In a recent study published in Biochimica et Biophysica Acta (BBA)-Bioenergetics researchers introduced a fluorescence microscopy technique called single-pixel reconstruction imaging (SPiRI) to visualize the three-dimensional (3D) structure of the thylakoid membrane in intact plant chloroplasts. The goal was to gain new insights into the photosynthetic membrane's structure and its response to environmental changes.
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Background
Photosynthesis is the process by which plants convert light energy into chemical energy for growth and metabolism. The thylakoid membrane, located inside the chloroplast, contains proteins and pigments that capture light and convert it into chemical energy. This membrane has two main parts: grana, rich in photosystem II (PSII), and stromal lamellae, which mainly contain photosystem I (PSI) and adenosine triphosphate (ATP) synthase.
Previous studies used 3D models based on electron microscopy (EM) data to understand how these domains form a continuous membrane system. However, EM does not show the functional organization of photosynthetic systems under natural conditions. Fluorescence imaging of chlorophyll pigments can reveal this, but most techniques have limited resolution and struggle to provide precise 3D recreation of the thylakoid membrane.
About the Research
In this paper, the authors developed and applied SPiRI, a type of confocal microscopy with a smaller detector than the point fluorophore’s image. This design allows SPiRI to achieve a 1.4-time improvement in lateral and axial resolution compared to traditional confocal microscopy, depending on the emission and excitation wavelengths.
Using a 488 nm laser to excite the sample and record signals in the 660-700 nm range (the fluorescence emission of PSII), SPiRI achieves axial and lateral resolutions of 365 nm and 140 nm, respectively, providing a 1.25-time improvement in lateral resolution.
The researchers applied SPiRI to capture two-dimensional (2D) images and 3D reconstructions of chloroplasts from peas (Pisum sativum), spinach (Spinacia oleracea), and Arabidopsis thaliana, grown under low or high light conditions.
They compared the grana diameter and fluorescence intensity of the grana and stroma lamellae across species and light conditions. They also visualized the complete 3D thylakoid network in intact, non-chemically-fixed chloroplasts.
Research Findings
SPiRI images clearly displayed grana stacks and stromal structures, similar to those seen with SIM or EM. The grana diameter varied with species and light conditions, with larger grana in chloroplasts from plants grown in low light. Increasing light intensity by five times reduced grana diameter by about 14 %.
SPiRI also provided 3D visualizations of chloroplasts that could be viewed from any angle. The grana appeared as elongated cylinders, mainly aligned parallel to the microscope's optical axis. The stacked areas were irregular and sometimes merged. Most stromal connections between grana were at an angle of about 155 °, while the rest were at about 135 °.
The researchers measured fluorescence intensity in the stroma lamellae and found it significantly lower than in the proximal grana, indicating less PSII in the stroma lamellae. The intensity was variable but consistent along the lamellae axis, suggesting a varied but even distribution of PSII within the stroma lamellae.
Applications
The authors demonstrated that SPiRI is a powerful and simple technique for studying the structure and function of the thylakoid membrane in plant chloroplasts. SPiRI provides high-resolution images and 3D reconstructions, revealing the complex network of grana and stroma lamellae and their responses to different light conditions.
It also offers insights into the functional organization of photosynthetic systems by measuring the fluorescence intensity of PSII in the grana and stroma lamellae. SPiRI can be used to study the photosynthetic membrane in vivo or conditions close to the native state, without specific staining or fluorescent tagging.
SPiRI can investigate how different scales of the photosynthetic membrane organization affect the regulation of energy flow during photosynthesis. It can explore the dynamics and adaptation of the thylakoid membrane in challenging environments, such as drought, heat, or salinity stress.
Additionally, SPiRI can compare the structure and function of the thylakoid membrane among different plant species and identify the genetic and molecular factors that control its organization.
Conclusion
The novel SPiRI technique effectively visualized the 3D architecture of the thylakoid membrane. It provided high-resolution images and 3D reconstructions, revealing the complex network of grana and stroma lamellae and their responses to different light conditions. SPiRI proved to be a promising method for studying the structure and function of the photosynthetic membrane under physiological conditions.
Journal Reference
Streckaite, S., et al. (2024). Functional organization of 3D plant thylakoid membranes as seen by high-resolution microscopy. Biochimica et Biophysica Acta (BBA) - Bioenergetics. DOI: 10.1016/j.bbabio.2024.14949
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