Discussion
Here we document the occurrence of p-type ABA dynamics in an angiosperm
species outside of Fabaceae, the evergreen Lauraceae species U.
californica . In U. californica foliage levels of ABA during long
term drought showed a highly similar pattern as Ψldeclined to that of the classical model species that initially
characterized p-type responses of foliage ABA levels during long term
drought, the conifer species C. rhomboidea (Brodribb & McAdam,
2013). This result, coupled with previous reports of p-type ABA dynamics
in species from two genera in Fabaceae (Nolan et al. , 2017; Yaoet al. , 2021a; Yao et al. , 2021b), all adapted to
seasonally dry or arid environments and with highly embolism resistant
xylem (no leaf death was seen in our drought experiment in either
species experienced a Ψl of -6 MPa), suggests that the
evolution of the p-type ABA response to long-term drought is linked to
the evolution of highly resistant xylem, and is not just a conifer and
Fabaceae specific phenomenon. Highly resistant xylem has evolved
frequently across angiosperm species (McAdam & Cardoso, 2018)
suggesting that if the two are linked, highly embolism resistant xylem
and the p-type ABA dynamic (Brodribb et al. , 2014), this ABA
response to drought could be quite commonly observed across angiosperms.
Taken together our results demonstrate the occurrence of a p-type ABA
response now in two highly divergent angiosperm families, the early
diverging Lauraceae and the Fabaceae (Nolan et al. , 2017; Yaoet al. , 2021a; Yao et al. , 2021b), future studies are
needed to investigate whether this response is common across angiosperms
with highly resistant xylem. The absence of high levels of ABA under
long term drought in angiosperm implies that, like in conifers, the
stomata of all p-type species may be closed passively by low
Ψl under long term drought (Brodribb & McAdam, 2013;
McAdam & Brodribb, 2015). This is a controversial hypothesis for
angiosperm stomatal biology (Franks, 2013; Merilo et al. , 2017),
especially given that angiosperm ABA biosynthetic and signalling mutants
have stomata that are insensitive to changes in leaf water status
(McAdam et al. , 2016; Cernusak et al. , 2019; Brodribbet al. , 2021). It has been suggested that passive regulation of
stomatal aperture in response to changes in leaf water status is absent
from this group of land plants (McAdam & Sussmilch, 2020). Recently in
characterizing p-type ABA dynamics in Caragana , Yao et al.(2021a) suggested that ethylene might be closing stomata during drought
and on recovery from drought when ABA levels are low but stomata are not
yet open to maximum apertures. Further work is required to address
whether the stomata of p-type angiosperms are closed at low
Ψl passively via low cell turgor pressure, or via an
alternative metabolic signal such as ethylene (Hasan et al. ,
2021).
Similarities in the dynamics of ABA, catabolite and conjugate levels, as
well as the inhibition of dehydration-induced ABA biosynthesis during
drought between the p-type angiosperm and conifer species suggests that
there is a shared mechanism driving the decline in ABA levels under long
term drought stress in seed plants. By rapidly dehydrating branches on
the bench we could assess the ability of leaf tissue to rapidly
synthesize ABA in response to dehydration (Wright & Hiron, 1969). This
technique allowed us to study ABA biosynthetic capacity without the need
to quantify the expression of key ABA biosynthetic genes, which can be
costly and time consuming and requires a detailed understanding of the
homologues of key genes (Sussmilch et al. , 2019). Despite
considerable investment in genome sequencing in the past decade (Kresset al. , 2022), we still do not have for any species with highly
resistant xylem to embolism. We show that ABA biosynthesis is highly
active and rapid in unstressed branches that are rapidly dehydrated on
the bench, like numerous early studies into ABA biosynthesis in plants
(Wright & Hiron, 1969; Pierce & Raschke, 1980; Davies et al. ,
1981). This ability is eliminated in branches that are taken from plants
when ABA levels are low under long-term drought and rehydrated overnight
on the bench before dehydration (Figure 4). A key limitation to this
method is that it relies on quantifying a change in ABA level which is
only possible if there are low levels of the hormone at the start of the
experiment, hence we are unable to use this method to determine
precisely when dehydration induced ABA biosynthesis was deactivated
during drought, because for much of a drought ABA levels were high
(Figure 1). We could speculate that the point at which ABA biosynthesis
was deactivated corresponded to the Ψl close to when ABA
levels peaked during drought. Coincidently, we found that the
Ψl at which peak ABA levels occurred was very similar to
the Ψtlp (Figure 8). Work is required to confirm that
the expression of key ABA biosynthetic genes such as
Nine-cis-epoxycarotenoid deoxygenase (NCED ) genes are no longer
upregulated on rapid dehydration in branches that do not rapidly
synthesize ABA when dehydrated on the bench (Hasan et al. , 2021).
