Sucrose-metabolizing enzymes in transport tissues and adjacent sink structures in developing citrus fruit

Juice tissues of citrus lack phloem; therefore, photosynthates enroute to juice sacs exit the vascular system on the surface of each segment. Areas of extensive phloem unloading and transport (vascular bundles + segment epidermis) can thus be separated from those of assimilate storage (juice sacs) and adjacent tissues where both processes occur (peel). Sugar composition, dry weight accumulation, and activities of four sucrose-metabolizing enzymes (soluble and cell-wall-bound acid invertase, alkaline invertase, sucrose synthase, and sucrose phosphate synthase) were measured in these transport and sink tissues of grapefruit (Citrus paradisi Macf.) to determine more clearly whether a given enzyme appeared to be more directly associated with assimilate transport versus deposition or utilization. Results were compared at three developmental stages. Activity of sucrose (per gram fresh weight and per milligram protein) extracted from zones of extensive phloem unloading and transport was significantly greater than from adjacent sink tissues during the stages (II and III) when juice sacs grow most rapidly. In stage II fruit, activity of sucrose synthase also significantly surpassed that of all other sucrose-metabolizing enzymes in extracts from the transport tissues (vascular bundles + segment epidermis). In contrast, sucrose phosphate synthase and alkaline invertase at this stage of growth were the most active enzymes from adjacent, rapidly growing, phloem-free sink tissues (juice sacs). Activity of these two enzymes in extracts from juice sacs was significantly greater than that form the transport tissues (vascular bundles + segment epidermis). Soluble acid invertase was the most active enzyme in extracts from all tissues of very young fruit (stage I), including nonvascular regions, but nearly disappeared prior to the onset of juice sac sugar accumulation. The physiological function of high sucrose synthase activity in the transport tissues during rapid sucrose import remains to be determined.     

AtRBOH I confers submergence tolerance and is involved in auxin-mediated signaling pathways under hypoxic stress

Satsuma mandarin fruit (Citrus unshiu Mark.) photosynthesizes as comparable to leaf at about 100 days after full bloom (DAFB). In this study, translocation and accumulation of fruit-fixed photosynthate were investigated by using ¹⁴CO₂. When fruit at 108 DAFB was exposed to ¹⁴CO₂ for 48 h under 135 photosynthetic photon flux density (PPFD), ¹⁴C-sucrose, ¹⁴C-glucose and ¹⁴C-fructose were detected not only in flavedo but juice sac; more than 50?% of fruit assimilated ¹⁴C-sugars were present in juice sac. Thus, majority of rind-fixed photosynthate are infiltrated into juice sac and accumulated there within 48 h after assimilation. Although ¹⁴C-sucrose was predominant at flavedo where high SS (sucrose synthase) activity toward synthesis was present, the amount decreased gradually from the outside (flavedo) to the inside (juice sac) of fruit. In vascular bundle, strong SS toward cleavage and soluble acid invertase activities were involved, and ¹⁴C-fructose was predominant in juice sac. Accordingly, rind-fixed photosynthate is once converted to sucrose, the translocated sugar in Citrus, at flavedo by SS toward synthesis, and loaded on vascular bundle through symplastic and/or apoplastic movement in the albedo tissue. In the vascular bundle, sucrose may be degraded by SS toward cleavage and invertase, and resulting hexoses transported symplastically to the juice sac through juice stalk.     

Sucrose synthase and invertase in isolated vascular bundles

Vascular bundles were isolated from grapefruit (Citrus paradisi Macf.) during periods of rapid sucrose translocation into fruit. Invertase and sucrose synthase activities were assayed in these strands and compared with immediately adjacent tissues (inner most peel and segment epidermis) and phloem-free juice sacs during four growing seasons. Although sucrose synthase was present in sink cells, the significantly greater activity in vascular strands (per unit fresh weight and protein) indicated that the role of this enzyme in translocation may include a vascular function in addition to its proposed involvement in metabolism of importing cells.     

