灵武长枣果实维管束韧皮部及周围细胞的超微结构特征

为了探讨灵武长枣果实光合同化物韧皮部卸载和运输的途径,该研究采用透射电镜技术,对不同发育时期灵武长枣果实维管束韧皮部及其周围薄壁细胞的超微结构特征进行了分析.结果表明:筛管/伴胞复合体及其周围韧皮薄壁细胞间在果实膨大前期富含胞间连丝,而韧皮薄壁细胞与周围库细胞以及相邻库细胞间几乎不存在胞间连丝,形成共质体隔离;筛管/伴...     

宁夏枸杞果实韧皮部及其周围细胞超微结构研究

应用透射电镜技术研究了宁夏枸杞果实韧皮部细胞的超微结构变化。结果表明:(1)随着枸杞果实的发育成熟,果实维管组织中的韧皮部筛分子筛域逐渐变宽,筛孔大而多,通过筛孔的物质运输十分活跃;筛分子和伴胞间有胞间连丝联系,伴胞属传递细胞类型,与其相邻韧皮薄壁细胞和果肉薄壁细胞连接处的细胞界面发生质膜内突,整个筛分子/伴胞复合体与韧皮薄壁细胞之间形成共质体隔离,韧皮部糖分的卸载方式主要以质外体途径进行。(2)韧皮薄壁细胞间的胞间连丝较多,而韧皮薄壁细胞与果肉薄壁细胞的胞间连丝相对较少,但果肉薄壁细胞间几乎无胞间连丝;果肉薄壁细胞之间胞间隙较大,细胞壁和质膜内突间形成较大的质外体空间,为质外体的糖分运输创造了条件。(3)筛管、伴胞、韧皮薄壁细胞和果肉薄壁细胞中丰富的囊泡以及活跃的囊泡运输现象,暗示囊泡也参与了果实糖分的运输过程。研究推测,枸杞果实韧皮部同化物的卸载方式以及卸载后的同化物运输主要以质外体途径为主。     

Acid invertase is predominantly localized to cell walls of both the practically symplasmically isolated sieve element/companion cell complex and parenchyma cells in developing apple fruits

The acid invertase (β‐fructosidase, EC 3·2·1·26) was localized at subcellular level via immunogold electron microscopy in the phloem‐unloading zone of developing apple fruit. The enzyme (immunogold particles) was found to reside predominantly in the cell walls of the sieve element/companion cell (SE/CC) complex, phloem parenchyma cells and other parenchyma cells. There was almost no gold particle found in cytoplasm and vacuole. This distribution pattern remained unchanged throughout the growing season, but the enzyme numbers varied. The density of immunogold particles increased during fruit development. The immunoblotting of soluble and insoluble acid invertases provided a supporting proof for the assays of immunolocalization. The biochemical analysis showed a predominantly cell‐wall‐distributed activity of acid invertase that corresponds essentially with its amount distribution. The ultrastructural observations showed that there were numerous plasmodesmata between the parenchyma cells, but almost no plasmodesmium between the SE/CC complex and its surrounding parenchyma cells, practically resulting in the symplasmic isolation of the SE/CC complex. It is therefore suggested that the unloading pathway of sucrose from the SE/CC complex may be predominantly apoplasmic in the developing apple fruit, and that the unloaded sucrose may be hydrolysed by the functional acid invertase localized in the cell wall before it is loaded in sink cells.     

Structure and Development of the Endophyte in the Parasitic Angiosperm <Emphasis Type="Italic">Cuscuta japonica</Emphasis>

