作者感谢欧盟 H2020 计划（项目 GAIN4CROPS，GA 编号 862087）。AgroCropFuture 农业生态学和未来气候中的新作物卓越中心由爱沙尼亚教育和研究部资助。我们要感谢 Evelin Loit-Harro 教授提供 T. aestivum 和 Z. mays 的种子，感谢 Paula Palmet 和 Vaiko Vainola 在准备叶横截面方面的帮助，以及 João Paulo de Silva Souza 在分析方面的帮助。所有图像均来自爱沙尼亚生命科学大学的显微镜部门，在各种项目下。

1. 植物生长条件
<ol><li>分别选择小麦 （Triticum aestivum L. Var. 冬小麦， “Fredis”） 和玉米 （Zea mays L. Var. Saccharata， “Golden Bantam”） 作为代表性的 C3 和 C4 植物。在环境受控的生长室中用种子培养这两种植物。</li>
	<li>将相对湿度保持在 55%，空气温度为 25 °C/18 °C（昼/夜），光照时间为 12 小时。将植物表面的光合光子通量密度 （Q） 保持在 1200 μmol/m<sup...

提高 C4 作物光合效率的显微镜分析

在本文中，我们讨论了测量叶片解剖结构的定量和定性方法以及优化它们的方法。此外，该方法应用于代表性作物物种，以确定哪些解剖性状在区分 C3 和 C4 横截面时最有用。了解这些特征至关重要，因为被称为 C2 光合作用的杂交物种正在成为一种更有前途的研究途径。截至目前，只有一种作物物种 Diplotaxis tenuifolia （芝麻菜）被确定使用 C2 光合作用，但?...

