Drought Tolerance In Rice Plants Research Articles

POsHAK1:OsSUT1 Promotes Sugar Transport and Enhances Drought Tolerance in Rice.

Plant cells accumulate osmotic substances (e.g., sugar) to protect cell components and maintain osmotic balance under drought stress conditions. Previous studies found that pOsHAK1:OsFLN2 promotes sugar metabolism and improves the drought tolerance of rice plants under drought stress. This study further evaluated the effect of the ectopic expression of the OsSUT1 gene driven by the OsHAK1 promoter on the sugar transport and drought tolerance of rice. The results showed that the net photosynthetic rate and sucrose phosphate synthase activity of plants expressing the OsSUT1 gene were not significantly different from those of wild-type (WT) rice plants under drought conditions. However, the sucrose transport rate in the phloem increased in the transgenic plants, and the sucrose contents were significantly lower in the leaves but significantly higher in the roots of transgenic plants than those in WT plants. The pOsHAK1:OsSUT1 and pOsHAK1:OsFLN2 transgenic lines had similar rates of long-distance sucrose transport and drought tolerance, which were higher than those of the WT plants. The relative water content of the transgenic plants was higher, while their water loss rate, hydrogen peroxide (H2O2), and malondialdehyde (MDA) contents were lower than those of the WT plants. The stress-responsive gene OsbZIP23 and the antioxidant-related gene OsCATB were significantly upregulated in the drought-treated transgenic lines, while the senescence indicator gene SGR and the stress-responsive gene OsNAC2 were down-regulated compared to WT plants. These results showed that promoting the long-distance sugar transport through the expression of pOsHAK1:OsSUT1 could produce an improved drought tolerance effect similar to that of pOsHAK1:OsFLN2, providing an effective way to improve the drought tolerance of cereal crops at the seedling stage.

Exogenous melatonin induces salt and drought stress tolerance in rice by promoting plant growth and defense system

Due to global climate change, crops are certainly confronted with a lot of abiotic and biotic stress factors during their growth that cause a serious threat to their development and overall productivity. Among different abiotic stresses, salt and drought are considered the most devastating stressors with serious impact on crop’s yield stability. Here, the current study aimed to elucidate how melatonin works in regulating plant biomass, oxidative stress, antioxidant defense system, as well as the expression of genes related to salt and drought stress in rice plants. Eight groups of rice plants (3 replicates, 5 plants each) underwent varied treatments: control, melatonin, salt, drought, salt + drought, salt + melatonin, drought + melatonin, and salt + drought + melatonin. Melatonin (100 µM) was alternately applied a week before stress exposure; salt stress received 100 mM NaCl every 3 days for 3 weeks, and drought stress involved 10% PEG. Young leaves were randomly sampled from each group. The results showed that melatonin treatment markedly reduces salt and drought stress damage by promoting root, shoot length, fresh and dry weight, increasing chlorophyll contents, and inhibiting excessive production of oxidative stress markers. Salt and drought stress significantly decreased the water balance, and damaged cell membrane by reducing relative water contents and increasing electrolyte leakage. However, melatonin treated rice plants showed high relative water contents and low electrolyte leakage. Under salt and drought stress conditions, exogenous application of melatonin boosted the expression level of salt and drought stress responsive genes like OsSOS, OsNHX, OsHSF and OsDREB in rice plants. Taken together, our results reveal that melatonin treatment significantly increases salt and drought tolerance of rice plants, by increasing plant biomass, suppressing ROS accumulation, elevating antioxidants defense efficiency, and up-regulating the expression of salt and drought stress responsive genes.

