Miniature Tomato Research Articles

The SlSHN2 transcription factor contributes to cuticle formation and epidermal patterning in tomato fruit

Tomato (Solanum lycopersicum) is an established model for studying plant cuticle because of its thick cuticle covering and embedding the epidermal cells of the fruit. In this study, we screened an EMS mutant collection of the miniature tomato cultivar Micro-Tom for fruit cracking mutants and found a mutant displaying a glossy fruit phenotype. By using an established mapping-by-sequencing strategy, we identified the causal mutation in the SlSHN2 transcription factor that is specifically expressed in outer epidermis of growing fruit. The point mutation in the shn2 mutant introduces a K to N amino acid change in the highly conserved ‘mm’ domain of SHN proteins. The cuticle from shn2 fruit showed a ~ fivefold reduction in cutin while abundance and composition of waxes were barely affected. In addition to alterations in cuticle thickness and properties, epidermal patterning and polysaccharide composition of the cuticle were changed. RNAseq analysis further highlighted the altered expression of hundreds of genes in the fruit exocarp of shn2, including genes associated with cuticle and cell wall formation, hormone signaling and response, and transcriptional regulation. In conclusion, we showed that a point mutation in the transcriptional regulator SlSHN2 causes major changes in fruit cuticle formation and its coordination with epidermal patterning.

A co-silencing system for functional analysis of genes without visible phenotype in tomato plant development and fruit ripening using tobacco rattle virus

Abstract Virus-induced gene silencing (VIGS) is known as a powerful tool for identifying gene functions during plant growth and development. Tobacco rattle virus (TRV) based VIGS has been successfully employed in many plants. However, there have been few reports that VIGS can be used to study the function of gene at fruit level, especially the genes without visible silencing phenotype. Here we used miniature tomato cultivar Micro-Tom (Solanum lycopersicum L.) as material to silence phytoene desaturase (SlPDS) and SlARG2, an important gene encoding arginase in arginine metabolism, separately or simultaneously with TRV agroinoculated by sprout vacuum-infiltration to test the utility of concurrently silencing two genes. When SlPDS and SlARG2 were co-silenced in the same tissue at the same time, there are no compromises in silencing efficiency and silencing efficacy compared with SlPDS or SlARG2 silenced separately. Moreover, the silence of SlPDS and SlARG2 with TRV agroinoculated by sprout vacuum-infiltration occurred at the early stage of tomato plant, and the silence can extend to full red stage of fruit. Therefore, our results provide a means for functional analysis of genes, particularly the genes with no obvious visible phenotype after silencing, involved in all of the metabolic networks during fruit development and ripening.

Bioactive components and antioxidant capacities of different miniature tomato cultivars grown by altered fertilizer applications

This study investigated the organic acid and phenolic compound levels, total phenolic (TP) and antioxidant capacity (TEAC) of three miniature tomato cultivars grown on a farmer’s field with three different fertilizer applications. Analysis of phenolic compound (protocatechuic, vanillic, gallic, chlorogenic, caffeic, syringic, p-coumaric and ferulic acid, rutin, quercetin, catechin and phloridzine) organic acid (citric, tartaric, malic, succinic and fumaric acid), TP and TEAC levels in fruit samples showed statistically significant (p < 0.05) differences between tomato cultivars and fertilizer applications. Rutin and chlorogenic acid were the predominant phenolic compounds found in all three cultivars. The highest rutin value (50.48 mg kg−1 FW) was found in the Black Zebra fertilized with DAP plus organic and biochemical fertilizers and the lowest (20.52 mg kg−1 FW) in the Black Zebra fertilized with DAP only. The highest chlorogenic acid value (63.31 mg kg−1 FW) was found in the sweet pea currant fertilized with DAP only and the lowest (21.06 mg kg−1 FW) in the Black Zebra fertilized with DAP plus chemical fertilizer. Citric acid content was the dominant organic acid in all three cultivars, with the highest citric acid value (6439.50 mg kg−1 FW) found in the Sweet Pea Currant cherry tomato fertilized with DAP plus organic and biochemical fertilizers and the lowest (2435.20 mg kg−1 FW) in the Window Box Yellow fertilized with DAP plus chemical fertilizer. Total phenolic and antioxidant levels in the window box yellow were significantly lower as compared to the Black Zebra and sweet pea currant varieties for all three fertilizer applications.

