Mind the leaf anatomy while taking ground truth with portable chlorophyll meters.

Measurements of four chlorophyll meters — three transmittance-based (SPAD-502, Dualex-4 Scientific, and MultispeQ 2.0) and one fluorescence-based (CCM-300), were calibrated against biochemically assessed chlorophyll content (Chl) on three distinctive common leaf types differing in leaf anatomy: laminar (i.e., broadleaved woody species with different anthocyanin content verified by biochemical assay) dorsiventral leaves, narrow grass leaves, and conifer needles. Reflectance in the 400-2500 nm range was measured on the laminar leaf samples using a contact probe. Methods In the present study we investigated three distinctive leaf anatomical types: laminar (i.e., deciduous woody species) leaves, grass leaves, and needles. The three groups are defined as follows: 1) laminar leaves of woody angiosperms dorsiventrally flattened (i.e., bifacial) leaves with differentiated mesophyll to palisade and spongy parenchyma and reticulate anastomosing vasculature (Laminar leaves). We further distinguished three subtypes of laminar leaves 1a) deciduous tree species, 1b) evergreens trees and shrubs, and 1c) leaves with multi-layered epidermis represented by Ficus species. 2) The second group included C3 grasses with bifacial strap-like leaves with undifferentiated mesophyll with longitudinally arranged vasculature (Grass leaves), and 3) gymnosperm equilateral needle-like leaves without differentiated mesophyll and vascular bundle in the central cylinder (Needles) represented only by Norway spruce (Picea abies) though with irradiance induced differentiation into sun and shaded ecotypes. Collection of leaf samples Leaves were collected at four different locations in the Czech Republic during the growing seasons 2019, 2020, and 2021. The dates (as DOY - day of the year are indicated in particular datasets). Woody plants with laminar bifacial leaves with differentiated mesophyll were collected in the Botanical Garden of Charles University in Prague (50.072N, 14.424E). Plants were selected to correspond to one of the following groups: 1) deciduous species, 2) evergreens and 3) leaves represented by indoor grown Ficus species with a focus on thick leaves and multi-layered epidermis. Usually, shaded leaves were sampled from the ground. For independent verification of the relationship of Chl content to chlorophyll meter reading, leaves were sampled in the floodplain forest at the confluence of the rivers Morava and Dyje, near the town of Lanžhot (48.682N, 16.946E) using deciduous woody plants with laminar bifacial leaves and differentiated mesophyll. Sunlit and shaded branches were cut by a tree climber from mature trees of Acer campestre L., Carpinus betulus L., Fraxinus angustifolia Vahl., Populus alba L., Quercus cerris L., Quercus robur L. and Tilia cordata Mill. Grass leaves were represented by four coexisting wild species from Poaceae family (Calamagrostis villosa (Chaix) J.F.Gmel., Deschampsia cespitosa (L.) P.Beauv., Molinia caerulea (L.) Moench and Nardus stricta L.) and were collected in relict alpine-arctic grass tundra in the Krkonoše (Giant Mountains) (50.734N, 15.696E). For each species, six plots with homogeneous canopy cover of the species were sampled. Needle leaves were represented by mature trees of Norway spruce (Picea abies (L.) H. Karst.) collected