Internal ammonium excess induces ROS-mediated reaction and causes carbon scarcity in rice

doi:10.21203/rs.2.17135/v1

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Internal ammonium excess induces ROS-mediated reaction and causes carbon scarcity in rice

https://doi.org/10.21203/rs.2.17135/v1

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published 07 Apr, 2020

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Background: Overuse of nitrogen fertilizers is often applied to satisfy strong nitrogen demand of high-yielding rice, leading to persistent NH 4 + excess in the plant. However, the mechanisms constraining the effectiveness of elevated plant NH 4 + in plant growth and grain yield of rice are not sufficiently addressed. In the current study, we attempt to real the nature or mode-of-action of such internal NH 4 + excess in rice, and the efficient coordination measure with current high N fertilizer cropping systems is investigated.

Results: By mimicking a rapid accumulation of plant NH 4 + and an RNA-Seq analysis, the present work reveals that internal NH 4 + excess in rice plant initiates a burst of reactive oxygen species (ROS) and triggers probably specifically the activation of glutathione transferase (GST)-mediated glutathione cycling for ROS cleavage. Meanwhile, the suppression of the expression of genes involved in photon caption and the activity of primary CO 2 fixation enzymes (Rubisco), provides implications of a reduction in photosynthetic carbon income. Along the progress of NH 4 + / ROS stresses, enhanced energy-producing processes (carbon breakdown) take place as indicated by strong induction of glycolysis related genes to meet the demand of energy consuming activation of ROS-cleavaging systems. The development of these defensive reactions causes a sugar scarcity in the plant that accumulatively leads to growth inhibition. To the opposite direction, a sucrose feeding treatment to the roots renders the accumulation of free NH 4 + and ROS, partly restores the activities of photosynthetic CO 2 fixation and thus alleviates the scarcity in active carbon source. Conclusion: Our results demonstrate the necessity of efficient carbon coordination, aiming at improving the nitrogen performances under current N fertilizer overuse circumstances.

Plant Physiology and Morphology

rice

NH4+ excess

ROS

GSH cycle

carbon scarcity

sucrose feeding

Nitrogen (N) limitation was a leading constraint on the grain yield of rice [1, 2]. Leaf N accounted for the largest N sink of rice plant, ca. 80% of which was distributed in the chloroplast and stored as Ribulose–1,5-bisphosphate carboxylase⁄oxygenases (Rubisco), the primary carbon ﬁxation enzymes of C₃ plants [3]. Photosynthesis was tightly correlated with leaf N content [4], and more than 80% of grain N was derived from leaves in rice [5]. Hence, insufficient leaf N storage would lead to reduction of photosynthetic carbon fixation efficiency and was therefore considered as a major limitation to biomass and grain production of cereal ecosystems [6–9].

In China’s rice farming, to satisfy the N demand of higher grain yield (6.4 t/hm² or above), the average N input was normally over 180 kg/hm²[10]. In the high-yielding rice farming areas, the N input could even reach to 300 kg/hm² and this was particularly the case for recent super-hybrid rice cultivars that achieved as high as >10 t/hm² of grain yields [11]. In soils, applied N fertilizers (e.g. urea form accounted for the majority of current N fertilizers) would rapidly convert to ammonium with the potent reactions of ureases. And in well-ventilated dryland soils, ammonium could convert to another root available nitrogen form-nitrate through the actions of nitrification microbes. However, in rice paddy soils, ca. 70–80% of the growth period was water flooded, causing an anaerobic environment that largely prevented the process of nitrification. To this end, retention of higher concentration of ammonium around the roots is a generalized fact in rice paddies. Recent results showed that over-dosed N fertilizers strengthened excessive N accumulation in rice plant that however did not accordingly result in increased grain yields [12]. Therefore, low N use efficiency and its agronomic benefits were major problems of high N input rice farming.