トマトMicro-Tomのトレハロース合成酵素系,およびストレス応答性SNF1関連キナーゼ(SnRK)の解析(セミナー「生体の温度・水分センサーの機能と構造」)

トマトMicro-Tomのトレハロース合成酵素系,およびストレス応答性SNF1関連キナーゼ(SnRK)の解析(セミナー「生体の温度・水分センサーの機能と構造」)

Selection of Reference Genes for Quantitative Real-Time RT-PCR Studies in Tomato Fruit of the Genotype MT-Rg1.

Quantitative real-time RT-PCR (qRT-PCR) has become one of the most widely used methods for accurate quantification of gene expression. Since there are no universal reference genes for normalization, the optimal strategy to normalize raw qRT-PCR data is to perform an initial comparison of a set of independent reference genes to assess the most stable ones in each biological model. Normalization of a qRT-PCR experiment helps to ensure that the results are both statistically significant and biologically meaningful. Tomato is the model of choice to study fleshy fruit development. The miniature tomato (Solanum lycopersicum L.) cultivar Micro-Tom (MT) is considered a model system for tomato genetics and functional genomics. A new genotype, containing the Rg1 allele, improves tomato in vitro regeneration. In this work, we evaluated the expression stability of four tomato reference genes, namely CAC, SAND, Expressed, and ACTIN2. We showed that the genes CAC and Exp are the best reference genes of the four we tested during fruit development in the MT-Rg1 genotype. Furthermore, we validated the reference genes by showing that the expression profiles of the transcription factors FRUITFULL1 and APETALA2c during fruit development are comparable to previous reports using other tomato cultivars.

The Glycerol-3-Phosphate Acyltransferase GPAT6 from Tomato Plays a Central Role in Fruit Cutin Biosynthesis.

The thick cuticle covering and embedding the epidermal cells of tomato (Solanum lycopersicum) fruit acts not only as a protective barrier against pathogens and water loss but also influences quality traits such as brightness and postharvest shelf-life. In a recent study, we screened a mutant collection of the miniature tomato cultivar Micro-Tom and isolated several glossy fruit mutants in which the abundance of cutin, the polyester component of the cuticle, was strongly reduced. We employed a newly developed mapping-by-sequencing strategy to identify the causal mutation underlying the cutin deficiency in a mutant thereafter named gpat6-a (for glycerol-3-phosphate acyltransferase6). To this end, a backcross population (BC1F2) segregating for the glossy trait was phenotyped. Individuals displaying either a wild-type or a glossy fruit trait were then pooled into bulked populations and submitted to whole-genome sequencing prior to mutation frequency analysis. This revealed that the causal point mutation in the gpat6-a mutant introduces a charged amino acid adjacent to the active site of a GPAT6 enzyme. We further showed that this mutation completely abolished the GPAT activity of the recombinant protein. The gpat6-a mutant showed perturbed pollen formation but, unlike a gpat6 mutant of Arabidopsis (Arabidopsis thaliana), was not male sterile. The most striking phenotype was observed in the mutant fruit, where cuticle thickness, composition, and properties were altered. RNA sequencing analysis highlighted the main processes and pathways that were affected by the mutation at the transcriptional level, which included those associated with lipid, secondary metabolite, and cell wall biosynthesis.

