A single amino acid at position 31 in the N-terminus of the coat protein of cucumber mosaic virus determines its avirulence function for RCY1-conferred virus resistance.

The coat protein (CP) of the cucumber mosaic virus (CMV) yellow strain [CMV(Y)], but not the CMV B2 strain [CMV(B2)], serves as an avirulence determinant against the NB-LRR class RCY1 of Arabidopsis thaliana. To investigate the avirulence function, a series of binary vectors were constructed by partially exchanging the CP coding sequence between CMV(Y) and CMV(B2) or introducing nucleotide substitutions. These vectors were transiently expressed in Nicotiana benthamiana leaves transformed with modified RCY1 cDNA. Analysis of hypersensitive resistance-cell death (HCD), CP accumulation, and defense gene expression at leaf sites infiltrated with Agrobacterium indicated that a single amino acid at position 31 of the CP seems to determine the avirulence function.

Effect of Light Quality on Metabolomic, Ionomic, and Transcriptomic Profiles in Tomato Fruit.

Light quality affects plant growth and the functional component accumulation of fruits. However, there is little knowledge of the effects of light quality based on multiomics profiles. This study combined transcriptomic, ionomic, and metabolomic analyses to elucidate the effects of light quality on metabolism and gene expression in tomato fruit. Micro-Tom plants were grown under blue or red light-emitting diode light for 16 h daily after anthesis. White fluorescent light was used as a reference. The metabolite and element concentrations and the expression of genes markedly changed in response to blue and red light. Based on the metabolomic analysis, amino acid metabolism and secondary metabolite biosynthesis were active in blue light treatment. According to transcriptomic analysis, differentially expressed genes in blue and red light treatments were enriched in the pathways of secondary metabolite biosynthesis, carbon fixation, and glycine, serine, and threonine metabolism, supporting the results of the metabolomic analysis. Ionomic analysis indicated that the element levels in fruits were more susceptible to changes in light quality than in leaves. The concentration of some ions containing Fe in fruits increased under red light compared to under blue light. The altered expression level of genes encoding metal ion-binding proteins, metal tolerance proteins, and metal transporters in response to blue and red light in the transcriptomic analysis contributes to changes in the ionomic profiles of tomato fruit.

High-Resolution X-ray Phase-Contrast Imaging and Sensory and Rheometer Tests in Cooked Edamame.

Although several reports exist on the use of X-ray analysis in vegetables and fruits to examine internal disorders, cavities, and porosity, information on X-ray analysis of qualities, such as texture, is lacking as well as information on X-ray analysis of legumes. Therefore, this study aimed to perform X-ray analysis with sensory and rheometer tests in cooked vegetable soybean (edamame). Edamame is popular worldwide due to its deliciousness and nutritional value. Vascular structures and cracks around them were clearly visualized using X-ray phase-contrast computed tomography (CT) imaging. In addition, we observed the fine structure of the seed coat, which could be important for seed development, germination, and processing. The density in the edamame beans declined as the boiling time increased, promoting a reduction in hardness described in sensory and rheometer tests. The reduction in density proceeded from the gap between cotyledons, the opposite side of the hypocotyl, and the crack. Collectively, the findings show that the high-resolution X-ray phase-contrast CT imaging conducted in a nondestructive manner may help in effectively evaluating the quality of vegetables and in observing the internal structures related to plant development.

Characterization of the FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 Homolog SlFKF1 in Tomato as a Model for Plants with Fleshy Fruit.

FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1) is a blue-light receptor whose function is related to flowering promotion under long-day conditions in Arabidopsis thaliana. However, information about the physiological role of FKF1 in day-neutral plants and even the physiological role other than photoperiodic flowering is lacking. Thus, the FKF1 homolog SlFKF1 was investigated in tomato, a day-neutral plant and a useful model for plants with fleshy fruit. It was confirmed that SlFKF1 belongs to the FKF1 group by phylogenetic tree analysis. The high sequence identity with A. thaliana FKF1, the conserved amino acids essential for function, and the similarity in the diurnal change in expression suggested that SlFKF1 may have similar functions to A. thaliana FKF1. CONSTANS (CO) is a transcription factor regulated by FKF1 and is responsible for the transcription of genes downstream of CO. cis-Regulatory elements targeted by CO were found in the promoter region of SINGLE FLOWER TRUSS (SFT) and RIN, which are involved in the regulation of flowering and fruit ripening, respectively. The blue-light effects on SlFKF1 expression, flowering, and fruit lycopene concentration have been observed in this study and previous studies. It was confirmed in RNA interference lines that the low expression of SlFKF1 is associated with late flowering with increased leaflets and low lycopene concentrations. This study sheds light on the various physiological roles of FKF1 in plants.

