Rin Fruit Research Articles

Semi-dominant effects of a novel ripening inhibitor (rin) locus allele on tomato fruit ripening.

The tomato (Solanum lycopersicum) ripening inhibitor (rin) mutation completely represses fruit ripening, as rin fruits fail to express ripening-associated genes and remain green and firm. Moreover, heterozygous rin fruits (rin/+) ripen normally but have extended shelf life, an important consideration for this perishable fruit crop; therefore, heterozygous rin has been widely used to breed varieties that produce red tomatoes with improved shelf life. We previously used CRISPR/Cas9 to produce novel alleles at the rin locus. The wild-type allele RIN encodes a MADS-box transcription factor and the novel allele, named as rinG2, generates an early stop codon, resulting in C-terminal truncation of the transcription factor. Like rin fruits, rinG2 fruits exhibit extended shelf life, but unlike rin fruits, which remain yellow-green even after long-term storage, rinG2 fruits turn orange due to ripening-associated carotenoid production. Here, to explore the potential of the rinG2 mutation for breeding, we characterized the effects of rinG2 in the heterozygous state (rinG2/+) compared to the effects of rin/+. The softening of rinG2/+ fruits was delayed compared to the wild type but to a lesser degree than rin/+ fruits. Lycopene and β-carotene levels in rinG2/+ fruits were similar to those of the wild type, whereas rin/+ fruits accumulated half the amount of β-carotene compared to the wild type. The rinG2/+ fruits produced lower levels of ethylene than wild-type and rin/+ fruits. Expression analysis revealed that in rinG2/+ fruits, the rinG2 mutation (like rin) partially inhibited the expression of ripening-associated genes. The small differences in the inhibitory effects of rinG2 vs. rin coincided with small differences in phenotypes, such as ethylene production, softening, and carotenoid accumulation. Therefore, rinG2 represents a promising genetic resource for developing tomato cultivars with extended shelf life.

Allelic Mutations in the Ripening -Inhibitor Locus Generate Extensive Variation in Tomato Ripening.

RIPENING INHIBITOR (RIN) is a transcription factor with transcriptional activator activity that plays a major role in regulating fruit ripening in tomato (Solanum lycopersicum). Recent studies have revealed that (1) RIN is indispensable for full ripening but not for the induction of ripening; and (2) the rin mutation, which produces nonripening fruits that never turn red or soften, is not a null mutation but instead converts the encoded transcriptional activator into a repressor. Here, we have uncovered aspects of RIN function by characterizing a series of allelic mutations within this locus that were produced by CRISPR/Cas9. Fruits of RIN-knockout plants, which are characterized by partial ripening and low levels of lycopene but never turn fully red, showed excess flesh softening compared to the wild type. The knockout mutant fruits also showed accelerated cell wall degradation, suggesting that, contrary to the conventional view, RIN represses over-ripening in addition to facilitating ripening. A C-terminal domain-truncated RIN protein, encoded by another allele of the RIN locus (rinG2), did not activate transcription but formed transcription factor complexes that bound to target genomic regions in a manner similar to that observed for wild-type RIN protein. Fruits expressing this truncated RIN protein exhibited extended shelf life, but unlike rin fruits, they accumulated lycopene and appeared orange. The diverse ripening properties of the RIN allelic mutants suggest that substantial phenotypic variation can be produced by tuning the activity of a transcription factor.

Seed development and viviparous germination in one accession of a tomato rin mutant.

In an experimental field, seed vivipary occurred in one accession of tomato rin mutant fruit at approximately 45–50 days after pollination (DAP). In this study, the possible contributory factors to this viviparous germination were investigated. Firstly, developing seeds were freshly excised from the fruit tissue every 5 days from 25–60 DAP. Germination occurred when isolated seeds were incubated on water, but was inhibited when they remained ex situ in fruit mucilage gel. The effect of abscisic acid (ABA) and osmoticum, separate and together, on germination of developing seeds was investigated. Additionally, ABA content in the seed and mucilage gel, as well as fruit osmolality were measured. The results showed that ABA concentrations in seeds were low during early development and increased later, peaking at about 50 DAP. ABA concentrations in rin accession were similar to those of the control cultivar and thus are not directly associated with the occurrence of vivipary. Developing seeds of rin accession are more sensitive than control seeds to all inhibitory compounds. However, osmolality in rin fruit at later developmental stages becomes less negative that is required to permit germination of developing seeds. Hence, hypo-osmolality in rin fruit may be an important factor in permitting limited viviparous germination.

