The males of plants as diverse as cabbage, cauliflower, broccoli, tomato, and rice can be made sterile by deleting a very small part of their genome’s DNA. This is the take-home message of a paper published in the journal Nature Communications in October by researchers at the State Key Laboratory of Vegetable Biobreeding of the Chinese Academy of Agricultural Sciences, Beijing.
The simple deletion resulting in such a drastic outcome brings to mind the story of a kingdom that was lost for want of a horseshoe nail. But here, instead of loss, the researchers assure us of a gain: that the deletion could lead to an abundant harvest of these plants, thanks to a process called heterosis.
Genes and promoters
The DNA molecule consists of two long strands. Each strand is composed of four compounds called nucleotide bases. They are designated A, C, G, and T for simplicity (for adenine, cytosine, guanine, and thymine respectively). An A on one strand makes chemical bonds, called hydrogen bonds, with a T on the other and a C on one strand makes hydrogen bonds with a G on the other.
The bonds between As and Ts and the bonds between Gs and Cs hold the two DNA strands together. A base-pair, or bp for short, is a single A-T or G-C pair between the two strands, with the dash denoting the bond.
The genome of the cabbage plant (Brassica oleracea) consists of around 1.06 billion base-pairs organised in 18 chromosomes, which every cell holds in nine pairs of two each. In each pair of two chromosomes, one chromosome comes from the pollen and the other comes from the egg. The DNA (which is all the base-pairs together) in every chromosome pair share a mostly identical sequence of base-pairs.
A gene is a well-defined sequence of typically a few thousand base-pairs, or a few kilo base-pairs (kbp), in the DNA molecule. When a gene is expressed, it means a segment of the base sequence on one of its strands is copied into the sequence of bases in a related molecule, called RNA.
DNA and RNA are the master and working copies of a gene. The RNA is loaded into a cellular machinery called the ribosome. The ribosome uses the base sequence of RNA to specify the sequence in which amino acids are linked together to create the protein encoded by the gene.
In short, a gene’s DNA sequence defines the amino acid sequence of its encoded protein and the protein’s structure. The cell uses the RNA and the ribosome to manufacture this entity.
Pollen loss promotes heterosis
Around 44 years ago, people found a cabbage plant that contained a natural mutation. As a result of this mutation, they found that the plant had lost the ability to make pollen.
At first, scientists didn’t know which particular gene in the plant had been mutated. They only named the altered gene, whichever it was, Ms-cd1.
The mutation’s effect was to make the plant male-sterile, but they had no other defects. In fact, the eggs of the mutant plant could be fertilised by pollen from a normal plant, and the fertilised eggs would go on to make normal seeds.
In other words, all the seeds from the mutant plants were the result of the plants’ eggs being fertilised by pollen from plants of other strains – a process called out-crossing. None of their seeds came from self-crossing. (In a self-cross, an egg is fertilised by pollen of the same strain.)
Out-cross seeds – which are also called hybrid seeds – germinate to produce more robust plants than self-cross seeds. This is because of a phenomenon called hybrid vigour or, in technical terms, heterosis.
The missing base-pair
The researchers who conducted the new study showed that seeds from the male-sterile plant consistently made bigger cabbages.
Specifically, they found that the Ms-cd1 mutation was dominant – meaning that if the mutant gene was present in only one of the chromosomes of the pair, the plant wouldn’t be able to make pollen. It didn’t matter if the other chromosome had a non-mutated gene.
Dominant mutations are relatively rare. Mutations are commonly recessive, meaning the same gene has to be mutated in both chromosomes for its effects to be expressed. Fortunately for us, it is easier to scale up the production of hybrid seeds using dominant male-sterile mutations. One part of this process has to do with fine-tuning protein levels when making pollen.
As it happens, it’s not necessary that all of a gene’s DNA sequence is copied into RNA. Some sequences aren’t copied, and one of them is the promoter. This sequence binds to regulatory proteins that determine when and in which cells a DNA sequence is copied to RNA.
The Ms-cd1 gene of cabbage is about 6 kbp long. Its promoter binds to a regulatory protein called ERF. This binding keeps the Ms-cd1 gene from being expressed.
Using an approach called genetic mapping, the researchers found that the only difference between a mutated Ms-cd1 gene and a non-mutated Ms-cd1 gene was that the promoter in the former was missing one DNA base-pair. This minuscule absence destroyed its ability to bind to ERF. As a result, the Ms-cd1 protein continued to be expressed when in fact it should have been repressed.
The eventual outcome: the plant couldn’t produce pollen and became male-sterile.
A fine balance
The researchers also induced mutations of their own in mutated and non-mutated copies of the Ms-cd1 genes, and in both cases the mutant became recessive. That is, plants with one (additionally) mutated copy and one normal copy were male-fertile.
The team also found that the dominant and the recessive mutations derailed pollen development in different ways – which was an indication that the plant makes pollen properly only if it can make the Ms-cd1 protein in copious amounts at some stages, while in other stages its levels are repressed by ERF binding to the gene’s promoter.
If either the protein is not made at all or isn’t repressed in a timely manner, the plant loses the ability to make pollen and becomes male-sterile. So as such, proper pollen development depends on a fine balance of the Ms-cd1 protein’s levels.
The researchers also introduced the (dominant) mutant gene into other plant species, such as rice, tomato, and arabidopsis (Arabidopsis thaliana, a favourite of plant biologists for research). In every case the recipient plants failed to make pollen. This showed that the pollen development pathway, along with the ways in which a plant can fine-tune it, works similarly across plant species.
So deleting the base-pair offers us a new tool to produce hybrid seeds in these and other crops.
That’s one small deletion for a genome, one giant cornucopia for humankind.
The author is a retired scientist.
