Real-time imaging of phloem unloading in the root tip of Arabidopsis

Confocal laser scanning microscopy (CLSM) has been used to image phloem transport and unloading in the root tip of Arabidopsis. The fluorescent probe 5(6) carboxyfluorescein (CF) was ester loaded into a single cotyledon and the entire seedling placed within an observation chamber under the microscope. Translocation of CF to the root tip was rapid, followed by unloading into discrete concentric files of cells. The position of the prominent unloading ‘zone’ corresponded precisely with that of the two protophloem files of sieve elements, demonstrating a functional role of these cells in symplastic sieve-element unloading. Symplastic transport following unloading was confined to the elongating zone of the root with little basipetal transport to more mature cells. Following photobleaching of the unloading zone, phloem transport was restored immediately into the protophloem sieve elements, followed rapidly by lateral, symplastic sieve-element unloading. The results demonstrate that phloem transport processes can now be imaged in real time, and non-invasively, within an intact plant system.     

Metabolic inhibitors induce symplastic movement of solutes from the transport phloem of Arabidopsis roots

The distribution of the phloem-mobile fluorescent probe carboxyfluorescein(CF) within the primary root of Arabidopsis thaliana was imagedusing a confocal laser scanning microscope (CLSM) and the tissueand subcellular distribution of the probe was shown to be influencedby treatment with a number of metabolic inhibitors. Sodium azidecompletely inhibited the phloem transport of CF into the treatedregion of root. Treatment with both CCCP and probenecid inducedthe lateral movement of CF from the transport phloem to theadjacent cell layers, and the probe accumulated in the cytoplasmof the pericycle, endodermis, cortex, and epidermis. This lateraltransfer of CF was restricted to the pericycle in the presenceof plasmolysing concentrations of sorbitol. Ultrastructuralinvestigations demonstrated the presence of a plasm odesmatalpathway leading from the sieve elementcompanion cell complex(SE-CC) out into the cortex. The results are consistent withthe operation of this symplastic pathway under conditions ofmetabolic energy reduction and are discussed in relation tothe regulation of plasmodesmatal conductance in the transportphloem. Key words: Arabidopsis, confocal laser scanning microscopy (CLSM), metabolic inhibitors, phloem transport, symplastic phloem unloading     

Symplastic communication between primary and developing lateral roots of Arabidopsis thaliana

The functional symplastic connections between primary and developinglateral roots of Arabidopsis were studied non-invasively usingconfocal laser scanning microscopy (CLSM), following ester-loadingof the phloem with carboxyfluorescein (CF). Prior to the formationof lateral primordia in the pericycle, the phloem of the primaryroot behaved as an isolated conducting domain. However, thedifferentiation of phloem connector elements within the dividingpericycle allowed the rapid establishment of intercellular communicationbetween the phloem and the cells of the lateral primordium.This communication was often established prior to the completeemergence of the lateral root from the parent root. Shortlyafter its emergence, functional conducting phloem became differentiatedwithin the developing lateral root. A progressive isolationbetween the phloem and surrounding cells at the base of thelateral root was observed as the lateral continued to grow;the new phloem conducting CF to the elongation zone where itwas unloaded symplastically from the protophloem into surroundingcells of the cortex and stele, a feature mirroring the patternfound near the apex of growing primary roots. Anomalous patternsof intercellular communication were found which indicated thatpreviously functional symplastic pathways may have become sealedoff following the emergence of some of the lateral roots. Key words: Arabidopsis, carboxyfluorescein, confocal laser scanning microscopy (CLSM), intercellular transport, lateral roots, phloem (unloading), symplast     

Living sieve cells of conifers as visualized by confocal,laser-scanning fluorescence microscopy

