Delayed Leaf Senescence by Upregulation of Cytokinin Biosynthesis Specifically in Tomato Roots.

Cytokinins (CKs) regulate numerous plant developmental processes, including photosynthesis and leaf senescence. Isopentenyltransferase (IPT) is a rate-limiting enzyme in the CK-biosynthesis pathway. We overexpressed ipt under tissue-specific promoters to study the long-range effect of CK on the functioning of tomato source leaves. Photosynthetic activity over time provided the measure for leaf aging. Significantly delayed leaf senescence was observed in plants expressing ipt under a root-specific promoter, but not in those expressing the gene under a source leaf-specific promoter. The root-derived influence on leaf aging was further confirmed by grafting experiments. CK concentration in source leaves of both transgenic lines increased significantly, with different proportions of its various derivatives. On the other hand, root CK concentration was only slightly elevated. Nevertheless, the significant change in the proportion of CK derivatives in the root indicated that CK biosynthesis and metabolism were altered. Partial leaf defoliation upregulates photosynthetic rate in the remaining leaf; however, overexpression of ipt in either tissues eliminated this response. Interestingly, stem girdling also eliminated the photosynthetic response. Taken together, our findings suggest that leaf senescence is regulated by a CK-mediated root-shoot communication network. We propose that CK-mediated signal is translocated to the leaf via the xylem where it alters CK biosynthesis, resulting in delayed senescence.

Long-distance regulation of shoot gravitropism by Cyclophilin 1 in tomato (Solanum lycopersicum) plants.

MAIN CONCLUSION: The phloem-mobile protein SlCyp1 traffics to distant parts of the shoot to regulate its gravitropic response. In addition, SlCyp1 targets specific cells in the root to promote lateral root development. The tomato (Solanum lycopersicum) Cyclophilin 1 (SlCyp1) gene encodes a peptidyl-prolyl isomerase required for auxin response, lateral root development and gravitropic growth. The SlCyp1 protein is a phloem-mobile signal that moves from shoot to root to regulate lateral root development (Spiegelman et al., Plant J 83:853-863, 2015; J Exp Bot 68:953-964, 2017a). Here, we explored the mechanism of SlCyp1 movement by fusing it to the fluorescent protein mCherry. We found that, once trafficked to the root, SlCyp1 is unloaded from the phloem to the surrounding tissues, including the pericycle and lateral root primordia. Interestingly, SlCyp1 not only moves to the root system, but also to distant parts of the shoot. Grafting of the SlCyp1 mutant diageotropica (dgt) scions on VFN8 control rootstocks resulted in recovery of dgt shoot gravitropism, which was associated with the restoration of auxin-response capacity. Application of the cyclophilin inhibitor cyclosporine A suppressed gravitropic recovery, indicating that SlCyp1 must be active in the target tissue to affect the gravitropic response. These results provide new insights on the mechanism of SlCyp1 transport and functioning as a long-distance signal regulating shoot gravitropism.

Upregulation of photosynthesis in mineral nutrition-deficient tomato plants by reduced source-to-sink ratio.

Photosynthetic activity is affected by environmental factors and endogenous signals controlled by the source-sink relationship. We recently showed upregulated photosynthetic rate following partial defoliation under favorable environmental conditions. Here, we examined the influence of partial defoliation on the remaining leaves' function in tomato plants under nutrient deficiency. The effect of partial defoliation was more pronounced under limited mineral supply vs. favorable conditions. Reduced source-sink ratio resulted in increased stomatal conductance and transpiration rate, as well as higher photosystem II efficiency. Although chlorophyll concentration was significantly reduced under limited nutrient supply, the photosynthetic rate in the remaining leaf was similar to that measured under normal fertilization. Expression of genes involved in the phloem loading of assimilated sugars was downregulated in the remaining source leaf of unfertilized plants, 15 d after partial defoliation; in fertilized plants, these genes' expression was similar in control and partially defoliated plants. We propose that at early stage, the additional carbon assimilated in the remaining leaf is devoted to increasing source size rather than sink growth. The size increase of the remaining leaf in unfertilized plants was not sufficient to rebalance the source-sink ratio, resulting in inhibited sugar export and further carbohydrate allocation in the remaining leaf.

