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Sex without Seed

Mouse-ear hawkweed can clone itself through its seeds.

Mouse-ear hawkweed can clone itself through its seeds.

By Dyani Lewis

Plant biologists are finding ways to retain hybrid vigour in important crops by generating clonal seed.

On the alpine slopes of New Zealand’s South Island, an innocuous-looking yellow flower, not dissimilar to a daisy in appearance, has invaded the terrain. A native of Europe and the central Asian steppe, mouse-ear hawkweed (Hieracium pilosella) has successfully colonised disturbed landscapes around the globe, contaminating pastures and out-competing native plants for ecological real estate. In New Zealand, Australia and many states of the US, it is classified as a noxious and invasive weed.

As much as hawkweed may be the scourge of pastoralists and environmentalists, some biologists are eagerly studying hawkweed to unlock an important quirk of its reproduction. Hawkweed’s allure comes from its ability to clone itself. Many plants can be propagated through cloning – grasses send out runners, and many others are helped along by gardeners carefully tending cuttings. But hawkweed’s trick is to clone itself through its seeds – a phenomenon known as apomixis – and this has caught the attention of scientists.

Plant breeders spend many years crossing varieties and looking for the next elite plant that will give them an edge against their competitors and the environment. Breeding is a vital weapon in the arms race agriculturalists are engaged in against viral and fungal pathogens. It can also increase crop yield, tolerance to drought and frost, and improve texture, flavour and processing properties.

Often the best seed is obtained by crossing two elite varieties to produce hybrids that are superior to either of their parents. Rice, corn, broccoli and spinach are all grown from hybrid seed.

But save seed from the hybrids, and you will find that the next generation isn’t so robust. Some plants will have some of the traits you want, but very few will bear much resemblance to the star hybrids from which they came.

This is because the hybrids have undergone run-of-the-mill sexual reproduction, where genes are mixed and matched into new combinations for the next generation. The “hybrid vigour” of the hybrid generation is lost in the process.

Developing apomixis in crop plants is high on the wish-list of plant breeders because it presents the tantalising opportunity to fix the hybrid vigour required by modern agriculture. “The dream is to make hybrid seeds and keep the hybrids going forever,” says Dr Anna Koltunow, a plant reproductive biologist who works on hawkweed at CSIRO Plant Industry in Adelaide.

Apomixis would allow crops to circumvent sexual reproduction, producing seed clones containing the exact genetic material that made them so desirable in the first place.

To understand how future crops might one day be able to produce seed without sex, we need to visit the glasshouses and petri dishes of scientists working on species far removed from the well-bred varieties we see on our supermarket shelves. Some of the latest insights into apomixis come from weeds like hawkweed, as well as Arabidopsis (thale cress), the workhorse for plant geneticists.

But intriguing findings from the humble moss – a plant that doesn’t itself have the ability to produce seeds – are also shedding light on how crop plants might one day reproduce without sex.

Alternation of Generations

Around the same time as amphibians were finding their legs and walking onto dry land in the Paleozoic era 450 million years ago, plants were also evolving from their aquatic green algal ancestors and venturing onto firm ground. As they evolved from their single-celled algal forebears, plants become multicellular.

Early land plants were small liverworts and mosses that required a moist environment to survive, but by 360 million years ago some plants had evolved more sophisticated vascular systems that allowed transport of water and nutrients, enabling them to grow larger and more complex. These vascular plants evolved into the plants that now dominate the Earth’s biosphere – the flowering plants that colour the landscapes with seasonal blooms, and the conifers whose majestic limbs waft fragrant perfumes over alpine mountains.

In the evolutionary transition from unicellular to multi­cellular organisms, plant life cycles became complex – in many ways, more complex than our own.

In humans, as in other animals, our life cycle is dominated by a multicellular body where cells contain two copies of each chromosome. The term for this – having two sets of chromosomes – is “diploid”.

When we reproduce, we produce single-celled gametes – eggs and sperm cells – that each carry a single set of chromosomes: half the genetic material that other cells in our body contain. With just one set of chromosomes, sperm and eggs are said to be “haploid”. When an egg and sperm fuse during fertilisation, they combine their genetic material and the embryo that results contains two sets of chromosomes, making it and the person it will become diploid.

In plants, both the haploid and the diploid life stages are able to form complex multicellular bodies. The two phases in the plant life cycle can be so distinct from each other that early botanists thought that some plants were two separate species. Because plants cycle between haploid and diploid phases, they are said to undergo an alternation of generations. Which generation is dominant depends on the plant.

The velvety carpet-like growth for which mosses are renowned is produced by a forest of tiny haploid moss gametophytes. In Physcomitrella patens, a moss that plant biologists study widely, each gametophyte can produce both male and female sex organs that generate male sperm and female eggs. When conditions are wet, the sperm are able to swim from the male sex organ to find and fertilise an egg housed in a female sex organ. The resulting embryo is diploid and develops into a multicellular sporophyte.

The moss sporophyte is a peculiar structure that is smaller and less impressive than the gametophyte it relies on. Standing up from the matt of carpeted gametophytes, sporophytes stick out like tiny brown submarine periscopes.

While the spindly stalk is growing skyward, tiny spores are being produced inside the capsule at its head. Each spore that the sporophyte produces contains just a single set of chromosomes, the process of meiosis having recombined and then halved the genetic material that the sporophyte received from its parental sperm and egg. The haploid spores are released from their elevated cradle, ready to be caught by the wind to make the next generation of gametophyte carpet growth.

In flowering plants – the plants that we rely on for the vast majority of our food – the diploid sporophyte is the dominant generation. The haploid gametophytes, although still multi­cellular, are reduced down to just three cells in the male pollen and eight cells in the female embryo sac. The embryo sac, containing the egg cell, is buried deep in the floral tissue where it awaits fertilisation by sperm delivered by the male pollen. Once the female gametophyte is fertilised, it and the surrounding tissue develop into a seed containing the diploid embryo for the next generation.

