Effect of Exogenous Nitrate on Anaerobic Metabolism in Excised Rice Roots: II. FERMENTATIVE ACTIVITY AND ADENYLIC ENERGY CHARGE

Reggiani, R., Brambilla, I. and Bertani, A. 1985. Effect ofexogenous nitrate on anaerobic metabolism in excised rice roots.II Fermentative activity and adenylic energy charge.—J.exp. Bot 36: 1698–1704. The presence of nitrate in the culture medium of excised sterilerice roots stimulated CO₂ and ethanol evolution and, to a smallerextent, alanine accumulation. The increased anaerobic carbohydrateconsumption observed in roots grown on nitrate is consistentwith the constantly higher level of adenylic energy charge.An hypothesis serving to explain the evidence concerning theeffect of nitrate on anaerobically grown rice roots is proposed. Key words: Anaerobiosis, ethanol, nitrate, alanine, adenylic energy charge     

Effect of Exogenous Nitrate on Anaerobic Metabolism in Excised Rice Roots: I. NITRATE REDUCTION AND PYRIDINE NUCLEOTIDE POOLS

Reggiani, R., Brambilla, I. and Bertani, A. 1985. Effect ofexogenous nitrate on anaerobic metabolism in excised rice roots.I. Nitrate reduction and pyridine nucleotide pools.— J.exp.Bot 36:1193-1199. In apical segments of sterile rice roots, reduction of nitratein the absence of oxygen is promoted by the presence of exogenousnitrate in the growth medium in the first 3 h of oxygen deficiency.Anaerobic treatment also increased the concentration of NADH.When nitrate was made available for reduction, smaller accumulationsof NADH were observed. Oxidation of reduced nudeotides associatedwith the conversion of nitrate to nitrite is suggested as beingresponsible for this behaviour. Key words: Anaerobiosis, nitrate, pyridine nucleotides     

The Effect of Exogenous IAA and Kinetin on Nitrate Reductase,Nitrite Reductase and Glutamate Dehydrogenase Activities in Excised Pea Roots

Nitrate reductase (NO₃R) activity, nitrite reductase (NO₂R) activity and NADH₂ dependent glutamate dehydrogenase (GDH) activity were followed in extracts from excised pea roots incubated under aseptic conditions for 9 and 24 h in nitrate containing nutrient medium to which IAA was added in concentrations promoting lateral root formation (1 × 10^?5; 3 × 10^?5; 5 × 10^?5 M) and kinetin in concentrations which reduce lateral root formation (0.1; 1; 5 mg 1^?1, that is 4.65 × 10^?7;4.65 × 10^?6 and 2.3 × 10^?5 M). NO₃R activity was not influenced by IAA, NO₂R activity was slightly depressed by IAA after 24 h incubation and GDH activity was slightly increased after 24 h incubation in the presence of IAA. Kinetin decreased NO₃R activity significantly both after 9 h and 24 h incubation, slightly increased NO₂R activity after 9 h incubation but slightly decreased it after 24 h incubation, and did not affect GDH activity after 24 h incubation. However, when applied together with IAA, kinetin abolished the promoting effect of IAA on GDH activity. IAA neither reversed nor accentuated the effect of kinetin on NO₂R activity. Nevertheless the depressing effect of kinetin on NO₃R activity was emphasized by the presence of IAA after 9 h incubation. The results obtained indicate that reduced nitrate assimilation due to the depression of nitrate reductase activity caused by kinetin probably contributes to the negative growth effect of kinetin in pea root segments grown in nitrate medium.     

Metabolism of Some Indole Auxins in Excised Tomato Roots

Indolylacetylaspartic acid (IAAsp) and possibly indolylacetylglutamicacid (IAG) are formed by exposure of excised tomato roots toIAA. Little ‘free’ IAA accumulates in the tissue.An unidentified substance reacting pink with nitricnitrite reagentis also formed. These substances are metabolized when IAA-treatedroots are transferred to auxin-free medium. IAAsp and IAA aresimilarly inhibitory to the growth of excised tomato roots.Excised tomato roots do not interconvert IAA and IAN. IAN-feedingleads to IAN accumulation and the appearance of indolylcarboxylicacid (ICA); transference to auxin-free medium causes a declinein the IAN activity but the ICA spot persists. The inhibitoryactivity of IAN is not due to its conversion to ICA. Excisedtomato and wheat roots respond very differently to externall-tryptophane but in neither case is there evidence of the conversionof tryptophane to ethyl acetate-soluble auxins.     

