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

Plant growth together with stress tolerance and flowering traits are known to be orchestrated in a complex and interdependent molecular manner. Water supply, temperature and soil quality have been shown to be the most relevant abiotic factors which significantly affect these traits [1]. During the last decade, naturally occurring genetic and phenotypic variation of Arabidopsis thaliana has been shown to be a promising tool for studying the molecular architecture of such physiological traits. On the cellular level, abiotic stress affects the integrity of membrane systems, transport proteins, metabolic enzymes and signalling compounds, ultimately leading to disfunctions in cellular metabolism which directly impair plant growth and development. Previous studies have shown and discussed significant differences in naturally occurring stress tolerance, morphology, developmental programming and flowering of Arabidopsis thaliana [2–9].

Low temperature belongs to one of the most important abiotic factors limiting the geographic distribution of plants. In many temperate species, the exposure of plants to low but non-freezing temperatures initiates a process termed cold acclimation resulting in increased freezing tolerance [10]. The process of cold acclimation is a multigenic trait being characterized by a comprehensive reprogramming of the transcriptome, proteome and the metabolome, but also of enzyme activities and the composition of membranes [3, 11–17]. Particularly, reprogramming of primary metabolism plays a crucial role during cold acclimation leading to a changed photosynthetic activity and the accumulation of soluble sugars, amino acids and polyamines. Concentrations of the di- and trisaccharide sucrose and raffinose, respectively, have been shown to correlate well with winter hardiness in several plant species [18, 19]. Further, several roles for sugars in protecting cells from freezing injury have been proposed [10]. Yet, soluble carbohydrates have been shown to be insufficient to fully describe the development of freezing tolerance [20]. While sugar levels are often found to positively correlate with freezing tolerance, the underlying regulatory mechanisms are poorly understood. On a whole plant level, it remains elusive whether sugar accumulation may result from reduced sink activity, because growth retardation at low temperatures is stronger than the reduction of photosynthetic activity [21]. Additionally, it is not clear whether sugars function as cryoprotective substances or because they are substrates for the cryoprotectant synthesis [19].

Together with sugars, also pools of organic and amino acids are significantly affected during cold-induced metabolic reprogramming. Aspartate, ornithine and citrulline were found to increase during cold exposure of Arabidopsis thaliana indicating the reprogramming of the urea cycle [14]. Beyond, the authors observed a cold-induced increase in levels of alpha-ketoglutarate, fumarate, malate and citrate which they suggested to result from an up-regulation of the citric acid cycle. Although many observations revealed an increase of metabolite levels to be characteristic for cold acclimation, the magnitude of changes in the metabolome does not necessarily indicate the capacity of Arabidopsis to increase its freezing tolerance [12]. A prominent example which shows the possible discrepancy between metabolic reprogramming and gain of freezing tolerance is the comparison of the freezing sensitive natural accessions Cvi, which originates from Cape Verde Islands, and C24, originating from the Iberian Peninsula. Both accessions similarly increase their freezing tolerance during cold acclimation while concomitant metabolome changes were found to differ dramatically [3]. It might not be surprising that the coordination of a complex trait like freezing tolerance cannot be directly related to one certain metabolic output, but, simultaneously, this observation indicates a high level of plasticity which is characteristic for intraspecific molecular responses to environmental cues. In this context, most of the naturally occurring biochemical mechanisms and metabolic regulatory strategies to acclimate to low temperature still remain elusive.

Plant growth is significantly reduced due to cold exposure. Although low temperature significantly affects metabolic processes and resource allocation, growth is not necessarily limited by photosynthetic activity. Following a period of 1 to 3 days after exposure to low temperature, during which cold stress is sensed and acclimation is initiated, rates of photosynthetic carbon assimilation can be almost fully recovered [22]. Together with the finding that growth is affected more significantly than photosynthesis during exposure to water deficit [23], this indicates that growth during stress exposure might rather be limited by sinks than sources. Such a cold-induced sink limitation has been discussed to be the reason for the characteristic accumulation of sugars during cold exposure. Although high levels of sugars have been shown to potentially repress the expression of photosynthetic genes [24, 25], cold acclimation and development at low temperature was found to reduce or even fully revert this effect [26–28]. Additionally, cold acclimation was found to have a significant effect on leaf respiration of Arabidopsis thaliana [29]. Both respiration rates in the light and in the dark were described to increase significantly during cold acclimation, while the more pronounced effect was found for respiration in darkness. Moreover, although cytosolic hexose phosphate concentrations increased dramatically, there was no significant correlation observed with respiration in the light indicating that respiration is not limited by substrate availability under low temperature stress [29].

