Misleading conclusions from exogenous ABA application: a cautionary tale about the evolution of stomatal responses to changes in leaf water status

ABSTRACT

Stomatal responses to changes in leaf water status are critical for minimizing excessive water loss during soil drought. A major debate has surrounded the evolution of stomatal responses to water status and this debate has particularly focused on the evolution of the regulatory role of the drought hormone abscisic acid (ABA). Studies relying on the application of high levels of exogenous ABA have occasionally concluded that all stomata respond to ABA and that stomatal regulation in response to this hormone has not evolved over the past 450 million years. In contrast, studies which have investigated stomatal function in intact plants, as well as the role of endogenous ABA in regulating stomatal aperture, have found major evolutionary transitions in the functional regulation of stomata across land plant lineages. We show that endogenous ABA plays no role in closing the stomata of the fern Nephrolepis exaltata during natural soil drought, in contrast to a recent finding using isolated epidermis and exceptionally high levels of exogenous ABA. We conclude that stomatal behavior in intact plants has evolved over time, and may have shaped the evolutionary and ecological success of successive land plant lineages.

KEYWORDS:

• ABA signaling
• drought stress
• floating epidermis
• leaf gas exchange
• leaf hydraulics
• stomatal evolution and behavior

Leaves continuously regulate the rate of water loss to the atmosphere by modifying stomatal aperture in response to both environmental and endogenous signals. During periods of water limitation in particular, stomatal closure is a critical tool for vascular plant survival, minimizing transpiration and preventing plant dehydration.^1,2 In angiosperms, stomatal closure during water stress is mediated by the phytohormone ABA;^3–5 however, contradictory results fuel an ongoing debate about the evolutionary origin of this hormonal regulation of stomata.^6–8

In contrast to vascular land plants, in which stomata mainly occur on leaves, in two lineages of bryophytes (the earliest diverging clades of land plants), the mosses and hornworts, stomata are restricted to the sporophyte.⁹ An early report of stomata on the sporophyte in the moss Physcomitrella closing in response to exogenous ABA suggested an early evolutionary acquisition of stomatal control by this hormone.⁸ A number of more recent studies on stomata of hornworts and mosses, however, demonstrate no stomatal responses to exogenous ABA.^10–12 Furthermore, the stomata of hornworts and mosses are anatomically and physiologically constrained such that they lack the capacity to alter stomatal aperture once the pore opens.^10,11,13,14 There is elegant evidence showing that the stomata of bryophytes open once at a single developmental stage and that this opening plays a primary role in sporophyte desiccation and spore discharge. This is the opposite function of vascular plant stomata, which close to prevent desiccation. The opening of some bryophyte stomata may even be by an opposite mechanism to that of vascular plant stomata, opening when the guard cells loose turgor.¹⁵

The major evolutionary transition in stomatal function between bryophytes and tracheophytes makes the ferns and lycophytes which are the earliest diverging, extant lineages of vascular land plants, the most important groups of plants for reconstructing physiological evolution in the tracheophytes.¹⁶ Understanding functional stomatal behavior in species from these early diverging lineages is essential for identifying the major evolutionary transition(s) that must have occurred in guard cells to cause the macro-shift in stomatal function between bryophytes and tracheophytes. Given the corresponding major shifts in ecological niche over time (from perennial swamp to seasonally dry uplands) and the association of these shifts with the emergence of new lineages of vascular plant,¹⁷ physiological explanations for these transitions remain surprisingly limited.¹⁸ It is tempting to hypothesize that the evolution of stomatal function, which is so critical for angiosperm survival in dry environments,² may have played a role over evolutionary time in the invasion of increasingly drier habitats by terrestrial plants.¹⁹ It seems that the evolution of the unique dumbbell stomata of grasses is associated with increased stomatal speed and responsiveness,²⁰ suggesting that there is indeed a possibility that stomatal function has evolved across the tracheophytes. Furthermore, there is evidence of major changes across tracheophyte lineages in stomatal regulation in response to light and as well as CO₂ concentration when altered in the dark,²¹ these changes have been associated with adaptations to light environments.

