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

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

已有少量研究证据表明，通过树皮吸收大气水分的过程被称为树皮吸水（bark water uptake, BWU），该过程可减轻木质部栓塞（tracheal embolism）、部分恢复导水率（hydraulic conductivity）并提升茎水势（stem water potential）（Katz等，1989；Earles等，2016；Liu等，2019）。据此，树皮吸水（BWU）在维持植物活组织与水力功能两方面均发挥重要作用，因此对于理解植物存活机制至关重要。然而，直接针对BWU开展的研究仍较为匮乏。本数据集以分布广泛、可耐受高盐环境且根系水分供应受限的红树林物种澳洲白骨壤（Avicenna marina subsp. australasica (Walp.) J.Everett）为研究对象，探讨了BWU的路径与水分运移过程、光照与脱水对BWU动态的影响，以及BWU对茎部肿胀的贡献。白骨壤（Avicenna marina）作为该亚种的原物种，在高盐生境中根系水分可及性有限，因此是研究BWU的理想材料。

本研究提出以下假说：1）树皮外表面的皮孔（lenticels）是BWU的主要通道；2）初始茎部脱水程度越高，BWU速率越高，这一现象反映了更大的水势梯度；3）光照条件下的BWU水平高于黑暗环境；4）BWU发生后，内部树皮与皮层组织会出现肿胀。

完整实验方法详见论文：Holly A. A. Beckett、Daryl Webb、Michael Turner、Adrian Sheppard与Marilyn C. Ball，2023年。《通过皮孔的树皮吸水过程提升茎部水分并促进茎肿胀》。《植物、细胞与环境》（Plant, Cell & Environment）。