Cucumber Mosaic Virus Infection in Arabidopsis: A Conditional Mutualistic Symbiont?

A cucumber mosaic virus isolate, named Ho [CMV(Ho)], was isolated from a symptomless Arabidopsis halleri field sample containing low virus titers. An analysis of CMV(Ho) RNA molecules indicated that the virus isolate, besides the usual cucumovirus tripartite RNA genome, additionally contained defective RNA3 molecules and a satellite RNA. To study the underlying mechanism of the persistent CMV(Ho) infection in perennial A. halleri, infectious cDNA clones were generated for all its genetic elements. CMV, which consists of synthetic transcripts from the infectious tripartite RNA genomes, and designated CMV(Ho)tr, multiplied in A. halleri and annual Arabidopsis thaliana Col-0 to a similar level as the virulent strain CMV(Y), but did not induce any symptoms in them. The response of Col-0 to a series of reassortant CMVs between CMV(Ho)tr and CMV(Y) suggested that the establishment of an asymptomatic phenotype of CMV(Ho) infection was due to the 2b gene of CMV RNA2, but not due to the presence of the defective RNA3 and satellite RNA. The accumulation of CMV(Ho) 2b protein tagged with the FLAG epitope (2b.Ho-FLAG) in 2b.Ho-FLAG-transformed Col-0 did not induce any symptoms, suggesting a 2b-dependent persistency of CMV(Ho)tr infection in Arabidopsis. The 2b protein interacted with Argonaute 4, which is known to regulate the cytosine methylation levels of host genomic DNA. Whole genomic bisulfite sequencing analysis of CMV(Ho)tr- and mock-inoculated Col-0 revealed that cytosine hypomethylation in the promoter regions of 82 genes, including two genes encoding transcriptional regulators (DOF1.7 and CBP1), was induced in response to CMV(Ho)tr infection. Moreover, the increased levels of hypomethylation in the promoter region of both genes, during CMV(Ho)tr infection, were correlated with the up- or down-regulation of their expression. Taken altogether, the results indicate that during persistent CMV(Ho) infection in Arabidopsis, host gene expression may be epigenetically modulated resulting from a 2b-mediated cytosine hypomethylation of host genomic DNA.

Effects of abiotic stresses on sorbitol biosynthesis and metabolism in tomato (Solanum lycopersicum).

Polyols such as sorbitol and ribitol are a class of compatible solutes in plants that may play roles in tolerance to abiotic stresses. This study investigated the effects of water stress on sorbitol biosynthesis and metabolism and sorbitol and ribitol accumulation in tomato (Solanum lycopersicum L.). Water stress induced by withholding water and by using polyethylene glycol as a root incubation solution to mimic water stress, and NaCl stress were applied to wild-type (WT) and three genetically-modified lines of tomato (cv. Ailsa Craig), a control vector line TR22, and 2 sorbitol dehydrogenase (sdh) antisense lines TR45 and TR49. Sorbitol and ribitol content, as well as the enzymatic activities, protein accumulation, and gene expression patterns of the key sorbitol cycle enzymes aldose-6-phosphate reductase (A6PR), aldose reductase (AR), and sorbitol dehydrogenase (SDH), were measured in mature leaves. In response to the stresses, both sorbitol and ribitol accumulated in leaf tissue, most significantly in the sdh antisense lines. A6PR, characterised for the first time in this work, and AR both exhibited increased enzymatic activity correlated with sorbitol accumulation during the stress treatments, with SDH also increasing in WT and TR22 to metabolise sorbitol, reducing the content to control levels within 3 days after re-watering. In the sdh antisense lines, the lack of significant SDH activity resulted in the increased sorbitol and ribitol content above WT levels. The results highlighted a role for both A6PR and AR in biosynthesis of sorbitol in tomato where the high activity of both enzymes was associated with sorbitol accumulation. Although both A6PR and AR are aldo-keto reductases and use NADPH as a co-factor, the AR-specific inhibitor sorbinil inhibited AR only indicating that they are different enzymes. The determination that sorbitol, and perhaps ribitol as well, plays a role in abiotic responses in tomato provides a cornerstone for future studies examining how they impact tomato tolerance to abiotic stresses, and if their alteration could improve stress tolerance.

