The cell cycle-associated protein kinase WEE1 regulates cell size in relation to endoreduplication in developing tomato fruit

Tomato fruit size results from the combination of cell number and cell size which are respectively determined by cell division and cell expansion processes. As fruit growth is mainly sustained by cell expansion, the development of pericarp and locular tissues is characterized by the concomitant arrest of mitotic activity, inhibition of cyclin-dependent kinase (CDK) activity, and numerous rounds of endoreduplication inducing a spectacular increase in DNA ploidy and mean cell size. To decipher the molecular basis of the endoreduplication-associated cell growth in fruit, we investigated the putative involvement of the WEE1 kinase (Solly;WEE1). We here report a functional analysis of Solly;WEE1 in tomato. Impairing the expression of Solly;WEE1 in transgenic tomato plants resulted in a reduction of plant size and fruit size. In the most altered phenotypes, fruits displayed a reduced number of seeds without embryo development. The reduction of plant-, fruit- and seed size originated from a reduction in cell size which could be correlated with a decrease of the DNA ploidy levels. At the molecular level downregulating Solly;WEE1 in planta resulted in the increase of CDKA activity levels originating from a decrease of the amount of Y15-phosphorylated CDKA, thus indicating a release of the negative regulation on CDK activity exerted by WEE1. Our data indicated that Solly;WEE1 participates in the control of cell size and/or the onset of the endoreduplication process putatively driving cell expansion.     

Cell expansion and endoreduplication show a large genetic variability in pericarp and contribute strongly to tomato fruit growth

Postanthesis growth of tomato (Solanum lycopersicon) as of many types of fruit relies on cell division and cell expansion, so that some of the largest cells to be found in plants occur in fleshy fruit. Endoreduplication is known to occur in such materials, which suggests its involvement in cell expansion, although no data have demonstrated this hypothesis as yet. We have analyzed pattern formation, cell size, and ploidy in tomato fruit pericarp. A first set of data was collected in one cherry tomato line throughout fruit development. A second set of data was obtained from 20 tomato lines displaying a large weight range in fruit, which were compared as ovaries at anthesis and as fully grown fruit at breaker stage. A remarkable conservation of pericarp pattern, including cell layer number and cell size, is observed in all of the 20 tomato lines at anthesis, whereas large variations of growth occur afterward. A strong, positive correlation, combining development and genetic diversity, is demonstrated between mean cell size and ploidy, which holds for mean cell diameters from 10 to 350 microm (i.e. a 32,000-times volume variation) and for mean ploidy levels from 3 to 80 C. Fruit weight appears also significantly correlated with cell size and ploidy. These data provide a framework of pericarp patterning and growth. They strongly suggest the quantitative importance of polyploidy-associated cell expansion as a determinant of fruit weight in tomato.     

The specific overexpression of a cyclin-dependent kinase inhibitor in tomato fruit mesocarp cells uncouples endoreduplication and cell growth

The size of tomato fruit results from the combination of cell number and cell size, which are respectively determined by the cell division and cell expansion processes. As fruit growth is mainly sustained by cell expansion, the development of fleshy pericarp tissue is characterized by numerous rounds of endoreduplication inducing a spectacular increase in DNA ploidy and mean cell size. Although a clear relationship exists between endoreduplication and cell growth in plants, the exact role of endoreduplication has not been clearly elucidated. To decipher the molecular basis of endoreduplication-associated cell growth in fruit, we investigated the putative involvement of the tomato cyclin-dependent kinase inhibitor SlKRP1. We studied the kinetics of pericarp development in tomato fruit at the morphological and cytological levels, and demonstrated that endoreduplication is directly proportional to cell and fruit diameter. We established a mathematical model for tissue growth according to the number of divisions and endocycles. This model was tested in fruits where we managed to decrease the extent of endoreduplication by over-expressing SlKRP1 under the control of a fruit-specific promoter expressed during early development. Despite the fact that endoreduplication was affected, we could not observe any morphological, cytological or metabolic phenotypes, indicating that determination of cell and fruit size can be, at least conditionally, uncoupled from endoreduplication.     