Summary Confocal laser scanning microscopy (CLSM) and fluorochromes were used to visualize the assimilate-conducting sieve cells of conifers in vivo. When still nucleate, the cytoplasm of these cells shows streaming and occupies the cell periphery including the pitlike, thin wall regions where sieve areas would develop. During differentiation the nuclear fluorescence and the central vacuoles disappear. At maturity and after ER-specific staining the sieve areas are the most conspicuous character of sieve cells. Those linking two sieve cells are covered on either side with prominent amounts of ER, while those leading to a Strasburger (=albuminous) cell show fluorescence on the sieve-cell side only. Within the sieve-area wall fluorescence appears also in the common median cavity which is part of the symplastic path between sieve cells. Electron microscopy (EM) depicts the ER as complexes of densely convoluted tubules of smooth ER, equally on either side of a sieve area, provided that the fixation of this sensitive tissue is appropriate. Purposeful wounding causes a swelling and vesiculation of the ER-tubules which is visible in both CLSM and EM. Electron micrographs of ER-complexes at sieve areas -in this paper demonstrated in vivo -have often been argued to be artefacts, since they should raise flow resistance considerably and are not consistent with the Münch hypothesis on phloem transport. The implications of this location for phloem transport are discussed.Abbreviations CLSM confocal laser scanning microscopy - DiOC 3,3-dioxacarbocyanine iodide - EM electron microscopy - ER endoplasmic reticulum - FDA fluorescein diacetate     

Temporal and spatial regulation of symplastic trafficking during development in Arabidopsis thaliana apices

Plasmodesmata provide symplastic continuity linking individual plant cells. However, specialized cells may be isolated, either by the absence of plasmodesmata or by down regulation of the cytoplasmic flux through these channels, resulting in the formation of symplastic domains. Maintenance of these domains may be essential for the co-ordination of growth and development. While cells in the center of the meristem divide slowly and remain undifferentiated, cells on the meristem periphery divide more frequently and respond to signals determining organ fate. Such symplastic domains were visualized within shoot apices of Arabidopsis, by monitoring fluorescent symplastic tracers (HPTS: 8-hydroxypyrene 1,3,6 trisulfonic acid and CF: carboxy fluorescein). Tracers were loaded through cut leaves and distributed throughout the whole plant. Confocal laser scanning microscopy on living Arabidopsis plants indicates that HPTS moves via the vascular tissue from leaves to the apex where the tracer exits the phloem and moves symplastically into surrounding cells. The distribution of HPTS was monitored in vegetative apices, and just prior to, during, and after the switch to production of flowers. The apices of vegetative plants loaded with HPTS had detectable amounts of tracer in the tunica layer of the meristem and in very young primordia, whereas the corpus of the meristem excluded tracer uptake. Fluorescence signal intensity decreased prior to the onset of flowering. Moreover, at approximately the time the plants were committed to flowering, HPTS was undetectable in the inflorescence meristem or young primordia. Later in development, after several secondary inflorescences and mature siliques appeared, inflorescence apices again showed tracer loading at levels comparable to that of vegetative apices. Thus, analysis of fluorescent tracer movement via plasmodesmata reveals there is distinct temporal and spatial regulation of symplastic domains at the apex, dependent on the developmental stage of the plant.     

Induction of phloem unloading in Arabidopsis thaliana roots by the parasitic nematode Heterodera schachtii.

Phloem unloading of both the fluorescent probe carboxyfluorescein (CF) and 14C-labeled solutes was induced in Arabidopsis thaliana L. roots by the parasitic nematode Heterodera schachtii Schmidt. Confocal laser scanning microscopy demonstrated that anomalous unloading of CF from the sieve element companion cell complexes occurred specifically into the syncytium, the nematode-induced feeding structure located within the stele of the root. From this syncytial complex of modified root cells, both fluorescent and radioactive labels were withdrawn by feeding nematodes. Movement of CF was unidirectional from the phloem to the syncytium. A range of low-molecular-weight fluorescent probes (including CF) microinjected into the syncytium stayed in this structure, demonstrating that it is symplastically isolated from the surrounding root tissue. The mechanism of unloading in this host-pathogen relationship therefore appears to be apoplastic. Our results provide unequivocal evidence that sedentary cyst-forming nematodes have direct access to phloem-derived solutes.     

