Endosperm Mutants Research Articles

Segregation for endosperm lysine in F2, F3 and F4 progeny from a cross of in vitro-selected and unselected cultivar of rice

Lysine is a limiting amino acid for optimal nutritional quality in rice grain. In vitro selections using inhibitory levels of lysine plus threonine or s-aminoethylcysteine allow the predictable recovery of variants with elevated levels of lysine and protein. These methods may generate useful starting germplasm for plant breeders. This study was conducted to define the genetics of lysine mutants in progeny from crosses of mutants derived from cells cultured in vitro in the presence of inhibitory levels of lysine plus threonine and s-(2-aminoethyl)-cysteine. In vitro selections produce a wide range of mutants, including endosperm mutants with elevated lysine and protein levels as well as mutants for high and low seed weights. Mutants were analyzed for lysine content by the endosperm half-seed method in which the halves without the embryo were ground and acid hydrolyzed for amino acid determinations. The halves with the embryos were preserved for later germination. In two different F2 populations derived from a cross of a selected mutant x M-101, a parental marker, there was an inverse relationship between seed weight and percent lysine in endosperm protein (R(2) 0.52 and 0.56). The F2 segregation patterns show that elevated lysine is inherited as a recessive gene and that increased lysine is correlated with decreased seed size. F3 and F4 data provide evidence for the transmission of high lysine genes to advanced germplasm in rice. This work supports our earlier conclusions that high lysine phenotypes can be recovered predictably from in vitro selections. The elevated lysine phenotypes are frequently, but not exclusively, associated with opaque seed. Some segregants from crosses produced increased lysine in plants with near normal seed weight and good fertility.

Glutinous (waxy) endosperm starch mutant of Triticum monococcum L.

Cultivars or mutant lines which contain glutinous endosperm starch have been developed in seven crops, including rice, maize, barley, etc. However, reports on Triticum species with glutinous endosperm are not available. Attempts were made to induce the mutation of endosperm glutinousness in Triticum monococcum L. by treatment with EMS. Four hundred seeds of T. Inonococcum were pre-soaked for 20h and treated with 0.4% (v/v) EMS for 24h at room temperataure. Germination percentage was 42.0%After transplanting of the M1 seedlings to a field, 99M1 plants died partly due to late transplanting and severe winter weather conditions. However, 2, 016 M2 seeds were harvested from the fertile 59M1 plants which survived. By staining the endosperm starch of the distal part of the seeds with a KI-I2 solution, six out of 2, 016 seeds showed a red brown color, which is the typical reaction of glutinous endosperm starch to KI-I2. Consequently, these six M2 seeds were considered to be glutinous endosperm mutants. The appearance of these seeds was not different from that of the seeds of normal T. monococcum. Two M2 seeds were also obtained, in which the endosperm starch showed a reddish purple color rather than the typical purple color of non-glutinous starch. The present results indicate that glutinous endosperm mutants can be obtained in Triticum species.

Molecular cloning of two isoinhibitor forms of chymotrypsin inhibitor 1 (CI-1) from barley endosperm and their expression in normal and mutant barleys

Full-length cDNA clones for barley chymotrypsin inhibitor 1 (CI-1) have been isolated from an endosperm-specific cDNA library. Hybridization and nucleotide sequence analyses indicate that these cDNAs represent two distinct types of CI-1 mRNA which we have called CI-1A and CI-1B. Both mRNAs encode polypeptides of 83 residues (M r=8790 and 8960) which differ at eleven positions. The full-length cDNA sequences do not predict N-terminal signal peptide extensions indicating that CI-1 is synthesized in the mature form in contrast to the homologous proteinase inhibitors of tomato and potato. Northern hybridization experiments show that the CI-1 genes are under strict developmental and organ-specific control. CI-1 transcripts were first detected in the developing barley endosperm between 12 and 14 days after anthesis but no CI-1-related sequences were detected in the RNA preparations from shoots, leaves or roots. The expression of CI-1 was also studied in the high-lysine barley mutants Hiproly, Risø 56 and Risø 1508. Approximately 15-fold (Hiproly) and 4-fold (Risø 56 and 1508) higher levels of CI-1 mRNA were detected in the mutant endosperms compared to normal barley. These results correlate well with the increased deposition of CI-1 in the high-lysine lines and show that the differential expression is controlled mainly at the level of transcription or stability of the mRNA. Using Southern-blots of barley DNA we estimate that there are three copies of CI-1 per haploid genome in both normal and mutant barley lines.

