Morphological and physiological changes in response to drought stress under two phosphorus levels
The phenotype of the wheat plant is an outward manifestation of all biological processes. In this study, short term drought (3d) had little effect on dry and fresh weight of wheat plants under the two conditions of phosphorus application. However, starting from 3d of drought stress, the influence of drought stress on normal phosphorus application is greater than that of low phosphorus treatment. From 5D to R3, CP treatment presents basically the same or gradually decreasing trend of dry and fresh weight of plants, while LP treatment presents a gradually increasing trend of dry and fresh weight of plants. Morphological changes of the root system were consistent with the above results. The root system at 0d and 7d under drought stress in LP was dense, and the total volume was significantly higher than that under CP. The experiment of soil cultivation also confirmed the promoting effect of stress environment on the growth and development of wheat root hairs [32]. Studies in common beans also showed that the ratio of the root to shoot increased in phosphorus-insufficient plants as compared to phosphorus-sufficient plants. It was speculated that the proliferation of lateral roots may partially contribute the variation of the ratio under phosphorus stress [33]. Moreover, the studies in Arabidopsis also showed that phosphate starvation can induce determinant root development [34]. Quantitative trait loci of root architecture traits associated with phosphorus acquisition in soybean have been identified, which further indicates the importance of root structure for low phosphorus adaptation in soybean [35]. The change of root architecture under phosphorus deficiency is the result of the internal balance of auxin, cytokinin and ethylene [36–41]. As one would expect, in this study, we found several differential metabolic pathways and metabolites involving plant hormone synthesis and phosphorus reactivity, such as: plant hormone signal transduction, indole alkaloid biosynthesis, phosphatidylinositol signaling system, and abscisic acid, in wheat root. With the increase of lateral root growth, the response of genes related to cell wall synthesis and growth to phosphorus was accelerated. For instance, the expression of the gene coding cell wall invertase was significantly increased [42].
Phosphorus participates in glycolysis process, which can enhance respiration action, make saccharide forms a variety of organic acids as ammonia receptor and form amino acid. Phosphorus is indispensable in the process of protein synthesis and enzyme activity regulation [33]. In addition, it is irreplaceable in the phosphorylation and dephosphorylation of proteins and the energy transfer of ATP in the process of information transmission. When phosphorus is deficient, protein synthesis is blocked, which affects the cell cycle [43]. Studies have shown that zeatin can affect cell cycle by regulating CYCD3 gene [44]. At the same time, phosphorus stress can also affect the cell cycle and induce the changes of endoploidy in plants [45]. As expected, zeatin biosynthesis related differential metabolites treated with LP were significantly reduced in this study, suggesting changes in the polyploidy level in root cells induced by LP. In addition, limited nutrition is an important factor in limiting cell division. Sucrose is a major transport carbon source in plants and plays an important regulatory role in cell cycle progression [46]. In this study, under LP treatment, sucrose metabolic pathways were inhibited, thus affecting sugar production and inhibiting cell division process, which may induce endoploidy changes in root cells. In present study, the phenomenon of root tip enlargement at D7 was more obvious in CP compared to LP. This phenomenon may be related to endoploidy of root cells, and the level of endoploidy of LP may be lower than that of CP treatment. Previous studies showed that under the stress of low phosphorus, the polyploidy level in both the mature area of the main root and the lateral root of barley plants decreased significantly, and the extent of the polyploidy level in the lateral root decreased more than that in the main root [45], which may consistent with the results of this study. Previous studies reported that the decrease of endoploidy occurred in both root and leaf under different stress conditions [47]. The reduction of endopolyploidy is helpful to reduce the input of carbon source and other nutrient elements in the process of effective utilization of water and mineral nutrients, while increasing the photosynthetic area. This is especially important for plants under abiotic stress, such as phosphorus stress, because photosynthesis is greatly affected, but more photosynthetic products are transported to the roots [48]. In addition, based on the important role of phosphorus in C, N and other metabolic pathways, plant cells often need to maintain phosphorus concentration within a certain range even under the condition of phosphorus deficiency [49]. Therefore, in the case of limited phosphorus content, saving the consumption of phosphorus is very important for plant survival. Studies have shown that 59–64% of the total phosphorus content in the roots of plants growing under low phosphorus conditions comes from nucleic acid substances [50]. High endopolyploidy levels increase the amount of rRNA, one of the main components of nucleic acids. Therefore, under the condition of low phosphorus, the reduction of polyploidy level in lateral roots contributes to the reduction of phosphorus consumption in the synthesis process of genomic DNA, RNA and other phosphorylated metabolites, and ultimately contributes to the better environmental adaptation of plants. In addition, DNA ladder detection of root tips also showed that,although the roots cultured by two phosphorus levels underwent the process of program of cell death under drought stress, the genomic DNA degradation level of the roots in LP was significantly weaker than that in CP. It indicates that the recovery ability of wheat plant under LP is higher than that in CP treatment after rehydration. This is consistent with root morphology and weight accumulation.
