Introduction
The xylem system of vascular plants generally features a “tip-to-base” widening with the maximal number of the narrowest conduits in the terminal parts; the size of these terminal conduits should not vary with plant size (or leaf size) (Lechthaler, Colangeli, Gazzabin & Anfodillo, 2019, Rosell & Olson, 2019, West, Brown & Enquist, 1999). With this hierarchical and basipetally widening xylem architecture, the energy cost of long-distance water transport is minimized (Anfodillo, Carraro, Carrer, Fior & Rossi, 2006, Shinozaki, Yoda, Hozumi & Kira, 1964, Westet al. , 1999). Under the negative pressure created by stomatal transpiration, water ascends from the soil, progressively through stem and leaf xylem vessels, all the way up to the terminal stomata. Covariation of stomatal and xylem traits in leaves is required to maintain a balance in water exchange between the liquid (water delivery) and the vapor (water loss) phase (Brodribb, McAdam & Carins Murphy, 2017, Carins Murphy, Jordan & Brodribb, 2014, Zhang, Carins Murphy, Cardoso, Jordan & Brodribb, 2018).
There is mounting evidence that vein density is proportional to stomatal density in leaves, and this pattern is applicable to diverse plants within and across species (Brodribb et al. , 2017, Carins Murphy, Jordan & Brodribb, 2016, Fiorin, Brodribb & Anfodillo, 2016). However, the causality of this relationship is difficult to interpret for three reasons. Firstly, leaf vein traits have been proposed to be proxies for leaf xylem properties (Blonder, Violle, Bentley & Enquist, 2011, Sack, Scoffoni, McKown, Frole, Rawls, Havran, Tran & Tran, 2012). Veins consist of more than xylem (e.g. they also host phloem), so simply considering vein density will ignore xylem vessel number and vessel lumen diameter, which have been deemed the predictors of conductive path length and leaf area respectively (Echeverría, Anfodillo, Soriano, Rosell & Olson, 2019, Rosell & Olson, 2019). We are not aware of any studies linking stomatal traits to xylem traits per se (i.e. size covariation of stomata and xylem vessels) (but see Meinzer and Grantz (1990) about xylem-stomatal conductance relationships) within and across species. Secondly, stomatal and vein densities reflect leaf water relations in terms of a leaf plane, while it is a system of conduits within a three-dimensional system, obviously finely tuned by natural selection in a way that directs water nearly optimally given carbon costs, conductance, and embolism resistance (Enquist, 2002, Westet al. , 1999). Thirdly, these vein-stomatal density studies (Brodribb et al. , 2017, Carins Murphy et al. , 2014, Sack, Dietrich, Streeter, Sanchez-Gomez & Holbrook, 2008) use the water balance of single leaves to implicate the whole-plant water balance. This approach might be an oversimplification for understanding the entire liquid phase and the vapour phase relation, even though leaf area has been proved to predict photosynthetic productivity precisely, from the single leaf, to the branch, to the whole-tree, to the forest level (Li, Reich, Schmid, Shrestha, Feng, Lyu, Maitner, Xu, Li & Zou, 2020).
We address this knowledge gap with a laboratory growth experiment that enabled us to obtain xylem and stomatal traits both at leaf and whole-plant level. We grew seedlings of 53 diverse woody species from cool-temperate and Mediterranean Europe in a standard growing environment (Cornelissen, Castro-Díez & Hunt, 1996, Zhong, Castro-Diez, Puyravaud, Sterck & Cornelissen, 2019).
We examined relations between xylem dimensions (Zhong et al. , 2019) and stomatal dimensions of these seedlings both at whole-plant level and at leaf level. Specifically, this study presents, for the first time, the allometric scaling relationships at two scales: (i) between stem xylem cross-sectional area (as well as stem xylem conductance area) and total stomatal area at the whole-plant level, and (ii) between leaf midvein xylem area and leaf total stomatal area at single leaf level. We hereby introduce uniformity in the analyzed pairwise traits as they are expressed in the same physical units, which helps to represent more directly the selection effect on the water flux and enlightens our understanding of the whole-plant water balance.
