A Cell Wall Extract from Piriformospora indica ...

Appl Biochem Biotechnol DOI 10.1007/s12010-013-0231-1

A Cell Wall Extract from Piriformospora indica Promotes Tuberization in Potato (Solanum tuberosum L.) Via Enhanced Expression of Ca+2 Signaling Pathway and Lipoxygenase Gene Chandrama Prakash Upadhyaya & Mayank Anand Gururani & Ram Prasad & Ajit Verma

Received: 20 December 2012 / Accepted: 7 April 2013 # Springer Science+Business Media New York 2013

Abstract Piriformospora indica is an axenically cultivable phytopromotional endosymbiont that mimics capabilities of arbuscular mycorrhizal fungi. This is a basidiomycete of the Sebacinaceae family, which promotes growth, development, and seed production in a variety of plant species. We report that the cell wall extract (CWE) from P. indica induces tuberization in vitro and promotes tuber growth and yield in potato. The CWE altered the calcium signaling pathway that regulates tuberization process. An increase in tuber number and size was correlated with increased transcript expression of the two Ca 2+-dependant proteins (CaM1 and St-CDPK1) and the lipoxygenase (LOX) mRNA, which are known to play distinct roles in potato tuberization. External supplementation of Ca 2+ ions induced a similar set of tuberization pathway genes, indicating presence of an active Ca2+ in the CWE of P. indica. Since potato tuberization is directly influenced by the presence of microflora in nature, the present study provides an insight into the novel mechanism of potato tuberization in relation to plant–microbe association. Ours is the first report on an in vitro tuber-inducing beneficial fungus. Keywords Calcium signaling pathway . Lipoxygenase . Piriformospora indica . Solanum tuberosum . Tuberization

C. P. Upadhyaya (*) Department of Botany, Guru Ghasidas Central University, Bilaspur 495009, India e-mail: [email protected] M. A. Gururani Subtropical Horticulture Research Institute, Faculty of Biotechnology, Jeju National University, Jeju 690756, South Korea R. Prasad : A. Verma Amity Institute of Microbial Technology, Amity University, Noida 201303, India

Appl Biochem Biotechnol

Abbreviations CaM1 Calmodulin CWE Cell wall extract GA Gibberellic acid LOX Lipoxygenase StCDPK1 Solanum tuberosum calcium-dependent protein kinase

Introduction Tuberization in potato (Solanum tuberosum L.) is a complex event consisting of a series of biochemical and morphological processes at the stolon tip [1–5]. Several intrinsic and extrinsic factors such as temperature [6], light, hormones, photoperiod [7, 8], N nutrition [9, 10], Ca 2+ [11], and soil microorganisms [12, 13] were reported to influence tuberization in potato. Several genes specifically expressed during early tuber formation have been identified including the tubulins [14], Sadenosylmethionine decarboxylase [14], MADS box genes [15], acyl carrier protein thioesterase [16], and lipoxygenases (LOXs) [1]. Potato StCDPK1 encoding a calcium-dependent protein kinase (CDPK) is reported to be induced upon tuberization at stolon tips [2]. An important aspect in plant survival is the early detection of and rapid response to specific stimuli. Calcium (Ca2+) signaling, which can be activated within seconds or minutes in response to quite diverse sets of stimuli [17–19], is an important part of the early signaling system including tuberization. Ca2+ plays an important role in physiology and many plant cell signaling pathways involving a transient increase in its cytosolic levels [20, 21]. An increase in free cytosolic Ca2+ concentration is one of the primary events in the transduction of many signals that can alter various biochemical processes in plants by activating particular enzymes [22]. The importance of the intracellular Ca2+ in tuber induction was also reported in potato [11]. Tuberization was inhibited in single leaf cuttings by the Ca2+ chelator (EGTA) and a Ca2+ ionophore (A 23287) but was restored by the addition of CaCl2 to the medium. It has been suggested that Ca 2+ and calmodulin are somehow involved in the tuberization process through gibberellic acid (GA) metabolism [23]. Similarly, studies on barley endosperm during seed germination indicated an evidence for the modulation of GA by Ca/calmodulin pathway [24]. Piriformospora indica is an endophytic fungus of the Sebacinaceae family that colonizes roots of several plants and promotes their growth, development, and seed production. It stimulates nutrient uptake and confers resistance to various biotic and abiotic stresses [25–31]. This cultivable fungus can grow on synthetic or complex media without a host [26, 28]. Since P. indica can colonize the roots of many plant species including trees, agricultural, horticultural, and medicinal plants; monocots and dicots; and even mosses [26, 29, 30, 32–36], the interaction between the symbiotic partners is thought to be based on general recognition and signaling processes. We report here the isolation of a fraction from the cell wall of P. indica, which could mimic presence of the fungus in the initial stages and induce tuberization in potato. The fraction was able to induce an elevation of [Ca+2]cyt in stoloniferous shoots similar to externally supplemented Ca+2 in the tuber-inducing medium that induced the Ca+2-dependent gene expression.