光合作用是将光能转化为化学能的基本过程，是陆地营养网络的基石。大多数植物遵循光合作用的 C3 途径，其中主要的光合作用产物是三碳化合物甘油酸酯 3-磷酸盐。C3 光合作用是在 20 多亿年前在大气中进化而来的，其中 CO2 含量丰富，O21 含量较低。在这些条件下进化的关键光合酶核酮糖 1,5 二磷酸羧化酶/加氧酶 （Rubisco） 在当前低 CO2 高 O2 条件下是次优的，因为它与 O2 竞争性反应，启动光呼吸2。光呼吸是一种浪费的途径，它消耗能量而不是产生能量并释放 CO2 作为副产品。因此，在叶绿体中 Rubisco 周围保持高 CO2 浓度以防止氧合至关重要 3,4。由于 C3 植物无法浓缩 CO2，因此 CO2 从环境空气中大量吸收到叶绿体中，抑制光合作用并影响植物生长和生物质生产 2,5,6。
在 C3 植物中，光合作用受到 CO2 通过气孔进入、通过叶肉扩散以及光合酶的生化活性的限制。CO2 的进入首先受到气孔导度的限制，气孔导度受空气温度和湿度等环境条件控制。然后 CO2 通过叶子的内部气相和液相扩散到 Rubisco7。在 C3 植物中，光合作用的所有阶段都发生在叶肉细胞的叶绿体中，植物需要保持 CO2 从大气中持续流入叶绿体。叶绿体中 CO2 的可用性取决于气孔开放性、叶肉结构以及单个细胞和叶绿体特性，这使得植物容易受到环境限制的影响，最终影响光合作用，如低水可用性和高温 7,8,9,10，特别突出了它们对气候变化条件的脆弱性11。
鉴于 C3 途径的低效率带来的挑战，以及维持最佳 CO2 水平和对环境因素的敏感性的限制，在某些植物中，另一种途径，即 C4 光合作用途径，已经进化出来。特征性地，C4 植物具有两条空间上分离的生化途径;磷酸烯醇式丙酮酸羧化酶在叶肉细胞中发生初始 CO2 固定，磷酸烯醇式丙酮酸羧化酶对 CO2 的亲和力高于 Rubisco，并且也缺乏氧合活性。形成的 C4 产物进一步转运到束状鞘细胞，在那里脱羧，CO2 再次被 Rubisco（C3 光合作用）释放和固定12,13,14。PEP 羧化酶对 CO2 的亲和力更大，束鞘细胞的厚细胞壁允许 CO2 在束鞘细胞中浓缩，因此，C4 植物通过空间分离 CO2 固定和卡尔文循环来最大限度地减少光呼吸。C4 途径的采用展示了大自然对环境限制的适应性反应，为在不断变化的气候条件下提高作物生产力和复原力的潜在策略提供了见解15。
C4 植物叶结构的专门解剖结构的特征是叶脉被含有叶绿体的扩大的维管束鞘细胞包围，并且叶肉细胞呈放射状排列，外环围绕束鞘细胞。叶肉细胞靠近束鞘细胞，这使得代谢物能够在两种细胞类型之间快速连续地运输。该细胞的排列是典型的 C4 植物，称为 Kranz 解剖学16。在 C3 物种中，叶肉细胞的特化和处置可能会有所不同，但束鞘细胞明显较小，并且有一些叶绿体或根本没有叶绿体。特异性 Kranz 解剖结构允许将 CO2 集中在 C3-羧化酶 Rubisco 所在的束鞘细胞中的叶绿体中，从而有效阻碍光呼吸 4,17,18。尽管其看似复杂的排列，但这些变化在被子植物的进化中独立地多次发生，表明它是一个相对可行的进化途径 19,20,21，并且各种分类群已被证明处于 C 3 和 C4 碳代谢之间的中间阶段，称为 C3-C 4 或 C 2， 具有浓缩和重新吸收 CO2 22,23,24,25 的能力。
许多 C4 植物是具有高度经济重要性的作物，对 C3 作物（如水稻）进行基因工程改造以提高其气候适应力并确保产量一直是过去几十年来感兴趣的话题26,27。然而，工程工作需要详细了解 C4 特化解剖结构及其如何控制光合作用 2,28。
建立 C4 Kranz 解剖结构是实现将 C4 光合作用工程化为 C3 作物的雄心勃勃目标的先决条件25。然而，目前对 Kranz 解剖学的调节和快速筛选其关键解剖特征的方法的理解是有限的，因此很难识别杂交物种。先前的研究表明，调节 C3 和 C4 植物光合效率的关键性状包括脉间距离、束鞘复合体的直径和束鞘细胞的大小14,29。这些性状可以使用徒手切片轻松筛选，并使用半薄切片进行定量分析。在这里，我们描述了通过徒手交叉和光学显微镜评估允许 C3 和 C4 解剖区分诊断的性状的方法，即束鞘面积、静脉间距离和静脉频率。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Disodium hydrogen phosphate dihydrate (Na2HPO42H2O) pure</td>
			<td>PENTA, CZ</td>
			<td>10028-24-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Embedding Film, 7.8 mil Thick, 8 x 12.5, (203 x 318mm)</td>
			<td>ACLAR, US</td>
			<td>10501-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol, abs. 100% a.r.</td>
			<td>Chem-Lab NV, BE</td>
			<td>CL00.0505.1000</td>
			<td>Danger: Highly inflammable liquid and vapour.</td>
		</tr>
		<tr>
			<td>EVOS Invitrogen FL Auto 2 Imaging System</td>
			<td>Thermo Fisher Scientific, US</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flat Embedding PTFE Mold with Metal Frame, 16 cavities</td>
			<td>PELCO, US</td>
			<td>10501</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass vial 2 ml</td>
			<td>VWR Life Science, US</td>
			<td>548-0045</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde 50% solution</td>
			<td>VWR Life Science, US</td>
			<td>23H2856331</td>
			<td>Danger: Fatal if inhaled. Toxic if swallowed. Causes severe skin burns and eye damage. May cause respiratory irritation. Wear protective gloves, protective clothing, eyes and face protection.</td>
		</tr>
		<tr>
			<td>Histo diamond knife</td>
			<td>Diatome, US</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LEICA EM UC7</td>
			<td>Leica Vienna, AT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LR White resin hard grade</td>
			<td>Electron Microscopy Sciences, US</td>
			<td>14383</td>
			<td>Danger: Causes skin irritation. Causes severe eye irritation May cause respiratory irritation. May cause drowsiness or dizziness Wear protective gloves, protective clothing, eyes and face protection.</td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Normax, PT</td>
			<td>5470308A</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Eclipse E600 and Nikon DS0Fi1 5 MP</td>
			<td>Nikon Corporation, JP</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Osmium Tetroxide (OsO4)</td>
			<td>Agar Scientific Ltd, GB</td>
			<td>R1019</td>
			<td>Danger: Fatal if swallowed, in contact with skin or if inhaled. Causes severe skin burns and eye damage Wear double protective gloves, protective clothing, eyes and face protection.</td>
		</tr>
		<tr>
			<td>Pipette and pipette tips</td>
			<td>Thermo Scientific, FI</td>
			<td>KJ16047</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dihydrogen phosphate dihydrate (NaH2PO4 . 2H2O) pure</td>
			<td>PENTA, CZ</td>
			<td>13472-35-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe 10 ml</td>
			<td>Ecoject, DE</td>
			<td>20010</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluidine blue, general purpose grade</td>
			<td>Fisher Scientific, GB</td>
			<td>2045836</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessing structural traits in triticum aestivum and zea mays for c3 and c4 photosynthetic differentiation using free-hand and semi-thin sections