Furthermore, as was proposed by the West-Brown-Enquist (WBE) model, the terminal vessels should be ‘invariant’ with plant size (or leaf size) along with plant growth for a given individual (Roddy, Théroux-Rancourt, Abbo, Benedetti, Brodersen, Castro, Castro, Gilbride, Jensen & Jiang, 2020, Simonin & Roddy, 2018, West et al. , 1999). We tested the relations between terminal vessel traits (i.e. minor vessel number and individual minor vessel area) and plant size (represented by stem xylem area and/or total leaf area per plant) across these 53 species, which were grown in a similar environment, in order to understand whether the terminal vessels ‘depended’ on plant size across diverse species. Additionally, the relations between stomatal traits (i.e. stomatal number and individual stomatal area) and leaf size (represented by midvein xylem area and/or average leaf area) were tested empirically, in order to link with the large body of studies on the allocation mechanism of leaf surface to stomata (Boer, Price, Wagner-Cremer, Dekker, Franks & Veneklaas, 2016, Franks & Farquhar, 2007, Parlange & Waggoner, 1970). Additionally, as stomatal allocation at the leaf surface tends to simultaneously minimize water loss (e.g. water exchange from the minor vessels to stomata) while maximizing gas exchange to maintain a constant photosynthetic productivity per unit leaf area (Boer et al. , 2016), we expect that the number of stomata should scale linearly with the number of minor vessels (Fig. 1).
Fig.1 Conceptual framework of this research concerning allometric relations of plant hydraulics across woody species. Natural selection acts on heritable variation between individuals within the same species. Individuals with vessels that do not widen with height growth, or widen little, will experience continual declines in leaf-specific conductance with height growth and therefore declining growth and reproductive output per unit leaf area. Individuals with vessels that widen very markedly would have conduits of low resistance, in contrast to the high-resistance variants with conduits that are ‘too narrow’, but they would have their own set of disadvantages. For example, for a given leaf area and transpirational demand, the wider conduits cost more for the same service provided. Each unit of carbon invested in excessively wide vessels is a unit that is not invested in further growth or reproduction, and so these variants should be at a selective disadvantage (Banavar, Cooke, Rinaldo & Maritan, 2014). Moreover, wider vessels are more vulnerable to gas embolisms that obstruct conductance, both from freezing ((Sevanto, Holbrook & Ball, 2012, Zanne, Tank, Cornwell, Eastman, Smith, FitzJohn, McGlinn, O’Meara, Moles, Reich, Royer, Soltis, Stevens, Westoby, Wright, Aarssen, Bertin, Calaminus, Govaerts, Hemmings, Leishman, Oleksyn, Soltis, Swenson, Warman & Beaulieu, 2014) and likely drought as well (Cai & Tyree, 2010, Jacobsen, Pratt, Venturas & Hacke, 2019, Liu, Gleason, Hao, Hua, He, Goldstein & Ye, 2019). As a result, plants with vessels that are ‘too wide’ would also be at a selective disadvantage (Zhong et al.(2019)). The variants that should have the largest amounts of surplus carbon to devote to growth and reproduction are those in the intermediate zone, in which conduits widen just enough that conductance remains constant per unit leaf area, but not so much as to incur excessive carbon costs and embolism vulnerability.
In our previous study, based on the same woody seedling populations, we found that, at the whole-plant level, the stem xylem cross-sectional area (Xstem ) of stem medium (a) closely scales with stem height (H ) and total leaf area per plant (LA ) asXstemH 1.52 andXstemLA 0.75 across all the studied species. For individual leaves, vessel diameter (D leaf) in the medium of leaf midvein (b) closely scales with average leaf area (MLA ) asD leafMLA 0.21 (Zhonget al. (2019). In this study, we test the poorly understood xylem-stomata covariation from the size perspective We ask: are there scaling relationships between total stomatal area and xylem cross-sectional area across species, at the entire plant and at individual leaf level? Specifically, we zoom in on the terminal part of water exchange (from minor vessels to stomata), and ask: does the minor vessel number (which scales with leaf area; see (Lechthaler et al. , 2019, Rosell & Olson, 2019)) scale with stomatal number per leaf and per plant across these woody seedlings? The conceptual picture should deepen our understanding of plants’ water transport system and have broad implications for integrating xylem architecture and stomatal distribution.
Based on the expectations above, we test the hypothesis that, despite large interspecific differences in leaf-habit, growth-form and relative growth rate (RGR), similar scaling should exist between total stomatal area and xylem area across woody seedlings, both at the entire plant and at individual leaf level, to ensure the balance between liquid- and vapour-phase water conductance. We also expect that the hypothesized scaling of total stomatal area to xylem area should be driven by the number covariation of stomatal and minor vessel elements; we also expect a scaling relation between mean stomatal area and mean minor vessel area as we presumed the distal element size for a given plant to be limited under long-term nature selection.