Plant responses to toxic concentrations of NH₄⁺ supplied exogenously had been extensively demonstrated centering the (molecular) mechanisms involved in the configuration adjustment of root architectures. In Arabidopsis, root tip contact was essential for triggering the inhibitory growth of primary roots subjected to high NH₄⁺ treatments [13]. Whereas leaf contact and absorption of toxic NH₄⁺obstructed primary polar transportation of IAA to the roots through the pathway of AUX1 thereby inhibited the emergence of lateral roots [14]. In rice, high concentrations of NH₄⁺ supplied to roots strongly inhibited seminal root elongation and led to reduction of plant growth [15–19]. Mechanisms of plant responses to external NH₄⁺ stresses had been summarized as the accumulative consequences of ion imbalance, intracellular pH disturbance, carbon limitation, charge/hormone imbalance and oxidative stresses [20–24]. The work on Arabidopsis hsn/vtc mutants provided further molecular evidence that GDP-mannose pyrophosphorylases were involved in protein N-glycosylation modulated inhibition of root elongation under NH₄⁺ stresses [25–27]. Morever, phytohormone signals were reported to interact with NH₄⁺ supply thus to regulate plant metabolism, growth and development [13, 14, 24, 25, 27, 28]. And these responsive components are closely related to high external NH₄⁺ supply but seemingly independent of internal NH₄⁺accumulation.

Due to the long-time cultivation in paddy soils wherein NH₄⁺ as the main N source, rice has been hailed as an uncommon NH₄⁺-tolerant species. Researches on (molecular) mechanism of rice response to excessive NH₄⁺ are ongoing. Under condition of high NH₄⁺supply, GABA alleviates NH₄⁺ toxicity through reducing NH₄⁺ accumulation and assimilation capacity for a energy saving [29]; OsPTR6 could promote rice root growth by enhancing OsAMT1 expression and GS activity but at the expense of decreasing NUE[30]; OsSE5 that encodes the heme-heme oxygenase 1 dedicates to relieve NH₄⁺ toxicity by reinforcing antioxidant defense system [18]. The transcriptome analysis illustrates that the phenylpropanoid and amino acid metabolism show importance in shoot while defense and signaling pathways play the leading role in rice root when stressed by excessive NH₄⁺ [19].

In general, the current recognitions on plant responses to NH₄⁺ stresses mainly emphasized their physiological and molecular mechanisms in remodeling the (root) architectures that depended on the occurrence of measurable (root) phenotypes accumulated within a relatively longer time course of exposure to NH₄⁺ stresses. To this respect, ‘mixed’ influences between specific NH₄⁺ stress responses and endogenous changes along the course of plant growth and development seemed inevitable. Therefore, to one hand, the nature of high NH₄⁺ stresses and the primary responses remained to be specifically clarified; to the other hand, the actions of internal NH₄⁺excess resulted from over-dosed application of N fertilizers needed to be addressed in rice plant without or beyond the frame of a root phenotype.

A previous study implied that the adjustment of carbohydrate metabolisms could be a notable feature in responding NH₄⁺ status in rice in a short time period [31]. Environmental stress stimuli such as salinity, drought etc. could induce overproduction of reactive oxygenspecies (ROS) and promptly trigger oxidative defense responses [32–33]. Meanwhile, the reduced photosynthetic CO₂ fixation efficiency and alteration of carbohydrate metabolism were speculatively major causes leading to compromised carbon gain and growth retardance [32, 34].

Based on the above descriptions, the present work aimed at revealing the specific nature and mode-of–action of internal NH₄⁺ excess stresses in rice plant which represented a practical problem of high-yielding rice farming. Our physiological assessments, gene expression and enzyme activity analyses demonstrated that internal NH₄⁺excess triggered ROS-induced reactions and activated GST-mediated GSH cycles for active radical scavenging that consumed carbohydrates for engergization and resulted in carbon scarcity in rice plants. A sucrose feeding treatments effectively alleviated NH₄⁺excess and ROS-induced frustrations supporting that the carbon scarcity was the major constraint of rice plant in dealing with NH₄⁺excess.

Growth inhibition under over-dosed NH₄⁺correlates to an NH₄⁺ excess induced ROS burst in rice seedlings

