[Development and structure defense strategies on the fruit of Chaenomeles speciosa].

OBJECTIVE: To study the development rule and structure defense strategies on the fruit of Chaenomeles speciosa. METHODS: Anatomical and histochemical methods were used to investigate the structure and localization of tannin of the fruit of Chaenomeles speciosa. RESULTS: The development of the fruit of Chaenomeles speciosa were divided into two periods: cells reproduced rapidly from fluorescence to 40 - 50 days after fluorescence, and cells augmented after this. The pericarp and seeds were protected by beceptacle which was formed by cuticle, epidermis, taniniferous cells layer, parenchyma cells which contained tannin and stone cells zones. Tannin was distributed in parenchyma cells of beceptacle mostly and in pericarp barely. CONCLUSION: The development and structure of fruit of were related with its defense strategies.

[Effects of irrigation amount on leaf structure, photosynthetic physiology, and fruit yield of Lycium barbarum in arid area].

Lycium barbarum is an important traditional medicinal plant in China. Under controlled condition, a field experiment was conducted to study the effects of different monthly irrigation quota on the leaf structure, photosynthetic physiology, and fruit yield of L. barbarum, aimed to determine an appropriate irrigation amount for the plant. When the monthly irrigation quota was less than 900 m3 x hm(-2), the leaf area, leaf thickness, palisade tissue thickness, cell tense ratio (CTR), net photosynthetic rate (Pn), intrinsic water use efficiency (WUE), stomatal limitation value (Ls), and fruit yield of L. barbarum all increased significantly with monthly irrigation quota, while leaf stoma density and intercellular CO2 concentration (Ci) showed a reverse trend. When the irrigation quota was more than 900 m3 x hm(-2), the Ci increased with irrigation quota, the leaf area, stoma density, and fruit yield had no obvious change, whereas the other indices showed a reverse trend. The leaf transpiration rate and Gs were the highest at irrigation quota 450 m3 x hm(-2), being 8.02 and 324 mmol x m(-2) x s(-1), respectively; whereas at other irrigation quota, these two indices were lower than the control. In terms of saving water, the monthly irrigation quota 900 m3 x hm(-2) was more appropriate for Lycium barbarum.

Localization and dynamic change of saponin in vegetative organs of Polygala tenuifolia.

Anatomical, histochemical and phytochemical methods were used to investigate the structure, localization and dynamic changes of total saponin and senegenin of vegetative organs in Polygala tenuifolia Willd. Histochemical localization results showed that saponin accumulated mainly in parenchyma cells of vegetative organs. The phytochemical results also showed that the saponin accumulated in the vegetative organs of P. tenuifolia, with higher content in roots and lower content in the aerial parts that included stems and leaves. The saponin content and dry weight of the vegetative organs of P. tenuifolia had dynamic variance at the developmental stages and all reached the highest level in the post-fruit period. Hence, the roots and aerial parts should be gathered in August to make full use of the plant. As the root is the main medicinal organ of P. tenuifolia, the content of total saponin and senegenin of different aged and different parts of the root were determined. The content of total saponin and senegenin exhibited a sustained decreasing trend with increasing root age; therefore, the annual roots had high quality. The content of total saponin and senegenin in different parts of the root showed obvious variation. The content in the "skin areas" was much higher than that of xylem. The results offer a theoretical basis for determining the appropriate harvesting stage and a reasonable harvest of P. tenuifolia.

[Localization and dynamic change of saponin in root tuber of cultivated Pseudostellaria heterophylla].

