Dynamics of plant DNA replication based on PCNA visualization

Abstract

DNA replication is an essential process for the copying of genomic information in living organisms. Imaging of DNA replication in tissues and organs is mainly performed using fixed cells after incorporation of thymidine analogs. To establish a useful marker line to measure the duration of DNA replication and analyze the dynamics of DNA replication, we focused on the proliferating cell nuclear antigen (PCNA), which functions as a DNA sliding clamp for replicative DNA polymerases and is an essential component of replisomes. In this study we produced an Arabidopsis thaliana line expressing PCNA1 fused with the green fluorescent protein under the control of its own promoter (pAtPCNA1::AtPCNA1-sGFP). The duration of the S phase measured using the expression line was consistent with that measured after incorporation of a thymidine analog. Live cell imaging revealed that three distinct nuclear localization patterns (whole, dotted, and speckled) were sequentially observable. These whole, dotted, and speckled patterns of subnuclear AtPCNA1 signals were indicative of the G1 or G2 phase, early S phase and late S phase, respectively. The results indicate that the pAtPCNA1::AtPCNA1-sGFP line is a useful marker line for visualization of S-phase progression in live plant organs.

Figure 1. Expression pattern of AtPCNA1-EGFP in A. thaliana.

(a,b) Leaf epidermis (a) and trichome on a leaf (b) of plants expressing AtPCNA1-EGFP at 11 days after sowing (DAS). (c–g) Hypocotyl (c), root meristematic zone (d), root elongation zone (e), root differentiation zone (f), and a lateral root primordium (g) of plants expressing AtPCNA1-EGFP at 12 DAS. Cell walls stained with propidium iodide and AtPCNA1-EGFP signals are shown in magenta and green, respectively. Bars = 50 μm.

Figure 2. Subcellular localization and dynamics of AtPCNA1-EGFP signals in A. thaliana roots.

(a) AtPCNA1-EGFP signals in the root meristematic zone. Blue, yellow and red boxes indicate nuclei with whole, dotted, and speckled patterns of AtPCNA1-EGFP signals, respectively. Bar = 20 μm. (b) Magnified images of nuclei with whole, dotted, and speckled patterns of AtPCNA1-EGFP signals. These are Z-stack maximum-intensity projection images. Bar = 3 μm. (c) Surface plots of fluorescence images shown in (b). (d) Optical slices of each AtPCNA1 signal pattern. Images show a series of Z-slices at each 0.5 μm interval. Bar = 3 μm. (e) Subcellular localization of AtPCNA1 (green) in nuclei stained with DAPI (magenta) in the root meristematic zone. AtPCNA1 was localized in interphase nuclei, but not mitotic chromosomes at prophase (Pro), metaphase (Meta), anaphase (Ana) and telophase (Telo). Bar = 50 μm. (f) Subcellular localization of AtPCNA1 (green) in nuclei stained with DAPI (magenta) in the root elongation zone. Chromocenters were detected in the cells that showed the whole pattern of AtPCNA1-EGFP signals (white arrowheads), but not in cells exhibiting the speckled pattern of AtPCNA1-EGFP signals (yellow arrowhead). The right panel is a high-magnification image of the left panel. Bar = 50 μm (left panel) and 10 μm (right panel).

Figure 3. Live imaging of AtPCNA1-EGFP through the cell-cycle progression.

(a) Images taken at 5 min intervals in the meristematic zone of a root expressing AtPCNA1-EGFP. The pattern of AtPCNA1 signals changed sequentially from whole to dotted, speckled and whole through the cell-cycle progression. The numbers indicate duration from time 0 in minutes. The yellow and red outer frames indicate that signals in the nucleus indicated by a white arrowhead exhibit dotted and speckled patterns, respectively. Bar = 5 μm. (b) Duration of dotted and speckled patterns of AtPCNA1-EGFP signals (n = 7 for dotted pattern, n = 8 for speckled pattern). Error bars denote the SE.

Figure 4. Relationship between subnuclear localization patterns of AtPCNA1 and DNA replication.

(a) EdU signals (magenta) in the meristematic zone of a root expressing AtPCNA1-EGFP (green). Bar = 50 μm. (b) Types of subnuclear localization patterns of AtPCNA1-EGFP and EdU signals. The classification of types is shown in Supplementary Table 1. Bar = 5 μm. (c) Frequency of each EdU signal pattern in nuclei with whole, dotted and speckled patterns of AtPCNA1 signals in epidermal cells of the root meristematic zone (n = 231 [whole], n = 24 [dotted] and n = 52 [speckled] nuclei from three individual plants). The blue graph (Type III) shows the frequency of EdU-negative cells. Yellow, orange and purple graphs indicate the frequency of the speckled, mixed and whole patterns of EdU signals, respectively. (d) Timeline of AtPCNA1 dynamics in the cell-cycle progression. Whole, dotted, and speckled patterns of AtPCNA1 signals are indicative of the G1 or G2 phase, early S phase and late S phase, respectively.

Figure 5. Effect of inhibitors of cell-cycle progression on the frequency of AtPCNA1 localization patterns.

(a) AtPCNA1 signals in the meristematic zone of roots treated for 24 h with 12 μg/mL aphidicolin (Aph) or 5 nM camptothecin (Cpt). Bar = 50 μm. (b) Frequency of each AtPCNA1 signal pattern in epidermal cells of the root meristematic zone. n ≥ 287 nuclei from 3–5 individual plants. Error bars indicate the SD.