Research progresses on <Emphasis Type="Italic">GH3s</Emphasis>, one family of primary auxin-responsive genes

Auxin plays a very important role in plant growth and development. Those genes that are specifically induced by auxin within minutes of exposure to the hormone are referred to as early/primary auxin-responsive genes, mainly including the auxin/indole-3-acetic acid (Aux/IAA), the small auxin-up RNA (SAUR), and the GH3 gene families. So far, GH3 genes have been identified in various plant species including soybean, Arabidopsis, rice, tobacco, pungent pepper, sweet orange, pine, and moss. Twenty members of GH3 family were identified in Arabidopsis and these genes were classified into three groups (Group I–III) based on their sequence similarities and substrate specificities. GH3s belong to acyl adenylate-forming firefly luciferase superfamily and can catalyze adenylation of specific substrates. Group I adenylates jasmonic acid (JA), and Group II adenylates indole-3-acetic acid (IAA) and salicylic acid (SA), respectively. Because of the presence of Auxin-Responsive Elements (AuxRE) in the GH3s’ promoter regions, Auxin Response Factors (ARFs) are able to bind to the AuxRE and regulate expression of some GH3s, which in turn modulate the auxin homeostasis. Identification of GH3 mutants in Arabidopsis reveals the function of GH3s in hypocotyl elongation under different light conditions, root growth, stress adaptation, sensitivity to MeJA, or susceptibility to P. syringae. Taken together, GH3s may be linkers among auxin, JA, SA and light signal transduction pathways.     

Co-suppression of the <Emphasis Type="Italic">EIN2</Emphasis>-homology gene <Emphasis Type="Italic">LeEIN2</Emphasis> inhibits fruit ripening and reduces ethylene sensitivity in tomato

EIN2 (ethylene insensitive 2) is a very important component in the ethylene signal transduction pathway. Recently, the genomic DNA and full-length cDNA of LeEIN2 (tomato EIN2) have been isolated in our laboratory. To reveal the function of LeEIN2, transgenic tomato plants with reduced expression levels of LeEIN2 were produced. The fruit ripening and expressions of ripening-related genes encoding polygalacturonase and TomLoxB were inhibited in the LeEIN2-silenced transgenic plants compared to the wild-type Ailsa Craig. In the seedling ethylene response assay, the transgenic tomato plants with reduced LeEIN2 expression exhibited ethylene insensitivity. These results indicate that LeEIN2 plays a critical role in regulating tomato fruit ripening and is a positive regulator in ethylene signal transduction pathway.     

Characterization and phylogenetic analysis of environmental stress-responsive <Emphasis Type="Italic">SAP</Emphasis> gene family encoding A20/AN1 zinc finger proteins in tomato

Characterization of genes responsive to stress is important for efforts on improving stress tolerance of plants. To address components involved in stress tolerance of tomato (Solanum lycopersicum), a stress-responsive gene family encoding A20/AN1 zinc finger proteins was characterized. In the present study, 13 members of this gene family were cloned from tomato cultivar Pusa Ruby and named as Stress Associated Protein (SAP) genes. Out of 13 genes, 12 have been mapped on their respective chromosomes. Expression of these genes in response to cold, heat, salt, desiccation, wounding, abscisic acid, oxidative and submergence stresses was analysed. All tomato SAP genes were found to be responsive to one or other type of environmental stress. The phylogenetic analysis of these genes, along with their orthologs from Solanaceae species suggests the presence of a common set of SAP genes in the studied Solanaceae species. The present study characterizes a SAP gene family, which encodes A20/AN1 zinc finger containing proteins from tomato for the first time. Genes showing high expression in response to a particular stress can be exploited for improving stress tolerance of tomato and other Solanaceae members. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

<Emphasis Type="Italic">Gr</Emphasis> and <Emphasis Type="Italic">hp</Emphasis>-<Emphasis Type="Italic">1</Emphasis> tomato mutants unveil unprecedented interactions between arbuscular mycorrhizal symbiosis and fruit ripening

