Expression of Human BMP-2 Gene in Different Tissues of Tobacco Plants

The bone morphogenetic proteins (BMPs) are a family of growth factors that regulate the development of bone. BMP-2 is the most effective in the induction of bone tissue. A large amount of BMP-2 is needed for both bone tissue engineering research and clinical application. Thus, an effective way is necessary to produce sufficient BMP-2 protein. With the advance in plant biotechnology, transgenic plants have been targeted as a bioreactor to produce desired recombinant proteins. Here, the expression of recombinant human bmp-2 gene (rhbmp-2) was studied in tobacco plants using gus as a reporter gene. The difference of expression levels in root, stem and leaf tissues was analyzed by GUS activity assay, semi-quantitive RT-PCR and western blotting. The results indicated that the expression levels of fusion protein in root and stem tissues were significantly higher than those in leaf tissue. For the protein compositions in root and stem tissues were simpler than those in leaf tissue, this suggested that the purification process with root and stem tissues would potentially be easier.     

Plant Leaf and Stem Proteins. II. Isozymes and Environmental Cabbage

The activity of 10 enzymes separated by acrylamide disc gel electrophoresis of leaf and stem extracts from Dianthus grown under summer and winter conditions was studied. While banding was constant and highly reproducible under each environment, differences between the 3 cultivars and between the tissues were evident. No significant differences in the isozyme patterns of glutamate dehydrogenase, 6-phosphogluconate dehydrogenase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, and catalase were observed between the 2 environments. Loss of activity was observed under winter conditions with amylase and lactate dehydrogenase and loss of certain isozymic components was evident with acid phosphatase and esterase. Prominent changes were observed in peroxidase isozymes, the hardy cultivars developing additional isozymic components under winter conditions. Only minor changes in the total protein banding were seen. The enzymes showed considerable stability in those tissues killed by the freezing conditions.     

濒危植物革苞菊营养器官的解剖学研究

革苞菊为菊科多年生强旱生草本植物,是蒙古高原植物区系的特有种。本文通过常规石蜡切片法,对革苞菊营养器官进行了形态结构的观察。结果表明：革苞菊叶为等面叶,表皮由1层排列紧密的细胞组成,上下表皮均具气孔,为不规则形;栅栏组织位于上下表皮的内侧,由1层细胞组成;海绵组织由2~3层细胞构成,细胞排列疏松。茎为外韧维管束,可分为表皮、皮层和维管柱三部分,髓发达,髓射线为4~6列细胞。根可分为周皮、皮层、维管柱三部分,根中无髓,导管由中心向四周呈束状辐射排列。在茎与根的薄壁组织中分布着排列整齐的分泌结构。用PAS反应鉴定多糖、考马斯亮蓝鉴定蛋白质后观察了革苞菊的根茎叶中蛋白质和多糖的分布。本文同时讨论了革苞菊营养器官的内部构造与其所处生态环境相适应的特性。     

Genotype,development and tissue-derived variation of cell-wall properties in the lignocellulosic energy crop Miscanthus

Background and Aims

Species and hybrids of the genus Miscanthus contain attributes that make them front-runners among current selections of dedicated bioenergy crops. A key trait for plant biomass conversion to biofuels and biomaterials is cell-wall quality; however, knowledge of cell-wall composition and biology in Miscanthus species is limited. This study presents data on cell-wall compositional changes as a function of development and tissue type across selected genotypes, and considers implications for the development of miscanthus as a sustainable and renewable bioenergy feedstock.

Methods

Cell-wall biomass was analysed for 25 genotypes, considering different developmental stages and stem vs. leaf compositional variability, by Fourier transform mid-infrared spectroscopy and lignin determination. In addition, a Clostridium phytofermentans bioassay was used to assess cell-wall digestibility and conversion to ethanol.

Key Results

Important cell-wall compositional differences between miscanthus stem and leaf samples were found to be predominantly associated with structural carbohydrates. Lignin content increased as plants matured and was higher in stem tissues. Although stem lignin concentration correlated inversely with ethanol production, no such correlation was observed for leaves. Leaf tissue contributed significantly to total above-ground biomass at all stages, although the extent of this contribution was genotype-dependent.

Conclusions

It is hypothesized that divergent carbohydrate compositions and modifications in stem and leaf tissues are major determinants for observed differences in cell-wall quality. The findings indicate that improvement of lignocellulosic feedstocks should encompass tissue-dependent variation as it affects amenability to biological conversion. For gene–trait associations relating to cell-wall quality, the data support the separate examination of leaf and stem composition, as tissue-specific traits may be masked by considering only total above-ground biomass samples, and sample variability could be mostly due to varying tissue contributions to total biomass.     

