Introduction
Drought is a leading cause of plant mortality (Brodribb et al. ,
2020) with the severity and frequency of droughts driving plant
evolution and species distributions (Engelbrecht et al. , 2007;
Bowles et al. , 2021). Death caused by drought is largely due to
the formation of embolism in the xylem which blocks water transport
leading to hydraulic failure and tissue desiccation (Brodribb &
Cochard, 2009; Urli et al. , 2013; Cardoso et al. , 2020;
Brodribb et al. , 2021). A key adaptation in vascular plants that
prevents declines in water potential and thus embolism formation during
drought is the closure of stomata (Martin-StPaul et al. , 2017;
Brodribb et al. , 2021). Stomata are dynamic valves on the surface
of leaves that open and close in response to environmental and
endogenous signals (Raschke, 1975). During drought stomata close to
prevent excessive evaporation. The mechanisms that drive stomatal
closure during drought have long been debated (Tardieu & Davies, 1993;
McAdam & Brodribb, 2014). In seed plants the phytohormone abscisic acid
(ABA) plays a critical role in closing stomata during drought stress
(Mittelheuser & Van Steveninck, 1969; Jones & Mansfield, 1970; McAdam
& Brodribb, 2012). ABA biosynthesis during drought is believed to be
triggered by a loss of cell turgor as leaves dehydrate, with peak ABA
biosynthesis occurring at a Ψl that is close to turgor
loss point (Ψtlp) in herbaceous species (Pierce &
Raschke, 1980; Davies et al. , 1981; Creelman & Mullet, 1991;
McAdam & Brodribb, 2016).
In most herbaceous species, as well as tree species with relatively
vulnerable xylem, as drought progresses ABA levels increase and continue
to do so until embolism forms (Zeevaart, 1980; Brodribb et al. ,
2014). Brodribb and McAdam (2013) investigating ABA dynamics under long
term drought in conifers discovered a divergent strategy in ABA
dynamics. In the highly drought tolerant Cupressaceae speciesCallitris rhomboidea R. Br. ex Rich. & A. Rich.(Cupressaceae) native to arid regions of southeastern Australia (Crispet al. , 2019), stomata closed at the onset of drought stress
driven by an increase in ABA levels (Brodribb & McAdam, 2013). However,
once plants were dehydrated to –4 MPa, ABA levels stopped increasing,
and over the subsequent 10 days of soil drought declined to prestress
levels (Brodribb & McAdam, 2013). The reduction in ABA levels under
long-term drought in Callitris meant that stomata transitioned
from closure being driven by ABA to closure being the result of a
passive reduction in guard cell turgor, similar to the mechanism of
stomatal closure under drought in ferns and lycophytes which have
stomata that are insensitive to ABA (McAdam & Brodribb, 2012). This
dynamic of ABA levels during drought was termed a “peaking-type”
(p-type) ABA dynamic and has subsequently been well characterized across
the conifer phylogeny, being associated with the evolution of highly
resistant xylem, defined as xylem requiring at least –4 MPa of tension
to induce embolism in at least 50% of the xylem (Brodribb et
al. , 2014). Conifer species from both the derived Cupressaceae
(including species from both the Southern Hemisphere callitroid and
sister Northern Hemisphere cupressoid clades) as well as Taxaceae have
evolved a p-type ABA dynamic under long-term drought (Brodribb et
al. , 2014). P-type ABA dynamics have been observed in the field inC. columellaris F.Muell in which six months of no rainfall each
year in the dry season in Northern Australia leads to a seasonal p-type
ABA dynamic, such that at the end of the dry season stomata are closed
yet ABA levels are as low as plants in the middle of the wet season when
Ψl are highest (McAdam & Brodribb, 2015). Three studies
so far have documented a p-type ABA dynamic in angiosperm species (as
recently reviewed by Hasan et al. (2021)). One study documented
this response in the considerably drought tolerant Central Australian
native tree Acacia aptaneura Maslin and J.E.Reid (Fabaceae)
(Nolan et al. , 2017) while two studies have documented the
response across six species of arid adapted Caragana (Fabaceae)
native to Inner Mongolia, China (Yao et al. , 2021a; Yao et
al. , 2021b). All of the angiosperm species in which a p-type ABA
dynamic during drought has been observed have highly resistant xylem to
embolism formation with the Ψl of peak ABA occurring
between –3.5 and –4 MPa (Nolan et al. , 2017; Yao et al. ,
2021a; Yao et al. , 2021b). From these observations we would
hypothesize that highly resistant xylem is required for the evolution of
a p-type ABA dynamic across seed plants, and not just in gymnosperms
(Brodribb et al. , 2014). Resolving the mechanistic unknown
driving the p-type ABA dynamic during drought remains challenging
because, while highly resistant xylem has evolved independently in at
least 130 species from 62 genera and 20 orders of seed plants (McAdam &
Cardoso, 2018), there still remains no species with resistant xylem that
yet has a sequenced genome. This lack of genetic information means that
resolving the mechanism driving ABA level decline under long term
drought requires a more classical physiological and biochemical approach
(Hasan et al. , 2021).