Drought stress tolerance in rice: a critical insight

Drought is currently a serious threat for farming especially in rice cultivation, due to its substantial water requirements throughout its lifecycle. Drought is one of the major environmental constraints disrupting the growth and yield of rice plants, affecting them at physiological, morphological, biochemical and molecular levels. Global climate change exacerbates this issue, leading to substantial economic losses. As rice is a major food crop worldwide, the demand for rice production is increasing in tandem with the expanding human population. Consequently, it has become imperative to utilize drought-prone areas for agriculture and develop drought-tolerant rice genotypes. In addition to conventional breeding methods, the application of multi-omics approaches proves most effective in meeting the need to enhance drought tolerance in rice plants. Protective mechanisms, such as morphological adaptation, physiological acclimatization, cellular adjustments and antioxidant defense, play pivotal roles in helping plants overcome drought stress. Plant-microbial interactions are important for plants to overcome drought-induced adversities. Furthermore, applications of conventional approaches, omics approaches and nanotechnology are very promising for generating climate smart agriculture. Our aim in this review is to focus on drought stress tolerance in rice including drought-tolerant rice genotypes, their adaptation mechanisms, the unveiling the genes, transcription factors, microRNAs (miRNA) involved, microbial assistance and exploring approaches to mitigate drought stress in rice plants. The present review might throw some light on understanding the mechanism of drought stress tolerance in rice, including its molecular crosstalk and biochemical dynamics, for future researchers.

Maize GOLDEN2-LIKE proteins enhance drought tolerance in rice by promoting stomatal closure.

Drought has become one of the most severe abiotic stresses experienced in agricultural production across the world. Plants respond to water deficit via stomatal movements in the leaves, which are mainly regulated by abscisic acid (ABA). A previous study from our lab showed that constitutive expression of maize (Zea mays L.) GOLDEN2-LIKE (GLK) transcription factors in rice (Oryza sativa L.) can improve stomatal conductance and plant photosynthetic capacity under field conditions. In the present study, we uncovered a function of ZmGLK regulation of stomatal movement in rice during drought stress. We found that elevated drought tolerance in rice plants overexpressing ZmGLK1 or GOLDEN2 (ZmG2) was conferred by rapid ABA-mediated stomatal closure. Comparative analysis of RNA-sequencing (RNA-seq) data from the rice leaves and DNA affinity purification sequencing (DAP-seq) results obtained in vitro revealed that ZmGLKs played roles in regulating ABA-related and stress-responsive pathways. Four upregulated genes closely functioning in abiotic stress tolerance with strong binding peaks in the DAP-seq data were identified as putative target genes of ZmGLK1 and ZmG2 in rice. These results demonstrated that maize GLKs play an important role in regulating stomatal movements to coordinate photosynthesis and stress tolerance. This trait is a valuable target for breeding drought-tolerant crop plants without compromising photosynthetic capacity.

Enhancing rice resilience to drought by applying biochar–compost mixture in low-fertile sandy soil

BackgroundClimate change alters modern drought episode patterns by making them longer, more frequent and more severe, in particular in arid and semi-arid agroecosystems. Amending soil properties and enhancing its fertility is a needed sustainable strategy for mitigating drought’s damaging effects on crop production and food security. Here, we planned to investigate the potential benefits of biochar–compost mixture (B×C) as a biochar-based fertilizer (BCF) in enhancing the drought tolerance of rice plants cultivated in low-fertile sandy soil.ResultsUnder drought stress, rice plants cultivated in unamended soil (no B×C) exhibited severely wilted, rolled and discolored shoots. Furthermore, the shoot dry biomass reduction ratio was 73.3% compared to 44.2 and 27.6% for plants treated with 5 and 15% B×C, respectively. Root anatomical and architectural traits were significantly less impaired in B×C plants and reflected better performance under drought compared to no B×C plants. During the induced drought episode, soil moisture content was enhanced by 2.5-fold through adding B×C, compared to unamended soil, thereby reducing the negative impact of drought stress. Moreover, the less drought-stressed rice plants (B×C-treated) rapidly recovered after rewatering and displayed the unwinding of previously rolled leaves and reproduced panicles. On the other hand, no B×C plants failed to recover and eventually perished completely. The expression profiles of several drought responsive genes suggest that leaves of more stressed rice plants (no B×C) significantly accumulated more cytosolic free calcium (OsCML3) and apoplastic H2O2 (OsOXO4) which eventually may trigger fast and prolonged stomatal closure (OsSRO1c). In addition, more drought-stressed plants (no B×C) may over-produce the reactive oxygen species (ROS) superoxide anion molecules (OsRbohB), the negative situation that has been further complicated by a possible reduction in the activity of the antioxidative enzyme SOD (OsSOD), and thus more lipid peroxidation (3.5-fold increase MDA) in drought-stressed (no B×C) plant shoots compared to B×C plants.ConclusionIt is suggested that soil amendment B×C (biochar–compost mixture) could promote drought stress tolerance in rice plants by retaining more soil moisture content, thereby mitigating the negative effects of drought stress, such as the over-production of ROS in leaves, and thus eventually facilitating recovery after rewatering.

