pH Changes Associated with Iron-Stress Response

When Fe-inefficient T3238fer and Fe-efficient T3238FER tomatoes were supplied iron, and nitrogen as nitrate, they increased the pH of the nutrient culture. When they were supplied nitrogen as ammonium, they decreased the pH. When Fe supply was limited, Fe-stress response developed in T3238FER that opposed the usual nitrate response and decreased, rather than increased, the pH. A “reductant” which reduced Fe³⁺ to Fe²⁺ was released from the roots of these plants and lowered the pH; and there was a tremendous increase in the uptake of Fe. T3238fer did not produce “reductant” in response to Fe-stress; the pH increased, and the plants developed Fe-deficiency when nitrogen was supplied as nitrate. Nitrogen nutrition and iron-stress response are important factors associated with iron chlorosis in plants. Release of hydrogen ions from roots of Fe-stressed plants is caused by more than response to imbalanced uptake of cations and anions.     

Anion channel SLAH3 functions in nitrate‐dependent alleviation of ammonium toxicity in Arabidopsis

Slow anion channels (SLAC/SLAH) are efflux channels previously shown to be critical for stomatal regulation. However, detailed analysis using the β‐glucuronidase reporter gene showed that members of the SLAC/SLAH gene family are predominantly expressed in roots, in addition to stomatal guard cells, implicating distinct function(s) of SLAC/SLAH in the roots. Comprehensive mutant analyses of all slac/slah mutants indicated that slah3 plants showed a greater growth defect than wild‐type plants when ammonium was supplied as the sole nitrogen source. Ammonium toxicity was mimicked by acidic pH in nitrogen‐free external medium, suggesting that medium acidification by ammonium‐fed plants may underlie ammonium toxicity. Interestingly, such toxicity was more severe in slah3 mutants and, particularly in wild‐type plants, was alleviated by supplementing the media with micromolar levels of nitrate. These data thus provide evidence that SLAH3, a nitrate efflux channel, plays a role in nitrate‐dependent alleviation of ammonium toxicity in plants.     

The use of different soil nitrogen sources by young Norway spruce plants

 Three-year-old Norway spruce trees were planted into a low-nitrogen mineral forest soil and supplied either with two different levels of mineral nitrogen (NH₄NO₃) or with a slow-release form of organic nitrogen (keratin). Supply of mineral nitrogen increased the concentrations of ammonium and nitrate in the soil solution and in CaCl₂-extracts of the rhizosphere and bulk soil. In the soil solution, in all treatments nitrate concentrations were higher than ammonium concentrations, while in the soil extracts ammonium concentrations were often higher than nitrate concentrations. After 7 months of growth, ¹⁵N labelled ammonium or nitrate was added to the soil. Plants were harvested 2 weeks later. Keratin supply to the soil did not affect growth and nitrogen accumulation of the trees. In contrast, supply of mineral nitrogen increased shoot growth and increased the ratio of above-ground to below-ground growth. The proportion of needle biomass to total above-ground biomass was not increased by mineral N supply. The atom-% ¹⁵N was higher in younger needles than in older needles, and in younger needles higher in plants supplied with ¹⁵N-nitrate than in plants supplied with ¹⁵N-ammonium. The present data show that young Norway spruce plants take up nitrate even under conditions of high plant internal N levels. Received: 1 April 1998 / Accepted: 9 October 1998     

The metabolic flux phenotype of heterotrophic Arabidopsis cells reveals a complex response to changes in nitrogen supply

The extent to which individual plants utilise nitrate and ammonium, the two principal nitrogen sources in the rhizosphere, is variable and many species require a balance between the two forms for optimal growth. The effects of nitrate and ammonium on gene expression, enzyme activity and metabolite composition have been documented extensively with the aim of understanding the way in which plant cells respond to the different forms of nitrogen, but ultimately the impact of these changes on the organisation and operation of the central metabolic network can only be addressed by analysing the fluxes supported by the network. Accordingly steady‐state metabolic flux analysis was used to define the metabolic phenotype of a heterotrophic Arabidopsis thaliana cell culture grown in Murashige and Skoog and ammonium‐free media, treatments that influenced growth and biomass composition. Fluxes through the central metabolic network were deduced from the redistribution of label into metabolic intermediates and end products observed when cells were labelled with [1‐¹³C]‐, [2‐¹³C]‐ or [¹³C₆]glucose, in tandem with ¹⁴C‐measurements of the net accumulation of biomass. Analysis of the flux maps showed that: (i) flux through the oxidative pentose phosphate pathway varied independently of the reductant demand for biosynthesis, (ii) non‐plastidic processes made a significant and variable contribution to the provision of reducing power for the plastid, and (iii) the inclusion of ammonium in the growth medium increased cell maintenance costs, in agreement with the futile cycling model of ammonium toxicity. These conclusions highlight the complexity of the metabolic response to a change in nitrogen nutrition.     

