Distribution of linker histone variants during plant cell differentiation in the developmental zones of the maize root, dedifferentiation in callus culture after auxin treatment

Abstract

Although several linker histone variants have been studied in both animal and plant organisms, little is known about their distribution during processes that involve alterations in chromatin function, such as differentiation, dedifferentiation and hormone treatment. In this study, we identified linker histone variants by using specific anti-histone H1 antibodies. Each variant's ratio to total H1 in the three developmental zones of maize (Zea mays L.) root and in callus cultures derived from them was estimated in order to define possible alterations either during plant cell differentiation or during their dedifferentiation. We also evaluated linker histone variants' ratios in the developmental zones of maize roots treated with auxin in order to examine the effects of exogenous applied auxin to linker histone variant distribution. Finally, immunohistochemical detection was used to identify the root tissues containing each variant and correlate them with the physiological status of the plant cells. According to the results presented in this study, linker histone variants' ratios are altered in the developmental zones of maize root, while they are similar to the meristematic zone in samples from callus cultures and to the differentiation zone in samples from roots treated with auxin. We propose that the alterations in linker histone variants' ratios are correlated with plant cell differentiation and dedifferentiation.

Alterations in core histone variant ratios during maize root differentiation, callus formation and in response to plant hormone treatment

“…In samples derived from roots treated with auxin, we found alterations in histone variants' ratios to the level similar to that obtained in differentiation zone of control roots, regardless the developmental zone the samples derived from. In particular, histone H1 0 variant was increased to above 30% of total H1 in all of the three developmental zones (Alatzas et al, 2008), while the ratios of ubiquitinated H2A and acetylated H4 (Figures 5 and 8 respectively) were decreased to 13% and 37% respectively, a pattern observed in samples from differentiation zone of controls. An exception to these observations was the increase in histone variant H3.1 (Fig.…”

“…In this case, we found alterations in histone variants' ratios to the level similar to that obtained either in differentiation or in meristematic zone of control roots. In general, a decrease in histone modified forms' ratios (Figures 5,7 and 8) and in H1 0 variant ratio (Alatzas et al, 2008) was observed in all samples, while H3.1 variant ratio (Fig. 7) was similar to meristematic zone of controls.…”

“…3B), histone H2A and the ubiquitinated form of H2A (Fig. 3C) has been described in detail elsewhere (Alatzas & Foundouli, 2006, Alatzas et al, 2008. A more detailed and representative twodimensional separation of each core histone variant in samples derived from either meristematic or differentiation zones is presented in Figure 4.…”

“…The antibodies employed were against histone H3 (monoclonal, a generous gift by Jockers-Wretou and co-workers, diluted 1: 5000) (Muller et al, 1985), histone H2B (polyclonal, a generous gift by Sekeri-Pataryas and co-workers, diluted 1: 100) (Sourlingas et al, 2003), histone H4 (polyclonal, Cell Signaling Technology, diluted 1: 2000), the phosphorylated and the acetylated forms of histone H3 (Cell Signaling Technology and Upstate Biotechnology respectively, both diluted 1: 2000) and the acetylated form of histone H4 (Serotek, diluted 1: 2000). Immunodetection of histone H2A and histone H1 have been described in detail elsewhere (Alatzas & Foundouli, 2006, Alatzas et al, 2008. The reaction was carried out at room temperature for 2 h. Following removal of excess antibody by washing three times with PBST, the membranes were incubated with the second antibody (horseradish peroxidase labeled goat anti-rabbit, diluted 1: 2000).…”

“…After identifying histone variants by western blotting, the Coomasie-or silver nitratestained gels were scanned and analyzed by means of Gel Pro Analyzer 3.1 software (Media Cybernetics) as described elsewhere (Alatzas et al, 2008). The optical density of each spot representing histone variant was measured and its portion of total histone class in each sample was evaluated.…”