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.     

Storage Protein Synthesis in Maize: Isolation of Zein-synthesizing Polyribosomes

Undegraded free and membrane-bound polysomes were isolated from developing kernels of Zea mays L. frozen in liquid nitrogen. Freezing in liquid nitrogen was a prerequisite for preserving polysome structure in stored kernels. Membrane-bound polysomes from 22-day post-pollination kernels ground in high pH buffers containing 50 mm Mg(2+) contained unique classes of large polysomes. These large polysomes were sensitive to ribonuclease, and electron micrographs verified that they were not formed by aggregation. The membrane-bound polysomes were the principal site of zein synthesis, since the major protein synthesized in vitro was similar to purified zein in its ethanol solubility and mobility on sodium dodecyl sulfate polyacrylamide gels.     

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.     

在条锈菌侵染早期小麦初生叶内多聚核糖体水平与蛋白质合成能力的变化

小麦初生叶接种条锈菌毒性生理小种（ＣＹ２９）及其弱毒突变菌系（ＣＹ２９－ｍｕｔ３）后,呈不亲和反应的寄主叶片可溶性蛋白质合成能力在接种后２４ｈ显著高于未接种对照,但其后逐渐降低,直至接近对照;而呈亲和性反应的寄主叶片可溶性蛋白质合成在侵染早期与对照相近,但与膜结合蛋白质在９６ｈ时大大高于对照。对接种叶中核糖体的密度梯度分析证实：呈不亲和反应寄主叶片游离多聚核糖体及亲和反应的寄主内与膜结合多聚核糖体均有特异性增加。上述结果表明寄主的抗病和感病反应均与蛋白质合成能力的变化有关。     

Developmental Changes in Ganglioside Composition and Synthesis in Embryonic Rat Brain

Developmental changes in ganglioside composition and biosynthesis was studied in rat brain between embryonic day (E) 14 and birth. In E14 brains, GM3 and GD3 were predominant. At E16, "b" series gangliosides, such as GD1b, GT1b, and GQ1b, increased in content. After E18, "a" series gangliosides such as GM1, GD1a, and GT1a increased in content, and the content of GM3 and GD3 markedly decreased. Because of these changes in composition, we determined the activities, in homogenates of embryonic brains, of two key enzymes of ganglioside synthesis: sialyltransferase for the synthesis of GD3 from GM3 and N-acetylgalactosaminyltransferase for GM2 synthesis from GM3. The sialyltransferase activity (GM3----GD3) was constant between E14 and E18 but decreased rapidly from E18 to birth. In contrast, the N-acetylgalactosaminyltransferase activity (GM3----GM2) increased between E14 and E18 but was constant from E18 to birth. These changes in ganglioside composition and enzymatic activities indicate that during development there is a shift from synthesis of the simplest gangliosides of the "a" and "b" pathways to synthesis of the more complex gangliosides.     

Polyribosomes from Peas: II. Polyribosome Metabolism during Normal and Hormone-induced Growth

Polyribosomes as large as 10-mers (strands of messenger RNA bearing 10 ribosomes) were isolated from etiolated pea (Pisum sativum L. var. Alaska) stem tissue during all stages of development when methods were used which essentially eliminated ribonuclease activity during extraction. Actively growing tissue, harvested from the apical 10 mm, yielded many large polyribosomes and a low (<20%) proportion of monosomes. Similar tissue, allowed to age by applying lanolin to decapitated apices, showed a progressive decrease in number of larger polyribosomes and an increase in the proportion of monosomes. Hormone treatments, which prolonged growth and delayed aging, delayed the loss in large polyribosomes and the increase in proportion of monosomes. Growth-stimulating hormones, added to previously aged tissue, stimulated the production of many large polyribosomes in pre-existing cells.     

