Endosperm Mutants Research Articles

Waxy Chlamydomonas reinhardtii: monocellular algal mutants defective in amylose biosynthesis and granule-bound starch synthase activity accumulate a structurally modified amylopectin.

Amylose-defective mutants were selected after UV mutagenesis of Chlamydomonas reinhardtii cells. Two recessive nuclear alleles of the ST-2 gene led to the disappearance not only of amylose but also of a fraction of the amylopectin. Granule-bound starch synthase activities were markedly reduced in strains carrying either st-2-1 or st-2-2, as is the case for amylose-deficient (waxy) endosperm mutants of higher plants. The main 76-kDa protein associated with the starch granule was either missing or greatly diminished in both mutants, while st-2-1-carrying strains displayed a novel 56-kDa major protein. Methylation and nuclear magnetic resonance analysis of wild-type algal storage polysaccharide revealed a structure identical to that of higher-plant starch, while amylose-defective mutants retained a modified amylopectin fraction. We thus propose that the waxy gene product conditions not only the synthesis of amylose from endosperm storage tissue in higher-plant amyloplasts but also that of amylose and a fraction of amylopectin in all starch-accumulating plastids. The nature of the ST-2 (waxy) gene product with respect to the granule-bound starch synthase activities is discussed.

Enhanced Emergence and Seedling Vigor in shrunken-2 Sweet Corn via Seed Disinfection and Solid Matrix Priming

Presowing seed treatments were devised to improve emergence and crop uniformity of two sweet corn (Zea mays L.) cultivars [`Crisp N' Sweet 711' (CNS-711) and `How Sweet It Is' (HSII)] that carry shrunken-2 (sh2) mutant endosperm. The treatments included a fungicide combination, sodium hypochlorite (SH), solid matrix priming (SMP), and SMP combined with SH during treatment (SMPSH). Seed germination was tested in a laboratory cold test. Emergence percentage, emergence rate index (ERI), and seedling dry weight were calculated from field trials. CNS-711, in the cold test and field trials, had a higher germination rate, ERI, final emergence, and seedling dry weight than HSII. In both cultivars, SMPSH significantly improved germination in the cold test and final emergence and ERI in the field trials for HSII compared to nontreated seeds. There was no significant difference between the fungicide and SMPSH treatments regardless of cultivar. These results suggest that the combination of SMP and disinfection with SH can be an alternative seed treatment to fungicides to improve uniformity and stand establishment in sh2 sweet corns.

Infusion of shrunken-2 Sweet Corn Seed with Organic Solvents: Effects on Germination and Vigor

Sweet corn (Zea mays L. var. rugosa Bonaf.) seed carrying the mutant endosperm gene shrunken-2 (sh2) are very susceptible to seed rot and pre- and post-emergence damping off. Experiments were conducted to determine if selected organic solvents were suitable carriers for fungicide infusion of sh2 sweet corn seed for improved germination and stand establishment. Seed of `Florida Staysweet' and `Crisp-n-Sweet 710' were immersed in acetone, cyclohexane, decahydronaphthalene (Decalin), dimethylsulfoxide (DMSO), ethanol, or xylene for 5 seconds, 0.25, 0.50, 1.0, 2.0, 4.0, or 8.0 hours, air-dried, and subjected to a cold-stress test. Total germination and percentage of normal seedlings in both cultivars were significantly decreased after 8 hours of immersion in acetone. Average seedling dry weight, however, did not decrease. DMSO was highly toxic to both cultivars. Ethanol increased seed mortality with increasing immersion times. Cyclohexane, Decalin, and xylene caused erratic responses in all measured variables as immersion time increased. In a second experiment, the effects of immersion time up to 4 hours in acetone on germination and vigor of 11 sh2 cultivars were compared. There was no correlation between cultivar germination or vigor and immersion in acetone. Results indicate acetone could be used to infuse fungicides into the seed of some sh2 cultivars without compromising seed germination or vigor. However, each sh2 cultivar must be screened individually to determine if it is a suitable candidate for organic solvent infusion of fungicides.

