Chua Alysha, Sherwood Orla L, Fitzhenry Laurence, Ng Carl K-Y, McCabe Paul F, Daly Cara T
Department of Science, Waterford Institute of Technology, Waterford, Ireland.
Pharmaceutical and Molecular Biotechnology Research Centre (PMBRC), Waterford Institute of Technology, Waterford, Ireland.
Front Plant Sci. 2020 Dec 14;11:490075. doi: 10.3389/fpls.2020.490075. eCollection 2020.
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 conditioned medium (CM) on root hair stress tolerance. We found that heat-stressed pre-treated with 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 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 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 CM treatment, with comparable PCD suppression levels and insignificant necrosis changes. To verify proline as one of the biologically active compounds in CM, we used three mutant . lines with proline transporter mutations (, and ). Compared with the wild-type seedlings, PCD-suppression in and mutants was significantly reduced when supplied with low proline (1-5 μM) levels. Similarly, pre-treatment with 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.
固氮异形胞蓝细菌被用作生物肥料接种剂以刺激植物生长，但也可通过分泌胞外代谢产物来缓解植物胁迫。然而，由于分泌的化合物种类繁多，只有少数研究专注于阐明这些生物活性物质的特性。在此，我们使用根毛测定法（RHA）作为一种快速的程序性细胞死亡（PCD）筛选工具，来表征蓝细菌条件培养基（CM）对根毛胁迫耐受性的生物活性。我们发现，与未处理的幼苗相比，用CM组分预处理的热胁迫幼苗的根毛PCD水平显著降低。用CM处理通过抑制根毛中的PCD而非坏死来提高胁迫耐受性，这表明生物活性化合物特异性地调节PCD途径而非一般的胁迫反应。基于已记录的胞外代谢产物，我们确定应激反应性脯氨酸是一种感兴趣的化合物，茚三酮测定和高效液相色谱的有力证据表明脯氨酸存在于CM中。为了确定脯氨酸是否能够抑制PCD，我们进行了脯氨酸补充实验。我们的结果表明，外源脯氨酸对根毛的作用与CM处理相似，具有相当的PCD抑制水平且坏死变化不显著。为了验证脯氨酸是CM中的生物活性化合物之一，我们使用了三个脯氨酸转运体突变体品系（、和）。与野生型幼苗相比，在低脯氨酸（1 - 5 μM）水平下供应时，和突变体中的PCD抑制作用显著降低。同样，与野生型幼苗相比，用CM预处理导致所有三个突变体品系中的PCD水平升高。我们的结果表明，植物对蓝细菌衍生脯氨酸的吸收改变了它们根毛PCD的敏感性阈值。这为一种新的生物肥料机制提供了证据，即降低胁迫诱导的PCD水平，这与文献中记录的现有机制无关。
