Wim H. Vriezen

The Solanum lycopersicum auxin response factor 7 (SlARF7) regulates auxin signaling during tomato fruit set and development

Auxin response factors (ARFs) are encoded by a gene family of transcription factors that specifically control auxin-dependent developmental processes. A tomato ARF gene, homologous to Arabidopsis NPH4/ARF7 and therefore designated as Solanum lycopersicum ARF7 (SlARF7), was found to be expressed at a high level in unpollinated mature ovaries. More detailed analysis of tomato ovaries showed that the level of SlARF7 transcript increases during flower development, remains at a constant high level in mature flowers, and is down-regulated within 48 h after pollination. Transgenic plants with decreased SlARF7 mRNA levels formed seedless (parthenocarpic) fruits. These fruits were heart-shaped and had a rather thick pericarp due to increased cell expansion, compared with the pericarp of wild-type fruits. The expression analysis, together with the parthenocarpic fruit phenotype of the transgenic lines, suggests that, in tomato, SlARF7 acts as a negative regulator of fruit set until pollination and fertilization have taken place, and moderates the auxin response during fruit growth.

The role of auxin and gibberellin in tomato fruit set

The initiation of tomato fruit growth, fruit set, is very sensitive to environmental conditions. Therefore, an understanding of the mechanisms that regulate this process can facilitate the production of this agriculturally valuable fruit crop. Over the years, it has been well established that tomato fruit set depends on successful pollination and fertilization, which trigger the fruit developmental programme through the activation of the auxin and gibberellin signalling pathways. However, the exact role of each of these two hormones is still poorly understood, probably because only few of the signalling components involved have been identified so far. Recent research on fruit set induced by hormone applications has led to new insights into hormone biosynthesis and signalling. The aim of this review is to consolidate the current knowledge on the role of auxin and gibberellin in tomato fruit set.

Exploring genetic variation in the tomato (Solanum section Lycopersicon) clade by whole-genome sequencing.

We explored genetic variation by sequencing a selection of 84 tomato accessions and related wild species representative of the Lycopersicon, Arcanum, Eriopersicon and Neolycopersicon groups, which has yielded a huge amount of precious data on sequence diversity in the tomato clade. Three new reference genomes were reconstructed to support our comparative genome analyses. Comparative sequence alignment revealed group-, species- and accession-specific polymorphisms, explaining characteristic fruit traits and growth habits in the various cultivars. Using gene models from the annotated Heinz 1706 reference genome, we observed differences in the ratio between non-synonymous and synonymous SNPs (dN/dS) in fruit diversification and plant growth genes compared to a random set of genes, indicating positive selection and differences in selection pressure between crop accessions and wild species. In wild species, the number of single-nucleotide polymorphisms (SNPs) exceeds 10 million, i.e. 20-fold higher than found in most of the crop accessions, indicating dramatic genetic erosion of crop and heirloom tomatoes. In addition, the highest levels of heterozygosity were found for allogamous self-incompatible wild species, while facultative and autogamous self-compatible species display a lower heterozygosity level. Using whole-genome SNP information for maximum-likelihood analysis, we achieved complete tree resolution, whereas maximum-likelihood trees based on SNPs from ten fruit and growth genes show incomplete resolution for the crop accessions, partly due to the effect of heterozygous SNPs. Finally, results suggest that phylogenetic relationships are correlated with habitat, indicating the occurrence of geographical races within these groups, which is of practical importance for Solanum genome evolution studies.

Perspectives on deciphering mechanisms underlying plant heat stress response and thermotolerance

Global warming is a major threat for agriculture and food safety and in many cases the negative effects are already apparent. The current challenge of basic and applied plant science is to decipher the molecular mechanisms of heat stress response (HSR) and thermotolerance in detail and use this information to identify genotypes that will withstand unfavorable environmental conditions. Nowadays X-omics approaches complement the findings of previous targeted studies and highlight the complexity of HSR mechanisms giving information for so far unrecognized genes, proteins and metabolites as potential key players of thermotolerance. Even more, roles of epigenetic mechanisms and the involvement of small RNAs in thermotolerance are currently emerging and thus open new directions of yet unexplored areas of plant HSR. In parallel it is emerging that although the whole plant is vulnerable to heat, specific organs are particularly sensitive to elevated temperatures. This has redirected research from the vegetative to generative tissues. The sexual reproduction phase is considered as the most sensitive to heat and specifically pollen exhibits the highest sensitivity and frequently an elevation of the temperature just a few degrees above the optimum during pollen development can have detrimental effects for crop production. Compared to our knowledge on HSR of vegetative tissues, the information on pollen is still scarce. Nowadays, several techniques for high-throughput X-omics approaches provide major tools to explore the principles of pollen HSR and thermotolerance mechanisms in specific genotypes. The collection of such information will provide an excellent support for improvement of breeding programs to facilitate the development of tolerant cultivars. The review aims at describing the current knowledge of thermotolerance mechanisms and the technical advances which will foster new insights into this process.

Developmental and heat stress-regulated expression of HsfA2 and small heat shock proteins in tomato anthers

The high sensitivity of male reproductive cells to high temperatures may be due to an inadequate heat stress response. The results of a comprehensive expression analysis of HsfA2 and Hsp17-CII, two important members of the heat stress system, in the developing anthers of a heat-tolerant tomato genotype are reported here. A transcriptional analysis at different developmental anther/pollen stages was performed using semi-quantitative and real-time PCR. The messengers were localized using in situ RNA hybridization, and protein accumulation was monitored using immunoblot analysis. Based on the analysis of the gene and protein expression profiles, HsfA2 and Hsp17-CII are finely regulated during anther development and are further induced under both short and prolonged heat stress conditions. These data suggest that HsfA2 may be directly involved in the activation of protection mechanisms in the tomato anther during heat stress and, thereby, may contribute to tomato fruit set under adverse temperatures.

