ERECTA Modulates Seed Germination and Fruit Development via Auxin Signaling in Tomato.

Tomato (Solanum lycopersicum) breeding for improved fruit quality emphasizes selecting for desirable taste and characteristics, as well as enhancing disease resistance and yield. Seed germination is the initial step in the plant life cycle and directly affects crop productivity and yield. ERECTA (ER) is a receptor-like kinase (RLK) family protein known for its involvement in diverse developmental processes. We characterized a Micro-Tom EMS mutant designated as a knock-out mutant of sler. Our research reveals that SlER plays a central role in controlling critical traits such as inflorescence development, seed number, and seed germination. The elevation in auxin levels and alterations in the expression of ABSCISIC ACID INSENSITIVE 3 (ABI3) and ABI5 in sler seeds compared to the WT indicate that SlER modulates seed germination via auxin and abscisic acid (ABA) signaling. Additionally, we detected an increase in auxin content in the sler ovary and changes in the expression of auxin synthesis genes YUCCA flavin monooxygenases 1 (YUC1), YUC4, YUC5, and YUC6 as well as auxin response genes AUXIN RESPONSE FACTOR 5 (ARF5) and ARF7, suggesting that SlER regulates fruit development via auxin signaling.

Fruit setting rewires central metabolism via gibberellin cascades.

Fruit set is the process whereby ovaries develop into fruits after pollination and fertilization. The process is induced by the phytohormone gibberellin (GA) in tomatoes, as determined by the constitutive GA response mutant procera However, the role of GA on the metabolic behavior in fruit-setting ovaries remains largely unknown. This study explored the biochemical mechanisms of fruit set using a network analysis of integrated transcriptome, proteome, metabolome, and enzyme activity data. Our results revealed that fruit set involves the activation of central carbon metabolism, with increased hexoses, hexose phosphates, and downstream metabolites, including intermediates and derivatives of glycolysis, the tricarboxylic acid cycle, and associated organic and amino acids. The network analysis also identified the transcriptional hub gene SlHB15A, that coordinated metabolic activation. Furthermore, a kinetic model of sucrose metabolism predicted that the sucrose cycle had high activity levels in unpollinated ovaries, whereas it was shut down when sugars rapidly accumulated in vacuoles in fruit-setting ovaries, in a time-dependent manner via tonoplastic sugar carriers. Moreover, fruit set at least partly required the activity of fructokinase, which may pull fructose out of the vacuole, and this could feed the downstream pathways. Collectively, our results indicate that GA cascades enhance sink capacities, by up-regulating central metabolic enzyme capacities at both transcriptional and posttranscriptional levels. This leads to increased sucrose uptake and carbon fluxes for the production of the constituents of biomass and energy that are essential for rapid ovary growth during the initiation of fruit set.

Stimulation of Tomato Drought Tolerance by PHYTOCHROME A and B1B2 Mutations.

Drought stress is a severe environmental issue that threatens agriculture at a large scale. PHYTOCHROMES (PHYs) are important photoreceptors in plants that control plant growth and development and are involved in plant stress response. The aim of this study was to identify the role of PHYs in the tomato cv. 'Moneymaker' under drought conditions. The tomato genome contains five PHYs, among which mutant lines in tomato PHYA and PHYB (B1 and B2) were used. Compared to the WT, phyA and phyB1B2 mutants exhibited drought tolerance and showed inhibition of electrolyte leakage and malondialdehyde accumulation, indicating decreased membrane damage in the leaves. Both phy mutants also inhibited oxidative damage by enhancing the expression of reactive oxygen species (ROS) scavenger genes, inhibiting hydrogen peroxide (H2O2) accumulation, and enhancing the percentage of antioxidant activities via DPPH test. Moreover, expression levels of several aquaporins were significantly higher in phyA and phyB1B2, and the relative water content (RWC) in leaves was higher than the RWC in the WT under drought stress, suggesting the enhancement of hydration status in the phy mutants. Therefore, inhibition of oxidative damage in phyA and phyB1B2 mutants may mitigate the harmful effects of drought by preventing membrane damage and conserving the plant hydrostatus.

SlMBP3 Knockout/down in Tomato: Normal-Sized Fruit with Increased Dry Matter Content through Non-Liquefied Locular Tissue by Altered Cell Wall Formation.