Although the above-mentioned findings only represent an excerpt from current findings about growth regulation and cold acclimation strategies in Arabidopsis, it clearly indicates a highly complex and interlaced relationship between metabolic and physiological consequences of low temperature. Systems biology focuses on such complex questions and has become a rapidly expanding and attractive research area during the last decade [30]. In a systems biology approach, elements of an interaction network, e.g. a metabolic map, are rather analysed and discussed as interacting components than isolated parts in order to improve the understanding of how a complex biological system is organized and regulated [31].

Research on plant freezing tolerance, growth regulation and plant systems biology has largely been driven by studies in Arabidopsis thaliana. The species is native to Europe and central Asia, its biogeography was described in detail, and it was shown that climate on a global scale is sufficient for shaping the range boundaries [32]. When compared to other Brassicaceae species, Arabidopsis has a wide climatic amplitude and shows a latitudinal range from 68 to 0°N, which makes it suitable for the analysis of variation in adaptive traits [33]. Arabidopsis represents a predominantly selfing species, and, hence, most of the individual Arabidopsis plants collected in nature represent homozygous inbred lines [34]. These homozygous lines are commonly referred to as accessions, representing genetically distinct natural populations that are specialized to particular sets of environmental conditions. The variation of morphological and physiological phenotypes enables the differentiation of most of the collected Arabidopsis accessions from others. In particular, considering the tolerance to abiotic factors, e.g. low temperature, a large variation has been reported (e.g. [33]), making Arabidopsis an attractive system to study plant-environment interactions.

In the present study, two of these Arabidopsis accessions were analysed with respect to naturally occurring variation in the traits of growth regulation and freezing tolerance. The selection of the two accessions, Cvi (origin: Cape Verde Islands) and Rschew (origin: Western Russia), was based on findings of previous studies which have shown that Cvi represents a freezing sensitive accession while Rsch is freezing tolerant (e.g. [35]). Based on this finding and due to their large distance with respect to geographical origin, cold acclimation capacity and cold-induced gene regulation [3], the molecular and biochemical study of both accessions can be expected to provide a suitable approach to quantify strategies of growth maintenance during environmental fluctuations. As previous work has already indicated, a multi-layered design of molecular physiological studies was necessary in order to derive coherent conclusions on a genome-wide level [11, 36]. Thus, the present study aimed at a comprehensive characterization of metabolomic, proteomic and phosphoproteomic levels of both natural accessions to unravel differential strategies of growth regulation in a changing environment.

Cold acclimation of plants represents a multifaceted and multigenic process affecting various levels of molecular organisation, e.g. gene expression, RNA processing or post-translational regulation [44, 45]. Hence, although numerous comprehensive studies have unravelled many crucial processes being involved in the acclimation process (for an overview see e.g. [46]), it is not surprising that many gaps still exist in our understanding of how metabolism is reprogrammed, and how the metabolic output is linked to the observed physiological output, e.g. changes in growth and yield. In general, plant growth requires a sufficient supply with energy, water and nutrients and is regulated in response to environmental changes. These environmental cues are sensed and integrated by a highly complex and conserved signalling network [47].