A multilevel analysis of fruit growth of two tomato cultivars in response to fruit temperature

Fruit phenotype is a resultant of inherent genetic potential in interaction with impact of environment experienced during crop and fruit growth. The aim of this study was to analyze the genetic and physiological basis for the difference in fruit size between a small (‘Brioso’) and intermediate (‘Cappricia’) sized tomato cultivar exposed to different fruit temperatures. It was hypothesized that fruit heating enhances expression of cell cycle and expansion genes, rates of carbon import, cell division and expansion, and shortens growth duration, whereas increase in cell number intensifies competition for assimilates among cells. Unlike previous studies in which whole‐plant and fruit responses cannot be separated, we investigated the temperature response by varying fruit temperature using climate‐controlled cuvettes, while keeping plant temperature the same. Fruit phenotype was assessed at different levels of aggregation (whole fruit, cell and gene) between anthesis and breaker stage. We showed that: (1) final fruit fresh weight was larger in ‘Cappricia’ owing to more and larger pericarp cells, (2) heated fruits were smaller because their mesocarp cells were smaller than those of control fruits and (3) no significant differences in pericarp carbohydrate concentration were detected between heated and control fruits nor between cultivars at breaker stage. At the gene level, expression of cell division promoters (CDKB2, CycA1 and E2Fe‐like) was higher while that of the inhibitory fw2.2 was lower in ‘Cappricia’. Fruit heating increased expression of fw2.2 and three cell division promoters (CDKB1, CDKB2 and CycA1). Expression of cell expansion genes did not corroborate cell size observations.     

Xyloglucan endotransglycosylase activity during fruit development and ripening of apple and kiwifruit

Xyloglucan endotransglycosylase (XET) activity was measured in apple (Malus domestica Borkh. cv. Braeburn) pericarp and kiwifruit (Actinidia deliciosa [A. Chev.] C. F. Liang et A. R. Ferguson var. deliciosa cv. Hayward) outer pericarp and core tissues in order to establish whether a correlation exists between the activity of the enzyme and different stages of fruit development Whereas the growth rate of kiwifruit paralleled changes in XET activity throughout fruit growth, that of apple did not. Both fruits showed the highest XET activity, on a fresh weight basis, in the first two weeks after anthesis when cell division was at its highest. XET activity then decreased sharply, but as the fruit increased in size (4–8 weeks after anthesis) there was a concomitant increase in XET activity in both fruits. In the latter stage of fruit development (16–26 weeks after anthesis) XET activity increased to peak at harvest in apple fruit. During this time there was relatively little increase in fruit size and presumably therefore minimal cell expansion. XET activity then declined as fruit softened after harvest. In core tissue from kiwifruit, XET activity increased throughout the later stages of fruit growth to harvest maturity in a similar manner to apple, but continued to increase after harvest until fruit were ripe. In contrast, XET activity in the outer pericarp of kiwifruit did not increase until ripening after harvest. In apple tissue up to 30% of the XET activity was cell wall bound and could not be solubilised, even in buffer containing 2 M NaCl. The results implicate XET in cell wall assembly during cell division and expansion early in apple and kiwifruit growth. However, the disparity between apple and kiwifruit with respect to XET activity late in fruit development and ripening and the different affinities of the enzyme for the cell wall in each fruit, suggest that XET has several roles in plant development, not all of which are related to cell wall loosening during periods of accelerated growth.     

Relationship between cell number,cell size and fruit size of seeded fruits of tomato (Lycopersicon esculentum Mill.), and those induced parthenocarpically by the application of plant growth regulators

Application of GA₃, IAA or 4-CPA to tomato ovaries induced the development of parthenocarpic fruit, which showed different growth rates. In the pericarp cell division and cell enlargement was affected differentially. GA₃-induced fruits had considerably less but larger cells than seeded control fruits, IAA treatment resulted in the same number of cells but these were smaller and 4-CPA treatment induced fruits with about 20% more cells. Reduction in cell number had a similar effect on final fruit size as diminution of cell size. A reduction in the number of cell division centres (area around vascular bundles) as well as changes in the degree of endoploidy are possible reasons for the observed reductions in cell numbers. Hormonal causes for the different number and size of pericarp cells after the various treatments are discussed.     

