Using a Live Analysis System to Study Amyloplast Replication in Arabidopsis Ovule Integuments

Materials and reagents

Biological materials

1. Seeds of transgenic Arabidopsis lines with stable expression of stroma-targeted fluorescent proteins (see General note 1). This protocol presents data from transgenic lines in the Col background [8] and minE [9], and ftsZ triple [10] (hereafter, ftsZ) mutants, in which a transit peptide (the N-terminal 90 aa of AtFtsZ1-1 [8])-fused YFP is driven by the constitutive CaMV35S promoter (see Figure 1)

Figure 1. Reproductive stages of Arabidopsis suitable for analysis of amyloplast replication. (A) Primary inflorescence. (B) Flower stages 14–17 [11] used for ovule extraction. (C) Expression pattern of CaMV35S promoter-driven stroma-targeted YFP in a transgenic Arabidopsis line. Images of brightfield (BF) and yellow fluorescent protein (YFP) signals are shown. Scale bar, 5 mm.

Reagents

1. Murashige and Skoog plant salt mixture (Wako Pure Chemical Industries, catalog number: 392-00591)

2. Sucrose (Wako Pure Chemical Industries, catalog number: 196-00015)

3. MES (Nacalai Tesque, catalog number: 21623-26)

4. Phyto agar (Duchefa Biochemie, catalog number: P1003.1000)

5. Jiffy-7 peat pellets, 44 × 42 mm (Jiffy International)

Laboratory supplies

1. Slide glass (Matsunami Glass, catalog number: S-2215)

2. Cover glass, 24 × 32 No. 1, thickness 0.13–0.17 mm (Matsunami Glass, catalog number: C024321)

3. Micropore surgical tape (3M, catalog number: 1530SP-0)

4. Paper tape (As One, catalog number: 6-691-01)

Equipment

1. Stereomicroscope with 0.63× objective lens (Leica Microsystems, model: MZ10 F)

2. CCD camera (Olympus, model: DP26) with 0.63× adaptor, attached to Leica MZ10 F

3. Inverted fluorescence microscope with 10×, 20×, and 60× objective lenses (Olympus, model: IX71)

4. CMOS camera (Hamamatsu Photonics, model: ORCA-flash2.8) with a 1.0× adaptor, attached to Olympus IX71

5. Tweezers 1 (Fontax, No. 5 Taxal)

6. Tweezers 2 (Dumont, No. DU-55 Dumoxel)

Software and datasets

1. Imaging software CellSens (Olympus, controller for DP26 camera)

2. Imaging software HCImage (Hamamatsu Photonics, controller for ORCA-flash2.8 camera)

Procedure

A. Plant cultivation and extraction of ovules for microscopy

1. Sow seeds on Murashige and Skoog medium (pH 5.7) supplemented with 0.1% MES, 2% sucrose, and 0.7% agar (see General note 2). Treat the seeds in the dark at 4 °C for several days [9]. Germination occurs in one or several days after irradiation at 23 °C. After germination, transfer seedlings to Jiffy-7 peat pellets pre-soaked in water and grow under a 16/8 light/dark cycle of white light illumination at 23 °C for 5–6 weeks (see General note 2; Figure 1).

2. Prepare two glass slides. One (slide 1) is used for ovule extraction. The other (slide 2), together with 1–3 layers of colored tape as a spacer, is used for microscopy analysis (see General note 3).

3. Excise an intact flower or silique from an inflorescence stem of an adult plant (see General note 4).

4. Place the flower or silique (segment) on slide 1. Fix the organ in place using surgical tape by securing the pedicle at one point so that it does not move during dissection (Figure 2A–E).

Figure 2. Extraction of ovules for microscopic analysis. (A,B) Sampling and dissection of a silique. (C,D) Sampling and dissection of a pistil from an open flower. (E) Slides used for dissection (top) and microscopy (bottom). (F) An extracted septum–ovule association (arrow) mounted in water. (G) 60× objective used for microscopy. (H, I) Ovules observed under 10× (H) and 20× (I) objectives. Scale bars, 2 mm (A–D, F) and 100 μm (H, I).

5. To extract ovules from a silique at flower stages 16–17, remove sepals, petals, and stamens from the silique, if attached, using basic tweezers (tweezers 1) under a stereomicroscope at zoom 2.0–3.2×. Peel off either or both valves (this is optional) from the silique (Figure 2A, B).

