The Tomato Fruit Cell Wall : II. Polyuronide Metabolism in a Nonsoftening Tomato Mutant

A nonsoftening tomato (Lycopersicon esculentum L.) variety, dg, was examined to assess the physiological basis for its inability to soften during ripening. Total uronic acid levels, 18 milligrams uronic acid/100 milligrams wall, and the extent of pectin esterification, 60 mole%, remained constant throughout fruit development in this mutant. The proportion of uronic acid susceptible to polygalacturonase in vitro also remained constant. Pretreatment of heat-inactivated dg fruit cell walls with tomato pectinmethylesterase enhances polygalacturonase susceptibility at all ripening stages. Pectinesterase activity of cell wall protein extracts from red ripe dg fruit was half that in extracts from analogous tissue of VF145B. Polygalacturonase activities of cell wall extracts, however, were similar in both varieties. Diffusion of uronic acid from tissue discs of both varieties increased beginning at the turning stage to a maximum of 2.0 milligrams uronic acid released/gram fresh weight at the ripe stage. The increased quantity of hydrolytic products released during ripening suggests the presence of in situ polygalacturonase activity. Low speed centrifugation was employed to induce efflux of uronide components from the cell wall tree space. In normal fruit, at the turning stage, 2.1 micrograms uronic acid/gram fresh weight was present in the eluant after 1 hour, and this value increased to a maximum of 8.2 micrograms uronic acid/gram fresh weight at the red ripe stage. However, centrifuge-aided extraction of hydrolytic products failed to provide evidence for in situ polygalacturonase activity in dg fruit. We conclude that pectinesterase and polygalacturonase enzymes are not active in situ during the ripening of dg fruit. This could account for the maintenance of firmness in ripe fruit tissue.     

Pectin methylesterase activity in vivo differs from activity in vitro and enhances polygalacturonase-mediated pectin degradation in tabasco pepper

Polygalacturonase (PG) and pectin methylesterase (PME) activities were analyzed in ripening fruits of two tabasco pepper (Capsicum frutescens) lines that differ in the extent of pectin degradation (depolymerization and dissolution). Ripe 'Easy Pick' fruit is characterized by pectin ultra-degradation and easy fruit detachment from the calyx (deciduous trait), while pectin depolymerization and dissolution in ripe 'Hard Pick' fruit is limited. PG activity in protein extracts increased similarly in both lines during fruit ripening. PME activity in vivo assessed by methanol production, however, was detected only in fruit of the 'Easy Pick' line and was associated with decreased pectin methyl-esterification. In contrast, methanol production in vivo was not detected in fruits of the 'Hard Pick' line and the degree of pectin esterification remained the same throughout ripening. Consequently, a ripening specific PME that is active in vivo appears to enhance PG-mediated pectin ultra-degradation resulting in cell wall dissolution and the deciduous fruit trait. PME activity in vitro, however, was detected in protein extracts from both lines at all ripening stages. This indicates that some PME isozymes are apparently inactive in vivo, particularly in green fruit and throughout ripening in the 'Hard Pick' line, limiting PG-mediated pectin depolymerization which results in moderately difficult fruit separation from the calyx.     

Predispersal reproductive biology of female Osyris quadripartita (Santalaceae), a hemiparasitic dioecious shrub of Mediterranean scrublands

Female Osyris quadripartita plants exhibit uninterrupted reproductive activity throughout the year, due to the long duration of successive stages in the cycle and marked within-crop developmental asynchrony. Cycles corresponding to the flowering seasons of consecutive years overlap in each individual. Flowering takes place in spring, and fruits develop in the dry summer season and ripen at any time of the year. Variation in flowering time explains a negligible proportion of variation in ripening time. The greatest reproductive losses are incurred in the phase extending from closed flowers through unripe fruits, mostly due to ovary abortion. Only 30% of closed flowers eventually reach this latter stage. In contrast, 75% of unripe fruits complete their development, with subsequent dispersal of seeds. The probability of the setting of ripe fruit steadily decreases from early to late season flowers, due to increased ovary abortion rates. Resource limitation in the dry summer season seems responsible for this pattern of selective fruit maturation.     