Partitioning of (13)C-photosynthate from spur leaves during fruit growth of three Japanese pear (Pyrus pyrifolia) cultivars differing in maturation date

BACKGROUND AND AIMS: In fruit crops, fruit size at harvest is an important aspect of quality. With Japanese pears (Pyrus pyrifolia), later maturing cultivars usually have larger fruits than earlier maturing cultivars. It is considered that the supply of photosynthate during fruit development is a critical determinant of size. To assess the interaction of assimilate supply and early/late maturity of cultivars and its effect on final fruit size, the pattern of carbon assimilate partitioning from spur leaves (source) to fruit and other organs (sinks) during fruit growth was investigated using three genotypes differing in maturation date. METHODS: Partitioning of photosynthate from spur leaves during fruit growth was investigated by exposure of spurs to (13)CO(2) and measurement of the change in (13)C abundance in dry matter with time. Leaf number and leaf area per spur, fresh fruit weight, cell number and cell size of the mesocarp were measured and used to model the development of the spur leaf and fruit. KEY RESULTS: Compared with the earlier-maturing cultivars 'Shinsui' and 'Kousui', the larger-fruited, later-maturing cultivar 'Shinsetsu' had a greater total leaf area per spur, greater source strength (source weight x source specific activity), with more (13)C assimilated per spur and allocated to fruit, smaller loss of (13)C in respiration and export over the season, and longer duration of cell division and enlargement. Histology shows that cultivar differences in final fruit size were mainly attributable to the number of cells in the mesocarp. CONCLUSIONS: Assimilate availability during the period of cell division was crucial for early fruit growth and closely correlated with final fruit size. Early fruit growth of the earlier-maturing cultivars, but not the later-maturing ones, was severely restrained by assimilate supply rather than by sink limitation.     

Functional analysis of the anaphase promoting complex activator CCS52A highlights the crucial role of endo‐reduplication for fruit growth in tomato

Tomato fruit growth is characterized by the occurrence of numerous rounds of DNA endo‐reduplication in connection with cell expansion and final fruit size determination. Endo‐reduplication is an impairment of mitosis that originates from the selective degradation of M phase‐specific cyclins via the ubiquitin‐mediated proteolytic pathway, requiring the E3 ubiquitin ligase anaphase promoting complex/cyclosome (APC/C). Two types of APC/C activators, namely CCS52 and CDC20 proteins, exist in plants. We report here the molecular characterization of such APC/C activators during fruit development, and provide an in planta functional analysis of SlCCS52A, a gene that is specifically associated with endo‐reduplication in tomato. Altering SlCCS52A expression in either a negative or positive manner had an impact on the extent of endo‐reduplication in fruit, and fruit size was reduced in both cases. In SlCCS52A over‐expressing fruits, endo‐reduplication was initially delayed, accounting for the altered final fruit size, but resumed and was even enhanced at 15 days post anthesis (dpa), leading to fruit growth recovery. This induction of growth mediated by endo‐reduplication had a considerable impact on nitrogen metabolism in developing fruits. Our data contribute to unravelling of the physiological role of endo‐reduplication in growth induction during tomato fruit development.     

The role of ethylene and cell wall modifying enzymes in raspberry (Rubus idaeus) fruit ripening

This study focuses on four raspberry ( Rubus idaeus ) genotypes from two different genetic backgrounds: cvs Glen Prosen and Glen Clova, bred at the Scottish Crop Research Institute (SCRI) and genotypes bred at Horticulture Research International (HRI), East Malling (EM), EM 4997 and EM 5007. The ripe fruit of each genotype pair were characterised subjectively by raspberry breeders as relatively firm or soft, respectively. Different stages of fruit development from each genotype were used to quantify fruit firmness, rates of ethylene evolution and ripening rate. Penetrometry data confirmed suspected firmness differences. Firmness correlated with rates of ethylene evolution. Rates of ethylene production also correlated with receptacle size. Storage of green fruits in 20 μl l⁻¹ ethylene reduced fruit firmness, enhanced respiration rate and colour (anthocyanin) development and stimulated the development of cell wall hydrolase activities. However, during natural ripening in the field, fruit respiration rate declined, which indicates a non-climacteric ripening pattern. In drupelets, the activities of polygalacturonase (PG), pectin methylesterase (PME), C x -cellulase (C x ) and β -galactosidase ( β -gal.) increased substantially as ripening progressed. More detailed studies with ripe fruit of cv. Glen Clova indicated major isoforms of PG at pIs 3.3, 8.6 and 10.1; of PME at pIs 7.2, 8.5, 8.7, 8.8; of C x at pI 2.4; and of β -gal. at pIs 6.3 and 6.7.     