The phenotypic effect of the knockdown/out of AGAMOUS clade MADS-box gene SlMBP3 in tomato was evaluated using a transferred DNA (T-DNA)-tagged mutant of SlMBP3 and SlMBP3-RNA interference lines. SlMBP3 was preferentially expressed in the locular tissue of fruit and the seed coat combined with the endoderm. Consistent with where SlMBP3 is expressed, the SlMBP3-knockout/down lines showed non-liquefied locular tissues and increased number of seed hairs than the wild type (WT). The early cell degradation of the locular tissue was not observed in the fruits of the SlMBP3-knockout/down lines, and the cells were elongated like placental cells resulting in non-liquefied locular tissues. As the result, the fruits of the SlMBP3-knockout/down lines exhibited higher dry matter contents and titratable acidity than those of the WT. During locular tissue cell development under the SlMBP3 knockout/down, the expression of cell-enlargement-related genes (beta-expansin gene SlEXPB1 and endo-beta-1,4-D-glucanase gene Cel8) and pectinase-inhibitor-related genes (pectin esterase inhibitor gene PE inhibitor and polygalacturonase inhibitor gene PG inhibitor) was upregulated and that of pectinase-encoding genes (polygalacturonase gene QRT3-like and pectin lyase gene PL2) was downregulated. In the seed coat of the SlMBP3-knockout/down lines, tomato trichome-formation-related genes such as MYB genes containing R2 and R3 repeats (R2R3-MYB) transcription factor SlMYB75, B-type cyclin SlCycB2 and Homeodomain Leucine Zipper (HD-Zip) IV transcription factor Woolly were downregulated. Our results demonstrate that SlMBP3 is involved in the liquefaction of the locular tissue through the modification of cell development and degradation processes and seed hair formation in tomato fruits, and the SlMBP3 knockout/down results in normal-sized fruit with increased dry matter content.

A Chimeric TGA Repressor Slows Down Fruit Maturation and Ripening in Tomato.

The bZIP transcription factor (TF) SlTGA2.2 was previously highlighted as a possible hub in a network regulating fruit growth and transition to ripening (maturation phase). It belongs to a clade of TFs well known for their involvement in the regulation of the salicylic acid-dependent systemic acquired resistance. To investigate if this TGA TF plays a role in tomato fruit growth and maturation, we took advantage of the fruit-specific SlPPC2 promoter (PPC2pro) to target the expression of a SlTGA2.2-SRDX chimeric repressor in a developmental window restricted to early fruit growth and maturation. Here, we show that this SlTGA2.2-SRDX repressor alters early fruit development and metabolism, including chloroplast number and structure, considerably extends the time necessary to reach the mature green stage and slows down fruit ripening. RNA sequencing and plant hormone analyses reveal that PPC2pro:SlTGA2.2-SRDX fruits are maintained in an immature stage as long as PPC2pro is active, through early modifications of plant hormonal signaling and down-regulation of MADS-RIN and NAC-NOR ripening regulators. Once PPC2pro becomes inactive and therefore SlTGA2.2-SRDX expression is reduced, ripening can proceed, albeit at a slower pace than normal. Altogether, this work emphasizes the developmental continuum between fruit growth, maturation and ripening and provides a useful tool to alter and study the molecular bases of tomato fruit transition to ripening.

Tomato MYB21 Acts in Ovules to Mediate Jasmonate-Regulated Fertility.

The function of the plant hormone jasmonic acid (JA) in the development of tomato (Solanum lycopersicum) flowers was analyzed with a mutant defective in JA perception (jasmonate-insensitive1-1, jai1-1). In contrast with Arabidopsis (Arabidopsis thaliana) JA-insensitive plants, which are male sterile, the tomato jai1-1 mutant is female sterile, with major defects in female development. To identify putative JA-dependent regulatory components, we performed transcriptomics on ovules from flowers at three developmental stages from wild type and jai1-1 mutants. One of the strongly downregulated genes in jai1-1 encodes the MYB transcription factor SlMYB21. Its Arabidopsis ortholog plays a crucial role in JA-regulated stamen development. SlMYB21 was shown here to exhibit transcription factor activity in yeast, to interact with SlJAZ9 in yeast and in planta, and to complement Arabidopsis myb21-5 To analyze SlMYB21 function, we generated clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR associated protein 9 (Cas9) mutants and identified a mutant by Targeting Induced Local Lesions in Genomes (TILLING). These mutants showed female sterility, corroborating a function of MYB21 in tomato ovule development. Transcriptomics analysis of wild type, jai1-1, and myb21-2 carpels revealed processes that might be controlled by SlMYB21. The data suggest positive regulation of JA biosynthesis by SlMYB21, but negative regulation of auxin and gibberellins. The results demonstrate that SlMYB21 mediates at least partially the action of JA and might control the flower-to-fruit transition..

