Spatial and temporal localization of SPIRRIG and WAVE/SCAR reveal roles for these proteins in actin-mediated root hair development.

Root hairs are single-cell protrusions that enable roots to optimize nutrient and water acquisition. These structures attain their tubular shapes by confining growth to the cell apex, a process called tip growth. The actin cytoskeleton and endomembrane systems are essential for tip growth; however, little is known about how these cellular components coordinate their activities during this process. Here, we show that SPIRRIG (SPI), a beige and Chediak Higashi domain-containing protein involved in membrane trafficking, and BRK1 and SCAR2, subunits of the WAVE/SCAR (W/SC) actin nucleating promoting complex, display polarized localizations in Arabidopsis thaliana root hairs during distinct developmental stages. SPI accumulates at the root hair apex via post-Golgi compartments and positively regulates tip growth by maintaining tip-focused vesicle secretion and filamentous-actin integrity. BRK1 and SCAR2 on the other hand, mark the root hair initiation domain to specify the position of root hair emergence. Consistent with the localization data, tip growth was reduced in spi and the position of root hair emergence was disrupted in brk1 and scar1234. BRK1 depletion coincided with SPI accumulation as root hairs transitioned from initiation to tip growth. Taken together, our work uncovers a role for SPI in facilitating actin-dependent root hair development in Arabidopsis through pathways that might intersect with W/SC.

Cell Type-Specific Imaging of Calcium Signaling in Arabidopsis thaliana Seedling Roots Using GCaMP3.

Cytoplasmic calcium ([Ca2+]cyt) is a well-characterized second messenger in eukaryotic cells. An elevation in [Ca2+]cyt levels is one of the earliest responses in plant cells after exposure to a range of environmental stimuli. Advances in understanding the role of [Ca2+]cyt in plant development has been facilitated by the use of genetically-encoded reporters such as GCaMP. Most of these studies have relied on promoters such as Cauliflower Mosaic Virus (35S) and Ubiquitin10 (UBQ10) to drive expression of GCaMP in all cell/tissue types. Plant organs such as roots consist of various cell types that likely exhibit unique [Ca2+]cyt responses to exogenous and endogenous signals. However, few studies have addressed this question. Here, we introduce a set of Arabidopsis thaliana lines expressing GCaMP3 in five root cell types including the columella, endodermis, cortex, epidermis, and trichoblasts. We found similarities and differences in the [Ca2+]cyt signature among these root cell types when exposed to adenosine tri-phosphate (ATP), glutamate, aluminum, and salt, which are known to trigger [Ca2+]cyt increases in root cells. These cell type-targeted GCaMP3 lines provide a new resource that should enable more in depth studies that address how a particular environmental stimulus is linked to specific root developmental pathways via [Ca2+]cyt.

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 two-dimensional 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.

TDNAscan: A Software to Identify Complete and Truncated T-DNA Insertions.

Transfer (T)-DNA insertions in mutants isolated from forward genetic screens are typically identified through thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR), inverse PCR, or plasmid rescue. Despite the popularity and success of these methods, they have limited capabilities, particularly in situations in which the T-DNA is truncated. Here, we present a next generation sequencing (NGS)-based platform to facilitate the identification of complete and truncated T-DNA insertions. Our method enables the detection of the corresponding T-DNA insertion orientation and zygosity as well as insertion annotation. This method, called TDNAscan, was developed to be an open source software. We expect that TDNAscan will be a valuable addition to forward genetics toolkits because it provides a solution to the problem of causal gene identification, particularly genes disrupted by truncated T-DNA insertions. We present a case study in which TDNAscan was used to determine that the recessive Arabidopsis thaliana hypersensitive to latrunculin B (hlb3) mutant isolated in a forward genetic screen of T-DNA mutagenized plants encodes a class II FORMIN.

Deletion analysis of AGD1 reveals domains crucial for plasma membrane recruitment and function in root hair polarity.

