Evidence of the functional role of the ethylene receptor genes SlETR4 and SlETR5 in ethylene signal transduction in tomato.

Ethylene receptors are key factors for ethylene signal transduction. In tomato, six ethylene receptor genes (SlETR1-SlETR6) have been identified. Mutations in different ethylene receptor genes result in different phenotypes that are useful for elucidating the roles of each gene. In this study, we screened mutants of two ethylene receptor genes, SLETR4 and SLETR5, from a Micro-Tom mutant library generated by TILLING. We identified two ethylene receptor mutants with altered phenotypes and named them Sletr4-1 and Sletr5-1. Sletr4-1 has a mutation between the transmembrane and GAF domains, while Sletr5-1 has a mutation within the GAF domain. Sletr4-1 showed increased hypocotyl and root lengths, compared to those of wild type plants, under ethylene exposure. Moreover, the fruit shelf life of this mutant was extended, titratable acidity was increased and total soluble solids were decreased, suggesting a reduced ethylene sensitivity. In contrast, in the absence of exogenous ethylene, the hypocotyl and root lengths of Sletr5-1 were shorter than those of the wild type, and the fruit shelf life was shorter, suggesting that these mutants have increased ethylene sensitivity. Gene expression analysis showed that SlNR was up-regulated in the Sletr5-1 mutant line, in contrast to the down-regulation observed in the Sletr4-1 mutant line, while the down-regulation of SlCTR1, SlEIN2, SlEIL1, SlEIL3, and SlERF.E4 was observed in Sletr4-1 mutant allele, suggesting that these two ethylene receptors have functional roles in ethylene signalling and demonstrating, for the first time, a function of the GAF domain of ethylene receptors. These results suggest that the Sletr4-1 and Sletr5-1 mutants are useful for elucidating the complex mechanisms of ethylene signalling through the analysis of ethylene receptors in tomato.

Efficient transient protein expression in tomato cultivars and wild species using agroinfiltration-mediated high expression system.

KEY MESSAGE: The new transient protein expression system using the pBYR2HS vector is applicable to several tomato cultivars and wild species with high level of protein expression. Innovation and improvement of effective tools for transient protein expression in plant cells is critical for the development of plant biotechnology. We have created the new transient protein expression system using the pBYR2HS vector that led to about 4 mg/g fresh weight of protein expression in Nicotiana benthamiana. In this study, we validated the adaptability of this transient protein expression system by agroinfiltration to leaves and fruits of several tomato cultivars and wild species. Although the GFP protein was transiently expressed in the leaves and fruits of all tomato cultivars and wild species, we observed species-specific differences in protein expression. In particular, GFP protein expression was higher in the leaves and fruits of Micro-Tom, Solanum pimpinellifolium (0043) and S. pimpinellifolium (0049-w1) than in those of cultivars and wild species. Furthermore, Agrobacterium with GABA transaminase enhanced transient expression in tomato fruits of Micro-Tom. Taken together with these results, our system is applicable to several tomato cultivars and species as well as a model tomato, even though characteristics are often different among tomato cultivars or species. Thus, the system is an effective, simple, and valuable tool to achieve rapid transgene expression to examine gene function in tomato plant cells.

TOMATOMA Update: Phenotypic and Metabolite Information in the Micro-Tom Mutant Resource.

TOMATOMA (http://tomatoma.nbrp.jp/) is a tomato mutant database providing visible phenotypic data of tomato mutant lines generated by ethylmethane sulfonate (EMS) treatment or γ-ray irradiation in the genetic background of Micro-Tom, a small and rapidly growing variety. To increase mutation efficiency further, mutagenized M3 seeds were subjected to a second round of EMS treatment; M3M1 populations were generated. These plants were self-pollinated, and 4,952 lines of M3M2 mutagenized seeds were generated. We checked for visible phenotypes in the M3M2 plants, and 618 mutant lines with 1,194 phenotypic categories were identified. In addition to the phenotypic information, we investigated Brix values and carotenoid contents in the fruits of individual mutants. Of 466 samples from 171 mutant lines, Brix values and carotenoid contents were between 3.2% and 11.6% and 6.9 and 37.3 µg g(-1) FW, respectively. This metabolite information concerning the mutant fruits would be useful in breeding programs as well as for the elucidation of metabolic regulation. Researchers are able to browse and search this phenotypic and metabolite information and order seeds of individual mutants via TOMATOMA. Our new Micro-Tom double-mutagenized populations and the metabolic information could provide a valuable genetic toolkit to accelerate tomato research and potential breeding programs.

