Effect of increased temperature in apical regions of maize ears on starch-synthesis enzymes and accumulation of sugars and starch

Apical florets of maize (Zea mays L.) ears differentiate later than basal florets and form kernels which have lower dry matter accumulation rates. The purpose of this study was to determine whether increasing the temperature of apical kernels during the dry matter accumulation period would alter the difference in growth rate between apical and basal kernels. Apical regions of field-grown maize (cultivar Cornell 175) ears were heated to 25 ± 3°C from 7 days after pollination to maturity (tip-heated ears) and compared with unheated ears (control). In controls, apical-kernel endosperm had 24% smaller dry weight at maturity, lower concentration of sucrose, and lower activity of ADP-Glc starch synthase than basal-kernel endosperm, whereas ADP-Glc-pyrophosphorylase (ADPG-PPase) activities were similar. In tip-heated ears apical-kernel endosperm had the same growth rate and final weight as basal-kernel endosperm and apical kernels had higher sucrose concentrations, higher ADP-Glc starch synthase activity, and similar ADPG-PPase activity. Total grain weight per ear was not increased by tip-heating because the increase in size of apical kernels was partially offset by a slight decrease in size of the basal- and middle-position kernels. Tip-heating hastened some of the developmental events in apical kernels. ADPG-PPase and ADP-Glc starch synthase activities reached peak levels and starch concentration began rising earlier in apical kernels. However, tip-heating did not shorten the period of starch accumulation in apical kernels. The results indicate that the lower growth rate and smaller size of apical kernels are not solely determined by differences in prepollination floret development.     

Localization of sucrose synthase activity in developing maize kernels by in situ enzyme histochemistry

Sucrose synthase is usually localized by immunocytochemistry, but this method does not show the actual activity of the localized enzyme. A histochemical assay is presented here showing the activity of sucrose synthase by tetrazolium salt precipitation on sections of developing maize kernels. The advantages of the assay are a high sensitivity for low amounts of active sucrose synthase and the independence of specific antibodies.In this study the activity of endosperm sucrose synthase is shown to move gradually from the apical part of the endosperm to the basal endosperm during kernel development. This shift in sucrose synthase activity correlates well with the localization of starch synthesis during kernel development. The assay also shows the early loss of activity in the aleurone layer bordering the embryo, and a loss of activity in the apical aleurone during the final stage of kernel development while the enzyme was still found by immunocytochemistry. This is in contrast to a high sucrose synthase activity in the epithelium of the scutellum, where hardly any labelling was found with antibodies against maize sucrose synthase. Low sucrose synthase activities were found in the pericarp and pedicel parenchyma.Possible functions of the high and low activity patterns in the developing maize kernels and differences between the enzyme assay and immunocytochemistry are discussed.     

Relationship Between Photosynthate Supply and Endosperm Development in Maize

Maize (Zea mays L., cultivar Pioneer 3925) plants were givenshaded, thinned and control light treatments during 10 d or20 d periods surrounding pollination. Glucose, sucrose, starch,and dry matter (DM) contents were measured at intervals in compositesamples of pericarp/nucellus (PN), and in endosperms taken fromdeveloping kernels. Total kernel DM per ear at maturity washigher in the thinned treatment than control and shaded treatmentsdue to higher kernel set in apical regions of ears. In PNs at11 d after pollination (DAP), DM and sucrose contents were slightlygreater in thinned than control and shaded plants. Glucose contentswere substantially greater than controls in PNs of thinned plantsand were less than controls in shaded plants. In endospermsfrom apical kernels at 8 to 12 DAP (during cell division), DM,glucose and sucrose contents were substantially less in shadedthan control and thinned plants. Sucrose contents were greaterin endosperms of thinned than control plants. Sugar contentsin endosperms from basal kernels were nearly the same in thethree light treatments. At 12 DAP, apical and basal endospermsin shaded plants had fewer nuclei than those of the other lighttreatments. The light treatments appeared to effect apical kernelgrowth by influencing the extent of cell division. Zea mays L, maize, light treatment, endosperm, cell division, glucose, sucrose, starch     