不同倍性罗汉果果实的生长与甙类含量动态变化规律的研究

对不同倍性罗汉果果实的生长动态、甙类含量动态及其变化规律的研究。结果表明:多倍体与二倍体果实的生长和甙类动态变化规律基本一致。果实的生长动态均可分为3个时期:迅速生长期(1～20 d)、缓慢生长期(20～30 d)、停止生长期(30～90 d),80 d时果实的形态达到最大值,4x>2x>3x,多倍体表现出巨大性。而甙类含量的动态随日龄的增长均表现为:苦味的甙ⅡE转化为甙Ⅲ,两者依次出现和消失;最终都转化为高甜度的甙Ⅴ,即成熟罗汉果以甙Ⅴ为主;80 d时甙Ⅴ的含量达到最大,4x>3x>2x:多倍体的甜甙含量显著提高,特别是三倍体还具有无籽的特性。     

Cell number, cell size and hormone levels in semi-isogenic mutants of Lycopersicon pimpinellifolium differing in fruit size

Tomato fruit growth parameters, cell number and cell size, and hormone levels [IAA, abscisic acid (ABA), zeatin (Z)/zeatin riboside (ZR), isopentenyladenosine (i-Ado)/isopentenlyadenine (i-Ade)], in the wild-type ( Lycopersicon pimpinellifolium Mill.) and a semi-isogenic mutant (mutant III) differing in fruit size were investigated during fruit development. An image-processing system was used for the determination of cell number and single cell size per fruit and hormone levels were measured by radioimmuno-assay (RIA). The bigger fruits of mutant III showed higher cell numbers throughout fruit development and cells enlarged faster than in wild-type fruits. During the first 10 days of fruit growth, the main cell division period after fertilization, high concentrations of cytokinins were found, these being correlated with high cell division activity. There were only slight differences in IAA and ABA levels in the different sized fruits. The results emphasized the importance of the cell number per fruit at anthesis as a determining factor of final fruit size in tomatoes. A possible relationship between cytokinins and subsequent fruit development is discussed.     

Do Genetic Make-up and Growth Manipulation Affect Tomato Fruit Size by Cell Number,or Cell Size and DNA Endoreduplication?

This work investigated the link between genetic and developmental controls of fruit size and composition. On two isogenic lines (CF12-C and CF14-L), differing by fruit weight and sugar content quantitative trait loci (QTLs) identified previously, basal and tip fruits were characterized at anthesis and at maturity through their growth, dry matter and sugar content, number and size of cells and nuclei DNA content. The influence of competition was assessed by removing either basal or tip ovaries at anthesis. On an intact inflorescence, CF12-C fruits grew less than CF14-L fruits, with 1.67 fewer cell layers and similar cell size, suggesting that genes controlling cell division may be responsible for this fruit size variation. Truss thinning masked the QTL effect on fruit size, mainly by reducing the difference in cell number between the two lines and by promoting cell expansion in tip fruits, so that fruit growth was similar at both positions and for both lines. Thus, in these lines, cell number exerts a control on final fruit size only when there is competition among fruits. Different responses of basal and tip fruits after flower removal suggested that this treatment induced changes in hormonal relationships within the truss. No fixed relationship between DNA endoreduplication and cell size was found, as while cell size and dry matter and sugar contents differed with tomato lines, fruit position and truss size, endoreduplication patterns were the same. CF12-C fruits had a higher dry matter (+0.3% of fresh weight) and carbohydrates (+8% of dry matter) content than CF14-L fruits. The percentage dry matter was independent of truss size but decreased slightly from basal to tip fruits.     