A non-climacteric fruit gene CaMADS-RIN regulates fruit ripening and ethylene biosynthesis in climacteric fruit.

MADS-box genes have been reported to play a major role in the molecular circuit of developmental regulation. Especially, SEPALLATA (SEP) group genes play a central role in the developmental regulation of ripening in both climacteric and non-climacteric fruits. However, the mechanisms underlying the regulation of SEP genes to non-climacteric fruits ripening are still unclear. Here a SEP gene of pepper, CaMADS-RIN, has been cloned and exhibited elevated expression at the onset of ripening of pepper. To further explore the function of CaMADS-RIN, an overexpressed construct was created and transformed into ripening inhibitor (rin) mutant tomato plants. Broad ripening phenotypes were observed in CaMADS-RIN overexpressed rin fruits. The accumulation of carotenoid and expression of PDS and ZDS were enhanced in overexpressed fruits compared with rin mutant. The transcripts of cell wall metabolism genes (PG, EXP1 and TBG4) and lipoxygenase genes (TomloxB and TomloxC) accumulated more abundant compared to rin mutant. Besides, both ethylene-dependent genes including ACS2, ACO1, E4 and E8 and ethylene-independent genes such as HDC and Nor were also up-regulated in transgenic fruits at different levels. Moreover, transgenic fruits showed approximately 1–3 times increase in ethylene production compared with rin mutant fruits. Yeast two-hybrid screen results indicated that CaMADS-RIN could interact with TAGL1, FUL1 and itself respectively as SlMADS-RIN did in vitro. These results suggest that CaMADS-RIN affects fruit ripening of tomato both in ethylene-dependent and ethylene-independent aspects, which will provide a set of significant data to explore the role of SEP genes in ripening of non-climacteric fruits.

Proteomic Analysis of Ripening Tomato Fruit Infected by Botrytis cinerea

Botrytis cinerea, a model necrotrophic fungal pathogen that causes gray mold as it infects different organs on more than 200 plant species, is a significant contributor to postharvest rot in fresh fruit and vegetables, including tomatoes. By describing host and pathogen proteomes simultaneously in infected tissues, the plant proteins that provide resistance and allow susceptibility and the pathogen proteins that promote colonization and facilitate quiescence can be identified. This study characterizes fruit and fungal proteins solubilized in the B. cinerea-tomato interaction using shotgun proteomics. Mature green, red ripe wild type and ripening inhibited (rin) mutant tomato fruit were infected with B. cinerea B05.10, and the fruit and fungal proteomes were identified concurrently 3 days postinfection. One hundred eighty-six tomato proteins were identified in common among red ripe and red ripe-equivalent ripening inhibited (rin) mutant tomato fruit infected by B. cinerea. However, the limited infections by B. cinerea of mature green wild type fruit resulted in 25 and 33% fewer defense-related tomato proteins than in red and rin fruit, respectively. In contrast, the ripening stage of genotype of the fruit infected did not affect the secreted proteomes of B. cinerea. The composition of the collected proteins populations and the putative functions of the identified proteins argue for their role in plant-pathogen interactions.

Transcriptome analysis of rin mutant fruit and in silico analysis of promoters of differentially regulated genes provides insight into LeMADS-RIN-regulated ethylene-dependent as well as ethylene-independent aspects of ripening in tomato

A thorough understanding of molecular mechanisms underlying ripening is the prerequisite for genetic manipulation of fruits for better shelf-life and nutritional quality. Mutation in LeMADS-RIN, a MADS-box gene, leads to non-ripening phenotype of rin fruits in tomato. Characterization of ripening-inhibitor (rin) mutant has elucidated important role of ethylene in the regulation of climacteric fruit ripening. A complete understanding of this mutation will unravel novel genetic regulatory mechanisms involved in fruit ripening. In this study, fruit transcriptomes of two genotypes, including a cultivated Indian cultivar Solanum lycopersicum cv. Pusa Ruby and a homozygous line harboring the rin mutation (LA1795) were compared to get better insight into RIN-regulated ethylene-dependent and ethylene-independent events during ripening. Cluster analysis of ripening-related genes indicated a major shift in their expression profiles in rin mutant fruit. A total of 112 genes, exhibiting expression patterns similar to that of LeMADS-RIN in wild-type fruits, showed down regulation of expression in the rin mutant. In silico analysis of putative promoters of these genes for the presence of CArG box along with ERE and ethylene inducibility of these genes revealed that genes lacking CArG box in their regulatory regions could be indirectly regulated by LeMADS-RIN. New regulators of ethylene-dependent aspect of ripening were also identified. In this study, we have made an attempt to distinguish between ethylene-dependent and ethylene-independent aspects of ripening, which will be useful for developing strategies to improve fruit-related agronomic traits in tomato and other crops.