Under persistent treatment with high NH₄⁺ (20 mM) for 14d, a significant growth inhibition was observed compared to the control condition (1 mM NH₄⁺)(Fig. 1a). The inhibition was more profound in roots showing a biomass reduction of up to 67% (Fig. 1a)and the root/shoot ratio was significantly lowered from approximately 0.5 down to 0.2 (Fig. 1b). Meanwhile, 7 and 5 folds higher concentrations of free NH₄⁺were measured in roots and shoots, respectively (Fig. 1c).The inhibitory effect by high NH₄⁺ subjected to plant roots was however, a well known phenomenon as described by numerous reports elsewhere (see Introduction). Efforts have be extensively made on the elucidation of molecular mechanisms involved in root architecture adjustments in response to the accumulation of relatively long-term (several days or longer) stress effects impended by high NH₄⁺ treatments. Here to reveal early responsive reactions that could be the trigger of the accumulative responses (growth modifications), a prompt status of internal NH₄⁺ excess is necessarily to be established without causing visible changes in plant growth (especially roots). Therefore, L-methionine-D,L-sulfoximine (MSX), a potent inhibitor of the primary NH₄⁺ assimilation pathway mediated by the activity of glutamine synthetases [35] was applied (1 mM) for 4 h in the presence of high NH₄⁺(20 mM). This treatment resulted in similar increases in free NH₄⁺ and rapidly achieved ‘saturable’ NH₄⁺ excess in both roots and shoots (Fig. 1d), providing a facilitated approach for subsequent identification of genes or processes involved in this response. In line with the accumulation of free NH₄⁺, bursts of reactive oxygen species (ROS) were observed (Fig. 1e), implying possible occurrence of oxidative injuries or ROS-induced reactions triggered by internal NH₄⁺ excess.

RNA-Seq analysis for preliminary identification of genes modulated by NH₄⁺excess

According to above description, rice seedlings were treated with high NH₄⁺ in the presence of 1mM MSX for 4 h to establish an internal environment of NH₄⁺excess. Then RNA-Seq analyses were carried out to seek for molecular responses related to this circumstance. Respectively 1077 and 1040 differentially expressed genes (DEGs) were obtained from roots and shoots, with > 2 fold changes in their transcriptional levels (Additional file1). Based on the GO classiﬁcation, these genes mainly belonged to “metabolic process”, “molecular function”, “binding” and “biological process” (Additional file 2). Further KEGG pathway analysis revealed possible involvements of the responsive genes (DEGs) in stress response, photosynthetic adjustment, carbohydrate and amino acid metabolisms, preparation of hormone signaling pathways and re-adjustment of NH₄⁺ transport (Additional file 3). The significantly regulated genes were further summarized below within the framework of major processes they participate.

Activation of GSH cycle for ROS scavenging

Following the acute plant NH₄⁺ excess and the bursts of ROS (Fig. 1c, d, e), a most remarkable response was the strong induction of glutathione S-transferases (GST) genes (Fig. 2). Eleven GST genes were typically upregulated for >7 or even some tens to hundreds fold both in roots and shoots (Fig. 2a, b, genes#1–11). Among those GSTs, a OsGSTU4 (Os10g0528300, Fig. 2a, gene#11) was the most severely induced by >300 and >600 fold in roots and shoots respectively, followed by 2 putative GST genes (Os10g0481300 and Os10g0527800) that were upregulated by 50–100 fold in both parts. Whereas Os10g0525500 (77 fold) and Os03g0785900 (90 fold) showed strong induction in roots and shoots respectively (Fig. 2a, b). Since GSTs catalyze the transfer of superoxide free radicals to reductive glutathione (GSH) that leads to the detoxification of the oxidants, these changes in GST gene expression provide indications for the critical involvement of the GSH cycle in scavenging the NH₄⁺ excess induced ROS.

The enhanced GST activity accelerates the consumption and conversion of GSH to its oxidized form (GSSG). In line with strengthened demand of reducing power, a putative glutathione reductase gene (Os10g0415300) responsible for the recruitment of GSH was moderately upregulated (~ 8 fold) in roots and vigorously enhanced by 70 fold in shoots (Fig. 2a). Meanwhile, a NADH dehydrogenase gene (Os07g0564500) was stimulated by 127 folds in shoots, partly reflecting the coupling of energization and reducing power with the operation of the GSH cycle (Fig. 2a).

In addition to profound changes related to the GSH cycle, 7 peroxidase genes were suppressed in roots whereas a putative 1-Cys peroxiredoxin B gene (Os07g0638400) was significantly induced in both roots (19 fold) and shoots (179 fold) (Fig. 2a), corresponding to the contradictory roles of peroxidases in the cleavage / homeostasis maintenance of ROS [36].

