A Highly Efficient System for Separating Glandular and Non-glandular Trichome of Cucumber Fruit for Transcriptomic and Metabolomic Analysis

Materials and reagents

Biological materials

1. North China type cucumber fruits on the day of flowering (Figure 1)

Figure 1. North China type cucumber fruit on the day of flowering

Reagents

1. Sorbitol (Sigma-Aldrich, CAS number: 50-70-4)

2. Tris-HCl (Sigma-Aldrich, CAS number: 1185-53-1)

3. Sucrose (Sigma-Aldrich, CAS number: 57-50-1)

4. KCl (Sigma-Aldrich, CAS number: 7447-40-7)

5. MgCl₂ (Sigma-Aldrich, CAS number: 7786-30-3)

6. Succinic acid (Sigma-Aldrich, CAS number: 110-15-6)

7. EGTA (Sigma-Aldrich, CAS number: 67-42-5)

8. K₂HPO₄ (Sigma-Aldrich, CAS number: 7758-11-4)

9. Triton X-100 (Sigma-Aldrich, CAS number: 9036-19-5)

10. Anhydrous ethanol (Sigma-Aldrich, CAS number: 64-17-5)

11. RNase remover I (Huayueyang Biotechnology, catalog number: 0416-100)

Solutions

1. Sorbitol buffer (see Recipes)

2. 70% (v/v) ethanol (see Recipes)

Recipes

1. Sorbitol buffer

2. 70% (v/v) ethanol

Laboratory supplies

1. Reagent bottles (Fisher Scientific, catalog number: FB800500)

2. Glass beads (Sangon Biotech, catalog number: A500478)

3. 900 μm steel sieves (Shaoxing Shangyu Shengchao Instrument Equipment, catalog number: 20 Mu)

4. 60 μm steel sieves (Shaoxing Shangyu Shengchao Instrument Equipment, catalog number: 250 Mu)

5. 45 μm steel sieves (Shaoxing Shangyu Shengchao Instrument Equipment, catalog number: 325 Mu)

6. Tissue grinder (Sangon Biotech, catalog number: F519062)

7. 50 mL centrifuge tubes (Sangon Biotech, catalog number: F607788)

8. Petri dishes (Sigma-Aldrich, catalog number: BR455751)

Equipment

1. High-speed refrigerated centrifuge (Bioridge, model: TGL-18M)

2. Optical microscope (OLYMPUS, model: CX23)

Procedure

A. Buffer pre-cooling

1. If the samples are obtained for RNA extraction, prepare the 70% (v/v) ethanol solution in advance and pre-cool at -20 °C.

2. If the samples are obtained for metabolite determination, prepare the sorbitol buffer in advance and pre-cool at 4 °C.

B. Pretreatment of laboratory supplies

1. For RNA extraction, perform RNA-free treatment on all experimental supplies involved. Dilute RNase remover I with deionized water at a ratio of 1:1,000. Subsequently, immerse reagent bottles, glass beads, steel sieves, tissue grinder, 50 mL centrifuge tubes, and Petri dishes in this diluted solution for 30 min. Repeat this procedure once with a fresh RNase remover I dilution.

2. After immersing, wrap the supplies in tin foil and autoclave them at 121 °C for 15 min.

3. Dry the laboratory supplies and pre-cool at -20 °C.

C. Trichome harvest

1. Prepare glass bottles containing 30 g of glass beads and 250 mL of pre-cooling buffer. Samples are obtained for RNA extraction using 70% (v/v) ethanol and for metabolite determination using sorbitol solution.

2. On the day of flowering, harvest 25–30 developing ovaries from cucumber plants (Figure 2A). Cut the middle section to a length of approximately 1 cm (Figure 2B) and collect them in glass bottles.

Figure 2. Details of cutting and shaking operation. (A) Developing cucumber ovary on the day of flowering. Bar represents 1 cm. (B) Cucumber ovary after cutting. Bar represents 1 cm. (C) A cut cucumber ovary before shaking. (D) A cut cucumber ovary after shaking.

3. Shake the glass bottles vigorously for 10–15 min until the non-glandular trichomes are almost completely removed from the fruit peels, as visible to the naked eye (Figure 2C and D). Keep the samples at ice-cold temperatures.

