Biochemical and genomic analysis of sucrose metabolism during coffee (Coffea arabica) fruit development

Sucrose metabolism and the role of sucrose synthase were investigated in the fruit tissues (pericarp, perisperm, and endosperm) of Coffea arabica during development. Acid invertase, sucrose phosphate synthase, and sucrose synthase activities were monitored and compared with the levels of sucrose and reducing sugars. Among these enzymes, sucrose synthase showed the highest activities during the last stage of endosperm and pericarp development and this activity paralleled closely the accumulation of sucrose in these tissues at this stage. Carbon partitioning in fruits was studied by pulse-chase experiments with (14)C-sugars and revealed high rates of sucrose turnover in perisperm and endosperm tissues. Additional feeding experiments with (14)CO(2) showed that leaf photosynthesis contributed more to seed development than the pericarp in terms of photosynthate supply to the endosperm. Sugar analysis, feeding experiments, and histological studies indicated that the perisperm plays an important role in this downloading process. It was observed that the perisperm presents a transient accumulation of starch which is degraded as the seed develops. Two full-length cDNAs (CaSUS1 and CaSUS2) and the complete gene sequence of the latter were also isolated. They encode sucrose synthase isoforms that are phylogenetically distinct, indicating their involvement in different physiological functions during cherry development. Contrasting expression patterns were observed for CaSUS1 and CaSUS2 in perisperm, endosperm, and pericarp tissues: CaSUS1 mRNAs accumulated mainly during the early development of perisperm and endosperm, as well as during pericarp growing phases, whereas those of CaSUS2 paralleled sucrose synthase activity in the last weeks of pericarp and endosperm development. Taken together, these results indicate that sucrose synthase plays an important role in sugar metabolism during sucrose accumulation in the coffee fruit.     

ATP-induced Changes in Energy Status and Membrane Integrity of Harvested Litchi Fruit and its Relation to Pathogen Resistance

Litchi is a subtropical fruit of high commercial value on the international market but the fruit deteriorates rapidly after harvest due to rot development caused by Peronophythora litchii. To investigate the role of energy metabolism during disease development on harvested litchi fruit, fruits were dipped into solutions of either 0 or 1.0 mm adenosine triphosphate (ATP) for 3 min before being inoculated with Peronophythora litchii or not. Fruit were then stored for 6 days at 25°C and 90–100% relative humidity. Significant reductions in pericarp browning and disease severity and significant delays in membrane permeability and malondialdehyde (MDA) content were found in ATP‐treated and P. litchii‐inoculated fruit. Higher ATP concentrations and adenylate energy charge (EC) were observed in ATP‐treated fruit. In addition, lower activities of phospholipase D, acid phosphatase and lipoxygenase enzymes involved in membrane lipid peroxidation and hydrolysis were recorded in ATP‐treated fruit. Thus, treatment with ATP maintained higher energy levels, inhibited activities of the membrane hydrolysis‐related enzymes, reduced membrane lipid peroxidation and helped maintain membrane integrity of the harvested litchi fruit at the early stage of storage, which could account for the inhibition of disease development of P. litchii‐inoculated fruit.     

Regulation of tomato fruit pericarp development by an interplay between CDKB and CDKA1 cell cycle genes

Growth of tomato fruits is determined by cell division and cell expansion, which are tightly controlled by factors that drive the core cell cycle. The cyclin-dependent kinases (CDKs) and their interacting partners, the cyclins, play a key role in the progression of the cell cycle. In this study the role of CDKA1, CDKB1, and CDKB2 in fruit development was characterized by fruit-specific overexpression and down-regulation. CDKA1 is expressed in the pericarp throughout development, but is strongly up-regulated in the outer pericarp cell layers at the end of the growth period, when CDKB gene expression has ceased. Overexpression of the CDKB genes at later stages of development and the down-regulation of CDKA1 result in a very similar fruit phenotype, showing a reduction in the number of cell layers in the pericarp and alterations in the desiccation of the fruits. Expression studies revealed that CDKA1 is down-regulated by the expression of CDKB1/2 in CDKB1 and CDKB2 overexpression mutants, suggesting opposite roles for these types of CDK proteins in tomato pericarp development.     

