Plant root plasticity during drought and recovery: What do we know and where to go?

Aims: Drought stress is one of the most limiting factors for agriculture and ecosystem productivity. Climate change exacerbates this threat by inducing increasingly intense and frequent drought events. Root plasticity during both drought and post-drought recovery is regarded as fundamental to understanding plant climate resilience and maximizing production. We mapped the different research areas and trends that focus on the role of roots in plant response to drought and rewatering and asked if important topics were overlooked. Methods: We performed a comprehensive bibliometric analysis based on journal articles indexed in the Web of Science platform from 1900-2022. We evaluated a) research areas and temporal evolution of keyword frequencies, b) temporal evolution and scientific mapping of the outputs over time, c) trends in the research topics analysis, d) marked journals and citation analysis, and e) competitive countries and dominant institutions to understand the temporal trends of root plasticity during both drought and recovery in the past 120 years. Results: Plant physiological factors, especially in the aboveground part (such as "photosynthesis", "gas-exchange", "abscisic-acid") in model plants Arabidopsis, crops such as wheat and maize, and trees were found to be the most popular study areas; they were also combined with other abiotic factors such as salinity, nitrogen, and climate change, while dynamic root growth and root system architecture responses received less attention. Co-occurrence network analysis showed that three clusters were classified for the keywords including 1) photosynthesis response; 2) physiological traits tolerance (e.g. abscisic acid); 3) root hydraulic transport. Thematically, themes evolved from classical agricultural and ecological research via molecular physiology to root plasticity during drought and recovery. The most productive (number of publications) and cited countries and institutions were situated on drylands in the USA, China, and Australia. In the past decades, scientists approached the topic mostly from a soil-plant hydraulic perspective and strongly focused on aboveground physiological regulation, whereas the actual belowground processes seemed to have been the elephant in the room. There is a strong need for better investigation into root and rhizosphere traits during drought and recovery using novel root phenotyping methods and mathematical modeling.

Comparing anatomy, chemical composition, and water permeability of suberized organs in five plant species: wax makes the difference.

MAIN CONCLUSION: The efficiency of suberized plant/environment interfaces as transpiration barriers is not established by the suberin polymer but by the wax molecules sorbed to the suberin polymer. Suberized cell walls formed as barriers at the plant/soil or plant/atmosphere interface in various plant organs (soil-grown roots, aerial roots, tubers, and bark) were enzymatically isolated from five different plant species (Clivia miniata, Monstera deliciosa, Solanum tuberosum, Manihot esculenta, and Malus domestica). Anatomy, chemical composition and efficiency as transpiration barriers (water loss in m s-1) of the different suberized cell wall samples were quantified. Results clearly indicated that there was no correlation between barrier properties of the suberized interfaces and the number of suberized cell layers, the amount of soluble wax and the amounts of suberin. Suberized interfaces of C. miniata roots, M. esculenta roots, and M. domestica bark periderms formed poor or hardly any transpiration barrier. Permeances varying between 1.1 and 5.1 × 10-8 m s-1 were very close to the permeance of water (7.4 × 10-8 m s-1) evaporating from a water/atmosphere interface. Suberized interfaces of aerial roots of M. deliciosa and tubers of S. tuberosum formed reasonable transpiration barriers with permeances varying between 7.4 × 10-10 and 4.2 × 10-9 m s-1, which were similar to the upper range of permeances measured with isolated cuticles (about 10-9 m s-1). Upon wax extraction, permeances of M. deliciosa and S. tuberosum increased nearly tenfold, which proves the importance of wax establishing a transpiration barrier. Finally, highly opposite results obtained with M. esculenta and S. tuberosum periderms are discussed in relation to their agronomical importance for postharvest losses and tuber storage.

Effective Metabolic Carbon Utilization and Shoot-to-Root Partitioning Modulate Distinctive Yield in High Yielding Cassava Variety.

