Changes in isoenzymes in embryo and endosperm of normal and opaque-2 Zea mays during imbibition

Electrophoretic patterns of soluble proteins, peroxidase, esterase, alcohol dehydrogenase (ADH) and glutamic dehydrogenase (GDR) from embryos and endosperm of normal and opaque-2 maize were studied after different periods of imbibition. The soluble protein pattern from endosperm of normal and opaque-2 differed both qualitatively as well as quantitatively. The embryo protein patterns were identical. Multiple forms (isoenzymes) were found for all the enzymes studied. The enzyme patterns changed during imbibition. Peroxidase and GDH patterns from embryos of normal and opaque-2 showed considerable differences during imbibition. Esterase and ADH pattern from embryo and endosperm of normal and opaque-2 showed few differences.     

Effect of drought on proteins and isoenzymes in rice during germination

Two drought tolerant varieties TKM-1 and TKM-2 and two drought susceptible varieties Jaya and Improved Sabarmati of rice were studied for soluble protein pattern and isoenzymes of malate dehydrogenase, glutamate dehydrogenase, esterase and peroxidase during germination at different water stress. MDH, GDH and esterase patterns were not affected, but the soluble proteins were changed. Peroxidase isoenzyme pattern from drought tolerant and susceptible varieties showed characteristic differences. The intensity of bands with higher electrophoretic mobility decreased in Jaya and Improved Sabarmati while in TKM-1 and TKM-2 the intensity of these bands did not change much after 72 hr water stress. In shoots of Jaya and Improved Sabarmati, the activity of the peroxidase isoenzymes decreased more than in TKM-1 and TKM-2 shoots with increase in water stress.     

Ribonuclease Activity in Normal, opaque-2, and floury-2 Maize Endosperm during Development

The elevated ribonuclease activity produced in the endosperm of a maize (Zea mays L.) inbred, W64A, by homozygous opaque-2, results from a more than doubled rate of ribonuclease accumulation occurring prior to 16 days post-pollination; after 16 days the rates in opaque-2 and normal are the same, suggesting that opaque-2 is no longer active. The pattern of ribonuclease increase in the opaque-2 dosage series indicates that opaque-2 is not fully recessive. Ribonuclease accumulation is not affected by floury-2 in a second inbred, B14. The results are discussed with reference to other proteins, notably zein, the net synthesis of which is affected by opaque-2.     

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.     

Characterization of polysomes and incorporation in vitro of leucine and lysine in normal and opaque-2 zea mays endosperm during development

Polysome preparations obtained from opaque-2 and normal maize endosperms during development did not show any significant difference in sedimentation coefficient or nucleotide composition. The pattern of incorporation in vitro of lysine and leucine, however, differed quite distinctly in these two preparations. During early stages of maturity the polysomes from opaque-2 incorporated substantially more lysine and less leucine as compared with those from normal maize. Although the trend was reversed at 25 days post-pollination, this did not result in any significant zein accumulation since very little total protein was synthesized after that stage in opaque-2 maize endosperm. It is, therefore, suggested that the opaque-2 gene exerts a regulatory control on mRNA synthesis, required for zein formation at early stages of maturation.     

Metabolism of acetate-14C in normal and opaque-2 Zea mays endosperm during development

Acetate-[2-¹⁴C]metabolism by developing normal and opaque-2 maize endosperms showed considerable differences in incorporation of label into organic     

离子注入对萝卜过氧化物酶、淀粉酶和蛋白酶同工酶的影响

研究了氮离子和氩离子注入萝卜种子对萝卜幼苗蛋白含量、过氧化物酶活性及过氧化物酶、淀粉酶和蛋白酶同工酶的影响。结果表明 :离子注入后 ,减低萝卜过氧化物酶活性和蛋白含量。萝卜不同生长时期同工酶变化不一样。在子叶时期 ,过氧化物酶同工酶谱带无明显变化 ,淀粉酶同工酶有酶带的消失 ;而在真叶时期 ,过氧化物酶在负极区减少一条酶带 ,Rf为 0 .2 2 ,正极区增加一条酶带 ,Rf为 0 .6 ,且随剂量增加 ,酶带着色增强 ;淀粉酶同工酶在注入剂量为 5× 10 5N+ / cm2 )时 ,有同工酶带增加 ,Rf为 0 .6 1。低剂量时蛋白酶活性增强 ,谱带增多 ,大剂量则减弱。因此 ,N+和 Ar+注入后 ,可影响萝卜过氧化物酶和淀粉酶的表达及蛋白质的合成或降解。     