Characterization of PcLEA14, a Group 5 Late Embryogenesis Abundant Protein Gene from Pear (Pyrus communis).

Fruit trees need to overcome harsh winter climates to ensure perennially; therefore, they are strongly influenced by environmental stress. In the present study, we focused on the pear homolog PcLEA14 belonging to the unique 5C late embryogenesis abundant (LEA) protein group for which information is limited on fruit trees. PcLEA14 was confirmed to belong to this protein group using phylogenetic tree analysis, and its expression was induced by low-temperature stress. The seasonal fluctuation in its expression was considered to be related to its role in enduring overwinter temperatures, which is particularly important in perennially. Moreover, the function of PcLEA14 in low-temperature stress tolerance was revealed in transgenic Arabidopsis. Subsequently, the pear homolog of dehydration-responsive element-binding protein/C-repeat binding factor1 (DREB1), which is an important transcription factor in low-temperature stress tolerance and is uncharacterized in pear, was analyzed after bioinformatics analysis revealed the presence of DREB cis-regulatory elements in PcLEA14 and the dormancy-related gene, both of which are also expressed during low temperatures. Among the five PcDREBs, PcDREB1A and PcDREB1C exhibited similar expression patterns to PcLEA14 whereas the other PcDREBs were not expressed in winter, suggesting their different physiological roles. Our findings suggest that the low-temperature tolerance mechanism in overwintering trees is associated with group 5C LEA proteins and DREB1.

Characterization of FLOWERING LOCUS C Homologs in Apple as a Model for Fruit Trees.

To elucidate the molecular mechanism of juvenility and annual flowering of fruit trees, FLOWERING LOCUS C (FLC), an integrator of flowering signals, was investigated in apple as a model. We performed sequence and expression analyses and transgenic experiments related to juvenility with annual flowering to characterize the apple FLC homologs MdFLC. The phylogenetic tree analysis, which included other MADS-box genes, showed that both MdFLC1 and MdFLC3 belong to the same FLC group. MdFLC1c from one of the MdFLC1 splice variants and MdFLC3 contain the four conserved motives of an MIKC-type MADS protein. The mRNA of variants MdFLC1a and MdFLC1b contain intron sequences, and their deduced amino acid sequences lack K- and C-domains. The expression levels of MdFLC1a, MdFLC1b, and MdFLC1c decreased during the flowering induction period in a seasonal expression pattern in the adult trees, whereas the expression level of MdFLC3 did not decrease during that period. This suggests that MdFLC1 is involved in flowering induction in the annual growth cycle of adult trees. In apple seedlings, because phase change can be observed in individuals, seedlings can be used for analysis of expression during phase transition. The expression levels of MdFLC1b, MdFLC1c, and MdFLC3 were high during the juvenile phase and low during the transitional and adult phases. Because the expression pattern of MdFLC3 suggests that it plays a specific role in juvenility, MdFLC3 was subjected to functional analysis by transformation of Arabidopsis. The results revealed the function of MdFLC3 as a floral repressor. In addition, MdFT had CArG box-like sequences, putative targets for the suppression of flowering by MdFLC binding, in the introns and promoter regions. These results indicate that apple homologs of FLC, which might play a role upstream of the flowering signals, could be involved in juvenility as well as in annual flowering. Apples with sufficient genome-related information are useful as a model for studying phenomena unique to woody plants such as juvenility and annual flowering.

Characterization and Expression Analysis of the Ca2+/Cation Antiporter Gene Family in Tomatoes.

The Ca2+/cation antiporter (CaCA) superfamily plays an important role in the regulation of the essential element Ca2+ and cation concentrations. Characterization and expression analyses of CaCA superfamily genes were performed in the tomato (Solanum lycopersicum) as a representative of dicotyledonous plants and fruit crops. Sixteen CaCA candidate genes were found and identified as tomato CaCA, SlCaCA, by a domain search. In a phylogenetic analysis of the SlCaCA superfamily, the 16 genes were classified into SlCAX, SlNCL, SlCCX, and SlMHX families. Among them, Solyc12g011070, belonging to the SlCAX family, had four splice variants, three of which were predicted to be nonfunctional because of a lack of important motifs. EF-hand domains were only found in SlNCL, in addition to consensus Na_Ca_ex domains, and the region containing EF-hand domains was characteristically long in some members of SlNCL. Furthermore, four genes of the SlCCX family were found to be intronless. As for intracellular localization, one SlCCX member was predicted to be localized to the plasma membrane, while other SlCCXs, SlCAXs, and SlMHXs were predicted to be localized to the vacuolar membrane. The expression patterns of SlCaCAs in various organs, including during several developmental stages of fruit, were classified into four groups. Genes involved in each of the SlCAX, SlNCL, and SlCCX gene families were categorized into three or four groups according to expression patterns, suggesting role sharing within each family. The main member in each subfamily and the members with characteristic fruit expression patterns included genes whose expression was regulated by sugar or auxin and that were highly expressed in a line having metabolite-rich fruit.