Differences in the Responses to Water Stress of Growing and Non-Growing Regions of Maize Mesocotyls: Protein Synthesis on Total, Free and Membrane-Bound Polyribosome Fractions

The growing and non-growing regions of the mesocotyl of youngmaize seedlings show different responses to water stress withrespect to their ability to retain polyribosomes. The growingregion shows a marked reduction in total polyribosomes, althoughsome are retained within the stressed tissue. In the non-growingregion there is little stress-induced change to the total polyribosomecontent. These results support the contention that physiologicallyyounger, growing tissues are more sensitive to water stressthan more mature tissues that have ceased to grow. The free(FP) and membrane-bound (MBP) polyribosome content of the growingmesocotyl region is considerably greater than that of the non-growingregion, which is indicative of their more active metabolism.Both the MBP and FP fractions decline when the cells of thegrowing region are subjected to stress: no differential sensitivityis evident. Hence any qualitative changes in protein synthesisinduced by stress must be expected to result from changes inactivity of both polyribosome fractions. In the stressed non-growingregion, FP decline slightly, but MBP exhibit no consistent changes.     

Increasing Lysine Content of Waxy Maize through Introgression of Opaque-2 and Opaque-16 Genes Using Molecular Assisted and Biochemical Development

The low lysine content of waxy maize cannot meet the nutritional requirements of humans, livestock, or poultry. In the present study, the high-lysine genes o2 and o16 were backcrossed into wx lines using the maize high-lysine inbreds TAIXI19 (o2o2) and QCL3021 (o16o16) as donors and the waxy maize inbred line QCL5019 (wxwx) as a receptor. In the triple-cross F₁, backcross, and inbred generations, the SSR markers phi027 and phi112 within the wx and o2 genes and the SSR marker umc1121 linked to the o16 gene were used for foreground selection. Background selection of the whole-genome SSR markers was performed for the selected individuals. The grain lysine content was determined using the dye-binding lysine method. The waxiness of the grain was determined with the I₂-KI staining and dual-wavelength spectrophotometric analysis. The BC₂F₂ generation included 7 plants of genotype wxwxo2o2O16_, 19 plants of genotype wxwxo16o16O2_, and 3 plants of genotype wxwxo2o2o16o16. In these seeds, the average amylopectin content was 96.67%, 96.87%, and 96.62%, respectively, which is similar to that of QCL5019. The average lysine content was 0.555%, 0.380%, and 0.616%, respectively, representing increases of 75.1%, 19.9%, 94.3%, respectively, over QCL5019. The average genetic background recovery rate of the BC₂F₃ families was 95.3%, 94.3%, 94.2%, respectively. Among these 3 wxwxo2o2O16O16 families, 4 wxwxo2o2O16o16 families, and 3 wxwxo2o2o16o16 families, the longest imported parent donor fragment was 113.35 cM and the shortest fragment was 11.75 cM. No significant differences in lysine content were found between the BC₂F₄ seeds and the BC₂F₃ seeds in these 10 families. This allowed us to increase the lysine content of waxy corn and produce seeds with excellent nutritional characteristics suitable for human consumption, animal feed, and food processing. This may be of significance in the breeding of high-quality corn and in improvement of the nutrition of humans, livestock, and poultry.     

Endosperm Protein Synthesis and l-[S]Methionine Incorporation in Maize Kernels Cultured In Vitro

This study was conducted to examine protein synthesis and l-[³⁵S] methionine incorporation into the endosperm of Zea mays L. kernels developing in vitro. Two-day-old kernels of the inbred line W64A were placed in culture on a defined medium containing 10 microCuries l-[³⁵S] methionine per milliliter (13 milliCuries per millimole) and harvested at 10, 15, 20, 25, 30, 35, and 40 days after pollination. Cultured kernels attained a final endosperm mass of 120 milligrams compared to 175 milligrams for field-grown controls. Field and cultured kernels had similar concentrations (microgram per milligram endospern) for total protein, albumin plus globulin, zein, and glutelin fractions at most kernel ages.     