Inducing Drought Tolerance in Rice Plants by Presoaking with Salicylic Acid

Rice needs more water, and therefore any water stress results in a reduction in overall yield and productivity. The purpose of this study was to examine the effects of salicylic acid (SA) 0.5 mM on the morphological and physiological traits of rice plants during the tillering stage under drought stress. Rice seeds presoaked with SA showed better growth in 7 days old rice seedlings in terms of germination percentage, root length, shoot length and fresh weight compared to the control. Drought conditions were applied to rice plants after 35 days of transplantation in two groups, SA treated and SA untreated, for 7 and 14 days of drought stress conditions, respectively. All experiments were conducted in a totally randomised manner with three replicates per treatment. Leaf area and relative water content (RWC) reduction were significantly prevented in salicylic acid-treated plants under drought conditions. Our data suggest that, using exogenous SA as a presoaking treatment on rice seeds improved drought tolerance in the later stages of rice plants in coping with drought stress by preserving water content in rice leaves. This method is low-cost and accessible to farmers for protecting plants during the seedling and tillering stages, which also affect rice yield at the end.

Drought Tolerance Enhancement with Co-Overexpression of DREB2A and APX in Indica Rice (<i>Oryza sativa</i> L.)

Rice is a major cereal crop providing food and energy to more than half of world’s population and drought is a challenging abiotic stress limiting rice production. Engineering drought tolerance trait is a major bottle neck because of multigenic control and complex nature. Two promising candidate genes utilized in engineering drought tolerance include DREB2A transcription factor (a master regulator of downstream stress inducible genes) and APX (an important ROS scavenging enzyme). Overexpression of DREB genes has shown encouraging results but with a negative impact on plant morphology and production. Moreover, co-expression of DREB2A and APX genes’ influence on drought stress has not been studied. Hence, in the present study, overexpression of single genes DREB2A or APX and co-expression of these genes were studied for enhancement of drought tolerance in indica rice. Both genes under control of CaMV 35S promoter were transferred by Agrobacterium transformation into rice variety BPT5204 popular for slender grains in South India. Confirmation of T-DNA integration into rice genome was done with PCR analysis of transgenes. Homozygous transgenic lines of DREB2A, APX and DREB2A-APX generated in T3 generation were evaluated for drought tolerance during seed germination, vegetative and reproductive stages. In seed germination stage, transgenic lines exhibited higher germination rates on 200 mM mannitol MS medium in comparison to WT. In vegetative stage, with-holding water for 7 days transgenic lines exhibited higher chlorophyll, proline, reducing sugars and enhanced activities of APX, SOD and catalase enzymes as well as reduced MDA content. The qRT-PCR analysis revealed higher relative transgene expression under drought stress. In reproductive stage, before maturity with-holding water for 7 days and restoring normal conditions, transgenic lines developed longer panicles and a higher number of grains/plant compared to WT. The overall results indicate that co-expression of DREB2A and APX can provide enhanced drought tolerance in rice plants to combat climate change conditions.

Overexpression of OsC3H10, a CCCH-Zinc Finger, Improves Drought Tolerance in Rice by Regulating Stress-Related Genes.