Transaminase GOT and GPT activity in extirped sprouts of normal and opaque-2 Maize (Zea mays L.) seedlings

The increased activity of GOT (E.C.2.6.1.1.) and GPT (E.C.2.6.1.2.) transaminases in maize seedlings found as a marker of genotype opaque-2, was investigated in extirped sprouts of both genotypes, normal and opaque-2. The enzymatic activity was determined in three maize samples from breeding experiments, each sample consisting of a genotype pair, normal and opaque-2, collected from segregating ears of maize plants in the S₁ generation. The seedlings were aseptically grown for 7 days in two variants of cultivation, intact seedlings and sprouts extirped after 4 days of germination. In the intact seedlings of genotype opaque-2 an increased activity of GOT and GPT, as compared to the intact normal plants, was observed. The extirpation of the sprouts enhanced GOT and GPT activity in the sprouts of both genotypes. However, in extirped sprouts the GOT and GPT activity was found to be still higher in the genotype opaque-2 as compared with the sprouts of normal genotype. Thus it seems that the increased transaminase activity in the sprouts of genotype opaque-2 is genetically determined. The increase does not result from an induction of enzyme synthesis through the supply of amino acids translocated from the endosperm to the sprouts. The absolute level of transaminases in the different breeding samples is dependent on the parenteral lines, the relative level of GOT and GPT activities is higher in the genotype opaque-2.     

大葱组织培养中玻璃苗特性研究

以章丘大葱为试验材料,从形态组织学及生理生化等方面探讨了大葱试管苗玻璃化发生的可能机理。结果表明,玻璃苗形态组织结构与正常苗有很大差异,玻璃苗组织含水量增加,过氧化物酶(POD)活性增强,叶绿素总量、叶绿素a、叶绿素b、过氧化氢酶(CAT)的活性、可溶性蛋白含量均降低。玻璃苗酯酶同工酶谱带与正常苗相比,增加了一条带,同时缺少了两条带;可溶性蛋白电泳分析表明,玻璃苗与正常苗间也存在明显差异。可见,当外界环境条件不适宜植物生长需要时,植物体在基因表达水平上将会受到影响,表达产物的差异可引起生理代谢功能上的紊乱,从而导致形态结构的畸型,进而产生玻璃化苗。     

Genetic regulation of storage protein content in maize endosperm

Sodium dodecylsulfate-polyacrylamide gel electrophoresis reveals that zein prepared from normal maize inbred (Zea mays L.) contains six separable components. Z1 and Z2 are the predominant species, with molecular weights of 21,800 and 19,000 daltons. Amino acid analysis of these two components shows that both are rich in glutamic acid, leucine, and proline, but low in lysine. Of the four minor bands, Z3, Z4, Z5, and Z6, the latter two exist only in trace amounts. A mutation at the opaque-2 locus severely suppresses the synthesis of Z1. The nonallelic mutant, opaque-7, strongly suppresses the synthesis of Z3 and Z4, while slightly reducing Z2. On the other hand, the floury-2 mutant appears to reduce the synthesis of these six proteins in the same relative proportion. In the double mutant combinations, opaque-2 apparently is epistatic to opaque-7 and floury-2 in the synthesis of zein components. The glutelin fraction shows a more complex banding pattern; however, qualitative differences are not apparent among the mutant lines examined.This research was supported in part by a grant from the Lilly Endowment.Journal Paper No. 6100 of the Purdue University Agricultural Experimental Station.     