The endophyte, that is, the haustorial part within the tissues of the host plant Impatiens balsamina, of the parasitic angiosperm Cuscuta japonica was studied with light and electron microscopy. The endophyte consisted mainly of vacuolated parenchymatous axial cells and elongate, superficial (epidermal) cells. Then the elongate, epidermal cells separated from each other and transformed into filamentous cells, called searching hyphae. The hyphae grew independently either intercellularly or intracellularly in the host parenchyma. The apical end of the hyphal cells was characterized by conspicuous, large nuclei with enlarged nucleoli and very dense cytoplasm with abundant organelles, suggesting that the hyphal cells penetrating host tissue were metabolically very active. Numerous osmiophilic particles and chloroplasts were noted in the hyphae. The osmiophilic particles were assumed to be associated with elongation of the growing hyphe. Plasmodemata connections between the searching hyphal cells of the parasite and the host parenchyma cells were not detected. Hyphal cells that reached the host xylem differentiated into water-conducting xylic hyphae by thickening of the secondary walls. A xylem bridge connecting the parasite and the host was confirmed from serial sections. Some hyphal cells that reached the host phloem differentiated into nutrient-conducting phloic hyphae. Phloic hyphae had a thin layer of peripheral cytoplasm with typical features of sieve-tube members in autotrophic angiosperms, i.e., parallel arrays of smooth endoplasmic reticulum, mitochondria, and plastids with starch granules. Interspecific open connections via the sieve pores of the host sieve elements and plasmodesmata of the parasite phloic hyphae were very rarely observed, indicating that the symplastic translocation of assimilate to the parasite from the host occurred.     

Subcellular concentrations of sugar alcohols and sugars in relation to phloem translocation in <Emphasis Type="Italic">Plantago major,Plantago maritima</Emphasis>, <Emphasis Type="Italic">Prunus persica</Emphasis>, and <Emphasis Type="Italic">Apium graveolens</Emphasis>

Sugar and sugar alcohol concentrations were analyzed in subcellular compartments of mesophyll cells, in the apoplast, and in the phloem sap of leaves of Plantago major (common plantain), Plantago maritima (sea plantain), Prunus persica (peach) and Apium graveolens (celery). In addition to sucrose, common plantain, sea plantain, and peach also translocated substantial amounts of sorbitol, whereas celery translocated mannitol as well. Sucrose was always present in vacuole and cytosol of mesophyll cells, whereas sorbitol and mannitol were found in vacuole, stroma, and cytosol in all cases except for sea plantain. The concentration of sorbitol, mannitol and sucrose in phloem sap was 2- to 40-fold higher than that in the cytosol of mesophyll cells. Apoplastic carbohydrate concentrations in all species tested were in the low millimolar range versus high millimolar concentrations in symplastic compartments. Therefore, the concentration ratios between the apoplast and the phloem were very strong, ranging between 20- to 100-fold for sorbitol and mannitol, and between 200- and 2000-fold for sucrose. The woody species, peach, showed the smallest concentration ratios between the cytosol of mesophyll cells and the phloem as well as between the apoplast and the phloem, suggesting a mixture of apoplastic and symplastic phloem loading, in contrast to the herbal plant species (common plantain, sea plantain, celery) which likely exhibit an active loading mode for sorbitol and mannitol as well as sucrose from the apoplast into the phloem.     

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.     

<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> is a susceptible host plant for the holoparasite <Emphasis Type="Italic">Cuscuta </Emphasis>spec

Arabidopsis thaliana and Cuscuta spec. represent a compatible host–parasite combination. Cuscuta produces a haustorium that penetrates the host tissue. In early stages of development the searching hyphae on the tip of the haustorial cone are connected to the host tissue by interspecific plasmodesmata. Ten days after infection, translocation of the fluorescent dyes, Texas Red (TR) and 5,6-carboxyfluorescein (CF), demonstrates the existence of a continuous connection between xylem and phloem of the host and parasite. Cuscuta becomes the dominant sink in this host–parasite system. Transgenic Arabidopsis plants expressing genes encoding the green fluorescent protein (GFP; 27 kDa) or a GFP–ubiquitin fusion (36 kDa), respectively, under the companion cell (CC)-specific AtSUC2 promoter were used to monitor the transfer of these proteins from the host sieve elements to those of Cuscuta. Although GFP is transferred unimpedly to the parasite, the GFP–ubiquitin fusion could not be detected in Cuscuta. A translocation of the GFP–ubiquitin fusion protein was found to be restricted to the phloem of the host, although a functional symplastic pathway exists between the host and parasite, as demonstrated by the transport of CF. These results indicate a peripheral size exclusion limit (SEL) between 27 and 36 kDa for the symplastic connections between host and Cuscuta sieve elements. Forty-six accessions of A. thaliana covering the entire range of its genetic diversity, as well as Arabidopsis halleri, were found to be susceptible towards Cuscuta reflexa.     

