Maize Endosperm Mutants Research Articles

Genetic control of lysine metabolism in maize endosperm mutants.

The capacity of three maize endosperm opaque mutants (o10, o11 and o13) to accumulate soluble lysine in the seed in relation to their wildtype counterpart, W22+, was investigated. The W22o13 and W22o11 mutants exhibited 278% and 186% increases in soluble lysine, respectively, while for W22o10, a 36% decrease was observed, compared with the wildtype. A quantitative and qualitative study of the N constituents of the endosperm has been conducted and data obtained for the total protein, non-protein N, soluble amino acids, albumins / globulins, zeins and glutelins present in the seed of the mutants. Following 2D-PAGE, a total of 38 different forms of zein polypeptides were detected and considerable differences were noted between the three mutant lines. The metabolism of lysine was also studied by analysis of the enzymes aspartate kinase, homoserine dehydrogenase, lysine 2-oxoglutarate reductase and saccharopine dehydrogenase, which exhibited major changes in activity, depending on the genotype, suggesting that the mutant genes may have distinct regulatory activities.

Genic and genotypic frequencies of endosperm mutants in maize populations under natural selection

The genic and genotypic frequencies of four maize endosperm mutants, opaque 2 (o2), shrunken 2 (sh2), sugary (su), and waxy (wx), which alter protein, sugar, soluble polysaccharides, and starchy fractions, respectively, were monitored throughout five successive generations in synthetic populations of the same genetic background under natural selection. The effect of selection was studied in the vegetative and reproductive phases through the comparison of the recessive homozygous frequencies in the seeds and in the adult plants. Data showed that natural selection acted diversely, in direction and intensity, on the mutants and in the phases of the plant life cycle. The genic frequencies indicated the occurrence of stabilizing selection over o2 and wx, and directional selection against sh2 and su.

Changes in carbohydrate composition and α-amylase expression during germination and seedling growth of starch-deficient endosperm mutants of maize

Kernels of 28 different maize ( Zea mays L.) genotypes consisting of several inbreds isogenic for the starch-deficient endosperm mutations sugary 1 ( su1), sugary1 sugary enhancer 1 ( su1se1), and shrunken2 ( sh2) were used to study the effect of these mutations on carbohydrate composition and α-amylase expression during germination and seedling growth in comparison to normal starchy kernels ( Su). Sugar (fructose, glucose, sucrose, and maltose), WSP (water-soluble polysaccharide), and insoluble starch contents were determined for endosperm and scutellar tissues for each of the genotypes at four stages of seedling development. While initial levels of endosperm sucrose were higher in the endosperm mutants, they rapidly declined by radicle emergence and remained low thereafter. Mobilization of carbohydrate reserves was most reduced in the sh2 genotypes. Collectively, these factors represent a limited carbon supply for the developing sh2 seedling which may impact seedling vigor. A more significant negative correlation was observed between percentage germination and endosperm sugar content than between percentage germination and scutellar sugar content. High initial levels of endosperm sucrose in the mutants correlate with a severe reduction of α-amylase (EC 3.2.1.1) transcript abundance. The same correlation does not hold for glucose and maltose, as at later stages of germination, levels were significantly higher in the Su genotype while the α-amylase mRNA levels remained high. Furthermore, the ratio of α-amylase enzyme activity to transcript abundance was considerably lower in the sh2 aleurone compared to the Su aleurone at radicle emergence. Genotype specific differences in α-amylase gene expression strongly correlate with endosperm sugar content and composition during germination.

Gene Expression in Developing Kernels of Some Endosperm Mutants of Maize

A number of endosperm mutants of maize (Zea mays L.) have altered kernel composition as the result of single locus mutations. We have examined eight different mutants to determine how these mutations might affect expression of eight different genes in developing kernels. We found that the abundance of aldolase, Shrunken-2, Brittle-2, Opaque-2 and Waxy transcripts were stim­ ulated by the mutations. The abundance of all of these transcripts, except for Shrunken-2, was correlated with the sucrose concentration in the kernel. Sucrose may modulate expression of these genes. Transcript abundance of the zein genes was lower in most of the mutants. A correlation be­ tween a-zein transcript abundance and mature kernel zein protein content suggested that zein accumulation is controlled by transcript abundance. A correlation between a-zein transcript abun­ dance and mature kernel dry weight suggested that zein gene expression is in some way linked with carbohydrate formation. The inverse relationship between sugar concentration in immature kernels and mature kernel dry weight supports the concept that the failure of developing kernels to utilize nutrients in the biosynthesis of storage products results in the accumulation of these nutrients. The accumulation of sugars and amino acids may then contribute to the observed changes in overall gene expression.