Intercellular Symplastic Connection and Isolation of the Unloading Zone in Flesh of the Developing Grape Berry

The uhrastructure and intercellular connection of the sugar unloading zone (i. e. the phloem in the dorsal vascular bundle and the phloem-surrounding the assimilate sink-cells) of grape ( Vitis vinifera x V. labrusca cv. Jingchao) berry was observed via transmission electron microscopy. The results showed that during the early developmental stages of grape berry, numerous plasmodesmata were found in the phloem between sieve element (SE) and companion cell (CC), between SE/CC complexes, between SE/CC complex and phloem parenchyma cell and in between phloem parenchyma cells, which made the phloem a symplastic integration, facilitating sugar unloading from sieve elements into both companion cells and phloem parenchyma cells via a symplastic pathway. On the contrary, there was almost no plasmodesma between phloem and its surrounding flesh photoassimilate sink-cells, neither in between the flesh photoassimilate sink-cells giving rise to a symplastic isolation both between phloem and its surrounding flesh photoassimilate sink-cells, as well as among the flesh photoassimilate sink-cells. This indicated that both the sugar unloading from phloem and pestphloem transport of sugars should be mainly via an apoplastic pathway. Dining the ripening stage, most of the plasmodesmata between SE/CC complex and the surrounding phloem parenchyma cells were shown to be blocked by the electron-opaque globules, and a phenomenon of plasmolysis was found in a number of companion cells, indicating a symplastic isolation between SE/CC complex and its surrounding parenchynm cells during this phase. The symplastic isolation between the whole phloem and its surrounding photoassimilate sink-cells during the early developmental stages shifted to a symplastic isolation within the phloem during the ripening phase, and thus the symplastic pathway of sugar unloading from SE/CC complex during the early development stages should be replaced by a dominant apoplastic unloading pathway from SE/CC complex in concordance.     

蒜鳞片休眠进程中胞间联络的变化及类外连丝结构与功能的研究

利用电子显微镜术,对蒜休眠进程中鳞片薄壁细胞间胞间联络的特征进行了实验观察,发现不同时期胞间联络具有随细胞间生理关系密切程度而呈现相应结构变化的特点。并观察到萌芽期鳞片中衰败细胞与存活细胞之间有类外连丝型胞间连丝的存在;以激光共聚焦荧光显微镜结合荧光标记物示踪检测,发现不透膜荧光物质分子量为457Da的萤黄(Lucifer yellow,LYCH),可以共质体运输方式进入存活细胞内,论证了类外连丝这一胞间连丝特定修饰态的存在,并可在一段时间内继续保持生理活性,起到进行物质共质运输的功能。     

Localization and movement of mineral oil in plants by fluorescence and confocal microscopy

Fluorescence and confocal laser scanning microscopy were explored to investigate the movement and localization of mineral oils in citrus. In a laboratory experiment, fluorescence microscopy observation indicated that when a 'narrow' distillation fraction of an nC23 horticultural mineral oil was applied to adaxial and opposing abaxial leaf surfaces of potted orange [Citrus x aurantium L. (Sapindales: Rutaceae)] trees, oil penetrated steadily into treated leaves and, subsequently, moved to untreated petioles of the leaves and adjacent untreated stems. In another experiment, confocal laser scanning microscopy was used to visualize the penetration into, and the subsequent cellular distribution of, an nC24 agricultural mineral oil in C. trifoliata L. seedlings. Oil droplets penetrated or diffused into plants via both stomata and the cuticle of leaves and stems, and then moved within intercellular spaces and into various cells including phloem and xylem. Oil accumulated in droplets in intercellular spaces and within cells near the cell membrane. Oil entered cells without visibly damaging membranes or causing cell death. In a field experiment with mature orange trees, droplets of an nC23 horticultural mineral oil were observed, by fluorescence microscopy, in phloem sieve elements in spring flush growth produced 4-5 months and 16-17 months after the trees were sprayed with oil. These results suggest that movement of mineral oil in plants is both apoplastic via intercellular spaces and symplastic via plasmodesmata. The putative pattern of the translocation of mineral oil in plants and its relevance to oil-induced chronic phytotoxicity are discussed.     