Root-shoot communication in tomato plants: cytokinin as a signal molecule modulating leaf photosynthetic activity.

Photosynthetic activity is affected by exogenous and endogenous inputs, including source-sink balance. Reducing the source to sink ratio by partial defoliation or heavy shading resulted in significant elevation of the photosynthetic rate in the remaining leaf of tomato plants within 3 d. The remaining leaf turned deep green, and its area increased by almost 3-fold within 7 d. Analyses of photosynthetic activity established up-regulation due to increased carbon fixation activity in the remaining leaf, rather than due to altered water balance. Moreover, senescence of the remaining leaf was significantly inhibited. As expected, carbohydrate concentration was lower in the remaining leaf than in the control leaves; however, expression of genes involved in sucrose export was significantly lower. These results suggest that the accumulated fixed carbohydrates were primarily devoted to increasing the size of the remaining leaf. Detailed analyses of the cytokinin content indicated that partial defoliation alters cytokinin biosynthesis in the roots, resulting in a higher concentration of trans-zeatin riboside, the major xylem-translocated molecule, and a higher concentration of total cytokinin in the remaining leaf. Together, our findings suggest that trans-zeatin riboside acts as a signal molecule that traffics from the root to the remaining leaf to alter gene expression and elevate photosynthetic activity.

Down-regulation of SlCyp1 in the phloem reduces auxin response and photosynthetic rate in tomato (Solanum lycopersicum) plants.

The tomato dgt mutant, containing a single mutation in the Cyclophilin1 (SlCyp1) gene, is auxin insensitive and exhibits a pleotropic phenotype that includes lack of lateral roots, malformed xylem structure and reduced root-to-shoot ratio. Recently, we found that the SlCyp1 protein is phloem-mobile and traffic from shoot to root to induce lateral root formation. These processes are achieved through activation of auxin-mediated developmental programs. Inhibition of the trafficked SlCyp1 activity at the target site resulted in inhibition of the auxin response, supporting the hypothesis that this protein is indeed a mobile signal. Here, we show that partial silencing of SlCyp1 in the phloem only resulted in perturbed auxin response in the roots and reduced photosynthetic and transpiration rates. The presented data suggests that expression of SlCyp1 in the phloem is essential for proper auxin response at the whole plant level. We, therefore, propose that this protein acts as a long-distance signaling molecule acting as coordinator between roots and shoot activities.

Secondary Effects of Glyphosate Action in Phelipanche aegyptiaca: Inhibition of Solute Transport from the Host Plant to the Parasite.

It is currently held that glyphosate efficiently controls the obligate holoparasite Phelipanche aegyptiaca (Egyptian broomrape) by inhibiting its endogenous shikimate pathway, thereby causing a deficiency in aromatic amino acids (AAA). While there is no argument regarding the shikimate pathway being the primary site of the herbicide's action, the fact that the parasite receives a constant supply of nutrients, including proteins and amino acids, from the host does not fit with an AAA deficiency. This apparent contradiction implies that glyphosate mechanism of action in P. aegyptiaca is probably more complex and does not end with the inhibition of the AAA biosynthetic pathway alone. A possible explanation would lie in a limitation of the translocation of solutes from the host as a secondary effect. We examined the following hypotheses: (a) glyphosate does not affects P. aegyptiaca during its independent phase and (b) glyphosate has a secondary effect on the ability of P. aegyptiaca to attract nutrients, limiting the translocation to the parasite. By using a glyphosate-resistant host plant expressing the "phloem-mobile" green fluorescent protein (GFP), it was shown that glyphosate interacts specifically with P. aegyptiaca, initiating a deceleration of GFP translocation to the parasite within 24 h of treatment. Additionally, changes in the entire sugars profile (together with that of other metabolites) of P. aegyptiaca were induced by glyphosate. In addition, glyphosate did not impair germination or seedling development of P. aegyptiaca but begun to exert its action only after the parasite has established a connection to the host vascular system and became exposed to the herbicide. Our findings thus indicate that glyphosate does indeed have a secondary effect in P. aegyptiaca, probably as a consequence of its primary target inhibition-via inhibition of the translocation of phloem-mobile solutes to the parasite, as was simulated by the mobile GFP. The observed disruption in the metabolism of major sugars that are abundant in P. aegyptiaca within 48 h after glyphosate treatment provides a possible explanation for this inhibition of translocation and might reflect a critical secondary effect of the herbicide's primary action that results in loss of the parasite's superior sink for solutes.