An efficient balancing of photosynthesis and respiration was shown to be a prerequisite for plant growth [48] and cold acclimation [29]. With regard to these two central processes, our findings revealed a more complex cold-induced metabolic reprogramming in the cold tolerant Arabidopsis accession Rsch which also showed a significantly higher shoot fresh weight than both non-acclimated and acclimated plants of Cvi (see Fig. 1). In addition, also glycolysis, TCA cycle and pathways of amino acid biosynthesis were found to be differentially affected by low temperature in both accessions. Together with the observed levels of sugars, organic and amino acids, which were, on an average, significantly higher in acclimated plants of Rsch, this points to a differential cold-induced redirection of carbon equivalents in both accessions. While we cannot experimentally exclude a limitation of CO₂ uptake as a reason for the lower metabolite levels in cold-acclimated plants of Cvi, there are several indications which rather suggest a differential regulation of carbon allocation to be the reason for the observed phenotype. First, on the level of the total proteome, we could observe a separation of acclimation states but not of accessions by cold-induced protein dynamics related to photosynthetic dark and light reactions (Additional file 11: Table S9). Second, in a former study, the analysis of the photosynthetic carbon uptake was found to be similar in cold-acclimated plants of cold sensitive and tolerant accessions [49]. While Nägele and colleagues did not analyse the Cape Verde accession Cvi but the cold-sensitive accession C24 originating from the Iberian Peninsula, further support of this hypothesis is provided by another study in which photosynthetic acclimation of Cvi was compared to the Finnish accession Hel-1, originating from Helsinki [50]. There, the author found that both accessions, originating from contrasting climates, showed a highly similar capability to acclimate to a broad regime of temperature and irradiance. Another indication for a non-limited CO₂-uptake is provided by the starch levels which were found to increase to a significantly higher level in Cvi than in Rsch (see Fig. 3). This agrees with the findings of Guy and co-workers who also described a significantly higher starch level in Cvi compared to Rsch after cold acclimation [12]. Based on this observation, Guy and co-workers suggested that, following a sufficiently long acclimation period, even in poorly acclimating accessions like Cvi energy constraints do not seem to limit the acquisition of freezing tolerance. Although our growth conditions (5 °C/7d of acclimation/125 μmol m⁻² s⁻¹) do not exactly reflect the growth conditions applied in the study of Guy and co-workers (4 °C/14d acclimation/90 μmol m⁻² s⁻¹), we still observed a similar output of starch metabolism.

To derive an explanation for the observed differences in starch metabolism, which has previously been suggested to be a major regulator of plant growth [51], the regulation of both starch synthesis and degradation has to be considered. While our study does not account for enzymatic activity, our proteomic results provide evidence for a different regulation of starch metabolism in cold acclimated plants of Cvi and Rsch. While, independently from cold exposure, levels of alpha-amylase AMY3 were found to be constitutively higher in Rsch than in Cvi, a cold-induced significant reduction in the level of beta-amylase BAM3 could only be observed for Cvi, while isoamylase 3, ISA3, was significantly increased only in cold-acclimated plants of Rsch. Alpha-, beta- and isoamylases play crucial roles in starch degradation [52–54], and, hence, these findings hint towards a distinct regulation of starch degradation which was previously discussed to play a decisive role in the process of cold acclimation [55, 56]. Starch molecules consist of mostly unbranched amylose (alpha-1,4-linked glucosyl moieties) and branched amylopectin (alpha-1,6-linked moieties). While alpha-amylase, hydrolysing the alpha-1,4-glucosidic linkages of starch, plays a central role in the degradation of storage starch in endosperm of germinating cereal seeds [57], a disruption of AtAMY3 by insertional mutagenesis did not affect starch degradation in Arabidopsis leaves [58]. However, removal of AMY3 in addition to the debranching, alpha-1,6-linkage hydrolysing, enzyme ISA3 was shown to lead to a strong starch excess phenotype [54]. A triple mutant with an additional removal of limit dextrinase, LDA, which represents another debranching enzyme, was finally shown to result in an effective block of starch breakdown accumulating even higher levels of starch than observed before in the double mutant [54]. While our presented shotgun proteomics approach could not resolve the cold-induced effect on LDA in either of both accessions, our findings indicate that the combination of constantly lower AMY3-levels in Cvi and a cold-induced increase in ISA3-levels in Rsch might provide an explanation for the higher starch levels observed in cold-acclimated plants of Cvi.

The complete process of (transitory) starch breakdown from the insoluble granule to the soluble compounds maltose and glucose comprises numerous additional steps and classes of enzymes, finally resulting in a complex and tightly (redox) regulated pathway [52, 59]. Beta-amylases (BAMs) primarily hydrolyse glucan chains, which have been previously released and linearized, liberating maltose [52]. The multigene family of BAMs in Arabidopsis thaliana comprises nine genes, and BAM3 was shown to encode a catalytically active plastidial enzyme playing a central role in leaf starch degradation at night in mesophyll cells [60, 61]. Hence, our finding of a significant decrease of BAM3 protein levels in cold acclimated plants of Cvi provides a further explanation for the strong increase of starch levels. The observation of a decrease in BAM3 protein levels contrasts the finding of a cold induced increase of BAM3 expression [56]. However, in a recent publication Monroe and co-workers derived a more complex picture in which the authors observed a decline in BAM3 activity after 2d and 4d of cold stress while BAM3 mRNA levels clearly increased [62]. Although these results were derived from studies within the genetic background of the Arabidopsis accession Columbia-0, and, hence, might not directly be comparable to the background of Cvi, they indicate the complex interplay of molecular levels of organization during exposure to a fluctuating environment. Such an adaptive and differential regulation of starch metabolism in response to cold was also exemplified in a previous study on the starchless Arabidopsis thaliana pgm mutant being deficient in a phosho-glucomutase activity [63]. In this study, the cold/heat-stress-induced increase of raffinose-family-oligosaccharide levels in the pgm mutant plants revealed an unexpected flexibility to adjust central metabolism to temperature stress in the absence of transitory starch.

