Effect of Bacillus subtilis WM13-24 on seedling growth of Arabidopsis and H. ammodendron and root development of Arabidopsis
Inoculation with strain WM13-24 was found to promote biomass production of Arabidopsis in vitro and in soil. Strain WM13-24 at three concentrations (104, 108 and 2×108 CFU mL− 1) significantly increased shoot dry weight (1.2-fold, 1.6-fold and 1.9-fold, respectively), root dry weight (93%, 1.2-fold and 1.5-fold, respectively), leaf chlorophyll a (14%, 32% and 43%, respectively) and chlorophyll b content (12%, 28% and 39%, respectively) (P < 0.05) of Arabidopsis compared to control (Fig. 1A, B, C). Correspondingly, strain WM13-24 at the concentration of 104 and 2×108 CFU mL− 1 significantly (P < 0.05) increased plant photosynthetic rate by 33% and 51%, respectively (Fig. 1D). In addition, strain WM13-24 significantly (P < 0.05) increased total fresh weight of H. ammodendron seedlings by 17% and 35% and total dry weight by 30% and 25% 14 and 40 days after inoculation of strain WM13-24 at 108 CFU mL− 1, respectively, compared to control (Fig. 2).
Then, the effects of WM13-24 on Arabidopsis seedling growth and root development were evaluated in sterile plates (Fig. 3A). Strain WM13-24 significantly increased the number of lateral root by 41% (P < 0.05) 14 days after inoculation, compared to control (Fig. 3B). In addition to positive effects on LR formation, WM13-24 had a strong impact on root hair development (Fig. 3C). B. subtilis WM13-24 showed the similar growth promotion effect on Arabidopsis compared to B. amyloliquefaciens GB03 as shown in Fig. S1.
Screening Arabidopsis-signaling pathway mutants for regulatory control of root development
In order to probe the mechanism by which bacterial volatiles can regulate root development, PGPR strain WM13-24 was tested against a series of Arabidopsis mutants defective in specific regulatory pathways, and total root length, LR number and LR density were analyzed at 14 days. The mutants included ein3-1 and etr1 for ethylene, cre1 for cytokinin, gai for gibberellin acid, jar and coi1 for jasmonic acid, cbb1 for brassinosteroid, NahG for salicylic acid. As showed in Fig. 4, these mutants (ein3-1, etr1, cre1, gai, jar, coi1, cbb1 and NahG) responded to VCs like Col-0 by increasing total root length (1.9-fold, 63%, 30%, 1.2-fold, 38%, 1.6-fold, 1.2-fold and 77%, respectively) (Fig. 4B), LR number (77%, 73%, 28%, 94%, 41%, 43%, 47% and 32%, respectively) (Fig. 4C) and lateral root density (73%, 67%, 29%, 64%, 56%, 35%, 59% and 33%, respectively) (P < 0.01) (Fig. 4D) 14 days after inoculation. Although strain WM13-24 also promoted root development in the cytokinin receptor mutant cre1 to a certain extent, the effect was much lower than that of the wild type Col-0, indicating that cytokinin was involved in the process of root development promoted by WM13-24, and the role of cytokinin in this process needs to be further investigated.
To further address the role of auxin signaling in WM13-24-induced root development, we analyzed the responses of Col-0 and Arabidopsis single or double mutants (arf7, arf19, arf7arf19, axr1, axr2, axr4 and eir1) defective in auxin signaling to VCs emitted from strain WM13-24 (Fig. 5). The arf19, axr1, axr4 and eir1 mutants, which formed a significantly reduced total root length under control treatment, responded to VCs emitted from strain WM13-24 with increased total root length, but only approximately 45%, 40%, 46% and 58% of that of WM13-24-treated Col-0, respectively (Fig. 5B). However, the double mutant arf7arf19 did not respond to the VCs without increased total root length (Fig. 5B). The VCs increased LR number of arf7, arf19, axr1, axr2, axr4 and eir1 mutants by 2-fold, 88%, 25%, 21%, 39% and 11% (P < 0.01), respectively, compared to their individual control, however, was not capable of rescuing the LR-defective phenotype of the arf7arf19 mutant, whose lateral root formation is completely abolished (Fig. 5C).
