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.     

Identification of granule-bound starch synthase in potato tubers

Starch granules isolated from potato (Solanum tuberosum L.) tubers were extracted with sodium dodecyl sulfate and the extract was analyzed. A major protein with a molecular weight of 60,000 daltons was detected. This protein was purified by preparative sodium dodecyl sulfate-gel electrophoresis and specific antibodies were prepared. The anti-60-kilodalton antibodies obtained (a) cross-reacted with the waxy proteins of both maize (Zea mays L.) and grain amaranth (Amaranthus hypochondriacus L.), and (b) inhibited starch synthase activity in partially digested starch granules of the grain amaranth. This evidence strongly suggests that the major 60-kilodalton protein present in potato starch granules represents the granule-bound starch synthase.     

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.     

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.     

Over-expression of AGPase genes enhances seed weight and starch content in transgenic maize

Cereal crops accumulate starch in the seed endosperm as an energy reserve. ADP-glucose pyrophosphorylase (AGPase) plays a key role in regulating starch biosynthesis in cereal seeds. The AGPase in the maize endosperm is a heterotetramer of two small subunits, encoded by Brittle2 (Bt2) gene, and two large subunits, encoded by the Shrunken2 (Sh2) gene. The two genes (Bt2, Sh2) from maize were introduced into two elite maize inbred lines, solely and in tandem, and under the control of endosperm-specific promoters for over-expression. PCR, Southern blotting, and real-time RT-PCR analysis indicated that the transgenes were integrated into the genome of transgenic plants and were over-expressed in their progeny. The over-expression of either gene enhanced AGPase activity, seed weight and starch content compared with the WT, but the amounts were lower than plants with over-expression of both Bt2 and Sh2. Developing seeds from co-expression transgenic maize plants had higher cytoplasmic AGPase activity: the 100-grain weight increased 15% over the wild type (WT), and the starch content increased to over 74% compared with the WT of 65%. These results indicate that over-expression of the genes in transgenic maize plants could improve kernel traits. This report provides a feasible approach for increasing starch content and seed weight in maize.     

Solubilization of the starch-granule-bound starch synthase of normal maize kernels

The starch-granule-bound starch synthase from Zea mays has been solubilized with a recovery of between 50 and 84%. Chromatography of the solubilized enzyme on DEAE-Sepharose resolves two fractions of activity which may be distinguished by their response to citrate. Neither solubilized isoenzyme displays any significant activity with UDPglucose.     

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.     

Genetic evidence that the two isozymes of sucrose synthase present in developing maize endosperm are critical, one for cell wall integrity and the other for starch biosynthesis

In maize, two paralogous genes, Sh1 and Sus1, encode two biochemically similar isozymes of sucrose synthase, SS1 and SS2, respectively. Previous studies have attributed the mild starch deficiency of the shrunken1 (sh1) endosperm to the loss of the SS1 isozyme in the mutant. Here we describe the first mutation in the sucrose synthase1 (Sus1) gene, sus1-1, and the isolation of a double recessive genotype, sh1 sus1-1. Combined data from diverse studies, including Northern and Western analyses, RT-PCR and genomic PCR, cloning and sequencing data for the 3′ region, show that the mutant sus1-1 gene has a complex pattern of expression, albeit at much reduced levels as compared to the Sus1 gene. Endosperm sucrose synthase activity in sh1 sus1-1 was barely 0.5% of the total activity in the Sh1 Sus1 genotype. Significantly, comparative analyses of Sh1 Sus1, sh1 Sus1 and sh1 sus1-1 genotypes have, for the first time, allowed us to dissect the relative contributions of each isozyme to endosperm development. Starch contents in endosperm of the three related genotypes were 100, 78 and 53%, respectively. Anatomical analyses, which confirmed the previously described early cell degeneration phenotype unique to the sh1 Sus1 endosperm, revealed no detectable difference between the two sh1 genotypes. We conclude that the SS1 isozyme plays the dominant role in providing the substrate for cellulose biosynthesis, whereas the SS2 protein is needed mainly for generating precursors for starch biosynthesis.     