There are a number of possible drivers for the p-type dynamic in ABA
levels during long term drought. Given that more than 90% of
accumulated ABA synthesized under drought is catabolized into the
primary catabolite phaseic acid (PA) when plants are rewatered
(Milborrow, 1974), one explanation for the decline in ABA levels during
long term drought in p-type species could be activated ABA catabolism.
ABA is catabolized into PA by two biochemical steps encoded by
cytochrome P450 CYP707A genes, the expression of these genes is
upregulated when plants are rewatered during drought stress, and when
plants are exposed to high humidity (Kushiro et al. , 2004;
Okamoto et al. , 2009). ABA can also be reversibly inactivated by
conjugation with UPD-glucose to abscisic acid-glucose ester (ABA-GE)
(Milborrow, 1970; Lee et al. , 2006). ABA-GE can be stored in the
vacuole (Burla et al. , 2013), as well as exported from the leaf
in the phloem (Zeevaart & Boyer, 1984). Conjugation occurs by a single
biochemical step, catalysed by two isoforms of β-glucosidase (Leeet al. , 2006; Xu et al. , 2012). There could be an enhanced
rate of conjugation of ABA to inactive forms under long term drought
that explains the decline in ABA levels. An additional explanation for
the decline in ABA levels under long term drought could be the cessation
of de novo biosynthesis of ABA. The loss of cell turgor is a
well-described trigger for increasing the expression the gene encoding
the rate limiting step in ABA biosynthesis in angiosperms,
9-cis -epoxycarotenoid deoxygenase (NCED3 inArabidopsis ) (Qin & Zeevaart, 1999; Sussmilch et al. ,
2017; Bacete et al. , 2022), only a relief of low cell turgor, via
rehydration, is known to decrease the expression of this rate limiting
step gene in the ABA biosynthetic pathway (Qin & Zeevaart, 1999). While
never described the cessation of de novo ABA biosynthesis at a
threshold Ψl under drought would lead to a decrease in
ABA levels that is independent of changes in the rate of ABA catabolism
or conjugation. A final, but least likely explanation, is an increase in
the rate of ABA export from the leaf via the phloem, phloem flux from
the leaf is a major sink for foliage derived ABA (Jeschke et al. ,
1997; Castro et al. , 2019). Given that the rate of phloem flux is
presumably low or non-existent when assimilation has ceased during
drought (Sevanto, 2014), a common occurrence when stomata are closed,
this seems the least likely driver for a decline in ABA levels under
drought.
Here we sought to characterize the mechanism driving declines in ABA
levels under long term drought in p-type seed plant species. We
conducted experiments on two species, the model system for
characterizing p-type ABA dynamics, the gymnosperm species C.
rhomboidea (Cupressaceae) native to South Eastern Australia and the
highly drought resistant evergreen angiosperm species Umbellularia
californica (Hook. & Arn.) Nutt. (Lauraceae) native to coastal forests
and the foothills of the Sierra Nevada in Western North America (DiLeoet al. , 2014). Ψl, canopy conductance
(gc ), foliage ABA, PA and conjugate levels were
measured in potted plants of each species through a prolonged drought
treatment until a non-fatal Ψl was reached. We tested
four mechanistic hypotheses for the p-type ABA dynamic: (1) increased
catabolism of ABA into either PA or (2) conjugation, occurs at a
threshold Ψl, (3) a cessation of ABA biosynthesis,
driven by osmotic adjustment or turgor loss, and/or (4) the phloem
export of ABA from leaves. The role of export was assessed by girdling
branches in drought stressed plants. We developed a novel technique
based on bench dehydration to assess the ability of shoots to rapidly
synthesize ABA to assess whether ABA biosynthesis was deactivated under
long-term drought.