Tracy A. Valentine

Root elongation, water stress, and mechanical impedance: a review of limiting stresses and beneficial root tip traits

Root elongation in drying soil is generally limited by a combination of mechanical impedance and water stress. Relationships between root elongation rate, water stress (matric potential), and mechanical impedance (penetration resistance) are reviewed, detailing the interactions between these closely related stresses. Root elongation is typically halved in repacked soils with penetrometer resistances >0.8-2 MPa, in the absence of water stress. Root elongation is halved by matric potentials drier than about -0.5 MPa in the absence of mechanical impedance. The likelihood of each stress limiting root elongation is discussed in relation to the soil strength characteristics of arable soils. A survey of 19 soils, with textures ranging from loamy sand to silty clay loam, found that ∼10% of penetration resistances were >2 MPa at a matric potential of -10 kPa, rising to nearly 50% >2 MPa at - 200 kPa. This suggests that mechanical impedance is often a major limitation to root elongation in these soils even under moderately wet conditions, and is important to consider in breeding programmes for drought-resistant crops. Root tip traits that may improve root penetration are considered with respect to overcoming the external (soil) and internal (cell wall) pressures resisting elongation. The potential role of root hairs in mechanically anchoring root tips is considered theoretically, and is judged particularly relevant to roots growing in biopores or from a loose seed bed into a compacted layer of soil.

Functional Analysis of a DNA-Shuffled Movement Protein Reveals That Microtubules Are Dispensable for the Cell-to-Cell Movement of Tobacco mosaic virus

Microtubules interact strongly with the viral movement protein (MP) of Tobacco mosaic virus (TMV) and are thought to transport the viral genome between plant cells. We describe a functionally enhanced DNA-shuffled movement protein (MPR3) that remained bound to the vertices of the cortical endoplasmic reticulum, showing limited affinity for microtubules. A single amino acid change was shown to confer the MPR3 phenotype. Disruption of the microtubule cytoskeleton in situ with pharmacological agents, or by silencing of the α-tubulin gene, had no significant effect on the spread of TMV vectors expressing wild-type MP (MPWT) and did not prevent the accumulation of MPWT in plasmodesmata. Thus, cell-to-cell trafficking of TMV can occur independently of microtubules. The MPR3 phenotype was reproduced when infection sites expressing MPWT were treated with a specific proteasome inhibitor, indicating that the degradation of MPR3 is impaired. We suggest that the improved viral transport functions of MPR3 arise from evasion of a host degradation pathway.

Efficient Virus-Induced Gene Silencing in Roots Using a Modified Tobacco Rattle Virus Vector

Due to their capability of eliciting a form of posttranscriptional gene silencing (termed virus-induced gene silencing or VIGS), plant viruses are increasingly used as reverse-genetics tools for functional characterization of plant genes. RNA viruses have been shown to trigger silencing in a variety of host plants, including members of Solanacae and Arabidopsis (Arabidopsis thaliana). Several factors affect the silencing response, including host range and viral tropism within the plant. The work presented here demonstrates that a modified tobacco rattle virus (TRV) vector retaining the helper protein 2b, required for transmission by a specific vector nematode, not only invades and replicates extensively in whole plants, including meristems, but also triggers a pervasive systemic VIGS response in the roots of Nicotiana benthamiana, Arabidopsis, and tomato (Lycopersicon esculentum). This sustained VIGS response was exemplified by the silencing of genes involved in root development (IRT1, TTG1 [transparent testa glabra], RHL1 [root hairless1], and β-tubulin), lateral root-meristem function (RML1 [root meristemless1]), and nematode resistance (Mi). Roots of silenced plants exhibit reduced levels of target mRNA and phenocopy previously described mutant alleles. The TRV-2b vector displays increased infectivity and meristem invasion, both key requirements for efficient VIGS-based functional characterization of genes in root tissues. Our data suggest that the TRV helper protein 2b may have an essential role in the host regulatory mechanisms that control TRV invasion.