Exogenous sucrose can decrease <Emphasis Type="Italic">in vitro</Emphasis> photosynthesis but improve field survival and growth of coconut (<Emphasis Type="Italic">Cocos nucifera</Emphasis> L.) <Emphasis Type="Italic">in vitro</Emphasis> plantlets

Summary Coconut (Cocos nucifera L.) plantlets grown in vitro often grow slowly when transferred to the field possibly, due to a limited photosynthetic capacity of in vitro-cultured plantlets, apparently caused by the sucrose added to growth medium causing negative feedback for photosynthesis. In this paper, we tested the hypothesis that high exogenous sucrose will decrease ribulose 1,5-bisphosphate carboxylase (Rubisco) activity and photosynthesis resulting in limited ex vitro growth. Plantlets grown with high exogenous sucrose (90 gl⁻¹) had reduced photosynthetic activity that resulted in a poor photosynthetic response to high levels of light and CO₂. These plantlets also had low amounts of Rubisco protein, low Rubisco activity, and reduced growth despite showing high survival when transferred to the field. Decreasing the medium’s sucrose concentration from 90 to 22.5 gl⁻¹ or 0 gl⁻¹ resulted in increased photosynthetic response to light and CO₂ along with increased Rubisco and phosphoenolpyruvate carboxylase (PEPC) activities and proteins. However, plantlets grown in vitro without exogenous sucrose died when transferred ex vitro, whereas those grown with intermediate exogenous sucrose showed intermediate photosynthetic response, high survival, fast growth, and ex vitro photosynthesis. Thus, exogenous sucrose at moderate concentration decreased photosynthesis but increased survival, suggesting that both in vitro photosynthesis and exogenous sucrose reserves contribute to field establisment and growth of coconut plantlets cultured in vitro.     

The small subunit ADP-glucose pyrophosphorylase (<Emphasis Type="Italic">ApS</Emphasis>) promoter mediates okadaic acid-sensitive<Emphasis Type="Italic"> uidA</Emphasis> expression in starch-synthesizing tissues and cells in<Emphasis Type="Italic"> Arabidopsis</Emphasis>

Transgenic plants of Arabidopsis thaliana Heynh., transformed with a bacterial beta-glucuronidase (GUS) gene under the control of the promoter of the small subunit (ApS) of ADP-glucose pyrophosphorylase (AGPase), exhibited GUS staining in leaves (including stomata), stems, roots and flowers. Cross-sections of stems revealed GUS staining in protoxylem parenchyma, primary phloem and cortex. In young roots, the staining was found in the root tips, including the root cap, and in vascular tissue, while the older root-hypocotyl axis showed prominent staining in the secondary phloem and paratracheary parenchyma of secondary xylem. The GUS staining co-localized with ApS protein, as found by tissue printing using antibodies against ApS. Starch was found only in cell and tissue types exhibiting GUS staining and ApS labelling, but not in all of them. For example, starch was lacking in the xylem parenchyma and secondary phloem of the root-hypocotyl axis. Sucrose potently activated ApS gene expression in leaves of wild-type (wt) plants, and in transgenic seedlings grown on sucrose medium where GUS activity was quantified with 4-methylumbelliferyl-beta-glucuronide as substrate. Okadaic acid, an inhibitor of protein phosphatases 1 and 2A, completely blocked expression of ApS in mature leaves of wt plants and prevented GUS staining in root tips and flowers of the transgenic plants, suggesting a similar signal transduction mechanism for ApS expression in various tissues. The data support the key role of AGPase in starch synthesis, but they also underlie the ubiquitous importance of the ApS gene for AGPase function in all organs/tissues of Arabidopsis.     

Molecular and phylogenetic characterization of the sieve element occlusion gene family in <Emphasis Type="Italic">Fabaceae</Emphasis> and non-<Emphasis Type="Italic">Fabaceae</Emphasis>plants

Background  

The phloem of dicotyledonous plants contains specialized P-proteins (phloem proteins) that accumulate during sieve element differentiation and remain parietally associated with the cisternae of the endoplasmic reticulum in mature sieve elements. Wounding causes P-protein filaments to accumulate at the sieve plates and block the translocation of photosynthate. Specialized, spindle-shaped P-proteins known as forisomes that undergo reversible calcium-dependent conformational changes have evolved exclusively in the Fabaceae. Recently, the molecular characterization of three genes encoding forisome components in the model legume Medicago truncatula (MtSEO1, MtSEO2 and MtSEO3; SEO = sieve element occlusion) was reported, but little is known about the molecular characteristics of P-proteins in non-Fabaceae.     

