Mutation of OsALDH7 causes a yellow-colored endosperm associated with accumulation of oryzamutaic acid A in rice

Aldehyde dehydrogenase proteins consist of a superfamily and the family 7 (ALDH7) is a typical group with highly conserved proteins across species. It catalyzes oxidation of α-aminoadipic semialdehyde (AASA) in lysine degradation, participates in protection against hyperosmotic stress, and detoxifies aldehydes in human; however, its function in plants has been much less documented. Here we reported a mutant with yellow-colored endosperm in rice, and showed that the yellow endosperm was caused by mutation of OsALDH7. OsALDH7 is expressed in all tissues detected, with the highest level in mature seeds. We found that oryzamutaic acid A accumulated during late seed development and after a year-long storage in the colored endosperm, whereas it was undetectable in the wild type endosperm. Moreover, lysine degradation was enhanced in yeast over-expressing OsALDH7 and as a result, content of lysine, glutamate and saccharopine was changed, suggesting a role of OsALDH7 in lysine catabolism.     

Genetic manipulation of lysine catabolism in maize kernels

In plants, lysine catabolism is thought to be controlled by a bifunctional enzyme, lysine ketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH). Lysine is converted to saccharopine, through condensation with alpha-ketoglutarate, by LKR, and subsequently to glutamate and alpha-aminoadipate-delta-semialdehyde by SDH. To investigate lysine catabolism in maize kernels, we generated transgenic plants with suppressed LKR/SDH activity in either endosperm or embryo. We found that the suppression of LKR/SDH in endosperm induced an increase in free lysine in developing endosperm, which peaked at 32 days after pollination. At later stages of kernel development, most of the free lysine was found in the embryo along with an elevated level of saccharopine. By combining endosperm LKR/SDH suppression with embryo LKR/SDH suppression through crosses, the saccharopine level in embryo was reduced and resulted in higher lysine accumulation in mature kernels. These results reveal new insights into how free lysine level is regulated and distributed in developing maize kernels and demonstrate the possibility of engineering high lysine corn via the suppression of lysine catabolism.     

Relationship between structural and biochemical characteristics and texture of corn grains

The texture of corn grains is a fundamental characteristic for the industry as well as for grain producers and processors. To further understand the mechanisms involved in grain hardness, contrasting corn cultivars for grain hardness and protein quality were evaluated. The cultivars were Cateto L237/67 (hard endosperm and low protein value), QPM BR 451 (semi-hard endosperm and high protein value); Bolivia-2 (floury endosperm and low protein value), and Opaque-2 (floury endosperm and high protein value). Evaluations were carried out at 10, 20, 30, 40, 50, and 60 days after pollination in developing corn grains and in the mature grain. In developing grains, evaluation consisted in the determination of the area, percentage of starch granules, distribution of starch granules, and protein bodies in the endosperm. In mature corn grains, the parameters evaluated were: density, percentage of total proteins, levels of lysine and tryptophan, and banding pattern of zeins. The results indicate that the higher physical resistance of corn grains from the cultivars analyzed is influenced by the high percentage of total proteins, high synthesis of 27-kDa zeins, presence of protein bodies, and perfect organization of starch granules in the endosperm, independent of their sizes.     

Evidence for the genetic control of lysine catabolism in maize endosperm

A split pollination was used to produce normal (Su su su O2 o2 o2) and high lysine double mutant sugary opaque-2 (su su su o2 o2 o2) endosperms on the same ear of sugary opaque-2 maize plants. Amino acids were determined in the vascular sap of the ear peduncle. Lysine content in the sap was compared with lysine stored in both normal and sugary opaque-2 endosperm during kernel filling. Lysine content in the ear peduncle sap could account for all lysine found in both endosperms. Preformed lysine is highly catabolized in the normal endosperm, but not in the high lysine sugary opaque-2 endosperm. The rate of lysine breakdown appears to be an important mechanism by which the high lysine mutant controls lysine level in maize endosperm.     

Tissue-specific and light-dependent changes of chromatin organization in barley (Hordeum vulgare)

The DNase I sensitivity of the nuclear genes encoding the NADPH-protochlorophyllide oxidoreductase, the light-harvesting chlorophyll a/b protein (LHCP), the hordeins and a 15-kDa protein of unknown function was assayed in chromatin of etiolated and green leaves and endosperm tissue of barley (Hordeum vulgare L.). A tissue-specific differentiation of chromatin structure was found for the LHCP, hordein and 15-kDa protein genes. The genes for the LHCP and the 15-kDa protein, which are expressed in leaf tissue, display DNase I sensitivity in leaves but not in endosperm. Hordein genes which are expressed solely in endosperm, were insensitive to low levels of digestion with DNase I in leaves but sensitive in endosperm. The effect of light on chromatin structure was determined by comparing leaves of etiolated plants and plants which had been grown under a day/night cycle. Only in the case of the 15-kDa protein is there a remarkable change from a DNAse-I-sensitive configuration in etiolated leaves to a more resistant one in leaves from illuminated plants. The gene for the NADPH-protochlorophyllide oxidoreductase was found to be equally sensitive to DNase I in leaves and endosperm.     

