Does stomatal patterning in amphistomatous leaves minimize the CO2 diffusion path length within leaves?

Photosynthesis is co-limited by multiple factors depending on the plant and its environment. These include biochemical rate limitations, internal and external water potentials, temperature, irradiance, and carbon dioxide (CO2). Amphistomatous leaves have stomata on both abaxial and adaxial leaf surfaces. This feature is considered an adaptation to alleviate CO2 diffusion limitations in productive environments as the diffusion path length from stomate to chloroplast is effectively halved in amphistomatous leaves. Plants may also reduce CO2 limitations through other aspects of optimal stomatal anatomy: stomatal density, distribution, patterning, and size. A number of studies have demonstrated that stomata are overdispersed compared to a random distribution on a single leaf surface; however, despite their prevelance in nature and near ubiquity among crop species, much less is known about stomatal anatomy in amphistomatous leaves, especially the coordination between leaf surfaces. Here we use novel spatial statistics based on simulations and photosynthesis modeling to test hypotheses about how amphistomatous plants may optimize CO2 diffusion in the model angiosperm Arabidopsis thaliana grown in different light environments. We find that 1) stomata are overdispersed, but not ideally dispersed, on both leaf surfaces across all light treatments; 2) the patterning of stomata on abaxial and adaxial leaf surfaces is independent; and 3) the theoretical improvements to photosynthesis from abaxial-adaxial stomatal coordination are miniscule (≪ 1%) across the range of feasible parameter space. However, we also find that 4) stomatal size is correlated with the mesophyll volume that it supplies with CO2, suggesting that plants may optimize CO2 diffusion limitations through alternative pathways other than ideal, uniform stomatal spacing. We discuss the developmental, physical, and evolutionary constraits which may prohibit plants from reaching this theoretical adaptive peak of uniform stomatal spacing and inter-surface stomatal coordination. These findings contribute to our understanding of variation in the anatomy of amphistomatous leaves.

Methods
Plants from the Columbia (Col-0) ecotype of Arabidopsis thaliana (L.) Heynh. were grown in three different light environments: low light (PAR = 50 μmol m-2  s-1), medium light (100 μmol m-2  s-1), and high light (200 μmol m-2  s-1). PAR stands for photosynthetically active radiation. Seeds were surface-sterilized and stratified at 4 °C for 3–5 d in 0.15% agarose solution and then sown directly into Pro-Mix HP soil (Premier Horticulture; Quakerstown, PA, USA) and supplemented with Scott's Osmocote Classic 14-14-14 fertilizer (Scotts-Sierra, Marysville, OH, USA). At 10–14 d, seedlings were thinned so only one seedling per container remained. Plants were grown to maturity in growth chambers where the conditions were as follows: 16 : 8 h, 22 : 20°C, day : night cycle. Imaging of the epidermis and internal leaf structures was performed using a Leica SP5 confocal microscope (Leica Microsystems, Wetzlar, Germany) with the protocol developed by Wuyts et al. (2010) with additional modification described in Dow et al. (2017). We captured 132 images in total, making 66 abaxial-adaxial image pairs. Images were square with an area of 0.386 mm2. We measured stomatal position and length using ImageJ (Schneider et al. 2012).

References

G. J. Dow, J. A. Berry, and D. C. Bergmann. Disruption of stomatal lineage signaling or transcriptional regulators has differential effects on mesophyll development, but maintains coordination of gas exchange. New Phytologist, 216(1):69–75, Oct. 2017. ISSN 0028-646X, 1469-8137. doi: 10.1111/nph.14746. URL https://nph.onlinelibrary.wiley.com/doi/10.1111/nph.14746.

C. A. Schneider, W. S. Rasband, and K. W. Eliceiri. NIH Image to ImageJ: 25 years of image analysis. Nature Methods, 9(7):671–675, July 2012. ISSN 1548-7091, 1548-7105. doi: 10.1038/nmeth.2089. URL http://www.nature.com/articles/nmeth.2089.

N. Wuyts, J.-C. Palauqui, G. Conejero, J.-L. Verdeil, C. Granier, and C. Massonnet. High-contrast three-dimensional imaging of the Arabidopsis leaf enables the analysis of cell dimensions in the epidermis and mesophyll. Plant Methods, 6(1):17, Dec. 2010. ISSN 1746-4811. doi: 10.1186/1746-4811-6-17. URL https://plantmethods.biomedcentral.com/articles/10.1186/1746-4811-6-17.