Postphloem, nonvascular transfer in citrus: kinetics, metabolism, and sugar gradients

Postphloem, nonvascular assimilate transport occurs over an unusually long area in citrus fruit and thus facilitates investigation of this process relative to sugar entry into many sink structures. Labeled photosynthates moving into juice tissues of grapefruit (Citrus paradisi Macf.) slowed dramatically after entering the postphloem transport path (parenchyma cells, narrow portions of segment epidermis, and hair-like, parenchymatous stalks of juice sacs). Kinetic, metabolic, and compositional data indicated that transfer through the nonvascular area was delayed many hours by temporary storage and/or equilibration with sugars in compartments along the postphloem path. Labeled assimilates were generally recovered as sucrose throughout the path, and extent of hexose formation enroute bore no apparent relationship to the assimilate transfer process. Even after 24 hours, radiolabel was restricted to discrete, highly localized areas directly between vascular bundles and juice sacs. Postphloem transfer occurred against an ascending sucrose concentration gradient in young fruit, whereas a descending gradient (favoring diffusion/cytoplasmic streaming) developed only later in maturation. Involvement of a postphloem bulk flow is complicated in the present instance by the extremely limited water loss from juice sacs either via transpiration or fluid backflow. Nonetheless, tissue expansion can account for a collective water inflow of at least 1.0 milliliter per day throughout the majority of juice sac development, thus providing a modest, but potentially important means of nonvascular solution flow. Overall, data indicate postphloem transfer (a) can follow highly localized paths through sizable nonvascular areas (up to 3.0 centimeters total), (b) appears to involve temporary storage and/or equilibration with compartmentalized sugars enroute, (c) can occur either against an overall up-hill sugar gradient (young tissues) or along a descending gradient (near full expansion), and (d) appears to involve at least some contribution by nonvascular mass flow accommodated by tissue expansion.     

Sugar utilization by citrus juice cells as determined by [14C]-sucrose and [14C]-fructose feeding analyses

Sugar utilization by mature citrus juice cells was investigated in light of previous reports suggesting the inability of these cells to phosphorylate hexoses. Grapefruit juice sac cells were incubated in solutions of [¹⁴C]-sucrose or [¹⁴C]-fructose for 16 h during which ¹⁴CO₂ evolution was measured by trapping into soluene. After the incubation period, tissue was extracted in 5 % trichloroacetic acid or 80 % ethanol and extracts separated and identified by thin layer chromatography. Fructose was taken up and metabolized more rapidly than sucrose. In both cases, significant amounts of ¹⁴CO₂, [¹⁴C]-pyruvic and [¹⁴C]-citric acid were recovered after incubation. In separate experiments, hexokinase activity in tissue extracts was found to co-sediment with mitochondrial fractions but was not detected in the soluble fractions as previously reported. The data indicated that, contrary to earlier observations, mature citrus fruit juice cells contain the enzymatic machinery to metabolize soluble sugars. This is consistent with the glycolytic utilization of sugars in cells undergoing anaerobic respiration.     

Translocation pathways in the petioles and stem between source and sink leaves of Populus deltoides Bartr. ex Marsh.

Microautoradiography was used to follow the translocation pathways of ¹⁴C-labeled photosynthate from mature source leaves, through the stem, to immature sink leaves three nodes above. Translocation occurred in specific bundles of the midveins and petioles of both the source and sink leaves and in the interjacent internodes. When each of six major veins in the lamina of an exporting leaf was independently spot-fed ¹⁴CO₂, label was exported through specific bundles in the petiole associated with that vein. When the whole lamina of a mature source leaf was fed ¹⁴CO₂, export occurred through all bundles of the lamina, but acropetal export in the stem was confined to bundles serving certain immature sink leaves. Cross-transfer occurred within the stem via phloem bridges. Leaves approaching maturity translocated photosynthate bidirectionally in adjacent subsidiary bundles of the petiole. That is, petiolar bundles serving the lamina apex were exporting unlabeled photosynthate while those serving the lamina base were simultaneously importing labeled photosynthate. The petioles and midveins of maturing leaves were strong sinks for photosynthate, which was diverted from the export front to differentiating structural tissues. The data support the idea of bidirectional transport in adjacent bundles of the petiole and possibly in adjacent sieve tubes within an individual bundle.Abbreviations C central leaf trace - L left leaf trace - LPI leaf plastochron index - R right leaf trace     