Partial purification and characterization of lysine-ketoglutarate reductase in normal and opaque-2 maize endosperms

Lysine-ketoglutarate reductase catalyzes the first step of lysine catabolism in maize (Zea mays L.) endosperm. The enzyme condenses l-lysine and α-ketoglutarate into saccharopine using NADPH as cofactor. It is endosperm-specific and has a temporal pattern of activity, increasing with the onset of kernel development, reaching a peak 20 to 25 days after pollination, and there-after decreasing as the kernel approaches maturity. The enzyme was extracted from the developing maize endosperm and partially purified by ammonium-sulfate precipitation, anion-exchange chromatography on DEAE-cellulose, and affinity chromatography on Blue-Sepharose CL-6B. The preparation obtained from affinity chromatography was enriched 275-fold and had a specific activity of 411 nanomoles per minute per milligram protein. The native and denaturated enzyme is a 140 kilodalton protein as determined by polyacrylamide gel electrophoresis. The enzyme showed specificity for its substrates and was not inhibited by either aminoethyl-cysteine or glutamate. Steady-state product-inhibition studies revealed that saccharopine was a noncompetitive inhibitor with respect to α-ketoglutarate and a competitive inhibitor with respect to lysine. This is suggestive of a rapid equilibrium-ordered binding mechanism with a binding order of lysine, α-ketoglutarate, NADPH. The enzyme activity was investigated in two maize inbred lines with homozygous normal and opaque-2 endosperms. The pattern of lysine-ketoglutarate reductase activity is coordinated with the rate of zein accumulation during endosperm development. A coordinated regulation of enzyme activity and zein accumulation was observed in the opaque-2 endosperm as the activity and zein levels were two to three times lower than in the normal endosperm. Enzyme extracted from L1038 normal and opaque-2 20 days after pollination was partially purified by DEAE-cellulose chromatography. Both genotypes showed a similar elution pattern with a single activity peak eluted at approximately 0.2 molar KCL. The molecular weight and physical properties of the normal and opaque-2 enzymes were essentially the same. We suggest that the Opaque-2 gene, which is a transactivator of the 22 kilodalton zein genes, may be involved in the regulation of the lysine-ketoglutarate reductase gene in maize endosperm. In addition, the decreased reductase activity caused by the opaque-2 mutation may explain, at least in part, the elevated concentration of lysine found in the opaque-2 endosperm.     

Some Aspects of Nitrate Assimilation in the Seedlings of Normal and Opaque-2 Mutant of Maize

The effects of 10 mM nitrate on the growth and nitrogenous componentsof Zea mays L. var. W64A wild type (normal) were compared tothose on its opaque-2 (high lysine) mutant during the first10 d of seedling growth at a constant temperature of 26 °Cand with a 16 h photoperiod. Nitrate supply had no effect onthe growth of embryonic axes in both lines till day 6. Growthof both lines was enhanced slightly after that time, however.Increases in 80% (v/v) ethanolsoluble and protein nitrogen werealso observed only after day 4 when the supply of nitrogen fromthe storage proteins in the endosperm was limiting. Nitratehad no effect on the synthesis of chlorophyll during leaf developmentbut it did increase the total chlorophyll in mature and senescingprimary leaves. The increase in nitrogenous components or chlorophyllin opaque-2 was more pronounced than in the normal type. Itmight be related to the lower proline or higher lysine in themutant.     

Protein and free amino acids in a high lysine maize double mutant

A comparative study of free amino acids and protein fractions of normal with a double mutant (su₁ o₂) was made, during endosperm development in segregating ears of a maize synthetic. Zein content showed striking differences in the two genotypes, being 7.7 and 6 times greater in the normal endosperm at 24 and 47 days after pollination respectively. This observed decrease in zein synthesis, coded by sugary-1/opaque-2 genes, causes an accumulation of alanine, glutamic and aspartic acids, glutamine and asparagine in the high lysine endosperm mutant.     

