Session Y03: Plant Physics

The multi-scale nature of leaf growth - fluctuations, timescales and adaptation

A growing leaf is a fascinating system. It increases its area by orders of magnitudes while its elements – the cells- are not controlled by a central control system. Under these conditions it is highly nontrivial that leaves succeed in growing "properly" to the desired shape (in most cases flat). In order to achieve this, growth must be regulated locally, i.e., by some effective rheology.

We try to expose the characteristics of the growth field dynamics by measuring the evolving leaf surface with high spatio-temporal resolution. We find that the field is not smooth. It consists of sharp variation in growth rate and directionality, which include extensive shrinkage events as part of normal growth. We identify dominant time and length scales as well as qualitative differences between growth during day and night.

We monitor giant variations in the growth rate due to environmental changes, such as transitions in illumination. These variations most likely result from stomatal activity, which involves overshooting and non-uniform spatial distribution. We, thus, suggest that stomatal activity is a central element in growth regulation.

In another set of experiments, we measure the effect of mechanical stress on leaf deformation and growth. The measured effective rheology is viscoelastic with time varying parameters. We provide evidences for the effect of stress on growth at different time scales. The effects include passive viscoelastic deformation at short times, growth correlated with stress during intermediated times and indications for tissue remodeling in response to extended application of mechanical stress.   

Benchmarking growth - how fast do plant cells expand during development?

Quantifying cell growth is essential for understanding the biophysics of development. Yet, we do not have a simple answer to basic questions, such as "How fast can a plant cell grow?; How does it compare to animal cells?; What growth should we expect for a given organ at a given developmental stage?".  For over a hundred years, the rate of plant growth has been  analysed, from measuring dry biomass increase to the expansion of a single cell. The majority of this data is not easily directly comparable, even between articles from the same research group. In our meta-analysis, we aim to standardise the growth rate data presented in several articles in order to make a direct comparison between studies of the same plant organ. We then extend this approach to distinguish the differences in growth pattern of different organs including the apparent juxtaposition of exponential versus linear growth in certain organs. We collate this data and present it as a reference for expected growth rates with controlled growth conditions, to serve as a benchmark for experimentalists and modellers. Finally, we propose a template for calculating and presenting growth rates and a simple method for comparing different types of data.   

The Impact of Pathogenic and Abiotic Stress on Nutrient Cycling

Plants lie at the interface between living and nonliving matter. With their ability to incorporate C, N, and P into biomass, they are the primary producers in many ecosystems as well as regulators of global biogeochemical cycles. As such, they are at the center of urgent challenges in food and climate. How is plant nutrient cycling shaped by biotic and abiotic stress? One important biotic stress is infectious disease driven by pathogenic microbes. However, it remains unclear how host-pathogen interactions are modulated by host genetics and microbe-microbe interactions in the wider microbiota. In Part 1 of this talk, we describe ongoing efforts to characterize the relationship between pathogens, hosts, and the microbiome using genomics and ecological modeling.

Plants also face abiotic stresses such as nutrient limitation, physical disturbance, and chemical shocks. Because plants are sessile, their repertoires of behavioral responses are limited. Instead, they rely upon developmental plasticity to adapt their morphologies to the environment. In Part 2, we analyze how morphological responses emerge from cellular/molecular events. We conclude by discussing the impacts of pathogens and stress on nutrient cycling.   

Leaf voltage differences induced by fluctuating light: a proxy for the electron transport?

Light induced voltage changes in leaves can provide information on plant physiological mechanisms. Nevertheless, available articles mostly related these electrical responses to the propagation of signals in terms of plant communication. Here instead we investigate the physical and biochemical processes involved in the generation of leaf electric potentials induced by light fluctuations. The aim of this study is to correlate these voltage differences with the photosynthetic electron transport, as a new direct method for carbon assimilation measurements of several plant organs and to unravel plant interactions with the natural environment. By inserting recording electrodes in different leaves of several higher plants, we found consistent short-term responses for all plants analysed and most likely correlated to the light phase of photosynthesis. This was confirmed by applying a specific inhibitor of the electron transport (DCMU) and by inducing fast fluctuations of light since electron transport is known to operate in this fast time scales. Once validated, this method will allow simultaneous carbon assimilation measurements of several plants and organs interacting with each other.   

