Qirong Shen

Spatial expression and regulation of rice high-affinity nitrate transporters by nitrogen and carbon status

The high affinity nitrate transport system (HATS) plays an important role in rice nitrogen acquisition because, even under flooded anaerobic cultivation when NH(4)(+) dominates, significant nitrification occurs on the root surface. In the rice genome, four NRT2 and two NAR2 genes encoding HATS components have been identified. One gene OsNRT2.3 was mRNA spliced into OsNRT2.3a and OsNRT2.3b and OsNAR2.1 interacts with OsNRT2.1/2.2 and OsNRT2.3a to provide nitrate uptake. Using promoter-GUS reporter plants and semi-quantitative RT-PCR analyses, it was observed that OsNAR2.1 was expressed mainly in the root epidermal cells, differently from the five OsNRT2 genes. OsNAR2.1, OsNRT2.1, OsNRT2.2, and OsNRT2.3a were up-regulated by nitrate and suppressed by NH(4)(+) and high root temperature (37 °C). Expression of all these genes was increased by light or external sugar supply. Root transcripts of OsNRT2.3b and OsNRT2.4 were much less abundant and not affected by temperature. Expression of OsNRT2.3b was insensitive to the form of N supply. Expression of OsNRT2.4 responded to changes in auxin supply unlike all the other NRT2 genes. A region from position -311 to -1, relative to the translation start site in the promoter region of OsNAR2.1, was found to contain the cis-element(s) necessary for the nitrate-, but not light- and sugar-dependent activation. However, it was difficult to define a conserved cis-element in the promoters of the nitrate-regulated OsNRT2/OsNAR2 genes. The results imply distinct physiological functions for each OsNRT2 transporter, and differential regulation pathways by N and C status.

Rice OsNAR2.1 interacts with OsNRT2.1, OsNRT2.2 and OsNRT2.3a nitrate transporters to provide uptake over high and low concentration ranges

Plants take up both nitrate and ammonium as main nitrogen (N) sources. Although ammonium is the predominant form in anaerobic-flooded paddy soil, it has been proposed that rice and other wetland plants may take up significant amounts of nitrate formed by nitrification of ammonium in the rhizosphere. A two-component system for nitrate transport including NRT2s with a partner protein (NAR2 or NRT3.1) has been identified in Arabidopsis. We report the physiological function of another member of the NAR2 family, OsNAR2.1 in rice (Oryza sativa, ssp. Japonica, cv. Nipponbare). OsNAR2.1 was mainly expressed in roots and induced by nitrate and suppressed by ammonium and some amino acids. Knockdown of OsNAR2.1 by RNA interference synchronously suppressed expression of OsNRT2.1, OsNRT2.2 and OsNRT2.3a in the osnar2.1mutants. Both high- and low-affinity nitrate transports were greatly impaired by OsNAR2.1 knockdown. Yeast two hybridization showed that OsNAR2.1 not only interacted with OsNRT2.1/OsNRT2.2, but also with OsNRT2.3a. Taken together, the data demonstrate that OsNAR2.1 plays a key role in enabling the plant to cope with variable nitrate supply.

Adaptation of plasma membrane H+‐ATPase of rice roots to low pH as related to ammonium nutrition

The preference of paddy rice for NH(4)(+) rather than NO(3)(-) is associated with its tolerance to low pH since a rhizosphere acidification occurs during NH(4)(+) absorption. However, the adaptation of rice root to low pH has not been fully elucidated. This study investigated the acclimation of plasma membrane H(+)-ATPase of rice root to low pH. Rice seedlings were grown either with NH(4)(+) or NO(3)(-). For both nitrogen forms, the pH value of nutrient solutions was gradually adjusted to pH 6.5 or 3.0. After 4 d cultivation, hydrolytic H(+)-ATPase activity, V(max), K(m), H(+)-pumping activity, H(+) permeability and pH gradient across the plasma membrane were significantly higher in rice roots grown at pH 3.0 than at 6.5, irrespective of the nitrogen forms supplied. The higher activity of plasma membrane H(+)-ATPase of adapted rice roots was attributed to the increase in expression of OSA1, OSA3, OSA7, OSA8 and OSA9 genes, which resulted in an increase of H(+)-ATPase protein concentration. In conclusion, a high regulation of various plasma membrane H(+)-ATPase genes is responsible for the adaptation of rice roots to low pH. This mechanism may be partly responsible for the preference of rice plants to NH(4)(+) nutrition.