Function of Cyclophilin1 as a long-distance signal molecule in the phloem of tomato plants.

Tomato (Solanum lycopersicum) diageotropica (dgt) mutants, containing a single mutation in the Cyclophilin1 (SlCyp1) gene, are auxin-insensitive, exhibiting a pleiotropic phenotype including lack of geotropism, abnormal xylem structure, lack of lateral roots (LRs), and elevated shoot-to-root ratio. SlCyp1 is a putative peptidyl-prolyl isomerase that can traffic from shoot to root, where it induces changes in auxin response, LR formation, and xylem development, suggesting it has a role as a long-distance signaling molecule. Here, we explored the mechanism underlying SlCyp1 function in the phloem. Expression of SlCyp1 under a phloem-specific (AtSuc2) promoter in dgt plants partially restored the wild-type phenotype, including lateral root development, root branching, and xylem morphology. The observed developmental changes were associated with physiological alternations at the whole-plant level, including a reduction in shoot-to-root ratio, enhanced transpiration, and elevated photosynthetic rates. Conversely, phloem-specific expression of SlCyp1 active-site mutants did not restore the wild-type phenotype. Local inhibition of cyclophilin functioning in the target tissue reduced auxin sensitivity, suggesting that its enzymatic activity in the distant organ is required for its action as a long-distance signalling agent. The data presented suggest that SlCyp1 is a signal molecule trafficking from shoot to root where its activity is required for auxin-mediated lateral root development.

Mechanism of glyphosate control of Phelipanche aegyptiaca.

MAIN CONCLUSION: Despite its total reliance on its host plant, the holoparasite Phelipanche aegyptiaca suffers from a deficiency of aromatic amino acids upon exposure to glyphosate. The herbicide glyphosate inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), a key enzyme in the biosynthesis of aromatic amino acids. However, the functionality of the EPSPS pathway in the obligate root holoparasite Phelipanche aegyptiaca is not straightforward because of the parasite's total dependence on the host plant. Despite the importance of glyphosate as a means of controlling P. aegyptiaca, the mechanism of action of the herbicide in this parasite is not clearly understood. We characterized glyphosate control of P. aegyptiaca by using a glyphosate-resistant tomato (GRT) genotype as the host plant and evaluating the activity of EPSPS and the levels of free aromatic amino acids in the parasite. The viability of the parasite's tissues deteriorated within the first 40 h after treatment (HAT) with glyphosate. In parallel, shikimate accumulation in the parasite was first detected at 24 HAT and increased over time. However, shikimate levels in the GRT host did not increase, indicating that the host was indeed glyphosate tolerant. Free phenylalanine and tyrosine levels decreased by 48 HAT in the parasite, indicating a deficiency of aromatic amino acids. The use of GRT as the host enabled us to observe, in an in situ experimental system, both endogenous EPSPS inhibition and a deficiency of aromatic amino acids in the parasite. We thus provided evidence for the presence of an active EPSPS and aromatic amino acid biosynthesis pathway in P. aegyptiaca and pinpointed this pathway as the target of glyphosate action in this parasite.

A tomato phloem-mobile protein regulates the shoot-to-root ratio by mediating the auxin response in distant organs.

