SUMMARYThe starch-statolith hypothesis proposes that starch-filled amyloplasts act as statoliths in plant gravisensing, moving in response to the gravity vector and signaling its direction. However, recent studies suggest that amyloplasts show continuous, complex movements in Arabidopsis shoots, contradicting the idea of a so-called 'static' or 'settled' statolith. Here, we show that amyloplast movement underlies shoot gravisensing by using a custom-designed centrifuge microscope in combination with analysis of gravitropic mutants. The centrifuge microscope revealed that sedimentary movements of amyloplasts under hypergravity conditions are linearly correlated with gravitropic curvature in wild-type stems. We next analyzed the hypergravity response in the shoot gravitropism 2 (sgr2) mutant, which exhibits neither a shoot gravitropic response nor amyloplast sedimentation at 1 g. sgr2 mutants were able to sense and respond to gravity under 30 g conditions, during which the amyloplasts sedimented. These findings are consistent with amyloplast redistribution resulting from gravity-driven movements triggering shoot gravisensing. To further support this idea, we examined two additional gravitropic mutants, phosphoglucomutase (pgm) and sgr9, which show abnormal amyloplast distribution and reduced gravitropism at 1 g. We found that the correlation between hypergravity-induced amyloplast sedimentation and gravitropic curvature of these mutants was identical to that of wild-type plants. These observations suggest that Arabidopsis shoots have a gravisensing mechanism that linearly converts the number of amyloplasts that settle to the 'bottom' of the cell into gravitropic signals. Further, the restoration of the gravitropic response by hypergravity in the gravitropic mutants that we tested indicates that these lines probably have a functional gravisensing mechanism that is not triggered at 1 g.

A newly designed microfluidic chip with a pinched-channel structure and two pairs of electrodes has been developed to enable easier single-cell capture and lysis. The function of the chip was evaluated by introducing zucchini protoplast cells into the channel. In the first experiment, we attempted to break a cell using the through force of a triangular pinched structure via electroosmotic flow generated by outer electrodes. The pinched structure appeared to break the cell without applying the electric field to the cell directly; however, in this case, the breakable size of the cell was limited by the width of the pinched structure. The next attempt was to break cells regardless of their sizes using a pair of inner electrodes located under the pinched structure. The inner electrodes generated a gradient electric field around the captured cell by applying an alternative voltage to the electrodes. Captured cells with a diameter from 40 to 85 mm could be broken using the inner electrodes with a trapezoidal pinched structure, and the cells were successfully broken at 10 V pp or less at a frequency of 1 MHz.

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Gravity is a ubiquitous force on the earth and a crucial environmental signal for living organisms. To adapt and survive in the gravitational field, plants sense the gravity vector (magnitude and direction) and change their morphology accordingly. These are widespread phenomena known as 'gravity resistance' and 'gravitropism'. We have studied the early process of shoot gravitropism, gravity sensing, using molecular genetic techniques in combination with two novel microscopes, a vertical-stage confocal microscope and a centrifuge microscope. The vertical-stage confocal microscope is a vertically oriented microscope equipped with a rotatable stage, a spinning-disk confocal scanning unit and a back-illuminated EM-CCD camera, which allows fluorescence imaging of the Arabidopsis stem specimen before and after gravistimulation (changes in the direction of gravity). The centrifuge microscope is a spinning upright microscope equipped with a radio system, which allows bright-field imaging during centrifugation (changes in the magnitude of gravity). In this review, we will introduce the recently developed microscopes that are essential to gain new insights into gravity sensing mechanisms in Arabidopsis inflorescence stems.

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