As overall invertases activities were considerably higher than SuSy activities, and assuming that the extractable activities of these enzymes in the total protein extracts paralleled their in vivo activities, our results would suggest that invertases provided the major route for Suc breakdown, as suggested in peach and tomato . Moreover, the reduction in invertase activities in both cultivars throughout the ripening-related stages, in agreement with the increase in transcript levels of INVINH was also observed in peach underlining the key role invertases play in determining overall fruit sugar composition . During ripening, Glu and Fru contents were higher in Santa Rosa fruits . Although VINV activity could be contributing to the higher hexose amounts in Santa Rosa fruits, the enhanced Sor breakdown in Santa Rosa fruits was a significant source of Glu and Fru in Santa Rosa. This conclusion is supported by the higher NAD+-SDH and SOX enzymatic activities and NAD+-SDH transcripts, and lower NAD+ levels in Santa Rosa fruits and by the report showing that NAD+-SDH is the key enzyme determining Fru concentrations in peach fruits . An additional observation supporting the above described scenario comes from both the HK transcript levels and HK enzyme activity. HK, which phosphorylates Glu into G6P, the precursor of Sor, displayed both lower activity and low HK transcript levels in Santa Rosa than in Sweet Miriam fruits . In apples, a decreased HK activity was observed throughout ripening, stacking flower pot tower suggesting a lower glucose metabolism similar to what was observed in Santa Rosa fruits.A noteworthy difference between Santa Rosa and Sweet Miriam fruits was their altered UDP-Gal metabolism.

These differences were indicated by the higher levels of the polyols Gol and Ino and the oligosaccharide Raf, and the lower contents of Gal in Sweet Miriam . UDP-Gal can be targeted towards cell wall biosynthesis through galactosyltransferases , can be used as a substrate for Suc synthesis through itsinterconversion to UDP-Glu , or can be used as a substrate for Raf biosynthesis via GolS . Our results suggested that in Santa Rosa, UDP-Gal was targeted towards Gal, while in Sweet Miriam it was targeted towards Raf . In Santa Rosa, these results were probably a consequence of the increased cleavage of galactosyl residues from cell wall polysaccharides through β-GAL, especially during the fruit softening stage , and increased cleavage of Raf into Gal and Suc through α-GAL . In Sweet Miriam fruits and leaves, UDP-Gal seemed to be targeted towards Raf biosynthesis via increased GolS and RS transcript levels, as overall Raf contents were higher in Sweet Miriam . In addition, Ino, a substrate for GolS and released by RS , provides a cycle which was overall increased in Sweet Miriam as compared with Santa Rosa . What might be the roles of the elevated contents of Gal in Santa Rosa and Gol, Raf, and Ino in Sweet Miriam? Free Gal, higher in Santa Rosa fruits, has been shown to increase ethylene production and induce earlier ripening in mature green tomatoes . This promotion of ripening due to the Gal-induced increase in ethylene production has been associated with the stimulation of 1-aminocyclopropane-1-carboxylic acid synthase activity, the rate-limiting step in ethylene biosynthesis, as well as to a transient increase in 1-aminocyclopropane-1-carboxylic acid . Thus, the higher levels of Gal in Santa Rosa  would suggest a link with the climacteric behavior of this cultivar, that could be further explored. Regarding Gol, Raf, and Ino, several reports have indicated that these metabolites are associated with protection against stresses due to their high antioxidant capacities . The fruit ripening process comprises a series of oxidative activities , specifically during cell wall breakdown and overall fruit softening .

Gol and Raf were dramatically elevated in peach fruits exposed to heat and cold stresses during post-harvest storage . Therefore, it is possible that the higher contents of Gol, Ino, and Raf in Sweet Miriam fruits improve their ability to cope with the oxidative processes occurring during ripening, as suggested by Aizat et al. in non-climacteric Capsicum. During ripening in post-harvest storage, these compounds were also higher in Sweet Miriam fruits, supporting their role as oxidative stress protectants . In addition to Gol, Ino, and Raf, Sweet Miriam fruits and leaves also displayed higher Tre contents, that were well correlated with lower TRE transcript levels . Tre has also been reported to play signaling/regulatory roles in plant stress responses , suggesting that the increase in Tre contents in Sweet Miriam could also contribute to cope with ripening-associated oxidative stress conditions. While in our previous publication we concentrated on characterizing sugar contents and some of their corresponding enzymes using two phenological stages and only in fruits, here we used a systems biology approach, combing gene expression, metabolomics, and biochemical analyses to show a reprograming of metabolism of major and minor sugars occurring in fruits and leaves of a non-climacteric bud mutant plum cultivar at four developmental stages. Non-climacteric plums accumulated higher amounts of Sor and lower amounts of Suc, Glu, and Fru than climacteric plums, and the higher amounts of Sor were a consequence of both increased synthesis, mediated by S6PDH, and decreased breakdown, mediated by NAD+-SDH and SOX. The non-climacteric behavior was also associated with a shift of UDP-Gal metabolism towards Raf and Gol, as well as the increase in Tre, probably playing a role in improving the overall ability of non-climacteric fruits to cope with oxidative processes associated with fruit ripening.