Tomato cv. ‘Micro-Tom’ as a model system to study postharvest chilling tolerance

Storage at low temperature is a common practice to extend the market life of many vegetables. Among other horticultural crops, tomato fruit suffers chilling injury when it is storage under refrigerated conditions. Much effort has been made to understand the mechanisms of generation of this physiologic disturbance, but many aspects need to be clarified yet. Tomato (Solanum lycopersicum) cv. “Micro-Tom” is a miniature tomato plant with various properties that make it useful as a model system in plant biology. In this work, the potential of tomato cv. “Micro-Tom” fruit as a model to study chilling injury was investigated. The effect of postharvest chilling was compared on cvs. “Micro-Tom” and “Minitomato”, another variety with fruit of similar size. Green mature fruits cvs. “Micro-Tom” and “Minitomato” were harvested and stored during 4 weeks at 4°C. It was observed that tomato cv. “Micro-Tom” fruit was clearly tolerant to chilling while tomato cv. “Minitomato” fruit developed severe chilling injury symptoms and avoided ripening. Harvest and chilling altered the length of time between the different ripening stages, the development of red full color and smell. Also, it was shown that harvesting fruits by visual appreciation is a rapid and useful method for distinguishing the different ripening stages during tomato cv. “Micro-Tom” fruit ripening. These results showed that tomato cv. “Micro-Tom” fruit was able to counteract the strain resulting from the imposed chilling stress. Altogether these data indicate that tomato cv. “Micro-Tom” fruit is a good model to study the mechanism of postharvest chilling response and tolerance in tomato.

Measurement of Loading Induced Acoustic Emissions at Stem of Miniature Tomato Plants

Measurement of Loading Induced Acoustic Emissions at Stem of Miniature Tomato Plants

Interaction of Moniliophthora perniciosa biotypes with Micro‐Tom tomato: a model system to investigate the witches' broom disease of Theobroma cacao

The miniature tomato (Solanum lycopersicum) cultivar Micro‐Tom (MT) has become an important platform to investigate plant–pathogen interactions. In the case of the witches' broom disease of Theobroma cacao (cacao), the existence of Moniliophthora perniciosa isolates pathogenic to Solanaceae (S‐biotype) may enable the use of MT to circumvent limitations of the cacao host, whereas the availability of a non‐infective cacao C‐biotype allows the evaluation of contrasting responses of MT. Infection of MT by the S‐biotype led to stem swelling and axillary shoot growth to form broom‐like symptoms similar to the biotrophic phase in cacao, but the infected tissues did not progress to necrosis. Conversely, inoculation with the C‐biotype did not cause typical symptoms, but reduced plant height, appearing as a non‐host interaction. Histopathological characterization of the S‐biotype infection of MT by light and electron microscopy indicated limited germ tube penetration, preferentially through wounds at the base of trichomes or actively through the epidermis. No intracellular mycelium was observed, corroborating the lack of the necrotrophic stage of the pathogen. The analysis of gene expression during the interaction between the S‐ or C‐biotype with MT indicated that expression of plant defence‐associated genes differs for kinetics and intensity between a compatible or incompatible M. perniciosa–MT interaction. The pattern of spore germination and low rate of mycelia penetration suggests that the S‐biotype is not a fully adapted tomato pathogen, but possibly a case of broken non‐host resistance, and evidence suggests the occurrence of a non‐host MT response against the C‐biotype.

Deficit Irrigation of Miniature Tomato Based on Estimation of Embolism Risk by Measurements of Acoustic Emission and Stress Wave at Stem

Deficit Irrigation of Miniature Tomato Based on Estimation of Embolism Risk by Measurements of Acoustic Emission and Stress Wave at Stem

Analyses of Tomato Fruit Brightness Mutants Uncover Both Cutin-Deficient and Cutin-Abundant Mutants and a New Hypomorphic Allele of GDSL Lipase

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.

Comparative analysis of common genes involved in early fruit development in tomato and grape

Fleshy fruits are important worldwide crops that are rich sources of useful and functional compounds in the human diet. Although fruit ripening has been extensively studied, early fruit development has not been paid much attention despite its contribution to the sensorial and nutritional quality of the fruit. This study aimed at identifying candidate genes involved in early fleshy fruit development that can contribute to the control of final fruit size and composition by comparative analysis of tomato and grape genes. By mining public sequences and microarray database, we identified 23 transcription factors belonging to 14 classes (AP2-EREBP, ARF, bHLH, bZIP, C2C2-GATA, FHA, GeBP, GRAS, HB, LIM, MYB, PBF-2-like, SBP and WRKY) as candidate regulatory genes for early fruit development. The function of these candidate genes will be confirmed by several reverse genetic approaches using the miniature tomato cv. Micro-Tom.