Sugar uptake by the grape berry: A note on the absorption pathway

Summary ³H-glucose was fed to excised Sultana grape berries via their pedicels for up to 5 hours. Autoradigraphy showed that the label was distributed throughout the fruit within 1 hour. Microautoradiography of tissue sections taken at a number of points showed that within the pedicel the walls of cortical cells had become heavily labelled, suggesting that the cortical cell walls offered a diffusion pathway for the solutes entering the vascular system from the external aqueous solution. Transport along the pedicel was confined to the central vascular tissue with little radioactivity occurring in the cortical cells. Within the pericarp, the vascular bundles and walls of nearby parenchyma cells had become heavily labelled, indicating that the labelled solute was present within the vicinity of cell walls. The general pattern of ³H-glucose accumulation by excised berries was similar to the deposition pattern of ²⁴C-labelled photosynthate within attached fruit.     

Temperature and oxygen effects on C-photosynthate unloading and accumulation in developing soybean seeds

The environmental sensitivity of the processes associated with the import of photosynthate by developing soybean seeds was investigated within intact fruit and with excised, immature embryos. Intact pods of field-grown (Glycine max [L.] Merr.) Amsoy 71 soybeans were subjected to localized regimes of 0, 21, or 100% O₂ and 15, 25, or 35°C during pulsechase translocation experiments and, 2.5 hours later, the uptake and distribution of ¹⁴C-photosynthate among dissected fruit tissues determined. In other experiments, excised embryos were incubated in [¹⁴C]sucrose solutions under various experimental conditions to separate the effects of these treatments on accumulation by the embryos from those which may operate on phloem unloading in the maternal seedcoat.     

Occurrence and Structure of Epicuticular Wax on the Juice Vesicles of Citrus Fruits

The surface of the juice vesicles of some citrus fruits wasstudied with the aid of the transmission and scanning electronmicroscopes. The epidermis of the vesicles is covered with athin cuticle on top of which are found wax secretions of variousstructural patterns. It is assumed that the secreted epicuticularwax serves as an adhesive which holds the juice vesicles ineach fruit segment together.     

Distribution of imported 14C in developing leaves of eastern cottonwood according to phyllotaxy

Summary Individual leaves of eastern cottonwood (Populus deltoides Bartr.), representing an ontogenetic series from leaf plastochron index (LPI) 3.0 to 8.0, were fed ¹⁴CO₂ and harvested after 2–24 h. Importing leaves from LPI-1.0 through 8.0 on each plant were sectioned into 9 parts, and each part was quantitatively assayed for ¹⁴C activity. The highest level of ¹⁴C import was by leaves from LPI 1.0 to 3.0, irrespective of source-leaf age. ¹⁴C was translocated preferentially to either the right or left lamina-half depending on the position of the importing leaf in the phyllotactic sequence and its stage of development. For example, import was high when the importing leaf and the source leaf had two vascular bundles in common, moderately high with one bundle in common, and low with no bundles in common. The distribution of ¹⁴C within young importing leaves was highest in the lamina tip and decreased toward the base. With increasing leaf age, incorporation declined in the lamina tip and increased in the base.It may be concluded that each cottonwood leaf progresses through a continuum of importing and exporting stages as its lamina expands. The photosynthate imported by a given leaf is compartmentalized, with different exporting leaves supplying photosynthate to rather restricted regions of the lamina. Such localization within the importing leaf depends on its vascular connections with each of the exporting leaves, and these are predictable from a knowledge of the phyllotaxy.Plant Physiologists.     

Observation of water and Na+ in tissues of theBruguiera gymnorrhiza by1H-and23Na-NMR imaging

Distributions of free water, which is called water in this investigation, in mangrove (Bruguiera gymnorrhiza (L.) Lam.) tissues were examined by using¹H-NMR imaging, and accumulation of Na⁺ in hypocotyls was examined by using high resolution²³Na-NMR and²³Na-NMR imaging in relation to their morphology. Water located preferentially in the epidermis and the outer layer of cortex adjacent to the epidermis, and around vascular bundles of a root, a branch stem, and hypocotyls. Amount of water detected in the middle parts of cortex and pith was small unlikeAucuba japonica branch tissue. On the other hand, relatively high concentration of Na⁺ was detected in the pith besides the epidermis and the outer layer of the cortex adjacent to the epidermis, and around vascular bundles of the hypocotyl. The localization of Na⁺ did not correspond to that of water. Concentrations of Na⁺ accumulated (up to 22mM) in the hypocotyl were approximately 10 times higher than those observed in tissues of ordinary plants. The characteristic water distribution and accumulation of Na⁺ in the mangrove are considered to relate to their ecological nature for the adaptation to saline environments.     

