The starchy endosperm (SE) of the developing grain (caryopsis) of barley (Hordeum vulgare L.) cv Himalaya, as well as that of other barley cultivars examined, acidifies during maturation. The major decrease in pH begins with the attainment of maximum grain dry weight, onset of dehydration, and completion of chlorophyll loss. Acidification is correlated with the accumulation of malate and lesser amounts of citrate and lactate, produced and probably secreted by the pericarp/testa/aleurone (PTA). It is accompanied by large concurrent rises in phosphoenolpyruvate carboxylase and alcohol dehydrogenase (ADH) activity in the PTA. The activity of seven other enzymes of oxaloacetate and pyruvate metabolism was found to fall or rise only slightly during acidification. Sequential changes in relative amount of ADH isozymes were found in both PTA and SE. The PTA maintained a high respiration rate and adenylate energy charge (AEC) throughout acidification, whereas the SE showed a low respiration rate and rising AEC. The data are consistent with the occurrence of hypoxia in the SE. It is suggested that the above enzyme changes are required for the development of a malate/ethanol fermentation (i.e. a mixed metabolism) in the aleurone layer during maturation.The mature endosperm (comprising the SE' and surrounding aleurone layer) ofbarley and other cereals has been studied extensively with regard to its structure, composition, and function during germination (reviewed in ref. 14). However, much less is known about endosperm development, despite an accumulation of data on morphology, starch synthesis, storage protein deposition, and enzyme activities (5,12,31). This is especially true of the maturation process, which involves transition from grain-filling to preparation for quiescence, dormancy, survival under adverse conditions, and finally germination. Despite the charting of enzyme activities related to starch degradation, glycolysis, and the pentose phosphate pathway (reviewed in ref. 13), no integrated picture of endosperm metabolism, or of the relative roles of the aleurone and SE, is available.It has been observed that ADH accumulates in the outer layers of the barley grain during development and that the (19). This suggested the possibility of hypoxic conditions within the developing grain. Because an early response of plant cells to hypoxia is acidification of the cytoplasm caused by lactic acid production (1 1, 30), we asked whether the endosperm of the developing barley grain also acidifies.In this report, we show that a marked acidification does in fact occur in the SE, due chiefly to production and secretion of protons and malate by the aleurone tissue. A number of biochemical changes are found that shed light on the mechanism of this process, the possible role of hypoxia, and the development of fermentative pathways. MATERIALS AND METHODS Plant MaterialGrain of barley (Hordeum vulgare cv Himalaya) was incubated at room temperature for 2 d on filter paper in 9 cm Petri dishes containing 5 mL water. The germi...

The amino acid composition of endosperm cavity sap and of sieve tube saps from the flag leaf, peduncle, rachis, grain pedicel, and grain were determined for wheat plants just past the mid-half of grain filling. On a mole percent basis, glutamine accounted for almost half of the amino acids in sieve tube sap from the peduncle and ear. Other protein amino acids, plug gamma-aminobutyrate, were present in varying, but mostly low (a few mole percent) proportions. The amino acid composition of phloem exudate resembled that of the mature wheat grain. The proportions of amino acids in the endosperm cavity were generally similar to those of the sieve tube sap supplying the grain. Cysteine, however, while virtually absent from sieve tube sap, comprised 1 to 2 mole percent of amino acids in the endosperm cavity, suggesting it is transported in a different form. Also, alanine and, to a lesser extent, glutamate were relatively more prominent in endosperm cavity sap than in the sieve tube sap. Thus, while most amino acids were more concentrated in the sieve tube sap than in the endosperm cavity sap, alanine and glutamate appeared to be moving from the sieve tube to the endosperm cavity in the absence of, or perhaps even against, their concentration gradients.

Lemnaperpusilla 6746, grown photoautotrophically at a series of sulfate concentrations ranging from 0.32 to 1,000 pm, was labeled to radioisotopic equilibrium with 36SO42-. Sulfur-containing compounds were isolated and purified from the colonies. Radioactivity in each compound was a measure of the amount of that compound present in the tissue. ,The following compounds were identified and quantitated: inorganic sulfate, glutathione, homocyst(e)ine, cyst(e)ine, methionine, S-methylmethionine sulfonium, Sadenosylmethionine, S-adenosylhomocysteine, cystathionine, chloroformsoluble (presumed to be sulfolipid), protein cyst(e)ine, and protein methionine. y-Glutamylcyst(e)ine, erythro-and threo-thiothreonine, and S-methylcysteine were not detected. No volatile 35S compounds were formed during plant growth at 1,000 isM sulfate, nor were significant amounts of 35S compounds excreted into the medium.The amount of each component present in colonies grown over the 3,000-fold range of medium sulfate was relatively constant except for inorganic sulfate. This increased about 30-fold from the lowest to the highest medium sulfate concentration. The total soluble sulfur amino acids increased about 1.5-to 2-fold, due primarily to an increased amount of glutathione. Protein cyst(e)ine and protein methionine were the major organic sulfur compounds in Lemna, and the amounts of these compounds remained virtually constant despite the variation in external sulfate concentration.Procedures for the analysis of S-adenosylmethionine, S-methylmethionine sulfonium, and S-adenosylhomocysteine are presented. MATERIALSBiological Materials. The conditions for maintenance of stock cultures of L. perpusilla 6746 and for the photoautotrophic growth ofexperimental colonies in both batch and semicontinuous culture in the phytostat are described in the accompanying paper (3). Cultures were examined for the possible presence of contaminating microorganisms (1) at the time of harvest. No contaminants were found in the cultures used here.Chemicals. The method of preparation of 5'-methylthioadenosine, either from S-adenosyl-L-methionine or from S-adenosyl-L-[methyl-3Hlmethionine (Amersham/Searle) has been described (5). Sodium [35SJthiosulfate (outer S labeled) was from New England Nuclear and S-[methyl-'4CJmethylmethionine sulfonium was from ICN. The sources ofother chemicals have been described (5). METHODSMethods for chromatography on Dowex 50-H+, paper chromatography, and paper electrophoresis have been described (1,5,6). Solvents used for paper chromatography included solvent A, 2-propanol-88% formic acid-H20 (7:1:2, v/v/v); solvent B, I-butanol-acetic acid-H20 (12:3:5, v/v/v); solvent C, methanol-pyri- v/v/v); solvent D, 2-propanol-88% formic acid (6:4, v/v); solvent E, isobutyric acid-0.5 N NH40H (5:3, v/v); and solvent F, v/v/v/v). Solvents for electrophoresis included solvent G, 0.05 M pyridine-0.32 M formic acid (pH 2.9); and solvent H, 0.46 M formic acid (pH 1.9).Recent work in this laboratory has led to the development of a phytosta...

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