Fungicides and pesticides were sprayed at regular intervals throughout the growing season

In addition, as we strictly controlled cropload to a similar level each year by thinning flowers and young fruits, dry matter accumulation was not drastically different between the transgenic lines and the CK in our experiment.In response to a decreased sorbitol supply from source leaves, both the transcript level and the activity of SDH decreased in the transgenic fruit, which is consistent with previous findings in apple fruit cortex tissues fed with sorbitol and in shoot tips and fruit of the transgenic trees. As most of the fructose in apple fruit is converted from sorbitol by SDH, a significantly lower fructose level had been predicted in the transgenic fruit based on dramatically reduced import of sorbitol into the transgenic fruit and the associated lower SDH activity. However, the fructose level in the transgenic fruit was remarkably similar to that in the untransformed CK: no difference before 74 DAB and at harvest with only a slight difference detected at rapid fruit expansion between the transgenic fruit and the CK . This near homeostasis of fructose level in the transgenic fruit has clearly resulted from the response of the Sucrose cycle and the associated sugar transport system to increased availability of sucrose in the transgenic fruit, specifically, more sucrose is taken up into parenchyma cells in fruit after phloem unloading; more fructose is generated from sucrose breakdown by NINV and sucrose synthase and less fructose is phosphorylated by FK in the cytosol; and tonoplast sugar transporters are upregulated to take up more hexoses into the vacuole. 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,draining plant pots 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 , 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 antisense 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.

The uptake of these sugars into the vacuole is carried out by sugar transporters located on the tonoplast. 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,drainage gutter 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 antiport activity for loading sucrose into the vacuole, but AtTMT1/2 probably represents a proton-coupled antiporter 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 antisense 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 down regulated, 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, 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.Five-year-old trees of the untransformed CK and transgenic lines of “Greensleeves” apple with antisense suppression of A6PR expression were used. A6PR activity in mature leaves of A27 and A04 was decreased to about 30% and 15% of that of CK, respectively. All trees were grafted onto M.26 root stocks and grown outdoors at Ithaca, NY, USA, under natural conditions, in 55-L plastic pots containing a sand:MetroMix 360 medium . There were five replicates for each genotype with three trees each arranged in a completely randomized design. The trees were trained as a spindle system at a density of 1.5 × 3.5 m2 . They were moved into a screen house for the entire bloom period to prevent pollen escape and hand-pollinated using mixed pollen of crab apple and several commercial varieties. The cropload of these trees was adjusted by hand-thinning to four fruits per cm2 trunk cross-sectional area at 10-mm king fruit size. During the growing season, the trees were supplied twice weekly with 15 mM N using Plantex® NPK with micro-nutrients .At 40 DAB , 74 DAB , 108 DAB , and 134 DAB , fruits were sampled from the south side of the tree canopy between 12 noon and 2:00 P.M. under full sun exposure. On each sampling date, five replicates per genotype with at least six fruits each from three trees were harvested. The fruits were immediately weighed, cut into small pieces after removing the core, and frozen in liquid nitrogen on-site. The entire process took approximately 2 min. To estimate the levels of transport carbohydrates, five replicates of leaf petioles and fruit pedicels with four each were covered with aluminum foil for 7 days prior to sampling on 75 DAB and were frozen in liquid nitrogen along with mature leaves. All samples were stored at −80 °C.Net CO2 assimilation rates of bourse shoot leaves were measured using a CIRAS-1 portable photosynthesis system with a broad leaf chamber at 5 tree developmental stages from 30 DAB to fruit harvest. On each sampling date, two bourse shoot leaves per replicate were measured at mid-day under full sun exposure and ambient temperature and relative humidity conditions. Fruit respiration was measured with the CIRAS-1 gas exchange system connected to a custom-made chamber, which accommodated the entire fruit for each sample. In the early afternoon on each of those four sampling dates, fruits under full sun exposure were detached from the trees and dark-adapted at 25 °C for 30 min before taking respiration measurements, and one fruit per replicate was measured.Although a large literature describes how recessions affect non-agricultural labor markets, few studies examine the effects of recessions in the seasonal agricultural labor market.