Continuous,high-resolution biospeckle imaging reveals a discrete zone of activity at the root apex that responds to contact with obstacles

Background and Aims

Shining a laser onto biological material produces light speckles termed biospeckles. Patterns of biospeckle activity reflect changes in cell biochemistry, developmental processes and responses to the environment. The aim of this work was to develop methods to investigate the biospeckle activity in roots and to characterize the distribution of its intensity and response to thigmostimuli.

Methods

Biospeckle activity in roots of Zea mays, and also Jatropha curcas and Citrus limonia, was imaged live and in situ using a portable laser and a digital microscope with a spatial resolution of 10 μm per pixel and the ability to capture images every 0·080 s. A procedure incorporating a Fujii algorithm, image restoration using median and Gaussian filters, image segmentation using maximum-entropy threshold methods and the extraction of features using a tracing algorithm followed by spline fitting were developed to obtain quantitative information from images of biospeckle activity. A wavelet transform algorithm was used for spectral decomposition of biospeckle activity and generalized additive models were used to attribute statistical significance to changes in patterns of biospeckle activity.

Key Results

The intensity of biospeckle activity was greatest close to the root apex. Higher frequencies (3–6 Hz) contributed most to the total intensity of biospeckle activity. When a root encountered an obstacle, the intensity of biospeckle activity decreased abruptly throughout the root system. The response became attenuated with repeated thigmostimuli.

Conclusions

The data suggest that at least one component of root biospeckle activity resulted from a biological process, which is located in the zone of cell division and responds to thigmostimuli. However, neither individual cell division events nor root elongation is likely to be responsible for the patterns of biospeckle activity.     

Biospeckle‐characterization of hairy root cultures using laser speckle photometry

Monitoring is indispensable for the optimization and simulation of biotechnological processes. Hairy roots (hr, plant tissue cultures) are producers of valuable relevant secondary metabolites. The genetically stable cultures are characterized by a rapid filamentous growth, making monitoring difficult with standard methods. This article focuses on the application of laser speckle photometry (LSP) as an innovative, non‐invasive method to characterize Beta vulgaris (hr). LSP is based on the analysis of time‐resolved interference patterns. Speckle interference patterns of a biological object, known as biospeckles, are characterized by a dynamic behavior that is induced by physical and biological phenomena related to the object. Speckle contrast, a means of measuring the dynamic behavior of biospeckles, was used to assess the biospeckle activity. The biospeckle activity corresponds to processes modifying the object and correlates with the biomass growth. Furthermore, the stage of the cultures’ physiological development was assessed by speckle contrast due to the differentiation between active and low active behavior. This method is a new means of monitoring and evaluating the biomass growth of filamentous cultures in real time. As a potential tool to characterize hairy roots, LSP is non‐invasive, time‐saving, can be used online and stands out for its simple, low‐cost setup.     

A new tool for analysis of root growth in the spatio-temporal continuum

? Quantification of overall growth and local growth zones in root system development is key to understanding the biology of plant growth, and thus to exploring the effects of environmental, genotypic and mutational variations on plant development and productivity. ? We introduce a methodology for analyzing growth patterns of plant roots from two-dimensional time series images, treating them as a spatio-temporal three-dimensional (3D) image volume. The roots are segmented from the images and then two types of analysis are performed: 3D spatio-temporal reconstruction analysis for simultaneous assessment of initiation and growth of multiple roots; and spatio-temporal pixel intensity analysis along root midlines for quantification of the growth zones. ? The test measurements show simultaneous emergence of basal roots but sequential emergence of lateral roots in Phaseolus vulgaris, while lateral roots of Cicer arietinum emerge in a rhythmic pattern. Local growth analysis reveals multimodal transient growth zone in basal roots. At the initial stages after emergence, the roots oscillate rapidly, which slows down with time. ? The methodology presented here allows detailed characterization of the phenomenology of roots, providing valuable information of spatio-temporal development, with applications in a wide range of growing plant organs.     

