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Construction of spr2 mutants using CRISPR/Cas9-based gene editing. (A) Schematic outline of CRISPR-mediated mutagenesis of SPR2a, b, c, d genes. Imperfect repair of CRISPR-induced DSBs produces indels, where non-triple basepair indels result in frameshift mutations. (B) Genomic SPR2 sequences before and after CRISPR-mediated mutation of two individual mutant lines used in this study, #7 and #21-2. Blue line, gRNA target sequence. (C) Gametophore and rhizoid of control and spr2 mutants were not dramatically different, except for a consistent undulation in the gametophore edge of spr2 mutants (inset). Dotted line, inset outline. Bars, 1 mm (left) and 0.5 mm (right).
Source publication
Stabilisation of minus ends of microtubules (MTs) is critical for organising MT networks in land plant cells, in which all MTs are nucleated independent of centrosomes. Recently, Arabidopsis SPIRAL2 (SPR2) protein was shown to localise to plus and minus ends of cortical MTs, and increase stability of both ends. Here, we report molecular and functio...
Contexts in source publication
Context 1
... generated a loss-of-function mutant of SPR2 by means of CRISPR/Cas9-mediated gene editing ( Lopez-Obando et al., 2016) in a line stably expressing mCherry-tubulin and MT plus end-tracking EB1-Citrine (Fig. 4A). We isolated two lines, in which small indels caused frameshifts to all four SPR2 genes (lines #7 and #21-2) (Fig. 4B). Both lines developed protonemata (filamentous cells that extend from germinated spores by serial division of their apical cells), gametophores (leafy shoot-like structures that house game- tangia differentiated off protonemata), and rhizoids (root- like filamentous cells differentiated from the base of gametophores) (Cove, 2005;Kofuji and Hasebe, 2014;Menand et al., 2007). Despite SPR2 being expressed in all moss organs (Ortiz-Ramirez et al., 2016), the protonema and rhizoid of spr2 mutants were not observably different from control. However, we observed undulation in the gametophore edge of spr2 mutants, suggesting that SPR2 plays a role in gametophore development (Fig. ...
Context 2
... generated a loss-of-function mutant of SPR2 by means of CRISPR/Cas9-mediated gene editing ( Lopez-Obando et al., 2016) in a line stably expressing mCherry-tubulin and MT plus end-tracking EB1-Citrine (Fig. 4A). We isolated two lines, in which small indels caused frameshifts to all four SPR2 genes (lines #7 and #21-2) (Fig. 4B). Both lines developed protonemata (filamentous cells that extend from germinated spores by serial division of their apical cells), gametophores (leafy shoot-like structures that house game- tangia differentiated off protonemata), and rhizoids (root- like filamentous cells differentiated from the base of gametophores) (Cove, 2005;Kofuji and Hasebe, 2014;Menand et al., 2007). Despite SPR2 being expressed in all moss organs (Ortiz-Ramirez et al., 2016), the protonema and rhizoid of spr2 mutants were not observably different from control. However, we observed undulation in the gametophore edge of spr2 mutants, suggesting that SPR2 plays a role in gametophore development (Fig. ...
Context 3
... generated a loss-of-function mutant of SPR2 by means of CRISPR/Cas9-mediated gene editing ( Lopez-Obando et al., 2016) in a line stably expressing mCherry-tubulin and MT plus end-tracking EB1-Citrine (Fig. 4A). We isolated two lines, in which small indels caused frameshifts to all four SPR2 genes (lines #7 and #21-2) (Fig. 4B). Both lines developed protonemata (filamentous cells that extend from germinated spores by serial division of their apical cells), gametophores (leafy shoot-like structures that house game- tangia differentiated off protonemata), and rhizoids (root- like filamentous cells differentiated from the base of gametophores) (Cove, 2005;Kofuji and Hasebe, 2014;Menand et al., 2007). Despite SPR2 being expressed in all moss organs (Ortiz-Ramirez et al., 2016), the protonema and rhizoid of spr2 mutants were not observably different from control. However, we observed undulation in the gametophore edge of spr2 mutants, suggesting that SPR2 plays a role in gametophore development (Fig. ...
Citations
... The orthogonal positioning of microtubules yields microtubule intersections termed crossover sites. The microtubule severing enzyme, katanin, is recruited to nucleation and crossover sites, where the nascent/longitudinally-oriented microtubule is severed, and its minus end stabilized by the protein SPIRAL2 (SPR2) [7][8][9][10][11]. The preferential severing and minus-end stabilization of longitudinal microtubules leads to their polymerization and amplification over the parental lateral array. ...
