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ABSTRACT: 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.
Frontiers in plant science. 01/2012; 3:111.
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ABSTRACT: The Arabidopsis thaliana AGD1 gene encodes a class 1 adenosine diphosphate ribosylation factor-gtpase-activating protein (ARF-GAP). Previously, we found that agd1 mutants have root hairs that exhibit wavy growth and have two tips that originate from a single initiation point. To gain new insights into how AGD1 modulates root hair polarity we analyzed double mutants of agd1 and other loci involved in root hair development, and evaluated dynamics of various components of root hair tip growth in agd1 by live cell microscopy. Because AGD1 contains a phosphoinositide (PI) binding pleckstrin homology (PH) domain, we focused on genetic interactions between agd1 and root hair mutants altered in PI metabolism. Rhd4, which is knocked-out in a gene encoding a phosphatidylinositol-4-phosphate (PI-4P) phosphatase, was epistatic to agd1. In contrast, mutations to PIP5K3 and COW1, which encode a type B phosphatidylinositol-4-phosphate 5-kinase 3 and a phosphatidylinositol transfer protein, respectively, enhanced the root hair defects of agd1. Enhanced root hair defects were also observed in double mutants to AGD1 and ACT2, a root hair-expressed vegetative actin isoform. Consistent with our double-mutant studies, targeting of tip growth components involved in PI signaling (PI-4P), secretion (RABA4b) and actin regulation (ROP2), were altered in agd1 root hairs. Furthermore, tip cytosolic calcium ([Ca²⁺](cyt) ) oscillations were disrupted in root hairs of agd1. Taken together, our results indicate that AGD1 links PI signaling to cytoskeletal-, [Ca²⁺](cyt-) , ROP2-, and RABA4b-mediated root hair development.
The Plant Journal 11/2011; 69(6):1064-76. · 6.16 Impact Factor
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ABSTRACT: During the past decade the use of live cytoskeletal probes has increased dramatically due to the introduction of the green fluorescent protein. However, to make full use of these live cell reporters it is necessary to implement simple methods to maintain plant specimens in optimal growing conditions during imaging. To image the cytoskeleton in living Arabidopsis root cells, we rely on a system involving coverslips coated with nutrient supplemented agar where the seeds are directly germinated. This coverslip system can be conveniently transferred to the stage of a confocal microscope with minimal disturbance to the growth of the seedling. Parallel to our live cell imaging approaches, we routinely process fixed plant material via indirect immunofluorescence. For these methods we typically use nonembedded vibratome-sectioned and whole mount permeabilized root tissue. The clearly defined developmental regions of the root provide us with an elegant system to further understand the cytoskeletal basis of plant development.
Methods in molecular biology (Clifton, N.J.) 01/2009; 586:157-69.
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ABSTRACT: A reverse genetic approach was used to investigate the functions of three members of the cellulose synthase superfamily in Arabidopsis (Arabidopsis thaliana), CELLULOSE SYNTHASE-LIKE D1 (CSLD1), CSLD2, and CSLD4. CSLD2 is required for normal root hair growth but has a different role from that previously described for CSLD3 (KOJAK). CSLD2 is required during a later stage of hair development than CSLD3, and CSLD2 mutants produce root hairs with a range of abnormalities, with many root hairs rupturing late in development. Remarkably, though, it was often the case that in CSLD2 mutants, tip growth would resume after rupturing of root hairs. In silico, semiquantitative reverse transcription-polymerase chain reaction, and promoter-reporter construct analyses indicated that the expression of both CSLD2 and CSLD3 is elevated at reduced temperatures, and the phenotypes of mutants homozygous for insertions in these genes were partially rescued by reduced temperature growth. However, this was not the case for a double mutant homozygous for insertions in both CSLD2 and CSLD3, suggesting that there may be partial redundancy in the functions of these genes. Mutants in CSLD1 and CSLD4 had a defect in male transmission, and plants heterozygous for insertions in CSLD1 or CSLD4 were defective in their ability to produce pollen tubes, although the number and morphology of pollen grains was normal. We propose that the CSLD family of putative glycosyltransferases synthesize a polysaccharide that has a specialized structural role in the cell walls of tip-growing cells.