Genomic variation across distribution of Micro-Tom, a model cultivar of tomato (Solanum lycopersicum).

Micro-Tom is a cultivar of tomato (Solanum lycopersicum), which is known as a major crop and model plant in Solanaceae. Micro-Tom has phenotypic traits such as dwarfism, and substantial EMS-mutagenized lines have been reported. After Micro-Tom was generated in Florida, USA, it was distributed to research institutes worldwide and used as a genetic resource. In Japan, the Micro-Tom lines have been genetically fixed; currently three lines have been re-distributed from three institutes, but many phenotypes among the lines have been observed. We have determined the genome sequence de novo of the Micro-Tom KDRI line, one of the Micro-Tom lines distributed from Kazusa DNA Research Institute (KDRI) in Japan, and have built chromosome-scale pseudomolecules. Genotypes among six Micro-Tom lines, including three in Japan, one in the United States, one in France, and one in Brazil showed phenotypic alternation. Here, we unveiled the swift emergence of genetic diversity in both phenotypes and genotypes within the Micro-Tom genome sequence during its propagation. These findings offer valuable insights crucial for the management of bioresources.

Molecular Bases of Heat Stress Responses in Vegetable Crops With Focusing on Heat Shock Factors and Heat Shock Proteins.

The effects of the climate change including an increase in the average global temperatures, and abnormal weather events such as frequent and severe heatwaves are emerging as a worldwide ecological concern due to their impacts on plant vegetation and crop productivity. In this review, the molecular processes of plants in response to heat stress-from the sensing of heat stress, the subsequent molecular cascades associated with the activation of heat shock factors and their primary targets (heat shock proteins), to the cellular responses-have been summarized with an emphasis on the classification and functions of heat shock proteins. Vegetables contain many essential vitamins, minerals, antioxidants, and fibers that provide many critical health benefits to humans. The adverse effects of heat stress on vegetable growth can be alleviated by developing vegetable crops with enhanced thermotolerance with the aid of various genetic tools. To achieve this goal, a solid understanding of the molecular and/or cellular mechanisms underlying various responses of vegetables to high temperature is imperative. Therefore, efforts to identify heat stress-responsive genes including those that code for heat shock factors and heat shock proteins, their functional roles in vegetable crops, and also their application to developing vegetables tolerant to heat stress are discussed.

Prospects and potentials of underutilized leafy Amaranths as vegetable use for health-promotion.

Climate change causes environmental variation worldwide, which is one of the most serious threats to global food security. In addition, more than 2 billion people in the world are reported to suffer from serious malnutrition, referred to as 'hidden hunger.' Dependence on only a few crops could lead to the loss of genetic diversity and high fragility of crop breeding in systems adapting to global scale climate change. The exploitation of underutilized species and genetic resources, referred to as orphan crops, could be a useful approach for resolving the issue of adaptability to environmental alteration, biodiversity preservation, and improvement of nutrient quality and quantity to ensure food security. Moreover, the use of these alternative crops will help to increase the human health benefits and the income of farmers in developing countries. In this review, we highlight the potential of orphan crops, especially amaranths, for use as vegetables and health-promoting nutritional components. This review highlights promising diversified sources of amaranth germplasms, their tolerance to abiotic stresses, and their nutritional, phytochemical, and antioxidant values for vegetable purposes. Betalains (betacyanins and betaxanthins), unique antioxidant components in amaranth vegetables, are also highlighted regarding their chemodiversity across amaranth germplasms and their stability and degradation. In addition, we discuss the physiological functions, antioxidant, antilipidemic, anticancer, and antimicrobial activities, as well as the biosynthesis pathway, molecular, biochemical, genetics, and genomic mechanisms of betalains in detail.

