Abstract
Nitrogen-fixing heterocystous cyanobacteria are used as biofertilizer inoculants for stimulating plant growth but can also alleviate plant stress by exometabolite secretion. However, only a small number of studies have focused on elucidating the identity of said bioactives because of the wide array of exuded compounds. Here, we used the root hair assay (RHA) as a rapid programmed cell death (PCD) screening tool for characterizing the bioactivity of cyanobacteria Nostoc muscorum conditioned medium (CM) on Arabidopsis thaliana root hair stress tolerance. We found that heat-stressed A. thaliana pre-treated with N. muscorum CM fractions exhibited significantly lower root hair PCD levels compared to untreated seedlings. Treatment with CM increased stress tolerance by suppressing PCD in root hairs but not necrosis, indicating the bioactive compound was specifically modulating the PCD pathway and not a general stress response. Based on documented N. muscorum exometabolites, we identified the stress-responsive proline as a compound of interest and strong evidence from the ninhydrin assay and HPLC indicate that proline is present in N. muscorum CM. To establish whether proline was capable of suppressing PCD, we conducted proline supplementation experiments. Our results showed that exogenous proline had a similar effect on root hairs as N. muscorum CM treatment, with comparable PCD suppression levels and insignificant necrosis changes. To verify proline as one of the biologically active compounds in N. muscorum CM, we used three mutant A. thaliana lines with proline transporter mutations (lht1, aap1 and atprot1-1::atprot2-3::atprot3-2). Compared with the wild-type seedlings, PCD-suppression in lht1and aap1 mutants was significantly reduced when supplied with low proline (1–5 μM) levels. Similarly, pre-treatment with N. muscorum CM resulted in elevated PCD levels in all three mutant lines compared to wild-type seedlings. Our results show that plant uptake of cyanobacteria-derived proline alters their root hair PCD sensitivity threshold. This offers evidence of a novel biofertilizer mechanism for reducing stress-induced PCD levels, independent of the existing mechanisms documented in the literature.
Highlights
Cyanobacteria are adaptable organisms found in aquatic and terrestrial ecosystems
N. muscorum was cultured in a closed batch system and the harvested conditioned medium (CM) diluted with fresh BG11 to generate a concentration range (20–100%) to determine the optimum CM% for the strongest programmed cell death (PCD)-suppressing effect at the 50◦C inflection point
By using the root hair assay (RHA) to characterize N. muscorum CM bioactivity, we found that a major bioactive compound was thermostable and directly affecting PCD levels but not necrosis
Summary
Cyanobacteria are adaptable organisms found in aquatic and terrestrial ecosystems. Their ability to inhabit most environments is attributed to the diverse range of exuded metabolites, termed exometabolites that can have antiviral, antibacterial, antifungal, antitumoral and anti-inflammatory properties (Singh et al, 2005; Jaiswal et al, 2008; Prasanna et al, 2010). In agriculture, nitrogenfixing heterocystous cyanobacteria are often used as biofertiliser inoculants to stimulate plant growth. Biofertiliser inoculants contain little macro- and micronutrients as they are catalysts for mobilizing nutrients into metabolically accessible forms that are otherwise unavailable to plants (Kennedy, 2008). Direct benefits make essential macronutrients available for plant growth via nitrogen fixation and phosphate solubilization, while indirect benefits rely on assorted mechanisms to safeguard against abiotic and biotic stresses (Barreto et al, 2011)
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