Indoor-Biofilter Growth and Exposure to Airborne Chemicals Drive Similar Changes in Plant Root Bacterial Communities

Due to the long durations spent inside by many humans, indoor air quality has become a growing concern. Biofiltration has emerged as a potential mechanism to clean indoor air of harmful volatile organic compounds (VOCs), which are typically found at concentrations higher indoors than outdoors. Root-associated microbes are thought to drive the functioning of plant-based biofilters, or biowalls, converting VOCs into biomass, energy, and carbon dioxide, but little is known about the root microbial communities of such artificially grown plants, how or whether they differ from those of plants grown in soil, and whether any changes in composition are driven by VOCs. In this study, we investigated how bacterial communities on biofilter plant roots change over time and in response to VOC exposure. Through 16S rRNA amplicon sequencing, we compared root bacterial communities from soil-grown plants with those from two biowalls, while also comparing communities from roots exposed to clean versus VOC-laden air in a laboratory biofiltration system. The results showed differences in bacterial communities between soil-grown and biowall-grown plants and between bacterial communities from plant roots exposed to clean air and those from VOC-exposed plant roots. Both biowall-grown and VOC-exposed roots harbored enriched levels of bacteria from the genus Hyphomicrobium. Given their known capacities to break down aromatic and halogenated compounds, we hypothesize that these bacteria are important VOC degraders. While different strains of Hyphomicrobium proliferated in the two studied biowalls and our lab experiment, strains were shared across plant species, suggesting that a wide range of ornamental houseplants harbor similar microbes of potential use in living biofilters.     

Interspecific interactions and biomass allocation among grassland plant species

Although a handful of studies have shown how interspecific interactions may influence plant shoot to root ratios, the issue of how these interactions influence biomass partitioning among coexisting plant species remains largely unexplored. In this study, we determined whether a given plant species could induce other plant species to allocate relative biomass to each of four zones (aboveground, and three soil depth layers) in a different manner to what they would otherwise, and whether this may influence the nature of competitive or facilitative interactions amongst coexisting plant species. We used a glasshouse study in which mixtures and monocultures of ten grassland plant species were grown in cylindrical pots to determine the effects of plant species mixtures versus monocultures on the production of shoots and of roots of other species for each of three soil depths. Across all experiments, stimulation of production in mixtures was far less common than suppression of production. Different plant species shifted their allocation to shoots or roots at different depths, suggesting that interspecific interactions can either: (1) increase the ratio of deep to shallow roots, perhaps because competition reduces root growth in the uppermost part of the soil profile; or (2) decrease this ratio by reducing plant vigour to such an extent that the plant cannot produce roots that can reach deep enough to exploit resources at lower depths. Further, these results suggest that there are instances in which competition may have the potential to enforce resource partitioning between coexisting plant species by inducing different species to root at different depths to each other.     

New insights into leaf and fine‐root trait relationships: implications of resource acquisition among 23 xerophytic woody species

Functional traits of leaves and fine root vary broadly among different species, but little is known about how these interspecific variations are coordinated between the two organs. This study aims to determine the interspecific relationships between corresponding leaf and fine‐root traits to better understand plant strategies of resource acquisition. SLA (Specific leaf area), SRL (specific root length), mass‐based N (nitrogen) and P (phosphorus) concentrations of leaves and fine roots, root system, and plant sizes were measured in 23 woody species grown together in a common garden setting. SLA and SRL exhibited a strong negative relationship. There were no significant relationships between corresponding leaf and fine‐root nutrient concentrations. The interspecific variations in plant height and biomass were tightly correlated with root system size characteristics, including root depth and total root length. These results demonstrate a coordinated plant size‐dependent variation between shoots and roots, but for efficiency, plant resource acquisition appears to be uncoupled between the leaves and fine roots. The different patterns of leaf and fine‐root traits suggest different strategies for resource acquisition between the two organs. This provides insights into the linkage between above‐ and belowground subsystems in carbon and nutrient economy.     

The rhizosphere microbiome and plant health

The diversity of microbes associated with plant roots is enormous, in the order of tens of thousands of species. This complex plant-associated microbial community, also referred to as the second genome of the plant, is crucial for plant health. Recent advances in plant-microbe interactions research revealed that plants are able to shape their rhizosphere microbiome, as evidenced by the fact that different plant species host specific microbial communities when grown on the same soil. In this review, we discuss evidence that upon pathogen or insect attack, plants are able to recruit protective microorganisms, and enhance microbial activity to suppress pathogens in the rhizosphere. A comprehensive understanding of the mechanisms that govern selection and activity of microbial communities by plant roots will provide new opportunities to increase crop production.     

