Differential Expression of Proteins and mRNAs from Border Cells and Root Tips of Pea

Many plants release large numbers of metabolically active root border cells into the rhizosphere. We have proposed that border cells, cells produced by the root cap meristem that separate from the rest of the root upon reaching the periphery of the cap, are a singularly differentiated part of the root system that modulates the environment of the plant root by producing specific substances to be released into the rhizosphere. Proteins synthesized in border cells exhibit profiles that are very distinct from those of the root tip (root cap, root meristem, and adjacent cells). In vivo-labeling experiments demonstrate that 13% of the proteins that are abundant in preparations from border cells are undetectable in root tip preparations. Twenty-five percent of the proteins synthesized by border cells in a 1-h period are rapidly excreted into the incubation medium. Quantitative variation in levels of specific marker proteins, including glutamine synthetase, heat-shock protein 70, and isoflavone reductase, also occurs between border cells and cells in the root tip. mRNA differential-display assays demonstrate that these large qualitative and quantitative differences in protein expression are correlated with similarly distinct patterns of gene expression. These observations are consistent with the hypothesis that a major switch in gene expression accompanies differentiation into root border cells, as expected for cells with specialized functions in plant development.     

Development of Vacuolar Volume in the Root Tips of Pea

Cell and vacuole areas were measured by light microscopy inlongitudinal and transverse sections cut at 0.4-mm intervalsalong the apical 7.2 mm of the primary root of pea. The vacuolararea (or volume) fraction — that is, vacuole area (orvolume) divided by cell area (or volume) — increased fromabout 15 % in cells 0.4 mm from the distal boundary of the apicalmeristem (the cap /root junction), to about 85% in cells situated6.8–7.2 mm from that boundary. At each distance, vacuoledevelopment tended to be greater in the cortex than in the stele.Vacuoles occupied about 22% of the tissue volume in the first1 mm length of root (measured from the cap/root junction), about31 % of the tissue volume in the first 2 mm, and about 45% whensummed over the apical 5-mm length of root. Phosphorus supplyor deprivation produced only minor and non-significant changesin vacuole development. The results have implications affectingprevious estimates of cytoplasmic and vacuolar phosphate concentrationsin pea root tips. Pisum sativum L., pea, root, vacuole, volume     

Histone H2A phosphorylation in DNA double-strand break repair

DNA repair must take place within the context of chromatin, and it is therefore not surprising that many aspects of both chromatin components and proteins that modify chromatin have been implicated in this process. One of the best-characterized chromatin modification events in DNA-damage responses is the phosphorylation of the SQ motif found in histone H2A or the H2AX histone variant in higher eukaryotes. This modification is an early response to the induction of DNA damage, and occurs in a wide range of eukaryotic organisms, suggesting an important conserved function. One function that histone modifications can have is to provide a unique binding site for interacting factors. Here, we review the proteins and protein complexes that have been identified as H2AS129ph (budding yeast) or H2AXS139ph (human) binding partners and discuss the implications of these interactions.     

Histone H2A significantly enhances in vitro DNA transfection.

BACKGROUND: Gene transfer is a potential treatment modality of genetic disease. Efficient, practical methods of DNA transfection are currently under investigation. MATERIALS AND METHODS: A beta-galactosidase reporter plasmid interacted electrostatically with histones, poly-L-Lys, poly-L-Arg, and a combination of poly-L-Lys and poly-L-Arg. This complex was then used to transfect COS-7 cells. beta-galactosidase activity was quantified and used to compare the efficiency of gene transfection in vitro. A comparison was also made of DNA transfection with the most active histone subclass, i.e., histone H2A, in the absence and presence of an anionic liposome. RESULTS: There was a marked increase in DNA transfection in the presence of histone H2A when compared with the control, whereas each of the other histones and polycations showed little, if any, effect. The extent of activation depends strongly on the DNA/histone ratio and is also a function of the molarity of the final Tris-acetate, pH 8, solution. The anionic liposomes used demonstrated an inhibitory effect. CONCLUSIONS: Histone H2A significantly enhances in vitro DNA transfection whereas other histones and anionic liposomes do not. A study of the difference between histone H2A and other histone subclasses may serve to clarify some of the mechanisms and the essential components of efficient gene delivery.     

Effects of Cycloheximide on Indoleacetic Acid-induced Ethylene Production in Pea Root Tips

Cycloheximide inhibited ethylene production in excised pea root tips treated with high levels of indoleacetic acid (100 μm and 10 μm). In contrast, cycloheximide did not inhibit ethylene production induced by a lower concentration (1 μm) of indoleacetic acid unless it was added 2 hours before the indoleacetic acid treatment. These observations suggest that indoleacetic acid has two effects on the enzyme system involved in ethylene synthesis. At low concentrations (1 μm) indoleacetic acid increases ethylene production without protein synthesis, whereas at the higher concentrations, the synthesis of new protein is associated with increased ethylene production.     