To address whether differences in the regulation of stomatal responses to leaf water status occur across the lineages of vascular plants, in an early study Brodribb and McAdam²² found that the stomata of a diversity of fern and lycophyte species were unresponsive (as measured by leaf gas exchange) to the feeding of exogenous ABA into the transpiration stream. Subsequently, in other species of fern and lycophyte, a lack of stomatal responses to ABA application has been found.^23–25 The most convincing data indicating that stomatal responses to changes in leaf water status in fern and lycophyte species are not regulated by ABA is the observation that the stomata of drought-stressed fern and lycophyte species rapidly reopen to pre-drought levels when instantaneously rehydrated, despite high endogenous levels of foliar ABA accumulated during drought.^26–28 This rapid reopening of stomata when endogenous ABA levels are high is never observed in seed plants, in which the accumulated endogenous ABA keeps stomata closed.^26,29,30 Instead of the metabolic regulation of stomatal responses to changes in leaf water status via ABA as typically observed in angiosperms,^3,31,32 the stomatal responses to changes in leaf water status in a diversity of fern and lycophyte species can be predicted accurately by a simple model that assumes guard cell turgor changes passively with leaf turgor.^22,33,34 The classical ABA signaling pathway responsible for closing stomata in angiosperms has also been found to regulate gametophyte sex determination and spore dormancy in the fern Ceratopteris richardii and not stomata, suggesting alternative ancestral functions for this plant hormone signaling pathway.³⁵

The conclusions from these data have, however, received continual criticism from studies in which stomatal aperture or leaf gas exchange are monitored in some fern and lycophyte species following the application of high levels of exogenous ABA.^6,23,24,36 These contrasting results are championed as evidence that stomata from all species of land plant are functionally responsive to ABA, and consequently that stomatal evolution has played no role the ecological differentiation or evolution of vascular plant lineages.⁶ Our recent work sought to investigate these conclusions by comprehensively examining the stomatal responses to changes in leaf water status in the fern species Athyrium filix-femina, which has been shown to have stomata that are closed by less than 20% following the spraying of leaves with exogenous ABA as high as 10 μM.²⁴ This applied concentration is an ideal standard treatment to elicit a saturating stomatal response to ABA in Arabidopsis and is widely used by cell and molecular biologists for this purpose. By virtue of this, it is indeed a high concentration, especially when considering the sensitivity of angiosperm stomata to ABA (Table 1), the levels of ABA synthesized by plants during drought stress,^48–51 and the levels of ABA measured in closed stomata during drought stress in angiosperms,⁵² which may even be synthesized in the guard cells.⁵³ We utilized a unique variety of this fern species as well as the wild-type to examine whether stomatal responses could be predicted by the simple assumption that guard cell turgor changes in unison with leaf turgor when leaf water status changes. We found that stomatal responses to changes in leaf water status in A. filix-femina were highly predictable, not requiring any metabolic input to explain responses to changes in vapor pressure difference. Furthermore, we found that the high levels of exogenous ABA required to close stomata by the reported small degree in this species²⁴ do not accumulate in planta when naturally water stressed.²⁸ During water stress A. filix-femina leaves start synthesizing ABA after stomata are closed, and this endogenous ABA does not restrict stomatal reopening when plants or leaves are rehydrated.²⁸

Table 1. The sensitivity of stomatal aperture in isolated epidermis of angiosperms exposed to exogenous ABA levels equal or lower than 10 μM.

Angiosperm species	Applied ABA (μM)	Decline in stomatal aperture (%)	Reference
Commelina communis	0.1	63	De silva et al.^Citation37
Commelina communis	0.1	53	Raschke^Citation38
Commelina communis	0.1	66	Wilkinson^Citation32
Commelina benghalensis	0.1	67	Raghavendra and Reddy^Citation39
Hordeum vulgare	0.1	54	Shen et al.^Citation40
Amborella trichopoda	0.3	57	McAdam and Brodribb^Citation41
Dahlia hybrida	0.7	48	McAdam and Brodribb^Citation41
Pisum sativum	0.7	62	McAdam and Brodribb^Citation41
Brachypodium distachyon	1.0	61	Shen et al.^Citation40
Hordeum vulgare	1.0	42	Shen et al.^Citation40
Triticum aestivum	1.0	60	Shen et al.^Citation40
Arabidopsis thaliana	2.0	66	Mustilli et al.^Citation42
Arabidopsis thaliana	10.0	44	Liang et al.^Citation43
Arabidopsis thaliana	10.0	33	Jung et al.^Citation44
Brassica napus	10.0	54	Savchenko et al.^Citation45
Delphinium spp.	10.0	66	Santamaria et al.^Citation46
Solanum lycopersicum	10.0	52	Savchenko et al.^Citation45
Vicia faba	10.0	71	Garcia-Mata and Lamattina^Citation47