The plant vascular system serves as a conduit for delivery of both nutrients and signaling molecules to various distantly located organs. The anucleate sieve tube system of the angiosperm phloem delivers sugars and amino acids to developing organs, and has recently been shown to contain a unique population of RNA and proteins. Grafting studies have established that a number of these macromolecules are capable of moving long distances between tissues, thus providing support for operation of a phloem-mediated inter-organ communication network. Currently, our knowledge of the roles played by such phloem-borne macromolecules is in its infancy. Here, we show that, in tomato, translocation of a phloem-mobile cyclophilin, SlCyp1, from a wild-type scion into a mutant rootstock results in restoration of vascular development and lateral root initiation. This process occurs through reactivation of auxin response pathways and reprogramming of the root transcriptome. Moreover, we show that long-distance trafficking of SlCyp1 is associated with regulation of the shoot-to-root ratio in response to changing light intensities, by modulating root growth. We conclude that long-distance trafficking of SlCyp1 acts as a rheostat to control the shoot-to-root ratio, by mediating root development to integrate photosynthesis and light intensity with requirements for access to water and mineral nutrients.

Don't kill the messenger: Long-distance trafficking of mRNA molecules.

The phloem sap contains numerous macromolecules such as proteins and RNAs, in addition to photoassimilates, amino acids and other small molecules. The transcription profile of messenger RNA (mRNA) molecules in the sieve tubes is unique and does not reflect the transcript profile in the neighboring companion cells. This discovery suggests tight regulation on cell-to-cell movement of mRNA molecules from the companion cells into the sieve tube. Heterografting experiments and RNA-detection methods have provided unequivocal evidence for the trafficking of several specific mRNA molecules between distant organs. Detection of various plant transcripts in their respective plant parasites further confirms this long-distance movement. The finding that several of these trafficked transcripts are involved in the control of developmental processes as well as responses to growth substances or environmental cues has led to a new paradigm that mRNA molecules act as non-cell-autonomous signaling agents operating in the vascular system. Trafficking of these molecules creates a communication network between distant organs that is required for coordinated development of the whole plant under adverse conditions. The generality of this concept, however, is still under debate, because the raison d'être for long-distance movement of mRNA is not clear. In this review we discuss the identity and potential function of phloem-sap mRNA molecules, the factors facilitating RNA transport, and the rationale for their action as long-distance signaling agents in the control of developmental processes.

Phloem-specific expression of a melon Aux/IAA in tomato plants alters auxin sensitivity and plant development.

Phloem sap contains a large repertoire of macromolecules in addition to sugars, amino acids, growth substances and ions. The transcription profile of melon phloem sap contains over 1000 mRNA molecules, most of them associated with signal transduction, transcriptional control, and stress and defense responses. Heterografting experiments have established the long-distance trafficking of numerous mRNA molecules. Interestingly, several trafficking transcripts are involved in the auxin response, including two molecules coding for auxin/indole acetic acid (Aux/IAA). To further explore the biological role of the melon Aux/IAA transcript CmF-308 in the vascular tissue, a cassette containing the coding sequence of this gene under a phloem-specific promoter was introduced into tomato plants. The number of lateral roots was significantly higher in transgenic plants expressing CmF-308 under the AtSUC2 promoter than in controls. A similar effect on root development was obtained after transient expression of CmF-308 in source leaves of N. benthamiana plants. An auxin-response assay showed that CmF-308-transgenic roots are more sensitive to auxin than control roots. In addition to the altered root development, phloem-specific expression of CmF-308 resulted in shorter plants, a higher number of lateral shoots and delayed flowering, a phenotype resembling reduced apical dominance. In contrast to the root response, cotyledons of the transgenic plants were less sensitive to auxin than control cotyledons. The reduced auxin sensitivity in the shoot tissue was confirmed by lower relative expression of several Aux/IAA genes in leaves and an increase in the relative expression of a cytokinin-response regulator, TRR8/9b. The accumulated data suggest that expression of Aux/IAA in the phloem modifies auxin sensitivity in a tissue-specific manner, thereby altering plant development.

Phloem-mobile Aux/IAA transcripts target to the root tip and modify root architecture.