The Regulation of Nitrate Reductase,Nitrite Reductase,and Glutamate Dehydrogenase in Excised Pea Roots by Some Exogenous Amino Acids

The effect of growth retarding amino acids (L-aspartic acid,L-leucine,L-methionine, andL-threonine) on nitrate reductase (NO₃R), nitrite reductase (NO₂R), and NADH₂ dependent glutamate dehydrogenase (GDH) in excised pea roots was followed,L-methionine andL-threonine slightly depressed NO₃R activity,L-aspartic acid enhanced NO₂R and GDH activities.L-methionine andL-threonine also slightly decreased nitrate uptake. The results obtained are discussed in connection with the growth effects of the amino acids investigated.     

The Carbohydrate Nutrition of Tomato Roots: III. The Effects of. External Sucrose Concentration on the Growth and Anatomy of Excised Roots

Variation of the concentration of sucrose in White‘s mediummarkedly affects the increase in length of the main axis, andthe number and total length of the laterals developed by excisedtomato roots. Highest values for these three features are obtainedwith medium containing 1.5 per cent, sucrose. Root materialhas been analysed anatomically. Root diameter, cortical thickness,number of xylem vessels, and development of the cortical air-spacesystem increase with increase in sucrose concentration. Thedegree of vacuolation of extra-stelar meristematic cells increases,and protophloem differentiates further from the promeristemwith rising sucrose concentrations. The interpretation of theseresults is discussed.     

Varietal Differences in the Kinetics of Iron Uptake by Excised Rice Roots

Iron uptake by excised rice roots can be described in kineticterms of active absorption and the formation of ion-carriercomplexes. Conventional interpretation indicates two carriers,one of which is responsible for iron absorption at low concentrations,with the second primarily functioning at high iron levels. Themaximum absorption of both carriers vanes greatly between varieties,the variety Pebifun having much greater absorptive capacitythan either Siam-29 or Paldal. Varietal differences in uptakecapacity primarily depend on total carrier concentration, 0.93,0.40,and 0.31 µ moles per g roots of Pebifun, Siam-29, andPaldal respectively. Although the kinetic treatment suggeststwo carriers, the chromatographic evidence indicates the presenceof the iron primarily in one major initial complex, with suggestionsof accumulation rather than turnover. An alternative to thecarrier hypothesis is therefore put forward which regards theaccumulation of iron as binding initially on to one or severalclosely related substances, followed by incorporation into secondaryproducts when a critical level of the initial product is exceeded.Manganese was found to competitively inhibit iron uptake byrice roots. Inhibition was more severe in Siam-29 than in Paldaland can be explained on the basis of carrier concentration andcompetition for the same carrier site. Copper was found notto be competitive for the iron absorption sites. The effectof copper was not significant with the low levels of iron butonly with the 5 x 10^–4 M Fe level. The effect varied betweenvarieties, in Pebifun both 5 x 10^–5 and 5 x 10^–6M Cu inhibited iron uptake compared with the control. In thecase of Siam-29 both copper levels accelerated iron uptake,5 x 10^–5 M Cu giving the greatest uptake. With 5 x 10^–5M Cu Pebifun took up much less iron than Siam-29. The mecharosmof copper inhibition/stimulation is highly speculative.     

Potassium Transport in Corn Roots : III. Perturbation by Exogenous NADH and Ferricyanide

It has recently been reported that plasmalemma electron transport may be involved in the generation of H⁺ gradients and the uptake of ions into root tissue. We report here on the influence of extracellular NADH and ferricyanide on K⁺ (⁸⁶Rb⁺) influx, K⁺ (⁸⁶Rb⁺) efflux, net apparent H⁺ efflux, and O₂ consumption in 2-centimeter corn (Zea mays [A632 × Oh43]) root segments and intact corn roots. In freshly excised root segments, NADH had no effect on O₂ consumption and K⁺ uptake. However, after the root segments were given a 4-hour wash in aerated salt solution, NADH elicited a moderate stimulation in O₂ consumption but caused a dramatic inhibition of K⁺ influx. Moreover, net apparent H⁺ efflux was significantly inhibited following NADH exposure in 4-hour washed root segments.