Effect of Bacillus subtilis WM13-24 VCs on auxin response of Arabidopsis
LR formation is tightly correlated with auxin signaling (Fukaki et al. 2007). To test the role of auxin response in WM13-24 VCs-induced LR formation, transgenic plants harbouring DR5::GUS and auxin-responsive marker DR5::GFP were used. WM13-24 VCs enhanced the expression of the GUS in the entire root, especially in the lateral root primordia, compared with control (Fig. 6A). Similarly, the VCs also increased the green fluorescence level of GFP in the apical meristem and lateral root (Fig. 6D). To probe the stages of LRP that are affected by strain WM13-24, the developmental stage of each LRP on WM13-24-treated roots was classified according to Malamy and Benfey (1997). LRP stages Ⅰ-Ⅲ and Ⅵ-Ⅶ were significantly increased in strain WM13-24 VCs treated seedlings (Fig. 6B). Strain WM13-24 VCs significantly increased the number of total LRP by 180%, compared to control (Fig. 6C). Furthermore, the expression levels of genes related to LR formation were analyzed using qRT-PCR. The expression levels of LATERAL ORGAN BOUNDARIES DOMAIN16 (LBD16) and LBD29 were induced significantly by WM13-24 VCs, although those of ARF7 and ARF19 were not altered (Fig. 6E).
Therefore, we further measured root IAA level and the expression levels of genes related to IAA biosynthesis, ASA1 (ANTHRANILATE SYNTHASE SUBUNIT 1), TAA1, YUC5, YUC8, YUC9, NIT1 (NITRILASE 1) and NIT2, and auxin dynamic balance maintenance, GH3.1 (GRETCHEN HAGEN 3.1), GH3.5 and GH3.6 (Staswick et al. 2005), using qRT-PCR. WM13-24 VCs enhanced root IAA level by 73% compared with control (Fig. 7A). WM13-24 VCs significantly increased the expression levels of ASA1, YUC5, YUC8, YUC9, NIT1, NIT2, GH3.1, GH3.5 and GH3.6 by 25%, 43%, 17%, 28%, 21%, 37%, 27%, 17% and 39%, compared with their individual controls (P < 0.05) (Fig. 7B). The major route of IAA biosynthesis is the indole-3-puruvate pathway (Kasahara 2016). To test whether the increase in LR formation was caused by auxin biosynthesis under strain WM13-24 VCs treatment. The involvement of the indole-3-puruvate pathway was evaluated using 0.5 and 1 µM L-kynurenine and 4-phenoxyphenyl boronic acid, which inhibit TAA1/TARs and YUCs (Inaji et al. 2020), respectively (Fig. S2A). In the presence of 0.5 and 1 µM 4-phenoxyphenyl boronic acid, WM13-24 VCs still stimulate LR formation with increased LR number, whereas in the presence of 1 µM L-kynurenine, LR formation was inhibited (Fig. S2B). The results shown that TAA1-mediated auxin biosynthesis may require for strain WM13-24 VCs-promoted LR formation.
Effect of Bacillus subtilis WM13-24 VCs on auxin polar transport of Arabidopsis
In order to investigate the role of auxin transport in WM13-24 VCs-induced lateral root formation, the LR phenotype of the auxin influx mutant aux1-7 was used (Fig. 8A). The LR number of aux1-7 was less than that of Col-0 under control condition, but WM13-24 VCs could still increase LR number of aux1-7 by 47% (similarly by 41% for Col-0) (P < 0.05) (Fig. 8B). Furtherly, to test the possible role of auxin polar transport in WM13-24 VCs-induced lateral root formation, the auxin efflux inhibitor 2-((1 naphthalenylamino)-carbonyl) benzoic acid (NPA) was supplemented to the plant growth media. In the presence of 1 µM NPA, WM13-24 VCs still stimulate LR formation with increased LR number, whereas in the presence of 5 µM NPA, LR formation was completely inhibited and WM13-24 VCs could not stimulate LR formation (Fig. 8C). Above results suggested auxin efflux was required for WM13-24 VCs-stimulated LR formation. Thus, we analyzed the levels of four auxin efflux carriers, PIN1, PIN2, PIN4 and PIN7, in WM13-24 VCs exposed transgenic lines expressing PIN1::PIN1::GFP, PIN2::PIN2::GFP, PIN4::PIN4::GFP and PIN7::PIN7::GFP. As shown in Fig. 9, WM13-24 VCs significantly reduced the green fluorescence level of PIN4 and slightly reduced those of PIN1 and PIN2, whereas significantly induced that of PIN7 compared with their individual controls (Fig. 9A, B). Subsequently, the gene expression levels of above four auxin efflux carriers and auxin influx carriers, AUX1, LAX1, LAX2 and LAX3, were analyzed using qRT-PCR. The expression levels of PIN1, PIN2 and PIN4 were reduced, whereas PIN7 was induced significantly by WM13-24 VCs, compared with their individual controls (Fig. 9C), which was consistent with the changes of their green fluorescence levels. The expression levels of AUX1, LAX1 and LAX2 were significantly reduced by WM13-24 VCs (Fig. 9C).