UV-B-induced Inhibition of Stem Elongation and Leaf Expansion in Pea Depends on Modulation of Gibberellin Metabolism and Intact Gibberellin Signalling

Information on the involvement of elongation-controlling hormones, particularly gibberellin (GA), in UV-B modulation of stem elongation and leaf growth, is limited. We aimed to study the effect of UV-B on levels of GA and indole-3-acetic acid (IAA) as well as involvement of GA in UV-B inhibition of stem elongation and leaf expansion in pea. Reduced shoot elongation (13%) and leaf area (37%) in pea in response to a 6-h daily UV-B (0.45 W m^?2) exposure in the middle of the light period for 10 days were associated with decreased levels of the bioactive GA₁ in apical stem tissue (59%) and young leaves (69%). UV-B also reduced the content of IAA in young leaves (35%). The importance of modulation of GA metabolism for inhibition of stem elongation in pea by UV-B was confirmed by the lack of effect of UV-B in the le GA biosynthesis mutant. No UV-B effect on stem elongation in the la cry-s (della) pea mutant demonstrates that intact GA signalling is required. In conclusion, UV-B inhibition of shoot elongation and leaf expansion in pea depends on UV-B modulation of GA metabolism in shoot apices and young leaves and GA signalling through DELLA proteins. UV-B also affects the IAA content in pea leaves.     

Proteomic analysis of rice leaf, stem and root tissues during growth course

Rice proteins were isolated from leaf, stem and root tissues, harvesting at 1, 2, 4, 8 and 10 weeks after budding. Each tissue of each age was separately pulverized in liquid nitrogen, and the resulted tissue powders were suspended in 10% TCA-acetone and followed by acetone suspension to precipitate at low temperature, which resulted in the tissue-specific and age-specific protein mixture. The protein mixtures were separated by 2-DE using polyacrylamide gels (26 x 20 cm). The protein spots were identified by N-terminal sequence analysis and by MALDI and LC-MS/MS analyses after in-gel tryptic digestion. From a total of 4532 spots, 676 unique proteins were identified, of which 80 proteins (12%) were observed in all three tissues: leaf, stem and root. In addition, 45 (7%) were common in leaf and stem, 57 (8%) in stem and root, and 10 (2%) proteins in root and leaf. Also 141 unique proteins (21%) were observed only for leaf, 96 (14%) for stem, and 247 (36%) for root tissue. Proteins playing a role for photosynthesis and energy production were most abundant in leaf and stem, and those for cell defense were rich in roots.     

Internal and external leaf flavonoids of Pericome caudata

Pericome caudata accumulates four flavonol aglycones externally on leaf and stem surfaces. The glycosides present in the leaf tissue are based on eight further flavonols. This preponderance of tissue flavonoids over exudate flavonoids is unusual.     

Comparative proteomic analysis of leaves between photoperiod-sensitive and photoperiod-insensitive maize inbred seedlings under long day treatments

Day length is an important environmental factor affecting the growth and development of maize (Zea mays), a short day (SD) plant grown in different latitudes. Leaf has been recognized as the light perceiving and signal producing organ. Under long day (LD) conditions, photoperiod-sensitive induction phase in maize begins at the fourth fully expanded leaf stage. However, the changes of maize leaf proteome in response to LD are largely unknown. To reveal maize proteome response to LD, proteins extracted from newly expanded fifth, sixth and seventh leaves from maize inbred line 496-10 (photoperiod sensitive) and Huangzao4 (HZ4, photoperiod insensitive) under LD treatments were compared via gel-based proteomic approach. As a result, eleven differentially expressed proteins were identified between 496-10 and HZ4 by mass spectrometry. This difference in protein accumulation was highly reproducible during the fifth to seventh leaf stages and most obvious at the seventh leaf stage. The identified proteins are mainly involved in circadian clock or iron metabolism, light harvesting and photosynthesis, nucleic acid metabolism and carbon fixation or energy metabolism. This study provides new insight into the influences of LD treatment on SD plants, such as maize, at proteome level.     

Subcellular localization of proteins of Oryza sativa L. in the model tobacco and tomato plants

The cellular localization and molecular interactions are indicative of functions of a protein. The development of a simple and efficient method for subcellular localization of a protein is indispensable to elucidate gene function in plants. In this study, we assessed the feasibility of Agrobacterium-mediated transformation (agroinfiltration) of tobacco and tomato leaf tissue to follow intracellular targeting of proteins from rice fused to green fluorescent protein (GFP). For this, a simple in planta assay for subcellular localization of rice proteins in the heterologous host systems of tobacco and tomato leaf via transient transformation was developed. We have tested the applicability of this method by expressing GFP fusions of the putative antiphagocytic protein 1 (APP1) (OsAPP, LOC_Os03g56930) and ZOS3-18-C2H2 zinc-finger protein (OsZF1, LOC_Os03g55540) from Oryza sativa L. subsp. japonica in tobacco and tomato leaf tissues. Our results demonstrate the suitability of GFP as a reporter in gene expression studies in tomato cv. MicroTom. The use of GFP-fused proteins from rice for subcellular targeting in the heterologous hosts of tobacco and tomato plant systems has been confirmed.Key words: agroinfiltration, confocal microscopy, GFP fusion protein, tomato cv, microtom     

QUANTITATIVE LAWS IN REGENERATION. I

1. Equal masses of sister leaves of Bryophyllum calycinum produce equal masses of shoots and roots in equal time and under equal conditions. 2. The mass of shoots and roots produced by different masses of sister leaves in equal time and under equal conditions is approximately in direct proportion to the masses of the leaves. 3. When a piece of stem inhibits the production of shoots and roots in a leaf of Bryophyllum connected with it, the stem gains in mass and this gain in mass equals approximately the mass of shoots and roots the leaf would have produced if it had been detached from the stem. 4. This suggests that the inhibitory influence of the stem upon the formation of shoots and roots in the leaf is due to the fact that the material available for this process naturally flows into the stem.     

Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm·cm² on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 ± 0.11 x 10^-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.     

Organic Acid contents of soybean: age and source of nitrogen

The organic acid content of soybean (Glycine max v. Hodgson) root, stem, and leaf tissue was followed for 33 days after germination. Malonate was the predominant acid in leaf and root tissue, whereas fumarate was predominant in the stem. The malonate concentrations of the stem and root showed similar variations with time, but the leaf response was quite different. In nodules from 33-day-old plants, malonate was the predominant acid. Malonate levels in root and nodule tissue of 33-day-old plants were depressed in response to the addition of either nitrate or ammonia. Nodule tissue had a higher malonate concentration on nitrate nitrogen than it did on ammonium nitrogen, whereas root tissue had the higher malonate concentration on ammonium nitrogen. Analysis of organic acid concentrations of roots as a function of age and distance from the root tip in young soybean seedlings revealed a zone consistently high in malate. The malonate level in the entire root rose dramatically in tissue of age 96 to 120 hours.     

Anatomical adaptations of Astragalus gombiformis Pomel. under drought stress

The present study was designed to study the effect of drought on root, stem and leaf anatomy of Astragalus gombiformis Pomel. Several root, stem and leaf anatomical parameters (cross section diameter, cortex, root cortical cells, pith, leaf lamina and mesophyll thickness) were reduced under moderate to severe water deficit (20–30 days of withheld irrigation). The stele/cross section root ratio increased under moderate water deficit. The root’s and stems vascular systems showed reduced xylem vessel diameter and increased wall thickness under water deficit. In addition, the root xylem vessel density was increased in these drought conditions while it was unchanged in the stems. The stomata density was increased under prolonged drought conditions whereas the stomata size was untouched. The leaf vascular system showed reduced xylem and phloem tissue thickness in the main vein under moderate to severe water deficit. However, in the lamina the vascular tissue and the distance between vascular bundle were unaffected. Our findings suggest a complex network of anatomical adaptations such as a reduced vessel size with increased wall thickness, lesser cortical and mesophyll parenchyma formation and increased stomata density. These proprieties are required for the maintenance of water potential and energy storage under water stress which can improve the resistance of A. gombiformis to survive in arid areas.     

Leaf production and nitrogen and phosphorus tissue content of Littorella uniflora (L.) Aschers. In relation to nitrogen and phosphorus enrichment of the sediment in oligotrophic Lake Hampen,Denmark

Littoral sediments with a dense population of Littorella uniflora (L.) Ascers. were artificially enriched with nitrogen and phosphorus. Responses to increasing levels of nutrients were recorded as leaf production and tissue nutrient content. Phosphorus enrichment resulted in increasing P concentrations in all plant fractions (leaves, stem and roots), whereas only leaves and roots showed increasing N concentrations when inorganic N was added. Leaf production increased significantly after increasing the sediment P level 2, 3 and 5 times. Nitrogen did not affect leaf production. The tissue content of total N and P in control plants averaged 3.29 and 0.28%, respectively. Thus the critical P level in L. uniflora seems to be about 0.28%, which is higher than previously published values. It was also found that L. uniflora translocates phosphorus from old to younger leaves.     

Direct shoot regeneration from excised leaf explants of in vitro grown seedlings of Alstroemeria L.

A two-step protocol for the induction of shoots from Alstroemeria leaf explants has been developed. Leaf explants with stem node tissue attached were incubated on shoot induction medium for 10 days, and then transferred to regeneration medium. Shoots from the area adjacent to the region between the leaf base and node tissue regenerated within 3 weeks after transfer to the regeneration medium, without a callus phase. The best induction was obtained with Murashige and Skoog medium containing 10 μm thidiazuron and 0.5 μm indole butyric acid. The regeneration medium contained 2.2 μm 6-benzylaminopurine. After several subcultures of the leaf explants with induced shoots, normal plantlets with rhizome were formed. In Alstroemeria, the percentage of responding leaf explants is more important than the number of shoots regenerated per leaf explant, because rhizome formation is the most important factor for micropropagation. The effect of other compounds in the induction medium, including glucose, sucrose, silver nitrate, and ancymidol, on regeneration was also investigated. Received: 14 June 1996 / Revision received: 27 September 1996 / Accepted: 20 October 1996