Main conclusion

Systemic responses to an arbuscular mycorrhizal fungus reveal opposite phenological patterns in two tomato ripening mutants depending whether ethylene or light reception is involved. The availability of tomato ripening mutants has revealed many aspects of the genetics behind fleshy fruit ripening, plant hormones and light signal reception. Since previous analyses revealed that arbuscular mycorrhizal symbiosis influences tomato berry ripening, we wanted to test the hypothesis that an interplay might occur between root symbiosis and fruit ripening. With this aim, we screened seven tomato mutants affected in the ripening process for their responsiveness to the arbuscular mycorrhizal fungus Funneliformis mosseae. Following their phenological responses we selected two mutants for a deeper analysis: Green ripe (Gr), deficient in fruit ethylene perception and high-pigment-1 (hp-1), displaying enhanced light signal perception throughout the plant. We investigated the putative interactions between ripening processes, mycorrhizal establishment and systemic effects using biochemical and gene expression tools. Our experiments showed that both mutants, notwithstanding a normal mycorrhizal phenotype at root level, exhibit altered arbuscule functionality. Furthermore, in contrast to wild type, mycorrhization did not lead to a higher phosphate concentration in berries of both mutants. These results suggest that the mutations considered interfere with arbuscular mycorrhiza inducing systemic changes in plant phenology and fruits metabolism. We hypothesize a cross talk mechanism between AM and ripening processes that involves genes related to ethylene and light signaling.

     

Abscisic acid levels in tomato ovaries are regulated by <Emphasis Type="Italic">LeNCED1</Emphasis> and <Emphasis Type="Italic">SlCYP707A1</Emphasis>

Although the hormones, gibberellin and auxin, are known to play a role in the initiation of fruits, no such function has yet been demonstrated for abscisic acid (ABA). However, ABA signaling and ABA responses are high in tomato (Solanum lycopersicum L.) ovaries before pollination and decrease thereafter (Vriezen et al. in New Phytol 177:60–76, 2008). As a first step to understanding the role of ABA in ovary development and fruit set in tomato, we analyzed ABA content and the expression of genes involved in its metabolism in relation to pollination. We show that ABA levels are relatively high in mature ovaries and decrease directly after pollination, while an increase in the ABA metabolite dihydrophaseic acid was measured. An important regulator of ABA biosynthesis in tomato is 9-cis-epoxy-carotenoid dioxygenase (LeNCED1), whose mRNA level in ovaries is reduced after pollination. The increased catabolism is likely caused by strong induction of one of four newly identified putative (+)ABA 8′-hydroxylase genes. This gene was named SlCYP707A1 and is expressed specifically in ovules and placenta. Transgenic plants, overexpressing SlCYP707A1, have reduced ABA levels and exhibit ABA-deficient phenotypes suggesting that this gene encodes a functional ABA 8′-hydroxylase. Gibberellin and auxin application have different effects on the LeNCED1 and SlCYP707A1 gene expression. The crosstalk between auxins, gibberellins and ABA during fruit set is discussed.     

Functional analysis of <Emphasis Type="Italic">SlEZ1</Emphasis> a tomato <Emphasis Type="Italic">Enhancer of zeste</Emphasis> (<Emphasis Type="Italic">E(z))</Emphasis> gene demonstrates a role in flower development

The Enhancer of Zeste (E(z)) Polycomb group (PcG) proteins, which are encoded by a small gene family in Arabidopsis thaliana, have been shown to participate to the control of flowering and seed development. For the time being, little is known about the function of these proteins in other plants. In tomato E(z) proteins are encoded by at least two genes namely SlEZ1 and SlEZ2 while a third gene, SlEZ3, is likely to encode a truncated non-functional protein. The analysis of the corresponding mRNA demonstrates that these two genes are differentially regulated during plant and fruit development. We also show that SlEZ1 and SlEZ2 are targeted to the nuclei. These results together with protein sequence analysis makes it likely that both proteins are functional E(z) proteins. The characterisation of SlEZ1 RNAi lines suggests that although there might be some functional redundancy between SlEZ1 and SlEZ2 in most plant organs, the former protein is likely to play specific function in flower development.     