Analysis of Bacillus subtilis WM13-24 VCs and their effects on plant growth promotion
Solid-phase microextraction and GC-MS were used to identify volatile compounds produced by strain WM13-24 alone or WM13-24 in the interaction with Arabidopsis seedlings. A total of 18 kinds of VCs, including alcohols, esters and others, were detected in the headspace of plates with strain WM13-24 compared with control (1/2 MS medium) or Arabidopsis seedling only (Table 1). Among these 18 kinds of VCs, acetoin, 2,3-butanediol and benzyl alcohol were detected in the plates with WM13-24 alone (Fig. S3) and acetoin and 2,3-butanediol in the plates with WM13-24 in the interaction with Arabidopsis seedlings (Fig. S4).
Table 1
VCs produced by strain WM13-24 as measured by SPME-GC-MS
No.
|
VCs
|
Retention time
(min)
|
Molecular formula
|
Match
|
1
|
nitroso-methane,
|
1.381
|
CH3NO
|
875
|
2
|
Ethyl acetate
|
2.166
|
C4H8O2
|
882
|
3
|
Acetic acid ethenyl ester
|
2.243
|
C4H6O2
|
923
|
4
|
1-butanol
|
3.516
|
C4H10O
|
859
|
5
|
Acetic acid
|
4.206
|
C2H4O2
|
931
|
6
|
α-methyl-benzene ethanol
|
4.537
|
C9H12O
|
778
|
7
|
Acetoin
|
6.456
|
C4H8O2
|
889
|
8
|
[S-(R*, R*)]-2, 3-butanediol
|
10.160
|
C4H10O2
|
939
|
9
|
1-ethyl-2-methyl-benzene
|
11.956
|
C9H12
|
802
|
10
|
2-ethyl-butanoic acid
|
12.204
|
C5H10O2
|
837
|
11
|
α-Methylstyrene
|
12.398
|
C9H10
|
920
|
12
|
1-methyl-4-propyl-benzene
|
12.962
|
C10H14
|
890
|
13
|
Formic acid, heptyl ester
|
14.272
|
C8H16O2
|
872
|
14
|
1-octanol
|
18.915
|
C8H18O
|
922
|
15
|
Benzyl alcohol
|
20.054
|
C7H8O
|
935
|
16
|
Phenol
|
20.778
|
C6H6O
|
886
|
17
|
4-(1, 1-dimethylpropyl)-cyclohexanone
|
22.187
|
C11H20O
|
714
|
18
|
1-nonanol
|
23.415
|
C9H20O
|
946
|
2,3-butanediol has been reported to promote plant growth (Perez-Flores et al. 2017; Ryu et al. 2003; Li et al. 2021). Therefore, the effect of other compounds (α-methyl-benzeneethanol, 1-nonanol, 4-(1,1-dimethylpropyl)-cyclohexanone and 1-butanol), benzyl alcohol and mixtures of benzyl alcohol and 2, 3-butanediol on plant growth were examined in this study. To determine the growth promotion effects of benzyl alcohol, the compound at various concentrations were applied to Arabidopsis (petri dishes), respectively. The growth promotion effect of benzyl alcohol is dose-dependent (Fig. 10A). Benzyl alcohol at 50 and 100 µM significantly increased the total fresh weight of Arabidopsis seedlings by 21% and 24%, respectively, however, significantly reduced the total fresh weight by 35% and 59%, respectively, at 750 and 5000 µM (Fig. 10B). Benzyl alcohol at 50, 100, 200 and 500 µM significantly increased LR number by 43%, 68%, 52% and 22%, respectively (Fig. 10C). Benzyl alcohol at all concentrations tested significantly increased LR density (Fig. 10D).
Above result indicated that benzyl alcohol at 50 and 100 µM had the best growth promotion effect. 50 or 100 µM of benzyl alcohol mixed with 2,3-butanediol at 50, 100 or 200 µM were applied to Arabidopsis, respectively (Fig. 11A). Mixture of benzyl alcohol and 2,3-butanediol at 50 + 50, 50 + 100, 50 + 200, 100 + 50, 100 + 100 and 100 + 200 µM significantly increased LR number by 22%, 31%, 29%, 44%, 50% and 50% (Fig. 11C), and lateral root density by 25%, 44%, 23%, 23%, 49% and 51%, respectively (P < 0.05) (Fig. 11D). Moreover, other compounds such as α-methyl-benzeneethanol, 1-nonanol and 4-(1,1-dimethylpropyl)-cyclohexanone, detected in strain WM13-24 VCs cannot induce LR formation tested by those pure compounds (Fig. S6, S7). 1-butanol at 100 µM increased the total fresh weight and lateral root density of Arabidopsis seedlings (Fig. S5).
Benzyl alcohol, 2, 3-butanediol and their mixture were also applied to Bacillus subtilis WM13-24’s host plant H. ammodendron to determine their growth promotion effects. Benzyl alcohol at 100 µM, 2,3-butanediol at 500 µM and their mixture at 100 + 500 µM significantly increased the total fresh weight by 13%, 9% and 7%, and total dry weight by 20%, 15% and 10%, respectively (P < 0.05) (Fig. S8).