Discussion
SLs fulfil critical functions in regulating plant growth and development, but the roles of these hormones in plant/insect interactions and their responses to changing atmospheric [CO2] had not been characterised. The data presented here show that growth under high (750±50 ppm) CO2significantly increases shoot height, biomass accumulation and branching in all genotypes. While all lines showed increased dry matter accumulation (by 40% when measured 32 days after germination), elevated [CO2] significantly decreased shoot water content of wild type but not the SL mutants. This finding is surprising sinceA. thaliana SL mutants had higher stomatal conductance than wild type plants, and their stomatal responses to high CO2were impaired (Kalliola et al., 2020). Moreover, such mutants have increased sensitivity to drought stress (Ha et al ., 2014). Substantial crosstalk between cytokinins (CKs) and SL signalling pathways in plant drought acclimation has been proposed, with CKs and SLs acting as negative and positive regulators of plant drought responses respectively (Li et al., 2019).
Although SL-deficient plants are often drought-sensitive due to their constitutively higher stomatal conductance (Arabidopsis – Ha et al. 2014; tomato - Visentin et al. 2016) and relative stomatal insensitivity to ABA (Arabidopsis – Ha et al. 2014), the rms pea mutants maintain normal stomatal conductance under optimal conditions (Dodd et al. 2008; Cooper et al. 2018). Impaired stomatal responses to high CO2 in SL-signalling and -biosynthesis Arabidopsis mutants (Kalliola et al., 2020) may have compromised stomatal regulation of plant water relations, but paradoxically the rms lines maintained shoot FW/DW ratio under elevated CO2 here unlike WT plants (Fig. 4C). Since this ratio indicates plant succulence (Mantovani 1999), the high proportion of plant biomass incorporated as young, succulent branching shoots in the rms lines probably accounts for this divergent phenotype under elevated CO2. Even though lignification of plant tissues (which would decrease FW/DW ratio) has not been investigated in SL mutants, impacts of elevated CO2 on xylem anatomy (Liu et al. 2019) and lignification (Richet et al. 2012), may involve SLs, especially since at least some SL-signalling mutants show diminished xylem development (Dodd et al. 2008).
Shoot branching is regulated by CK, SL and auxin crosstalk in axillary buds. The data presented here demonstrate the negative effect of SL on pea axillary bud outgrowth i.e. branching under both ambient and high CO2 conditions. In peas, regulation of SL and CKs converge on the BRANCHED1 (BRC1) transcription factor, which represses bud outgrowth, independent of auxin signals (Brewer et al., 2015). Long-distance transport of SL occurs in the xylem sap (Kohlen et al., 2011), with regulation of SL loading and unloading that regulates bud suppression apparently occurring very close to buds. SLs regulate rice shoot architecture through enhanced cytokinin catabolism (Duan et al., 2020). SL modulates the expression of the OsCKX9 gene, which encodes a CK oxidase, in the regulation of rice tillering, plant height, and panicle size (Duan et al., 2020). CK such as trans-zeatin also play an important role in plant immunity. The data presented show that the levels of trans-zeatin, which is considered to induce resistance to biotrophic pathogens, were differentially changed in response to high [CO2] in the rms3-1 and rms4-1mutants. While the aphid- infested rms4-1 mutants had similar trans-zeatin levels to the wild type, the rms3-1 mutants had significantly less trans-zeatin under both growth conditions. Therms3-1 mutant is defective in the pea orthologue of the rice D14 SL receptor (de Saint Germain et al ., 2016), and hence may be more receptive to the aphid-induced signalling pathways thanrms4-1 mutants, particularly in air. Hence, SL-dependent activation of CK signalling may be important in the resistance of peas to aphid infestation.
Plant defences against root knot nematodes involves the activation of JA- and ABA-mediated defences, which are suppressed in the absence of SL (Xu et al., 2019). The data presented here show that ABA levels were similar in all the aphid-infested lines. Moreover, JA levels were significantly increased by high [CO2] in the wild type and rms3-1 mutants. However, high [CO2] had no significant effect on aphid fecundity in any of the lines, suggesting that these changes in JA levels were not important in aphid resistance. In contrast, the levels of gibberellic acid were lower at high [CO2] in all lines. Moreover, gibberellic acid levels were significantly lower in the rms4-1 mutants, than the wild type peas under both growth conditions. These data not only show that gibberellic acid levels are decreased in the aphid-infested SL mutants. Cross talk between SL and gibberellic acid signalling has been reported in the responses of rice plants to Striga infection (Ito et al., 2017). Gibberellic acid was shown to be a regulator of the expression of rice SL biosynthesis genes, while gibberellic acid -treated rice showed reduced Striga infection (Ito et al., 2017). Moreover, the SL receptor, D14, interacts with the gibberellic acid signalling repressor, SLR1 (Nakamura et al., 2013). While there have been very few reports of the role of gibberellic acid in plant/aphid interactions, the levels of this phytohormone were decreased in response to aphids (Wang et al., 2016).
In summary, the data presented here demonstrates that growth under high CO2 does not alter the fecundity of the pea aphid infesting pea plants grown with a full nitrogen supply. Growth under eCO2 decreased the levels of SA and increased JA levels in the wild type peas, without any significant effect on aphid fecundity. However, the absence of SL-mediated defences led to a significant increase in aphid fecundity. This analysis also linked aphid performance to the levels of gibberellic acid in the infested plants. Taken together, these data support the view that plants perceive high CO2 as a stress, as evidenced by changes in phytohormones (Foyer and Noctor, 2020). How far eCO2stress impacts on plant insect interactions is variable but it likely to depend on other environmental factors as well as the species involved e.g. generalist feeders verses specialists.