In Vitro Study of Mitochondrial Protein Synthesis during Mitochondrial Biogenesis in Excised Plant Storage Tissue

Mitochondrial biogenesis was induced in Jerusalem artichoke (Helianthus tuberosus) tuber by aging tissue discs in distilled water for up to 26 hours. Changes in the purified mitochondrial fraction during aging included an increase in both protein content and specific respiratory activity. Using intact isolated mitochondria, conditions were optimized for incorporation of radioactive amino acid into protein. Incorporation was dependent upon the supply of an oxidizable substrate or an external ATP-generating system and showed characteristic sensitivity to inhibitors of protein synthesis. Aging of the tissue resulted in a 3-fold increase in the rate of in vitro incorporation of [³⁵S]methionine into mitochondrial protein. An analysis of the free amino acid pool in the mitochondrial fraction showed that the decrease in methionine level during aging of intact tissue was sufficient to account for the increased rate of protein labeling. The activation of mitochondrial biogenesis which occurs after slicing is not dependent on an increase in the capacity of mitochondria to synthesize protein as assayed in vitro.     

Within-day Changes in the Polyribosome Content and in Synthesis of Proteins in Leaves of Capsicum annuum L

Capsicum annuum cv. California Wonder was grown in controlled environment with a 12-hour photoperiod. The polyribosome content of leaves varied from 60 to 72% of total ribosomes with the highest level occurring in the middle of the photoperiod and the lowest in the middle of the dark period. The variation was accounted for by changes in the content of large polyribosomes (hexamers and larger). There was no indication of an immediate effect on polyribosome content of light-on or light-off.

The synthesis of proteins at two times in the 24-hour cycle was compared using a dual isotope technique. Statistically significant results were obtained that suggested that protein(s) with molecular weights of 26,000 daltons were preferentially synthesized in the photoperiod compared to the dark period. No evidence was found for the differential synthesis of proteins within the photoperiod.

     

Protein Metabolism in Cultured Plant Tissues: III. Changes in the Rate of Protein Synthesis, Accumulation, and Degradation in Cultured Pith Tissue

During the transition of tobacco (Nicotiana tabacum) pith tissue to callus tissue, there were changes in the composition of the soluble amino acid pools, in the distribution of amino acids between pool and protein, and in the synthesis, accumulation, and degradation of proteins. The size of the leucine pool decreased from 90 nanomoles per gram fresh weight in fresh pith to 20 nanomoles in 24-hour cultured pith, followed by a return to 90 nmoles in pith cultured longer than 5 days. The latter value is the same as that reported for exponentially growing callus cells. Many other pool amino acids changed as dramatically. However, they always approached callus levels after 5 days of culturing. The total amino acid content of pith tissue (the sum of both pool and protein) remained unchanged during culturing. The value for total amino acid content (34 to 42 nanomoles per gram fresh weight) was also similar to that found in callus. The distribution of amino acids between pool and protein did change during culturing. The transition of pith tissue with 88% of its total amino acids free in the soluble pool to callus with 92% of its amino acids in protein was further characterized by changes in protein metabolism. Both protein synthesis and accumulation increased over the first 50 hours in culture to a maximum rate of 45 milligrams protein synthesized gram protein⁻¹ hour⁻¹. After 50 hours in culture, the rate of protein accumulation decreased to equal the rate of fresh weight accumulation (10 mg g⁻¹ hour⁻¹). However, protein synthesis continued at a high rate for several days, suggesting protein degradation was turned on by this time. By 5 days protein synthesis had decreased to a rate similar to that of callus.     

