Arabidopsis Qa-SNARE SYP2 proteins localized to different subcellular regions function redundantly in vacuolar protein sorting and plant development

SYP2 proteins are a sub-family of Qa-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) that may be responsible for protein trafficking between pre-vacuolar compartments (PVC) and vacuoles. Arabidopsis thaliana SYP22/VAM3/SGR3 and SYP21/PEP12 proteins function independently, but are both reported to be essential for male gametophytic viability. Here, we systematically examined the redundancy of three SYP2 paralogs (i.e. SYP21, 22 and 23) using a Col-0 ecotype harboring a SYP2 paralog (SYP23/PLP) that lacked a transmembrane domain. Surprisingly, no visible phenotypes were observed, even in the double knockout syp21/pep12 syp23/plp. Deficiency of either SYP21/PEP12 or SYP23/PLP in the syp22 background resulted in a defect in vacuolar protein sorting, characterized by abnormal accumulation of protein precursors in seeds. SYP21/PEP12 knockdown enhanced the syp22 phenotype (i.e. semi-dwarfism, poor leaf vein development and abnormal development of myrosin cells), and additional knockout of SYP23/PLP further aggravated the phenotype. A GFP-SYP23/PLP fusion localized to the cytosol, but not to the PVC or vacuolar membrane, where SYP21/PEP12 or SYP22/VAM3, respectively, were localized. Immunoprecipitation analysis showed that SYP23/PLP interacted with the vacuolar Qb- and Qc-SNAREs, VTI11 and SYP5, respectively, suggesting that SYP23/PLP is able to form a SNARE complex anchoring the membrane. Unexpectedly, we found that expression of multiple copies of a genomic fragment of SYP23/PLP suppressed the abnormal syp22-3 phenotype. Thus, SYP2 proteins, including cytosolic SYP23/PLP, appear to function redundantly in vacuolar trafficking and plant development.     

Direct interaction of actin filaments with F‐BAR protein pacsin2

Two mechanisms have emerged as major regulators of membrane shape: BAR domain‐containing proteins, which induce invaginations and protrusions, and nuclear promoting factors, which cause generation of branched actin filaments that exert mechanical forces on membranes. While a large body of information exists on interactions of BAR proteins with membranes and regulatory proteins of the cytoskeleton, little is known about connections between these two processes. Here, we show that the F‐BAR domain protein pacsin2 is able to associate with actin filaments using the same concave surface employed to bind to membranes, while some other tested N‐BAR and F‐BAR proteins (endophilin, CIP4 and FCHO2) do not associate with actin. This finding reveals a new level of complexity in membrane remodeling processes.     

Non-specific phospholipases C2 and C6 redundantly function in pollen tube growth via triacylglycerol production in Arabidopsis

Non-specific phospholipase Cs (NPCs) are responsible for membrane lipid remodeling that involves hydrolysis of the polar head group of membrane phospholipids. Arabidopsis NPC2 and NPC6 are essential in gametogenesis, but their underlying role in the lipid remodeling remains elusive. Here, we show that these NPCs are required for triacylglycerol (TAG) production in pollen tube growth. NPC2 and NPC6 are highly expressed in developing pollen tubes and are localized at the endoplasmic reticulum. Mutants of NPC2 and NPC6 showed reduced rate of pollen germination, length of pollen tube and amount of lipid droplets (LDs). Overexpression of NPC2 or NPC6 induced LD accumulation, which suggests that these NPCs are involved in LD production. Furthermore, mutants defective in the biosynthesis of TAG, a major component of LDs, showed defective pollen tube growth. These results suggest that NPC2 and NPC6 are essential in gametogenesis for a role in hydrolyzing phospholipids and producing TAG required for pollen tube growth. Thus, lipid remodeling from phospholipids to TAG during pollen tube growth represents an emerging role for the NPC family in plant developmental control.     