The Ionomic Study of Vegetable Crops.

Soil contains various essential and nonessential elements, all of which can be absorbed by plants. Plant ionomics is the study of the accumulation of these elements (the ionome) in plants. The ionomic profile of a plant is affected by various factors, including species, variety, organ, and environment. In this study, we cultivated various vegetable crop species and cultivars under the same field conditions and analyzed the level of accumulation of each element in the edible and nonedible parts using ionomic techniques. The concentration of each element in the edible parts differed between species, which could be partly explained by differences in the types of edible organs (root, leaf, seed, and fruit). For example, the calcium concentration was lower in seeds and fruit than in other organs because of the higher dependency of calcium accumulation on xylem transfer. The concentration of several essential microelements and nonessential elements in the edible parts also varied greatly between cultivars of the same species, knowledge of which will help in the breeding of vegetables that are biofortified or contain lower concentrations of toxic elements. Comparison of the ionomes of the fruit and leaves of tomato (Solanum lycopersicum) and eggplant (S. melongena) indicated that cadmium and boron had higher levels of accumulation in eggplant fruit, likely because of their effective transport in the phloem. We also found that homologous elements that have been reported to share the same uptake/transport system often showed significant correlation only in a few families and that the slopes of these relationships differed between families. Therefore, these differences in the characteristics of mineral accumulation are likely to affect the ionomic profiles of different families.

Dynamic Metabolic Regulation by a Chromosome Segment from a Wild Relative During Fruit Development in a Tomato Introgression Line, IL8-3.

We performed comparative metabolome and transcriptome analyses throughout fruit development using the tomato cultivar M82 and its near-isogenic line IL8-3, with interesting and useful traits such as a high content of soluble solids. Marked differences between M82 and IL8-3 were found not only in ripe fruits but also at 20 days after flowering (DAF) in the hierarchical clustering analysis of the metabolome, whereas patterns were similar between the two genotypes at 10 and 30 DAF. Our metabolome analysis conclusively showed that 20 DAF is an important stage of fruit metabolism and that the Solanum pennellii introgressed region in IL8-3 plays a key role in metabolic changes at this stage. Carbohydrate and amino acid metabolism were found to be promoted in IL8-3 at 20 DAF and the ripening stage, respectively, whereas transcriptome analysis showed no marked differences between the two genotypes, indicating that dynamic metabolic regulation at 20 DAF and the ripening stage was controlled by relatively few genes. The transcript levels of the cell wall invertase (LIN6) and sucrose synthase (TOMSSF) genes in starch and sucrose metabolic pathway and that of the glutamate synthase (SlGOGAT) gene in the amino acid metabolic pathway in IL8-3 fruit were higher than those in M82, and SlGOGAT expression was enhanced under high-sugar conditions. The results suggest that the promotion of carbohydrate metabolism by LIN6 and TOMSSF in IL8-3 fruit at 20 DAF affects SlGOGAT expression and amino acid accumulation via higher sugar concentration at the late stage of fruit development.

Expression of an extracellular ribonuclease gene increases resistance to Cucumber mosaic virus in tobacco.

