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.     

STARCH GRANULE (AMYLOPLAST) DEVELOPMENT IN ENDOSPERM OF SEVERAL ZEA MAYS L. GENOTYPES AFFECTING KERNEL POLYSACCHARIDES

Endosperm cell and starch granule (amyloplast) development of six maize (Zea mays L.) genotypes, normal, amylose-extender (ae), sugary (su), waxy (wx), amylose-extender sugary (ae su), and amylose-extender waxy (ae wx), was compared. Endosperms of all genotypes were indistinguishable at 14 days after pollination. Cells were highly vacuolated and those in the central crown area of the kernel contained small starch granules in close association with the nucleus. Cellular and nuclear enlargement occurred during endosperm development in all genotypes, and major and minor gradients in physiological age of endosperm cells were observed in all kernels. Amyloplast development varied with genotype. Plastid development in normal and wx cells was characterized by an initial starch granule formation followed by granule enlargement to cell maturity. Endosperms homozygous for ae (ae, ae su, and ae wx) developed abnormal plastid-granules. Secondary granule formations preceded development of abnormality in ae and ae su, but not in ae wx endosperms. In contrast to ae and ae su starch granules, ae wx granules were highly birefringent indicating a high degree of crystallinity. In all three ae genotypes, abnormality increased as a function of kernel and physiological cell age. The su mutant had two distinct effects on amyloplast development. First, a mobilization of the initially formed starch, and second a synthesis and accumulation of phytoglycogen and the formation of large rounded plastids. In ae su plastid development, there was a mobilization of the starch initially formed (resulting in irregularly shaped, nonbirefringent granules) but only small amounts of phytoglycogen were produced.     

Analysis of Endosperm Sugars in a Sweet Corn Inbred (Illinois 677a) Which Contains the Sugary Enhancer (se) Gene and Comparison of se with Other Corn Genotypes

The endosperm sugars of a new corn (Zea mays L.) mutant, sugary enhancer (se), were analyzed by gas-liquid chromatography and were compared with sugars of other genotypes. Illinois 677a, a sugary (su) inbred containing the se gene, was high in sucrose and was distinguished from all of the other genotypes by its high maltose content. During kernel development, the maltose content of IL677a increased to 3.28% dry weight at 40 days postpollination and remained high at the dry mature stage, whereas `Silver Queen,' a high quality sugary (su) hybrid not possessing the se gene, showed no such trend in maltose accumulation. Sucrose, fructose, and glucose decreased during kernel development in `Silver Queen' and IL677a from 19 days postpollination until the dry mature stage. The slow drying characteristic and the reduced starch content previously reported for maturing seeds of IL677a may be related to the maltose accumulation reported here.     

Analysis of genetic mapping in a waxy/dent maize RIL population using SSR and SNP markers

A maize genetic linkage map was generated using SSR and SNP markers in a F_7:8 recombinant inbred line (RIL) population derived from a cross of waxy corn (KW7) and dent corn (Mo17). A total of 465 markers, including 459 SSR and 6 SNP markers, were assigned to 10 linkage groups which spanned 2,656.5 cM with an average genetic distance between markers of 5.7 cM, and the number of loci per linkage group ranged from 39 to 55. The SSR (85.4%) and SNP (83.3%) markers showed Mendelian segregation ratios in the RIL population at a 5% significance threshold. In linkage analysis of six SNP loci associated with kernel starch synthesis genes (ae1, bt2, sh1, sh2, su1, and wx1), all six loci were successfully mapped and are closely linked with SSR markers in chromosomes 3 (sh2), 4 (su1 and bt2), 5 (ae1), and 9 (sh1 and wx1). The SSR markers linked with genes in starch synthesis may be utilized in marker assisted breeding programs. The resulting genetic map will be useful in dissection of quantitative traits and the identification of superior QTLs from the waxy hybrid corn. Additionally, these data support further genetic analysis and development of maize breeding programs.     

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.     

Source-sink relations in maize mutants with starch-deficient endosperms

Partitioning and translocation of photosynthates were compared between a nonmutant genotype (Oh 43) of corn (Zea mays L.) and two starch-deficient endosperm mutants, shruken-2 (sh2) and brittle-1 (bt1), with similar genetic backgrounds. Steady-state levels of ¹⁴CO₂ were supplied to source leaf blades for 2-hour periods, followed by separation and identification of ¹⁴C-assimilates in the leaf, kernel, and along the translocation path. An average of 14.1% of the total ¹⁴C assimilated was translocated to normal kernels, versus 0.9% in sh2 kernels and 2.6% in btl kernels. Over 98% of the kernel ¹⁴C was in free sugars, and further analysis of nonmutant kernels showed 46% of this label in glucose and fructose. Source leaves of mutant plants exported significantly less total photosynthate (24.0% and 36.3% in sh2 and bt1 compared to 48.0% in the normal plants) and accumulated greater portions of label in the insoluble (starch) fraction. Mutant plants also showed lower percentages of photosynthate in the leaf blade and sheath below the exposed blade area. The starch-deficient endosperm mutants influence the partitioning and translocation of photosynthates and provide a valuable tool for the study of source-sink relations.     