The dynamic gene expression from different genetic systems for protein and lysine contents of indica rice

The dynamic expression of genes for protein and lysine contents of rice grain under different environments was carried out with time-dependent measures by using the developmental genetic models for quantitative traits of triploid endosperm in cereal crops. The results showed that the genetic effects, especially genotype × environment (GE) interaction effects from the genes expression of different genetic systems including triploid endosperm, cytoplasm and diploid maternal plant were important for the performance of both nutrient quality traits at all developmental times/stages of rice grain. The conditional genetic variance analysis found that the activation of quantitative genes especially from endosperm and maternal plant genetic systems for protein and lysine contents was gradually carried through the developmental process of rice grain. The net genetic effects showed that the new expression of quantitative genes for protein and lysine contents was more active at late filling stage (15–21 days after flowering) and maturity stage (22–28 days) of rice grain. Also the sequential expression of cytoplasmic genes cannot be ignorable for the development of nutrient quality traits. The phenomena that some genes could continuously express for several developmental stages or the genes expression could be interrupted among developmental stages of rice grain was detected especially for net endosperm additive main effects or maternal additive main effects. The differences of genetic relationships from different genetic systems were found for protein and lysine contents among developmental times of rice grain.     

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

Summary 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 (R² 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.Research done under the auspices of the USDA, ARS, Plant Sciences Institute, Plant Molecular Biology Laboratory, Beltsville, MD 20705, USA     

Synthesis and degradation of proteins during wheat endosperm development

Synthesis of proteins rich in lysine declines progressively with endosperm development and these proteins appear to be degraded preferentially at later stages. The proteolytic enzymes in extracts of endosperms at a late stage of development release considerably more lysine radioactivity from labelled endosperm proteins as compared with the enzymes in endosperms at an early stage.     

Regulation of maize lysine metabolism and endosperm protein synthesis by opaque and floury mutations.

The capacity of two maize opaque endosperm mutants (o1 and o2) and two floury (fl1 and fl2) to accumulate lysine in the seed in relation to their wild type counterparts Oh43+ was examined. The highest total lysine content was 3.78% in the o2 mutant and the lowest 1.87% in fl1, as compared with the wild type (1.49%). For soluble lysine, o2 exhibited over a 700% increase, whilst for fl3 a 28% decrease was encountered, as compared with the wild type. In order to understand the mechanisms causing these large variations in both total and soluble lysine content, a quantitative and qualitative study of the N constituents of the endosperm has been carried out and data obtained for the total protein, nonprotein N, soluble amino acids, albumins/globulins, zeins and glutelins present in the seed of the mutants. Following two-dimensional PAGE separation, a total of 35 different forms of zein polypeptides were detected and considerable differences were noted between the five different lines. In addition, two enzymes of the aspartate biosynthetic pathway, aspartate kinase and homoserine dehydrogenase were analyzed with respect to feedback inhibition by lysine and threonine. The activities of the enzymes lysine 2-oxoglutate reductase and saccharopine dehydrogenase, both involved in lysine degradation in the maize endosperm were also determined and shown to be reduced several fold with the introduction of the o2, fl1 and fl2 mutations in the Oh43+ inbred line, whereas wild-type activity levels were verified in the Oh43o1 mutant.     

Induction of Nitrate Reductase and Peroxidase Activity in the Seedlings of Normal and High Lysine Maize

Steady state levels of in vivo nitrate reductase activity in the endosperm, scutella, roots and shoots of maize seedlings were higher in normal maize than those in high lysine maize. Activity of peroxidase in the roots, however, was higher in the high lysine cultivar. The nitrate reductase activity increased with the supply of nitrate in all parts of the seedlings of both cultivars, although the rates of increment in the endosperm were lower than those in scutella, roots and shoots. In relation to substrate concentration, a saturation was achieved at 5 to 10 mM of nitrate except in the endosperm, where activity increased slowly up to 100 mM at least. Final levels of enzyme activity were significantly higher in the scutella of normal than in that of high lysine seedlings. In vitro enzyme activity in the roots also increased with the supply of nitrate in both cultivars, reaching maximum at 5 to 10 mM nitrate.     

玉米赖氨酸相关基因opaque-2及其修饰基因研究进展

玉米隐性突变o2基因能通过减少醇溶蛋白的合成来显著提高赖氨酸含量,为培育高赖氨酸含量的优质蛋白玉米（quality protein maize, QPM）提供了良好的基因资源。对玉米o2基因的发现、研究现状及其修饰基因的研究进展,以及当前育种家利用这两种基因相互作用培育优质蛋白玉米的研究进展进行了综述,以期为高赖氨酸玉米育种提供参考。     

Increased lysine content in rice grains by over-accumulation of BiP in the endosperm

Over-accumulation of lysine-rich binding protein (BiP) in the rice endosperm caused strong endoplasmic reticulum (ER) stress and reduced seed storage proteins, resulting in a relative increase in nutritionally balanced non-seed storage proteins. We show that transgenic rice with over-accumulated BiP was a high-lysine rice germplasm and that the over-accumulation of BiP in the endosperm offered a unique strategy to improve the lysine content of cereal grains.     

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.