ABSTRACUChlorsulfuron, an inhibitor of acetolactate synthase (EC 4.13.18) (TB Ray 1984 Plant Physiol 75: 827-831), markedly inhibited the growth of Lemma minor at concentrations of 10' molar and above, but had no inhibitory effects on growth at 10' molar. At growth inhibitory concentrations, chlorsulfuron caused a pronounced increase in total free amino acid levels within 24 hours. Valine, leucine, and isoleucine, however, became smaller percentages of the total free amino acid pool as the concentration of chlorsulfuron was increased. At concentrations of chlorsulfuron of 10'8 molar and above, a new amino acid was accumulated in the free pool. This amino acid was identified as a-amino-n-butyrate by chemical ionization and electron impact gas chromatography-mass spectrometry. The amount of a-amino-n-butyrate increased from undetectable levels in untreated plants, to as high as 840 nanomoles per gram fresh weight (2.44% of the total free pool) in plants treated with 10 molar chlorsulfuron for 24 hours. The accumulation of this amino acid was completely inhibited by methionine sulfoximine. Chlorsulfuron did not inhibit the methionine sulfoximine induced accumulations of valine, leucine, and isoleucine, supporting the idea that the accumulation of the branched-chain amino acids in methionine sulfoximine treated plants is the result of protein turnover rather than enhanced synthesis. Protein turnover may be primarily responsible for the failure to achieve complete depletion of valine, leucine, and isoleucine even at concentrations of chlorsulfuron some 104 times greater than that required to inhibit growth. Tracer studies with "N demonstrate that chlorsulfuron inhibits the incorporation of '"N into valine, leucine, and isoleucine. The a-amino-nbutyrate accumulated in the presence of chlorsulfuron and ["NjR.+ was heavily labeled with "N at early time points and appeared to be derived by transamination from a rapidly labeled amino acid such as glutamate or alanine. We propose that chlorsulfuron inhibition of acetolactate synthase may lead to accumulation of 2-oxobutyrate in the isolencine branch of the pathway, and transamination of 2-oxobutyrate to a-aminon-butyrate by a constitutive transaminase utilizing either glutamate or alanine as a-amino-N donors.

When Lemna minor L. is supplied with the potent inhibitor of glutamine synthetase, methionine sulfoximine, rapid changes in free amino acid levels occur. Glutamine, glutamate, asparagine, aspartate, alanine, and serine levels decline concomitantly with ammonia accumulation. However, not all free amino acid pools deplete in response to this inhibitor. Several free amino acids including proline, valine, leucine, isoleucine, threonine, lysine, phenylalanine, tyrosine, histidine, and methionine exhibit severalfold accumulations within 24 hours of methionine sulfoximine treatment. To investigate whether these latter amino acid accumulations result from de novo synthesis via a methionine sulfoximine insensitive pathway of ammonia assimilation (e.g. glutamate dehydrogenase) or from protein turnover, fronds of Lemna minor were prelabeled with I'5NIH.4, prior to supplying the inhibitor. Analyses of the 'IN abundance of free amino acids suggest that protein turnover is the major source of these methionine sulfoximine induced amino acid accumulations. Thus, the pools of valine, leucine, isoleucine, proline, and threonine accumulated in response to the inhibitor in the presence of [I5NIH4', are '4N enriched and are not apparently derived from '5N-labeled precursors. To account for the selective accumulation of amino acids, such as valine, leucine, isoleucine, proline, and threonine, it is necessary to envisage that these free amino acids are relatively poorly catabolized in rivo. The amino acids which deplete in response to methionine sulfoximine (i.e. glutamate, glutamine, alanine, aspartate, asparagine, and serine) are all presumably rapidly catabolized to ammonia, either in the photorespiratory pathway or by alternative routes.It is now well established that GS2 occupies a central position in plant N metabolism (12, 13). The GS-GOGAT cycle is thought to be responsible for the assimilation of most, if not all, of the ammonia derived from nitrate reduction and photorespiration (2-4, 10, 18, 19, 21, 23, 25). Studies with the potent inhibitor of GS, MSO, appear to rule out any major contribution of GDH to ammonia assimilation (1,3,8,19,21,23). However, recent investigations with isolated plant mitochondria suggest that a small fraction of the ammonia generated from glycine decarboxylation can be directly reassimilated into glutamic acid via a mitochondrial GDH (26). The quantitative significance of this latter pathway in vivo still remains obscure. '

The effects of varying concentrations and duration of alachlor [2-chloro-2′,6′diethyl-N-(methoxymethyl)acetanilide] and metolachlor [2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl)acetamide] treatment on root growth, cell division, and cell enlargement were studied. Peas (Pisum sativumL. ‘Alaska’) and oats (Avena sativaL. ‘Victory’) were treated from 0 to 48 h with concentrations ranging from 1 × 10-8to 1 × 10-3M of each herbicide. After 48 h, average growth rates were significantly inhibited at concentrations of 1 × 10-7M alachlor and 5 × 10−8M metolachlor, and 5 × 10−7M alachlor and 1 × 10-6M metolachlor for peas and oats, respectively. When growth inhibitions were examined across time at concentrations greater than these, the degree of growth inhibition was a function of both concentration and duration of treatment. Often the greatest decrease in growth occurred between 0 and 12 h. Mitotic indices of root tip squashes from pea roots and paraffin sections from oat roots were determined. There was a significant reduction in the mitotic indices of pea roots treated for 48 h with 5 × 10−6M alachlor or 1 × 10-5M metolachlor. After a 30-h treatment, the mitotic indices of oat roots were significantly reduced by 1 × 10−7M metolachlor and 1 × 10−6M alachlor. Significant inhibition of elongation of etiolated oat coleoptiles were observed at 5 × 10−6M alachlor (27%) and 5 × 10−5M metolachlor (30%). Inhibition of pea hypocotyl elongation did not occur at concentrations below 5 × 10−4M. It was concluded that the growth inhibition of plants caused by alachlor and metolachlor results from both an inhibition of cell division and cell enlargement.

The effects of chloracetamides on protein synthesis were studied both in vivo and in vitro. Four chloracetamide herbicides, alachlor [2-chloro-2′,6′-diethyl-N-(methoxymethyl)acetanilide], metolachlor [2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl)acetamide], CDAA (N–N-diallyl-2-chloroacetamide), and propachlor (2-chloro-N-isopropylacetanilide) were tested for inhibition of [3H]-leucine incorporation into protein. Incorporation of3H-leucine into trichloroacetic acid (TCA)-insoluble protein was inhibited in oat (Avena sativaL. ‘Victory’) seedlings grown in sand culture and treated 12 h at 1 × 10−4M with these chloracetamides. The herbicides were also tested in a cell-free protein synthesizing system containing polyribosomes purified from oat root cytoplasm. These herbicides had no effect on the rates of polypeptide elongation nor on the synthesis of specific polypeptides when herbicides (1 × 10−4M) were added directly to the system. Polypeptide formation was inhibited 89% when 1 × 10−4M cycloheximide was added during translation. Cytoplasmic polyribosomes were isolated from oat roots treated 12 h with 1 × 10−4M herbicide. Translation rates and products were not altered when these polyribosomes were added to the in vitro system. Protein synthesis is inhibited when tested in an in vivo system; however, the inhibition does not occur during the translation of mRNA into protein.