Does the Response of Perennial Ryegrass to Elevated CO2 Concentration Depend on the Form of the Supplied Nitrogen?

To test whether different nitrogen form (nitrate or ammonium) in substrate can alter the response to elevated partial pressure of CO₂ (pCO₂) plants of perennial ryegrass (Lolium perenne cv. Bastion) were grown from seeds in growth chambers under pCO₂ of either 35 Pa (ambient, CA) or 70 Pa (elevated, CE) in a hydroponic system (with nutrient and pH control) for 24 d. Nitrogen was supplied as ammonium, nitrate or an equimolar mixture of both N forms. Under CE plants grew faster than their counterparts under CA during the first 14 d but after 23 d of cultivation stimulation disappeared. Despite the strong positive effect of mixed forms of N on plant growth, the beneficial effect of CE was similar to that in the other two N treatments. However, the almost alike final growth response to CE had different underlying mechanisms in different N treatments. Plants supplied with nitrate as a sole source of nitrogen had lower leaf mass ratio but much higher specific leaf area compared to plants supplied with ammonium. The decrease in the content of leaf organic N (per unit of structural dry mass) under CE was found only in leaves of plants supplied with ammonium on day 14. Nevertheless, the available form of N evidently contributes to changes of leaf N content under CE. The high levels of N and non-structural saccharides in plants supplied with ammonium at CE suggest that the CO₂ response of these plants was controlled by factors other than amount of available carbon and nitrogen.     

NADH‐dependent glutamate synthase plays a crucial role in assimilating ammonium in the Arabidopsis root

Plant roots under nitrogen deficient conditions with access to both ammonium and nitrate ions, will take up ammonium first. This preference for ammonium rather than nitrate emphasizes the importance of ammonium assimilation machinery in roots. Glutamine synthetase (GS) and glutamate synthase (GOGAT) catalyze the conversion of ammonium and 2‐oxoglutarate to glutamine and glutamate. Higher plants have two GOGAT species, ferredoxin‐dependent glutamate synthase (Fd‐GOGAT) and nicotinamide adenine dinucleotide (NADH)‐GOGAT. While Fd‐GOGAT participates in the assimilation of ammonium, which is derived from photorespiration in leaves, NADH‐GOGAT is highly expressed in roots and its importance needs to be elucidated. While ammonium as a minor nitrogen form in most soils is directly taken up, nitrate as the major nitrogen source needs to be converted to ammonium prior to uptake. The aim of this study was to investigate and quantify the contribution of NADH‐GOGAT to the ammonium assimilation in Arabidopsis (Arabidopsis thaliana Columbia) roots. Quantitative real‐time polymerase chain reaction (PCR) and protein gel blot analysis showed an accumulation of NADH‐GOGAT in response to ammonium supplied to the roots. In addition the localization of NADH‐GOGAT and Fd‐GOGAT did not fully overlap. Promoter–β‐glucuronidase (GUS) fusion analysis and immunohistochemistry showed that NADH‐GOGAT was highly accumulated in non‐green tissue like vascular bundles, shoot apical meristem, pollen, stigma and roots. Reverse genetic approaches suggested a reduction in glutamate production and biomass accumulation in NADH‐GOGAT transfer DNA (T‐DNA) insertion lines under normal CO₂ condition. The data emphasize the importance of NADH‐GOGAT in the ammonium assimilation in Arabidopsis roots.     

