Enhanced Labeling Techniques to Study the Cytoskeleton During Root Growth and Gravitropism

Gravity effects the growth and development of all living organisms. One of the most obvious manifestations of gravity's effects on biological systems lies in the ability of plants to direct their growth along a path that is dictated by the gravity vector (called gravitropism). When positioned horizontally, in florescence stems and hypocotyls in dicots, and pulvini in monocots, respond by bending upward whereas roots typically bend downward. Gravitropism allows plants to readjust their growth to maximize light absorption for photosynthesis and to more efficiently acquire water and nutrients form the soil. Despite its significance for plant survival, there are still major gaps in understanding the cellular and molecular processes by which plants respond to gravity. The major aim of this proposal was to develop improved fluorescence labeling techniques to aid in understanding how the cytoskeleton modulated plant responses to gravity

Divergence and Redundancy in CSLD2 and CSLD3 Function During Arabidopsis Thaliana Root Hair and Female Gametophyte Development

The Arabidopsis cellulose synthase-like D (CSLD) 2 and 3 genes are known to function in root hair development. Here, we show that these genes also play a role in female gametophyte development because csld2 csld3 double mutants were observed to have low seed set that could be traced to defects in female transmission efficiency. Cell biological studies of csld2 csld3 ovules showed synergid cell degeneration during megagametogenesis and reduced pollen tube penetration during fertilization. Although CSLD2 and CSLD3 function redundantly in female gametophyte development, detailed analyses of root hair phenotypes of progeny from genetic crosses between csld2 and csld3, suggest that CSLD3 might play a more prominent role than CSLD2 in root hair development. Phylogenetic and gene duplication studies of CSLD2 and CSLD3 homologs in Arabidopsis lyrata, Populus, Medicago, maize, and Physcomitrella were further performed to investigate the course of evolution for these genes. Our analyses indicate that the ancestor of land plants possibly contained two copies of CSLD genes, one of which developed into the CSLD5 lineage in flowering plants, and the other formed the CSLD1/2/3/4 clade. In addition, CSLD2 and CSLD3 likely originated from a recent genome-wide duplication event explaining their redundancy. Moreover, sliding-window dN/dS analysis showed that most of the coding regions of CSLD2 and CSLD3 have been under strong purifying selection pressure. However, the region that encodes the N-terminus of CSLD3 has been under relatively relaxed selection pressure as indicated by its high dN/dS value, suggesting that CSLD3 might have gained additional functions through more frequent non-synonymous sequence changes at the N-terminus, which could partly explain the more prominent role of CSLD3 during root hair development compared to CSLD2

Medicago truncatula and Glomus intraradices gene expression in cortical cells harboring arbuscules in the arbuscular mycorrhizal symbiosis

<p>Abstract</p> <p>Background</p> <p>Most vascular flowering plants have the capacity to form symbiotic associations with arbuscular mycorrhizal (AM) fungi. The symbiosis develops in the roots where AM fungi colonize the root cortex and form arbuscules within the cortical cells. Arbuscules are enveloped in a novel plant membrane and their establishment requires the coordinated cellular activities of both symbiotic partners. The arbuscule-cortical cell interface is the primary functional interface of the symbiosis and is of central importance in nutrient exchange. To determine the molecular events the underlie arbuscule development and function, it is first necessary to identify genes that may play a role in this process. Toward this goal we used the Affymetrix GeneChip^®Medicago Genome Array to document the <it>M. truncatula </it>transcript profiles associated with AM symbiosis, and then developed laser microdissection (LM) of <it>M. truncatula </it>root cortical cells to enable analyses of gene expression in individual cell types by RT-PCR.</p> <p>Results</p> <p>This approach led to the identification of novel <it>M. truncatula </it>and <it>G. intraradices </it>genes expressed in colonized cortical cells and in arbuscules. Within the arbuscule, expression of genes associated with the urea cycle, amino acid biosynthesis and cellular autophagy was detected. Analysis of gene expression in the colonized cortical cell revealed up-regulation of a lysine motif (LysM)-receptor like kinase, members of the GRAS transcription factor family and a symbiosis-specific ammonium transporter that is a likely candidate for mediating ammonium transport in the AM symbiosis.</p> <p>Conclusion</p> <p>Transcript profiling using the Affymetrix GeneChip^®Medicago Genome Array provided new insights into gene expression in <it>M. truncatula </it>roots during AM symbiosis and revealed the existence of several <it>G. intraradices </it>genes on the <it>M. truncatula </it>GeneChip^®. A laser microdissection protocol that incorporates low-melting temperature Steedman's wax, was developed to enable laser microdissection of <it>M. truncatula </it>root cortical cells. LM coupled with RT-PCR provided spatial gene expression information for both symbionts and expanded current information available for gene expression in cortical cells containing arbuscules.</p