BACKGROUND: The apoplast plays an important role in plant defense against pathogens. Some extracellular PR-4 proteins possess ribonuclease activity and may directly inhibit the growth of pathogenic fungi. It is likely that extracellular RNases can also protect plants against some viruses with RNA genomes. However, many plant RNases are multifunctional and the direct link between their ribonucleolytic activity and antiviral defense still needs to be clarified. In this study, we evaluated the resistance of Nicotiana tabacum plants expressing a non-plant single-strand-specific extracellular RNase against Cucumber mosaic virus. RESULTS: Severe mosaic symptoms and shrinkage were observed in the control non-transgenic plants 10 days after inoculation with Cucumber mosaic virus (CMV), whereas such disease symptoms were suppressed in the transgenic plants expressing the RNase gene. In a Western blot analysis, viral proliferation was observed in the uninoculated upper leaves of control plants, whereas virus levels were very low in those of transgenic plants. These results suggest that resistance against CMV was increased by the expression of the heterologous RNase gene. CONCLUSION: We have previously shown that tobacco plants expressing heterologous RNases are characterized by high resistance to Tobacco mosaic virus. In this study, we demonstrated that elevated levels of extracellular RNase activity resulted in increased resistance to a virus with a different genome organization and life cycle. Thus, we conclude that the pathogen-induced expression of plant apoplastic RNases may increase non-specific resistance against viruses with RNA genomes.

A novel strategy to produce sweeter tomato fruits with high sugar contents by fruit-specific expression of a single bZIP transcription factor gene.

Enhancement of sugar content and sweetness is desirable in some vegetables and in almost all fruits; however, biotechnological methods to increase sugar content are limited. Here, a completely novel methodological approach is presented that produces sweeter tomato fruits but does not have any negative effects on plant growth. Sucrose-induced repression of translation (SIRT), which is mediated by upstream open reading frames (uORFs), was initially reported in Arabidopsis AtbZIP11, a class S basic region leucine zipper (bZIP) transcription factor gene. Here, two AtbZIP11 orthologous genes, SlbZIP1 and SlbZIP2, were identified in tomato (Solanum lycopersicum). SlbZIP1 and SlbZIP2 contained four and three uORFs, respectively, in the cDNA 5'-leader regions. The second uORFs from the 5' cDNA end were conserved and involved in SIRT. Tomato plants were transformed with binary vectors in which only the main open reading frames (ORFs) of SlbZIP1 and SlbZIP2, without the SIRT-responsive uORFs, were placed under the control of the fruit-specific E8 promoter. Growth and morphology of the resulting transgenic tomato plants were comparable to those of wild-type plants. Transgenic fruits were approximately 1.5-fold higher in sugar content (sucrose/glucose/fructose) than nontransgenic tomato fruits. In addition, the levels of several amino acids, such as asparagine and glutamine, were higher in transgenic fruits than in wild-type fruits. This was expected because SlbZIP transactivates the asparagine synthase and proline dehydrogenase genes. This 'sweetening' technology is broadly applicable to other plants that utilize sucrose as a major translocation sugar.

Constitutively overexpressing a tomato fructokinase gene (LeFRK1) in cotton (Gossypium hirsutum L. cv. Coker 312) positively affects plant vegetative growth, boll number and seed cotton yield.

Increasing fructokinase (FRK) activity in cotton (Gossypium hirsutum L.) plants may reduce fructose inhibition of sucrose synthase (Sus) and lead to improved fibre yield and quality. Cotton was transformed with a tomato (Solanum lycopersicum L.) fructokinase gene (LeFRK1) under the control of the CMV 35S promoter. In a greenhouse, the LeFRK1 plants had increased fibre and leaf FRK activity over nonexpressing nulls, but not improved fibre length and strength. Compared with the nulls, LeFRK1 plants yielded 13-100% more seed-cotton mass per boll and more bolls per plant, and therefore more seed cotton and fibre yield per plant. The enhanced yield was related to a greater seed number per boll for LeFRK1 plants. Photosynthetic rates were not appreciably different among genotypes. However, more area per leaf and leaf number (in some instances) for LeFRK1 plants than for nulls enhanced the capacity for C gain. Larger leaf areas for LeFRK1 plants were associated with larger stem diameters. Lower sucrose levels in developing leaves of LeFRK1 plants suggest that LeFRK1 overexpression leads to improved in vivo Sus activity in developing leaves and possibly in developing seeds. The improvement in yield for LeFRK1 plants may also be the result of improvements in photosynthate supply as a consequence of greater leaf area.

Characterization of an uncharacterized aldo-keto reductase gene from peach and its role in abiotic stress tolerance.

