Fernández González Alma, Fang Ze Tian, Sen Dipankar, Henrich Brian, Nagashima Yukihiro, Sokolov Alexei V, Okumoto Sakiko, Verhoef Aart J
Department of Soil and Crop Sciences, Texas A&M University and Texas A&M AgriLife Research, College Station, TX, USA.
Institute of Quantum Science and Engineering, Texas A&M University, College Station, TX, USA.
Plant Methods. 2024 Dec 18;20(1):185. doi: 10.1186/s13007-024-01302-3.
Nitrate (NO) is one of the two major forms of inorganic nitrogen absorbed by plant roots, and the tissue nitrate concentration in roots is considered important for optimizing developmental programs. Technologies to quantify the expression levels of nitrate transporters and assimilating enzymes at the cellular level have improved drastically in the past decade. However, a technological gap remains for detecting nitrate at a high spatial resolution. Using extraction-based methods, it is challenging to reliably estimate nitrate concentration from a small volume of cells (i.e., with high spatial resolution), since targeting a small or specific group of cells is physically difficult. Alternatively, nitrate detection with microelectrodes offers subcellular resolution with high cell specificity, but this method has some limitations on cell accessibility and detection speed. Finally, optical nitrate biosensors have very good (in-vivo) sensitivity (below 1 mM) and cellular-level spatial resolution, but require plant transformation, limiting their applicability. In this work, we apply Raman microspectroscopy for high-dynamic range in-vivo mapping of nitrate in different developmental zones of Arabidopsis thaliana roots in-situ.
As a proof of concept, we have used Raman microspectroscopy for in-vivo mapping of nitrate content in roots of Arabidopsis seedlings grown on agar media with different nitrate concentrations. Our results revealed that the root nitrate concentration increases gradually from the meristematic zone (~ 250 µm from the root cap) to the maturation zone (~ 3 mm from the root cap) in roots grown under typical growth conditions used for Arabidopsis, a trend that has not been previously reported. This trend was observed for plants grown in agar media with different nitrate concentrations (0.5-10 mM). These results were validated through destructive measurement of nitrate concentration.
We present a methodology based on Raman microspectroscopy for in-vivo label-free mapping of nitrate within small root tissue volumes in Arabidopsis. Measurements are done in-situ without additional sample preparation. Our measurements revealed nitrate concentration changes from lower to higher concentration from tip to mature root tissue. Accumulation of nitrate in the maturation zone tissue shows a saturation behavior. The presented Raman-based approach allows for in-situ non-destructive measurements of Raman-active compounds.
硝酸盐（NO）是植物根系吸收的两种主要无机氮形式之一，根系中的组织硝酸盐浓度被认为对优化发育程序很重要。在过去十年中，在细胞水平上量化硝酸盐转运蛋白和同化酶表达水平的技术有了大幅改进。然而，在高空间分辨率下检测硝酸盐仍存在技术差距。使用基于提取的方法，从小体积细胞（即具有高空间分辨率）可靠估计硝酸盐浓度具有挑战性，因为针对小的或特定的细胞群在物理上很困难。另外，用微电极检测硝酸盐可提供亚细胞分辨率和高细胞特异性，但该方法在细胞可及性和检测速度方面有一些限制。最后，光学硝酸盐生物传感器具有非常好的（体内）灵敏度（低于1 mM）和细胞水平的空间分辨率，但需要植物转化，限制了它们的适用性。在这项工作中，我们应用拉曼显微光谱对拟南芥根不同发育区的硝酸盐进行高动态范围的原位体内成像。
作为概念验证，我们使用拉曼显微光谱对在不同硝酸盐浓度的琼脂培养基上生长的拟南芥幼苗根系中的硝酸盐含量进行了原位体内成像。我们的结果表明，在用于拟南芥的典型生长条件下生长的根系中，根硝酸盐浓度从分生组织区（距根冠约250 µm）到成熟区（距根冠约3 mm）逐渐增加，这一趋势此前尚未见报道。在不同硝酸盐浓度（0.5 - 10 mM）的琼脂培养基中生长的植物中都观察到了这一趋势。这些结果通过对硝酸盐浓度的破坏性测量得到了验证。
我们提出了一种基于拉曼显微光谱的方法，用于对拟南芥小根组织体积内的硝酸盐进行原位无标记成像。测量是在原位进行的，无需额外的样品制备。我们的测量揭示了从根尖到成熟根组织硝酸盐浓度从低到高的变化。硝酸盐在成熟区组织中的积累表现出饱和行为。所提出的基于拉曼的方法允许对拉曼活性化合物进行原位无损测量。