Agronomical performance and breeding value of selected strains of diploid wheat, Triticum monococcum

Twenty-one germplasm accessions and breeding lines of einkorn wheat (Triticum monococcum ssp. monococcum and ssp. sinskajae) were grown at two sites in Italy and evaluated for various field and seed characteristics. Grain yields of germplasm accessions were relatively high (317–3238 kg/ha), but distinctly lower than those of four modern cultivars of tetraploid (T. turgidum ssp. durum) and hexaploid wheat (T. aestivum ssp. Aestivum) included in the experiments as controls. As expected, all Einkorns-including some substantially higher yielding crossbred lines (3415–4362 kg/ha)-were defective for one or more agronomically relevant features. However, a few of the accessions examined were found to contain, as a group, practically all the genes needed to breed monococcums having the main field attributes of a modern wheat cultivar: high yielding capacity, good threshability, large kernel size, earliness, short stature and adequate lodging resistance. Still higher yielding diploid wheats, more responsive to improved growing conditions and of better seed quality, could probably be obtained from crosses with wild monococcums bearing mostly two-seeded florets and with accessions producing less slender-shaped kernels. Some of the Einkorns examined were found to carry minor genes for easy threshing which might enhance the efficacy of the major gene for soft glumes carried by T. monococcum ssp. sinskajae, a partially free-threshing diploid wheat taxon. Seed protein percentage of monococcums was markedly higher than that of durum and bread wheat cultivars even in those cases where their grain yields surpassed those of the polyploid checks. The possibilities offered by diploid wheat in the exploitation of novel endosperm mutants and F1 hybrid vigour, as well as in the fields of celiac disease, crop diversification and resistance to agro-biological stresses are discussed. Breeding priorities and strategies are also proposed.

The Maize Invertase-Deficient miniature-1 Seed Mutation Is Associated with Aberrant Pedicel and Endosperm Development.

Genetic evidence is presented to show that the developmental stability of maternal cells in the pedicel at the base of maize seeds is determined by the genotype of the developing endosperm. An early degeneration and withdrawal of maternal cells from the endosperm of homozygous miniature (mn mn) seed mutants were arrested if mn plants were pollinated by the wild-type Mn pollen. Similarly, the stability of the wild-type, Mn mn, maternal cells was also dependent on whether or not these cells were associated with the normal (Mn) or the mutant (mn) endosperm on the same ear. Biochemical and cellular analyses indicated that developing mn kernels have extremely low (<0.5% of the wild type) to undetectable levels of both soluble and wall-bound invertase activities. Extracts from endosperm with a single copy of the Mn gene showed a significant increase in both forms of invertases, and we suggest it is the causal basis of the wild-type seed phenotype. Collectively, these data provide evidence that invertase-mediated maintenance of a physiological gradient of photosynthate between pedicel and endosperm constitutes the rate-limiting step in structural stability of maternal cells as well as normal development of endosperm and seed.

Sweet Corn Cultivars Respond Differentially to the Herbicide Nicosulfuron

Twelve sweet corn (Zea mays L. var. rugosa Bonaf.) cultivars were tested for response to nicosulfuron at rates of 0, 18, 36, and 72 g a.i./ha. Weight of marketable ears indicated that five cultivars were intolerant to the herbicide. Three of the cultivars that were intolerant contained the shrunken-2 endosperm mutant (sh) and two contained the sugary enhancer endosperm mutant (se). Cultivars that were most tolerant of nicosulfuron contained the sh, gene. Incorporation of terbufos insecticide before planting led to decreased marketable yield when nicosulfuron was applied at 36 g·ha in all cultivars tested. Chlorpyrifos insecticide incorporated before planting did not affect tolerance to nicosulfuron. Neither soil-applied insecticide affected yield when nicosulfuron was not applied. Chemical names used: 2-[[[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]amino]sulfony]-N,N-dimethyl-3-pyridinecarboxamide (nicosulfuron); S-[[(1,1-dimethylethyl)thio]methyl] O,O-diethylphosphorodithioate (terbufos); O,O-diethyl O-(3,5,6-trichloro-2-pyridyl)phosphorothioate (chlorpyrifos).

Genetic analysis of endosperm mutants in rice Oryza sativa L.

Sugary, shrunken, floury, white core, amylose extender and dull mutants induced in japonica varieties were used in this study. The results of an allelic analysis conducted in japonica background indicated that the two sugary mutants 82GF and EM5 are allelic. The two amylose extender mutants 2064 and EM16 are also allelic. The opaque mutant ESD7-3(0) and floury mutants 2047, EM17 and EM28 are allelic as well and have the flo-1 gene. The three white core mutants EM3, EM24 and EM66 were found to be non-allelic. Eleven dull mutants were investigated. Dull mutants 2057, 2083, 2091 and EM15 were found to be allelic to each other. Similarly, dull mutants 2077, 2078 and 2120 have allelic genes. Dull mutants 2035, EM12, EM47, and EM98 are non-allelic to the above loci. Dull genes in EM12, EM15, and EM98 were designated earlier as du-1, du-2 and du-4, respectively.The mutant genes were transferred to indica background by two backcrosses to IR36. Some of the mutant genes were located to respective chromosomes through trisomic analysis using primary trisomics of IR36. In this way the amylose extender gene ae was located to chromosome 2, the flo-1 was located to chromosome 5 and the flo-2 to chromosome 4. Dull genes of EM47, 2120, and 2035 were assigned to chromosomes 6, 9, and 6, respectively.