Sucrose and Nitrogen Supplies Regulate Growth of Maize Kernels

The growth of maize (Zea mays L.) kernels depends on the availabilityof carbon (C) and nitrogen (N) assimilates supplied by the motherplant and the capacity of the kernel to use them. Our objectiveswere to study the effects of N and sucrose supply levels ongrowth and metabolism of maize kernels. Kernel explants of Pioneer34RO6 were culturedin vitro with varying combinations of N (5to 30 m M) and sucrose (117 to 467 m M). Maximum kernel growthwas obtained with 10 m M N and 292 m M sucrose in the medium,and a deficiency of one assimilate could not be overcome bya sufficiency of the other. Increasing the N supply led to increasesin the kernel sink capacity (number of cells and starch granulesin the endosperm), activity of certain enzymes (soluble andbound invertases, sucrose synthase, and aspartate aminotransaminase),starch, and the levels of N compounds (total-N, soluble protein,and free amino acids), and decreased the levels of C metabolites(sucrose and reducing sugars). Conversely, increasing the sucrosesupply increased the level of endosperm C metabolites, freeamino acids, and ADPG-PPase and alanine transaminase activities,but decreased the activity of soluble invertase and concentrationsof soluble protein and total-N. Thus, while C and N are interdependentand essential for accumulation of maximum kernel weight, theyappear to regulate growth by different means. Nitrogen supplyaids the establishment of kernel sink capacity, and promotesactivity of enzymes relating to sucrose and nitrogen uptake,while sucrose regulates the activities of invertase and ADPG-PPase.Copyright 1999 Annals of Botany Company Zea mays, maize,, invertase, ADPG-PPase, media composition, sucrose, nitrogen, C/N.     

Heat stress effects on sink activity of developing maize kernels grown in vitro

The objective of this study was to determine the effect of short-term (4 days) and long-term (8 days) heat stress (35°C) on sink activity of maize (Zea mays L.) kernels. Beginning at 3 days after pollination (DAP) kernels were grown in vitro at 25°C and 24 h later were transferred to 35°C for either 4 or 8 days. Each treatment had a control that was maintained continously at 25°C. Two experiments were designed to examine the uptake and distribution of ¹⁴C among hexoses, sucrose and starch in the pedicel placento-chalazal (pedicel/p-c). endosperm, and pericarp tissues of kernels exposed to heat stress for 4 or 8 days. Kernels cultured in vitro were placed in ¹⁴C-sucrose medium either during the period of heat stress (experiment 1; 5 to 13 DAP) or immediately following heat-stress treatments (experiment 2; 10 to 22 DAP). In both experiments no significant effect of heat stress was observed on the total radioactivity accumulated in the kernels until about 17 DAP, after which heat-stressed kernels accumulated less ¹⁴C than the control. During the linear fill period, the endosperm of kernels exposed to heat stress accumulated more radioactivity associated with hexoses and sucrose and less radioactivity incorporated into starch, as compared to the control. Kernels heat stressed for 4 days showed a partial recovery in starch synthesis by 21 DAP, but to levels of only 65% of that of the control. Kernels heat stressed for 8 days did not recover. When ¹⁴C-sucrose was supplied during the heat stress period (5–13 DAP). kernels from all treatments accumulated more hexoses that sucrose in the pedicel/p-c. However, during the period following heat stress (10–22 DAP), pedicel/p-c accumulated sucrose, but only in kernels exposed to long-term heat stress. Soluble invertase activity was inhibited by both short-term and long-term heat stress, whereas the activity of insoluble invertase was affected only by long-term heat stress. These results support the hypothesis that the disruption of kernel growth and more particularly endosperm starch biosynthesis, in response to heat stress, is mainly associated with changes in carbon utilization and partitioning between the different nonstructural carbohydrates within the endosperm rather than with a limitation in carbon supply to the kernel. Therefore, the effect on sink activity does not seem to be attributable to a thermal disruption of kernel uptake of sugars, but rather it is a consequence of heat perturbation of other physiological processes such as endosperm sugar metabolism and starch biosynthesis.     

Enzymes of sucrose and hexose metabolism in developing kernels of two inbreds of maize

