Cell type distinct accumulations of mRNA and protein for NADH-dependent glutamate synthase in rice roots in response to the supply of NH4+

Transient and NH₄⁺-inducible accumulation of the mRNA for NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14) in the roots of rice seedlings was analyzed in situ to identify the cell types responsible for the induction. The mRNA was detected specifically in sclerenchyma cells (the third cell-layer from the root surface), and the maximal accumulation was seen at 3–6 h following the supply of NH₄⁺ ions. Expression of the NADH-GOGAT gene in sclerenchyma cells was also confirmed using transgenic rice plants expressing GUS reporter gene under the control of rice NADH-GOGAT promoter. On the other hand, clear signals for the NADH-GOGAT protein were detected in epidermial cells and exodermal cells (the first and second cell layers from the root surface) at 12 h, following the supply of NH₄⁺ ions. The distinct localization of mRNA and protein for NADH-GOGAT suggests that either the mRNA or the translated protein in the sclerenchyma cells is migrated to the root surface. In contrast to NADH-GOGAT protein, Fd-GOGAT (EC 1.4.7.1) protein was detected in sclerenchyma cells, cortex cells, and stele in the rice roots. The distinct localization of the two GOGAT species indicates that they have different roles in the nitrogen metabolism in rice roots.     

Evidence of ammonium assimilation via the glutamine synthetase-glutamate synthase system in rice seedling roots

When rice seedling roots were fed ¹⁵N-ammonium for 1 hr, theamide nitrogen of glutamine showed the highest ¹⁵N abundance.Moreover, glutamine amino, glutamic acid, aspartic acid andalanine showed higher ¹⁵N abundance than ammonium did. In roots whose GS activity was inhibited with MS, both the amountof ammonium and its ¹⁵N abundance were increased. In contrast,both the amount of all examined amino acids containing glutamicacid and their ¹⁵N abundance decreased in roots whose GS activitywas inhibited. From these results, it could be concluded thatthe first step of ammonium assimilation in rice seedling rootswas mainly glutamine synthesis by GS and the second was glutamicacid formation by the GOGAT system. The results of an experiment using ¹⁵N glutamine also supportedthis conclusion. (Received February 23, 1977; )     

Increase in glutamate synthase (NADH) activity in maize seedlings in response to nitrate and ammonium nitrogen

Roots and leaves of Zea mays L. cv. Ganga Safed-2 seedlings grown with nutrient solution containing either 10 m M KNO₃ or NH₄Cl or 5 m M NH₄NO₃ had considerably higher glutamate synthase (NADH, EC 1.4.1.14) activity than the corresponding organs from seedlings grown without any nitrogen. The supply of inorganic nitrogen for a short time, i.e. 3 h, to roots and leaves excised from seedlings grown without nitrogen also increased the enzyme activity in these organs. This increase was more pronounced with nitrate than with ammonium nitrogen. When excised roots and leaves from NH₄NO₃-grown seedlings were incubated in a minus nitrogen medium for 24 h, the enzyme activity declined considerably. This decline was inhibited to some extent by nitrogen, especially by nitrate. Inorganic nitrogen prevented similarly the decline in in vitro enzyme activity during 24 h storage at 25°C, more regularly for the root than for the leaf enzyme. The experiments demonstrate the role of inorganic nitrogen in the regulation of glutamate synthase activity.     

Oxygen supply from shoots to roots relative to the total oxygen consumption in rice roots

A new simplified mathematical model was introduced to assess the rate of oxygen supply to plant roots, and to evaluate separately the longitudinal flux through the aerenchymatous system and the transversal flux from the root medium. The parameters were derived from the experiemntal results with decapitated rice seedlings solution-cultured with aeration. With decreasing oxygen concentration in the root medium, the transversal oxygen flux decreased and the longitudinal flux increased so as to compensate for the decrease of the former, but in some older rice seedlings there was observed a growing insufficiency in oxygen supply to the roots.     