A novel morphological response of maize (Zea mays) adult roots to heterogeneous nitrate supply revealed by a split‐root experiment

Approximately 35–55% of total nitrogen (N) in maize plants is taken up by the root at the reproductive stage. Little is known about how the root of an adult plant responds to heterogeneous nutrient supply. In this study, root morphological and physiological adaptations to nitrate‐rich and nitrate‐poor patches and corresponding gene expression of ZmNrt2.1 and ZmNrt2.2 of maize seedlings and adult plants were characterized. Local high nitrate (LoHN) supply increased both lateral root length (LRL) and density of the treated nodal roots of adult maize plants, but only increased LRL of the treated primary roots of seedlings. LoHN also increased plant total N acquisition but not N influx rate of the treated roots, when expressed as per unit of root length. Furthermore, LoHN markedly increased specific root length (m g^?1) of the treated roots but significantly inhibited the growth of the lateral roots outside of the nitrate‐rich patches, suggesting a systemic carbon saving strategy within a whole root system. Surprisingly, local low nitrate (LoLN) supply stimulated nodal root growth of adult plants although LoLN inhibited growth of primary roots of seedlings. LoLN inhibited the N influx rate of the treated roots and did not change plant total N content. The gene expression of ZmNrt2.1 and ZmNrt2.2 of the treated roots of seedlings and adult plants was inhibited by LoHN but enhanced by LoLN. In conclusion, maize adult roots responded to nitrate‐rich and nitrate‐poor patches by adaptive morphological alterations and displayed carbon saving strategies in response to heterogeneous nitrate supply.     

Influence of ammonium and nitrate nitrogen on nitrogenase activity of pea plants as affected by light intensity and sugar addition

Summary Addition of ammonium chloride or potassium nitrate to nodulated pea plants resulted in a decrease in acetylene-reducing activity. Both nodule growth and specific activity of the nodules were diminished. Acetylene-reducing activity of isolated bacteroids, treated with EDTA-toluene and supplied with ATP and dithionite, had not decreased after a 3-day treatment of the plants with NH₄Cl or KNO₃. The effect of combined nitrogen could be counteracted by raising the light intensity or by the addition of sucrose to the growth medium. The latter treatment reduced the nitrogen uptake by the plants. It is concluded that combined nitrogen affects symbiotic nitrogen fixation via the carbohydrate supply to the bacteroids.     

The NIN‐like protein 5 (ZmNLP5) transcription factor is involved in modulating the nitrogen response in maize

Maize exhibits marked growth and yield response to supplemental nitrogen (N). Here, we report the functional characterization of a maize NIN‐like protein ZmNLP5 as a central hub in a molecular network associated with N metabolism. Predominantly expressed and accumulated in roots and vascular tissues, ZmNLP5 was shown to rapidly respond to nitrate treatment. Under limited N supply, compared with that of wild‐type (WT) seedlings, the zmnlp5 mutant seedlings accumulated less nitrate and nitrite in the root tissues and ammonium in the shoot tissues. The zmnlp5 mutant plants accumulated less nitrogen than the WT plants in the ear leaves and seed kernels. Furthermore, the mutants carrying the transgenic ZmNLP5 cDNA fragment significantly increased the nitrate content in the root tissues compared with that of the zmnlp5 mutants. In the zmnlp5 mutant plants, loss of the ZmNLP5 function led to changes in expression for a significant number of genes involved in N signalling and metabolism. We further show that ZmNLP5 directly regulates the expression of nitrite reductase 1.1 (ZmNIR1.1) by binding to the nitrate‐responsive cis‐element at the 5′ UTR of the gene. Interestingly, a natural loss‐of‐function allele of ZmNLP5 in Mo17 conferred less N accumulation in the ear leaves and seed kernels resembling that of the zmnlp5 mutant plants. Our findings show that ZmNLP5 is involved in mediating the plant response to N in maize.     

Effects of Nitrate and Ammonium on Gene Expression of Phosphoenolpyruvate Carboxylase and Nitrogen Metabolism in Maize Leaf Tissue during Recovery from Nitrogen Stress

