Developmental Regulation of Intercellular Protein Trafficking through Plasmodesmata in Tobacco Leaf Epidermis

Plasmodesmata mediate direct cell-to-cell communication in plants. One of their significant features is that primary plasmodesmata formed at the time of cytokinesis often undergo structural modifications, by the de novo addition of cytoplasmic strands across cell walls, to become complex secondary plasmodesmata during plant development. Whether such modifications allow plasmodesmata to gain special transport functions has been an outstanding issue in plant biology. Here we present data showing that the cucumber mosaic virus 3a movement protein (MP):green fluorescent protein (GFP) fusion was not targeted to primary plasmodesmata in the epidermis of young or mature leaves in transgenic tobacco (Nicotiana tabacum) plants constitutively expressing the 3a:GFP fusion gene. Furthermore, the cucumber mosaic virus 3a MP:GFP fusion protein produced in planta by biolistic bombardment of the 3a:GFP fusion gene did not traffic between cells interconnected by primary plasmodesmata in the epidermis of a young leaf. In contrast, the 3a MP:GFP was targeted to complex secondary plasmodesmata and trafficked from cell to cell when a leaf reached a certain developmental stage. These data provide the first experimental evidence, to our knowledge, that primary and complex secondary plasmodesmata have different protein-trafficking functions and suggest that complex secondary plasmodesmata may be formed to traffic specific macromolecules that are important for certain stages of leaf development.     

Studies on Plasmodesmata in the Trichomes and Leaf Epidermis of some Asclepiadaceae

The occurrence of plasmodesmata has been studied in the trichomesand leaf epidermis of 12 species of the Asclepiadaceae. Plasmodesmataare observed in the walls of hair cells, epidermal cells, andstomatal apparatus. Plasmodesmata are present between adjacenthair cells from base to the apex; epidermal and basal hair cells;epidermal cells; epidermal and subsidiary cells; subsidiarycells, and subsidiary and guard cells, thus establishing mutualsymplasmatic contacts.     

Putting the Squeeze on Plasmodesmata: A Role for Reticulons in Primary Plasmodesmata Formation

