to be responsible for the observed changes in the inflammatory response of AGS (stomach adenocarcinoma) cells to tumor necrosis factor alpha (TNFα) when pretreated with model onion juices. A preliminary rat feeding trial indicated that the tearless onions may also play a key role in reducing weight gain.

Construction of reference chromosome-scale pseudomolecules for potato: integrating the potato genome with genetic and physical maps.

The genome of potato, a major global food crop, was recently sequenced. The work presented here details the integration of the potato reference genome (DM) with a new sequence-tagged site marker-based linkage map and other physical and genetic maps of potato and the closely related species tomato. Primary anchoring of the DM genome assembly was accomplished by the use of a diploid segregating population, which was genotyped with several types of molecular genetic markers to construct a new ~936 cM linkage map comprising 2469 marker loci. In silico anchoring approaches used genetic and physical maps from the diploid potato genotype RH89-039-16 (RH) and tomato. This combined approach has allowed 951 superscaffolds to be ordered into pseudomolecules corresponding to the 12 potato chromosomes. These pseudomolecules represent 674 Mb (~93%) of the 723 Mb genome assembly and 37,482 (~96%) of the 39,031 predicted genes. The superscaffold order and orientation within the pseudomolecules are closely collinear with independently constructed high density linkage maps. Comparisons between marker distribution and physical location reveal regions of greater and lesser recombination, as well as regions exhibiting significant segregation distortion. The work presented here has led to a greatly improved ordering of the potato reference genome superscaffolds into chromosomal "pseudomolecules".

Allele diversity for the apoplastic invertase inhibitor gene from potato.

In planta the enzymatic activity of apoplastic and vacuolar invertases is controlled by inhibitory proteins. Although these invertase inhibitors (apoplastic and vacuolar forms) have been implicated as contributing to resistance to cold-induced sweetening (CIS) in tubers of potato (Solanum tuberosum L.), there is a lack of information on the structure and allelic diversity of the apoplastic invertase inhibitor genes. We have PCR-isolated and sequenced the alleles of the apoplastic invertase inhibitor gene (Stinh1) from three tetraploid potato genotypes: 1021/1 (a genotype with very high tolerance to CIS), 'Karaka' and 'Summer Delight' (two cultivars that are highly susceptible to CIS). In total, five alleles were identified in these genotypes, of which four (Stinh1-c, Stinh1-d, Stinh1-e, Stinh1-f) were novel. An analysis of allele diversity was conducted by incorporating previously published sequences of apoplastic invertase inhibitors from potato. Eight alleles were assessed for sequence polymorphism in the two exons and the single hypervariable intron. Contrary to the hypervariable intron, only 65 single nucleotide polymorphisms were observed in the exons, of which 42 confer amino acid substitutions. Phylogenetic analysis of amino acid sequences indicates that the alleles of the invertase inhibitor are highly conserved amongst members of the Solanaceae family.