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.     

Enzyme activities of starch and sucrose pathways and growth of apical and Basal maize kernels

Apical kernels of maize (Zea mays L.) ears have smaller size and lower growth rates than basal kernels. To improve our understanding of this difference, the developmental patterns of starch-synthesis-pathway enzyme activities and accumulation of sugars and starch was determined in apical- and basal-kernel endosperm of greenhouse-grown maize (cultivar Cornell 175) plants. Plants were synchronously pollinated, kernels were sampled from apical and basal ear positions throughout kernel development, and enzyme activities were measured in crude preparations. Several factors were correlated with the higher dry matter accumulation rate and larger mature kernel size of basal-kernel endosperm. During the period of cell expansion (7 to 19 days after pollination), the activity of insoluble (acid) invertase and sucose concentration in endosperm of basal kernels exceeded that in apical kernels. Soluble (alkaline) invertase was also high during this stage but was the same in endosperm of basal and apical kernels, while glucose concentration was higher in apical-kernel endosperm. During the period of maximal starch synthesis, the activities of sucrose synthase, ADP-Glc-pyrophosphorylase, and insoluble (granule-bound) ADP-Glc-starch synthase were higher in endosperm of basal than apical kernels. Soluble ADP-Glc-starch synthase, which was maximal during the early stage before starch accumulated, was the same in endosperm from apical and basal kernels. It appeared that differences in metabolic potential between apical and basal kernels were established at an early stage in kernel development.     

Transport and Metabolism of a Sucrose Analog (1'-Fluorosucrose) into Zea mays L. Endosperm without Invertase Hydrolysis

1′-Fluorosucrose (FS), a sucrose analog resistant to hydrolysis by invertase, was transported from husk leaves into maize (Zea mays L., Pioneer Hybrid 3320) kernels with the same magnitude and kinetics as sucrose. ¹⁴C-Label from [¹⁴C]FS and [¹⁴C]sucrose in separate experiments was distributed similarly between the pedicel, endosperm, and embryo with time. FS passed through maternal tissue and was absorbed intact into the endosperm where it was metabolized and used in synthesis of sucrose and methanol-chloroform-water insolubles. Accumulation of [¹⁴C] sucrose from supplied [¹⁴C]glucosyl-FS indicated that the glucose moiety from the breakdown of sucrose (here FS), which normally occurs in the process of starch synthesis in maize endosperm, was available to the pool of substrates for resynthesis of sucrose. Uptake of FS into maize endosperm without hydrolysis suggests that despite the presence of invertase in maternal tissues and the hydrolysis of a large percentage of sucrose unloaded from the phloem, hexoses are not specifically needed for uptake into maize endosperm.     

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.     

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     

The Effects of Modifying Sucrose Concentration on the Development of Maize Kernels Grown in vitro

Intact Zea mays L. kernels attached to cob tissue develop tomaturity when grown in vitro. This experiment was designed todetermine if it is possible to prolong kernel growth by refreshingthe culture medium. Blocks of maize kernels were grown in vitroon media containing several concentrations of sucrose. Kernels,at all concentrations of sucrose, developed to maturity at 30–35d post-pollination, indicating that it is not possible to extendthe kernel growth phase by supplying a carbohydrate source.Kernels grown on media containing 80 g 1^–1 or higher sucroseconcentration had a significantly greater percentage of kernelsthat developed to maturity, and had greater weight and starchcontent per seed. Zea mays, kernel culture, seed development, starch     

A proposed role of zein and glutelin as N sinks in maize

Zea mays grown with high levels of N fertilizer transports more sucrose into kernels than with low N. Sucrose translocation was greatest in genotypes with the highest capacity to deposit nitrogenous compounds as zein and glutelin in the kernel. These two proteins combined contain about 80% of the total N in the kernel and about 60% of the total N in the plant at maturity. They appear to serve as a functional N sink for the deposition of nitrogenous compounds. As the N sink capacity increases with additional available N fertilizer, more sucrose is transported into the kernel, resulting in increased kernel weight and grain yield. Zein functions as a more dynamic N sink than glutelin because the synthesis of zein is readily manipulated by N fertilization and genetic means. Increases in N deposition in the normal endosperm induced by N fertilizer are confined primarily to zein. Early termination of zein accumulation in the opaque-2 mutant results in a reduction of sucrose movement into kernels. By using plants heterozygous for normal and opaque-2 in these studies, interplant variability was eliminated and the hypothesis relating the kernel N sink capacity to productivity was strengthened.     

