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Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the starchy endosperm protein of rice (Oryza sativa L. Japonica cv Koshihikari) during seed development confirmed that storage protein begins to accumulate about 5 days after flowering. Two polypeptide groups, 22 to 23 and 37 to 39 kilodaltons, the components of glutelin, the major storage protein in rice seed, appeared 5 days after flowering. A 26-kilodalton polypeptide, the globulin component, also appeared 5 days after flowering. Smaller polypeptides (10-to 16-kilodaltons) including prolamin components, appeared about 10 days after flowering. In contrast, the levels of the 76-and 57-kilodalton polypeptides were fairly constant throughout seed development. Transmission electron microscopy and fractionation by sucrose density gradient centrifugation of the starchy endosperms at various stages of development showed that protein body type II, the accumulation site of glutelin and globulin, was formed faster than protein body type I, the accumulation site of prolamin.The 57-kilodalton polypeptide but not the glutelin subunits was labeled in a 2-hour treatment with I14CIleucine given between 4 and 12 days after flowering to developing ears. In vivo pulse-chase labeling studies showed the 57-kllodalton polypeptide to be a precursor of the 22 to 23 and 37 to 39 kilodalton subunits. The 57-kilodalton polypeptide was salt-soluble, but the mature glutelin subunits were almost salt insoluble.In vitro protein synthesis also showed that the mRNAs directly coding the 22 to 23 and 37 to 39 kilodalton components were absent in developing seeds and that the 57-kilodalton polypeptide was the major product. Thus, it was concluded that the two subunits of rice glutelin are formed through post-translational cleavage of the 57-kilodalton polypeptide.The major storage proteins of most cereal grains are glutelin and prolamin. In rice grains, however, glutelin is the major protein of the starchy endosperm, constituting at least 80%o of the total protein, prolamin accounting for less than 5% (12). Rice glutelin has a mol wt of 6 x I05 according to Tecson et al. (27), but Takeda et a!. (25) demonstrated that it has a heterogeneous mol wt. The increase in glutelin in the developing rice grain coincides with the appearance of PB' in the starchy endosperm 7 to 8 DAF (7,29), and glutelin is found exclusively in PB (7,26,30). In a previous paper (26), we reported two types of PB in the starchy endosperm of rice grains and described their isolation. One (type I, PB-I) is spherical with a concentric ring structure, whereas the other (type II, PB-II) is stained homogeneously by osmium tetroxide and does not have this structure. PB-I contains prolamin, and PB-II is rich in glutelin and globulin. The glutelin in PB-II is composed of two principal subunits, the 22-to 23-and 37-to 39-kD complexes, and the prolamin in PB-I is composed mainly of 13-kD polypeptide.

Three signal transduction pathways, dependent on cGMP and/or calcium, are utilized by phytochrome to control the expression of genes required for chloroplast development and anthocyanin biosynthesis in plant cells. For example, cbs is controlled by a cGMP-dependent pathway, cab is controlled by a calcium-dependent pathway, and far is regulated by a pathway that requires both cGMP and calcium. Using a soybean photomixotrophic cell culture and microinjection into the cells of a phytochrome-deficient tomato mutant, we have studied the regulatory mechanisms acting within and between these three signaling pathways. We provide evidence that changes in cGMP levels mediate the observed induction and desensitization of cbs gene expression in response to light and demonstrate that high cGMP concentrations cause negative regulation of both the calcium-and the calcium/cGMP-dependent pathways. Conversely, high activity of the calcium-dependent pathway can negatively regulate the cGMP-dependent pathway. We have termed these opposing regulatory mechanisms reciprocal control. In all cases, the molecules that are involved appear to be downstream components of the signal transduction pathways, rather than calcium and cGMP themselves. Furthermore, we have found that the calcium/cGMP-dependent pathway has a lower requirement for cGMP than does the cGMP-dependent pathway. The role of these phenomena in the regulation of plant photoresponses is discussed.