Exogenous ferricyanide inhibited K⁺ influx in a similar fashion to that caused by NADH, but caused a moderate stimulation of net H⁺ efflux. Additionally, both reagents substantially altered K⁺ efflux at both the plasmalemma and tonoplast.

These complex results do not lend themselves to straightforward interpretation and are in contradiction with previously published results. They suggest that the interaction between cell surface redox reactions and membrane transport are more complex than previously considered. Indeed, more than one electron transport system may operate in the plasmalemma to influence, or regulate, a number of transport functions and other cellular processes. The results presented here suggest that plasmalemma redox reactions may be involved in the regulation of ion uptake and the `wound response' exhibited by corn roots.

     

Some Effects of Light on Excised Wheat Roots with Special Reference to Peroxide Metabolism

When excised wheat roots are exposed to blue light, catalase activity changes in a way to he expected as a result of photodestruction with first order kinetics. Wheat root catalase in vivo is less light sensitive than animal catalase in vitro, possibly due to internal screening. Illumination of the roots with red light causes some increase in catalasc activity. Peroxidase activity is much less affected by light. No relation has been found between catalase destruction and chlorophyll formation. The ability of roots to oxidize pyrogallol to purpurogallin (by other workers interpreted as peroxide production) is decreased by light, especially blue light. On the contrary, one peroxide producing enzyme, glycolic acid oxidase, was detected only in roots grown in blue light. The total flavin content, or the fraction present as FAD, is not affected by light. The ascorbic acid content is low, but slightly increased by blue light.     

Effect of l-Canavanine on Nitrate Reductase in Corn Roots

l-Canavanine inhibits the appearance of nitrate reductase (NADH-nitrate oxidoreductase, EC 1.6.6.1) in both root tips and mature root sections of corn (Zea mays L.). Ten-fold more canavanine was required to cause a 50% reduction in the level of nitrate reductase activity (NRA) in root tips than in mature root sections. For example with one particular batch of seeds 500 μm canavanine was effective in root tips whereas only 50 μm was required in mature root sections. In root tips arginine (1 mm) completely reversed the effect of 1 mm canavanine. In mature root sections higher concentrations of arginine (approximately 5 mm) were required for a complete reversal of the canavanine effect. Additions of canavanine to roots after a period of 3 hours with 5 mm KNO₃ resulted in a loss of NRA. NO₃⁻ protected nitrate reductase from this inactivation in both root tip and mature root sections.     

Effect of Various Light Treatments on Chlorophyll Formation in Excised Potato Roots

Potato root tips were grown in cultures and the effect of blue, red, and white light on chlorophyll formation was studied. The roots grown in white light turned green in 4–6 weeks, whereas in blue or red light, green colour occasionally appeared at places. The chlorophyll contents, as determined by the spectrophotometeric method, were found to be maximum in unfiltered light followed by blue and red light. In white and blue light treatments chlorophyll a contents were higher than chlorophyll b, however in red light this was reversed. The results are compared with earlier experiments on chlorophyll formation in excised roots.     

Anaerobic Metabolism in the N-Limited Green Alga Selenastrum minutum: III. Alanine Is the Product of Anaerobic Ammonium Assimilation

We have determined the flow of ¹⁵N into free amino acids of the N-limited green alga Selenastrum minutum (Naeg.) Collins after addition of ¹⁵NH₄⁺ to aerobic or anaerobic cells. Under aerobic conditions, only a small proportion of the N assimilated was retained in the free amino acid pool. However, under anaerobic conditions almost all assimilated NH₄⁺ accumulates in alanine. This is a unique feature of anaerobic NH₄⁺ assimilation. The pathway of carbon flow to alanine results in the production of ATP and reductant which matches exactly the requirements of NH₄⁺ assimilation. Alanine synthesis is therefore an excellent strategy to maintain energy and redox balance during anaerobic NH₄⁺ assimilation.