Quantification and cluster analysis of actin cytoskeletal structures in plant cells: role of actin bundling in stomatal movement during diurnal cycles in Arabidopsis guard cells

Manual evaluation of cellular structures is a popular approach in cell biological studies. However, such approaches are laborious and are prone to error, especially when large quantities of image data need to be analyzed. Here, we introduce an image analysis framework that overcomes these limitations by semi-automatic quantification and clustering of cytoskeletal structures. In our framework, cytoskeletal orientation, bundling and density are quantified by measurement of newly-developed, robust metric parameters from microscopic images. Thereafter, the microscopic images are classified without supervision by clustering based on the metric patterns. Clustering allows us to collectively investigate the large number of cytoskeletal structure images without laborious inspection. Application of this framework to images of GFP-actin binding domain 2 (GFP-ABD2)-labeled actin cytoskeletons in Arabidopsis guard cells determined that microfilaments (MFs) are radially oriented and transiently bundled in the process of diurnal stomatal opening. The framework also revealed that the expression of mouse talin GFP-ABD (GFP-mTn) continuously induced MF bundling and suppressed the diurnal patterns of stomatal opening, suggesting that changes in the level of MF bundling are crucial for promoting stomatal opening. These results clearly demonstrate the utility of our image analysis framework.     

The reorganization of actin filaments is required for vacuolar fusion of guard cells during stomatal opening in Arabidopsis

The reorganization of actin filaments (AFs) and vacuoles in guard cells is involved in the regulation of stomatal movement. However, it remains unclear whether there is any interaction between the reorganization of AFs and vacuolar changes during stomatal movement. Here, we report the relationship between the reorganization of AFs and vacuolar fusion revealed in pharmacological experiments, and characterizing stomatal opening in actin‐related protein 2 (arp2) and arp3 mutants. Our results show that cytochalasin‐D‐induced depolymerization or phalloidin‐induced stabilization of AFs leads to an increase in small unfused vacuoles during stomatal opening in wild‐type (WT) Arabidopsis plants. Light‐induced stomatal opening is retarded and vacuolar fusion in guard cells is impaired in the mutants, in which the reorganization and the dynamic parameters of AFs are aberrant compared with those of the WT. In WT, AFs tightly surround the small separated vacuoles, forming a ring that encircles the boundary membranes of vacuoles partly fused during stomatal opening. In contrast, in the mutants, most AFs and actin patches accumulate abnormally around the nuclei of the guard cells, which probably further impair vacuolar fusion and retard stomatal opening. Our results suggest that the reorganization of AFs regulates vacuolar fusion in guard cells during stomatal opening.     

Monomeric G‐actin is uniformly distributed in pollen tubes and is rapidly redistributed via cytoplasmic streaming during pollen tube growth

Dynamic assembly and disassembly of the actin cytoskeleton has been implicated in the regulation of pollen germination and subsequent tube growth. It is widely accepted that actin filaments are arrayed into distinct structures within different regions of the pollen tube. Maintenance of the equilibrium between monomeric globular actin (G‐actin) and filamentous actin (F‐actin) is crucial for actin assembly and array construction, and the local concentration of G‐actin thus directly impacts actin assembly. The localization and dynamics of G‐actin in the pollen tube, however, remain to be determined conclusively. To address this question, we created a series of fusion proteins between green fluorescent protein (GFP) and the Arabidopsis reproductive actin ACT11. Expression of a fusion protein with GFP inserted after methionine at position 49 within the DNase I‐binding loop of ACT11 (GFP_Met49–ACT11) rescued the phenotypes in act11 mutants. Consistent with the notion that the majority of actin is in its monomeric form, GFP_Met49–ACT11 and GFP fusion proteins of four other reproductive actins generated with the same strategy do not obviously label filamentous structures. In further support of the functionality of these fusion proteins, we found that they can be incorporated into filamentous structures in jasplakinolide (Jasp)‐treated pollen tubes. Careful observations showed that G‐actin is distributed uniformly in the pollen tube and is rapidly redistributed via cytoplasmic streaming during pollen tube growth. Our study suggests that G‐actin is readily available in the cytoplasm to support continuous actin polymerization during rapid pollen tube growth.     

Improved imaging of actin filaments in transgenic Arabidopsis plants expressing a green fluorescent protein fusion to the C- and N-termini of the fimbrin actin-binding domain 2

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.     

