RNase activity requires formation of disulfide bonds and is regulated by the redox state

The activity of many RNases requires the formation of one or more disulfide bonds which can contribute to their stability. In this study, we show that RNase activity and, to a much lesser extent, nuclease activity, are redox regulated. Intracellular RNase activity was altered in vitroby changes in the glutathione redox state. Moreover, RNase activity was abolished following exposure to reducing agents such as -ME or DTT. Following reduction with glutathione (GSH), RNase activity could be fully reactivated with oxidized glutathione (GSSG). In contrast, RNase activity could not be reactivated when reduced with DTT. Decreasing the level of glutathione in vivoin wheat increased RNase activity. Tobacco engineered to have an increased glutathione redox state exhibited substantially lower RNase activity during dark-induced senescence. These results suggest that RNase activity requires the presence of one or more disulfide bonds that are regulated by glutathione and demonstrate for the first time that RNase activity can be altered with an alteration in cellular redox state.     

Isolation and sugar uptake characteristics of protoplasts from maize endosperm suspension cultures

The isolation and sugar uptake characteristics of protoplasts from maize ( Zea mays L.) endosperm-derived suspension cultures are described. In contrast with protoplasts from intact developing endosperm, which by virtue of their large size and high starch content are too fragile for sugar uptake experiments, suspension cultures yielded protoplasts capable of withstanding the necessary handling and centrifugations. Intactness of the protoplasts was demonstrated by dye exclusion or accumulation and latency of malate dehydrogenase activity. Uptake of radioactivity from [³H]-inulin did not increase with time, but that from [¹⁴C]-sugars increased over a wide range of external concentrations. Kinetics of fructose, glucose and sucrose uptake were biphasic, and the saturable components of uptake were eliminated by p -chloromercuribenzene sulfonate (PCMBS). Rates of uptake of sucrose and 1'-fluorosucrose were similar, confirming that hydrolysis by cell wall invertase contributes to sucrose uptake by the suspension cultures. The isolation of protoplasts from this tissue source will enable experimental access to plasma membrane sugar carriers which may exist in the intact maize endosperm.     

Dinitrophenol-induced efflux of sucrose from maize scutellum cells

Maize scutellum slices accumulated sucrose during incubation in glucose, fructose or sucrose. Sucrose was accumulated in two compartments, tentatively     

Renaturation, activation, and practical use of recombinant duplex-specific nuclease from Kamchatka crab

We overexpressed duplex-specific nuclease (DSN) from Kamchatka crab in Escherichia coli cells and developed procedures for purification, renaturation, and activation of this protein. We demonstrated identity of the properties of the native and recombinant DSN. We also successfully applied the recombinant DSN for full-length cDNA library normalization.     

Tissue-specific expression in transgenic maize of four endosperm promoters from maize and rice

The tissue-specific, developmental, and genetic control of four endosperm-active genes was studied via expression of GUS reporter genes in transgenic maize plants. The transgenes included promoters from the maize granule-bound starch synthase (Waxy) gene (zmGBS), a maize 27 kDa zein gene (zmZ27), a rice small subunit ADP-glucose pyrophosphorylase gene (osAGP) and the rice glutelin 1 gene (osGT1). Most plants had a transgene expression profile similar to that of the endogenous gene: expression in the pollen and endosperm for the zmGBS transgene, and endosperm only for the others. Histological analysis indicated expression initiated at the periphery of the endosperm for zmGBS, zmZ27 and osGT1, while osAGP transgene activity tended to start in the lower portion of the seed. Transgene expression at the RNA level was proportional to GUS activity, and did not influence endogenous gene expression. Genetic analysis showed that there was a positive dosage response with most lines. Activity of the zmGBS transgene was threefold higher in a low starch (shrunken2) genetic background. This effect was not seen with zmZ27 or osGT1 transgenes. The expression of the transgenes is discussed relative to the known behaviour of the endogenous genes, and the developmental programme of the maize endosperm     

The induction of α-amylase activity by sucrose starvation in suspension-cultured rice cells is regulated by polyamines

When suspension-cultured rice ( Oryza sativa L. cv. Tainan 5) cells were deprived of sucrose, α-amylase (EC 3.2.1.1) activity in the cells and the culture medium increased markedly. The increase in activity of α-amylase caused by sucrose starvation in the cells and the medium was strongly reduced in the presence of exogenously added spermine. Putrescine and spermidine also inhibited, though only slightly, the increase in α-amylase activity caused by sucrose starvation. Preincubation of the enzyme extract or enzyme in the medium with polyamines had no effect on α-amylase activity. Sucrose starvation resulted in lower polyamine levels in rice suspension cells. D-Arginine and α-methylomithine, inhibitors of polyamine biosynthesis, caused reduced levels of polyamines and increased activity of α-amylase in rice suspension cells cultured in the presence of sucrose. Our results indicate that the induction of α-amylase activity by sucrose starvation in rice suspension cells is mediated, at least partly, through the internal level of polyamines.     

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.     