Expression of A/B zeins in single and double maize endosperm mutants

Zeins, the major endosperm proteins in maize (Zea mays L.), are deficient in the essential amino acids lysine and tryptophan. Some mutant genes, like opaque-2 (o2) and floury-2 (fl2), reduce the levels of A- and B-zeins, thereby improving maize's nutritional value. Other mutants, such as amylose-extender (ae), floury-1 (fl1), soft starch (h), dull-1 (du), shrunken-1 (sh1), sugary-1 (su1), sugary-2 (su2), and waxy (wx), primarily affect starch composition, but also alter zein composition. We undertook this study to examine the effects of some of these mutant genes on A/B-zein composition and to study the interactions of these genes in double-mutant combinations. Endosperm prolamins were extracted from inbred B37, ten near-isogenic single mutants (ae, du, fl1, fl2, h, o2, sh1, su1, su2, and wx), and most double-mutant combinations. Zeins in these extracts were fractionated by reversed-phase highperformance liquid chromatography (RP-HPLC) into 22-24 peaks. Of the resulting 22 major peaks the areas of 16 (per milligram endosperm) were significantly affected by individual mutant genes relative to the zein composition of the normal inbred. In combination these genes exhibited significant epistatic interactions in regulating the expression of individual A/B zeins. Epistatic interactions were judged to be significant when the amount of a peak in a double mutant differed from the averages for the peak in the two respective single mutants. The o2 gene, alone and in combination with other mutant genes, significantly decreased the amounts of many individual zeins. The effect of the o2 gene was the greatest of all the genes examined. Various clustering techniques were used to see if mutant effects could be grouped; among these was principal component analysis, a multivariate statistical technique that analyzes all peak sizes simultaneously. Three-dimensional scatter graphs were constructed based on the first three principal components. For the single mutants, these showed no relationships to gene actions; for the double mutants, however, this technique showed that four single mutants, o2, sh1, su1 and su2, had the greatest effects on zein composition when combined with each other and with the remaining six single mutants.

Increases in binding protein (BiP) accompany changes in protein body morphology in three high-lysine mutants of maize

A maize 75 kDa protein recently has been identified as a plant homolog of the mammalian binding protein (BiP). To better understand the function of BiP in protein body formation in maize endosperm, immunomicroscopy studies were conducted on three maize endosperm mutants, floury-2, Mucronate, and Defective endosperm-B 30, in which the level of BiP is highly elevated. Our results showed that protein body morphology in all three mutants was altered. In addition, BiP was localized in both the ER and peripheral regions of the abnormal protein bodies. The degree to which protein body morphology differed from normal was positively correlated with increased amounts of BiP. In addition, the accumulation of BiP in abnormal protein bodies increased with protein body maturation. In the three endosperm mutants, the arrangement of zeins within protein bodies had been perturbed, yet none of the specific zein subclasses exhibited the staining pattern found for BiP. The association of BiP with abnormal packaging of proteins in protein bodies may reflect a biological function to mediate protein folding and assembly in maize endosperm.

Regulatory mechanisms involved in the biosynthesis of starch

Abstract

Sugar Metabolism in Developing Kernels of Starch-Deficient Endosperm Mutants of Maize

Sugar metabolism in kernels of starch-deficient endosperm mutants of maize (Zea mays L.) was examined to determine how single locus mutations of carbohydrate metabolism affect carbohydrate metabolism as a whole. Activities of 14 enyzmes were measured in extracts from endosperms from isogenic lines of normal, shrunken, shrunken-2, shrunken-4, brittle-1, and brittle-2 maize in an OH43 background. Nearly every enzyme activity examined was affected in some or all of the mutants. Sucrose synthase and aldolase activities were lower in all mutants compared to normal. ADP-Glc pyrophosphorylase activity in immature kernels was much higher in brittle endosperms than in normal, but absent in brittle-2 and shrunken-2 endosperms. The activity in those genotypes exhibiting activity was positively correlated with sucrose concentration in the kernels. Sucrose may be modulating the coarse control of ADP-Glc pyrophosphorylase activity by affecting the genetic transcription of message for this enzyme. Sorbitol dehydrogenase activity was negatively correlated with its substrate, fructose, supporting the hypothesis that sorbitol dehydrogenase converts fructose produced during sucrose degradation into sorbitol. Glucokinase activity was positively correlated with mature kernel dry weight. This supports the hypothesis that glucokinase activity may limit sucrose utilization. Shrunken-4 extracts had lower activities for a number of enzymes, supporting the view that this mutant may have an impediment to protein synthesis. Elevated sucrose levels were evenly distributed throughout 20-day postpollination shrunken-2 kernels, whereas a sucrose concentration gradient existed in normal kernels between the basal region and the upper endosperm. This gradient is apparently generated by the utilization of sugars and may facilitate the movement of sugars into developing corn kernels.

