These results suggest that molecular events triggering the low invertase activity were not fully reversed by feeding sucrose and might be responsible for the incomplete recovery from abortion with fed sucrose and thus the key to abortion itself. Moreover, in leaves and roots of young maize, Pelleschi et al. (1997), Kim et al. (2000), and Trouverie et al. (2003) report that invertase activity increased at low w. This marked contrast with the ovaries implies that the signal to abort development might be lacking (or undetected) in leaves and roots, most of which survive at low w, and present in ovaries, which abort.
Given these differences, possible changes in gene expression become of interest. In the maize leaves and roots at low w, increased invertase activity was accompanied by increases in mRNA abundance of Ivr2, a soluble acid invertase gene (Kim et al., 2000; Trouverie et al., 2003). By contrast, in maize ovaries, low activities of soluble and cell wall-bound acid invertases (Zinselmeier et al., 1995b, 1999; Andersen et al., 2002) were accompanied by lower mRNA abundance for Ivr2 and Incw2 (Andersen et al., 2002). This correlation between enzyme activity and mRNA abundance is consistent with the contrasting development between leaves and ovaries at low w. If the lower invertase activity triggers abortion, these results indicate that regulation occurs at least in part at the transcriptional level.
Nevertheless, the rescue of the ovaries by feeding sugar to the parent plant suggests that these transcriptional changes are either inconsequential for abortion or are responding to the sucrose. Koch (1996) and Sheen et al. (1999) reviewed the genes known to be sugar-responsive, and some invertases are in that group. Soluble acid invertase Ivr2 was reported to be up-regulated in response to increasing sugar levels, for example. Although the sugar-responsiveness for Incw2 and Ivr2 was not measured in the studies of Kim et al. (2000), Trouverie et al. (2003), or Andersen et al. (2002), the latter authors observed that lower Incw2 and Ivr2 mRNA levels corresponded to higher sucrose concentrations in the ovaries. They suggested that the invertases might respond to the quality of the sugar pool rather than its concentration, or possibly to other undetermined regulatory factors at low w. By contrast, working with leaves, Kim et al. (2000) reported increased sugar concentrations when the message increased for Ivr2, and Trouverie et al. (2003) could increase the mRNA for this gene by feeding abscisic acid to cut leaves. In view of the problems of working with bulk ovaries described above, further work seems necessary before the regulatory significance of these changes is understood.
Some evidence also supports a role for post-transcriptional regulation. A peptide in maize can act as an invertase inhibitor (Jaynes and Nelson, 1971; Bate et al., 2004) by binding non-competitively to acid invertase at low sucrose concentrations (about 1–2 mM in tobacco (Nicotiana tabacum L.) (Weil et al., 1994). When invertase is extracted without sucrose, it becomes susceptible to the inhibitor in vitro (Jaynes and Nelson, 1971; Pressey, 1967). It is conceivable that low sugar concentrations in vivo might have the same effect. Because the inhibitor is present in maize (Jaynes and Nelson, 1971; Bate et al., 2004), its action could prevent a close correlation between transcriptional activity and the in vivo activity of the enzyme in some tissues or treatments.
It may be important that low w also affects cell division. Setter and Flannigan (2001) found decreased transcript abundance for genes encoding enzymes for cell proliferation and endoreduplication in maize endosperm when the plants were subjected to low w a few days after pollination. DNA synthesis was markedly reduced, suggesting that cell division was inhibited. If abortion involves an irreversible block in ovary development, decreased cell division would be expected and might involve down-regulation of cell-cycle genes at the transcriptional level.
Zinselmeier et al. (2002) used cDNA microarrays to measure differential gene expression in maize ovaries and pedicel tissues when low w occurred around the time of pollination. More than 1500 genes representing 27 regulatory and metabolic pathways were investigated. Of these, 1–3% showed differential expression 4 d after moderate water stress was imposed, with some genes down-regulated and others up-regulated. A greater number of genes were differentially expressed as the stress intensified. Interestingly, more genes showed differential expression in the pedicel tissues than in the ovaries themselves. Zinselmeier et al. (2002) hypothesized that this difference might be due to a hydraulic isolation of the ovary from the rest of the plant, buffering it against large changes in w. However, measurements of ovary w in water-deficient plants indicate that ovary water status responds directly to changes in plant water status (Westgate and Boyer, 1986a; Westgate and Thomson Grant, 1989; Zinselmeier et al., 1999). Relatively small changes in ovary w can have a large impact on kernel set.
