Temperature alters the response of hydraulic architecture to CO2 in cotton plants (Gossypium hirsutum)

Journal Pre-proof Temperature alters the response of hydraulic architecture to CO2 in cotton plants (Gossypium hirsutum) Ximeng Li (Conceptualization) (Methodology) (Investigation) (Validation) (Visualization) (Writing - original draft), Xin He (Investigation) (Formal analysis) (Writing - review and editing), Renee Smith (Investigation) (Formal analysis) (Writing - review and editing), Brendan Choat (Conceptualization) (Methodology) (Supervision) (Writing - review and editing), David Tissue (Conceptualization) (Supervision) (Writing - review and editing) (Project administration) (Funding acquisition)

PII:

S0098-8472(20)30030-7

DOI:

https://doi.org/10.1016/j.envexpbot.2020.104004

Reference:

EEB 104004

To appear in:

Environmental and Experimental Botany

Received Date:

12 November 2019

Revised Date:

1 February 2020

Accepted Date:

2 February 2020

Please cite this article as: Li X, He X, Smith R, Choat B, Tissue D, Temperature alters the response of hydraulic architecture to CO2 in cotton plants (Gossypium hirsutum), Environmental and Experimental Botany (2020), doi: https://doi.org/10.1016/j.envexpbot.2020.104004

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.

Temperature alters the response of hydraulic architecture to CO2 in cotton plants (Gossypium hirsutum) Authors: Ximeng Li1, Xin He1,2, Renee Smith1, Brendan Choat1, David Tissue1 Affiliations: 1Hawkesbury

Institute for the Environment, Western Sydney University, Locked Bag 1797,

Penrith, NSW 2751, Australia 2Institute

of Food Safety and Nutrition, Jiangsu Academy of Agricultural Science, Nanjing,

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People’s Republic of China Author for correspondence: Hawkesbury Institute for the Environment, Western Sydney University, Locked Bag 1797, Penrith, NSW 2751, Australia

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Email: [email protected]

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Highlights:

Impacts of climate change on plant water relations are currently unclear

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Hydraulic traits were examined on cotton plants exposed to elevated CO2 and temperature

Interactive effects of CO2 and temperature were observed on traits conferring drought tolerance

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Elevated temperature mitigated the weakening effect of CO2 on drought tolerance

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Complex, interactive environmental factor experiments are required to assess climate

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change impacts on plant hydraulics

Abstract: Elevated CO2 and temperature are expected to result in drought stress with increased intensity and frequency, yet our understanding of subsequent plant response is generally limited. The objective of this study was to investigate the impacts of elevated CO2 and temperature on physiological traits affecting drought tolerance of cotton plants (Gossypium hirsutum). We grew cotton plants in the glasshouse under two CO2 treatments (Ca: 420 ppm; Ce: 640 ppm) and two temperature treatments (Ta: 32/24 oC; day/night; Te: 1

36/28 oC; day/night) with adequate irrigation and fertilization. Plant allometry, leaf gas exchange and a suite of hydraulic characteristics (xylem resistance to drought-induced embolism in the leaf and stem, leaf tolerance to dehydration-induced loss of rehydration capacity, and water transport capacity of stem) were examined. Xylem anatomical traits of the leaf and stem were also examined to elucidate the structural basis for potential physiological adjustments. Ce increased canopy leaf area and decreased leaf level water loss at Ta, and decreased stomatal conductance and transpiration at both temperatures. Moreover, Ce significantly increased stem conductivity at Ta, but xylem tissue was less resistant to drought induced embolism. Te altered the pattern of xylem conductivity and

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embolism resistance response to CO2, with the stem less hydraulically conductive while the xylem was more tolerant to embolism under Ce. The variation of stem conductivity and

embolism resistance of the stem across CO2 and temperature treatments was likely to be

partially explained by xylem anatomy. Overall, CO2 and temperature had interactive effects

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on traits associated with water relations of cotton, such that elevated CO2 compromised

mitigated by elevated temperature.

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drought tolerance under ambient temperature, but these negative impacts were partially

Key words: drought, climate change, embolism resistance, rehydration capacity, drought

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tolerance, Gossypium hirsutum

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1. Introduction Global atmospheric CO2 concentration fluctuated between 180 parts per million (ppm) during the strongest glacial periods and 280 ppm during the interglacial period (Barnola et al., 1987; Jouzel et al., 1993), and has risen rapidly since the industrial revolution (Monnin et al., 2001) to ca. 410 ppm and continues increasing ca. 2 ppm per year. During the same time period, global mean temperature has risen by 1oC, with up to 5oC rise projected by climate models by the end of this century, depending on emission scenarios (Pachauri et al., 2014). Given tight coupling between the global energy balance and hydrological cycle, one

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consequence of climate change is increased aridity in some environments, which is often the most limiting factor to the distribution and productivity of terrestrial vegetation (Dai,

2011; Diffenbaugh et al., 2015; Engelbrecht et al., 2007; Stocker et al., 2019). Widespread

drought stress with increased frequency and intensity in the past few decades has incurred

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large-scale forest dieback and crop yield loss (Allen et al., 2015; Allen et al., 2010; Dai, 2011). The capacity for plants to cope with drought is therefore key for forest and crop productivity

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in future climates.