Increasing cassava production could mitigate one of the global food insecurity challenges by providing a sustainable food source. To improve the yield potential, physiological strategies (i.e., the photosynthetic efficiency, source-to-sink carbon partitioning, and intracellular carbon metabolism) can be applied in breeding to screen for superior genotypes. However, the influences of source-to-sink carbon partitioning and carbon metabolism on the storage root development of cassava are relatively little understood. We hypothesized that carbon partitioning and utilization vary modulating the distinctive storage root yields of high and low-yielding cassava varieties, represented in this study by varieties Kasetsart 50 (KU50) and Hanatee (HN), respectively. Plant growth, photosynthesis measurements, soluble sugars, and starch contents of individual tissues were analyzed at different developmental stages. Also, the diurnal patterns of starch accumulation and degradation in leaves were investigated through iodine staining. Despite a comparable photosynthetic rate, KU50 grew better and yielded greater storage roots than HN. Interestingly, both varieties differed in their carbon partitioning strategies. KU50 had a high photosynthetic capacity and was better efficient in converting photoassimilates to carbon substrates and allocating them to sink organs for their growth. In contrast, HN utilized the photoassimilates at a high metabolic cost, in terms of respiration, and inefficiently allocated carbon to stems rather than storage roots. These results highlighted that carbon assimilation and allocation are genetic potential characteristics of individual varieties, which in effect determine plant growth and storage root yield of cassava. The knowledge gained from this study sheds light on potential strategies for developing new high-yielding genotypes in cassava breeding programs.

Assessing the Storage Root Development of Cassava with a New Analysis Tool.

Storage roots of cassava plants crops are one of the main providers of starch in many South American, African, and Asian countries. Finding varieties with high yields is crucial for growing and breeding. This requires a better understanding of the dynamics of storage root formation, which is usually done by repeated manual evaluation of root types, diameters, and their distribution in excavated roots. We introduce a newly developed method that is capable to analyze the distribution of root diameters automatically, even if root systems display strong variations in root widths and clustering in high numbers. An application study was conducted with cassava roots imaged in a video acquisition box. The root diameter distribution was quantified automatically using an iterative ridge detection approach, which can cope with a wide span of root diameters and clustering. The approach was validated with virtual root models of known geometries and then tested with a time-series of excavated root systems. Based on the retrieved diameter classes, we show plausibly that the dynamics of root type formation can be monitored qualitatively and quantitatively. We conclude that this new method reliably determines important phenotypic traits from storage root crop images. The method is fast and robustly analyses complex root systems and thereby applicable in high-throughput phenotyping and future breeding.

Genetic components of root architecture and anatomy adjustments to water-deficit stress in spring barley.

Roots perform vital roles for adaptation and productivity under water-deficit stress, even though their specific functions are poorly understood. In this study, the genetic control of the nodal-root architectural and anatomical response to water deficit were investigated among diverse spring barley accessions. Water deficit induced substantial variations in the nodal root traits. The cortical, stele, and total root cross-sectional areas of the main-shoot nodal roots decreased under water deficit, but increased in the tiller nodal roots. Root xylem density and arrested nodal roots increased under water deficit, with the formation of root suberization/lignification and large cortical aerenchyma. Genome-wide association study implicated 11 QTL intervals in the architectural and anatomical nodal root response to water deficit. Among them, three and four QTL intervals had strong effects across seasons and on both root architectural and anatomical traits, respectively. Genome-wide epistasis analysis revealed 44 epistatically interacting SNP loci. Further analyses showed that these QTL intervals contain important candidate genes, including ZIFL2, MATE, and PPIB, whose functions are shown to be related to the root adaptive response to water deprivation in plants. These results give novel insight into the genetic architectures of barley nodal root response to soil water deficit stress in the fields, and thus offer useful resources for root-targeted marker-assisted selection.

Soil compaction and the architectural plasticity of root systems.

