In apple fruit, sucrose as well as sorbitol enters the parenchyma cells via the apoplastic pathway after being released from SE-CC complex. In many species that employ apoplastic unloading for sucrose in sink cells, sucrose is mainly converted to glucose and fructose by CWINV in the cell wall space and then transported into the parenchyma cells by hexose transporters. CWINV is typically considered as a sink-specific enzyme and its activity is usually very low in source leaves. However, we found that, except for MdCWINV3 in 40- DAB fruit, the expression of MdCWINVs was much lower in the fruit than in the shoot tips where sucrose unloading is symplastic. In yeast cells expressing apple SOTs, sorbitol uptake is competitively inhibited by glucose and fructose but not by sucrose. So we postulate that most sucrose is directly transported into the parenchyma cells by plasma membrane-bound SUCs in apple fruit to avoid inhibition of sorbitol uptake by sucrose-derived glucose and fructose. Increased sucrose import into transgenic fruit did not alter the activity of CWINV but significantly elevated the transcript levels of both MdSUC1 and MdSUC4 , indicating that more sucrose is taken up into the parenchyma cells in the transgenic fruit. There are two pathways for sucrose breakdown in the cytosol of fruit parenchyma cells: conversion to fructose and glucose by NINV or to fructose and UDP-glucose by SUSY . The upregulation of transcript levels of MdNINV1, MdNINV3, and MdSUSY1-3 and activities of NINV and SUSY in the transgenic fruit , which is indicative of higher availability of sucrose in the cytosol, generates more fructose. This, combined with a lower FK2 transcript level and a lower FK activity , plastic pots large makes enough fructose available in the cytosol for accumulation in the vacuole of the transgenic fruit to largely compensate for the reduced level of sorbitolderived fructose.
The higher NINV activity is also expected to elevate the glucose level in the cytosol, which may have led to higher transcript levels of MdHKs and a higher HK activity through glucose signaling and a higher dark respiration rate in the transgenic fruit . The higher HK activity detected in the transgenic fruit is similar to that of rice leaves in response to glucose manipulation. However, we found that increases in both HK activity and the glucose concentration did not enhance, but rather diminished, the accumulation of G6P in the transgenic fruit. This is likely due to a decrease in F6P flux from phosphorylation of fructose along with an increase in dark respiration such that more G6P was reversibly converted to F6P. Our result is consistent with the finding that glucose derived from sucrose contributes to the hexose phosphate pool more than fructose derived from sorbitol or sucrose in the apple fruit. Despite a higher glucose flux going through dark respiration in the transgenic fruit, more glucose is still available for transport into vacuole for accumulation as indicated by the 3–6-fold increase in glucose concentration in the transgenic fruit at harvest. Higher fruit glucose levels have also been reported for these anti-sense plants by Teo et al. but to a lesser degree. It is interesting that greater import of sucrose did not significantly increase its concentration in transgenic fruit except at 74 DAB. We think that two factors may have contributed to this near homeostasis of sucrose in the transgenic fruit. First, upregulation of sucrose breakdown described above uses more sucrose. Second, downregulation of MdSPS3 and MdSPS6 transcript levels and SPS activity in the transgenic fruit makes less sucrose re-synthesized from F6P and UDP-glucose.
While upregulation of SUSY in response to increased sucrose supply was observed in both fruit and shoot tips of the transgenic plants, NINV responded in the transgenic fruit but not in the shoot tips. The exact reason for this difference is not known, but differences in sucrose concentration and/or presence of different isoforms of NINV between fruit parenchyma cells and shoot tips might exist. Our findings on activities of SUSY, NINV, FK, HK, and SPS are not in agreement with those reported by Teo et al.. We believe that the discrepancy might be related to the difference in the way fruit samples were taken. In our study, it took about 2 min to cut and freeze fruit samples on site in the orchard, but in Teo et al.all harvested fruits were placed on ice before being transported to the laboratory and it was only after several quality indices were measured that the cortical tissues were frozen for further analysis. Because import of sorbitol and sucrose into fruit stops upon detachment from the tree, both enzyme activity and gene expression may be altered if they are not frozen in liquid nitrogen in a very short period of time. In addition, strict cropload CK in our study as reflected in much larger fruit might have made the difference between the transgenic fruit and the CK easier to be detected. Most of the hexoses and sucrose in fruit parenchyma cells are stored in the central vacuole that occupies >80% of the cell volume. Transcript levels of MdvGT1 and MdvGT2, both of which are vacuolar glucose transporters encoded by two Malus orthologs of AtvGT, were higher in the transgenic fruit than in the CK , suggesting that more glucose is transported into the vacuole of the transgenic fruit. This is consistent with the glucose concentration measured on bulk fruit samples .