Transient protein expression systems in plants and their applications.

The production of recombinant proteins is important in academic research to identify protein functions. Moreover, recombinant enzymes are used in the food and chemical industries, and high-quality proteins are required for diagnostic, therapeutic, and pharmaceutical applications. Though many recombinant proteins are produced by microbial or mammalian cell-based expression systems, plants have been promoted as alternative, cost-effective, scalable, safe, and sustainable expression systems. The development and improvement of transient expression systems have significantly reduced the period of protein production and increased the yield of recombinant proteins in plants. In this review, we consider the importance of plant-based expression systems for recombinant protein production and as genetic engineering tools.

Genetic and Molecular Mechanisms Conferring Heat Stress Tolerance in Tomato Plants.

Climate change is a major threat to global food security. Changes in climate can directly impact food systems by reducing the production and genetic diversity of crops and their wild relatives, thereby restricting future options for breeding improved varieties and reducing the ability to adapt crops to future challenges. The global surface temperature is predicted to rise by an average of 0.3°C during the next decade, and the Paris Agreement (Paris Climate Accords) aims to limit global warming to below an average of 2°C, preferably to 1.5°C compared to pre-industrial levels. Even if the goal of the Paris Agreement can be met, the predicted rise in temperatures will increase the likelihood of extreme weather events, including heatwaves, making heat stress (HS) a major global abiotic stress factor for many crops. HS can have adverse effects on plant morphology, physiology, and biochemistry during all stages of vegetative and reproductive development. In fruiting vegetables, even moderate HS reduces fruit set and yields, and high temperatures may result in poor fruit quality. In this review, we emphasize the effects of abiotic stress, especially at high temperatures, on crop plants, such as tomatoes, touching upon key processes determining plant growth and yield. Specifically, we investigated the molecular mechanisms involved in HS tolerance and the challenges of developing heat-tolerant tomato varieties. Finally, we discuss a strategy for effectively improving the heat tolerance of vegetable crops.

Multiplex exome sequencing reveals genome-wide frequency and distribution of mutations in the 'Micro-Tom' Targeting Induced Local Lesions in Genomes (TILLING) mutant library.

While the 'Micro-Tom' TILLING mutant library is used for a wide range of purposes, including both basic research of gene function and breeding of commercial cultivars, genome-wide distribution and frequency of mutations have not yet been thoroughly elucidated on a population scale. In this study, we developed a 96-plex exome sequencing method to identify and analyze mutations within the TILLING mutants that were developed in the University of Tsukuba. First, an Illumina paired-end sequencing coupled with 96-plex exome capture resulted in the acquisition of an exome sequence dataset with an average read count of 5.6 million for the 95 mutants. Over 98% of the capture target region could be covered by the short reads with an averaged read depth of 12.8, which enabled us to identify single nucleotide polymorphisms and Indels in a genome-wide manner. By subtracting intra-cultivar DNA variations that are present between wild-type 'Micro-Tom' lines, we identified 241,391 mutation candidates in 95 mutant individuals. Of these, 64,319 and 6,480 mutations were expected to cause protein amino acid substitutions or premature stop codon, respectively. Based on the exome mutation dataset, a mutant line designated 'TOMJPW601' was found to carry a premature stop codon mutation (W261*) in a putative auxin influx carrier gene SlLAX1 (Solyc09G014380), consistent with our previous report of its curly leaf phenotype. Our results suggested that a population-scale mutation database developed by multiplexed exome sequencing could be used for in silico mutant screening, which in turn could contribute to both gene function research and breeding programs.

Functional Disruption of the Tomato Putative Ortholog of HAWAIIAN SKIRT Results in Facultative Parthenocarpy, Reduced Fertility and Leaf Morphological Defects.