Soil compaction is a serious global problem, and is a major cause of inadequate rooting and poor yield in crops around the world. Root system architecture (RSA) describes the spatial arrangement of root components within the soil and determines the plant's exploration of the soil. Soil strength restricts root growth and may slow down root system development. RSA plasticity may have an adaptive value, providing environmental tolerance to soil compaction. However, it is challenging to distinguish developmental retardation (apparent plasticity) or responses to severe stress from those root architectural changes that may provide an actual environmental tolerance (adaptive plasticity). In this review, we outline the consequences of soil compaction on the rooting environment and extensively review the various root responses reported in the literature. Finally, we discuss which responses enhance root exploration capabilities in tolerant genotypes, and to what extent these responses might be useful for breeding. We conclude that RSA plasticity in response to soil compaction is complex and can be targeted in breeding to increase the performance of crops under specific agronomical conditions.

Plant Screen Mobile: an open-source mobile device app for plant trait analysis.

BACKGROUND: The development of leaf area is one of the fundamental variables to quantify plant growth and physiological function and is therefore widely used to characterize genotypes and their interaction with the environment. To date, analysis of leaf area often requires elaborate and destructive measurements or imaging-based methods accompanied by automation that may result in costly solutions. Consequently in recent years there is an increasing trend towards simple and affordable sensor solutions and methodologies. A major focus is currently on harnessing the potential of applications developed for smartphones that provide access to analysis tools to a wide user basis. However, most existing applications entail significant manual effort during data acquisition and analysis. RESULTS: With the development of Plant Screen Mobile we provide a suitable smartphone solution for estimating digital proxies of leaf area and biomass in various imaging scenarios in the lab, greenhouse and in the field. To distinguish between plant tissue and background the core of the application comprises different classification approaches that can be parametrized by users delivering results on-the-fly. We demonstrate the practical applications of computing projected leaf area based on two case studies with Eragrostis and Musa plants. These studies showed highly significant correlations with destructive measurements of leaf area and biomass from both ground truth measurements and estimations from well-established screening systems. CONCLUSIONS: We show that a smartphone together with our analysis tool Plant Screen Mobile is a suitable platform for rapid quantification of leaf and shoot development of various plant architectures. Beyond the estimation of projected leaf area the app can also be used to quantify color and shape parameters of other plant material including seeds and flowers.

Ethylene modulates root cortical senescence in barley.

Background and Aims: Root cortical senescence (RCS) is a poorly understood phenomenon with implications for adaptation to edaphic stress. It was hypothesized that RCS in barley (Hordeum vulgare L.) is (1) accelerated by exogenous ethylene exposure; (2) accompanied by differential expression of ethylene synthesis and signalling genes; and (3) associated with differential expression of programmed cell death (PCD) genes. Methods: Gene expression of root segments from four barley genotypes with and without RCS was evaluated using quantitative real-time PCR (qRT-PCR). The progression of RCS was manipulated with root zone ethylene and ethylene inhibitor applications. Key Results: The results demonstrate that ethylene modulates RCS. Four genes related to ethylene synthesis and signalling were upregulated during RCS in optimal, low nitrogen and low phosphorus nutrient regimes. RCS was accelerated by root zone ethylene treatment, and this effect was reversed by an ethylene action inhibitor. Roots treated with exogenous ethylene had 35 and 46 % more cortical senescence compared with the control aeration treatment in seminal and nodal roots, respectively. RCS was correlated with expression of two genes related to programmed cell death (PCD). Conclusions: The development of RCS is similar to root cortical aerenchyma formation with respect to ethylene modulation of the PCD process.

Rapid seedling establishment and a narrow root stele promotes waterlogging tolerance in spring wheat.

