Effect of peanut root exudates on substance transport and energy metabolism of P9
Microbe–plant interactions involve consumption of a large amount of energy. Fan et al. reported that maize root exudates promote significant up-regulation of the glycolytic pathway and tricarboxylic acid (TCA) cycle-related genes in Bacillus amyloliquefaciens FBZ42 [18]. In the present study, the expression of genes associated with glycolysis, TCA cycle, and pentose phosphate pathways were up-regulated in response to peanut root exudates, and the P9 strain adopted a more efficient and productive approach for biosynthesis and metabolism. The up-regulated genes included fruK encoding 1-phosphofructokinase in the glycolytic pathway, aceA encoding isocitrate lyase (ICL) in the TCA cycle, garR encoding the NAD-binding domain of 6-phosphogluconate dehydrogenase in the pentose phosphate pathway, and gcl encoding lyoxylate carboligase that catalyzes decarboxylation of glyoxylate. High concentrations of acetyl coenzyme A (enoyl-CoA) are an indication of excellent cellular nutritional status [19] as well as an important intermediate in metabolic networks, such as glycolysis, the TCA cycle, glyoxalate bypass, and acetate metabolism, and ultimately ATP synthesis [20]. The enoyl-CoA hydratase gene echA and the enoyl-CoA acetyltransferase gene ACAT were up-regulated in P9; these enzymes together catalyze β-oxidation of fatty acids to synthesize enoyl-CoA. The alanine dehydrogenase gene ald was up-regulated and encodes a protein that catalyzes the production of pyruvate from alanine, which can be further oxidized to acetyl CoA and participates in the TCA cycle [21]. In addition, the alkane 1-monooxygenase gene alkM, which encodes a protein that oxidizes alkanes to alcohols and further oxidizes alcohols and aldehydes to fatty acids [22], and ppk2, a polyphosphate kinase 2 gene involved in the oxidative phosphorylation pathway, were up-regulated and may lead to enhanced synthesis of GTP. Thus, root exudates of peanut were indicated to promote synthesis of enoyl-CoA, ATP, and GTP in P9, as well as increased regeneration and storage of fatty acids, which may produce sufficient energy for enhanced metabolism and substance synthesis. The corresponding up-regulation of the MFS superfamily transporter protein gene smvA and ABC transporter genes in P9 resulted in enhanced uptake and transport of exogenous nutrients, which was similar to the increased uptake of hexose by Burkholderia cepacia Q208 induced by sugarcane root exudates [23].
The expression of bldC, a member of the bldC family of transcription regulators, was significantly up-regulated, whereas iclR, a gene of the IclR transcription regulator family associated with spore formation, was down-regulated. Up-regulated expression of bldC and down-regulated expression of iclR can prevent premature development of actinomycete spores, and are important for maintenance of normal development during the vegetative growth period [24, 25]. Potato root exudates cause differential expression of certain genes encoding membrane proteins and genes associated with spore formation or germination in Mycobacterium mycoides [26]. Thus, we speculate that peanut root exudates may also be involved in mediating the normal biosynthesis and development of bacterial membranes and spores.
Effects of Peanut Root Exudates on the Stress Tolerance and Growth-promoting Characteristics of P9 Strain
Siderophores are chelating agents synthesized by microorganisms in a low iron environment that can specifically chelate Fe3+. The siderophores produced by PGPR can compete with plant rhizosphere pathogens for limited iron ions, and inhibit their growth and reproduction [1, 27]. Klonowska et al. reported that Mimosa pundica root exudates promoted significant up-regulation of the siderophore biosynthetic pathway in symbiotic nitrogen-fixing bacteria [28]. In the current study, after addition of peanut root exudates, all seven genes associated with siderophore synthesis and utilization were up-regulated in P9, specifically, mbtH (MbtH protein family gene), fagD (iron siderophore-binding protein gene), fepB (encoding iron-enterobacterin binding protein), fepD (encoding iron-siderophore transport system permease protein), yqjH (encoding NADPH-dependent ferric siderophore reductase/siderophore-interacting protein, SIP), and hpaC (flavin reductase gene). The MbtH proteins are associated with the biosynthesis of oxyximate-type and catechol-type siderophores in many actinomycetes [29], and up-regulation of mbtH is consistent with root exudates causing a marked increase in siderophore content in P9 (Table 1). Of interest, proteins encoded by fagD, fepB, and fepD are normally membrane proteins involved in the uptake transport of Fe3+-siderophores by G+ bacteria, a class of periplasmic-binding proteins that bind Fe3+ to siderophores and transport them into the cytoplasm via the siderophore–permease(s)–ATPase system [30]; significant up-regulation of these three genes significantly improved the transport efficiency of Fe3+-siderophores by P9. NADPH-dependent ferric siderophore reductase, known as SIP, is a flavin protein containing a specific siderophore-binding site that reduces and releases Fe3+ obtained from siderophores into highly soluble Fe2+ [31]. Up-regulated expression of yqjH and hpaC promotes efficient utilization of Fe3+. Thus, the peanut root exudates can induce an increase in the biosynthesis and utilization of siderophores, and improve the uptake of Fe3+ by P9, which would not only promote growth, but also enhance resistance to pathogens, which plays an important role in the growth-promoting effects of P9.
