Quantitative phase tomography of Arabidopsis seeds reveals intercellular void network

Abstract

We used quantitative phase tomography with synchrotron radiation to elucidate the 3D structure of Arabidopsis seeds in their native state. The cells are clearly distinguished, and their internal structure is revealed through local variations in electron density. We visualized a 3D network of intercellular air space that might allow immediate gas exchange for energy supply during germination and͞or serve for rapid water uptake and distribution during imbibition.mature seed structure ͉ synchrotron radiation ͉ x-ray imaging ͉ embryo organs ͉ cell components O ur understanding of seed structure and germination depends critically on insights about the structural arrangement of organs and tissues within the seed. Germination starts with water uptake by the dry seed and is complete when the elongating radicle traverses the seed coat. One of the first changes during imbibition is reestablishment of respiration in mitochondria. This activity is connected to a remarkable initial rise in oxygen consumption that declines afterward until germination is complete; i.e., the radicle traverses the seed coat, and atmospheric oxygen can enter the seed and reach the young growing plantlet (1-3). In seeds, limited seed coat permeability is a strong obstacle to gas exchange (4, 5). Consequently, the oxygen needed for germination to start should be rapidly available within the seed. Oxygen might originate from inside the seed and͞or from water entering the seed at the beginning of imbibition. At present, no data are available concerning the internal oxygen content in mature seeds because the rigidity of the seed coat at that stage precludes measuring this content. However, stored internal oxygen should exist and be important for germination because oxygen production by endogenous photosynthesis is known to influence seed viability and germination. Unlike animals, plants lack specialized circulation systems for oxygen transport, and the question arises whether air (and oxygen) is stored in seeds and by which means it circulates.To answer these questions it is important to know the fine structure of the mature seeds. Histology of seeds is usually based on optical or electron microscopy of slices, the results from adjacent sections being then combined. The cutting and fixation steps alter the tissues, and only one-directional (i.e., 2D) processing is possible. Thus, methods based on slicing and fixation procedures cannot give reliable visualization of small cellular interspaces or indicate the existence of networks of spaces. We therefore looked for noninvasive tomographic techniques that could improve the visualization of cell-to-cell organization in the seed tissues. The tomographic approach consists of acquiring projections of an object along different directions and combining them computationally to obtain a 3D reconstruction of the object. This approach has been successfully used in soft x-ray microscopy, with Fresnel zone plates as objective lenses, providing unique visualization of a whole cell at 60-nm resolution (6, 7). The emer...

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“…These air networks are known to be specially useful during the imbibition process when an entry of water triggers the transition from an inert dry seed into a living plant. An imbibition process is expected to be faster when the air networks interconnect all the parts of the seed with each other [4]. The inspection of this connectivity can be done directly from the images.…”