Exogenous application of γ-PGA enhanced drought resistance of maize
In order to investigate the effect of exogenous application of γ-PGA on maize under drought stress, the drought-resistant phenotype of maize with different concentrations of γ-PGA (0, 50, 70, 100mg/L) were examined (Additional file 1: Fig. S1). The results showed that the addition of γ-PGA could significantly enhance the drought resistance of maize, even at a lower concentration (50mg/L), and could regenerate maize rapidly after rewatering, while most of the control maize showed severe wilting and could not grow again after rewatering. 50mg/L γ-PGA treatment was used for the subsequent experiments.
Maize treated with 50mg/L γ-PGA exhibited a better phenotype after 7 days of drought stress (Fig. 1A). The dry weight, content of ABA, soluble sugar, proline, chlorophyll and the photosynthetic parameters of maize seedlings after 5 days drought treatment were determined. As shown in Fig. 1B, under drought condition, the dry weight of maize treated with γ-PGA (0.96g) was significantly higher than that of control maize (0.39g), indicating that γ-PGA could alleviate the inhibition of drought stress on the growth of maize seedlings. In addition, compared with the control group, the contents of ABA, soluble sugar, proline and chlorophyll in γ-PGA treatment group were 27.46%, 43.61%, 108% and 51.51% higher, respectively (Fig. 1B). This indicated that γ-PGA could promote the accumulation of ABA, soluble sugar, proline and the chlorophyll in maize under drought stress. The photosynthetic parameters of the maize under drought for 5d were also measured, the results showed that both the net photosynthetic rate and stomatal conductance of the maize added γ-PGA were significantly higher than the control maize under drought stress (Fig. 1B).
In order to observe the effect of γ-PGA on maize growth under drought stress more directly, the simulated drought experiment with 18% PEG6000 solution was performed. It was found that the fresh weight of leaves and roots in γ-PGA treatment group was higher than that of the control group (Fig. 2), indicating that the drought resistance of leaves and roots in γ-PGA + PEG group was significantly higher than that of the control group.
γ-PGA significantly improved roots development, urease activity and ABA contents of maize rhizospheric soil under drought stress
It was found that γ-PGA significantly improved the roots development both under the normal condition and drought stress (Fig. 2A). Under normal growing conditions, the maize treated with γ-PGA had a better developed root system, and the fresh weight of roots was significantly increased than that of the control group. Under PEG simulate drought stress, the roots growth of the control group was significantly inhibited, however, the roots of the maize treated with γ-PGA were little affected by drought stressand the roots fresh weight was significantly higher than that of control group. Since maize rhizospheric soil was closely contacted with the roots, the urease activity (closely related to soil nitrogen transformation) and ABA contents (closely related to the drought resistance) of the maize rhizospheric soil under the severe drought stress were also detected. It was observed that the urease activity of rhizospheric soil of γ-PGA treatments was increased by 27.74%, while the ABA contents of γ-PGA treatments soil was also increased by 21.70% (Table 1).
Effect of γ-PGA on the contents of ABA and Urease activity of maize rhizospheric soil under severe drought stress
ABA (µg/g DW)
1.479 ± 0.011
767.583 ± 124.714
1.800 ± 0.002**
980.524 ± 46.475**
|Values are means ± sd (n ≥ 3 repeats). Significant differences are indicated by asterisks (**, P ≤ 0.01).|
Differentially expressed genes (DEGs) between maize with γ-PGA addition and control under drought stress
In order to explain the mechanism of γ-PGA in improving the drought resistance of maize, the leaves of γ-PGA treatment and the control maize under drought condition for 5 days were used for RNA sequencing to identify the DEGs and pathways in response to drought stress. The total raw reads, clean reads, genome mapping ratio, and uniquely mapping ratio were listed in Additional file 10: Table S1. 16126 DEGs were identified and the distribution of the DEGs was illustrated in Fig. 3A. These DEGs were subjected to enrichment analysis of KEGG pathways and Gene Ontology (GO) functions. Based on KEGG pathway analysis, all DEGs were significantly enriched into 6 pathways (Q value ≤ 0.05), namely photosynthesis-antenna proteins (31 DEGs), photosynthesis (105 DEGs), glyosylate dicarboxylate metabolism (102 DEGs), oxidative phosphorylation (156 DEGs), alanine, aspartate and glutamate metabolism (73 DEGs) and carotenoid biosynthesis pathway (65 DEGs) (Fig. 3B). The results of GO annotation functions enrichment analysis also showed that GO terms such as photosynthesis and photosystem, response to abiotic stimulus, chlorophyll metabolic process, response to biotic stimulus, electron transport chain and so on were significantly enriched (Additional file 2: Fig. S2B). A more detailed classification of the terms of response to abiotic stimulus showed that these DEGs were mainly related to the response to stress (osmotic stress, salt, heat, cold, reactive oxygen species, and hydrogen peroxide), the response to hormone (ABA, JA, and SA), ABA biosynthetic process, chlorophyll metabolic process, proline biosynthetic process, protein folding, and so on (Additional file 2: Fig. S2B).
