Daniel Just

Silencing of the mitochondrial ascorbate synthesizing enzyme L-galactono-1,4-lactone dehydrogenase affects plant and fruit development in tomato.

l-Galactono-1,4-lactone dehydrogenase (EC 1.3.2.3) catalyzes the last step in the main pathway of vitamin C (l-ascorbic acid) biosynthesis in higher plants. In this study, we first characterized the spatial and temporal expression of SlGalLDH in several organs of tomato (Solanum lycopersicum) plants in parallel with the ascorbate content. P35S:SlgalldhRNAi silenced transgenic tomato lines were then generated using an RNAi strategy to evaluate the effect of any resulting modification of the ascorbate pool on plant and fruit development. In all P35S:SlgalldhRNAi plants with reduced SlGalLDH transcript and activity, plant growth rate was decreased. Plants displaying the most severe effects (dwarf plants with no fruit) were excluded from further analysis. The most affected lines studied exhibited up to an 80% reduction in SlGalLDH activity and showed a strong reduction in leaf and fruit size, mainly as a consequence of reduced cell expansion. This was accompanied by significant changes in mitochondrial function and altered ascorbate redox state despite the fact that the total ascorbate content remained unchanged. By using a combination of transcriptomic and metabolomic approaches, we further demonstrated that several primary, like the tricarboxylic acid cycle, as well as secondary metabolic pathways related to stress response were modified in leaves and fruit of P35S:SlgalldhRNAi plants. When taken together, this work confirms the complexity of ascorbate regulation and its link with plant metabolism. Moreover, it strongly suggests that, in addition to ascorbate synthesis, GalLDH could play an important role in the regulation of cell growth-related processes in plants.

Changes in Transcriptional Profiles Are Associated with Early Fruit Tissue Specialization in Tomato

The cell expansion phase contributes in determining the major characteristics of a fleshy fruit and represents two-thirds of the total fruit development in tomato (Solanum lycopersicum). So far, it has received very little attention. To evaluate the interest of a genomic scale approach, we performed an initial sequencing of approximately 1,200 cell expansion stage-related sequence tags from tomato fruit at 8, 12, and 15 d post anthesis. Interestingly, up to approximately 35% of the expressed sequence tags showed no homology with available tomato expressed sequence tags and up to approximately 21% with any known gene. Microarrays spotted with expansion phase-related cDNAs and other fruit cDNAs involved in various developmental processes were used (1) to profile gene expression in developing fruit and other plant organs and (2) to compare two growing fruit tissues engaged mostly in cell division (exocarp) or in cell expansion (locular tissue surrounding the seeds). Reverse transcription-polymerase chain reaction analysis was further used to confirm microarray results and to specify expression profiles of selected genes (24) in various tissues from expanding fruit. The wide range of genes expressed in the exocarp is consistent with a protective function and with a high metabolic activity of this tissue. In addition, our data show that the expansion of locular cells is concomitant with the expression of genes controlling water flow, organic acid synthesis, sugar storage, and photosynthesis and suggest that hormones (auxin and gibberellin) regulate this process. The data presented provide a basis for tissue-specific analyses of gene function in growing tomato fruit.

SNP Discovery and Linkage Map Construction in Cultivated Tomato

Few intraspecific genetic linkage maps have been reported for cultivated tomato, mainly because genetic diversity within Solanum lycopersicum is much less than that between tomato species. Single nucleotide polymorphisms (SNPs), the most abundant source of genomic variation, are the most promising source of polymorphisms for the construction of linkage maps for closely related intraspecific lines. In this study, we developed SNP markers based on expressed sequence tags for the construction of intraspecific linkage maps in tomato. Out of the 5607 SNP positions detected through in silico analysis, 1536 were selected for high-throughput genotyping of two mapping populations derived from crosses between ‘Micro-Tom’ and either ‘Ailsa Craig’ or ‘M82’. A total of 1137 markers, including 793 out of the 1338 successfully genotyped SNPs, along with 344 simple sequence repeat and intronic polymorphism markers, were mapped onto two linkage maps, which covered 1467.8 and 1422.7 cM, respectively. The SNP markers developed were then screened against cultivated tomato lines in order to estimate the transferability of these SNPs to other breeding materials. The molecular markers and linkage maps represent a milestone in the genomics and genetics, and are the first step toward molecular breeding of cultivated tomato. Information on the DNA markers, linkage maps, and SNP genotypes for these tomato lines is available at http://www.kazusa.or.jp/tomato/.

