The precursor of trehalose biosynthesis, trehalose-6-phosphate (T6P), is essential for development and controls carbon utilization in Arabidopsis thaliana seedlings. Furthermore, T6P accumulation in the absence of available carbon causes growth arrest when Arabidopsis seedlings are supplied with 100 mM trehalose. But what are the mechanisms involved?
In 2008, we had been able to establish a medium throughput method to determine T6P levels in plant extracts that has since evolved. The method combined liquid and solid-phase extractions before separation and detection using HPLC/MS, and more recently CE/MS. In 2012 we have published an improved method to achieve high throughput HILIC-UPLC/MS detection of phosphorylated intermediates of metabolism. We currently are working on a simplified pre-purification of the extracts destined to the determination of phosphorylated sugars from plant extracts.
Through collaboration with Dr. Matthew Paul at Rothamstead Research (UK) we report T6P inhibition of SnRK1 kinase activity showing for the first time that T6P controls growth and carbon utilization by way of a central kinase signaling network in 2009. The collaboration further uncovered a remarkable accumulation of T6P during wheat embryo development in 2011. In 2012, the collaboration with Dr. Astrid Wingler University College London (UK) led to a publication on the role of T6P in senescence that is independent of the accumulation of soluble sugars.
Feeding trehalose to Arabidopsis seedlings causes a reversal of carbon allocation with starch accumulating in cotyledons and no starch and growth arrest at the apical meristems (Figure 1). To understand this phenomenon we are using a genetic approach. We have isolated and characterized mutants that overcome the growth inhibition on 100 mM trehalose. A subset of these mutants is affected in genes known to be otherwise involved in nutrient stress responses and these include mutants with altered SnRK1 activity and bZIP11 expression: we conclude and publish in 2011 that on trehalose medium, T6P accumulation inhibits SnRK1 activity and thence growth in Arabidopsis. Our latest interest has turned to the question in how far the sugar and T6P control are gated by the circadian rhythms and so we are left searching in the dark.
Figure 1: Feeding trehalose to Arabidopsis seedlings causes reversal of carbon allocation. Seedlings were grown on 100 mM trehalose for 14 days, destained, then starch stained using Lugol. wt, wild type seedlings accumulate large quantities of starch in the cotyledons, the apical meristems are growth arrested and no starch is seen in the columella of the roots. pgm1, mutants of the chloroplastic phosphoglucomutase lack the precursor of starch synthesis and so are unable to make starch yet also stop growing on trehalose. Reversal of carbon allocation by trehalose is therefore not caused by induced starch accumulation.