Anatomical, histochemical and phytochemical methods were used to investigate the structure, the localization and content changes of total saponin of the root tuber of Pseudostellaria heterophylla on different developmental stages. Results showed that the primary structure and secondary structure of P. heterophylla adventitious root were similar to that of other herbaceous dicotyledons. 80% of mature root tuber were secondary xylem which consisted of most parenchyma cells and less vessels. The secondary phloem were composed of parenchyma cells too. The results of histochemical methods showed that saponin distributed in pericycle and parenchyma cells of primary phloem in the primary structure of root. In secondary structure and mature root tuber, saponin distributed in periderm and secondary vascular structure except cork layer and vessel. Besides, the colors of the secondary phloem were darker. The results of phytochemical test showed that the content of saponin in skin areas was higher than that in xylems in February and July. The result was consistent with that of histochemistry. The content of saponin in the head of the root was higher than that in the end of the root which was in turn higher than that in the middle part of the root. In the developmental process of root tuber, the content of saponin showed a dynamic tendency of high-low-high. This characteristics were related to the development of root system.

[The structure of vegetative organs, and saponins histochemical localization and content comparization in Polygala sibirica L].

Anatomical, histochemical and phytochemistry methods were used to investigate the structure of vegetative organs, and saponins localization and dynamic changes in Polygala sibirica L. The root consisted of developed periderm and secondary vascular. The secondary phloem was thick, and mainly composed of parenchyma. There were well-developed vessels and fibers in the secondary xylem. The stem was composed of epidermis, cortex and vascular bundle. The ring of sclerenchymatous cells lied between cortex and phloem might be the apoplastic protective screen which could protect the stem from drought. The leaf was bifacial one. The root and stem possessed characteristics adapting to arid environment. Histochemical localization results showed that saponins distributed in secondary phloem and phelloderm of root, in epidermis, cortex and phloem of stem, mainly in mesophyll of leaf. It displayed that saponins accumulated mainly in parenchyma cells of vegetative organs, among of which, the secondary phloem was the main storage site. The HPLC results also showed that the saponins accumulated in all the vegetative organs of Polygala sibirica L., with higher content in roots and lower content in the aerial part that included stems and leaves. The study indicated the aerial part of Polygala sibirica L. also had medicinal value. The saponins content had dynamic variance at the developmental stage, the crude drug should be gathered at period from April to May.

Vegetative storage protein with trypsin inhibitor activity occurs in Sapindus mukorassi, a sapindaceae deciduous tree.

A vegetative storage protein (VSP) with trypsin inhibitor activity in a deciduous tree, Sapindus mukorassi, was characterized by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western-blot, immuno-histochemical localization, light- and electro-microscopy, together with analysis of proteinase inhibitor activity of the purified VSP in vitro. There were two proteins with molecular masses of about 23 and 27 kDa in a relatively high content in the bark tissues of terminal branches of S. mukorassi in leafless periods. The proteins decreased markedly during young shoot development, indicating their role in seasonal nitrogen storage. Immuno-histochemical localization with the polyclonal antibodies raised against the 23 kDa protein demonstrated that the 23 kDa protein was the major component of protein inclusions in protein-storing cells. The protein inclusions were identified by protein-specific staining and should correspond to the electron-dense materials in different forms in the vacuoles of phloem parenchyma cells and phloem ray parenchyma cells under an electron microscope. So, the 23 kDa protein was a typical VSP in S. mukorassi. The 23 and 27 kDa proteins shared no immuno-relatedness, whereas the 23 kDa protein was immuno-related with the 22 kDa VSP in lychee and possessed trypsin inhibitor activity. The 23 kDa protein may confer dual functions: nitrogen storage and defense.

Localization and dynamic change of saikosaponin in root of Bupleurum chinense.

Anatomical, histochemical and phytochemical methods were used to investigate the structure, the localization and content changes of total saikosaponin and saikosaponin-a of the roots of Bupleurum chinense DC. at different developmental stages. Results showed that saikosaponin was mainly distributed in pericycle and primary phloem in the young root; but in the mature root, it was mainly distributed in vascular cambium and secondary phloem. During the whole growth period from the pre-blossom, blossom, fruit, and fruit mature periods until the pre-withering period, it was in the fruit mature period that both the total saikosaponin content and the saikosaponin-a content reached the highest level. So the last 20 d of October was considered as the right collecting season for the drug of B. chinense. In addition, the quality of 1-year-old drug was better than that of 2-year-old drug due to its higher saikosaponin content. On the other hand, judging from the external characteristics of the drug, the one with an acerose taproot and more lateral roots was of better quality. The results offered theoretical bases for selecting medicinal material of high quality and determining the most appropriate harvesting stage and part of B. chinense.