We previously showed that the selective accumulation of phosphoenolpyruvate carboxylase (PEPC) in photosynthetically maturing maize (Zea mays L.) leaf cells induced by nitrate supply to nitrogen-starved plants was primarily a consequence of the level of its mRNA (B Sugiharto, K Miyata, H Nakamoto, H Sasakawa, T Sugiyama [1990] Plant Physiol 92: 963-969). To determine the specificity of inorganic nitrogen sources for the regulation of PEPC gene expression, nitrate (16 millimolar) or ammonium (6 millimolar) was supplied to plants grown previously in low nitrate (0.8 millimolar), and changes in the level of PEPC and its mRNA were measured in the basal region of the youngest, fully developed leaves of plants during recovery from nitrogen stress. The exogenous supply of nitrogen selectively increased the levels of protein and mRNA for PEPC. This increase was more pronounced in plants supplemented with ammonium than with nitrate. The accumulation of PEPC during nitrogen recovery increased in parallel with the increase in the activity of glutamine synthetase and/or ferredoxin-dependent glutamate synthase. Among the major amino acids, glutamine was the most influenced during recovery, and its level increased in parallel with the steady-state level of PEPC mRNA for 7 hours after nitrogen supply. The administration of glutamine (12 millimolar) to nitrogen-starved plants increased the steady-state level of PEPC mRNA 7 hours after administration, whereas 12 millimolar glutamate decreased the level of PEPC mRNA. The results indicate that glutamine and/or its metabolite(s) can be a positive control on the nitrogen-dependent regulation of PEPC gene expression in maize leaf cells.     

Differential Effects of Ammonium and Nitrate on Growth Performance of <i>Glechoma longituba</i> under Heterogeneous Cd Stress

Water, minerals, nutrients, etc., can be shared by physiological integration among inter-connected ramets of clonal plants. Nitrogen plays an important role in alleviating cadmium (Cd) stress for clonal plants. But how different forms of nitrogen affect growth performance of clonal plants subjected to heterogeneous Cd stress still remains poorly understood. A pot experiment was conducted to investigate the differential effects of ammonium and nitrate on growth performance of Glechoma longituba under heterogeneous Cd stress. In the experiment, parent ramets of Glechoma longituba clonal fragments were respectively supplied with modified Hoagland solution containing 7.5 mM ammonium, 7.5 mM nitrate or the same volume of nutrient solution without nitrogen. Cd solution with different concentrations (0, 0.1 or 2.0 mM) was applied to offspring ramets of the clonal fragments. Compared with control (N-free), nitrogen addition to parent ramets, especially ammonium, significantly improved antioxidant capacity [glutathione (GSH), proline (Pro), peroxidase (POD,) superoxide dismutase (SOD) and catalase (CAT)], PSII activity [maximum quantum yield of PSII (F_v/F_m) and effective quantum yield of PSII (ΦPSII)], chlorophyll content and biomass accumulation of the offspring ramets suffering from Cd stress. In addition, negative effects of nitrate on growth performance of whole clonal fragments were observed under Cd stress with high concentration (2.0 mM). Transportation or sharing of nitrogen, especially ammonium, can improve growth performance of clonal plants under heterogeneous Cd stress. The experiment provides insight into transmission mechanism of nitrogen among ramets of clonal plants suffering from heterogeneous nutrient supply. Physiological integration might be an important ecological strategy for clonal plants adapting to heterogeneous environment stress conditions.     

Humic acid differentially improves nitrate kinetics under low‐ and high‐affinity systems and alters the expression of plasma membrane H+‐ATPases and nitrate transporters in rice

Humic acids (HAs) have a major effect on nutrient uptake, metabolism, growth and development in plants. Here, we evaluated the effect of HA pretreatment applied with a nutrient solution on the uptake kinetics of nitrate nitrogen (N‐NO₃^?) and the metabolism of nitrogen (N) in rice under conditions of high and low NO₃^? supply. In addition, the kinetic parameters of NO₃^? uptake, N metabolites, and nitrate transporters (NRTs) and the plasma membrane (PM) H⁺‐ATPase gene expression were examined. The plants were grown in a growth chamber with modified Hoagland and Arnon solution until 21 days after germination (DAG), and they were then transferred to a solution without N for 48 h and then to another solution without N and with and without the addition of HAs for another 48 h. After this period of N deprivation, the plants received new nutrient solutions containing 0.2 and 2.0 mM N‐NO₃^?. Treatment of rice plants with HA promoted the induction of the genes OsNRT2.1‐2.2/OsNAR2.1 and some isoforms PM H⁺‐ATPase in roots. The application of HAs differentially modified the parameters of the uptake kinetics of NO₃^? under both concentrations. When grown with 0.2 mM NO₃^?, the plants pretreated with HA had lower K_m and C_min values as well as a higher V_max/K_m ratio. When grown with 2 mM NO₃^?, the plants pretreated with HA had a higher V_max value, a greater root and shoot mass, and a lower root/shoot ratio. The N fractions were also altered by pretreatment with HA, and a greater accumulation of NO₃^? and N‐amino was observed in the roots and shoots, respectively, of plants pretreated with HA. The results suggest that pretreatment with HA modifies root morphology and gene expression of PM H⁺‐ATPases and NO₃^? transporters, resulting in a greater efficiency of NO₃^? acquisition by high‐ and low‐affinity systems.     

