Central metabolite and sterol profiling divides tobacco male gametophyte development and pollen tube growth into eight metabolic phases

Abstract

While changes in the transcriptome and proteome of developing pollen have been investigated in tobacco and other species, the metabolic consequences remain rather unclear. Here, a broad range of metabolites was investigated in close succession of developmental stages. Thirteen stages of tobacco male gametophyte development were collected, ranging from tetrads to pollen tubes. Subsequently, the central metabolome and sterol composition were analyzed by GC-mass spectrometry (MS), monitoring 77 metabolites and 29 non-identified analytes. The overall results showed that development and tube growth could be divided into eight metabolic phases with the phase including mitosis I being most distinct. During maturation, compounds such as sucrose and proline accumulated. These were degraded after rehydration, while γ-aminobutyrate transiently increased, possibly deriving from proline breakdown. Sterol analysis revealed that tetrads harbor similar sterols as leaves, but throughout maturation unusual sterols increased. Lastly, two further sterols exclusively accumulated in pollen tubes. This study allows a deeper look into metabolic changes during the development of a quasi-single cell type. Metabolites accumulating during maturation might accelerate pollen germination and tube growth, protect from desiccation, and feed pollinators. Future studies of the underlying processes orchestrating the changes in metabolite levels might give valuable insights into cellular regulation of plant metabolism.

Characterization of the enzymatic activity and physiological function of the lipid droplet‐associated triacylglycerol lipase AtOBL1

“…Pollen grains of tobacco contain 16 mg TAG g À1 DW (Rotsch et al, 2017), much less than concentrations commonly found in dicotyledon seeds but much more than those in nonstressed (Mueller et al, 2015). Therefore, TAG might represent an important energy storage for pollen to drive pollen tube growth.…”

“…Tobacco plants (Nicotiana tabacum L. variety Samsun NN) were grown in a glasshouse as described (Rotsch et al, 2017). Arabidopsis (Arabidopsis thaliana (L.) Heynh.…”

“…Pollen tubes were cultivated from 10 mg pollen per sample for 14.5 h on cellophane layered on solid medium as described (Rotsch et al, 2017). Pollen tubes were flash-frozen in liquid nitrogen and lyophilized.…”

“…Pollen tubes were cultivated from 100 mg pollen per sample for 14.5 h on cellophane layered on solid medium as described (Rotsch et al, 2017). All plant material (tobacco pollen tubes, N. benthamiana leaves, imbibed Arabidopsis seeds) was pestled on ice in grinding medium (0.4 M sucrose (in the case of pollen tubes without sucrose), 10 mM KCl, 1 mM MgCl 2 , 1 mM EDTA, 100 mM HEPES/KOH pH 7.5, 5 mM DTT) and a small amount of sea sand.…”

“…1d), which at least does not oppose a potential flux of FAs between the TAG and phospholipid pools. Also the sterol esters contain all of the sterol species found as free sterols including the ones formed only during pollen tube growth and not present in mature pollen (cycloeucalenol and the nonidentified sterol U5; Rotsch et al, 2017;Fig. 1e).…”