J041013 植物水ポテンシャルの非破壊評価を目的としたミニトマト茎部の超音波測定([J041-01]超音波計測・解析法の新展開(1))

J041013 植物水ポテンシャルの非破壊評価を目的としたミニトマト茎部の超音波測定([J041-01]超音波計測・解析法の新展開(1))

From randomly to inevitable: Accelerating tomato breeding by comprehensive tools and information

Tomato (Solanum lycopersicum L.) is an important crop in the fresh produce and food-processing industry, with one of the highest production yields for edible crops in the world. Due to the variation in consumer palatability and preference, tomato cultivars are diverse in terms of shape, color, taste and functional materials compared to major cereal crops. Accordingly, rapid and efficient breeding technologies for tomato are required. Tomato belongs to the Solanaceae family and is a model species for investigating fleshy fruit development. In 2003, the International Solanaceae Project (SOL), comprising over 30 countries, was formed to address important questions in plant biology using plant species in the Solanaceae family. The consortium initiated the full-genome sequencing of tomato using the inbred tomato cultivar Heinz 1706, and the high-quality genome sequence was recently published (The Tomato Genome Consortium 2012). The Japanese SOL (JSOL), co-chaired by Dr. Hiroshi Ezura at the University of Tsukuba and Dr. Daisuke Shibata at the Kazusa DNA Research Institute, was established in March 2004 and began developing unique and comprehensive tools and resources to accelerate tomato research and breeding. JSOL has used an inbred miniature tomato cultivar, Micro-Tom, as the core genetic background due to its many advantages as an experimental material. The key activities of JSOL include the following: collection and sequencing of tomato ESTs, including full-length cDNAs; building databases for tomato genomics; establishing extensive databases of phenotypic information for tomato populations mutagenized with EMS and gamma-irradiation (Saito et al. 2011); developing tools for such tomato functional genomics as high-throughput genetic transformation protocol (Sun et al. 2006), a TILLING platform (Okabe et al. 2011) and a T-DNA tagline-based mapping platform; establishing a platform for comprehensive metabolite analysis and providing technical workshops for the Solanaceae research community. In 2007, a portion of the JSOL activities was funded by the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan (Yamazaki et al. 2010) as the National BioResource Project (NBRP) tomato, with aims to collect, propagate, maintain and distribute tomato bio-resources to promote studies of tomato functional genomics (Ariizumi et al. 2011). Under NBRP tomato, the sequencing of full-length cDNAs (Aoki et al. 2010) and BAC end-sequencing (Asamizu et al. 2012) have been performed, and the information is available for the research community. In addition, the resequencing of the Micro-Tom genome has been completed, and the high-quality genome sequence will soon be released. In association with these NBRP tomato activities, the development of other tools and information useful for tomato research and breeding is in progress by the tomato research community. These tools and resources will contribute to the rapid and efficient breeding of tomato and also to fleshy fruit research. This special issue of Breeding Science highlights some of the recent progress and current challenges in accelerating tomato research and breeding. Five review articles of note represent the exciting frontier of the development of comprehensive tools and information for tomato research and breeding. These review articles cover the discovery of genes responsible for tomato yield (Ariizumi et al. 2013), tomato functional genomics (Aoki et al. 2013), DNA markers (Shirasawa et al. 2013), omic approaches for tomato breeding (Kusano et al. 2013) and tomato mutant resources and the TILLING platform (Okabe et al. 2013). Taken together, these review articles report the comprehensive tools and information that are changing tomato breeding from a random effort to inevitable research. I appreciate the authors of the articles featured in this issue for their contributions and thoughtful insight on the current developments in this field of research. I hope that these review articles will serve as a valuable resource for advancing our technical knowledge on tomato research and breeding. Finally, I would like to thank Dr. Masaru Iwanaga, the Editor-in-Chief, and Dr. Kenji Katayama, the Managing Editor of Breeding Science, for providing the timely opportunity to prepare this special issue.

J041024 広帯域加速度センサを用いたミニトマト茎部の超音波AE測定([J041-02]超音波計測・解析法の新展開(2))

Ultrasonic acoustic emission (AE) can be detected by an AE sensor at a stem of a plant when cavitation occurs in the xylem. Wideband acceleration sensor with high sensitivity was used for the measurement of AE at stem of miniature tomato in this study because of low cost compared to AE sensor. The waveforms obtained by acceleration sensor exhibited the center frequency which coincided with the resonant frequency of the sensor while the AE sensor delivered various waveforms. On the other hand, the sensitivity of AE detection by the acceleration sensor was almost same as that by the AE sensor. The both sensors showed that AE occurrence rate was higher at daytime than that at nighttime because the cavitation tended to occur at daytime. The both sensors exhibited the similar behavior of AE occurrence for 20 days. The low-cost acceleration sensor, therefore, has the possibility to apply AE measurement for health monitoring of tomato cultivation.