光合作用受多种因子共同限制，具体依赖于植物自身与所处环境。这些限制因子包括生化速率限制、内外水势、温度、辐照度与二氧化碳（carbon dioxide, CO₂）。两面气孔叶（amphistomatous leaf）在远轴面（abaxial leaf surface）与近轴面（adaxial leaf surface）均分布有气孔。该特征被认为是植物对高生产力环境的适应性演化：相较于仅单一面分布气孔的叶片，两面气孔叶中气孔到叶绿体的二氧化碳扩散路径长度可有效缩短一半，从而缓解扩散限制。植物还可通过优化气孔解剖结构的其他维度降低二氧化碳扩散限制，包括气孔密度、分布格局、排布模式与尺寸。多项研究已证实，单一叶面上的气孔分布相较于随机分布呈现过离散（overdispersed）特征；然而，尽管这类叶片在自然界中广泛存在、且在作物物种中近乎普遍分布，学界对两面气孔叶的气孔解剖结构的认知仍十分有限，尤其是不同叶面间气孔排布的协同关系。本研究基于模拟与光合模型构建新型空间统计方法，旨在检验相关假说：即在不同光环境下生长的模式被子植物拟南芥（Arabidopsis thaliana）中，两面气孔植物如何优化二氧化碳扩散过程。本研究获得以下结果：1）所有光处理组的两面叶面的气孔均呈现过离散特征，但未达到理想离散状态；2）远轴面与近轴面的气孔排布相互独立；3）在可行参数空间范围内，通过远轴-近轴气孔协同作用带来的光合理论提升幅度极小（远小于1%）。此外，我们还发现4）气孔尺寸与其为二氧化碳供应的叶肉体积呈显著相关，这表明植物或可通过理想均匀气孔间距之外的其他途径优化二氧化碳扩散限制。我们进一步讨论了可能阻碍植物达到均匀气孔间距与跨叶面气孔协同这一理论适应性峰值的发育、物理与演化约束。本研究结果有助于深化我们对两面气孔叶解剖结构变异的理解。

材料与方法
本研究使用哥伦比亚（Col-0）生态型拟南芥（Arabidopsis thaliana (L.) Heynh.），将其种植于三种不同光环境中：低光（光合有效辐射（photosynthetically active radiation, PAR）= 50 μmol·m⁻²·s⁻¹）、中光（100 μmol·m⁻²·s⁻¹）与高光（200 μmol·m⁻²·s⁻¹）。将种子进行表面灭菌后，置于0.15%琼脂糖溶液中于4 ℃下层积处理3~5天，随后直接点播于Pro-Mix HP营养土（Premier Horticulture，美国宾夕法尼亚州贵格敦），并施加Scott's Osmocote Classic 14-14-14复合肥（Scotts-Sierra，美国俄亥俄州马里斯维尔）。播种后10~14天，对幼苗进行间苗，每个容器仅保留1株幼苗。将植物置于生长箱中培养至成熟，生长箱条件设置为：光周期16小时光照/8小时黑暗，昼温22 ℃/夜温20 ℃。采用Leica SP5共聚焦显微镜（Leica Microsystems，德国韦茨拉尔）对表皮与叶片内部结构进行成像，成像方案基于Wuyts等人（2010）的方法，并结合Dow等人（2017）的改进方案。本研究共采集132张图像，对应66对远轴-近轴叶面图像。所有图像均为正方形，单张面积为0.386 mm²。使用ImageJ软件（Schneider等人，2012）测量气孔位置与气孔长度。

参考文献
1. G. J. Dow, J. A. Berry, D. C. Bergmann. 气孔谱系信号或转录调控因子的破坏对叶肉发育具有差异化影响，但维持了气体交换的协调性. *New Phytologist*, 216(1):69–75, 2017年10月. ISSN 0028-646X, 1469-8137. doi: 10.1111/nph.14746. 链接：https://nph.onlinelibrary.wiley.com/doi/10.1111/nph.14746.
2. C. A. Schneider, W. S. Rasband, K. W. Eliceiri. NIH Image到ImageJ：图像分析25年历程. *Nature Methods*, 9(7):671–675, 2012年7月. ISSN 1548-7091, 1548-7105. doi: 10.1038/nmeth.2089. 链接：http://www.nature.com/articles/nmeth.2089.
3. N. Wuyts, J.-C. Palauqui, G. Conejero, J.-L. Verdeil, C. Granier, C. Massonnet. 拟南芥叶片高对比度三维成像实现表皮与叶肉细胞尺寸分析. *Plant Methods*, 6(1):17, 2010年12月. ISSN 1746-4811. doi: 10.1186/1746-4811-6-17. 链接：https://plantmethods.biomedcentral.com/articles/10.1186/1746-4811-6-17.