Vitor Amorim-Silva

The SUD1 Gene Encodes a Putative E3 Ubiquitin Ligase and Is a Positive Regulator of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Activity in Arabidopsis

In contrast with animals, little is known about the regulation of HMGR, the rate-limiting enzyme of isoprenoid biosynthesis, in plants. Through the identification of second-site suppressors of the Arabidopsis dry2/sqe1-5 mutant, we found that the putative E3 ubiquitin ligase SUD1, likely involved in endoplasmic reticulum–associated degradation, is a regulator of HMGR activity. The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) enzyme catalyzes the major rate-limiting step of the mevalonic acid (MVA) pathway from which sterols and other isoprenoids are synthesized. In contrast with our extensive knowledge of the regulation of HMGR in yeast and animals, little is known about this process in plants. To identify regulatory components of the MVA pathway in plants, we performed a genetic screen for second-site suppressor mutations of the Arabidopsis thaliana highly drought-sensitive drought hypersensitive2 (dry2) mutant that shows decreased squalene epoxidase activity. We show that mutations in SUPPRESSOR OF DRY2 DEFECTS1 (SUD1) gene recover most developmental defects in dry2 through changes in HMGR activity. SUD1 encodes a putative E3 ubiquitin ligase that shows sequence and structural similarity to yeast Degradation of α factor (Doα10) and human TEB4, components of the endoplasmic reticulum–associated degradation C (ERAD-C) pathway. While in yeast and animals, the alternative ERAD-L/ERAD-M pathway regulates HMGR activity by controlling protein stability, SUD1 regulates HMGR activity without apparent changes in protein content. These results highlight similarities, as well as important mechanistic differences, among the components involved in HMGR regulation in plants, yeast, and animals.

Genetic and genome-wide transcriptomic analyses identify co-regulation of oxidative response and hormone transcript abundance with vitamin C content in tomato fruit

BackgroundL-ascorbic acid (AsA; vitamin C) is essential for all living plants where it functions as the main hydrosoluble antioxidant. It has diverse roles in the regulation of plant cell growth and expansion, photosynthesis, and hormone-regulated processes. AsA is also an essential component of the human diet, being tomato fruit one of the main sources of this vitamin. To identify genes responsible for AsA content in tomato fruit, transcriptomic studies followed by clustering analysis were applied to two groups of fruits with contrasting AsA content. These fruits were identified after AsA profiling of an F8 Recombinant Inbred Line (RIL) population generated from a cross between the domesticated species Solanum lycopersicum and the wild relative Solanum pimpinellifollium.ResultsWe found large variability in AsA content within the RIL population with individual RILs with up to 4-fold difference in AsA content. Transcriptomic analysis identified genes whose expression correlated either positively (PVC genes) or negatively (NVC genes) with the AsA content of the fruits. Cluster analysis using SOTA allowed the identification of subsets of co-regulated genes mainly involved in hormones signaling, such as ethylene, ABA, gibberellin and auxin, rather than any of the known AsA biosynthetic genes. Data mining of the corresponding PVC and NVC orthologs in Arabidopis databases identified flagellin and other ROS-producing processes as cues resulting in differential regulation of a high percentage of the genes from both groups of co-regulated genes; more specifically, 26.6% of the orthologous PVC genes, and 15.5% of the orthologous NVC genes were induced and repressed, respectively, under flagellin22 treatment in Arabidopsis thaliana.ConclusionResults here reported indicate that the content of AsA in red tomato fruit from our selected RILs are not correlated with the expression of genes involved in its biosynthesis. On the contrary, the data presented here supports that AsA content in tomato fruit co-regulates with genes involved in hormone signaling and they are dependent on the oxidative status of the fruit.

Effect of salt on ROS homeostasis, lipid peroxidation and antioxidant mechanisms in Pinus pinaster suspension cells