Short‐term ammonium supply induces cellular defence to prevent oxidative stress in Arabidopsis leaves

Plants can assimilate nitrogen from soil pools of both ammonium and nitrate, and the relative levels of these two nitrogen sources are highly variable in soil. Long‐term ammonium nutrition is known to cause damage to Arabidopsis that has been linked to mitochondrial oxidative stress. Using hydroponic cultures, we analysed the consequences of rapid shifts between nitrate and ammonium nutrition. This did not induce growth retardation, showing that Arabidopsis can compensate for the changes in redox metabolism associated with the variations in nitrogen redox status. During the first 3 h of ammonium treatment, we observed distinct transient shifts in reactive oxygen species (ROS), low‐mass antioxidants, ROS‐scavenging enzymes, and mitochondrial alternative electron transport pathways, indicating rapid but temporally separated changes in chloroplastic, mitochondrial and cytosolic ROS metabolism. The fast induction of antioxidant defences significantly lowered intracellular H₂O₂ levels, and thus protected Arabidopsis leaves from oxidative stress. On the other hand elevated extracellular ROS production in response to ammonium supply may be involved in signalling. The response pattern displays an intricate plasticity of Arabidopsis redox metabolism to minimise stress in responses to nutrient changes.     

The interaction between elevated carbon dioxide and nitrogen nutrition: the physiological and molecular background

AGPase, ADP glucose pyrophosphorylase
GS, glutamine synthetase
GOGAT, glutamate : oxoglutarate amino transferase
NADP-ICDH, NADP-dependent isocitrate dehydrogenase
NR, nitrate reductase
OPPP, oxidative pentose phosphate pathway
3PGA, glycerate-3-phosphate
PEPCase, phosphoenolpyruvate carboxylase
Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase
SPS, sucrose phosphate-synthase

This review first summarizes the numerous studies that have described the interaction between the nitrogen supply and the response of photosynthesis, metabolism and growth to elevated [CO₂]. The initial stimulation of photosynthesis in elevated [CO₂] is often followed by a decline of photosynthesis, that is typically accompanied by a decrease of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), an accumulation of carbohydrate especially starch, and a decrease of the nitrogen concentration in the plant. These changes are particularly marked when the nitrogen supply is low, whereas when the nitrogen supply is adequate there is no acclimation of photosynthesis, no major decrease in the internal concentration of nitrogen or the levels of nitrogen metabolites, and growth is stimulated markedly. Second, emerging evidence is discussed that signals derived from nitrate and nitrogen metabolites such as glutamine act to regulate the expression of genes involved in nitrate and ammonium uptake and assimilation, organic acid synthesis and starch accumulation, to modulate the sugar-mediated repression of the expression of genes involved in photosynthesis, and to modulate whole plant events including shoot–root allocation, root architecture and flowering. Third, increased rates of growth in elevated [CO₂] will require higher rates of inorganic nitrogen uptake and assimilation. Recent evidence is discussed that an increased supply of sugars can increase the rates of nitrate and ammonium uptake and assimilation, the synthesis of organic acid acceptors, and the synthesis of amino acids. Fourth, interpretation of experiments in elevated [CO₂] requires that the nitrogen status of the plants is monitored. The suitability of different criteria to assess the plant nitrogen status is critically discussed. Finally the review returns to experiments with elevated [CO₂] and discusses the following topics: is, and if so how, are nitrate and ammonium uptake and metabolism stimulated in elevated [CO₂], and does the result depend on the nitrogen supply? Is acclimation of photosynthesis the result of sugar-mediated repression of gene expression, end-product feedback of photosynthesis, nitrogen-induced senescence, or ontogenetic drift? Is the accumulation of starch a passive response to increased carbohydrate formation, or is it triggered by changes in the nutrient status? How do changes in sugar production and inorganic nitrogen assimilation interact in different conditions and at different stages of the life history to determine the response of whole plant growth and allocation to elevated [CO₂]?     

