Ray C. Huffaker

Differential synthesis of ribulosediphosphate carboxylase subunits

Abstract Chloramphenicol specifically inhibited the synthesis of the large subunit of ribulosediphosphate carboxylase. Cycloheximide exerted a primary effect upon synthesis of the smaller subunit and influenced production of the larger subunit by rapidly inhibiting total protein synthesis.

Nitrogen fixation and delayed leaf senescence in soybeans.

Delayed leaf senescence has been found in a soybean population which maintains its chlorophyll and ribulosebisphosphate carboxylase activity in leaves and nitrogen fixation (acetylene reduction) activity in root nodules throughout seed maturation. Incorporation of delayed leaf senescence into an agronomically desirable genetic background may help to increase seed yield and symbiotic nitrogen fixation during seed development.

Latent nitrate reductase activity is associated with the plasma membrane of corn roots.

Latent nitrate reductase activity (NRA) was detected in corn (Zea mays L., Golden Jubilee) root microsome fractions. Microsome-associated NRA was stimulated up to 20-fold by Triton X-100 (octylphenoxy polyethoxyethanol) whereas soluble NRA was only increased up to 1.2-fold. Microsome-associated NRA represented up to 19% of the total root NRA. Analysis of microsomal fractions by aqueous two-phase partitioning showed that the membrane-associated NRA was localized in the second upper phase (U2). Analysis with marker enzymes indicated that the U2 fraction was plasma membrane (PM). The PM-associated NRA was not removed by washing vesicles with up to 1.0 M NACl but was solubilized from the PM with 0.05% Triton X-100. In contrast, vanadate-sensitive ATPase activity was not solubilized from the PM by treatment with 0.1% Triton X-100. The results show that a protein capable of reducing nitrate is embedded in the hydrophobic region of the PM of corn roots.

Evidence for a plasma-membrane-bound nitrate reductase involved in nitrate uptake of Chlorella sorokiniana

Anti-nitrate-reductase (NR) immunoglobulin-G (IgG) fragments inhibited nitrate uptake into Chlorella cells but had no affect on nitrite uptake. Intact anti-NR serum and preimmune IgG fragments had no affect on nitrate uptake. Membrane-associated NR was detected in plasma-membrane (PM) fractions isolated by aqueous two-phase partitioning. The PM-associated NR was not removed by sonicating PM vesicles in 500 mM NaCl and 1 mM ethylenediaminetetraacetic acid and represented up to 0.8% of the total Chlorella NR activity. The PM NR was solubilized by Triton X-100 and inactivated by Chlorella NR antiserum. Plasma-membrane NR was present in ammonium-grown Chlorella cells that completely lacked soluble NR activity. The subunit sizes of the PM and soluble NRs were 60 and 95 kDa, respectively, as determined by sodium-dodecyl-sulfate electrophoresis and western blotting.

Comparative Induction of Nitrate and Nitrite Uptake and Reduction Systems by Ambient Nitrate and Nitrite in Intact Roots of Barley (Hordeum vulgare L.) Seedlings

The induction by ambient NO3- and NO2- of the NO3- and NO2- uptake and reduction systems in roots of 8-d-old intact barley (Hordeum vulgare L.) seedlings was studied. Seedlings were induced with concentrations of NaNO3 or NaNO2 ranging from 0.25 to 1000 [mu]M. Uptake was determined by measuring the depletion of either NO3- or NO2- from uptake solutions. Enzyme activities were assayed in vitro using cell-free extracts. Uptake and reduction systems for both NO3- and NO2- were induced by either ion. The Km values for NO3- and NO2- uptake induced by NO2- were similar to those for uptake induced by NO3-. Induction of both the uptake and reduction systems was detected well before any NO3- or NO2- was found in the roots. At lower substrate concentrations of both NO3- and NO2- (5–)10 [mu]M), the durations of the lag periods preceding induction were similar. Induction of uptake, as a function of concentration, proceeded linearly and similarly for both ions up to about 10 [mu]M. Then, while induction by NO3- continued to increase more slowly, induction by NO2- sharply decreased between 10 and 1000 [mu]M, apparently due to NO2- toxicity. In contrast, induction of NO3- reductase (NR) and NO2- reductase (NiR) by NO2- did not decrease above 10 [mu]M but rather continued to increase up to a substrate concentration of 1000 [mu]M. NO3- was a more effective inducer of NR than was NO2-; however, both ions equally induced NiR. Cycloheximide inhibited the induction of both uptake systems as well as NR and NiR activities whether induced by NO3- or NO2-. The results indicate that in situ NO3- and NO2- induce both uptake and reduction systems, and the accumulation of the substrates per se is not obligatory.

Evidence for the Existence of Peptide Hydrolase Activity Associated with Chloroplasts Isolated From Barley Mesophyll Protoplasts

Summary Peptide hydrolase activity was assessed by following the loss of the large subunit (LSU) of endogenous ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) in a reaction mixture prepared from chloroplasts isolated from barley (Hordeum vulgare L.) mesophyll protoplasts. The activity was assayed by measuring the relative density of the LSU protein band obtained following polyacrylamide gel electrophoresis in the presence of SDS. An assay of the enzyme activity over a pH range of 3 to 9 indicated a pH optimum of 4.5 and an inhibition by sulfhydryl group inhibitors. The peptide hydrolase activity in chloroplasts treated with 0.1% SDS had a higher pH optimum of 6.5 and was about four-fold greater than the activity with pH 4.5 optimum. The SDS-stimulated activity was also decreased by sulfhydryl inhibitors but in addition was sensitive to the serine protease inhibitor, phenylmethylsulfonylfluoride.