6. To extract ovules from a pistil at flower stages 13–16, remove sepals, petals, and stamens from an open flower using extra-fine tweezers (tweezers 2) under a stereomicroscope at zoom 6.3×. Peel off either or both valves (this is optional) from the pistil (Figure 2C, D).

7. Apply 40–70 µL of deionized water or liquid Murashige and Skoog medium onto slide 2. Transfer a septum–ovule association obtained by dissection to the water drop (Figure 2F).

8. Mount a coverslip carefully onto the slide (see General note 5). Gently apply the mounting medium between the coverslip and the slide, so that it just fills the space between them (Figure 2E).

9. Confirm the extraction of intact ovules by low-magnification light microscopy (Figure 2H, I).

10. Observe amyloplasts in outer ovule integument (epidermal or oi2 [12]) cells by high-magnification fluorescence microscopy using a 60×/1.20 NA water-immersion objective (Figure 2G; see section B).

B. Observation of amyloplasts by conventional fluorescence microscopy

1. Turn on the halogen lamp, mercury lamp, camera controller (computer), and digital camera.

2. Place slide 2 on the stage of the inverted fluorescence microscope.

3. To perform counting and morphological characterization of amyloplasts, use a 60× water-immersion objective (Figure 2G). Microscope settings for the detection of YFP are described in Table 1.

4. Start the camera controller software HCImage (Hamamatsu Photonics). The settings of the mercury lamp and camera for the detection of YFP fluorescence are described in Table 2.

5. Explore and focus on individual ovules at 60× through eyepieces using differential interference contrast (DIC) optics.

6. Perform image acquisition (Figures 3–5). For time-lapse fluorescence microscopy, we manually focus on individual cells and capture images for up to 60 min. Each image is captured every 30 s or 1 min.

Table 1. Settings for standard epifluorescence microscopy

Item	Products/values (maker)
Objective	10× dry (N.A. 0.40), 20× dry (N.A. 0.75) 60× water-immersion lens (N.A. 1.20)
Light source for DIC observation	Halogen lamp at 10%–50% output (TH4-100, Olympus)
Light source for fluorescence observation	Mercury lamp at 3%–12% output (U-HGLGPS, Olympus) Plus ND filter 25
Optical filters for YFP	BP490-500HQ (ex) (Olympus) FF506-DiO3 (di) (Semrock) FF01-545/55 (em) (Semrock)

Table 2. Settings of the camera and software for fluorescence detection

Item	Products/values
Image	12 bit, 1,920 × 1,440 pixel
Binning	1
HCImage software for single image capture: Gain (range: 0–255) Exposure	100 500 ms
HCImage software for time-lapse microscopy: Gain (range: 0–255) Exposure	150 500 ms

Data analysis

A. Morphological characterization and counting of mature amyloplasts in WT

1. Single and compound starch granules in amyloplasts can be distinguished using both brightfield (DIC) and YFP fluorescence imaging (Figure 3A, WT). Stromal YFP does not enter the interior of starch grains but accumulates in a small space(s) between grains.

2. The amyloplast distribution from the top to the bottom of a tile-formed cell is checked using both DIC and stromal YFP, or by DIC imaging alone. The amyloplast number per cell is obtained with relative ease.

B. Morphological characterization and counting of mature amyloplasts in plastid-division mutants

1. The observation points essentially resemble those of WT amyloplasts, but the following observations are specific to plastid-division mutants. Integument cells of chloroplast division-inhibition mutants often contain YFP-labeled and starchless plastids. Such starchless plastids occur occasionally in WT but frequently appear in severe plastid-division inhibition mutants.

2. In general, an unusually elevated number of starch granules occurs inside giant amyloplasts.

3. Another structural factor in amyloplasts of plastid-division mutants is the activation of stromule formation in cells. Starch grains are often detected within stromules. In particular, ftsZ mutants tend to show extended stromule formation until late Phase III or early Phase IV amyloplasts. In such cases, a stromule-starch grain(s)-giant amyloplast association should be counted as a single amyloplast (Figure 3A, minE and ftsZ).

4. In ftsZ mutants, activated stromules disappear in late Phase III or early Phase IV (Figure 3B, C). Although amyloplast counting is more difficult at early Phase IV than at late Phase III, because of limited cytoplasmic space, the final amyloplast number per cell is revealed.