Robustness of central carbohydrate metabolism in developing maize kernels

The central carbohydrate metabolism provides the precursors for the syntheses of various storage products in seeds. While the underlying biochemical map is well established, little is known about the organization and flexibility of carbohydrate metabolic fluxes in the face of changing biosynthetic demands or other perturbations. This question was addressed in developing kernels of maize (Zea mays L.), a model system for the study of starch and sugar metabolism. ¹³C-labeling experiments were carried out with inbred lines, heterotic hybrids, and starch-deficient mutants that were selected to cover a wide range of performances and kernel phenotypes. In total, 46 labeling experiments were carried out using either [U-¹³C₆]glucose or [U-¹³C₁₂]sucrose and up to three stages of kernel development. Carbohydrate flux distributions were estimated based on glucose isotopologue abundances, which were determined in hydrolysates of starch by using quantitative ¹³C-NMR and GC-MS. Similar labeling patterns in all samples indicated robustness of carbohydrate fluxes in maize endosperm, and fluxes were rather stable in response to glucose or sucrose feeding and during development. A lack of ADP-glucose pyrophosphorylase in the bt2 and sh2 mutants triggered significantly increased hexose cycling. In contrast, other mutations with similar kernel phenotypes had no effect. Thus, the distribution of carbohydrate fluxes is stable and not determined by sink strength in maize kernels.     

Starch and water-soluble polysaccharides from sugary endosperm of sorghum

The starch water-soluble polysaccharides from sugary (su) endosperm of sorghum were isolated and characterized. Starch granule structure and co     

Shrunken-1 encoded sucrose synthase is not required for sucrose synthesis in the maize endosperm

Kernels of wild-type maize (Zea mays L.) shrunken-1 (sh1), deficient in the predominant form of endosperm sucrose synthase and shrunken-2 (sh2), deficient in 95% of the endosperm ADP-glucose pyrophosphorylase were grown in culture on sucrose, glucose, or fructose as the carbon source. Analysis of the endosperm extracts by gas-liquid chromatography revealed that sucrose was present in the endosperms of all genotypes, regardless of carbon supply, indicating that all three genotypes are capable of synthesizing sucrose from reducing sugars. The finding that sucrose was present in sh1 kernels grown on reducing sugars is evidence that shrunken-1 encoded sucrose synthase is not necessary for sucrose synthesis. Shrunken-1 kernels developed to maturity and produced viable seeds on all carbon sources, but unlike wild-type and sh2 kernels grown in vitro, sucrose was not the superior carbon source. This latter result provides further evidence that the role of sucrose synthase in maize endosperm is primarily that of sucrose degradation.     

Dissection of maize kernel composition and starch production by candidate gene association

Cereal starch production forms the basis of subsistence for much of the world's human and domesticated animal populations. Starch concentration and composition in the maize (Zea mays ssp mays) kernel are complex traits controlled by many genes. In this study, an association approach was used to evaluate six maize candidate genes involved in kernel starch biosynthesis: amylose extender1 (ae1), brittle endosperm2 (bt2), shrunken1 (sh1), sh2, sugary1, and waxy1. Major kernel composition traits, such as protein, oil, and starch concentration, were assessed as well as important starch composition quality traits, including pasting properties and amylose levels. Overall, bt2, sh1, and sh2 showed significant associations for kernel composition traits, whereas ae1 and sh2 showed significant associations for starch pasting properties. ae1 and sh1 both associated with amylose levels. Additionally, haplotype analysis of sh2 suggested this gene is involved in starch viscosity properties and amylose content. Despite starch concentration being only moderately heritable for this particular panel of diverse maize inbreds, high resolution was achieved when evaluating these starch candidate genes, and diverse alleles for breeding and further molecular analysis were identified.     

