Building better bread: Using genetics to study senescence and nutrient content in wheat.
Wheat provides over 20% of the calories consumed worldwide, the second most of any crop after rice (1). Nearly all of us will eat wheat in one form or another every day—staple foods like bread and pasta as well as our favourite treats, from cake and biscuits to certain types of beer. For many cultures, wheat has been essential for thousands of years – it was originally domesticated around 10,000 years ago. The wheat we eat today is descended from 3 different kinds of wild grasses which crossed together at different times to produce the wild ancestor of wheat (Figure 1)(2). Some of us can take it for granted now that we’ll be able to pop down to the corner shop and pick up a loaf of bread at a moment’s notice, but it took thousands of years of selection by farmers to get to the wheat that we’d recognise today.
Figure 1: Wheat originated from two separate crosses between wild grasses. The first occurred around 400,000 years ago, producing wild emmer. Wild emmer then crossed with a different grass around 10,000 years ago. This final cross produced Triticum aestivum, which would be domesticated into bread wheat by humans. At each cross, the genomes of the wild grasses were combined, resulting in Triticum aestivum containing 3 separate genomes (shown as “AABBDD”, with each letter corresponding to one of the ancestral genomes). Figure courtesy of Dr. Cristobal Uauy.
This process of selection was accelerated in the mid-1900s, during the period called the “Green Revolution.” A combination of research into better breeding techniques and new chemical fertilizers, among other factors, contributed to the substantial increase in yield seen during this period. One critical change involved reducing the height of wheat plants which allowed more energy from photosynthesis to be moved into the grain rather than being stored in the leaves and stems of the plants. The yield increases that came about due to the Green Revolution were essential to keep up with the demands of the growing world population.
Most of the work during the Green Revolution was focused on increasing yield alone, boosting the calories that could be extracted from a single field of wheat. But the benefits of wheat extend far beyond calories along. Perhaps surprisingly, wheat provides 25% of the global protein intake (1). Most of us would think of meat or beans as our main sources of protein, but as a staple crop wheat is essential for our protein intake. The nutrients present in the wheat grain, like iron and zinc, are also essential in our diet.
Campaigns to eradicate hunger have had unprecedented success in recent years, and over 89% of the world’s population are able to obtain enough calories for their basic needs (3). Yet increasingly it is the nutrient content of our diets that is leading to the growing health crises globally. At one extreme, malnutrition, defined as the lack of essential nutrients in a diet that has sufficient calories, is one of the leading causes of childhood stunting (3). At the other extreme, obesity in both childhood and adulthood is more common, partly a result of highly calorific food with poor nutritional value becoming so easily available.
During the development of wheat, the period of growth known as “senescence” is critical in regulating the amounts of proteins and nutrients in the developing grain. This is the period where wheat changes from its living, green state to the dead, yellowing state that is so familiar to us at the end of summer. As the leaves die, the molecules in the leaf start to break down and the elements that make up these molecules are transported from the leaves into the developing grain. At the same time, proteins and carbohydrates are also being remobilised from the leaves and moved to the grain. It’s this movement of nutrients and protein that is essential in establishing the quality of the grain. Different levels of protein determine what the grain can be used for. Bread making requires high-protein flour—this protein makes gluten which creates the structure of bread. At the bottom end of the scale, lower quality wheat can be used as feed for livestock and poultry. However, while increased quality is desired, historically a trade-off has been seen between wheat quality and yield (Figure 2).
Figure 2: Increasing quality and yield often leads to a trade-off. As senescence moves later, yield tends to increase, while quality (such as protein and nutrient levels) tends to decrease. The reverse is found with earlier senescence. This leads to a balancing act with the timing of senescence—how can you maximise both yield and quality?
My research is focused on understanding how the process of senescence is controlled in wheat in the hope that we can use this knowledge to increase the nutritional quality of wheat grains. I’m particularly interested in studying genes that are involved in regulating senescence. These genes are called transcription factors, and they act as master regulators in the cell. Transcription factors are able to bind to DNA and influence the expression of other genes. Oftentimes, changing how a transcription factor is expressed can have a large impact on many other downstream targets.
