Identification of novel plant cysteine oxidase inhibitors from a yeast chemical genetic screen.

Hypoxic responses in plants involve Plant Cysteine Oxidases (PCOs). They catalyze the N-terminal cysteine oxidation of Ethylene Response Factors VII (ERF-VII) in an oxygen-dependent manner, leading to their degradation via the cysteine N-degron pathway (Cys-NDP) in normoxia. In hypoxia, PCO activity drops, leading to the stabilization of ERF-VIIs and subsequent hypoxic gene upregulation. Thus far, no chemicals have been described to specifically inhibit PCO enzymes. In this work, we devised an in vivo pipeline to discover Cys-NDP effector molecules. Budding yeast expressing AtPCO4 and plant-based ERF-VII reporters was deployed to screen a library of natural-like chemical scaffolds and was further combined with an Arabidopsis Cys-NDP reporter line. This strategy allowed us to identify three PCO inhibitors, two of which were shown to affect PCO activity in vitro. Application of these molecules to Arabidopsis seedlings led to an increase in ERF-VII stability, induction of anaerobic gene expression, and improvement of tolerance to anoxia. By combining a high-throughput heterologous platform and the plant model Arabidopsis, our synthetic pipeline provides a versatile system to study how the Cys-NDP is modulated. Its first application here led to the discovery of at least two hypoxia-mimicking molecules with the potential to impact plant tolerance to low oxygen stress.

Novel R2R3 MYB transcription factors regulate anthocyanin synthesis in Aubergine tomato plants.

BACKGROUND: A high content in anthocyanins, for their health beneficial properties, represents an added value for fruits and vegetables. Tomato (Solanum lycopersicum) is one of the most consumed vegetables worldwide and is rich in vitamins and carotenoids. In recent years, purple-skinned tomatoes, enriched of anthocyanins, were produced recovering allelic variants from wild Solanum species. The molecular basis of the Anthocyanin fruit (Aft) locus, exploited by breeders to activate the anthocyanin synthesis in tomato epicarp, has been recently identified in the correct splicing of the R2R3 MYB gene AN2like. Aubergine (Abg) is a tomato accession which introgressed from Solanum lycopersicoides a locus activating the synthesis of anthocyanins in the fruit. The Abg locus was mapped in the region of chromosome 10 containing Aft and the possibility that Abg and Aft represented alleles of the same gene was hypothesized. RESULTS: We dissected the R2R3 MYB gene cluster located in the Abg genomic introgression and demonstrated that AN2like is correctly spliced in Abg plants and is expressed in the fruit epicarp. Moreover, its silencing specifically inhibits the anthocyanin synthesis. The Abg allele of AN2like undergoes alternative splicing and produces two proteins with different activities. Furthermore, in Abg the master regulator of the anthocyanin synthesis in tomato vegetative tissues, AN2, is very poorly expressed. Finally, a novel R2R3 MYB gene was identified: it encodes another positive regulator of the pathway, whose activity was lost in tomato and in its closest relatives. CONCLUSION: In this study, we propose that AN2like is responsible of the anthocyanin production in Abg fruits. Unlike wild type tomato, the Abg allele of AN2like is active and able to regulate its targets. Furthermore, in Abg alternative splicing leads to two forms of AN2like with different activities, likely representing a novel type of regulation of anthocyanin synthesis in tomato.

Effect of Iodine treatments on Ocimum basilicum L.: Biofortification, phenolics production and essential oil composition.

Iodine biofortification has been gaining interest in recent years as a sustainable and innovative approach to eradicate iodine deficiency disorders. Studying the impact of iodine biofortification on plant phenotype, biochemical and physiological parameters is crucial to leverage the expertise and best practices for the agro-food industry and human health. The aim of this study was to evaluate iodine biofortification on the main quantitative and qualitative traits of basil (Ocimum basilicum L.) plants cultivated both in open field and in growth chamber. The impact of KI and KIO3 treatments was evaluated on biomass production, as well as on the synthesis of phenolic compounds, especially rosmarinic acid and other caffeic acid derivatives, and on the essential oil (EO) composition. These compounds are typically accumulated in basil leaves and strongly contribute to the plant nutraceutical value and aroma. In open field, the use of increasing concentrations of both iodine salts gradually enhanced iodine accumulation in leaves, also determining an increase of the antioxidant power, total phenolics, rosmarinic acid and cinnamic acid accumulation. The composition of EO was only slightly affected by the treatments, as all the samples were characterized by a linalool chemotype and a minor alteration in their relative content was observed. A growth chamber experiment was performed to test EO variation in controlled conditions, broadening the range of iodine concentrations. In this case, plant chemotype was significantly affected by the treatments and large EO variability was observed, suggesting that iodine form and concentration can potentially influence the EO composition but that in open field this effect is overcome by environmental factors.

