SA improvement of hyperhydricity reversion in Thymus daenensis shoots culture may be associated with polyamines changes

In shoot cultures of Thymus daenensis, hyperhydricity syndrome promoted by benzyladenine (BA) is characterised by the development of chlorophyll-deficient shoots with a high water content and reduced growth that is less differentiated. By removing the BA from the culture medium, the hyperhydricity was reversed, and the reversion toward a normal growth in vitro was more efficient in shoots treated with 5 μM of salicylic acid (SA), showing a significant increase in chlorophyll b after 4 weeks of culture. In the present study, the effect of salicylic acid on the reversion of shoot hyperhydricity was investigated at the level of the free, soluble and insoluble conjugated polyamine content. In T. daenensis micropropagated shoots, the level of polyamines was high, with a predominance of putrescine. BA, which triggered hyperhydricity, caused a reduction of the polyamine (PA) content by one-half due to a decrease in the putrescine content and insoluble conjugated PAs that were not detected in the hyperhydric shoots. In the reverted shoots, changes of the free polyamines, spermidine and, more notably, spermine, were shown. The spermine content doubled after 4 weeks of culture, and its amount was the same as that found in normal shoots, suggesting that free spermine could be particularly involved in the reversion of hyperhydricity. In the SA-reverted tissues, the PA pattern was marked with a transient increase of free putrescine, spermidine and spermine and an enhancement of soluble conjugated spermine. This transitory SA-dependent amplification of PAs was concomitant with a remarkable transient increase of H2O2, suggesting that SA may be implicated in PA signalling pathways for tissue differentiation during the reversion of hyperhydricity in T. daenensis.