Transcriptome Responses of Ripe Cherry Tomato Fruit Exposed to Chilling and Rewarming Identify Reversible and Irreversible Gene Expression Changes.

Tomato fruit stored below 12°C lose quality and can develop chilling injury upon subsequent transfer to a shelf temperature of 20°C. The more severe symptoms of altered fruit softening, uneven ripening and susceptibility to rots can cause postharvest losses. We compared the effects of exposure to mild (10°C) and severe chilling (4°C) on the fruit quality and transcriptome of 'Angelle', a cherry-type tomato, harvested at the red ripe stage. Storage at 4°C (but not at 10°C) for 27 days plus an additional 6 days at 20°C caused accelerated softening and the development of mealiness, both of which are commonly related to cell wall metabolism. Transcriptome analysis using RNA-Seq identified a range of transcripts encoding enzymes putatively involved in cell wall disassembly whose expression was strongly down-regulated at both 10 and 4°C, suggesting that accelerated softening at 4°C was due to factors unrelated to cell wall disassembly, such as reductions in turgor. In fruit exposed to severe chilling, the reduced transcript abundances of genes related to cell wall modification were predominantly irreversible and only partially restored upon rewarming of the fruit. Within 1 day of exposure to 4°C, large increases occurred in the expression of alternative oxidase, superoxide dismutase and several glutathione S-transferases, enzymes that protect cell contents from oxidative damage. Numerous heat shock proteins and chaperonins also showed large increases in expression, with genes showing peak transcript accumulation after different times of chilling exposure. These changes in transcript abundance were not induced at 10°C, and were reversible upon transfer of the fruit from 4 to 20°C. The data show that genes involved in cell wall modification and cellular protection have differential sensitivity to chilling temperatures, and exhibit different capacities for recovery upon rewarming of the fruit.

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.

Mechanisms of Selenium Enrichment and Measurement in Brassicaceous Vegetables, and Their Application to Human Health.

Selenium (Se) is an essential micronutrient for human health. Se deficiency affects hundreds of millions of people worldwide, particularly in developing countries, and there is increasing awareness that suboptimal supply of Se can also negatively affect human health. Selenium enters the diet primarily through the ingestion of plant and animal products. Although, plants are not dependent on Se they take it up from the soil through the sulphur (S) uptake and assimilation pathways. Therefore, geographic differences in the availability of soil Se and agricultural practices have a profound influence on the Se content of many foods, and there are increasing efforts to biofortify crop plants with Se. Plants from the Brassicales are of particular interest as they accumulate and synthesize Se into forms with additional health benefits, such as methylselenocysteine (MeSeCys). The Brassicaceae are also well-known to produce the glucosinolates; S-containing compounds with demonstrated human health value. Furthermore, the recent discovery of the selenoglucosinolates in the Brassicaceae raises questions regarding their potential bioefficacy. In this review we focus on Se uptake and metabolism in the Brassicaceae in the context of human health, particularly cancer prevention and immunity. We investigate the close relationship between Se and S metabolism in this plant family, with particular emphasis on the selenoglucosinolates, and consider the methodologies available for identifying and quantifying further novel Se-containing compounds in plants. Finally, we summarize the research of multiple groups investigating biofortification of the Brassicaceae and discuss which approaches might be most successful for supplying Se deficient populations in the future.

Preferentially enhancing anti-cancer isothiocyanates over glucosinolates in broccoli sprouts: How NaCl and salicylic acid affect their formation.

Broccoli (Brassica oleracea L. var. italica) sprouts contain glucosinolates (GLs) that when hydrolysed yield health promoting isothiocyanates such as sulforaphane (SF). SF content can be increased by salt (NaCl) stress, although high salt concentrations negatively impact plant growth. Salicylic acid (SA) treatments can attenuate the negative effects of salt on growth. To test whether sprout isothiocyanate content could be elevated without sprout growth being compromised, broccoli seed were germinated and grown for seven days in salt (0, 80 and 160 mM) alone and in combination with 100 µM SA. Increasing concentrations of salt lowered transcript accumulation of GL biosynthetic genes which was reflected in lowered content of Gluconapin, 4-methoxyglucobrassicin and neoglucobrassicin glucosinolates. Other glucosinolates such as glucoraphanin did not alter significantly. Salt (160 mM) increased transcript abundance of the GL hydrolytic gene MYROSINASE (BoMYO) and its cofactor EPITHIOSPECIFIER MODIFIER1 (BoESM1) whose encoded product directs MYROSINASE to produce isothiocyanate rather than nitrile forms. SF content was increased 6-fold by the 160 mM salt treatment, but the salt treatment reduced percentage seed germination, slowed seed germination, and reduced sprout hypocotyl elongation. This growth inhibition was prevented if 100 µM SA was included with the salt treatment. These findings suggest that the increase in SF production by salt occurs in part because of increased transcript abundance of genes in the hydrolytic pathway, which occurs independently of the negative impact of salt on sprout growth.