A recent study has reported that the stomata of N. exaltata are not closed by exogenous ABA application, even when levels as high as 100 µM are applied to leaves.²⁵ These results are in direct conflict with those presented recently by Cai et al.⁶ in which stomatal aperture in isolated epidermis of the same species was reported to close by approximately 25% in response to exposures ranging from 50 to 200 μM exogenous ABA. How can these two contrary results in the same species be reconciled? Despite differences in methodology, neither of these two recent and conflicting studies conducted experiments to examine the effect of endogenous ABA synthesized by the plant on stomatal function in this species. To address this particular limitation, we specifically examined the role of endogenous ABA synthesized by the plant in controlling stomatal responses to changes in leaf water status in N. exaltata. Like other fern species, endogenous ABA levels in N. exaltata were found to be very low under well-watered conditions (Table 2).²⁶ During natural soil water deficit, stomata closed and the level of endogenous ABA increased. To test whether this enhanced endogenous level of ABA played any role in closing stomata during drought stress, as it does in seed plants, we employed a simple experiment based on strong inference.⁵⁴ Two alternative hypotheses for the mechanism driving stomatal closure during drought are high levels of ABA (or other metabolic signals) that are synthesized by the plant during drought, or low turgor, which biophysically closes stomata. An experiment to systematically test which of these alternative possibilities are closing the stomata during drought is the monitoring of leaf gas exchange when a drought-stressed stem is excised under water, relaxing the tension on the water column. Instantaneous rehydration from a water stressed state rapidly increases cell turgor and therefore immediately eliminates the biophysical loss of cell turgor as a driving mechanism of stomatal closure. When instantaneously rehydrated, the stomata of N. exaltata rapidly reopened to predrought levels (Figure 1), despite the maintenance of high levels of naturally synthesized, endogenous ABA accumulated in the leaves (Table 2). This result completely excludes the hypothesis that endogenous ABA accumulated naturally by the plant during drought (or indeed any other metabolic signal) plays a role in closing stomata during water stress in this species. A similar response has been observed in a number of other fern and lycophyte species.^26–28 Our results rule out any role of endogenous ABA functionally regulating stomatal closure during soil drought in N. exaltata. Whether endogenous ABA changes the rate of stomatal responses to fluctuating light intensity as recently hypothesized by Grantz et al.²⁵ remains untested. It should be noted though that this novel and alternative role for ABA in regulating stomatal responses is very different from the effect of ABA driving stomatal closure.

Table 2. Leaf water potential and foliar ABA level for the control plants and two drought-stressed and rehydrated individuals of Nephrolepis exaltata from Figure 1.

Plant	Leaf water potential (MPa)		Foliar ABA level (ng g^−1 DW)
Control	−0.21		22
	Before rehydration	After rehydration
Plant 1 (black)	−0.76	−0.26	542.17
Plant 2 (white)	−0.78	−0.31	617.95

Figure 1. Highest endogenous ABA levels synthesized naturally by the fern Nephrolepis exaltata do not play a role in closing stomata during water stress.

The response of stomatal conductance to leaf rehydration through the stipe in two drought stressed individuals of N. exaltata (black and white circles). Dotted lines represent the stomatal conductance (mean ± SD) of three well-watered individuals of N. exaltata. For leaf water potential and foliar ABA levels (see Table 2).

Our results indicating no role of endogenous ABA driving stomatal closure during water stress call into question the proposition by Cai et al.⁶ of a meaningful physiological function of ABA in regulating stomatal aperture during drought in N. exaltata. Like other ferns, the ABA naturally synthesized by N. exaltata is neither responsible for stomatal closure under drought conditions, nor prevents stomatal reopening following rehydration.^26–28 Both of these effects of ABA on stomata have been well-characterized physiologically and at a molecular level in angiosperms.^{3,26,29–32,55} We believe that the two contrasting theories on stomatal evolution proposed over the past decade stem from a very different approach to investigating this question, particularly the use of experimental designs by one camp that cannot make a strong inference about stomatal function in intact plants. A common method employed by studies which conclude that stomatal function is unchanged across land plant lineages is the use of solutions of unnatural and extremely high levels of exogenous ABA and isolated epidermis floated in these solutions to determine the effect of this hormone on stomata (Figure 2). This is despite a long and well-documented history of artifacts when working with epidermal peels, resulting from the physical stress of removing epidermal strips, mesophyll contamination, epidermal cell death,⁵⁸ and lack of replication with studies observing stomatal function in intact leaves or plants.^59,60 In contrast, most studies which have employed gas exchange or direct stomatal observation in live and intact leaves, as well as experiments testing the effect of endogenous ABA on stomatal aperture during drought, have found little or no functional effect of ABA on stomatal responses to changes in leaf water status in fern and lycophyte species.

Figure 2. Comparison between the endogenous levels of ABA found in plants of Nephrolepis exaltata and the exogenous levels of ABA applied in two different studies using the same species.