A number of plant microRNAs have been demonstrated to regulate developmental processes by integrating internal and environmental cues. Recently, the Arabidopsis thaliana F-box protein HAWAIIAN SKIRT (HWS) gene has been described for its role in miRNA biogenesis. We have isolated in a forward genetic screen a tomato (Solanum lycopersicum) line mutated in the putative ortholog of HWS. We show that the tomato hws-1 mutant exhibits reduction in leaflet serration, leaflet fusion, some degree of floral organ fusion, and alteration in miRNA levels, similarly to the original A. thaliana hws-1 mutant. We also describe novel phenotypes for hws such as facultative parthenocarpy, reduction in fertility and flowering delay. In slhws-1, the parthenocarpy trait is influenced by temperature, with higher parthenocarpy rate in warmer environmental conditions. Conversely, slhws-1 is able to produce seeds when grown in cooler environment. We show that the reduction in seed production in the mutant is mainly due to a defective male function and that the levels of several miRNAs are increased, in accordance with previous HWS studies, accounting for the abnormal leaf and floral phenotypes as well as the altered flowering and fruit development processes. This is the first study of HWS in fleshy fruit plant, providing new insights in the function of this gene in fruit development.

Cloning, expression and purification of cytochrome c(6) from the brown alga Hizikia fusiformis and complete X-ray diffraction analysis of the structure.

The primary sequence of cytochrome c(6) from the brown alga Hizikia fusiformis has been determined by cDNA cloning and the crystal structure has been solved at 1.6 A resolution. The crystal belonged to the tetragonal space group P4(1)2(1)2, with unit-cell parameters a = b = 84.58, c = 232.91 A and six molecules per asymmetric unit. The genome code, amino-acid sequence and crystal structure of H. fusiformis cytochrome c(6) were most similar to those of red algal cytochrome c(6). These results support the hypothesis that brown algae acquired their chloroplasts via secondary endosymbiosis involving a red algal endosymbiont and a eukaryote host.

Evaluation of internal control genes for quantitative realtime PCR analyses for studying fruit development of dwarf tomato cultivar 'Micro-Tom'.

Quantitative real-time PCR (qRT-PCR) is widely used to analyze the expression profiles of the genes of interest. In order to obtain accurate quantification data, normalization by using reliable internal control genes is essential. In this study, we evaluated the stability and applicability of eight internal control gene candidates for analyzing gene expression during fruit development in dwarf tomato cultivar Micro-Tom. We collected seventeen different samples from flowers and fruits at different developmental stages, and estimated the expression stability of the candidate genes by two statistical algorithms, geNorm and NormFinder. The combined ranking order and qRT-PCR analyses for expression profiles of SlYABBY2a, SlYABBY1a, FRUITFULL1 and APETALA2c suggested that EXPRESSED was the most stable and reliable internal control gene among the candidates. Our analysis also suggested that RPL8 was also suitable if the sample group is limited to fruits at different maturation stages. In addition to EXPRESSED, GAPDH was also applicable for relative quantitation to monitor gene expression profiles through fruit development from pistil to pericarp.

Improvement of the transient expression system for production of recombinant proteins in plants.

An efficient and high yielding expression system is required to produce recombinant proteins. Furthermore, the transient expression system can be used to identify the localization of proteins in plant cells. In this study, we demonstrated that combination of a geminiviral replication and a double terminator dramatically enhanced the transient protein expression level in plants. The GFP protein was expressed transiently in lettuce, Nicotiana benthamiana, tomatoes, eggplants, hot peppers, melons, and orchids with agroinfiltration. Compared to a single terminator, a double terminator enhanced the expression level. A heat shock protein terminator combined with an extensin terminator resulted in the highest protein expression. Transiently expressed GFP was confirmed by immunoblot analysis with anti-GFP antibodies. Quantitative analysis revealed that the geminiviral vector with a double terminator resulted in the expression of at least 3.7 mg/g fresh weight of GFP in Nicotiana benthamiana, approximately 2-fold that of the geminiviral vector with a single terminator. These results indicated that combination of the geminiviral replication and a double terminator is a useful tool for transient expression of the gene of interest in plant cells.