... Initial investigations demonstrated that SPR2 colocalizes with cortical microtubules, has in vitro microtubule binding activity, affects microtubule dynamics and microtubule array reorientation, and modulates microtubule severing [13][14][15][16]. Subsequent investigations found that SPR2 family members bind and stabilize the microtubule minus end, both in vivo and when examined using in vitro microtubule dynamics reconstitution assays [9][10][11]. In metazoans, CAMSAP protein family members bind and regulate microtubule minus ends using a CKK domain [17][18][19][20][21]. Higher plants lack CAMSAP proteins, but have members of the plant-specific SPR2 family [22]. ...
... Katanin is recruited to microtubule crossover sites, where it severs microtubules oriented in the longitudinal array, thereby amplifying the number of microtubules in the longitudinal array [7]. SPR2 recognizes and stabilizes microtubule minus ends, which is critical to prevent depolymerization of the longitudinal array [9][10][11]. How katanin is recruited to microtubule crossover sites and specifically cleaves the longitudinally-oriented microtubule remains to be fully determined [8], as is the mechanism by which SPR2 specifically binds and stabilizes the microtubule minus end. ...
Epidermal cells of dark-grown plant seedlings reorient their cortical microtubule arrays in response to blue light from a net lateral orientation to a net longitudinal orientation with respect to the long axis of cells. The molecular mechanism underlying this microtubule array reorientation involves katanin, a microtubule severing enzyme, and a plant-specific microtubule associated protein called SPIRAL2. Katanin preferentially severs longitudinal microtubules, generating seeds that amplify the longitudinal array. Upon severing, SPIRAL2 binds nascent microtubule minus ends and limits their dynamics, thereby stabilizing the longitudinal array while the lateral array undergoes net depolymerization. To date, no experimental structural information is available for SPIRAL2 to help inform its mechanism. To gain insight into SPIRAL2 structure and function, we determined a 1.8 Å resolution crystal structure of the Arabidopsis thaliana SPIRAL2 C-terminal domain. The domain is composed of seven core α-helices, arranged in an α-solenoid. Amino-acid sequence conservation maps primarily to one face of the domain involving helices α1, α3, α5, and an extended loop, the α6-α7 loop. The domain fold is similar to, yet structurally distinct from the C-terminal domain of Ge-1 (an mRNA decapping complex factor involved in P-body localization) and, surprisingly, the C-terminal domain of the katanin p80 regulatory subunit. The katanin p80 C-terminal domain heterodimerizes with the MIT domain of the katanin p60 catalytic subunit, and in metazoans, binds the microtubule minus-end factors CAMSAP3 and ASPM. Structural analysis predicts that SPIRAL2 does not engage katanin p60 in a mode homologous to katanin p80. The SPIRAL2 structure highlights an interesting evolutionary convergence of domain architecture and microtubule minus-end localization between SPIRAL2 and katanin complexes, and establishes a foundation upon which structure-function analysis can be conducted to elucidate the role of this domain in the regulation of plant microtubule arrays.
... Plants lack CAMSAP/Patronin/Nezha proteins but instead have SPIRAL2 (SPR2). SPR2 tracks and stabilizes the microtubule minus-ends by reducing the depolymerization rate, thereby facilitating light-induced reorientation of the microtubule arrays (Fan et al. 2018, Leong et al. 2018, Nakamura et al. 2018). However, the structural features of SPR2 that determine its regulatory role at the microtubule minus-end remained unknown. ...
... The molecular mechanism of plant twisted growth has been gradually revealed in the past two decades. The majority of plant organ twisted growth is associated with microtubule changes, and mutations in microtubulerelated genes cause twisted growth on the left or right hand [14][15][16][17][18][19][20][21][22][23][24][25][26][27][28]. The SPIRAL1 and SPIRAL2 genes can cause twisted growth in Arabidopsis by affecting microtubules [14]. ...
... The changes in microtubule composition and mutations in microtubule-associated proteins can lead to twisted growth of plant organs [29]. IQD family members of microtubule-associated proteins, as different signal integrators, can connect auxin signals to the downstream microtubule regulator SPR2, while IQD proteins bind to SPR2, thus inhibiting the function of microtubule end stabilization [26,27,32]. In this study, the expression of microtubule-related gene IQD1 was significantly downregulated in the twisted branch of 'Dongzao' mutant. ...
Background
Plant organs grow in a certain direction and organ twisted growth, a rare and distinctive trait, is associated with internal structure changes and special genes. The twisted branch mutant of Chinese jujube jujube, an important fruit tree native to China and introduced to nearly 50 countries, provides new typical materials for exploration of plant twisted growth.
