Previous work had shown a role for jasmonate signalling in anthocyanin accumulation [19, 28, 29] and for freezing tolerance [10, 11]. It was also shown that jasmonate-induced anthocyanin formation is sucrose dependent [19] and that cold treatment results in sucrose accumulation [2, 8]. Anthocyanin synthesis, in turn, has been related to freezing tolerance [15], suggesting that the jasmonate and sucrose signalling pathways interact. Here, we analysed the interactive effects of sucrose and cold (chilling) stress on anthocyanin formation and hormone contents in wild-type Arabidopsis and in jasmonate signalling mutants.
We found clear impacts of medium type, sucrose availability and temperature on hormone, anthocyanin and sugar contents (Fig. 5). Although the jar1–1 mutant showed the expected impairment in JA-Ile accumulation (Fig. 3) and compost-grown coi1–16 plants had increased sugar contents (Fig. 2) and altered hormone responses to temperature (Figs. 3 and 4), disruptions in the cold acclimation pathways were temporary (Fig. 6) and the mutants were able to cold acclimate and accumulate anthocyanins (Fig. 1).
Hormone contents are affected by the growth medium and temperature
One of the most striking finding was that contents of several stress hormones, JA, JA-Ile, JA-Leu, SA and ABA, were substantially higher in compost than on agar medium (Figs. 3 and 4). Jasmonate synthesis is associated with biotic stress [9], but no signs of pathogen infection or insect infestation were observed, and the plants looked healthy. The formation of defence hormones (jasmonates and SA) could be a response to the microbial environment, which suggests that the contents of these hormones may be considerably higher in nature than under sterile conditions on agar plates. However, other factors (development or nutrient availability) than exposure to microbes may have contributed to differences between agar- and compost-grown plants.
In addition, sucrose addition to the growth medium affected hormone contents, e.g. increasing the contents of JA-Leu, ABA, SA and IAA (Additional file 4). Research on jasmonate signalling has often been carried out using seedlings cultivated with routine addition of sucrose to the growth medium [e.g. 28, 29, 35, 38], which does not allow differentiation between sucrose-dependent and -independent jasmonate responses. At the sucrose concentration of 55 mM (equivalent to 1.88% w/v) that was used in our experiments, sucrose contents exceeded those in plants grown under more natural conditions in compost (Fig. 2). Routine addition of similar concentrations of sucrose to growth medium (e.g. 2% [38] or 3% [29]) may result in unnaturally high sugar contents in the plants that can affect plant responses through interactions between sugar with hormone signalling. In addition, the induction of jasmonates, SA and ABA by sucrose on agar medium may have been related to sucrose-induced senescence, as indicated by a drop in Fv/Fm in the presence of sucrose (Fig. 1c and Additional file 2).
Temperature also has an effect on hormone contents, partially in interaction with sucrose availability. In the alpine perennial, Arabis alpina, JA and zeatin contents were increased at low temperature, while IAA content was reduced; however, these effects were genotype-specific [8]. Here, JA-Ile (with and without sucrose addition) and JA-Leu (only with sucrose addition) contents were decreased by the cold treatment (Fig. 3), whereas there was an overall increase in SA and IAA contents in compost in response to cold treatment (Fig. 4). While JA conjugates were not measured in A. alpina [8], the effect on IAA content suggests a difference in the response in Arabidopsis compared to the alpine perennial. Differences in changes in hormone contents in response to cold treatment were also found between a winter and spring cultivars of wheat, including a high IAA content in the spring cultivar after long-term cold treatment, which may be linked to lower freezing tolerance [39].
Interactions between temperature and sucrose treatments were observed for GA3 which increased in response to sucrose, but only at warm temperature. This is consistent with the role of gibberellins in growth and a reduction in bioactive GAs by the CBF-dependent cold acclimation pathway [40].