CCCH zinc finger proteins are members of the zinc finger protein family, and are known to participate in the regulation of development and stress responses via the posttranscriptional regulation of messenger RNA in animals and yeast. However, the molecular mechanism of CCCHZF-mediated drought tolerance is not well understood. We analyzed the functions of OsC3H10, a member of the rice CCCHZF family. OsC3H10 is predominantly expressed in seeds, and its expression levels rapidly declined during seed imbibition. The expression of OsC3H10 was induced by drought, high salinity and abscisic acid (ABA). Subcellular localization analysis revealed that OsC3H10 localized not only in the nucleus but also to the processing bodies and stress granules upon stress treatment. Root-specific overexpression of OsC3H10 was insufficient to induce drought tolerance, while the overexpression of OsC3H10 throughout the entire plant enhanced the drought tolerance of rice plants. Transcriptome analysis revealed that OsC3H10 overexpression elevated the expression levels of genes involved in stress responses, including LATE EMBRYOGENESIS ABUNDANT PROTEINs (LEAs), PATHOGENESIS RELATED GENEs (PRs) and GERMIN-LIKE PROTEINs (GLPs). Our results demonstrated that OsC3H10 is involved in the regulation of the drought tolerance pathway by modulating the expression of stress-related genes.

Exogenous Glutathione-Mediated Drought Stress Tolerance in Rice (Oryza sativa L.) is Associated with Lower Oxidative Damage and Favorable Ionic Homeostasis

Drought stress is a major concern for current agriculture as it limits plant growth and yield. An experiment was conducted with a drought susceptible rice (Oryza sativa L. cv. BRRI dhan29) to explore the potential functions and possible mechanisms of exogenous glutathione (GSH)-induced drought stress tolerance. Fifteen-day-old seedlings, GSH-pretreated or non-pretreated (0.2 mM GSH for 72 h), were subjected to PEG-induced (15% polyethylene glycol 6000) drought stress for 4 days. Rice plants exposed to drought stress displayed reduced growth, which was correlated with reduced chlorophyll content, water balance, antioxidant enzymes activities and mineral contents (K+, Ca2+ and Mg2+), as evidenced by principle component analysis (PCA) and heatmap clustering. The PCA also revealed that hydrogen peroxide (H2O2) and malondialdehyde (MDA) accumulations were strongly linked with the declined growth and development of rice plants under drought stress. Importantly, pretreatment with GSH improved the growth responses of rice plants to drought stress. GSH strengthened numerous physio-biochemical processes which were coupled with increased uptake of K+, Ca2+ and Mg2+, enhanced accumulation of soluble sugars, higher activities of enzymatic and non-enzymatic antioxidants. Moreover, GSH-pretreated seedlings under drought stress conditions had lower levels of O2·− , H2O2 and MDA which indicates successful adaptation of rice seedlings to drought stress. The results of PCA strongly supported that GSH conferred drought tolerance mostly by enhancing antioxidant activities and mineral homeostasis. Collectively, our findings provide an important prospect for use of GSH in modulating drought tolerance in rice plants. Breeding strategies should therefore be undertaken to increase the GSH content of rice plants as a means to increase drought tolerance.

The NAD kinase OsNADK1 affects the intracellular redox balance and enhances the tolerance of rice to drought

BackgroundNAD kinases (NADKs) are the only known enzymes that directly phosphorylate NAD(H) to generate NADP(H) in different subcellular compartments. They participate in multiple life activities, such as modulating the NADP/NAD ratio, maintaining the intracellular redox balance and responding to environmental stresses. However, the functions of individual NADK in plants are still under investigation. Here, a rice NADK, namely, OsNADK1, was identified, and its functions in plant growth regulation and stress tolerance were analysed by employing a series of transgenic plant lines.ResultsOsNADK1 is a cytosol-localized NADK in rice. It was expressed in all rice tissues examined, and its transcriptional expression could be stimulated by a number of environmental stress treatments. Compared with wild-type (WT) rice, the mutant plant osnadk1 in which OsNADK1 was knocked out was a dwarf at the heading stage and had decreased NADP(H)/NAD(H), ascorbic acid (ASA)/dehydroascorbate (DHA) and reduced glutathione (GSH)/oxidized glutathione (GSSG) ratios, which led to increased oxidation states in the rice cells and sensitivity to drought. Moreover, certain stress-related genes showed differential expression patterns in osnadk1 under both normal growth and drought-stress conditions compared with WT. Among these genes, OsDREB1B and several WRKY family transcription factors, e.g., OsWRKY21 and OsWRKY42, showed correlated co-expression patterns with OsNADK1 in osnadk1 and the plants overexpressing or underexpressing OsNADK1, implying roles for these transcription factors in OsNADK1-mediated processes. In addition, overexpression of OsNADK1 enhanced the drought tolerance of rice plants, whereas loss of function of the gene reduced the tolerance. Furthermore, the proline content was dramatically increased in the leaves of the OsNADK1-overexpressing lines under drought conditions.ConclusionsAltogether, the results suggest that an OsNADK1-mediated intracellular redox balance is involved in the tolerance of rice plants to drought.