Ripening-regulated susceptibility of tomato fruit to Botrytis cinerea requires NOR but not RIN or ethylene.

Fruit ripening is a developmental process that is associated with increased susceptibility to the necrotrophic pathogen Botrytis cinerea. Histochemical observations demonstrate that unripe tomato (Solanum lycopersicum) fruit activate pathogen defense responses, but these responses are attenuated in ripe fruit infected by B. cinerea. Tomato fruit ripening is regulated independently and cooperatively by ethylene and transcription factors, including NON-RIPENING (NOR) and RIPENING-INHIBITOR (RIN). Mutations in NOR or RIN or interference with ethylene perception prevent fruit from ripening and, thereby, would be expected to influence susceptibility. We show, however, that the susceptibility of ripe fruit is dependent on NOR but not on RIN and only partially on ethylene perception, leading to the conclusion that not all of the pathways and events that constitute ripening render fruit susceptible. Additionally, on unripe fruit, B. cinerea induces the expression of genes also expressed as uninfected fruit ripen. Among the ripening-associated genes induced by B. cinerea are LePG (for polygalacturonase) and LeExp1 (for expansin), which encode cell wall-modifying proteins and have been shown to facilitate susceptibility. LePG and LeExp1 are induced only in susceptible rin fruit and not in resistant nor fruit. Thus, to infect fruit, B. cinerea relies on some of the processes and events that occur during ripening, and the fungus induces these pathways in unripe fruit, suggesting that the pathogen itself can initiate the induction of susceptibility by exploiting endogenous developmental programs. These results demonstrate the developmental plasticity of plant responses to the fungus and indicate how known regulators of fruit ripening participate in regulating ripening-associated pathogen susceptibility.

Ripening-Specific Stigmasterol Increase in Tomato Fruit Is Associated with Increased Sterol C-22 Desaturase (CYP710A11) Gene Expression

Phytosterol content and composition and sterol C-22 desaturase (LeSD1; CYP710A11) transcript levels in pericarp tissue of 'Rutgers' tomato fruit were compared in the wild-type (wt) and isogenic lines of the nonripening mutants nor and rin at four stages of ripening/aging. Wild-type fruit were harvested at the mature-green (MG), breaker (BK), breaker plus 3 days (B + 3), and breaker plus 6 days (B + 6) stages, whereas nor and rin fruits were harvested at comparable chronological ages (days after pollination). At the MG stage, wt and mutant fruits had closely similar sterol contents, compositions, and conjugations, with >91% of the total sterols in the acylated steryl glycoside plus steryl glycoside (ASG + SG) fraction. During ripening/aging, there were substantial increases in total sterols and the percentage of sterols in the free plus esterified (FS + SE) fraction. Both changes were greater in wt than in nor or rin. In fruit of wt, rin, and nor, respectively, the increases in total sterols between MG and B + 6 were 2.1-, 1.9-, and 1.5-fold, and at B + 6 the percentages of total sterols in FS + SE were 42, 21, and 24. Among all sterol lipids (ASG, SG, FS, and SE), the ratio of stigmasterol (stigmasta-5,22-dien-3beta-ol) to beta-sitosterol (stigmast-5-en-3beta-ol), the two major sterols in tomato, increased 2.3-fold during ripening of wt fruit but declined slightly during comparable aging of nor and rin fruits. In accord with these changes, the abundance of LeSD1 transcript increased 4-fold in pericarp of ripening wt fruit, peaking at B + 3, whereas transcript levels in nor and rin fruits fluctuated but never exceeded the abundance in wt fruit at the MG stage. These findings indicate that the ripening-specific increase in stigmasterol in wt fruit results from a marked increase in LeSD1 transcription and translation, which accelerates C-22 desaturation of the precursor sterol, beta-sitosterol.

