Bark water uptake in the mangrove Avicenna marina: pathways, the effects of light and dehydration, and the contribution to stem swelling ARC DP180102969

Full description

A small body of evidence suggests that absorption of atmospheric water through the bark, referred to as bark water uptake (BWU), may decrease tracheal embolism, partially re-establish hydraulic conductivity and increase stem water potential (Katz et al., 1989; Earles et al., 2016; Liu et al., 2019). Consequently, BWU may play significant roles in the maintenance of both living plant tissues and hydraulic function, and is therefore critical to understanding plant survival. Yet, few studies have directly investigated BWU. This dataset investigates pathways for BWU and movement, the effects of light and dehydration on dynamics of BWU, and the contribution of BWU to stem swelling in the mangrove Avicenna marina subsp. australasica (Walp.) J.Everett. Avicenna marina is a widely distributed mangrove tolerant of hypersaline conditions where water availability at the roots is limited, making it a likely candidate for BWU.

We hypothesised that 1) lenticels on the outer bark surface are the primary pathway for BWU, 2) increasing initial stem dehydration would enhance BWU reflecting greater water potential gradients, 3) BWU under light will be greater than in dark conditions, and 4) inner bark and cortex tissues will swell following BWU.

For full methods see publication: Holly A. A. Beckett, Daryl Webb, Michael Turner, Adrian Sheppard, and Marilyn C. Ball, 2023. Bark water uptake through lenticels increases stem hydration and contributes to stem swelling. Plant, Cell & Environment.

Abridged methods:

1. Species: Avicenna marina subsp. australasica (Walp.) J.Everett. saplings were grown hydroponically under glasshouse conditions in 50% seawater from propagules collected from the Clyde River, New South Wales, Australia in 2019 as per Fuenzalida et al. (2022). Saplings were cut at the interface between stem and root growth. The most mature 6-9 cm length of stem at the bottom of the severed shoots was cut from each shoot and used for all BWU experiments.

2. Water potential: Stem water potential was measured prior to BWU were indicated below. Shoots were equilibrated in black plastic bags for 30 minutes. Water potential for each individual was measured on a fully expanded leaf cut at the petiole using a Scholander pressure chamber (Pressure chamber model 1050D, PMS Instrument, Albany, USA).

3. Stem sealing: Stem cut ends were sealed prior to measurement of BWU. Cut ends and 5 mm of bark from the cut end were coated with Vaseline and wrapped tightly with Parafilm. The interface between the exposed bark and the Parafilm wrap was sealed with orthodontic wax.

4. Stem characteristics: Stem length, stem length exposed for BWU, and stem diameter were measured for calculation of stem surface area and volume assuming a cylindrical shape. Stems were oven dried for four days at 60°C (LABEC Laboratory Equipment, Pty. Ltd. Australia) for stem dry mass.

5. Measurement of BWU: Initial stem mass was taken (XP 205 Metter Toledo balance, Mettler – Toledo Ltd., Greifensee, Switzerland) and stems were placed in individual wetting chambers consisting of 50ml FALCON Conical Centrifuge Tubes with plastic mesh fitted inside to prevent stem contact with pooled water on the bottom of the chamber. A nasal spray atomiser filled with tap water was used to spray stems until wetted, mimicking small rain, fog or dew water droplets. Wetting chambers were sealed with lids and placed under full spectrum grow lights in a growth chamber (TRIL-1175-SD-1SL Thermoline Scientific Growth Chamber – Thermoline Scientific Equipment Pty. Ltd. Australia) at 22.1°C. Stem mass was measured at 0.5, 1, 2, 4, 6, 9, and 12 hours of BWU treatment. Prior to each mass measurement stems were patted dry with paper towel and once returned to the wetting chamber post mass measurement, were again sprayed liberally with tap water.

6. Fluorescent microscopy: Stems from three individuals were cut into 3 cm long segments and sealed as per section 3. Using a nasal spray atomiser the three segments from each individual were sprayed with 1% w/v fluorescein and assigned to one of 0.5, 1 or 2 hours of incubation in a Petri dish. Stems were blotted dry and seals were removed for imaging of the bark surface confirming seal efficacy and identifying bark features of note under a Zeiss Axiostar Plus epifluorescence microscope with filter cube Dapi/Fitc/Tritc (Chroma 96000, Chroma, Vermont USA) and illuminated with a CoolLED PE300-W at the lowest blue light intensity. Segments were carefully rinsed with water at the middle of the segment and patted dry. A sledge microtome G.S.L.1 (S.Lucchinetti, Schenkung Dapples, Zurich, Switzerland) was used to cut 100 µm and 200 µm thick transverse slices. Slices were observed dry on a slide under a coverslip using the above Zeiss Axiostar Plus epifluorescence microscope under both fluorescence and brightfield. The Halide Mark II Pro Camera app on an iPhone XR (iOS15.6.1) was used to capture RAW format images. The procedure was then repeated for stems from three additional individuals, cut into two segments and assigned to either 4 or 6 hours of incubation. Fiji imaging software (Schindelin et al., 2012) was used to analyse images of three transverse slices from each segment to measure fluorescein area in the stem, depth of penetration from bark to pith and dye entry points through the bark.