Divergent selection in a maize population for germination at low temperature in controlled environment: study of the direct response, of the trait inheritance and of correlated responses in the field

Improving cold tolerance in maize (Zea mays L.) is an important breeding objective, allowing early sowings which result in many agronomic advantages. Using as source the F₂ population of B73 × IABO78 single cross, we previously conducted four cycles of divergent recurrent selection for high (H) and low (L) cold tolerance level, evaluated as the difference (DG) between germination at 9.5 °C and at 25 °C in the germinator. Then, we pursued the divergent selection in inbreeding from S₁ to S₄. This research was conducted to study (1) the direct response to selection (by testing ten S₄ L and ten S₄ H lines), (2) the trait inheritance (in a complete diallel scheme involving four L and four H lines), (3) the associated responses for cold tolerance in the field (at early and delayed sowings) and (4) the responses for other traits, by testing the ten L and the ten H lines at usual sowing. Selection was effective, leading to appreciable and symmetric responses for DG. Variation among crosses was mainly due to additive effects and the ability to predict hybrid DG based on parental lines DG was appreciable. Associated responses for cold tolerance traits in the field were noticeable, though the relationship between DG and these traits was not outstanding. High tolerance was also associated with early flowering, short plants, less leaves, low kernel moisture, red and thin cob, and flint kernels. These divergently selected lines can represent valuable materials for undertaking basic studies and breeding works concerning cold tolerance.     

QTL mapping for maize resistance and yield under infestation with Sesamia nonagrioides

The Mediterranean corn borer (MCB) is the most important maize insect pest in the Mediterranean region. The main objective was to map quantitative trait loci (QTL) for yield performance under infestation with MCB, resistance and agronomic traits in a maize RIL population derived from an inbred cross European flint × Reid. Six QTL for resistance traits were located: one QTL for tunnel length (bin 9.03; p = 19.8 %), one QTL for stalk lodging (bin 3.07, p = 11.5 %), and four QTL for ear resistance (bins 1.07, 5.03/5.05, and 8.04; p = 25–63 %). Twelve QTL for agronomic traits were located: a QTL for yield under infestation (bin 5.03, p = 15 %); two QTL for grain moisture (bins 1.07 and 8.05); two QTL for days to anthesis (bin 1.07 and 8.05); two QTL for days to silking (bins 8.04 and 10.02); three QTL for plant height (bins 5.04, 8.05 and 9.03); and two QTL for ear height (bins 8.05 and 9.03). No genetic correlations between yield and other traits were observed. The cross validation (CV) approach showed that the estimation biases for QTL for resistance traits were higher than those for agronomic traits. This work stresses the importance of the region 9.03 for controlling corn borer resistance and suggests the presence of QTL with small effect on ear-resistance traits. At the same genomic region, there are also genes that control plant and ear height and future works could elucidate whether these genes are the same or are closely linked. The QTL for yield seem to play an important role in MCB tolerance in this genetic background. Large biases observed for QTL effects by CV were mainly due to the small sample size used and were higher for resistance traits due to their larger genetic complexity. We consider that it is more appropriate to select for grain yield under infestation instead of selecting for resistance traits because resistance to MCB could have unfavorable associations with agronomic traits.     

The citrate-stimulated starch synthase of starchy maize kernels: Purification and properties

Chromatography of maize kernel extracts on DEAE-cellulose resolves two fractions of starch synthase activity, one of which (starch synthase 1) is capable of synthesizing α-glucan in the absence of exogenous primer and the presence of 0.5 m citrate (J. L. Ozbun, J. S. Hawker, and J. Preiss, Plant Physiol. (1971) 48, 765–769). This starch synthase has been purified 200-fold from developing kernels of normal maize, and shown to have no detectable activities of branching enzyme, amylase, pullulanase, phosphorylase, and D enzyme. The preparation, however, was not electrophoretically homogeneous. This preparation had a K_m value of 0.033 mm for ADPglucose in the presence of 0.5 m citrate. The reaction in the presence of citrate was stimulated 10-fold by the addition of excess purified branching enzyme. This stimulation is higher than those reported previously (C. D. Boyer and J. Preiss, Plant Physiol. (1979) 64, 1039–1042) but is consistent with the predicted effects of removal of amylase activity. The effects of salts other than citrate on activity in the absence of exogenous primer were small, but the stimulation could be restored by the addition of excess purified branching enzyme. Citrate increased the affinity of the enzyme for the endogenous primer present to such a level that no effect of exogenous primer on reaction rate could be observed in the presence of 0.5 m citrate. Analysis of the glucan/iodine complex and the enzymatic breakdown products patterns from the products of the starch synthase reaction indicates a high degree of linearity. The results obtained are discussed in relation to the biosynthesis of starch in vivo.     

