Identification of Genes Involved in Indole-3-Acetic Acid Biosynthesis by Gluconacetobacter diazotrophicus PAL5 Strain Using Transposon Mutagenesis

Abstract

Gluconacetobacter diazotrophicus is a beneficial nitrogen-fixing endophyte found in association with sugarcane plants and other important crops. Beneficial effects of G. diazotrophicus on sugarcane growth and productivity have been attributed to biological nitrogen fixation process and production of phytohormones especially indole-3-acetic acid (IAA); however, information about the biosynthesis and function of IAA in G. diazotrophicus is still scarce. Therefore, the aim of this work was to identify genes and pathways involved in IAA biosynthesis in this bacterium. In our study, the screening of two independent Tn5 mutant libraries of PAL5T strain using the Salkowski colorimetric assay revealed two mutants (Gdiaa34 and Gdiaa01), which exhibited 95% less indolic compounds than the parental strain when grown in LGIP medium supplemented with L-tryptophan. HPLC chromatograms of the wild-type strain revealed the presence of IAA and of the biosynthetic intermediates indole-3-pyruvic acid (IPyA) and indole-3-lactate (ILA). In contrast, the HPLC profiles of both mutants showed no IAA but only a large peak of non-metabolized tryptophan and low levels of IPyA and ILA were detected. Molecular characterization revealed that Gdiaa01 and Gdiaa34 mutants had unique Tn5 insertions at different sites within the GDI2456 open read frame, which is predicted to encode a L-amino acid oxidase (LAAO). GDI2456 (lao gene) forms a cluster with GDI2455 and GDI2454 ORFs, which are predicted to encode a cytochrome C and an RidA protein, respectively. RT-qPCR showed that transcript levels of lao. cccA, and ridA genes were reduced in the Gdiaa01 as compared to PAL5T. In addition, rice plants inoculated with Gdiaa01 showed significantly smaller root development (length, surface area, number of forks and tips) than those plants inoculated with PAL5T. In conclusion, our study demonstrated that G. diazotrophicus PAL5T produces IAA via the IPyA pathway in cultures supplemented with tryptophan and provides evidence for the involvement of an L-amino acid oxidase gene cluster in the biosynthesis of IAA. Furthermore, we showed that the mutant strains with reduction in IAA biosynthesis ability, in consequence of the lower transcription levels of genes of the lao cluster, had remarkable effects on development of rice roots.

Daucus carota L. Seed Inoculation with a Consortium of Bacteria Improves Plant Growth, Soil Fertility Status and Microbial Community

“…Pedraza [54] also found that in G. diazotrophicus, IAM is not always active. Also, the recent work by Rodrigues et al [55] demonstrated that G. diazotrophicus cultures supplemented with Trp produce IAA via the indole-3-pyruvic acid (IPyA) pathway (Trp → IPyA → indoleacetaldehyde (IAAld) → IAA); in this study, the involvement of an L-amino acid oxidase gene cluster in the biosynthesis of IAA was also shown. In H. seropedicae the difficult discrimination is similar to the case of Arthrobacter spp.…”

Humic Substances in Combination With Plant Growth-Promoting Bacteria as an Alternative for Sustainable Agriculture

“…One of the main impacts of PGPB and HSs on plant growth is the improvement in the acquisition of nutrients and stimulation of the root system. Frequently, these effects are related to auxin or the "auxin-like effect" (Canellas et al, 2002;Zandonadi et al, 2007;Spaepen et al, 2014;Rodrigues et al, 2016). It has been proposed that the role of auxin in plant growth is related to the induction of (PM) H+-ATPase activity, which generates apoplastic acidification and thus provides loosening of the cell wall, allowing cell elongation (Hager et al, 1991;Frías et al, 1996).…”

Auxin production in the green alga Chlamydomonas involves an extracellular L-amino acid oxidase and supports algal-bacterial mutualism with methylobacteria

“…We provide genetic evidence that supports IAA production by the model alga Chlamydomonas via an unexpected LAAO-mediated pathway, which has thus far been restricted to a single bacterial species (Rodrigues et al, 2016). This finding supports a new role for microbial LAAOs in auxin production that may be widespread in other algal lineages (Calatrava et al, 2019; Labeeuw et al, 2016).…”

“…For these new applications, understanding how and under what environmental conditions IAA is produced by rhizospheric and phyllospheric algae is of great interest. Bacterial degradation of IAA has been shown to be essential for rhizosphere colonization and plant growth promotion (Zúñiga et al, 2013), and similar to how LAAO-mediated auxin production is involved in plant growth promotion by G. diazotrophicus (Rodrigues et al, 2016), auxin production by other LAAO-containing microbes in the rhizosphere like Chlamydomonas could have an impact on plant growth and fitness. Our findings suggest that future studies should carefully consider the role that terrestrial algae play in plant microbiomes (Lee and Ryu, 2021) and in the historical record of land plant evolution (Puginier et al, 2022).…”