1-Deoxy-D-xylulose 5-phosphate reductoisomerase and plastid isoprenoid biosynthesis during tomato fruit ripening

The recently discovered 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for the biosynthesis of plastid isoprenoids (including carotenoids) is not fully elucidated yet despite its central importance for plant life. It is known, however, that the first reaction completely specific to the pathway is the conversion of 1-deoxy-D-xylulose 5-phosphate (DXP) into MEP by the enzyme DXP reductoisomerase (DXR). We have identified a tomato cDNA encoding a protein with homology to DXR and in vivo activity, and show that the levels of the corresponding DXR mRNA and encoded protein in fruit tissues are similar before and during the massive accumulation of carotenoids characteristic of fruit ripening. The results are consistent with a non-limiting role of DXR, and support previous work proposing DXP synthase (DXS) as the first regulatory enzyme for plastid isoprenoid biosynthesis in tomato fruit. Inhibition of DXR activity by fosmidomycin showed that plastid isoprenoid biosynthesis is required for tomato fruit carotenogenesis but not for other ripening processes. In addition, dormancy was reduced in seeds from fosmidomycin-treated fruit but not in seeds from the tomato yellow ripe mutant (defective in phytoene synthase-1, PSY1), suggesting that the isoform PSY2 might channel the production of carotenoids for abscisic acid biosynthesis. Furthermore, the complete arrest of tomato seedling development using fosmidomycin confirms a key role of the MEP pathway in plant development.     

Coexpression of a defensin gene and a thionin-like gene via different signal transduction pathways in pepper and Colletotrichum gloeosporioides interactions

The anthracnose fungus, Colletotrichum gloeosporioides, interacts incompatibly with the ripe fruit of pepper (Capsicum annuum). It interacts compatibly with the unripe-mature fruit. We isolated a defensin gene, j1-1, and a thionin-like gene, PepThi, expressed in the incompatible interaction by using an mRNA differential display method. Both genes were developmentally regulated during fruit ripening, organ-specifically regulated, and differentially induced during the compatible and incompatible interactions. Expression of the PepThi gene was rapidly induced in the incompatible-ripe fruit upon fungal infection. The fungus-inducible PepThi gene is highly inducible only in the unripe fruit by salicylic acid. In both ripe and unripe fruit, it was induced by wounding, but not by jasmonic acid. Expression of the j1-1 gene is enhanced by jasmonic acid in the unripe fruit but suppressed in the ripe fruit. These results suggest that both small and cysteine-rich protein genes are induced via different signal transduction pathways during fruit ripening to protect the reproductive organs against biotic and abiotic stresses.     

Rainfall seasonality predicts the germination behavior of a tropical dry‐forest vine

Seed dormancy is considered to be an adaptive strategy in seasonal and/or unpredictable environments because it prevents germination during climatically favorable periods that are too short for seedling establishment. Tropical dry forests are seasonal environments where seed dormancy may play an important role in plant resilience and resistance to changing precipitation patterns. We studied the germination behavior of seeds from six populations of the Neotropical vine Dalechampia scandens (Euphorbiaceae) originating from environments of contrasting rainfall seasonality. Seeds produced by second greenhouse‐generation plants were measured and exposed to a favorable wet environment at different time intervals after capsule dehiscence and seed dispersal. We recorded the success and the timing of germination. All populations produced at least some dormant seeds, but seeds of populations originating from more seasonal environments required longer periods of after‐ripening before germinating. Within populations, larger seeds tended to require longer after‐ripening periods than did smaller seeds. These results indicate among‐population genetic differences in germination behavior and suggest that these populations are adapted to local environmental conditions. They also suggest that seed size may influence germination timing within populations. Ongoing changes in seasonality patterns in tropical dry forests may impose strong selection on these traits.     

Detection of expansin proteins and activity during tomato fruit ontogeny

Expansins are plant proteins that have the capacity to induce extension in isolated cell walls and are thought to mediate pH-dependent cell expansion. J.K.C. Rose, H.H. Lee, and A.B. Bennett ([1997] Proc Natl Acad Sci USA 94: 5955-5960) reported the identification of an expansin gene (LeExp1) that is specifically expressed in ripening tomato (Lycopersicon esculentum) fruit where cell wall disassembly, but not cell expansion, is prominent. Expansin expression during fruit ontogeny was examined using antibodies raised to recombinant LeExp1 or a cell elongation-related expansin from cucumber (CsExp1). The LeExp1 antiserum detected expansins in extracts from ripe, but not preripe tomato fruit, in agreement with the pattern of LeExp1 mRNA accumulation. In contrast, antibodies to CsExp1 cross-reacted with expansins in early fruit development and the onset of ripening, but not at a later ripening stage. These data suggest that ripening-related and expansion-related expansin proteins have distinct antigenic epitopes despite overall high sequence identity. Expansin proteins were detected in a range of fruit species and showed considerable variation in abundance; however, appreciable levels of expansin were not present in fruit of the rin or Nr tomato mutants that exhibit delayed and reduced softening. LeExp1 protein accumulation was ethylene-regulated and matched the previously described expression of mRNA, suggesting that expression is not regulated at the level of translation. We report the first detection of expansin activity in several stages of fruit development and while characteristic creep activity was detected in young and developing tomato fruit and in ripe pear, avocado, and pepper, creep activity in ripe tomato showed qualitative differences, suggesting both hydrolytic and expansin activities.     