Electromicrographic Analysis Storage Protein Accumulation in Endosperms of Maize with Qualified Protein

The characteristics of storage protein accumulation of maize with qualified protein (MQP) and 02 maize were analysed basing on the genetical and biochemical point of views. The 22 kD and 20 kD zeins in the developing endosperms of maize accumulated 15 days after pollination. The structural genes encoding 22 kD and 20 kD zeins in the developing endosperms were simutaneously expressed. In the endosperms of MPQ and o2 maize the synthesis of 22 kD and 20 kD zeins was suppressed. That is to say, o2 gene negatively regulated the synthesis of 22 kD and 20 kD zeins. Two-dimentional electrophoretic analysis of zeins in the maize endosperms further revealed the effects of o2 gene and its modifiers on the synthesis of zeins. In Mol7 and Mo17/o2 endosperms the synthesis of 27 kD, 22 kD, 20 kD and 15 kD zeins was severely suppressed. In 041/oz and 040/o2 endosperms little difference existed SDS-PAGE analysis of the soluble proteins of Mol 7 and Mo17/o2 endosperms revealted that two bands with molecular weight (MW) of 38.7 kD and 26.7 kD were present in wild type but absent in o2 mutant, while two bands with MW 27.2 kD and 26.1 kD were present in o2 mutant but absent in wild type. These differences were resulted from the effect of o2 gene. In 040/02 and 041/o2 endosperms two bands with MW 18.6 kD and 17.6 kD were present in 041/o2 but absent in 040/02 while one band with MW 40. 2 kD was present in 040/02 and absent in 041/o2, which was closely related to the effects of the modifiers of o2 gene.     

微管解聚剂和光合作用抑制型除草剂诱导的黑麦和玉米中蛋白质组分变化的比较研究

研究了黑麦、玉米在经甲基胺草膦和Atrazine两种除草剂处理后叶绿素含量、叶绿体和根尖分生组织内的蛋白质组份变化,实验结果表明,0.1 mg·L^-1的Atrazine可使黑麦中的叶绿素含量下降（分别从对照的1.72±0.034 mg·g^-1 FW下降到1.62±0.05 mg·g^-1 FW、1.25±0.015 mg·g^-1 FW）。2种除草剂均可使黑麦、玉米中的蛋白质组份产生改变,如当分别用0.1 mg·L^-1的Atrazine处理时,黑麦分生组织中,有4个蛋白质斑点,斑点7、斑点18~20被诱导产生,12个蛋白质斑点,斑点6、斑点8~17、斑点21消失。玉米分生组织中,有4个蛋白质斑点,斑点5、斑点14~16被诱导产生,4个蛋白质斑点,斑点17~20消失。黑麦叶绿体中,有两个蛋白质斑点被诱导产生,13个蛋白质斑点,斑点1~13消失,但Atrazine处理不引起玉米叶绿素含量和叶绿体蛋白质组份的变化。4 mg·L^-1 APM可引起黑麦和玉米分生组织蛋白质组份变化,在黑麦中,1个蛋白质斑点被诱导产生,4个蛋白质斑点,斑点2~5消失。玉米分生组织中,15个蛋白质斑点,斑点4~5、斑点7~16、斑点21~23被诱导产生,4个蛋白质斑点,斑点1~3、斑点6消失。APM均不能引起2种作物中叶绿素含量和叶绿体蛋白质组份的变化。     

Activities of Transaminases, Amylases and Proteases during Endosperm Degradation in Normal and Opaque-2 Zea mays L. cv. Maya

Transaminase, amylase and protease activities were comparedin seedlings of normal and Opaque-2 (o2) maize. Transaminaseactivity, greater in normal maize, was highest in the scutellumfrom which it decreased rapidly in activity from day 2 afterimbibition; only low activity was observed in endosperm andaxis tissue. Amylolytic activity, optimal around pH 5, was greater in normalmaize at all stages of endosperm degradation. Activity whichwas low on day 2, rose to a peak on day 6 and declined afterwards.The level of free sugars in the endosperm of normal was higherthan in o2 maize, and in both varieties was highly correlatedwith amylolysis. Protease activity, optimal at pH 3.6, was also greater in normalendosperms and increased up to day 6 and activity was maintainedat this level until around day 14. Although the activity ofall three enzyme systems examined was greater in normal maizethere were no apparent differences in the overall growth ofnormal and o2 seedlings during this period. Zea mays L, maize, corn, endosperm, enzyme activity, transaminase, amylase, protease     