Plant physiology 10/2008; 148(3):1238-53. · 6.53 Impact Factor
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ABSTRACT: Membrane trafficking and cytoskeletal dynamics are important cellular processes that drive tip growth in root hairs. These processes interact with a multitude of signaling pathways that allow for the efficient transfer of information to specify the direction in which tip growth occurs. Here, we show that AGD1, a class I ADP ribosylation factor GTPase-activating protein, is important for maintaining straight growth in Arabidopsis (Arabidopsis thaliana) root hairs, since mutations in the AGD1 gene resulted in wavy root hair growth. Live cell imaging of growing agd1 root hairs revealed bundles of endoplasmic microtubules and actin filaments extending into the extreme tip. The wavy phenotype and pattern of cytoskeletal distribution in root hairs of agd1 partially resembled that of mutants in an armadillo repeat-containing kinesin (ARK1). Root hairs of double agd1 ark1 mutants were more severely deformed compared with single mutants. Organelle trafficking as revealed by a fluorescent Golgi marker was slightly inhibited, and Golgi stacks frequently protruded into the extreme root hair apex of agd1 mutants. Transient expression of green fluorescent protein-AGD1 in tobacco (Nicotiana tabacum) epidermal cells labeled punctate bodies that partially colocalized with the endocytic marker FM4-64, while ARK1-yellow fluorescent protein associated with microtubules. Brefeldin A rescued the phenotype of agd1, indicating that the altered activity of an AGD1-dependent ADP ribosylation factor contributes to the defective growth, organelle trafficking, and cytoskeletal organization of agd1 root hairs. We propose that AGD1, a regulator of membrane trafficking, and ARK1, a microtubule motor, are components of converging signaling pathways that affect cytoskeletal organization to specify growth orientation in Arabidopsis root hairs.
Plant physiology 07/2008; 147(4):1659-74. · 6.53 Impact Factor
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ABSTRACT: The role of the actin cytoskeleton in plant development is intimately linked to its dynamic behavior. Therefore it is essential to continue refining methods for studying actin organization in living plant cells. The discovery of green fluorescent protein (GFP) has popularized the use of translational fusions of GFP with actin filament (F-actin) side-binding proteins to visualize in vivo actin organization in plants. The most recent of these live cell F-actin reporters are GFP fusions to the actin-binding domain 2 (ABD2) of Arabidopsis fimbrin 1 (ABD2-GFP). To improve ABD2-GFP fluorescence for enhanced in vivo F-actin imaging, transgenic Arabidopsis plants were generated expressing a construct with GFP fused to both the C- and N-termini of ABD2 under the control of the CaMV 35S promoter (35S::GFP-ABD2-GFP). The 35S::GFP-ABD2-GFP lines had significantly increased fluorescence compared with the original 35S::ABD2-GFP lines. The enhanced fluorescence of the 35S::GFP-ABD2-GFP-expressing lines allowed the acquisition of highly resolved images of F-actin in different plant organs and stages of development because of the reduced confocal microscope excitation settings needed for data collection. This simple modification to the ABD2-GFP construct presents an important tool for studying actin function during plant development.
New Phytologist 02/2008; 177(2):525-36. · 6.64 Impact Factor
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ABSTRACT: Plant development is regulated by numerous chemicals derived from a multitude of metabolic pathways. However, we know very little about the biological effects and functions of many of these metabolites in the cell. N-Acylethanolamines (NAEs) are a group of lipid mediators that play important roles in mammalian physiology. Despite the intriguing similarities between animals and plants in NAE metabolism and perception, not much is known about the precise function of these metabolites in plant physiology. In plants, NAEs have been shown to inhibit phospholipase Dalpha (PLDalpha) activity, interfere with abscisic acid-induced stomatal closure, and retard Arabidopsis seedling development. 1-Butanol, an antagonist of PLD-dependent phosphatidic acid production, was reported to induce defects in Arabidopsis seedling development that were somewhat similar to effects induced by elevated levels of NAE. This raised the possibility that the impact of NAE on seedling growth could be mediated in part via its influence on PLD activity. To begin to address this possibility, we conducted a detailed, comparative analysis of the effects of 1-butanol and N-lauroylethanolamine (NAE 12:0) on Arabidopsis root cell division, in vivo cytoskeletal organization, seed germination, and seedling growth. Although both NAE 12:0 and 1-butanol induced profound cytoskeletal and morphological alterations in seedlings, there were distinct differences in their overall effects. 1-Butanol induced more pronounced modifications in cytoskeletal organization, seedling growth, and cell division at concentrations severalfold higher than NAE 12:0. We propose that these compounds mediate their differential effects on cellular organization and seedling growth, in part through the differential modulation of specific PLD isoforms.
Protoplasma 01/2006; 226(3-4):109-23. · 1.92 Impact Factor
