Preparation of Intact Tissue for Microscopic Analysis of the Endosperm Cell Layer in Developing and Mature Arabidopsis Seeds

Summary

This protocol describes the preparation of intact samples of the endosperm cell layer in Arabidopsis thaliana seeds. The method requires only common laboratory equipment, such as an injection needle and precision forceps, and enables high-resolution fluorescent live-cell imaging of endosperm cells in both developing and mature seeds.

Abstract

In Arabidopsis seeds, the endosperm, a single layer of living cells located between the embryo and the testa, plays a critical role in regulating seed maturation, dormancy, and germination. Microscopic analysis of intact endosperm cells is essential for understanding the physiological functions of the endosperm at cellular and molecular levels. However, sample preparation has been challenging due to the small size of Arabidopsis seeds and the location of the endosperm cell layer beneath the testa. This article details the preparation of intact endosperm cell layer samples suitable for microscopic observation and analysis in both developing and mature seeds. This method enables the observation of large areas and numerous intact endosperm cells without requiring fixation or sectioning. Additionally, the protocol utilizes only standard laboratory equipment, such as injection needles, precision forceps, and stereo microscopes. This approach successfully enables high-resolution live-cell imaging of fluorescent signals, such as green fluorescent protein (GFP), in intact endosperm cells. This method allows for the observation of intracellular localization and movement of various proteins, as well as the morphology of organelles, in the endosperm cells of different Arabidopsis mutants. This protocol contributes to the elucidation of novel endosperm functions and expands the potential for cellular and molecular studies of this essential tissue.

Introduction

Because plants are sessile organisms, seed germination is a crucial event that determines their fate. The decision to germinate is strictly regulated by both internal and environmental factors, such as primary seed dormancy levels, temperature, light intensity and wavelength, and nitrogen concentration^1,2,3,4,5,6. Seeds have complex structures consisting of multiple tissue types⁷. In Arabidopsis dry seeds, the embryo, which develops into a seedling, is surrounded by a single layer of endosperm and the outermost layers, the testa. The testa is composed of multiple layers of dead cells, whereas the embryo and endosperm remain alive even in dry seeds. The endosperm is commonly regarded as a storage tissue that provides nutrients for embryo growth and, together with the testa, confers mechanical resistance to radicle protrusion^{8,9,10,11,12,13}.

Several recent studies have demonstrated that the endosperm plays an essential role in regulating optimal seed germination^14,15,16,17. For instance, the photoreceptor phytochrome B (PHYB) in endosperm cells detects either red (R) or far-red (FR) light, regulating germination responses¹⁵. The endosperm also functions as a temperature-sensing tissue, suppressing germination responses under high temperatures¹⁶. Quality control of the endosperm is critical for optimal seed germination, particularly in long-term stored seeds¹⁷.

Live-cell imaging is now necessary to further elucidate the physiological functions of the endosperm. Microscopic analysis of intact endosperm cells expressing fluorescent-tagged proteins allows the investigation of the molecular mechanisms by which the endosperm regulates seed germination. However, preparing intact endosperm cells for microscopic observation is challenging, particularly in Arabidopsis seeds. The seeds are approximately 0.4 mm in diameter, and the endosperm is a single-cell layer located between the embryo and the testa, making precise manipulation difficult. Consequently, despite its important physiological roles, the endosperm has rarely been observed using live-cell imaging.

This article presents a protocol for the rapid preparation of intact endosperm cell layer samples suitable for live-cell imaging in both developing and mature seeds.

Protocol

In this study, two different procedures were established for the preparation of living endosperm cell layer samples: one for developing seeds and one for mature seeds. Slightly different approaches are required depending on the solidity of the testa. The details of the reagents and equipment used are listed in the Table of Materials.

1. Preparation of intact endosperm samples from developing seeds

1. Collection of siliques
   1. Grow Arabidopsis plants on soil or rockwool until blooming.
   2. Mark fully opened flowers with colored threads (0 days after flowering, 0 DAF).
      NOTE: Avoid using green, yellow, or brown threads to mark flowers, as these colors are difficult to distinguish from growing or mature plants and siliques.
   3. Cut off the marked developing siliques at the pedicel (indicated in Figure 1(1)) and collect them in 1.5 mL tubes.
      NOTE: Developing siliques from 12-16 DAF are suitable for preparation using this protocol.
2. Dissection of developing seeds
   NOTE: The following steps must be performed on wet filter paper to protect the samples from desiccation. Manipulations should be done under a stereo microscope.
   1. Split a valve (indicated in Figure 1(2)) from the replum (indicated in Figure 1(1)) using two forceps: one with thick tips for holding and the other with sharp tips for tearing).
   2. Gently pick up developing seeds from the siliques using forceps with the tips closed to avoid damaging the seeds (Figure 1(2)).
   3. Holding the seed with forceps without crashing the seed, make a scar approximately 0.2 mm in size on the testa and endosperm surrounding the embryo using an injection needle (27 G, 0.40 mm × 19 mm) (Figure 1(3)).
      NOTE: The optimal location for making the scar is at the junction of the cotyledons and radicle.
   4. Push out the embryo by pinching the seed with forceps (Figure 1(4)). Do not crush the empty seed envelope, which consists of the testa and endosperm, and try to maintain its round shape.
   5. Insert the injection needle into the empty seed envelope at the scar, piercing it from the inside out (Figure 1(5)).
   6. Keep the needle in position, scratch the surface of the testa using forceps with the tips closed, and cut one side of the empty seed envelope to allow it to open (Figure 1(6)).
   7. Open the empty seed envelope into a sheet using forceps with sharp tips (Figure 1(7)). The sample should now be isolated as a bilayer sheet consisting of the endosperm and testa layers (Figure 1(8)).
      NOTE: If the sample tends to curl up when water is used as the mounting medium in step 3 below, divide the sheet-form sample into two pieces. Seeds harvested from siliques at around 18 DAF (at this stage, the testa is brown but not yet completely dry) can also be processed using this protocol, although the seeds must be imbibed for several minutes before preparation.