Ultrastructure and secretion of secretory canals in vegetative organs of Bupleurum chinense DC.

The development of secretory canals in vegetative organs of Bupleurum chinense DC. and the accumulation of essential oils were investigated by transmission electron microscopy (TEM) analysis. The secretion mechanism of the essential oil was also discussed. The results indicate that the plastid, ground substances of cytoplasm and mitochondria took part in the biosynthesis of oil or oil precursor. The endoplasmic reticulum involved in the transport of essential oil to the secretory lumen. At latter stages of development of secretory cells, numerous different sized vesicles fused with the plasmalemma along the boundary between two neighbor secretory cells and secreted the substance into the wall. Thus, the wall between two neighbor secretory cells near the lumen became loosely structured. Then, the wall lined the lumen near two neighbor secretory cells extruded numerous grey vesicles with various sizes on the side facing the lumen, and released these vesicles into the lumen. As a result, the manner of secretion in secretory canals of Bupleurum chinense DC. appeared to be exocytosis.

[Histochemecal localization and the content compare of main medicinal components of vegetative organs in Bupleurum chinense DC].

The paraffin sectioning, histochemistry and phytochemistry methods were applied in the study of the localization and the content changes of saponins and flavonoids in the vegetative organs in Bupleurum Chinense DC. The results showed that the saponins distributed in vascular cambium and secondary phloem of the root; In the stem, they distributed mainly in epiderm,collenchyma and the epithelial cells of the secretory canals which lied in cortex and pith; In the leaf, they distributed in the epiderm and spongy and palisade. However, flavonoids distributed in epiderm, collenchyma, cortex, pith path and myelin shealth cells of the stem; In the leaf, they were located mainly in the epiderm and the collenchyma. Meanwhile, the content of saponins in vegetative organs showed a changing law that the accumulation of them in the root occupied first place, the leaf came second and the stem was the lowest. But the content of flavonoids in leaf was higher than that in stem, and the content of them in stem was higher than that in root. Besides, the content of flavonoids in leaf was considerably high, thus it could offer basis for comprehensive utilization of Bupleurum Chinense DC. and made sense for both exploiting legitimately drug and conserving resource of Bupleurum Chinense DC.

Vegetative storage protein in Litchi chinensis, a subtropical evergreen fruit tree, possesses trypsin inhibitor activity.

BACKGROUND AND AIMS: Vegetative storage proteins (VSPs) are commonly bioactive in herbaceous plants but few VSPs with bioactivity have been identified in trees. In addition, information on the characterization of VSPs in evergreen trees is limited. The objective of this study was to characterize the VSPs with bioactivity in evergreen trees. Methods The VSP in lychee (Litchi chinensis), an evergreen fruit tree, was characterized by a combination of cytological, biochemical and molecular biological techniques. KEY RESULTS: The VSP in lychee was a 22-kDa protein. It accumulated in the large central vacuoles of protein-storing cells (PSCs) in two distinguishable forms, granular and floccular. The PSCs were of a novel type. The 22-kDa protein is distributed in mature leaves, bark tissues of branches, trunk and large roots, paralleling the distribution of PSCs. Its homologues were present in mature seed. During young shoot development and fruiting, the 22-kDa protein decreased apparently, suggesting a nitrogen-storage function. The 22-kDa protein had several isoforms encoded by a small multigene family. One gene member, LcVSP1, was cloned. The LcVSP1 had no intron and contained a 675 bp open reading frame encoding a putative protein of 225 amino acids. LcVSP1 was homologous to Kunitz trypsin inhibitors. The 22-kDa protein inhibited trypsin and chymotrypsin, but had no inhibitory effect on subtilisin. CONCLUSIONS: Lychee is rich in a 22-kDa VSP with trypsin inhibitor activity. The VSP plays an important role in nitrogen storage while its possible defensive function remains to be elucidated.