Endopolygalacturonase: a Candidate Gene for <Emphasis Type="Italic">Freestone</Emphasis> and <Emphasis Type="Italic">Melting Flesh</Emphasis>in Peach

Peach fruit are handled, processed, and marketed according to their stone adhesion and fruit softening type. Uncertainty exists over whether these simply inherited traits are controlled by two linked loci, Freestone (F) and Melting flesh (M) or one multi-allelic locus, and whether M is controlled by the cell wall degrading enzyme, endopolygalacturonase. From morphological and molecular analysis of two related segregating populations of peach, we conclude that a single locus containing at least one gene for endopolygalacturonase, controls both F and M with at least three effective alleles. A simple diagnostic PCR test is now available for the three major phenotypes of freestone melting flesh (FMF), clingstone melting flesh (CMF), and clingstone non-melting flesh (CNMF).     

<Emphasis Type="Italic">Amborella trichopoda</Emphasis>, plasmodesmata,and the evolution of phloem loading

Phloem loading is the process by which photoassimilates synthesized in the mesophyll cells of leaves enter the sieve elements and companion cells of minor veins in preparation for long distance transport to sink organs. Three loading strategies have been described: active loading from the apoplast, passive loading via the symplast, and passive symplastic transfer followed by polymer trapping of raffinose and stachyose. We studied phloem loading in Amborella trichopoda, a premontane shrub that may be sister to all other flowering plants. The minor veins of A. trichopoda contain intermediary cells, indicative of the polymer trap mechanism, forming an arc on the abaxial side and subtending a cluster of ordinary companion cells in the interior of the veins. Intermediary cells are linked to bundle sheath cells by highly abundant plasmodesmata whereas ordinary companion cells have few plasmodesmata, characteristic of phloem that loads from the apoplast. Intermediary cells, ordinary companion cells, and sieve elements form symplastically connected complexes. Leaves provided with ¹⁴CO₂ translocate radiolabeled sucrose, raffinose, and stachyose. Therefore, structural and physiological evidence suggests that both apoplastic and polymer trapping mechanisms of phloem loading operate in A. trichopoda. The evolution of phloem loading strategies is complex and may be difficult to resolve.     

Cloning and expression of the sucrose phosphorylase gene from <Emphasis Type="Italic">Leuconostoc </Emphasis><Emphasis Type="Italic">mesenteroides</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The gene encoding sucrose phosphorylase (742sp) in Leuconostoc mesenteroides NRRL B-742 was cloned and expressed in Escherichia coli. The nucleotide sequence of the transformed 742sp comprised an ORF of 1,458 bp giving a protein with calculated molecular mass of 55.3 kDa. 742SPase contains a C-terminal amino acid sequence that is significantly different from those of other Leu. mesenteroides SPases. The purified 742SPase had a specific activity of 1.8 U/mg with a K _m of 3 mM with sucrose as a substrate; optimum activity was at 37°C and pH 6.7. The purified 742SPase transferred the glucosyl moiety of sucrose to cytosine monophosphate (CMP). Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The cellular pathway and enzymatic activity for phloem-unloading transition in developing <Emphasis Type="Italic">Camellia oleifera</Emphasis> Abel. Fruit

The phloem-unloading pathway of sucrose and mechanism of sugar-to-oil transition are still unknown in Camellia oleifera Fruit. Here, transmission electronic microscopy (TEM) and confocal laser-scanning microscopy (CLSM) were used to observe the cellular structure of vascular bundles and symplastic tracer, carboxyfluorescein (CF), transport in phloem zone. The results showed that sucrose was transported via symplast system in the early and late phases, whereas apoplast system exerted the function in middle stage. Moreover, enzymatic assays showed that acid invertase had a higher activity at the transition stage during the whole fruit development. The cell wall bound invertase (CWI) activity reached the highest at the middle stage of fruit development and the switch in phloem-unloading coincided with fruit developmental phase change and oil accumulation. Correlation analysis showed that the oil accumulation was significantly negatively correlated with content of soluble sugar at P < 0.05 level. However, the soluble acid invertase (SAI), CWI, and neutral invertase showed a significant positive correlation with oil accumulation at P < 0.01 level. In summary, our data provide new cytological insights into the transition of unloading transfer between symplasmic and apoplasmic patterns in C. oleifera fruit and suggest that invertases are positively involved in sugar–oil transition process.     