GIWAXS reveals interactions between cellulose and matrix polysaccharides in plant primary cell walls

The spatial organization and interactions of the cell wall components determine growth, and physical and chemical properties of the cell wall. Elucidating the interactions between wall biopolymers is crucial for understanding how cell walls are assembled and for designing efficient approaches to digest cell walls for renewable energy and biomaterials. Here, we used grazing incidence wide angle X-ray scattering (GIWAXS) to investigate the effect of defective cell wall biosynthesis on the nanoscale organization of cellulose in primary cell walls. GIWAXS reveals that cellulose crystals have a preferred alignment in primary cell walls.  X-ray pole figures constructed using GIWAXS were used to quantify the degree of preferred orientation (texture) of cellulose crystals. Comparing pole figures from pectin and xyloglucan deficient mutants to that of wild type Arabidopsis thaliana reveals that cellulose texture is disrupted in pectin mutants, but not in xyloglucan mutants. Our results indicate that an absence of normal pectin during cell wall biosynthesis alters cellulose patterning in plant cell walls.   

In vitro experiments and kinetic models of pollen hydration show that MSL8 is not a simple tension-gated osmoregulator

Pollen, a neighbor-less cell that contains the male gametes, undergoes multiple mechanical challenges during plant sexual reproduction, including desiccation and rehydration. It was previously showed that the pollen-specific mechanosensitive ion channel MscS-Like(MSL)8 is essential for pollen survival during hydration. Here we test our previous hypothesis that MSL8 functions as a tension-gated osmoregulator with a combination of mathematical modeling and laboratory experiments. Time-lapse imaging revealed that wild-type pollen grains swell and then stabilize in volume rapidly during hydration. msl8 mutant pollen grains, however, continue to expand and eventually burst. We found that a mathematical model wherein MSL8 acts as a simple tension-gated osmoregulator does not replicate this behavior. A better fit was obtained from variations of the model wherein MSL8 inactivation is independent of its membrane tension gating threshold or MSL8 strengthens the cell wall without osmotic regulation. Experimental and computational testing of several perturbations, including hydration in an osmolyte-rich solution, hyper-desiccation of the grains, and MSL8-YFP overexpression, indicated that the Cell Wall Strengthening Model best simulated experimental responses. Expression of a non-conducting MSL8 variant did not complement the msl8overexpansion phenotype. These data indicate that, contrary to our hypothesis and to known MS ion channel function in single-cell systems, MSL8 does not act as a simple membrane tension-gated osmoregulator. Instead, they support a model wherein ion flux through MSL8 is required to alter pollen cell wall properties. These results demonstrate the utility of pollen as a cellular-scale model system and illustrate how mathematical models can correct intuitive hypotheses.   

Parallelized plant morphogenesis in a controlled microfluidic environement.

Plant morphogenesis is a variable yet highly robust process. A same species growing in different conditions will have the same general aspect. But growing in the same conditions will not produce the same final individuals. To quantify this process we developed a microfluidic system to follow several individuals growing in parallel under the same controlled conditions.

We use the plant model Marchantia polymorpha, capable of growing in aqueous media, whose vegetative reproductive cycle produces numerous, genetically identical, gemmae that will each grow to form a new plant. Gemmae are close to flat, and during the first days of growth they are millimetric in size. This makes them suited for observation in a microfluidic chip. Our system has traps to isolate individuals and keep them in place for observation over a few days. The flow inside the chip can be controlled thus allowing for continuous or fast changes in the surrounding media.

With this system we will better uderstand the mechanisms behind plant growth. By using different osmolarity conditions and variations, we will investigate the force balance between turgor pressure and cell wall rigidity that drives growth. Additionally the use of drugs andt mutants will help identify the relevant actors of the morphogenesis process.   

