Many microorganisms associated with plants are active in the rhizosphere; those that promote plant growth, control diseases, and increase crop yield are collectively referred to as plant growth-promoting rhizobacteria (PGPR).
Previous studies have demonstrated that nitrogen fertilizers cannot completely replace the beneficial rotation effect of legumes. The 75% increase in the yield of soybean cannot be explained by existing theories (Heaterman et al. 1986). The nodules on legumes release large amounts of H2 during nitrogen fixation. Approximately 1.5 mol of H2 is produced for every unit of N2 fixed. Soybeans with nitrogen-fixing capacity can produce approximately 5000 l of H2 per hectare per day at peak growth and 2.4 × 105 l H2 per hectare per the growing season. The production of H2 requires 5–6% of the net energy produced during photosynthesis (Dong et al. 2003). From a metabolic point of view, the release of H2 from the root is a waste of energy; however, neither long-term natural evolution nor manual screening strategies have successfully reduced this energy loss.
Most of the hydrogen released from legume nodules is absorbed by the soil (La Favre et al. 1983). A large amount of hydrogen is released during the nitrogen fixation process, but only a small amount is released into the atmosphere because of the actions of soil microorganisms that harvest the produced H2 for energy. Kärst et al. (1987) demonstrated that hydrogen-utilizing bacteria could promote plant growth because they possess hydrogenase, a metal-containing protein that catalyzes the reduction of protons to produce hydrogen as an end product of the electron transport chain (Kärst et al. 1987). The reversible reaction of catalytic protons to electron production is as follows: 2H+ + 2e– ↔ H2.
This study aimed to provide a new understanding of the role of hydrogen in the plant-rhizobacterium interaction and gain a better understanding of metabolic processes underlying these relationships using
5’-GGCACAATCGTGCGTGTCCTTCAAC-3’ and 5’-GCCATTCGCCCATCTCAT-3’;
We used 16S rRNA as an internal reference gene to calculate normalized gene expression intensity using the Q-gene software (Muller et al. 2002).
The content of 17 amino acids was determined according to the method described by Sokolova et al. (2007) in an automatic amino acid analyzer (Hitachi 835–50 amino acid analyzer; Tokyo, Japan) with the measurement repeated three times for each sample. The
Similarly, 50 μM 2,6-dichlorophenol indophenol, 2 mM NaN3, 2 μg/ml rotenone, 2 μg/ml antimycin A, and 25 μM benzoquinone (10 μl each) were added to the buffer to which 20 μl of the bacterial suspension was added, and after mixing well, the mixture was incubated in a 28°C-water bath for 3 min, followed by the addition of 10 μl 20 μM sodium succinate to start the reaction. Immediately afterward, the OD value was measured with a standard quartz cuvette at 600 nm in a UV spectrophotometer. The CII activity was calculated from the standard curve.
As indicated above, 10 μl of 2 mM NaN3 and 10 μl of 50 μM cytochrome c reductase were added sequentially to the buffer to which 20 μl of the bacterial suspension was added, and the mixture was incubated in a 28°C-water bath for 3 min, followed by the addition of 10 μl of 80 μM coenzyme Q to start the reaction. Immediately afterward, the OD value was measured with a standard quartz cuvette at 550 nm using a UV spectrophotometer. The CIII activity was determined from the standard curve.
Similarly, 10 μl of 0.025%
Differential gene clustering. Red dots indicate downregulated genes and blue dots indicate upregulated genes in comparison with the control.
Differentially expressed genes – change with hydrogen treatment.