Opaque-2 modifiers increase gamma-zein synthesis and alter its spatial distribution in maize endosperm.

Through the action of opaque-2 modifier genes, the soft, floury endosperm of opaque-2 mutants is converted to a vitreous phenotype. This change in endosperm texture is associated with a twofold to threefold increase in gamma-zein content. To investigate the effect of opaque-2 modifiers on the expression of gamma-zein genes, we analyzed the synthesis and distribution of gamma-zein protein and the level of gamma-zein mRNAs in developing endosperms of the inbreds W64A and W64Ao2, a modified opaque-2 mutant Pool 34 QPM, and their reciprocal F1 hybrids. We also characterized the number and organization of gamma-zein genes in these and related maize genotypes. Our studies show that opaque-2 modifiers are semidominant genes, resulting in a twofold to threefold increase in gamma-zein gene expression in both opaque-2 and normal genetic backgrounds. The increase in gene expression appears to be a consequence of enhanced mRNA transcription or stability rather than gene amplification because gamma-zein genes occur in one or two copies in modified as well as nonmodified genetic backgrounds. Ultrastructural studies showed that gamma-zein occurs in high concentrations in the first few subaleurone cells of nonmodified endosperms, but high concentrations of gamma-zein occur in the subaleurone and central endosperm cells of modified opaque-2 mutants. The increased concentration and distribution of gamma-zein in modified endosperms are highly correlated with the activity of opaque-2 modifier genes.

Characterization of an immunoglobulin binding protein homolog in the maize floury-2 endosperm mutant.

The maize b-70 protein is an endoplasmic reticulum protein overproduced in the floury-2 (fl2) endosperm mutant. The increase in b-70 levels in fl2 plants occurs during seed maturation and is endosperm specific. We have used amino acid sequence homology to identify b-70 as a homolog of mammalian immunoglobulin binding protein (BiP). Purified b-70 fractions contain two 75-kilodalton polypeptides with pl values of 5.3 and 5.4. Both 75-kilodalton polypeptides share several properties with BiP, including the ability to bind ATP and localization within the lumen of the endoplasmic reticulum. In addition, both b-70 polypeptides can be induced in maize cell cultures with tunicamycin treatment. Like BiP, the pl 5.3 form of b-70 is post-translationally modified by phosphorylation and ADP-ribosylation. However, modification of the pl 5.4 species was not detected in vitro or in vivo. Although the b-70 gene is unlinked to fl2, b-70 overproduction is positively correlated with the fl2 gene and is regulated at the mRNA level. In contrast, the fl2 allele negatively affects the accumulation of the major endosperm storage proteins. The physical similarity of b-70 to BiP and its association with abnormal protein accumulation in fl2 endoplasmic reticulum may reflect a biological function to mediate protein folding and assembly in maize endosperm.

Characterization of an Immunoglobulin Binding Protein Homolog in the Maize floury-2 Endosperm Mutant

Characterization of an Immunoglobulin Binding Protein Homolog in the Maize floury-2 Endosperm Mutant

Regulatory mechanisms involved in the biosynthesis of starch

Abstract

Molecular Characterization of the Brittle-2 Gene Effect on Maize Endosperm ADPglucose Pyrophosphorylase Subunits