Hydraulic characteristics provide valuable insight into plant drought tolerance

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(Brodribb et al., 2019; Choat et al., 2018), including xylem vulnerability to embolism, which prevents bulk water transport from the soil to the plant canopy (Adams et al., 2017; Choat et al., 2018). Plants with embolism resistant xylem can operate at more negative water

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potentials, thereby maintaining gas exchange and avoiding desiccation during chronic drought stress (Blackman et al., 2019). This capacity is commonly quantified by P50, which refers to the water potential threshold triggering 50% loss of xylem conductivity (Brodribb,

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2017; Choat et al., 2012). As a mechanistic trait, species P50 is well coupled with habitat aridity at both global and regional scales, indicating its adaptive significance to the

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colonization and survivorship of plants (Brodribb, 2017; Engelbrecht et al., 2007; Li et al., 2018; Trueba et al., 2017). Furthermore, leaf turgor loss point (TLP) is an important indicator of plant drought tolerance (Bartlett et al., 2012; Blackman, 2018). Leaves with low (i.e. more negative) TLP are able to maintain stomatal openness, diverse cell metabolic function and consequently growth by maintaining cell turgidity when soil water is limiting (Bartlett et al., 2012; Farrell et al., 2017). Leaf TLP has also been shown to vary systematically with habitat precipitation within and across biomes (Bartlett et al., 2012; Lenz et al., 2006; Zhu et al., 3

2018), integrates different aspects of hydraulics, and acts as a potent proxy of the overall operating range of water potential (Fu et al., 2019; Li et al., 2019a; Meinzer et al., 2016). Moreover, leaf rehydration capacity has recently been proposed to be an important characteristic indicative of drought tolerance (John et al., 2018). In particular, leaf relative water content at 10% loss of rehydration capacity (PLRC10) has been designated as the “permanent turgor loss point”, beyond which the complete recovery of bulk cell hydration is impossible, and thus sets the lower bound of leaf water status for the full recovery of leaf gas exchange after drought stress (Oppenheimer and Leshem, 1966; Trueba et al., 2019). The variation of traits conferring drought tolerance (including P50, TLP and PLRC) across

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species has been examined extensively, yet our knowledge regarding the plasticity of these traits is relatively limited.

Elevated CO2 may alter drought tolerance traits of plants through its effect on water

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balance. Reductions in leaf stomatal conductance and transpiration rate are often observed in plants grown at elevated CO2, regardless of the duration of exposure (Ainsworth and Long, 2005; Ainsworth and Rogers, 2007; Field et al., 1995; Medlyn et al., 2001). However,

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for plants grown under elevated CO2, leaf level water savings may be counterbalanced at the canopy scale by increased canopy leaf area (Ainsworth and Long, 2005; Leuzinger and

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Körner, 2007; Pataki et al., 1998; Way, 2013). Depending on adjustments in sapwood area (i.e. conducting xylem relative to canopy leaf area), alterations in the supply-demand

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relationship can affect water status within organs, which may act as a driving force for the variation of physiological drought tolerance characteristics. Moreover, it has been shown that elevated CO2 can influence the anatomical structure of xylem, such that plants grown

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under higher CO2 often exhibit larger conduits (Atkinson and Taylor, 1996; Atwell et al., 2009; Domec et al., 2010; Domec et al., 2017; Maherali and DeLucia, 2000). Widened vessel

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cross-sectional area would contribute to substantially increased conductivity given that flow rate is scaled to the fourth power of vessel diameter (Hagen-Poiseuille’s law). Meanwhile, large conduits are thought to be less resistant to embolism, as evidenced by the trade-offs between maximum stem conductivity (Ks) and P50 across species (Li et al., 2018), although these relationships are weak at the global scale or may not exist in some cases (Gleason et al., 2016; Santiago et al., 2018). Early studies have shown that the response of xylem embolism resistance to elevated CO2 is highly variable, with some species showing less 4

negative air entry threshold in response to elevated CO2 while others do not (Domec et al., 2010; Hao et al., 2018; Warren et al., 2011). Elevated temperature may also lead to shifts in plant drought tolerance via its impacts on water balance and xylem anatomical features. Warming may increase the canopy demand for water either by increasing the canopy leaf area or by increasing leaf-toair water vapor gradient (Poorter et al., 2012; Way and Oren, 2010). Alone or in combination, these shifts can result in more negative canopy water potential unless plant allometric (e.g. increased sapwood area or root-to-shoot ratio) and/or physiological

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attributes (e.g. more stringent stomatal regulation) can be adjusted accordingly. Moreover, plants grown under warming conditions can also develop larger conduits (Maherali and DeLucia, 2000; Way et al., 2013), which may affect conductivity, but not necessarily

embolism resistance. The effects of warming on plant hydraulics have been investigated by

