Characterization of four defense-related genes up-regulated in root nodules of Casuarina glauca

Abstract

Actinorhizal plants are capable of high rates of nitrogen fixation, due to their capacity to establish a rootnodule symbiosis with N 2 -fixing actinomycetes of the genus Frankia. Nodulation is an ontogenic process which requires a sequence of highly coordinated events. One of these mechanisms is the induction of defense-related events, whose precise role during nodulation is largely unknown. In order to contribute to the clarification of the involvement of defense-related genes during actinorhizal root-nodule symbiosis, we have analysed the differential expression of several genes with putative defense-related functions in Casuarina glauca nodules versus noninoculated roots. Four genes encoding a chitinase (CgChi1), a glutathione S-transferase (CgGst), a hairpin-inducible protein (CgHin1) and a peroxidase (CgPox4) were found to be up-regulated in mature nodules compared to roots. In order to find out to which extend were the encoded proteins involved in nodule protection, development or both, gene regulation studies in response to SA and wounding as well as phylogenetic analysis of the protein sequences were performed. These were further characterized through expression studies after SA-treatment and wounding, and by phylogenetic analysis. We suggest that CgChi1 and CgGst are involved in defense or microsymbiont control and CgPox4 is involved in nodule development. For CgHin1 the question "defense, development or both" remains open.

Comparative Proteomic Analysis of Nodulated and Non-Nodulated Casuarina glauca Sieb. ex Spreng. Grown under Salinity Conditions Using Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS)

“…Additionally, such a low number of differentially expressed proteins (DEPs) might be explained by the assumption that C. glauca has constitutive defense mechanisms against various stresses and that defense responses are activated during the early stages of stress imposition [21]. Moreover, the difference between KNO 3 + and NOD + plants regarding the number of DEPs might be associated with the fact that the process of nodulation in C. glauca leads to a set of defense-related events, such as the transcriptional activation of defense-related genes encoding proteins involved in ROS-detoxification [2,3], or the protection of nodules against pathogenic microorganisms [3,38], probably triggering a systemic defense response. On the other hand, the fact that at 200 mM NaCl Frankia was no longer fixing nitrogen in C. glauca nodules, and thus at this salt concentration the plants were subjected to both salt and mineral stresses [12], may explain the slightly higher induction of DEPs in NOD + than in KNO 3 + at 200 (24% vs. 16%) and 400 mM (56% vs. 44%), which was then similar at 600 mM NaCl (93%) when major physiological changes related to photosynthesis, membrane stability and osmoprotection mechanisms are induced [4,7,8,13,21,22].…”

“…It is plausible to postulate that in nodulated plants, osmotic stress defense mechanisms are induced earlier than in control plants. Firstly, nodulated plants activate anti-pathogen defense mechanisms when they develop nodules, which are likely to include systemic responses [2,3,37,[40][41][42]. Secondly, since Frankia nitrogenase activity is reduced dramatically from 200 mM NaCl upwards [12,13], and thus the nodulated plants are exposed to N-deficiency at this point, not only to salinity stress.…”

“…However, broad, interdisciplinary and integrate research to ascertain the contribution of symbiotic Frankia to the environmental plasticity of actinorhizal species has emerged only recently, mostly focusing on salt stress in Casuarinaceae, particularly in the model species Casuarina glauca Sieb. ex Spreng e.g., Santos et al, Ribeiro et al, Batista-Santos et al, Mansour et al, Ribeiro-Barros et al, Jorge et al, and Ngom et al [2][3][4][5][6][7][8][9]. C. glauca is a tropical tree widely used to prevent desertification and erosion, as well as to rehabilitate poor and degraded soils [1,10].…”

Validation of candidate reference genes for qRT-PCR studies in symbiotic and non-symbiotic Casuarina glauca Sieb. ex Spreng. under salinity conditions

“…Moreover, the choice of the reference genes is organ-dependent: Ap47 and PP2A for branchlets, S23 and GAPDH for roots, and S23 and Ap47 for nodules. We highlight the significance of our study in C. glauca where most of the expression studies have been conducted with one reference gene (CgUbi) (e.g., PerrinWalker et al 2010;Santos et al 2010;Hocher et al 2011;Diédhiou et al 2014). …”

“…have become increasingly important as climate changes threaten to reshape the global landscape over the next decades (Santos et al 2010). Interestingly, for several reasons C. glauca is also considered the model of actinorhizal plants (Zhong et al 2013).…”