Purification and properties of an endospermic protein of maize associated with the Opaque-2 and Opaque-6 genes

Maize endosperms accumulate during development a large amount of storage proteins (zeins). The rate of zein accumulation is under the control of several regulatory genes. Two of these, the opaque-2 and opaque-6 mutants, lower the zein level, thus improving the nutritional quality of maize meals. An endosperm protein of Mr 32 000 (b-32) appears to be correlated with the zein level. The b-32 protein is encoded by the opaque-6 gene which, in turn, is activated by opaque-2. We report the purification, amino-acid composition and peptide map of b-32 protein. Furthermore we demonstrate that the protein exists as a monomer likely located in the soluble cytoplasm. As a step towards the isolation of a complementary-DNA clone for b-32 protein, the purification of its corresponding mRNA is described.Abbreviations b-32 endosperm protein of M_r 32000 - cDNA complementary DNA - EDTA ethylenediaminetetraacetic acid - O2, O6 opaque 2, opaque-6 genes - PMSF phenylmethylsulfonylfluoride - RSP reduced soluble proteins - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Genomic Organization of α-Amylase Gene in High Lysine Opaque-2 Maize

Genomic DNA was isolated from 7-day old etiolated seedlings of normal and high lysine opaque-2 maize and purified by CsCl gradient. Purified DNA was found to be ～48.5 kb in size. Restriction analysis of genomic DNA with EcoRI and HindIII did not reveal any noticeable difference between normal and opaque-2 DNA. Southern blot analysis, using α-amylase probe, showed multiple bands. One of the hybridizing bands (～4 kb) of genomic DNA was more intense in opaque-2 than in normal. This DNA was cloned into pUC 18 plasmid and presence of α-amylase was confirmed by Southern hybridization using α-amylase probe.     

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.     

Changes in peroxidase activity and isoenzymes in spruce needles after exposure to different concentrations of cadmium

We studied the guaiacol peroxidase activity, isoenzyme pattern and metal content in the needles of 2-year-old spruce grown on soils supplemented with cadmium concentrations from 1 to 21 mg kg(-1). Following exposure to cadmium, an initial increase and subsequent decrease in the activity of the soluble fraction was observed. A parallel change of their isoenzyme pattern occurred. An increase of the cell wall-bound peroxidase activity under prolonged metal treatment was evident. The results obtained show that peroxidase activity and isoenzyme pattern could be used to evaluate the capacity of one part of the defense system in spruce seedlings to withstand metal stress.     

Storage Protein Synthesis in Maize: II. Reduced Synthesis of a Major Zein Component by the Opaque-2 Mutant of Maize

Two zein proteins (Z1 and Z2) represent the majority of the protein synthesized during maize endosperm development. Undegraded membrane-bound polysomes isolated from normal maize synthesized these proteins when incubated in a cell-free protein-synthesizing system from wheat germ. The proteins synthesized in vitro were similar to authentic zein in ethanol solubility and electrophoretic mobility. Zein synthesis was associated with large size classes of membrane bound polysomes in normal maize.Membrane-bound polysomes isolated from developing kernels of opaque-2 mutant synthesized less total zein in vitro, and dramatically reduced incorporation into the Z1 component. The reduction in total zein corresponded to a 50% reduction in the level of membrane-bound polysomes in opaque-2, and the near absence of the large polysome size classes, which synthesized zein in normal maize. We concluded that the opaque-2 mutation results in a decreased "availability" of the zein mRNAs, reflected in a reduced level of membrane-bound polysomes.     

四个普通小麦同核异质系的生化标记研究

以４上个普通小麦同核异质系－可育系Ｄ^2-CA8057(BC14),D^2型不育系msD^2－ＣＡ８０５７（ＢＣ１１）、ＹＡ型不育系msA-CA8057(BC12)、CA8057(核亲本）为材料,采用ＲＡＧＥ方法比较了４个系灌浆期种子胚乳和“花粉双核期”花药的过氧化物酶同工酶,采用梯度ＳＤＳ－ＰＡＧＥ方法比较了４个系不同发育时期（干种子胚乳、越冬前和越冬后苗期叶片、“花粉双核期”旗叶、“花粉双核期”花药）可溶性蛋白质的组份,发现可育系之间差异较小、不育系与可育系之间,D^2型不育系与ＹＡ型不育系之间的存在显著差异,表明细胞质（不育）基因的表达具有时空性和特异性,这些差异（表达）带作为细胞质（不育）基因对核基因的特异调控表达产物,可以作为４个系的生化标记,同时也表明msD^2-CA8057和msA-CA8057是２个不同的新不育类型。     

