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.     

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.     

硫营养对小麦籽粒淀粉合成及相关酶活性的影响

在田间条件下,研究了施硫对小麦籽粒淀粉合成及相关酶活性的影响.结果表明:在0～20 cm土层土壤有效硫含量为5.84 mg/kg的地块上施硫不仅提高了小麦籽粒中蔗糖的含量,而且催化蔗糖降解代谢的蔗糖合成酶(SS)活性提高,利于籽粒蔗糖的降解.施硫显著提高了灌浆期间籽粒可溶性淀粉合成酶(SSS)活性,并使腺苷二磷酸葡萄糖焦磷酸化酶(ADPGPPase)和束缚态淀粉合成酶(GBSS)活性在灌浆中、后期维持在较高水平,对直链和支链淀粉的合成都起促进作用, 使总淀粉积累增加,千粒重提高,产量增加.     

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.     

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.     

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.     

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.     

钾对小麦旗叶蔗糖和籽粒淀粉积累的影响

 利用冬小麦品种‘鲁麦22’(Triticum aestivum cv. `Lumai 22’)在大田条件下研究了钾素对小麦旗叶蔗糖和籽粒淀粉积累及其有关酶活性的影响。结果表明,钾素有利于提高旗叶光合速率,增强开花后旗叶磷酸蔗糖合成酶活性,提高旗叶中蔗糖的含量;从而提高了灌浆期间籽粒中蔗糖的供应,增强了籽粒中蔗糖合成酶和腺苷二磷酸葡萄糖焦磷酸化酶的活性,加速了淀粉积累速率,提高了粒重和产量。     

Physiology of kernel shrivelling and its reduction in triticale

The present paper elucidates the basic physiological attributesresponsible for kernel shrivelling in triticale. High alphaamylase activity with low endosperm starch and loss of kernelmoisture not in harmony with the increase in dry weight, weresome of the physiological attributes observed to be associatedwith kernel shrivelling in triticale. Seed vernalization for60 days at 4? 1?C, with a critical stage of 5 hr imbibition,caused a reduction in kernel shrivelling, to a maximum of 30%as compared with 80% in the control. Alpha amylase activitywas relatively low widi high endosperm starch content in allstages of developing kernels of the vernalized crop. In thevernalized crops, loss of kernel moisture was gradual and inharmony with the increase in dry weight throughout developmenttill maturity. Other improvements were also observed. (Received July 28, 1977; )     

Kernel Abortion in Maize : II. Distribution of C among Kernel Carbohydrates

This study was designed to compare the uptake and distribution of ¹⁴C among fructose, glucose, sucrose, and starch in the cob, pedicel, and endosperm tissues of maize (Zea mays L.) kernels induced to abort by high temperature with those that develop normally. Kernels cultured in vitro at 30 and 35°C were transferred to [¹⁴C]sucrose media 10 days after pollination. Kernels cultured at 35°C aborted prior to the onset of linear dry matter accumulation. Significant uptake into the cob, pedicel, and endosperm of radioactivity associated with the soluble and starch fractions of the tissues was detected after 24 hours in culture on labeled media. After 8 days in culture on [¹⁴C]sucrose media, 48 and 40% of the radioactivity associated with the cob carbohydrates was found in the reducing sugars at 30 and 35°C, respectively. This indicates that some of the sucrose taken up by the cob tissue was cleaved to fructose and glucose in the cob. Of the total carbohydrates, a higher percentage of label was associated with sucrose and a lower percentage with fructose and glucose in pedicel tissue of kernels cultured at 35°C compared to kernels cultured at 30°C. These results indicate that sucrose was not cleaved to fructose and glucose as rapidly during the unloading process in the pedicel of kernels induced to abort by high temperature. Kernels cultured at 35°C had a much lower proportion of label associated with endosperm starch (29%) than did kernels cultured at 30°C (89%). Kernels cultured at 35°C had a correspondingly higher proportion of ¹⁴C in endosperm fructose, glucose, and sucrose. These results indicate that starch synthesis in the endosperm is strongly inhibited in kernels induced to abort by high temperature even though there is an adequate supply of sugar.     