Products Released from Enzymically Active Cell Wall Stimulate Ethylene Production and Ripening in Preclimacteric Tomato (Lycopersicon esculentum Mill.) Fruit

Enzymically active cell wall from ripe tomato (Lycopersicon esculentum Mill.) fruit pericarp release uronic acids through the action of wall-bound polygalacturonase. The potential involvement of products of wall hydrolysis in the induction of ethylene synthesis during tomato ripening was investigated by vacuum infiltrating preclimacteric (green) fruit with solutions containing pectin fragments enzymically released from cell wall from ripe fruit. Ripening initiation was accelerated in pectin-infiltrated fruit compared to control (buffer-infiltrated) fruit as measured by initiation of climacteric CO₂ and ethylene production and appearance of red color. The response to infiltration was maximum at a concentration of 25 micrograms pectin per fruit; higher concentrations (up to 125 micrograms per fruit) had no additional effect. When products released from isolated cell wall from ripe pericarp were separated on Bio-Gel P-2 and specific size classes infiltrated into preclimacteric fruit, ripening-promotive activity was found only in the larger (degree of polymerization >8) fragments. Products released from pectin derived from preclimacteric pericarp upon treatment with polygalacturonase from ripe pericarp did not stimulate ripening when infiltrated into preclimacteric fruit.     

Effect of Fruit Load on Partitioning of Dry Matter and Energy in Cantaloupe (Cucumis melo L.)

Cantaloupe (Cucumis melo L.) plants set groups of fruits whichgenerate large variations in the reproductive:vegetative dryweight balance. We studied the influence of fruit number onthe partitioning of dry matter and energy between the vegetativeand reproductive organs and among the seeds and the variousfruit tissues during the development of the first fruits. Over2 years and on two Charentais cantaloupe cultivars, fruit numberwas either limited to one or left unrestricted, which led tothe setting of two to six fruits. Because of the high lipidcontent in seeds, the distribution of assimilates was studiedin terms of energy equivalent as well as dry weight. Measureddry weights were converted into energy equivalents by calculatingthe construction cost of tissues from their elemental composition.Seeds differed from other tissues in showing an increase inconstruction cost, from 1.1 to 1.8 g CH₂O g^-1d. wt between 10and 30 d after pollination. For this reason, during the secondhalf of fruit development on plants with unrestricted fruitload, they made up to 31% of the fruit and 12% of the aerialpart of the whole plant in terms of dry weight, but 39 and 18%in terms of energy (glucose equivalents). The fraction of assimilatesallocated to the fruits showed a saturation-type response tothe number of fruits per plant. It did not increase in cultivarTalma above two fruits per plant, which could be due to a decreasingsink strength with fruit rank, whereas cultivar Galoubet maintaineda more homogeneous fruit size within plants. At a similar fruitload, the reproductive:vegetative dry weight balance differedbetween the 2 years of the experiment, probably because of variationin the fruit sink strength. Copyright 1999 Annals of BotanyCompany Charentais cantaloupe, Cucumis melo L., assimilate distribution, construction cost, development, dry matter partitioning, fruit load, seeds, sink strength.     

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.     

Proanthocyanidin synthesis and expression of genes encoding leucoanthocyanidin reductase and anthocyanidin reductase in developing grape berries and grapevine leaves

Proanthocyanidins (PAs), also called condensed tannins, can protect plants against herbivores and are important quality components of many fruits. Two enzymes, leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR), can produce the flavan-3-ol monomers required for formation of PA polymers. We isolated and functionally characterized genes encoding both enzymes from grapevine (Vitis vinifera L. cv Shiraz). ANR was encoded by a single gene, but we found two highly related genes encoding LAR. We measured PA content and expression of genes encoding ANR, LAR, and leucoanthocyanidin dioxygenase in grape berries during development and in grapevine leaves, which accumulated PA throughout leaf expansion. Grape flowers had high levels of PA, and accumulation continued in skin and seeds from fruit set until the onset of ripening. VvANR was expressed throughout early flower and berry development, with expression increasing after fertilization. It was expressed in berry skin and seeds until the onset of ripening, and in expanding leaves. The genes encoding LAR were expressed in developing fruit, particularly in seeds, but had low expression in leaves. The two LAR genes had different patterns of expression in skin and seeds. During grape ripening, PA levels decreased in both skin and seeds, and expression of genes encoding ANR and LAR were no longer detected. The results indicate that PA accumulation occurs early in grape development and is completed when ripening starts. Both ANR and LAR contribute to PA synthesis in fruit, and the tissue and temporal-specific regulation of the genes encoding ANR and LAR determines PA accumulation and composition during grape berry development.     