γ-PGA improved drought resistance of maize by affecting the expression of photosynthesis-related genes
As known, drought could significantly reduce the photosynthesis capability of plants. However, KEGG analysis showed that under drought stress, compared with the control maize, the photosynthesis related genes of maize treated with γ-PGA were significantly enriched (Fig. 3B), with most of related genes were dramatically upregulated. As shown in Fig. 4 and Additional file 10: Table S1, most genes in DEGs of photosystem II complex were upregulated, except PsbA, PsbB, PsbC, PsbE, PsbF and 1 for PsbP, which were downregulated. In photosystem I complex, all of the DEGs were upregulated. In cytochrome b6/f complex, 7 genes encoding PetA, 2 genes encoding PetC and 1 genes encoding PetG were upregulated, while only 1 gene encoding PetD and 1 gene encoding PetA were downregulated. In photosynthetic electron transport, other 16 genes encoding PetE, PetF, PetH and PetJ were all up-regulated except 3 genes encoding PetF and 2 for PetH,. In F-type ATPase complex, except 1 gene encoding beta, 1 for gamma and 1 b which were downregulated, the other 14 genes encoding alpha, beta, gamma, delta, epsilon, a, b and c subunits respectively were upregulated. Additionally, all DEGs (67 genes) encoding antenna proteins were also up-regulated (Additional file 3: Fig. S3). To confirm the results, 14 genes with different transcript abundances were validated by real-time RT-PCR (Additional file 4: Fig. S4). The expression of these genes showed good consistency between the two detection methods. Meanwhile, the motifs in the promoter region of these genes were analyzed, higher percentage of drought, low-temperature, salicylic stress and ABA response elements were found (Additional file 5: Fig. S5, Additional file 6: Fig. S6).
γ-PGA promoted ABA accumulation and affected ABA signaling to improve drought resistance of maize
ABA, as an important drought response hormone, plays an important role in the response of maize to abiotic stress. Based on KEGG pathway analysis, it was found that DEGs related with carotenoid biosynthesis pathway which contains ABA biosynthesis pathway were significantly enriched (Fig. 3B), γ-PGA could promote ABA accumulation under drought condition (Fig. 1B). CHY2, ABA1, NCED, ABA2 and AAO3 were reported to be involved in ABA biosynthesis [35–38], 8'-hydroxyase was reported to play an important role in the catabolism of ABA . RNAseq results showed that 2 genes encoding CHY2, 7 genes encoding ABA1, 3 genes encoding NCED, 2 genes encoding ABA2, and 1 genes encoding AAO3 were significantly upregulated, while 2 genes encoding 8'-hydroxyase were downregulated. In addition, ABA signaling pathway related genes, including ABA receptor (PYR/PYL), PP2C, SnRK2 and ABFs were also differentially expressed. Among these DEGs, 3 for PYL, 4 for SnRK2, and 4 for ABF were upregulated, 10 for PP2C were downregulated (Additional file 7: Fig. S7).
γ-PGA affected the bacterial community diversity and structure of rhizospheric soil
In order to study the influence of γ-PGA on bacterial community diversity under drought stress, the relative abundance and diversity of maize rhizospheric soil bacteria were analyzed by high-throughput sequencing of 16S rRNA. The species curve showed that the samples were representative enough to obtain a true bacterial community (Additional file 8: Fig. S8). NMDS (stress = 0.00422) of the weighted UniFrac distance ordinations were conducted (Fig. 5A), the results indicated that the bacterial community composition of the soil with γ-PGA application brought shifts compared with that of the soil without γ-PGA under the drought stress, the communities in maize rhizospheric soil with γ-PGA were grouped together and significantly separated from those in soil without γ-PGA under the drought stress. The obtained high-quality sequences were belonged to 36 phylum,among which the main phylum was Proteobacteria, followed by Actinobacteria, Chloroflexi, Bacteroidetes, and Acidobacteria. Although the diversity of bacterial community changed after the addition of γ-PGA under drought stress༌the predominant phylum were similar. There was no difference in species composition among these samples, but the relative abundances of some species changed (Fig. 5B). Compared to the control, the relative abundance of Actinobacteria and Chloroflexi were higher in soil added γ-PGA under drought stress. LEfSe analysis (LDA ≥ 3) showed the species with the most significant variation (Fig. 5C). Under drought stress, the application of γ-PGA could significantly enrich Actinobacteria, Chloroflexi and Cyanobacteria at phylum level, while Alphaproteobacteria and Deltaproteobacteria were enriched at class level. At the genus level, bacteria such as Rhodobacter༌Sphingobium, Sphingomonas, Sphingopyxis, Haliangium, Methylibium, Lysobacter, Azoarcus and Arenimonas of Proteobacteria, Aeromicrobium, Lechevalieria and Streptomyces of Actinobacteria, Subgroup_10 of Acidobacteria, Clostridium and Pelotomaculum of Firmicutes, Chloronema, A4b and KD4-96 of Chloroflexi were dominant in γ-PGA added rhizosphere soil under the persistent severe drought condition. The abundances of these genera in maize rhizospheric soil with γ-PGA addition were all higher than that of control (Additional file 9: Fig. S9), while Bacillus of Proteobacteria was dominated in control (Fig. 5C).