Improvement of recombinant miraculin production in transgenic tomato by crossbreeding-based genetic background modification.

An important optimization step in plant-based recombinant protein production systems is the selection of an appropriate cultivar after a potential host has been determined. Previously, we have shown that transgenic tomatoes of the variety 'Micro-Tom' accumulate incredibly high levels of miraculin (MIR) due to the introduction of MIR gene controlled by a CaMV35S promoter and a heat-shock protein terminator. However, 'Micro-Tom' is unsuitable for commercial production of MIR as it is a dwarf cultivar characterized by small-sized fruit and poor yield. Here, we used the crossbreeding approach to transfer the high MIR accumulation trait of transgenic 'Micro-Tom' tomatoes to 'Natsunokoma' and 'Aichi First', two commercial cultivars producing medium and large fruit sizes, respectively. Fruits of the resultant crossbred lines were larger (~ 95 times), but their miraculin accumulation levels (~ 1,062 µg/g fresh mass) were comparable to the donor cultivar, indicating that the high miraculin accumulation trait was preserved regardless of fruit size or cultivar. Further, the transferred trait resulted in a 3-4 fold increase in overall miraculin production than that of the previously reported line 5B. These findings demonstrate the effectiveness of crossbreeding in improving MIR production in tomatoes and could pave the way for a more efficient production of recombinant proteins in other plants.

Increased ACS Enzyme Dosage Causes Initiation of Climacteric Ethylene Production in Tomato.

Fruits of wild tomato species show different ethylene-dependent ripening characteristics, such as variations in fruit color and whether they exhibit a climacteric or nonclimacteric ripening transition. 1-Aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) and ACC oxidase (ACO) are key enzymes in the ethylene biosynthetic pathway encoded by multigene families. Gene duplication is a primary driver of plant diversification and angiosperm evolution. Here, interspecific variations in the molecular regulation of ethylene biosynthesis and perception during fruit ripening in domesticated and wild tomatoes were investigated. Results showed that the activated ACS genes were increased in number in red-ripe tomato fruits than in green-ripe tomato fruits; therefore, elevated dosage of ACS enzyme promoted ripening ethylene production. Results showed that the expression of three ACS isogenes ACS1A, ACS2, and ACS4, which are involved in autocatalytic ethylene production, was higher in red-ripe tomato fruits than in green-ripe tomato fruits. Elevated ACS enzyme dosage promoted ethylene production, which corresponded to the climacteric response of red-ripe tomato fruits. The data suggest that autoinhibitory ethylene production is common to all tomato species, while autocatalytic ethylene production is specific to red-ripe species. The essential regulators Non-ripening (NOR) and Ripening-Inhibitor (RIN) have experienced gene activation and overlapped with increasing ACS enzyme dosage. These complex levels of transcript regulation link higher ethylene production with spatiotemporal modulation of gene expression in red-ripe tomato species. Taken together, this study shows that bursts in ethylene production that accompany fruit color changes in red-ripe tomatoes are likely to be an evolutionary adaptation for seed dispersal.

Functional Characterization of Tomato Phytochrome A and B1B2 Mutants in Response to Heat Stress.

Heat stress (HS) is a prevalent negative factor affecting plant growth and development, as it is predominant worldwide and threatens agriculture on a large scale. PHYTOCHROMES (PHYs) are photoreceptors that control plant growth and development, and the stress signaling response partially interferes with their activity. PHYA, B1, and B2 are the most well-known PHY types in tomatoes. Our study aimed to identify the role of tomato 'Money Maker' phyA and phyB1B2 mutants in stable and fluctuating high temperatures at different growth stages. In the seed germination and vegetative growth stages, the phy mutants were HS tolerant, while during the flowering stage the phy mutants revealed two opposing roles depending on the HS exposure period. The response of the phy mutants to HS during the fruiting stage showed similarity to WT. The most obvious stage that demonstrated phy mutants' tolerance was the vegetative growth stage, in which a high degree of membrane stability and enhanced water preservation were achieved by the regulation of stomatal closure. In addition, both mutants upregulated the expression of heat-responsive genes related to heat tolerance. In addition to lower malondialdehyde accumulation, the phyA mutant enhanced proline levels. These results clarified the response of tomato phyA and phyB1B2 mutants to HS.

The conserved brassinosteroid-related transcription factor BIM1a negatively regulates fruit growth in tomato.