2. Preparation of intact endosperm samples from mature seeds

1. Imbibition of mature seeds
   1. Add 1 mL of double-distilled water to a 1.5 mL tube containing dry Arabidopsis seeds.
   2. Keep the seeds imbibed for at least 40 min at room temperature (Figure 2(1)).
      NOTE: Dry seeds and seeds within 40 min of imbibition are difficult to scar at the testa and endosperm and to remove the embryo from inside the seed without damaging the empty seed envelope in steps 2.2.1 and 2.2.2, whereas a longer imbibition time facilitates manipulation.
   3. Use a 1000 µL micropipette to transfer the imbibed seeds onto wet filter paper.
2. Dissection of mature seeds
   NOTE: The following steps must be performed on wet filter paper to protect the samples from desiccation. Manipulations should be conducted under a stereo microscope.
   1. Holding the seed with forceps without crashing the seed, make a scar approximately 0.2 mm in size on the testa and endosperm surrounding the embryo using an injection needle (Figure 2(2)). 
   2. Push out the embryo by pinching the seed with forceps (Figure 2(3)). Do not crush the empty seed envelope, which includes the testa and endosperm. Try to maintain its round shape.
   3. Cut the upper and lower sides of the empty seed envelope with an injection needle to shape it into a cylinder (Figure 2(4)).
   4. Cut the cylindrical-shaped empty seed envelope along its central axis to separate it into two pieces (Figure 2(5)). The samples should be isolated as bilayer sheets consisting of the endosperm layer and the testa layer (Figure 2(6)).

3. Microscopic observation

1. Place the endosperm samples in sheet form on a glass slide and mount them in water or perfluorodecalin (PFD).
   NOTE: If air bubbles remain between the sample and the coverslip, perfluorodecalin (PFD), which has been reported to be particularly useful for imaging samples containing air pockets, such as leaves^18,19, would be useful. PFD is known to have the lowest surface tension, allowing it to easily fill the air spaces on the sample surface. For time-lapse imaging, however, the use of water as a mounting medium is recommended, as the water content in mature seeds should be abundant to maintain cellular liquidity.
2. Place a coverslip gently over the sample. Ensure that the endosperm layer is facing the coverslip.
   NOTE: Nail polish or grease can be used to seal the edges of the coverslip to prevent desiccation of the sample and the mounting medium.

Results

Using the protocol shown in Figure 1, endosperm samples were prepared from developing seeds harvested from siliques at 14 DAF (at this stage, the testa is still green). Numerous endosperm cells across a large area and their intracellular structures were observed (Figure 3A). In this experiment, seeds expressing PHYB fused with GFP at the C-terminus (PHYB-GFP) were used. It is well known that PHYB translocates to the nucleus upon activation by red light and forms...

Discussion

Roles of the endosperm in seed germination have been revealed through genetic and biochemical analyses using separated seed tissues, such as gene expression analysis and the quantification of lipids and phytohormones^{9,14,25,26,27}. An in vitro seed coat bedding assay, combining the empty seed envelope (endosperm and testa) with an embryo isolated from ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.5 mL Microcentrifuge Tubes	Watoson Bio Lab	131-815C
Coverslip (18 x 18 mm)	Matsunami Glass Ind.,Ltd.	C218181
DDW			Water for mountting
Filter Paper No.526 (400 x 400 mm)	ADVANTEC VIETNAM CO., LTD.	02453400
Genki-kun Seru Senyo yodo kopu N-150 (55 L)	Katakura & Co-op Agri Corporation		Soils for Plant Growth
Glass slide (26mm x 76 mm)	Matsunami Glass Ind.,Ltd.	S1215
Grodan AO 36 x 36 x 40 mm Cubes	Grodan		Rockwools for Plant Growth
Iris Scissors	Premium Plus Japan Co.,Ltd.	FC-0212
Jewelers forceps, Dumont No. 5 (4 1/4 in.)	Dumont	F6521	Forceps for Tearing
Leica Application Suite X (LAS X)	Leica		Software for Sterallis 8
Leica Microsystems Immersion Oil for Microscopes	Very Low Autofluorescence Immersion Oil	THMOIL-10LF
LIOR precision forceps 110mm  SL-14	KENIS Ltd.	KN33450438	Forceps for Holding
NAIL HOLIC	KOSE		Nail polish
Needls 27G 3/4 (19 mm) RB	Misawa Medical Industry Co., Ltd.		A Ingection Needle for Cutting
Nichipet Air 1000 uL	Nichiryo	00-NAR-1000	A 1000 µL Micropipette
Perfluorodecalin	APOLLO SCIENTIFIC	PC5960	Reagents for mounting
Red light/far-red light LED panel	TOKYO RIKAKIKAI CO., LTD.	10147599
Schappe Spun #60	Fujix Co., Ltd.		Thread
SPINKOTE Lubricant 2 oz	BECKMAN COULTER	306812	Grease
Sterallis 8	Leica		Confocal Laser Scanning Microscopy
Stereomicroscope Stemi 305 cam W	Carl Zeiss NTS Ltd.	491903-0017-000
White light LED	PANASONIC	FL40SSW/37