A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

Summary

We present the procedure to assemble a seed coat bedding assay (SCBA) from Arabidopsis thaliana seeds. The SCBA was shown to be a powerful tool to explore genetically and in vitro how the endosperm controls seed germination in dormant seeds and in response to light cues. The SCBA is in principle applicable under any situation where the endosperm is suspected to influence embryonic growth.

Abstract

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of dormant seeds or that of nondormant seeds irradiated by a far red (FR) light pulse. In order to further gain insight into the molecular genetic mechanisms underlying the germination repressive activity exerted by the endosperm, a "seed coat bedding" assay (SCBA) was devised. The SCBA is a dissection procedure physically separating seed coats and embryos from seeds, which allows monitoring the growth of embryos on an underlying layer of seed coats. Remarkably, the SCBA reconstitutes the germination repressive activities of the seed coat in the context of seed dormancy and FR-dependent control of seed germination. Since the SCBA allows the combinatorial use of dormant, nondormant and genetically modified seed coat and embryonic materials, the genetic pathways controlling germination and specifically operating in the endosperm and embryo can be dissected. Here we detail the procedure to assemble a SCBA.

Introduction

In Arabidopsis mature seeds, the seed coat is composed of the testa, an external layer of dead tissue of maternal origin, and the endosperm, a single cell layer of live tissue directly surrounding the embryo¹. The endosperm and the embryo are derived from separate fertilization events: the endosperm is a triploid tissue with two maternal and one paternal genome whereas the embryo is a diploid tissue with one maternal and one paternal genome².

The main function traditionally assigned to the endosperm is that of a nutritive tissue. However, it is becoming increasingly evident that the endosperm also plays a central role to control seed germination. This notion became first apparent in the case of dormancy, a trait exhibited by newly produced seeds. Dormant seeds fail to germinate despite the presence of favorable germination conditions. Seeds lose their dormancy after a ripening period and become nondormant, i.e. they will germinate when exposed to favorable germination conditions. In many plant species, including the model plant Arabidopsis, the seed coat is absolutely required to prevent the germination of dormant seeds since seed coat removal triggers embryonic growth and greening^3,4. In Arabidopsis, Bethke et al. observed that germination remained repressed after removing the testa while maintaining the endosperm surrounding the endosperm⁵. These observations strongly indicated that the endosperm is the tissue within the seed coat exerting a repressive activity on the embryo. However, seed coat removal experiments do not necessarily help clarifying the nature of the germination repressive activity provided by the seed coat nor identifying the genes that implement it.

We recently introduced a seed coat bedding assay (SCBA) where seed coats and embryos are physically separated but kept in close proximity so that the germination repressive activity provided by the endosperm is maintained⁶. The SCBA allows the combinatorial use of dormant, nondormant, and genetically modified seed coat and embryonic materials. As a result, the genetic pathways controlling germination and specifically operating in the endosperm and embryo can be dissected. The SCBA was used in the context of dormancy to show that the endosperm releases the phytohormone abscisic acid (ABA) towards the embryo to repress its growth⁶. Furthermore we could use the SCBA to identify the signaling pathways operating in endosperm and embryonic tissues to promote dormancy.

The role of the endosperm to control germination was further strengthened by considering the case of nondormant seeds exposed to a pulse of far red (FR) light. Early upon seed imbibition a FR light pulse is known to inhibit germination^7,8. When seed coats were removed from seeds a pulse of FR light was unable to inhibit germination, strongly suggesting that the endosperm can also repress the germination of nondormant seeds ⁹. Remarkably, the SCBA could also be used to recapitulate FR-dependent inhibition of germination. This allowed to show that that FR-dependent inhibition of seed germination is also a process involving ABA release from the endosperm⁹. Furthermore, the SCBA allowed identifying the different light-signaling pathways operating in the endosperm and the embryo to control nondormant seed germination in response to light cues^9,10.

The SCBA appears therefore to be a reliable technique to explore the function of the endosperm in the context of the control of seed germination. It is also a powerful tool to assess in vitro whether genes suspected to control germination operate in the endosperm, the embryo or both tissues. Here we detail the various steps required to assemble a SCBA.