Ionomic changes in responses to drought stress under two phosphorus levels
Under drought stress, water metabolism in plants was seriously affected, and metal ion metabolism closely related to water metabolism was also affected. In this study, under the drought stress and rehydration condition, different phosphorus supply has a great influence on mineral elements contents. At D7 and R3, the concentrations of most elements measured in the roots significantly decreased under LP treamemt, however, the contents of Fe and S significantly increased at D7, and the levels of Mn and Fe significantly increased at R3. Meanwhile, it was observed that the roots of wheat seedlings at D7 were yellow, which might be the manifestation of root damage. Watt et al. found that the accumulation of hydrophobic substances on the cell wall of plant root epidermal cells under drought stress may affect the absorption and transmembrane transport of mineral elements in root extracellular bodies [51]. In addition, due to drought, wheat seedlings lost water and transpiration tension increased. Consequently, the transport rate was increased and the metal elements contents in the plant increased [52].It is also possible that drought stress may lead to inhibition of growth in the shoot and decrease of the pool of photo-compounds. Then the cumulative assimilate transport to the root system was increased and the root osmotic regulation was enhanced. Subsequently, root cells were expanded and the root system proportion were increased [53]. Ultimately, the absorption capacity of root system was enhanced and the plant metal contents were increased.
Li et al. reported that drought reduced Cu contents in all parts of the plant, and the greater the degree of drought, the greater the decrease. However, Mn contents in all parts increased under both drought treatment conditions, and the greater the drought degree, the more the increase [54]. Tan et al. showed that mild drought increased Cu and Mn levels in wheat seedlings, while severe drought reduced Cu and Mn contents [55]. In this study, there was no significant difference in Cu content in the roots at D7 under the condition of two levels of phosphorus supply. However, compared with CP, the Cu content was significantly reduced in LP at R3,which indicating that LP treatment under the superimposed stress of phosphorus and drought reduced the absorption capacity of Cu. After rehydration, the change of Mn content was just opposite to that of Cu content. LP treatment promoted the increase of Mn content, which may be related to the mechanism of Mn absorption by roots and the antagonism between elements. In this study, compared with D7, the contents of eight elements were significantly increased at R3 in LP, while only four elements were significantly increased under CP treatment. This indicated that LP treatment has a significantly higher recovery ability than CP after rehydration, although most element contents in LP treatment were significantly lower than that in CP. Previous studies have shown that plants deficient in phosphorus produce a series of traits similar to those adapted to water. Therefore, in the case of water stress, due to the adaptability to drought, the sensitivity to water stress is reduced, and the final result is that the yield reduction is significantly less than that of phosphorus application. Based on this, as mentioned above, the degradation level of root genomic DNA under LP treatment was significantly weaker than that under CP treatment at R3, which also confirmed that the recovery ability of LP was higher than CP treatment after rehydration, promoting the absorption and transport of related elements.