与 C3 机制相比，C4 光合作用的效率更高，源于其将 CO2 浓缩在束鞘细胞中的能力。C4 光合作用的有效性和内在水分利用效率与叶肉和束状叶细胞的份额、束状叶鞘的大小和密度以及束状鞘状细胞的大小、密度和细胞壁厚度直接相关。可以使用常规光学显微镜对徒手和半薄切片进行这些性状的快速显微镜分析，通过识别和检查特定细胞类型提供有关 C4 作物光合效率的宝贵信息。此外，还显示了影响解剖测量和细胞类型诊断的手绘和半薄片制备错误，以及如何避免这些错误。这种显微镜方法提供了一种有效的方法来收集对环境变化的光合适应的见解，并有助于快速筛选作物以适应未来的气候。

Photosynthesis is a fundamental process where light energy is converted into chemical energy, serving as the cornerstone of terrestrial trophic networks. The majority of plants follow the C3 pathway of photosynthesis, where the primary photosynthetic product is the three-carbon compound glycerate 3-phosphate. C3 photosynthesis evolved over 2 billion years ago in the atmosphere abundant in CO2 and low in O21. The key photosynthetic enzyme ribulose 1,5 bisphosphate carboxylase/oxygenase (Rubisco), which evolved under these conditions, is suboptimal for current low CO2 high O2 conditions as it competitively reacts with O2, initiating photorespiration2. Photorespiration is a wasteful pathway that consumes energy instead of producing it and releasing CO2 as a byproduct. Consequently, it is crucial to maintain high CO2 concentration around Rubisco in chloroplasts to prevent oxygenation3,4. Due to the inability of C3 plants to concentrate CO2, there is a significant CO2 drawdown from ambient air to chloroplasts, curbing photosynthesis and affecting plant growth and biomass production2,5,6.
In C3 plants, photosynthesis is limited by the entrance of CO2 through stomata, its diffusion through the mesophyll, and the biochemical activity of photosynthetic enzymes. The entry of CO2 is first limited by stomatal conductance, which is controlled by environmental conditions such as air temperature and humidity. Then CO2 diffuses through the leaf&#39;s internal gaseous and liquid phase to Rubisco7. In C3 plants, all stages of photosynthesis occur in the chloroplasts of mesophyll cells, and plants need to maintain a constant influx of CO2 from the atmosphere into chloroplasts. The dependence of CO2 availability in chloroplasts on stomatal openness, mesophyll architecture, and individual cell and chloroplast characteristics leaves plants susceptible to environmental constraints that eventually affect photosynthesis, like low water availability and high temperatures7,8,9,10, particularly highlighting their vulnerability to climate change conditions11.
Given the challenges posed by the inefficiencies of the C3 pathway, as well as limitations in maintaining optimal CO2 levels and susceptibility to environmental factors, in certain plants, another pathway, the C4 photosynthesis pathway, has evolved. Characteristically, C4 plants have two spatially separated biochemical pathways; the initial CO2 fixation occurs in mesophyll cells by phosphoenolpyruvate carboxylase, which has a higher affinity for CO2 than Rubisco and also lacks oxygenation activity. The formed C4 product is further transported to bundle sheath cells, where it is decarboxylated, and CO2 is again released and fixed by Rubisco (C3 photosynthesis)12,13,14. The greater affinity of PEP carboxylase to CO2 and thick cell walls of bundle sheath cells allows CO2 concentration in bundle sheath cells, and thus, C4 plants minimize the photorespiration by spatially segregating CO2 fixation and the Calvin cycle. The adoption of the C4 pathway showcases nature&#39;s adaptive response to environmental constraints, offering insights into potential strategies for improving crop productivity and resilience in changing climate conditions15.
The specialized anatomy of the leaf structure in C4 plants is characterized by veins surrounded by enlarged vascular bundle sheath cells containing chloroplasts and with a radial arrangement of mesophyll cells in an outer ring patterning around bundle sheath cells. The mesophyll cells are in close proximity to the bundle sheath cells, which enables a rapid and continuous transport of metabolites between the two cell types. This cell&#39;s arrangement is typical of C4 plants and is referred to as Kranz anatomy16. In C3 species, mesophyll cell specialization and disposition can vary, but bundle sheath cells are distinctly smaller and have a few chloroplasts or no chloroplasts at all. Specific Kranz anatomy allows concentrating CO2 in chloroplasts in bundle sheath cells where the C3-carboxylating enzyme Rubisco is located, effectively hindering photorespiration4, 17, 18. Despite its seemingly complex arrangement, these changes have occurred independently multiple times in the evolution of angiosperms, indicating that it is a relatively feasible evolutionary pathway19,20,21, and various taxa have been shown to be at an intermediate stage between C3 and C4 carbon metabolism, referred to as C3-C4 or C2, having abilities to concentrate and re-assimilate CO222,23,24,25.
Many C4 plants are crops with high economic importance, and genetically engineering C3 crops, like rice, to improve their climate resilience and secure the yield has been a topic of interest in the last decades26,27. However, the engineering efforts call for a detailed understanding of C4 specialized anatomy and how it controls photosynthesis2,28.
Establishing the C4 Kranz anatomy is a prerequisite to achieving the ambitious goal of engineering C4 photosynthesis into C3 crops25. However, the current understanding of the regulation of Kranz anatomy and methods for quickly screening its key anatomical traits is limited, making it difficult to identify hybrid species. Previous studies have shown that key traits regulating photosynthetic efficiency in C3 and C4 plants include the interveinal distance, the diameter of the bundle sheath complex, and the size of bundle sheath cells14,29. These traits can be easily screened using free-hand sections and quantitatively analyzed using semi-thin sections. Here, we describe the method of assessing the traits that allow for C3 and C4 anatomical differentiation diagnostics through free-hand cross and light microscopy, namely bundle sheath area, interveinal distance, and vein frequency.

