Genome-wide investigation and expression analysis suggest diverse roles of auxin-responsive <Emphasis Type="Italic">GH3</Emphasis> genes during development and response to different stimuli in tomato (<Emphasis Type="Italic">Solanum lycopersicum</Emphasis>)

In plants, auxin-mediated responses are regulated by diverse proteins. One such class of proteins, i.e. GH3, is involved in the conjugation of IAA to amino acids and provides a negative feedback loop to control auxin homoeostasis. In order to have a better understanding of the mechanism of the auxin action, 15 genes encoding GH3 members were identified using existing EST databases of tomato. Their orthologs were identified from tobacco, potato, N. benthemiana, pepper, and petunia. Phylogenetic analysis of AtGH3, SlGH3, and their Solanaceae orthologs provided insights into various orthologous relationships among these proteins. These genes were found to be responsive to a variety of signals including, phytohormones and environmental stresses. Analysis of AuxRE elements in their promoters showed variability in the sequence as well as number of this element. Up-regulation of only 11 SlGH3 genes, in response to exogenous auxin, suggested possible relationship between the diversity in the sequence and number of AuxRE element with the auxin inducibility. Expression analysis of SlGH3 genes in different vegetative and reproductive tissues/stages suggested limited or no role for most of the SlGH3 genes at the initiation of fruit ripening. However, up-regulation of SlGH3-1 and -2 at the onset of fruit ripening indicates that these genes could have a role in fruit ripening. The present study characterizes GH3 gene family of tomato and its evolutionary relationship with members of this family from other Solanaceae species and Arabidopsis. It could help in the identification of GH3 genes and revelation of their function during vegetative/reproductive development stages from other Solanaceae members.     

<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.

     

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.     

Regulation of the expression of lipoxygenase genes in <Emphasis Type="Italic">Prunus persica</Emphasis> fruit ripening

Three genes of the lipoxygenase (LOX) family in peach (Prunus persica var. compressa cv. Ruipan 4) were cloned, and their expression patterns during fruit ripening were analyzed using real-time quantitative PCR. All of the three peach LOX genes had been expressed during fruit ripening; however, their expression patterns were significantly different. During the normal ripening of peach fruits, the expression levels of PpLox1, PpLox2 and PpLox3 increased in varying degrees accompanying upsurge of ethylene evolution. After treated by methyl jasmonic acid (MeJA), the peak of ethylene releasing occurred in advance, and the declining rate of fruit hardness was accelerated, the expression level of the three peach LOX genes in fruits markedly enhanced at the early stage of storage, but significantly decreased at the late storage stage. So, it could be suggested that all three LOXs relate to fruit ripening; however, their functions might be different. PpLox1 expression increase along with the upsurge of ethylene evolution in both control and MeJA-treated peach fruits suggested that PpLox1 probably played a major role in the peach fruit ripening. Expression peak of PpLox2 appeared at the 1 DAH (days after harvest) in both control and MeJA-treated peach fruits, while obvious changes in ethylene evolution and fruit hardness was not observed, which suggested that the rise of PpLox2 expression can be induced by certain stimulation related to ripening, such as harvesting stress and MeJA treatment. The expression of PpLox3 kept a lower level in the natural ripening fruits, whereas raced up at the early stage of storage in the fruits treated with MeJA, which indicated that PpLox3 was expressed inductively and had minor roles during the normal ripening of peach fruits, but when encountered with external stimulation, its expression level would rapidly enhance and accelerate the ripening of peach fruit.     

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.     

Cytokinin and Ethylene Affect Auxin Transport-Dependent Rhizogenesis in Hypocotyls of Common Ice Plant (<Emphasis Type="Italic">Mesembryanthemum crystallinum</Emphasis> L<Emphasis Type="Italic">.</Emphasis>)

Hypocotyl explants of Mesembryanthemum crystallinum regenerated roots when cultured vertically with either the apical end (AE) or basal end (BE) in media containing indole-3-acetic acid (IAA). IAA alone induced roots regularly from the basal end of the explants, either from the cut surface immersed in the medium or from the opposite side. The inhibitors of auxin efflux carriers, α-naphthylphthalamic acid (NPA) and 2,3,5-triiodobenzoic acid (TIBA), inhibited rhizogenesis only from AE-cultured explants, indicating the role of polar auxin transport in root regeneration in this system. Cytokinin (zeatin, kinetin, BAP) added to auxin-containing medium reduced rhizogenesis from the explants maintained with BE and AE and additionally changed the IAA-induced pattern of rooting in AE-cultured explants by favoring rooting from the apical end and middle part of the hypocotyl with its concomitant reduction from the basal end. The addition of kinetin did not influence the content of IAA in the explants maintained with AE, suggesting that the cytokinin effect on root patterning was not dependent on auxin biosynthesis. Kinetin, however, strongly enhanced ethylene production. The importance of ethylene in regulating PAT-dependent rhizogenesis was tested by using an ethylene antagonist AgNO₃, an inhibitor of ethylene synthesis aminoethoxyvinylglycine (AVG), and a precursor of ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC). AgNO₃ applied together with IAA or with IAA and kinetin strongly reduced the production of ethylene, inhibited rhizogenesis, and induced nonregenerative callus from BE, suggesting the need for ethylene signaling to elicit the rhizogenic action of auxin. A reduction of rhizogenesis and decrease of ethylene biosynthesis was also caused by AVG. In addition, AVG at 10 μM reversed the effect of cytokinin on root patterning, resulting in roots emerging only from BE on the medium with IAA and kinetin. Conversely, ACC at 200 μM markedly enhanced the production of ethylene and partly mimicked the effect of cytokinin when applied with IAA alone, thus confirming that in cultured hypocotyls of ice plant, cytokinin affects IAA-induced rhizogenesis through an ethylene-dependent pathway.     

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.     

Genomewide analysis of <Emphasis Type="Italic">ABCB</Emphasis>s with a focus on <Emphasis Type="Italic">ABCB1</Emphasis> and <Emphasis Type="Italic">ABCB19</Emphasis> in <Emphasis Type="Italic">Malus domestica</Emphasis>

The B subfamily of ATP-binding cassette (ABC) proteins (ABCB) plays a vital role in auxin efflux. However, no systematic study has been done in apple. In this study, we performed genomewide identification and expression analyses of the ABCB family in Malus domestica for the first time. We identified a total of 25 apple ABCBs that were divided into three clusters based on the phylogenetic analysis. Most ABCBs within the same cluster demonstrated a similar exon–intron organization. Additionally, the digital expression profiles of ABCB genes shed light on their functional divergence. ABCB1 and ABCB19 are two well-studied auxin efflux carrier genes, and we found that their expression levels are higher in young shoots of M106 than in young shoots of M9. Since young shoots are the main source of auxin synthesis and auxin efflux involves in tree height control. This suggests that ABCB1 and ABCB19 may also take a part in the auxin efflux and tree height control in apple.