Results
In this study, the cytological characteristics and related genes of twisted branches in Chinese jujube were revealed by microscopy observation and transcriptome analysis. The unique coexistence of primary and secondary structures appeared in the twisted parts of branches, and special structures such as collateral bundle, cortical bundles, and internal phloem were formed. Ninety differentially expressed genes of ‘Dongzao’ and its twisted mutant were observed, in which ZjTBL43, ZjFLA11, ZjFLA12 and ZjIQD1 were selected as candidate genes. ZjTBL43 was homologous to AtTBL43 in Arabidopsis, which was involved in the synthesis and deposition of cellular secondary wall cellulose. The attbl43 mutant showed significant inflorescence stem bending growth. The transgenic lines of attbl43 with overexpression of ZjTBL43 were phenotypically normal.The branch twisted growth may be caused by mutations in ZjTBL43 in Chinese jujube. AtIQD10, AtFLA11 and AtFLA12 were homologous to ZjIQD1, ZjFLA11 and ZjFLA12, respectively. However, the phenotype of their function defect mutants was normal.
Conclusion
In summary, these findings will provide new insights into the plant organ twisted growth and a reference for investigation of controlling mechanisms of plant growth direction.
... Whereas SPR2 is strongly expressed in various tissues, its close homolog SP2L is weakly expressed but relatively abundant in the hydathodes and roots (Yao et al., 2008). SPR2 binds MT plus and minus ends and MT intersections (Fan et al., 2018;Leong et al., 2018;Nakamura et al., 2018;Wightman et al., 2013) and is uniquely tracking with and stabilizing the treadmilling MT minus ends in land plants (Fan et al., 2018;Leong et al., 2018;Nakamura et al., 2018). SP2L and SPR2 have overlapping functions and regulate MT-end dynamics and anisotropic organ growth (Buschmann et al., 2004;Shoji et al., 2004;Yao et al., 2008). ...
... Whereas SPR2 is strongly expressed in various tissues, its close homolog SP2L is weakly expressed but relatively abundant in the hydathodes and roots (Yao et al., 2008). SPR2 binds MT plus and minus ends and MT intersections (Fan et al., 2018;Leong et al., 2018;Nakamura et al., 2018;Wightman et al., 2013) and is uniquely tracking with and stabilizing the treadmilling MT minus ends in land plants (Fan et al., 2018;Leong et al., 2018;Nakamura et al., 2018). SP2L and SPR2 have overlapping functions and regulate MT-end dynamics and anisotropic organ growth (Buschmann et al., 2004;Shoji et al., 2004;Yao et al., 2008). ...
Plants have evolved signaling mechanisms that guide growth away from adverse environments that can cause yield losses. Root halotropism is a sodium-specific negative tropism that is crucial for surviving and thriving under high salinity. Although root halotropism was discovered some years ago, the underlying molecular and cellular mechanisms remain unknown. Here, we show that abscisic acid (ABA)-mediated root twisting determines halotropism in Arabidopsis. An ABA-activated SnRK2 protein kinase (SnRK2.6) phosphorylates the microtubule-associated protein SP2L at Ser406, which induces a change in the anisotropic cell expansion at the root transition zone and is required for root twisting during halotropism. Salt stress triggers SP2L-mediated cortical microtubule reorientation, which guides cellulose microfibril patterns. Our findings thus outline the molecular mechanism of root halotropism and indicate that anisotropic cell expansion through microtubule reorientation and microfibril deposition has a central role in mediating tropic responses.
... The similarity in amino acid sequences suggested that TPX2-1 to −4 have redundant functions. Therefore, we simultaneously targeted these genes using a previously established CRISPR/Cas9 protocol 23 . We isolated a line, named TPX2 1-4Δ, in which frameshifts were introduced to all four TPX2 genes in the exons present in all transcript variants identified in the Phytozome database (Supplementary Fig. 1a). ...
... A detailed protocol for endogenous gene tagging and knockouts in P. patens has been previously published 17 . The CRISPR protocol has been described in detail in 23 . In brief, CRISPR gRNAs targeting one of the exons were designed using the online tool, CRISPOR (http://crispor.tefor.net/), ...
Asymmetric cell division (ACD) underlies the development of multicellular organisms. In animal ACD, the cell division site is determined by active spindle-positioning mechanisms. In contrast, it is considered that the division site in plants is determined prior to mitosis by the microtubule-actin belt known as the preprophase band (PPB) and that the localization of the mitotic spindle is typically static and does not govern the division plane. However, in some plant species, ACD occurs in the absence of PPB. Here, we isolate a hypomorphic mutant of the conserved microtubule-associated protein TPX2 in the moss Physcomitrium patens (Physcomitrella) and observe spindle motility during PPB-independent cell division. This defect compromises the position of the division site and produces inverted daughter cell sizes in the first ACD of gametophore (leafy shoot) development. The phenotype is rescued by restoring endogenous TPX2 function and, unexpectedly, by depolymerizing actin filaments. Thus, we identify an active spindle-positioning mechanism that, reminiscent of acentrosomal ACD in animals, involves microtubules and actin filaments, and sets the division site in plants.