Primary plasmodesmata (PD) arise at cytokinesis when the new cell plate forms. During this process, fine strands of endoplasmic reticulum (ER) are laid down between enlarging Golgi-derived vesicles to form nascent PD, each pore containing a desmotubule, a membranous rod derived from the cortical ER. Little is known about the forces that model the ER during cell plate formation. Here, we show that members of the reticulon (RTNLB) family of ER-tubulating proteins in Arabidopsis (Arabidopsis thaliana) may play a role in the formation of the desmotubule. RTNLB3 and RTNLB6, two RTNLBs present in the PD proteome, are recruited to the cell plate at late telophase, when primary PD are formed, and remain associated with primary PD in the mature cell wall. Both RTNLBs showed significant colocalization at PD with the viral movement protein of Tobacco mosaic virus, while superresolution imaging (three-dimensional structured illumination microscopy) of primary PD revealed the central desmotubule to be labeled by RTNLB6. Fluorescence recovery after photobleaching studies showed that these RTNLBs are mobile at the edge of the developing cell plate, where new wall materials are being delivered, but significantly less mobile at its center, where PD are forming. A truncated RTNLB3, unable to constrict the ER, was not recruited to the cell plate at cytokinesis. We discuss the potential roles of RTNLBs in desmotubule formation.Plasmodesmata (PD), the small pores that connect higher plant cells, are complex structures of about 50 nm in diameter. Each PD pore is lined by the plasma membrane and contains an axial endoplasmic reticulum (ER)-derived structure known as the desmotubule (Overall and Blackman, 1996; Maule, 2008; Tilsner et al., 2011). The desmotubule is an enigmatic structure whose function has not been fully elucidated. The small spiraling space between the desmotubule and the plasma membrane, known as the cytoplasmic sleeve, is almost certainly a conduit for the movement of small molecules (Oparka et al., 1999). Some reports, however, suggest that the desmotubule may also function in cell-to-cell trafficking, providing an ER-derived pathway between cells along which macromolecules may diffuse (Cantrill et al., 1999). The desmotubule is one of the most tightly constricted membrane structures found in nature (Tilsner et al., 2011), but the forces that generate its intense curvature are not understood. In most PD, the desmotubule is a tightly furled tube of about 15 nm in diameter in which the membranes of the ER are in close contact along its length. The desmotubule may balloon out in the region of the middle lamella into a central cavity, but at the neck regions of the PD pore it is tightly constricted (Robinson-Beers and Evert, 1991; Ding et al., 1992; Glockmann and Kollmann, 1996; Overall and Blackman, 1996; Ehlers and Kollmann, 2001). Studies of PD using GFP targeted to the ER lumen (e.g. GFP-HDEL) have shown that GFP is excluded from the desmotubule due to the constriction of ER membranes in this structure (Oparka et al., 1999; Crawford and Zambryski, 2000; Martens et al., 2006; Guenoune-Gelbart et al., 2008). Therefore, lumenal GFP is unable to move between plant cells unless the membranes of the desmotubule become relaxed in some way. On the other hand, dyes and some proteins inserted into the ER membrane can apparently move through the desmotubule, either along the membrane or through the lumen, at least under some conditions (Grabski et al., 1993; Cantrill et al., 1999; Martens et al., 2006; Guenoune-Gelbart et al., 2008).Recently, a number of proteins have been described in mammalian, yeast, and plant systems that induce extreme membrane curvature. Among these are the RETICULONS (RTNs), integral membrane proteins that induce curvature of the ER to form tubules (Voeltz et al., 2006; Hu et al., 2008; Tolley et al., 2008, 2010; Sparkes et al., 2010). In animals, RTNs have been shown to be involved in a wide array of endomembrane-related processes, including intracellular transport and vesicle formation, and as RTNs can also influence axonal growth, they may have roles in neurodegenerative disorders such as Alzheimer’s disease (Yang and Strittmatter, 2007). Arabidopsis (Arabidopsis thaliana) has 21 RTN homologs, known as RTNLBs (Nziengui et al., 2007; Sparkes et al., 2010), considerably more than in yeast or mammals, but most have not been examined. RTNLBs contain two unusually long hydrophobic helices that form reentrant loops (Voeltz et al., 2006; Hu et al., 2008; Sparkes et al., 2010; Tolley et al., 2010). These are thought to induce membrane curvature by the molecular wedge principle (Hu et al., 2008; Shibata et al., 2009). When RTNLBs are overexpressed transiently in cells expressing GFP-HDEL, the ER becomes tightly constricted and GFP-HDEL is excluded from the lumen of the constricted ER tubules (Tolley et al., 2008, 2010), a situation similar to that which occurs in desmotubules (Oparka et al., 1999; Crawford and Zambryski, 2000; Martens et al., 2006). In vitro studies with isolated membranes have shown that the degree of tubulation is proportional to the number and spacing of RTNLB proteins in the membrane (Hu et al., 2008). For example, to constrict the ER membrane into a structure of 15 nm, the diameter of a desmotubule, would require RTNLBs to be inserted every 2 nm or less along the desmotubule axis (Hu et al., 2008), potentially making the desmotubule an extremely protein-rich structure (Tilney et al., 1991). Interestingly, a number of RTNLB proteins appear in the recently described PD proteome (Fernandez-Calvino et al., 2011), suggesting that RTNLBs are good candidates for proteins that model the cortical ER into desmotubules.Primary PD form at cytokinesis during the assembly of the cell plate (Hawes et al., 1981; Hepler, 1982). Of the numerous studies devoted to the structure of the cell plate, very few have examined the behavior of the ER during cytokinesis. During mitosis, elements of the ER are located in the spindle apparatus, separated from the cytoplasm (Hepler, 1980). Just prior to cytokinesis, there is a relative paucity of ER in the region destined to become the cell plate (Hepler, 1980; Hawes et al., 1981). The studies of Hawes et al. (1981) and Hepler (1982), exploiting heavy-metal impregnation of the ER, showed that during the formation of the new cell plate, strands of cortical ER are inserted across the developing wall, between the Golgi-derived vesicles that deposit wall materials. These ER strands become increasingly thinner during formation of the desmotubule, eventually excluding heavy metal stains from the ER lumen (Hepler, 1982). The center of the desmotubule often appears electron opaque in transmission electron microscopy images and has been referred to as the central rod (Overall and Blackman, 1996). This structure may consist of proteins that extend from the inner ER leaflets or may correspond to head groups of the membrane lipids themselves. In the fully formed primary PD, the desmotubule remains continuous with the cortical ER that runs close to the new cell wall (Hawes et al., 1981; Hepler, 1982; Oparka et al., 1994).Here, we show that two of the RTNLBs present in the PD proteome, RTNLB3 and RTNLB6, become localized to the cell plate during the formation of primary PD. These RTNLBs remain associated with the desmotubule in fully formed PD and are immobile, as evidenced by fluorescence recovery after photobleaching (FRAP) studies. A truncated version of RTNLB3, in which the second hydrophobic region was deleted (Sparkes et al., 2010), was not recruited to the cell plate at cytokinesis. We suggest that RTNLBs play an important role in the formation of primary PD and discuss mechanisms by which these proteins may model the ER into desmotubules.     