Iodine fortification of vegetables improves human iodine nutrition: in vivo evidence for a new model of iodine prophylaxis.

BACKGROUND: Iodine deficiency is the result of insufficient intake of dietary iodine and as a consequence causes multiple adverse effects. About 2 billion individuals in the world are affected by iodine deficiency. It has been found that the most effective way to control iodine deficiency is through the universal salt iodization. However, salt iodization alone may not be sufficient to assure adequate iodine nutrition. In most industrialized countries, excess consumption of salt has become recognized as a health risk. Therefore, biofortification of vegetables with iodine offers an excellent opportunity to increase iodine intake. AIM AND METHODS: The aim of this study was to test the efficiency of a new model of iodine prophylaxis in a group of 50 healthy volunteers through the intake of vegetables (potatoes, cherry tomatoes, carrots, and green salad) fortified with iodine. Each serving of vegetables consisted of 100 g of potatoes, carrots, tomatoes, or salad containing 45 mg of iodine (30% of the Recommended Daily Allowance), and the volunteers consumed a single serving of vegetables, as preferred, each day for 2 weeks. Urinary iodine (UI) excretion was measured before and after intake of vegetables. RESULTS: The UI concentration measured in volunteers before the intake of vegetables was 98.3 mg/L (basal value), increasing to 117.5 mg/L during the intake of vegetables. Seven days after the discontinuation of vegetable intake, UI was 85 mg/L. UI concentration increment was 19.6% compared with the basal value; therefore, the difference was statistically significant (P = .035). CONCLUSIONS: Biofortification of vegetables with iodine provides a mild but significative increase in UI concentration and, together with the habitual use of iodized salt, may contribute to improve the iodine nutritional status of the population without risks of iodine excess.

HRE-type genes are regulated by growth-related changes in internal oxygen concentrations during the normal development of potato (Solanum tuberosum) tubers.

The occurrence of hypoxic conditions in plants not only represents a stress condition but is also associated with the normal development and growth of many organs, leading to adaptive changes in metabolism and growth to prevent internal anoxia. Internal oxygen concentrations decrease inside growing potato tubers, due to their active metabolism and increased resistance to gas diffusion as tubers grow. In the present work, we identified three hypoxia-responsive ERF (StHRE) genes whose expression is regulated by the gradual decrease in oxygen tensions that occur when potato tubers grow larger. Increasing the external oxygen concentration counteracted the modification of StHRE expression during tuber growth, supporting the idea that the actual oxygen levels inside the organs, rather than development itself, are responsible for the regulation of StHRE genes. We identified several sugar metabolism-related genes co-regulated with StHRE genes during tuber development and possibly involved in starch accumulation. All together, our data suggest a possible role for low oxygen in the regulation of sugar metabolism in the potato tuber, similar to what happens in storage tissues during seed development.

Heterologous microarray experiments allow the identification of the early events associated with potato tuber cold sweetening.

BACKGROUND: Since its discovery more than 100 years ago, potato (Solanum tuberosum) tuber cold-induced sweetening (CIS) has been extensively investigated. Several carbohydrate-associated genes would seem to be involved in the process. However, many uncertainties still exist, as the relative contribution of each gene to the process is often unclear, possibly as the consequence of the heterogeneity of experimental systems. Some enzymes associated with CIS, such as beta-amylases and invertases, have still to be identified at a sequence level. In addition, little is known about the early events that trigger CIS and on the involvement/association with CIS of genes different from carbohydrate-associated genes. Many of these uncertainties could be resolved by profiling experiments, but no GeneChip is available for the potato, and the production of the potato cDNA spotted array (TIGR) has recently been discontinued. In order to obtain an overall picture of early transcriptional events associated with CIS, we investigated whether the commercially-available tomato Affymetrix GeneChip could be used to identify which potato cold-responsive gene family members should be further studied in detail by Real-Time (RT)-PCR (qPCR). RESULTS: A tomato-potato Global Match File was generated for the interpretation of various aspects of the heterologous dataset, including the retrieval of best matching potato counterparts and annotation, and the establishment of a core set of highly homologous genes. Several cold-responsive genes were identified, and their expression pattern was studied in detail by qPCR over 26 days. We detected biphasic behaviour of mRNA accumulation for carbohydrate-associated genes and our combined GeneChip-qPCR data identified, at a sequence level, enzymatic activities such as beta-amylases and invertases previously reported as being involved in CIS. The GeneChip data also unveiled important processes accompanying CIS, such as the induction of redox- and ethylene-associated genes. CONCLUSION: Our Global Match File strategy proved critical for accurately interpretating heterologous datasets, and suggests that similar approaches may be fruitful for other species. Transcript profiling of early events associated with CIS revealed a complex network of events involving sugars, redox and hormone signalling which may be either linked serially or act in parallel. The identification, at a sequence level, of various enzymes long known as having a role in CIS provides molecular tools for further understanding the phenomenon.