The Effects of Endosperm Mutations and Environmental Temperature during Development on the Distribution of Molecular Weight of Amylose in Rice Endosperms

The Effects of Endosperm Mutations and Environmental Temperature during Development on the Distribution of Molecular Weight of Amylose in Rice Endosperms

Effect of Endosperm Type on Pericarp Thickness in Sweet Corn Inbreds1

Sweet corn (Zea mays L.) quality is adversely affected by thick pericarp. Most cultivars of sweet corn in the USA have had sugary (su) endosperm. Recently, other endosperm mutants have become important in sweet corn improvement. Given the close relationship between endosperm and pericarp, it would be useful to determine if endosperm type has any affect on pericarp thickness. The objective of this research was to determine the effects of 10 combinations of endosperm mutants on pericarp thickness in seven sweet corn inbred backgrounds. Endosperm types that may be useful in sweet corn improvement were included. Pericarp thickness of seven sets of near‐isogenic lines that differed in endosperm composition was measured using a pressure micrometer. The endosperm mutants studied were sugary (su), dull (du), waxy (wx), sugary‐2 (su2), and shrunken‐2 (sh2). Pericarp thickness was significantly affected by inbreds, endosperm types, endosperm by inbred interactions, ear, and position of measurement on the kernel. Pericarp thickness of inbreds averaged across endosperm types ranged from 53.3 üm to 33.9 üm. Averaged across inbreds, the endosperm combination Su su2 had significantly thicker pericarp, 48 üm, than all other endosperm types. Endosperm combination su su2 had the thinnest pericarp, 38.5 üm, but was not significantly different from Su sh2 (38.7 üm), su (39.1 üm), or three of the other endosperm combinations. The thicker pericarp associated with Su su2 could adversely affect tenderness. However, the average pericarp thickness of the most widely used new endosperm type, sh2, was not significantly different than the pericarp thickness of standard (su) types, and thus would not be expected to alter pericarp tenderness of converted inbreds.

Morphology and ultrastructure of 11 barley shrunken endosperm mutants

Eleven Na-azide induced barley shrunken endosperm mutants expressing xenia (sex) were characterized genetically and histologically. All mutants have reduced kernel size with kernel weights ranging from 11 to 57% of the wild type. With one exception, the mutant phenotypes are ascribable to single recessive mutant alleles, giving rise to a ratio of 3∶1 of normal and shrunken kernels on heterozygous plants. One mutant (B10), also monofactorially inherited, shows a gene dosage dependent pattern of expression in the endosperm. Among the 8 mutants tested for allelism, no allelic mutant genes were discovered. By means of translocation mapping, the mutant gene of B10 was localized to the short arm of chromosome 7, and that of B9 to the short arm of chromosome 1. Based on microscopy studies, the mutant kernel phenotypes fall into three classes, viz. mutants with both endosperm and embryo affected and with a non-viable embryo, mutants with both endosperm and embryo affected and with a viable embryo giving rise to plants with a clearly mutant phenotype, and finally mutants with only the endosperm affected and with a normal embryo giving rise to plants with normal phenotype. The mutant collection covers mutations in genes participating in all of the developmental phases of the endosperm, i.e. the passage from syncytial to the cellular endosperm, total lack of aleurone cell formation and disturbance in the pattern of aleurone cell formation. In the starchy endosperm, varying degrees of cell differentiation occur, ranging from slight deviations from wild type to complete loss of starchy endosperm traits. In the embryo, blocks in the major developmental phases are represented in the mutant collection, including arrest at the proembryo stage, continued cell divisions but no differentiation, and embryos deviating only slightly from the wild type.

