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 14 d, 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). However, the strong inhibition of root growth under high NH4+ supplement was a well-defined issue as demonstrated by numerous reports. 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.
To further demonstrate the involvement of radical species in the acute response to NH4+ excess, we carried out respectively DAB (3,3′-diaminobenzidine) and NBT: (nitroblue tetrazolium) histochemical staining to trace the occurrence of H2O2 and O2- in newly-born roots and the 2nd leaves of the above treated rice plants. Results showed that upon the acute exposure to high NH4+, significant accumulation of H2O2 in both leaves and roots was detected with strong colored staining (Additional file 1, Figure S1, a & b). The stains were readily faded to close to the control levels following a feeding of 1% sucrose (Additional file 1, Figure S1, a & b), indicating the fallback of the H2O2 burst to the normal levels. Consistent with the observation of H2O2, the NBT stained O2- showed closely similar changes (Additional file 2, Figure S2, a & b). Thus set of data rose questions that the burst of ROS (probably independent of their composition species) was an initiation step of the toxicity mediated by NH4+ excess. Consequently, a set of ROS–triggered reactions or responses would be expected to take place as extensively described for abiotic stress responses. Indeed, according to the measurements of free amino acid contents (Additional file 3, Figure S3), high NH4+ also caused a significant accumulation of free amino acids in both roots and leaves, resembling a common protective response of that of a drought or salinity stress.
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 file 4). Based on the GO classification, these genes mainly belonged to “metabolic process”, “molecularfunction”, “binding” and “biological process” (Additional file 5).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(Additionalfile 6). 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 (4h) 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 ofNH4+ 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).
Under high NH4+ conditions, the expression levels of 3 AMT1 genes (OsAMT1;1–Os04g0509600, OsAMT1;2–Os02G0620500 and OsAMT1;3–Os02G0620600) were suppressed respectively by 3, 67 and 6 fold in roots, implying a reduction in NH4+ uptake activity. With the supplement of sucrose (1%) to the high NH4+ hydroponics (Fig. 5c), their expression levels restored to close to the ‘normal’ levels (at 1 mM NH4+). . This implied a release of ammonium transporting activity from suppression by NH4+ excess, thus contributed to enhanced NH4+ accumulation in roots under high NH4+plus sucrose condition. Whereas the reduced free 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.