Changes in Enzyme Regulation during Growth of Maize: II. Relationships among Multiple Molecular Forms of Homoserine Dehydrogenase

The relative contribution of each of several forms of homoserine dehydrogenase (EC 1.1.1.3) to the total enzyme population in etiolated shoots and in roots of Zea mays L. var. earliking was examined by the use of gel filtration chromatography and disc gel electrophoresis. In enzyme preparations derived from shoots of seedlings grown for 72, 120, or 168 hours, two molecular forms, II and III, which have the same apparent molecular weight but differ in net charge, contributed 75 to 80% of the total enzyme activity. A lower molecular weight species, form I, contributed 20 to 25% of the activity from 72-hour shoots, but was found to decrease concomitantly with a proportional increase in activity contributed by aggregated enzyme form(s) during shoot development. Form I contributed a comparatively larger fraction of the total enzyme activity in preparations of roots of 72-hour seedlings.     

Quantitative estimates of the distribution of homoserine dehydrogenase isozymes in maize tissues

The low molecular weight threonine-resistant (class I) and the higher molecular weight threonine-sensitive (class II/III) isozymes of homoserine dehydrogenase (EC 1.1.1.3) isolated from Zea mays L. were shown to differ in stability during incubations in the presence of urea. Class II/III was inactivated by urea in a time- and concentration-dependent manner, with complete inactivation occurring within 24 hours at 5 degrees C in 4.0 m urea. Under identical conditions, neither the activity nor the properties of class I were affected. Therefore, it was possible to estimate the amounts and properties of both maize isozymes in crude mixtures by measurements of enzyme activity before and after treatment with urea.The relative amounts of the two isozymes proved to be tissue-specific. When shoots of etiolated seedlings were extracted under optimum conditions, the resultant preparations contained about 16% class I and 84% class II/III. This distribution of isozymes, as well as the regulatory properties of class II/III, were constant during growth of the seedlings between 4 and 13 days. Enzyme preparations isolated from shoots of light-grown plants contained higher proportions of class I. The two isozymes were not uniformly distributed within leaves, as the basal meristematic region contained high levels of II/III and small amounts of I. During leaf maturation, the amount of II/III declined while the level of I remained constant or increased slightly. As a result, nearly half of the enzyme extracted from leaf tips was class I. The synthesis of specific members of the aspartate family of amino acids might be expected to differ when the ratio of threonine-sensitive to threonine-resistant homoserine dehydrogenase is altered. However, additional information on the subcellular localization and the catalytic characteristics of the two enzymes is required for evaluation of this possibility.     

Effects of plant age and extraction conditions on the properties of homoserine dehydrogenase isolated from maize seedlings

Homoserine dehydrogenase (EC 1.1.1.3) was extracted from shoots of etiolated seedlings of Zea mays L. which had been grown for periods ranging from three to thirteen days. Both the amount of enzyme extracted and its regulatory properties, as measured by the sensitivity of the enzyme to inhibition by the feedback modulator, l-threonine, were found to be a function of seedling age and extraction conditions. Equivalent amounts of enzyme with similar properties could be isolated from young seedlings under a variety of conditions. Extraction media containing comparatively low concentrations of the buffer component and a high concentration of dithioerythritol were found to be required for optimum extraction of the enzyme from shoots of seedlings grown longer than four days and from leaves of light-grown plants. In the absence of dithioerythritol, diminished regulatory control was observed to be a direct function of seedling age. Evidence of rapid desensitization of the enzyme during extraction was obtained from experiments in which dithioerythritol was added to extracts prepared in the absence of a thiol compound. Therefore, previous observations of growth-dependent desensitization in a number of plants could be due to incomplete extraction or to changes in cellular factors which inactivate and/or alter the enzyme. Whether the enzyme itself becomes increasingly susceptible to alteration during seedling growth remains to be established.     