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a handful of studies, but no consistent results have been found (Blackman et al., 2017; Maherali and DeLucia, 2000; Thomas et al., 2004; Way et al., 2013). Notably, although

carbon trait response to climate change factors has been comprehensively and elegantly

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elucidated, little information exists with respect to the interactive effect of elevated CO 2 and warming on plant water relations. Given the strong link between gas exchange and

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hydraulics (Brodribb and Feild, 2000; Liu et al., 2019; Santiago et al., 2004; Zhu et al., 2018), knowledge regarding the impact of elevated CO2 and temperature on plant hydraulics will

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help to predict the carbon dynamics of vegetation under global climate change. This study investigated the impacts of elevated CO2 and warming on plant hydraulic traits related to drought tolerance on cotton (Gossypium hirsutum), which is cultivated in

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regions characterized by dry climates, where drought stress may become more intense and frequent due to global climate change. Despite being a woody perennial, cotton plants are

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commonly grown as an annual crop. Photosynthesis of cotton plants are known to be resilient to short-term, mild water stress, whereas growth and yield can be greatly compromised if drought stress persists, especially in the presence of other stressors such as high temperature (Broughton et al., 2017; Chastain et al., 2014; Yi et al., 2016a, b). Furthermore, recent studies of the hydraulic strategy of cotton found that hydraulic transport is generally resistant to embolism (Li et al., 2019b), yet it is unclear if this feature will be altered by climate change factors. We grew cotton under two CO2 (Ambient and 5

Elevated) and two temperature (Ambient and Ambient +4oC) treatments under wellwatered and fertilized conditions for ten weeks. A suite of hydraulic traits, including xylem vulnerability to drought in leaves and stems, leaf turgor loss point, rehydration capacity in response to dehydration, and stem hydraulic conductivity were measured. Plant allometry, gas exchange characteristics and xylem anatomical traits were examined to assess potential mechanisms underpinning variation in physiology across treatments. We hypothesised that elevated CO2 would decrease drought tolerance through its effects on canopy water balance and xylem anatomical structure, and these negative effects will be exacerbated by elevated temperature.

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2. Material and methods 2.1 Plant material and growth conditions

Cotton (Gossypium hirsutum L. Cv, 71BRF [Bollgard II® Roundup Ready Flex®]) seeds were

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sown into 25 litre plastic pots filled with a mixture of composted potting mix (Australian

Native Landscapes) and slow release fertilizer (Osmocota, Scotts Australia, Bella Vista, NSW)

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in late April 2019. Germination commenced approximately two weeks after sowing, and plants were then thinned to one plant per pot. The plants were evenly allocated to four CO 2

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and temperature controlled chambers in a naturally sunlit glasshouse facility on the Hawkesbury campus of Western Sydney University, Richmond, NSW, Australia. Specificity regarding the glasshouse facility has been described in detail in Ghannoum et al. (2010). A

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2×2 factorial design was adopted to investigate the main and interactive effects of CO2 and temperature on physiological and anatomical traits of cotton plants. Of the four glasshouse

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chambers, two were set to ambient CO2 concentration (Ca: 420 ppm) and two were set to elevated CO2 concentration (Ce: 640 ppm). Temperature of the two chambers within each

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CO2 treatment was either set to ambient (Ta: 32/24 oC; day/night), representing the mean growth season temperature for cotton plants in Australia or to elevated (Te: 36/28 oC; day/night) representing future temperatures. Pots were watered manually, daily or every other day, to field water capacity to ensure an absence of water stress over the developmental stage. Experiments started approximately 70 days after planting when plants were ca. 1.2 m tall. 2.2 Gas exchange measurements 6

Leaf gas exchange characteristics were measured between 10-11 am on two consecutive sunny days for three individuals within each treatment combination using two crosscalibrated portable photosynthesis systems (Model 6400XT, Li-Cor, Lincoln, NE, USA) equipped with 2×3 cm red-blue LED light source (6400-02B) and external CO2 injector (640001). For each plant, three upper canopy, fully expanded leaves were selected. Gas exchange, including stomatal conductance (gs, mol H2O m-2 s-1) and transpiration rate (E, mmol H2O m-2 s-1), was measured by inserting leaf lamina into the cuvette supplied with saturated irradiance (i.e. 1500 µmol m-2 s-1 photo flux density). Data were logged when the real-time reading was visually stable and the total ecoefficiency of variation 1, which was typically

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achieved in 10 min. During measurement, CO2 and temperature in the cuvette were set to match the growth values of each glasshouse chamber, and air flow rate was set to 500 ml

min-1. To gain the maximum rate of gas exchange, a custom-made humidifier was attached to the gas inlet of the Licor to lower the leaf-to-air vapor pressure deficit (VPD, kPa) when

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necessary such that VPD inside the cuvette varied between 1.6-2.1 kPa. Immediately

following gas exchange measurements, leaves were excised and leaf water potential (Ψleaf)

USA).

