Changes in Pool Sizes of Free Amino Acids and Amides in Leaves and Plastids of Zea mays during Leaf Development

Homoserine dehydrogenase is associated with the multibranched pathway of amino acid biosynthesis originating with aspartic acid. Like most of the related pathway enzymes, this enzyme is localized in chloroplasts. The activity and regulatory properties of the threonine-sensitive isozyme of homoserine dehydrogenase isolated from Zea mays var earliking were examined under variable conditions that could exist within chloroplasts. Catalytic activity is not significantly altered within the range of pHs that occur within these organelles, but inhibition of the enzyme by the pathway product, L-threonine, is markedly diminished at the alkaline pHs characteristic of illuminated chloroplasts. Inhibition by threonine is also subject to modulation by physiological levels of NADPH. Under conditions considered to represent the environment within unilluminated chloroplasts, the enzyme is severely inhibited by micromolar concentrations of threonine, but significant enzyme activity is retained under conditions that are likely to occur during illumination, even in the presence of millimolar levels of threonine. These results indicate that homosenne dehydrogenase may be subject to environmentally mediated regulation in vivo. Other observations support this concept and suggest that the intrinsic catalytic and regulatory properties of key enzymes could facilitate a direct link between light-dependent carbon and nitrogen assimilation and amino acid biosynthesis in chloroplasts of higher plants.Aspartic acid serves as a common precursor of several nutritionally essential amino acids, including lysine, methionine, threonine, and isoleucine, with most of the requisite enzymes being localized in chloroplasts of higher plants (8). Homoserine dehydrogenase (EC 1.1.1.3) is necessary for the synthesis of methionine, threonine and isoleucine, and catalyzes the NAD(P)H-dependent reduction of aspartic semialdehyde to homoserine (7). Of the two isozymes of HSDH2 that have been identified in several plant species, HSDH-II is localized within chloroplasts and is clearly involved in amino acid biosynthesis, while the physiological function of the cytoplasmic, isozyme, HSDH-I, remains unclear (8). HSDH-II isolated from Zea mays L. has been purified to homogeneity and studied in some detail (22,29). It is characterized by the ability to undergo ligand-induced hysteretic transitions among four physically distinct states, each of which exhibits different catalytic and regulatory properties (20,21 (19,26,28). Using this and other information, the present study was conducted to evaluate the extent to which variable physiological conditions might influence the properties of maize HSDH-II. The results suggest that changes in pH and substrate concentrations that could occur in vivo can have a profound influence on the regulatory properties of this enzyme. These results differ from those obtained under conventional assay conditions and suggest a novel regulatory role of the enzyme within the context of amino acid biosynthesis in higher plants. MATERIA...

Section: Other Physiological Factorssupporting

confidence: 80%

Differential Regulation of Maize Homoserine Dehydrogenase under Physiological Conditions

Bryan

1990

“…In spinach and other C3 plants, the proportion of total-cell glutamate located in the chloroplast was found to be greater than that of alanine (1). In the C4 plant corn, the distribution was the reverse (6). It could be assumed that the alanine and glutamate distribution in oat is similar to that of C3 plants investigated and not to that of the C4 plant corn.…”

Section: Discussionmentioning

confidence: 99%

“…alanine and glutamate) between the chloroplast and the other compartments was studied by Aach and Heber (1) and Chapman and Leech (6). In spinach and other C3 plants, the proportion of total-cell glutamate located in the chloroplast was found to be greater than that of alanine (1).…”

Section: Discussionmentioning

confidence: 99%

Aminotransfer from Alanine and Glutamate to Glycine and Serine during Photorespiration in Oat Leaves