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.     

Multiple forms of starch branching enzyme of maize: Evidence for independent genetic control

Purification of starch branching enzymes from kernels of two nonlinked mutants of maize, $\text{sugary}$ and $\text{amylose-extender}$, showed the basis of the two mutations to be associated with branching enzymes I and IIb, respectively. Branching enzyme I from $\text{sugary}$ kernels purified as nonmutant branching enzyme I, but had an altered pattern of activity when amylose was used as a substrate. In addition to the typical fall in absorbance at high wavelengths (550–700 nm) of the amylose-iodine complex, branching of amylose by $\text{sugary}$ branching enzyme I caused an increase in absorbance at low wavelengths (400–550 nm). Branching enzyme IIb was undetected in extracts of $\text{amylose-extender}$ kernels, while branching enzymes I and IIa appeared unaltered. Low umprimed starch synthase activity was also observed in DEAE-cellulose fractions of $\text{amylose-extender}$ maize, but this activity was regenerated by the addition of any branching enzyme.     

Characterization of cell growth and starch production in the marine green microalga Tetraselmis subcordiformis under extracellular phosphorus-deprived and sequentially phosphorus-replete conditions

Microalgal starch is a potential feedstock for biofuel production. Nutrient stress is widely used to stimulate starch accumulation in microalgae. Cell growth and starch accumulation in the marine green microalga Tetraselmis subcordiformis were evaluated under extracellular phosphorus deprivation with initial cell densities (ICD) of 1.5, 3.0, 6.0, and 9.0?×?10⁶ cells mL^?1. The intracellular stored phosphorus supported cell growth when extracellular phosphorus was absent. The maximum starch content of 44.1 % was achieved in the lowest ICD culture, while the maximum biomass productivity of 0.71 g L^?1 day^?1, starch concentration of 1.6 g L^?1, and starch productivity of 0.30 g L^?1 day^?1 were all obtained in the culture with the ICD of 3.0?×?10⁶ cells mL^?1. Appropriate ICD could be used to regulate the intracellular phosphorus concentration and maintain adequate photosynthetic activity to achieve the highest starch productivity, along with biomass and starch concentration. The recovery of phosphorus-deprived T. subcordiformis in medium containing 0.5, 1.0, or 6.0 mM KH₂PO₄ was also tested. Cell growth and starch accumulation ability could be recovered completely. A phosphorus pool in T. subcordiformis was shown to manipulate its metabolic activity under different environmental phosphorus availability. Though lower starch productivity and starch content were achieved under phosphorus deprivation compared with nitrogen- or sulfur-deprived conditions, the higher biomass and starch concentration make T. subcordiformis a good candidate for biomass and starch production under extracellular phosphorus deprivation.     

Genetic analysis and major QTL detection for maize kernel size and weight in multi-environments

Key Message

Twelve major QTL in five optimal clusters and several epistatic QTL are identified for maize kernel size and weight, some with pleiotropic will be promising for fine-mapping and yield improvement.

