The plant growth and P-associated traits, photosynthetic parameters, and yields
The growth and P-associated traits as well as the photosynthetic parameters at various growth stages during two growth seasons are shown in Figs. 1a-1h. Compared with sufficient-P (SP), deficient-P treatment (DP) decreased the biomass, P concentrations, P accumulative amounts of plants (Figs. 1a-1c), deteriorated behaviors of Pn, gs, Ci, ΨPSII, and enhanced NPQ (Figs. 1d-1h) in tested cultivar plants managed by deficit irrigation. Although Shixin 828 (high NUE cultivar) was comparable on the above growth, P-associated and photosynthetic traits under SP conditions, it was more improved on the traits mentioned than Jimai 518 under DP (Figs. 1a-1h). The yields in the cultivars under contrasting P input treatments are consistent with above growth, P-associated, and the photosynthetic traits (Table 1), with higher shown in Shixin 828 than Jimai 518 under DP. These results suggested that the high PUE cultivars possess enhanced yield formation capacity under P deprivation condition, which is associated with the improved P accumulation, photosynthetic function, and plant biomass production.
The transcript datasets generated under P deprivation condition
The transcript datasets in Shixin 828 treated with both SP and DP were established based on RNA-seq analyses. Among the raw base pairs yielded over 58 millons each, more than 51 million were identified to be clean reads. Of which, 88.36-90.22% were mapped to the reference genome (c.v. Chinese spring), with 75.33-76.42% were suggested to be unique and 14.75-15.08% to share a nature of multi mapping characterization (Table 2). The transcript datasets obtained in this study suggested the feasibility using RNA-seq approach for detection of transcriptome in wheat under modified P input conditions.
The DE genes identified under P deprivation conditions
A large quantity of genes in transcript datasets derived from the contrasting P input treatments were differentially expressed (DE) under DP, including 2948 of upregulated and 1844 of downregulated (Datasets 1 and 2). The scatter plot showing expression patterns of the DE genes are shown in Fig. 2. The DE genes detected by RNA-seq analysis indicates that the plant P deprivation response is comprehensively determined by modified transcription of genes at global level.
The expression patterns of randomly selected DE genes
Ten of DE genes were randomly selected from the modified transcript datasets under DP and subjected to expression level evaluation. As expected, the five genes with upregulated expression pattern elevated the transcripts under DP with respect to SP, with comparable folds of modified expression as shown in transcript datasets (Figs. 3a-3e). Likewise, the five genes with downregulated pattern lowered the expression levels in plants treated with DP compared with those with SP, all of them being similar on modified transcripts as shown in the RNA-seq analysis (Figs. 3f-3j). These results together validated the reproducibility of the transcriptome results generated under high throughput RNA-seq evaluation.
The functional classifications of DE genes
The DE genes were categorized into three functional groups based on GO assignment analysis, including ‘biological process’, ‘cellular components’, and ‘molecular function’ (Fig. 4a). Among them, the genes in the ‘biological process’ group are enriched by following processes: metabolic process, cellular process, single-organism process, response to stimulus, cellular component organization or biogenesis, biological regulation, regulation of biological process, localization, developmental process, and multicellular organisamal process; the genes in the ‘cellular components’ group are overrepresented by following constituents: cell, cell part, organelle, organelle part, membrane, macromolecular complex, membrane part, and extracellular region; the genes in ‘molecular function’ group are closely associated with molecule binding and catalytic activity (Fig. 4a). These results suggested that plant P deprivation acclimation is underlying modulation of numerous modified biological processes regulated by the DE genes, which act in coordination to mediate plant P starvation response.
The Pi deprivation-associated biological processes overrepresented by the DE genes were defined based on KEGG analysis, which included those associated with photosynthesis-antenna proteins, carbon fixation in photosynthetic organisms, glyoxylate and dicarboxylate metabolism, phenylalanine metabolism, porphyrin and chlorophyll metabolism, alanine, aspartate and glutamate metabolism, cysteine and methionine metabolism, arginine biosynthetsis, DNA replication, taurine and hypotaurine metabolism, and fructose and mannose metabolism, etc. (Fig. 4b). Therefore, these processes and related biochemical pathways were suggested to exert essential roles in modulating plant P starvation adaptation in high PUE wheat cultivar, through improving P uptake, internal P translocation across tissues, photosynthetic function, and biomass production.
The phytohormone signaling-associated genes from DE genes
The phytohormones, such as auxin, cytokinin, gibberellin, abscisic acid, ethylene, salicylic acid, and jasmonic acid, are critical regulators and involved in modulating wide ranges of physiological processes. Among the DE genes identified, a large set of them were shown to be phytohormone signaling-associated ones, including those to be associated with responses to auxin, cytokinin, gibberellin, abscisic acid (ABA), ethylene, salicylic acid (SA), and jasmonic acid (Table 2). These results suggested the crucial roles of the phytohormone signals in plant P deprivation response through the cooperate mediation of diverse stress-associated biological processes.
The function of TaZFP1 in regulating P starvation tolerance
Two lines overexpressing TaZFP1 (i.e., Line 2 and Line 3) with more target transcripts (Fig. S1) together with wild type (WT) were cultivated under two P contrasting treatments, to address the function of target genes in regulating Pi deprivation response. Under SP condition, Line 2 and Line 3 were comparable on biomass, P concentrations, P accumulative amounts, and photosynthetic traits with the WT plants (Figs. 5a-5h). Under DP, however, the transgenic lines displayed more improved biomass, P concentrations, P accumulative amounts, and photosynthetic traits (i.e., Pn, gs, Ci, ΨPSII, and NPQ) of plants than WT (Figs. 5a-5h). These results suggested that TaZFP1, one of the significantly upregulated DE genes under DP, acts as a crucial regulator in plant P deprivation adaptation. The TaZFP1-improved low-P tolerance was associated with the gene function in enhancing P uptake in plants once challenged by low-P stress.