Brassinosteroids (BRs) are steroid hormones that play key roles in plant development and defense. Our goal is to harness the extensive knowledge of the Arabidopsis BR signaling network to improve productivity in crop species. This first requires identifying components of the conserved network and their function in the target species. Here, we investigated the function of SlBIM1a, the closest tomato homolog of AtBIM1, which is highly expressed in fruit. SlBIM1a-overexpressing lines displayed severe plant and fruit dwarfism, and histological characterization of different transgenic lines revealed that SlBIM1a expression negatively correlated with fruit pericarp cell size, resulting in fruit size modifications. These growth phenotypes were in contrast to those found in Arabidopsis, and this was confirmed by the reciprocal ectopic expression of SlBIM1a/b in Arabidopsis and of AtBIM1 in tomato. These results determined that BIM1 function depends more on the recipient species than on its primary sequence. Yeast two-hybrid interaction studies and transcriptomic analyses of SlBIM1a-overexpressing fruit further suggested that SlBIM1a acts through its interaction with SlBZH1 to govern the transcriptional regulation of growth-related BR target genes. Together, these results suggest that SlBIM1a is a negative regulator of pericarp cell expansion, possibly at the crossroads with auxin and light signaling.

Overproduction of ascorbic acid impairs pollen fertility in tomato.

Ascorbate is a major antioxidant buffer in plants. Several approaches have been used to increase the ascorbate content of fruits and vegetables. Here, we combined forward genetics with mapping-by-sequencing approaches using an ethyl methanesulfonate (EMS)-mutagenized Micro-Tom population to identify putative regulators underlying a high-ascorbate phenotype in tomato fruits. Among the ascorbate-enriched mutants, the family with the highest fruit ascorbate level (P17C5, up to 5-fold wild-type level) had strongly impaired flower development and produced seedless fruit. Genetic characterization was performed by outcrossing P17C5 with cv. M82. We identified the mutation responsible for the ascorbate-enriched trait in a cis-acting upstream open reading frame (uORF) involved in the downstream regulation of GDP-l-galactose phosphorylase (GGP). Using a specific CRISPR strategy, we generated uORF-GGP1 mutants and confirmed the ascorbate-enriched phenotype. We further investigated the impact of the ascorbate-enriched trait in tomato plants by phenotyping the original P17C5 EMS mutant, the population of outcrossed P17C5 × M82 plants, and the CRISPR-mutated line. These studies revealed that high ascorbate content is linked to impaired floral organ architecture, particularly anther and pollen development, leading to male sterility. RNA-seq analysis suggested that uORF-GGP1 acts as a regulator of ascorbate synthesis that maintains redox homeostasis to allow appropriate plant development.

Efficient base editing in tomato using a highly expressed transient system.

KEY MESSAGE: Base editing in tomatoes was achieved by transient expression. The Solanaceae plants, particularly the tomato (Solanum lycopersicum), is of huge economic value worldwide. The tomato is a unique model plant for studying the functions of genes related to fruit ripening. Deeper understanding of tomatoes is of great importance for both plant research and the economy. Genome editing technology, such as CRISPR/Cas9, has been used for functional genetic research. However, some challenges, such as low transformation efficiency, remain with this technology. Moreover, the foreign Cas9 and gRNA expression cassettes must be removed to obtain null-segregants In this study, we used a high-level transient expression system to improve the base editing technology. A high-level transient expression system has been established previously using geminiviral replication and a double terminator. The pBYR2HS vector was used for this transient expression system. nCas9-CDA and sgRNA-SlHWS were introduced into this vector, and the protein and RNA were then transiently expressed in tomato tissues by agroinfiltration. The homozygous mutant produced by base editing was obtained in the next generation with an efficiency of about 18%. nCas9-free next-generation plants were 71%. All the homozygous base-edited plants in next generation are nCas9-free. These findings show that the high-level transient expression system is useful for base editing in tomatoes.

The effect of low ascorbic acid content on tomato fruit ripening.

