Faithful editing of a tomato-specific mRNA editing site in transgenic tobacco chloroplasts

RNA editing sites and their site-specific trans-acting recognition factors are thought to have coevolved. Hence, evolutionary loss of an editing site by a genomic mutation is normally followed by the loss of the specific recognition factor for this site, due to the absence of selective pressure for its maintenance. Here, we have tested this scenario for the only tomato-specific plastid RNA editing site. A single C-to-U editing site in the tomato rps12 gene is absent from the tobacco and nightshade plastid genomes, where the presence of a genomic T nucleotide obviates the need for editing of the rps12 mRNA. We have introduced the tomato editing site into the tobacco rps12 gene by plastid transformation and find that, surprisingly, this heterologous site is efficiently edited in the transplastomic plants. This suggests that the trans-acting recognition factor for the rps12 editing site has been maintained, presumably because it serves another function in tobacco plastids. Bioinformatics analyses identified an editing site in the rpoB gene of tobacco and tomato whose sequence context exhibits striking similarity to that of the tomato rps12 editing site. This may suggest that requirement for rpoB editing resulted in maintenance of the rps12 editing activity or, alternatively, the pre-existing rpoB editing activity facilitated the evolution of a novel editing site in rps12.     

Elucidating the functional role of endoreduplication in tomato fruit development

Background

Endoreduplication is the major source of endopolyploidy in higher plants. The process of endoreduplication results from the ability of cells to modify their classical cell cycle into a partial cell cycle where DNA synthesis occurs independently from mitosis. Despite the ubiquitous occurrence of the phenomenon in eukaryotic cells, the physiological meaning of endoreduplication remains vague,although several roles during plant development have been proposed, mostly related to cell differentiation and cell size determination.

Scope

Here recent advances in the knowledge of endoreduplication and fruit organogenesis are reviewed, focusing on tomato (Solanum lycopersicum) as a model, and the functional analyses of endoreduplication-associated regulatory genes in tomato fruit are described.

Conclusions

The cyclin-dependent kinase inhibitory kinase WEE1 and the anaphase promoting complex activator CCS52A both participate in the control of cell size and the endoreduplication process driving cell expansion during early fruit development in tomato. Moreover the fruit-specific functional analysis of the tomato CDK inhibitor KRP1 reveals that cell size and fruit size determination can be uncoupled from DNA ploidy levels, indicating that endoreduplication acts rather as a limiting factor for cell growth. The overall functional data contribute to unravelling the physiological role of endoreduplication in growth induction of fleshy fruits.     

In silico prediction of proteins related to xyloglucan fucosyltransferases in Solanaceae genomes

Two independent studies have shown that the cell wall of pollen tubes from tobacco and tomato species contained fucosylated xyloglucan (XyG). These findings are intriguing as many reports have shown that XyG of somatic cells of these species is not fucosylated but instead is arabinosylated. In order to produce fucosylated XyG, plants must express a functional galactoside α-2-fucosyltransferase. Here, using a bioinformatics approach, we show that several candidate genes coding for XyG fucosyltransferases are present in the genome of coffee and several Solanaceae species including tomato, tobacco, potato, eggplant and pepper. BLAST and protein alignments with the 2 well-characterized XyG fucosyltransferases from Arabidopsis thaliana and Pisum sativum revealed that at least 6 proteins from different Solanaceae species and from coffee displayed the 3 conserved motifs required for XyG fucosyltransferase activity.     

Genomic variation in tomato,from wild ancestors to contemporary breeding accessions

Background

Domestication modifies the genomic variation of species. Quantifying this variation provides insights into the domestication process, facilitates the management of resources used by breeders and germplasm centers, and enables the design of experiments to associate traits with genes. We described and analyzed the genetic diversity of 1,008 tomato accessions including Solanum lycopersicum var. lycopersicum (SLL), S. lycopersicum var. cerasiforme (SLC), and S. pimpinellifolium (SP) that were genotyped using 7,720 SNPs. Additionally, we explored the allelic frequency of six loci affecting fruit weight and shape to infer patterns of selection.

