Abiotic stress induces change in Cinnamoyl CoA Reductase (CCR) protein abundance and lignin deposition in developing seedlings of Leucaena leucocephala

Abstract

Aboitic stress such as drought and salinity are class of major threats, which plants undergo through their lifetime. Lignin deposition is one of the responses to such abiotic stresses. The gene encoding Cinnamoyl CoA Reductase (CCR) is a key gene for lignin biosynthesis, which has been shown to be over-expressed under stress conditions. In the present study, developing seedlings of Leucaena leucocephala (Vernacular name: Subabul, White popinac) were treated with 1 % mannitol and 200 mM NaCl to mimic drought and salinity stress conditions, respectively. Enzyme linked immunosorbant assay (ELISA) based expression pattern of CCR protein was monitored coupled with Phlorogucinol/HCl activity staining of lignin in transverse sections of developing L. leucocephala seedlings under stress. Our result suggests a differential lignification pattern in developing root and stem under stress conditions. Increase in lignification was observed in mannitol treated stems and corresponding CCR protein accumulation was also higher than control and salt stress treated samples. On the contrary CCR protein was lower in NaCl treated stems and corresponding lignin deposition was also low. Developing root tissue showed a high level of CCR content and lignin deposition than stem samples under all conditions tested. Overall result suggested that lignin accumulation was not affected much in case of developing root however developing stems were significantly affected under drought and salinity stress condition.

Keywords: Abiotic stress; Cinnamoyl CoA reductase; Developing seedlings; Leucaena leucocephala.

Fig. 1

ELISA profile of CCR protein Expression: a Stem b Root. The CCR protein was quantified in normal as well as stress treated samples using recombinant CCR protein standard graph. Two factor ANOVA was performed. (p value for Fig. 1a: 0.380158; Fig. 1b: 0. 0.672243)

Fig. 2

Immuno-cytolocalization of CCR protein in 12d stem and root tissues under normal as well as stressed conditions. a Control 12 day stem samples, b Control 12 day root samples, c Manitol (1 %) treated 12 days stem sample, d Manitol (1 %) treated 12 days root sample. e NaCl (200 mM) treated 12 days stem sample, f: NaCl (200 mM) treated 12 days root sample. All pictures were taken at 40× magnification

Fig. 3

Lignin staining (Phluoroglucinol) of developing stem under normal as well as stressed conditions. (a, b, c). Control 8, 12 and 15 days samples, respectively. (d, e, f). Mannitol (1 %) treated 8, 12 and 15 days samples, respectively. (f, g, h). NaCl (200 mM) treated 8, 12 and 15 days samples, respectively. All pictures were taken at 40×magnification

Fig. 4

Lignin staining (Phluoroglucinol) of developing root under normal as well as stressed conditions. (a, b, c). Control 8, 12 and 15 days samples, respectively. (d, e, f). Mannitol (1 %) treated 8, 12 and 15 days samples, respectively. (f, g, h). NaCl (200 mM) treated 8, 12 and 15 days samples, respectively. All pictures were taken at 40× magnification except Fig. 4d and g which were taken at 100× magnification

Fig. 5

Upper panel: FTIR analysis of normal 10d old and 15 d old stem samples. Lower panel: FTIR analysis of 10 d and 15 d old normal root samples