at the experimental station Bílý Kříž, Beskydy Mountains, Czech Republic (49.503N, 18.539E). Sunlit and shaded branches were cut by a tree climber, and samples were taken from the current year's needles, the previous year's needles, and four-year-old needles. Leaf sampling Laminar leaves: leaves were measured immediately after being detached from the branch or stored in a refrigerator for no more than 30 minutes before processing. First, the reflectance of the leaves was measured using a spectroradiometer and a contact probe. Second, readings from all portable chlorophyll meters were recorded. Third, one disk (area = 68 mm2) was cut from each leaf for Chl and anthocyanin extraction. Finally, a square segment of the leaf was cut out and immersed in fixative solution for anatomical analysis. A second leaf of similar size, colour, position in the canopy, and developmental stage was removed from the branch, weighed, scanned, and later dried and weighed again. This "twin" was used to assess leaf mass per area (LMA), equivalent water thickness (EWT). Grass leaves: chlorophyll meter readings were taken on grass leaves attached to the plant using a chlorophyll meter (CCMCFR,) then leaves were collected immediately in the field for Chl extraction. A 2 cm long leaf segment was cut, flattened under a microscope glass, photographed for area assessment, and stored in plastic vials in a refrigerator before freezing. A subsample was weighed fresh, scanned, and dried for calculation of LMA and EWT. Needles: Shoots were separated from the branch, sorted by age, and stored in a refrigerator for no longer than 24 hours before processing. First, CCMCFR were taken from the middle part of three needles and the same needles were used for Chl extraction. A second parallel set of needles was immersed in fixative solution for anatomical analysis. The third set of needles was weighed fresh, scanned, and dried for calculation of LMA and EWT. Optical assessment of Chl content using portable chlorophyll meters Three transmittance-based chlorophyll meters: SPAD-502 SPAD), Dualex-4 Scientific (Dx) and MultispeQ (MSPQ), and one fluorescence chlorophyll meter: CCM-300 (CCM), were used for optical assessment of Chl content in leaves. For laminar leaves, three readings were taken on each leaf with each instrument from the adaxial leaf side. Measurements were taken in the central part of the leaf, avoiding the midrib and main veins. The three measurements were averaged, and the average was used as a representative value for the leaf. Measurements with all four chlorophyll meters (SPADvalues, Dxvalues, MSPQvalues, CCMCFR) were obtained for laminar leaves. For grass leaves, Chl values were measured at a single location in the apical third of the leaf blade. All four grass species were measured by CCM (CCMCFR), and three species with a wide enough lamina to cover the SPAD measurement area (Calamagrostis villosa, Deschampsia cespitosa, and Molinia careulea) were also measured by SPAD and SPADvalues detected. For the spruce needles, three needles were measured only once with the CCM, always taking a reading in the central part of the needle. The average of these three needle measurements was used to relate to Chl. Reflectance measurements and spectral processing Reflectance was measured for laminar leaves collected in Botanical Garden of Charles University in Prague