Proteomic and physiological approaches reveal new insights for uncover the role of rice thylakoidal APX in response to drought stress

Chloroplast APX isoforms display controversial roles as H2O2 scavengers and signaling players in response to abiotic stress and conclusive results are lacking. We tested the hypothesis that thylakoidal APX displays an important role for drought tolerance, especially by regulating abundance of essential protein species. For this, OsApx8 RNAi-silenced rice (apx8) and non-transformed plants (NT) were exposed to mild water deficit. The drought-sensitivity in apx8 plants was revealed by decreases in shoot growth, relative water content and photosynthesis, which was accompanied by increased membrane damage, all compared to NT plants. This higher sensitivity of apx8 plants to mild drought stress was also related to a lower accumulation of important protein species involved in several metabolic processes, especially photosynthesis, photorespiration and redox metabolism. Despite apx8 plants have displayed an effective induction of compensatory antioxidant mechanisms in well-watered conditions, it was not enough to maintain H2O2 homeostasis and avoid oxidative and physiological disturbances under mild drought conditions. Thus, thylakoidal APX is involved in several phenotypic modifications at proteomic profile level, possibly via a H2O2-induced signaling mechanism. Consequently, this APX isoform is crucial for rice plants effectively cope with a mild drought condition. Biological significanceThis work provides for the first time an integrative study involving proteomic, physiological and biochemical analyses directed to elucidation of thylakoidal APX roles for drought tolerance in rice plants. Our data reveal that this enzyme is crucial for maintaining of growth and photosynthesis under mild water deficit conditions. This essential role is related to maintaining of H2O2 homeostasis and accumulation of essential proteins involved in several important metabolic pathways. Remarkably, for drought resistance was essential the accumulation of proteins involved with metabolism of photosynthesis, signaling, carbohydrates, protein synthesis/degradation and stress. These results can contribute to understand the role of chloroplast ascorbate peroxidases in drought tolerance, highlighting the physiological importance of key proteins in this process.

Rice OVATE family protein 6 regulates plant development and confers resistance to drought and cold stresses.

OVATE family proteins (OFPs) regulate multiple aspects of plant growth and development; however, their roles in responses to abiotic stresses have not yet been studied. Here, we report that rice OsOFP6 regulates growth and development, and alters responses to drought and cold stresses. OsOFP6 is expressed in various tissues and developmental stages. Knock-down of OsOFP6 by RNA interference (RNAi) resulted in semi-dwarf stature, altered grain shape, and shorter lateral roots. OsOFP6 was shown to interfere with auxin in regulating lateral root growth and initiation, as RNAi plants had increased density of lateral roots under IAA (indole-3-acetic acid) treatment. OsOFP6 also affects polar auxin transport, which was demonstrated by the different responses of RNAi and OsOFP6-overexpression (OE) plants to treatment with N-1-naphthylphalamic acid (NPA). Furthermore, under drought conditions OE lines showed slower water loss and less accumulation of H2O2 compared with RNAi plants, which displayed faster water loss and higher H2O2, implying that OsOFP6 may confer both drought avoidance and drought tolerance in rice plants. In addition, under cold treatment OE and RNAi plants exhibited lower and higher relative electrical conductivity (REC), respectively, suggesting an important role of OsOFP6 in response to cold stress. These results provide new insights into the multiple functions of OVATE family proteins.

