In the current study, we elucidated the mechanism through which B. aryabhattai promotes the growth of Arabidopsis and N. tabacum plants. N. tabacum plants were included to evaluate their influence on other plant species. The bacterium increases the plant size and fresh and dry weights. Notably, although this endophytic bacterium was isolated from wild plant species, it exerts a robust impact on the growth of nonhost plants. The findings indicate that B. aryabhattai can stimulate growth in a range of plant species. However, these effects should be studied in a natural context and with other crop species. Regardless of the context, based on the effect of B. aryabhattai on plants, this bacterium could be used as a biofertilizer to boost growth and agricultural production.
The beneficial effect of different Bacillus species on various plant species was previously established [11, 13, 15]. Bacillus species are the most common type of growth-promoting bacterium, and these bacterial usually promote growth via growth-responsive genes, proteins, phytohormones, and metabolites . Additionally, Bacillus is reportedly effective in increasing the biomass and height of important crops through increases in plant N uptake, phosphate solubilization and root-promoting phytohormones [36-38]. Additionally, most of these effects are associated with the production of phytohormones such as indole-3-acetic acid (IAA), cytokinins, gibberellic acid (GA), and spermidines [15, 19, 39]. Furthermore, the induction of endogenous proteins, amino acids, and minerals by Bacillus species could promote plant growth [15, 40].
Beneficial plant-bacteria interactions have been extensively analyzed. However, the specific molecular pathways that are associated with these interactions are unclear. This knowledge is critical for maximizing the potential of these microbes in the field. In the current study, we done RNA sequencing to analyze the genes expressed during the Arabidopsis-B. aryabhattai interaction. We conducted an analysis of the RNA-seq dataset and found various types of genes that might be ideal candidates for characterizing the host pathway governing this essential plant-microbe relationship. A high number of novel genes involved in metabolite biosynthesis were differentially expressed in our dataset. The results reveal new insights into plant and bacterial gene expression and improve our understanding of the molecular events involved in the Arabidopsis-B. aryabhattai interaction. Notably, GO and KEGG analyses revealed significant changes between treated and nontreated plants. Our data indicate that B. aryabhattai triggers important molecular pathways related to plant growth.
Interestingly, the KEGG pathway analysis revealed that the most significant transcripts were involved in the indole alkaloid biosynthesis and linoleic acid metabolism pathways. These pathways have been related to resistance and tolerance to biotic and abiotic factors [41, 42]. Most likely, the bacterium can induce plant protection against pathogens and abiotic factors through the production of jasmonic acid and secondary metabolites associated with the defense responses of plants. The synthesis of indole-alkaloids in plants causes consistent alterations of the microbiota surviving at the barley root-soil interface while having no discernible detrimental influence on plant growth performance in two elite barley types . These results indicate that the application of indole-alkaloids modulates the proliferation of a subset of soil microbes with relatively broad phylogenetic assignments .
In contrast, bacteria can undergo symbiotic or pathogenic interactions with plants. Membrane lipids and lipid-derived molecules from the plant or microbial organism play important roles during the infection process . For example, lipids are involved in establishing the membrane interface between the two organisms . Furthermore, lipid-derived molecules are crucial for intracellular signaling in plant cells, and lipids serve as signals during plant-microbe communication . Linolenic acid is released from several complex fatty acids mainly located in the membranes of organelles such as chloroplasts and is a precursor of jasmonic acid [45, 46]. The involvement of this hormone in different biological processes of plants, such as responses to biotic and abiotic stress conditions, indicates the possible use of this bacterium to activate plant defense.
Curiously, the expression of the genes encoding cinnamyl alcohol dehydrogenase, apyrase, thioredoxin H8, benzaldehyde dehydrogenase, indoleacetaldoxime dehydratase, berberine bridge enzyme-like, gibberellin-regulated protein, maturase K, tetratricopeptide repeat (TPR)-like superfamily protein, BTB/POZ and TAZ domain-containing protein and auxin-responsive GH3 family protein genes was highly induced during the Arabidopsis-B. aryabhattai interaction. Some identified genes from our dataset are addressed here, as well as their roles in other plant-microbe relationships. B. aryabhattai most likely promotes the synthesis of lignin during the growing phase of Arabidopsis and N. tabacum plants, which results in the robust phenotype observed in the plants treated with the bacterium. Cinnamyl alcohol dehydrogenase is a key enzyme involved in lignin synthesis and is closely related to plant growth and development. This enzyme is expressed in the lateral roots and root tips of sweet potato, and its activity is induced by abscisic acid .
Similarly, apyrases play a crucial role in regulating the growth of Arabidopsis plants treated with the bacterium. Specifically, apyrases influence auxin transport and stomatal aperture, and the removal of apyrase activity can lead to growth inhibition . A previous study revealed that silencing of the apyrase gene induces significant phenotypic changes, growth retardation, an increase in the tuber number per plant, and an effect on the tuber morphology of potato plants [48. Moreover, the expression of apyrase genes in Arabidopsis plants exerts a marked effect on the growth of plant tissues and the accumulation of auxin [50, 51].
Interestingly, B. aryabhattai can indirectly induce plant growth through benzaldehyde dehydrogenase, which plays a key role in the benzoic acid pathway. In addition, benzaldehyde dehydrogenase is involved in the processing of benzaldehyde to benzoic acid. The growth, mineral composition, and chlorophyll content of soybean plants are influenced by benzoic acid . Herein, benzoic acid exerts a marked effect on the growth and yield of tomato plants. Similarly, benzoic acid exerts a positive effect on fruit yield .