Gene ID | Gene name | Change with hydrogen treatment | Gene description |
---|---|---|---|
Gene1418 | sugE | Down | Quaternary ammonium compound efflux SMR transporter SugE |
Gene1984 | msbB | Down | Lipid A biosynthesis (KDO)2-(lauroyl)-lipid IVA acyltransferase |
Gene2119 | SMc04350 | Down | Multidrug efflux system transmembrane protein |
Gene2120 | SMc04351 | Down | Transmembrane ATP-binding ABC transporter protein |
Gene2494 | mtlK | Down | Oxygen-independent coproporphyrinogen III oxidase |
Gene2940 | SMc02983 | Down | Arginine decarboxylase |
Gene3019 | SMc03139 | Ups | Hypothetical protein |
Gene3089 | SMc02519 | Down | ABC transporter ATP-binding protein |
Gene3090 | SMc02518 | Down | ABC transporter ATP-binding protein |
Gene3090 | SMc02518 | Down | ABC transporter ATP-binding protein |
Gene3091 | SMc02517 | Down | ABC transporter permease |
Gene3471 | SMa0081 | Ups | ABC transporter permease |
Gene361 | iolB | Down | 5-deoxy-glucuronate isomerase |
Gene362 | iolE | Down | Myo-inosose-2 dehydratase |
Gene4065 | SMa1163 | Down | Cation transport P-type ATPase |
Gene4073 | SMa1176 | Down | Hypothetical protein |
Gene4075 | nosR | Down | NosR regulatory protein for N2O reductase |
Gene4076 | nosZ | Down | Nitrous-oxide reductase |
Gene4078 | nosF | Down | NosF ATPase |
Gene4079 | nosY | Down | NosY permease |
Gene4082 | fhp | Down | Nitric oxide dioxygenase |
Gene4092 | fixI1 | Down | ATPase |
Gene4093 | fixH | Down | Nitrogen fixation protein FixH |
Gene4094 | fixG | Down | FixG iron sulfur membrane protein |
Gene4095 | fixP1 | Down | FixP1 di-heme cytochrome c |
Gene4097 | fixO1 | Down | Cbb3-type cytochrome c oxidase subunit II |
Gene4123 | hemN | Down | Oxygen-independent coproporphyrinogen III oxidase |
Gene4550 | SMa2051 | Down | Desaturase |
Gene4551 | SMa2053 | Down | MocE-like protein |
Gene5192 | SM_b20487 | Down | Sugar ABC transporter permease |
Gene5193 | SM_b20488 | Down | Hypothetical protein |
Gene5194 | SM_b20489 | Down | Carbohydrate kinase |
Gene5727 | groEL | Down | Chaperonin GroEL |
Gene5728 | groES5 | Down | Molecular chaperone GroES |
Gene6038 | cyoB | Down | Cytochrome O ubiquinol oxidase subunit I |
Gene6039 | cyoC | Down | Cytochrome O ubiquinol oxidase subunit III |
Gene6040 | cyoD | Down | Cytochrome O ubiquinol oxidase CyoD |
Gene6083 | SM_b20654 | Ups | Hypothetical protein |
Gene6166 | SM_b20753 | Ups | Acyl-CoA dehydrogenase |
Gene6288 | agaL1 | Down | Alpha-galactosidase (melibiase) protein |
Gene811 | groES | Down | Co-chaperone GroES (Cpn10) binds to Cpn60 in the presence of Mg-ATP and suppresses the ATPase activity of the latte |
Gene968 | betA | Down | Choline dehydrogenase |
Gene969 | betB | Down | Betaine aldehyde dehydrogenase |
Gene970 | betC | Down | Choline-sulfatase |
Histogram of GO terms associated with differentially expressed genes; the GO term is on the ordinate; the number of differentially expressed genes associated with each term is on the abscissa.
Differential gene bubble map in the KEGG metabolic pathway.
During the KEGG pathway analysis, we noted that the arginine and proline metabolism pathway (ko00330) was downregulated in the hydrogen treatment group. To clarify this pathway’s connection with hydrogen treatment, we checked if the pathway involves any hydrogenases. There are 3896 hydrogenases (IUBMB database, last accessed on January 2019). We shortlisted the hydrogenases (Table II) involved in the arginine and proline metabolism pathway to determine the effect of hydrogen treatment.
Hydrogenases involved in arginine and proline metabolism.
EC number | Name |
---|---|
1.2.1.19 | Aminobutyraldehyde dehydrogenase |
1.2.1.3 | Aldehyde dehydrogenase (NAD+) |
1.4.3.22 | Diamine oxidase |
1.2.1.71 | Succinylglutamate-semialdehyde dehydrogenase |
1.2.1.88 | L-glutamate gamma-semialdehyde dehydrogenase |
1.5.1.19 | D-nopaline dehydrogenase |
1.5.1.11 | D-octopine dehydrogenase |
Hydrogen treatment caused a decrease in the expression of the gene encoding aminobutyraldehyde dehydrogenase (EC: 1.2.1.19), which is an important hydrogenase in the synthesis of 4-amino-butanoate and in β-alanine metabolism. Moreover, butanoate, alanine, glutamate, and ADC activities were also decreased under hydrogen treatment, which is the same outcome observed during the synthesis of β-alanine (ko00410).