Overexpression of a pH-sensitive nitrate transporter in rice increases crop yields

Significance Significant progress has been made in our understanding of plant adaptive responses to maintain cellular pH under varied N supply forms. Rice is a plant adapted to grow in waterlogged or dryland environments, in contrast to other crops, such as wheat, soybean, and maize. The nitrate transporter OsNRT2.3b provides a molecular mechanism explaining plant adaptation to the ammonium-nitrate supply shift between the waterlogged and drained soil environments. The sensing of cytosolic pH by OsNRT2.3b can function to improve rice nitrogen use efficiency and pH balance, providing an explanation for plant adaptation to changes in the form of N supply. Cellular pH homeostasis is fundamental for life, and all cells adapt to maintain this balance. In plants, the chemical form of nitrogen supply, nitrate and ammonium, is one of the cellular pH dominators. We report that the rice nitrate transporter OsNRT2.3 is transcribed into two spliced isoforms with a natural variation in expression ratio. One splice form, OsNRT2.3b is located on the plasma membrane, is expressed mainly in the phloem, and has a regulatory motif on the cytosolic side that acts to switch nitrate transport activity on or off by a pH-sensing mechanism. High OsNRT2.3b expression in rice enhances the pH-buffering capacity of the plant, increasing N, Fe, and P uptake. In field trials, increased expression of OsNRT2.3b improved grain yield and nitrogen use efficiency (NUE) by 40%. These results indicate that pH sensing by the rice nitrate transporter OsNRT2.3b is important for plant adaption to varied N supply forms and can provide a target for improving NUE.

Ammonium nutrition increases water absorption in rice seedlings (Oryza sativa L.) under water stress

Water stress is a primary limitation on plant growth. In previous studies, it has been found that ammonium enhances the tolerance of rice plants to water stress, but how water is related to nitrogen form and water stress remains unknown. To study the effects of nitrogen form (NH4+, NO3−, and a mixture of NH4+ and NO3−) on the growth and water absorption of rice (Oryza sativa L.) seedlings, a hydroponic experiment with water stress, simulated by the addition of polyethylene glycol (PEG, 10% w/v, MW 6000), was conducted in a greenhouse. The results showed that, compared with non-water stress, under water stress, the fresh weight of rice seedlings increased by 14% with NH4+ nutrition, whereas it had decreased by about 20% with either NO3− or mixed nitrogen nutrition. No significant difference was found in the transpiration rate of excised shoots or in xylem exudation of excised roots in NH4+ supply between the two water situations, whereas xylem flow decreased by 57% and 24% under water stress in NO3− and mixed nutrition, and root hydraulic conductivity decreased by 29% and 54% in plants in NH4+ and NO3− nutrition conditions, respectively. Although water absorption ability decreased in both NH4+ and NO3− nutrition, aquaporin activity was higher in NH4+ than in NO3− nutrition, regardless of water stress. We conclude that NH4+ nutrition can improve water handling in rice seedlings and subsequently enhance their resistance to drought.

Drought-Induced Root Aerenchyma Formation Restricts Water Uptake in Rice Seedlings Supplied with Nitrate

Previous studies demonstrated that ammonium nutrition results in higher water uptake rate than does nitrate nutrition under water stress, and thus enhances the tolerance of rice plants to water stress. However, the process by which water uptake is related to nitrogen form under water stress remains unknown. A hydroponic experiment with simulated water stress induced by polyethylene glycol (PEG6000) was conducted in a greenhouse to study the relationship between root aerenchyma formation and water uptake rate, such as xylem sap flow rate and hydraulic conductance, in two different rice cultivars (cv. Shanyou 63 hybrid indica and cv. Yangdao 6 indica, China). The results showed that root aerenchyma tissue increased in water-stressed plants of both cultivars fed by nitrate. No significant difference was found in root hydraulic conductivity and/or xylem sap flow rate between the two rice cultivars fed by ammonium regardless of water status, whereas these parameters decreased significantly in water-stressed plants fed by nitrate. It was concluded that aerenchyma that formed in the root cortex impeded the radial transport of water in the root cylinder and decreased water uptake in water-stressed rice plants fed by nitrate. Water transport occurred mainly through Hg-sensitive water channels in rice roots supplied with ammonium.

New Insights into the Nitrogen Form Effect on Photosynthesis and Photorespiration

Abstract Under high light conditions, ammonium nutrition has a negative effect on plant growth. This suggests that the adverse effects of ammonium nutrition on plant growth may be related to carbon gain, photosynthesis, and photorespiration. However, there is no consistent evidence of a specific mechanism that could explain the plant growth reduction under ammonium supply. It is generally accepted that during the light reaction, a surplus of nicotinamide adenine dinucleotide hydrogen phosphate (NADPH) is produced, which is not completely used during the assimilation of CO2. Nitrate reduction in the leaf represents an additional sink for NADPH that is not available to ammonium-grown plants. Nitrate and ammonium nutrition may use different pathways for NADPH consumption, which leads to differences in photosynthesis and photorespiration. The morphological (i.e., cell size, mesophyll thickness, and chloroplast volume) and enzymic (i.e., ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), phosphoenolpyruvate carboxylase (PEPCase), and glutamine synthetase/glutamate synthetase (GS/GOGAT)) differences that develop when plants are treated with either nitrate or ammonium nitrogen forms are related to photosynthesis and photorespiration. The differences in photorespiration rate for plants treated with nitrate or ammonium are related to the conversion of citrate to 2-oxoglutarate (2-OG) and photorespiratory CO2 refixation.