The role of the persistent fruit wall in seed water regulation in Raphanus raphanistrum (Brassicaceae)

Background and Aims

Dry fruits remain around the seeds at dispersal in a number of species, especially the Brassicaceae. Explanations for this vary, but usually involve mechanisms of innate dormancy. We speculate that, instead, a persistent fruit may give additional protection through control of dehydration, to species growing in arid or Mediterranean environments where water is sporadic.

Methods

X-rays and weight measurements were used to determine the extent to which Raphanus raphanistrum seeds within mature fruits imbibe water, and germination tests determined the roles of the fruit and seed coat in seed dormancy. Rates of water uptake and desiccation, and seedling emergence were compared with and without the fruit. Finally, germinability of seeds extracted from fruits was determined after various periods of moist conditions followed by a range of dry conditions.

Key Results

Most seeds rapidly take up water within the fruit, but they do not fully imbibe when compared with naked seeds. The seed coat is more important than the dry fruit wall in maintaining seed dormancy. The presence of a dry fruit slows emergence from the soil by up to 6–8 weeks. The fruit slows the rate of desiccation of the seed to a limited extent. The presence of the fruit for a few days during imbibition somehow primes more seeds to germinate than if the fruit is absent; longer moist periods within the pod appear to induce dormancy.

Conclusions

The fruit certainly modifies the seed environment as external conditions change between wet and dry, but not to a great extent. The major role seems to be: (a) the physical restriction of imbibition and germination; and (b) the release and then re-imposition of dormancy within the seed. The ecological significance of the results requires more research under field conditions.     

Tissue-specific organic acid metabolism in reproductive and non-reproductive parts of the fig fruit is partially induced by pollination

Organic acids are important components of overall fruit quality through flavor, taste, nutritional and medicinal values. Pollinated fig (Ficus carica L.) fruit quality is enhanced by increased acidity. We quantified the major organic acids and characterized the expression pattern of organic acid metabolic pathway-related genes in the reproductive part – inflorescence and non-reproductive part – receptacle of parthenocarpic and pollinated fig fruit during ripening. Essentially, pollinated fruit contains seeds in the inflorescence, as opposed to no seeds in the parthenocarpic inflorescence. The major organic acids – citrate and malate – were found in relatively high quantities in the inflorescence compared to the receptacle of both parthenocarpic and pollinated fig fruit. Notably, pollination increased citric acid content significantly in both inflorescence and receptacle. Genes related to the phosphoenolpyruvate carboxylase (PEPC) cycle, tricarboxylic acid cycle, citrate catabolism and glyoxylate cycle were identified in fig fruit. Expression levels of most of these genes were higher in inflorescences than in receptacles. In particular, FcPEPC and FcFUM (encoding fumarase) had significantly higher expression in the inflorescence of pollinated fruit. Most importantly, expression of the glyoxylate cycle genes FcMLS and FcICL (encoding malate synthase and isocitrate lyase, respectively) was induced to strikingly high levels in the inflorescence by pollination, and their expression level was highly positively correlated with the contents of all organic acids. Therefore, the glyoxylate cycle may be responsible for altering the accumulation of organic acids to upgrade the fruit taste during ripening, especially in the pollinated, seeded inflorescence.     

Seeds of tomato (Lycopersicon esculentum Mill.) which develop in a fully hydrated environment in the fruit switch from a developmental to a germinative mode without a requirement for desiccation