Symplastic continuity in Agrobacterium tumefaciens-induced tumours

The Agrobacterium tumefaciens-induced plant tumour is regarded as a strong sink, containing a well-developed vascular system that guarantees an efficient supply of water and nutrients from the host plant into the tumour. The phloem transport and unloading of the fluorescent dye carboxyfluorescein (CF) was studied to examine the potential pathways for unloading of a low-molecular-mass solute, and was compared with the symplastic movement of potato virus X expressing a green fluorescent protein-coat protein fusion (PVX.GFP-CP). The distribution of both CF and PVX.GFP-CP in the host plant, Nicotiana benthamiana, demonstrated a clear symplastic pathway between the phloem of the host stem and the cells of the tumour, and also a considerable capacity for subsequent cell-to-cell transport between tumour cells. This same pattern of CF transport was also demonstrated independently for the host species Cucurbita maxima and Ricinus communis. In addition to entering the tumour, CF and PVX both moved through the vascular rays of the host stem towards the stele. The results confirm that host and tumour tissues in the Agrobacterium gall are in direct symplastic continuity and emphasize an important symplastic pathway for radial solute transport in stems.Key words: Agrobacterium tumefaciens, carboxyfluorescein, GFP, symplastic phloem unloading, plant tumour, vascular rays      

Phloem unloading in developing walnut fruit is symplasmic in the seed pericarp and apoplasmic in the fleshy pericarp

The sieve element-companion cell (SE-CC) complex of the sepal bundles feeding the fleshy pericarp of developing walnut (Juglans regia L.) fruit is structurally symplasmically isolated, but the SE-CC complex of the minor ventral carpellary bundles located in the seed pericarp and feeding the seed is structurally symplasmically connected to its adjacent parenchyma cells. 14C-autoradiography indicated that the phloem of both the sepal and carpellary bundles was functional for unloading. Confocal laser scanning microscopy imaging of carboxyfluorescein unloading showed that the dye is confined to the phloem strands of the sepal bundles in the fleshy pericarp, but released from the phloem strands of the minor ventral carpellary bundles into the surrounding parenchyma cells in the seed pericarp. A 60-kDa acid invertase was immunolocalized to the cell wall of SE-CC complex and parenchyma cells in both the fleshy and seed pericarp. These data provide clear evidence for an apoplasmic phloem unloading pathway in the fleshy pericarp and a predominant symplasmic phloem unloading pathway parallel with a possible apoplasmic path as suggested by the presence of the extracellular invertase in the seed pericarp. A model of complex phloem unloading pathways in developing walnut fruit has been proposed.     

Transfer of phloem-mobile substances from the host plants to the holoparasite Cuscuta sp

During the development of the haustorium, searching hyphae of the parasite and the host parenchyma cells are connected by plasmodesmata. Using transgenic tobacco plants expressing a GFP-labelled movement protein of the tobacco mosaic virus, it was demonstrated that the interspecific plasmodesmata are open. The transfer of substances in the phloem from host to the parasite is not selective. After simultaneous application of (3)H-sucrose and (14)C-labelled phloem-mobile amino acids, phytohormones, and xenobiotica to the host, corresponding percentages of the translocated compounds are found in the parasite. An open continuity between the host phloem and the Cuscuta phloem via the haustorium was demonstrated in CLSM pictures after application of the phloem-mobile fluorescent probes, carboxyfluorescein (CF) and hydroxypyrene trisulphonic acid (HPTS), to the host. Using a Cuscuta bridge (14)C-sucrose and the virus PVY(N) were transferred from one host plant to the another. The results of translocation experiments with labelled compounds, phloem-mobile dyes and the virus should be considered as unequivocal evidence for a symplastic transfer of phloem solutes between Cuscuta species and their compatible hosts.     