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2.3 Xylem vulnerability to embolism

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was measured using a Scholander-type pressure chamber (PMS Instruments, Corvalis, OR,

Xylem vulnerability to embolism for leaves and stems was measured on the same plant

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using the optical visualization (OV) method (Brodribb et al., 2016). VCs measured by the OV technique has been shown to agree well with those generated by other methods (Brodribb et al., 2017; Brodribb et al., 2016; Skelton et al., 2017). Detailed information regarding the

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principles of the OV technique can be found in www.opensource.org.

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Three individuals within each treatment combination were used for VCs measurement. Prior to the day of measurement, plants were bagged with opaque material to ensure maximum stomatal closure and equilibrium of water potential across organs. Sampling was carried out at predawn when xylem tension was minimal. Upon harvest, plants were uprooted and immediately placed into bags humidified with moist paper towels, and transported to the laboratory within 15 min. For each individual, an upper canopy, recently-matured leaf was selected for imaging. In addition, an approximately 2 cm 2

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section of bark was carefully removed from the mid-portion of the stem using a razor blade to expose the xylem tissue. Images were taken with two custom-made clamps integrated with a 20× magnification hand lens and single-board microcomputer (Raspberry Pi Zero, Raspberry Pi Foundation, ENG, UK). Following installation of the imaging system, the plant was protected from ambient light to minimize the noise-to-signal ratio and was allowed to dehydrate under lab conditions. Xylem tissue of the plant was photographed at 10 min intervals upon illumination. A stem psychrometer (PSY1, ICT International, Armidale, NSW, Australia) was used to measure the water potential of the plant. A piece of bark similar to the size of the psychrometer chamber was removed from the main stem. The chamber was

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pressed to the exposed window to ensure that the thermocouple touched the surface of the xylem, and was then held in place using a clamp. The psychrometer measured the plant water potential at 10 min intervals.

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Obtained images were processed using ImageJ software (Schindelin et al., 2012).

Protocols for image processing have been detailed elsewhere (e.g. Skelton et al., 2017). In brief, the difference between two consecutive images was revealed as black or white pixels

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using Image J, which represent embolized regions. Pixel area on each image was then summed and the percentage of embolism over time was calculated as the ratio of pixel area

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at each time-step to the total pixel area over the dehydration period. Plant water potential at each time-step was calculated from the timestamp of images with the coefficient of the

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regression between psychrometer readings and time, which typically shows a linear relationship after stomatal closure (Skelton et al., 2017). VCs was determined by plotting percentage of embolism against corresponding water potential.

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2.4 Leaf rehydration capacity

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Leaf rehydration capacity was assessed by the percentage loss of rehydration capacity (PLRC) curve modified by John et al. (2018). Within each treatment combination, three or four plants were harvested at predawn by cutting the basal portion of the main stem, quickly transferring it to a bucket with clean water, and then recutting the stem underwater. Plant material was transported to the laboratory and allowed to rehydrate in darkness for up to 3 hours. Thereafter, leaves were randomly collected from the plants and immediately weighed for fresh mass (FWsat, g), and then were bench-dried for up to two weeks. For each treatment combination, a total of 25-40 leaves were sampled. Over the course of 8

dehydration, 2-4 leaves were randomly selected each day to represent a gradual dehydration gradient. Leaves were weighed (FWdehy, g) and then rehydrated by immersing the petiole under water for 8 hours (John et al. 2018). Thereafter, leaves were weighed again for mass after rehydration (FWrehy, g), placed in paper bags, and oven-dried at 70oC for at least 72 hours until constant mass (DW, g). Leaf PLRC was calculated as: SWCrehy ) SWCsat

(1)

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PLRC = 100 × (1 −

where SWCrehy and SWCsat are the water content (g g-1) of rehydrated and saturated leaves, which were estimated as follows: FWsat − DW DW

(2)

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SWCsat =

FWrehy − DW DW

(3)

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SWCrehy =

where FWsat, FWrehy and DW are the mass of the saturated, rehydrated and oven-

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dried leaf, respectively. In addition, the relative water content (RWC, %) of dehydrated leaf (i.e. before rehydration) was calculated as:

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RWC = 100 × (

FWdehy − DW ) FWsat − DW

(4)

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where FWdehy is the mass of dehydrated leaf. PLRC curves were generated by plotting

PLRC against corresponding RWC. 2.5 Stem hydraulic conductivity Following sampling for leaf rehydration capacity, an ca. 10 cm segment was collected from the basal portion of the main stem for each individual by alternately trimming it under water. The stem sample was then attached to a pressure tank through silicon tubing and flushed by 2 mmol KCL solution under high pressure (0.2 MPa) for at least 30 min to remove 9

any possible embolism. Thereafter, the sample was connected to a pressure head (0.002 MPa) generated gravitationally and flow rate (K) was measured using a digital liquid flow meter (Liqui-Flow L10, Bronkhorst High-Tech BV, Ruurlo, Gelderland, Netherlands) connected to flow analysis programs FlowDDE and FlowPlot (Version 4.69 and 3.34, respectively, Bronkhorst, FlowWare, http://downloads.bronhorst.com). Measurement was started when the real-time K was visually stable and was typically finished in 5 min, thus avoiding overestimation of K due to radial flow (De Baerdemaeker et al., 2019). Stem specific hydraulic conductivity (Ks, kg m-1 s-1 MPa-1) was calculated by normalizing K with sapwood area and length of the segment.