Materials and Methods Culture Conditions and Preparation of the Cell Wall Extract from P. indica P. indica was cultured as described previously [28, 29, 37] on Aspergillus-minimal medium. For solid medium, 1 % (w/v) agar was used. The cell wall extract (CWE) was prepared using the protocol of Anderson-Prouty and Albersheim [38], with minor modifications as reported by Vadassery et al. [37]. Mycelia from 14-day-old liquid cultures were homogenized using mortar and pestle in 10 ml water (10 g ml−1 mycelia). The homogenate was filtered using a coarse sintered glass funnel. The residue was washed three to five times with water, once with chloroform/methanol (1:1), and, finally, in acetone. This preparation was air dried for 2 h and the mycelial cell wall material was recovered. Elicitor fractions were prepared from the mycelial cell wall by suspending 10 g of cell wall in 50 ml water and autoclaving for 20 min at 121 °C. Autoclaving releases the active fraction of the cell wall. The suspension was centrifuged at 20,000×g for 15 min, filter-sterilized using a 0.22-μM filter, and used for further assay. In Vitro Tuberization with Supplemented Cell Wall Extract of P. indica and Ca2+ and/or Calcium Chelators/Antagonists S. tuberosum cv. Desiree was raised and multiplied in vitro through single-node cutting (SNCs) on MS [39] medium containing sucrose (3 %, w/v) and 0.8 % phyto agar. SNCs from 30-dayold plants were used as explants (Fig. 1). These explants were cultured in magenta boxes (120 mm diameter×200 mm height) containing 50 ml of tuber induction medium [MS+sucrose (7 %, w/v) and 0.8 % phyto agar] supplemented with the CWE (2.5 ml and 5.0 ml) of P. indica. The MS medium devoid of Ca2+ and CWE was used as a control. Cultures were incubated at 20 °C temperature with 65 % relative humidity under continuous dark. Each treatment contained a total of 25 explants, and the experiment was repeated thrice. SNCs were also cultured on tuber-inducing medium [MS with sucrose (7 % w/v) and 0.8 % phyto agar] supplemented with Ca2+ in the form of CaCl2 (3, 6, and 9 mM). In the initial screening, it was found that MS medium supplemented with 6 mM CaCl2 and 7 % sucrose proved better for obtaining the maximum number of the tubers. MS medium devoid of Ca2+ served as control. Cultures were incubated at 20 °C temperature and 65 % relative humidity under continuous dark. Nodal segments were inoculated on tuber-inducing medium [MS+sucrose (7 %, w/v) and 0.8 % phyto agar] supplemented with CaCl2 or CWE and the Ca2+ chelator, 1-2-bis(o-aminophenoxy) ethane-N,N,N,N′-tetraacetic acid (BAPTA; Sigma-Aldrich, St. Louis, MO, USA; dissolved in water) or LaCl3 (inhibitor of Ca+2 import

Fig. 1 Influence of P. indica cell wall extract (CWE) on in vitro tuberization in Potato cv. Desiree nodal segments used for in vitro tuberization studies. Early tuber formations through the subapical swelling of the nodal region in the treated explants were observed. C control, T treated


across membrane). Observations on tuber induction, growth, and yield were taken after 30 days’ culture. The treatment contained a total of 75 explants, and the experiment was repeated thrice. All the experiments were independently carried out at least three times and each time, with at least three replicates for all measurements. Data were analyzed with Origin 8.1 data analysis and graphing software program for calculating the statistical differences. Standard error was calculated using the n values of each experiment. Expression Analysis of Calcium Signaling Pathway Genes Affecting Tuberization The tubers were developed on the tuber induction medium treated with P. indica CWE, CaCl2, calcium chelators, and antagonists. Total RNA was isolated from three independent replicates of initial tubers (