As cells loose turgor or lose volume ABA biosynthesis is triggered
(Pierce & Raschke, 1980; Davies et al. , 1981; Creelman &
Mullet, 1991; McAdam & Brodribb, 2016), yet there has been very little
work conducted on plant tissue that has been dehydrated to a
Ψl more negative than
Ψtlp.. Consequently, the potential
causes of ABA biosynthesis cessation at a Ψl more
negative than Ψtlp are highly speculative. Explanations
range from an absent trigger for NCED expression once membrane
pressure on the cell wall ceases (Bacete et al. , 2022); cellular
processes such as transcription and translation of RNA ceasing at a
Ψl more negative than Ψtlp (Dhindsa &
Cleland, 1975); or carotenoid precursors for ABA biosynthesis, often
sorted in chloroplasts, may be depleted because of increased in the
de-epoxidation state of the xanthophyll cycle (Munné-Bosch & Alegre,
2000), reducing availability for conversion to ABA. Munné-Bosch and
Alegre (2000) found in the extremely drought resistant Rosmarinus
officinalis , in which 50% of the xylem experiences embolism at a
Ψl at -8 MPa (Brodribb et al. , 2017) during a
severe summer drought the levels of ABA carotenoid precursors
violaxanthin and neoxanthin declined by more than 85% maximum levels.
We show that the ability to recover ABA biosynthesis can occur, with ABA
levels accumulating, but not to levels in never-before stressed branches
4 days after rewatering. This recovery might reflect the rapid recovery
of carotenoid levels upon rehydration (Munné-Bosch & Alegre, 2000).
Future studies in herbaceous species at a Ψl more
negative than Ψtlp could help elucidate whether the
mechanism driving ABA biosynthesis cessation after Ψtlpis universal to all plants. These experiments can only be done in
strictly controlled environments since a slight decrease in
Ψl once turgor is lost could trigger lethal embolism as
the two occur at very similar Ψl in herbaceous plants
(Skelton et al. , 2017). There is a hypothesis that ABA
biosynthesis is triggered by a loss of cell volume and not a change in
cell turgor (Sack et al. , 2018), our results refute the
hypothesis ABA biosynthesis ceases in p-type species yet cell volume
presumably continues to decline at a fairly linear rate during drought.
This corroborates recent data that indicates an intact cell wall is
essential for the biosynthesis of ABA when cells are exposed to
solutions of high osmotic potential (Bacete et al. , 2022).
Once ABA biosynthesis ceases at a Ψl more negative than
Ψtlp our results suggest that continual conjugation of
ABA, presumably into ABA-GE, which is the primary, if not only,
conjugate for ABA (Milborrow, 1970), and not catabolism of the remaining
is the main driver for a decline in foliage ABA levels once ABA
biosynthesis ceases. It is believed that phloem flux is greatly reduced
during drought (Hartmann et al. , 2013; Sevanto, 2014), and our
results from a girdling experiment in C. rhomboidea demonstrate
that girdling the phloem does not change the p-type ABA dynamic during
drought or the accumulation of ABA conjugates. By quantifying the levels
of PA during long term drought, we are able to categorically rule out
catabolism was the primary driver of a decrease in ABA levels after
Ψtlp. Our results
demonstrate that catabolism of ABA into PA did not significantly
increase after peak ABA levels. Interestingly, the dynamic of PA levels
and ABA levels during drought suggests that PA levels and the rate of
ABA catabolism could be a simple function of current ABA level.