Tissue distribution and activity of enzymes involved in sucrose and hexose metabolism were examined in kernels of two inbreds of maize (Zea mays L.) at progressive stages of development. Levels of sugars and starch were also quantitated throughout development. Enzyme activities studied were: ATP-linked fructokinase, UTP-linked fructokinase, ATP-linked glucokinase, sucrose synthase, UDP-Glc pyrophosphorylase, UDP-Glc dehydrogenase, PPi-linked phosphofructokinase, ATP-linked phosphofructokinase, NAD-dependent sorbitol dehydrogenase, NADP-dependent 6-P-gluconate dehydrogenase, NADP-dependent Glc-6-P dehydrogenase, aldolase, phosphoglucoisomerase, and phosphoglucomutase. Distribution of invertase activity was examined histochemically. Hexokinase and ATP-linked phosphofructokinase activities were the lowest among these enzymes and it is likely that these enzymes may regulate the utilization of sucrose in developing maize kernels. Most of the hexokinase activity was found in the endosperm, but the embryo had high activity on a dry weight basis. The endosperm, which stores primarily starch, contained high PPi-linked phosphofructokinase and low ATP-linked phosphofructokinase activities, whereas the embryo, which stores primarily lipids, had much higher ATP-linked phosphofructokinase activity than did the endosperm. It is suggested that PPi required by UDP-Glc pyrophosphorylase and PPi-linked phosphofructokinase in the endosperm may be supplied by starch synthesis. Sorbitol dehydrogenase activity was largely restricted to the endosperm, whereas 6-P-gluconate and Glc-6-P dehydrogenase activities were highest in the base and pericarp. A possible metabolic pathway by which sucrose is converted into starch is proposed.     

Distribution of enzyme activities within the developing maize (Zea mays) kernel in relation to starch, oil and protein accumulation

The association of enzyme activities in developing kernels with specific storage product accumulation at maturity was analyzed in different parts of Zea mays inbred OH43 kernels. Maize kernels were harvested at 20 and 55 days post-pollination and dissected into basal region, pericarp, embryo, lower endosperm, middle endosperm and upper endosperm. Mature (55 days pos(-pollination) kernel parts were analyzed for starch, total protein, zein and oil content. Immature (20 days post-pollination) kernel parts were assayed for activities of 15 enzymes of sugar and amino acid metabolism. Statistical analyses of the data suggested that glucokinase (EC 2.7.1.2) fructokinase (EC 2.7.1.4) and phosphofructokinase (EC 2.7.1.1 11) activities were primarily associated with oil accumulation, whereas ADP'-glueose pyrophosphorylasc (EC 2.7.7.27) and sucrose synthase (EC 2.4.1.13) activities were associated with starch accumulation. The results suggest that oil biosynthesis utilizes inveitase-mediated sucrose degradation in a pathway not requiring pyrophosphatc. whereas starch biosynthesis utilizes a sucrose synthase-mediated pathway of sucrose degradation in a pathway requiring pyrophosphatc. Additional groups of enzyme activities were associated with each oilier but not with any specific storage product and appeared to be associated with general metabolic activity.     

Nitrogen-induced changes in the growth and metabolism of developing maize kernels grown in vitro

Cereal kernel growth and grain yield are functions of endosperm starch accumulation. The objective of this study was to examine how various metabolic factors in developing maize (Zea mays L.) endosperm influence starch deposition. Kernels were grown in vitro on medium with: (a) zero N (−N), (b) optimum N (+N), or (c) −N from 3 to 20 days after pollination followed by +N until maturity (±N) to produce different degrees of endosperm growth and to promote an enhancement of starch synthesis midway through development. At intervals, kernels were harvested and levels of enzyme activities and carbohydrate and N constituents examined. Endosperm starch and protein accumulation were decreased in −N compared to +N kernels, but relief of N starvation increased both constituents. With greater movement of N into ±N kernels, endosperm sugar concentrations declined suggesting an inverse relationship between C and N transport. Unusually high concentrations of sugar in N stressed kernels did not appear to limit or enhance starch production. Rather, increased accumulation of starch in ±N endosperm was correlated with significant increases in the enzymatic activities of sucrose synthase and PPi-linked phosphofructokinase, and to a lessor extent hexokinase. In addition, the occurrence of specific proteins of the albumin/globulin fraction either increased, decreased, or remained unchanged in relation to starch synthesis. These data suggest that lack of N limits starch deposition in maize endosperm primarily through an influence on synthesis of key proteins.     

Sucrose synthase activity in developing wheat endosperms differing in maximum weight

Past research on kernel growth in wheat (Triticum aestivum) has shown that the kernel itself largely regulates the influx of sucrose for consequent starch synthesis in the endosperm of the grain. The first step in the conversion of sucrose to starch is catalyzed by sucrose synthase (EC 2.4.13). Sucrose synthase activity was assayed in developing endosperms from kernels differing in growth rate and in maximum dry weight accumulation. From 10 to 22 days after anthesis, sucrose synthase activity per wheat endosperm remained constant with respect to time in all grains. However, kernels which had higher rates of kernel growth and which achieved greatest maximum weight had consistently and significantly higher sucrose synthase activities at any point in time than did kernels with slower rates of dry matter accumulation and lower maximum weight. In addition, larger kernels had a significantly greater amount of water in which this activity could be expressed. Although the results do not implicate sucrose synthase as the “rate limiting” enzyme in wheat kernel growth, they do emphasize the importance of sucrose synthase activity in larger or more rapidly growing kernels, as compared to smaller slower growing kernels.     