Overexpression of STARCH BRANCHING ENZYME II increases short-chain branching of amylopectin and alters the physicochemical properties of starch from potato tuber.

BACKGROUND: Starch is biosynthesised by a complex of enzymes including various starch synthases and starch branching and debranching enzymes, amongst others. The role of all these enzymes has been investigated using gene silencing or genetic knockouts, but there are few examples of overexpression due to the problems of either cloning large genomic fragments or the toxicity of functional cDNAs to bacteria during cloning. The aim of this study was to investigate the function of potato STARCH BRANCHING ENZYME II (SBEII) using overexpression in potato tubers. RESULTS: A hybrid SBEII intragene consisting of potato cDNA containing a fragment of potato genomic DNA that included a single intron was used in order to prevent bacterial translation during cloning. A population of 20 transgenic potato plants exhibiting SBEII overexpression was generated. Compared with wild-type, starch from these tubers possessed an increased degree of amylopectin branching, with more short chains of degree of polymerisation (DP) 6-12 and particularly of DP6. Transgenic lines expressing a GRANULE-BOUND STARCH SYNTHASE (GBSS) RNAi construct were also generated for comparison and exhibited post-transcriptional gene silencing of GBSS and reduced amylose content in the starch. Both transgenic modifications did not affect granule morphology but reduced starch peak viscosity. In starch from SBEII-overexpressing lines, the increased ratio of short to long amylopectin branches facilitated gelatinisation, which occurred at a reduced temperature (by up to 3°C) or lower urea concentration. In contrast, silencing of GBSS increased the gelatinisation temperature by 4°C, and starch required a higher urea concentration for gelatinisation. In lines with a range of SBEII overexpression, the magnitude of the increase in SBEII activity, reduction in onset of gelatinisation temperature and increase in starch swollen pellet volume were highly correlated, consistent with reports that starch swelling is greatly dependent upon the amylopectin branching pattern. CONCLUSION: This work reports the first time that overexpression of SBEII has been achieved in a non-cereal plant. The data show that overexpression of SBEII using a simple single-intron hybrid intragene is an effective way to modify potato starch physicochemical properties, and indicate that an increased ratio of short to long amylopectin branches produces commercially beneficial changes in starch properties such as reduced gelatinisation temperature, reduced viscosity and increased swelling volume.

Distribution of selenoglucosinolates and their metabolites in Brassica treated with sodium selenate.

In Brassica species, hydrolysis of (methylthio)glucosinolates produces sulfur-containing aglycons which have demonstrated anticancer benefits. Selenized Brassicaceae contain (methylseleno)glucosinolates and their selenium-containing aglycons. As a prelude to biological testing, broccoli, cauliflower, and forage rape plants were treated with sodium selenate and their tap roots, stems, leaves, and florets analyzed for selenoglucosinolates and their Se aglycons. Two new selenoglucosinolates were identified: glucoselenoraphanin in broccoli florets and glucoselenonasturtiin in forage rape roots. A new aglycon, selenoberteroin nitrile, was identified in forage rape. The major selenoglucosinolates were glucoselenoerucin in broccoli, glucoselenoiberverin in cauliflower, and glucoselenoerucin and glucoselenoberteroin in forage rape roots. In broccoli florets, the concentrations of selenglucosinolates exceeded those of their sulfur analogues. Fertilization with selenium slightly reduced (methylthio)glucosinolates and aglycons in the roots, but increased them in the florets, the leaves, and sometimes the stems. These discoveries provide a new avenue for investigating how consumption of Brassica vegetables and their organoselenides may promote human health.

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.

Selenoglucosinolates and their metabolites produced in Brassica spp. fertilised with sodium selenate.