Polyamine oxidase activity and polyamine content in maize during seed germination

Polyamine oxidase (PAO, EC 1.5.3.3) activity and polyamine content in the cell wall and soluble fractions obtained from embryos, endosperms and shoots and roots of etiolated or green seedlings of maize ( Zea mays L. cv. WF9) during the first 7 days of germination were investigated. Polyamine content was also determined in the trichloroacetic acid-soluble (free polyamines) and trichloroacetic acid insoluble (bound polyamines) fraction obtained from the same tissues. PAO activity, determined by the radiometric method based on the recovery of the labelled reaction product 1-pyrroline, was mostly localized in the cell wall fraction. The activity was very low in embryos and endosperms and present in traces in roots. In etiolated shoots PAO activity increased sharply, while in green shoots it was low and increased slowly. No polyamines were found in the cell wall fraction and only putrescine was detected in the soluble fraction, with the exception of the embryo, where spermidine and spermine were also present. In the TCA-soluble fraction of embryos, putrescine increased during imbibition, while spermidine and spermine decreased; in the endosperm no relevant changes in polyamines occurred. In the same fraction of green and etiolated seedlings, putrescine increased, giving a peak at days 3–5, while spermidine decreased to very low levels. The amount of bound polyamines was 1–4% of the free ones. The pattern of PAO activity seems to be unrelated to endogenous free polyamine content, which is the same in shoots and roots of etiolated and green seedlings. Enzyme activity, very low in ungerminated seeds, increased continuously during the progression of germination, especially in etiolated shoots, indicating a possible involvement in cell wall formation.     

Changes in Enzyme Regulation during Growth of Maize: III. Intracellular Localization of Homoserine Dehydrogenase in Chloroplasts

Extracts of leaf tissue of Zea mays L. seedlings were fractionated on nonlinear sucrose gradients to separate subcellular organelles. Homoserine dehydrogenase (EC 1.1.1.3) was identified in those fractions containing intact chloroplasts, as judged by the presence of chlorophyll and triosephosphate isomerase activity. Neither enzyme activity was detected in fractions containing ruptured chloroplasts, mitochondria, or microbodies. Quantitative measurements of enzyme activity and chlorophyll, and electron microscopic analysis of plastid preparations support the conclusion that maize mesophyll chloroplasts contain a significant fraction of the total cellular content of homoserine dehydrogenase.     

Identification and expression of a cDNA from Daucus carota encoding a bifunctional aspartokinase-homoserine dehydrogenase

Aspartokinase (EC 2.7.2.4) and homoserine dehydrogenase (EC 1.1.1.3) catalyze steps in the pathway for the synthesis of lysine, threonine, and methionine from aspartate. Homoserine dehydrogenase was purified from carrot (Daucus carota L.) cell cultures and portions of it were subjected to amino acid sequencing. Oligonucleotides deduced from the amino acid sequences were used as primers in a polymerase chain reaction to amplify a DNA fragment using DNA derived from carrot cell culture mRNA as template. The amplification product was radiolabelled and used as a probe to identify cDNA clones from libraries derived from carrot cell culture and root RNA. Two overlapping clones were isolated. Together the cDNA clones delineate a 3089 bp long sequence encompassing an open reading frame encoding 921 amino acids, including the mature protein and a long chloroplast transit peptide. The deduced amino acid sequence has high homology with the Escherichia coli proteins aspartokinase I-homoserine dehydrogenase I and aspartokinase II-homoserine dehydrogenase II. Like the E. coli genes the isolated carrot cDNA appears to encode a bifunctional aspartokinase-homoserine dehydrogenase enzyme.Abbreviations AK aspartokinase - HSDH homoserine dehydrogenase - PCR polymerase chain reaction - SDS sodium dodecyl sulfate The mention of vendor or product does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over vendors of similar products not mentioned.     

Comparison of sensitive and desensitized forms of maize homoserine dehydrogenase

The properties of homoserine dehydrogenase (EC 1.1.1.3) isolated from shoots of young etiolated seedlings of Zea mays L. var. earliking can be reversibly altered by dialysis against an appropriate buffer. Treatment with 500 millimolar potassium phosphate buffer (pH 7.5) in the absence of l-threonine results in diminished regulatory control such that the enzyme becomes less sensitive to feedback inhibition. The physical and regulatory properties of experimentally altered and unaltered enzymes are compared with those of enzyme isolated from shoots of older seedlings. Multiple forms of both sensitive and insensitive enzymes are identified, and a model which is consistent with the observed isozymes and the difference in regulatory properties of enzymes obtained from seedlings of different ages is proposed. The initially sensitive enzyme is postulated to undergo a conformational change followed by formation of insensitive multimeric aggregated forms. The experimental conditions which facilitate alteration of the enzyme are discussed in relation to conditions which could occur in vivo.     