作者聚焦：了解气候适应型作物植物结构-功能关系的创新方法

使用徒手和半薄切片评估 小麦和 Zea mays 的 C3 和 C4 光合作用分化的结构特征

Our research focuses on plant structure, function relationships. We aim to better understand the limitations to photosynthesis across plant functional types and how they can be improved for climate-resilient crops. New technology and software are expensive and often inaccessible, which requires a degree of trial and innovation to accomplish the same resolutions in imaging different plant functional types.The identification of anatomical structures that differentiate C3 and C4 leaves, particularly in as well as troubleshooting the preparative steps for microscopy.

图 1A 显示了用于新鲜切片和光学显微镜检查的叶子切片的正确方向。使用单面剃须刀和牙科蜡片切割新鲜切片的方法如图 1B 所示。生成的部分如图 1C 所示。
图 2 显示了 C3 植物 T. aestivum 和 C4 植物 Z. mays 的代表性叶子的徒手切片。在叶脉周围可以看到束鞘?...

评估 C3 和 C4 叶横截面之间的解剖学差异有助于了解光合作用效率。本文描述了徒手和半薄叶横截面的制备和检查，以及作物物种 Triticum aestivum 和 Zea mays 的准备注意事项。

The enhanced efficiency of C4 photosynthesis, compared to the C3 mechanism, arises from its ability to concentrate CO2 in bundle sheath cells. The ...

assessing-structural-traits-triticum-aestivum-zea-mays-for-c3-c4

Research

JoVE Journal

Biology

Assessing Structural Traits in Triticum aestivum and Zea mays for C3 and C4 Photosynthetic Differentiation Using Free-hand and Semi-thin Sections

使用徒手和半薄切片评估小麦和 Zea mays 的 C₃ 和 C₄ 光合作用分化的结构特征

Abstract

使用徒手和半薄切片评估 小麦和 Zea mays 的 C3 和 C4 光合作用分化的结构特征

Abstract

使用徒手和半薄切片评估小麦和 Zea mays 的 C₃ 和 C₄ 光合作用分化的结构特征