Cytological development, calcium dynamic and metabolite profile of stress-induced tobacco (Nicotiana tabacum L.) microspore under calcium chloride treatment.

Abstract

Exogenous calcium is pivotal in activating microspore embryogenesis via calcium signaling. Altering the external calcium levels in the medium has been documented to impact the success rate of microspore embryogenesis initiation. However, the cellular effects on the formation of embryogenic tobacco microspore and its cellular changes during stress treatment remain elusive. We examined microspore cytological development, specifically vacuolation types and first division pattern. We also used Fluo 3-AM staining to observe the calcium dynamics, including accumulation and distribution of intracellular calcium ions (Ca2+) under fluorescent microscope. Metabolomic analysis of induced microspores was carried out using gas chromatography-mass spectrometry (GC-MS). The result showed that at 2.0 mM CaCl2.2H2O treatment, 57.06 ± 6.95% of microspores exhibited star-like structures, and 47.39 ± 8.84% underwent symmetric first division, twice as effective as 1.0 mM. Fluo 3-AM staining revealed increased Ca2+ fluorescence on day one, persisting in 2.0 mM treatment by day three. By day six, fluorescence in 1.0 mM and 2.0 mM treatments significantly differed. Intracellular Ca2+ signals rose with 1.0 mM CaCl2.2H2O and fell with 2.0 mM CaCl2.2H2O. The metabolomic analysis detected 34 metabolites, including amino acids, organic acids, and fatty acids, with predominant oleic acid, palmitic acid, and stearic acids. We conclude that two millimolar of calcium chloride was optimum for inducing embryogenic microspore formation in tobacco. Our findings underscore the significant role of exogenous calcium in microspore embryogenesis, elucidating its impact on cellular dynamics and metabolite profiles. These insights contribute to hold potential implications for enhancing crop breeding strategies.