Micromanipulation of amyloplasts with optical tweezers in <i>Arabidopsis</i> stems
Y. AbeKeisuke MeguriyaTakahisa MatsuzakiTeruki SugiyamaHiroshi YoshikawaMiyo Terao MoritaMasatsugu Toyota
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Abstract:
Intracellular sedimentation of highly dense, starch-filled amyloplasts toward the gravity vector is likely a key initial step for gravity sensing in plants. However, recent live-cell imaging technology revealed that most amyloplasts continuously exhibit dynamic, saltatory movements in the endodermal cells of Arabidopsis stems. These complicated movements led to questions about what type of amyloplast movement triggers gravity sensing. Here we show that a confocal microscope equipped with optical tweezers can be a powerful tool to trap and manipulate amyloplasts noninvasively, while simultaneously observing cellular responses such as vacuolar dynamics in living cells. A near-infrared (λ=1064 nm) laser that was focused into the endodermal cells at 1 mW of laser power attracted and captured amyloplasts at the laser focus. The optical force exerted on the amyloplasts was theoretically estimated to be up to 1 pN. Interestingly, endosomes and trans-Golgi network were trapped at 30 mW but not at 1 mW, which is probably due to lower refractive indices of these organelles than that of the amyloplasts. Because amyloplasts are in close proximity to vacuolar membranes in endodermal cells, their physical interaction could be visualized in real time. The vacuolar membranes drastically stretched and deformed in response to the manipulated movements of amyloplasts by optical tweezers. Our new method provides deep insights into the biophysical properties of plant organelles in vivo and opens a new avenue for studying gravity-sensing mechanisms in plants.Keywords:
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Cells of land plants contain several kinds of plastids such as chloroplasts, etioplasts, proplastids, leucoplasts, and amyloplasts. Among them, the chloroplast, the most well characterized type of plastid, relies on expression of plastid-encoded photosynthetic genes. The genome of plastids encodes many photosynthetic genes that are mainly transcribed by the plastid-encoded plastid RNA polymerase (PEP). Transcriptional activity of PEP is controlled by nuclear-encoded sigma factors that are important for transcription initiation and promoter selectivity. Arabidopsis thaliana possesses 6 sigma factor genes, SIG1 to 6. Here, we analyzed the function of SIG6, a gene related to chloroplast differentiation. The null mutant (sig6-1) of the SIG6 gene exhibited a pale green phenotype in the cotyledons and in the basal part of emerging true leaves at early stages of development. Interestingly, as leaves matured, the color of cotyledons and true leaves changed to green. In the wild-type and sig6-1 mutant, plastids visualized by green fluorescent protein (GFP) were observed under the epifluorescence microscope. In cotyledons of 3-day-old seedlings, chloroplasts of the sig6-1 mutant showed small and irregular morphology compared with that of the wild-type chloroplasts. However, amyloplasts and leucoplasts in root tissues showed no obvious differences between the wild-type and the sig6-1 mutant. These results suggest that SIG6 plays a key role during the early stages of chloroplast differentiation, but not in differentiation into other types of plastids such as leucoplasts and amyloplasts.
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Changes in the structure of plastids and mitochondria were observed with the electron microscope during the microsporogenesis and pollen development of Tradescantia paludosa. It was found that either organelle develops more or less synchronously and divides simultaneously within certain limited periods. Generation of the organelles is, thus, recognized. The plastids develop from proplastids to amyloplasts in each generation. The mitochondria take characteristic morphology for each generation (Tex-fig. 1). On the contrary to the Golgi body, the plastids and the mitochondria keep their continuity through alteration of generation; namely, at the division periods amyloplasts turn to proplastids by diminution of starch in the case of the plastids, and the meta-morphosis of forms or internal structures takes place in the case of the mito-chondria.
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When tobacco (Nicotiana tabacum L.) cultured cells (line BY-2) at the stationary phase were transferred to culture medium that contained cytokinin (benzyladenine) instead of auxin (2,4-dichlorophenoxyacetic acid), proplastids in the BY-2 cells were converted to amyloplasts within 48 h. The data obtained from in vitro transcription assays using isolated plastid-nucleoids (nuclei) strongly suggested that amyloplast formation in BY-2 cells was accompanied by changes in the transcriptional activities of plastid genes.
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Summary Starch granule size is an important parameter for starch applications in industry. Starch granules are formed in amyloplasts, which are, like chloroplasts, derived from proplastids. Division processes and associated machinery are likely to be similar for all plastids. Essential roles for FtsZ proteins in plastid division in land plants have been revealed. FtsZ forms the so‐called Z ring which, together with inner and outer plastid division rings, brings about constriction of the plastid. It has been shown that modulation of the expression level of FtsZ may result in altered chloroplast size and number. To test whether FtsZ is also involved in amyloplast division and whether this, in turn, may affect the starch granule size in crop plants, FtsZ protein levels were either reduced or increased in potato. As shown previously in other plant species, decreased StFtsZ1 protein levels in leaves resulted in a decrease in the number of chloroplasts in guard cells. More interestingly, plants with increased StFtsZ1 protein levels in tubers resulted in less, but larger, starch granules. This suggests that the stoichiometry between StFtsZ1 and other components of the plastid division machinery is important for its function. Starch from these tubers also had altered pasting properties and phosphate content. The importance of our results for the starch industry is discussed.
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