Physiological investigation of C4-phosphoenolpyruvate-carboxylase-introduced rice line shows that sucrose metabolism is involved in the improved drought tolerance

We compared the drought tolerance of wild-type (WT) and transgenic rice plants (PC) over-expressing the maize C4PEPC gene, which encodes phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) gene, and evaluated the roles of saccharide and sugar-related enzymes in the drought response. Pot-grown seedlings were subjected to real drought conditions outdoors, and the yield components were compared between PC and untransformed wild-type (WT) plants. The stable yield from PC plants was associated with higher net photosynthetic rate under the real drought treatment. The physiological characters of WT and PC seedlings under a simulated drought treatment (25% (w/v) polyethylene glycol-6000 for 3 h; PEG 6000 treatment) were analyzed in detail for the early response of drought. The relative water content was higher in PC than in WT, and PEPC activity and the C4-PEPC transcript level in PC were elevated under the simulated drought conditions. The endogenous saccharide responses also differed between PC and WT under simulated drought stress. The higher sugar decomposition rate in PC than in WT under drought analog stress was related to the increased activities of sucrose phosphate synthase, sucrose synthase, acid invertase, and neutral invertase, increased transcript levels of VIN1, CIN1, NIN1, SUT2, SUT4, and SUT5, and increased activities of superoxide dismutase and peroxidase in the leaves. The greater antioxidant defense capacity of PC and its relationship with saccharide metabolism was one of the reasons for the improved drought tolerance. In conclusion, PEPC effectively alleviated oxidative damage and enhanced the drought tolerance in rice plants, which were more related to the increase of the endogenous saccharide decomposition. These findings show that components of C4 photosynthesis can be used to increase the yield of rice under drought conditions.

Overexpression of the OsbZIP66 transcription factor enhances drought tolerance of rice plants

Drought stress is a major constraint of crop development and productivity. Plants have evolutionally developed several mechanisms at the molecular, cellular, and physiological levels to overcome drought stress. The basic leucine zipper (bZIP) transcription factor (TF) family members are starting to be concerned about their roles in drought stress responses. In this study, we functionally characterized OsbZIP66, a rice group-E bZIP TF, to be associated with rice drought tolerance mechanisms. Expression of OsbZIP66 was significantly induced upon treatments of rice plants with drought, high salinity, and ABA. These observations and the fact that the OsbZIP66 promoter contains ten ABA-responsive cis-elements suggest that OsbZIP66 is up-regulated by drought stress in an ABA-dependent manner. Overexpression of OsbZIP66 both in a whole plant body and specifically in roots enhanced drought tolerance of rice plants, indicating that the rice drought tolerance positively correlates with the expression levels of OsbZIP66. Thus, our results demonstrated that OsbZIP66 has a potential for use in biotechnological development of high-yielding rice plants under drought conditions.

Rice G-protein subunits qPE9-1 and RGB1 play distinct roles in abscisic acid responses and drought adaptation.

Heterotrimeric GTP-binding protein (G-protein)-mediated abscisic acid (ABA) and drought-stress responses have been documented in numerous plant species. However, our understanding of the function of rice G-protein subunits in ABA signalling and drought tolerance is limited. In this study, the function of G-protein subunits in ABA response and drought resistance in rice plants was explored. It was found that the transcription level of qPE9-1 (rice Gγ subunit) gradually decreased with increasing ABA concentration and the lack of qPE9-1 showed an enhanced drought tolerance in rice plants. In contrast, mRNA levels of RGB1 (rice Gβ subunit) were significantly upregulated by ABA treatment and the lack of RGB1 led to reduced drought tolerance. Furthermore, the results suggested that qPE9-1 negatively regulates the ABA response by suppressing the expression of key transcription factors involved in ABA and stress responses, while RGB1 positively regulates ABA biosynthesis by upregulating NCED gene expression under both normal and drought stress conditions. Taken together, it is proposed that RGB1 is a positive regulator of the ABA response and drought adaption in rice plants, whereas qPE9-1 is modulated by RGB1 and functions as a negative regulator in the ABA-dependent drought-stress responses.

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+.