Influence of mutation in the gene Sym26 of the garden pea (Pisum sativum L.) on the organization of tubulin cytoskeleton in nodules

Symbiotic nodule is a unique organ forming on legume roots. Indeterminate nodules (with prolonged meristem activity) (F. Guinel, 2009) are characterized by differentiation of both the nodule cells and the bacteria that infect nodule and are converted into a form specialized for nitrogen fixation - bacteroids. Bacteroids surrounded by a membrane of plant origin, form organelle-like symbiosomes (A. Tsyganova et al., 2018; T. Coba de la Peña et al., 2018). Cell differentiation leads to appearance of uninfected (free of bacteria) and infected cells filled with many thousands of symbiosomes formed in the central part of nodule (A. Tsyganova et al., 2018). A prolonged activity of the meristem results in histological zonation of the indeterminate nodule. A meristem, an infection zone, a nitrogen fixation zone are distinguished, and a senescence zone appears in the basal part of a mature nodule (F. Guinel, 2009). Obviously, the tubulin cytoskeleton plays an important role in the development of a nodule, but until now researchers had a focus on the early stages of nodule development (A. Timmers, 2008). Only recently it was revealed that the tubulin cytoskeleton plays a key role in the differentiation of nodule cells (A. Kitaeva et al., 2016). It was shown that in nodules of garden pea ( Pisum sativum L.) and barrel medic ( Medicago truncatula Gaertn.) the release of bacteria into the cytoplasm of a plant cell prevents the formation of a regular pattern of cortical microtubules, oriented parallel to each other and perpendicular to the longitudinal axis of the cell, typical for uninfected cells. This leads to an irregular pattern of cortical microtubules, the appearance of which contributes to the transition of infected cells to isodiametric growth (A. Kitaera et al., 2016). Endoplasmic microtubules build a mold for the growth of infection threads, and support the location of infection droplets and symbiosomes in infected cells (A. Kitaeva et al., 2016). However, changes in the organization of the tubulin cytoskeleton during senescence of nodule cells have not been studied. In this study, using immunocytochemical analysis and confocal laser scanning microscopy, the organization of the tubulin cytoskeleton in the nodules of the pea mutant SGEFix--3 ( sym26 ) (V. Tsyganov et al., 2000) was studied. This mutant is characterized by the formation of ineffective nodules with premature degradation of symbiotic structures (T. Serova et al., 2018). It was shown that in the mutant line, the formed patterns of cortical and endoplasmic microtubules did not differ from those of the initial line SGE. Cortical microtubules formed an irregular pattern in meristematic and infected cells and regular pattern in uninfected and colonized cells. Endoplasmic microtubules surrounded the nucleus in interphase cells, formed spindles and preprophase bands during mitosis, and also surrounded infection threads. At the same time, in the senescence zone in degrading cells, complete depolymerization of the tubulin cytoskeleton occurred in both infected and uninfected cells. In the initial line, senescence was induced only in four-week-old nodules, and microtubule depolymerization was also observed in senescent cells. Thus, the complete depolymerization of microtubules in various types of nodule cells can be a cytological marker of its senescence.