Evidence for independent genetic control of the multiple forms of maize endosperm branching enzymes and starch synthases

Soluble starch synthase and starch-branching enzymes in extracts from kernels of four maize genotypes were compared. Extracts from normal (nonmutant) maize were found to contain two starch synthases and three branching enzyme fractions. The different fractions could be distinguished by chromatographic properties and kinetic properties under various assay conditions. Kernels homozygous for the recessive amylose-extender (ae) allele were missing branching enzyme IIb. In addition, the citrate-stimulated activity of starch synthase I was reduced. This activity could be regenerated by the addition of branching enzyme to this fraction. No other starch synthase fractions were different from normal enzymes. Extracts from kernels homozygous for the recessive dull (du) allele were found to contain lower branching enzyme IIa and starch synthase II activities. Other fractions were not different from the normal enzymes. Analysis of extracts from kernels of the double mutant ae du indicated that the two mutants act independently. Branching enzyme IIb was absent and the citrate-stimulated reaction of starch synthase I was reduced but could be regenerated by the addition of branching enzyme (ae properties) and both branching enzyme IIa and starch synthase II were greatly reduced (du properties). Starch from ae and du endosperms contains higher amylose (66 and 42%, respectively) than normal endosperm (26%). In addition, the amylopectin fraction of ae starch is less highly branched than amylopectin from normal or du starch. The above observations suggest that the alterations of the starch may be accounted for by changes in the soluble synthase and branching enzyme fractions.     

Response of enzymes and storage proteins of maize endosperm to nitrogen supply

To examine the effects of N nutrition upon endosperm development, maize (Zea mays) kernels were grown in vitro with either 0, 3.6, 7.1, 14.3, or 35.7 millimolar N. Kernels were harvested at 20 days after pollination for determination of enzyme activities and again at maturity for quantification of storage products and electrophoretic separation of zeins. Endosperm dry weight, starch, zein-N, and nonzein-N all increased in mature kernels as N supply increased from zero to 14.3 millimolar. The activities of sucrose synthase, aldolase, phosphoglucomutase, glutamate-pyruvate transaminase, glutamate-oxaloacetate transaminase, and acetolactate synthase increased from 1- to 2.5-fold with increasing N supply. Adenosine diphosphate-glucose pyrophosphorylase and both ATP- and PPi-dependent phosphofructokinases increased to lesser extents, while no significant response was detected for hexose kinases and glutamine synthetase. Nitrogen-induced changes in enzyme activities were often highly correlated with changes in final starch and/or zein-N contents. Separation of zeins indicated that these peptides were proportionately enhanced by N supply, with the exception of C-zein, which was relatively insensitive to N. These data indicate that at least a portion of the yield increase in maize produced by N fertilization is induced by a modification of kernel metabolism in response to N supply.     

Sugar Uptake and Metabolism in the Developing Endosperm of Tassel-seed Tunicate (Ts-5 Tu) Maize

Factors regulating assimilate transport into developing maize (Zea mays L.) kernels have been difficult to determine because of the structural complexity of basal kernel tissues and the damage that results from tissue dissection. The sensitivity of maize kernels to experimental manipulation is such that substantial maternal tissue is required to support kernel growth in vitro. Consequently, sugar transport experiments with isolated seed tissues or detached kernels have not unequivocally demonstrated how sugar transport occurs. In the present study, Tassel-seed Tunicate (Ts-5 Tu) maize kernels were investigated as a model system for introducing test solutions into the pedicel apoplast with minimal wounding. Transpiration in leafy glumes drew ¹⁴C-sugar solutions up the 8- to 10-millimeter-long pedicel stalks into the basal endosperm transfer cell region. ¹⁴C from fructose was incorporated into starch for 8 days. Sugar uptake into endosperm and embryo tissue showed specificity and inhibitor sensitivity. In particular, p-chloromercuribenzene sulfonate partially inhibited fructose uptake into the endosperm but had no effect on the metabolic conversion of that fructose that entered the endosperm. These results are consistent with active, carrier-mediated sugar transport, but a definitive determination would require more detailed tissue analysis. We propose that further refinement of the incubation solution may allow long-term kernel growth without cob tissue and thus provide a more precise determination of which maternal factors influence seed development.     