7. Stem anatomy: Transverse slices 100 µm and 200 µm thick were taken from five stems and imaged as described in section 6 under a Zeiss Axiostar Plus epifluorescence microscope under both brightfield to characterise stem anatomy, and under blue light for comparison of fluorescence with and without exposure to fluorescein. Tissue layer thickness and area, and vessel size and density (measured in 1 mm² of vascular tissue) was measured on images of two slices from each individual using Fiji imaging software.

8. BWU under varying levels of dehydration: 20 saplings were assigned to one of four dehydration treatments: no dehydration, 0.5 hours, 1 hour and 4.5 hours of dehydration. Saplings in the greenhouse were wrapped loosely with damp paper towel, clingfilm and covered with a bag overnight to approximately equilibrate stems with root water. Saplings were unwrapped and shoots were cut. No dehydration shoots were immediately equilibrated and measured for water potential as described in section 2. Shoots assigned to 0.5, 1 and 4.5 hours of dehydration were placed uncovered in the growth chamber described in section 5 before equilibration and measurement of water potential as described in section 2. The stem was cut from each shoot, sealed as per section 3, measured for stem characteristics as per section 4, and measured for BWU as described in section 5.

9. BWU under light or dark conditions: 15 saplings were assigned to one of dark, light or bare stem treatments, wrapped with damp paper towel and clingfilm, covered by a black plastic bag and a thick paper bag to prevent light exposure prior to the treatment and left overnight. Shoots were cut while remaining covered. The following procedure was carried out in low light conditions to prevent light exposure outside of the assigned treatment. Shoots were measured for water potential as per section 2. Stems were cut and cut ends of stems assigned to light and dark treatments were sealed as per section 3. Bare stems were left unsealed to compare water uptake when vascular tissue was exposed. Stem characteristics were measured as per section 4. The wetting chambers assigned to dark stem treatments were wrapped with foil to prevent light exposure, chambers were left uncovered for those assigned to light and bare treatments. BWU was measured as described in section 5.

10. Stem swelling following BWU measured using X-ray micro-computed tomography (XMCT): One sapling with a stem diameter less than 10 mm was equilibrated with root water overnight as per section 8. The stem was cut from the sapling, cut ends were sealed as per section 3 and stem characteristics were measured as per section 4. Two replicate stems were treated as above but were left uncovered overnight and shoots were dehydrated in the growth chamber described in section 5 for one hour. Stems were wrapped tightly with dry paper towel and placed in a glass tube 10 mm in diameter and sealed with a thin silicone bung. Stems were scanned at an energy of 60 kV and a current of 60 μA at the National Laboratory for X-ray Micro Computed Tomography (CTLab, ANU), using a HeliScan MicroCT system with an optimized space-filling trajectory (Kingston et al., 2018) to yield sharp images (Latham et al., 2008; Myers et al., 2011). The first stem was scanned twice prior to the addition of water, both using a space-filling helical-scanning trajectory (Varslot et al., 2011) comprising 1.94 revolutions; the first was a 70-minute high-definition high-fidelity scan; the second was a faster, 17-minute lower-definition scan. The paper towel around the stem was saturated with 1.5 ml tap water introduced using a syringe with a needle inserted through the silicone bung. The stem was imaged using the lower-definition scan parameters for the first two hours before switching to the high-definition scan parameters for 12 hours with a final scan made at 24 hours of BWU exposure. Analysis revealed no rapid changes in the first two hours of BWU and so replicate stems were imaged using the high-definition parameters alone. Stems were removed, patted dry, weighed and dried for dry mass as described in section 4.

Reconstructed tomograms had dimensions of c. 2480 x 2480 x 2160 voxels with a voxel size of c. 4.2 μm. Details about the reconstruction process can be found in Kingston et al. (2018). Three scan slices from the same point in the stem were isolated dry and following 6, 12 and 24 hours of BWU. Each slice was analysed using Fiji imaging software. Tissue layer thickness was measured from pith to bark at the same three points around the stem circumference with no lenticels dry and following 6, 12 and 24 hours of BWU to control for variation in stem diameter and uneven swelling. Tissue layer thickness was also measured from pith to the centre of each lenticel in each slice where present dry and following 6, 12 and 24 hours of BWU to characterise and isolate effects of lenticel swelling. Lenticel area, inner and outer pore width and height were measured dry and following 6, 12 and 24 hours of BWU.

11. Statistical analysis: R statistical software package (R Core Team, 2023 version 4.3.1) was used for all statistical analysis. To test the effect of dehydration and light or dark on BWU a linear model with treatment as a fixed effect was used. The Anova() function in the car R package (Fox & Weisberg, 2019) was used to assess the significance of main effects at the p<0.05 level. A linear mixed effects model was used to asses stem swelling dry and following 24 hours of BWU, with treatment as a fixed effect and random intercepts for each individual, slice analysed and measurement number within the slice using the lmer() function in the lmerTest R package (Kuznetsova et al., 2017). The anova() function was used to assess the significance of main effects and interactions at the p<0.05 level. Model fit was assessed using diagnostic residual plots and the Shapiro-Wilk test. Dependent variables were log transformed where necessary to improve model fit. Model estimated marginal means, standard error (SE) and mean difference were produced using the emmeans R package (Lenth, 2020).

Significance statement

Understanding the contribution of bark water uptake to the maintenance of whole plant hydration is essential to understanding how plants respond when water availability declines and demand for water increases. This dataset shows that bark water uptake through lenticels increases hydration in the stem, and therefore may play a pivotal role in maintaining turgor and reducing hydraulic vulnerability during water stress.