Figure 3. Amyloplast count and morphology in integument cells of wildtype (WT), minE, and ftsZ plants. (A) Integument cells of WT, minE, and ftsZ plants with developed amyloplasts (Phase III). (B) Integument cells of ftsZ with fully mature amyloplasts (late Phase III). (C) Integument cells of ftsZ upon entry into the cell death stage (early Phase IV). (A–C) show images of stroma-targeted YFP (top) and DIC (bottom). Single and double yellow arrowheads indicate single and compound starch grains, respectively. Cyan single arrowheads indicate the accumulation of stromal YFP in amyloplasts. Numbers indicate amyloplast counts per cell. Scale bars, 10 µm.

C. Morphological characterization and counting of amyloplasts during middle development in WT and plastid-division mutants

1. During the middle development of amyloplasts, the number as well as the starch grain morphology in each amyloplast from both WT and plastid-division mutants should be judged using both DIC and stromal YFP images. Amyloplast counting is difficult when using only DIC images (Figure 4, WT). The distribution patterns of stromal fluorescent proteins, the position and shape of starch grains, and their temporal change during the observation enable these characterizations.

2. If starch grain-containing stromules are not separated from the main body of amyloplasts, they are regarded as a single amyloplast (Figure 4, minE).

Figure 4. Count and morphology of middle-developed amyloplasts in wildtype (WT) and minE integument cells. Images show stroma-targeted yellow fluorescent protein (YFP) and differential interference contrast (DIC). Single and double arrowheads indicate the position of stroma-accumulated YFP signals and compound starch grains, respectively. Numbers indicate amyloplast counts per cell. Scale bars, 10 μm.

D. Morphological characterization and counting of proplastids or early differentiating amyloplasts in WT and plastid-division mutants

1. To assess amyloplast proliferation phenotypes during ovule development in WT and plastid-division mutants, information on the proplastid number per cell is critical. The following four factors are essential for this analysis:

a. A high level of stromal fluorescence signal for proplastid labeling.

b. Clear-cut fluorescence detection conditions to capture the proplastid configuration.

c. Counting by live observation, rather than by using static fluorescence images only.

d. Counts that recognize the entire proplastid configuration within a given cell by shifting the focus from the top to the bottom of the cell.

If one or more of the above factors are unsatisfactory, precise counting of proplastids may become difficult.

2. Selected captured images of proplastids in integument cells of WT (Figure 5A) and ftsZ (Figure 5B) are presented as examples. In WT, proplastids are often distributed at the top and bottom surfaces of integument cells and assume a dumbbell shape. In ftsZ, proplastids extend stromules in three dimensions. The extent of stromule elongation and branching is relatively modest in young cells compared with that in more mature and expanded cells. The small volumes of integument cells in mature ovules, prior to their development after fertilization [13,14], enable proplastid counting in ftsZ.

3. Counting and analysis of early differentiating (Phase I) amyloplasts are most difficult, as indicated in Figure 5C. Integument cells begin to expand, and differentiating amyloplasts also grow larger. In addition, individual amyloplasts are often hard to distinguish.

Figure 5. Count and morphology of proplastids and early differentiating (Phase I) amyloplasts in integument cells of wild-type and ftsZ plants. (A–C) Images of stroma-targeted yellow fluorescent protein (YFP) and differential interference contrast (DIC). Single arrowheads indicate the location of an extending stromule along the z-axis. Numbers indicate amyloplast counts per cell. Scale bars, 10 μm.

Validation of protocol

This protocol, or parts of it, has been used and validated in the following research article:

1. Fujiwara et al. [7]. Principles of amyloplast replication in the ovule integuments of Arabidopsis thaliana. Plant Physiology (Figure 1F–K, M–O; Figure 2A–D; Figure 4A–D; Figure 5A–E; and Supplementary figures).

General notes and troubleshooting

General notes

1. This system is compatible with commonly used fluorescent proteins, including GFP, CFP, YFP, and RFP.

2. Sowing seeds on 1%–3% sucrose-containing Murashige and Skoog media or directly into Jiffy-7 resulted in similar amyloplast development and proliferation results. We cultivated one plant per Jiffy-7. We do not recommend using >7-week-old plants for analysis.

3. Flower stages 13–14 [11] allow for the analysis of proplastids in integument cells. Flower stages 15–17 are suitable for examining developing or mature amyloplasts.

4. Selection of inflorescence stems: We primarily use primary to third inflorescence stems for analysis.

5. Take special care when extracting ovules and mounting samples and coverslips.

Acknowledgments

We thank Prof. Katherine W. Osteryoung (Michigan State University) for providing seeds of the ftsZ triple mutant, INRA (France) for seeds of minE, and Dr. Hiroaki Ichikawa (NIAS) for the original T-DNA vector. We also thank Mr. Ichiro Hamano for supporting the microscope setting. This study was supported by Grants-in-Aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (26440152 to R.D.I.) and by grants from Sophia University (to M.T.F.). Contributions for each author: Conceptualization, M.T.F.; Investigation, M.T.F., R.A. Writing—Original Draft, M.T.F., R.A.; Writing—Review & Editing, M.T.F., T.A., R.D.I.

