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.