Abstract

Kernel size and weight are important target traits in maize (Zea mays L.) breeding programs. Here, we report a set of quantitative trait loci (QTL) scattered through the genome and significantly controlled the performance of four kernel traits including length, width, thickness and weight. From the cross V671 (large kernel) × Mc (small kernel), 270 derived F_2:3 families were used to identify QTL of maize kernel-size traits and kernel weight in five environments, using composite interval mapping (CIM) for single-environment analysis along with mixed linear model-based CIM for joint analysis. These two mapping strategies identified 55 and 28 QTL, respectively. Among them, 6 of 23 coincident were detected as interacting with environment. Single-environment analysis showed that 8 genetic regions on chromosomes 1, 2, 4, 5 and 9 clustered more than 60 % of the identified QTL. Twelve stable major QTLs accounting for over 10 % of phenotypic variation were included in five optimal clusters on the genetic region of bins 1.02–1.03, 1.04–1.06, 2.05–2.07, 4.07–4.08 and 9.03–9.04; the addition and partial dominance effects of significant QTL play an important role in controlling the development of maize kernel. These putative QTL may have great promising for further fine-mapping with more markers, and genetic improvement of maize kernel size and weight through marker-assisted breeding.     

Nucleotide sugars and starch synthesis in spadix of Arum maculatum and suspension cultures of Glycine max

The aim of this work was to determine the relative contributions of ADPglucose and UDPglucose to starch synthesis in two non-photosynthetic tissues, the developing club of the spadix of Arum maculatum and suspension cultures of Glycine max. Rates of starch accumulation during growth are compared with estimates of the maximum catalytic activities in vitro of ADPglucose starch synthase, ADPglucose pyrophosphorylase, UDPglucose pyrophosphorylase and UDPglucose starch synthase. The latter could only be measured at high concentrations (10–30 mM) of UDPglucose. Clubs of Arum and cells of Glycine contained 292 and 6.8 nmol UDPglucose per gram fresh weight, respectively. The corresponding figures for ADPglucose were 29 and 0.4. From the above data it is argued that in both Arum club and Glycine cells the activity of UDPglucose starch synthase is too low to make any quantitatively significant contribution to starch synthesis. The activities of ADPglucose starch synthase and pyrophosphorylase were high enough to mediate the observed rates of starch accumulation. It is suggested that starch synthesis in these tissues is via ADPglucose.     

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     

Enhancement of starch accumulation in plants by exogenously applied methyl jasmonate

Increasing starch production is a central issue in plant biology and applied biotechnology. Although genetic engineering has been applied to produce plants containing much starch, chemicals that promote starch accumulation have not been well studied. Here, we report that exogenously applied methyl jasmonate (MeJA) enhanced the leaf starch content of Arabidopsis thaliana. A significant increase in starch production was detected during the light period after Arabidopsis was treated with high doses of MeJA (100–1,000 μM). The MeJA application influenced starch production rather than starch degradation because the expression of starch biosynthetic genes was upregulated by MeJA. The promotion of starch accumulation by MeJA was demonstrated not only in Arabidopsis but also in tobacco and spinach. These results suggest that the promotion of starch accumulation by MeJA is a common response found in a variety of plants.     

Selective measurement of starch synthesizing enzymes in permeabilized potato tuber slices

Osmotically permeabilized potato (Solanum tuberosum L.) tuber slices were used to study the biosynthesis of starch under semi in vivo conditions. Criteria to distinguish the various enzymes involved in starch biosynthesis were developed based on the characteristics of the enzymes in in vitro experiments. Branching enzyme activity was inhibited at pH 8.5 or higher, while the starch synthases functioned optimally between pH 8.8 and 9.1. Unprimed soluble starch synthase activity was only apparent in the presence of sodium citrate (0.4 molar or higher). Granulebound and primed soluble starch synthase were active in the absence of sodium citrate. Primed soluble starch synthase activity was susceptible to inhibition by 10 millimolar zinc sulfate, while granule-bound starch synthase activity was not. The incorporation of the Glc moiety of ADP-Glc into starch in tissue slices by the various starch synthases was consistent with in vitro data with respect to the affinity of the enzymes for substrate, the pH profile, the stimulation by citrate, and the inhibition by zinc sulfate. These data were used to determine the activity of each of the starch synthases in tissue slices: granule-bound and soluble starch synthase transferred 37 and 55 picomoles ADP-Glc per hour per milligram fresh weight into starch of permeabilized tissue slices at 30°C and pH 9.1. In the presence of 0.5 molar sodium citrate, at least 40 picomoles ADP-Glc per hour per milligram fresh weight as transferred into starch by unprimed soluble starch synthase activity.