Starch characteristics in cultures of normal and mutant maize endosperm

Vigorously growing suspension cultures of 'normal', amylose-extender (ae) and waxy (wx) maize endosperm were established from near isogenic lines of maize inbred A636. The recovery of the ability to produce vigorous cultures of ae and wx endosperm by backcrossing demonstrate the genetic control of endosperm growth in vitro. Phenotypic expression of the endosperm mutants in culture was studied by examining the properties of starch accumulated in endosperm cultures and starch from developing and mature kernels of the same genotype. After 9 months in culture, the amylose contents of the starch in normal callus tissue and normal endosperm tissue were not significantly different, 28.2% and 31.7%, respectively. Starch granules from normal cultures and endosperm stained blue-black with iodine and were round to polygonal in shape. The starches of wx endosperm and callus cultures contained no amylose, and wx starch granules stained brown-orange with iodine. Although, wx starch granules were primarily round, a few granules with "jagged edges" were observed in starch samples isolated from cultures and kernels. The percent amylose in starch from ae callus was significantly lower than the amylose content of starch from ae endosperm tissue, 39.9% and 67.7%, respectively. Starch granules from ae endosperm and cultures were smaller than normal and wx starch granules. Irregular starch granules which are typical of ae endosperm were present in ae callus tissue, but were less frequently observed. We conclude that specific endosperm mutant phenotypes are expressed in vitro.

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.

The effects of endosperm mutations and environmental temperature during development on the distribution of molecular weight of amylose in rice endosperms.

The effects of endosperm mutations and environmental temperature during development on the distribution of molecular weight of amylose in rice endosperms.

Differential gene expression in the developing barley endosperm

Barley prolamin storage proteins account for 50% of the seed proteins. They are encoded by small multigene families that are only expressed in the developing endosperm. Previous work has shown that the major prolamins in barley are characterized by the presence of two or more unrelated structural domains, one of which contains repeated sequences. The non-repetitive domain is homologous with sequences present in other seed proteins found in the seed of mono- and dicotyledonous plants. Comparison of the 5' flanking region of a B1 hordein storage protein gene of barley with those of other prolamin genes (zeins and α-gliadins) reveals short sequences within 600 base pairs (bp) of the translation initiation codon that are strongly conserved. A short sequence at —300 bp seems to be unique to the prolamin genes and is possibly involved in the control of gene expression in the developing cereal endosperm. Six DNA-binding proteins have been identified that might recognize and interact with the putative regulatory sequences identified in the B1 hordein gene. Protease inhibitors account for a large proportion of the salt-soluble proteins of the barley seed, and contain up to 10% lysine. Cloned cDNAs for chymotrypsin inhibitors 1 and 2 have been isolated and characterized. All contain ochre stop condons in the sequences encoding a putative signal peptide. The two inhibitors are encoded by small multigene families that specify several subfamilies of mRNAs. The accumulation of chymotrypsin inhibitors in normal and mutant endosperms of barley is related to the abundances of their mRNAs.

Effects of the barley mutants Risø 1508 and 527 high lysine genes on the cellular development of the endosperm

The dry grain weight of the Risø barley (Hordeum vulgare L. var. disticum) high lysine mutants 1508 and 527 at maturity was 32 and 37% lower, respectively, than the grains of the cultivar Bomi. Dry grain weight of the double mutant 527/1508 was reduced by 57%. Total number of endosperm nuclei from cv. Bomi, mutants 1508, 527 and 527/1508 at 24 days after anthesis was 173 000,156 000,121 000 and 111 000, respectively. Transverse mid‐grain sections from mutants 1508 and 527 contained fewer aleurone cells and approximately the same number of starchy endosperm cells as cv. Bomi. The cellular organization of the endosperm of the double mutant deviated substantially from the normal. Cell volume in the central starchy endosperm of cv. Bomi, mutants 1508 and 527 at 33 days after anthesis averaged 390 000, 270 000 and 180 000 um5, respectively. Cell volume in the double mutant was smaller than in 527, but could not be accurately estimated due to the irregular shape of the cells. The mean section area of individual large starch granules in the central endosperm of mutants 1508, 527 and 527/1508 at 33 days after anthesis was 30, 48 and 72% smaller, respectively, than those of cv. Bomi. The average aleurone cell volume in cv. Bomi at 33 days after anthesis was 6 200 μm3.