Fe-6mass%Ni-(0.0008ϳ0.29)mass%C alloys were hot-deformed in torsion at 600-720°C (above the cooling transformation start temperatures A r3 ) after austenitization. An in-situ X-ray diffraction study revealed that g→a transformation occurred during deformation in a wide range of condition, even above A 3 p (paraequilibrium g→a transformation temperature). Corresponding to this transformation, apparent decrease in deformation stress from that expected for austenite was observed. Microstructural study of the specimens quenched after the deformation showed that a large amount of fine-grained ferrite was formed due to the deformation. The analysis of deformation stress and chemical driving-force of the transformation indicated that the transformation occurred in order to reduce the total energy of deformed material since the deformation of energy of a was revealed to be considerably smaller than that of g and the amount of deformation energy saved by the transformation was shown to be much greater than the chemical energy consumed by the transformation at the tested temperatures.KEY WORDS: low carbon steels; nickel steels; hot deformation; grain refinement; thermomechanical heat treatment; strain-induced transformation. study during hot deformation has been undertaken in low carbon Fe-Ni alloys. The in-situ X-ray diffraction technique had been successfully applied to find dynamic recrystallization of aluminum. 7) The experimental results of the in-situ X-ray study have been already reported. [8][9][10] This report puts together all the results of the in-situ X-ray study done in these reports and also includes the results of deformation stress measurement and microstructural study. In the deformation stress measurement, it was aimed to determine the difference in deformation stress between g and a more accurately. The cause of DT will then be discussed on the basis of these three kinds of experimental results. Experimental MaterialsThe compositions of the alloys used in this study are shown in Table 1. Nickel was added to reduce transformation temperature so as to make high temperature X-ray studies easier. It also enhances the hardenability of the alloys. Table 2 gives the transformation temperatures of the alloys. Equilibrium transformation temperatures were calculated by using Thermo-Calc. In this alloy system transformation during cooling was known to start from their paraequilibrium transformation start temperature, A 3 p , 11) not from orthoequilibrium transformation start temperature, A o 3 . Experimental cooling transformation start temperatures, A r3 , and transformation heating finish temperatures, A c3 , and a martensite start temperature, Ms, were adopted from preceding work, 12) in which alloys of the compositions close to the present ones were used. The alloys were vacuum-melted and hot-rolled to 3-mm thick plates. They were sliced and drawn into wires of 2-mm diameter, which were cut to 42 mm in length for torsion test. Apparatus and ProceduresThe schematic setup of X-ray diffraction experiment during t...

From the soybean cDNA library, we isolated and analyzed the chlH gene encoding a subunit of Mg-chelatase. The subunit was a polypeptide of 1,383 amino acids with a molecular mass of 153,491 Da, which shared 90% identity with the olive gene from Antirrhinum majus. The regulation of the expression of chlH was investigated in photomix-otrophic soybean suspension cells (SB-P). The expression was light-inducible, and the induction was more rapid than those of chlI and cab2. Furthermore, the levels of the transcripts and products of chlH appeared to be regulated by a circadian oscillation. The subchloroplastic localization of ChlH was investigated by immunoblot analyses with antiserum against recombinant ChlH. Depending on the concentration of Mg2+ in the lysis buffer, the localization of ChlH protein migrated between the stroma and the envelope membrane; ChlH was localized on the envelope membrane, a major site of chlorophyll biosynthesis, when the Mg2+ concentration of the lysis buffer was high (above 5 mM). These results indicated that the activity of Mg-chelatase was regulated by modulation of the expression and subchloroplastic localization of ChlH protein.

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Cucumisin, a subtilisin-like serine protease, is expressed at high levels in the fruit of melon (Cucumis melo L.) and accumulates in the juice. We investigated roles of the promoter regions and DNA-protein interactions in fruit-specific expression of the cucumisin gene. In transient expression analysis, a chimeric gene construct containing a 1.2-kb cucumisin promoter fused to a ␤-glucuronidase (GUS) reporter gene was expressed in fruit tissues at high levels, but the promoter activities in leaves and stems were very low. Deletion analysis indicated that a positive regulatory region is located between nucleotides ؊234 and ؊214 relative to the transcriptional initiation site. Gain-of-function experiments revealed that this 20-bp sequence conferred fruit specificity and contained a regulatory enhancer. Gel mobility shift experiments demonstrated the presence of fruit nuclear factors that interact with the cucumisin promoter. A typical G-box (GACACGTGTC) present in the 20-bp sequence did not bind fruit protein, but two possible cis-elements, an I-box-like sequence (AGATAT-GATAAAA) and an odd base palindromic TGTCACA motif, were identified in the promoter region between positions ؊254 and ؊215. The I-box-like sequence bound more tightly to fruit nuclear protein than the TGTCACA motif. The I-box-like sequence functions as a negative regulatory element, and the TGTCACA motif is a novel enhancer element necessary for fruit-specific expression of the cucumisin gene. Specific nucleotides responsible for the binding of fruit nuclear protein in these two elements were also determined.Timing and levels of gene expression are critical to the proper development of eukaryotic organisms. Regulation of the expression pattern of a particular gene can involve the specific binding of trans-acting factors to the cognate cis-elements, constituting a crucial step in transcriptional initiation and, in turn, on the spatial and temporal expression of genes. Plant genes that show tissue specificity, developmental specificity, and a wide range of expression levels have been characterized, whereas their expression patterns are also influenced by environmental stimuli. A family of genes for fruit proteins provide a model system for the study of the regulatory mechanisms of plant genes, since their expression is restricted to a specific tissue and stage during fruit development (1). A number of fruit-specific genes that are activated during ripening have been isolated from tomato and other fruits, and genes responding to ethylene and nonethylene signals have been identified (1, 2). The promoters of fruit-specific genes would also be of great interest for use in strategies to manipulate fruit metabolism and produce valuable proteins such as antibody, biopharmaceuticals, and edible vaccines through methods of genetic engineering (3-5). However, the detailed mechanisms by which the expression of fruit protein genes are regulated are poorly understood, as many of the essential cis-elements have not been identified.Melon cucumisin, an extracellular...

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