油茶扦插生根的生化特性

以4年生‘岑软2号’油茶(Camellia oleifera ‘Cenruan 2’)的半木质化枝条为材料,采用不同生长调节剂处理插穗,测定扦插生根过程中插穗过氧化物酶(POD)、可溶性蛋白质及叶绿素含量的动态变化。结果表明,扦插后不同时间的POD活性、可溶性蛋白质和叶绿素含量均有极显著差异(P<0.01),且呈规律性变化,POD活性呈“上升–下降–上升–下降”双峰变化趋势,可溶性蛋白质呈“上升–下降–上升”变化趋势,叶绿素含量呈“下降–上升–下降–上升”变化趋势;与对照相比,经生长调节剂处理的插穗POD活性、可溶性蛋白质和叶绿素含量的峰值显著提高,说明其促进油茶插穗生根。     

Some genetic and biochemical characteristics of a new source of maize opaque-2 mutant

Summary The opaque kernels separated from the F₁ of crosses of our opaque-2 strains with normal inbred lines contained 45, 50 and 74 percent more lysine in the whole kernel than the translucent kernels from the same ear. The opaque kernels from these crosses contained almost the same level of lysine as the parental opaque-2 strains.Some of our opaque-2 strains contain 63 to 122 percent more lysine than the tested dent or flint normal inbred lines. In comparison with opaque-2 Purdue, four opaque-2 strains had almost the same lysine content and a strain labelled as SP-1 No. 15 contained 25 percent more lysine and 73 percent more tryptophan. Some of our opaque-2 strains contained 55 to 100 percent more tryptophan in the whole kernel than opaque-2 Purdue.In contrast to lysine content, the transmission of the tryptophan content from opaque-2 strains to opaque kernels from their crosses shows variability.We found weak positive correlation (r = +0,3057) between lysine and tryptophan content in opaque-2 kernels.Our opaque-2 strains had a higher lysine content in the endosperm than did opaque-2 Purdue 22.1 – 36.6% more, and compared with normal lines they had 109.5 – 142.6% more.It is apparent that a new source of opaque-2 mutant gene, which has the same genetic and biochemical characteristics as the opaque-2 mutant discovered by Mertz, Bates and Nelson (1964), has been found in a completely new, genetically divergent, strain derived from a large number of populations.

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Zusammenfassung Die vorgenommenen Prüfungen haben gezeigt, daß undurchsichtige Körner, die nach F₁-Kreuzung unserer Opaque 2-Linien mit Normallinien ausgelesen wurden, 45, 50 und sogar 74% mehr Lysin im ganzen Korn als normale durchsichtige Körner desselben Maiskolbens enthalten. Der Lysingehalt der undurchsichtigen Körner aus diesen Kreuzungen entspricht annähernd dem der Opaque 2-Elternlinien.Einige unserer Opaque 2-Linien enthalten 63 bis 122% mehr Lysin als die untersuchten normalen Inzuchtlinien. Im Vergleich zu Opaque 2 — Purdue weisen unsere 4 Opaque 2-Linien einen etwa gleichen Lysingehalt auf, während die SP-1 No. 15 25% mehr Lysin und 73% Tryptophan enthält. Unsere Opaque 2-Linien zeigen im ganzen Korn 55 bis 100% mehr Tryptophan als Opaque 2 — Purdue.Im Unterschied zum Lysingehalt zeigt die Übertragung des Tryptophangehalts von Opaque 2-Linien auf undurchsichtige Körner bei den Kreuzungen Variabilität.Wir fanden eine schwach positive Korrelation (r = + 0,3057) zwischen dem Lysin- und Tryptophangehalt bei den Opaque 2-Köinern.Unsere Opaque 2-Linien hatten mehr Lysin im Endosperm als Opaque 2-Purdue, und zwar 22,1 bis 36,6% bzw. 109,5 bis 142,6% im Vergleich zu den normalen Linien.Offensichtlich ist eine neue Quelle für das mutante Opaque 2-Gen in einer ganz neuen, genetisch unterschiedlichen Synthese einer großen Anzahl von Populationen entdeckt worden. Die neue Quelle hat dieselben genetischen und biochemischen Merkmale wie die Opaque 2-Mutante, die von Mertz, Bates und Nelson (1964) aufgefunden wurde.