and deciduous trees from floodplain forest. Leaf reflectance from the adaxial side of the leaves was measured with an ASD FieldSpec 4 Wide-Res spectroradiometer with attached contact probe (ASD Inc., Boulder, CO, USA). Three measurements per leaf were always taken, when leaf size allowed. Measurements were placed at the same locations where chlorophyll meter readings were taken. Leaf reflectance spectra ranging from 350 to 2500 nm were normalized against a white reference spectrum (99% Spectralon white panel) to obtain relative reflectance spectra. The median of the spectral curve from three measurements was used as a representative value for the leaf.本研究针对四款叶绿素仪开展校准工作：三款为基于透射法的叶绿素仪（transmittance-based chlorophyll meters），即SPAD-502、Dualex-4 Scientific与MultispeQ 2.0，另一款为基于荧光法的CCM-300（fluorescence-based chlorophyll meter）。校准以三种不同叶片解剖结构类型的常见叶片的生化测定叶绿素含量（Chl）为参照：分别为背腹扁平的层状叶片（即通过生化实验验证了花青素含量差异的阔叶木本植物叶片）、窄型禾本科叶片以及针叶。此外，使用接触式探头对层状叶片样品开展了400~2500 nm波段范围的反射率测量。 ## 研究方法 本研究共考察三类具有独特叶片解剖结构的叶片类型：层状叶片、禾本科叶片与针叶，并定义如下： 1. 层状叶片：木本被子植物的背腹扁平（即两面型）叶片，其叶肉分化为栅栏组织与海绵组织，维管束呈网状吻合结构（层状叶片）。本研究进一步将层状叶片划分为三个亚型：1a）落叶乔木物种，1b）常绿乔木与灌木，1c）以榕属（Ficus）物种为代表的具多层表皮的叶片。 2. 禾本科叶片：具有两面型带状结构的C3禾草叶片，叶肉未分化，维管束纵向排列（禾本科叶片）。 3. 针叶：裸子植物的等轴针状叶片，中央维管束柱中无分化的叶肉与维管束，本研究仅以挪威云杉（Picea abies）为代表，其可根据光照诱导分化为阳生与阴生生态型。 ## 叶片样品采集 叶片样品采集于捷克共和国境内的四个不同样点，采样时间覆盖2019、2020及2021年的生长季，具体日期以各数据集标注的日序（DOY, day of the year）为准。 1. 具背腹扁平层状叶片的木本被子植物：采集于布拉格查理大学植物园（50.072°N，14.424°E）。供试植物分为三类：1）落叶物种，2）常绿物种，3）以室内栽培榕属植物为代表的厚叶片、多层表皮叶片。采样通常选取植株基部的阴生叶片。 2. 独立验证叶绿素含量与叶绿素仪读数相关性的样品：采集于摩拉瓦河与迪耶河汇流处的漫滩林，靠近兰日霍特镇（48.682°N，16.946°E），供试植物为具背腹扁平层状叶片且叶肉已分化的落叶木本植物。由攀树人员从成熟的栓皮槭（Acer campestre L.）、欧洲鹅耳枥（Carpinus betulus L.）、狭叶白蜡（Fraxinus angustifolia Vahl.）、银白杨（Populus alba L.）、土耳其栎（Quercus cerris L.）、夏栎（Quercus robur L.）以及心叶椴（Tilia cordata Mill.）的植株上截取阳生与阴生枝条。 3. 禾本科叶片：采集于克尔科诺谢山（巨人山）的残遗高山-北极草甸苔原（50.734°N，15.696°E），供试物种为禾本科的4种野生共存物种：绒毛拂子茅（Calamagrostis villosa (Chaix) J.F.Gmel.）、丛毛发草（Deschampsia cespitosa (L.) P.Beauv.）、蓝茎茅（Molinia caerulea (L.) Moench）以及欧洲异燕麦（Nardus stricta L.）。针对每个物种，选取6个冠层覆盖均匀的样地开展采样。 4. 针叶叶片：采集于捷克共和国贝斯基迪山脉的比利克里日实验站（49.503°N，18.539°E）的成熟挪威云杉（Picea abies (L.) H. Karst.）植株。由攀树人员截取阳生与阴生枝条，分别采集当年生、前一年生以及四年生的针叶样品。 ## 叶片采样流程 ### 层状叶片 叶片从枝条上剪下后立即开展测量，或置于冰箱冷藏不超过30分钟后再进行处理。实验流程依次为：首先使用分光辐射度计与接触式探头测量叶片反射率；其次记录四款便携式叶绿素仪的读数；随后从每片叶片上切取面积为68 mm²的圆片，用于叶绿素与花青素提取；最后切取方形叶片片段，浸入固定液中以备解剖学分析。另外，从同一枝条上采集一片尺寸、颜色、冠层位置以及发育阶段均相似的“配对叶片”，称量鲜重、扫描成像，后续经干燥后再称量干重，以此计算单位面积叶质量（LMA）与等效水厚度（EWT）。 ### 禾本科叶片 使用叶绿素仪（CCMCFR）对植株上的活体禾本科叶片开展读数测定，随后立即在野外采集叶片用于叶绿素提取。切取2 cm长的叶片切段，置于载玻片下压平后拍照以评估叶面积，随后装入塑料瓶置于冰箱冷藏直至冷冻保存。取部分样品称量鲜重、扫描成像并干燥，用于计算单位面积叶质量与等效水厚度。 ### 针叶叶片 将枝条剪下后分离出针叶，按叶龄分组，置于冰箱冷藏不超过24小时后再开展处理。首先，从3根针叶的中部位置获取CCMCFR读数，随后使用同一根针叶开展叶绿素提取；另外取一组平行的针叶样品浸入固定液中以备解剖学分析；第三组针叶用于称量鲜重、扫描成像并干燥，以计算单位面积叶质量与等效水厚度。 ## 便携式叶绿素仪的叶绿素含量光学测定 本研究使用四款叶绿素仪开展光学测定：三款基于透射法的SPAD-502（SPAD）、Dualex-4 Scientific（Dx）与MultispeQ（MSPQ），以及一款基于荧光法的CCM-300（CCM）。 针对层状叶片，每款仪器均从叶片正面的中央区域（避开主脉与主要侧脉）选取3个测点开展读数，取平均值作为该叶片的代表性数值。最终获取了所有四款叶绿素仪的测定值（SPAD值、Dx值、MSPQ值与CCMCFR值）。 针对禾本科叶片，叶绿素仪读数选取在叶片叶尖1/3区域的单个测点完成。所有4种禾草均使用CCM（CCMCFR）开展测定，其中叶宽足够覆盖SPAD测量区域的3个物种（绒毛拂子茅、丛毛发草与蓝茎茅）额外使用SPAD开展测定并获取SPAD值。 针对云杉针叶，仅使用CCM对3根针叶的中部位置各测定一次，取这3根针叶的测定平均值以关联叶绿素含量。 ## 反射率测量与光谱处理 本研究针对采集于布拉格查理大学植物园的层状叶片以及漫滩林的落叶树种开展反射率测量。使用搭载接触式探头的ASD FieldSpec 4 Wide-Res分光辐射度仪（ASD公司，美国科罗拉多州博尔德）测量叶片正面的反射率。当叶片尺寸允许时，每片叶片开展3次测量，测点位置与叶绿素仪读数的测点位置保持一致。测量光谱范围为350~2500 nm，以99%反射率的Spectralon白色标准板作为参照进行归一化，得到相对反射率光谱。取3次测量光谱曲线的中位数作为该叶片的代表性反射率光谱。