The intricate spotted patterns dappling the bright blooms of the monkeyflower plant may be a delight to humans, but they also serve a key function for the plant. These patterns function as landing pads for bees, attracting nearby pollinators to the flower and signaling the best approach to access the sweet nectar inside.
“They are like runway landing lights, helping the bees orient so they come in right side up instead of upside down,” said Benjamin Blackman, assistant professor of plant and molecular biology at the University of California, Berkeley.
Program for patterned flowers
In a new paper, Blackman and his group at UC Berkeley, in collaboration with Yaowu Yuan and his group at the University of Connecticut, reveal for the first time the genetic programming that helps Erythranthe guttata — and likely other patterned flowers — achieve their spotted glory. The study was published online in the journal Current Biology.
“While we know a good deal about how hue is specified in flower petals — whether it is red or orange or blue, for instance — we don’t know a lot about how those pigments are then painted into patterns on petals during development to give rise to these spots and stripes that are often critical for interacting with pollinators,” Blackman said. “Our lab, in collaboration with others, has developed the genetic tools to be able to identify the genes related to these patterns and perturb them so that we can confirm what’s actually going on.”
How to make distinct dots
The positions of petals’ spots aren’t mapped out ahead of time, like submarines in a game of Battleship, Blackman said. Instead, scientists have long theorized that they could come about through the workings of an activator-repressor system. The system follows what is known as a reaction-diffusion model, in which an activator molecule stimulates a cell to produce the red-colored pigment, resulting in a spot. At the same time, a repressor molecule is expressed and sent to neighboring cells to instruct them not to produce the red pigment.
The results are small, dispersed bunches of red cells surrounded by cells that keep the background yellow color.
“By tweaking the parameters — how strongly a cell turns on an inhibitor, how strongly the inhibitor can inhibit the activator, how quickly it moves between cells — it can lead to big spots, small spots, striped patterns, really interesting periodic patterns,” Blackman said.
In the study, UC Berkeley postdoctoral researcher Srinidhi Holalu and research associate Erin Patterson identified two natural varieties of the yellow monkeyflower — one type with the typical red spots in the throat of the flower and a second type with an all-red throat — appearing in multiple natural populations in California and Oregon, including at the UC Natural Reserve System’s McLaughlin Natural Reserve near Clear Lake. In parallel, UConn postdoctoral researcher Baoqing Ding worked with a very similar plant with fully red-throated flowers found when surveying a population of Lewis’s monkeyflower that had induced DNA mutations.
In a previous study, the Yuan lab had found that a gene called NEGAN (nectar guide anthocyanin) acts as an activator in the monkeyflower petals, signaling the cells to produce the red pigment. Through detailed genomic analysis in both monkeyflower species, the two groups were able to pinpoint that a gene called RTO, short for red tongue, acts as the inhibitor.
Knocking out the spot gene
The red-throated forms of the monkeyflower have defective RTO inhibitor genes, resulting in a characteristic all-red throat, rather than red spots. To confirm their findings, Holalu used the CRISPR-Cas9 gene editing system to knock out the RTO gene in spotted variants of the flower. The result was flowers with a flashy red throat. Further experiments revealed how the functional form of the RTO protein moves to neighboring cells and represses NEGAN to prevent the spread of pigmentation beyond the local spots. This study is the first reported use of CRISPR-Cas9 editing to research the biology of monkeyflowers.
The team also collaborated with Michael Blinov at the UConn School of Medicine to develop a mathematical model to explain how different self-organized patterns might arise from this genetic system.
“This work is the simplest demonstration of the reaction-diffusion theory of how patterns arise in biological systems,” said Yaowu Yuan, associate professor of ecology and evolutionary biology at UConn. “We are closer to understanding how these patterns arise throughout nature.”