Molecular cloning of a homologue of dad-1 gene in citrus: distinctive expression during fruit development

A cDNA homologue to the human defender against apoptotic death gene (dad-1), which is involved in programmed cell death, was isolated from satsuma mandarin (Citrus unshiu Marc.) fruit. It (Citdad-1-1) was 345 bp long, with a deduced protein sequence of 115 amino acids. Southern hybridization suggests that dad-1-related sequences are present as a small gene family in the citrus genome. Expression of Citdad-1-1 was progressively down-regulated in leaves as they matured, but not in juice sac/segment epidermis (edible part) towards fruit ripening. The role of dad-1 during citrus development is also discussed.     

Biosynthesis and degradation of α-tomatine in developing tomato fruits

Fruits of tomato incorporated [2-¹⁴C]mevalonic acid lactone into the steroidal glycoalkaloid α-tomatine. Young fruits showed the greatest alkaloid-synthesizing ability but this decreased as the fruits developed. Analysis of sap exuded from fruit stalks and also application of[4-¹⁴C]cholesterol to leaves confirmed that tomatine is not transported into fruits from vegetative organs. Accumulation of this alkaloid in fruits thus appears entirely due to synthesis. Excised fruits of all developmental stages degraded injected [¹⁴C]tomatine and rates were directly related to fruit age. The pattern of accumulation/decline in fruit tomatine may be explicable on the basis of changing capacity for synthesis/degradation during development. Label from injected [¹⁴C]tomatine was present mainly in chlorophylls and carotenoids where it increased with time as that in tomatine decreased. The significance of the relationship between tomatine disappearance and carotenoid development is briefly discussed. The aglycone tomatidine was not detected in green fruits but a Δ¹⁶-5α-pregnenolone-like compound was.     

Transport of Radioactivity in Relation to Bracts in the Cotton Plant

[¹⁴C]sucrose was applied to the leaf subtending 14-d-old boll(cotton fruit) with and without bracts, and to bracts alonein var. Suvin (Gossypium barbadense L.). The removal of bractssubstantially enhanced the transport of radioactivity from theleaf to the carpels and seed cotton. When bracts alone weretreated, still larger quantities of radioactivity were incorporatedin the carpels and seed cotton. The bracts appeared to nourishthe developing boll through their own photosynthate and regulatethe transport of assimilate from the leaf. Radioactivity, transport, bracts, boll, carpels, seed cotton     

红地球葡萄延后栽培成熟期水分运输研究

以3年生延后栽培的红地球葡萄为材料,在生育后期设置土壤水分处理控制在0.20(T_1)、0.25(T_2)和0.30 m~3·m~(-3)(T_3)左右,以果农常用的管理方法为对照(CK),在果实成熟期采用切片和田间树体茎流测定方法,研究果实水分运输结构变化和树体耗水状况。结果表明:(1)葡萄果实转色初期,果梗维管束的木质部结构完整且导管壁清晰可见;转色中期,果梗木质部部分导管壁变模糊,果刷中央维管束横切面积较转色前期变小;转色后期,果刷和果梗木质部导管壁模糊且破裂。(2)当葡萄果实可溶性固形物为7%时,果梗表皮细胞排列紧密且细胞间隙小,果刷维管束导管清晰可见;当可溶性固形物含量达到11%时,果刷中央维管束导管数量大于胚珠维管束,且分布不同;当可溶性固形物含量增加至15%时,果梗表皮细胞排列疏松,表皮细胞拉长,部分细胞发生破裂,果刷中央维管束和胚珠维管束的导管均出现解体。(3)9月11日左右,不同土壤水分处理(CK、T_1、T_2和T_3)茎液流达到最大值,峰值分别为3.51、3.95、4.37和4.59 d·L~(-1);葡萄转色中期,在低温影响下T_1的茎液流量整体显著低于T_2、T_3(P0.05),但与CK无显著差异;10月15日至10月26日无明显极端低温发生,不同处理树体茎液流量均呈下降趋势;11月下旬随着温度持续降低,树体各处理茎液流量迅速下降。研究发现,土壤含水量过高,低温发生时会造成树体茎液流量降低,增加土壤供水能够延缓葡萄可溶性固形物的积累,从而延缓葡萄成熟。     