Suppression of photosynthesis components and contrasting regulation of energy producing carbohydrate metabolism

The chlorophyll a/b binding proteins of light-harvesting complexes (LHCs), also known as antenna proteins, are involved in gathering light energy (photons) of the primary reaction of photosynthesis [37]. Then trapped photons and electrons are transported to reaction center for further photochemical reactions. Disruption of these processes by photodamage, herbicides, or accumulation of highly active radicals will obviously hinder the progress of photosynthesis. Upon a prompt (4 h) NH₄⁺ excess treatment, 6 genes coding for the LHC antenna proteins (4 LHC II and 2 LHC I, respectively), a PS I and a PS II reaction center genes were almost evenly suppressed by approximately 5 fold (Fig. 3), indicating the onset of the reduction of efficiencies of photon gathering and transfer. It would be easily supposed that apparent suppression of photosynthesis would accumulate along the progress of NH₄⁺ excess stress and growth inhibition would consequently occur. Meanwhile, Os12G0292400 coding for the small chain of Rubisco, the key enzyme catalyzes the fixation / assimilation of CO₂, was downregulated by ~ 5 fold (Fig. 3), providing further indication of compromised photosynthetic carbon production. Therefore, plant NH₄⁺ excess initiates and probably also develops the disruption of photosynthesis by interfering in the primary reaction and the Calvin Cycle.

Radical scavenging enzymes are activated and energized by the ATP producing processes including glycolysis and the TCA pathways. However, several genes involved in glycolysis and the TCA cycle were contrastingly regulated in roots and shoots (Fig. 4). In roots, genes coding for 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (Os05g0482700, gene#33) and fructose-bisphosphate aldolases(Os08g0120600, gene#34 and Os01g0905800, gene#35) of glycolysis, isocitrate dehydrogenase (Os05g0573200, gene#36) and malate dehydrogenase (Os05g0574400, gene#37) of the TCA cycle were down-regulated by 6–10 fold following 4h of NH₄⁺excess treatments (Fig. 4a). Meanwhile genes involved in glycogen breakdown were suppressed in roots (Fig. 4a): phosphoenolpyruvate carboxykinase (Os10g0204400, gene#32, –19 fold), beta-glucosidase (Os09g0491100, gene# 40, –11 fold), beta-glucosidase (Os02g0131400, foldgene#41,–15 fold), beta-D-xylosidase 4 (Os04g0640700, gene#42, –7 fold), sucrose synthase (Os03g0401300, gene#43, –8 fold), beta-fructofuranosidase (Os02g0106100, gene#44, –11 fold). To the contrary, enhanced glycolysis / glycogen breakdown in shoots could be indicated by the upregulation of related genes (Fig. 4b): glucose–6-phosphate 1-dehydrogenase (Os02g0600400, gene#39, +5 fold), inorganic pyrophosphatase (Os05g0438500, gene#49, +18 fold), phosphoenolpyruvate carboxykinase (Os10g0204400, gene#32, +34 fold), beta-glucosidase (Os05g0366600, gene#47, +12 fold), beta-glucosidase (Os09g0511600, gene#48, +20 fold). Notably, a pyruvate decarboxylase gene (Os05g0469600, gene #38) of glycolysis, was specifically induced in shoots (Fig. 4b). In addition, two genes Os06g0222100 and Os08g0445700 coding for trehalose 6-phosphate synthase/phosphatases were induced by respectively 15 and 13 fold in roots (Fig. 4a, genes #45, 46), suggesting enhanced biosynthesis of the ‘survival substance’ [32] trehalose induced by NH₄⁺ excess stress.

Sucrose feeding alleviates NH₄⁺ excess stress responses

The above analyses revealed rather frustrating responses to NH₄⁺ excess stress in rice plant that closely associated with the consumption of carbohydrates for energy demand. Hence a sugar scarcity could accumulatively (to a longer time course) result in growth inhibition. To test this hypothesis, we fed 1% of sucrose as a sugar compensation to the high NH₄⁺ (20 mM) hydroponics for 24 h. This treatment compensated the sucrose consumption at high NH₄⁺ and allowed the sucrose contents in roots and shoots to restore to equivalent levels of the control (1 mM NH₄⁺) conditions (Fig. 5a). The sucrose feeding treatments further increased the free NH₄⁺ contents in roots, but significantly reduced NH₄⁺ accumulation to the shoots (Fig. 5b).