Evidence for the expression of morphological and biochemical characteristics of C3-photosynthesis in chlorohyllous callus cultures of Zea mays

A Zea mays callus culture containing chlorophyll was established and grown photomixotrophically. Cell chloroplast structure, and pigment and soluble protein contents were examined. Expression of some key enzymes of C₄ carbon metabolism was compared with that of etiolated (heterotrophic) and green photoautotrophic leaves. Chlorophyll content of the callus was 15–20% that of green leaves. Soluble protein content of callus was half that of leaf cells. Electron microscopic observations showed that green callus cells contained only typical granal chloroplasts. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.38) activities in green callus were ca 30% those of green leaves but 2–3 times higher than in etiolated leaves. Quantitative enzyme protein determination, using antibodies specific to maize leaf Rubisco showed that the chloroplastic carboxylase represented about 7% of total soluble protein in green callus, in parallel to its low chlorophyll content. The specific activity of Rubisco in callus and leaves was unchanged. Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) activity in green callus was about 20% that of green leaves and similar to that measured in etiolated leaves. Apparent K_m (PEP) values (0.08 mM) for PEPC isolated from green callus and etiolated leaves were very different from values (0.5 mM) obtained with PEPC from green leaves. These kinetic characteristics together with the absence of inhibition by malate and activation by glucose-6-phosphate suggest that the properties of PEPC isolated from green callus and etiolated maize leaves are very similar to those of PEPPC from C₃ plants. Using PEPC antibodies specific to green maize leaf enzyme, immunotitration of PEPC preparations containing identical enzyme units allowed complete precipitation of the green leaf enzyme with increasing antibody volumes. In contrast, 60–70% of the activity of PEPC from etiolated and green callus was inhibited, suggesting low affinity for the maize green leaf PEPC antiserum (typical C₄ form). Ouchterlony double diffusion tests revealed only partial recognition of PEPC in green callus and etiolated leaves. NAD-malate dehydrogenase (NAD-MDH, EC 1.1.1.37) activity in callus was 2 and 3 times higher, respectively, than in etiolated and green leaves. NADP-malic enzyme (NADP-ME, EC 1.1.1.40) activity in callus cultures was much lower than in green leaves. All our data support the hypothesis that cultures of fully dedifferentiated chlorophyllous tissues of Zea mays possess a C₃-like metabolism.     

beta-Aspartokinase from developing endosperm of maize.

β-aspartokinase (EC 2.7.2.4.) has been isolated from the developing endosperm (30 days post-pollination) of Zea mays (cv. Pioneer 3145). Enzyme activity was dependent upon ATP, Mg⁺⁺ or Mn⁺⁺, aspartate, and protein concentration. Double reciprocal plots of velocity vs. aspartate concentrations deviated from a straight line at low aspartate concentration indicating two apparent Km's of 0.5 and 6.6 mM. Enzyme activity was inhibited by lysine but not by methionine or threonine. The endosperm-derived β-aspartokinase behaved similarly to enzyme isolated from 6-day-old etiolated shoot tissue. The presence of β-aspartokinase in developing endosperm provides new insight into the source of the aspartate-derived amino acids in maize endosperm.     

Use of monoclonal antibodies for the purification and characterization of the threonine-sensitive isozyme of maize homoserine dehydrogenase

Monoclonal antibodies, highly specific for the threonine-sensitive isozyme of maize homoserine dehydrogenase, have been prepared and utilized to purify the enzyme to homogeneity. The results of one- and two-dimensional polyacrylamide gel electrophoresis under denaturing conditions indicate that the enzyme is composed of subunits of identical molecular weight. Apparent microheterogeneity of the subunits was observed during isoelectric focusing, but peptide maps generated by partial cleavage with three different chemical reagents did not reveal any differences among the proteins separated by isoelectric focusing. It is concluded that the subunits of the active dimeric and tetrameric configurations of the maize enzyme are identical or very similar. Evidence is presented which indicates that the enzyme purified by immunoaffinity chromatography retains all of the properties of freshly isolated enzyme, including the ability to undergo several ligand-induced slow transitions among four unique states and complex kinetic responses to physiological substrates. Two monoclonal antibodies are shown to interact differently with the purified enzyme. One, MC-11, reacts with all enzyme molecules, while the other, MC-3, is able to resolve two antigenically distinct subpopulations. These populations are present in approximately equal amounts in etiolated shoots and leaves of light-grown seedlings. However, the results of kinetic and hysteretic studies indicate that they are functionally indistinguishable. The antibodies appear to recognize a structural difference between the enzyme populations which does not result in detectable alterations in their catalytic or regulatory properties.     

