Jeffrey Perkel has been a scientific writer and editor since 2000. He holds a PhD in Cell and Molecular Biology from the University of Pennsylvania, and did postdoctoral work at the University of Pennsylvania and at Harvard Medical School.
You don’t need to be a plant geneticist to know plants are different from most eukaryotes. Every high-school biology textbook should note that, unlike mammalian cells, plant cells are surrounded by tough cell walls and contain an extra organelle (the chloroplast) for harvesting sunlight.
What those books likely won’t tell you is those differences can make studying plants—or at least, their biomolecular components—considerably more complicated. Researchers who focus on mammalian systems can use essentially the same methods whether they study the muscles of mice or the neurons of men. But methods that work on the model plant, Arabidopsis thaliana, may not work on corn. And methods that successfully extract nucleic acids from leaves may fail when applied to flower petals from the same species.
“There’s much greater variability from sample type to sample type than with mammalian cells,” says Eric Vincent, product manager in the genomics group at Promega.
Fundamentally, says Vincent, plants present two significant problems for nucleic acid and protein extraction. First, their fibrous, cellulose-based structure is much harder to crack open than animal tissues. Secondly, plant tissues contain components that can inhibit downstream processes and reactions. Fortunately, researchers and tool developers have largely worked out methods to address these issues. Just be prepared to do a little optimization.
Tissue disruption
Researchers have several options to overcome the cell wall and fibrous nature of plant materials, but all involve some form of manual disruption. One approach is to freeze the material in liquid nitrogen and grind it to powder. Researchers can grind the tissue manually in a mortar and pestle, mash it in a dounce homogenizer or pulverize it in a bead mill, like a paint mixer.
Thermo Fisher Scientific’s P-PER® (Plant Protein Extraction Reagent) takes a different approach. Users place the plant material and extraction reagent inside a mesh insert within a plastic bag, like the resealable zipper bags commonly used to store food. Then, as per the instructions, the user should:
Use a hard, round-tipped object, such as a capped marking pen, and from the outside of the bag, rub and massage the plant tissue making sure the WS [working solution] and the tissue are thoroughly mixed and no visibly intact tissue remains. For hard seeds, such as soybeans, a ceramic pestle works well for smashing and mixing tissue into the WS. (Emphasis added.)
Not exactly your run-of-the-mill extraction protocol, but that’s plants for you.
According to Monica O’Hara, market segment manager for protein research products at Thermo Fisher Scientific, the P-PER system works on leaves, roots, flowers and seeds, and it has been tested in Arabidopsis, tobacco, spinach, peas and soybean, producing “equivalent or higher levels” of protein compared to “traditional methods.” But, she adds, the method is definitely not high throughput, as each piece of tissue must be processed individually and by hand.
Dealing with inhibitors
Keenan Amundsen, assistant professor at the University of Nebraska–Lincoln, uses both liquid nitrogen and bead beaters to effect cell disruption in his work with turfgrasses, such as buffalograss. He follows that treatment with centrifugation and phenol/chloroform extraction to isolate nucleic acids.
Plants, Amundsen says, contain differing levels of polysaccharides and polyphenols, both of which can complicate downstream experiments. Polyphenols, for instance, can inhibit enzymatic reactions; polysaccharides coprecipitate with DNA “and create a big gooey mess.”
Amundsen generally uses homebrew methods for his extractions. Specifically, he uses an extraction method based on the ionic detergent CTAB, cetyltrimethylammonium bromide, for DNA, and lithium chloride for RNA. For quick-and-dirty assays, he sometimes opts for commercial kits from Qiagen, though in his experience homebrew methods produce higher quality material and more of it, he says. In either case, he typically adds polyethylene glycol (PEG) to remove polysaccharides and PVPP (polyvinylpolypyrrolidone) to remove phenolics.
Some researchers use cellulases or other enzymes in their preps to remove polysaccharides and other fibrous materials.
Plants also contain pigments and enzymes that can gum up purification strategies. Steven Spoel, a group leader at the University of Edinburgh, studies transcriptional regulation of the Arabidopsis “immune response” to pathogens using chromatin immunoprecipitation (ChIP). His team typically ignores chlorophyll, he says, but sometimes they need to eliminate it, such as during ChIP. “We can also do gel filtration or ion exchange column runs, but the columns are basically useless afterwards,” he says.
Likewise, they mostly ignore the enzyme Rubisco, which mediates carbon fixation; it constitutes “a significant amount of biomass on the planet, and getting rid of a protein as abundant as Rubisco is a challenge,” Spoel says. Protein fractionation and commercially available immunodepletion methods “offer solutions to this challenge.”
Spoel says he has to run a differential centrifugation of plant extracts to isolate nuclei away from chloroplasts and other organelles prior to ChIP. Otherwise, he explains, “if you break open the nucleus, you will break open the chloroplasts, as well, and that seems to add a lot of noise to the assay.”
Tips and tricks
Unfortunately, given the diversity of plants and plant tissues, no one protocol will serve in every case. But there are a few guidelines you can follow.
First, as always consult the literature. Even if your specific species is relatively new, others may have experience with something similar—a related species, perhaps.
Try using relatively young, tender tissues, like young leaves. These will be easier to work with than hard, woodier materials like bark or seeds, says Trista Schagat, who manages the scientific applications support team at Promega.
For tissue disruption, Schagat suggests bead-beating for a first pass, as liquid nitrogen (which must be poured into a mortar and pestle) can be challenging for newcomers to work with.
Next, pick up a commercial kit, says Amundsen—“They’re not terribly expensive,” he says, “some are just a couple dollars a sample”—and start with its basic protocol. From there, you can optimize. For instance, says Schagat, if you see intact tissue after your disruption method, “you’re not being aggressive enough.” On the other hand, if what you have at the end of your purification is “cloudy and green,” you may have to dial back the amount of input material.
Companies will generally tell you what species the method has been tested on, and their tech-support team may well be aware of other species in which it has or hasn’t worked. “That standard protocol is going to cover the majority of plant types,” Schagat explains. “It’s going to be the most generic you can do and it’s a great place to start. And you begin modifying from there as needed.”