ABNORMAL DEVELOPMENT OF THE SUSPENSOR IN AN EMBRYO‐LETHAL MUTANT OF ARABIDOPSIS THALIANA

Developmental arrest of the embryo proper in aborted seeds from mutant 50B, a recessive embryo‐lethal mutant of Arabidopsis thaliana, was shown to be followed by abnormal growth of the suspensor. Each of the 12 aborted seeds examined in sectioned material contained an abnormally large suspensor and an embryo proper arrested at a preglobular stage of development. Analysis of serial sections revealed that mutant suspensors contained 15–150 cells whereas wild‐type suspensors were composed of only six to eight cells. Development of the mutant endosperm continued to a late nuclear or early cellular stage even in the absence of further development of the embryo proper. These results suggest that the missing gene product in mutant 50B is required for development of the embryo proper but not for continued growth of the suspensor or endosperm tissue. The pattern of abnormal development observed in this mutant provides further evidence that continued growth of the suspensor during normal development is inhibited by the developing embryo proper and that the full developmental potential of cells in the suspensor is expressed only when this inhibitory effect is removed through a mutation or experimental treatment that is lethal only to cells of the embryo proper.

Abnormal Development of the Suspensor in an Embryo-Lethal Mutant of Arabidopsis thaliana

Developmental arrest of the embryo proper in aborted seeds from mutant 50B, a recessive embryo-lethal mutant of Arabidopsis thaliana, was shown to be followed by abnormal growth of the suspensor. Each of the 12 aborted seeds examined in sectioned material contained an abnormally large suspensor and an embryo proper arrested at a preglobular stage of development. Analysis of serial sections revealed that mutant suspensors contained 15–150 cells whereas wild-type suspensors were composed of only six to eight cells. Development of the mutant endosperm continued to a late nuclear or early cellular stage even in the absence of further development of the embryo proper. These results suggest that the missing gene product in mutant 50B is required for development of the embryo proper but not for continued growth of the suspensor or endosperm tissue. The pattern of abnormal development observed in this mutant provides further evidence that continued growth of the suspensor during normal development is inhibited by the developing embryo proper and that the full developmental potential of cells in the suspensor is expressed only when this inhibitory effect is removed through a mutation or experimental treatment that is lethal only to cells of the embryo proper.

DNA amplification patterns in maize endosperm nuclei during kernel development

Increased DNA levels in centrally located endosperm nuclei are shown to be related to endosperm development in Zea mays. Mitotic activity sharply decreases in endosperm cells 10-12 days after pollination. At this time nuclear size and DNA content per nucleus (where C = haploid content) sharply increase until peak levels are reached at about 14-18 days after pollination. Mean DNA content per endosperm nucleus in strain A188 was shown by Feulgen cytophotometry to increase to about 90C by this peak stage, with the pattern being remarkably consistent over four consecutive growing seasons. Some individual nuclei achieved levels of >200C. Most other strains compared during one growing season averaged even higher peak levels of DNA per nucleus than did A188. Individual nuclei in those strains reached levels as high as 690C. A decrease in DNA level was observed in older endosperms with most strains. Endosperm mutant strains did not show a significant reduction in DNA. Opaque-2 mutants in several backgrounds achieved higher levels of DNA per nucleus. DNA levels from F(1) endosperms did not indicate heterosis. Regardless of differences in DNA content, the pattern of DNA increasing as development proceeds followed by a DNA decrease was observed for most strains. Cytological studies reveal much variation in chromatin strandedness, a maximum of three nucleoli, a maximum of three nucleolar organizer regions, and approximately 30 diffuse chromatin masses in older endosperm tissue. A form of DNA amplification, perhaps polytenization, appears to be occurring during endosperm development.

Effect of sucrose accumulation on zein synthesis in maize starch-deficient mutants

Starch-deficient maize ( Zea mays) mutants, brittle-2 ( bt2), brittle-1 ( bt), and shrunken-2 ( sh2), which accumulated large quantities of sucrose, had less than normal amounts of zein (the major storage protein) in the endosperm. Reduction of zein synthesis in the starch-deficient mutants was negatively correlated with the accumulation of sucrose and low osmotic potential in the developing endosperms. When radioactive amino acids were injected into the shank below ears that segregated for the starch-deficient mutant and normal kernels at 28 days post-pollination, mutant kernels absorbed only ca 22–36% of the labelled amino acids found in their normal controls. Thus, a low osmotic potential in the mutant endosperm may favour water movement but reduce solute movement. The inability of amino acids to move into the mutant endosperms, therefore, in part explains the reduction of zein accumulation in starch-deficient mutant endosperms.