Rice Chalky Ring Formation Caused by Temporal Reduction in Starch Biosynthesis during Osmotic Adjustment under Foehn-Induced Dry Wind

Foehn-like extreme hot and dry wind conditions (34°C, >2.5 kPa vapor pressure deficit, and 7 m s⁻¹) strongly affect grain quality in rice (Oryza sativa L.). This is a current concern because of the increasing frequency and intensity of combined heat and water-deficit stress under climate change. Foehn-induced dry wind conditions during the grain-filling stage increase ring-shaped chalkiness as a result of spatiotemporal reduction in starch accumulation in the endosperm, but kernel growth is sometimes maintained by osmotic adjustment. Here, we assess the effects of dry wind on chalky ring formation in environmentally controlled growth chambers. Our results showed that hot and dry wind conditions that lasted for >24 h dramatically increased chalky ring formation. Hot and dry wind conditions temporarily reduced panicle water potential to –0.65 MPa; however, kernel growth was maintained by osmotic adjustment at control levels with increased transport of assimilate to the growing kernels. Dynamic tracer analysis with a nano-electrospray-ionization Orbitrap mass spectrometer and quantitative polymerase chain reaction analysis revealed that starch degradation was negligible in the short-term treatment. Overall expression of starch synthesis-related genes was found to be down-regulated at moderately low water potential. Because the events observed at low water potential preceded the packing of starch granules in cells, we concluded that reduced rates of starch biosynthesis play a central role in the events of cellular metabolism that are altered at osmotic adjustment, which leads to chalky ring formation under short-term hot and dry wind conditions.     

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.     

Abscisic Acid inhibition of endosperm cell division in cultured maize kernels

The response of developing maize (Zea mays L.) endosperm to elevated levels of abscisic acid (ABA) was investigated. Maize kernels and subtending cob sections were excised at 5 days after pollination (DAP) and placed in culture with or without 90 micromolar (±)-ABA in the medium. A decreased number of cells per endosperm was observed at 10 DAP (and later sampling times) in kernels cultured in medium containing ABA from 5 DAP, and in kernels transferred at 8 DAP to medium containing ABA, but not in kernels transferred at 11 DAP to medium containing ABA. The number of starch granules per endosperm was decreased in some treatments, but the reduction, when apparent, was comparable to the decreased number of endosperm cells. The effect on endosperm fresh weight was slight, transient, and appeared to be secondary to the effect on cell number. Mature endosperm dry weight was reduced when kernels were cultured continuously in medium containing ABA. Endosperm (+)-ABA content of kernels cultured in 0, 3, 10, 30, 100, or 300 micromolar (±)-ABA was measured at 10 DAP by indirect ELISA using a monoclonal antibody. Content of (+)-ABA in endosperms correlated negatively (R = −0.92) with endosperm cell number. On the basis of these studies we propose that during early kernel development, elevated levels of ABA decrease the rate of cell division in maize endosperm which, in turn, could limit the storage capacity of the kernel.     

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.     

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.     

Nucleotides and Nucleotide Sugars in Developing Maize Endosperms (Synthesis of ADP-Glucose in brittle-1)

As part of an in vivo study of carbohydrate metabolism during development of Zea mays L. kernels, quantities of nucleotides and nucleotide sugars were measured in endosperm extracts from normal, the single-mutant genotypes shrunken-1 (sh1), shrunken-2 (sh2), and brittle-1 (btl}, and the multiple-mutant genotypes sh1bt1, sh2bt1, and sh1sh2bt1. Results showed that bt1 kernels accumulated more than 13 times as much adenosine 5[prime] diphospho-glucose (ADP-Glc) as normal kernels. Activity of starch synthase in bt1 endosperm was equal to that in endosperm extracts from normal kernels. Thus the ADP-Glc accumulation in bt1 endosperm cells was not due to a deficiency in starch synthase. ADP-Glc content in extracts of sh1bt1 endosperms was similar to that in bt1, but in extracts of the sh2bt1 mutant kernels ADP-Glc content was much reduced compared to bt1 (about 3 times higher than that in normal). Endosperm extracts from sh1sh2bt1, kernels that are deficient in both ADP-Glc pyrophosphorylase (AGPase) and sucrose synthase, had quantities of ADP-Glc much lower than in normal kernels. These results clearly indicate that AGPase is the predominant enzyme responsible for the in vivo synthesis of ADP-Glc in bt1 mutant kernels, but Suc synthase may also contribute to the synthesis of ADP-Glc in kernels deficient in AGPase.