Cyanobacteria-Derived Proline Increases Stress Tolerance in Arabidopsis thaliana Root Hairs by Suppressing Programmed Cell Death.

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.

Sorting the Wheat From the Chaff: Programmed Cell Death as a Marker of Stress Tolerance in Agriculturally Important Cereals.

Conventional methods for screening for stress-tolerant cereal varieties rely on expensive, labour-intensive field testing and molecular biology techniques. Here, we use the root hair assay (RHA) as a rapid screening tool to identify stress-tolerant varieties at the early seedling stage. Wheat and barley seedlings had stress applied, and the response quantified in terms of programmed cell death (PCD), viability and necrosis. Heat shock experiments of seven barley varieties showed that winter and spring barley varieties could be partitioned into their two distinct seasonal groups based on their PCD susceptibility, allowing quick data-driven evaluation of their thermotolerance at an early seedling stage. In addition, evaluating the response of eight wheat varieties to heat and salt stress allowed identification of their PCD inflection points (35°C and 150 mM NaCl), where the largest differences in PCD levels arise. Using the PCD inflection points as a reference, we compared different stress effects and found that heat-susceptible wheat varieties displayed similar vulnerabilities to salt stress. Stress-induced PCD levels also facilitated the assessment of the basal, induced and cross-stress tolerance of wheat varieties using single, combined and multiple individual stress exposures by applying concurrent heat and salt stress in a time-course experiment. Two stress-susceptible varieties were found to have low constitutive resistance as illustrated by their high PCD levels in response to single and combined stress exposure. However, both varieties had a fast, adaptive response as PCD levels declined at the other time-points, showing that even with low constitutive resistance, the initial stress cue primes cross-stress tolerance adaptations for enhanced resistance even to a second, different stress type. Here, we demonstrate the RHA's suitability for high-throughput analysis (∼4 days from germination to data collection) of multiple cereal varieties and stress treatments. We also showed the versatility of using stress-induced PCD levels to investigate the role of constitutive and adaptive resistance by exploring the temporal progression of cross-stress tolerance. Our results show that by identifying suboptimal PCD levels in vivo in a laboratory setting, we can preliminarily identify stress-susceptible cereal varieties and this information can guide further, more efficiently targeted, field-scale experimental testing.