Glyceraldehyde-3-Phosphate Dehydrogenase in Greening Zea mays L. Leaves

Nicotinamide adenine dinucleotide phosphate (NADP)-dependent glyceraldehyde-3-phosphate dehydrogenase (GPDH) (EC 1.2.1.13), a chloroplast enzyme, had low activity in etioplasts of maize leaves. A light dependent increase of enzyme activity of 7-day-old etiolated seedlings showed a lag period of about 2.5 hours followed by a rapid increase in activity during the next 10 hours. The chlorophyll content followed a similar pattern of increasing concentration, but its formation was not directly related to NADP-GPDH formation. The specific activity of NADP-GPDH was lowest in the morphologically youngest tissue near the base of the lamina. The increase in NADP-GPDH was inhibited by cycloheximide but not by chloramphenicol. This indicates that at least some of the enzyme polypeptides are synthesized by 80S ribosomes in the cytoplasm, transported into chloroplasts and become active in chloroplasts. In etiolated maize shoots subjected to a combination of both 3-(p-chlorophenyl)-1,1-dimethylurea, monuron at 7 x 10(-5)m and far red light treatment for 15 hours, the NADP-GPDH activity increased 42% over the dark control compared to 70% increase for the light control. It is concluded that NADPH is not absolutely required for the activation of NADP-GPDH in maize leaves under physiological conditions.     

Comparative Enzymology of the Glyceraldehyde 3-Phosphate Dehydrogenases from Pisum sativum

Glyceraldehyde 3-phosphate dehydrogenases (EC 1.2.1.12 and 1.2.1.13) have been purified from the seed, root, etiolated, and green shoot of peas (Pisum sativum). These enzymes are tetramers of 140,000 daltons, with subunits of 35,000 daltons. The enzymes differ in isoelectric point. The seed enzyme has a pI of 5.1, and the root enzyme has a pI of 4.5. The cytoplasmic enzyme from etiolated shoots is slightly acidic with a pI of 5.7 to 6.1 and is found in two separable forms. The chloroplast enzyme (from green shoots) is most basic with a pI of 8.0.     

Evidence for asparagusate and asparagusate dehydrogenase localized near the apices of asparagus shoots

A method was developed for high-speed liquid chromatographicdetermination of asparagusate in asparagus shoot. The in vivo concentration of asparagusate and the activitiesof asparagusate dehydrogenase and lipoyl dehydrogenase (EC 1.6.4.3 [EC] )in etiolated and green shoots of asparagus (Asparagus officinalisL.) were measured. The region around the apices of the shootshad higher levels of asparagusate, asparagusate dehydrogenase,and lipoyl dehydrogenase than other regions. The level of asparagusatein the etiolated shoots was lower than that in the green shoots,whereas the levels of asparagusate dehydrogenase and lipoyldehydrogenase in the etiolated shoots were higher than thosein the green shoots. Asparagusate was localized in the solublefraction and asparagusate dehydrogenase and lipoyl dehydrogenaseactivities were localized in the mitochondrial fraction. Furthermore,asparagusate was found in all the various asparagus speciestested. (Received May 4, 1976; )     

Nonreversible d-Glyceraldehyde 3-Phosphate Dehydrogenase of Plant Tissues

Preparations of TPN-linked nonreversible d-glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.9), free of TPN-linked reversible d-glyceraldehyde 3-phosphate dehydrogenase, have been obtained from green shoots, etiolated shoots, and cotyledons of pea (Pisum sativum), cotyledons of peanut (Arachis hypogea), and leaves of maize (Zea mays). The properties of the enzyme were similar from each of these sources: the Km values for d-glyceraldehyde 3-phosphate and TPN were about 20 μm and 3 μm, respectively. The enzyme activity was inhibited by l-glyceraldehyde 3-phosphate, d-erythrose 4-phosphate, and phosphohydroxypyruvate. Activity was found predominantly in photosynthetic and gluconeogenic tissues of higher plants. A light-induced, phytochrome-mediated increase of enzyme activity in a photosynthetic tissue (pea shoots) was demonstrated. Appearance of enzyme activity in a gluconeogenic tissue (endosperm of castor bean, Ricinus communis) coincided with the conversion of fat to carbohydrate during germination. In photosynthetic tissue, the enzyme is located outside the chloroplast, and at in vivo levels of triose-phosphates and pyridine nucleotides, the activity is probably greater than that of DPN-linked reversible d-glyceraldehyde 3-phosphate dehydrogenase. Several possible roles for the enzyme in plant carbohydrate metabolism are considered.     