Results

Our results revealed a pattern of variation that strongly supported a two-step domestication process, occasional hybridization in the wild, and differentiation through human selection. These interpretations were consistent with the observed allele frequencies for the six loci affecting fruit weight and shape. Fruit weight was strongly selected in SLC in the Andean region of Ecuador and Northern Peru prior to the domestication of tomato in Mesoamerica. Alleles affecting fruit shape were differentially selected among SLL genetic subgroups. Our results also clarified the biological status of SLC. True SLC was phylogenetically positioned between SP and SLL and its fruit morphology was diverse. SLC and “cherry tomato” are not synonymous terms. The morphologically-based term “cherry tomato” included some SLC, contemporary varieties, as well as many admixtures between SP and SLL. Contemporary SLL showed a moderate increase in nucleotide diversity, when compared with vintage groups.

Conclusions

This study presents a broad and detailed representation of the genomic variation in tomato. Tomato domestication seems to have followed a two step-process; a first domestication in South America and a second step in Mesoamerica. The distribution of fruit weight and shape alleles supports that domestication of SLC occurred in the Andean region. Our results also clarify the biological status of SLC as true phylogenetic group within tomato. We detect Ecuadorian and Peruvian accessions that may represent a pool of unexplored variation that could be of interest for crop improvement.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1444-1) contains supplementary material, which is available to authorized users.     

Genotoxicity of Nicotiana tabacum leaves on Helix aspersa

Tobacco farmers are routinely exposed to complex mixtures of inorganic and organic chemicals present in tobacco leaves. In this study, we examined the genotoxicity of tobacco leaves in the snail Helix aspersa as a measure of the risk to human health. DNA damage was evaluated using the micronucleus test and the Comet assay and the concentration of cytochrome P450 enzymes was estimated. Two groups of snails were studied: one fed on tobacco leaves and one fed on lettuce (Lactuca sativa L) leaves (control group). All of the snails received leaves (tobacco and lettuce leaves were the only food provided) and water ad libitum. Hemolymph cells were collected after 0, 24, 48 and 72 h. The Comet assay and micronucleus test showed that exposure to tobacco leaves for different periods of time caused significant DNA damage. Inhibition of cytochrome P450 enzymes occurred only in the tobacco group. Chemical analysis indicated the presence of the alkaloid nicotine, coumarins, saponins, flavonoids and various metals. These results show that tobacco leaves are genotoxic in H. aspersa and inhibit cytochrome P450 activity, probably through the action of the complex chemical mixture present in the plant.     

Trap Crops and Population Management of Globodera tabacum tabacum

Tobacco, eastern black nightshade, and tomato were grown for 3 to 13 weeks to assess differences in invasion, development, and soil density of Globodera tabacum tabacum (tobacco cyst nematode) in field plots and microplots over three seasons. Tobacco cyst nematodes invaded roots of resistant and susceptible tobacco, nightshade, and tomato. Nematode development was fastest in nightshade and slowest in tomato, and few adults developed in roots of nematode-resistant tobacco. Soil populations of tobacco cyst nematodes were reduced up to 80% by destroying nightshade or susceptible tobacco grown for 3 to 6 weeks. Nematode populations were reduced up to 96% by destroying tomato or resistant tobacco grown for 3 to 6 weeks. Timing of crop destruction was less critical with tomato and resistant tobacco, as nematode populations did not increase after 13 weeks of growth. These studies demonstrate that trap cropping, through crop destruction, can significantly reduce G. t. tabacum populations.     

In situ analysis of foliar zinc absorption and short-distance movement in fresh and hydrated leaves of tomato and citrus using synchrotron-based X-ray fluorescence microscopy

Background and Aims Globally, zinc deficiency is one of the most important nutritional factors limiting crop yield and quality. Despite widespread use of foliar-applied zinc fertilizers, much remains unknown regarding the movement of zinc from the foliar surface into the vascular structure for translocation into other tissues and the key factors affecting this diffusion.Methods Using synchrotron-based X-ray fluorescence microscopy (µ-XRF), absorption of foliar-applied zinc nitrate or zinc hydroxide nitrate was examined in fresh leaves of tomato (Solanum lycopersicum) and citrus (Citrus reticulatus).Key Results The foliar absorption of zinc increased concentrations in the underlying tissues by up to 600-fold in tomato but only up to 5-fold in citrus. The magnitude of this absorption was influenced by the form of zinc applied, the zinc status of the treated leaf and the leaf surface to which it was applied (abaxial or adaxial). Once the zinc had moved through the leaf surface it appeared to bind strongly, with limited further redistribution. Regardless of this, in these underlying tissues zinc moved into the lower-order veins, with concentrations 2- to 10-fold higher than in the adjacent tissues. However, even once in higher-order veins, the movement of zinc was still comparatively limited, with concentrations decreasing to levels similar to the background within 1–10 mm.Conclusions The results advance our understanding of the factors that influence the efficacy of foliar zinc fertilizers and demonstrate the merits of an innovative methodology for studying foliar zinc translocation mechanisms.     