Improving the waterlogging tolerance of wheat varieties could alleviate yield constraints caused by excessive rain and poor soil drainage. In this study, we investigated root and shoot growth as well as anatomical traits of six spring wheat genotypes with contrasting waterlogging tolerance properties. Our aim was to identify root traits that differentiate tolerant from sensitive genotypes. Two experiments were conducted using rhizoboxes and photography for data acquisition. In experiment one, root growth of the genotypes was studied during seedling establishment and a subsequent waterlogging treatment, starting at the 3-leaf stage and maintained for seven days. In the second experiment, root and shoot growth of previously waterlogged plants was compared between the genotypes during seven days of recovery. At harvest of experiment two, root segments were sampled to investigate genotype differences of root cross sectional area, root cortex area, stele area and percentage of aerenchyma. The results show that tolerant, in contrast to sensitive genotypes, developed seminal roots faster in the seedling establishment phase and more nodal roots during the waterlogging treatment. NK93602 and Bjarne were the best performing genotypes. Bjarne in particular had a narrower relative stele size of nodal (13.4%) and seminal roots (11.7%) compared to other genotypes (e.g. 16.3% in nodal roots and 13.9% in seminal roots of sensitive Quarna). The results from this study suggests that early vigor is an important trait for waterlogging tolerance in the field. Anatomical root traits, such as a narrow stele and aerenchyma may contribute to improving waterlogging tolerance furthermore.

Root Cortical Senescence Improves Growth under Suboptimal Availability of N, P, and K.

Root cortical senescence (RCS) in Triticeae reduces nutrient uptake, nutrient content, respiration, and radial hydraulic conductance of root tissue. We used the functional-structural model SimRoot to evaluate the functional implications of RCS in barley (Hordeum vulgare) under suboptimal nitrate, phosphorus, and potassium availability. The utility of RCS was evaluated using sensitivity analyses in contrasting nutrient regimes. At flowering (80 d), RCS increased simulated plant growth by up to 52%, 73%, and 41% in nitrate-, phosphorus-, and potassium-limiting conditions, respectively. Plants with RCS had reduced nutrient requirement of root tissue for optimal plant growth, reduced total cumulative cortical respiration, and increased total carbon reserves. Nutrient reallocation during RCS had a greater effect on simulated plant growth than reduced respiration or nutrient uptake. Under low nutrient availability, RCS had greater benefit in plants with fewer tillers. RCS had greater benefit in phenotypes with fewer lateral roots at low nitrate availability, but the opposite was true in low phosphorus or potassium availability. Additionally, RCS was quantified in field-grown barley in different nitrogen regimes. Field and virtual soil coring simulation results demonstrated that living cortical volume per root length (an indicator of RCS) decreased with depth in younger plants, while roots of older plants had very little living cortical volume per root length. RCS may be an adaptive trait for nutrient acquisition by reallocating nutrients from senescing tissue and secondarily by reducing root respiration. These simulated results suggest that RCS merits investigation as a breeding target for enhanced soil resource acquisition and edaphic stress tolerance.

Root cortical senescence decreases root respiration, nutrient content and radial water and nutrient transport in barley.

The functional implications of root cortical senescence (RCS) are poorly understood. We tested the hypotheses that RCS in barley (1) reduces the respiration and nutrient content of root tissue; (2) decreases radial water and nutrient transport; and (3) is accompanied by increased suberization to protect the stele. Genetic variation for RCS exists between modern germplasm and landraces. Nitrogen and phosphorus deficiency increased the rate of RCS. Maximal RCS, defined as the disappearance of the entire root cortex, reduced root nitrogen content by 66%, phosphorus content by 63% and respiration by 87% compared with root segments with no RCS. Roots with maximal RCS had 90, 92 and 84% less radial water, nitrate and phosphorus transport, respectively, compared with segments with no RCS. The onset of RCS coincided with 30% greater aliphatic suberin in the endodermis. These results support the hypothesis that RCS reduces root carbon and nutrient costs and may therefore have adaptive significance for soil resource acquisition. By reducing root respiration and nutrient content, RCS could permit greater root growth, soil resource acquisition and resource allocation to other plant processes. RCS merits investigation as a trait for improving the performance of barley, wheat, triticale and rye under edaphic stress.