Submergence induces expansin gene expression in flooding-tolerant Rumex palustris and not in flooding-intolerant R. acetosa

Abstract. Ethylene-enhanced leaf elongation upon submergence is part of the survival mechanism of Rumex palustris Sm. plants that grow in frequently flooded areas. Other Rumex species, like R. acetosa L., do not possess this ability and can therefore only survive in habitats that are not frequently inundated. Expansins are proteins that induce extension of isolated cell walls, and therefore might play a role in the stimulation of petiole elongation, also in Rumex. We report here on the identification of several gene sequences encoding for α-expansins in R. palustris and R. acetosa plants. The pattern of transcript accumulation of one of these genes, Rp-EXP1, could be correlated with the pattern of leaf elongation in R. palustris after submergence or ethylene treatment. Induction of expansin gene activity was not found in R. acetosa upon these treatments, indicating that ethylene induces the expression of expansin genes in leaves of species that exhibit flooding-induced shoot elongation.

The Solanum lycopersicum AUXIN RESPONSE FACTOR 7 (SlARF7) mediates cross-talk between auxin and gibberellin signalling during tomato fruit set and development

Transgenic tomato plants (Solanum lycopersicum L.) with reduced mRNA levels of AUXIN RESPONSE FACTOR 7 (SlARF7) form parthenocarpic fruits with morphological characteristics that seem to be the result of both increased auxin and gibberellin (GA) responses during fruit growth. This paper presents a more detailed analysis of these transgenic lines. Gene expression analysis of auxin-responsive genes show that SlARF7 may regulate only part of the auxin signalling pathway involved in tomato fruit set and development. Also, part of the GA signalling pathway was affected by the reduced levels of SlARF7 mRNA, as morphological and molecular analyses display similarities between GA-induced fruits and fruits formed by the RNAi SlARF7 lines. Nevertheless, the levels of GAs were strongly reduced compared with that in seeded fruits. These findings indicate that SlARF7 acts as a modifier of both auxin and gibberellin responses during tomato fruit set and development.

Abscisic acid levels in tomato ovaries are regulated by LeNCED1 and SlCYP707A1

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.

Temperature stress differentially modulates transcription in meiotic anthers of heat-tolerant and heat-sensitive tomato plants

BackgroundFluctuations in temperature occur naturally during plant growth and reproduction. However, in the hot summers this variation may become stressful and damaging for the molecular mechanisms involved in proper cell growth, impairing thus plant development and particularly fruit-set in many crop plants. Tolerance to such a stress can be achieved by constitutive gene expression or by rapid changes in gene expression, which ultimately leads to protection against thermal damage. We have used cDNA-AFLP and microarray analyses to compare the early response of the tomato meiotic anther transcriptome to moderate heat stress conditions (32°C) in a heat-tolerant and a heat-sensitive tomato genotype. In the light of the expected global temperature increases, elucidating such protective mechanisms and identifying candidate tolerance genes can be used to improve breeding strategies for crop tolerance to heat stress.ResultsThe cDNA-AFLP analysis shows that 30 h of moderate heat stress (MHS) alter the expression of approximately 1% of the studied transcript-derived fragments in a heat-sensitive genotype. The major effect is gene down-regulation after the first 2 h of stress. The microarray analysis subsequently applied to elucidate early responses of a heat-tolerant and a heat-sensitive tomato genotype, also shows about 1% of the genes having significant changes in expression after the 2 h of stress. The tolerant genotype not only reacts with moderate transcriptomic changes but also exhibits constitutively higher expression levels of genes involved in protection and thermotolerance.ConclusionIn contrast to the heat-sensitive genotype, the heat-tolerant genotype exhibits moderate transcriptional changes under moderate heat stress. Moreover, the heat-tolerant genotype also shows a different constitutive gene expression profile compared to the heat-sensitive genotype, indicating genetic differences in adaptation to increased temperatures. In the heat-tolerant genotype, the majority of changes in gene expression is represented by up-regulation, while in the heat-sensitive genotype there is a general trend to down-regulate gene expression upon MHS. The putative functions associated with the genes identified by cDNA-AFLP or microarray indicate the involvement of heat shock, metabolism, antioxidant and development pathways. Based on the observed differences in response to MHS and on literature sources, we identified a number of candidate transcripts involved in heat-tolerance.

Isolation and characterization of male-germ-cell transcripts in Nicotiana tabacum

Abstract. Until recently, little knowledge existed about the molecular mechanisms regulating male gamete development. This was mainly due to the low transcriptional activity and the cellular inaccessibility of the generative and sperm cells that are enclosed by the vegetative cell in pollen. In order to study sperm cell development and possible preferential fusion during double fertilization, we have constructed a cDNA library of mRNA isolated from pure tobacco sperm cells. An initial screen of 396 clones from this library has yielded 2 cDNAs representing sperm-cell-expressed transcripts, designated NtS1 and NtS2. A preliminary characterization of these two clones showed that they accumulate in both the generative and sperm cells (i.e. the male gamete) indicating that gene expression programs between these two cell types overlap. In addition, we found that NtS1 codes for a polygalacturonase suggesting a role for this enzyme in wall degradation during differentiation of the male germ cells in tobacco. Together, these results show that with the construction of this sperm-cell cDNA library we now have a powerful tool to investigate male gamete development and function.