Postharvest heat and cold treatment to control Ceratitis capitata on cactus pear fruit

In order to work out a quarantine treatment for cactus pear fruit a factorial experimental plan was carried combining postharvest water dips at 20, 50, 54, 58 and 60 degrees C and storage at 1 degrees C for 3, 6 and 10 days. Cactus pear fruit cv 'Rossa' were artificially infested with Med. fly eggs (at least 20 eggs per fruit) then left in the lab at 25 degrees C for 4 days. Treatments took place by dipping the fruit at each water temperature for 2 mm. At each established time fruit was picked and checked for vital larvae and degree of chilling injury (CI). Probit 9 requirements were achieved in all cases when fruit was cold-stored for 10 days. When fruit was kept for 6 days the quarantine requirement was achieved only by dipping the fruit at 58 and 60 degrees C while none dip treatment was effective if fruit was stored for 3 days at 1 degrees C. All Fruit stored for 10 days at 1 degrees C showed severe CI symptoms and when kept for 6 days the same degree of CI was found on fruit dipped in water at 20, 58 and 60 degrees C. No CI was observed after 3 days at 1 degrees C. In conclusion only when fruit was dipped at 50-54 degrees C and stored for 10 days at 1 degrees C the probit 9 condition was attained with acceptable CI symptoms.     

Growth and proteomic analysis of tomato fruit under partial root-zone drying

The effects of partial root-zone drying (PRD) on tomato fruit growth and proteome in the pericarp of cultivar Ailsa Craig were investigated. The PRD treatment was 70% of water applied to fully irrigated (FI) plants. PRD reduced the fruit number and slightly increased the fruit diameter, whereas the total fruit fresh weight (FW) and dry weight (DW) per plant did not change. Although the growth rate was higher in FI than in PRD fruits, the longer period of cell expansion resulted in bigger PRD fruits. Proteins were extracted from pericarp tissue at two fruit growth stages (15 and 30 days post-anthesis [dpa]), and submitted to proteomic analysis including two-dimensional gel electrophoresis and mass spectrometry for identification. Proteins related to carbon and amino acid metabolism indicated that slower metabolic flux in PRD fruits may be the cause of a slower growth rate compared to FI fruits. The increase in expression of the proteins related to cell wall, energy, and stress defense could allow PRD fruits to increase the duration of fruit growth compared to FI fruits. Upregulation of some of the antioxidative enzymes during the cell expansion phase of PRD fruits appears to be related to their role in protecting fruits against the mild stress induced by PRD.     

Morphological characteristics of leaf epidermis and size variation of leaf, flower and fruit in different ploidy levels in Buddleja macrostachya (Buddlejaceae)

Buddleja macrostachya (Buddlejaceae) is a widespread shrub native to the Sino-Himalayan mountains and beyond. It has been found to occur at two ploidy levels, hexaploid, 2n=6x=114 and dodecaploid, 2n=12x=228. To determine if morphological characters might be used as indicators of ploidy levels, we measured floral and fruit length, relative and absolute leaf size, trichome density on both leaf surfaces, and stomatal density and length in different populations of B. macrostachya. In general, flower and fruit length, absolute leaf size, and stomatal length increased with an increase at ploidy level (P<0.01), whereas adaxial cell and stomatal density decreased with an increase at ploidy level (P<0.01). We found no conspicuous differences in relative leaf size (P>0.05) in different populations. Other characters studied such as trichome type, cuticular membrane and ornamentation of stomata, cell and stomatal shape, and anticlinal wall pattern were quite constant in this species. Thus it appears that flower and fruit length, absolute leaf size, and stomatal frequency and length can be used to distinguish hexaploid from dodecaploid cytotypes either in the field or in herbarium specimens.     

Changes in the distribution of cell wall polysaccharides in early fruit pericarp and ovule, from fruit set to early fruit development, in tomato (Solanum lycopersicum)