In addition, tonoplast monosaccharide transporters can transport both glucose and fructose into the vacuoles, and Arabidopsis TMT1 activity for fructose is approximately 30% of that for glucose. In the five Malus orthologs of TMT, it is possible that proteins encoded by MdTMT1 and/or MdTMT2 have high ability to transport fructose, and the enhanced expression by MdTMT1 in the transgenic fruit might indicate a regulatory response to the reduced flux of fructose derived from sorbitol. Alternatively, as fructose-specific TMTs have not been identified in fructose-accumulating fleshy fruits, the upregulation of MdTMT1 could be triggered by higher levels of glucose derived from sucrose in the transgenic fruit. In addition to hexoses, the vacuoles in ripening apple fruit accumulate a high concentration of sucrose. So far, no SUC has been identified to have proton-coupled anti-port activity for loading sucrose into the vacuole, but AtTMT1/2 probably represents a proton-coupled anti-porter capable of transporting both glucose and sucrose into the vacuole. A recent report on TMTs in sugar beet indicates that one of the two TMT2 proteins has developed specific affinity to sucrose and is responsible for sucrose accumulation in the taproots. The expression patterns of both MdTMT1 and MdTMT2 are in general agreement with that of sucrose accumulation in our apple fruit. It has been demonstrated that interruption of carbohydrate import into fruit by girdling or adjustment of cropload did not alter the fructose level in apple fruit. Contrasting light exposure did not appear to affect peel fructose level either. The data obtained from the transgenic fruit in this study provides further evidence for supporting the idea that the Sucrose cycle and the associated transport system operates to maintain the homeostasis of fructose in the apple fruit. From an evolutionary perspective, having fructose homeostasis in the apple fruit may help seed dispersal for this species because fructose is the sweetest among all the soluble sugars present in fleshy fruits. In conclusion, when sorbitol synthesis is decreased by anti-sense suppression of A6PR in the source leaves of apple trees, less sorbitol but more sucrose is transported from the leaves to the fruit. In response to the lower sorbitol/higher sucrose supply, sorbitol metabolism is downregulated, whereas breakdown of sucrose is upregulated in the transgenic fruit to compensate for the decreased flux of fructose derived from sorbitol. This altered sugar metabolism, together with corresponding changes in the sugar transport system, black plastic nursery pots leads to near homeostasis of fructose and sucrose and much higher levels of glucose and galactose in the transgenic fruit. This study clearly demonstrates the metabolic flexibility and the advantages of having two transport carbohydrates in sorbitol-synthesizing Rosaceae tree fruit species and the central role of the Sucrose cycle and the sugar transport system in determining sugar metabolism and accumulation in fleshy fruits.The global fruit market has grown appreciably in the past decade. The absence of seeds from fruit that is consumed has become one of the most appreciated traits by consumers. Reducing seed content without changing the size of the fruit is one of the main objectives of the cultivation of many fruit trees. The goal is to enhance the experience of fruit consumption by consumers and improve the quality of fruits for food processing . Sugar apple is a tropical fruit species in the family Annonaceae. Seedless fruits have been described for some spontaneous mutants of A. squamosa whose origin is still unknown .
They include the Cuban cultivar Cuban seedless , with good fruit characteristics but lower productivity than fertile cultivars ; Brazilian seedless originally identified in northeast Brazil, which produces small, asymmetric fruits that frequently perish ; the Thai seedless mutant that produces normal size fruits among other types with apparently similar fruits, such as in the Philippines and Hawaii . Lora et al. were among the first researchers to examine details of the seedless trait in A. squamosa in Ts. This variety produces fruit following pollination and fertilization. The authors demonstrated that seedlessness results from a deffect in ovule development where the outer of the two integuments sheathing the nucellus fails to form. This deffect directly mirrors the effect of INNER NO OUTER loss of function mutants in Arabidopsis thaliana . INO encodes a putative transcription factor belonging to the YABBY family. Members of this family are involved in the determination of abaxial identity in a variety of plant organs . Lora et al. isolated an A. squamosa INO ortholog and demonstrated the association of the Ts mutant with an apparent deletion of the INO gene, indicating a candidate gene for the seedless trait. The Bs variety was also evaluated in a breeding program undertaken at the State University of Montes Claros. Results showed that the absence of seeds was also associated with failure in the development of the external integument of the ovule and in the development of seeds, similar to that observed in Ts . In addition, preliminary studies of inheritance of the stenospermocarpic absence of seeds involving F1 progenies in Bs also indicated the same expected recessive nature for the mutation as in Ts . However, it was not known whether the molecular basis of the INO deletion described is widespread in A. squamosa and may be responsible for the case of aspermia described in Bs. In Arabidopsis ino mutants, the absence of the outer integument leads to failure in development of the embryo sac so that the ovules do not attract pollen tubes and degenerate . In contrast, Lora et al. found that the embryo sac fully formed in the Ts line, and was successfully fertilized, but this fertilization did not lead to the formation of seed. Santos et al. went further in analysis of Bs and showed that 72 h after pollination the embryos formed, but after seven days a degeneration of embryos and endosperm was observed, forming the sterile aborted seed with a whitish color and smooth consistency. It has been hypothesized that the more robust ovules of A. squamosa, with a much thicker inner integument than is found in Arabidopsis, can better support the development of the embryo sac in the absence of the outer integument and that this is the reason for the further embryo sac development in A. squamosa . The resulting initial stage of seed formation triggers the initiation of fruit development, explaining the stenospermocarpy. The present study addressed the following questions: Is the inheritance of the presence/absence of seeds in A. squamosa monogenic?; What are the molecular details of the INO gene deletion?; Is there a complete correspondence of homozygosity for the deletion with the seedless phenotype; What is the relationship between the known seedless accessions, and Can codominant molecular markers specific to INO be designed for use in assisted selection?Wild-types M1, M2 and M3 and seedless Bs lines were previously described by de Souza et al. , and Ts was described by Lora et al. .