The aldo-keto reductase (AKR) superfamily is a large enzyme group of NADP-dependent oxidoreductases with numerous roles in metabolism, but many members in this superfamily remain uncharacterized. Here, PpAKR1, which was cloned from the rosaceous peach tree (Prunus persica), was investigated as a member of the superfamily. While PpAKR1 had amino acids that are important in AKRs and which belonged to the AKR4 group, PpAKR1 did not seem to belong to any of the AKR4 subgroups. PpAKR1 mRNA abundance increased with abscisic acid, oxidative stress, and cold and salt stress treatments in peach. NADP-dependent polyol dehydrogenase activity was increased in Arabidopsis thaliana transformed with PpAKR1. Salt tolerance increased in Arabidopsis transformed with PpAKR1. PpAKR1, which was a previously uncharacterized member of the AKR superfamily, could be involved in the abiotic stress tolerance.

Transcriptional profile of tomato roots exhibiting Bacillus thuringiensis-induced resistance to Ralstonia solanacearum.

KEY MESSAGE: Activation of SA-dependent signaling pathway and suppression of JA-dependent signaling pathway seem to play key roles inB. thuringiensis-induced resistance toR. solanacearumin tomato plants. Bacillus thuringiensis, a well-known and effective bio-insecticide, has attracted considerable attention as a potential biological control agent for the suppression of plant diseases. Treatment of tomato roots with a filter-sterilized cell-free filtrate (CF) of B. thuringiensis systemically suppresses bacterial wilt caused by Ralstonia solanacearum through systemic activation of the plant defense system. Comparative analysis of the expression of the Pathogenesis-Related 1(P6) gene, a marker for induced resistance to pathogens, in various tissues of tomato plants treated with CF on their roots suggested that the B. thuringiensis-induced defense system was activated in the leaf, stem, and main root tissues, but not in the lateral root tissue. At the same time, the growth of R. solanacearum was significantly suppressed in the CF-treated main roots but not in the CF-treated lateral roots. This distinct activation of the defense reaction and suppression of R. solanacearum were reflected by the differences in the transcriptional profiles of the main and lateral tissues in response to the CF. In CF-treated main roots, but not CF-treated lateral roots, the expression of several salicylic acid (SA)-responsive defense-related genes was specifically induced, whereas jasmonic acid (JA)-related gene expression was either down-regulated or not induced in response to the CF. On the other hand, genes encoding ethylene (ET)-related proteins were induced equally in both the main and lateral root tissues. Taken together, the co-activation of SA-dependent signaling pathway with ET-dependent signaling pathway and suppression of JA-dependent signaling pathway may play key roles in B. thuringiensis-induced resistance to R. solanacearum in tomato.

Heterologous expression of tomato glycoside hydrolase family 3 α-L-arabinofuranosidase/ß-xylosidases in tobacco suspension cultured cells and synergic action of a family 51 isozyme under antisense suppression of the enzyme.

Four cDNA clones (SlArf/Xyl1-4) encoding α-l-arabinofuranosidase/ß-xylosidase belonging to glycoside hydrolase family 3 were obtained from tomato (Solanum lycopersicum) fruit. SlArf/Xyl1 was expressed in various organs. Its level was particularly high in flower and leaves but low in fruit. SlArf/Xyl3 was highly expressed in flower. On the contrary, SlArf/Xyl2 and 4 were expressed in early developmental stage in various organs. Comparison with SlArf/Xyl4, SlArf/Xyl2 expression was observed in earlier stages. The active recombinant proteins were obtained by using BY-2 tobacco (Nicotiana tabacum) suspension cultured cells. The SlArf/Xyl1 and 2 recombinant proteins showed a bi-functional activity of α-l-arabinofuranosidase/ß-xylosidase while the SlArf/Xyl4 protein possessed a ß-xylosidase activity predominantly. Neither enzyme activities were detected for the SlArf/Xyl3 protein under the same conditions. Although SlArf/Xyl2 possessed a bi-functional activity, it preferentially hydrolyzed arabinosyl residues from tomato hemicellulosic polysaccharides. Antisense suppression of SlArf/Xyl2 resulted in no apparent changes in the enzyme activities, monosaccharide composition or fruit phenotype. Increment of a family 51 α-l-arabinofuranosidase expression rather than that of family 3 resulted in a restoring the activity in SlArf/Xyl2-suppressed fruit. The ability of recombinant SlArf/Xyl2 to hydrolyze both arabinan and arabinoxylan is nearly identical to that of α-l-arabinofuranosidases belonging to family 51. Our results suggested that BY-2 cells are a useful expression system for obtaining active cell wall hydrolyzing enzymes. In addition, an α-l-arabinofuranosidase activity derived from SlArf/Xyl2 would be essential in young organ development and the action of the enzyme could be restored by the other enzyme belonging to a different family under a defective condition.