Movement of C-Labeled Assimilates into Kernels of Zea mays L: II. Invertase Activity of the Pedicel and Placento-Chalazal Tissues

Invertases of the placento-chalazal and pedicel tissues are much more active than invertase from the pericarp of Zea mays L. kernels 12 to 40 days after pollination. Sucrose synthetase was not detected in the pedicel or placento-chalazal tissues. Sucrose content and percentage increased in the pedicel with advancing kernel age. Hexoses accounted for over half of the sugars extracted from the placento-chalazal tissues. These data are consistent with the hypothesis that sucrose translocated to the pedicel is hydrolyzed by acid invertase(s) prior to entry of sugar into the endosperm tissue. The placentochalazal tissue appears to be the primary site of sucrose inversion with the pedicel invertase contributing more or less to this process depending on kernel age.     

Pollen source effects on growth of kernel structures and embryo chemical compounds in maize

Background and Aims

Previous studies have reported effects of pollen source on the oil concentration of maize (Zea mays) kernels through modifications to both the embryo/kernel ratio and embryo oil concentration. The present study expands upon previous analyses by addressing pollen source effects on the growth of kernel structures (i.e. pericarp, endosperm and embryo), allocation of embryo chemical constituents (i.e. oil, protein, starch and soluble sugars), and the anatomy and histology of the embryos.

Methods

Maize kernels with different oil concentration were obtained from pollinations with two parental genotypes of contrasting oil concentration. The dynamics of the growth of kernel structures and allocation of embryo chemical constituents were analysed during the post-flowering period. Mature kernels were dissected to study the anatomy (embryonic axis and scutellum) and histology [cell number and cell size of the scutellums, presence of sub-cellular structures in scutellum tissue (starch granules, oil and protein bodies)] of the embryos.

Key Results

Plants of all crosses exhibited a similar kernel number and kernel weight. Pollen source modified neither the growth period of kernel structures, nor pericarp growth rate. By contrast, pollen source determined a trade-off between embryo and endosperm growth rates, which impacted on the embryo/kernel ratio of mature kernels. Modifications to the embryo size were mediated by scutellum cell number. Pollen source also affected (P < 0·01) allocation of embryo chemical compounds. Negative correlations among embryo oil concentration and those of starch (r = 0·98, P < 0·01) and soluble sugars (r = 0·95, P < 0·05) were found. Coincidently, embryos with low oil concentration had an increased (P < 0·05–0·10) scutellum cell area occupied by starch granules and fewer oil bodies.

Conclusions

The effects of pollen source on both embryo/kernel ratio and allocation of embryo chemicals seems to be related to the early established sink strength (i.e. sink size and sink activity) of the embryos.Key words: Zea mays, maize, pollen, kernel, embryo, endosperm, oil, protein, starch, soluble sugars     

Sugar Efflux from Maize (Zea mays L.) Pedicel Tissue

Sugar release from the pedicel tissue of maize (Zea mays L.) kernels was studied by removing the distal portion of the kernel and the lower endosperm, followed by replacement of the endosperm with an agar solute trap. Sugars were unloaded into the apoplast of the pedicel and accumulated in the agar trap while the ear remained attached to the maize plant. The kinetics of ¹⁴C-assimilate movement into treated versus intact kernels were comparable. The rate of unloading declined with time, but sugar efflux from the pedicel continued for at least 6 hours and in most experiments the unloading rates approximated those necessary to support normal kernel growth rates. The unloading process was challenged with a variety of buffers, inhibitors, and solutes in order to characterize sugar unloading from this tissue.

