Invertase inhibitors in potato: towards a biochemical and molecular understanding of cold-induced sweetening.

Invertase inhibitors classified as cell wall/apoplastic and vacuolar belonging to the pectin methylesterase family, play a major role in cold-induced sweetening (CIS) process of potato tubers. The CIS process is controlled at the post-translational level via an interaction between invertase (cell wall/apoplastic and vacuolar) by their compartment-specific inhibitors (cell wall/apoplastic and vacuolar). Invertase inhibitors have been cloned, sequenced and functionally characterized from potato cultivars differing in their CIS ability. The secondary structure of the invertase inhibitors consisted of seven alpha-helices and four conserved cysteine residues. The well-conserved three amino acids i.e. Pro-Lys-Phe are known to interact with invertase. Location of the genes encoding cell wall/apoplastic and vacuolar invertase inhibitors on potato chromosome number twelve in a tandem orientation without any intervening genes suggest their divergence into the cell wall and vacuole forms following the event of gene duplication. Under cold storage conditions, the vacuolar invertase inhibitor gene showed developmentally regulated alternative splicing and produce hybrid mRNAs which were the result of mRNA splicing of an upstream region of vacuolar invertase inhibitor gene to a downstream region of the apoplastic invertase inhibitor gene. Transgenic potato tubers overexpressing invertase inhibitors resulted in decreased invertase activity, low reducing sugars and improved processing quality making invertase inhibitors highly potential candidate genes for overcoming CIS. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene-editing technology offers transgene-free breeding for developing CIS resistant potato cultivars. Moreover, the post-transcriptional regulation of invertase inhibitors during cold storage can be warranted. This review summarizes progress and current knowledge on biochemical and molecular approaches used for the understanding of invertase inhibitors with special reference to key findings in potato.

Cold storage reveals distinct metabolic perturbations in processing and non-processing cultivars of potato (Solanum tuberosum L.).

Cold-induced sweetening (CIS) causes considerable losses to the potato processing industry wherein the selection of potato genotypes using biochemical information has found to be advantageous. Here, 1H NMR spectroscopy was performed to identify metabolic perturbations from tubers of five potato cultivars (Atlantic, Frito Lay-1533, Kufri Jyoti, Kufri Pukhraj, and PU1) differing in their CIS ability and processing characteristics at harvest and after cold storage (4 °C). Thirty-nine water-soluble metabolites were detected wherein significantly affected metabolites after cold storage were categorized into sugars, sugar alcohols, amino acids, and organic acids. Multivariate statistical analysis indicated significant differences in the metabolic profiles among the potato cultivars. Pathway enrichment analysis revealed that carbohydrates, amino acids, and organic acids are the key players in CIS. Interestingly, one of the processing cultivars, FL-1533, exhibited a unique combination of metabolites represented by low levels of glucose, fructose, and asparagine accompanied by high citrate levels. Conversely, non-processing cultivars (Kufri Pukhraj and Kufri Jyoti) showed elevated glucose, fructose, and malate levels. Our results indicate that metabolites such as glucose, fructose, sucrose, asparagine, glutamine, citrate, malate, proline, 4-aminobutyrate can be potentially utilized for the prediction, selection, and development of potato cultivars for long-term storage, nutritional, as well as processing attributes.

Sequence diversity and in silico structure prediction of the vacuolar invertase inhibitor gene from potato (Solanum tuberosum L.) cultivars differing in sugar content.

The aim of this study was to examine the variations in sugar content and identify the polymorphism in vacuolar invertase inhibitor (INH2) gene from Indian non-processing (Kufri Jyoti, Kufri Pukhraj and PU1) and exotic processing (Atlantic and Frito Lay-1533) potato cultivars. Upon cold storage (4 °C) processing cultivars maintained low reducing sugars as compared to non-processing cultivars. Sequencing of the INH2 gene identified four alleles of which three identified as novel alleles. A total twelve SNPs resulted in silent mutations, with five conferring the amino acid substitutions. Phylogenetic analysis suggests a highly conserved nature of the INH2 gene. The 3D predicted structures generated for all the alleles revealed slight variations in the orientation of the helices (α1-3) in N-terminal region. Sequence polymorphism observed in INH2 alleles in processing and non-processing potato cultivars can be correlated with the observed variations in the sugar content suggesting a possible role in cold-induced sweetening.

De novo root transcriptome of a medicinally important rare tree Oroxylum indicum for characterization of the flavonoid biosynthesis pathway.

Oroxylum indicum (L.) Kurz is a medicinally important and rare tree species of the family Bignoniaceae. It is rich in flavonoid content and its mature roots are extensively used in Ayurvedic formulations. O. indicum specific flavonoids like oroxylin B, prunetin and oroxindin possess antibacterial, antiproliferative, antioxidant and anticancerous properties, signifying its importance in modern medicine. In the present study, de novo transcriptome analysis of O. indicum root was performed to elucidate the genes involved in flavonoid metabolism. A total of 24,625,398 high quality reads were assembled into 121,286 transcripts with N50 value 1783. The BLASTx search of 81,002 clustered transcripts against Viridiplantae Uniprot database led to annotation of 46,517 transcripts. Furthermore, Gene ontology (GO) revealed that 34,231 transcripts mapped to 3049 GO terms and KEGG analysis demonstrated that 4570 transcripts plausibly involved in 132 biosynthetic pathways. The transcriptome data indicated that cinnamyl-alcohol dehydrogenase (OinCAD) was abundant in phenylpropanoid pathway genes while; naringenin chalcone synthase (OinCHS), flavone synthase (OinFNS) and flavonoid 3', 5'-methyltransferase (OinF35 MT) were abundant in flavonoid, isoflavonoid, flavone and flavonol biosynthesis pathways, respectively. Transcription factor analysis demonstrated the abundance of MYB, bHLH and WD40 transcription factor families, which regulate the flavonoid biosynthesis. Flavonoid pathway genes displayed differential expression in young and old roots of O. indicum. The transcriptome led to the identification of 31 diverse full length Cytochrome P450 (CYP450) genes which may be involved in biosynthesis of specialized metabolites and flavonoids like baicalein and baicalin. Thus, the information obtained in this study will be a valuable tool for identifying genes and developing system biology approaches for in vitro synthesis of specialized O. indicum metabolites.

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.