Fructan Accumulation and Sucrose Metabolism in Transgenic Maize Endosperm Expressing a Bacillus amyloliquefaciens SacB Gene

Over 40,000 species of plants accumulate fructan, [beta]-2-1- and [beta]-2-6-linked polymers of fructose as a storage reserve. Due to their high fructose content, several commercial applications for fructans have been proposed. However, plants that accumulate these polymers are not agronomically suited for large-scale cultivation or processing. This study describes the transformation of a Bacillus amyloliquefaciens SacB gene into maize (Zea mays L.) callus by particle bombardment. Tissue-specific expression and targeting of the SacB protein to endosperm vacuoles resulted in stable accumulation of high-molecular-weight fructan in mature seeds. Accumulation of fructan in the vacuole had no detectable effect on kernel development or germination. Fructan levels were found to be approximately 9-fold higher in sh2 mutants compared to wild-type maize kernels. In contrast to vacuole-targeted expression, starch synthesis and endosperm development in mature seeds containing a cytosolically expressed SacB gene were severely affected. The data demonstrate that hexose resulting from cytosolic SacB activity was not utilized for starch synthesis. Transgenic seeds containing a chimeric SacB gene provide further evidence that the dominant pathway for starch synthesis in maize endosperm is through uridine diphosphoglucose catalyzed by the enzyme sucrose synthase.     

Purification and characterization of soluble starch synthases from maize endosperm

This study identified and characterized the soluble starch synthase of maize endosperm that was initially revealed as the SSII activity peak in anion exchange chromatography (J. L. Ozbun et al. (1971) Plant Physiol. 48, 765-769). At least six different genes coding for starch synthases are expressed in maize, although previously it was not known which of these is responsible for the SSII activity peak. The enzyme activity in the SSII peak was neutralized to a large extent by antibodies raised against the product of the Du1 gene, but was not affected by antibodies specific for the other highly expressed soluble starch synthase, zSSI, or for the zSSIIa or zSSIIb isoforms. These data provide direct evidence that Du1 codes for the starch synthase responsible for the SSII activity peak. This starch synthase was purified approximately 350-fold from endosperm extracts. The following enzymatic properties of the SSII activity were determined: temperature optimum, thermostability, pH effects, K(m) for different glucan primers and the glucosyl unit donor ADPGlc, V(max) using various primers, and stimulation by citrate. These properties were compared to those of zSSI purified over 1600-fold from maize endosperm by a parallel procedure. The major differences between the two enzymes were that the SSII activity displayed higher K(m) values for ADPGlc, a distinct temperature range for maximal activity, and different relative activities toward specific exogenous substrates. The purified SSI and SSII activities both were shown to be capable of elongating maltooligosaccharide primers in vitro.     

Enzymes Catalyzing the Reversible Conversion of Fructose-6-Phosphate and Fructose-1,6-Bisphosphate in Maize (Zea mays L.) Kernels

The significance of the glycolytic and gluconeogenic conversion of fructose-6-phosphate and fructose-1,6-bisphosphate on sugar metabolism was investigated in maize (Zea mays L.) kernels. Maximum extractable activities of the pyrophosphate (PPi) dependent phosphofructokinase, fructose-1,6-bisphosphatase, and the ATP-dependent phosphofructokinase were measured in normal and four maize genotypes, which accumulate relatively more sugars and less starch, to determine how these enzymes are affected by the genetic lesions. Normal endosperm accumulated more dry matter than the high sugar/low starch genotypes, but protein contents did not differ greatly among the genotypes. Mutation of several starch biosynthetic enzymes had little impact on the activities of PPi-dependent phosphofructokinase, fructose-1,6-bisphosphatase, and ATP-dependent phosphofructokinase, despite the altered capacity of the cell to synthesize starch. The PPi-dependent phosphofructokinase appeared to be more active toward glycolysis in all genotypes studied. Activity of the PPi-dependent phosphofructokinase in shrunken (low sucrose synthase genotype) did not differ from the activity in other genotypes, suggesting that the gluconeogenic production of PPi may not be the primary role of the enzyme. As expected, shrunken kernels contained more sugars and less starch than normal kernels throughout kernel development except at the very early stages. Developmental profiles of normal kernels also showed marked changes in the PPi-dependent phosphofructokinase activity, whereas the level of ATP-dependent phosphofructokinase activity remained relatively steady during kernel development. In addition, the ATP-dependent phosphofructokinase, and not the PPi-dependent phosphofructokinase, appeared to correlate more closely with respiration rate. These findings suggest that glycolysis catalyzed by the ATP-dependent phosphofructokinase may serve primarily to support energy production, and glycolysis catalyzed by the PPi-dependent phosphofructokinase may contribute mainly to generation of biosynthetic intermediates.     