Root exudates are a carbon and energy source for PGPR, and provide a precursor library for biotransformation of PGPR [32]. After adding peanut root exudates and components of proline, alanine, and glycine, the ability of P9 to secrete IAA was significantly improved. In addition to tryptophan as a precursor for IAA biosynthesis, other amino acid components in peanut root exudates also promoted IAA biosynthesis as a carbon source (Fig. 4). Tyrosine, phenylalanine, and aspartate acid have been reported to be carbon sources to promote IAA biosynthesis in some bacteria to varying degrees [33].
In B. amyloliquefaciens FBZ42 certain genes involved in antibiotic biosynthesis are significantly up-regulated under the influence of maize root exudates [18]. In the present study, up-regulated expression of mbtH is associated with the biosynthesis of peptide antibiotics [34]. The expression of the histidine kinase gene ASU32_16205 was up-regulated, which can act as a sensor to induce antibiotic production [35]. The lipid transfer protein encoded by the up-regulated gene SCP2 is considered to be a protein that inhibits and kills pathogens [36].
Effect of Peanut Root Exudates on Biofilm Formation and Colonization by P9
When PGPR enter the soil, their ability to colonize and interact with plants are crucial to their growth-promoting properties, and biofilm-related pathways and protein adhesion to roots are associated with rhizosphere colonization [37]. In the present study, ata, a gene that encodes a trimeric autotransporter adhesin (TAA), was significantly up-regulated (9.29-fold, the highest). TAA proteins mediate G− bacterial adhesion to host tissues [38]. We hypothesized that up-regulated expression of ata also enhanced the adhesion of P9, which laid the foundation for its effective colonization. Lin et al. observed that a ferric ABC transporter positively regulates biofilm formation [39]. In addition to significant up-regulation of iron ABC transporter genes (fagD, fepB, and fepD), the MCE protein family gene mlaD and polyphosphate kinase 2 gene ppk2 were significantly up-regulated under the influence of peanut root exudates. The encoded product of mlaD has a MCE domain that binds lipids, and ppk2 is associated with polyphosphate homeostasis in phospholipid synthesis [40], all of which suggest that peanut root exudates promote the biofilm formation of P9. As components of root exudates, oxalic acid, citric acid, and malic acid promoted biofilm formation of P9, which is consistent with the reported biofilm-inducing effects on strains of Bacillus spp. [41].
The intrinsic antibiotic resistance of PGPR is critical for the adaptation and survival of strains in the presence of other aggressive microorganisms [42]. The MarR and WhiB transcription regulator families, and ABC transporters are associated with the development of bacterial antibiotic resistance [43, 44]. The expression of marR, whiB1_2_3_4, ABC, and ASU32_16205 genes were all up-regulated in P9 under the influence of peanut root exudates, giving the strain a stronger competitive ecological niche. Genes lacking crucial virulence or virulence-related factors have been detected in some PGPR, thus ensuring a mutually beneficial relationship between PGPR and plants [45]. In the current study, the expression of the IclR transcription regulator gene IclR and branched-chain amino acid ABC transporter gene Liv were down-regulated; the former is associated with certain bacterial virulence and plant pathogenicity [46], whereas the latter is associated with virulence integrity [47]. It is speculated that down-regulation of these genes may be more conducive to establishment of interaction between P9 and peanut roots.
Effects on Arginine Biosynthesis Pathway and Urea Cycle
The arginine biosynthesis pathway was the only significantly down-regulated pathway in the present investigation. Three genes associated with the urea cycle, namely argG (encoding argininosuccinate synthase), argH (encoding argininosuccinate lyase), and argF (encoding ornithine carbamoyl- transferase), and an allophanate hydrolase gene (atzF) and urease accessory protein gene (ureD) involved in urea decomposition, were significantly down-regulated in P9. The pH was lower in the initial LB medium supplemented with root exudates and at the beginning of the logarithmic growth phase of P9 compared with that of the control, whereas the pH of the two cultures all increased to the same level at 22 h (Table 1). We tentatively speculated that the control P9 group without the addition of root exudates required the enhancement of this pathway to decompose a greater amount of urea, and the ammonia produced led to the increase in pH of the culture, whereas the urea in the root exudate was directly supplied to P9 in the treated group, but this hypothesis needs to be studied further. In addition, the expression of genes involved in the urea cycle and decomposition was down-regulated overall, indicating a decrease in the biosynthesis of the metabolites citrulline and arginine. Previous studies have reported that citrulline performs roles such as scavenging hydroxyl groups and protecting DNA from oxidative reactions, and arginine is a source of polyamines involved in scavenging reactive oxygen species (ROS) and protecting membranes from lipid peroxidation via the arginine deiminase pathway [48], which indirectly suggests that ROS contents of the P9 strain are lower under the influence of peanut root exudates.