冷藏条件下白沙枇杷与红沙枇杷果皮活性氧代谢研究

为探讨红沙枇杷与白沙枇杷冷藏耐储性差异的原因,为枇杷采后生理和保鲜技术研究提供参考,以白沙枇杷"白玉"和红沙枇杷"鸡蛋红"为材料,在6℃的冷藏条件下,测定了果皮中氧自由基产生速率(oxygenfree radical production rate, SPR)、超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)等保护酶活性,以及膜脂过氧化伤害产物(MDA)的含量等活性氧(reactive oxygen species, ROS)代谢相关指标的变化规律。表明,随着冷藏的进程,冷藏的前10 d,红沙枇杷果皮SPR高于白沙枇杷,10 d后红沙枇杷果皮SPR有所下降而白沙枇杷果皮则大幅上升,导致10 d后红沙枇杷果皮SPR低于白沙枇杷;冷藏后红沙枇杷果皮ROS相关酶(SOD, POD和CAT)活性高于白沙枇杷果皮,即其ROS清除能力高于白沙枇杷果皮;在冷藏5 d后白沙枇杷果皮膜脂过氧化伤害产物MDA含量持续上升,而红沙枇杷果皮则维持在较低水平并低于白沙枇杷果皮,说明白沙枇杷果皮膜脂过氧化程度较高。分析认为冷藏初期红沙枇杷果皮较高的氧自由基产生速率激活了ROS清除系统,导致红沙枇杷果皮具有更高的ROS清除能力;冷藏后期白沙枇杷果皮则出现了氧自由基和MDA的积累,暗示其膜脂过氧化的发生和果实内外环境的恶化。红沙枇杷和白沙枇杷果皮ROS代谢的差异与冷藏耐储性相关。     

Pericarp development and fruit structure in borassoid palms (Arecaceae-Coryphoideae-Borasseae)

Background and Aims

The Borasseae form a highly supported monophyletic clade in the Arecaceae–Coryphoideae. The fruits of Coryphoideae are small, drupaceous with specialized anatomical structure of the pericarp and berries. The large fruits of borassoid palms contain massive pyrenes, which develop from the middle zone of the mesocarp. The pericarp structure and mode of its development in Borasseae are similar to those of Eugeissona and Nypa. A developmental carpological study of borassoid palms will allow us to describe the process of pericarp development and reveal the diagnostic fruit features of borassoid palms, determine the morphogenetic fruit type in Borasseae genera, and describe similarities in fruit structure and pericarp development with other groups of palms.

Methods

The pericarp anatomy was studied during development with light microscopy based on the anatomical sections of fruits of all eight Borasseae genera.

Key Results

The following general features of pericarp structure in Borasseae were revealed: (1) differentiation of the pericarp starts at early developmental stages; (2) the exocarp is represented by a specialized epidermis; (3) the mesocarp is extremely multilayered and is differentiated into several topographical zones – a peripheral parenchymatous zone(s) with scattered sclerenchymatous elements and vascular bundles, a middle zone (the stony pyrene comprising networks of elongated sclereids and vascular bundles) and an inner parenchymatous zone(s); (4) differentiation and growth of the pyrene tissue starts at early developmental stages and ends long before maturation of the seed; (5) the inner parenchymatous zone(s) of the mesocarp is dramatically compressed by the mature seed; (6) the endocarp (unspecialized epidermis) is not involved in pyrene formation; and (7) the spermoderm is multilayered in Hyphaeninae and obliterated in Lataniinae.

Conclusions

The fruits of Borasseae are pyrenaria of Latania-type. This type of pericarp differentiation is also found only in Eugeissona and Nypa. The fruits of other Coryphoideae dramatically differ from Borasseae by the pericarp anatomical structure and the mode of its development.     

外源脱落酸对富士苹果果实膨大后期光合产物向果实运输的影响

为了明确脱落酸(ABA)对苹果果实糖分积累的影响机理,本试验以5年生‘烟富3’/M26/平邑甜茶为试材,采用¹³C同位素标记技术,研究在果实膨大后期用不同浓度(0、50、100和150 mg·L^-1)脱落酸溶液及氟啶酮(ABA生物合成抑制剂)处理果实对光合产物向果实运输及糖代谢的影响.结果表明: 随着ABA浓度的增加,糖代谢相关酶活性、蔗糖转运蛋白基因MdSUT1、MdSUT2.2和山梨醇转运蛋白基因MdSUT3相对表达量呈先升高后降低趋势,均以100 mg·L^-1ABA处理时最高.氟啶酮处理显著抑制了糖代谢酶活性和糖转运蛋白基因相对表达量.与其他处理相比,100 mg·L^-1ABA处理显著减少了叶片¹³C含量,增加了果实¹³C含量,提升了光合产物由叶片向果实的运输速率.表明外源ABA通过增强果实库强,促进更多的光合产物向果实运输,提高了成熟期果实可溶性糖含量.     

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.     

Modelling central metabolic fluxes by constraint‐based optimization reveals metabolic reprogramming of developing Solanum lycopersicum (tomato) fruit

Modelling of metabolic networks is a powerful tool to analyse the behaviour of developing plant organs, including fruits. Guided by our current understanding of heterotrophic metabolism of plant cells, a medium‐scale stoichiometric model, including the balance of co–factors and energy, was constructed in order to describe metabolic shifts that occur through the nine sequential stages of Solanum lycopersicum (tomato) fruit development. The measured concentrations of the main biomass components and the accumulated metabolites in the pericarp, determined at each stage, were fitted in order to calculate, by derivation, the corresponding external fluxes. They were used as constraints to solve the model by minimizing the internal fluxes. The distribution of the calculated fluxes of central metabolism were then analysed and compared with known metabolic behaviours. For instance, the partition of the main metabolic pathways (glycolysis, pentose phosphate pathway, etc.) was relevant throughout fruit development. We also predicted a valid import of carbon and nitrogen by the fruit, as well as a consistent CO₂ release. Interestingly, the energetic balance indicates that excess ATP is dissipated just before the onset of ripening, supporting the concept of the climacteric crisis. Finally, the apparent contradiction between calculated fluxes with low values compared with measured enzyme capacities suggest a complex reprogramming of the metabolic machinery during fruit development. With a powerful set of experimental data and an accurate definition of the metabolic system, this work provides important insight into the metabolic and physiological requirements of the developing tomato fruits.     