Starch biosynthesis of amylomaize during endosperm development

In an attempt to investigate starch biosynthesis of amylomaize, the endosperm starches from four genotypes with different doses of amylose-extender (ae) between 0 and 3 were isolated at 14, 18, 22, 28, and 36 days after pollination, and their properties were examined. Gene effects of ae on the endosperm starches were found in the apparent amylose content from measurements of iodine binding capacity and in the elution pattern from fractionation of starches debranched by isoamylase on Sephadex G-75. With increasing doses of ae, the lengths of two side-chain fractions of amylopectin in the original starch granule and the carbohydrate content of shorter side-chain fraction both increased. However, the length of each side chain fraction varied little between 14 and 36 days in all genotypes. In addition, the amylose content in ae homozygous at the 14-day stage was ca 24 % lower than the value of amylose and intermediate fractions in the original starch granule.     

Amylolytic activity and carbohydrate levels in relation to coleoptile anoxic elongation in Oryza sativa genotypes

Among starchy seeds, rice has the unique capacity to germinate successfully under complete anaerobiosis. In this conditions, starch degradation is supported by a complete set of starch-degrading enzymes that are absent or inactive in cereals except rice. A characterization of carbohydrate metabolism and starch-degrading enzyme activity across twenty-nine genotypes of Oryza sativa L. is presented here. The zymogram of amylolytic activities present in rice embryos and endosperms under anaerobic conditions seven days after sowing (DAS) revealed marked differences among cultivars. Coleoptile elongation was positively correlated with total amylolytic activities and α-amylase activity in embryos, and negatively correlated with α-amylase activity in endosperm. Moreover, carbohydrate content in embryos was found to be positively correlated with total amylolytic activities under anaerobic conditions, while a negative relationship was recorded in the endosperm. Carbohydrate status in rice seedlings has a primary importance in sustaining coleoptile elongation towards the surface. The relationship between carbohydrate level in embryo and anoxic germination, as well as with total amylolytic activities present in rice embryo under anaerobic condition 7 DAS, is consistent with the role of sugar metabolism to support rice germination under oxygen-deprived environment.     

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     

C]Sucrose Uptake and Labeling of Starch in Developing Grains of Normal and segl Barley

Previous work showed that the segl mutant of barley (Hordeum vulgare cv Betzes) did not differ from normal Betzes in plant growth, photosynthesis, or fertility, but it produced only shrunken seeds regardless of pollen source. To determine whether defects in sucrose uptake or starch synthesis resulted in the shrunken condition, developing grains of Betzes and segl were cultured in [¹⁴C]sucrose solutions after slicing transversely to expose the endosperm cavity and free space. In both young grains (before genotypes differed in dry weight) and older grains (17 days after anthesis, when segl grains were smaller than Betzes), sucrose uptake and starch synthesis were similar in both genotypes on a dry weight basis. To determine if sucrose was hydrolyzed during uptake, spikes of Betzes and segl were allowed to take up [fructose-U-¹⁴C]sucrose 14 days after anthesis and the radioactivity of endosperm sugars was examined during 3 hours of incubation. Whereas less total radioactivity entered the endosperm and the endosperm cavity (free space) of segl, in both genotypes over 96% of the label of endosperm sugars was in sucrose, and there was no apparent initial or progressive randomization of label among hexose moieties of sucrose as compared to the free space sampled after 1 hour of incubation. We conclude that segl endosperms are capable of normal sucrose uptake and starch synthesis and that hydrolysis of sucrose is not required for uptake in either genotype. Evidence suggests abnormal development of grain tissue of maternal origin during growth of segl grains.     

Identification and Genetic Analysis of a Novel Allelic Variation of <i>Brittle-1</i> with Endosperm Mutant in Maize

Endosperm mutants are critical to the studies on both starch synthesis and metabolism and genetic improvement of starch quality in maize. In the present study, a novel maize endosperm mutant A0178 of natural variation was used as the experimental material and identified and then characterized. Through phenotypic identification, genetic analysis, main ingredients measurement and embryo rescue, development of genetic mapping population from A0178, the endosperm mutant gene was located. The results showed that the mutant exhibited extremely low germination ability as attributed to the inhibited embryo development, and amounts of sugars were accumulated in the mutant seeds and more sugars content was detected at 23 days after pollination (DAP) in A0178 than B73. Employing genetic linkage analysis, the mutant trait was mapped in the bin 5.04 on chromosome 5. Sequence analysis showed that two sites of base transversion and insertion presented in the protein coding region and non-coding region of the mutant brittle-1 (bt1), the adenylate translocator encoding gene involved in the starch synthesis. The single base insertion in the coding region cause frameshift mutation, early termination and lose of function of Brittle-1 (BT1). All results suggested that bt1 is a novel allelic gene and the causal gene of this endosperm mutant, providing insights on the mechanism of endosperm formation in maize.