The lower Gal contents in Sweet Miriam could also play a role in its non-climacteric behavior due to the reported capacity of free Gal to induce ethylene production through stimulating ACS activity. Whether the differences in ethylene and ripening behavior between the two cultivars are also dependent on changes of other hormones is currently under investigation.Most flowering plants are perfect-flowered hermaphrodites , but hermaphrodites are not necessarily equisexual . Instead, pollen and ovule counts reveal continuous variation from predominantly male to predominantly female gamete production, both among plants in a population and among the flowers produced by an individual . This variation invites the question, why should one hermaphroditic individual be more or less female than another? Evolutionary stable strategy models indentify several factors that together determine a plant’s optimal sex allocation . These models find the sex allocation strategy that, if adopted by all members of the population, cannot be invaded by an alternative, initially rare, strategy. Factors affecting sex allocation optima include the shapes of male and female gain curves , the strength of competition among related pollen grains and related seeds, and the rate of self-fertilization . When the environment is assumed to be homogenous, ESS models find a single optimum for individuals in a population. If, however, the environment is heterogeneous, sex allocation optima may differ among plants for two reasons. First, environmental heterogeneity can alter the shape of gain curves. In wind-pollinated species, for example, the male gain curve may decelerate more rapidly in closed habitatsthan in open habitats . Second, environmental heterogeneity can induce variation in plant size, which may alter the shape of gain curves , and/or lead to variation in the size of reproductive investment made. Plants making a larger absolute investment are expected to allocate relatively more to the function whose gain curve decelerates slower . Optima can also differ among sequentially opening flowers on a plant. Like individuals, danish trolley flowers that differ in reproductive investment will vary in sex allocation optima if male and female gain curves are not identical . Variation in sex allocation optima also occurs when flowers vary in selfing rates: flowers with a higher selfing rate are expected to allocate more to female function than those with a lower selfing rate when inbreeding depression , 0.5; the prediction is reversed when d . 0.5 . The strongest driver of among-flower variation in allocation optima, however, is among-flower variation in pollen transfer probability. This can be caused by temporal shifts in the mating environment arising from flower dichogamy . Other causes of variation in pollen transfer probability include the tendency of pollinators to visit inflorescences from bottom-to-top , and varying pollinator attraction as display size changes . All else being equal, flowers with the greatest pollen transfer probability are expected to allocate relatively more to male function . Empirical study of sex allocation variation in hermaphroditic plants has focused largely on systems exhibiting dichogamy, or, to a lesserextent, pollinator directionality or variable selfing rates . Less attention has been paid to adichogamous, self-incompatible species lacking pollinator directionality, even though sex allocation variation can occur in such species if plants or flowers vary in reproductive investment .

Moreover, a particular type of variation in investment – declining fruit-set from first to last flowers on plants – might cause variation in the mating environment of adichogamous plants by altering the quality of siring opportunity over time. In their ESS model, Brunet & Charlesworth assumed a constant probability of fruit-set across all flowers, despite among-flower variation in reproductive investment. Fruit-set probability, however, frequently declines from first to last flowers on plants , and this decline is often attributed to resource pre-emption by first flowers . This post-fertilization decline in resources clearly lowers the expected female reproductive success of last flowers on plants. Less obvious are the effects on expected male mating opportunity. If first flowers are more likely to set fruit, then those flowers and plants that transfer pollen to the first flowers of others are more likely to fertilize ovules that successfully mature into seed. This siring advantage should fall to first flowers on plants, and to early-flowering plants in a population, because these are more likely to temporally coincide with the first flowers of others. Brunet recognized that declining fruit-set reduces the expected male success of last flowers on plants, and Weis and Kossler predicted that declining fruit-set leads to decreasing male success from early- to late- flowering plants . The consequences of this temporal shift in mate quality for relative mating success through the two genders have not, however, been fully examined. We asked the following questions. Does functional gender vary within plants when fruit-set declines? Does the predicted among-plant decline in male success cause appreciable variation in functional gender? Are within- and among-plant trends in expected functional gender matched by within- and among-plant trends in relative male and female investment? To answer these questions, we developed a numerical model examining effects of declining fruit-set probability on expected male success and functional gender. Informed by model results, we characterized within- and among-plant variation in pollen, ovule and fruit production in Brassica rapa . We contrast temporal trends in relative male and female investment in B. rapa to temporal trends in functional gender predicted by the model.The numerical model confirmed that declining fruit-set probability from first to last flowers on plants can, under a wide range of conditions, induce temporal variation in expected male success and functional femaleness . In most cases examined, average functional femaleness decreased sharply in the last flower class on plants. In contrast, at the among-plant level, functional femaleness increased substantially from early- to late-flowering plants in all cases where fruit-set declined . Temporal trends in phenotypic femaleness in B. rapa opposed expected trends in functional femaleness: phenotypic femaleness increased from first to last flowers on plants , and decreased from early- to late-flowering plants . Below, we discuss the numerical model in relation to previous ESS models, and examine the discrepancy between predicted trends in functional femaleness and observed trends in phenotypic femaleness.Variation in the mating environment of sequentially opening flowers on plants can lead to variation in their sex allocation optima . Effects of dichogamy are particularly well studied. Brunet and Charlesworth reasoned that dichogamy creates a temporal shift in the quantity of male- versus female-phase flowers , and therefore generates a temporal trend in total expected pollen transfer probability from first to last flowers on plants. This shift in the mating environment causes variation in sex allocation optima . This ESS model prediction is well supported by data from several dichogamous species . Dichogamy was not, however, the only scenario examined in this ESS model. Brunet and Charlesworth also reasoned that when resources decline from first to last flowers on plants, first flowers offer a higher quality siring opportunity than last flowers .