A Key Transition Gene

Given the vast differences between mosses and flowering plants regarding which life stage is dominant, and how they reproduce, it may seem odd that a key discovery with implications for apomixis comes from moss research. It was also a surprise for one of the people who made the discovery, Professor John Bowman of Monash University.

Bowman, an evolutionary developmental biologist whose discovery was published in the journal Science earlier this year, wasn’t looking to solve the mysteries of apomixis at all. “My whole goal was to understand how plants evolved, how their forms evolved,” he says.

Bowman cut his teeth investigating plant development in the model plant, Arabidopsis. In recent years he has broadened his research palette to study more ancient lineages of land plants, including mosses and liverworts, “to trace the evolutionary history of genes”.

By looking at the diversity in plant form that exists now, and investigating what closely related genes are doing in very distantly related plants, Bowman is able to peer back through time to understand how genes have evolved and adapted through evolutionary history. His questions are both simple, yet complex: “When were genes born? What were they doing originally? How have they evolved to do other things?”

When a postdoctoral researcher in his lab, Dr Keiko Sakakibara, suggested that they investigate what a gene called KNOX2 is doing in mosses, Bowman conceded that the results could be interesting. Although the related KNOX1 genes are known to be essential developmental genes in flowering plants, nothing much was known about KNOX2 genes – in flowering plants or in mosses.

To discover what their function might be, Sakakibara took the standard approach of investigating what happens when the genes are knocked out. When Sakakibara genetically modified moss plants to delete their KNOX2 genes, the tiny gametophytes that grew in her petri dishes looked much like their unadulterated wild-type counterparts. But the KNOX2 mutants failed to produce mature sporophytes – instead their sporophytes arrested after 4 weeks, never getting old enough for meiosis or spore formation.

When she extracted and then carefully cultured the stunted sporophyte embryos that she found, Sakakibara noticed that the sporophytes were doing something that only gametophytes should normally do. They were producing fine filaments.

When it first germinates, a moss spore grows more like a fungus than a mature moss gametophyte. It produces a fine felt-like matt of spidery filaments called protonemata. Buds will eventually form on these filaments, and an upright gametophyte, complete with stem, leaves and root-like rhizoids, will grow from each bud.

The mutant KNOX2 sporophytes were somehow able to grow protonemata and, when cultured for long enough, went on to bud and form leafy gametophyte-like plants. This shouldn’t have been possible – the tissue was sporophytic, after all, and analysis of the chromosome number confirmed that the mutant “gametophytes” had two copies of each chromosome. They were indeed diploid.

It was as though the brakes on the gametophytic genetic program had been released when the KNOX2 genes were removed, revealing an important role for the genes. “We discovered that they are involved in the regulation of the alternation of generations,” Bowman says.

It’s not the first time that this kind of out-of-sync transition has been described. In 1876, German botanist Nathanael Pringsheim conducted seminal experiments showing that gametophytes could be induced from sporophytes by severing the sporophyte stalk. Somehow, this simple act alters the cellular program in the moss sporophyte tissue, so that protonemata and then leafy gametophytes can form without the need for meiosis and spore formation.

And herein lies the relevance of this new discovery in moss to apomixis in crop plants. The ultimate goal of apomixis is to bypass meiosis, which is exactly what the KNOX2 moss mutants were able to do. Sakakibara and Bowman’s discovery has peeled back the cover on the genetics – and therefore the molecular machinery – behind a phenomenon first described nearly 140 years ago. It has also simultaneously revealed a pathway that could be exploited in the quest for sex-free seed in crop plants.

“We were interested in basic science and what these genes might do,” Bowman says, “but it could also be related to applications in agriculture because apomixis is just a skipping of a generation.”

How Would It Work?

Although more than 400 species of flowering plants are apomictic, no important crop species reproduce in this way, and efforts to breed apomixis into crops have been unsuccessful. Several labs around the world are looking at how plants can avoid meiosis and produce clonal seed, both in apomicts like hawkweed that do it naturally and in Arabidopsis and corn mutants that usually only manage the first step in the process.

Koltunow has focused her efforts on hawkweed, a natural apomict that produces only 3–5% of its seeds sexually. “The whole process of seed development is fully autonomous, which means that you don’t need the male at all,” Koltunow says.

In hawkweed, the genes responsible for this intriguing trait have not yet been identified, although Koltunow is searching for them. “If we find apomixis genes,” she says, “we’ll know a lot more about how it works”. Her lab has narrowed their search to three distinct genetic loci that are required for apomixis.

What this will mean for apomixis in crops remains to be seen. “The question of whether it’s actually transferable to a crop – those genes themselves – we won’t know until we find out what they are,” she says.

Professor Ueli Grossniklaus of the University of Zurich, who has been working on apomixis since the 1990s, agrees that the path to apomictic crop plants is far from a simple one. “Until you have an apomictic crop in the field,” he says, “it is a long way away”.

Given the results in moss, Bowman, Koltunow and Grossniklaus are all now curious to see what role KNOX2 genes might play in flowering plants. Koltunow is looking to see whether the genes are active in hawkweed embryo sacs, and Bowman and Grossniklaus are teaming up to see what effect tinkering with KNOX2 genes has in Arabidopsis.

Despite the challenges, Grossniklaus remains confident that this basic research into plant reproduction and the alternation of generations will bear fruit in the coming decades. “I always was hoping I would see that before my retirement, 15 or 16 years from now,” he says. “I think that’s still possible.”

Dyani Lewis is a freelance science writer.