巴西固氮螺菌Yu62在玉米根的定植

将GFPmut2质粒中的gfp基因(编码绿色荧光蛋白)克隆到载体pVK100中,构建成重组质粒pVK1001。将pVK1001通过电转化方法导入到联合固氮菌巴西固氮螺菌Yu62中,获得GFP)标记的巴西固氮螺菌Yu62菌株。用标记菌株接种限菌培养条件下生长的玉米(农大3318)幼苗,在接种后8d、12d,用激光共聚焦扫描显微镜进行观测,结果表明巴西固氮螺菌Yu62菌株能定植于玉米根部皮层的薄壁细胞间隙。用扫描电镜和超薄切片电镜观察表明,大多数细菌主要定植于根表,少数菌可进入玉米根组织内。     

Uptake of Lucifer yellow CH in leaves ofCommelina communis is mediated by endocytosis

Summary Lucifer yellow CH (LY) uptake into intact leaves ofCommelina communis has been studied with conventional fluorescence microscopy as well as confocal laser scanning microscopy. LY, a highly fluorescent tracer for apoplastic transport in plants and fluid phase endocytosis in animal cells, accumulates in the vacuole of leaf cells. However, considerable differences in the ability to take up LY were observed among the various cell types. Mesophyll cells take up large amounts of the dye whereas epidermal cells, including guard and subsidiary cells, showed no fluorescence in their vacuoles. An exception to this are trichome cells which show considerable accumulation of LY. When introduced into the cytoplasm of mesophyll protoplasts ofC. communis by means of a patch-clamp pipette, LY does not enter the vacuole. This supports the contention that exogenous LY can only gain access to the vacuole via endocytosis. Differences in the capacity for LY uptake may therefore reflect differences in endocytotic activity.Abbreviations CLSM Confocal laser scanning microscopy - DIC differential interference contrast - LY Lucifer yellow CH - PM plasma membrane     

巴西固氮螺菌Yu62在玉米根的定植

将GFPmut2质粒中的gfp基因 (编码绿色荧光蛋白)克隆到载体pVK100中,构建成重组质粒pVK1001.将pVK1001通过电转化方法导入到联合固氮菌巴西固氮螺菌Yu62中,获得GFP标记的巴西固氮螺菌Yu62菌株.用标记菌株接种限菌培养条件下生长的玉米(农大3318)幼苗,在接种后8 d、12 d,用激光共聚焦扫描显微镜进行观测,结果表明巴西固氮螺菌Yu62菌株能定植于玉米根部皮层的薄壁细胞间隙.用扫描电镜和超薄切片电镜观察表明,大多数细菌主要定植于根表,少数菌可进入玉米根组织内.     

Phloem mobility of fluorescent xenobiotics in Arabidopsis in relation to their physicochemical properties

The transport of a range of fluorescent probes within the rootphloem of Arabidopsis thaliana has been imaged using the confocallaser scanning microscope. The phloem mobility of these probesand their subsequent subcellular distribution in the cells ofthe ‘unloading zone’, close to the root tip, arediscussed in relation to a structure-activity relations (SAR)model. This is a generalized model describing the interactionof low molecular weight xenobiotics with living cells, basedon the physicochemical properties of the former. The work demonstratesthat the model can be used to predict the phloem mobility ofxenobiotics, but only partly predicts the subcellular distributionof phloem-mobile probes following unloading. The potential useof phloem-mobile fluorescent probes as physiological indicatorsis discussed. Key words: Arabidopsis, confocal laser scanning microscopy (CLSM), fluorescent probes, phloem transport, xenobiotic transport     

Quantifying anisotropic solute transport in protein crystals using 3-D laser scanning confocal microscopy visualization

The diffusion of a solute, fluorescein into lysozyme protein crystals has been studied by confocal laser scanning microscopy (CLSM). Confocal laser scanning microscopy makes it possible to non-invasively obtain high-resolution three-dimensional (3-D) images of spatial distribution of fluorescein in lysozyme crystals at various time steps. Confocal laser scanning microscopy gives the fluorescence intensity profiles across horizontal planes at several depths of the crystal representing the concentration profiles during diffusion into the crystal. These intensity profiles were fitted with an anisotropic model to determine the diffusivity tensor. Effective diffusion coefficients obtained range from 6.2 x 10(-15) to 120 x 10(-15) m2/s depending on the lysozyme crystal morphology. The diffusion process is found to be anisotropic, and the level of anisotropy depends on the crystal morphology. The packing of the protein molecules in the crystal seems to be the major factor that determines the anisotropy.     