Abstract• In the Pinus genus, information on the effectiveness of oxidative defence mechanisms during exposure to salt is lacking. The effect of salt stress imposition on ROS homeostasis was investigated using maritime pine (Pinus pinaster Ait.) suspension cells as a model system.• Cells were maintained in MS-based medium, exposed to salt (50, 100 and 150 mM NaCl) and analysed for biomass production, evidencing a decreasing growth capacity. Use of 100 mM NaCl imposed severe salt stress without affecting cell viability, being chosen for subsequent studies on the ROS homeostasis of salt shock-treated suspension cells.• Increased total ROS levels were evident on the second day of salt exposure, but a superoxide ion transient burst was immediately noticeable. Additionally, lipid peroxide formation seemed to correlate with superoxide ion breakdown. In-gel superoxide dismutase activity evidenced a FeSOD homodimer with strongly increasing activity between hours 12–48 of salt stress imposition. Subsequently, P. pinaster Fe-Sod1 and csApx1 genes were isolated from a cDNA library and expression was shown to increase within 12–24 h.• Results show that severe salt treatment generates oxidative stress in P. pinaster cells despite the induction of antioxidant systems, and suggest a putative involvement of ROS in salt stress signalling.Résumé• Les informations sur les mécanismes de défense oxydative du pin en réponse à un stress salin sont rares. L’effet d’une exposition au sel sur l’homéostasie des formes réactives d’oxygène (FRO) a été étudié en utilisant une suspension cellulaire de pin maritime (Pinus pinaster Ait.) comme modèle.• Les cellules cultivées dans un milieu MS modifié ont été exposées au sel (50, 100 et 150 mM NaCl) et l’analyse de la production de biomasse a révélé une réduction de leur croissance. Une concentration de 100 mM NaCl, stress sévère qui n’affecte cependant pas la viabilité cellulaire, a été choisie pour les études suivantes.• L’augmentation des teneurs en FRO est évidente le jour suivant l’enrichissement du milieu en sels mais une production transitoire d’ions superoxyde est immédiatement constatée. De plus, l’apparition de produits issus de la peroxydation des lipides semble concomitante à la disparition des ions superoxyde. La mesure par tests in-gel de l’activité de la superoxyde dismutase supporte l’implication d’un homodimère de FeSOD dont l’activité augmente fortement au bout de 12 et jusqu’à 48 h d’exposition au sel. Les gènes Fe-Sod1 et csApx1, isolés d’une banque d’ADNc de P. pinaster, voient leur expression augmenter au bout de 12 h et jusqu’à 24 h de traitement.• Les résultats montrent que de fortes concentrations de sels provoquent un stress oxydatif dans les cellules de P. pinaster malgré l’induction de réponses antioxydantes et suggèrent l’implication des ERO dans les voies de transduction du stress salin.

Arabidopsis Squalene Epoxidase 3 (SQE3) Complements SQE1 and Is Important for Embryo Development and Bulk Squalene Epoxidase Activity

The existence of multigenic families in the mevalonate pathway suggests divergent functional roles for pathway components involved in the biosynthesis of plant sterols. Squalene epoxidases (SQEs) are key components of this pathway, and Squalene Epoxidase 1 (SQE1) has been identified as a fundamental enzyme in this biosynthetic step. In the present work, we extended the characterization of the remaining SQE family members, phylogenetically resolving between true SQEs and a subfamily of SQE-like proteins that is exclusive to Brassicaceae. Functional characterization of true SQE family members, Squalene Epoxidase 2 (SQE2) and Squalene Epoxidase 3 (SQE3), indicates that SQE3, but not SQE2, contributes to the bulk SQE activity in Arabidopsis, with sqe3-1 mutants accumulating squalene and displaying sensitivity to terbinafine. We genetically demonstrated that SQE3 seems to play a particularly significant role in embryo development. Also, SQE1 and SQE3 both localize in the endoplasmic reticulum, and SQE3 can functionally complement SQE1. Thus, SQE1 and SQE3 seem to be two functionally unequal redundant genes in the promotion of plant SQE activity in Arabidopsis.

A Strategy for the Identification of New Abiotic Stress Determinants in Arabidopsis Using Web-Based Data Mining and Reverse Genetics

Since the sequencing of the Arabidopsis thaliana genome in 2000, plant researchers have faced the complex challenge of assigning function to thousands of genes. Functional discovery by in silico prediction or homology search resolved a significant number of genes, but only a minor part has been experimentally validated. Arabidopsis entry into the post-genomic era signified a massive increase in high-throughput approaches to functional discovery, which have since become available through publicly-available web-based resources. The present work focuses on an easy and straightforward strategy that couples data-mining to reverse genetics principles, to allow for the identification of new abiotic stress determinant genes. The strategy explores systematic microarray-based transcriptomics experiments, involving Arabidopsis abiotic stress responses. An overview of the most significant resources and databases for functional discovery in Arabidopsis is presented. The successful application of the outlined strategy is illustrated by the identification of a new abiotic stress determinant gene, HRR, which displays a heat-stress-related phenotype after a loss-of-function reverse genetics approach.