Properties of Residual Maize Starches Following Acid Hydrolysis

AbstractThe properties of residual starches following initial acid hydrolysis were investigated. Starch granules of several endosperm mutants of maize, namely opaque‐2 (o2), amylose‐extender; opaque‐2 (ae o2), waxy; opaque‐2 (wx o2), dull; opaque‐2 (du o2), and sugary‐2; opaque‐2 (su2 o2) were used. Initial rate of hydrolysis for su2o2 was fastest and that for aeo2 was slowest. Following the acid hydrolysis by the enzymatic and chromatographic method, in the initial phase of hydrolysis amylose was preferentially hydrolyzed in the starches, except in the case of wx o2. In the case of wx o2 the long B chains of amylopectin were preferentially hydrolyzed. The rate of decrease in chain length of ae o2 was relatively fast and that of su2o2 was slow. Fine structures of amorphous regions in these starch granules are discussed.

Dependence of Seed Vigor during Germination on Carbohydrate Source in Endosperm Mutants of Maize

Differences in seed vigor of four genotypes of maize (Zea mays L.), brittle-1 (bt1), shrunken-2 (sh2), sugary (su), and normal, in an isogenic background, were investigated. Excised whole embryos and axes were grown on Murashige and Skoog (MS) media containing various carbohydrate sources. Of the four genotypes examined, sh2 seeds had the lowest vigor, especially under germination stress conditions. Embryo dry weights of sh2 were less than su and normal but equal to bt1 and made up nearly 25% of the whole seed weight. The sh2 seeds and whole embryos had low starch levels compared with the other three genotypes. Sugar levels were comparable in the three endosperm mutants, which were two times higher than normal. Optimum growth of excised embryos of all genotypes was obtained on MS medium containing 5% sucrose. However, this concentration did not totally overcome poor germination and growth of sh2 embryos and axes. Axes of su and normal had greater growth rates than sh2 and bt1 on sucrose-free medium, although the difference between genotypes decreased when whole embryos were used. When ground endosperm was employed as the carbohydrate source, sh2 embryos germinated and grew poorly, particularly on normal endosperm. With a commercial corn starch as the carbohydrate source, sh2 germlings were shorter in length and displayed a greater loss in dry weight than the other genotypes. The poor growth of sh2 embryos on ground endosperm and starch media may indicate a dysfunction of the scutellum or axis in relation to carbohydrate metabolism and utilization.

Developmental Changes in Starch Properties of Several Endosperm Mutants of Maize

AbstractThe properties of starches during development of several endosperm mutants of maize were investigated. Starch contents of normal and waxy (wx) kernels were higher than those of dull (du), sugary‐2 (su2), and amylose‐extender (ae) kernels, and in general the greater increase in starch contents occurred earlier in kernel development. The normal, du, su2, and ae mutants in that order, respectively, increased the apparent amylose concentration of the starches as measured by the “blue value” method. Within each of these genotypes, absorption intensity increased and λ max. of absorption curves shifted to a longer wavelength region with increasing maturity. Waxy starches did not change in either of these properties at any of the developmental stages. Few detectable differences were observed in X‐ray diffraction intensity, gelatinization temperature, heat of gelatinization, and the photopastegrams within these genotypes during development. Exhaustive degradation of starch granules by glucoamylase showed that ae starches were more resistant to amylase than the other starches. Except for wx, the starches tended to be more resistant to amylase with increasing maturity.

Effects of Two Maize Endosperm Mutants on Kernel Maturity, Carbohydrates, and Germination1

The shrunken‐2 (sh2) and sugary enhancer (se) genes which modify endosperm carbohydrates in maize (Zea mays L.) show promise as a means of improving quality for human consumption. A major factor limiting their use is the associated low germination of the seed and low seedling vigor. This research was undertaken to measure germination of sh2 and se hybrids as compared to their respective standard counterparts, Sh2 and Se, when seed was harvested at successive stages of maturity, and to examine germination differences as related to corresponding carbohydrate fractions. Hybrids were hand pollinated on the same day, and ears were harvested at approximately weekly intervals on six successive dates beginning 32 days after pollination. Kernel weight, moisture, reducing sugars (RS), total sugars (TS), water soluble polysaccharides (WSP), total soluble carbohydrates (TSC), water insoluble polysaccharides (WlP), tal non‐structural carbohydrates (TNC), cold and warm temperature germination, and seedling vigor were determined for seed at each harvest. Although the sh2 gene markedly increased kernel sugars and reduced kernel weight, seed of the hybrid, sh2 (2 ✕ 3), had satisfactory cold.temperature germination when harvested after mid‐September, when its moisture content was approximately 50%. Seed of the hybrid, se (8 ✕ 10), also higher in sugar than its standard counterpart, germinated satisfactorily at cold‐temperature when harvested at a moisture content of 60%. Cold‐temperature germination was negatively associated with kernel moisture, RS, TS, WSP, TSC, and TNC and positively associated with WlP. It usually was not significantly related to kernel weight. Root, shoot, and total seedling length were positively correlated with cold temperature germination. These results indicate that germination at low temperatures simulating those in the field can often be satisfactory for hybrids carrying the sh2 or se genes.