For the shoots, the contents of most elements in different comparison groups showed a downward trend, indicating that the influence of phosphorus supply and drought stress on the shoots were greater than that on the roots. In this study, it was observed that the leaves of wheat seedlings with severe drought were yellow and even dry. Phosphorus deficiency directly affects and destroys cell membrane structure, inhibits cell division, reduces chlorophyll content in leaves, and decreases photosynthesis. Compared with the elements contents at D7, seven elements in LP were significantly lower at R3, while the contents of four elements in CP increased significantly, and the difference of six elements were not significant. It was showed that drought can enhance the stress effect of low phosphorus, showing obvious superposition of stress [30–31]. However, CP treatment significantly improved the drought resistance of wheat because it satisfied the phosphorus demand of wheat. It was indicated that there are differences between the shoots and the roots in response to the stress environment.
As mentioned above, long-term phosphorus insufficient has provided wheat with certain adaptability and established a new ion balance. When water stress occurs, the ion balance is destroyed but the sensitivity is reduced. After rehydration, the content of a batch of elements, such as K, P, Si, Mn, Mg, Zn, Ca and Fe in the root, related to stress resistance increases significantly. Likewise, the contents of K and Ca in shoots increased significantly. Potassium can activate most enzymes in plant metabolism. Calcium can improve osmotic pressure of plants. The uptake and accumulation of K and Ca under water stress can reduce the adverse effects on plant growth and enhance the tolerance of plants to water stress [20]. Under two phosphorus condition, the content of silicon increased significantly at R3, which played an important role in promoting the growth of roots, improving the activity of plant roots, and promoting the absorption of water and nutrients by plants. It was worth noting that, except for the LP/CP comparison group in the root at R3, the content of Na in the other comparison groups significantly decreased. LP and CP treatments showed a decreasing trend from D7 to R3, but the decrease multiple of LP was lower than that of CP. This may be related to the increase of protons outside the root system after rehydration, which enhanced the exchange power of Na+ reverse transport in the root plasma membrane, and increased the discharge of Na+ in the root system [56].The change of Na content in LP treatment was lower than that in CP treatment. It may be related to the decrease of drought sensitivity in LP treatment.
Metabolic changes in response to drought stress under two phosphorus levels
In this study, differential metabolites in root and shoot of wheat cultured by two phosphorus levels at D3 and D7 were studied by metabonomic methods. It was found that there were significant differences in metabolites in root and shoot of wheat induced by drought stress under two phosphorus supply. As indicated above, under the supply of two levels phosphorus, drought stress had a greater influence on CP treatment than LP, among which the influences on CP shoots were greater than that on roots. While the effects of drought stress on LP roots were greater than that on shoots. Taking the ABC transporter pathway shared by LP and CP treatment groups between D3 and D7 as an example, CP treatment mainly involved sugars, especially maltose and maltodextrin. And LP mainly involved sugars were xylose, arabinose and ribose, as well as arginine. In plants, sugars are usually produced through photosynthesis, degradation of polysaccharides, and gluconeogenesis. The changes of sugar and other sugar content in plants under drought stress are considered as metabolic signals in drought-stress environment [18]. Sugar is the main participant in regulating osmotic in wheat leaves. Compared with sucrose synthesis, starch synthesis was more easily inhibited under short drought conditions. In this study, the starch and sucrose metabolic pathways in wheat roots cultured by CP between D3 and D7 were significantly changed, while no significant changes in the starch and sucrose metabolic pathways were detected under LP treatment. Likewise, we found that the amount of differential metabolites related to glucose metabolism pathways under CP treatment were higher than that of LP, further indicating that CP treatment was more sensitive to drought stress. In addition, compared with CP, LP treatment did not involve sugar metabolism at D3. While the pathways related to sugar metabolism, involving galactose metabolism, pentose and glucuronate interconversions, starch and sucrose metabolism, glycolysis/gluconeogenesis, fructose and mannose metabolism, were detected the significant difference at D7. It indicated that with the extension of drought stress period, the effect on sugar metabolism was greater. LP had the stronger adaptability to drought stress than CP, which probably is based on the comprehensive mobilization of sugar metabolism to adjust osmotic balance and cope with double stress.