Stimulation of Nitrate and Nitrite Efflux by Ammonium in Barley (Hordeum vulgare L.) Seedlings

The inhibitory effect of NH4+ on net NO3- uptake has been attributed to an enhancement of efflux and, recently, to an inhibition of influx. To study this controversy, we devised treatments to distinguish the effects of NH4+ on these two processes. Roots of intact barley (Hordeum vulgare L.) seedlings, uninduced or induced with NO3- or NO2-, were used. Net uptake and efflux, respectively, were determined by following the depletion and accumulation in the external solutions. In roots of both uninduced and NO2- -induced seedlings, NO3- efflux was negligible; hence, the initial uptake rates were equivalent to influx. Under these conditions, NH4+ had little effect on NO3- uptake (influx) rates by either the low- or high-Km uptake systems. In contrast, in plants preloaded with NO3-, NH4+ and its analog CH3NH3+ decreased net uptake, presumably by enhancing NO3- efflux. The stimulatory effect of NH4+ on NO3- efflux was a function of external NH4+ and internal NO3- concentration. These results were corroborated by the absence of any effect of NH4+ on NO2- uptake unless the roots were preloaded with NO2-. In this case NH4+ increased efflux and decreased net uptake. Hence, the main effect of NH4+ on net NO3- and NO2- uptake appears to be due to enhancement of efflux and not to inhibition of influx.

Effect of ammonium on the regulation of nitrate and nitrite transport systems in roots of intact barley (Hordeum vulgare L.) seedlings

The effect of NH4+on the regulation of NO3−and NO2−transport systems in roots of intact barley (Hordeum vulgareL.) seedlings grown in NO3−or NO2−was studied. Ammonium partially inhibited “induction” of both transport systems. The inhibition was less severe in NO2−-fed than in NO3−-fed seedlings, presumably due to lower uptake of NH4+in the presence of NO2−. In seedlings pretreated with NH4+subsequent “induction” was inhibited only when NH4+was also present during “induction”, even though pretreated roots accumulated high levels of NH4+. This indicates that inhibition may be regulated by NH4+concentration in the cytoplasm rather than its total accumulation in roots. L-Methionine sulfoximine did not relieve the inhibition by NH4+, suggesting that inhibition is caused by NH4+itself rather than by its assimilation product(s). Ammonium inhibited subsequent expression of NO3−transport activity similarly in roots grown in 0.1, 1.0, or 10 mM NO3−for 24 h (steady-state phase) or 4 d (decline phase), indicating that it has a direct, rather than general feedback effect. “Induction” of the NO3−transport system was about twice as sensitive to NH4+as compared to the NO2−transport system. This may relate to higher turnover rates of membraneassociated NO3−-transport proteins.

Ascorbate induced zeaxanthin formation in wheat leaves and photoprotection of pigment and photochemical activities during aging of chloroplasts in light

Summary Mechanisms by which higher levels of Zeaxanthin (Zx) in detached wheat leaves, induced by ascorbate in vivo treatment, photoprotect isolated chloroplasts aging in light were investigated. Leaves were incubated for 3 h in light at 400 µM −2 S −1 in the following media: ascorbate (Asc), Asc plus dithiotreitol (DTT) and DTT. Pigments were assayed using a reverse phase HPLC, and photochemical activity (PCA) was measured by an oxygen electrode. The Zx level increased by three times when the leaves were incubated in ascorbate for 3 h. When leaves were treated with Asc plus DTT, Zx accumulation that was stimulated by Asc disappeared. Chloroplasts isolated from untreated leaves, followed by in Vitro incubation in light for 4 h, significantly lost pigments and PCA, including whole chain (H 2 O to methyl viologen) electron transport and photosystem II (PS II). Chloroplasts isolated from Asc treated leaves showed relatively less chlorophyll and carotenoid loss, and PCA loss. The photoprotection was nullified in chloroplasts isolated from Asc plus DTT treated leaves, which showed lower levels of Zx. Chloroplasts isolated from the DTT treated leaves showed higher pigment loss and PCA loss as compared with the control. These results clearly show that Zx is responsible for the photoprotection of chloroplasts aging in a cell free condition in light.

Effect of pH and Calcium on Short-Term NO3- Fluxes in Roots of Barley Seedlings

The effect of pH and Ca2+ on net NO3- uptake, influx, and efflux by intact roots of barley (Hordeum vulgare L.) seedlings was studied. Seedlings were induced with NO3- or NO2-. Net NO3- uptake and efflux, respectively, were determined by following its depletion from, and accumulation in, the external solution. Since roots of both uninduced and NO2--induced seedlings contain little internal NO3-, initial net uptake rates are equivalent to influx (M. Aslam, R.L. Travis, R.C. Huffaker [1994] Plant Physiol 106: 1293–1301). NO3- uptake (influx) by these roots was little affected at acidic pH. In contrast, in NO3--induced roots, which accumulate NO3-, net uptake rates decreased in response to acidic pH. Under these conditions, NO3- efflux was stimulated and was a function of root NO3- concentration. Conversely, at basic pH, NO3- uptake by NO3-- and NO2--induced and uninduced roots decreased, apparently because of the inhibition of influx. Calcium had little effect on NO3- uptake (influx) by NO2-- induced roots at either pH 3 or 6. However, in NO3--induced roots, lack of Ca2+ at pH 3 significantly decreased net NO3- uptake and stimulated efflux. The results indicate that at acidic pH the decrease in net NO3- uptake is due to the stimulation of efflux, whereas at basic pH, it is due to the inhibition of influx.