拟南芥ABA不敏感型突变体及其ABA结合特性

拟南芥的脱落醚（ABA）不敏感型突变体abi2,在对ABA的敏感性、气孔开度及种子休眠方面,与野生型有明显差异。通过3H－ABA与野生型对的亚细胞组分的结合分析,表明38000×g组分特异结合活性最高,结合最适温度为20℃,最适保温时间：20℃时为70min;0℃时为90min。由饱和曲线的Scatchard分析表明：abi2存在一种ABA结合位点,野生型有两种ABA结合位点。对3H－ABA结合的38000×g组分的SDS－PAGE电泳分析表明：野生型有3个结合活性峰,而abi2只有1个结合活性峰。     

Metal binding affinity and structural properties of calmodulin‐like protein 14 from Arabidopsis thaliana

In addition to the well‐known Ca²⁺ sensor calmodulin, plants possess many calmodulin‐like proteins (CMLs) that are predicted to have specific roles in the cell. Herein, we described the biochemical and biophysical characterization of recombinant Arabidopsis thaliana CML14. We applied isothermal titration calorimetry to analyze the energetics of Ca²⁺ and Mg²⁺ binding to CML14, and nuclear magnetic resonance spectroscopy, together with intrinsic and ANS‐based fluorescence, to evaluate the structural effects of metal binding and metal‐induced conformational changes. Furthermore, differential scanning calorimetry and limited proteolysis were used to characterize protein thermal and local stability. Our data demonstrate that CML14 binds one Ca²⁺ ion with micromolar affinity (K_d ～ 12 µM) and the presence of 10 mM Mg²⁺ decreases the Ca²⁺ affinity by ～5‐fold. Although binding of Ca²⁺ to CML14 increases protein stability, it does not result in a more hydrophobic protein surface and does not induce the large conformational rearrangement typical of Ca²⁺ sensors, but causes only localized structural changes in the unique functional EF‐hand. Our data, together with a molecular modelling prediction, provide interesting insights into the biochemical properties of Arabidopsis CML14 and may be useful to direct additional studies aimed at understanding its physiological role.     

A lysine-rich cluster in the N-BAR domain of ARF GTPase-activating protein ASAP1 is necessary for binding and bundling actin filaments

Actin filament maintenance is critical for both normal cell homeostasis and events associated with malignant transformation. The ADP-ribosylation factor GTPase-activating protein ASAP1 regulates the dynamics of filamentous actin-based structures, including stress fibers, focal adhesions, and circular dorsal ruffles. Here, we have examined the molecular basis for ASAP1 association with actin. Using a combination of structural modeling, mutagenesis, and in vitro and cell-based assays, we identify a putative-binding interface between the N-Bin-Amphiphysin-Rvs (BAR) domain of ASAP1 and actin filaments. We found that neutralization of charges and charge reversal at positions 75, 76, and 79 of ASAP1 reduced the binding of ASAP1 BAR-pleckstrin homology tandem to actin filaments and abrogated actin bundle formation in vitro. In addition, overexpression of actin-binding defective ASAP1 BAR-pleckstrin homology [K75, K76, K79] mutants prevented cellular actin remodeling in U2OS cells. Exogenous expression of [K75E, K76E, K79E] mutant of full-length ASAP1 did not rescue the reduction of cellular actin fibers consequent to knockdown of endogenous ASAP1. Taken together, our results support the hypothesis that the lysine-rich cluster in the N-BAR domain of ASAP1 is important for regulating actin filament organization.     

Phosphorylated Glycans Produced from Nonreducing Mono- and Oligosaccharides by the Action of P2O5 in Dimethyl Sulfoxide,and Their Interactions with Concanavalin A

The action of P₂O₅ in DMSO on methyl α-d-glucopyranoside, sucrose and trehalose afforded nondializable, phosphorylated glycans in 6~34% yields. Polysucrose has a molecular weight of ~9,500. The synthetic glycans consist of carbohydrate (46~59%) and P (11.4~13.1%) and show reducing sugar values (5.0~30.8%). The alkaline hydrolysis of polysucrose was accompanied with a depolymerization and afforded sugar phosphates and oligosaccharides. The periodate oxidation gave formic acid (0.15~0.34 mole) and formaldehyde (0.07~0.17 mole/monosaccharide residue). The methylation study indicated their variously branched structures. 2,3,4,6-Tetra-O-methyl-d-glucose was found in only 0.7 ~3.2% yields, and this is in agreement with their weak precipitation reactions with Con A. It is considered that the glycans are produced from nonreducing mono- and oligosaccharides by dehydration, transglycosidation and esterification with phosphate.     