文冠果果实韧皮部及其周围薄壁细胞的超微结构观察及功能分析

该研究应用透射电镜技术,对生长发育过程中的文冠果果实的韧皮部及其周围薄壁细胞的超微结构进行了观察,以探讨文冠果果实同化物韧皮部卸载的细胞学路径及其机理。结果显示:(1)文冠果果实发育过程中,筛分子细胞胞腔较空,几乎没有细胞器,但有类似于囊泡的丝状不定型物存在;伴胞胞质浓密且细胞器丰富,液泡化程度不一,大多数存在多个小液泡;薄壁细胞具有中央大液泡,发育中期富含线粒体、高尔基体、内质网等细胞器,并存在囊泡运输现象,发育后期细胞器发生降解,说明随着果实生长发育,果实内物质代谢和转运活跃程度逐渐下降。(2)果实发育过程中筛分子和伴胞之间始终有胞间连丝,薄壁细胞之间也一直存在大量的胞间连丝,而筛分子-伴胞复合体与薄壁细胞之间只有在果实发育前期和后期存在一定数量的胞间连丝,发育中期却几乎没有胞间连丝。研究结果表明,文冠果果实发育过程中同化物韧皮部卸载路径可能发生了共质体途径-质外体途径-共质体途径的转变。     

灵武长枣果实ATPase超微细胞化学定位和功能研究

采用ATPase超微细胞化学定位技术,研究灵武长枣果实不同发育阶段韧皮部和果肉库薄壁细胞ATPase分布特征,以明确灵武长枣果实ATPase超微细胞化学定位特征和功能。结果显示:(1)第一次快速生长期SE/CC复合体与周围的薄壁细胞有丰富的胞间连丝,形成共质体连续,韧皮部薄壁细胞之间有丰富的胞间连丝,ATPase反应物在韧皮部各细胞分布较少。(2)缓慢生长期ATPase反应物在韧皮部各细胞分布逐渐增加。(3)第二次快速生长期SE/CC复合体与周围的薄壁细胞缺乏胞间连丝,形成共质体隔离,韧皮薄壁细胞及果肉库薄壁细胞的胞间连丝较少,囊泡和膜泡在筛管、韧皮薄壁细胞和库薄壁细胞中很丰富,质膜、液泡膜、囊泡膜、细胞壁和胞间隙的ATPase活性较高。研究表明,果实在第一次快速生长期同化物从筛分子的卸出主要采取共质体途径,缓慢生长期同化物卸出时可能为共质体和质外体途径共存,第二次快速生长期则主要以质外体途径为主,证明果实不同发育阶段韧皮部同化物卸出路径存在差异。     

Seasonal changes in plasma membrane H<Superscript>+</Superscript>-ATPase activity of <Emphasis Type="Italic">Vaccinium myrtillus</Emphasis> (L.) and <Emphasis Type="Italic">Pinus sylvestris</Emphasis> (L.)

Seasonal changes in vanadate sensitive plasma membrane H⁺-ATPase activity of bilberry (Vaccinium myrtillus L.) and Scots pine (Pinus sylvestris L.) were studied in a period from February to August in northern Finland. The plasma membrane isolation was performed by sucrose gradient centrifugation, and the H⁺-ATPase activity was assayed by spectrophotometrical determination of released inorganic phosphate. The studied species showed seasonal changes from high winter to low spring activity, indicating probable physiological changes between hardened and dehardened tissue. ATPase activity of bilberry peaked up at the beginning of the growth period, obviously due to active phloem loading of photosynthates.     

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.     

Cryopreservation of <Emphasis Type="Italic">Robinia</Emphasis><Emphasis Type="Italic">pseudoacacia</Emphasis>

Cryopreservation of Robinia pseudoacacia explants by vitrification achieved 78% survival following the stepwise preculture of shoot tips in (0.3 + 0.5 + 0.7 M) sucrose with a 80 min incubation in PVS2; compared to 87% survival after desiccation of explants to 30% water content, following 3 days alginate bead (with glycerol and sucrose treatments) preculture in 0.7 M sucrose.