Pattern Formation in the Arabidopsis Embryo Revealed by Position-Specific Lipid Transfer Protein Gene Expression

During Arabidopsis embryogenesis, the zygote divides asymmetrically in the future apical-basal axis; however, a radial axis is initiated only within the eight-celled embryo. Mutations in the GNOM, KNOLLE, and KEULE genes affect these processes: gnom zygotes tend to divide symmetrically; knolle embryos lack oriented cell divisions that initiate protoderm formation; and in keule embryos, an outer cell layer is present that consists of abnormally enlarged cells from early development. Pattern formation along the two axes is reflected by the position-specific expression of the Arabidopsis lipid transfer protein (AtLTP1) gene. In wild-type embryos, the AtLTP1 gene is expressed in the protoderm and initially in all protodermal cells; later, AtLTP1 expression is confined to the cotyledons and the upper end of the hypocotyl. Analysis of AtLTP1 expression in gnom, knolle, and keule embryos showed that gnom embryos also can have no or reversed apical-basal polarity, whereas radial polarity is unaffected. knolle embryos initially lack but eventually form a radial pattern, and keule embryos are affected in protoderm cell morphology rather than in the establishment of the radial pattern.     

Preparation of Leaf Epidermis for Topographic Study

Epidermal strips of leaves of the Gramineae can be prepared using the following technique: The mature leaf is dipped in boiling water to kill the cells, and decolorized in boiling 70% alcohol. It is cleared and softened in 88% lactic acid. Epidermal, mesophyll and vascular tissue is removed from a selected constant area of the leaf leaving an epidermal strip 1-3 cm in length. This is inverted on a slide, stained in lactopheno-cotton blue, and destained in 88% lactic acid. Transverse and longitudinal sections of the strip are obtained at this stage. The epidermal strip is finally mounted on a slide in 88% lactic acid. The preparation is photographed with a 35 mm camera using transmitted light, and a yellow filter in the microscope lamp. Photomicrographs of known enlargement are then prepared from which accurate measurements can be recorded. The technique is applicable to both fresh and herbarium material.     

Spatiotemporal Developmental Trajectories in the Arabidopsis Root Revealed Using High-Throughput Single-Cell RNA Sequencing

1. Download high-res image (327KB)
2. Download full-size image

     

一种用透明胶带粘取叶片表皮观察气孔的方法

用普通塑料透明胶带粘取叶片下表皮观察植物的气孔,比印迹法和撕取下表皮观察气孔的方法具有操作简单、速度快、真实性强的优点,尤其适宜于试管苗等幼嫩材料的气孔变化动态和形态学指标的研究.此法可以防止因保卫细胞失水而导致气孔开张度的变化,能真实地观察植物不同阶段和时期气孔的变化动态.     