Analysis of softening in air- and ethylene-treated rin, nor and wild-type tomato fruit

Rin; ripening- inhibitor and nor; non- ripening are previously identified spontaneous mutations that affect the primary regulation of tomato fruit ripening. Mutations at these loci result in fruit that are either partially or completely inhibited in their ability to ripen. Internal and whole fruit firmness of air- and ethylene-treated wild-type fruit declined by 59–87%. Firmness of whole rin and nor fruit decreased slightly after 9 days of air-treatment and internal fruit firmness did not change. Ethylene-treated rin and nor whole fruit softened over 9 days, but substantially less than wild type. At harvest and after air or ethylene treatment, firmness was greater in nor than in rin fruit. Surprisingly, slices from ethylene-treated rin and nor fruit softened much more than whole fruit, and softening of slices was similar for fruit of rin and nor.

Comparison of ethylene- and wound-induced responses in fruit of wild-type, rin and nor tomatoes

Abstract Responses of wild-type, rin , and no r tomato ( Lycopersicon esculentum Mill.) fruit to exogenous ethylene and wounding were compared to identify the events directly controlled by each mutation. When rin and nor fruit were exposed to exogenous ethylene, respiration and accumulation of E4 , E8 , and LE - ACO1 mRNAs increased to levels similar to those observed in the wild-type fruit, indicating that expression of these genes is independent of either mutations. In contrast, accumulation of LE - ACS4 and PG mRNAs was not restored by exogenous ethylene treatment in both mutants, indicating that the expression of both genes requires both RIN and NOR . Interestingly accumulation of LE - ACS2 mRNA was stimulated by ethylene treatment in rin fruit but not in nor fruit. This suggests that the signaling pathways of RIN and NOR affect the regulation of ripening-associated genes in distinct ways, at least with respect to the expression of LE - ACS2 . Wounding induced ethylene biosynthesis and increased accumulation of LE - ACS2 , LE - ACS6 , LE - ACO1 , and E4 mRNAs in both wild-type and the mutants to the same extent. Treatment with 1-methylcyclopropene (1-MCP), a potent inhibitor of ethylene perception, preceding wounding, inhibited the increase of E4 mRNA but did not affect the expression profile of other genes in all the lines tested. These results show that the wounding signal that controls LE - ACS2 , LE - ACS6 , and LE - ACO1 is independent of rin and nor mutations and ethylene signaling.

Senescence of rin, rin/rin, rin/+ and +/+ tomato fruits

Ripening inhibitor (rin) gene is a spontaneous recessive mutant which changes fruit ripening aspects (most important synthesis of carotene, especially lycopene). It also delays fruit senescence. Tomato is a vegetable crop with specific maturing climax (pik). Tomato genotypes homozygote for rin gene does not have this maturing climax, so the ethyl-ene production and red, lycopene colour does not appear. In order to research the maturing process material from the final tomato selection cycle we used: pure line S-49 (genetic constitution u/u, with uniform ripening), line hom 4 (rin/rin, homozygote with ripening inhibitor) and hybrid combination 449 Fl (u/rin, heterozygote for ripening inhibitor). Fruits with uniform ripening and hybrids ripened simultaneously, while homozygote with rin gene did not ripe at all. Ethylene stimulates the appearance of yellow colour in rin fruits and the lycopene production. After treatment with ETEPHONE (0.1%) (Ethylene) fruits with uniform ripening, senescence more quickly, while hybrids senescence slowly. Rin homozygotes did not change colors, but the fruits senescence more quickly comparing to control. Ethylene treatment speeded the maturing and senescence process in tomato fruits. Extended maturing process as a result influence of rin gene, makes the new created hybrids a late maturing ones. On the other hand, the firmness of fruits is improved as well as the "shelf life", which enables longer transportation and storing, coordinated maturing according to market demands. The aim was to research the maturing according to market demands. The aim was to research the maturing process of rin heteroyzgote, and the reaction of some genotypes to treatment with RT-REL (Ethzlene) in order to decrease extremely late maturing.