The enhanced drought tolerance of rice plants under ammonium is related to aquaporin (AQP)

Previously, we demonstrated that drought resistance in rice seedlings was increased by ammonium (NH4(+)) treatment, but not by nitrate (NO3(-)) treatment, and that the change was associated with root development. To study the effects of different forms of nitrogen on water uptake and root growth under drought conditions, we subjected two rice cultivars (cv. Shanyou 63 hybrid indica and cv. Yangdao 6 indica, China) to polyethylene glycol-induced drought stress in a glasshouse using hydroponic culture. Under drought conditions, NH4(+) significantly stimulated root growth compared to NO3(-), as indicated by the root length, surface area, volume, and numbers of lateral roots and root tips. Drought stress decreased the root elongation rate in both cultivars when they were supplied with NO3(-), while the rate was unaffected in the presence of NH4(+). Drought stress significantly increased root protoplast water permeability, root hydraulic conductivity, and the expression of root aquaporin (AQP) plasma intrinsic protein (PIP) genes in rice plants supplied with NH4(+); these changes were not observed in plants supplied with NO3(-). Additionally, ethylene, which is involved in the regulation of root growth, accumulated in rice roots supplied with NO3(-) under conditions of drought stress. We conclude that the increase in AQP expression and/or activity enhanced the root water uptake ability and the drought tolerance of rice plants supplied with NH4(+).

Chloroplast Downsizing Under Nitrate Nutrition Restrained Mesophyll Conductance and Photosynthesis in Rice (Oryza sativa L.) Under Drought Conditions

The phenomenon whereby ammonium enhances the tolerance of rice seedlings (Oryza sativa L., cv. Shanyou 63 hybrid indica China) to water stress has been reported in previous studies. To study the intrinsic mechanism of biomass synthesis related to photosynthesis, hydroponic experiments supplying different nitrogen (N) forms were conducted; water stress was simulated by the addition of polyethylene glycol. Water stress decreased leaf water potential (Ψ(leaf)) under nitrate nutrition, while it had no negative effect under ammonium nutrition. The decreased Ψ(leaf) under nitrate nutrition resulted in chloroplast downsizing and subsequently decreased mesophyll conductance to CO(2) (g(m)). The decreased g(m) and stomatal conductance (g(s)) under nitrate nutrition with water stress restrained the CO(2) supply to the chloroplast and Rubisco. The relatively higher distribution of leaf N to Rubisco under ammonium nutrition might also be of benefit for photosynthesis under water stress. In conclusion, chloroplast downsizing induced a decline in g(m), a relatively higher decrease in g(s) under nitrate nutrition with water stress, restrained the CO(2) supply to Rubisco and finally decreased the photosynthetic rate.

Thermographic visualization of leaf response in cucumber plants infected with the soil-borne pathogen Fusarium oxysporum f. sp. cucumerinum.

Infection with the soil-borne pathogen Fusarium oxysporum f. sp. cucumerinum (FOC), which causes Fusarium wilt of cucumber plants, might result in changes in plant transpiration and water status within leaves. To monitor leaf response in cucumber infected with FOC, digital infrared thermography (DIT) was employed to detect changes in leaf temperature. During the early stages of FOC infection, stomata closure was induced by ABA in leaves, resulting in a decreased transpiration rate and increased leaf temperature. Subsequently, cell death occurred, accompanied by water loss, resulting in a little decrease in leaf temperature. A negative correlation between transpiration rate and leaf temperature was existed. But leaf temperature exhibited a special pattern with different disease severity on light-dark cycle. Lightly wilted leaves had a higher temperature in light and a lower temperature in dark than did in healthy leaves. We identified that the water loss from wilted leaves was regulated not by stomata but rather by cells damage caused by pathogen infection. Finally, water balance in infected plants became disordered and dead tissue was dehydrated, so leaf temperature increased again. These data suggest that membrane injury caused by FOC infection induces uncontrolled water loss from damaged cells and an imbalance in leaf water status, and ultimately accelerate plant wilting. Combining detection of the temperature response of leaves to light-dark conditions, DIT not only permits noninvasive detection and indirect visualization of the development of the soil-borne disease Fusarium wilt, but also demonstrates certain internal metabolic processes correlative with water status.