A quantitative and statistically robust method for the determination of xylem conduit spatial distribution

? Premise of the study: Because of their limited length, xylem conduits need to connect to each other to maintain water transport from roots to leaves. Conduit spatial distribution in a cross section plays an important role in aiding this connectivity. While indices of conduit spatial distribution already exist, they are not well defined statistically. ? Methods: We used point pattern analysis to derive new spatial indices. One hundred and five cross-sectional images from different species were transformed into binary images. The resulting point patterns, based on the locations of the conduit centers-of-area, were analyzed to determine whether they departed from randomness. Conduit distribution was then modeled using a spatially explicit stochastic model. ? Key results: The presence of conduit randomness, uniformity, or aggregation depended on the spatial scale of the analysis. The large majority of the images showed patterns significantly different from randomness at least at one spatial scale. A strong phylogenetic signal was detected in the spatial variables. ? Conclusions: Conduit spatial arrangement has been largely conserved during evolution, especially at small spatial scales. Species in which conduits were aggregated in clusters had a lower conduit density compared to those with uniform distribution. Statistically sound spatial indices must be employed as an aid in the characterization of distributional patterns across species and in models of xylem water transport. Point pattern analysis is a very useful tool in identifying spatial patterns.     

Effects of physical connection and genetic identity of neighbouring ramets on root-placement patterns in two clonal species

Root-placement patterns were examined in the clonal species Glechoma hederacea and Fragaria vesca when grown with different types of neighbours. Three different patterns were predicted as consequences of different types of interactions between roots: the avoidance pattern if root growth decreases in the presence of neighbouring roots; the intrusive pattern if root growth increases towards neighbouring roots; and the unresponsive pattern if root growth is unaffected by neighbouring roots. Experiments were conducted in which physical connection between ramets, and the genetic identity of neighbouring ramets, were manipulated. The patterns of distribution of entire root systems and elongation rates of individual roots were measured. Root systems and individual roots of G. hederacea avoided contact with roots of neighbouring ramets, irrespective of connection to the neighbour and its genetic or specific identity. In contrast, F. vesca roots grew equally towards and away from intraspecific ramet neighbours and their elongation was stimulated by contact with roots of G. hederacea ramets. These results demonstrate that root-placement patterns of plants grown with different types of neighbours vary between species, and suggest that factors additional to resource depletion could be involved in their development.     

Starch-related carbon fluxes in roots and leaves of Arabidopsis thaliana

Both photoautotrophic and heterotrophic tissues from plants are capable of synthesizing and degrading starch. To analyze starch metabolism in the two types of tissue from the same plant, several starch-related mutants from Arabidopsis thaliana were grown hydroponically together with the respective wild-type control. Starch contents, patterns of starch-related enzymes and the monomer patterns of the cytosolic starch-related heteroglycans were determined. Based on the phenotypical data obtained, three comparisons were made: First, data from leaves and roots of the mutants were compared with the respective wild-type controls. Secondly, data from leaves and roots from the same plant were compared. Third, we included data obtained from soil-grown plants and compared them with those from hydroponically grown plants. Thus, phenotypical features reflecting altered gene expression can be distinguished from those that are due to the specific growth conditions. Implications on the carbon fluxes in photoautotrophic and heterotrophic cells are discussed.Key words: starch metabolism, cytosolic heteroglycans, cytosolic glucosyl transferases, carbon fluxes     

Influence of three vesicular-arbuscular mycorrhizal fungi (Glomaceae) on the activity of specific enzymes in the root system of Cucumis sativus L.

The influence of three vesicular-arbuscular mycorrhizal (VAM) Glomus species on the activity of enzymes in the roots of Cucumis sativus was tested. Cucumber plants were grown in a split-root system, in which colonized and uncolonized roots of a single plant could be separated. The activity of the host root malate dehydrogenase (MDH), glucose 6-phosphate dehydrogenase (Gd), glutamate oxaloacetate transaminase (GOT) and glutamate dehydrogenase (GDH) was measured on a densitometer after separation of the host and fungal enzymes on polyacrylamide gels.The results showed that only minor changes in the activity of the host root enzymes occurred after VAM inoculation. Gd was stimulated by VAM and phosphorus, and one of the fungi decreased the activity of GDH in the host plant when both parts of the root system were colonized.     