Activity of the enzyme ADPglucose pyrophosphorylase is known to be reduced in maize (Zea mays L.) endosperm mutants at two independent loci, Shrunken-2 (Sh(2)) and Brittle-2 (Bt(2)). Spinach leaf ADPglucose pyrophosphorylase has previously been shown to comprise two subunits of 51 and 54 kilodaltons. Anti-bodies raised to each of the two subunits of spinach leaf ADPglucose pyrophosphorylase were found to cross-react to different bands on Western blots prepared from polyacrylamide gel electrophoresis separated wild-type maize endosperm proteins. The anti-spinach leaf 51 kilodalton subunit antibody cross-reacted with a 55 kilodalton maize endosperm protein and the anti-spinach leaf 54 kilodalton subunit antibody cross-reacted with a 60 kilodalton maize endosperm protein. These immunological reactions were observed in maize endosperm extracts and with a highly purified preparation of maize endosperm ADPglucose pyrophosphorylase. Mutant bt(2) endosperm lacked the 55 kilodalton subunit while mutant sh(2) endosperm lacked the 60 kilodalton subunit on Western blots. These results suggest that the maize endosperm ADPglucose pyrophosphorylase is made up of two immunologically dissimilar subunits and that the bt(2) and sh(2) mutations cause reduction in ADPglucose pyrophosphorylase activity through the lack of one of these two subunits. An ADPglucose pyrophosphorylase cDNA clone antigenically selected from a rice seed cDNA expression library was found to hybridize strongly with a cDNA corresponding to a maize endosperm transcript which is absent in a W64A bt(2) mutant. Thus, the bt(2) mutant causes the absence not only of the small subunit but of the corresponding transcript. Bt(2) is implicated as the structural gene for the small (54 kilodalton) subunit of maize endosperm ADPglucose pyrophosphorylase.

A novel shrunken endosperm mutant of barley

Although mutations affecting several enzymes of the starch synthetic pathway in developing cereal endosperm have been isolated, none has a major effect on soluble starch synthase We report a new recessive shrunken endosperm mutant in barley (Hordeum vulgare L. cv. Bomi‐like), shx, which has 25% of normal starch content. We have assayed the activity of sucrose synthase (EC 2.4.1.13), ADP and UDP‐glucose pyrophosphorylases (EC 2.7.7.27 and 2.7.7.9), branching enzyme (EC.2.4.1.18), and granule‐bound and soluble starch synthase (EC 2.4.1.21) in shx. Sucrose synthase activity is reduced by 49% and UDP‐glucose pyrrphosphorylase is 80% of the normal level. Branching enzyme and starch‐bound starch synthase activities are normal, but ADP‐glucose pyrophosphorylase activity is reduced by 72%. The soluble starch synthase that is primer‐independent in the presence of sodium citrate shows 14% of normal activity in shx. whereas the primer‐dependent form is unaffected. This lower starch synthase activity in shx cannot be explained by inhibition, substrate destruction or lack of primer. Although several starch‐synthetic enzymes are affected, it is suggested that the primer independent from of soluble starch synthase may be the primary‐site of the mutation in shx.

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.

A novel shrunken endosperm mutant of barley

A novel shrunken endosperm mutant of barley

Agronomic Performance of Sugary‐Brawn2 Maize: A Potential Additive for High‐Protein Silage Production

AbstractAlfalfa (Medicago sativa L.) is low in soluble carbohydrates, which are required for successful silage preservation. Storing alfalfa as silage could reduce quality losses due to weathering, however. Adding grain high in soluble carbohydrates, such as sugary‐Brawn2 (su‐Bn2) endosperm maize (Zea mays L.), improves silage preservation, and results in higher quality feed. However, certain endosperm mutants, including alleles at the sugary locus, have been shown to reduce grain yields, dry matter production, and possibly photosynthetic rates. Our objective was to determine if su‐Bn2 maize hybrids have acceptable yield, ear moisture levels, and stalk and root quality for on‐farm grain production in the northern Corn Belt. Twenty su‐Bn2 hybrids and two adapted dent hybrids were grown at three locations in Wisconsin in 1986 and 1987. Data were collected on ear yield, ear moisture, heat units to midpollen and midsilk, plant and ear height, and root and stalk lodging. Yield of the su‐Bn2 hybrids ranged from 7.26 to 9.41 Mg ha−1, as compared with 7.96 and 9.40 Mg ha−1, for the two checks. Ear moisture of the su‐Bn2 hybrids was higher than that of the checks, although a number of su‐Bn2 hybrids had acceptable moisture levels. The su‐Bn2 hybrids compared favorably with the checks for plant and ear height, and stalk and root lodging. Maize with su‐Bn2 endosperm can be grown successfully in the northern Corn Belt for use as an additive in the ensiling of alfalfa. Management practices and economic aspects need to be further investigated.

