Actinidin diversity: discovery of common and selective substrates for actinidin isoforms and Actinidia cultivars.

The actinidin proteinase family has a striking sequence diversity; isoelectric points range from 3.9 to 9.3. The biological drive for this variation is thought to be actinidin's role as a defense-related protein. In this study we map mutations in the primary sequence onto the 3D structure of the protein and show that the region with the highest diversity is close to the substrate binding groove. Non-conservative substitutions in the active site determine substrate preference and therefore create problems for quantification of actinidin activity. Here we use a peptide substrate library to compare two actinidin isoforms, one from the kiwiberry cultivar 'Hortgem Tahi' (Actinidia arguta), and the other from the familiar kiwifruit cultivar 'Hayward' (Actinidia chinensis var. deliciosa). Among 360 octamer substrates we find one substrate (RVAAGSPI) with the useful property of being readily cleaved by all the functionally active actinidins in a set of A. arguta and A. chinensis var. deliciosa isoforms. In addition, we find that two substrates (LPPKSQPP & ILRDKDNT) have the ability to differentiate different isoforms from a single fruit. We compare actinidins from 'Hayward' and A. arguta for their ability to digest the allergenic gluten peptide (PFPQPQLPY) but find the peptide to be indigestible by all sources of actinidin. The ability to inactivate salivary amylase is shown to be a common trait in Actinidia cultivars due to proteolysis by actinidin and is particularly strong in 'Hortgem Tahi'. A mixture of 10% 'Hortgem Tahi' extract with 90% saliva inactivates 100% of amylase activity within 5 minutes. Conceivably, 'Hortgem Tahi' might lower the glycaemic response in a meal rich in cooked starch.

Selenium treatment differentially affects sulfur metabolism in high and low glucosinolate producing cultivars of broccoli (Brassica oleracea L.).

The effect of selenium (Se) application on the sulfur (S)-rich glucosinolate (GSL)-containing plant, broccoli (Brassica oleracea L. var. italica) was examined with a view to producing germplasm with increased Se and GSL content for human health, and to understanding the influence of Se on the regulation of GSL production. Two cultivars differing in GSL content were compared. Increased Se application resulted in an increase in Se uptake in planta, but no significant change in total S or total GSL content in either cultivar. Also no significant change was observed in the activity of ATP sulfurylase (ATPS, EC 2.7.7.4) or O-acetylserine(thiol) lyase (OASTL, EC 2.5.1.47) with increased Se application. However, in the first investigation of APS kinase (APSK, EC 2.7.1.25) expression in response to Se fertilisation, an increase in transcript abundance of one variant of APS kinase 1 (BoAPSK1A) was observed in both cultivars, and an increase in BoAPSK2 transcript abundance was observed in the low GSL producing cultivar. A mechanism by which increased APSK transcription may provide a means of controlling the content of S-containing compounds, including GSLs, following Se uptake is proposed.

Consumption of selenium-enriched broccoli increases cytokine production in human peripheral blood mononuclear cells stimulated ex vivo, a preliminary human intervention study.

SCOPE: Selenium (Se) is a micronutrient essential for human health, including immune function. Previous research indicates that Se supplementation may cause a shift from T helper (Th)1- to Th2-type immune responses. We aim to test the potential health promoting effects of Se-enriched broccoli. METHODS AND RESULTS: In a human trial, 18 participants consumed control broccoli daily for 3 days. After a 3-day wash-out period, the participants were provided with Se-enriched broccoli containing 200 µg of Se per serving for 3 days. Plasma and peripheral blood mononuclear cell (PBMC) samples were collected at the start and end of each broccoli feeding period for analysis of total Se and measurement of cytokine production from PBMC stimulated with antigens ex vivo. Plasma Se content remained consistent throughout the control broccoli feeding period and the baseline of the Se-enriched broccoli period (1.22 µmol/L) and then significantly increased following 3 days of Se-enriched broccoli feeding. Interleukin (IL-2, IL-4, IL-5, IL-13, and IL-22) production from PBMC significantly increased after 3 days of Se-enriched broccoli feeding compared with baseline. CONCLUSION: This study indicates that consumption of Se-enriched broccoli may increase immune responses toward a range of immune challenges.

Post-translational regulation of acid invertase activity by vacuolar invertase inhibitor affects resistance to cold-induced sweetening of potato tubers.

Cold-induced sweetening (CIS) is a serious post-harvest problem for potato tubers, which need to be stored cold to prevent sprouting and pathogenesis in order to maintain supply throughout the year. During storage at cold temperatures (below 10 °C), many cultivars accumulate free reducing sugars derived from a breakdown of starch to sucrose that is ultimately cleaved by acid invertase to produce glucose and fructose. When affected tubers are processed by frying or roasting, these reducing sugars react with free asparagine by the Maillard reaction, resulting in unacceptably dark-coloured and bitter-tasting product and generating the probable carcinogen acrylamide as a by-product. We have previously identified a vacuolar invertase inhibitor (INH2) whose expression correlates both with low acid invertase activity and with resistance to CIS. Here we show that, during cold storage, overexpression of the INH2 vacuolar invertase inhibitor gene in CIS-susceptible potato tubers reduced acid invertase activity, the accumulation of reducing sugars and the generation of acrylamide in subsequent fry tests. Conversely, suppression of vacuolar invertase inhibitor expression in a CIS-resistant line increased susceptibility to CIS. The results show that post-translational regulation of acid invertase by the vacuolar invertase inhibitor is an important component of resistance to CIS.

Induction of vacuolar invertase inhibitor mRNA in potato tubers contributes to cold-induced sweetening resistance and includes spliced hybrid mRNA variants.

Cold storage of tubers of potato (Solanum tuberosum L.) compromises tuber quality in many cultivars by the accumulation of hexose sugars in a process called cold-induced sweetening. This is caused by the breakdown of starch to sucrose, which is cleaved to glucose and fructose by vacuolar acid invertase. During processing of affected tubers, the high temperatures involved in baking and frying cause the Maillard reaction between reducing sugars and free amino acids, resulting in the accumulation of acrylamide. cDNA clones with deduced proteins homologous to known invertase inhibitors were isolated and the two most abundant forms, termed INH1 and INH2, were shown to possess apoplastic and vacuolar localization, respectively. The INH2 gene showed developmentally regulated alternative splicing, so, in addition to the INH2α transcript encoding the full-length protein, two hybrid mRNAs (INH2ß*A and INH2ß*B) that encoded deduced vacuolar invertase inhibitors with divergent C-termini were detected, the result of mRNA splicing of an upstream region of INH2 to a downstream region of INH1. Hybrid RNAs are common in animals, where they may add to the diversity of the proteome, but are rarely described in plants. During cold storage, INH2α and the hybrid INH2ß mRNAs accumulated to higher abundance in cultivars resistant to cold-induced sweetening than in susceptible cultivars. Increased amounts of invertase inhibitor may contribute to the suppression of acid invertase activity and prevent cleavage of sucrose. Evidence for increased RNA splicing activity was detected in several resistant lines, a mechanism that in some circumstances may generate a range of proteins with additional functional capacity to aid adaptability.