简化实验方法：
1. 实验材料：按照Fuenzalida等（2022）的方法，于2019年从澳大利亚新南威尔士州克莱德河采集澳洲白骨壤（Avicenna marina subsp. australasica (Walp.) J.Everett）的繁殖体，随后在温室条件下以50%海水进行水培培育幼苗。将幼苗在茎与根的生长界面处切断，截取每段切下枝条基部最成熟的6~9 cm茎段，用于所有BWU实验。
2. 水势测定：在BWU实验开始前测定茎水势。将枝条置于黑色塑料袋中平衡30分钟。使用Scholander压力室（型号1050D，PMS仪器公司，美国奥尔巴尼），对从叶柄处剪下的完全展开叶片测定单株枝条的水势。
3. 茎段密封：在BWU测定前需密封茎段切口。将切口及切口处5 mm范围内的树皮涂抹凡士林（Vaseline），随后用封口膜（Parafilm）紧密缠绕。暴露的树皮与封口膜之间的界面使用正畸蜡密封。
4. 茎段参数测定：测量茎段长度、用于BWU实验的暴露茎段长度以及茎直径，基于圆柱形状假设计算茎段表面积与体积。将茎段置于60℃烘箱中干燥4天（LABEC实验室设备有限公司，澳大利亚）以测定茎干质量。
5. BWU测定：使用XP 205梅特勒托利多天平（Mettler-Toledo有限公司，瑞士格赖芬塞）测定初始茎段质量，随后将茎段置于独立的加湿舱中。加湿舱采用50 ml FALCON锥形离心管，内部安装塑料网以防止茎段接触舱底积水。使用装满自来水的鼻腔喷雾器喷洒茎段至完全湿润，模拟小雨、雾或露水滴。将加湿舱加盖密封，置于22.1℃的生长箱（TRIL-1175-SD-1SL Thermoline科学生长箱，Thermoline科学设备有限公司，澳大利亚）中的全光谱生长灯下。在BWU处理的0.5、1、2、4、6、9和12小时分别测定茎段质量。每次称量前，用纸巾将茎段轻轻拍干；称量后放回加湿舱前，再次用自来水充分喷洒茎段。
6. 荧光显微镜观测：选取3株幼苗的茎段，切成3 cm长的小段，按照第3节的方法进行密封。使用鼻腔喷雾器将每株幼苗的3个茎段喷洒1%重量体积比的荧光素溶液，随后将其置于培养皿中分别孵育0.5、1或2小时。将茎段吸干水分并移除密封装置，使用带有Dapi/Fitc/Tritc滤光块（Chroma 96000，Chroma公司，美国佛蒙特州）的Zeiss Axiostar Plus落射荧光显微镜（epifluorescence microscope），以最低蓝光强度的CoolLED PE300-W光源照明，对树皮表面进行成像，以验证密封效果并识别关键树皮结构。随后用清水仔细冲洗茎段中部并轻轻拍干。使用滑动式切片机G.S.L.1（S.Lucchinetti，Schenkung Dapples，瑞士苏黎世）切取100 µm和200 µm厚的横切片。将切片置于载玻片上，加盖盖玻片后，使用上述Zeiss Axiostar Plus落射荧光显微镜分别在荧光模式与明场模式下进行观测。使用iPhone XR（iOS15.6.1）上的Halide Mark II Pro相机应用程序拍摄RAW格式图像。上述流程重复操作：选取另外3株幼苗的茎段，切成2个小段，分别孵育4或6小时。使用Fiji成像软件（Schindelin等，2012）分析每个茎段的3个横切片图像，以测定茎内荧光素的面积、从树皮到髓心的渗透深度以及水分通过树皮进入的位点。
7. 茎部解剖结构观测：从5个茎段上切取100 µm和200 µm厚的横切片，按照第6节的方法使用Zeiss Axiostar Plus落射荧光显微镜进行成像：分别在明场模式下表征茎部解剖结构，在蓝光模式下对比施加与未施加荧光素时的荧光信号差异。使用Fiji成像软件分析每株幼苗的2个切片图像，测定组织层厚度与面积、导管尺寸与密度（在1 mm²的维管组织中计数）。
8. 不同脱水程度下的BWU测定：将20株幼苗随机分为4组，分别进行0小时（无脱水）、0.5小时、1小时和4.5小时的脱水处理。将温室内的幼苗用湿纸巾、保鲜膜松散包裹，再套上塑料袋放置过夜，使茎部与根系水分大致达到平衡。次日解开包裹并剪下枝条：无脱水组的枝条立即按照第2节的方法进行平衡并测定水势；0.5、1和4.5小时脱水组的枝条敞开放置于第5节所述的生长箱中，随后按照第2节的方法进行平衡并测定水势。将每个枝条的茎段剪下，按照第3节的方法密封，按照第4节的方法测定茎段参数，再按照第5节的方法测定BWU。
9. 光照与黑暗条件下的BWU测定：将15株幼苗随机分为黑暗组、光照组与裸露茎段组，先用湿纸巾和保鲜膜松散包裹，再套上黑色塑料袋与厚纸袋以避光，放置过夜。在保持包裹状态下剪下枝条，随后在弱光环境下开展后续操作以避免非实验处理的光照影响。按照第2节的方法测定枝条的水势，剪下茎段：光照组与黑暗组的茎段切口按照第3节的方法密封，裸露茎段组不做密封处理，以对比维管组织暴露时的水分吸收情况。按照第4节的方法测定茎段参数。黑暗组的加湿舱用铝箔包裹以避光，光照组与裸露茎段组的加湿舱保持敞口。按照第5节的方法测定BWU。
10. 基于X射线显微计算机断层扫描（XMCT）的BWU后茎肿胀测定：选取1株茎直径小于10 mm的幼苗，按照第8节的方法使其与根系水分平衡过夜。剪下其茎段，按照第3节密封切口，按照第4节测定茎段参数。另外2个重复茎段按照上述流程处理，但不包裹过夜，而是将枝条置于第5节所述的生长箱中脱水1小时。将茎段用干纸巾紧密包裹，放入直径10 mm的玻璃管中，用薄硅胶塞密封。在澳大利亚国立大学X射线显微计算机断层扫描国家实验室（CTLab, ANU），使用优化空间填充轨迹的HeliScan MicroCT系统（Kingston等，2018），以60 kV能量、60 μA电流进行扫描，以获取清晰图像（Latham等，2008；Myers等，2011）。在加水前，对第一个茎段进行两次扫描：均采用包含1.94圈的空间填充螺旋扫描轨迹（Varslot等，2011），第一次为70分钟的高清晰度高保真扫描，第二次为17分钟的快速低清晰度扫描。通过硅胶塞插入针头，用注射器向包裹茎段的纸巾注入1.5 ml自来水使其饱和。在BWU处理的前2小时，使用低清晰度扫描参数进行成像；随后切换为高清晰度扫描参数，持续12小时；最后在BWU处理24小时时进行最终扫描。前期分析显示BWU前2小时无快速变化，因此后续重复茎段仅使用高清晰度参数进行成像。将茎段取出、拍干、称重，按照第4节的方法测定干质量。
重建的断层扫描图像尺寸约为2480×2480×2160体素，体素大小约为4.2 μm。重建流程的详细信息详见Kingston等（2018）。选取茎部同一位点的3个扫描切片，分别在未处理（干燥状态）以及BWU处理6、12和24小时后进行分离。使用Fiji成像软件分析每个切片：在茎周无皮孔的3个相同位点，从髓心到树皮测量组织层厚度，以控制茎直径差异与肿胀不均的影响；在每个切片中存在皮孔的位置，从髓心到皮孔中心测量组织层厚度，以表征并分离皮孔肿胀的效应。同时测量干燥状态以及BWU处理6、12和24小时后的皮孔面积、内外孔隙宽度与高度。
11. 统计分析：所有统计分析均使用R统计软件包（R核心团队，2023年版本4.3.1）完成。为检验脱水程度与光照条件对BWU的影响，采用以处理为固定效应的线性模型。使用car R包中的Anova()函数（Fox与Weisberg，2019）评估主效应的显著性，显著性水平设为p<