Roots are fundamental to a plant’s survival, but some of their behaviour at a cellular level remains a mystery to scientists. Jess Wade talks to Giovanni Sena, who is particularly interested in how electric fields can affect root growth and regeneration
When I cycle through Hyde Park in London on my way to work, pausing as I cross the Serpentine to admire the view of the Houses of Parliament, I rarely think about what is going on beneath my tyres. There are just too many above-ground distractions to spend much time wondering about the subterranean network of plant roots weaving their way through the dark and mysterious soil below. And anyway, what have roots got to do with physics?
Roots are complex branched systems whose topology and structure determine the entire physiology of the plants above them. The tip of each branched root has distinct regions where different cell activities take place. At the very end there is the “meristem” where cell divisions occur. Moving up the root, an “elongation zone” comes next where the cells stop dividing and instead elongate. It is followed by a “differentiation zone” where the cells stop elongating and start differentiating.
The direction and rate of root tip growth are influenced by the signals they perceive from the environment, and the organization of the internal tissue within each root tip plays a big role in this root-soil interaction. As root tips explore, the signals help them seek out the most efficient paths to discover water and nutrients. The composition of the soil will influence the architecture of the root system, which ultimately determines the efficacy of the exploration. It seems that evolution has placed enormous pressure on root tips to understand their environments – and it has taken until now for us to start asking why.
A flower’s nano-powers
The physics of the soil–root interactions that optimize subterranean structures is crucial for plant survival. The roots sense what is going on around them with remarkable detail at the appropriate length scale. “We are not talking about the scale of quantum mechanics, but interactions described by classical physics,” says Giovanni Sena, a development biologist at Imperial College London in the UK, who has a strong background in theoretical physics.
Roots are constantly exploring their space, steering through natural force fields – gravitational and electromagnetic fields as well as magnetostatic and electrostatic – and experiencing the mechanical and chemical properties of the soil they live in. Gradients in light, chemicals, temperature and oxygen provide positional information that allows the root to navigate and forage efficiently. These kinds of root responses are known as a “tropisms”.
The impact of mechanical forces on roots is relatively intuitive – stress and strain can change the shape, number and organization of growing tissue – and the alignment of root tips with the gravitational field is also relatively well understood. However, there is still no model that can effectively describe the much more subtle interaction between an external electric field and the internal electric field of biological tissue.
Keeping a plant alive without damaging it during measurements is no mean feat, and near-on impossible without understanding instrumentation and optics
We know that living cells actively maintain a negatively charged cytoplasm – the material inside a cell excluding the nucleus – and this establishes a non-zero membrane potential. Oscillations in the membrane potential create a temporal pattern, while a spatial pattern is produced by the differences in potential between cells. Yet there are still many unanswered questions. Could these patterns, for example, be crucial to tissue organization and morphogenesis, the process that causes an organism to develop its shape? Is spatial variability just stochastic noise? Or is it an actual pattern that is used as “positional information” during morphogenesis? Until now, how bioelectricity in plant tissue impacts their shape and function has never been defined.
One of Sena’s collaborators, Michael Levin at Tufts University in the US, is studying the bioelectrical cues of membrane potentials and using them to describe cell behaviour. Levin argues that cracking “the bioelectrical code” – understanding how patterns are encoded into biological networks – would allow us to programme cell activity and transform synthetic bioengineering and cancer treatment (Mol. Biol. Cell 25 3835). Sena’s group is collaborating with Levin and partially funded by the Allen Discovery Center at Tufts University to develop models of bioelectric circuits within roots that store patterning information during morphogenesis.
Cut to regenerate
For a physicist, Arabidopsis – a small flowering plant also known as thale cress – is an ideal subject to observe and model because it has transparent, slow-growing cylindrical roots. It is now well established that if you cut the end off its root tip, it will regenerate and reform with no external influences, as shown in figure 1 (Nature 457 1150). However, like a lot of developmental biology, a comprehensive understanding is still missing.
Electrotropism – the alignment of a root with an electric field – has also been observed for decades, but not explained, and Sena’s lab further identified that if you place the cut root tip in a weak electric field, it is more likely to regenerate (Regeneration 3 156). The field impacts some hormones more than others but cause no obvious changes in tissue patterning. These are fascinating results that science still can’t explain: why does an external electric field make tissue regenerate faster? And why does a plant root align with an electric field in the first place?