In plants, the phloem is the component of the vascular system that delivers nutrients and transmits signals from mature leaves to developing sink tissues. Recent studies have identified proteins, mRNA, and small RNA within the phloem sap of several plant species. It is now of considerable interest to elucidate the biological functions of these potential long-distance signal agents, to further our understanding of how plants coordinate their developmental programs at the whole-plant level. In this study, we developed a strategy for the functional analysis of phloem-mobile mRNA by focusing on IAA transcripts, whose mobility has previously been reported in melon (Cucumis melo cv. Hale's Best Jumbo). Indoleacetic acid (IAA) proteins are key transcriptional regulators of auxin signaling, and are involved in a broad range of developmental processes including root development. We used a combination of vasculature-enriched sampling and hetero-grafting techniques to identify IAA18 and IAA28 as phloem-mobile transcripts in the model plant Arabidopsis thaliana. Micro-grafting experiments were used to confirm that these IAA transcripts, which are generated in vascular tissues of mature leaves, are then transported into the root system where they negatively regulate lateral root formation. Based on these findings, we present a model in which auxin distribution, in combination with phloem-mobile Aux/IAA transcripts, can determine the sites of auxin action.

Effect of CMV infection and high temperatures on the enzymes involved in raffinose family oligosaccharide biosynthesis in melon plants.

Ultrastructural and molecular studies have provided experimental evidence for the classification of cucurbits as symplastic loaders, mainly translocating the raffinose family oligosaccharides (RFOs) raffinose and stachyose. Earlier studies established that cucumber mosaic virus (CMV) infection causes a significant increase in the sucrose-to-RFO ratio in the phloem sap of melon plants. The alteration in phloem sap sugar composition was associated with upregulation of CmSUT1 transcript within the vascular bundles. The current research aimed to explore the effect of CMV infection on the enzymes involved in symplastic phloem loading and RFO biosynthesis. Viral infection did not affect the activity of either raffinose or stachyose synthases in source leaves, but caused upregulation of the respective transcripts. Interestingly, activity of galactinol synthase was higher in CMV-infected leaves, associated with upregulation of CmGAS2. A significant increase in CmGAS2 expression in source leaves of melon plants exposed to high temperatures indicated that this response is common for both biotic and abiotic stresses. However, the effect of CMV or heat stress on phloem sap sugar composition is not due to alteration in RFO biosynthesis.

Movement of protein and macromolecules between host plants and the parasitic weed Phelipanche aegyptiaca Pers.

Little is known about the translocation of proteins and other macromolecules from a host plant to the parasitic weed Phelipanche spp. Long-distance movement of proteins between host and parasite was explored using transgenic tomato plants expressing green fluorescent protein (GFP) in their companion cells. We further used fluorescent probes of differing molecular weights to trace vascular continuity between the host plant and the parasite. Accumulation of GFP was observed in the central vascular bundle of leaves and in the root phloem of transgenic tomato plants expressing GFP under the regulation of AtSUC2 promoter. When transgenic tomato plants expressing GFP were parasitized with P. aegyptiaca, extensive GFP was translocated from the host phloem to the parasite phloem and accumulated in both Phelipanche tubercles and shoots. No movement of GFP to the parasite was observed when tobacco plants expressing GFP targeted to the ER were parasitized with P. aegyptiaca. Experiments using fluorescent probes of differing molecular weights to trace vascular continuity between the host plant and the parasite demonstrated that Phelipanche absorbs dextrans up to 70 kDa in size from the host and that this movement can be bi-directional. In the present study, we prove for the first time delivery of proteins from host to the parasitic weed P. aegyptiaca via phloem connections, providing information for developing parasite resistance strategies.

Sucrose transporter plays a role in phloem loading in CMV-infected melon plants that are defined as symplastic loaders.

Based on the high density of plasmodesmata interconnecting the intermediary cells and their neighboring phloem parenchyma or bundle-sheath cells, and based on the insensitivity to the sucrose transport inhibitor p-chloromercuribenzenesulfonic acid (PCMBS), cucurbits have been concluded to be symplastic loaders. In the present study, we identified and characterized the full-length sequence of sucrose transporter gene (CmSUT1) from melon (Cucumis melo L. cv. Hale's best jumbo). In vitro experiments confirmed that the identified gene product has sucrose transporter activity in baker's yeast. Healthy and cucumber mosaic virus (CMV)-infected melon plants were employed to examine sucrose transporter activity in planta. Pretreatment with PCMBS inhibited loading of newly fixed ¹4CO2 into minor veins of CMV-infected plants. Moreover, CMV infection caused significant increase in CmSUT1 transcripts expression, mainly in vascular bundles of minor veins, which was associated with elevated sucrose content in phloem sap collected from source-leaf petioles. We propose that cucurbit plants contain the machinery for apoplastic phloem loading and that CMV infection causes a quantitative shift in the mode by which photoassimilates are loaded into the sieve tube.