Characterization of expression, and cloning, of beta-D-xylosidase and alpha-L-arabinofuranosidase in developing and ripening tomato (Lycopersicon esculentum Mill.) fruit.

Modifications to the cell wall of developing and ripening tomato fruit are mediated by cell wall-degrading enzymes, including a beta-d-xylosidase or alpha-l-arabinofuranosidase, which participate in the breakdown of xylans and/or arabinoxylans. The activity of both enzymes was highest during early fruit growth, before decreasing during later development and ripening. Two beta-d-xylosidase cDNAs, designated LeXYL1 and LeXYL2, and an alpha-l-arabinofuranosidase cDNA, designated LeARF1, were obtained. Accumulation of mRNAs for beta-d-xylosidase and alpha-l-arabinofuranosidase was examined during fruit development and ripening. LeARF1 and LeXYL2 genes were relatively highly expressed during fruit development and decreased after the onset of ripening. By contrast, LeXYL1 was not expressed during fruit development, but was expressed later, particularly during over-ripening. The expression of all three genes was also followed in ripening-impaired mutants, Nr, Nr2, nor, and rin of cv. Ailsa Craig fruit. LeXYL2 mRNA was detected in the ripe fruits of all the mutants and its abundance was similar to that in mature green wild-type fruit. By contrast, LEXYL1 mRNA was expressed only in the ripe fruits of the Nr mutant, suggesting that the two beta-d-xylosidase genes are subject to distinct regulatory control during fruit development and ripening. LeARF1 mRNA was detected in ripe fruits of Nr2, nor and rin, and not in ripe fruit of the Nr mutant. The accumulation of LeARF1 in ripe fruit was restored by 1-methylcyclopropene (1-MCP), an inhibitor of ethylene action, while 1-MCP had no effect on the expression of LeXYL1 or LeXYL2. This suggests that LeARF1 expression is subject to negative regulation by ethylene and that the two beta-d-xylosidase genes are independent of ethylene action.

Ethylene and developmental signals regulate expression of lipoxygenase genes during tomato fruit ripening

The role of ethylene in regulating the expression of three lipoxygenase (LOX) genes TomloxA, TomloxB and TomloxC during fruit ripening has been investigated. The study used a combination of low-ethylene fruit containing an ACC oxidase (ACO1) sense-suppressing transgene and tomato fruit ripening mutants deficient in ethylene perception in the case of Never-ripe (Nr) or ripening in the case of the ripening-inhibitor (rin). The results indicate that the three genes are differentially regulated during fruit ripening and that a combination of ethylene and developmental factors influence their expression. The expression of TomloxA declines during ripening and this is delayed in the ACO1 transgenic low-ethylene and Nr fruit indicating that this phenomenon is ethylene regulated. However, transcript abundance also declines during rin fruit development indicating that developmental factors also influence the expression of TomloxA. TomloxB expression increases during fruit ripening and expression is increased by ethylene as reduced accumulation is evident in transgenic and mutant fruit. The same ethylene up-regulated and ripening-related expression is shown by TomloxC, however, expression increases in rin fruit at the onset of colour change, indicating that there may also be a developmental component regulating TomloxC expression. These results indicate that individual LOX isoforms are differentially regulated and may have distinct functions during tomato fruit ripening.

Ethylene and developmental signals regulate expression of lipoxygenase genes during tomato fruit ripening

The role of ethylene in regulating the expression of three lipoxygenase (LOX) genes TomloxA, TomloxB and TomloxC during fruit ripening has been investigated. The study used a combination of low-ethylene fruit containing an ACC oxidase (ACO1) sense-suppressing transgene and tomato fruit ripening mutants deficient in ethylene perception in the case of Never-ripe (Nr) or ripening in the case of the ripening-inhibitor (rin). The results indicate that the three genes are differentially regulated during fruit ripening and that a combination of ethylene and developmental factors influence their expression. The expression of TomloxA declines during ripening and this is delayed in the ACO1 transgenic low-ethylene and Nr fruit indicating that this phenomenon is ethylene regulated. However, transcript abundance also declines during rin fruit development indicating that developmental factors also influence the expression of TomloxA. TomloxB expression increases during fruit ripening and expression is increased by ethylene as reduced accumulation is evident in transgenic and mutant fruit. The same ethylene up-regulated and ripening-related expression is shown by TomloxC, however, expression increases in rin fruit at the onset of colour change, indicating that there may also be a developmental component regulating TomloxC expression. These results indicate that individual LOX isoforms are differentially regulated and may have distinct functions during tomato fruit ripening.