Development Changes in Fine Structure of Starches of Several Endosperm Mutants of Maize

AbstractDistribution of components of maize starches from several endosperm mutants sampled at three stages of development were investigated by gel filtration after debranching by isoamylase. The waxy (wx) starches consisted of only amylopectin, and the distribution of components did not change during development. In other starches: normal, amylose‐extender (ae), dull (du), sugary‐1 (su1) and sugary‐2 (su2) starches, amylose contents increased during the 20–35 days after pollination (DAP) period of development. The ae, du, su1 and su2 mutants produced high‐amylose starches, while amylopectin of su2 was normal type and those of ae, du and su1 were novel type, and ae starches possessed longer average chain‐lengths of amylopectin. Phytoglycogens were prepared from su1 kernels during development and their characteristics were compared with those of su1 starches. The characteristics of phytoglycogens did not change during the developmental phase studied and the average chain‐lengths of phytoglycogens were shorter than those of amylopectin of su1 starches at each stage of development.

Characterization of Starch from Maize Endosperm Mutants

AbstractSome maize endosperm mutants have branched‐chain starch components with long exterior chains which make the blue‐value procedure for estimating amylose content questionable. Gel filtration was used to estimate starch composition of normal, high‐amylose, waxy and other maize endosperm mutants. Loosely‐branched polymers appeared responsible for the apparent amylose content. In some mutants high and intermediate loosely branched polysaccharides were found in addition to amylose. The sweet corn background of some endosperm genotypes influenced the types of polymers produced compared to the dent backgrounds used by previous researchers.

Susceptibility of Starch Granules from Maize Endosperm Mutants to Amylases1

Abstract Starch granules were extracted from maize (Zea mays L.) endosperm of mature F2 kernels of IA5125 × IA453 normal, amylose-extender (ae), dull (du) waxy (wx), and ae du wx and from ae du wx F2 kernels harvested at the fresh market stage of development. Samples were digested for 1.5 hours with hog pancreatic α- amylase and Rhizopus II amyloglucosidase. The mature and immature ae du wx samples were usually digested significantly more than the other 4 genotypes, while ae was digested significantly less than the others. Digestion of starch granules from mature and immature ae du wx kernels did not differ. The sweet corn background used had little effect, since our observations of the single mutant genotypes were similar to those in a dent background. Clearly, ae du wx starch granules do not reflect the reduced digestibility associated with ae, and thus cultivars homozygous for ae du wx are not inferior with respect to their ability to supply carbohydrates to the diet.

Scanning electron microscopic observations of degradation of starch granules in germinating kernels of several maize (Zea mays L.) endosperm mutants in four inbred and one hybrid background and their normal counterparts.

The objective of this study was to make SEM observations of starch granules of several different maize endosperm mutants following the early germination period and to compare the relative patterns and digestibility by starch-degrading enzymes. Mature kernels of the ae endosperm mutant in four inbred lines and in a single cross hybrid and of five endosperm mutants in an inbred and a single cross hybrid, and their normal counterparts were used. Enzymatic erosion of the starch granules was visible in the disected endosperm of the normal maize following germination 4, 6 and 8 days after planting. The types of the enzymatic erosion were similar to those of the in vitro attack by a-amylase on normal starch granules. Namely, numerous pin holes were visible on the surfaces of the granules and the pores penetrated into the granules and the inner portion, which appeared to be terraced or step-shaped indicative of layered internal structures. Some types of enzymatic erosion suggested the possibility of enzymatic attack other than that of a-amylase. Starch granules of the germinating Oh43, C103, W64A, and Oh43XC103 ae endosperm mutant lines scarcely showed enzymatic erosion on their surfaces. These observations were similar to the in vitro amylase attack of ae starch granules. On the other hand, enzymatic erosion of starch granules of the B37 ae endosperm was similar to the germinating kernels of the normal B37. The types of enzymatic erosion of endosperm starch granules of the germinating su2 su2o2, wx, and 02 mutants were similar to those of their normalcounterparts.

Comparisons of Lysine and Zein and Nonzein Protein Contents in Immature and Mature Maize Endosperm Mutants1

Maize (Zea mays L.) endosperm mutant shrunken‐4 (sh4) was compared with nine other starch mutants and three “high‐lysine” mutants for lysine and zein and nonzein protein content at 22 days after pollination and at maturity. The sh4 mutant was unique in showing no zein at 22 days and only 10% of the normal value at maturity. Nonzein protein and lysine/kernel were extremely low at maturity in sh4, showing almost no increase after 22 years. The effects of sh4 on protein content appear to be an exaggerated form of those produced by the highlysine mutant opaque‐7.