AGD1, a plant ACAP-type ADP-ribosylation factor-GTPase activating protein (ARF-GAP), functions in specifying root hair polarity in Arabidopsis thaliana To better understand how AGD1 modulates root hair growth, we generated full-length and domain-deleted AGD1-green fluorescent protein (GFP) constructs, and followed their localization during root hair development. AGD1-GFP localized to the cytoplasm and was recruited to specific regions of the root hair plasma membrane (PM). Distinct PM AGD1-GFP signal was first detected along the site of root hair bulge formation. The construct continued to mark the PM at the root hair apical dome, but only during periods of reduced growth. During rapid tip growth, AGD1-GFP labeled the PM of the lateral flanks and dissipated from the apical-most PM. Deletion analysis and a single domain GFP fusion revealed that the pleckstrin homology (PH) domain is the minimal unit required for recruitment of AGD1 to the PM. Our results indicate that differential recruitment of AGD1 to specific PM domains is an essential component of the membrane trafficking machinery that facilitates root hair developmental phase transitions and responses to changes in the root microenvironment.

HLB1 Is a Tetratricopeptide Repeat Domain-Containing Protein That Operates at the Intersection of the Exocytic and Endocytic Pathways at the TGN/EE in Arabidopsis.

The endomembrane system plays essential roles in plant development, but the proteome responsible for its function and organization remains largely uncharacterized in plants. Here, we identified and characterized the HYPERSENSITIVE TO LATRUNCULIN B1 (HLB1) protein isolated through a forward-genetic screen in Arabidopsis thaliana for mutants with heightened sensitivity to actin-disrupting drugs. HLB1 is a plant-specific tetratricopeptide repeat domain-containing protein of unknown function encoded by a single Arabidopsis gene. HLB1 associated with the trans-Golgi network (TGN)/early endosome (EE) and tracked along filamentous actin, indicating that it could link post-Golgi traffic with the actin cytoskeleton in plants. HLB1 was found to interact with the ADP-ribosylation-factor guanine nucleotide exchange factor, MIN7/BEN1 (HOPM INTERACTOR7/BREFELDIN A-VISUALIZED ENDOCYTIC TRAFFICKING DEFECTIVE1) by coimmunoprecipitation. The min7/ben1 mutant phenocopied the mild root developmental defects and latrunculin B hypersensitivity of hlb1, and analyses of ahlb1/ min7/ben1 double mutant showed that hlb1 and min7/ben1 operate in common genetic pathways. Based on these data, we propose that HLB1 together with MIN7/BEN1 form a complex with actin to modulate the function of the TGN/EE at the intersection of the exocytic and endocytic pathways in plants.

Transcriptional response of Arabidopsis seedlings during spaceflight reveals peroxidase and cell wall remodeling genes associated with root hair development.

UNLABELLED: • PREMISE OF THE STUDY: Plants will be an important component of advanced life support systems during space exploration missions. Therefore, understanding their biology in the spacecraft environment will be essential before they can be used for such systems.• METHODS: Seedlings of Arabidopsis thaliana were grown for 2 wk in the Biological Research in Canisters (BRIC) hardware on board the second to the last mission of the space shuttle Discovery (STS-131). Transcript profiles between ground controls and space-grown seedlings were compared using stringent selection criteria.• KEY RESULTS: Expression of transcripts associated with oxidative stress and cell wall remodeling was repressed in microgravity. These downregulated genes were previously shown to be enriched in root hairs consistent with seedling phenotypes observed in space. Mutations in genes that were downregulated in microgravity, including two uncharacterized root hair-expressed class III peroxidase genes (PRX44 and PRX57), led to defective polar root hair growth on Earth. PRX44 and PRX57 mutants had ruptured root hairs, which is a typical phenotype of tip-growing cells with defective cell walls and those subjected to stress.• CONCLUSIONS: Long-term exposure to microgravity negatively impacts tip growth by repressing expression of genes essential for normal root hair development. Whereas changes in peroxidase gene expression leading to reduced root hair growth in space are actin-independent, root hair development modulated by phosphoinositides could be dependent on the actin cytoskeleton. These results have profound implications for plant adaptation to microgravity given the importance of tip growing cells such as root hairs for efficient nutrient capture.