Numbers of nitrite-oxidizing bacteria in the root zone of grassland plants

Abstract The results of Most Probable Number determinations applying low and high concentrations of nitrite reveal the presence of at least two different communities of potential nitrite-oxidizing bacteria in a number of soil types. The effect of plant roots on these two communities was studied in pot experiments with soil from natural grassland in the presence or absence of either Festuca rubra or Plantago lanceolata . Both plant species are dominant on the grassland soil used in this study. Plant roots had a stimulating effect on the numbers of nitrite-oxidizing bacteria determined with 0.05 mM nitrite in the enumeration medium as well as on the potential nitrite-oxidizing activity. On the other hand, plants roots, especially in younger plants, repressed the numbers of nitrite-oxidizing bacteria enumerated with 5.0 mM nitrite in the counting medium. Pure culture studies with organotrophically grown Nitrobacter species clearly showed that this type of potential nitrite-oxidizing bacteria could not have been responsible for the relatively high Most Probable Numbers observed in the root zones when applying 0.05 mM nitrite in the enumeration medium.     

Size inequality and the tragedy of the commons phenomenon in plant competition

Game theory predicts that the evolutionarily stable level of root production is greater for plants grown with neighbours compared to plants grown alone, even when the available resources per plant are constant. This follows from the fact that for plants grown alone, new roots compete only with other roots on the same plant, whereas for multiple plants grown in a group, new roots can also compete with the roots of other plants, thereby potentially acquiring otherwise unavailable resources at their neighbours’ expense. This phenomenon, which results in plants grown with neighbours over-proliferating roots at the expense of above-ground biomass, has been described as a ‘tragedy of the commons’, and requires that plants can distinguish self from non-self tissues. While this game theoretical model predicts the evolutionarily stable strategies of individual plants, it has only been tested on average allocation patterns of groups of plants. This is problematic, because average patterns can appear to reflect a tragedy of the commons, even when none has occurred. In particular, assuming (1) a decelerating relationship between individual plant biomass and the amount of resources available, and (2) greater size inequality in plants grown with neighbours compared to plants grown alone (due to asymmetric competition), then plants grown with neighbours should, at least on average, be smaller than plants grown alone. This is a manifestation of ‘Jensen’s Inequality’, which states that for decelerating functions, the average value of the function is less than the function of the average value. We suggest that Jensen’s Inequality should serve as an appropriate null hypothesis for examining biologically-based explanations of changes in biomass allocation strategies.     

Vanadium distribution in roots and leaves of Phaseolus vulgaris: morphological and ultrastructural effects

In different plant species, vanadium has been considered either as beneficial or as a toxic element, or even as secondary metabolism elicitor, but the mechanisms involved are still not completely understood. In this study, the responses of Phaseolus vulgaris L. cv. Contender roots and leaves to different vanadyl sulfate concentrations were studied. The plants grown hydroponically with V had thicker roots, a less developed main root, and a smaller number of secondary roots than the control plants. The V content in roots and leaves was correlated with V supply concentration but the V content in leaf was always much lower than in the root, which leads us to conclusion that V accumulates in the roots and only small quantities are transferred to the leaves. However, thylakoid disorganisation was observed in the chloroplasts of plants grown with vanadyl sulphate.     

Variation and morphogenetic characteristics of different Stachys species during microclonal propagation

Morphogeneses of Stachys different species introduced in culturing in vitro have been compared. The frequency of altered forms have been demonstrated to be related to the plant genotype. All regenerants of S. sieboldii, which reproduces in vivo only vegetatively, are phenotypically normal, irrespective of the concentrations of plant growth regulators at which they have been obtained. Only changes in isozyme patterns have been observed in the regenerants grown in media containing at least 10 mg/l benzyl aminopurine (BAP); most of these changes are the absence of a particular component of the pattern. The cross-pollinating species Stachys ocymastrum, which typically reproduces by seeds, has yielded morphologically altered forms even in phytohormone-free media; its isozyme patterns often contained a new component. Analysis of the isoperoxidase patterns of regenerants of both Stachys species obtained with the use of high phytohormone concentrations has demonstrated qualitative and quantitative changes suggesting the appearance of somaclonal variants even in the course of plant regeneration directly from nodal segments, bypassing callus formation. Changes have also been found in Stachys plants regenerating from the callus tissue.     