During fruit development in tomato (Solanum lycopersicum), cell proliferation and rapid cell expansion occur after pollination. Cell wall synthesis, alteration, and degradation play important roles during early fruit formation, but cell wall composition and the extent of cell wall synthesis/degradation are poorly understood. In this study, we used immunolocalization with a range of specific monoclonal antibodies to examine the changes in cell wall composition during early fruit development in tomato. In exploring early fruit development, the ?1 day post-anthesis (DPA) ovary and fruits at 1, 3, and 5 DPA were sampled. Paraffin sections were prepared for staining and immunolabeling. The 5 DPA fruit showed rapid growth in size and an increase in both methyl-esterified pectin and de-methyl-esterified pectin content in the pericarp, suggesting rapid synthesis and de-methyl esterification of pectin during this growth period. Labeling of pectic arabinan with LM6 antibody and galactan with LM5 antibody revealed abundant amounts of both, with unique distribution patterns in the ovule and premature pericarp. These results suggest the presence of rapid pectin metabolism during the early stages of fruit development and indicate a unique distribution of pectic galactan and arabinan within the ovule, where they may be involved in embryogenesis.     

The contribution of fruit photosynthesis to the carbon requirement of cucumber fruits as affected by irradiance, temperature and ontogeny

The photosynthetic contribution of a fruit to its carbon requirement throughout ontogeny and under different growing conditions was quantified in cucumber ( Cucumis sativus L. cv. Corona). In addition, the effects of shading on fruit dry matter accumulation and the diurnal course of the elongation rate were studied. Fruit darkening had no photomorphogenic effect on fruit growth, while the cumulative photosynthetic contribution of a fruit to its own carbon requirement ranged from 1 to 5%. During the day there was always a net CO₂ efflux. The photosynthetic rate per fruit, calculated as the difference between rates of CO₂ exchange in light and dark, increased during fruit ontogeny, while the photosynthetic rate per unit fruit surface area declined. The latter was not dependent on fruit size. The photosynthetic activity per unit surface area of fruits was estimated to be about 20–30% as efficient as that of leaves. The rate of calculated photosynthesis was reduced by 60–65% when the photosynthetically active radiation incident on the fruit decreased from 200 to 50 μmol m⁻² s⁻¹. Temperature (20–30°C) had no pronounced effect on the rate of calculated fruit photosynthesis when fruits of the same developmental stage (temperature sum) were compared. However, the relative photosynthetic contribution of a fruit to its carbon requirement increased when temperature decreased. Moreover, this contribution increased when irradiance increased or fruit growth was reduced by competing fruits. During fruit ontogeny the daily photosynthetic contribution was highest (up to 15%) in young and old fruits, with a small growth rate.     

Sucrolytic activities during fruit development of Lycopersicon genotypes differing in tolerance to salinity

The different growth responses under control and moderate salinity (70 mM NaCl) in relation to the carbon partitioning and sucrose metabolism in developing tomato fruits [20 days after anthesis (DAA), start of ripening and ripe stages] were studied in the cultivated tomato Lycopersicon esculentum Mill (cv. H-324-1), in the wild relative species L. cheesmanii (ac. LA-530) (hexose-accumulators), L. chmielewskii (ac. LA-1028) (sucrose-accumulator) and in two interspecific F1 hybrids (hexose-accumulators) (F1-530: H-324-1 x A-530, F1-1028: H-324-1 x A-1028). The higher salt-tolerance of the wild species and hybrids with respect to the domestic tomatoes was also observed at the fruit level because these genotypes were less affected in the assimilation of dry weight (DW) under salinity. With the exception of the wild tomatoes, the sink strength, evaluated as the dry matter accumulation rate (mg DW day-1) and the sink activity, evaluated as a relative growth rate (mg DW mg-1 day-1), were reduced during the early fruit growing period (20 DAA-start ripening). However, a total recovery of growth was registered in the salinized hybrid fruits during the late growing period (start of ripening-ripe fruits). The early reduction in sink activity in the hybrid and domestic fruits was related to a sucrose accumulation and a decrease in the total sucrolytic activity at 20 DAA, especially the cytoplasmic sucrolytic activities sucrose synthase (EC 2.4.1.13) and neutral invertase (EC 3.2.1.26). The further recovery in sink strength of the hybrid fruits was related to the maintenance of the insoluble acid invertase (EC 3.2.1.25) and the induction of the cytoplasmic sucrolytic activities, namely at the start of ripening stage, demonstrating the existence of an inverse relationship between these activities, which suggests a regulatory mechanism in order to maintain the sink capacity. The roles of different enzymes in the control of assimilate import under salinity in relation to the sucrose transport and possible regulatory mechanisms are discussed.