Anaerobic accumulation of amino acids in rice roots: role of the glutamine synthetase/glutamate synthase cycle

Summary. Accumulation of amino acids was studied in rice roots of 3-day-old seedlings subjected for 48 h to anaerobic conditions. Alanine and Gaba were the main amino acids accumulated under anoxia. Their synthesis was strongly inhibited by MSX and AZA, inhibitors of glutamine synthetase and glutamate synthase. These activities increased after 8 h of anaerobic treatment and, by immunoprecipitation of ³⁵S-labeled proteins, it was shown that glutamine synthetase and ferredoxin-dependent glutamate synthase were synthesized during the treatment. These findings indicate that the glutamine synthetase/glutamate synthase cycle play an important role in anaerobic amino acid accumulation. Received April 5, 1999     

Expression of asparagine synthetase in rice (Oryza sativa) roots in response to nitrogen

The expression of asparagine synthetase (AS; EC 6.3.5.4) in response to externally supplied nitrogen was investigated with respect to enzyme activity and protein levels as detected immunologically in rice ( Oryza sativa ) seedlings. The asparagine content was very low in leaves and roots of nitrogen-starved rice plants but increased significantly after the supply of 1 m M NH₄⁺ to the nutrient solution. While neither AS activity nor AS protein could be detected in leaves and roots prior to the supply of nitrogen, levels became detectable in roots but not in leaves within 12 h of the supply of 1 m M NH₄⁺ or 10 m M glutamine. Other nitrogen compounds, such as nitrate, glutamate, aspartate and asparagine had no effect. Methionine sulfoximine completely inhibited the NH₄⁺-induced accumulation of AS protein but did not affect the glutamine-induced accumulation of the enzyme. The results suggested that glutamine or glutamine-derived metabolites regulate AS expression in rice roots.     

The plasmalemma surface area exposed to the soil solution is markedly reduced by maturation of the exodermis and death of the epidermis in onion roots

The dimorphic exodermis of the root of onion (Allium cepa L.) consists of long and short cells, both of which have Casparian bands. The long cells and some of the short cells also have suberin lamellae. The proportion of short cells with lamellae increases with distance from the root tip and with plant age, but is not influenced by drought stress. In young regions of onion roots, characterized by a mature endodermis and an immature exodermis, the plasmalemma surface area that can be contacted by the soil solution is 90·9 mm² per mm length of root, i.e. the sum of the plasmalemma surface areas of the epidermis, immature exodermis, cortical parenchyma and endodermis external to the Casparian band. This is reduced to 14·5–14·7 mm² by the development of a Casparian band in the exodermis, which cuts off access to the cortical parenchyma, and by the development of suberin lamellae, which cut off access to the plasmalemmae of the long and some of the short cells of the exodermis. Death of all the epidermal cells, a consequence of drought, further reduces this area to 0·205–0·0183 mm², i.e. the area of the outer tangential plasmalemmae of the short cells without suberin lamellae. In this condition, the root's capacity for ion uptake should be reduced but its capacity to resist water loss to the soil should be increased.     

Functions of passage cells in the endodermis and exodermis of roots

Passage cells frequently occur in the endodermis and exodermis but are not ubiquitous in either layer. Passage cells occur in the form of short cells in the dimorphic type of exodermis. In both layers, Casparian bands are formed in all cells, but the subsequent development of suberin lamellae and thick, cellulosic walls are delayed or absent in the passage cells. Available evidence suggests that passage cells of the endodermis are important for the transfer of calcium and magnesium into the stele and thus into the transpiration stream. They become the only cells which present a plasmalemma surface to the soil solution (and are thus capable of ion uptake) when the epidermis and central cortex die. This occurs naturally in some herbaceous and woody species and is known to be promoted by drought. Most evidence indicates that the development of suberin lamellae in both the endodermis and exodermis increases the resistance of the root to the radial flow of water. Passage cells thus provide areas of low resistance for the movement of water, and the position of these cells in the endodermis (i.e., in close proximity to the xylem) is explained in terms of function. Exodermal passage cells have a cytoplasmic structure suggesting an active role in ion uptake. This may be related to the tendency of the epidermis to die, leaving the passage cells as the only ones with their membranes exposed to the soil solution. Passage cells in the exodermis attract endomycorrhizal fungi while those in the endodermis do not. It is clear that passage cells of the endodermis and exodermis play a variety of roles in the plant root system.     