Pampered inside, pestered outside? Differences and similarities between plants growing in controlled conditions and in the field.

I. 839 II. 839 III. 841 IV. 845 V. 847 VI. 848 VII. 849 VIII. 851 851 852 References 852 Appendix A1 854 SUMMARY: Plant biologists often grow plants in growth chambers or glasshouses with the ultimate aim to understand or improve plant performance in the field. What is often overlooked is how results from controlled conditions translate back to field situations. A meta-analysis showed that lab-grown plants had faster growth rates, higher nitrogen concentrations and different morphology. They remained smaller, however, because the lab plants had grown for a much shorter time. We compared glasshouse and growth chamber conditions with those in the field and found that the ratio between the daily amount of light and daily temperature (photothermal ratio) was consistently lower under controlled conditions. This may strongly affect a plant's source : sink ratio and hence its overall morphology and physiology. Plants in the field also grow at higher plant densities. A second meta-analysis showed that a doubling in density leads on average to 34% smaller plants with strong negative effects on tiller or side-shoot formation but little effect on plant height. We found the r2 between lab and field phenotypic data to be rather modest (0.26). Based on these insights, we discuss various alternatives to facilitate the translation from lab results to the field, including several options to apply growth regimes closer to field conditions.

Digital imaging of root traits (DIRT): a high-throughput computing and collaboration platform for field-based root phenomics.

BACKGROUND: Plant root systems are key drivers of plant function and yield. They are also under-explored targets to meet global food and energy demands. Many new technologies have been developed to characterize crop root system architecture (CRSA). These technologies have the potential to accelerate the progress in understanding the genetic control and environmental response of CRSA. Putting this potential into practice requires new methods and algorithms to analyze CRSA in digital images. Most prior approaches have solely focused on the estimation of root traits from images, yet no integrated platform exists that allows easy and intuitive access to trait extraction and analysis methods from images combined with storage solutions linked to metadata. Automated high-throughput phenotyping methods are increasingly used in laboratory-based efforts to link plant genotype with phenotype, whereas similar field-based studies remain predominantly manual low-throughput. DESCRIPTION: Here, we present an open-source phenomics platform "DIRT", as a means to integrate scalable supercomputing architectures into field experiments and analysis pipelines. DIRT is an online platform that enables researchers to store images of plant roots, measure dicot and monocot root traits under field conditions, and share data and results within collaborative teams and the broader community. The DIRT platform seamlessly connects end-users with large-scale compute "commons" enabling the estimation and analysis of root phenotypes from field experiments of unprecedented size. CONCLUSION: DIRT is an automated high-throughput computing and collaboration platform for field based crop root phenomics. The platform is accessible at http://www.dirt.iplantcollaborative.org/ and hosted on the iPlant cyber-infrastructure using high-throughput grid computing resources of the Texas Advanced Computing Center (TACC). DIRT is a high volume central depository and high-throughput RSA trait computation platform for plant scientists working on crop roots. It enables scientists to store, manage and share crop root images with metadata and compute RSA traits from thousands of images in parallel. It makes high-throughput RSA trait computation available to the community with just a few button clicks. As such it enables plant scientists to spend more time on science rather than on technology. All stored and computed data is easily accessible to the public and broader scientific community. We hope that easy data accessibility will attract new tool developers and spur creative data usage that may even be applied to other fields of science.

Cooperative action of the paralogous maize lateral organ boundaries (LOB) domain proteins RTCS and RTCL in shoot-borne root formation.