Auxin increases the hydrogen peroxide (H2O2) concentration in tomato (Solanum lycopersicum) root tips while inhibiting root growth

Background and Aims

The hormone auxin and reactive oxygen species (ROS) regulate root elongation, but the interactions between the two pathways are not well understood. The aim of this study was to investigate how auxin interacts with ROS in regulating root elongation in tomato, Solanum lycopersicum.

Methods

Wild-type and auxin-resistant mutant, diageotropica (dgt), of tomato (S. lycopersicum ‘Ailsa Craig’) were characterized in terms of root apical meristem and elongation zone histology, expression of the cell-cycle marker gene Sl-CycB1;1, accumulation of ROS, response to auxin and hydrogen peroxide (H₂O₂), and expression of ROS-related mRNAs.

Key Results

The dgt mutant exhibited histological defects in the root apical meristem and elongation zone and displayed a constitutively increased level of hydrogen peroxide (H₂O₂) in the root tip, part of which was detected in the apoplast. Treatments of wild-type with auxin increased the H₂O₂ concentration in the root tip in a dose-dependent manner. Auxin and H₂O₂ elicited similar inhibition of cell elongation while bringing forth differential responses in terms of meristem length and number of cells in the elongation zone. Auxin treatments affected the expression of mRNAs of ROS-scavenging enzymes and less significantly mRNAs related to antioxidant level. The dgt mutation resulted in resistance to both auxin and H₂O₂ and affected profoundly the expression of mRNAs related to antioxidant level.

Conclusions

The results indicate that auxin regulates the level of H₂O₂ in the root tip, so increasing the auxin level triggers accumulation of H₂O₂ leading to inhibition of root cell elongation and root growth. The dgt mutation affects this pathway by reducing the auxin responsiveness of tissues and by disrupting the H₂O₂ homeostasis in the root tip.     

Chloroplast to chromoplast transition in tomato fruit: spectral confocal microscopy analyses of carotenoids and chlorophylls in isolated plastids and time-lapse recording on intact live tissue

Background and Aims

There are several studies suggesting that tomato (Solanum lycopersicum) chromoplasts arise from chloroplasts, but there is still no report showing the fluorescence of both chlorophylls and carotenoids in an intermediate plastid, and no video showing this transition phase.

Methods

Pigment fluorescence within individual plastids, isolated from tomato fruit using sucrose gradients, was observed at different ripening stages, and an in situ real-time recording of pigment fluorescence was performed on live tomato fruit slices.

Key results

At the mature green and red stages, homogenous fractions of chloroplasts and chromoplasts were obtained, respectively. At the breaker stage, spectral confocal microscopy showed that intermediate plastids contained both chlorophylls and carotenoids. Furthermore, an in situ real-time recording (a) showed that the chloroplast to chromoplast transition was synchronous for all plastids of a single cell; and (b) confirmed that all chromoplasts derived from pre-existing chloroplasts.

Conclusions

These results give details of the early steps of tomato chromoplast biogenesis from chloroplasts, with the formation of intermediate plastids containing both carotenoids and chlorophylls. They provide information at the sub-cellular level on the synchronism of plastid transition and pigment changes.     

Nitric oxide is involved in light-specific responses of tomato during germination under normal and osmotic stress conditions