Abscisic acid catabolism in maize kernels in response to water deficit at early endosperm development

To further our understanding of the greater susceptibility of apical kernels in maize inflorescences to water stress, abscisic acid (ABA) catabolism activity was evaluated in developing kernels with chirally separated (+)-[(3)H]ABA. The predominant pathway of ABA catabolism was via 8'-hydroxylase to form phaseic acid, while conjugation to glucose was minor. In response to water deficit imposed on whole plants during kernel development, ABA accumulated to higher concentrations in apical than basal kernels, while both returned to control levels after rewatering. ABA catabolism activity per gram fresh weight increased about three-fold in response to water stress, but was about the same in apical and basal kernels on a fresh weight basis. ABA catabolism activity was three to four-fold higher in placenta than endosperm, and activity was higher in apical than basal kernels. In vitro incubation tests indicated that glucose did not affect ABA catabolism. We conclude that placenta tissue plays an important role in ABA catabolism, and together with ABA influx and compartmentation, determine the rate of ABA transport into endosperms.     

Intervening in sibling competition for assimilates by controlled pollination prevents seed abortion under postpollination drought in maize

During maize production, drought throughout the flowering stage usually induces seed abortion and yield losses. The influence of postpollination drought stress on seed abortion and its underlying mechanisms are not well characterized. By intervening in the competition for assimilates between kernel siblings under different degrees of postpollination drought stresses accompanied by synchronous pollination (SP) and incomplete pollination (ICP) approaches, the mechanisms of postpollination abortion were investigated at physiological and molecular levels. Upon SP treatment, up to 15% of the fertilized apical kernels were aborted in the drought-exacerbated competition for assimilates. The aborted kernels exhibited weak sucrose hydrolysis and starch synthesis but promoted the synthesis of trehalose-6-phosphate and ethylene. In ICP where basal pollination was prevented, apical kernel growth was restored with reinstated sucrose metabolism and starch synthesis and promoted sucrose and hexose levels under drought stress. In addition, the equilibrium between ethylene and polyamine in response to the drought and pollination treatments was associated with the abortion process. We conclude that competition for assimilates drives postpollination kernel abortion, whereas differences in sugar metabolism and the equilibrium between ethylene and polyamines may be relevant to the “live or die” choice of kernel siblings during this competition.     

Soluble invertase expression is an early target of drought stress during the critical,abortion-sensitive phase of young ovary development in maize

To distinguish their roles in early kernel development and stress, expression of soluble (Ivr2) and insoluble (Incw2) acid invertases was analyzed in young ovaries of maize (Zea mays) from 6 d before (-6 d) to 7 d after pollination (+7 d) and in response to perturbation by drought stress treatments. The Ivr2 soluble invertase mRNA was more abundant than the Incw2 mRNA throughout pre- and early post-pollination development (peaking at +3 d). In contrast, Incw2 mRNAs increased only after pollination. Drought repression of the Ivr2 soluble invertase also preceded changes in Incw2, with soluble activity responding before pollination (-4 d). Distinct profiles of Ivr2 and Incw2 mRNAs correlated with respective enzyme activities and indicated separate roles for these invertases during ovary development and stress. In addition, the drought-induced decrease and developmental changes of ovary hexose to sucrose ratio correlated with activity of soluble but not insoluble invertase. Ovary abscisic acid levels were increased by severe drought only at -6 d and did not appear to directly affect Ivr2 expression. In situ analysis showed localized activity and Ivr2 mRNA for soluble invertase at sites of phloem-unloading and expanding maternal tissues (greatest in terminal vascular zones and nearby cells of pericarp, pedicel, and basal nucellus). This early pattern of maternal invertase localization is clearly distinct from the well-characterized association of insoluble invertase with the basal endosperm later in development. This localization, the shifts in endogenous hexose to sucrose environment, and the distinct timing of soluble and insoluble invertase expression during development and stress collectively indicate a key role and critical sensitivity of the Ivr2 soluble invertase gene during the early, abortion-susceptible phase of development.