We observed high induction of maturase K gene expression during the interaction. Recent studies revealed that the maturase K gene is induced during the Arabidopsis-Bacillus altitudinis interaction . In addition, the maturase K gene is highly expressed in Anoectochilus roxburghii plants treated with endophytic fungi . Most likely, this gene could be related to the Bacillus-plant association, but a functional analysis of Arabidopsis mutants could provide a clear understanding of the real function of this gene during Bacillus-plant interactions.
Furthermore, tetratricopeptide repeat (TPR)-like superfamily protein genes might have indirect effects on the activation of phytohormones related to plant growth. This gene was found to be expressed during the interaction and constitutes a basic component of gibberellin and ethylene responses. The silencing of an Arabidopsis chloroplast-localized tetratricopeptide repeat protein gene affects plant growth, leaf greening, chloroplasts, and photosynthesis genes .
We also predicted that hormone-related genes and transcription factors would be modulated during this interaction. The bacterium B. aryabhattai induced plant growth by triggering key molecular pathways involved in the production of phytohormones and transcription factors. Root development, shoot growth, and fruit ripening are regulated by Aux/IAA family genes . Auxin influences numerous stages of plant development and growth by regulating the expression of auxin-activated genes , and auxin controls plant development and growth by altering the expression of different genes . This finding could be directly correlated with the phenotype observed in the Arabidopsis plants treated with the bacteria.
Many of the processes that occur in a plant during interactions with endophytic bacteria are known. It is also important to understand the processes that take place in a bacterium during its interaction with a plant, such as the specific genes that are expressed in the bacterium that may contribute to the growth phenotype of treated plants. This point is more complex, as demonstrated by the low proportion of bacterial transcripts found during the interaction. A functional analysis of these types of genes would elucidate the actual role of these genes during the interaction. Interestingly, the expression of arginine decarboxylase, D-hydantoinase, ATP synthase gamma chain and 2-hydroxyhexa-2,4-dienoate hydratase genes was highly induced in B. aryabhattai during its interaction with the plant, and this finding constitutes the first line of evidence indicating that these types of genes are expressed in this species. We speculate that the overexpression of these genes in B. aryabhattai might enhance plant growth. Based on the same principles, we discuss some bacterial genes expressed during the interaction.
For example, the activity of arginine decarboxylase has been implicated in the effect of hormones on plant growth . The expression of this enzyme is correlated with cell growth and stress responses in apple plants . Additionally, this enzyme is involved in efficient ROS elimination and its influence on root growth, which is conducive to drought tolerance . Most of the effects of these genes could be indirectly implicated in enhancement of the plant physiological status and thus in a better growing environment.
Contradictorily, the expression of the D-hydantoinase gene was induced in the bacterium during its interaction with the plant. These D-amino acids (D-AAs) exert a growth-inhibiting effect on plants [63-65]. The exogenous administration of various D-AAs to growth media has a deleterious or favorable effect on the growth and development of many plant species, and these effects depend on the D-AAs used [66, 67]. However, the mechanism responsible for the finding that its expression is correlated with growth stimulation but not growth inhibition. Although some D-AAs impede seedling growth, some lines of evidence show that D-AAs can also promote plant growth. Recent studies found that the application of 0.1 mM d-Leu, d-Val, and d-Cys increases the growth of pepper plants . Additionally, Arabidopsis seedlings treated with d-Lys and d-Ile at a range of 1-10 mM exhibit superior growth. This ostensible inconsistency has been resolved through functional studies. Another conclusion is that D-AAs should not be considered a class of chemicals with uniform features but rather that each D-AA should be evaluated separately.
Among the identified group of plant genes and those showing the most differential expression, the zinc finger C-x8-C-x5-C-x3-H type family protein, ankyrin repeat/KH domain protein, CAPRICE-like MYB3, HSP20-like chaperone superfamily protein and gibberellin-regulated protein were downregulated during the interaction. Additionally, bacterial genes such as putative universal stress protein, L-lactate dehydrogenase, and succinate dehydrogenase flavoprotein subunit showed the strongest repression during the interaction.
For example, the zinc finger C-x8-C-x5-C-x3-H type family protein functions as a transcriptional activator and is involved in secondary wall biosynthesis. Additionally, some evidence shows that this type of protein is involved in ABA-, GA- and phytochrome-mediated seed germination responses . Furthermore, the ankyrin repeat protein family plays a crucial role in plant growth and development and in the responses to biotic and abiotic stresses [70, 71]. In addition, CAPRICE-like MYB3 encodes a small protein with an R3 MYB motif and promotes root hair cell differentiation in Arabidopsis plants. The overexpression of this protein results in the suppression of trichomes and the overproduction of root hairs and exerts pleiotropic effects on flowering development, epidermal cell size and trichome branching [72, 73]. Similarly, HSP20-like chaperone superfamily proteins mediate protein folding and are associated with abiotic stresses and death . Gibberellin-regulated proteins are important endogenous plant growth regulators involved in different physiological processes, and these types of proteins are sometimes up- or downregulated depending on the developmental stages of the plants . Recent studies have shown that orange gibberellin-regulated proteins are involved in allergy reactions .
Among the downregulated bacterial genes, putative universal stress proteins are important elements for survival under anaerobic conditions and during persistent colonization and infection with pathogenic bacteria . In addition, lactate dehydrogenase plays an important role in the anaerobic metabolic pathway and catalyzes the reversible conversion of lactate to pyruvate with the reduction of NAD+ to NADH [78, 79]. Interestingly, the succinate dehydrogenase flavoprotein subunit is involved in cellular energetics and is needed for the virulence of many important bacterial pathogens . Certainly, most of these genes could play an important role during the interaction, but functional analyses are needed to draw a conclusion.