The results of the amino acid analysis showed that the content of arginine, glycine, proline, serine, and threonine decreased after hydrogen treatment, and the content of arginine and proline exhibited the most significant decrease (Table III). After 72 h, the arginine content decreased by 0.47 mg g–1, and the proline content decreased by 0.51 mg g–1. The content of some other amino acids was elevated, but the increase was small.
The content of amino acids after hydrogen treatment.
Amino acid | Concentration (mg/g) | |||
---|---|---|---|---|
Control group | Test group | |||
24 h | 48 h | 72 h | ||
Alanine | 0.32 ± 0.012 | 0.292 ± 0.03 | 0.342 ± 0.011 | 0.372 ± 0.05 |
Arginine | 0.0842 ± 0.006 | 0.0682 ± 0.002 | 0.0622 ± 0008 | 0.0372 ± 0.004 |
Aspartic acid | 1.822 ± 0.015 | 1.862 ± 0.3 | 1.882 ± 0.29 | 1.772 ± 0.24 |
Cysteine | 0.182 ± 0.07 | 0.152 ± 0.04 | 0.222 ± 0.06 | 0.142 ± 0.03 |
Glutamic acid | 2.12 ± 0.25 | 2.252 ± 0.16 | 2.242 ± 0.19 | 2.22 ± 0.11 |
Glycine | 0.0772 ± 0.004 | 0.0742 ± 0.006 | 0.0712 ± 0.003 | 0.0682 ± 0.009 |
Histidine | 0.162 ± 0.07 | 0.182 ± 0.09 | 0.172 ± 0.05 | 0.152 ± 0.02 |
Isoleucine | 0.72 ± 0.037 | 0.682 ± 0.02 | 0.772 ± 0.03 | 0.732 ± 0.08 |
Leucine | 0.252 ± 0.022 | 0.222 ± 0.03 | 0.262 ± 0.04 | 0.292 ± 0.05 |
Lysine | 3.62 ± 0.17 | 3.42 ± 0.11 | 3.32 ± 0.2 | 3.72 ± 0.19 |
Methionine | 0.092 ± 0.003 | 0.062 ± 0.001 | 0.092 ± 0.004 | 0.0122 ± 0.002 |
Phenylalanine | 1.52 ± 0.08 | 1.72 ± 0.05 | 1.622 ± 0.07 | 1.582 ± 0.03 |
Proline | 0.0732 ± 0.002 | 0.0642 ± 0.008 | 0.0262 ± 0.002 | 0.0222 ± 0.004 |
Serine | 0.0652 ± 0.007 | 0.0612 ± 0.002 | 0.062 ± 0.008 | 0.0552 ± 0.007 |
Threonine | 0.0782 ± 0.005 | 0.0772 ± 0.005 | 0.0542 ± 0.007 | 0.0582 ± 0.003 |
Tyrosine | None | None | None | None |
Valine | 0.062 ± 0.003 | 0.0622 ± 0.007 | 0.0642 ± 0.004 | 0.0672 ± 0.008 |
Oxidative phosphorylation (ko00190) is an important aspect of metabolism and is closely related to nitrogen fixation (Allen and Arnon 1955). Hydrogen treatment increased the expression of cytochrome c oxidase (EC:1.9.3.1), inhibiting the H+ consumption process in complex IV to reduce the intensity of oxidative phosphorylation.
Nitrogenase can capture large amounts of H+ for nitrogen fixation, thereby reducing the consumption of H+ in oxidative phosphorylation and providing energy for nitrogen fixation by nitrogenase (Millar et al. 1995).
Cytochrome O ubiquinol oxidase subunit I (
Fold change in the expression levels of SMc02983, SMc01578, SMc01656, SMc02677, SM_b20752, cyoD, cyoB, and cyoC after the hydrogen treatment as determined by real-time quantitative PCR.
The results of oxidative phosphorylation complex activity showed that hydrogen treatment reduced the activity of complex IV most significantly. There may also be other mechanisms underlying the role of CIV activity in reducing complex II activity by regulating the electron transfer between CIII and CIV (Fig. 5).
The oxidative phosphorylation complex activity map. Each experiment was performed three times.
The differential genes involved in the experiments were annotated in several aspects. Based on previous studies, we focused on hydrogenase pathways. These genes were verified by real-time quantitative PCR and were found to be consistent with RNA-Seq findings. Therefore, the research is somewhat reliable. Taken together, hydrogen affects
We determined the transcriptomic changes in