夹竹桃科植物叶表皮特征及其系统学研究

以萝藦科植物匙羹藤为对照,在扫描电镜下观察了传统上分属于夹竹桃科3亚科植物的叶下表皮特征。结果表明:(1)所观察的夹竹桃科25种植物叶下表皮特征具有一定程度的多样性,如少数物种具有表皮毛;部分角质层表面有丝状蜡质;叶下表皮纹饰有条纹状、脊状皱褶增厚、块状突起、花蕊形和较平滑等类型;气孔大小差异较大,气孔外拱盖和内缘形状分浅波状和较平滑2种类型,气孔保卫细胞极区有T型加厚和无T型加厚两种类型,气孔外拱盖与表皮细胞间分凹陷和连续两种类型等,这些叶表皮特征对夹竹桃科植物属内和属间类型的分类以及对疑难类群的划分具有重要的参考价值。(2)夹竹桃科25种植物的绝大部分种的叶表皮气孔类型、气孔外拱盖及其内缘形态基本一致,但叶表皮纹饰、气孔外缘角质层特征呈现出较高的多样性,在属内各种和属间有较大的差异。(3)萝藦科植物匙羹藤与夹竹桃科植物盆架树在气孔形状,叶片气孔外缘角质层特征、气孔外拱盖等叶表皮特征具有高度的相似性,推测这两种植物的亲缘关系可能较近,而且萝藦科与夹竹桃科中一些植物的系统位置需进一步的研究。(4)本研究结果支持仔榄树属应从山橙族中分离出来独立成族,海杧果属应从萝芙木族中独立出来的观点;但不支持新的分类方法中将糖胶树和盆架树同归属于鸡骨常山属的观点。     

一种禾本科植物鲜叶下表皮制片方法

介绍一种禾本科植物鲜叶下表皮制片的快速方法,该方法也能适用于多数单子叶植物鲜叶和经FAA固定液固定的单子叶植物叶片的气孔观察。该方法制片能有效防止叶表皮脱水、透明后发生卷曲,使保持完整的结构,便于准确的显微观察。具操作简单、材料易得、可靠性和稳定性高等优点。     

易卷曲叶表皮制片技术(NaOCl法)的改进

在利用NaOCl法制备用于光学显微镜观察的叶表皮过程中,一些科/属植物的叶表皮在100％乙醇脱水后遇二甲苯便发生卷曲,增加了叶表皮制片的难度,甚至无法进行。本文介绍一种简便易行的方法：在系列乙醇脱水后,加盖小块盖玻片,防止叶表皮脱水后卷曲,并使其保持平整,便于显微摄影。这种方法使得叶表皮显微制片技术更加完善。     

易卷曲叶表皮制片技术（NaOCl法）的改进

在利用NaOCl法制备用于光学显微镜观察的叶表皮过程中,一些科/属植物的叶表皮在100%乙醇脱水后遇二甲苯便发生卷曲,增加了叶表皮制片的难度, 甚至无法进行。本文介绍一种简便易行的方法：在系列乙醇脱水后,加盖小块盖玻片,防止叶表皮脱水后卷曲,并使其保持平整,便于显微摄影。这种方法使得叶表皮显微制片技术更加完善。     