Molecular computing with plant cell phenotype serving as quality controlled output.

The ability of autonomous biomolecular computing devices to interact directly with biological systems and even with living organisms without any interface represents their main advantage over the electronic computers. This study shows that the expression of fluorescent proteins in live plant cells can be utilized as a highly accurate visual output of DNA-based computing. Each of the two possible outputs of a 2-symbol 2-state finite automaton was represented here by either green or cyan fluorescence in eukaryotic cells. The automata were programmed by the choice of several molecules from a library of 8 transition molecules, each containing a recognition site for a type II endonuclease. Two enzymes, endonuclease and a DNA ligase, as well as ATP, represented the hardware. Each input molecule, in the form of a dsDNA, included a string of symbols, 6 bp each, and a 6 bp terminator. The two detection molecules were also dsDNA, each containing a 4-base sticky end, complementary to the appropriately restricted terminator and a gene encoding for a different fluorescent protein. Computation was carried out by mixing all components in a homogeneous solution, leading to autonomous processing of the input molecule via repetitive cycles of digestion, hybridization, and ligation. The output processing procedure involved the creation of a circular dsDNA that contained the gene of either green fluorescent protein or cyan fluorescent protein. Insertion of these plasmids into onion cells by particle bombardment resulted in either green fluorescent or cyan fluorescent live cells as phenotypical output signals. The plasmid formation was an important step because it served as a quality control gate that transformed a rather noisy output into a clean signal. This process of noise elimination allowed for clean and flawless outputs with high fidelity and zero noise.

Melon phloem-sap proteome: developmental control and response to viral infection.

In addition to small molecules such as sugars and amino acids, phloem sap contains macromolecules, including mRNA and proteins. It is generally assumed that all molecules in the phloem sap are on the move from source to sink, but recent evidence suggests that the macromolecules' direction of movement can be controlled by endogenous plant mechanisms. To test the hypothesis that the phloem-sap protein profile is affected by local metabolic activities, we analyzed the phloem-sap proteome in young and mature tissues of melon plants. We also examined the effect of cucumber mosaic virus (CMV) infection and expression of CMV movement protein in transgenic melon plants on the phloem protein profile. Sap collected from cut sections of young stems or petioles contained specific proteins that were absent from sap collected from mature stems or petioles. Most of these proteins were involved in defense response and protection from oxidative stress, suggesting that they play a role in maintaining safe activity of the sieve tubes in young tissues. Phloem sap collected from CMV-infected plants and transgenic plants expressing the CMV movement protein contained only a few additional proteins with molecular masses of 18 to 75 kDa. Here again, most of the additional proteins were associated with stress responses. Our study indicated that the proteome of phloem sap is dynamic and under developmental control. Entry and exit of proteins from the sieve tube can be regulated at the tissue level. Moreover, the plant can maintain regulation of protein trafficking from companion cells to sieve elements under viral infection or other perturbations in plasmodesmal function.

An orange ripening mutant links plastid NAD(P)H dehydrogenase complex activity to central and specialized metabolism during tomato fruit maturation.

In higher plants, the plastidial NADH dehydrogenase (Ndh) complex supports nonphotochemical electron fluxes from stromal electron donors to plastoquinones. Ndh functions in chloroplasts are not clearly established; however, its activity was linked to the prevention of the overreduction of stroma, especially under stress conditions. Here, we show by the characterization of Orr(Ds), a dominant transposon-tagged tomato (Solanum lycopersicum) mutant deficient in the NDH-M subunit, that this complex is also essential for the fruit ripening process. Alteration to the NDH complex in fruit changed the climacteric, ripening-associated metabolites and transcripts as well as fruit shelf life. Metabolic processes in chromoplasts of ripening tomato fruit were affected in Orr(Ds), as mutant fruit were yellow-orange and accumulated substantially less total carotenoids, mainly beta-carotene and lutein. The changes in carotenoids were largely influenced by environmental conditions and accompanied by modifications in levels of other fruit antioxidants, namely, flavonoids and tocopherols. In contrast with the pigmentation phenotype in mature mutant fruit, Orr(Ds) leaves and green fruits did not display a visible phenotype but exhibited reduced Ndh complex quantity and activity. This study therefore paves the way for further studies on the role of electron transport and redox reactions in the regulation of fruit ripening and its associated metabolism.