Glucosinolates are sulphur-containing glycosides found in many Brassica spp. that are important because their aglycone hydrolysis products protect the plant from herbivores and exhibit anti-cancer properties in humans. Recently, synthetically produced selenium analogues have been shown to be more effective at suppressing cancers than their sulphur counterparts. Although selenium is incorporated into a number of Brassica amino acids and peptides, firm evidence has yet to be presented for the presence of selenium in the glucosinolates and their aglycones in planta. In this study broccoli and cauliflower florets, and roots of forage rape, all obtained from plants treated with sodium selenate, were analysed for the presence of organoselenides. GC-MS analysis of pentane/ether extracts identified six organoselenium compounds including selenium analogues of known myrosinase-derived Brassica volatiles: 4-(methylseleno)butanenitrile, 5-(methylseleno)pentanenitrile, 3-(methylseleno)propylisothiocyanate, 4-(methylseleno)butylisothiocyanate, and 5-(methylseleno)pentylisothiocyanate. LC-MS analysis of ethanolic extracts identified three selenoglucosinolates: 3-(methylseleno)propylglucosinolate (glucoselenoiberverin), 4-(methylseleno)butylglucosinolate (glucoselenoerucin), and 5-(methylseleno)pentylglucosinolate (glucoselenoberteroin). LC-MS/MS analysis was used to locate the position of the selenium atom in the selenoglucosinolate and indicates preferential incorporation of selenium via selenomethionine into the methylselenyl moiety rather than into the sulphate or ß-thioglucose groups. In forage rape, selenoglucosinolates and their aglycones (mainly isothiocyanates), occurred at concentrations up to 10% and 70%, respectively, of their sulphur analogues. In broccoli, concentrations of the selenoglucosinolates and their aglycones (mainly nitriles) were up to 60% and 1300%, respectively of their sulphur analogues. These findings indicate the potential for the incorporation of high levels of selenium into Brassica glucosinolates.

Biofortification of tomato (Solanum lycopersicum) fruit with the anticancer compound methylselenocysteine using a selenocysteine methyltransferase from a selenium hyperaccumulator.

Methylselenocysteine (MeSeCys) is an amino acid derivative that possesses potent anticancer activity in animals. Plants that can tolerate growth on soils with high Se content, known as Se hyperaccumulators, do so by converting inorganic Se to MeSeCys by the enzyme selenocysteine methyltransferase (SMT). A cDNA encoding the SMT from a Se hyperaccumulator was overexpressed in tomato (Solanum lycopersicum). Transgenic plants were provided with selenite or selenate to the roots during fruit development, and liquid chromatography-mass spectrometry was used to show that MeSeCys accumulated in the fruit but not in the leaves. Depending on the transgenic line and Se treatment, up to 16% of the total Se in the fruit was present as MeSeCys. MeSeCys was produced more effectively from selenite on a percentage conversion basis, but greater accumulation of MeSeCys could be achieved from selenate due to its better translocation from the roots. MeSeCys was heat stable and survived processing of the fruit to tomato juice.

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.

Organoselenides from Nicotiana tabacum genetically modified to accumulate selenium.

Nicotiana tabacum L. (tobacco) plants were transformed to overexpress a selenocysteine methyltransferase gene from the selenium hyperaccumulator Astragalus bisulcatus (Hook.) A. Gray (two-grooved milkvetch), and an ATP-sulfurylase gene from Brassica oleracea L. var. italica (broccoli). Solvent extraction of leaves harvested from plants treated with selenate revealed five selenium-containing compounds, of which four were identified by chemical synthesis as 2-(methylseleno)acetaldehyde, 2,2-bis(methylseleno)acetaldehyde, 4-(methylseleno)-(2E)-nonenal, and 4-(methylseleno)-(2E,6Z)-nonadienal. These four compounds have not previously been reported in nature.

Accumulation of an organic anticancer selenium compound in a transgenic Solanaceous species shows wider applicability of the selenocysteine methyltransferase transgene from selenium hyperaccumulators.