Fluorescent protein-based reporters of the actin cytoskeleton in living plant cells: fluorophore variant, actin binding domain, and promoter considerations.

Genetically encoded filamentous actin (F-actin) reporters designed based on fluorescent protein fusions to F-actin binding domains of actin regulatory proteins have emerged as powerful tools to decipher the role of the actin cytoskeleton in plant growth and development. However, these probes could interfere with the function of endogenous actin binding proteins and in turn impact actin organization and plant growth. We therefore surveyed F-actin labeling and compared organ growth in Arabidopsis thaliana lines expressing a variety of F-actin markers. Here we show that the variant of fluorescent protein, type of actin binding domain, and the promoter that drives reporter expression can influence the quality of F-actin labeling particularly in stable plant lines. For example, older red fluorescent protein (RFP)-based probes such as DsRed2 and mOrange induced more aberrant labeling compared to the newer RFP-based, mCherry, GFP, and GFP-derived fluorophores such as YFP and CFP. Moreover, qualitative and quantitative analyses revealed differences in F-actin organization in seedlings expressing Talin- and Lifeact-based reporters in some cell types compared to the fimbrin actin binding domain 2 (ABD2)-based reporters. Finally, the use of the ubiquitin10 (UBQ10) promoter to drive expression of the GFP-ABD2-GFP probe minimized loss of fluorescence and growth defects observed in the 35S-driven version. Taken together, this study shows that care must be taken in the interpretation of data derived from stable expression of certain F-actin reporters and that using alternative promoters such as UBQ10 can overcome some of the pitfalls that accompany the use of in vivo F-actin probes in plants. © 2014 Wiley Periodicals, Inc.

Fluorescence resonance energy transfer-sensitized emission of yellow cameleon 3.60 reveals root zone-specific calcium signatures in Arabidopsis in response to aluminum and other trivalent cations.

Fluorescence resonance energy transfer-sensitized emission of the yellow cameleon 3.60 was used to study the dynamics of cytoplasmic calcium ([Ca(2+)](cyt)) in different zones of living Arabidopsis (Arabidopsis thaliana) roots. Transient elevations of [Ca(2+)](cyt) were observed in response to glutamic acid (Glu), ATP, and aluminum (Al(3+)). Each chemical induced a [Ca(2+)](cyt) signature that differed among the three treatments in regard to the onset, duration, and shape of the response. Glu and ATP triggered patterns of [Ca(2+)](cyt) increases that were similar among the different root zones, whereas Al(3+) evoked [Ca(2+)](cyt) transients that had monophasic and biphasic shapes, most notably in the root transition zone. The Al(3+)-induced [Ca(2+)](cyt) increases generally started in the maturation zone and propagated toward the cap, while the earliest [Ca(2+)](cyt) response after Glu or ATP treatment occurred in an area that encompassed the meristem and elongation zone. The biphasic [Ca(2+)](cyt) signature resulting from Al(3+) treatment originated mostly from cortical cells located at 300 to 500 mu m from the root tip, which could be triggered in part through ligand-gated Glu receptors. Lanthanum and gadolinium, cations commonly used as Ca(2+) channel blockers, elicited [Ca(2+)](cyt) responses similar to those induced by Al(3+). The trivalent ion-induced [Ca(2+)](cyt) signatures in roots of an Al(3+)-resistant and an Al(3+)-sensitive mutant were similar to those of wild-type plants, indicating that the early [Ca(2+)](cyt) changes we report here may not be tightly linked to Al(3+) toxicity but rather to a general response to trivalent cations.

A class I ADP-ribosylation factor GTPase-activating protein is critical for maintaining directional root hair growth in Arabidopsis.

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.