Unloading was not affected by apoplastic pH or a variety of metabolic inhibitors. Although p-chloromercuribenzene sulfonic acid (PCMBS), a nonpenetrating sulfhydryl group reagent, did not affect sugar unloading, it effectively inhibited extracellular acid invertase. When the pedicel cups were pretreated with PCMBS, at least 60% of sugars unloaded from the pedicel could be identified as sucrose. Unloading was inhibited up to 70% by 10 millimolar CaCl₂. Unloading was stimulated by 15 millimolar ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid which partially reversed the inhibitory effects of Ca²⁺. Based on these results, we suggest that passive efflux of sucrose occurs from the maize pedicel symplast followed by extracellular hydrolysis to hexoses.

     

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.     

Sugar utilization by developing wild type and shrunken-2 maize kernels

To characterize the movement of sugars during kernel development in maize, a newly devised in vitro kernel development scheme was utilized. Viable seeds of wild type maize (Zea mays L.) as well as the mutant shrunken-2 (sh2) were found to mature when grown in culture with reducing sugars or sucrose as the carbon source. However, wild type and sh2 kernels had greater germination, starch content, and seed weight when sucrose, rather than reducing sugars, was the carbon source. By the use of labeled sucrose it was shown that sucrose can move into endosperm tissue without intervening degradation and resynthesis. These results show that when grown in vitro the maize seed can utilize reducing sugars for development, but it prefers sucrose.     

Growth and composition of maize kernels cultured in vitro with varying supplies of carbon and nitrogen

This study employed in vitro seed culture to determine how C and N supply influence the growth (i.e. starch accumulation) and protein composition of maize (Zea mays L.) endosperm. Immature kernels were grown to maturity on liquid medium containing various concentrations of C (sucrose at 234 millimolar [low] and 468 millimolar [high]) and N (amino acid mixture ranging in N from 0 to 144 millimolar). Low C supply limited starch, but not N, accumulation in the endosperm. With high C, endosperm starch and protein content increased concomitantly as N supply increased from 0 to 13.4 millimolar. Endosperm growth was unaffected by additional N until concentrations exceeding approximately 72 millimolar reduced starch accumulation. A similar inhibition of starch deposition occurred with lower N concentrations when kernels were grown with low C. Endosperm total N content reached a point of saturation with approximately 36 millimolar N in the medium, regardless of C supply. Zein synthesis in the endosperm responded positively across all N levels, while glutelin content remained static and albumin/globulin proteins were reduced in amount when N supply was greater than 36 millimolar. A reciprocal, inverse relationship was observed in mature endosperm tissue between the concentrations of free amino acids and soluble sugars. Our data suggest that under N stress starch and protein accumulation in the endosperm are interdependent, at least in appearance, but are independent otherwise.     

Invertase Activity in Normal and Mutant Maize Endosperms during Development

A bound invertase and two soluble invertases are found in the developing endosperm of maize (Zea mays L.). The two soluble invertases can be separated on diethylaminoethyl-cellulose and Sephadex columns and distinguished by their kinetic constants. One soluble invertase, invertase I, is present from the 10- to 28-day stages of endosperm development with maximal activity per normal endosperm at the 12-day stage. In two endosperm mutant lines, shrunken-1 and shrunken-2, there is a second increase in invertase I activity later in development which could be a secondary effect caused by the abnormal metabolism in these lines. Another soluble invertase, invertase II, is present in the embryo upon germination and is also found in the very young developing endosperm (6-day stage). The third form of invertase, bound invertase, is present in the endosperm by the 6-day stage, and its activity remains approximately constant during development.     

微胚乳玉米籽粒发育期间非胚部位（胚乳）中的腺苷二磷酸-葡萄糖焦磷酸化酶活性变化

检测微胚乳玉米非胚部位（胚乳）中腺苷二磷酸-葡萄糖焦磷酸化酶（ADPG—PPase）的活性结果表明：微胚乳玉米的非胚部位（胚乳）中ADPG．PPase活性在授粉后21-28d达到峰值,而对照的玉米品种‘高油115’的ADPG—PPase活性在授粉后14-21d达到峰值,滞后约7d。‘高油115’非胚部位（胚乳）中ADPG—PPase活性最大值极显著高于微胚乳玉米,其单粒ADPG—PPase活性最大值为微胚乳玉米的2．2-3．6倍,其每克干重ADPG—PPase活性最大值则为微胚乳玉米的2．4—3．8倍。     