Endogenous levels of ABA (mean ± SD; log scale) found in plants of N. exaltata under well-watered (n = 3) and water stress conditions (n = 2) (data from Figure 1) and exogenous levels of ABA applied to individuals from the same species in the studies from Cai et al.⁶ (black solid and black dotted lines represent maximum concentration used in the dose–response curve and concentration chosen in further experiments, respectively) and Grantz et al.²⁵(gray solid line represents maximum concentration used in experiments). The red horizontal line represents 0.1 μM, corresponding to the cytosolic concentration of ABA required to fully activate ABA receptors in angiosperms.⁵⁶ To estimate the endogenous levels of ABA in terms of volume (molarity) rather than weight (gram), we utilized the same foliar ABA concentrations from Figure 1, but in terms of FW, and the relationship between RWC and water potential for this species (Equation 1). It should be noted that in a water-stressed plants 15% of the ABA in the leaf remains bound in the chloroplasts, the rest is assumed to be cytosolic,⁵⁷ meaning that we our calculated molar concentrations of ABA from foliar measurements are an overestimate of cytosolic levels.

The behavior of fern and lycophyte stomata in an intact plant is very different from the stomatal behavior of seed plants, particularly in terms of stomatal responses to changes in leaf water status.⁶¹ It is possible that this transition in stomatal behavior influenced major ecological transitions that occurred as the seed plant lineages first emerged during the late Carboniferous and early Permian, invading seasonally dry environments.⁶² Discovering the molecular mechanisms underpinning this major evolutionary transition in how stomata perceive and respond to water limitation is consequently a major future research challenge. Future work examining the presence and function of ABA receptors, signaling proteins and anion channels in fern guard cells, as well as the capacity of ferns to synthesize ABA during water stress will reveal critical insights into the evolution of the stomatal response to ABA.

Material and methods

Plant material

Individuals of N. exaltata were grown in a 2:1 mix of Indiana Miami topsoil and ground pine bark under greenhouse conditions set at a night/day temperatures of 16/23ºC, and natural photoperiod. Plants were grown under a 75% shade cloth resulting in light intensity of approximately 300 μmol m⁻² s^–1. They received daily watering and a weekly application of liquid fertilizer, and a misting fan was used to maintain relative humidity at or above 70% during the day.

Leaf gas exchange, water potential and foliar ABA

Fully hydrated plants were used to assess initial leaf gas exchange and collect samples for foliar ABA quantification. Leaf gas exchange was measured using an infrared gas analyzer (LI-6800, Licor Biosciences). Conditions in the leaf cuvette were controlled for the duration of the experiment; light intensity was set at 500 μmol m⁻² s⁻¹, temperature to 23°C, VPD at 1.0 kPa, and CO₂ concentration at 400 μmol mol⁻¹. Following leaf gas exchange assessment, measurements of the water potentials were performed immediately using a Scholander pressure chamber (615D, PMS Instrument Company), and leaves were then harvested for foliar ABA analysis. Samples were weighted, covered in cold 80% methanol in water (v v⁻¹), and immediately stored at −30°C. Samples were further purified, and ABA levels quantified by physicochemical methods with an added internal standard using HPLC-MS according to McAdam.⁶³ Foliar ABA levels were expressed in terms of DW, which was quantified after ABA determination by weighing the dry mass of the sample used for analysis.

After the initial characterization of plants, two individuals were drought stressed by withholding water under greenhouse conditions. One frond from each plant was then enclosed in the cuvette of the LI-6800; the terminal end of the frond was taken for water potential measurements. Using the LI-6800, we measured the kinetics of stomatal reopening following instantaneous rehydration thought the stipe, which took place by the excision of the stipe under water and recutting of the end for the duration of the experiment to ensure no blockage of the xylem occurred from mucilage. After leaf gas exchange stabilization, leaves were collected for final water potential measurements and foliar ABA quantification as described above.

Foliar ABA levels were calculated terms of molarity based on the water potential and relationship between relative water content and leaf water potential determined by pressure–volume curves according to the following equation:

$ABA\left(M\right)=\frac{\frac{ABAFW}{RWC}x{10}^{-6}}{MolarweightofABA}$

where ABA (M) is ABA on a molar basis, ABA FW unit is ng g⁻¹ FW, RWC ranges from 0 to 1.0, and the molar weight of ABA is 264.321 g mol⁻¹.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Abbreviations

Table

ABA	Abscisic acid
FW	Fresh weight
DW	Dry weight
HPLC-MS	High-performance liquid chromatography-mass spectrometry
SD	Standard deviation
RWC	Relative water content
VPD	Leaf-to-air vapor pressure difference

Additional information

Funding

This work was supported by the USDA National Institute of Food and Agriculture, Hatch Project 1014908 (S.A.M.M.).