Delta-Pyrroline-5-carboxylic Acid Dehydrogenase in Barley, a Proline-accumulating Species

The characteristics of the enzyme Delta(1)-pyrroline-5-carboxylic acid dehydrogenase from etiolated barley (Hordeum distichum) shoots have been examined. The bulk of the enzyme activity was found in the 10,000g pellet fraction, this activity being displayed only after detergent treatment of the suspended pellet. The enzyme was most active at pH 8, and activity was NAD-dependent. Enzyme activity was unaffected by either mannitol or sucrose in the reaction mixture up to a concentration of 0.45 m but was strongly inhibited by Cl(-) and, to a lesser extent, SO(4) (2-). The inhibition attributable to KCl was reversed by increasing the concentration of Delta(1)-pyrroline-5-carboxylic acid in the reaction mixture.     

Light induced changes in the polypeptide composition of Sorghum bicolor

Two dimensional gel electrophoresis resolved total protein extracted from Sorgum bicolor (L.) Moench shoots into 430 polypeptides detectable by silver staining. The relative amunts of 82 polypeptides increased and 192 decreased upon exposure of etiolated shoots to light. Total protein extracted from etiolated mesocotyls was resolved into 360 polypeptides. The relative amounts of 23 mesocotyl polypeptides increased and 5 decreased after light exposure. Of 274 polypeptides whose relative amounts was altered in shoots exposed to light, 189 were found in mesocotyls. The relative amounts of only 6 polypeptides showed the same response to light in mesocotyls as in shoots. Immunoblotting identified 2 subunits of phosphoenolpyruvate carboxylase (PEPCase) differing in isoelectric point and molecular weight. The 81 000 dalton subunit was the major subunit found in mesocotyls while the 95 000 dalton subunit was the major subunit found in green shoots suggesting that these subunits are derived from a non-photosynthetic and photosynthetic isozyme of PEPCase. In etiolated shoots but not in mesocotyls, light induced the 81 000 and 95 000 apparent molecular weight (MW) subunits of PEPCase, the subunit of pyruvate orthophosphate dikinase (PPDK) as well as the large and small subunits of ribulose-1,5-bisphosphate carboxylase (RuBPCase). Specific activity measurements indicated that light induced the accumulation of the peroxisomal enzyme hydroxypyruvate reductase and inhibited the accumulation of the mitochondrial enzyme serine hydroxymethyltransferase in both mesocotyls andshoots. NADP-glyceraldehyde-3-phosphate dehydrogenase activity was light induced in shoots but undetectable in mesocotyls. In shoots and meocotyls, light had little effect on the mitochondrial enzyme fumarase or the cytoplasmic enzyme NAD-glyceraldehyde-3-P-dehydrogenase.     

Activation of glutamate dehydrogenase by L-leucine

The activation of glutamate dehydrogenase (L-glutamate: NAD(P)+ oxidoreductase (deaminating), EC 1.4.1.3) by L-leucine has been studied. Apparently homogeneous preparations from ox liver and brain were found to respond similarly. Commercially obtained preparations of the enzyme, which had suffered limited proteolysis during the purification procedure, were shown to behave similarly to preparations which had not suffered such proteolysis when the effects of L-leucine on the oxidative deamination reaction were studied using either NAD+ or NADP+ as the coenzyme. There was also no significant difference in the responses when the reductive reaction was determined with NADPH or with 40 microM NADH. At higher concentrations of NADH (160 microM) the unproteolysed preparations were activated by L-leucine to a considerably greater extent than those which had suffered limited proteolysis. These results accord with the greater sensitivity of the former preparations to inhibition by high concentrations of NADH and the relief of such inhibition by L-leucine. This amino acid was also found to relieve the inhibition of the enzyme by GTP, resulting in an apparent increase in the activation observed in the presence of this nucleotide. In contrast, under the conditions used in this work, the apparent degree of activation by L-leucine was found to be decreased in the presence of the activators ATP or ADP. The presence of high concentrations of NADH (200 microM) potentiated the high substrate inhibition by 2-oxoglutarate, and L-leucine significantly reduced this effect. The effects of L-leucine on the activity of glutamate dehydrogenase thus appear to be composed of a direct effect on the activity of the enzyme together with a relief of high substrate inhibition. The effects of GTP and 2-oxoglutarate in potentiating inhibition by NADH can account for their effects in enhancing the apparent activation by L-leucine. The marked differences in the responses of proteolysed and unproteolysed preparations of the enzyme result from the effects of proteolysis in decreasing the sensitivity to high concentrations of NADH.     