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2.6 Pressure-volume curves

For each treatment, one upper canopy leaf per individual was collected from three or four plants and used for pressure-volume (PV) curve determination. Sampling was conducted

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before sunrise when the leaf water potential was highest (i.e. least negative). Excised leaves were allowed to rehydrate in darkness for up to 2 hours by submerging petioles under

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water. PV curves were measured following the protocol described by Tyree and Hammel (1972). In short, leaves were bench-dehydrated under lab conditions, while leaf water

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potential and fresh mass were determined periodically using the pressure chamber and digital analytical balance (weighed to 0.1 mg), respectively. Finally, leaves were oven-dried at 70oC until constant mass. Leaf PV traits were analysed following Lenz et al. (2006). To

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facilitate calculation, the inverse of leaf water potential was plotted against RWC, and leaf turgor loss point (TLP, -MPa) was taken as the inflection point where the line became non-

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linear.

2.7 Xylem anatomy

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Within each treatment, three individuals were harvested for anatomical analysis. For each individual, three fully developed leaves were collected from the 3rd to 5th node. In addition, an ca. 5 cm segment was excised from the basal portion of the main stem and stored in falcon tubes filled with distilled water after removing the bark. Histological slides were prepared by hand-cutting the sample into ca. 3 mm width sections. For leaf samples, the sections were always collected from the midrib at 1 cm above the joint of petiole and lamina. Selected sections with flat surface were submerged in distilled water for at least 20 10

min prior to measurement. For observation, samples were mounted under a glass cover slip in distilled water on a glass slide, and were then observed using a Leica TCS SP5 confocal inverted microscope (Leica Microsystems) equipped with a HCX PL APO CS × 20×0.70 IMM objective (image size: 775 × 775 µm). Confocal imaging used sample auto-fluorescence, excitation was conducted by 405 nm diode laser, and enabled maximum signal collection structure information emission ranged between 460 and 520 nm (emission maximum = 492 nm) for stem samples, between 448 and 538 nm (emission maximum = 473 nm) for midrib samples, and between 665 and 695 nm (emission maximum = 687 nm) for chlorophyll signals in both stem and midrib samples. A total of 20 serial optical sections of partially

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overlapping images were captured for each cross-section and merged into a single image using the “Z project” function of Image J; vessel diameter was measured with the same software.

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2.8 Morphological characteristics

Four to six leaves were collected per plant from four individuals within each treatment.

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Petioles of leaves were first removed and projected lamina area (LA, cm2) was measured using a rotating belt leaf area meter (Model 3100C, Li-Cor, Lincoln, NE, USA). Leaves were

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weighed for DW after being oven-dried at 70oC for 72 hours. Specific leaf area (SLA, m2 kg) was calculated as the ratio of LA to DM. In addition, following xylem VCs measurement, leaf lamina were carefully collected, oven-dried and weighed to determine DM of canopy leaf.

leaf DM and SLA.

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Canopy leaf area (CLA, m2) was estimated by extrapolating the relationship between canopy

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2.9 Statistical analysis

Stem and leaf VC curves, and leaf PLRC data, were fitted by sigmoidal model using the fitplc

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package in R (R Development Core Team, 2014). Water potential inducing 50% loss of xylem conductivity (P50, -MPa) were extracted for both leaves (PL50, -MPa) and stems (Px50) from the fitted VC curves. Similarly, relative water content at 10% and 50% loss of rehydration capacity (PLRC10 and PLRC50) were estimated from fitted PLRC curves. The impacts of CO2 and temperature on plant morphological, physiological and anatomical traits were analysed using a two-way ANOVA with the exception of parameters related to PLRC, which were analysed by comparing the width of confidence interval. The CO2 effects 11

within each temperature treatment were analysed using T-test. Statistically significance difference was considered if p≤0.05. All data and statistical analysis were performed in R 3.5.3 statistical computing environment. 3. Results 3.1 Impacts of elevated CO2 and temperature on plant water status There was a significant interaction between CO2 and temperature (p=0.03, Fig. 1,) on canopy leaf area (CLA). Ce increased CLA by 22.5% (p0.001) under Ta but not under Te. Plants grown under Ce showed considerably lower maximum stomatal conductance (gs) and

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consequently rates of transpiration (E). Across CO2 treatments, gs and E were reduced by 41.9% and 32.9% respectively under elevated CO2 (gs: p=0.01; E: p=0.03), while no

treatment effect of temperature on gs and E was detected. Significant interactive effects of CO2 and temperature were also found for leaf water potential (Ψleaf; p0.001). Under Ta, Ce

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reduced Ψleaf by 0.19 MPa but an inverse pattern was shown under Te, where Ψleaf was 0.3

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MPa more negative under Ce.

