Alfalfa is an important feed crop in Europe and across the world, accounting for more than USD 329 million in international exports alone in 2020, according to the World Bank. It’s a favourite for farmers and livestock alike since it’s nutrient-rich, hardy and helps restore soil quality as a nitrogen-fixing legume.
But like many cultivars, alfalfa has double the number of chromosomes humans do, making it difficult to improve through selective breeding. Current strains of alfalfa can be difficult for animals to digest, and early flowering reduces the plant’s ability to produce leafy greens.
Research from the University of Copenhagen’s NovoCrops Center proposes new genetic targets to delay flowering – and create strains of lusher, more digestible alfalfa. If successful, the technique could lead to improved produce at the grocery store, from plumper strawberries to cheaper quinoa.
A plant with three spare tyres
Michael Palmgren, Professor at the University of Copenhagen’s Department of Plant and Environmental Science, says developing better strains of crops is really the delicate art of sabotage. “Normally when you ‘improve’ crops, theory tells us that there’s a loss of function,” Palmgren explains. “You take out a function that is beneficial in nature but not so good for the farmer.”
Alfalfa is hard to sabotage due to tremendous genetic redundancy, he says. Most animals are diploid, meaning they have two sets of chromosomes, one from each parent. To introduce a recessive trait – for instance, curly hair in cats – you need two copies of the desired gene since one “normal” version can compensate for the dysfunction of an altered gene. In a diploid plant or animal, “I would call it spare tyres – if one wheel is punctured, then you have another one back here you put in instead.”
Due to a chromosomal doubling event in its hazy evolutionary past, alfalfa is autotetraploid – it has four near-identical copies of every gene. “That means it’s not enough to remove function of one allele; you need to alter a second allele, a third allele,” Palmgren says.
Another headache is that alfalfa is an obligate outcrossing plant, meaning it can’t be selfed – the process of fertilising a plant with its own pollen to ensure that a desired trait is passed on to the next generation.
Alfalfa’s genetic code wasn’t sequenced until 2020. “Nowadays, it costs only a couple thousand euros to sequence and entire complicated genome,” says co-author Stephan Wenkel, an Associate Professor of Plant Biochemistry at the University of Copenhagen. “Ten years ago, it would have cost maybe millions.”
Armed with the full alfalfa genome, the researchers considered how to improve alfalfa’s quality and yield by altering flowering patterns. “Farmers need the seeds after all, so completely removing flowering is not the direction to go,” says lead author of the study Maurizio Junior Chiurazzi, PhD Fellow in plant biochemistry at the University of Copenhagen.
To delay flowering, the scientists identified two potential cellular targets that could sabotage alfalfa in the farmer’s favour.
Chiurazzi and his team propose using CRISPR-Cas-9 genetic editing to disrupt specific microRNAs – tiny strands of RNA that “intercept” instructions from the cell’s nucleus.
DNA communicates with the cell via messenger RNAs, which instruct the machinery of the cell on which proteins to build. MicroRNAs flag certain messenger RNAs for destruction by attaching to binding sites on the messenger RNA that are mirror images of their own genetic code.
The researchers set their sights on the AP2 gene family, a group of genes that control flowering time in a variety of plants ranging from roses to the model organism rockcress. AP2 works as the brakes on flowering, suppressing other genes that will begin the process. But by slightly changing the binding sites for AP2’s messenger RNAs, the researchers believe they can turn the tide in favour of AP2, delaying flowering.
For their second target, the researchers homed in on microproteins called CONSTANS/CONSTANS-LIKE transcription factors, which are thought to be involved in regulating flowering time and branch length.
The CONSTANS/CONSTANS-LIKE microproteins work in pairs. Each one is “half of the transcription factor,” Wenkel explains. They bind together at one end – called the B-box domain – while the CCT domains latch onto a strand of DNA “like a clamp”.
Again using CRISPR-Cas-9, the researchers propose deleting the genes for the CCT domains, essentially lopping off half the microprotein. If one or more of the alfalfa plant’s four chromosomes are altered this way, some of the CONSTANS/CONSTANS-LIKE microproteins will be produced as B-box nubs – crucially, these nubs will still be able to bind to unaltered CONSTANS/CONSTANS-LIKE microproteins, rendering them useless too.
The ineffectual transcription factors would be like a pair of scissors with the handles intact but only one blade.
Impact for the greater field
The researchers are optimistic that these changes could produce dominant traits. If their theories are borne out in additional research, targeting microproteins and microRNA is “a strategy that could be universal for complicated genomes,” Wenkel says.
There’s no shortage of complicated genomes in the farmer’s field – like alfalfa, potatoes also have four copies of every gene, and strawberries and sugar cane have eight.
But the many meddlesome spare tyres of high-ploidy crops come with their advantages too. “With higher ploidy you also have more yield sometimes,” Wenkel explains, and the plant can overcome harmful mutations more easily. “You also have larger organ size,” Chiurazzi adds, referring to a plant’s foliage, flowers or fruit.
“You have an easy plant” in alfalfa, Palmgren chides Chiurazzi with a laugh. “We work with wheatgrass, and they’re hexaploid” – meaning they have triple the normal compliment of genetic material.