Saline growing conditions induce ripening of the non-ripening mutants nor and rin tomato fruits but not of Nr fruit

Tomato (Lycopersicon esculentum Mill.) plants cv. ‘Rutgers’, and their three nearly isogenic ripening mutant derivative lines nor, rin and Nr, were grown in sand culture irrigated with nutrient solution. Six-week-old plants were exposed to saline growing conditions (EC 15.5 mmohs:cm) by adding NaCl or NaCl CaCl2 to the nutrient solution. Ripening was induced in nor fruit under both saline conditions as indicated by fruit softness, red colour development, induction of a climacteric pattern of C2H4 production and an increase in 1-aminocyclopropane-1-carboxylic acid (ACC) content over that of the control. Similar stimulation of all ripening parameters was obtained in rin fruit, but only with NaCl treatment. It seems that the presence of Ca 2 in the saline solution blocked salt-induced rin fruit ripening. Neither saline treatment affected Nr fruit ripening. Furthermore, Nr fruit grown under non-saline conditions showed yellow colour development and produced C2H4 at a level comparable to that produced by ‘Rutgers’ fruit with no post-climacteric decline. The ACC content of Nr fruit was significantly higher than that of post-climacteric ‘Rutgers’ fruit. These data are discussed as to the possible role of saline treatment with or without Ca 2 in inducing ripening in the non-ripening mutants. © 1998 Elsevier Science B.V. All rights reserved.

EVALUATION OF RHEOLOGICAL PROPERTIES CONTRIBUTING TO FRUIT TEXTURE

Transgenic tomato isolines wild-type, antisense-PG, antisense-PME, rin and rin[(E8/PG)/(E8/PG)] have been created having reduced or enhanced levels of single pectin degrading enzymes, with the purpose of altering fruit texture. However, because of limitations in current methods of fruit textural analysis, it has been difficult to identify and characterize the often subtle effects of altered pectin chemistry on fruit texture. Stress relaxation measurements provided additional insights beyond that which could be achieved by conventional textural analysis. The methodology permits the acquisition of time-dependent as well as non-time-dependent parameters. We found, using stress-relaxation analysis, that important texture differences between transgenic genotypes could be identified. Antisense-PG fruit were significantly more resistant to initial load stress than wild-type isolines, but relaxation of tissue following stress application in antisense-PG fruit was not significantly altered. In contrast, antisense-PME fruit showed no alteration in resistance to initial load stress, but the time required for relaxation of tissue following stress was significantly lower in antisense-PME fruit than in wild-type isolines. In rin[(E8/PG)/(E8/PG)] fruit, resistance to initial load was lower and time of relaxation was longer than in wild-type fruit. In transformed rin fruit, the pericarp was softer but nevertheless the analysis suggested the development ofa higher tissue viscosity.

Effect of EDTA and low calcium fertility on pericarp cation levels and ripening of rin tomato fruit

Ripening inhibited ( rin ) tomato plants were grown in silicate sand and watered with half-strength Hoagland's solution until flowering. After flowering, plants were watered until run-off daily with full-strength Hoagland's solution containing 5 mM (control) or 1 mM calcium, or full-strength Hoagland's solution with 1.25 mM or 2.5 mM Na 2 EDTA. Fruit pericarp calcium levels were the same in Na 2 EDTA and control treatments at 90 days postpollination. The 1 mM Ca treatment significantly decreased total pericarp Ca levels compared to the control treatment. Sodium levels were higher in fruit from Na 2 EDTA-treated plants than controls. Fruit from control and 1 mM Ca treatments had significantly higher Zn than fruit from the Na 2 EDTA-treatments. None of the treatments induced increased ethylene production, redness, or increased soluble solids content of rin fruit.

Differential Expression of Two Endo-1,4-[beta]-Glucanase Genes in Pericarp and Locules of Wild-Type and Mutant Tomato Fruit.