3.2 Responses of hydraulic traits to elevated CO2 and temperature

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The spatiotemporal pattern for the propagation of embolism within the leaf and stem vein networks was clearly revealed by photographing these organs under intense illumination over the course of dehydration (Fig. 2). For leaves, embolisms tended to initiate in higher

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order veins (e.g. midrib) at less negative tension, while lower order veins appeared to be more resistant to embolism (Fig. 2a). No clear spatiotemporal pattern for the development

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of embolism was found for the xylem of stems (Fig. 2b). Across treatment, the mean water potential inducing 50% loss of xylem conductivity (P50) ranged from -3.3 MPa to -4.36 MPa

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in leaves (PL50), and from -2.91 MPa to -4.47 MPa in stems (Px50) (Fig. 3). Percentage loss of rehydration capacity (PLRC) of leaves showed a sigmoidal increase

as leaf relative water content (RWC) decreased (Fig. 4). The range of RWC threshold for 10% and 50% loss of rehydration capacity (PLRC10 and PLRC50, respectively) across treatment were 63-75% and 36-40%, respectively. Specifically, under Ca, leaves grown under Te exhibited significantly higher PLRC10 compared with those grown under Ta, but this

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difference was small. No treatment effect on PLRC50 was found. Additionally, PLRC10 was consistently lower than the RWC at TLP (data not shown) across treatment. Interactive effects were found between CO2 and temperature on hydraulic traits including PL50, Px50, leaf turgor loss point (TLP) and stem specific conductivity (Ks) (Fig. 5). Ce reduced PL50 under Ta but an opposite pattern was observed under Te (Fig. 5a, p=0.04). A similar response was found for Px50 (Fig. 5b, p=0.02). Ce increased resistance to wilting in leaves, represented by TLP, under Ta, while no such change was observed across CO2 treatments under Te (Fig. 5c, p=0.01). Moreover, stem specific hydraulic conductivity (Ks)

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was increased by Ce under Ta, but this pattern was reversed by Te (Fig. 5d, p=0.02). 3.3 Effects of elevated CO2 and temperature on anatomical traits

CO2 did not affect the vessel diameter of the leaf midrib (Dmidrib; Fig. 6a, p=0.22), but Dmidrib was decreased by 10% on average under Te (p<0.001). By contrast, CO2 and temperature

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had a significant interactive effect on the vessel diameter of stem (Dstem; Fig. 6b). Ce

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increased Dstem by 23.5% under Ta (p<0.01), but decreased Dstem by 6.8% under Te (p<0.01). 4. Discussion

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4.1 Impacts of CO2 and warming on allometry and water status Ce significantly increased the canopy leaf area (CLA) under Ta, which is consistent with the

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observations for many species grown under enriched CO2, including cotton (Ainsworth and Long, 2005; Broughton et al., 2017; Leakey et al., 2009). However, there was no effect in Te which contradicts the finding in some tree species (Duan et al., 2014; Ghannoum et al.,

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2010), but is similar to a previous study on cotton with a similar experimental design (Broughton et al., 2017). It is generally assumed that elevated CO2 will mitigate water or

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nutrient limitations on canopy development, thus allowing the construction of resource exchange surfaces that are proximate to its optimum under field conditions (Woodward, 1990). However, in the present study our plants were supplied with adequate water and nutrients during developmental stage. CLA is determined by both leaf area and number of leaves. Under T a, mean leaf area increased by 39% for plants grown under Ce, probably due to the stimulatory effect of CO2 on cell division (Ranasinghe and Taylor, 1996), but was reduced by 10% in Te; canopy leaf 13

number was not counted. Nonetheless, elevated leaf number under Ce may have also contributed to increased CLA, as reported in many previous studies (Atwell et al., 2009; Epron et al., 1996; Ghannoum et al., 2010). Consistent with the observation on many other plants (Ainsworth and Rogers, 2007; Medlyn et al., 2001), cotton exposed to Ce exhibited reductions in stomatal conductance (gs) and transpiration rate (E). Notably, both gs and E did not differ across temperature treatments, indicating that warming did not directly regulate leaf level gas exchange at these temperatures (Ghannoum et al., 2010; Way et al., 2013). Intuitively, decreased E will

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lead to reduced water use, thus creating a “water saving” effect, which can be especially advantageous when soil water is limited (Leakey et al., 2009). However, reduced leaf level water loss can often be offset by concurrently increased canopy leaf transpiring area,

resulting in similar amounts of water transpired under Ce (Broughton et al., 2017; Duan et

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al., 2014; Pataki et al., 1998). For example, neither rates of sap flow nor canopy level

stomatal conductance was affected by increased growth CO2 in Pinus taeda because of the proportionally increased leaf and sapwood area, despite the reduced leaf gs (Pataki et al.,

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1998; Tissue et al., 1997). Total plant canopy water use was not measured in the present study, but in a similar study, Ce increased cumulative plant water use of cotton plants by

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22% under ambient temperature (Broughton et al., 2017). These results highlight the importance of allometry when assessing the impacts of CO2 on plant water relations (Hao et

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al., 2018).