Understanding the regeneration of damaged tissues would be a game changer in controlling morphogenesis. For roots it can be modelled as a self-organizing 3D system of interacting and coupled cells, which divide with a certain probability – a complicated mathematical model. Instead of just being a cylinder, the root becomes a 3D complex system of interacting units, where the units respond to an electric field. Despite advances in the computational models for developmental biology, there is still no effective way to model the increased regeneration.
Unlike animal cells that can move and change shape because they do not have cell walls, plant cells have a rigid extracellular cellulose coat that restricts their ability to migrate when embedded in a tissue. The driving force to shape plant organs is instead cell division – exactly where and when cells divide will strongly influence the overall tissue shape. The dynamics of cell division events are entirely non-trivial during morphogenesis, and at present very little is known about their spatial and temporal correlations.
Physics tells us that if you have a complex system undergoing a transition there is a sudden change of the correlation length. Sena wants to identify how the spatial and temporal correlation of cell divisions change during regeneration, and how regeneration is influenced by the physical forces that the root tip is growing in.
Experiments with tiny plants
To investigate dynamic developmental processes, scientists need to image living tissues at cellular resolution regularly over an extended period of time. Keeping a plant alive without damaging it during measurements is no mean feat, and near-on impossible without an understanding of instrumentation and optics. To overcome these challenges, building on his previous experience as postdoc at Rockefeller University in New York (PLOS ONE 6 e21303), Sena’s team created its own automated light sheet fluorescence microscope (LSFM), combining optical sectioning with a miniaturized plant growth chamber.
At any given time, the root is illuminated by a thin laser sheet (about 4 µm across), reducing the incident photo-energy and acquisition time by avoiding the need for lateral scans. The plant is held within a cuvette that is continuously illuminated and held at a constant temperature, with oxygen and an actively circulated liquid nutrient solution to keep it growing. Cell divisions are identified by tagging a protein – that appears just before and is destroyed just after cell division – with a fluorescent marker.
Because plants are so sensitive to gravitational fields, Sena had to play with the geometry of the microscope, with the fluorescent signal collected by a long-working distance lens normal to the excitation light-sheet (generated by a laser beam through a cylindrical lens) and the cuvette positioned vertical in the microscope. The stage is automatically repositioned to keep the root tip within the focus of the objective. As even slow-growing Arabidopsis can extend by a few millimetres per day, Sena and his group wrote an algorithm to compare the most recent image with the one before and automatically reposition the stage in 3D.
Before Sena’s dynamic LSFM, confocal microscopy only let researchers take daily images of regenerating root tips. If you wanted to know whether the root reforms or not, confocal microscopy was fine, but if you wanted to investigate regeneration at high temporal resolution, it was not enough. The LSFM allows Sena to grow a root and observe in 3D every cell division event every 10 minutes for at least one week at a time – and by measuring both cut and uncut roots, his team can compare the dynamics of regeneration against normal growth.
A glowing worm
On a computer, the process looks like a glowing worm wiggling across the screen, but it’s really the meristem of a root tagged by fluorescent proteins being tracked by Sena’s automated microscope stage. Whenever and wherever the root blinks, a cell division event has occurred. The video represents a 2D projection of a week of LSFM data collected every 10 minutes. For a non-expert, watching cell division is breathtakingly beautiful, but for Sena, it is just more intriguing physics and biology. Alongside the sped-up video, he can track the number of cell divisions, analyse their temporal and spatial distributions, characterize the bursts of proliferation activity and the more quiet periods, and in general compare normal and regenerating growth. The data are messy – well, we are dealing with biology, not electrons – but the implications are profound.
There is no doubt that physics contributes significantly to Sena’s team’s understanding of plant morphogenesis. In his lab almost all the researchers are experimental physicists – one even used to build particle detectors at CERN – while the others are biologists who have seen the light. If you gave their data to pure biologists, they’d jump immediately to genetics, and start a quest for “mutants” – genetic defects sufficient to modify the observed cell behaviour. Conventional plant science is familiar with visualizing root tips as static slices, but Sena argues that the dynamics are much more important – abnormal processes might look perfectly normal when viewed as static images. Plant morphogenesis, influenced by cell division, is an outcome of evolution – so how and why natural selection has resulted in these division dynamics is an underpinning question for biosciences. And the answers could provide a whole new perspective of those subterranean networks that I cycle above every day.
- Enjoy the rest of the April 2018 issue of Physics World in our digital magazine or via the Physics World app for any iOS or Android smartphone or tablet. Membership of the Institute of Physics required