The Hidden Geometries of the Arabidopsis thaliana Epidermis

The quest for the discovery of mathematical principles that underlie biological phenomena is ancient and ongoing. We present a geometric analysis of the complex interdigitated pavement cells in the Arabidopsis thaliana (Col.) adaxial epidermis with a view to discovering some geometric characteristics that may govern the formation of this tissue. More than 2,400 pavement cells from 10, 17 and 24 day old leaves were analyzed. These interdigitated cells revealed a number of geometric properties that remained constant across the three age groups. In particular, the number of digits per cell rarely exceeded 15, irrespective of cell area. Digit numbers per 100 µm² cell area reduce with age and as cell area increases, suggesting early developmental programming of digits. Cell shape proportions as defined by length∶width ratios were highly conserved over time independent of the size and, interestingly, both the mean and the medians were close to the golden ratio 1.618034. With maturity, the cell area∶perimeter ratios increased from a mean of 2.0 to 2.4. Shape properties as defined by the medial axis transform (MAT) were calculated and revealed that branch points along the MAT typically comprise one large and two small angles. These showed consistency across the developmental stages considered here at 140° (± 5°) for the largest angles and 110° (± 5°) for the smaller angles. Voronoi diagram analyses of stomatal center coordinates revealed that giant pavement cells (≥500 µm²) tend to be arranged along Voronoi boundaries suggesting that they could function as a scaffold of the epidermis. In addition, we propose that pavement cells have a role in spacing and positioning of the stomata in the growing leaf and that they do so by growing within the limits of a set of ‘geometrical rules’.     

植物高度木质化叶片表皮细胞制备方法

以凤凰茶(Camellia sinensis)叶片为例,介绍一种获取高度木质化植物叶片表皮细胞的简易方法——叶片燃烧法.该方法根据高度木质化的叶片在燃烧、过水之后,其上下表皮自行分开、细胞透明化,从而获得材料上下表皮细胞的轮廓.该方法具有简单迅速、制片效率高、上下表皮易区分及可靠稳定等优点,非常适用于木质化叶片表皮特征的研究,也可为植物学及生态学的相关研究方法提供参考.     

9种梧桐科植物叶表皮特征的扫描电镜研究

用扫描电镜对9种梧桐科植物叶片进行观察研究以明确其叶表皮微形态特征差异.结果显示,绝大部分梧桐科植物的气孔类型和气孔形状相似,但其表皮的毛状体类型、形态以及表皮纹饰等方面有显著差异.结果表明9种梧桐科植物的叶表皮毛和表皮纹饰可作为梧桐科种间分类的特征,且研究结果支持将胖大海归属于胖大海属,将翅子树单独列为翅子树族的分类观点.     

Organization of the Human Frontal Pole Revealed by Large-Scale DTI-Based Connectivity: Implications for Control of Behavior

The goal of the current study was to examine the pattern of anatomical connectivity of the human frontal pole so as to inform theories of function of the frontal pole, perhaps one of the least understood region of the human brain. Rather than simply parcellating the frontal pole into subregions, we focused on examining the brain regions to which the frontal pole is anatomically and functionally connected. While the current findings provided support for previous work suggesting the frontal pole is connected to higher-order sensory association cortex, we found novel evidence suggesting that the frontal pole in humans is connected to posterior visual cortex. Furthermore, we propose a functional framework that incorporates these anatomical connections with existing cognitive theories of the functional organization of the frontal pole. In addition to a previously discussed medial-lateral distinction, we propose a dorsal-ventral gradient based on the information the frontal pole uses to guide behavior. We propose that dorsal regions are connected to other prefrontal regions that process goals and action plans, medial regions are connected to other brain regions that monitor action outcomes and motivate behaviors, and ventral regions connect to regions that process information about stimuli, values, and emotion. By incorporating information across these different levels of information, the frontal pole can effectively guide goal-directed behavior.     

中国兜被兰属植物叶表皮微形态特征的研究

利用光学显微镜和扫描电子显微镜观察了中国兜被兰属１２种植物叶表皮的微形态特征,结果表明：该属植物成熟叶片的上下表皮均具角质层,其表面纹饰有直线形、浅波形、深波形和网状４种类型;有的种表皮细胞垂周壁明显凸起;气孔不具副卫细胞,仅分布在叶片下表面,气孔的外拱盖单层,表面光滑。这些特征在属级水平上较为一致,但也表现出种间差异,可以作为种间分类的辅助特征