Protocol

Once the SCBA is assembled, the growth of embryos is monitored over several days. Therefore, before the seed dissection procedure and assembly of the SCBA, one needs to sterilize seeds to avoid future contaminations that could prevent proper assessment of the effect of seed coat material on embryonic growth.

1. Seed Sterilization

1. Pour 50-60 μl of mature and dry Arabidopsis seeds in a 1.5 ml microcentrifuge tube and prepare a 70% ethanol solution.
2. Add 1 ml of 70% ethanol solution to the microcentrifuge tube containing the seeds and shake at room temperature for 10 min at 1,200 rpm in a vortexer.
3. Centrifuge microcentrifuge tube for 3 sec at 4,000 x g to concentrate seeds at the bottom of the tube.
4. Aspirate the 70% ethanol solution using a vacuum suction tip carefully leaving the seeds at the bottom of the microcentrifuge tube.
5. Add 1 ml of sterile distilled water to the microcentrifuge tube still containing the seeds.
6. Shake at room temperature for 10 min at 1,200 rpm.
7. Centrifuge microcentrifuge tube for 3 sec at 4,000 x g to concentrate seeds at the bottom of the tube. Aspirate water supernatant using a vacuum suction tip carefully leaving the seeds at the bottom of the tube.
8. Add 1 ml of sterile water distilled water to the microcentrifuge tube still containing the seeds. Ensure that the flow of water homogenously suspends the seeds inside the tube. The goal here is to avoid further shaking so as to preserve the integrity of the seeds.
9. Let the seeds settle down by gravity at the bottom of the tube by leaving the tube still for 30 sec. Aspirate water supernatant using a vacuum suction tip carefully leaving the seeds at the bottom of the tube.
10. Repeat four times steps 1.8 - 1.9. Overall the sterilization procedure takes about 30 min.

2. Seed Plating

All subsequent steps are performed inside a laminar flow cabinet to preserve sterile conditions.

1. Prepare a Petri dish plate (100 mm diameter, 20 mm height) containing 30 ml of germination medium prepared by autoclaving a solution containing 4.3 g/L Murashige and Skoog medium in 2.5 mM 2-(N-morpholino)ethanesulfonic acid (MES-KOH; pH 5.7) and 0.8 g/L agar. Let the solution cool down in a 50 °C water bath before pouring it on the Petri dish plate.
2. Add 200 μl of sterile distilled water to the tube containing the seeds. Resuspend the seeds and transfer them on the surface of the germination medium using a standard pipette with a 1 ml tip.
3. Remove the water surrounding the seeds on the surface of the germination medium using a vacuum suction tip.
4. Leave the Petri dish containing the seeds without its lid inside the laminar flow cabinet for 2 hr for further drying. Close the Petri dish with the lid and let it stand under the laminar flow cabinet for about 90 min so that about 4 hr have passed since the initiation of the seed sterilization procedure (step 1.2).

3. Seed Dissection

The following steps necessitate working with a stereomicroscope placed inside the laminar flow hood cabinet. A Dumont forceps #5 with truncated (i.e. blunt) tips greatly facilitates the handling of the seeds (Figure 1A).

1. Prepare a new Petri dish plate containing 30 ml of germination medium. Place on the surface of the medium two juxtaposed rectangular sterile Whatman 3MM papers (1.5 cm x 1.0 cm; Figure 1B, step 1). Whatman 3MM paper is sterilized in an autoclave.
2. Transfer seeds on the Whatman 3MM papers using sterile forceps (Figure 1B, step 2).
3. Hold an individual seed against the Whatman 3MM paper by gently pressing the seed using the juxtaposed blunt tips of the truncated forceps (Figure 1B, step 3).
4. Cut the seed coat (testa and endosperm) of the seed using a syringe needle (e.g. U100 insulin syringe + 1 ml syringe, 0.33 mm (29 G) x 12.7 mm) (Figure 1B, step 4). The shape of Arabidopsis seed is ellipsoidal and it is recommended to cut the seed coat along the longest semi-principal axis as close as possible to the place where the cotyledons are joined to the radicle (i.e. away from the radical tip). This helps the safe release of the embryo in the next step of the dissection procedure.
5. Push the seed against the Whatman 3MM paper using the juxtaposed blunt tips of the truncated forceps (Figure 1B, step 5). This step releases the embryo out the seed coat through the opening created in step 3.4 (Figure 1B, step 6). It is recommended to apply the push on the seed where the tip of the cotyledons are in closest to the tip of the radicle (i.e. away from the opening).