Nonuniform Transport of Phosphorus from Single Roots to the Leaves of Zea mays

The postulate that single roots of Zea mays transport their absorbed phosphorus nonuniformly to the leaves was tested. Plants were grown under growth chamber conditions for three to four weeks in nutrient solution. At this stage of growth a series of plants was placed into a system in which two roots on each plant were allowed to absorb either ³³P or ³²P from uptake solutions for time intervals of up to 24 hours. Plants subsequently were harvested such that each leaf was partitioned into samples containing tissue from one side or the other of the midrib. All samples were assayed for ³³P and ³²P and the results were expressed as the amount of total P transported into different plant parts from a single root. Nonuniform P accumulation in the leaves occurred and different patterns of accumulation, dependent on the type of root chosen for uptake were observed. Nearly uniform P accumulation occurred between one side and the other of a given leaf when transport was from radicle roots. In marked contrast, transport from adventitious roots resulted in an alternating pattern of accumulation between one side and the other of each successive leaf up the stem. The seminal root system supplied more P to the older leaves than did the adventitious root system. The nature of these nonuniform P transport patterns is attributed to the vascular organization between roots and leaves.     

Assessment of Root System Dynamics of Species Grown in Mixtures under Field Conditions using Herbicide Injection and 15N Natural Abundance Methods: A Case Study with Pea, Barley and Mustard

Two methods were developed and used to study the root system dynamics of two species grown together or separately under field conditions. The first method, based on herbicide injection at different soil depths, was used to determine the rooting depth penetration rate of each species in pea–barley and pea–mustard mixtures. The roots absorbed the herbicide when they reached the treated zone leading to visible symptoms on the leaves which could be readily monitored. The second method used differences in ¹⁵N natural abundance and N concentration between legume and non-legume species to quantify the contribution of each species to root biomass of a pea–barley mixture. Each contribution was calculated using ¹⁵N abundance and N concentration of root mixtures and of subsamples of roots of individual species within mixtures. Both methods can indeed be used to distinguish roots of species in mixtures and thus to study belowground competition between associated species. The use of these methods demonstrated species differences in root system dynamics between species but also significant effects of interactions between species in mixtures. The rooting depth penetration rate was mainly species specific whereas root biomass was dependant on plant growth, allocation of dry matter between shoot and root components and growth factors such as N fertilization. Root biomass of each species may vary therefore with the level of competition between species.     

Succession of root-associated fungi in Pisum sativum during a plant growth cycle as examined by 454 pyrosequencing

Purpose

Roots are inhabited by a broad range of fungi, including pathogens and mycorrhizal fungi, with functional traits related to plant health and nutrition. Management of these fungi in agroecosystems requires profound knowledge about their ecology. The main objective of this study was to examine succession patterns of root-associated fungi in pea during a full plant growth cycle.

Methods

Plants were grown in pots with field soil in a growth chamber under controlled conditions. Fungal communities in pea roots were analyzed at different plant growth stages including the vegetative growth, flowering and senescence, using 454 pyrosequencing.

Results

One hundred and twenty one non-singleton operational taxonomic units (OTUs) representing fungal species were detected. Pathogenic and arbuscular mycorrhizal fungi dominated during the vegetative growth stage, whereas saprotrophic fungi dominated during plant senescence.

Conclusions

In conclusion, the results from the present study demonstrated highly diverse fungal communities in pea roots with clear succession patterns related to fungal traits.     

Sexual dimorphism in resource acquisition and deployment: both size and timing matter

Background and Aims

The males and females of many dioecious plant species differ from one another in important life-history traits, such as their size. If male and female reproductive functions draw on different resources, for example, one should expect males and females to display different allocation strategies as they grow. Importantly, these strategies may differ not only between the two sexes, but also between plants of different age and therefore size. Results are presented from an experiment that asks whether males and females of Mercurialis annua, an annual plant with indeterminate growth, differ over time in their allocation of two potentially limiting resources (carbon and nitrogen) to vegetative (below- and above-ground) and reproductive tissues.

Methods

Comparisons were made of the temporal patterns of biomass allocation to shoots, roots and reproduction and the nitrogen content in the leaves between the sexes of M. annua by harvesting plants of each sex after growth over different periods of time.

Key Results and Conclusions

Males and females differed in their temporal patterns of allocation. Males allocated more to reproduction than females at early stages, but this trend was reversed at later stages. Importantly, males allocated proportionally more of their biomass towards roots at later stages, but the roots of females were larger in absolute terms. The study points to the important role played by both the timing of resource deployment and the relative versus absolute sizes of the sinks and sources in sexual dimorphism of an annual plant.