Seed water content is high during early development of tomato seeds (10–30 d after pollination (DAP)), declines at 35 DAP, then increases slightly during fruit ripening (following 50 DAP). The seed does not undergo maturation drying. Protein content during seed development peaks at 35 DAP in the embryo, while in the endosperm it exhibits a triphasic accumulation pattern. Peaks in endosperm protein deposition correspond to changes in endosperm morphology (i.e. formation of the hard endosperm) and are largely the consequence of increases in storage proteins. Storage-protein deposition commences at 20 DAP in the embryo and endosperm; both tissues accumulate identical proteins. Embryo maturation is complete by 40 DAP, when maximum embryo protein content, size and seed dry weight are attained. Seeds are tolerant of premature drying (fast and slow drying) from 40 DAP.Thirty-and 35-DAP seeds when removed from the fruit tissue and imbibed on water, complete germination by 120 h after isolation. Only seeds which have developed to 35 DAP produce viable seedlings. The inability of isolated 30-DAP seed to form viable seedlings appears to be related to a lack of stored nutrients, since the germinability of excised embryos (20 DAP and onwards) placed on Murashige and Skoog (1962, Physiol. Plant. 15, 473–497) medium is high. The switch from a developmental to germinative mode in the excised 30- and 35-DAP imbibed seeds is reflected in the pattern of in-vivo protein synthesis. Developmental and germinative proteins are present in the embryo and endosperm of the 30- and 35-DAP seeds 12 h after their isolation from the fruit. The mature seed (60 DAP) exhibits germinative protein synthesis from the earliest time of imbibition. The fruit environment prevents precocious germination of developing seeds, since the switch from development to germination requires only their removal from the fruit tissue.Abbreviations DAP days after pollination - kDa kilodaltons - SP1-4 storage proteins 1–4 - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - HASI hours after seed isolation - MS medium Murashige and Skoog (1962) medium This work is supported by National Science and Engineering Research Council of Canada grant A2210 to J.D.B.     

香蕉果胶裂解酶基因的克隆

根据已经报告的香蕉果胶裂解酶基因序列,设计了特异引物,通过RT-PCR获得果胶裂解酶的cDNA,并克隆测序,与已报告的序列进行了比较,二者核苷酸序列的同源性达99.24%;推测的氨基酸序列也具有很高的同源性,达97.7%.通过RT-PCR的方法对香蕉不同组织和不同成熟度果实的果胶裂解酶基因的表达进行了研究.结果表明该基因只在果实中表达,具有组织特异性,而且只在果实的特定发育阶段表达.     

Expression of six expansin genes in relation to extension activity in developing strawberry fruit.

Expansins are proteins which have been demonstrated to induce cell wall extension in vitro. The identification and characterization of six expansin cDNAs from strawberry fruit, termed FaExp3 to FaExp7, as well as the previously identified FaExp2 is reported here. Analysis of expansin mRNAs during fruit development and in leaves, roots and stolons revealed a unique pattern of expression for each cDNA. FaExp3 mRNA was present at much lower levels than the other expansin mRNAs and was expressed in small green fruit and in ripe fruit. FaExp4 mRNA was present throughout fruit development, but was more strongly expressed during ripening. FaExp5 was the only clone to show fruit specific expression which was up-regulated at the onset of ripening. FaExp6 and FaExp7 mRNAs were present at low levels in the fruit with highest expression in stolon tissue. During fruit development FaExp6 had the highest expression at the white, turning and orange stages whereas expression of FaExp7 was highest in white fruit. The expression profiles of FaExp2 and FaExp5 in developing fruit were similar except that FaExp2 was induced at an earlier stage. Analysis of expansin protein by Western blotting using an antibody raised against CsExp1 from cucumber hypocotyls identified two bands of 29 and 31 kDa from developing fruit. Protein extracts from developing fruit were assayed for extension activity. Considerable rates of extension were observed with extracts from ripening fruit, but no extension was observed with protein from unripe green fruit. These results demonstrate the presence of at least six expansin genes in strawberry fruit and that during ripening the fruit acquires the ability to cause extension in vitro, characteristic of expansin action.     

柑橘果实的光合特性、产物运输及分配在糖分积累中的作用

测定了温州蜜柑 (CitrusunshiuMarc .cv .Miyagawawase)果实发育进程中干鲜重、果皮光合速率和叶绿素含量的变化 ,并用14 CO2 示踪技术研究了果皮和叶同化生成的光合产物在果实内的运输分配特性。结果表明 :果皮光合速率与叶绿素含量有关 ,随着叶绿素含量的下降 ,果实光合速率也快速下降。在果实完熟之前 ,即使是当果皮积累的干重超过汁囊时 ,叶同化产物仍主要分配到汁囊中 ;而在完熟阶段 ,果皮光合速率接近零 ,果皮成了叶同化产物的主要库。果皮的同化产物 ,主要保留在果皮中 ,输入到汁囊的比率随果实发育而下降 ,但高峰时也有 12 %输入汁囊。与对照相比 ,果实遮光处理后降低了果皮与汁囊的干重和含糖量。上述结果表明果皮光合产物主要用于果皮自身的发育并能减少对叶光合产物的依赖 ,同时也能部分增加汁囊糖的积累