Lemna synthesis

Experiments conducted with the tobacco hornworm, Manduca sexta and the aquatic plant, Lemna minor are consistent in finding that canavanine does not affect whole organism ability to incorporate [ Hjleucine into trichloroacetic acid-insoluble materials (see Table I). Such determinations evaluate the balance between reactions fostering protein synthesis and those responsible for the turnover and degradation of proteins. If the treatment time is reduced to 30 min, so as to accentuate synthetic reactions over those of catabolism, the result is the same. 

Figure 4. Enzymes of Rhizobium (a) and Lemna (b) proposed as sites of glyphosate inhibition of aromatic amino acid synthesis. Abbreviations CM, chorismate mutase PDH, prephenate dehydrogenase and PD, prephenate dehydratase.

Fig. 5. The pool sizes and rates of metabolism of key N containing intermediates during the assimilation of [ N]HJ by Lemna. The numbers in square boxes show the computed pool sizes (mmol/g fresh weight) and the annotations on the arrows the estimated transfer coefficients (nmol/min/g fresh weight). Quantities marked are estimates based on balancing synthesis and evolution rather than isotopic labeling. Arrows with broken lines indicate the rates required to maintain the pools in steady state growth. (Reproduced with permission from Rhodes et al. 1979.)...

The final reaction in the biosynthesis of threonine involves a /8-y rearrangement and the loss of phosphate from O-phosphohomoserine (Fig. 2). Threonine synthases have been isolated from Lemna (Schnyder et al., 1975) radish, sugarbeet (Madison and Thompson, 1975), peas (Schnyder et al., 1975 Thoen et al., 1978b), and barley (Aames, 1978). None of these enzymes has been extensively characterized but a requirement for pyridoxyl-5 -phosphate was demonstrated after partial purification of the barley and pea enzymes. Unlike several other enzymes associated with threonine synthesis, the activity of threonine synthase was not stimulated by monovalent cations. However, all of the plant enzymes are strongly activated by 5-adeno-sylmethionine (Section III,B,5). 

Most of the inorganic sulfate assimilated and reduced by plants appears ultimately in cysteine and methionine. These amino acids contain about 90% of the total sulfur in most plants (Allaway and Thompson, 1966). Nearly all of the cysteine and methionine is in protein. The typical dominance of protein cysteine and protein methionine in the total organic sulfur is illustrated in Table I by analyses of the sulfur components of a lower plant (Chlorella) and a higher plant (Lemna). Thede novo synthesis of cysteine and methionine is one of the key reactions in biology, comparable in importance to the reduction of carbon in photosynthesis (Allaway, 1970). This is so because all nonruminant animals studied require a dietary source of methionine or its precursor, homocysteine. Animals metabolize methionine via cysteine to inorganic sulfate. Plants complete the cycle of sulfur by reduction of inorganic sulfate back to cysteine and methionine, and are thus the ultimate source of the methionine in most animal diets (Siegel, 1975). 

Phosphohomoserine serves as a precursor of both threonine and methionine in higher plants, and regulation of its utilization in both branches of the pathway would be expected. This appears to occur, in part, by 5-adenosylmethio-nine activation of threonine synthase (5). Results obtained with partially purified Lemna threonine synthase (Giovanelli et al, 1984) indicate that the enzyme is essentially inactive in the absence of -adenosylmethionine, which cooperatively activates the enzyme at concentrations of less than 100 /iM. Conceptually, methionine could be synthesized and converted to S-adenosylmethionine prior to enzyme activation and the synthesis of threonine. Both orthophosphate and AMP inhibit Lemna threonine synthase in vitro, but the physiological significance of these effects is uncertain (Giovanelli et al, 1986). 

The level of sulfotransferase in Lemna and in Phaseolus vulgaris is also subject to strong inhibition by gaseousH2S(Brunoldand Schmidt, 1976,1978 Wyss and Brunold, 1979). However, the extractable acti vity of cysteine synthase is not similarly affected. Removal of H2S firom the gas phase results in rapid restoration of activity which, based on a study of labeling of the enzyme (von Arb and Brunold, 1980), was attributed to synthesis ofthe enzyme de novo. HjS also inhibits the level of APS sulfotransferase in cell suspension cultures of Nicotiana sylvestris in this tissue neither the ATP-sulfiirylase or cysteine synthase activity was affected by H2S or cysteine (Brunold etal., 9Sl). Importantly, the inhibition of APS sulfotransferase by H2S was correlated with an enhanced level of cysteine, suggesting that the H2S inhibition could have been mediated via this reaction product. Uptake of exogenous sulfate was also inhibited by H2S in this system (Brunold et al., 1981). 

In tobacco cell cultures the extractable levels of ATP-sulfurylase and cysteine synthase are very low when the cells are subject to nitrogen stress but increase rapidly upon alleviation of the stress, suggesting that a product of nitrogen assimilation derepresses the levels of these enzymes. In Lemna (and possibly in cultured Rosa cells) it appears that this role is fulfilled by APS sulfotransferase and that ATP-sulfurylase and cysteine play unimportant roles in coordinating the sulfate assimilation pathway with the nitrate assimilation pathway. A further regulatory mechanism known to occur in cultured tobacco cells is that excessively high concentrations of cysteine induce the synthesis of cysteine desulfiiydrase (see Section VI). 

The results of these studies (Giovanelli et al, 1985b) are given for Lemna in Fig. 8. They show that the rate of synthesis of methionine from homocysteine is about five times faster than the rate of incorporation of sulfate sulfur into homocysteine and that metabolism of methionine to SAM is about four times more rapid than the rate of incorporation of methionine into the methionyl residues of protein. By contrast, an analysis of the products of p S]sulfate incorporation (Section II) shows that protein methionine is the single most important sink for the incorporation of inorganic sulfur. The answer to this... 

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