Three‐channel electrical impedance spectroscopy for field‐scale root phenotyping

AbstractElectrical impedance spectroscopy has long been considered a promising technique for noninvasive, in‐situ root investigation because of its sensitivity to anatomy and physiology. However, the complexity of the root system and its coupling with stem and soil have hindered the signal interpretation and methodological upscaling to field applications. This study addresses these key issues by introducing three‐channel acquisitions and their interpretation through Cole–Cole fitting. This solution could successfully decouple the impedance response of stem, roots, and soil, as well as provide convenient parametrization and comparison of their impedance signals. The methodological solution was tested on 80 wheat (Triticum aestivum L.) and 10 pecan [Carya illinoensis (Wangenh.) K. Koch] plants, the first extensive and field investigation. The investigation provided evidence of (a) proximal current leakage in herbaceous root systems, extending recent laboratory results and previous indirect field studies. (b) Major role of the plant stem, which has been a substantial concern raised in numerous studies. (c) Minor contribution from the soil, addressing the doubts on the comparability of results obtained in different soil conditions. All together, these evidences lead to indirect correlations between impedance signals and root traits. The explored solution is expected to support the adoption of the impedance spectroscopy, in line with the diffusion of multichannel impedance meters and growing interest in root physiology and phenotyping

Brassinosteroids Inhibit Autotropic Root Straightening by Modifying Filamentous-Actin Organization and Dynamics

When positioned horizontally, roots grow down toward the direction of gravity. This phenomenon, called gravitropism, is influenced by most of the major plant hormones including brassinosteroids. Epi-brassinolide (eBL) was previously shown to enhance root gravitropism, a phenomenon similar to the response of roots exposed to the actin inhibitor, latrunculin B (LatB). This led us to hypothesize that eBL might enhance root gravitropism through its effects on filamentous-actin (F-actin). This hypothesis was tested by comparing gravitropic responses of maize (Zea mays) roots treated with eBL or LatB. LatB- and eBL-treated roots displayed similar enhanced downward growth compared with controls when vertical roots were oriented horizontally. Moreover, the effects of the two compounds on root growth directionality were more striking on a slowly-rotating twodimensional clinostat. Both compounds inhibited autotropism, a process in which the root straightened after the initial gravistimulus was withdrawn by clinorotation. Although eBL reduced F-actin density in chemically-fixed Z. mays roots, the impact was not as strong as that of LatB. Modification of F-actin organization after treatment with both compounds was also observed in living roots of barrel medic (Medicago truncatula) seedlings expressing genetically encoded F-actin reporters. Like in fixed Z. mays roots, eBL effects on F-actin in living M. truncatula roots were modest compared with those of LatB. Furthermore, live cell imaging revealed a decrease in global F-actin dynamics in hypocotyls of etiolated M. truncatula seedlings treated with eBL compared to controls. Collectively, our data indicate that eBL-and LatB-induced enhancement of root gravitropism can be explained by inhibited autotropic root straightening, and that eBL affects this process, in part, by modifying F-actin organization and dynamics