Betsche

1983

ABSTRACT"N-Labeled glutamate and alanine were used to examine the photorespiratory nitrogen metabolism in oat (A vena sativa L.) leaf slices. Glutamate and alanine supply amino groups for glycine formation during photorespiration. The nitrogen flux from alanine to glycine was estimated to be 3 times higher than that from glutamate. It is concluded from these results that alanine is a direct and important amino donor for photorespiratory glycine formation in oat leaves. The 'N labeling of serine was almost as high as that of glycine during the initial period of the labeling experiments. Thereafter, the ratio of "N label in serine to "N label in glycine declined substantially.During photorespiration glycine is formed from glyoxylate. This process occurs in the leaf peroxisomes. The glycine enters the mitochondria, where two glycine are converted into one serine (24). In this complex reaction, CO2 and NH3 are liberated. The serine reenters the leaf peroxisomes, where serine:glyoxylate aminotransferase mediates the formation of hydroxypyruvate and glycine. By this process, one amino-nitrogen is directly recycled for glycine formation. The nitrogen of the other amino group, liberated as NH3, is refixed to glutamate via the glutamine synthetase/glutamate synthase pathway (1 1, 13, 22, 30). The finding that leaf peroxisomes contain glutamate:glyoxylate aminotransferase leads to the suggestion that the additional amino nitrogen needed for the formation of the second glycine is provided by this glutamate (13). However, there are a number of reports indicating that leaf extracts, or leaf peroxisomes, have substantial alanine:glyoxylate aminotransferase activity that may even exceed the glutamate:glyoxylate aminotransferase activity (4,7,21,24,25,28). Alanine is the main substrate for the formation of glycine also in the microbodies (peroxisomes) of the alga Mougeotia (29). In higher plants, most of the alanine:glyoxylate aminotransferase activity is attributed to the glutamate:glyoxylate aminotransferase (21,24,25). Glutamate:glyoxylate aminotransferase is also responsible for the glutamate:pyruvate and the alanine:a-ketoglutarate aminotransferase activities of leaf peroxisomes (3, 12,19,20 Preparation of Leaf Slices. The leaves were cut into slices (1.5-2.0 mm) with a razor blade. Four g of the leaf slices were vacuuminfiltrated in 40 ml of an osmotic medium consisting of 0.3 M sorbitol, 2 nim KH2PO4, 2 mM MgCl2, 1 mim MnCl2, 1 mm EDTA, and 5 mm Hepes/KOH, pH 7.9. Care was taken to release the vacuum slowly. Before use, the leaf slices were rinsed in a sieve with 40 ml of the same medium.Labeling Experiments. The leaf slices were transferred into a cuvette (8 x 5 x 2 cm) filled with 55 ml of the osmotic medium described above. The latter was bubbled with air for 3 h before being used. The leaf slices were stirred gently from the top, and the cuvette was illuminated with slide projectors from both sides the leaf slices were quickly transferred into a sieve, rinsed with 30 ml of water, and dipped several times into a large...

“…The advantage of many monocotyledonous plants is that in the leaves of their young seedlings all cell divisions occur in a basal meristem, resulting in a developmental sequence of cells from the base to the tip of each leaf. This has already been exploited in maize (2,5,6,12) (27) to study plastid and cell differentiation.In the present paper we demonstrate how a similar sequential developmental system in wheat leaves can be used to investigate the replication process of young chloroplasts and the relationship between the timing of DNA synthesis and plastid division. Wheat was chosen because many varieties and special lines are available whose genetics are well understood, and the complication of chloroplast dimorphism, as found in maize, is avoided.…”

mentioning

confidence: 99%

mentioning

confidence: 99%

Chloroplast Division and DNA Synthesis in Light-grown Wheat Leaves

Boffey¹,

Ellis²,

Selldén³

et al. 1979

Self Cite

103

Light-grown 7-day-old wheat seedlgs (Trticum aestavum, var. Maris Dove) showed an increase of 200% in plastids per ceUl between 1.7 and 4.5 centimeters from the leaf base. This inacrease was the result of divisions of young chbiolasts at various stages of development, and was well separated in distance, and therefore in time from the region of ceO division in the basal meristem. 13HIThymide was incorporated into plastid DNA throughout the zone of plastid division, but not above it.During leaf development, proplastids differentiate into chloroplasts and their differentiation is accompanied by a large increase in the number of plastids per cell, caused by the division of both proplastids and young chloroplasts (11). In spinach leaves the division of young grana-containing, photosynthetic chloroplasts is the most important factor determining the number of chloroplasts per mature leaf cell: Possingham and Saurer (19) have shown that up to 90%o of the final number of plastids can be accounted for by the division of young chloroplasts. Chloroplast division has also been observed to occur in vitro (8,22) but no subsequent increase in size has been observed.For ease of experimentation, the effects of exogenous factors on chloroplast division have been examined mainly by using discs excised from leaf tissue (4,14,(16)(17)(18)20); but in view of the large biochemical changes which occur rapidly after the excision of leaf tissue (15), the results of investigations of the control of plastid division in leaf discs should be interpreted with some caution. The division of young chloroplasts and its significance in the development and function of the leaf would be examined least equivocally in an intact, growing plant.The aim of the present investigation was to use an intact plant in which a large increase in the number of plastids per cell occurred during leaf development and to examine the sequence of biochemical events associated with this increase. To follow the division process sequentially, we required plants from which large numbers of cells and plastids could be harvested at each stage of development. Dicotyledonous leaves are not an ideal tissue in which to study plastid division since the leaf is a mosaic of cells at different stages of differentiation. The advantage of many monocotyledonous plants is that in the leaves of their young seedlings all cell divisions occur in a basal meristem, resulting in a developmental sequence of cells from the base to the tip of each leaf. This has already been exploited in maize (2,5,6,12) (27) to study plastid and cell differentiation.In the present paper we demonstrate how a similar sequential developmental system in wheat leaves can be used to investigate the replication process of young chloroplasts and the relationship between the timing of DNA synthesis and plastid division. Wheat was chosen because many varieties and special lines are available whose genetics are well understood, and the complication of chloroplast dimorphism, as found in maize, is avoided. MATERIALS ...