Project description:Ultrathin sections of rice leaf blades observed two-dimensionally using a transmission electron microscope (TEM) show that the chlorenchyma is composed of lobed mesophyll cells, with intricate cell boundaries, and lined with chloroplasts. The lobed cell shape and chloroplast positioning are believed to enhance the area available for the gas exchange surface for photosynthesis in rice leaves. However, a cell image revealing the three-dimensional (3-D) ultrastructure of rice mesophyll cells has not been visualized. In this study, a whole rice mesophyll cell was observed using a focused ion beam scanning electron microscope (FIB-SEM), which provides many serial sections automatically, rapidly and correctly, thereby enabling 3-D cell structure reconstruction.Rice leaf blades were fixed chemically using the method for conventional TEM observation, embedded in resin and subsequently set in the FIB-SEM chamber. Specimen blocks were sectioned transversely using the FIB, and block-face images were captured using the SEM. The sectioning and imaging were repeated overnight for 200-500 slices (each 50?nm thick). The resultant large-volume image stacks ( x = 25??m, y = 25??m, z = 10-25??m) contained one or two whole mesophyll cells. The 3-D models of whole mesophyll cells were reconstructed using image processing software.The reconstructed cell models were discoid shaped with several lobes around the cell periphery. The cell shape increased the surface area, and the ratio of surface area to volume was twice that of a cylinder having the same volume. The chloroplasts occupied half the cell volume and spread as sheets along the cell lobes, covering most of the inner cell surface, with adjacent chloroplasts in close contact with each other.Cellular and sub-cellular ultrastructures of a whole mesophyll cell in a rice leaf blade are demonstrated three-dimensionally using a FIB-SEM. The 3-D models and numerical information support the hypothesis that rice mesophyll cells enhance their CO 2 absorption with increased cell surface and sheet-shaped chloroplasts.

Project description:Drought stress response involves vigorous reprogramming of transcriptome while the mechanism modulating this process remains elusive. The role of 3D-genome in the regulation of rice development has recently been unveiled in rice, which exhibited characteristics distinct from that in mammals and other plants. However, the relationship between spatial chromosome organization and drought responsive gene reprogramming is still poorly understood. In this study, we identified H3K9ac as an efficient histone mark that response dynamically to drought stress in rice and re-constructed high-resolution 3D genome contact maps based on these sites under the normal, drought, and recovery conditions. We discovered significant decondensation of chromatin contacts with over 10000 chromatin loops lost upon drought stress treatment while the recovery of 3D genome was limited after 4-day’s re-watering. We identified dominate promoter-promoter interacting (PPI) loops under each condition and identified their significant correlations to altered gene expressions in response to the corresponding condition. Based on the relative contact intensities of the PPI connections, we characterized super-promoter regions that integrate closer connections of genes with more vigorous inductions to condition shifts. Especially, about 75% of the drought stress-dominate PPI loops were associated to the binding by a well-defined drought stress-responsive transcription factor, OsbZIP23. The mutation of OsbZIP23 led to the diffuse of over 80% DS-dominate PPI loops and impaired the expression of the connected genes. As a case study, we showed how OsbZIP23 binding to a super-promoter region can integrate the PPI loop formation and transcriptional activation of four function-vital dehydrin genes upon drought stress. Our results shed light on the mechanisms of 3D genome dynamic in response to water supply variations in rice and highlight the role of OsbZIP23 in the regulation of chromatin loop formation under drought stress.

Project description:Many biological processes involve the collective generation and transmission of mechanical stresses across cell monolayers. In these processes, the monolayer undergoes lateral deformation and bending because of the tangential and normal components of the cell-generated stresses. Monolayer stress microscopy (MSM) methods have been developed to measure the intracellular stress distribution in cell monolayers. However, current methods assume plane monolayer geometry and neglect the contribution of bending to the intracellular stresses. This work introduces a three-dimensional (3D) MSM method that calculates monolayer stress from measurements of the 3D traction stresses exerted by the cells on a flexible substrate. The calculation is carried out by imposing equilibrium of forces and moments in the monolayer, subject to external loads given by the 3D traction stresses. The equilibrium equations are solved numerically, and the algorithm is validated for synthetic loads with known analytical solutions. We present 3D-MSM measurements of monolayer stress in micropatterned islands of endothelial cells of different sizes and shapes. These data indicate that intracellular stresses caused by lateral deformation emerge collectively over long distances; they increase with the distance from the island edge until they reach a constant value that is independent of island size. On the other hand, bending-induced intracellular stresses are more concentrated spatially and remain confined to within one to two cell lengths of bending sites. The magnitude of these bending stresses is highest at the edges of the cell islands, where they can exceed the intracellular stresses caused by lateral deformations. Our data from nonpatterned monolayers suggests that biomechanical perturbations far away from monolayer edges also cause significant localized alterations in bending tension. The localized effect of bending-induced stresses may be important in processes like cellular extravasation, which are accompanied by significant normal deflections of a cell monolayer (i.e., the endothelium) and require localized changes in monolayer permeability.

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Three-dimensional ultrastructural change of chloroplasts in rice mesophyll cells responding to salt stress.

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