[Morphological identification of 20 medicinal species in Hypericum].

OBJECTIVE: To provide anatomical evidences for the morphological and histological identification of 20 medicinal species in Hypericum. METHOD: Morphological and anatomical study on the organs of 20 medicinal species in Hypericum using tissue clearing, paraffin sectioning and thin sectioning. RESULT: According to their anatomical characteristics, the secretory structures can be divided into nodules, secretory cavities (canals) and tiny secretory tubes of 20 medicinal species in Hypericum. Hypericin was produced and stored in the nodules, while the volatile oil was produced and stored in the secretory cavities (canals) and tiny secretory tubes. The types differed markedly from each other in location, diameter and distributional density of leaf, and the anatomical structures differed from each other of stem, calyx, petal, anther and fruit among the 20 species in Hypericum. CONCLUSION: The secretory structures may be as anatomical evidences for the morphological and histological identification of 20 medicinal species in Hypericum.

[Structural development of root and their relationship to accumulation of triterpenoid saponins in Achyranthes bidentata Bl].

A study concerning the relationship of dynamic accumulation of triterpenoid saponins and anatomical characteristics of Achyranthes bidentata Bl roots was undertaken by anatomical, histochemical and phytochemical method respectively. Results revealed that the primary and secondary structures of the root resembled those of usual dicots. The continual thickening growth of root principally resulted from the differentiation and development of the tertiary structure. The first ring of supernumerary cambium originated from the parenchyma and vascular ray cells of secondary phloem and each of the followed rings initiated in the outmost foundamental parenchyma cells which were derived from the immediate preceding ring. In the supernumerary cambiums, there had not distinction between the fusiform initial and the ray initial. Its cells present stratifide arranged from a longitudinal section through root. Regular and concentric rings of tertiary vascular bundles who differentiate centrifugally were enclosed by the connective parenchyma. The number of the rings continually increase with the development of the root itself. Triterpenoid saponins accumulated mainly in pericycle, primary phloem and parenchyma between primary phloem and xylem in the primary structure of root but came into existence in cells of secondary phloem and phelloderm with secondary structure development of root, and as well as in supernumerary cambium and phloem of tertiary vascular bundle after the tertiary structure maturated gradually in the roots. The investigation provides indications that the tertiary structure were not only main parts in the roots of Achyranthes bidentata Bl, but also important storage region of triterpenoid saponins in its growth and development. In addition, the analysis of using the HPLC showed that dynamic increasing trend oleanolic acid was as "S" curve during the roots growth and development and up to the highest content of triterpenoid saponins after plants grew 120 days. Meanwhile, the number of the rings of tertiary vascular bundles, length and diameter of the roots were the same as the triterpenoid saponins increasing trend. It should be optimal season for harvest.

[Morphogenesis and histochemical investigation of peltate glandular trichomes on Glycyrrhiza uralensis Fisch. leaves].

Anatomical and histochemical investigation of the glandular trichomes on the leaves of Glycyrrhiza uralensis Fisch.were carried out by using scanning electronic microscopy (SEM), Light microscopy and fluorescence microscopy. Two types of peltate glandular trichomes can be distinguished: one occurred on the leaves abaxial and adaxial surface with a short stalk and the second on the calyx surface with a long stalk. Secretory trichomes started as outgrowth of epidermal cells, subsequent divisions gave rise to trichomes made up of a basal cell, stalk cells and a multicellular secretory head. The histochemical identification revealed that complex substance excreted by the secretory cells contained flavoniods, polysaccharides and lipophilic compounds. During the development of glandular trichomes, only mature trichomes with ten-celled secretory head or even more cells could be observed the flavonoids inside or in subcuticular space but before that stages the flavonoids were not found. The findings provided a scientific basis for understanding the flavonoids synthesis and biological function.

[Ultracytochemical studies of aloin in Aloe arborescens leaves].