In a parallel study, Zinselmeier et al. (2002) observed a ‘coordinate down-regulation of genes associated with starch synthesis’ in ovaries on well-watered plants covered with 95% shade cloth during silk emergence. The decrease in gene expression was substantial and consistent with the dramatic decrease in ovary starch content and inhibition of ovary growth caused by the shade treatment. But in contrast to the shade results, the expression profiles at low w showed no evidence of altered gene expression for starch synthesis.
Yu and Setter (2003) conducted a similar study but withheld water from plants 5–9 d after pollination. Gene expression profiles from endosperm and pedicel tissues showed the water deficit affected 79 genes in the pedicel and 56 in the endosperm. Most were up-regulated in the pedicel but down-regulated in the endosperm. Curiously, there were no changes in expression of starch synthesis genes reported in either tissue. Like Zinselmeier et al. (2002), these authors attribute the differences between the tissues to the hydraulic isolation of the endosperm from the pedicel, but suggest in addition that they may be related to differential ABA concentrations.
Both of these microarray studies involved sampling tissues after the stresses were imposed or relieved. In one situation, this was after moderate or severe water deficit developed (Zinselmeier et al., 2002), and in the other, kernels were harvested during severe stress and 3 d after rewatering (Yu and Setter, 2003). In view of the transient expression of many genes and limited lifetime of their mRNA (Seki et al., 2002), these sampling times might have missed the critical changes in gene expression that respond to changes in the sugar stream and trigger abortion. A larger number of sampling times would help with this problem. For example, it might be expected that changes in genes associated with rapid growth would be affected before senescence genes at low w. Sampling at only one or two times would be likely to miss this difference.
As with the invertase activities and gene expression, these results with microarrays are difficult to reconcile with those of the sucrose feeding experiments. Because 40–80% of the ovaries could be rescued by feeding sucrose to the parent at low w, the changes in gene expression must have been inconsequential for the rescued ovaries or reversed by sucrose feeding. It should be noted that feeding sucrose to the stems did not change the w of the parent or the ovaries. The plants experienced the same low w whether they were fed or not. Therefore, any changes in gene expression caused by low w should have persisted in the fed plants, unless they responded to the ingredients being fed.
If the genes responded to the feeding, more genes would be considered sugar-responsive than are now known (Koch, 1996; Sheen et al., 1999). The differential patterns would represent massive metabolic responses to a lack of substrate at low w. These are not starvation responses because the up-regulated genes in the microarrays suggest that there were active transcriptional responses to low w. This argues against abortion as a passive starvation response, as proposed by Zinselmeier et al. (1999).
Many passive responses are observed at the enzyme level at low w (Todd, 1972; Kramer and Boyer, 1995) and are not necessarily caused directly by the low w. Rather, many appear to be caused by changed availability of substrates, cofactors, or other features of the chemical environment immediately surrounding the enzymes (Kramer and Boyer, 1995). The chemical environment could be altered by water loss from the cells (Potter and Boyer, 1973; Kaiser, 1987) or depleted because of decreased photosynthesis (Westgate and Boyer, 1985; Boyle et al., 1991), or compartmentalized differently because of distorted or ruptured membranes (Fellows and Boyer, 1976, 1978), or other local effects (Shaner and Boyer, 1976; Huang et al., 1975; Pankhurst and Sprent, 1975). As a consequence, no single factor can account for the changed activities, and the control of activity often involves alterations in the particular cell environment that each enzyme requires, in addition to any transcriptional and translational regulation.
Likewise for gene expression, some of the microarray results might develop from the physiological changes occurring at low w. Some genes might respond passively while others are actively regulated by promoters or other features of the genome. A critical experiment will be to determine the effect of sucrose feeding on gene expression at low w. Those genes responding to the feeding would be implicated in ovary rescue from abortion. The others responding to low w but not sucrose would probably be passive or not limiting to ovary development.