Based on our investigation of the two natural accessions Rsch and Cvi during cold-acclimation, we suggest that the orchestration of growth and cold acclimation differs significantly in the redirection of photoassimilates between soluble metabolic compounds and the insoluble storage compound starch. In addition, the observation described in a previous study, that the biomass formation in the starchless pgm mutant is restricted by high respiratory losses in the root [48], allows us to hypothesise that the differences we observed in the fresh weight of Cvi and Rsch might also be due to a differential regulation of sink-source interaction both before and after the cold acclimation period. In future studies it would be interesting to analyse whether the observed differences in starch degradation are somehow related to resource allocation and root respiration in both accessions.

In context of Arabidopsis cold acclimation, the C-repeat binding factor (CBF) pathway belongs to one of the most intensively studied pathways which has a crucial role in the development of freezing tolerance [64]. Within minutes after transfer to low temperature, the CBF1-3 [65], i.e. DREB1a-c [66], expression is induced. They encode members of the AP2/ERF family of transcription factors recognizing the C-repeat (CRT)/dehydration-responsive element (DRE) being present in the promotors of CBF-targeted genes [66]. The constitutive overexpression of either CBF1, 2 or 3 alters the expression of cold-regulated (COR) genes resulting in an increase of freezing tolerance without exposure to low temperature [67, 68]. In the present study, the level of COR78 (AT5G52310) and its phosphorylation were observed to be positively correlated with the acclimation state of both accessions. Further, independent from the acclimation state, protein levels were found to be constitutively higher in Rsch than in Cvi. Interestingly, COR78 transcript abundance was previously discussed to be regulated by sucrose [69] which would explain our findings of higher protein abundance and sucrose levels in Rsch (see Figs. 3 and 7). In addition, these observations allow for the speculation about a link between sugar signalling networks and the cold responsive gene regulation which could probably comprise central conserved signalling compounds like the complex and antagonistic interaction network spanned by the kinases Sucrose-non-fermenting-1-Related Protein Kinase 1(SnRK1) and Target Of Rapamycin (TOR) [70].

Finally, the observation of differentially phosphorylated transcription factors, like the BASIC PENTACYSTEINE (BPC), but also membrane proteins, e.g. PIP2;3 aquaporins which are involved in numerous developmental and growth-regulatory processes [71, 72], clearly shows the wide range of cellular processes which might contribute to a systematic and differential stress acclimation output in naturally occurring accessions of Arabidopsis. Our results indicate that a comprehensive reprogramming not only of the process of protein synthesis, but also of metabolic pathways regulating the flux of photoassimilates to the TCA cycle and to pathways of amino acid biosynthesis, contributes to the stabilization of a metabolic homeostasis during cold acclimation. Together with previous studies on the stress-induced dynamics of protein phosphorylation patterns, which have, for example, revealed the central role of protein phosphorylation in cold-induced subcellular sugar allocation [73], and its applicability to crop science [74], this clearly indicates the necessity for integrative molecular profiling approaches to unravel a comprehensive picture of complex plant acclimation strategies.

The findings presented in this study provide evidence for a central role of the starch degradation pathway in the molecular orchestration of plant growth and abiotic plant-environment interactions in different natural Arabidopsis accessions. We conclude that manipulation of the starch degradation pathway represents a promising target for improving plant yield and stress tolerance. We hypothesise that stress-induced reprogramming of starch degradation plays a central role in the orchestration of photosynthetic metabolism rather than being a pure consequence from cold-induced metabolic changes. Together with reprogramming of translational regulation and protein synthesis it seems to differentially affect the cold-induced metabolic homeostasis which finally contributes to the observed acclimation output.