Protein Composition of PNS Myelin: Developmental Comparison of Control and Quaking Mice

Protein compositions were determined for sciatic nerve myelin isolated from young and adult control and quaking (Qk) mice. Age-related changes in the relative amounts of large (Pl) and small (Pr) basic proteins were found. In control animals, the ratio Pr/Pl increased with age, a change similar to that observed for the large (Bl) and small (Bs) CNS myelin basic proteins of adult mice. Pr/Pl also increased with age in the Qk mouse sciatic nerve, but only to the point that the value in the adult Qk mouse was similar to that observed for young control animals, a situation reminiscent of the effect of the Qk mutation on CNS basic proteins. Thus, our data suggest that the Qk mutation has a similar effect on peripheral nervous system (PNS) and CNS basic proteins. Our findings are consistent with recent electrophoretic and immunochemical data showing that PNS and CNS myelin basic proteins in rodents are analogous, and they suggest that the genetic program controlling basic protein expression is common to oligodendroglia and Schwann cells.     

Developmental Changes in the Oligosaccharide Composition of Synaptic Junctional Glycoproteins

Synaptic junctions (SJs) were isolated from the forebrains of rats ranging in postnatal age from 10 days to greater than 1 year. SJ glycoproteins that react with Concanavalin A (Con A) were isolated by chromatography on Con A-agarose and separated by gel electrophoresis. The concentrations of the major SJ Con A binding (Con A+) glycoproteins (apparent Mr 180,000, 130,000, and 110,000) increased between 10 and 28 days, with GP180 and GP110 showing greater relative increases than GP130. Con A binding oligosaccharides associated with 10-day SJs were sensitive to digestion with endoglycosidase C11 and alpha-mannosidase, indicating that they were of the high-mannose type, as previously shown for 28-day SJs. Con A+ oligosaccharides from rats of increasing postnatal age were analyzed by chromatography on Biogel P-4. Two major oligosaccharides, containing five and eight mannose residues, were present in SJs of all ages examined. During development the ratio of man5 to man8 oligosaccharides increased, so that man5 constituted the predominant species in 28-day and adult SJs. Peptide mapping experiments showed that GP180, GP130, and GP110 were each associated with a unique polypeptide composition. Little or no change in peptide composition of the major SJ glycoproteins occurred during development.     

Fat Storage-inducing Transmembrane Protein 2 Is Required for Normal Fat Storage in Adipose Tissue

Triglycerides within the cytosol of cells are stored in a phylogenetically conserved organelle called the lipid droplet (LD). LDs can be formed at the endoplasmic reticulum, but mechanisms that regulate the formation of LDs are incompletely understood. Adipose tissue has a high capacity to form lipid droplets and store triglycerides. Fat storage-inducing transmembrane protein 2 (FITM2/FIT2) is highly expressed in adipocytes, and data indicate that FIT2 has an important role in the formation of LDs in cells, but whether FIT2 has a physiological role in triglyceride storage in adipose tissue remains unproven. Here we show that adipose-specific deficiency of FIT2 (AF2KO) in mice results in progressive lipodystrophy of white adipose depots and metabolic dysfunction. In contrast, interscapular brown adipose tissue of AF2KO mice accumulated few but large LDs without changes in cellular triglyceride levels. High fat feeding of AF2KO mice or AF2KO mice on the genetically obese ob/ob background accelerated the onset of lipodystrophy. At the cellular level, primary adipocyte precursors of white and brown adipose tissue differentiated in vitro produced fewer but larger LDs without changes in total cellular triglyceride or triglyceride biosynthesis. These data support the conclusion that FIT2 plays an essential, physiological role in fat storage in vivo.