MAIN CONCLUSION: The oxidant/antioxidant balance affects the ripening time of tomato fruit. Ripening of tomato fruit is associated with several modifications such as loss of cell wall firmness and transformation of chloroplasts to chromoplasts. Besides a peak in H2O2, reactive oxygen species (ROS) are observed at the transition stage. However, the role of different components of oxidative stress metabolism in fruit ripening has been scarcely addressed. Two GDP-L-galactose phosphorylase (GGP) Solanum lycopersicum L. cv Micro-Tom mutants which have fruit with low ascorbic acid content (30% of wild type) were used in this work to unravel the participation of ascorbic acid and H2O2 in fruit maturation. Both GGP mutants show delayed fruit maturation with no peak of H2O2; treatment with ascorbic acid increases its own concentration and accelerates ripening only in mutants to become like wild type plants. Unexpectedly, the treatment with ascorbic acid increases H2O2 synthesis in both mutants resembling what is observed in wild type fruit. Exogenous supplementation with H2O2 decreases its own synthesis delaying fruit maturation in plants with low ascorbic acid content. The site of ROS production is localized in the chloroplasts of fruit of all genotypes as determined by confocal microscopy analysis. The results presented here demonstrate that both ascorbic acid and H2O2 actively participate in tomato fruit ripening.

Deficiency of GDP-L-galactose phosphorylase, an enzyme required for ascorbic acid synthesis, reduces tomato fruit yield.

MAIN CONCLUSION: Reduced GDP-L-galactose phosphorylase expression and deficiency of ascorbic acid content lead to decreased fruit set and yield in tomato plants. Reduced GDP-L-galactose phosphorylase expression and deficiency of ascorbic acid content lead to decreased fruit set and yield in tomato plants. GDP-L-galactose phosphorylase (GGP) catalyzes the first step committed to ascorbic acid synthesis. The participation of GDP-L-galactose phosphorylase and ascorbate in tomato fruit production and quality was studied in this work using two SlGGP1 deficient EMS Micro-Tom mutants. The SlGGP1 mutants display decreased concentrations of ascorbate in roots, leaves, flowers, and fruit. The initiation of anthesis is delayed in ggp1 plants but the number of flowers is similar to wild type. The number of fruits is reduced in ggp1 mutants with an increased individual weight. However, the whole fruit biomass accumulation is reduced in both mutant lines. Fruits of the ggp1 plants produce more ethylene and show higher firmness and soluble solids content than the wild type after the breaker stage. Leaf CO2 uptake decreases about 50% in both ggp1 mutants at saturating light conditions; however, O2 production in an enriched CO2 atmosphere is only 19% higher in wild type leaves. Leaf conductance that is largely reduced in both mutants may be the main limitation for photosynthesis. Sink-source assays and hormone concentration were measured to determine restrictions to fruit yield. Manipulation of leaf area/fruit number relationship demonstrates that the number of fruits and not the provision of photoassimilates from the source restricts biomass accumulation in the ggp1 lines. The lower gibberellins concentration measured in the flowers would contribute to the lower fruit set, thus impacting in tomato yield. Taken as a whole these results demonstrate that ascorbate biosynthetic pathway critically participates in tomato development and fruit production.

Evaluation of heritability of ß-eudesmol/hinesol content ratio in Atractylodes lancea De Candolle.

BACKGROUND: Atractylodes lancea De Candolle is a medicinal plant distributed in East Asia. Its rhizome has been used as an important crude drug in traditional Chinese and Japanese medicines for the treatment of numerous diseases and disorders. In recent years, the demand for mass production of the crude drug with a stable quality has increased. Its major active compounds are sesquiterpenoids, such as ß-eudesmol and hinesol that have closely related chemical structures with each other. As the criteria for evaluating the quality of A. lancea, the ß-eudesmol/hinesol content ratio is considered important. In A. lancea, the ratio could be considered to be influenced by genetic factors, geographical environment factors and these interactions. Few studies of a detail genetic analyses for ß-eudesmol/hinesol content ratio have been reported. Therefore, we evaluated the heritability and genotype-environment interaction on the ß-eudesmol/hinesol content ratio in A. lancea using clonal lines propagated with division of rhizome. RESULTS: The heritability of the ß-eudesmol/hinesol content ratio in A. lancea was evaluated through the cultivation of clonal lines of A. lancea in both different years (2016, 2017) and locations (Hokkaido, Ibaraki). Correlations between ß-eudesmol and hinesol contents were identified in all clonal lines, with high correlation coefficients (r = 0.73-0.99). The broad-sense heritability of the ß-eudesmol/hinesol content ratio was revealed to be high at 0.92. The effects of cultivation year were smaller than that of genotype, and few genotype-environment interactions were observed. In addition, the influence of cultivation location was also smaller than that of genotype, and the correlation between the two cultivation locations on the ß-eudesmol/hinesol content ratio was high. The results suggested that the ß-eudesmol/hinesol content ratio in A. lancea is highly dependent on genetic factors. CONCLUSION: We demonstrate that the heritability of ß-eudesmol/hinesol content ratio is high and that the effects of genetic factors were stronger than that of environmental factors such as cultivation location and year. Our findings suggested that selective breeding and clonal propagation are effective strategies for the production of A. lancea with stable qualities for use in the production of crude drugs.