As the AMT1 (OsAMT1;1(Os04g0509600/LOC_Os04g43070), OsAMT1;2(Os02G0620500/LOC_Os02g40710) and OsAMT1;3(Os02G0620600/LOC_Os02g40730)) expression level that previously suppressed by high NH₄⁺ in roots (respectively by 3, 67 and 6 fold) subsequently restored to close to their initial levels (at 1 mM NH₄⁺) with the supplement of sucrose to the high NH₄⁺ hydroponics (Fig. 5c). This implied a release of ammonium transporting activity facilitated by the supply of sucrose, thus to contribute to enhanced NH₄⁺ accumulation in roots under high NH₄⁺ plus sucrose condition, whereas the reduced NH₄⁺ content under the same condition in shoots indicated probably the efficient utilization of NH₄⁺upon the addition of sucrose (Fig. 5b). Meanwhile the GS (Fig. 5d) and GOGAT (Fig. 5e) activities were respectively enhanced by 17 % (GS) and 29% (GOGAT) in roots following the sucrose feeding treatments, indicating a restoration of NH₄⁺ assimilation activities from initial suppression by NH₄⁺ excess.

Upon the compensation of sucrose source, the total ROS contents in both roots and shoots were lowered down by 20–30%, close to the levels determined at control (1 mM NH₄⁺) conditions (Fig. 6a). Accordingly, the GSH content and GST activity were significantly reduced to the initial levels (at 1 mM NH₄⁺), no longer showing strong induction by NH₄⁺ excess (Fig. 6b, c). Unexpectedly, no significant changes were observed with the activities of classical defense enzymes CAT, POD and SOD under either treatment (Fig. 6d, e, f). Together with the gene expression analyses (Fig. 2), our results demonstrated that the activation of GSH reducing pathway is probably a featured response of rice in dealing with NH₄⁺ excess and ROS accumulation.Finally, in consistent with the decreased level of ROS, Rubisco activity was elevated by 24% (compared with high NH₄⁺) in shoots with the presence of sucrose feeding (Fig. 6g), suggesting enhanced efficiency of primary CO₂ fixation activity.

Taken together, this set of experiments indicated that sucrose feeding could effectively alleviate rice plant from carbon scarcities exerted by internal NH₄⁺ excess and ROS stresses.

Due to particular water-flooding and anaerobic growth environment, NH₄⁺ is the major form of N nutrient available to paddy rice. Current farming practice of the overuse of N fertilizers to satisfy the N demand of high-yielding rice creates a persistent circumstance of NH₄⁺excess around the roots and inside the plant for rice to cope with [10–12, 38, 39]. Therefore, a study focuses on such farming-intervened special circumstances would be helpful in discovering ‘bottlenecked’ constraints and adaptation strategies related to the (molecular) physiological and agronomic respects of N performances in rice.

NH₄⁺ stresses and toxicities have been considered as a major human-intervened environmental distress exerted on plants and attracted extensive research interests. Researches on these topics have focused on the identification of mechanisms or pathways that primarily modulate the biological modifications of root architectures [20–24]. Solid evidences have shown the re-configuration of plant root morphology in response to NH₄⁺ stresses is tightly controlled through the interactions with plant hormone signaling pathways [13, 14, 24, 25, 27]. Whereas NH₄⁺ toxicities could be attributed to ion imbalances [20], intracellular pH disturbance [40], energy consumption due to invalid NH₄⁺ cycles in roots [13, 41]. Assessments of NH₄⁺ stress responses in plant roots and its biological toxicities, to a great extent, rely on the establishment of measurable growth phenotypes that requires effects or reactions to accumulate for a desired time course. These analyses are obviously important in addressing the mode-of-action of physiological effects or processes developed along the progresses of the treatments. To the other hand, since plants grow and develop during the experimental periods, these accumulative observations might be not satisfactory for capturing the initial reactions or the nature of NH₄⁺ excess stresses. Therefore, in the present work, we established prompt methods for identification of the early responses especially to internal NH₄⁺ excess without causing visible growth changes by blocking the primary NH₄⁺ assimilation with MSX.

Our results demonstrate that internal NH₄⁺ excess (accumulation of free NH₄⁺) correlates to a burst of ROS radicals (Fig. 1c, d, e) and activates (may be specifically) the GST-mediated GSH cycling rather than classical antioxidant defense enzymes (such as CAT, POD and SOD) for ROS scavenging (Fig. 2, 6). Therefore, our results support the notion that the initial nature of NH₄⁺ excess in rice plant is probably the induction of ROS bursts that triggers the downstream reactions.