The unique transfusion tissue in conifer needles: the bridge between production and transport. II. Transport efficiency

The transfusion tissue occupies most of the central “stele” of conifer needles, bounded on the outside by the bundle sheath and on the inside by the axial xylem and- phloem cells (tracheids and sieve elements). It serves the dual purpose of 1. conducting water from the axial xylem via the transfusion tracheids outward through the bundle sheath to evaporate through the stomata or to enter the mesophyll to assist photosynthesis, and 2. to conduct sugars from the production sites in the mesophyll inward through the bundle sheath via transfusion parenchyma to the axial phloem. The structure resembles a sponge, where the parenchyma cells form the holes in the sea of tracheids - with one important modification: In some regions the parenchyma cells are strongly stretched in the axial direction, giving the tissue a significant, spatially varying anisotropy. This emphasizes the 3-dimensional nature of the osmotically driven sugar flow through the tissue, as opposed to a more intuitive radial inflow.  We argue that this is related to the way sugars are loaded into the phloem for efficient sugar transport. As described by Liesche et al., New Phytologist (2021) the sugar is only loaded at the flanks of the phloem to avoid blocking the sugar export in sieve elements further upstream.   

The unique transfusion tissue in conifer needles: the bridge between production and transport. I. Structure and function

Conifer needles have a unique transfusion tissue in which the axial vascular elements of xylem and phloem are imbedded. The existence of this tissue has been known for a long time, since the pioneering work of Strasburger (1891). The transfusion tissue fills most of the stele and consists of two cell types: dead transfusion tracheids and living transfusion parenchyma cells, bounded by the bundle sheath. These cells take care of the difficult task of transporting water for photosynthesis and transpiration outward from the xylem, and sugars, produced in the mesophyll, inward to the phloem. Details of this two-way traffic are not known, and it is complicated by the existence of a Casparian strip between the cells of the bundle sheath, forcing both water and sugar at this point to go symplasmically in opposite directions through the same cells. We have studied the needles of P. pinaster using X-ray tomography on living needles and by TEM. Our results reveal a surprising structure reminiscent of a sponge where the water moves out through the continuum and the sugar moves through the holes. We shall discuss main structural features of the tissue and how they relate to sugar transport and the harsh environmental conditions, in particular draught, which conifers are adapted to.   

How memory shapes shoot gravitropism

Tropisms are growth-driven motions by which plants reorient themselves in response to directional stimuli (eg. light or gravity). It has long been known that tropisms obey a dose-response relationship i.e. that plants respond to an integrated history of stimuli. Several recent studies have proposed an updated model of tropisms where the temporal integration behaviour is accounted for by a response function, or memory kernel.

Here, we aim to determine the memory kernel underlying shoot gravitropism. We conduct several kinds of transient gravistimulation experiments on wheat coleoptiles. The resulting data is then used to numerically invert the Volterra integral equation describing plant tropisms with memory.

Our approach reveals the complex shape of the memory kernel, displaying a striking negative lobe. We then show that this specific shape leads to previously unobserved regimes of tropic responses. Finally, we explore the processes underpinning the form of the memory kernel, showing that it depends on the stimulation time and the gravistimulation method.

Altogether, our findings provide a quantitative framework to further investigate behavioral processes in plant tropisms, as well as the microscopic origins of temporal integration.   

Probing time-dependent gravitropic response in rice roots

Plants navigate diverse environments via tropisms, or growth responses to external stimuli such as light (phototropism), touch (thigmotropism), or gravity (gravitropism). Experiments have long demonstrated that these tropisms act on an integrated history of stimulus instead of a current instantaneous stimulus, but a theoretical framework encapsulating this phenomenon has been proposed only recently [Meroz, JRSI 2019]. To test and build upon existing models, and better understand how plant roots respond to time-dependent stimuli, we developed an automated, rotating planter apparatus consisting of a stepper-motor/gear driven apparatus which can provide a programmable time-varying gravitational stimulus. We image root growth dynamics using a camera co-rotating with the device. We present results from experiments in which rice roots (O. sativa) are rotated between 0 and 90 degrees, with respect to gravity, at various frequencies. This allows us to probe timescales in the gravitropic response mechanism. We also compare our results to a cell-based Discrete Element Model simulation of growing roots, subjecting the simulation to similar frequency analysis.   