Role of Vacuolar H+-inorganic pyrophosphatase in tomato fruit development.

cDNA corresponding to two type-I vacuolar H(+)-inorganic pyrophosphatases (V-PPases) (SlVP1, SlVP2) and one type-II V-PPase (SlVP3) was isolated from tomato fruit to investigate their role in fruit development. Southern analysis revealed that type-I V-PPase genes form a multigene family, whereas there is only one type-II V-PPase gene in the tomato genome. Although SlVP1 and SlVP2 were differentially expressed in leaves and mature fruit, the highest levels of both SlVP1 and SlVP2 mRNA were observed in fruit at 2-4 days after anthesis. The expression pattern of type-II SlVP3 was similar to that of SlVP2, and the highest levels of SlVP3 mRNA were also observed in fruit at 2-4 days after anthesis, thus suggesting that SlVP3 plays a role in early fruit development. Because SlVP1 and SlVP2 mRNA was more abundant than SlVP3 mRNA, expression of type-I V-PPases was analysed further. Type-I V-PPase mRNA was localized in ovules and their vicinities and in vascular tissue at an early stage of fruit development. Tomato RNAi lines in which the expression of type-I V-PPase genes was repressed using the fruit-specific promoter TPRP-F1 exhibited fruit growth retardation at an early stage of development. Although the major function of V-PPases in fruit has been believed to be the accumulation of materials such as sugars and organic acids in the vacuole during cell expansion and ripening, these results show that specific localization of V-PPase mRNA induced by pollination has a novel role in the cell division stage.

RCY1-mediated resistance to Cucumber mosaic virus is regulated by LRR domain-mediated interaction with CMV(Y) following degradation of RCY1.

RCY1, which encodes a coiled coil nucleotide-binding site leucine-rich repeat (LRR) class R protein, confers the hypersensitive response (HR) to a yellow strain of Cucumber mosaic virus (CMV[Y]) in Arabidopsis thaliana. Nicotiana benthamiana transformed with hemagglutinin (HA) epitope-tagged RCY1 (RCY1-HA) also exhibited a defense response accompanied by HR cell death and induction of defense-related gene expression in response to CMV(Y). Following transient expression of RCY1-HA by agroinfiltration, the defense reaction was induced in N. benthamiana leaves infected with CMV(Y) but not in virulent CMV(B2)-infected N. benthamiana leaves transiently expressing RCY1-HA or CMV(Y)-infected N. benthamiana leaves transiently expressing HA-tagged RPP8 (RPP8-HA), which is allelic to RCY1. This result suggests that Arabidopsis RCY1-conferred resistance to CMV(Y) could be reproduced in N. benthamiana leaves in a gene-for-gene manner. Expression of a series of chimeric constructs between RCY1-HA and RPP8-HA in CMV(Y)-infected N. benthamiana indicated that induction of defense responses to CMV(Y) is regulated by the LRR domain of RCY1. Interestingly, in CMV(Y)-infected N. benthamiana manifesting the defense response, the levels of both RCY1 and chimeric proteins harboring the RCY1 LRR domain were significantly reduced. Taken together, these data indicate that the RCY1-conferred resistance response to CMV(Y) is regulated by an LRR domain-mediated interaction with CMV(Y) and seems to be tightly associated with the degradation of RCY1 in response to CMV(Y).

Suppression of telomere-binding protein gene expression represses seed and fruit development in tomato.

Tomato (Solanum lycopersicum L.) plants were transformed with an antisense construct of a cDNA encoding tomato telomere-binding protein (LeTBP1) to describe the role of a telomere-binding protein at the whole plant level. Fruit size decreased corresponding to the degree of suppression of LeTBP1 expression. This inhibition of fruit development was likely due to a decrease in the number of seeds in the LeTBP1 antisense plants. Pollen fertility and pollen germination rate decreased in accordance with the degree of suppression of LeTBP1 expression. Ovule viability was also reduced in the LeTBP1 antisense plants. Although plant height was somewhat reduced in the antisense plants compared to the control plants, the number and weight of leaves were unaffected by LeTBP1 suppression. The number and morphology of flowers were also normal in the antisense plants. These indicate that reduced fertility in the antisense plants is not an indirect effect of altered vegetative growth. LeTBP1 expression was sensitive to temperature stress in wild-type plants. We conclude that LeTBP1 plays a critical role in seed and fruit development rather than vegetative growth and flower formation.