The paralogous maize (Zea mays) LBD (Lateral Organ Boundaries Domain) genes rtcs (rootless concerning crown and seminal roots) and rtcl (rtcs-like) emerged from an ancient whole-genome duplication. RTCS is a key regulator of crown root initiation. The diversity of expression, molecular interaction and phenotype of rtcs and rtcl were investigated. The rtcs and rtcl genes display highly correlated spatio-temporal expression patterns in roots, despite the significantly higher expression of rtcs. Both RTCS and RTCL proteins bind to LBD downstream promoters and act as transcription factors. In line with its auxin inducibility and binding to auxin response elements of rtcs and rtcl promoters, ARF34 (AUXIN RESPONSE FACTOR 34) acts as transcriptional activator. Yeast two-hybrid screening combined with bimolecular fluorescence complementation (BiFC) experiments revealed conserved and unique interaction partners of RTCS and RTCL. The rtcl mutation leads to defective shoot-borne root elongation early in development. Cooperative action of RTCS and RTCL during shoot-borne root formation was demonstrated by rtcs-dependent repression of rtcl transcription in coleoptilar nodes. Although RTCS is instrumental in shoot-borne root initiation, RTCL controls shoot-borne root elongation early in development. Their conserved role in auxin signaling, but diverse function in shoot-borne root formation, is underscored by their conserved and unique interaction partners.

Opportunities and challenges in the subsoil: pathways to deeper rooted crops.

Greater exploitation of subsoil resources by annual crops would afford multiple benefits, including greater water and N acquisition in most agroecosystems, and greater sequestration of atmospheric C. Constraints to root growth in the subsoil include soil acidity (an edaphic stress complex consisting of toxic levels of Al, inadequate levels of P and Ca, and often toxic levels of Mn), soil compaction, hypoxia, and suboptimal temperature. Multiple root phenes under genetic control are associated with adaptation to these constraints, opening up the possibility of breeding annual crops with root traits improving subsoil exploration. Adaptation to Al toxicity, hypoxia, and P deficiency are intensively researched, adaptation to soil hardness and suboptimal temperature less so, and adaptations to Ca deficiency and Mn toxicity are poorly understood. The utility of specific phene states may vary among soil taxa and management scenarios, interactions which in general are poorly understood. These traits and issues merit research because of their potential value in developing more productive, sustainable, benign, and resilient agricultural systems.

Root phenomics of crops: opportunities and challenges.

Reliable techniques for screening large numbers of plants for root traits are still being developed, but include aeroponic, hydroponic and agar plate systems. Coupled with digital cameras and image analysis software, these systems permit the rapid measurement of root numbers, length and diameter in moderate (typically <1000) numbers of plants. Usually such systems are employed with relatively small seedlings, and information is recorded in 2D. Recent developments in X-ray microtomography have facilitated 3D non-invasive measurement of small root systems grown in solid media, allowing angular distributions to be obtained in addition to numbers and length. However, because of the time taken to scan samples, only a small number can be screened (typically <10 per day, not including analysis time of the large spatial datasets generated) and, depending on sample size, limited resolution may mean that fine roots remain unresolved. Although agar plates allow differences between lines and genotypes to be discerned in young seedlings, the rank order may not be the same when the same materials are grown in solid media. For example, root length of dwarfing wheat (Triticum aestivum L.) lines grown on agar plates was increased by ~40% relative to wild-type and semi-dwarfing lines, but in a sandy loam soil under well watered conditions it was decreased by 24-33%. Such differences in ranking suggest that significant soil environment-genotype interactions are occurring. Developments in instruments and software mean that a combination of high-throughput simple screens and more in-depth examination of root-soil interactions is becoming viable.

Cuticular permeance in relation to wax and cutin development along the growing barley (Hordeum vulgare) leaf.

