Integrative molecular profiling indicates a central role of transitory starch breakdown in establishing a stable C/N homeostasis during cold acclimation in two natural accessions of Arabidopsis thaliana

Abstract

BackgroundThe variation of growth and cold tolerance of two natural Arabidopsis accessions, Cvi (cold sensitive) and Rschew (cold tolerant), was analysed on a proteomic, phosphoproteomic and metabolomic level to derive characteristic information about genotypically distinct strategies of metabolic reprogramming and growth maintenance during cold acclimation.ResultsGrowth regulation before and after a cold acclimation period was monitored by recording fresh weight of leaf rosettes. Significant differences in the shoot fresh weight of Cvi and Rschew were detected both before and after acclimation to low temperature. During cold acclimation, starch levels were found to accumulate to a significantly higher level in Cvi compared to Rschew. Concomitantly, statistical analysis revealed a cold-induced decrease of beta-amylase 3 (BAM3; AT4G17090) in Cvi but not in Rschew. Further, only in Rschew we observed an increase of the protein level of the debranching enzyme isoamylase 3 (ISA3; AT4G09020). Additionally, the cold response of both accessions was observed to severely affect ribosomal complexes, but only Rschew showed a pronounced accumulation of carbon and nitrogen compounds. The abundance of the Cold Regulated (COR) protein COR78 (AT5G52310) as well as its phosphorylation was observed to be positively correlated with the acclimation state of both accessions. In addition, transcription factors being involved in growth and developmental regulation were found to characteristically separate the cold sensitive from the cold tolerant accession. Predicted protein-protein interaction networks (PPIN) of significantly changed proteins during cold acclimation allowed for a differentiation between both accessions. The PPIN revealed the central role of carbon/nitrogen allocation and ribosomal complex formation to establish a new cold-induced metabolic homeostasis as also observed on the level of the metabolome and proteome.ConclusionOur results provide evidence for a comprehensive multi-functional molecular interaction network orchestrating growth regulation and cold acclimation in two natural accessions of Arabidopsis thaliana. The differential abundance of beta-amylase 3 and isoamylase 3 indicates a central role of transitory starch degradation in the coordination of growth regulation and the development of stress tolerance. Finally, our study indicates naturally occurring differential patterns of C/N balance and protein synthesis during cold acclimation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0668-1) contains supplementary material, which is available to authorized users.

A Benchtop Fractionation Procedure for Subcellular Analysis of the Plant Metabolome

“…In general, and except for melibiose levels in Cvi, sugars were found to accumulate significantly during the acclimation process. Those differences between absolute levels in Cvi and Rsch have been described before for the same experimental setup (Nagler et al, 2015). Here, we now provide the relative distribution of metabolites among cellular compartments (for details see Supplementary Data 4) allowing for the calculation of absolute levels within these compartments (Table 2; Supplementary Data 5).…”

“…Hence, to summarize our findings about relative shifts of metabolite levels during cold acclimation with respect to the freezing tolerance of the considered accessions (Cvi < Col < Rsch, see e.g., Hannah et al, 2006) we suggest that the intensity of significant subcellular re-arrangement of primary metabolism is negatively correlated with freezing tolerance. In contrast, the absolute amount of substance of many sugars, amino acids and organic acids has been shown to positively correlate with freezing tolerance, not only on a cellular (Klotke et al, 2004; Nagler et al, 2015) but also on a subcellular level (Nägele and Heyer, 2013). These significant differences between absolute subcellular metabolite levels became also clear in the present study (Figure 5B; Table 2; Supplementary Data 5 Tables SIII, SIV).…”

Cold acclimation has a differential effect on leaf vascular bundle structure and carbon export rates in natural Arabidopsis accessions originating from southern and northern Europe

“…Although sucrose is transported in the phloem and xylem only indirectly contributes to leaf sucrose transport from source to sink tissue, the significance of the cold-induced negative correlation of xylem and petiole areas with sucrose exudation rates might indicate a functional role in cold acclimation of northern accessions. Previously, we found that the northern cold tolerant accession Rsch has a higher growth rate in terms of shoot fresh weight accumulation during cold acclimation than the sensitive accession Cvi (Nagler et al, 2015). Thus, a higher growth rate might also be reflected by increased petiole and xylem areas of northern accessions in the present study.…”

Protein sumoylation and phosphorylation intersect in Arabidopsis signaling

“…After discarding the last acetone wash, the protein pellet was air dried. Protein digestion was done as in Nagler et al (2015). The protein pellet was dissolved in 8 M urea, 0.1 M ammonium bicarbonate (AmBic; Merck, http://www.merckmillipore.com/product/Ammo nium-hydrogen-carbonate,MDA_CHEM-101131).…”