Mycorrhizal fungus Serendipita indica-associated acid phosphatase rescues the phosphate nutrition with reduced arsenic uptake in the host plant under arsenic stress.

Symbiotic interactions play a vital role in maintaining the phosphate (Pi) nutrient status of host plants and providing resilience during biotic and abiotic stresses. Serendipita indica, a mycorrhiza-like fungus, supports plant growth by transporting Pi to the plant. Despite the competitive behaviour of arsenate (AsV) with Pi, the association with S. indica promotes plant growth under arsenic (As) stress by reducing As bioavailability through adsorption, accumulation, and precipitation within the fungus. However, the capacity of S. indica to enhance Pi accumulation and utilization under As stress remains unexplored. Axenic studies revealed that As supply significantly reduces intracellular ACPase activity in S. indica, while extracellular ACPase remains unaffected. Further investigations using Native PAGE and gene expression studies confirmed that intracellular ACPase (isoform2) is sensitive to As, whereas extracellular ACPase (isoform1) is As-insensitive. Biochemical analysis showed that ACPase (isoform1) has a Km of 0.5977 µM and Vmax of 0.1945 Unit/min. In hydroponically cultured tomato seedlings, simultaneous inoculation of S. indica with As on the 14thday after seed germination led to hyper-colonization, increased root/shoot length, biomass, and induction of ACPase expression and secretion under As stress. Arsenic-treated S. indica colonized groups (13.33 µM As+Si and 26.67 µM As+Si) exhibited 8.28-19.14 and 1.71-3.45-fold activation of ACPase in both rhizospheric media and root samples, respectively, thereby enhancing Pi availability in the surrounding medium under As stress. Moreover, S. indica (13.33 µM As+Si and 26.67 µM As+Si) significantly improved Pi accumulation in roots by 7.26 and 9.46 times and in shoots by 4.36 and 8.85 times compared to the control. Additionally, S. indica induced the expression of SiPT under As stress, further improving Pi mobilization. Notably, fungal colonization also restricted As mobilization from the hydroponic medium to the shoot, with a higher amount of As (191.01 ppm As in the 26.67 µM As+Si group) accumulating in the plant's roots. The study demonstrates the performance of S. indica under As stress in enhancing Pi mobilization while limiting As uptake in the host plant. These findings provide the first evidence of the As-Pi interaction in the AM-like fungus S. indica, indicating reduced As uptake and regulation of PHO genes (ACPase and SiPT genes) to increase Pi acquisition. These data also lay the foundation for the rational use of S. indica in agricultural practices.

Impact of arsenic on phosphate solubilization, acquisition and poly-phosphate accumulation in endophytic fungus Serendipita indica.

Symbiotic interactions play a crucial role in the phosphate (Pi) nutrient status of the host plant and offer resilience during biotic and abiotic stresses. Despite a competitive behavior of arsenic (AsV) with Pi, Serendipita indica association promotes plant growth by reducing arsenic bioavailability in the rhizosphere. Reduced arsenic availability is due to the adsorption, accumulation, and precipitation of arsenic in the fungus. The present investigation focused on the fitness and performance of Pi acquisition and utilization in S. indica for growth and metabolism under arsenic stress. The fungus accumulates a massive amount of arsenic up to 2459.3 ppm at a tolerable limit of arsenic supply (1 mM) with a bioaccumulation factor (BAF) 32. Arsenic induces Pi transporter expression to stimulate the arsenic acquisition in the fungus. At the same time, Pi accumulation was also enhanced by 112.2 times higher than the control with an increase in poly-P (polyphosphate) content (6.69 times) of the cell. This result suggests arsenic does not hamper poly-P storage in the cell but shows a marked delocalization of stored poly-P from the vacuoles. Furthermore, an enhanced exopolyphosphatase activity and poly-P storage during arsenic stress suggest induction of cellular machinery for the utilization of Pi is required to deal with arsenic toxicity and competition. However, at high arsenic supply (2.5 and 5 mM), 14.55 and 22.07 times reduced Pi utilization, respectively, was observed during the Pi uptake by the fungus. The reduction of Pi uptake reduces the cell growth and biomass due to competition between arsenic and phosphate. The study suggests no negative impact of arsenic on the Pi acquisition, storage, and metabolism in symbiotic fungus, S. indica, under environmental arsenic contamination.

An effective in-gel assay protocol for the assessment of acid phosphatase (ACPase) isoform expression in the fungus Serendipita indica.

The induction of acid phosphatase (ACPase) in the mycorrhizal fungi is an adaptive survival mechanism to cope in a low-phosphate environment. A mycorrhizal fungi Serendipita indica can induce the ACPase enzyme and enhance the phosphate (Pi) level to the host plant through Pi-solubilization mechanism, both intracellular and extracellular (media) levels. The spectrophotometer technique has been widely and commonly used to measure the ACPase enzyme activity in all microorganisms and plants using pNPP as a substrate. However, this technique cannot be useful when studying the involvement of ACPase isoforms in Pi-solubilization. In this article, we developed a single method to identify and express the ACPase isoforms of S. indica that contribute to the Pi-nutrition in the plant. This is native-PAGE electrophoresis with the in-gel assay and staining to detect the isoforms of the ACPase enzyme. The dark red-brown color developed after staining indicates the non-denatured (native) ACPase enzyme. This method utilized a modified minimal media for the de-repression of P-responsive genes such as ACPases with minimum salt contamination in the samples. This method will be helpful for the characterization of secretory and intracellular ACPases in fungi.

Fungal mediated biotransformation reduces toxicity of arsenic to soil dwelling microorganism and plant.

Rhizospheric and plant root associated microbes generally play a protective role against arsenic toxicity in rhizosphere. Rhizospheric microbial interaction influences arsenic (As) detoxification/mobilization into crop plants and its level of toxicity and burden. In the present investigation, we have reported a rhizospheric fungi Aspergillus flavus from an As contaminated rice field, which has capability to grow at high As concentration and convert soluble As into As particles. These As particles showed a reduced toxicity to soil dwelling bacteria, fungi, plant and slime mold. It does not disrupt membrane potential, inner/outer membrane integrity and survival of the free N2 fixating bacteria. In arbuscular mycorrhiza like endophytic fungi Piriformospora indica, these As particles does not influence mycelial growth and plant beneficial parameters such as phosphate solubilizing enzyme rAPase secretion and plant root colonization. Similarly, it does not affect plant growth and chlorophyll content negatively in rice plant. However, these As particles showed a poor absorption and mobilization in plant. These As particle also does not affect attachment process and survival of amoeboid cells in slime mold, Dictyostelium discoideum. This study suggests that the process of conversion of physical and chemical properties of arsenic during transformation, decides the toxicity of arsenic particles in the rhizospheric environment. This phenomenon is of environmental significance, not only in reducing arsenic toxicity but also in the survival of healthy living organism in arsenic-contaminated rhizospheric environment.