Background and Aims Nitric oxide (NO) is involved in the signalling and regulation of plant growth and development and responses to biotic and abiotic stresses. The photoperiod-sensitive mutant 7B-1 in tomato (Solanum lycopersicum) showing abscisic acid (ABA) overproduction and blue light (BL)-specific tolerance to osmotic stress represents a valuable model to study the interaction between light, hormones and stress signalling. The role of NO as a regulator of seed germination and ABA-dependent responses to osmotic stress was explored in wild-type and 7B-1 tomato under white light (WL) and BL. Methods Germination data were obtained from the incubation of seeds on germinating media of different composition. Histochemical analysis of NO production in germinating seeds was performed by fluorescence microscopy using a cell-permeable NO probe, and endogenous ABA was analysed by mass spectrometry. Key Results The NO donor S-nitrosoglutathione stimulated seed germination, whereas the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) had an inhibitory effect. Under WL in both genotypes, PTIO strongly suppressed germination stimulated by fluridone, an ABA inhibitor. The stimulatory effect of the NO donor was also observed under osmotic stress for 7B-1 seeds under WL and BL. Seed germination inhibited by osmotic stress was restored by fluridone under WL, but less so under BL, in both genotypes. This effect of fluridone was further modulated by the NO donor and NO scavenger, but only to a minor extent. Fluorescence microscopy using the cell-permeable NO probe DAF-FM DA (4-amino-5-methylamino-2',7'-difluorofluorescein diacetate) revealed a higher level of NO in stressed 7B-1 compared with wild-type seeds. Conclusions As well as defective BL signalling, the differential NO-dependent responses of the 7B-1 mutant are probably associated with its high endogenous ABA concentration and related impact on hormonal cross-talk in germinating seeds. These data confirm that light-controlled seed germination and stress responses include NO-dependent signalling.     

Negative regulation of <Emphasis Type="Italic">KNOX</Emphasis> expression in tomato leaves

Leaves of seed plants can be described as simple, where the leaf blade is entire, or dissected, where the blade is divided into distinct leaflets. Both simple and dissected leaves are initiated at the flanks of a pluripotent structure termed the shoot apical meristem (SAM). In simple-leafed species, expression of class I KNOTTED1-like homeobox (KNOX) proteins is confined to the meristem while in many dissected leaf plants, including tomato, KNOX expression persists in leaf primordia. Elevation of KNOX expression in tomato leaves can result in increased leaflet number, indicating that tight regulation of KNOX expression may help define the degree of leaf dissection in this species. To test this hypothesis and understand the mechanisms controlling leaf dissection in tomato, we studied the clausa (clau) and tripinnate (tp) mutants both of which condition increased leaflet number phenotypes. We show that TRIPINNATE and CLAUSA act together, to restrict the expression level and domain of the KNOX genes Tkn1 and LeT6/Tkn2 during tomato leaf development. Because loss of CLAU or TP activity results in increased KNOX expression predominantly on the adaxial (upper) leaf domain, our observations indicate that CLAU and TP may participate in a domain-specific KNOX repressive system that delimits the ability of the tomato leaf to generate leaflets.     

Shade Tobacco Yield Loss and Globodera tabacum tabacum Population Changes in Relation to Initial Nematode Density

Field microplot experiments were conducted from 1987 to 1992 to determine the relationship between fresh weight leaf yield of shade tobacco (Nicotiana tabacum) and initial density of Globodera tabacum tabacum (encysted J2 per cm³ soil). Initial nematode densities of 0.1 to 1,097 J2/cm³ soil were negatively correlated with leaf yield, total shoot weight, and normalized plant height 5 to 6 weeks after transplanting (r = -0.73, -0.73, and -0.52, respectively). Nonlinear damage functions were used to relate initial G. t. tabacum densities to the yield and shoot weight data. The model described leaf yield losses of < 5 % for initial nematode densities of less than 100 J2/cm³ soil. Densities above 100 J2 resulted in yields decreasing exponentially to a maximum yield loss of >40% at 500 to 1,000 J2/cm³ soil. A similar initial density tolerance threshold relationship was observed for total shoot weight. No threshold effect was evident for standardized plant height, which was a poor predictor of leaf yield. Globodera tabacum tabacum population increase over a growing season was described by a linear relation on a log/log plot (R² = 0.73).     

Hatch and Reproduction of Globodera tabacum tabacum in Response to Tobacco,Tomato, or Black Nightshade

The effects of broadleaf tobacco, tomato, and black nightshade on juvenile hatch and reproduction of Globodera tabacum tabacum were determined in laboratory and greenhouse experiments. Root exudates from nightshade stimulated greater egg hatch than those from either ''Rutgers'' tomato or ''86-4'' tobacco. Hatch was greater at higher proportions of root exudates for all three plant species. Root exudates from plants greater than 3 weeks old stimulated more hatch than younger plants. No regression relationships existed between plant age and nematode batch. In other experiments, hatch from eggs in cysts was higher for tomato and nightshade after 10 weeks in greenhouse pots compared to tobacco and bare soil. Numbers of second-stage juveniles in eggs in cysts produced from a previous generation on the same host were highest on nightshade and less on tomato and tobacco. Cysts of variable age recovered from field soil had increased hatch in both root exudates or water compared to recently produced cysts from plants in growth chambers. Globodera t. tabacum may be subject to both host and environmentally mediated diapause.     