Biochemical genetics of the interaction of the lysine plus threonine resistant mutant Ltr ∗1 with opaque-2 maize mutant

The lysine plus threonine (LT) resistant maize mutant Ltr ∗1 selected by culturing maize cells in the presence of lysine plus threonine [1] was transferred by repeated back-crosses to maize inbred lines containing normal, britle (bt), shrunken-2 (sh2) and opaque-2 (o>2) endosperms. Genetic analysis located the Ltr ∗1 gene on the short arm of chromosome 7 at 10.6 centimorgans from the o2 gene. The presence of the Ltr ∗1 gene increased the level of soluble threonine in the normal endosperm by 8–18-fold. A synergistic effect on the increase of soluble threonine was observed when the Ltr ∗1 gene was combined with endosperm mutations. An increase of 45–144-fold in soluble threonine and 3–10-fold in total soluble amino acid pool was observed in the double mutant Ltr ∗1Ltr ∗1/o2o2 when compared with o2 and normal endosperms, respectively. In general it was observed that the Ltr ∗1 gene intensified the effect of the o2 gene on amino acid and protein synthesis in maize endosperm.

Characterization of sugary-1 (su-1) sugary enhancer (se) Kernels in Segregating Sweet Corn Populations

A single-kernel, sugar analysis technique was used to study the genetic relationship between morphological and metabolic traits previously associated with expression of the sugary enhancer (se) endosperm mutation in a su-1 sweet corn (Zea mays L.) background. Analysis of sucrose and total carotene content in su-1 kernel populations segregating for se showed that light-yellow kernel color was a reliable phenotypic indicator for kernels homozygous for the se gene. High levels of kernel maltose was not always indicative of su-1 se kernels in mature (55 days after pollination) kernel populations. Characteristic high levels of percent moisture in su-1 se kernels at 28 and 35 days post-pollination were identified as an expression of high sugar content. Kernels homozygous for su-1 se were also found to weigh less at maturity than su-1 Se kernels, and se was found to be partially expressed in a heterozygous condition.

Response of Sweet Corn (Zea mays) Endosperm Mutants to Chloracetamide and Thiocarbamate Herbicides

Cold stress and field studies were conducted to determine the effects of alachlor, metolachlor, butylate plus dichlormid, and EPTC plus dichlormid on germination and seedling vigor of 30 sweet corn cultivars with three commercially important endosperm mutants (su, se, andsh2). Seedling emergence from cool soils was best when treated with EPTC plus dichlormid and alachlor and was decreased 11% by metolachlor and butylate plus dichlormid. Theseandsh2endosperm mutants were injured the most by the herbicides. Plant size was reduced by the thiocarbamate herbicides but not by the chloracetamides.

Comparative Kernel Structure of Three Endosperm Mutants of Zea mays L. Relating to Seed Viability and Seedling Vigor

Comparative anatomical and histochemical observations were made of mature and germinating kernels of Zea mays `Funks G4646' (Su), `Midway' (su), and two high-sugar endosperm mutants, `Illinois 677a' (su se) and `Illini Xtra Sweet' (sh2), to further the investigation of reduced viability and seedling vigor in these mutants. The embryo of each cultivar was distinct morphologically, but no anatomical abnormalities were seen. The lengths of the scutellum/endosperm interface were reduced for `Illinois 677a' (su se) and `Illini Xtra Sweet' (sh2). `Funks G4646' (Su) and `Illini Xtra Sweet' (sh2) endosperms stained uniformly dense for insoluble carbohydrates, but `Midway' (su) and `Illinois 677a' (su se) stained less densely in the endosperm center. Protein assays showed reduced total protein in the endosperm of `Illini Xtra Sweet' (sh2) and the embryos of the three sugary cultivars. A reduction in starch breakdown was seen in the vicinity of the aleurone in `Illini Xtra Sweet' (sh2) and the scutellar epithelium in `Illinois 677a' (su se); whereas, similar patterns of protein depletion were seen in all the cultivars. The scutellar epithelial cell length of 7-d imbibed kernels of all corn cultivars increased in length but increased significantly more in `Illini Xtra Sweet' (sh2). Endosperm dry weights and volume measurements, as well as endosperm:embryo ratios were reduced in all the sugary cultivars. Significantly reduced lengths of scutellum/endosperm interface, endosperm dry weights and volumes, endosperm:embryo ratios, total protein in the embryos, and total protein in the endosperm of `Illini Xtra Sweet' (sh2) correlated with the reduction in seedling vigor of `Illinois 677a' (su se) and with both viability and seedling vigor in `Illini Xtra Sweet' (sh2). Epithelial cell length increase, as well as dry weights and volumes of the embryos, did not correlate with reduced vigor. The high-sugar endosperm mutants appear to be deficient in hydrolase production-`Illini Xtra Sweet' (sh2) from the aleurone and `Illinois 677a' (su se) from the scutellum.