Following the ROS radical bursts, genes involved in photosynthetic photon caption and the most important enzymes for primary CO₂ assimilation-Rubisco (gene expression and enzyme activity as well) were suppressed (Fig. 3, 6g), indicating the carbon income is reduced from its origin of production. Next, to support the sharp energy demand of ROS cleavage through the GSH cycling, we observe strong inductions especially in shoots, of genes involved in ATP-generating reactions (glycolysis and glycogen breakdown, Fig. 4) and enhanced gene expressions related to processes of gluconeogenesis and structural sugar breakdown to fill up the consumption of ‘active’ carbons (Fig. 4). To this end, the carbon consumption and thereby the carbon scarcity associated with NH₄⁺ excess in rice plant resemble the metabolism alterations caused by well known abiotic stresses, such as drought or salinity stresses described in other plant species [32, 34].

The oxidative inhibition of photosynthesis and the scarcity in active carbon source initiated by internal NH₄⁺ excess are further supported by the experiments of sucrose feeding (Fig. 5, 6). Sucrose is the major form of active carbon source in the phloem and can be transported and allocatedin plant tissues and organs [42]. Therefore we choose sucrose for carbon compensation experiments. Efficient NH₄⁺ uptake through AMT transporters is closely related to the removal of the substrate by GS-mediated assimilation processes, uneffective removal or accumulation of NH₄⁺ would hinder the uptake of the ions—a phenomenon of so-called feedback inhibition [43–45]. The strong suppression of root-specific AMT genes, OsAMT1;2 and OsAMT1;3 under rapid NH₄⁺ excess conditions (Fig. 5c) and reduction of GS and GOGAT activities (Fig. 5d, e) provide further evidence of such feedback regulation in rice. Here, upon the carbon compensation by sucrose feeding, the NH₄⁺ assimilation activities restore to normal rates (normal NH₄⁺, 1mM) and the AMT expression levels are accordingly enhanced (Fig. 5c, d, e), supportedly suggesting that carbon scarcity may be the major cause that leads to feedback inhibition of NH₄⁺ uptake. This speculation could find further supports by the reduction of free NH₄⁺ and ROS contents to the normal levels as observed in the control (1 mM NH₄⁺) conditions upon a 24 h of sucrose feeding (Fig. 5b, 6a). With the tranquilness of ROS burst, enhanced GSH content and GST activity are no longer essential (Fig. 6b, c) and the Rubisco CO₂ fixation enzymes restore to act at normal (or even facilitated) activity (Fig. 6g). Thus, the NH₄⁺ excess stress caused growth inhibition as evidenced elsewhere could be interpreted by the carbon scarcity resulted from the reduction of carbon synthesis and violent carbon consumption for energy production.

The present work reveals that the essential nature of internal NH₄⁺ excess stresses in rice plant is closely correlated to its accompanying ROS bursts. Coping with ROS bursts by energy and carbon consumption processes leads to carbon scarcity that accumulatively inhibits plant growth. Sucrose feeding alleviates stress responses, indicating that enhancement of carbon income processes could be a useful approach for further improving nitrogen performances of rice under current farming practice of N fertilizer overuse.

Plant growth and treatments

Rice seeds of Oryza. sativa ssp. Japonica Nipponbare were obtained from Prof. Yingguo Zhu’s group, College of Life Sciences, Wuhan University. The seeds were sterilized, germinated and seedlings were grown in a growth chamber according to previously described [31]. The growth chamber was set with 16/8 h day/night, 27/25℃,day/night; light intensity was 400 mol m^–2 s ^–1, relative humidity was set at 70%. Seedlings were grown in the IRRI solution [31]. The pH of hydroponics was buffered to 5.7 with 10 mM MES and renewed every 48h. For long-term growth tests, uniform seedlings of 7 d were treated with either 1 mM (control) or 20 mM (high NH₄⁺) NH₄Cl supplemented to nitrogen-free IRRI solutions for further 14 days with daily refreshment of the culture solutions. To achieve a rapid NH₄⁺excess in rice plants without causing a growth discrepancy, so that NH₄⁺ excess-responsive genes could be analyzed at the early stages of responses, 10-d old seedlings were promptly treated with ‘control’ or ‘high NH₄⁺' (see above) for 4 h in the presence of 1 mM methionine sulfoximine (MSX, a potent inhibitor of glutamine synthetase) to block the assimilation of NH₄⁺.