Differing substomatal and chloroplastic CO2 concentrations in water-stressed wheat

Gas exchanges of wheat (Triticum aestivum L. cv. Courtot) shoots were measured before and during a water stress. While photosynthesis, transpiration and dark respiration decreased because of the stress, photorespiration increased initially, up to a maximum of 50% above its initial value. The CO2 concentration in the intercellular space was calculated from gas-diffusion resistances, and remained approximately constant before and during the stress. On the other hand, the CO2 concentration in the chloroplast, in the vicinity of Ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco), was evaluated from the ratio of CO2 to O2 uptake, using the known kinetic constants of the oxygenation and carboxylation reactions which compete for Rubisco. In the well-watered plants, the calculated chloroplastic concentration was slightly smaller than the substomatal concentration. During water stress, this concentration decreased while the substomatal CO2 concentration remained constant. Hypotheses to explain this difference between substomatal and chloroplastic CO2 concentrations are discussed.

NMR study of low subcellular pH during the development of cherry tomato fruit

Changes in metabolites (organic acids, sugars and amino acids) and subcellular pH were studied during fruit development of cherry tomato (Lycopersicon esculentum Mill. var. cerasiformae). Fructose and glucose were the major sugars, whereas citrate and malate the two major organic acids. At different stages of fruit development, vacuolar and cytoplasmic pH changes were followed by in vivo 13C and 31P NMR spectroscopy. Fruit compartments had a cytoplasmic pH around 7.1 as early as the cell-divi-sion and -expansion stages. The vacuolar pH measured by in vivo 13C NMR spectroscopy decreased from 4.5 to 3.6. Concomitantly, strong accumulation of γ-aminobutyric acid (GABA) was observed during the first 15 days after anthesis and glutamate decarboxylase (GAD) activity increased 10-fold during the first 8 days of development. The relationships between organic acid biosynthesis and storage, GABA produc-tion, and subcellular pH changes during development of cherry tomato fruit are discussed.

Physiological effects of temporary immersion on Hevea brasiliensis callus

In vitro culture by temporary immersion generates potentially stressful conditions for explants that may differ from those associated with classic methods. In order to evaluate the effects of these conditions on physiological changes in explants, different parameters of metabolic activity were investigated for a friable embryogenic callus of Hevea brasiliensis (Müll. Arg.), in response to 1 min, and 1, 12 and 24 h per day of immersion, using semi-solid and agitated liquid media as controls. The relative growth rate of the callus was not significantly different for the 1 min immersion treatment and the controls, but it decreased by about 60% for the 1-, 12- and 24-h immersion treatments. During the immersed stage, the rate of respiration of the callus was comparable for all the treatments. However, during the emersed stage, the respiration rate increased by 140 and 164% for the 12- and 24-h immersion treatments, respectively. Meanwhile, the total adenylate nucleotide concentration and the ratio of ATP/ADP remained almost constant, or even decreased. The adenylate energy charge was comparable for all the treatments, averaging 0.88. The superoxide dismutase activity and the lipid peroxidation increased with the immersion duration, and were significantly higher for the 12- and 24-h immersion treatments than for controls. However, after 24 h in emersed stage, there was no lipid peroxidation, regardless of previous immersion duration. It appears from these results that the immersed stage induced a substantial oxidative stress, which was not associated with the callus immersion per se.

Analyses of tomato fruit brightness mutants uncover both cutin-deficient and cutin- abundant mutants and a new hypomorphic allele of GDSL lipase

Opposite changes in the cutin polyester component in tomato fruit cuticle mutants lead to increased fruit glossiness. The cuticle is a protective layer synthesized by epidermal cells of the plants and consisting of cutin covered and filled by waxes. In tomato (Solanum lycopersicum) fruit, the thick cuticle embedding epidermal cells has crucial roles in the control of pathogens, water loss, cracking, postharvest shelf-life, and brightness. To identify tomato mutants with modified cuticle composition and architecture and to further decipher the relationships between fruit brightness and cuticle in tomato, we screened an ethyl methanesulfonate mutant collection in the miniature tomato cultivar Micro-Tom for mutants with altered fruit brightness. Our screen resulted in the isolation of 16 glossy and 8 dull mutants displaying changes in the amount and/or composition of wax and cutin, cuticle thickness, and surface aspect of the fruit as characterized by optical and environmental scanning electron microscopy. The main conclusions on the relationships between fruit brightness and cuticle features were as follows: (1) screening for fruit brightness is an effective way to identify tomato cuticle mutants; (2) fruit brightness is independent from wax load variations; (3) glossy mutants show either reduced or increased cutin load; and (4) dull mutants display alterations in epidermal cell number and shape. Cuticle composition analyses further allowed the identification of groups of mutants displaying remarkable cuticle changes, such as mutants with increased dicarboxylic acids in cutin. Using genetic mapping of a strong cutin-deficient mutation, we discovered a novel hypomorphic allele of GDSL lipase carrying a splice junction mutation, thus highlighting the potential of tomato brightness mutants for advancing our understanding of cuticle formation in plants.