Characterization of regiospecific flavonoid 3′/5′-O-methyltransferase from tomato and its application in flavonoid biotransformation

Flavonoids are ubiquitous secondary metabolites in plants and possess a wide range of biological activities such as anti-oxidant, anti-microbial, and anti-cancer activities. As a food ingredient, flavonoids are also thought to have health-promoting and disease-preventing properties. Structural modifications of flavonoids can improve their biological activities. Methylation is a common flavonoid modification that is catalyzed by O-methyltransferases (OMTs). We isolated four putative OMTs from the miniature tomato cultivar Micro-Tom. One OMT (SlOMT3) was successfully expressed in E. coli and purified. Recombinant SlOMT3 was found to be a regiospecific flavonoid 3′/5′-O-methyltransferase. Biochemical parameters show that SlOMT3 has higher binding affinity and catalytic efficiency for quercetin and luteolin than for eriodictyol. This suggests that flavonols and flavones are preferable substrates for SlOMT3. Biotransformation is considered a promising method to modify flavonoid structures. Transgenic E. coli expressing SlOMT3 was used to modify flavonoid substrates and was found to efficiently convert quercetin, luteolin, and eriodictyol to isorhamnetin, chrysoeriol and homoeriodictyol, respectively.

Correlation of Stress Wave Velocity Transmitted Through Stem with Stem Structure of Miniature Tomato

Correlation of Stress Wave Velocity Transmitted Through Stem with Stem Structure of Miniature Tomato

J042022 ミニトマト栽培の灌水制御を目的とした変動荷重を受ける茎部でのAE測定

The acoustic emission (AE) was measured for 25 days on a stem of a miniature tomato while applying loading and unloading by an electric actuator. The sensor attached on the stem applying the loading and unloading detected more AEs than the other. Moreover, the number of AE events was often rapidly increased just after applying the loading and unloading to the stem. These results showed the possibility of the induced cavitations in the stem by the loading and unloading. Number of AE events was increased on the dark period as well as the light period by applying the loading and unloading. In addition, the number of AE events of the dark period was increased with the increase in drought stress. However, the correlation between the drought stress and the ratio of AE occurrence rate due to the loading and unloading was not found. The influence of the loading and unloading on the AE occurrence behavior, therefore, might be longer than the interval of loading and unloading.

BREEDING THE TOMATO MICRO-TOM MODEL SYSTEM FOR ORNAMENTAL VALUE

BREEDING THE TOMATO MICRO-TOM MODEL SYSTEM FOR ORNAMENTAL VALUE

Differential ultrastructural changes in tomato hormonal mutants exposed to cadmium

Cadmium (Cd) is a toxic heavy metal, which can cause severe damage to plant development. The aim of this work was to characterize ultrastructural changes induced by Cd in miniature tomato cultivar Micro-Tom (MT) mutants and their wild-type counterpart. Leaves of diageotropica ( dgt) and Never ripe ( Nr) tomato hormonal mutants and wild-type MT were analysed by light, scanning and transmission electron microscopy in order to characterize the structural changes caused by the exposure to 1 mM CdCl 2. The effect of Cd on leaf ultrastructure was observed most noticeably in the chloroplasts, which exhibited changes in organelle shape and internal organization, of the thylakoid membranes and stroma. Cd caused an increase in the intercellular spaces in Nr leaves, but a decrease in the intercellular spaces in dgt leaves, as well as a decrease in the size of mesophyll cells in the mutants. Roots of the tomato hormonal mutants, when analysed by light microscopy, exhibited alterations in root diameter and disintegration of the epidermis and the external layers of the cortex. A comparative analysis has allowed the identification of specific Cd-induced ultrastructural changes in wild-type tomato, the pattern of which was not always exhibited by the mutants.