Andrés A. Borges

Cloning and Biochemical Characterization of ToFZY, a Tomato Gene Encoding a Flavin Monooxygenase Involved in a Tryptophan-dependent Auxin Biosynthesis Pathway

Indole-3-acetic acid (IAA), the main endogenous auxin, has been known for decades to be a key regulator for plant growth and development. Multiple routes have been proposed for IAA biosynthesis but physiologic roles or relevance of the different routes are still unclear. Recently, four members of the Arabidopsis thaliana YUC gene family have been implicated in an additional requirement of IAA involved in floral organ and vascular tissue formation. The loss-of-function yuc1yuc4 double mutants in Arabidopsis displayed phenotypes similar to the previously described loss-of-function floozy mutants in petunia (fzy). Moreover, it has been demonstrated that YUC1 encodes a flavin monooxygenase (FMO) that catalyzes a rate-limiting step of a tryptophan-dependent auxin biosynthesis pathway: the conversion of tryptamine to N-hydroxyl-tryptamine. Here we report on the genetic study of ToFZY, the putative tomato ortholog of YUC4 and FZY, including gene and cDNA sequence comparison and a preliminary expression analysis. In addition, we describe a novel conserved amino acid motif that may be considered a hallmark potentially useful for the identification of new YUC-like FMOs. We also demonstrate that ToFZY encodes a protein with the same enzymatic activity as YUC1. Finally, we provide evidence suggesting that the ToFZY gene belongs to a multigenic family whose members may exhibit a temporal and spatial specialization similar to that described in A. thaliana.

Gene structure and spatiotemporal expression profile of tomato genes encoding YUCCA-like flavin monooxygenases: the ToFZY gene family.

The flavin monooxygenases (FMO) encoded by plant YUCCA genes are thought to catalyze a rate-limiting step in the tryptamine pathway for indole-3-acetic acid biosynthesis. Recent experiments with different plant models have indicate that YUCCA genes play essential roles in growth and development through their contribution to the local pool of free auxin. In this study we have characterized five new genes that encode YUCCA-like FMOs in the tomato genome (ToFZY2 to ToFZY6), including gene structure, conserved motifs and phylogenetic analyses. As a first step towards clarifying the individual functions of ToFZY genes, we have used quantitative real-time RT-PCR to conduct a systematic comparison of the steady-state mRNA levels of 6 ToFZY genes, in 33 samples representing major organs and the entire tomato life cycle. We followed an absolute quantification strategy which allowed us to cross-compare transcript levels among different ToFZY genes in a given spatiotemporal coordinate. Our results indicate that expression of ToFZY genes is temporally and spatially regulated, and that the distinctive expression pattern of each ToFZY gene partially overlaps with other members of the multigenic family. We compare our data with previous results in other plant species and make some predictions about the role of tryptamine pathway in tomato growth and development.

Priming crops against biotic and abiotic stresses: MSB as a tool for studying mechanisms

Biotic and abiotic stresses are the main problems affecting agricultural losses. Consequently, understanding the mechanisms underlying plant resistance or tolerance helps us to develop fruitful new agricultural strategies. These will allow us to face the challenges of producing food for a growing human population in a sustainable and environmentally friendly way. To compensate for their sessile life and face a broad range of biotic and abiotic stresses, plants have evolved a wide range of survival and adaptation strategies. Amongst them, higher plants are capable of inducing some stress “memory,” or “stress imprinting.” Bruce et al. (2007) define stress imprinting as genetic or biochemical modifications induced by a first stress exposure that leads to enhanced resistance to a later stress. This phenomenon also known as “priming” results in a faster and stronger induction of basal resistance mechanisms upon subsequent pathogen attack, or greater tolerance against abiotic stresses (Pastor et al., 2013). Basal resistance by itself is too weak to protect against virulent pathogens, since it constitutes a residual level of resistance after immune suppression by the pathogen through co-evolution (Walters and Heil, 2007; Conrath, 2011). However, Ahmad et al. (2010) proposed that priming-inducing stimuli can provide more effective basal resistance, particularly when an earlier defense response precedes immune suppression by the invading pathogen. Following perception of microbe-associated molecular patterns (MAMPs), recognition of pathogen-derived effectors or colonization by beneficial microbes, priming can also be induced by treatment with some natural or synthetic compounds or even by wounding (Conrath, 2011). Through priming plants are able to induce responses to a range of biotic and abiotic stresses, providing low-cost protection in relatively high stress-pressure conditions. Despite priming phenomena having been widely described, the molecular mechanisms of defense priming are still unclear. Such techniques are now starting to emerge as a promising alternative for sustainable modern pest management in the field, since some pesticides have been shown to actually exert their known plant health- and yield-increasing effects through priming (Beckers and Conrath, 2007). From an ecological point of view, the benefits of priming are clear: rather than leading to the costly and potentially wasteful activation of defenses, a metabolic state of alert is induced after an initial infection, enabling a rapid intense resistance response to subsequent attacks. Thus, this strategy appears promising for crop protection purposes (Walters and Heil, 2007).

Menadione Sodium Bisulphite (MSB) enhances the resistance response of tomato, leading to repel mollusc pests

BACKGROUND Snails and slugs are terrestrial gastropods representing an important biotic stress that adversely affects crop yields. These pests are typically controlled with molluscicides, which produce pollution and toxicity and further induce the evolution of resistance mechanisms, making pest management even more challenging. In our work, we have assessed the efficacy of two different plant defence activators, menadione sodium bisulphite (MSB) and 1,2,3-benzothiadiazole-7-thiocarboxylic acid S-methyl ester (BTH), as inducers of resistance mechanisms of the model plant for defence, Solanum lycopersicum, against the generalist mollusc Theba grasseti (Helicidae). The study was designed to test the feeding behaviour and choice of snails, and also to analyse the expression profile of different genes specifically involved in defence against herbivores and wounds. RESULTS Our data suggest that, through the downregulation of the terpene volatile genes and the production of proteinase inhibitors, treated MSB plants may be less apparent to herbivores that use herbivore-induced plant volatiles for host location. By contrast, BTH was not effective in the treatment of the pest, probably owing to an antagonistic effect derived from the induction of both salicylic-acid-dependent and jasmonic-acid-dependent pathways. CONCLUSIONS This information is crucial to determine the genetic basis of the choice of terrestrial gastropod herbivores in tomato, providing valuable insight into how the plant defence activators could control herbivore pests in plants. Our work not only reports for the first time the interaction between tomato and a mollusc pest but also presents the action of two plant defence inductors that seems to produce opposed responses by inducing resistance mechanisms through different defence pathways.