How plant architecture affects light absorption and photosynthesis in tomato: towards an ideotype for plant architecture using a functional-structural plant model

Background and Aims

Manipulation of plant structure can strongly affect light distribution in the canopy and photosynthesis. The aim of this paper is to find a plant ideotype for optimization of light absorption and canopy photosynthesis. Using a static functional structural plant model (FSPM), a range of different plant architectural characteristics was tested for two different seasons in order to find the optimal architecture with respect to light absorption and photosynthesis.

Methods

Simulations were performed with an FSPM of a greenhouse-grown tomato crop. Sensitivity analyses were carried out for leaf elevation angle, leaf phyllotaxis, leaflet angle, leaf shape, leaflet arrangement and internode length. From the results of this analysis two possible ideotypes were proposed. Four different vertical light distributions were also tested, while light absorption cumulated over the whole canopy was kept the same.

Key Results

Photosynthesis was augmented by 6 % in winter and reduced by 7 % in summer, when light absorption in the top part of the canopy was increased by 25 %, while not changing light absorption of the canopy as a whole. The measured plant structure was already optimal with respect to leaf elevation angle, leaflet angle and leaflet arrangement for both light absorption and photosynthesis while phyllotaxis had no effect. Increasing the length : width ratio of leaves by 1·5 or increasing internode length from 7 cm to 12 cm led to an increase of 6–10 % for light absorption and photosynthesis.

Conclusions

At high light intensities (summer) deeper penetration of light in the canopy improves crop photosynthesis, but not at low light intensities (winter). In particular, internode length and leaf shape affect the vertical distribution of light in the canopy. A new plant ideotype with more spacious canopy architecture due to long internodes and long and narrow leaves led to an increase in crop photosynthesis of up to 10 %.     

Early Crop Root Destruction for Management of Tobacco Cyst Nematodes

Prompt tillage after crop harvest was investigated as a cultural control for the tobacco cyst nematode, Globodera tabacum tabacum, on stalk-cut broadleaf cigar wrapper tobacco. Stalk stumps and roots remaining after harvest were destroyed by tilling immediately or from 2 to 6 wk after harvest in field experiments over 4 yr. Cyst nematode Pf/Pi ratios ranged from 0.65 to 1.62 when plants were tilled immediately after harvest and 1.13 to 5.88 when tillage was delayed. Nematode population development was monitored by inoculating plants in pots placed in fields with J2 in eggs and sampling over time (8 to 18 wk). Three generations per year were observed, and G. t. tabacum generation time was as short as 6 wk for each generation. Destroying stalks and root systems remaining after harvesting stalk-cut broadleaf cigar wrapper tobacco removes the host to preclude development of nematodes at the end of the second and entire third generation. Early tillage resulted in consistently lower tobacco cyst nematode populations than allowing viable roots to remain in fields for an additional 8 to 18 wk. This management tactic reduces the need for nematicide application to slow nematode population increases over time and can reduce losses due to infection by G. t. tabacum.     

Reference gene selection for quantitative real-time PCR normalization in tomato subjected to nitrogen, cold, and light stress

We examined eight putative consistently expressed genes—actin (ACT), β-tubulin, elongation factor 1α (EF1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), ribosomal protein L2 (RPL2), ubiquitin (UBI), and a catalytic subunit of protein phosphatase 2A (PP2Acs)—for their potential as references for the normalization of gene expression in tomato leaves. Expression stability of candidate reference genes was tested during growth conditions of nitrogen (N) starvation, low temperature, and suboptimal light. The geNorm algorithm, using reciprocal cross-validation among a larger group of candidate references, was applied for this purpose. The widely used reference genes GAPDH and PGK were top ranked during light stress but poorly ranked during N and cold stress. In contrast, EF1 was top ranked during N and cold stress but poorly ranked during light stress. The novel references RPL2 and PP2Acs, as well as the traditional references ACT and UBI, appeared to be stably expressed when looking at the data set as a whole. No gene was identified that exhibited such a constant level of expression as to outperform the other candidates under all experimental conditions. Thus, the results highlight the need for normalizing gene expression in tomato using the geometric average of multiple carefully selected reference genes.