D. Separating glandular and non-glandular trichome

1. Remove any remaining cucumber fruit tissues from the glass bottle.

2. Use a 900 μm steel sieve to filter the remaining solution (approximately 250 mL) from the glass bottle in step D1 into a Petri dish and collect the filtrate. Wash the glass bottle with 50 mL of the corresponding solution and collect the additional filtrate. The recovery volume of the filtrate should be higher than 95% of the volume of the added solution.

3. Use a 60 μm steel sieve to filter the filtrate (approximately 285 mL) obtained in step D2 into a new Petri dish and collect the filtrate. Wash the glass bottle with 50 mL of the corresponding solution and collect the additional filtrate. The recovery volume of the filtrate should be higher than 95% of the volume of the added solution.

4. Wash the residual materials on the 60 μm sieve with 50 mL of the corresponding solution and collect them.

5. Observe 20 μL of the recovered products from step D4 under an optical microscope and check that the collected products are non-glandular trichomes. Centrifuge the collected products (approximately 45 mL) in step D4 at 5,000× g for 5 min at 4 °C and remove all supernatants. The precipitate will consist of non-glandular trichomes (approximately 1.5 g).

6. Use a 45 μm steel sieve to filter the filtrate (approximately 320 mL) obtained in step D3 into a new Petri dish and collect the filtrate. Wash the glass bottle with 50 mL of the corresponding solution and collect the additional filtrate. The recovery volume of the filtrate should be higher than 95% of the volume of the added solution.

7. Observe 20 μL of the recovered products from step D6 under an optical microscope and check that the collected products are glandular trichomes. Centrifuge the filtrate (approximately 370 mL) collected in step D6 at 5,000× g for 5 min at 4 °C and remove all supernatants. The precipitate will consist of glandular trichomes (approximately 0.8 g) that are ready for subsequent use.

E. Grinding non-glandular trichome

1. Suspend the non-glandular trichome enrichment products in 800 μL of the corresponding solution and transfer to a tissue grinder. Keep the samples at ice-cold temperatures.

2. Wash the 50 mL centrifuge tube with 500 μL of the corresponding solution and transfer the wash to the tissue grinder. Keep the samples at ice-cold temperatures.

3. Grind the samples with the tissue grinder until no obvious particles remain, then transfer to a new 50 mL centrifuge tube. Keep the samples at ice-cold temperatures.

4. Centrifuge the ground product at 5,000× g for 5 min at 4 °C and remove the supernatant (the main component is the buffer used for extraction). The remaining precipitate consists of non-glandular trichomes ready for RNA extraction. Keep the samples at ice-cold temperatures.

Validation of protocol

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

• Feng et al. [10]. Novel players in organogenesis and flavonoid biosynthesis in cucumber glandular trichomes. Plant Physiology (Figures 1–3).

In this paper, we successfully separated glandular and non-glandular trichomes of cucumber fruits. The concentration of total RNAs prepared from glandular and non-glandular trichome samples reached 900 and 60 ng/μL, respectively. We conducted subsequent glandular trichome metabolome sequencing and glandular and non-glandular trichome transcriptome sequencing and mined many genes related to the development and metabolism of cucumber trichomes. The above experimental results prove that our protocol is reliable.

General notes and troubleshooting

General notes

1. RNA sample preparation: Ensure all pretreatments are completed and maintain an ice-cold environment throughout the process.

2. Filtrate collection: The recovery rate of the filtrate at each step should be higher than 95% of the volume of the added solution.

3. Protocol scope: This protocol is designed for obtaining one set of samples. Adjust the sample preparation based on actual requirements.

4. Storage: If the collected products are not used immediately, store them at -80 °C.

Troubleshooting

Problem 1: After shaking for 15 min, many non-glandular trichomes remained on the surface of the fruit peels.

Possible cause: The number of glass beads is too small, or the diameter of glass beads is too small.

Solution: Increase the glass beads to more than 30 g or replace them with glass beads with a diameter of more than 1 mm.

Acknowledgments

This study was supported by the National Key Research and Development Program "Strategic Science and Technology Innovation Cooperation" Key Special Project (2023YFE0206900) and "The 2115 talent development program" of China Agricultural University. A special thanks to the original research paper by Feng et al. [10] (DOI: 10.1093/plphys/kiad236), which described and validated the protocol used in our study, for their invaluable contributions to the field.

Competing interests

There are no conflicts of interest or competing interests.

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