Prediction of auxin response elements based on data fusion in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The plant hormone “auxin” is a key regulator of plant development and environmental responses. Many genes in Arabidopsis thaliana are known to be up-regulated in response to auxin. Auxin response factors activate or repress the expression of genes by binding at their promoter regions within auxin response elements (AuxRE) that are key regulatory cis-acting motives. Therefore, the identification of auxin-response elements is among the most important issues to understand the auxin regulation mechanisms. Thus, searching the TGTCTC motif is an unreliable method to identify AuxRE because many AuxRE variants may also be functional. In the present study, we perform an In-silico prediction of AuxREs in promoters of primary auxin responsive genes. We exploit microarray data of auxin response in Arabidopsis thaliana seedlings, in order to provide biological annotation to AuxRE. We apply a data fusion method based on the combined use of evidence theory and fuzzy sets to scan upstream sequences of response genes.     

Pro-apoptotic cell death genes, <Emphasis Type="Italic">hid</Emphasis> and <Emphasis Type="Italic">reaper</Emphasis>, from the tephritid pest species, <Emphasis Type="Italic">Anastrepha suspensa</Emphasis>

Pro-apoptotic proteins from the reaper, hid, grim (RHG) family are primary regulators of programmed cell death in Drosophila due to their antagonistic effect on inhibitor of apoptosis (IAP) proteins, thereby releasing IAP-inhibition of caspases that effect apoptosis. Using a degenerate PCR approach to conserved domains from the 12 Drosophila species, we have identified the first reaper and hid orthologs from a tephritid, the Caribfly Anastrepha suspensa. As-hid is the first identified non-drosophilid homolog of hid, and As-rpr is the second non-drosophilid rpr homolog. Both genes share more than 50% amino acid sequence identity with their Drosophila homologs, suggesting that insect pro-apoptotic peptides may be more conserved than previously anticipated. Importantly, both genes encode the conserved IBM and GH3 motifs that are key for IAP-inhibition and mitochondrial localization. Functional verification of both genes as cell death effectors was demonstrated by cell death assays in A. suspensa embryonic cell culture, as well as in heterologous Drosophila melanogaster S2 cells. Notably, heterologous cell death activity was found to be higher for Anastrepha genes than their Drosophila counterparts. In common with the Drosophila cognates, As-hid and As-rpr negatively regulated the Drosophila inhibitor of apoptosis (DIAP1) gene to promote apoptosis, and both genes when used together effected increased cell death activity, indicating a co-operative function for As-hid and As-rpr. We show that these tephritid cell death genes are functional and potent as cell death effectors, and could be used to design improved transgenic lethality systems for insect population control.     

Distribution of <Emphasis Type="Italic">Hordoindoline</Emphasis> genes in the genus <Emphasis Type="Italic">Hordeum</Emphasis>

Hordoindoline (Hin) genes, which are known to comprise Hina, Hinb-1, and Hinb-2, are associated with grain hardness in barley. However, the interspecific variation in the Hin genes in the genus Hordeum has not been studied in detail. We examined the variation in Hin genes and used it to infer the phylogenetic relationships between the genes found in two H. vulgare subspecies (cultivated barley and H. vulgare subsp. spontaneum) and 10 wild relatives (H. bogdanii, H. brachyantherum, H. bulbosum, H. chilense, H. comosum, H. marinum, H. murinum, H. patagonicum, H. pusillum, and H. roshevitzii). The Hina and Hinb genes of these species were amplified by PCR. We found two Hinb genes in three wild species (H. bogdanii, H. brachyantherum, and H. roshevitzii) and preliminarily named them Hinb-A and Hinb-B. Cluster analysis showed that the 17 Hinb genes present in Hordeum formed two distinct clusters (named A and B). Seven Hinb genes were included in Cluster-A, and 10 Hinb genes were included in Cluster-B. All Hinb-A genes were included in Cluster-A, while all of the Hinb-B genes were included in Cluster-B. In contrast, the Hinb-1 and Hinb-2 genes in H. vulgare were included in Cluster-B. These results suggest that the Hinb genes duplicated during the early stages of diversification in the genus Hordeum. On the other hand, the Hinb-1 and Hinb-2 genes in H. vulgare seem to have been generated by a duplication of the Hinb gene after the split of the lineages leading to H. vulgare and H. bulbosum.