Studies have shown that there are a large number of starches in plant root cap cells [57]. The starch is stable and does not hydrolyze even when the plant is hungry, but disappears under high temperature and dehydration, and the more resistant the plant is to drought, the slower the starch hydrolyzes. Therefore, the drought resistance of crop varieties can be judged by the amount of starch residue in root cap cells of seedlings after stress [57]. According to the results of gene chip in our laboratory, compared with CP, the gene of β-amylase involved in starch degradation was significantly up-regulated. While, the genes of coding starch branching enzyme III and glycogen (starch) synthase related to starch synthesas were significantly down-regulated at D7 in LP treatment [42]. Based on this, we speculated that the degradation rate of starch was stimulated and the synthesis rate of starch decreased under LP treatment. It was consistent with the results that the fold change of glucose-1-phosphate,as the precursor of starch synthesis, was less than 1 at D7 between LP and CP group, indicating a decrease in LP (Table 4). It probably suggested that the root starch begin to degrade and the root tissue is already in a state of dehydration, which was more serious under LP than CP treatment. This speculation were in agreement with the performance of DNA fragmentation and root water content [42].
The metabolic analysis of bean roots showed that several sugars, including xylo-galactose, fructose, mannose and sucrose, were more plentiful in phosphorus-insufficient roots than that in phosphorus-sufficient roots, indicating that sugars may preferentially distributed in phosphorus-deficient roots to support the expression of genes induced by phosphorus stress [33]. However, in this study, compared with CP, the expression of PT1 gene (high-affinity phosphate transporter) in the roots cultured by LP was significantly down-regulated, and the gene of phosphate transporter 8 was significantly up-regulated at D7. Among three MYB-related protein genes induced by low phosphorus, two were down-regulated and one was up-regulated [42]. In addition, most of the differential metabolites involved in sugar metabolism showed a response ratio lower than 1, indicating a decrease in phosphorus-deficient roots (Table 4). It was inconsistent with the results of studies in soybean [33]. However, the abundance of several substances related to sugar metabolism did change. The reason for the inconsistency might be related to species and stress intensity, or it might be the different influencing mechanism of double stress of low phosphorus and drought on phosphorus absorption and sugar metabolism in this study.
Most terpenoids were involved in the differential metabolites of the shoots. Terpenoids are important secondary metabolites in plant growth and development, and they are directly or indirectly involved in a series of biological processes such as plant hormone synthesis, cell membrane stability and photosynthesis. The level of plant nutrients is involved in the regulation of terpene metabolism [58]. The changes of terpenoids contribute to the self-protection and defensive response of plants. In this study, the contents of relevant terpenoids in the shoots treated by CP at D3 were significantly higher than that at D7. In addition, terpenoids were more involved in shoot differential metabolites than roots, indicating that drought stress under CP treatment had a greater influence on terpenoids synthesis in shoots than roots and were more affected by early drought.
Our metabolic analysis of wheat phosphorus-sufficient shoots indicated the osmotic regulation of short-term drought (drought for 3d) may be due to the greater effect of free amino acids and prolines than soluble sugars (Supplementary table 1). With the extension of drought stress period, photosynthesis decreased, amino acid synthesis and proline metabolism decreased, sugar metabolism and organic acid increased, at this moment, the osmotic regulation of soluble sugar may be stronger than that of proline (Supplementary table 1). Additionally, our ion contents analysis revealed that the content of cations such as K+, Ca2+ and Na+ in the shoots treated by CP was significantly higher than that of LP at D7. Previous studies have shown that the accumulation of organic acids in vacuoles may play a central role in the regulation of intracellular pH by neutralizing excess cations [61, 62].Therefore, it is necessary to accumulate various organic acids in plant cells, which is also the key adaptation mechanism to maintain intracellular ion homeostasis in seedlings under stress. These information investigated that the various osmotic regulatory substances play different roles during different drought periods. While, due to the decrease of sensitivity to drought, the changes of each osmotic regulatory substance in the shoots were not obvious under LP treatment.