Michael J. Scanlon, Plant Biology, spends his days looking for the answer to a fundamental scientific question: how do plant organs and plant parts become patterned, that is, distinct from each other? In his lab, Scanlon and his fellow researchers are studying the mechanisms that turn a plant’s undifferentiated embryonic stem cells into all the structures that make up the mature plant. They’re making some unexpected discoveries.
Much of the Scanlon lab’s work is focused on the shoot apical meristem (SAM), an area of undifferentiated stem cells that ultimately generates all the above ground organs of the plant shoot, including leaves, stems, and flowers. The SAM comprises just a single cell in the moss Physcomitrella patens, but in flowering plants such as maize (corn) the SAM may contain over 1,200 cells. Regardless of their ultimate size, all SAMs perform two strategic functions: to maintain the stem cell identity of the SAM and to generate differentiating cells that will form plant organs such as leaves, stems, and flowers.
“Even before the meristem cells differentiate into distinct cells that give rise to all the organs in the shoot, they demarcate boundaries between plant domains,” says Scanlon. “These are lines drawn between cells and tissues that will become different structures in the plant shoot.” Understanding the mechanism of boundary formation during plant development increases our basic understanding of nature and may ultimately lead to new agronomic strategies designed to increase plant production.
The Plant’s Toolkit of Genes
Scanlon is looking at the formation of two structures in maize: the ligule (a small fringe of epidermal tissue on the face of grass leaves) and the auricle (a hinge-like structure that functions to project the leaf away from the vertical axis of the plant). Both are located on the maize leaf at the juncture between the blade, which carries out photosynthesis, and the sheath, which wraps around the stem of the plant and gives the plant its strength.
“The ligule and the auricle form very early in the development of a leaf, when it is a tiny, six-millimeter primordium,” Scanlon says. “Before the ligule and auricle are formed, all the cells in the primordial leaf look pretty much identical, but then the ligule/auricle boundary is created, and the blade and the sheath soon develop distinctly different structure and function. The ligule/auricle can be removed by mutations, and plant viability is unaffected. This makes it a great system to study how plants make a boundary—how a line is drawn between two developmental fields with distinct fates.”
Scanlon and his coresearchers found that the genes expressed in the tiny leaf primordium at the ligule/auricle boundary are expressed at multiple boundaries throughout plant development, such as when the plant is starting to make a leaf from the meristem or a branch from the axis of the leaf and the stem. “This was a surprise,” Scanlon says. “The ligule doesn’t have vasculature. It doesn’t have multiple tissue layers. I didn’t think that ligule/auricle genes would have homology to the genes required to initiate a whole leaf or a branch, but those same genes are all there at the ligule. I think that’s because whenever you start to make a boundary, you’re using the same tool kit. Then later, after the boundary is formed, many additional genes are expressed in a developing leaf or a branch that we don’t see at the tiny ligule.”
“The major increases in maize yield over the past 50 to 60 years have not been achieved by increasing the amount of grain per plant, but by planting more maize plants per unit area.”
The Scanlon lab’s discoveries about the mechanisms of ligule/auricle development could lead to important practical applications. “The major increases in maize yield over the past 50 to 60 years have not been achieved by increasing the amount of grain per plant, but by planting more maize plants per unit area,” Scanlon explains. “Leaf angle is very much controlled by the ligule and auricle, so if you can decrease the leaf angle and still keep the plant productive, then you can pack more plants closer together in a maize field and increase grain yield.”
Johnson Grass, a Farmer’s Scourge
In another project with practical significance to farmers, Scanlon is working with geneticist Andrew Paterson at the University of Georgia to identify the genes involved in the formation of rhizomes, underground shoot meristems, in Sorghum halepense (Johnson Grass). A highly invasive weed, Johnson Grass is actually a hybrid of the common agricultural crop Sorghum bicolor, which does not propagate by rhizomes, and Sorghum propinquum, an Asian relative that does. Johnson Grass is so rhizomatous that it will take over fields of sorghum with ease, making it the bane of many farmers, especially in the southern United States. “Rhizomes are SAMs that grow laterally instead of vertically and send out invasive clonal shoots,” Scanlon says. “We’ve micro-dissected these tiny meristems from rhizomes, and also from tillers, which are branches that grow upright. We’re trying to identify the genes that are expressed specifically in rhizomes.”
Scanlon and his coresearchers used laser micro-dissection, a way to extract RNA from extremely small discrete units of tissue such as the SAM, and RNA-seq, a technique that can determine all of the genes expressed in a given tissue, organ, or plant at a given moment in time. They have now identified a number of genes in Johnson Grass that are important for making rhizomes versus upright shoots. “The cool thing is a lot of these genes are derived from the parent that didn’t make rhizomes, but they’re being used in a different way in the rhizomatous hybrid,” Scanlon says. “This was a big surprise. It means these genes from domesticated sorghum have acquired a new function in the hybrid Johnson Grass. It’s evolution in action.”
The project is currently focused on identifying the key genes controlling rhizomes. “If we can figure out which genes are needed to make a rhizome, then there’s direct application if we can augment their expression or gene activity,” Scanlon says. If the researchers can prevent these key genes from being expressed, Johnson Grass may have finally met its match.
Testing 10x Genomics Inc.’s Protocol
Lately, the Scanlon lab’s new focus is on using RNA-seq to look at the genes expressed in single cells comprising a plant’s meristem, instead of micro-dissecting all 1,200 meristem cells at one time. They are involved in testing a protocol invented by the company 10x Genomics Inc. to gather single-cell RNA-seq data from plant cells. Although 10x Genomics’ system has been used extensively on animals, Scanlon says plants pose more of a challenge because all plant cells have cell walls around them. In order to do single-cell sequencing, researchers need to generate protoplasts, plant cells from which the cell walls have been removed. The 10x Genomics protocol had never before been tested on plant protoplasts.
As a proof of concept, Scanlon and his lab have applied 10x Genomics’ system of single-cell RNA-seq to moss (Physcomitrella patens) because moss is easy to protoplast. “We’re generating transcriptomic profiles of the genes expressed in single cells,” Scanlon says. “We eventually want to use this in the maize SAM to try to understand how gene expression in individual cells is affected by positional cues from neighboring cells, from long-distance signaling within the plant, or by environmental signals.”
Scanlon has always loved genetics. He says, however, these days the field has become even more interesting with the invention of new tools for exploring the mysteries of heredity, gene expression, and pattern formation. “It’s been a fascinating ride so far, and now it’s really getting exciting because we are able to employ much more powerful methods. The ability to deduce gene expression and function in whole organs, tissues, or even single cells, has enabled us to do some very cool things.”