Rapid separation of developing Arabidopsis seeds from siliques for RNA or metabolite analysis
© Bates et al.; licensee BioMed Central Ltd. 2013
Received: 20 February 2013
Accepted: 20 March 2013
Published: 26 March 2013
Protein, starch and oil produced in plant seeds are major renewable sources of food, chemicals and biofuels. Developing Arabidopsis thaliana seeds are commonly utilized as a model for seed crop research. However, due to the very small size of Arabidopsis seeds efficient collection of large amounts of tissue for gene expression or metabolite analysis is very difficult and time consuming.
Here we describe a method that allows very rapid separation and collection of large amounts of developing Arabidopsis seeds from their encapsulating silique tissue after flash freezing whole siliques in liquid nitrogen. The efficient popping open of the frozen siliques on dry ice and filtering the seeds away from the silique tissue with liquid nitrogen cooled funnels and sieves allows large amounts of developing seeds to be quickly isolated while remaining frozen. This method increases the speed of developing seed collection approximately 10 fold over methods which dissect individual siliques one at a time.
Analysis of gene expression or metabolite levels in different tissues of a biological organism is essential to understanding how different tissues function independently from each other. In plants developing seed tissue is the major source of starch, protein, and oils utilized by humans for food, industrial feedstocks or fuel. Therefore accurate analysis of developing seed metabolism is required to understand how these valuable commodities are produced in plants. In the model plant species Arabidopsis thaliana, seeds develop in a non-fleshy fruit tissue known as a silique. The silique tissue has different metabolism and gene expression profiles from the enclosed developing seeds [1, 2], and the silique walls contain more tissue on a fresh weight basis than the developing seeds throughout most of silique/seed development. Therefore, to accurately analyze developing seed gene expression or low concentration metabolic intermediates (such as acyl-CoAs) the developing seeds must be removed from the silique. The very small size of developing Arabidopsis siliques and enclosed seeds (>0.5 mm, >20 μg fresh weight per seed)  makes rapid collection of adequate amounts of tissue for gene expression or metabolite analysis very difficult. Dissecting Arabidopsis seeds from siliques can take 1-3 min per silique, depending on the skill of the experimentalist. Therefore, collection of large amounts of developing seed tissue can be very time consuming. Here we present a method that allows efficient separation of the frozen and metabolically quenched seeds from silique tissue after rapidly flash freezing the siliques in liquid nitrogen. Seeds from many siliques may be collected at the same time greatly reducing the time required to collect large amounts of developing seed tissue.
Results and discussion
Additional file 1: Popping siliques as in protocol step 3 (Figure 1 B,C).(WMV 2 MB)
Our silique popping method greatly increases the speed (>10 fold) at which large amounts of developing Arabidopsis seeds can be collected for subsequent analysis of RNA or other low concentration metabolites. Additionally, the rapid quenching of silique/seed metabolism by harvesting the siliques directly into liquid nitrogen ensures minimal changes in metabolite pools due to tissue disruption during dissection.
Silique popping protocol
Liquid nitrogen, a “bed” of dry ice large enough to hold a plastic petri plate, a second dry ice container for 1.5 ml collection tubes, small glass funnel that will fit into 1.5 ml tube, forceps and glass rod, a sieve (or strainer) with pore size large enough for Arabidopsis seeds to fall through (small metal sieves used for straining tea leaves work well).
It is important that all normal safety precautions for using liquid nitrogen and dry ice as indicated by their respective MSDS are observed (http://hazard.com/msds/).
Harvest developing Arabidopsis siliques directly into liquid nitrogen. Let siliques sit in the liquid nitrogen for at least 5 minutes (Figure 1A).
Take care not to damage the silique tissue. Remove the siliques from the shoot by cutting the pedicel near the stem.
Siliques ages of ~8-12 days after flowering pop open very well.
Put a petri plate on a bed of dry ice and let cool for a few minutes, (while siliques are soaking).
As long as all materials that come in contact with the seeds/siliques are at dry ice temperature or below then the seeds/siliques will stay frozen through each step.
Using a liquid nitrogen cooled metal strainer (or forceps) transfer some frozen siliques from the liquid nitrogen to the petri plate on dry ice and immediately put the lid on (Figure 1B).
Once siliques have popped, remove the lid and repeat until all siliques are the 1.5 ml tube, transferred from the liquid nitrogen bath to the petri plate.
Any siliques that do not pop open can be quickly crushed with a liquid-nitrogen-cooled glass rod.
A thin pair of warm gloves under your lab gloves helps to keep your fingers from freezing while handling liquid nitrogen cooled utensils.
Separate the seeds from the silique tissue by filtering through a liquid nitrogen cooled sieve.
Place a 1.5 ml collection tube upright in a second container of dry ice.
Put sieve into funnel and dip both into liquid nitrogen together with large forceps (Figure 1D) until the liquid nitrogen stops bubbling rigorously (~20-30 seconds).
Immediately place the cooled sieve/funnel onto the 1.5 ml tube.
Immediately pour the frozen seeds/siliques from the petri plate into the sieve/funnel and tap a few times. Most of the seeds will flow into the 1.5 ml tube while most of the siliques will be caught in the sieve (Figure 1E).
A second straining of the collected seeds through the liquid nitrogen cooled sieve will help remove any silique bits that ended up with the seeds.
Once collected the seeds may be stored at -80°C until needed for further analysis.
Frozen seed samples were ground to a fine power with a bead beater and RNA extracted by , DNA was removed by the DNA-Free RNA Kit (Zymo Research). Total RNA was quantified on a nano-spectrophotometer, values reported are average and standard error. qRT-PCR followed standard procedures [8, 9] and was normalized to TIP41-like . Primers utilized for RT-PCR: qTCH1-f, ATTTGCATGATTGGTGGAGATATG; qTCH1-r, CCATCGGTTTCAATCCAACTTAC; qTCH2-f, GGAAGATTTCGCCGGAGATTAG; qTCH2-r, AGAGCAGAAGATATAGAAACAACCG; qTCH4-f, TCACAAGAGCTTGACTCAACAG; qTCH4-r, TCTTGTTCCTCTCTCAACTCTTTAC. RNA quality was measured on an Agilent 2100 bioanalyzer .
We would like to thank Henrik Tjellström (Michigan State University) for independently testing the method and giving helpful suggestions. This work was supported by the U.S. National Science Foundation (grant no. DBI–0701919) and by the Agricultural Research Center at Washington State University.
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