拟南芥雄性不育突变体ms1142的遗传定位与功能分析

经EMS诱变野生型拟南芥(Arabidopsis thaliana)群体筛选得到一株雄性不育突变体ms1142, 突变体的果荚短小, 不含种子。细胞学观察和扫描电镜结果表明, 突变体花药发育过程中, 花药中小孢子外壁异常、破裂, 最后没有花粉形成。遗传分析表明, 该突变体为隐性单核基因突变所致; 利用图位克隆的方法将MS1142基因定位于第1条染色体的BAC克隆F16P17上44 kb区间内, 目前尚未见该区间内有雄性不育基因的报道。以上结果结合生物信息学分析表明, MS1142是一个新的调控花药发育的关键基因。该工作为花药发育关键基因MS1142的克隆及功能分析奠定了基础。     

Molecular and biochemical characterization of a novel actin bundling protein in Acanthamoeba

Actin binding proteins play key roles in cell structure and movement particularly as regulators of the assembly, stability and localization of actin filaments in the cytoplasm. In the present study, a cDNA clone encoding an actin bundling protein named as AhABP was isolated from Acanthamoeba healyi, a causative agent of granulomatous amebic encephalitis. This clone exhibited high similarity with genes of Physarum polycephalum and Dictyostelium discoideum, which encode actin bundling proteins. Domain search analysis revealed the presence of essential conserved regions, i.e., an active actin binding site and 2 putative calcium binding EF-hands. Transfected amoeba cells demonstrated that AhABP is primarily localized in phagocytic cups, peripheral edges, pseudopods, and in cortical cytoplasm where actins are most abundant. Moreover, AhABP after the deletion of essential regions formed ellipsoidal inclusions within transfected cells. High-speed co-sedimentation assays revealed that AhABP directly interacted with actin in the presence of up to 10 microM of calcium. Under the electron microscope, thick parallel bundles were formed by full length AhABP, in contrast to the thin actin bundles formed by constructs with deletion sites. In the light of these results, we conclude that AhABP is a novel actin bundling protein that is importantly associated with actin filaments in the cytoplasm.     

Altered resource allocation during seed development in Arabidopsis caused by the abi3 mutation

The regulation of whole-plant resource allocation during seed development in Arabidopsis thaliana was investigated by examining growth rate and partitioning of ¹⁴CO₂ in wild-type plants and those carrying the abi3 mutation. Plants carrying the abi3 mutation partitioned more resources into seed development than the wild type. The extra resources were available as a result of delayed senescence of the cauline leaves in the mutant. After supply of ¹⁴CO₂ at later stages of reproductive development differences in patterns of ¹⁴C distribution between mutant and wild type were consistent with long-term changes in growth and allocation. The role of long-distance signals in the regulation of seed yield in Arabidopsis is discussed.     

Stochastic dynamics of actin filaments in guard cells regulating chloroplast localization during stomatal movement

Actin filaments and chloroplasts in guard cells play roles in stomatal function. However, detailed actin dynamics vary, and the roles that they play in chloroplast localization during stomatal movement remain to be determined. We examined the dynamics of actin filaments and chloroplast localization in transgenic tobacco expressing green fluorescent protein (GFP)-mouse talin in guard cells by time-lapse imaging. Actin filaments showed sliding, bundling and branching dynamics in moving guard cells. During stomatal movement, long filaments can be severed into small fragments, which can form longer filaments by end-joining activities. With chloroplast movement, actin filaments near chloroplasts showed severing and elongation activity in guard cells during stomatal movement. Cytochalasin B treatment abolished elongation, bundling and branching activities of actin filaments in guard cells, and these changes of actin filaments, and as a result, more chloroplasts were localized at the centre of guard cells. However, chloroplast turning to avoid high light, and sliding of actin fragments near the chloroplast, was unaffected following cytochalasin B treatment in guard cells. We suggest that the sliding dynamics of actin may play roles in chloroplast turning in guard cells. Our results indicate that the stochastic dynamics of actin filaments in guard cells regulate chloroplast localization during stomatal movement.