JRE4 is a master transcriptional regulator of defense-related steroidal glycoalkaloids in tomato.

Steroidal glycoalkaloids (SGAs) are specialized anti-nutritional metabolites that accumulate in Solanum lycopersicum (tomato) and Solanum tuberosum (potato). A series of SGA biosynthetic genes is known to be upregulated in Solanaceae species by jasmonate-responsive Ethylene Response Factor transcription factors, including JRE4 (otherwise known as GAME9), but the exact regulatory significance in planta of each factor has remained unaddressed. Here, via TILLING-based screening of an EMS-mutagenized tomato population, we isolated a JRE4 loss-of-function line that carries an amino acid residue missense change in a region of the protein important for DNA binding. In this jre4 mutant, we observed downregulated expression of SGA biosynthetic genes and decreased SGA accumulation. Moreover, JRE4 overexpression stimulated SGA production. Further characterization of jre4 plants revealed their increased susceptibility to the generalist herbivore Spodoptera litura larvae. This susceptibility illustrates that herbivory resistance is dependent on JRE4-mediated defense responses, which include SGA accumulation. Ethylene treatment attenuated the jasmonate-mediated JRE4 expression induction and downstream SGA biosynthesis in tomato leaves and hairy roots. Overall, this study indicated that JRE4 functions as a primary master regulator of SGA biosynthesis, and thereby contributes toward plant defense against chewing insects.

Aberrant Stamen Development is Associated with Parthenocarpic Fruit Set Through Up-Regulation of Gibberellin Biosynthesis in Tomato.

Parthenocarpy, a process in which fruit set occurs without fertilization, leads to the production of seedless fruit. A number of floral homeotic mutants with abnormal stamen development exhibit parthenocarpic fruit set. Flower development is thought to repress ovary growth before anthesis. However, the mechanism of parthenocarpic fruit development caused by aberrant flower formation is poorly understood. To investigate the molecular mechanism of parthenocarpic fruit development in floral homeotic mutants, we performed functional analysis of Tomato APETALA3 (TAP3) by loss-of-function approaches. Organ-specific promoter was used to induce organ-specific loss of function in stamen and ovary/fruit. We observed increased cell expansion in tap3 mutants and TAP3-RNAi lines during parthenocarpic fruit growth. These were predominantly accompanied by the up-regulation of GA biosynthesis genes, including SlGA20ox1, SlGA20ox2, and SlGA20ox3, as well as reduced expression of the GA-inactivating gene SlGA2ox1 and the auxin signaling gene SlARF7 involved in a crosstalk between GA and auxin. These transcriptional profiles are in agreement with the GA levels in these lines. These results suggest that stamen development negatively regulates fruit set by repressing the GA biosynthesis.

Agrobacterium tumefaciens Enhances Biosynthesis of Two Distinct Auxins in the Formation of Crown Galls.

The plant pathogen Agrobacterium tumefaciens infects plants and introduces the transferred-DNA (T-DNA) region of the Ti-plasmid into nuclear DNA of host plants to induce the formation of tumors (crown galls). The T-DNA region carries iaaM and iaaH genes for synthesis of the plant hormone auxin, indole-3-acetic acid (IAA). It has been demonstrated that the iaaM gene encodes a tryptophan 2-monooxygenase which catalyzes the conversion of tryptophan to indole-3-acetamide (IAM), and the iaaH gene encodes an amidase for subsequent conversion of IAM to IAA. In this article, we demonstrate that A. tumefaciens enhances the production of both IAA and phenylacetic acid (PAA), another auxin which does not show polar transport characteristics, in the formation of crown galls. Using liquid chromatography-tandem mass spectroscopy, we found that the endogenous levels of phenylacetamide (PAM) and PAA metabolites, as well as IAM and IAA metabolites, are remarkably increased in crown galls formed on the stem of tomato plants, implying that two distinct auxins are simultaneously synthesized via the IaaM-IaaH pathway. Moreover, we found that the induction of the iaaM gene dramatically elevated the levels of PAM, PAA and its metabolites, along with IAM, IAA and its metabolites, in Arabidopsis and barley. From these results, we conclude that A. tumefaciens enhances biosynthesis of two distinct auxins in the formation of crown galls.

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.