Pathways for the induction of anthocyanin formation by sucrose and cold treatment
Our analysis confirms a close relationship between sugar and anthocyanin contents across the treatments (Fig. 5 and Additional file 6). Since senescence typically results in anthocyanin accumulation, higher anthocyanin content may reflect the early senescence observed after growth on sucrose-containing medium at warm temperature. Sucrose induces anthocyanin accumulation via MYB75 [17, 18], while low temperature probably acts via MYB90 [15]. It was therefore suggested that anthocyanin accumulation at low temperature is not induced by sucrose. Here, cold treatment further increased anthocyanin accumulation in the presence of external sucrose in an additive manner (Fig. 1), despite the observation that there was no cold-related increase in sucrose content when sucrose was supplied externally (Fig. 2). This supports the view that the pathways for sucrose and cold-induced anthocyanin synthesis act independently.
The cold-induced accumulation of anthocyanins in the presence of sucrose could be explained with a decline in gibberellin signalling. Similar to degradation of JAZ proteins in the presence of JA-Ile, GAs induce the degradation of repressors, the DELLA proteins. During cold stress, DELLA proteins accumulate as gibberellins are degraded which results in growth inhibition [40]. A mechanism for crosstalk between the gibberellin and jasmonate signalling pathways that can explain the opposite effects of these groups of hormones in anthocyanin formation has been proposed [33]. By binding to JAZ proteins, DELLA proteins activate jasmonate-responsive genes. Degradation of DELLAs by GAs, on the other hand, represses jasmonate signalling. Moreover, it was recently proposed that GA and JA signalling in anthocyanin synthesis are integrated by the MBW transcriptional complex which includes MYB75 [35]. Sequestration of JAZ proteins by DELLA proteins thus results in MBW-dependent anthocyanin synthesis e.g. during cold stress. This crosstalk of gibberellins and jasmonates is supported by the finding that sucrose-dependent induction of MYB75 and anthocyanin synthesis were reduced in a quadruple della mutant of Arabidopsis [34]. The further induction of anthocyanin formation at cold temperature in the presence of sucrose could therefore be a result of lower contents of active GAs, such as GA3 at cold compared to warm temperature (Fig. 4). Such a mechanism could also explain how the DELLA pathway could activate jasmonate signalling downstream of the COI1/JA-Ile pathway in the mutants (see below).
The jar1–1 and coi1–16 mutants have altered hormone contents
Accumulation of ABA in response to water deficit was much lower in the jar1–1 and coi1–16 mutants than wild-type plants [31], suggesting that jasmonate signalling acts upstream of ABA. In contrast, it was proposed for tomato that JA acts downstream of ABA in cold signalling [41]. ABA contents were not reduced in the mutants in response to cold stress, although the coi1–16 mutant showed unusual patterns of ABA accumulation after growth on compost (Fig. 4). In addition to ABA, the coi1–16 mutant contained increased amounts of JA-Leu and JA-Ile compared to its wild type after growth in compost at warm temperature. SA and IAA contents, on the other hand, were reduced compared to wild type at low temperature. This suggests interactions of altered jasmonate signalling with hormone synthesis pathways. Moreover, sugar contents were significantly increased in the coi1–16 mutant in compost (Fig. 2).
The jar1–1 mutant was unable to accumulate JA-Ile and JA-Leu under any of the conditions tested, but did contain small amounts of JA-Ile (Fig. 3). This is in agreement with previous reports of some JA-Ile being formed in the jar1–1 mutant [21, 22] and indicates that there may be another enzyme that can catalyse the formation of JA conjugates. The Km value of the JAR1 enzyme for Ile is considerably lower than its Km for Leu, Val and Phe, suggesting that it primarily catalyses the conjugation of JA with Ile [22]. Our results do, however, suggest that JAR1 is also involved in JA-Leu synthesis. Accumulation of the precursor of jasmonates, OPDA, on agar medium at cold temperature (Fig. 3) indicates a feedback effect of lower contents of the JA conjugates in the jar1–1 mutant.