Rapid identification of causal mutations in tomato EMS populations via mapping-by-sequencing

The tomato is the model species of choice for fleshy fruit development and for the Solanaceae family. Ethyl methanesulfonate (EMS) mutants of tomato have already proven their utility for analysis of gene function in plants, leading to improved breeding stocks and superior tomato varieties. However, until recently, the identification of causal mutations that underlie particular phenotypes has been a very lengthy task that many laboratories could not afford because of spatial and technical limitations. Here, we describe a simple protocol for identifying causal mutations in tomato using a mapping-by-sequencing strategy. Plants displaying phenotypes of interest are first isolated by screening an EMS mutant collection generated in the miniature cultivar Micro-Tom. A recombinant F2 population is then produced by crossing the mutant with a wild-type (WT; non-mutagenized) genotype, and F2 segregants displaying the same phenotype are subsequently pooled. Finally, whole-genome sequencing and analysis of allele distributions in the pools allow for the identification of the causal mutation. The whole process, from the isolation of the tomato mutant to the identification of the causal mutation, takes 6–12 months. This strategy overcomes many previous limitations, is simple to use and can be applied in most laboratories with limited facilities for plant culture and genotyping.

Proteolysis and Proteolytic Activities in the Acclimation to Stress: The Case of Sugar Starvation in Maize Root Tips

The changes that stress induce in a plant may be either specific of the type of stress, or non-specific, ie, an effect of, or a response to, the stress situation itself. Several phenomena have been found to occur in a number of plant tissues under different stresses and are therefore of the non-specific type. This is the case of the production of toxic oxygen species (Foyer et al., 1991), the production of ethylene (Wang et al, 1990), increasing levels of polyamines (Flores, 1990), or the induction of heat-shockcognate proteins (Cabane et al., 1993) in response to temperature, osmotic, mineral or wounding stresses. It is not always clear whether these changes are part of an acclimation mechanism which would improve stress tolerance, or are symptoms of the cellular degradations caused by the environmental constraints. These observations however suggest that in spite of the diversity of stresses, similar mechanisms may be involved in the response to stress. In addition, modifications by stresses in the pattern of protein synthesis, in carbon allocation among plant organs, in maintenance respiration, or in developmental programs and senescence induction (Amthor and McCree, 1990) indicate that, at the same time as specific responses are induced, the metabolic background is profoundly modified. We believe that proteolysis is part of the common response system.

Chapter Nine - High-Throughput Biochemical Phenotyping for Plants

Abstract There is an urgent need in low cost and fast technologies that enable the exploration of natural or induced biodiversity in plants. Biochemical phenotyping is often considered as particularly promising to identify given analytes that are linked to desirable plant phenotypes and that could be used as markers for plant performance. Over the past 15 years, metabolomics and other omics approaches have been intensively used to study plant phenotypes. Still, they do not enable routine screens of very large populations in breeding programs, probably because this would represent unaffordable financial investments for equipment and specialised engineers. Along the last century, biochemical phenotyping and -screening have nevertheless been successfully exploited in medicine, pharmaceutical research and agro-industry. In medicine, over 6000 biochemical procedures exploiting a large variety of concepts and equipment are available for routine diagnosis and therapeutic monitoring. Pharmaceutical companies have spent massive investments to screen for biologically active molecules, whereas in the agro-industry increasingly sophisticated biochemical analysis has been developed to control the quality of raw and transformed products. Microplate technology is a well-established technology, which originated from the medical field and benefited from huge investments by the pharmaceutical industry. It offers the possibility to operate a wide range of analyses at very low costs per sample in plant research. To illustrate the potential of microplate technology as a high-throughput phenotyping tool, a screen for tomato metabolic mutants is shown where 24 enzymatic traits have been measured in more than 1500 samples within only 1 month with a relatively small investment.