Tuberization in potato involves a switch from apoplastic to symplastic phloem unloading

Phloem unloading was studied in potato plants in real time during the early stages of tuberization using carboxyfluorescein (CF) as a phloem-mobile tracer, and the unloading pattern was compared with autoradiography of tubers that had transported (14)C assimilates. In stolons undergoing extension growth, apoplastic phloem unloading predominated. However, during the first visible signs of tuberization, a transition occurred from apoplastic to symplastic transport, and both CF and (14)C assimilates subsequently followed identical patterns of phloem unloading. It is suggested that the switch to symplastic sucrose unloading may be responsible for the upregulation of several genes involved in sucrose metabolism. A detailed analysis of sugar levels and (14)C sugar partitioning in tuberizing stolons revealed a distinct difference between the apical region of the tuber and the subapical region. Analysis of invertase activity in nontuberizing and tuberizing stolons revealed a marked decline in soluble invertase in the subapical region of swelling stolons, consistent with the switch from apoplastic to symplastic unloading. However, cell wall-bound invertase activity remained high in the apical 1 to 2 mm of tuberizing stolons. Histochemical analysis of potato lines transformed with the promoter of an apoplastic invertase gene (invGE) linked to a reporter gene also revealed discrete gene expression in the apical bud region. Evidence is presented that the apical and lateral tuber buds function as isolated domains with respect to sucrose unloading and metabolism.     

Evidence for apoplasmic phloem unloading in developing apple fruit

The phloem unloading pathway remains unclear in fleshy fruits accumulating a high level of soluble sugars. A structural investigation in apple fruit (Malus domestica Borkh. cv Golden Delicious) showed that the sieve element-companion cell complex of the sepal bundles feeding the fruit flesh is symplasmically isolated over fruit development. 14C-autoradiography indicated that the phloem of the sepal bundles was functional for unloading. Confocal laser scanning microscopy imaging of carboxyfluorescein unloading showed that the dye remained confined to the phloem strands of the sepal bundles from the basal to the apical region of the fruit. A 52-kD putative monosaccharide transporter was immunolocalized predominantly in the plasma membrane of both the sieve elements and parenchyma cells and its amount increased during fruit development. A 90-kD plasma membrane H(+)-ATPase was also localized in the plasma membrane of the sieve element-companion cell complex. Studies of [14C]sorbitol unloading suggested that an energy-driven monosaccharide transporter may be functional in phloem unloading. These data provide clear evidence for an apoplasmic phloem unloading pathway in apple fruit and give information on the structural and molecular features involved in this process.     

A dual switch in phloem unloading during ovule development in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Developing flowers are important sinks in Arabidopsis thaliana. Their energy demand is covered by assimilates which are synthesized in source leaves and transported via the vasculature. Assimilates are unloaded either symplastically through plasmodesmata or apoplastically by specific transport proteins. Here we studied the pathway of phloem unloading and post-phloem transport in developing gynoecia. Using phloem-mobile fluorescent tracers, we show that phloem unloading into cells of ovule primordia followed a symplastic pathway. Subsequently, the same tracers could not move out of phloem cells into mature ovules anymore. A further change in the mode of phloem unloading occurred after anthesis. In open flowers as well as in outgrowing siliques, the phloem was again unloaded via the symplast. This observed onset of symplastic phloem unloading was accompanied by a change in frequency of MP17-GFP-labeled plasmodesmata. We could also show that the change in cell–cell connectivity was independent of fertilization and increasing sink demand. The presented results indicate that symplastic connectivity is highly regulated and varies not only between different sink tissues but also between different developmental stages.