Internal management of non-structural carbohydrate resources in apple leaves and branch wood under a broad range of sink and source manipulations.

Apple (Malus x domestica Borkh.) grown in a Mediterranean climate depends on regular irrigation throughout the growing season. The objective of the current study was to elucidate the changes in carbohydrate storage and utilization by mature, field-grown apple trees in response to water availability to the trees and to the level of cropping. Fourteen-year-old apple trees cv. 'Golden Delicious' were grown under various combinations of irrigation rate (11, 33 or 77 l day(-)(1) per tree) and crop level ( approximately 100, approximately 300 or >1000 fruits per tree) beginning 47 days after full bloom (DAFB). Non-structural carbohydrate concentrations were measured at 78 (leaves and branch wood), 102 (leaves), 183 (branch wood) and 214 (branch wood) DAFB. Midday stem water potential (SWP) was measured at 2-week intervals between June and October. Trunk cross-sectional area was measured 47 and 265 DAFB. At harvest, 139 DAFB, the fruits of each tree were counted and weighed. SWP at 102 DAFB ranged between -0.6 and -2.7 MPa. Fruit fresh weight at harvest was positively related to SWP measured 37 days before harvest with distinct slopes for light/intermediate and heavy crop levels. Leaf and branch wood starch concentrations 78 and 102 DAFB were positively related to irrigation rate and negatively related to crop level. Mean fruit weight at harvest was positively related to branch wood starch concentration and neared maximum at a concentration of 40 mg g(-)(1) dry weight. Branch wood starch concentration recovered after harvest, especially in water-stressed trees. Sorbitol concentration was negatively related to irrigation rate. The sorbitol-to-starch concentration ratio in leaves at 102 DAFB was closely proportional to SWP. It is suggested that branch wood starch concentration represents the overall balance between carbon sources and sinks and may therefore serve as a reliable indicator of photo-assimilate availability. In water-stressed trees, sorbitol is prioritized over starch, probably to support osmotic adjustment, thereby suppressing fruit growth even further.

Sucrose accumulation in watermelon fruits: genetic variation and biochemical analysis.

Sugar accumulation, the key process determining fruit quality, is controlled by both the translocation of sugars and their metabolism in developing fruits. Sugar composition in watermelon, as in all cucurbit fruits, includes sucrose, fructose and glucose. The proportions of these three sugars are determined primarily by three enzyme families: invertases, sucrose synthases (SuSys) and sucrose phosphate synthases (SPSs). The goal of the present research was to explore the process of sugar metabolism in watermelon fruits. Crosses between the domestic watermelon (Citrullus lanatus) and three wild species provided a wide germplasm to explore genetic variability in sugar composition and metabolism. This survey demonstrated great genetic variability in sugar content and in the proportions of sucrose, glucose and fructose in mature fruits. Genotypes accumulating high and low percentage of sucrose provided an experimental system to study sugar metabolism in developing fruits. Insoluble invertase activity was high and constant throughout fruit development in control lines and in genotypes accumulating low levels of sucrose, while in genotypes accumulating high levels of sucrose, activity declined sharply 4 weeks after pollination. Soluble acid invertase activity was significantly lower in genotypes accumulating high levels of sucrose than in low-sucrose-accumulating genotypes. Conversely, activities of SuSy and SPS were higher in the high-sucrose-accumulating genotypes. The present results establish that, within the genus Citrullus, there are genotypes that accumulate a high percentage of sucrose in the fruit, while others accumulate high percentages of glucose and fructose. The significant negative correlation between insoluble invertase activity and fruit sucrose level suggests that sucrose accumulation is affected by both phloem unloading and sugar metabolism.