Transparent Soil for Imaging the Rhizosphere

Understanding of soil processes is essential for addressing the global issues of food security, disease transmission and climate change. However, techniques for observing soil biology are lacking. We present a heterogeneous, porous, transparent substrate for in situ 3D imaging of living plants and root-associated microorganisms using particles of the transparent polymer, Nafion, and a solution with matching optical properties. Minerals and fluorescent dyes were adsorbed onto the Nafion particles for nutrient supply and imaging of pore size and geometry. Plant growth in transparent soil was similar to that in soil. We imaged colonization of lettuce roots by the human bacterial pathogen Escherichia coli O157:H7 showing micro-colony development. Micro-colonies may contribute to bacterial survival in soil. Transparent soil has applications in root biology, crop genetics and soil microbiology.

Soil strength and macropore volume limit root elongation rates in many UK agricultural soils

BACKGROUND AND AIMS Simple indicators of crop and cultivar performance across a range of soil types and management are needed for designing and testing sustainable cropping practices. This paper determined the extent to which soil chemical and physical properties, particularly soil strength and pore-size distribution influences root elongation in a wide range of agricultural top soils, using a seedling-based indicator. METHODS Intact soil cores were sampled from the topsoil of 59 agricultural fields in Scotland, representing a wide geographic spread, range of textures and management practices. Water release characteristics, dry bulk density and needle penetrometer resistance were measured on three cores from each field. Soil samples from the same locations were sieved, analysed for chemical characteristics, and packed to dry bulk density of 1.0 g cm(-3) to minimize physical constraints. Root elongation rates were determined for barley seedlings planted in both intact field and packed soil cores at a water content close to field capacity (-20 kPa matric potential). KEY RESULTS Root elongation in field soil was typically less than half of that in packed soils. Penetrometer resistance was typically between 1 and 3 MPa for field soils, indicating the soils were relatively hard, despite their moderately wet condition (compared with <0.2 MPa for packed soil). Root elongation was strongly linked to differences in physical rather than chemical properties. In field soil root elongation was related most closely to the volume of soil pores between 60 µm and 300 µm equivalent diameter, as estimated from water-release characteristics, accounting for 65.7 % of the variation in the elongation rates. CONCLUSIONS Root elongation rate in the majority of field soils was slower than half of the unimpeded (packed) rate. Such major reductions in root elongation rates will decrease rooting volumes and limit crop growth in soils where nutrients and water are scarce.

Root traits for infertile soils

Crop production is often restricted by the availability of essential mineral elements. For example, the availability of N, P, K, and S limits low-input agriculture, the phytoavailability of Fe, Zn, and Cu limits crop production on alkaline and calcareous soils, and P, Mo, Mg, Ca, and K deficiencies, together with proton, Al and Mn toxicities, limit crop production on acid soils. Since essential mineral elements are acquired by the root system, the development of crop genotypes with root traits increasing their acquisition should increase yields on infertile soils. This paper examines root traits likely to improve the acquisition of these elements and observes that, although the efficient acquisition of a particular element requires a specific set of root traits, suites of traits can be identified that benefit the acquisition of a group of mineral elements. Elements can be divided into three Groups based on common trait requirements. Group 1 comprises N, S, K, B, and P. Group 2 comprises Fe, Zn, Cu, Mn, and Ni. Group 3 contains mineral elements that rarely affect crop production. It is argued that breeding for a limited number of distinct root ideotypes, addressing particular combinations of mineral imbalances, should be pursued.

Challenges and opportunities for quantifying roots and rhizosphere interactions through imaging and image analysis

The morphology of roots and root systems influences the efficiency by which plants acquire nutrients and water, anchor themselves and provide stability to the surrounding soil. Plant genotype and the biotic and abiotic environment significantly influence root morphology, growth and ultimately crop yield. The challenge for researchers interested in phenotyping root systems is, therefore, not just to measure roots and link their phenotype to the plant genotype, but also to understand how the growth of roots is influenced by their environment. This review discusses progress in quantifying root system parameters (e.g. in terms of size, shape and dynamics) using imaging and image analysis technologies and also discusses their potential for providing a better understanding of root:soil interactions. Significant progress has been made in image acquisition techniques, however trade-offs exist between sample throughput, sample size, image resolution and information gained. All of these factors impact on downstream image analysis processes. While there have been significant advances in computation power, limitations still exist in statistical processes involved in image analysis. Utilizing and combining different imaging systems, integrating measurements and image analysis where possible, and amalgamating data will allow researchers to gain a better understanding of root:soil interactions.