Lead acetate precipitation method was used for ultracytochemical localization of aloin. The processes of aloin production, transport and storage were studied by transmission electron microscope. Results showed that aloin was produced in the plastids of the assimilating tissue. The aloin was transported through the plastid membrane to the surrounding endoplasmic reticulum and enveloped in the vesicles by the endoplasmic reticulum elements, the vesicles approached and later fused with the plasmalemma. Some vesicles of the plastid membrane directly fused with the plasmalemma. The vesicles released their contents into the apoplast through exocytosis, and finally reached the vascular bundle sheath by apoplastic translocation. Aloin was transported to the internal tangential wall of vascular bundle sheath cell through endoplasmic reticulum vesicles, and reached the cytoplasm of aloin cell by means of plasmodesmata. Finally, aloin was stored in the vacuoles of aloin cell.

[Effects of MG132 , an inhibitor of proteasome , on the pollen germination and tube growth of Pecea wilsonii].

The ubiquitin/proteasome system is regarded as a major pathway of proteolysis in eukaryotic cells, in which the proteasome acts as primary protease for its function of degrading substrate proteins to short peptides. In the present paper, cytological, statistical studies and Fourier transform infrared (FTIR) analysis on the effects of MG132, an inhibitor of proteasome, on the pollen germination and tube growth of Pecea wilsonii were carried out in an artificial experimental system. It is showed that MG132 significantly reduced the germination rate and tube growth. Furthermore, MG132 treatment lead to vacuolization occurred both in tube cytoplasm and generative cell. While DMSO and non-proteasome inhibitor E-64 do not have similar effects. FTIR analysis revealed that MG132 treatment markedly reduced the contents of wall-bound proteins and pectin at the apex of tube. Those findings provided evidence that by inhibiting the activity of proteasme, MG132 strongly affects pollen germination and tube growth of P. wilsonii, and that UPP plays an important role in organization and maintaining polarized growth of pollen tube. Inhibition of UPP will induce apoptosis of pollen tube.

[Histochemical localization of ginsenosides in Gynostemma pentaphyllum and the content changes of total gypenosides].

The light microscopy technique, histochemistry and phytochemistry methods were applied in the study of the localization of gingsenosides in the vegetative organs in Gynostemma pentaphyllum, and the content of total gypenosides related to different growing periods, organs and genders. The results showed that ginsenosides distributed mainly in the assimilating tissue and phloem parenchyma cells, very little in collenchyma, epidermis and phelloderm, and no coloration in xylem and pith parenchyma. The accumulation of gingsenosides in the leaf occupied first place, the stem came second, and the root was the lowest. The content of total gypenosides showed a changing trend from low to high, then to low, during the whole growing period from vegetative growth to florescence and fructescence, and withering period. The content in leaf is higher than that in stem, and of male is higher than of female. It is suggested that harvest the above-ground parts only and remaining the rhizome and root in florescence and fructescence (Sept. to Oct.) is of benefit to both improving herbal quality and quantity, and accelerating the sustainable utilization for wild resources.

[Development of aloin cells and accumulation of anthraquinone in aloe leaf].

The development of aloin cells and its relationship with the accumulation of anthraquinone in aloe leaf were investigated with the methods of paraffin section, semi-thin section, histochemistry and fluorescent microscopy. The results showed: cells rounded the procambium bundle differentiated into bundle sheath at the initial stage of procambium bundle developing into vascular bundle. When the sieve tube members appeared in protophloem, there were a lay of procambium bundle cells reserved between the sieve tube members and bundle sheath. These cells began to devise, then developed into aloin cells through enlargement of volume and vacuolization with the differentiation of metaphloem and metaxylem. So the aloin cells were special phloem parenchyma cells because they shared the same origin with the other phloem cells. The investigation of histochemistry reflected that there were aloin precipitate in the central vacuole of aloin cells after the material was soaked in the liquid of 1% lead acetate [Pb (CH3COO)2]. In addition, the yellow fluorescence was observed in aloin cells when the section of fresh material was investigated under the fluorescent microscope with blue light, which suggested the aloin cells of vascular bundles were the mainly storage site of anthraquinone.