A Mechano-Sensing Mechanism for Waving in Plant Roots

Arabidopsis roots grown on inclined agar surfaces exhibit unusual sinusoidal patterns known as root-waving. The origin of these patterns has been ascribed to both genetic and environmental factors. We propose a mechano-sensing model for root-waving, based on a combination of friction induced by gravitropism, the elasticity of the root and the anchoring of the root to the agar by thin hairs, and demonstrate its relevance to previously obtained experimental results. We further test the applicability of this model by performing experiments in which we measure the effect of gradually changing the inclination angles of the agar surfaces on the wavelength and other properties of the growing roots. We find that the observed dynamics is different than the dynamics reported in previous works, but that it can still be explained using the same mechano-sensing considerations. This is supported by the fact that a scaling relation derived from the model describes the observed dependence of the wavelength on the tilt angle for a large range of angles. The results indicate that waving can be explained using mechanics and gravitropism alone and that mechanics may play a greater role in root growth and form than was previously considered.   

Modeling plant root circumnutation using cellular simulation

Circumnutation, the helical motion of a growing plant root tip, is hypothesized to benefit plants by allowing them to penetrate heterogeneities in soil [Taylor and Lehner et al, PNAS 2021].  We develop a numerical simulation using a DEM (Discrete Element Modeling) approach to model plant roots to study circumnutation as an emergent property of cell level behaviors. The simulated root is treated as a growing array of cells, which divide close to the tip and enlarge in the elongation zone, implementing the meristematic and elongation zones found in real roots. Each cell is represented as a particle with variable length to allow for elongation during osmotic expansion. Circumnutation is generated by a 'nearest neighbor' coupling model that reproduces the delay in the onset of circumnutation observed in biological experiments. The simulation was tested for a range of parameters intended to capture laboratory experiments of rice roots grown in a gel-based media [Taylor and Lehner et al, PNAS 2021]. We compare the simulation to both wild-type circumnutating and mutant non-circumnutating biological roots and analyze their curvatures over time. We also note that the frequency of circumnutation decreases at 24 hours in rice roots and discuss hypotheses for why this might occur.     

Root growth response to rigid obstacles.

In the current context of climate change and the ensuing hardening of the soil because of a predicted increase of drought events, studying the effect of soil mechanical resistance on growing roots takes a whole new significance and importance. Our research aims at studying the impact of mechanical stress on a primary root growing inside a soft homogeneous soil and encountering a single rigid obstacle such as a stone or a hard pan.

To mimic such a system, maize seedlings were grown inside a soft gel (agarose gel 2% w/w) placed above a force sensor acting as an obstacle. The root was guided through a vertical cylindrical hole pre-formed inside the gel. The whole setup was illuminated with Infra-Red lighting, to perform time-lapse imaging of the root growth as well as detailed kinematic studies of the root growth zone through a PIV technique. Growth observation was coupled with force measurements when the root tip emerged from the gel and pushed against the force sensor.

Force-velocity relationships and strain rate profiles along the root axis were established to test the validity of Lockhart-type growth models under mechanical stress.   

Waving and skewing of Arabidopsis thaliana roots results from mechanical interactions with the substrate

We present a novel numerical solver based on a Cosserat rod integrator and a quasi-static integration scheme, which allows to quantitatively investigate how growing rod-like organs mechanically interact with their environment, for the first time.

We use our solver to shed new light on the well-known waving, coiling and skewing patterns presented by roots of Arabidopsis thaliana when grown on agar plates that are tilted with respect to gravity. Although we use simplified models for the roots and the experimental geometry, our simulations are able to test mechanistic assumptions regarding the development of these growth patterns that have been hard to verify until now.

Our simulations confirm previous theories suggesting that waving and coiling result from the combination of gravitropism and mechanical interactions of the root with the tilted plane, while skewing is related to an intrinsic twist profile. We develop analytic models relating skewing angles, and the transition between waving and coiling patterns, to the tilt angle of the substrate. Furthermore, we compare our simulation results regarding skewing angles to quantitative data from a number of published experimental papers and find a good agreement.