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.