Competing interests

The authors declare no conflicts of interest.

References

1. Kirk, J.T.O. and Tilney-Bassett, R. A. E. (1978). The plastids: their chemistry, structure, growth and inheritance. 2nd Ed. Elsevier/North- Holland Biomedical Press, Amsterdam.
2. Pyke, K. A. (2009). Plastid biology. Cambridge University Press, Cambridge. https://doi.org/10.1093/aob/mcq100
3. Possingham, J. and Lawrence, M. (1983). Controls to Plastid Division. Int Rev Cytol. 84: 1–56. https://doi.org/10.1016/s0074-7696(08)61014-1
4. Leech, R. M. and Pyke, K. A. (1988). Chloroplast division in higher plants with particular reference to wheat. In Boffey SA, Lloyd D (Eds). Division and Segregation of Organelles. Cambridge University Press, 39–62.
5. Osteryoung, K. W. and Pyke, K. A. (2014). Division and Dynamic Morphology of Plastids. Annu Rev Plant Biol. 65(1): 443–472. https://doi.org/10.1146/annurev-arplant-050213-035748
6. Chen, C., MacCready, J. S., Ducat, D. C. and Osteryoung, K. W. (2017). The Molecular Machinery of Chloroplast Division. Plant Physiol. 176(1): 138–151. https://doi.org/10.1104/pp.17.01272
7. Fujiwara, M. T., Yoshioka, Y., Kazama, Y., Hirano, T., Niwa, Y., Moriyama, T., Sato, N., Abe, T., Yoshida, S., Itoh, R. D., et al. (2024). Principles of amyloplast replication in the ovule integuments of Arabidopsis thaliana. Plant Physiol. 196(1): 137–152. https://doi.org/10.1093/plphys/kiae314
8. Chen, Y., Asano, T., Fujiwara, M. T., Yoshida, S., Machida, Y. and Yoshioka, Y. (2009). Plant Cells Without Detectable Plastids are Generated in the crumpled leaf Mutant of Arabidopsis thaliana. Plant Cell Physiol. 50(5): 956–969. https://doi.org/10.1093/pcp/pcp047
9. Fujiwara, M. T., Hashimoto, H., Kazama, Y., Abe, T., Yoshida, S., Sato, N. and Itoh, R. D. (2008). The Assembly of the FtsZ Ring at the Mid-Chloroplast Division Site Depends on a Balance Between the Activities of AtMinE1 and ARC11/AtMinD1. Plant Cell Physiol. 49(3): 345–361. https://doi.org/10.1093/pcp/pcn012
10. Schmitz, A. J., Glynn, J. M., Olson, B. J., Stokes, K. D. and Osteryoung, K. W. (2009). Arabidopsis FtsZ2-1 and FtsZ2-2 Are Functionally Redundant, But FtsZ-Based Plastid Division Is Not Essential for Chloroplast Partitioning or Plant Growth and Development. Mol Plant. 2(6): 1211–1222. https://doi.org/10.1093/mp/ssp077
11. Smyth, D. R., Bowman, J. L. and Meyerowitz, E. M. (1990). Early flower development in Arabidopsis. Plant Cell. 2(8): 755–767. https://doi.org/10.1105/tpc.2.8.755
12. Vijayan, A., Tofanelli, R., Strauss, S., Cerrone, L., Wolny, A., Strohmeier, J., Kreshuk, A., Hamprecht, F. A., Smith, R. S. and Schneitz, K. (2021). A digital 3D reference atlas reveals cellular growth patterns shaping the Arabidopsis ovule. eLife 10: e63262. https://doi.org/10.7554/eLife.63262
13. Windsor, J. B., Symonds, V. V., Mendenhall, J. and Lloyd, A. M. (2000). Arabidopsis seed coat development: morphological differentiation of the outer integument. Plant J. 22(6): 483–493. https://doi.org/10.1046/j.1365-313x.2000.00756.x
14. Western, T. L., Skinner, D. J. and Haughn, G. W. (2000). Differentiation of Mucilage Secretory Cells of the Arabidopsis Seed Coat. Plant Physiol. 122(2): 345–356. https://doi.org/10.1104/pp.122.2.345