The supply of nutrient ions by diffusion to plant roots in soil

Summary Observation of soil grown roots of rye-grass shows that an approximately cylindrical volume of soil, the root hair cylinder, is densely occupied by root hairs. Estimates are given of the concentration of labile and solution potassium within the root hair cylinder during experiments measuring potassium uptake from two soils by single roots. Calculations, using a diffusion model, suggest that labile potassium concentrations may be reduced to between 99.3 and 53 per cent of the initial, depending on the diffusion characteristics of the soil and nutrient demand by the root. Of the total potassium absorbed by a root in 4 days, the proportion which is supplied from within the root hair cylinder is small (0.8 to 6.3 per cent) indicating that diffusion to the root from the soil outside the root hair cylinder is of paramount importance. When root demand is high, diffusion appears to limit uptake to between 71 and 59 per cent of that which roots of comparable physiology would be expected to absorb from stirred solution of the same concentration. Nevertheless, the presence of root hairs is calculated to have enhanced uptake by up to 77 per cent compared with roots without hairs because they virtually increase the root diameter. Diffusion does not appear to be a limiting factor when root demand is low and hairs can then add little to the efficiency of the root system in potassium absorption.     

The supply of nutrient ions by diffusion to plant roots in soil

Summary Measurements were made of the diffusion of P³²-labelled phosphate to single roots of onion, leek and rye-grass growing in an Upper Greensand sandy loam (UGS) and a Coral Rag Clay (CRC) to which different amounts of phosphate had been added. Concentration-dependent diffusion coefficients for phosphate ions in the soils were calculated from phosphate desorption isotherms in calcium chloride. The experimental uptake by roots of known dimensions was compared with supply expected by diffusion to a cylindrical model root of the same dimensions. Allowance was made for absorption by the root hairs on rye-grass roots. Phosphate absorption by a cm length of intact root was found to continue for at least 16 days for onion, 10 days for leek and 5 days for rye-grass. Over a wide range of conditions (phosphate concentrations, soils, plant species), experimental uptake was close to the maximum calculated to be possible for the diffusion model except on one soil at a high level of phosphate. Although the concentration of phosphate in the soil solution at the root boundary appeared to be reduced to a small fraction of the initial concentration, because of the extreme non-linear form of the desorption isotherm less than 1/2 of the P³² exchangeable pool of P was considered to contribute to diffusion. Phosphate uptake by rye grass could only be accounted for if the root hairs were active. Although only a small fraction of the uptake is derived from inside the root hair cylinder, this increases the efficiency of the central root 2.3 fold by providing a zone close to the central root through which phosphate moves very readily.     

The supply of nutrient ions by diffusion to plant roots in soil

Summary A single-root technique is used to measure the rate of supply of potassium by diffusion to 1-cm portions of cylindrical roots of onion and leek plants growing in soils containing different levels of exchangeable potassium. The relation between uptake and characteristics of the plant and soil is interpreted on the basis of a diffusion supply model. Uptake is accounted for in terms of the geometry of the absorbing root surface, the physiologically controlled absorbing power of the root, and the diffusion through the soil. The different uptakes of potassium by roots of comparable absorbing power from different soils can be predicted with some success from calculations using the root dimensions and either diffusion coefficients of potassium in soil, derived from flux to a cation exchange resin paper, or the form of the potassium scorption isotherm relating the concentration of labile ions to those in the soil solution. It is calculated that diffusion through the soil has reduced potassium uptake by the roots to between 87 and 39 per cent of that expected for roots of the same absorbing power in a stirred culture solution at the same initial soil solution concentration.     

Location of two isoenzymes of NADH-dependent glutamate synthase in root nodules of Phaseolus vulgaris L.