The mRNA accumulation of two endo-1,4-[beta]-D-glucanase genes, Cel1 and Cel2, was examined in the pericarp and locules throughout the development of normal tomato (Lycopersicon esculentum) fruit and the ripening-impaired mutants rin and Nr. Both Cel1 and Cel2 were expressed transiently at the earliest stages of fruit development during a period corresponding to cell division and early cell expansion. In the pericarp, the mRNA abundance of both genes increased markedly at the breaker stage; the level of Cel1 mRNA decreased later in ripening, and that of Cel2 increased progressively. Cel2 mRNA levels also increased at the breaker stage in locules but after initial locule liquefaction was already complete. In rin fruit mRNA abundance of Cel1 was reduced and Cel2 was virtually absent, whereas in Nr Cel1 was expressed at wild-type levels and Cel2 was reduced. In wild-type fruit ethylene treatment slightly promoted the mRNA accumulation of both genes. In rin fruit ethylene treatment strongly increased the mRNA abundance of Cel1 to an extent greater than in wild-type fruit, but Cel2 mRNA was absent even after ethylene treatment. These two endo-1,4-[beta]-D-glucanase genes, therefore, do not show coordinated expression during fruit development and are subject to distinct regulatory control. These results suggest that the product of the Cel2 gene contributes to ripening-associated cell-wall changes.

Differential Expression of the Two Subunits of Tomato Polygalacturonase Isoenzyme 1 in Wild-Type and rin Tomato Fruit.

The [beta] subunit of tomato (Lycopersicon esculentum Mill.) fruit polygalacturonase 1 is a cell wall glycoprotein that binds to and apparently regulates the catalytic PG2 polypeptide in vivo. [beta] Subunit and polygalacturonase 2 (PG2) expression have been investigated in both wild-type and ripening inhibitor (rin) mutant fruit. During fruit development and ripening, [beta] subunit expression was unrelated to expression of the catalytic PG2 protein. In wild-type fruit, [beta] subunit mRNA and protein were first detected early in development and increased to maximal levels before PG2 mRNA and protein were detected. At the onset of ripening [beta] subunit mRNA decreased dramatically, but [beta] subunit protein levels remained stable. In rin fruit, which fail to ripen, [beta] subunit expression was similar to that in wild type, although PG2 mRNA and protein were not detected. These data suggest that [beta] subunit expression is ethylene independent and regulated primarily by developmental cues. This conclusion is supported by results from ethylene-treated immature (20 days after pollination) wild-type and rin fruit in which no significant differences were observed in [beta] subunit expression patterns in response to ethylene treatment. Surprisingly, RNA blot analysis indicated that catalytic PG2 mRNA was induced in immature rin fruit after 3 d of exogenous ethylene treatment. In addition, [beta] subunit mRNA and protein were also detected at lower levels in root, leaf, and flower tissues of both genotypes, suggesting a broader functional role for the protein.

CDNA cloning and characterisation of novel ripening-related mRNAs with altered patterns of accumulation in the ripening inhibitor (rin) tomato ripening mutant.

A cDNA library produced from mRNA isolated from the pericarp of wild-type tomato fruit (Lycopersicon esculentum Mill. cv Ailsa Craig) at the first visible sign of fruit ripening was differentially screened to identify clones whose homologous mRNAs were present at reduced levels in fruit of the tomato ripening mutant, ripening inhibitor,rin. Five clones were isolated (pERT 1, 10, 13, 14, 15). Accumulation of mRNA homologous to each of these clones increased during the ripening of wild-type fruit and showed reduced accumulation in ripening rin fruit. The levels of three of them (homologous to ERT 1, 13 and 14) were increased by ethylene treatment of the mutant fruit. A further clone, ERT 16 was identified for a mRNA present at a high level in both normal and mutant fruit at early stages of ripening. Database searches revealed no significant homology to the DNA sequence of ERT 14 and 15; however, DNA and derived amino acid sequence of ERT 1 both contain regions of homology with several reported UDP-glucosyl and glucuronosyl transferases (UDPGT) and with a conserved UDPGT motif. A derived amino acid sequence from the ERT 10 cDNA contains a perfect match to a consensus sequence present in a number of dehydrogenases. The ERT 13 DNA sequence has homology with an mRNA present during potato tuberisation. The presence of these mRNAs in tomato fruit is unreported and their role in ripening is unknown. The ERT 16 DNA sequence has homology with a ripening/stress-related cDNA isolated from tomato fruit pericarp.