Effects of CO 2 enrichment on photosynthesis,growth, and nitrogen metabolism of the seagrass Zostera noltii

Seagrass ecosystems are expected to benefit from the global increase in CO ₂ in the ocean because the photosynthetic rate of these plants may be C_i‐limited at the current CO ₂ level. As well, it is expected that lower external pH will facilitate the nitrate uptake of seagrasses if nitrate is cotransported with H⁺ across the membrane as in terrestrial plants. Here, we investigate the effects of CO ₂ enrichment on both carbon and nitrogen metabolism of the seagrass Zostera noltii in a mesocosm experiment where plants were exposed for 5 months to two experimental CO ₂ concentrations (360 and 700 ppm). Both the maximum photosynthetic rate (P_m) and photosynthetic efficiency (α) were higher (1.3‐ and 4.1‐fold, respectively) in plants exposed to CO ₂‐enriched conditions. On the other hand, no significant effects of CO ₂ enrichment on leaf growth rates were observed, probably due to nitrogen limitation as revealed by the low nitrogen content of leaves. The leaf ammonium uptake rate and glutamine synthetase activity were not significantly affected by increased CO ₂ concentrations. On the other hand, the leaf nitrate uptake rate of plants exposed to CO ₂‐enriched conditions was fourfold lower than the uptake of plants exposed to current CO ₂ level, suggesting that in the seagrass Z. noltii nitrate is not cotransported with H⁺ as in terrestrial plants. In contrast, the activity of nitrate reductase was threefold higher in plant leaves grown at high‐CO ₂ concentrations. Our results suggest that the global effects of CO ₂ on seagrass production may be spatially heterogeneous and depend on the specific nitrogen availability of each system. Under a CO ₂ increase scenario, the natural levels of nutrients will probably become limiting for Z. noltii. This potential limitation becomes more relevant because the expected positive effect of CO ₂ increase on nitrate uptake rate was not confirmed.     

Importance of seed Zn content for wheat growth on Zn-deficient soil

Eggplants (Solanum melongena L. cv. Bonica) were grown in a glasshouse during summer under natural light with one unbranched shoot or one shoot with 3 to 4 branches and with or without fruit in quartz sand buffered and not buffered with 0.5% CaCO₃ (w : v), respectively. Nutrient solutions supplied contained nitrate or ammonium as the sole nitrogen source. Compared with nutrient solutions containing nitrate (10 mM), solutions containing ammonium (10 mM) caused a decrease in net photosynthesis of eggplants during early stages of vegetative growth when grown in quartz sand not buffered with CaCO₃. The decrease was not observed before leaves showed interveinal chlorosis. In contrast, net photosynthesis after bloom at first increased more rapidly in eggplants supplied with ammonium than with nitrate nitrogen. However, even in this case, net photosynthesis decreased four weeks later when ammonium nutrition was continued. The decrease was accompanied by epinasty and interveinal chlorosis on the lower leaves and later by severe wilting, leaf drop, stem lesions, and hampered growth of stems, roots, and fruits. These symptoms appeared later on plants not bearing fruits than on plants bearing fruits. If nutrient solutions containing increasing concentrations of ammonium (0.5–30 mM) were supplied after the time of first fruit ripening, shoot growth and set of later flowers and fruits were promoted. In contrast, vegetative growth and reproduction was only slightly affected by increasing the concentration of nitrate in the nutrient solutions. In quartz sand buffered with CaCO₃ ammonium nutrition caused deleterious effects only under low light conditions (shade) and on young plants during rapid fruit growth. If eggplants were supplied with ammonium nitrogen before bloom, vegetative growth was promoted, and set of flowers and fruit occurred earlier than on plants supplied with nitrate. Furthermore, the number of flowers and fruit yield increased. These effects of ammonium nutrition were more pronounced when plants were grown with branched shoots than with unbranched shoots. The results indicate that vegetative and reproductive growth of eggplants may be manipulated without causing injury to the plants by supplying ammonium nitrogen as long as the age of the plants, carbohydrate reserves of the roots, quantity of ammonium nitrogen supplied, and pH of the growth medium are favourable. T W Rufty Section editor