The developing leaf three of barley provides an excellent model system for the direct determination of relationships between amounts of waxes and cutin and cuticular permeance. Permeance of the cuticle was assessed via the time-course of uptake of either toluidine blue or (14)C-labelled benzoic acid ([(14)C] BA) along the length of the developing leaf. Toluidine blue uptake only occurred within the region 0-25 mm from the point of leaf insertion (POLI). Resistance--the inverse of permeance--to uptake of [(14)C] BA was determined for four leaf regions and was lowest in the region 10-20 mm above POLI. At 20-30 and 50-60 mm above POLI, it increased by factors of 6 and a further 32, respectively. Above the point of emergence of leaf three from the sheath of leaf two, which was 76-80 mm above POLI, resistance was as high as at 50-60 mm above POLI. GC-FID/MS analyses of wax and cutin showed that: (1) the initial seven fold increase in cuticular resistance coincided with increase in cutin coverage and appearance of waxes; (2) the second, larger and final increase in cuticle resistance was accompanied by an increase in wax coverage, whereas cutin coverage remained unchanged; (3) cutin deposition in barley leaf epidermis occurred in parallel with cell elongation, whereas deposition of significant amounts of wax commenced as cells ceased to elongate.

The short-term growth response to salt of the developing barley leaf.

Recent results concerning the short-term growth response to salinity of the developing barley leaf are reviewed. Plants were grown hydroponically and the growth response of leaf 3 was studied between 10 min and 5 d following addition of 100 mM NaCl to the root medium. The aim of the experiments was to relate changes in variables that are likely to affect cell elongation to changes in leaf growth. Changes in hormone content (ABA, cytokinins), water and solute relationships (osmolality, turgor, water potential, solute concentrations), gene expression (water channel), cuticle deposition, membrane potential, and transpiration were followed, while leaf elongation velocity was monitored. Leaf elongation decreased close to zero within seconds following addition of NaCl. Between 20 and 30 min after exposure to salt, elongation velocity recovered rather abruptly, to about 46% of the pre-stress level, and remained at the reduced rate for the following 5 d, when it reached about 70% of the level in non-stressed plants. Biophysical and physiological analyses led to three major conclusions. (i) The immediate reduction and sudden recovery in elongation velocity is due to changes in the water potential gradient between leaf xylem and peripheral elongating cells. Changes in transpiration, ABA and cytokinin content, water channel expression, and plasma membrane potential are involved in this response. (ii) Significant solute accumulation, which aids growth recovery, is detectable from 1 h onwards; growing and non-growing leaf regions and mesophyll and epidermis differ in their solute response. (iii) Cuticular wax density is not affected by short-term exposure to salt; transpirational changes are due to stomatal control.

Apoplastic polyesters in Arabidopsis surface tissues--a typical suberin and a particular cutin.

Cutinized and suberized cell walls form physiological important plant-environment interfaces as they act as barriers limiting water and nutrient loss and protect from radiation and invasion by pathogens. Due to the lack of protocols for the isolation and analysis of cutin and suberin in Arabidopsis, the model plant for molecular biology, mutants and transgenic plants with a defined altered cutin or suberin composition are unavailable, causing that structure and function of these apoplastic barriers are still poorly understood. Transmission electron microscopy (TEM) revealed that Arabidopsis leaf cuticle thickness ranges from only 22 nm in leaf blades to 45 nm on petioles, causing the difficulty in cuticular membrane isolation. We report the use of polysaccharide hydrolases to isolate Arabidopsis cuticular membranes, suitable for depolymerization and subsequent compositional analysis. Although cutin characteristic omega-hydroxy acids (7%) and mid-chain hydroxylated fatty acids (8%) were detected, the discovery of alpha,omega-diacids (40%) and 2-hydroxy acids (14%) as major depolymerization products reveals a so far novel monomer composition in Arabidopsis cutin, but with chemical analogy to root suberin. Histochemical and TEM analysis revealed that suberin depositions were localized to the cell walls in the endodermis of primary roots and the periderm of mature roots of Arabidopsis. Enzyme digested and solvent extracted root cell walls when subjected to suberin depolymerization conditions released omega-hydroxy acids (43%) and alpha,omega-diacids (24%) as major components together with carboxylic acids (9%), alcohols (6%) and 2-hydroxyacids (0.1%). This similarity to suberin of other species indicates that Arabidopsis roots can serve as a model for suberized tissue in general.