Overexpression of Fatty Acid Amide Hydrolase Induces Early Flowering in Arabidopsis thaliana

N-Acylethanolamines (NAEs) are bioactive lipids derived from the hydrolysis of the membrane phospholipid N-acylphosphatidylethanolamine (NAPE). In animal systems this reaction is part of the “endocannabinoid” signaling pathway, which regulates a variety of physiological processes. The signaling function of NAE is terminated by fatty acid amide hydrolase (FAAH), which hydrolyzes NAE to ethanolamine and free fatty acid. Our previous work in Arabidopsis thaliana showed that overexpression of AtFAAH (At5g64440) lowered endogenous levels of NAEs in seeds, consistent with its role in NAE signal termination. Reduced NAE levels were accompanied by an accelerated growth phenotype, increased sensitivity to abscisic acid (ABA), enhanced susceptibility to bacterial pathogens, and early flowering. Here we investigated the nature of the early flowering phenotype of AtFAAH overexpression. AtFAAH overexpressors flowered several days earlier than wild type and AtFAAH knockouts under both non-inductive short day (SD) and inductive long day (LD) conditions. Microarray analysis revealed that the FLOWERING LOCUS T (FT) gene, which plays a major role in regulating flowering time, and one target MADS box transcription factor, SEPATALLA3 (SEP3), were elevated in AtFAAH overexpressors. Furthermore, AtFAAH overexpressors, with the early flowering phenotype had lower endogenous NAE levels in leaves compared to wild type prior to flowering. Exogenous application of NAE 12:0, which was reduced by up to 30% in AtFAAH overexpressors, delayed the onset of flowering in wild type plants. We conclude that the early flowering phenotype of AtFAAH overexpressors is, in part, explained by elevated FT gene expression resulting from the enhanced NAE hydrolase activity of AtFAAH, suggesting that NAE metabolism may participate in floral signaling pathways

Combining loss of function of FOLYLPOLYGLUTAMATE SYNTHETASE1 and CAFFEOYL-COA 3-O-METHYLTRANSFERASE1 for lignin reduction and improved saccharification efficiency in Arabidopsis thaliana

This article tests if lignin content can be further reduced by combining genetic mutations in C1 metabolism and the lignin biosynthetic pathway by generating and functionally characterizing fpgs1ccoaomt1 double mutants. The observations demonstrate that additional reduction in lignin content and improved sugar release can be achieved by simultaneous downregulation of a gene in the C1 (FPGS1) and lignin biosynthetic (CCOAOMT) pathways

Enhanced Gravitropism of Roots with a Disrupted Cap Actin Cytoskeleton

The actin cytoskeleton has been proposed to be a major player in plant gravitropism. However, understanding the role of actin in this process is far from complete. To address this problem, we conducted an analysis of the effect of Latrunculin B (Lat B), a potent actin-disrupting drug, on root gravitropism using various parameters that included detailed curvature kinetics, estimation of gravitropic sensitivity, and monitoring of curvature development after extended clinorotation. Lat B treatment resulted in a promotion of root curvature after a 90° reorientation in three plant species tested. More significantly, the sensitivity of maize (Zea mays) roots to gravity was enhanced after actin disruption, as determined from a comparison of presentation time of Lat B-treated versus untreated roots. A short 10-min gravistimulus followed by extended rotation on a 1-rpm clinostat resulted in extensive gravitropic responses, manifested as curvature that often exceeded 90°. Application of Lat B to the cap or elongation zone of maize roots resulted in the disruption of the actin cytoskeleton, which was confined to the area of localized Lat B application. Only roots with Lat B applied to the cap displayed the strong curvature responses after extended clinorotation. Our study demonstrates that disrupting the actin cytoskeleton in the cap leads to the persistence of a signal established by a previous gravistimulus. Therefore, actin could function in root gravitropism by providing a mechanism to regulate the proliferation of a gravitropic signal originating from the cap to allow the root to attain its correct orientation or set point angle