For carbon source feeding experiments, 14-d old seedlings were treated with control (1 mM NH₄⁺) or high NH₄⁺ (20 mM NH₄⁺) in IRRI solution in the presence of 1% (w/v) sucrose for 24 h. To avoid the burst of microbes associated with sucrose-containing hydroponics, antibiotics penicillin (50 mg L^–1) and chloramphenicol (25 mg L^–1) were included to the culturing solution according to Lejay’s description [46]. Also in order to prevent possibly undesired impacts, the treatment was limited to within 24 h.

RNA-Seq and quantitative real-time PCR analyses

Total RNAs from treated root or shoot samples was extracted with TRIzol total RNA extraction kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. For RNA-Seq analysis, RNAs from control (1mM NH₄⁺）or high NH₄⁺plus 1 mM MSX treated (4 h) tissue samples were used for library construction and sequencing. Data extraction, identification of differentially expressed genes (DEGs) and functional annotation were analyzed according to our previous work [31]. DEGs were designated with expression fold changes greater than 2 (p < 0.05) between the rapid NH₄⁺accumulation (high NH₄⁺ + MSX) and the control conditions.

Quantitative real-time PCR (qRT-PCR) analyses was carried out to reveal possible responses at the gene expression level related to special conditions such as NH₄⁺ excess stress or sucrose feeding treatments. About 1 μg of total RNA was used to synthesize ﬁrst-strand cDNA using thePrimeScript™ RT Master Mix (Perfect Real Time, TaKaRa, Japan) according to the manufacturer’s description. Primer sequences used for qRT-PCR were listed in Additional file4. Thermocycling and ﬂuorescence detection were performed with C1000 Thermal Cycler CFX96 Real-Time System (Bio-Rad) using the SYBR Premix Ex Taq (TaKaRa, Japan) as indicated by the manufacturer’s protocol. The reaction was performed under the following conditions: 95 °C for 30 s, followed by 44 cycles of 95 °C for10 s, 60 °C for 15 s and 72 °C for 15 s. For fold change analysis, gene expression abundance was quantiﬁed with –2^ΔΔCtand normalized against the internal OsActin gene. PCR ampliﬁcations were repeated three times using cDNA templates synthesized from three independent plant samples.

Tissue free NH₄⁺, GSH and sucrose content assays

Fresh root or shoot samples (0.2 g) were ground into fine powder in liquid N₂ and homogenized in 5 ml of 0.3 mM sulfuric acid. The supernatant was harvested by centrifugation with 20000 g at 4℃ for 20 min. Aliquots of supernatant (200 L) were mixed with 4.9 mL each of phenol-sodium nitroprusside solution and alkaline hydrochlorite solution according to the method of Weatherbur [47]. The color reaction was allowed to develop at room temperature for 1h before the colorimetric absorbance been measured at 625 nm. The tissue GSH content was measured according to Cheng’s method [48]. The tissue sucrose extractioon was carried out according to Sonnewald’s method [49], and the tissue sucrose contents determination was measured based on Stitt’s description [49, 50].

Determination of total ROS and ROS scavenging enzyme activities

Total reactive oxygen species (ROS) contents induced by internal NH₄⁺excess were assessed by 2′,7′-Dichlorofluoresceindiacetate (H₂DCF-DA) method [51]. Fresh root or shoot samples were first made into single cell suspensions with single cell suspension medimachine. Then H₂DCF-DA was added to 200 L single cell suspensions to a final concentration of 10 M, mixed and incubated at 37℃ for 30 min. Cells were pelleted by centrifugation at 1000 g for 10min, washed twice with PBS, and diluted with PBS for fluorescence assay. The absorbance was determined on a ﬂuorescence microplate reader (BioTek Instruments, Winooski, VT) at an excitation wavelength of 500 nm and an emission wavelength of 530 nm according to the descriptions of Karlsson and Sun [52, 53].

For antioxidative enzyme activity analyses, 0.2 g of fresh root or shoot samples were ground in liquid N₂, homogenized and crude extracts were used for the measurements of CAT, POD and SOD activities as previously described [32]. The specific activity of GST was assayed in the supernatant by following the increase of absorbance at 340 nm using GST Assay Kit according to the manufacturer instructions (CS0410, Sigma, USA). One unit of activity was deﬁned as the amount of enzyme required to form 1 M product per minute at 30℃.Enzyme activities were expressed as U. mg^–1FW.