根域限制对葡萄果实不同发育期维管束水分运输的影响

本文采用质外体染料示踪法研究了葡萄果实不同发育期根域限制和非根域限制（对照）处理果实维管束水分运输变化。结果表明,在葡萄果实发育的第一次快速生长期,根域限制和对照处理的果实周缘维管束被染色数量最多,染料溶液在葡萄果实中的运输速率也最高,分别为1.72和1.63cm·h-1,根域限制处理果实中周缘维管束和中央维管束中染料溶液的运输速度低于对照处理,但胚珠维管束中染料运输速度要高于对照,为1.32cm·h-1;进入生长停滞期后,根域限制和对照处理的葡萄果实中周缘维管束被染色数目降低,染料溶液在中央维管束中的运输速度分别降低为0.72和0.70cm·h-1;在果实的第二次快速生长期,根域限制和对照处理果实周缘维管束染色数目仍然低于果实发育的第一次快速生长期,根域限制果实周缘维管束染色范围和染料运输速率都高于对照处理。     

Isomerization of feruloylputrescine in orange juice by light exposure

Exposure to light may adversely affect the quality of foods. This investigation of how light exposure affects citrus (orange and mandarin blend) juice in polyethylene terephthalate (PET) bottles demonstrated that the isomeric form of a compound in the juice changed during storage. This compound was identified as feruloylputrescine (FP; CAS: 501-13-3; C₁₄H₂₀N₂O₃) using LC/MS (Q-TOF). LC/MS and NMR measurements showed that the content of the original form, trans-FP, decreased as it isomerized to cis-FP during storage. This phenomenon could be observed in citrus fruit juices containing FP, such orange and grapefruit juices. Therefore, determining the content of these two isomers of FP could be used to indicate the level of light exposure experienced by citrus fruit products.     

Influence of growth regulator treatments on dry matter production,fruit abscission,and14C-assimilate partitioning in citrus

Experiments were performed to monitor (1) uptake and translocation of foliar-applied microdroplets of¹⁴C hormones and (2) effects of multiple growth regulator sprays on foliar and fruit growth variables and photosynthate partitioning in Valencia orange trees (Citrus sinensis (L.) Osbeck). The uptake of¹⁴C-sucrose,¹⁴C-paclobutrazol (PP333), and¹⁴C-napthaleneacetic acid (NAA) in 6-month-old greenhouse-grown trees exceeded that of¹⁴C-abscisic acid (ABA) and¹⁴C-benzyladenine (BA) 48 h after microdroplet application.¹⁴C-sucrose transport from the application site was much greater than any other source, especially¹⁴C-BA. In a second study, 2-year-old Valencia orange trees were maintained under field conditions and were sprayed to foliar runoff (3 × /week for 3 weeks) with BA, NAA, ABA, PP333, and gibberellic acid (GA₃) at 100 M during flowering and early fruit set. Select branches were then briefly exposed to¹⁴CO₂ and harvested 24 h later. Both GA₃ and BA sprays promoted foliar growth. BA also stimulated fruit growth, whereas GA₃ sharply increased fruit dry weight while fruit number decreased. BA and GA₃ enhanced¹⁴C assimilate export by the foliage to the developing fruit, and GA₃ was especially active in promoting fruit sink intensity (¹⁴C/dry wt). The other compounds (NAA, ABA, PP333) restricted foliar and fruit growth. They also inhibited transport of¹⁴C assimilate from the leaves to the fruit. Results indicate that foliar-applied growth regulators can influence source-sink relations in citrus early in reproductive development by manipulating photoassimilate production and partitioning.