Tolerance to high selenium (Se) soils in Se-hyperaccumulating plant species is correlated with the ability to biosynthesise methylselenocysteine (MeSeCys), due to the activity of selenocysteine methyltransferase (SMT). In mammals, inclusion of MeSeCys in the diet reduces the incidence of certain cancers, so increasing the range of crop plants that can produce this compound is an attractive biotechnology target. However, in the non-Se accumulator Arabidopsis, overexpression of SMT does not result in biosynthesis of MeSeCys from selenate because the rate at which selenate is reduced to selenite by ATP sulfurylase (ATPS) is low. This limitation is less problematic in other species of the Brassicaceae that can produce MeSeCys naturally. We investigated the potential for biosynthesis of MeSeCys in other plant families using Nicotiana tabacum L., a member of the Solanaceae. When plants were watered with 200 microM selenate, overexpression of a SMT transgene caused a 2- to 4-fold increase in Se accumulation (resulting in increased numbers of leaf lesions and areas of necrosis), production of MeSeCys (up to 20% of total Se) and generation of volatile dimethyl diselenide derived directly from MeSeCys. Despite the greatly increased accumulation of total Se, this did not result in increased Se toxicity effects on growth. Overexpression of ATPS did not increase Se accumulation from selenate. Accordingly, lines overexpressing both ATPS and SMT did not show a further increase in total Se accumulation or in leaf toxicity symptoms relative to overexpression of SMT alone, but directed a greater proportion of Se into MeSeCys. This work demonstrates that the production of the cancer-preventing compound MeSeCys in plants outside the Brassicaceae is possible. We conclude that while the SMT gene from Se hyperaccumulators can probably be utilised universally to increase the metabolism of Se into MeSeCys, the effects of enhancing ATPS activity will vary depending on the species involved.

Senescence-associated down-regulation of 1-aminocyclopropane-1-carboxylate (ACC) oxidase delays harvest-induced senescence in broccoli.

To gain an in-depth understanding of the role of ethylene in post harvest senescence, we used broccoli (Brassica oleracea var. italica) as our model species. The senescence-associated asparagine synthetase (AS) promoter from asparagus was used to drive the expression of an antisense 1-aminocyclopropane-1-carboxylate oxidase (ACO) cDNA from broccoli, BoACO2, to reduce ethylene production following harvest. Physiological analyses revealed that transgenic broccoli lines harbouring the antisense BoACO2 gene construct (designated as AS-asACO) displayed delayed senescence in both detached leaves and detached heads as measured by hue angle. Harvested floret tissue from these plants also showed a delayed loss of chlorophyll, lower protease activity and higher total protein content, and changes in transcript levels of senescence marker genes when compared with wild type and transgenic lines transformed with an empty T-DNA. Genes that were down-regulated included those coding for cysteine protease (BoCP5), metallothionein-like protein (BoMT1), hexokinase (BoHK1), invertase (BoINV1) and sucrose transporters (BoSUC1 and BoSUC2). Northern analysis for BoACO1 and BoACO2, ACO assays and western analysis, revealed reduced ACO transcript, enzyme activity and protein accumulation, as well as reduced ethylene production in the transgenic AS-asACO lines when compared with controls, confirming that a key enzyme regulating ethylene biosynthesis was reduced in these plants. This, together with the changes observed in gene expression, confirm a significant role for ethylene in regulating the events leading to senescence in broccoli following harvest.

Flowering genes in Metrosideros fit a broad herbaceous model encompassing Arabidopsis and Antirrhinum.

Molecular studies were conducted on Metrosideros excelsa to determine if the current genetic models for flowering with regard to inflorescence and floral meristem identity genes in annual plants were applicable to a woody perennial. MEL, MESAP1 and METFL1, the fragments of LEAFY (LFY), APETALA1 (AP1) and TERMINAL FLOWER1 (TFL1) equivalents, respectively, were isolated from M. excelsa. Temporal expression patterns showed that MEL and MESAP1 exhibited a bimodal pattern of expression. Expression exhibited during early floral initiation in autumn was followed by down-regulation during winter, and up-regulation in spring as floral organogenesis occurred. Spatial expression patterns of MEL showed that it had greater similarity to FLORICAULA (FLO) than to LFY, whereas MESAP1 was more similar to AP1 than SQUAMOSA. The interaction between MEL and METFL1 was more similar to the interaction between FLO and CENTRORADIALIS than that between LFY and TFL1. Consequently, the three genes from M. excelsa fit a broader herbaceous model encompassing Antirrhinum as well as Arabidopsis, but with differences, such as the bimodal pattern of expression seen with MEL and MESAP1. In mid-winter, at the time when both MEL and MESAP1 were down-regulated, GA(1) was below the level of detection in M. excelsa buds. Even though application of gibberellin inhibits flowering in members of the Myrtaceae, MEL was responsive to gibberellin with expression in juvenile plants up-regulated by GA(3). However, MESAP1 was not up-regulated indicating that meristem competence was also probably required to promote flowering in M. excelsa.