4. Assembly of the Seed Coat Bedding Assay (SCBA)

See discussion for the proper choice of the number of seed coats and embryos.

1. Prepare a new Petri dish plate containing 30 ml of germination medium. Place on the surface of the medium a sterile rectangular piece (3.5 cm x 5.0 cm) of Nylon mesh (Figure 2, step 1).
2. To avoid damage, embryos and seed coats can be moved to the surface of the metallic edges of the forceps by gently pushing them using the syringe needle (Figure 2, step 2). Transfer embryos and seed coats on the Nylon mesh (Figure 2, step 3).
3. Assemble a circular and single layer of seed coats using the blunted forceps and the needle making sure that seeds coats are in as much closer proximity as possible (Figure 2, step 4). Try to expose as much as possible the seed coat opening generated by the needle upwards. This step was not systematically tested. However, it could increase the efficiency of the SCBA since diffusible substances inhibiting the growth of the embryo are expected to directly diffuse out towards the embryo rather than away from it.
4. Place the embryos as a circular single layer on the center of the seed coat bed assembled in step 4.3 (Figure 2, step 5). Ensure that the embryos are in as much closer proximity as possible.
5. Incubate the SCBAs under continuous light (40 μmol/m²sec) at 20-21 °C. In the case where a FR pulse is used to arrest germination, assemble the SCBA under normal light, apply the FR pulse as described⁹ and leave Petri dish in darkness.

Results

Previous work showed that mutant seeds unable to synthesize GA were unable to germinate as a result of high ABA accumulation in seeds ^11,12. However, inability to germinate requires the seed coat since its removal triggers embryonic growth¹³. This strongly indicated that the endosperm of seeds unable to synthesize GA is releasing ABA to block embryonic growth. We therefore expect seeds coats unable to synthesize GA to block the growth of embryos in a SCBA unlike seed coats unable to synthesize GA an...

Discussion

The seed coat bedding assay (SCBA) procedure described here is in principle applicable to any circumstance where Arabidopsis seed germination is blocked (or delayed) and where the endosperm is suspected to implement this arrest. The latter can be evidenced by removing the seed coat (testa and endosperm) and observing that embryonic growth proceeds faster relative to that observed when embryos are surrounded by the seed coat. Germination may be blocked in response to particular environmental physical parameters (...

Materials

Name	Company	Catalog Number	Comments
Thermomixer Comfort	Eppendorf AG	5355 000.011	Eppendorf AG, Hamburg, Germany
Vacusafe Comfort	INTEGRA Biosciences AG	158 310	Integra Biosciences AG, Zizers, Switzerland
Petri dish plate (100 mm x 20 mm)	Greiner Bio-One GmbH	664 102	Greiner Bio-One GmbH, Frickenhausen, Germany
Murashige and Skoog	Sigma-Aldrich	M5524	Sigma-Aldrich, St Louis, MO, USA
MES	Sigma-Aldrich	M3671	Sigma-Aldrich, St Louis, MO, USA
Agar (plant agar)	Duchefa Biochemie B.V.	P1001	Duchefa Biochemie, Haarlem, Netherlands
Dumont forceps #5	Fine Science Tools GmbH	11251-10	Fine Science Tools GmbH, Heidelberg Germany
Syringe needle	BD Micro-Fine	324827	BD, Franklin Lakes, NJ USA
Nylon mesh (SEFAR NYTEX)	SEFAR AG	03-50/31	Sefar AG, Heiden, Switzerland
Growth chamber	CLF Plant Climatics	Percival I-30BLLX	CLF plant Climatics, Wertingen, Germany
Paclobutrazol	Sigma-Aldrich	46046	Sigma-Aldrich, St Louis, MO, USA