Timing of Kernel Development in Water-stressed Maize: Water Potentials and Abscisic Acid Concentrations

Maize (Zea mays L. cv. Pioneer 3925) subjected to post-anthesiswater stress during the first 2 weeks of kernel developmenthad lower leaf-water potentials and higher leaf-ABA concentrationsthan well-watered controls. There was a concomitant rise inABA concentration in kernel tissues 3 and 7 d after pollination(DAP), after which the concentration decreased to control levelsby 13 DAP. Kernel water potential, however, remained unchangedby the water stress. Radiolabelled ABA, fed to a leaf, was translocatedto kernels, where free ABA as well as several ABA metaboliteswere the major labelled fractions. This suggested that the stress-inducedkernel ABA was of maternal origin. Since ABA plays a putativerole in seed maturation of several crop species, and appliedABA or water stress often hastens seed development, we expectedthat a water-stress-induced rise in kernel ABA concentrationearly in grain development may serve to prematurely induce storage-productaccumulation. Zein, starch and several enzymes key to the starchsynthesis pathway followed the same course of induction throughoutthe experiment, with no difference between treatments Henceit was concluded that although water stress increased kernelABA independent of kernel water status, there was no apparenteffect of water stress or ABA on timing of early kernel developmentalprocesses. Zea mays L. cv. Pioncer 3925, maize, water stress, abscisic acid, endosperm development     

Characterization and distribution of invertase activity in developing maize (Zea mays) kernels

Invertase ( β -fructofuranoside fructohydrolase, EC 3.2.1.26) activity in developing maize ( Zea mays L. inbred W64A) was separated into soluble and particulate forms. The particulate form was solubilized by treatment with 1 M NaCl or with other salts. However, CaCl₂ inhibited invertase activity, and neither detergents nor 0.5 M methyl mannoside were effective in solubilizing the invertase activity. The soluble and particulate invertases were both glycoproteins, both had pH optima of 5.0 and K_m values for sucrose of 2.83 and 1.84 m M , respectively. The apparent molecular weight of salt-solubilized invertase was 40 kDa. Gel filtration of the soluble invertase showed multiple peaks with apparent molecular weights ranging from 750 kDa to over 9 000 kDa. Histochemical staining of cell wall preparations for invertase activity suggested that the particulate invertase is associated with the cell wall. Also, nearly all the invertase activity was localized in the basal endosperm and pedicel tissues, which are sites of sugar transport. No invertase activity was found in the upper endosperm, the embryo or in the placento-chalazal tissue. In contrast, sucrose synthase (EC 2.4.1.13) activity was found primarily in the embryo and the upper endosperm, which are areas of active biosynthesis of storage compounds.     

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.     

An invertase inhibitor from maize localizes to the embryo surrounding region during early kernel development

Invertase activity is thought to play a regulatory role during early kernel development by converting sucrose originating from source leaves into hexoses to support cell division in the endosperm and embryo. Invertases are regulated at the posttranslational level by small protein inhibitors, INVINHs. We found that in maize (Zea mays), an invertase inhibitor homolog (ZM-INVINH1) is expressed early in kernel development, between 4 and 7 d after pollination. Invertase activity is reduced in vitro in the presence of recombinant ZM-INVINH1, and inhibition is attenuated by pre-incubation with sucrose. The presence of a putative signal peptide, fractionation experiments, and ZM-INVINH1::green fluorescent protein fusion experiments indicate that the protein is exported to the apoplast. Moreover, association of ZM-INVINH1 with the glycoprotein fraction by concanavalin A chromatogaphy suggests that ZM-INVINH1 interacts with an apoplastic invertase during early kernel development. ZM-INVINH1 was localized to the embryo surrounding region by in situ analysis, suggesting that this region forms a boundary, compartmentalizing apoplast invertase activity to allow different embryo and endosperm developmental rates.