The Interaction of Endosperm Genotype and Genetic Background. Part I. Differences in Chromatographic Profiles of Starches from Nonmutant and Mutant Endosperms

AbstractThe effect of maize endosperm genotype and genetic background on variation of endosperm starch properties has been examined by gel filtration. Nonmutant, and single, double and triple mutant combinations of the endosperm genes amylose‐extender (ae), dull (du), sugary (su), and waxy (wx) in four maize inbred lines were compared. The major effects of endosperm genes on starch properties did not vary as a result of genetic background. Starches from wx endosperms contained no amylose. The mutants ae, du and su resulted in starches with increased amylose content. Genetic background did affect starch properties in predictable ways. For example, the production of amylose in mutant endosperms was higher in the dent inbred background, followed by the sweet corn inbreds. However, the production of low molecular weight amylopectin and intermediate polysaccharide fractions was greatest in a sweet corn inbred background, We conclude that the material included in this study will be valuable in future investigations designed to delineate the interaction of genetic background and endosperm genotype in starch biosynthesis and starch properties.

High amylose mutants of rice,Oryza sativa L.

Five mutant lines of rice with increased amylose content in starch granules were identified among floury endosperm mutants. The amylose contents of the mutants ranged from 29.4% to 35.4% and were about twice as high as that of the normal counterpart. Starch properties of the high amylose mutants were analyzed by column chromatography, X-ray diffractometry, photopastegraphy and scanning electron microscopy. The high amylose mutants produced longer unit chains of amylopectin than those of the normal counterpart as well as an increased amount of amylose. A X-ray diffractogram of starch in the mutant was characterized by a type B pattern, while that in the normal counterpart showed a type A pattern which is typical for starches of common cereals. The temperatures at the initiation of gelatinization of the mutants were much higher than that for the normal counterpart. The endosperm cells of the mutant were loosely packed with irregular round-shaped starch granules, whereas those of the normal counterpart were densely packed with polyhedral starch granules. Judging from the results obtained, it was concluded that starch properties of the high amylose mutants of rice were similar to those of the amylose-extender (ae) mutant of maize.

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.

Effect of RNase on Zein Synthesis in Endosperms of brittle-2opaque-2 Maize Double Mutant

Posttranscriptional degradation of zein mRNAs in Zea mays, presumably caused by high RNase activity, may be involved in reducing zein synthesis in the endosperms of zein-deficient mutants. Zein proteins prepared from mature endosperms of zein-deficient double mutants such as brittle-2;opaque-2 showed an almost complete blockage of the synthesis of two major zein components, Z1 and Z2. However, these two components were present prior to 12 days postpollination. The changes in quantity and quality of zein synthesis in these double mutants correlated well with the rising RNase levels in the developing endosperm. In addition to its effect on zein synthesis, high RNase activity also influenced the stability of rRNAs in the brittle-2;opaque-2 double mutant at the later stages of endosperm development. These results suggest that RNase played a role in regulating zein synthesis in developing zein-deficient mutant endosperms.

Zein synthesis in the embryo and endosperm of maize mutants.

Zein synthesis in the developing (22 and 50 days postpollination) endosperm and embryo of maize (Zea mays L.) double mutants, brittle-1; opaque-2 and brittle-2;opaque-2, were compared and correlated with sucrose concentration and RNase activity in order to test the hypothesis that high sucrose concentrations may prevent the interaction between zein polyribosomes and endoplasmic reticulum and make the zein mRNAs more susceptible to hydrolysis by high RNase activity, resulting in a severe reduction in zein synthesis. The double-mutant combinations of opaque-2 with each of the starch-deficient mutants, brittle-1 and brittle-2, maintained not only a high sucrose concentration in the endosperm but also a higher RNase activity than either one of the single mutants alone. Consequently, these double mutants severely suppressed the synthesis of two major zein components in their endosperms. In contrast to the endosperm system, embryos of the double mutants produced amounts of zein (and electrophoretic patterns) similar to that of the opaque-2 embryo, and their embryos contained levels of sucrose and RNase activity comparable to that of the o2 and normal control. These results are consistent with the notion that a posttranscriptional degradation of zein mRNAs by RNase, rather than a specific transcriptional block, is involved in the endosperm to suppress zein synthesis in these double mutants.