... Microtubule severing probability at crossovers is influenced by factors other than katanin activities. SPIRAL2 is a minus-end tracking and stabilising protein (Fan et al., 2018;Leong et al., 2018;Nakamura et al., 2018). Suppressed minus-end depolymerisation of crossing microtubules increases lifetime of microtubule crossovers, resulting in a greater opportunity time for severing by katanin (Fan et al., 2018;Nakamura et al., 2018). ...
Microtubule severing by katanin plays key roles in generating various array patterns of dynamic microtubules, while also responding to developmental and environmental stimuli. Quantitative imaging and molecular genetic analyses have uncovered that dysfunction of microtubule severing in plant cells leads to defects in anisotropic growth, division and other cell processes. Katanin is targeted to several subcellular severing sites. Intersections of two crossing cortical microtubules attract katanin, possibly by using local lattice deformation as a landmark. Cortical microtubule nucleation sites on preexisting microtubules are targeted for katanin-mediated severing. An evolutionary conserved microtubule anchoring complex not only stabilises the nucleated site, but also subsequently recruits katanin for timely release of a daughter microtubule. During cytokinesis, phragmoplast microtubules are severed at distal zones by katanin, which is tethered there by plant-specific microtubule-associated proteins. Recruitment and activation of katanin are essential for maintenance and reorganisation of plant microtubule arrays.
... For timelapse microscopy, thalli were severed into < 2 mm pieces in the MSW and treated with Hoechst 33342 at 10 μg/mL (final) for DNA and cell wall staining. After 30 min incubation with the Hoechst dye, the thalli were injected into the microfluidic device which has previously been used for moss imaging [23,24]. In brief, a polydimethylsiloxane (PDMS) device was attached to a glass-bottom dish (dish diameter 35 mm; glass diameter 27 mm; glass thickness 0.16-0.19 ...
Regeneration is a widely observed phenomenon by which the integrity of an organism is recovered after damage. To date, studies on the molecular and cellular mechanisms of regeneration have been limited to a handful of model multicellular organisms. Here, the regeneration ability of marine macroalgae (Rhodophyta, Phaeophyceae, Chlorophyta) was systematically surveyed after thallus severing. Live cell imaging on severed thalli uncovered the cellular response to the damage. Three types of responses–budding, rhizoid formation, and/or sporulation–were observed in 25 species among 66 examined, proving the high potential of regeneration of macroalgae. The cellular and nuclear dynamics were monitored during cell repair or rhizoid formation of four phylogenetically diverged species, and the tip growth of the cells near the damaged site was observed as a common response. Nuclear translocation followed tip growth, enabling overall distribution of multinuclei or central positioning of the mononucleus. In contrast, the control of cell cycle events, such as nuclear division and septation, varied in these species. These observations showed that marine macroalgae utilise a variety of regeneration pathways, with some common features. This study also provides a novel methodology of live cell imaging in macroalgae.
... While 90% of the nucleating MTs had γ-tubulin signals at the minus ends, no signals were identified in the other 10% of wild-type cells. The stability of these MTs was explained by the identification of the plant-specific minus-end binding and stabilizing protein Spiral2 (Leong et al., 2018). Another notable system is the noncentrosomal fat body cell in Drosophila, where γ-tubulin is dispensable for perinuclear MTOC formation, despite being localized at the perinuclear region with the activators (Zheng et al., 2020). ...
The γ-tubulin complex acts as the predominant microtubule (MT) nucleator that initiates MT formation and is therefore an essential factor for cell proliferation. Nonetheless, cellular MTs are formed after experimental depletion of the γ-tubulin complex, suggesting that cells possess other factors that drive MT nucleation. Here, by combining gene knockout, auxin-inducible degron, RNA interference, MT depolymerization/regrowth assay, and live microscopy, we identified four microtubule-associated proteins (MAPs), ch-TOG, CLASP1, CAMSAPs, and TPX2, which are involved in γ-tubulin–independent MT generation in human colon cancer cells. In the mitotic MT regrowth assay, nucleated MTs organized noncentriolar MT organizing centers (ncMTOCs) in the absence of γ-tubulin. Depletion of CLASP1 or TPX2 substantially delayed ncMTOC formation, suggesting that these proteins might promote MT nucleation in the absence of γ-tubulin. In contrast, depletion of ch-TOG or CAMSAPs did not affect the timing of ncMTOC appearance. CLASP1 also accelerates γ-tubulin–independent MT regrowth during interphase. Thus, MT generation can be promoted by MAPs without the γ-tubulin template.