Ce resulted in higher (less negative) Ψleaf under Ta but not under Te. Studies examining the water status of plants exposed to elevated CO2 have generated variable

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results (Atwell et al., 2009; Domec et al., 2010; Tognetti et al., 1999a). Leaf water potential is dependent on the soil water availability, root-to-leaf conductivity and the rates of water

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loss at canopy level. As Ψleaf was measured under well-water conditions, it is unlikely that the variation of water status was related to water supply. In addition, allometric adjustment may not have contributed to the variation of Ψleaf across treatment, at least for plants grown under Te because no change in CLA in combination with decreased leaf level E, should result in less negative Ψleaf, which we did not observe. More likely, the variation of Ψleaf reflects changes in hydraulic conductivity, which governs the amount of water transported

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from soil to leaf, and driven the variation of embolism resistance across treatments given that plant drought tolerance is largely determined by water potential. 4.2 Elevated temperature alters the response of drought tolerance traits to CO2 We show that xylem vulnerability to embolism of leaf and stem was altered by Ce, with the direction of response depending on growth temperature. The reference P50 for cotton leaves and stems (i.e. P50 under CaTa) in this study was slightly less negative than for cotton in a separate study (Li et al., 2019b), reflecting differences in growth temperatures where warming may reduce embolism resistance in some species (Way et al., 2013). It is proposed

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that plants grown under Ce could be less resistant to embolism (Domec et al., 2017; Way, 2013), but this is not universally supported by experimental evidence. Rather, the response of xylem embolism threshold to Ce is highly diverse (Domec et al., 2010; Hao et al., 2018;

Tognetti et al., 1999b; Warren et al., 2011). For instance, in the study of Domec et al. (2010),

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Liquidambar styraciflua and Cornus florida grown under CO2 enriched environments

exhibited less negative Px50, yet this trait was unaffected by CO2 in Pinus taeda and Ulmus

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alata. In a recent study by Hao et al. (2018), Px50 of six species showed little variation in response to growth CO2. On the other hand, contrary to our expectation that T e would

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exacerbate the synergistic effect of Ce on xylem embolism resistance, both leaf and stem P50 were more negative when exposed to elevated CO2. The variation in xylem vulnerability to embolism under Ce may arise from alterations

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in xylem structure. It is hypothesized that increased photosynthesis and thus carbohydrate availability for plants under Ce can promote cell division and expansion, therefore producing

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vascular tissue characterized by increased size and number of conduits (Atkinson and Taylor, 1996; Atwell et al., 2009; Domec et al., 2010; Phillips et al., 2011). However, existing data

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regarding wood anatomy under elevated CO2 and temperature are sparse and inconsistent (Atkinson and Taylor, 1996; Luo et al., 2005; Maherali and DeLucia, 2000) indicating that the effect of CO2 and temperature on xylem anatomy is highly species-specific. We found that stem vessel diameter was increased by Ce under Ta, while an inverse pattern was observed under warming conditions. Such adjustments in anatomy might strongly affect hydraulic conductivity given the exponential relationship between vessel dimension and theoretical flow rate. It could also include embolism resistance, which potentially explains the variation of Ks and in turn Ψleaf, as well as Px50 across treatments, although a robust correlative test 15

was not possible due to insufficient numbers of treatments. Yet, it does not appear that embolism threshold and vessel dimension are mechanistically linked because embolism formation and spread within the vascular network during drought stress is primarily determined by the properties of inter-vessel pits (Choat et al., 2008; Gleason et al., 2016). Consistent with some previous studies, leaf water potential at zero turgor (TLP) was slightly higher in Ce under Ta (Morse et al., 1993), but not under Te. Given that stomatal behavior is related to cell turgor, increased TLP suggests that stomata can remain open, while leaves remain metabolically active, at more negative water potentials during early

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phases of drought stress, symbolizing a more profligate water use strategy (Meinzer et al., 2016). The variation of TLP across treatments may be associated with changes in leaf osmotic potential, which was regulated by leaf carbohydrate content and is a major

determinant of TLP (Bartlett et al., 2012). On the other hand, when grown in Ce, leaves were

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more susceptible during desiccation at Ta, as indicated by the higher RWC threshold at

PLRC10. However, the overall variation of PLRC thresholds across treatments was trivial, suggesting that this trait was only moderately responsive to environmental perturbations

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and therefore may be adaptive (John et al., 2018). Notably, when compared with TLP, PLRC10 invariably occurs at lower RWC across treatments, similar to Trueba et al. (2019),

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suggesting that stomatal closure protects the vitality of mesophyll cells during drought. Collectively, despite observed treatment effects on both TLP and PLRC, these changes were

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generally small, and hence may not generate significant biological impacts during drought stress.