Biosynthesis, accumulation and degradation of theobromine in developing Theobroma cacao fruits

We have studied the purine alkaloid content and purine metabolism in Theobroma cacao fruits at differing growth stages: Stage A (young small fruit, fresh weight, ca. 2 g); stage B (medium size fruit, fresh weight, ca. 100 g) and stage C (large size, fresh weight, ca. 500 g). The major purine alkaloid in stage A fruits (mainly pericarp) was theobromine (0.7 micromol g(-1) fresh weight), followed by caffeine (0.09 micromol g(-1) fresh weight). The theobromine content of the pericarp decreased sharply with tissue age, and the caffeine content decreased gradually. A large amount of theobromine (22 micromol g(-1) fresh weight) had accumulated in seeds (mainly cotyledons) of stage C fruits. Theobromine was found also in the seed coat and placenta. Tracer experiments with [8-(14)C]adenine show that the major sites of theobromine synthesis are the young pericarp and cotyledons of T. cacao fruits. Limited amounts of purine alkaloids may be transported from the pericarp to seed tissue, but most purine alkaloids that accumulated in seeds appeared to be synthesised in cotyledons. Degradation of [8-(14)C]theobromine and [8-(14)C]caffeine to CO2 via 3-methylxanthine and ureides (allantoin and allantoic acid) was detected only in the pericarp of stage C fruits.     

Activity and expression of polygalacturonase vary at different fruit ripening stages of sweet pepper cultivars

Activity and expression of polygalacturonase (PG), a hydrolytic enzyme involved in ultrastructural changes in the pericarp of sweet pepper (Capsicum annaum), were investigated at different ripening stages of the pepper cultivars Mandi and Talanduo. Molecular cloning of CaPG was carried out by constructing a cDNA library from three stages of fruit ripening. Morphological determination, PG assay, RT-PCR, and ultrastructural studies were used to quantify changes in CaPG gene expression in the pericarp from green, color change and fully ripened stages. We found that CaPG gene expression, PG activity and striking changes in the structure of the cell wall occurred with the transition of ripening stages. CaPG gene expression was high (obvious PCR products) in mature and ripened stages of both cultivars; however, the CaPG gene was not expressed in preclimacteric fruits or vegetative tissues. We conclude that developmental regulation of CaPG gene expression is instrumental for sweet pepper fruit ripening; its expression during development leads to dissolution of middle lamella and eventually disruption of the fully ripened cell wall.     

龙眼果实采后失水果皮褐变与活性氧及酚类代谢的关系

研究了(10±1)℃和50%相对湿度贮藏条件下"福眼"龙眼果实果皮褐变与活性氧和酚类代谢的关系.结果表明,采后失水导致龙眼果实果皮褐变,果皮活性氧清除酶SOD、CAT、APX、GR活性和内源抗氧化物质AsA、GSH含量下降,O-2产生速率和MDA含量增加,细胞膜透性迅速增大;PPO和POD活性增加,总酚和类黄酮含量明显下降.据此认为,果皮褐变可能是细胞的活性氧代谢失调,细胞膜结构破坏,使PPO、POD与酚类物质(含类黄酮)接触、酚类物质氧化的结果.     

Enzymic properties and capacities of developing tomato (Lycopersicon esculentum L.) fruit plastids

To characterize the developmental stage of tomato fruits, chlorophyll content, photosynthetic O2 evolution and CO2 fixation of pericarp slices were determined. During the first developmental stages a higher expression level of the triose phosphate translocator was detected. Transport measurements revealed that both the hexose phosphate and the triose phosphate translocator are very likely to be active at this time. Plastidic and cytosolic fructose-1,6-bisphosphatase are active in green fruit pericarp, whereas in red pericarp only the cytosolic form is present. Tomato fruit chloroplasts are able to synthesize starch from Glc6P. Starch synthesis is strongly dependent on the addition of 3PGA and ATP and on plastid illumination. Fruit chloroplasts exhibit very low CO2 fixation rates and so the capacities of green pericarp slices were investigated. In relation to chlorophyll content, pericarp slices show the same capacity of starch synthesis as spinach or potato leaves. To investigate the presence of further reactions consuming the products of photosynthetic electron transport, the GOGAT activity was measured. In the light, glutamine/2-oxoglutarate-dependent formation of glutamate occurred with a high activity. In the presence of Glc6P only 18% of the light activity was obtained. Since the Glc6P-dependent activity is rather low, the release of ¹⁴CO2 from labelled [1-¹⁴C]-Glc6P was also measured. In the dark, the formation of glutamate and oxidation of Glc6P are very tightly coupled to each other in fruit chloroplasts.