Protein breakdown and protease properties of germinating maize endosperm

Protein breakdown during germination of maize at 28° is closely correlated with the appearance of protease activity. In the first 2 days of germination, a slight disaggregation of only G₃ glutelins into more simple elements (albumin-globulins) can be observed. Between 2 and 2.5 days, there is extensive breakdown of all protein fractions, the rate of which coincides with the rate of appearance of proteolytic activity. After 2.5 days these phenomena slow down and the bulk of the endosperm proteins disappears. Three acid proteases in endosperm extracts of germinated grain (P₁₁, P₂₁ and P₂₂) have been isolated by affinity chromatography and gel filtration, and partially characterized. P₁₁ (MW 40 000) which is present in the ungerminated grain, cannot hydrolyse prolamins and is insensitive to reducing agents. P₂₁ (MW 36 000) and P₂₂ (MW 12 000), which appear on day 3 of germination, can degrade prolamins in vitro. Reducing agents enhance their activity and prevent their aggregation or denaturation. Comparative assays with different substrates suggest our enzyme preparations are principally endotype proteases with little contaminating carboxypeptidase activity.     

Influence of genotype and texture on zein content in endosperm of maize grains

The effect of genotypes and texture on the content of proteins in maize grains was examined by assessing absolute amounts of six protein fractions in the whole endosperms of four wild‐type lines with high protein content and four quality protein maize (QPM) varieties and for hand‐dissected hard and soft endosperm regions from eight other lines. As previously reported for six wild‐type lines and their opaque‐2(o2) versions, zeins were predominant for all genetic backgrounds and all types of endosperms. From these data and others the amounts of zeins and true proteins (crude proteins free of non‐protein nitrogen) in developing and mature endosperms of wild‐type lines were correlated. The data points for zeins from hard endosperms lay between the regression line and the upper limit of confidence area. Those for zeins from soft endosperms were located at the lower part of confidence area and on a level with the points corresponding to the most immature endosperms. Furthermore, some data points for zeins from o2 and QPM samples lay near the lower limit while the others were outside the confidence area. This suggested an initial zein accumulation dependent on the genotype at a low relative rate, followed by an accumulation at higher rate. The conditions used for isolating and quantitating zeins are discussed.     

Biosynthesis of lysine and other amino acids in the developing maize endosperm

Tracer studies with aspartic acid-[4-¹⁴C], alanine-[1-¹⁴C] acetate-[2-¹⁴C] and diaminopimelic acid-[1,(7)-¹⁴C] injected into the developing endosperm of maize revealed that the biosynthesis of lysine and other amino acids occurs in this organ. The data suggest that lysine is synthesized via the diaminopimelic acid pathway.     

Effect of zinc nutrition on sucrose biosynthesis in maize

Zinc deficiency caused an accumulation of ¹⁴C into malic acid, sugar phosphates, sugar nucleotides, glucose, fructose, phosphoenolpyruvate, glycine and alanine, whereas the ¹⁴C labelling in sucrose decreased. The activity of sucrose synthetase (EC 2.4.1.13) was unaffected up to the 15th day and thereafter it declined. Severe Zn deficiency reduced the biosynthesis of total protein and sucrose synthetase by 50 and 20%, respectively.     

Glucose control of sucrose synthase in the maize scutellum

The in vivo amounts of UDPG, UTP, UDP and UMP, metabolites known to influence the activity of sucrose phosphate synthase (SPS) and sucrose synthase (SS), were measured throughout 5 hr incubations of scutellum slices in fructose or water, i.e. under conditions of sucrose synthesis or breakdown. Cytosolic concentrations were estimated assuming that these metabolites were confined to the cytosol. Within the estimated in vivo concentration ranges, UDPG, UTP and UDP had little effect on the in vitro SS activity, but glucose (100 mM) inhibited SS in the synthesis direction by 63–70% and in the breakdown direction by 86–93%. Glucose inhibition of SS was considerably less when saturating levels of substrates were used. Sucrose did not inhibit SS. It is concluded that during germination the glucose produced from starch breakdown in the maize endosperm enters the scutellum and inhibits SS, preventing a futile cycle and limiting SS participation in sucrose synthesis.     

DNA endoreduplication in maize endosperm cells: the effect of exposure to short-term high temperature

DNA endoreduplication in Zea mays L. (cv. A619 × W64A) endosperm peaks between 16 and 18 d after pollination (DAP). The physiological function of DNA endoreduplication is not known but it is believed to be important in maize kernel development. In the present study, we investigated how 2, 4 or 6 d of high temperature (35 °C) affected DNA endoreduplication and maize kernel development in comparison with control kernels grown at 25 °C. Data were collected on fresh weight (FW), nuclei number, mitotic index, and DNA endoreduplication. Maize endosperm FW and nuclei number were reduced by exposure to 4 or 6 d of high temperature. At 18 DAP, the 2 d high temperature treatment (HTT) caused a reduction in FW and nuclei number, but had no effect on DNA endoreduplication and average DNA content per endosperm. However, when the exposure to high temperature was increased to 4 or 6 d, FW, nuclei number and the magnitude of DNA endoreduplication were progressively reduced, and the peak mitotic index was delayed compared with the control endosperm. At 18 DAP, the 4 d treatment showed 54·7% of the cells were 3 or 6 C, whereas only 41·2% were 12 C or higher. Six days of high temperature also resulted in a reduction in endosperm FW, nuclei number and a delay in the peak of mitotic index. DNA endoreduplication occurred in the kernels exposed to this treatment, although the magnitude was severely reduced compared with the control kernels. Nuclear DNA content was highly correlated (r = 0·93) with kernel FW, suggesting an important role of DNA endoreduplication in determining endosperm FW. The data suggest that high temperature during endosperm cell division exerted negative effects on DNA endoreduplication by dramatically reducing the nuclei number, leaving fewer nuclei available for DNA endoreduplication. However, the data also suggest that prolonged exposure to high temperature restricts entry of mitotic cells into the endoreduplication phase of the cell cycle.