Analysis of Cytosine Methylation Pattern in Response to Water Deficit in Pea Root Tips

Abstract: The correlation between environmental stress and DNA methylation has been studied by following the methylation status of cytosine residues in the DNA of pea root tips exposed to water deficit. DNA methylation was evaluated by two complementary approaches: (i) immunolabelling by means of a monoclonal antibody against 5-methylcytosine; (ii) MSAP (Methylation-Sensitive Amplified Polymorphism) to verify if methylation and de-methylation in response to water deficit may be related to specific DNA sequences. Immunolabelling showed that water stress induces cytosine hypermethylation in the pea genome. Regarding the CCGG target sequence, an increase in methylation specifically in the second cytosine (about 40 % of total site investigated) was revealed by MSAP analyses. In addition, MSAP band profile detected in three independent repetitions was highly reproducible suggesting that, at least for the CCGG target sequence, methylation was addressed to specific DNA sequences.     

H+-Independent Glutamine Transport in Plant Root Tips

Background

Glutamine is one of the primary amino acids in nitrogen assimilation and often the most abundant amino acid in plant roots. To monitor this important metabolite, a novel genetically encoded fluorescent FRET-reporter was constructed and expressed in Arabidopsis thaliana. As a candidate for the glutamine fluxes, the root tip localized, putative amino acid transporter CAT8 was analyzed and heterologously expressed in yeast and oocytes.

Principal Findings

Rapid and reversible in vivo fluorescence changes were observed in reporter-expressing root tips upon exposure and removal of glutamine. FRET changes were detected at acid and neutral pH and in the presence of a protonophore, suggesting that part of the glutamine fluxes were independent of the pH. The putative amino acid transporter CAT8 transported glutamine, had a half maximal activity at ∼100 µM and the transport was independent of external pH. CAT8 localized not only to the plasma membrane, but additionally to the tonoplast, when tagged with GFP. Ultrastructural analysis confirmed this dual localization and additionally identified CAT8 in membranes of autophagosomes. Loss-of function of CAT8 did not affect growth in various conditions, but over-expressor plants had increased sensitivity to a structural substrate analog, the glutamine synthetase inhibitor L-methionine sulfoximine.

Conclusions

The combined data suggest that proton-independent glutamine facilitators exist in root tips.     

Effect of Chloride and Sulphate Types of Salinity on the Nicotinamide-adenine-dinucleotides in Pea Root Tips

It was shown that in pea root tips grown in media salinatedwith NaCl the content of NAD + NADH decreased while the contentof NADPH+NADP increased with increasing salinity. In pea roottips, grown in sulphate salinated media, only the decrease inthe content of NAD+NADH was noted. The content of NADPH+NADPin such root tips remained more or less constant. The implicationof these results and of previous results for the explanationof the nature of salinity damage was discussed.     

组蛋白变异体H2A.Z的研究进展

H2A.Z是组蛋白H2A的变异体之一,是高度保守的组蛋白变异体,参与保护常染色体,防止形成异染色质;并且与转录调节、抗沉默、沉默和基因组稳定性有关。组蛋白变异体H2A.Z可能与染色体形成独立的结构域,从而调节染色质结构功能。但是,H2A.Z对染色体结构功能的作用机制还不是很清楚。组蛋白变异体H2A.Z和它的表观遗传修饰对染色体动态结构和功能起重要的作用。该文将对组蛋白变异体H2A.Z进行综述。     

Phosphorus Nutrition and the Intracellular Distribution of Inorganic Phosphate in Pea Root Tips: A Quantitative Study using31P-NMR

Pea plants (Pisum sativum L.) were supplied with external phosphatefor differing periods of time, so that their phosphorus statusvaried, and the intracellular distribution of inorganic phosphate(P₁) in the roots was examined by ³¹P nuclear magnetic resonance.Over the range of phosphorus nutrition investigated, the quantityof vacuolar P₁ per unit fresh weight of root tip changed considerably,whereas the quantity of cytoplasmic P₁ per unit fresh weightof root tip did not alter. The relative volumes of the cytoplasmand the vacuole in pea root tips seemed to be little affectedby differences in phosphorus nutrition, and this implied thatthe concentration of P₁ in the cytoplasm was kept almost constant,at a level estimated to be 18 mM. The rate of absorption of ³²P-labelled phosphate was negativelycorrelated with the vacuolar P₁ concentration, but there wasno clear correlation with the concentration of P₁ in the cytoplasm. Key words: Compartmentation, Cytoplasm, Vacuole, Concentration, Absorption     

Root Formation in Pea Cuttings

Cuttings were either decapitated or both decapitated and disbudded at different time intervals. Auxin, at different concentrations, was applied to the cuttings in lanoline. Auxin applied to decapitated and disbudded cuttings promoted root formation in the early stage of the initiation phase. Auxin treated cuttings, which were only decapitated, did not show an increase in number of roots per cutting. However, an increase in the root mass was found in the early stage of the initiation phase. The results seem to indicate that auxin is active only in the first part of the initiation phase. It is acting alone, not together with other substances synthesized in the shoot meristem.