No evidence for an essential role of jasmonate signalling in anthocyanin accumulation and cold acclimation
In addition to impaired ABA formation during drought stress [31], both the jar1–1 and coi1–16 mutants have been shown to be hypersensitive to ozone [32, 42], supporting the view that these mutant alleles are impaired in stress responses. Low temperature treatment results in JA accumulation, and application of external JA can increase freezing tolerance. In addition, jar and coi1 mutants showed decreased freezing tolerance with and without cold acclimation [10]. However, their physiological response to low, but above-freezing (chilling) temperatures was not explored. While we detected differences in the development of cold acclimation (Fig. 6), both mutants were able to acclimate and reached normal Fv/Fm values. In addition, anthocyanin accumulation in response to cold (Fig. 1) or sucrose treatment (Additional file 3) was not impaired. However, our results do not allow us to draw direct conclusions concerning freezing tolerance.
The ability of the jar1–1 mutant to accumulate anthocyanins is in agreement with previous findings for jasmonate and sucrose responses in this mutant [19, 30]. Normal sucrose-dependent induction of anthocyanin formation in jar1–1 could be explained with the presence of small amounts of JA-Ile (Fig. 3; see also [21, 22]). However, the inability of the jar1–1 mutant to accumulate JA-Ile in response to sucrose makes it unlikely that JA-Ile synthesis is required for increased anthocyanin content. In our experiments, JA-Ile and anthocyanin contents were negatively (and not positively) correlated (Additional file 6), supporting the view that JA-Ile accumulation is not required for the induction of anthocyanin formation in response to sucrose and low temperature.
Normal anthocyanin contents in the coi1–16 mutant contradict the previous view that, in contrast to the upstream signalling component JAR1, COI1 is required for sucrose-dependent anthocyanin formation [19]. The observation that anthocyanins accumulated in the coi1–16 mutant here can possibly be explained with the fact that coi1–16, in contrast to coi1–1, is leaky. While coi1–1 is male sterile, coi1–16 produces fertile pollen at a temperature of 16 °C (but not at 22 °C) and can therefore be maintained as a homozygous line [30]. To overcome the problem of male sterility, the previous study [19] selected homozygous coi1–1 seedlings on JA-containing medium and performed experiments on leaf strips, showing that expression of the dihydroflavonol reductase (DFR) gene for anthocyanin synthesis was not induced by sucrose, MeJA, or a combination of both, in coi1–1. However, anthocyanin content itself was not measured in coi1–1 and gene expression changes were reported after 24 h, whereas treatments were much longer here. It has been described that the coi1–1 mutant allele accumulates anthocyanins normally when plants are germinated under continuous light or exposed to water deficit [23], supporting our findings that jasmonate signalling does not play a major role in anthocyanin accumulation, as shown here for cold stress, despite effects on the regulation of genes involved in anthocyanin synthesis as demonstrated earlier [19].
Other experiments showing a role of jasmonate signalling in anthocyanin formation, e.g. using the leaky coi1–2 mutant allele, were mainly conducted in seedlings [28, 29]. Since the anthocyanin contents presented here are the endpoint of cold-treatment of mature plants for 15 (in compost) or 25 (on agar plates) days, even a lower initial rate of anthocyanin accumulation may have resulted in normal final contents in the coi1–16 mutant. In the presence of sucrose, when GA3 content was reduced by cold temperature (Fig. 4), antagonistic effects between gibberellin and jasmonate signalling through sequestration of JAZ proteins by DELLA [33] could activate jasmonate signalling. As this interaction occurs downstream of the JA-Ile/COI1 pathway, it could compensate for the jar1–1 and coi1–16 mutations under cold conditions, and thus result in anthocyanin accumulation in response to sucrose.
If JAR1 is required upstream of COI1, then jar1 mutants should show the same defects and increased stress responses as coi1 mutants. However, the jar1–1 mutant accumulated the precursor of JAs, OPDA, on agar medium at cold temperature (Fig. 3). OPDA is proposed to act independently of jasmonate signalling [26] which could potentially explain normal anthocyanin formation in this mutant. However, a new pathway of JA synthesis from OPDA was recently discovered, which supports the view that OPDA effects are mediated by JA formation and signalling through the JA-Ile/COI1 pathway [43]. Overall, it is unlikely that sugar-induced anthocyanin synthesis would require COI1, but not JA-Ile formation by JAR1.