Previous work found a specific transcription factor, known as NAM-B1, which promoted the onset of senescence (4). When this transcription factor wasn’t active, senescence in wheat was significantly delayed (Figure 3). This delayed senescence was also correlated to a drop in the nutritional content of the wheat grain. This suggested that the timing of senescence could directly influence the levels of nutrients and proteins in the grain. Notably, grain size was not affected by the change in nutrient content and senescence timing, suggesting that studying the NAM-B1 gene might provide insight into how to break the trade-off between quality and yield.
Figure 3: Reducing the action of NAM-B1 (left) leads to delayed senescence in wheat compared to the wild-type plant (right). Panel from (4).
I’m now trying to identify new transcription factors that also regulate the timing of senescence. One way that we’re approaching this question is to look for proteins that interact with NAM-B1. We know that the NAM-B1 transcription factor is only functional when it is bound to another transcription factor in the same family, called NACs. This partner might be another copy of itself, or it could possibly be a different NAC transcription factor entirely. We hypothesised that NAC transcription factors that bind NAM-B1 might also regulate senescence. To study this, we can use different experimental techniques in species as varied as yeast and Nicotiana benthamiana, a relative of tobacco, to look for proteins that can bind to NAM-B1.
Once I’ve identified proteins that bind to NAM-B1, the next question is what these proteins do in the wheat plant. A recently developed resource, the wheat TILLING population, has started to make this process much quicker and easier (5). This is a large set of different lines of wheat that have been mutated by a chemical known as ethyl methanesulfonate (or EMS). This chemical leads to specific single-base-pair changes in the DNA sequence. This means that, in at least one of the thousands of different wheat lines, you’re very likely to find a mutation that knocks-out the action of your favourite gene. All of the mutated wheat lines in this TILLING population have had their genes sequenced. This means that all of the mutations in the genes have been identified and catalogued. Now it’s very easy for us to search for mutations in a gene we’re interested in, and we can order the lines we want online.
After identifying mutations in the genes I’m interested in, I then need to start making crosses before I can look at the effect. This is because, unlike us, wheat is a polyploid. This means that wheat has three different genomes, a legacy of the way wheat was domesticated from three different wild grasses (Figure 1). One of the big effects of this is that there are usually at least 3 copies of each gene—one for each genome. So a mutation in one of the three genes may not actually make any difference to the plant, as the other two copies can compensate. As a result, it’s very important to make crosses so that all of the copies of the genes have mutations in them. Otherwise it would be very easy to think that a gene isn’t important as a single mutation doesn’t cause any change. This polyploidy is one of the reasons that breeding in wheat has historically been so difficult, as random mutations are unlikely to happen more than one copy and are thus often obscured—what can be called the “hidden variation” (2).
Once you’ve found your candidate genes, identified mutated lines, and made all of your crosses, you’re ready to see if your gene has an effect. I do most of my trials in the greenhouse, so that I can look at my plants on a smaller scale than you would need for the field. By scoring for senescence onset and progression in my mutant plants, I’m able to identify whether my mutants influence the timing of senescence (Figure 4). This is quite important as earlier senescence may lead to increased nutrient content, so it’s a useful proxy as it’s quick and cheap to study. After identifying mutant lines that have an interesting phenotype (in this case variation in senescence timing), I can directly measure the levels of nutrients such as iron and zinc in the grain. This is an essential final step to see how the variation in senescence timing correlates with the grain nutrient content.
Figure 4: Variation in chlorophyll breakdown in mutant plants. The mutant plant on the left has yellow leaves, indicating that the chlorophyll is being broken down much earlier than the wild-type plant on the right. This suggests that certain pathways associated with senescence are being activated earlier in the mutant plant.
Currently in my research, I’m still in the process of scoring my plants for senescence and identifying interesting mutants. Wheat takes quite a long time to grow in the greenhouse—about 4 months from seed to seed—so it takes quite an investment of time to get through the generations needed for crossing. A new technique for wheat growth called, appropriately, “Speed Breeding” is starting to change this (6). By growing wheat under special LED lighting for 22 hours a day in rooms where the environment is kept constant we can reduce the time for each generation to between 8 and 10 weeks. This is a significant time saving, and is incredibly powerful particularly for generation of new lines from crosses.
It still remains to be seen whether the proteins that I found to interact with NAM-B1 play a significant role in regulating senescence. There are some promising initial results from the mutants I’ve developed, but it will require another few sets of experiments in the glasshouse and the field before I’m sure we’ve honed in on good candidates. Watch this space!