Measurement of GS, GOGAT and Rubisco activities

To prepare the crude enzyme extracts, roots or shoots of each sample were ground into fine powder with liquid N₂ and homogenized with 50 mM Tris–HCl buffer (pH 7.6, containing 10 mM MgCl₂, 1mM EDTA, 1mM β-mercaptoethanoland 4% (w/v) polyvinylpolypyrrolidone–40) using a chilled pestle and mortar. The homogenate was centrifuged at 15000 g for 30 min at 4℃and the supernatants were used for the determination of enzyme activities. The glutamine synthetase (GS) activity was measured according to Sakurai’s description [54]. One unit of GS activity was expressed as the amount of enzyme catalyzing the formation of 1 mol γ-glutamyl hydroxamate per min at 37℃ [55]. The glutamate synthase (GOGAT) activity in the supernatants was determined by the conversion of 2-ketoglutarate to glutamate in a reaction mixture containing 200 mM KH₂PO-KOH pH 7.5,10 mM glutamine (Gln), 10 mM 2-ketoglutarate, 0.14 mM NADH [56], One unit of GOGAT activity was deﬁned as the oxidation rate of 1 nmol NADH per min at 30℃. And the Rubisco activity was measured according to [57]. One unit of Rubisco activity was defined as the oxidation rate of 1 nmol NADH per min at 25℃.

Statistical analysis

Experiment data were expressed as means ± S. E. M. of 3 independent replicates. Statistical differences were evaluated by Duncan’s or t-test with SPSS 13.0 and the level of statistically significant difference was set at p＜0.05.

Supplementary information

Additional files

Additional file 1: Table S1. Summary of total DEGs identifided in rice roots and shoots following a 4 h rapid NH₄⁺ accumulation treatment.

Additional file 2: Table S2. GO enrichment analysis of DEGs in rice roots and shoots following a 4 h rapid NH₄⁺ accumulation treatment.

Additional file 3: Table S3. KEGG Enrichment Analysis of DEGs in rice roots and shoots following a 4 h rapid NH₄⁺ accumulation treatment.

Additional file 4: Table S4. The sequences of primers used for real-time RT-PCR in this research.

Abbreviations

AMT: ammonium transporter; CAT: catalase; CO₂: carbon dioxide; DEGs: Differentially Expressed Genes; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; GOGAT: glutamate synthase; GS: Glutamine synthetase; GSH: Glutathione; GST: Glutathione S-transferase;

H₂DCF-DA: 2′,7′-Dichlorofluoresceindiacetate; LHCs: light-harvesting complexes; N, nitrogen; NH₄⁺: ammonium; qRT-PCR: Quantitative Reverse Transcription Polymerase Chain Reaction; POD: peroxidase; ROS: Reactive Oxygen Species; RNA-Seq: RNA sequencing; SOD: superoxiddismutase; Rubisco: Ribulose–1, 5-bisphosphate carboxylase/oxygenase； TCA cycle：Tricarboxylic acid cycle

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All data supporting the conclusions of this article are provided within the article (and its additional files).

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by the National Key Research & Development Program of China (2017YFD0200100, 2017YFD0200103), the National Science Foundation of China (Grant Nos. 31501826, 31701991) and the Project of Priority and Key Areas, ISSCAS (ISSASIP1609). The funders did not play any roles in the design of the study, collection, analysis and interpretation of the relevant data, and writing the manuscript.

Authors’ contributions

YS and SY conceived and designed the research. SY, DH, MJ prepared samples for RNA-Seq analysis, performed quantitative PCR validation and analyzed data. SY, DH, MJ, YL, ZL, YH and TC carried out physiological/biochemical assays and data analyses. SY and YS wrote the manuscript. All authors read and approved the manuscript.

Acknowledgements

We would like to express our sincere gratitude to the technical support staffs Rong Huang and Zuohao Ma from the Analytical and Testing Center, Institute of Soil Science (Nanjing), Chinese Academy of Sciences for their help in the tissue ROS determination.

Author details

* Correspondence: [email protected]

¹State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, No. 71, East Beijing Road, Nanjing 210008, China

²University of Chinese Academy of Sciences, Beijing 100049, China

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