Estimating the motion of plant root cells from in vivo confocal laser scanning microscopy images

Images of cellular structures in growing plant roots acquired using confocal laser scanning microscopy have some unusual properties that make motion estimation challenging. These include multiple motions, non-Gaussian noise and large regions with little spatial structure. In this paper, a method for motion estimation is described that uses a robust multi-frame likelihood model and a technique for estimating uncertainty. An efficient region-based matching approach was used followed by a forward projection method. Over small timescales the dynamics are simple (approximately locally constant) and the change in appearance small. Therefore, a constant local velocity model is used and the MAP estimate of the joint probability over a set of frames is recovered. Occurrences of multiple modes in the posterior are detected, and in the case of a single dominant mode, motion is inferred using Laplace’e method. The method was applied to several Arabidopsis thaliana root growth sequences with varying levels of success. In addition, comparative results are given for three alternative motion estimation approaches, the Kanade–Lucas–Tomasi tracker, Black and Anandan’s robust smoothing method, and Markov random field based methods.

PIV as a method for quantifying root cell growth and particle displacement in confocal images

Particle image velocimetry (PIV) quantifies displacement of patches of pixels between successive images. We evaluated PIV as a tool for microscopists by measuring displacements of cells and of a surrounding granular medium in confocal laser scanning microscopy images of Arabidopsis thaliana roots labeled with cell‐membrane targeted green fluorescent protein. Excellent accuracy (e.g., displacement standard deviation <0.006 pixels) was obtained for root images that had undergone rigid digital translations of up to 40 pixels. Analysis of zoomed images showed that magnifications of up to 5% maintained good linear relations between PIV‐predicted and actual displacements (r2 > 0.83). Root mean squared error for these distorted images was 0.4–1.1 pixels, increasing at higher magnification factors. Cell growth and rhizosphere deformation were tracked with good temporal (e.g., 1‐min interval) and spatial resolution, with PIV patches located on recognizable cell features being tracked more successfully. Appropriate choice of GFP‐label was important to decrease small‐scale biological noise due to intracellular motion. PIV of roots grown in stiff 2% versus 0.7% agar showed patterns of cell expansion consistent with physically impeded roots of other species. Roots in glass ballotini underwent rapid changes in growth direction on a timescale of minutes, associated with localized arching of ballotini. By tracking cell vertices, we monitored automatically cell length, width, and area every minute for 0.5 h for cells in different stages of development. In conclusion, PIV measured displacements successfully in images of living root cells and the external granular medium, revealing much potential for use by microscopists. Microsc. Res. Tech., 2009.

Performance of Low-Level Motion Estimation Methods for Confocal Microscopy of Plant Cells in vivo

The performance of various low-level motion estimation methods applied to fluorescence labelled growing cellular structures imaged using confocal laser scanning microscopy is investigated. This is a challenging and unusual domain for motion estimation methods. A selection of methods are discussed that can be contrasted in terms of how much spatial or temporal contextual information is used. The Lucas Kanade feature tracker, a spatially and temporally localised method, was, as one would expect, accurate around resolvable structure. It was not able to track the smaller, repetitive cell structure in the root tip and was somewhat prone to identifying spurious features. This approach is improved by developing a full multi-frame, robust, Bayesian method, and it is demonstrated that by using extra frames with motion constraints reduces such errors. Next, spatially global methods are discussed, including robust variational smoothing and Markov Random Field (MRF) modelling. A key conclusion that is drawn from investigation of these methods is that generic low-level (robust) smoothing functions do not provide good results in this application and that this is probably due to the large regions with little stable structure. Furthermore, contrary to recently reported successes, graph cuts and loopy belief propagation for MAP estimation of the MRF labels provided often poor and inconsistent estimates. The results suggest the need for greater emphasis on temporal smoothing for generic low-level motion estimation tools and more task specific, spatial constraints, perhaps in the form of high level models in order to accurately recover motion from such data. Finally, the form of the estimated growth is briefly discussed and related to contemporary biological models. We hope that this paper will assist non-specialists in applying state-of-the-art methods to this form of data.