The two isoenzymes of NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14), previously identified in root nodules of Phaseolus vulgaris L., have both been shown to be located in root-nodule plastids. The nodule specific NADH-GOGAT II accounts for the majority of the activity in root nodules, and is present almost exclusively in the central tissue of the nodule. However about 20% of NADH-GOGAT I activity is present in the nodule cortex, at about the same specific activity as this isoenzyme is found in the central tissue. Glutamine synthetase (GS; EC 6.3.1.2) occurs predominantly as the polypeptide in the central tissue, whereas in the cortex, the enzyme is represented mainly by the polypeptide. Over 90% of both GS and NADH-GOGAT activities are located in the central tissue of the nodule and GS activity exceeds NADH-GOGAT activity by about twofold in this region. Using the above information, a model for the subcellular location and stoichiometry of nitrogen metabolism in the central tissue of P. vulgaris root nodules is presented.Abbreviations Fd-GOGAT ferredoxin-dependent glutamate synthase - GOGAT glutamate synthase - GS glutamine synthetase - NADH-GOGAT NADH-dependent glutamate synthase - IEX-HPLC ion-exchange high-performance liquid chromatography     

Identification of microRNAs in rice root in response to nitrate and ammonium

Nitrate and ammonium are two major nitrogen(N) sources for higher plants,but they differ in utilization and signaling.Micro RNAs(mi RNAs) play an essential role in N signal transduction;however,knowledge remains limited about the regulatory role of mi RNAs responsive to different N sources,especially in crop plants.To get global overview on mi RNAs involved in N response in rice,we performed high-throughput small RNA-sequencing under different nitrate and ammonium treatments.The results demonstrated that only 16 and 11 mi RNAs were significantly induced by nitrate and ammonium under short-term treatment,respectively.However,60 differentially expressed mi RNAs were found between nitrate and ammonium under long-term cultivation.These results suggested that mi RNA response greatly differentiates between nitrate and ammonium treatments.Furthermore,44 mi RNAs were found to be differentially expressed between high-and low-N conditions.Our study reveals comprehensive expression profiling of mi RNAs responsive to different N sources and different N treatments,which advances our understanding on the regulation of different N signaling and homeostasis mediated by mi RNAs.     

A comparison of glutamate synthase obtained from maize endosperms and roots

Glutamate synthase (EC 2.6.1.53) has been examined in developing endosperms and roots of maize. KCl is required for maximum activity in each tissue. The effect with KCl is seen with buffer strength of 25 to 100 millimolar in the assay. The optimum concentration for the enzyme from endosperm is 20 millimolar and for the enzyme from root tissue the saturating concentration is about 20 millimolar. In root material the enzyme is labile but activity can be restored if KCl is added to the assay. Divalent cations such as Mg²⁺ or Mn²⁺ also activate the enzyme to some extent.     

Role of glutamate dehydrogenase and phosphoenolpyruvate carboxylase activity in ammonium nutrition tolerance in roots

Spinach (Spinacea oleracea L. “Correnta F1”) and pea (Pisum sativum L. “Macrocarpon”) plants were grown in a hydroponic culture with nitrate (5 mM), or ammonium (5 mM) as the nitrogen source. Dry matter accumulation declined dramatically in spinach plants fed with ammonium, whereas there was no change in pea plants when compared with nitrate-fed plants. Data obtained from δ¹⁵N, the organic nitrogen content, N-assimilation enzyme activity, glutamine synthetase (L-glutamate:ammonia-ligase; EC 6.3.1.2), glutamate dehydrogenase (L-glutamate:NAD⁺-oxidoreductase; EC 1.4.1.2) and enzymes from the tricarboxylic acid cycle suggest that ammonium incorporation into organic nitrogen is localized in the roots in pea plants and in the shoots in spinach plants. Distribution of incorporated ammonium (in shoots and roots) may determine ammonium tolerance. Our results show that unlike in spinach plants, in pea plants, an ammonium-tolerant species, GDH enzyme plays an important role in ammonium detoxification by its incorporation into amino acids. Furthermore, phosphoenolpyruvate carboxylase (phosphate:oxaloacetate-carboxy-lyase; EC 4.1.1.31) and pyruvate kinase (ATP:pyruvate-2-O-phosphotransferase; EC 2.7.1.40) activities reflect a major flow of carbon for ammonium assimilation through oxalacetate in pea plants and through pyruvate in spinach plants. The differences in the sensitivity to ammonium between the species are discussed in terms of differences in the site of ammonium assimilation as well as in the nitrogen assimilation ways.