... cytoskeleton or plasma membrane protein dynamics). In a pioneering study, researchers used shallow channels to increase the visible cell surface for HILO imaging (Leong et al. 2018). In a subsequent study, using an optimized channel depth for HILO imaging, cell viability was more stable in the microfluidic device compared to samples under coverslips. ...
Many plant processes occur in the context of and in interaction with a surrounding matrix such as soil (e.g. root growth and root–microbe interactions) or surrounding tissues (e.g. pollen tube growth through the pistil), making it difficult to study them with high-resolution optical microscopy. Over the past decade, microfabrication techniques have been developed to produce experimental systems that allow researchers to examine cell behavior in microstructured environments that mimic geometrical, physical and/or chemical aspects of the natural growth matrices and that cannot be generated using traditional agar plate assays. These microfabricated environments offer considerable design flexibility as well as the transparency required for high-resolution, light-based microscopy. In addition, microfluidic platforms have been used for various types of bioassays, including cellular force assays, chemoattraction assays, and electrotropism assays. Here, we review the recent use of microfluidic devices to study plant cells and organs, including plant roots, root hairs, moss protonemata, and pollen tubes. The increasing adoption of microfabrication techniques by the plant science community may transform our approaches to investigating how individual plant cells sense and respond to changes in the physical and chemical environment.
... In the interphase MT array, Kinesin-13s accumulated at the ends of growing MTs and disappeared from the ends when MTs switched to the shrink phase ( Figure 6B, C, Supplemental Figure 2C, Movie 5). Since MT minus-ends are stabilised and exhibit little to no dynamicity in this cell type ( Leong et al., 2018), we concluded that Kinesin-13 localises to the plusends of growing MTs. The plus-end tracking behaviour is reminiscent of human KIF2C/MCAK and Drosophila KLP10A, which are recruited by EB1 (End Binding 1) protein to growing plus-ends ( Mennella et al., 2005;Lee et al., 2008). ...
... ( Figure S1A). gRNAs were then integrated into a BsaI site of a vector carrying the U6 promoter and RNA scaffold (pCasGuide/pUC18) ( Lopez-Obando et al., 2016;Collonnier et al., 2017), and then the CRISPR cassettes were cloned into a vector carrying nourseothricin resistance (pSY034) with InFusion (Takara) to assemble multiple gRNA cassettes into a plasmid (pSY062) following methods previously described (Leong et al., 2018). Equal amounts of this circular multicassette plasmid and Streptococcus pyogenes Cas9 expression vector (pGenius, (Collonnier et al., 2017)) were transformed into the Kinesin-13b KO/EB1-Citrine/mCherry-tubulin background. ...
... Equal amounts of this circular multicassette plasmid and Streptococcus pyogenes Cas9 expression vector (pGenius, (Collonnier et al., 2017)) were transformed into the Kinesin-13b KO/EB1-Citrine/mCherry-tubulin background. Confirmation of Kinesin-13 and Kinesin-8 KO lines (Figure S1B-D) were carried out by PCR as previously described (Yamada et al., 2016;Leong et al., 2018). ...
Kinesin-13 and -8 are well-known microtubule (MT) depolymerases that regulate MT length and chromosome movement in animal mitosis. While much is unknown about plant Kinesin-8, Arabidopsis thaliana and rice (Oryza sativa) Kinesin-13 have been shown to depolymerise MTs in vitro. However, the mitotic function of both kinesins has yet to be determined in plants. Here, we generated complete null mutants of Kinesin-13 and -8 in the moss Physcomitrella patens. Both kinesins were found to be non-essential for viability, but the Kinesin-13 knockout (KO) line had increased mitotic duration and reduced spindle length, whereas the Kinesin-8 KO line did not display obvious mitotic defects. Surprisingly, spindle MT poleward flux, which is mediated by Kinesin-13 in animals, was retained in the absence of Kinesin-13. MT depolymerase activity was not detectable for either kinesin in vitro, while MT catastrophe inducing (Kinesin-13) or MT gliding (Kinesin-8) activity was observed. Interestingly, both KO lines showed waviness in their protonema filaments, which correlated with positional instability of the MT foci in their tip cells. Taken together, the results suggest that plant Kinesin-13 and -8 have diverged in both mitotic function and molecular activity, acquiring roles in regulating MT foci positioning for directed tip growth.