Light-mediated changes in the plastidic phosphorylase patterns in shoots of Pisum sativum

α-1,4-Glucan phosphorylase (EC 2.4.1.1) forms from light or dark grown shoots of Pisum sativum L. cv. 'Kleine Rheinländerin' have been studied using various electrophoretic techniques. The phosphorylase patterns of green and etiolated shoots differed. Etiolated shoots contained two enzyme forms, one residing inside and the other outside the etioplast; this was shown by electrophoresis of extracts of isolated etioplasts. Purity and intactness of the organelle preparation were ascertained by electron microscopy. Light-grown shoots contained, in addition to these two enzyme forms, a third phosphorylase which appears to be chloroplast-specific. The two plastidic phosphorylase forms differed slightly in their apparent molecular masses (as determined by non-denaturing polyacrylamide gel electrophoresis) and in their affinities towards branched polyglucans (as revealed by affinity electrophoresis). The apparent affinity of the extrachloroplastic phosphorylase form to these polyglucans was orders of magnitude higher than that of the two plastidic enzyme forms. The development of the chloroplast-specific phosphorylase pattern is under photocontrol. Investigations performed with red or far-red illuminated wild-type plants and with a pale mutant which has a highly reduced pigment and thylakoid content suggest that this photocontrol is mediated by phytochrome.     

Dehydrophylloquinone, α-dehydrotocopherolquinone and dehydrotocopherols from etiolated maize and barley shoots

Dehydrophylloquinone, α-dehydrotocopherolquinone, α-dehydrotocopherol and γ-dehydrotocopherol have been isolated from etiolated maize and barley shoots and excised etiolated maize shoots that have been exposed to light. They are not present, however, in green maize shoots, spinach leaves and etiolated bean leaves. Demethylphylloquinone was not detected in any of the tissues analysed.     

Solubilization of a Proline Dehydrogenase from Maize (Zea mays L.) Mitochondria

L-Proline is oxidized to pyrroline-5-carboxylic acid in intact plant mitochondria by a proline dehydrogenase (EC 1.4.3) that is bound to the matrix side of the inner mitochondrial membrane (TE Elthon, CR Stewart [1981] Plant Physiol 67: 780-784). This investigation reports the first solubilization of the L-proline dehydrogenase (PDH) from plant mitochondria. The supernatant from NP-40-treated etiolated shoot mitochondria of maize, Zea mays L., reduced iodonitrotetrazolium violet in a proline dependent manner. The pH optimum for this activity was 8. The apparent K_m for proline was 6.6 millimolar. When supplied with proline, this solubilized PDH activity also synthesized pyrroline-5-carboxylic acid. The PDH activity was inhibited in vitro by 300 millimolar potassium chloride but not by 300 millimolar potassium acetate. The PDH activity had a molecular mass that was greater than 150 kilodaltons. Mitochondria were prepared from etiolated shoots grown in 100% water-saturated vermiculite (control) and 16% water-saturated vermiculite (stress). The specific activity of solubilized PDH from the stress treatment was 11% of the same activity from the control treatment. Oxygen uptake in the presence of proline and ADP (state 3 proline oxidation) by mitochondria from the stress treatment was 25% of the same rate by mitochondria from the control treatment. Mitochondria were also prepared 16 hours after rewatering the seedlings growing in the stress treatment. Both the solubilized PDH specific activity and state 3 proline oxidation returned to the control levels. The specific activities of the NAD⁺-dependent pyrroline-5-carboxylic acid dehydrogenase and cytochrome c oxidase in the solubilized preparations were unaffected by these stress and recovery treatments. Oxygen uptake rates by intact mitochondria in the presence of ADP and NADH, succinate or malate-pyruvate were also unaffected by these treatments.