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4.3 Implications and conclusion

Assessing plant drought tolerance in response to multiple climate change factors is

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important to predict future performance of crops. Our results demonstrate that elevated CO2 can alter xylem water transport and embolism resistance, but the impact is dependent on temperature. Elevated CO2 increased stem-specific conductivity but reduced embolism resistance in Ta, while an opposite pattern was observed in Te. These results may be related to anatomical adjustments in the xylem, but the capacity of leaves to withstand wilting and loss of rehydration capacity showed little change in response to environmental alterations. In cotton farming, water potentials triggering leaf and stem embolism were uncommon in the field in the past, so embolism related mortality rarely occurred. However, as we have 16

reported previously, cotton plants protect the integrity of xylem in stem through massive leaf shedding during prolonged drought stress, which initially occurs shortly after stomatal closure (Li et al., 2019b). To sustain canopy photosynthesis, irrigation should be scheduled to ensure leaf RWC is above ca. 85% and not lower than 70%, which are the RWC thresholds for turgor loss and permanent mesophyll cell damage, respectively. If these results hold more widely, these physiological traits together with adjustments in allometry, can have profound consequences for plant performance during drought stress. For example, under field conditions where soil water is finite, canopy water loss is unchanged under elevated CO2 because reduced leaf level water loss is offset by increased canopy leaf area. If the

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stringency of stomatal regulation remains the same or becomes less, without other

compensatory water-saving strategies (i.e. leaf shedding), plants will be more prone to lose leaf vitality and succumb to embolism during drought because of reduced critical

thresholds. Warming is likely to mitigate the risk of hydraulic impairment, but may do so at

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the expense of carbon gain due to constrained canopy leaf area and xylem conductivity. This study highlights the importance of assessing the potential interactive effects of CO2 and

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warming on plant water relations, which is relatively scarce in the current hydraulic

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literature.

Author's contributions: X. L. designed the experiment. X. L. and X. H. collected and analyzed

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the data. X.L. led the writing of the manuscript. D. T. provided the funding, critical edits and revisions, and made final approval of the article. X. H., R. S. and B. C. contributed to

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manuscript revising and editing.

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Author statement

Ximeng Li: Conceptualization, Methodology, Investigation, Validation, Visualization and Writing Original Draft Xin He: Investigation, Formal analysis, Writing - Review & Editing Renee Smith: Investigation, Formal analysis, Writing - Review & Editing Brendan Choat: Conceptualization, Methodology, Supervision, Writing - Review & Editing

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David Tissue: Conceptualization, Supervision, Writing - Review & Editing, Project administration, Funding acquisition

Funding: This work was supported by the Cotton Research Development Corporation (CRDC; CSP1501) to DT.

Declaration of interests

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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement: The author appreciates the valuable suggestions provided by Dr.

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na

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Jinchao Feng from MUC.

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Figure captions Fig. 1 The impacts of CO2 and temperature (T) on canopy leaf area (CLA, panel a), leaf maximum stomatal conductance (gs, panel b), maximum transpiration rate (E, panel c) and water potential of transpiring leaves under well-watered conditions (Ψleaf, panel d) for cotton plants. Error bars represent standard error of mean (n=3-4). The results of two-way ANOVA are presented with statistically significant effects displayed in bold. Fig. 2 Images of the spatio-temporal occurrence of xylem embolism in leaf (panel a) and stem (panel b) over the course of dehydration. Color gradient indicates water potential,

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from high (i.e. less negative; cool color) to low (warm color), by which the entire vein network is colored accordingly depending on the conduit-specific water potential threshold for embolism. Numbers indicate the value of water potential at which first and last embolism events were observed.

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Fig. 3 Effects of CO2 (Ambient: Ca; Elevated: Ce) and temperature (Ambient: Ta; Elevated: Te) on xylem vulnerability to embolism in leaves (blue) and stems (red). Vulnerability to

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embolism is shown as the increasing percentage loss of xylem conductivity (%) with decreasing water potential (-MPa). Shaded region surrounding each curve represents the

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standard error of mean (SE) for water potential at given level of xylem embolism. Dashed vertical lines indicate the water potential at which 50% loss of xylem embolism was

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recorded (i.e. P50).

Fig. 4 The response of percentage loss of leaf rehydration capacity (PLRC, %) to leaf relative water content (RWC, %) under different CO2 (Ambient: Ca; Elevated: Ce) and temperature

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(Ambient: Ta; Elevated: Te) treatment combinations. Data were fitted using sigmoidal model, and grey shaded band represents the bootstrap confidence interval of fitted curve. Vertical

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lines encompassed by shaded regions are the RWC thresholds at which 10% (red) and 50% (blue) leaf rehydration capacity was lost, with the upper and lower bounds of confidence interval being represented by the edges of shaded regions. Fig. 5 The impacts of CO2 and temperature (T) on water potential threshold inducing 50% loss of xylem conductivity in leaves (PL50, panel a), stems (Px50, panel b), leaf turgor loss point (TLP, panel c) and maximum stem conductivity (Ks, panel d) for cotton plants. Error bars

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represent standard error of mean (n=3-4). The results of two-way ANOVA are presented with statistically significant effects displayed in bold. Fig. 6 The impacts of CO2 and temperature (T) on xylem vessel diameter of leaf midrib (panel a) and stem (panel b). Error bars stand for standard error of mean (n=4). Inset images show photos acquired using confocal microscope under ×20 magnification, with different tissue types appearing as different colors under native fluorescence. Scale bar within the inset images indicate 150 µm. The results of two-way ANOVA are presented with

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statistically significant effects displayed in bold.

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