Regulation of capsule spine formation in castor.

Castor (Ricinus communis L.) is a dicotyledonous oilseed crop that can have either spineless or spiny capsules. Spines are protuberant structures that differ from thorns or prickles. The developmental regulatory mechanisms governing spine formation in castor or other plants have remained largely unknown. Herein, using map-based cloning in 2 independent F2 populations, F2-LYY5/DL01 and F2-LYY9/DL01, we identified the RcMYB106 (myb domain protein 106) transcription factor as a key regulator of capsule spine development in castor. Haplotype analyses demonstrated that either a 4,353-bp deletion in the promoter or a single nucleotide polymorphism leading to a premature stop codon in the RcMYB106 gene could cause the spineless capsule phenotype in castor. Results of our experiments indicated that RcMYB106 might target the downstream gene RcWIN1 (WAX INDUCER1), which encodes an ethylene response factor known to be involved in trichome formation in Arabidopsis (Arabidopsis thaliana) to control capsule spine development in castor. This hypothesis, however, remains to be further tested. Nevertheless, our study reveals a potential molecular regulatory mechanism underlying the spine capsule trait in a nonmodel plant species.

Different acyl-CoA:diacylglycerol acyltransferases vary widely in function, and a targeted amino acid substitution enhances oil accumulation.

Triacylglycerols (TAGs) are the major component of plant storage lipids such as oils. Acyl-CoA:diacylglycerol acyltransferase (DGAT) catalyzes the final step of the Kennedy pathway, and is mainly responsible for plant oil accumulation. We previously found that the activity of Vernonia DGAT1 was distinctively higher than that of Arabidopsis and soybean DGAT1 in a yeast microsome assay. In this study, the DGAT1 cDNAs of Arabidopsis, Vernonia, soybean, and castor bean were introduced into Arabidopsis. All Vernonia DGAT1-expressing lines showed a significantly higher oil content (49% mean increase compared with the wild-type) followed by soybean and castor bean. Most Arabidopsis DGAT1-overexpressing lines did not show a significant increase. In addition to these four DGAT1 genes, sunflower, Jatropha, and sesame DGAT1 genes were introduced into a TAG biosynthesis-defective yeast mutant. In the yeast expression culture, DGAT1s from Arabidopsis, castor bean, and soybean only slightly increased the TAG content; however, DGAT1s from Vernonia, sunflower, Jatropha, and sesame increased TAG content >10-fold more than the former three DGAT1s. Three amino acid residues were characteristically common in the latter four DGAT1s. Using soybean DGAT1, these amino acid substitutions were created by site-directed mutagenesis and substantially increased the TAG content.

Autophosphorylation Inhibits RcCDPK1, a Dual-Specificity Kinase that Phosphorylates Bacterial-Type Phosphoenolpyruvate Carboxylase in Castor Oil Seeds.

Phosphoenolpyruvate carboxylase (PEPC) is a tightly regulated enzyme that plays a crucial anaplerotic role in central plant metabolism. Bacterial-type PEPC (BTPC) of developing castor oil seeds (COS) is highly expressed as a catalytic and regulatory subunit of a novel Class-2 PEPC heteromeric complex. Ricinus communis Ca2+-dependent protein kinase-1 (RcCDPK1) catalyzes in vivo inhibitory phosphorylation of COS BTPC at Ser451. Autokinase activity of recombinant RcCDPK1 was detected and 42 autophosphorylated Ser, Thr or Tyr residues were mapped via liquid chromatography-tandem mass spectrometry. Prior autophosphorylation markedly attenuated the ability of RcCDPK1 to transphosphorylate its BTPC substrate at Ser451. However, fully dephosphorylated RcCDPK1 rapidly autophosphorylated during the initial stages of a BTPC transphosphorylation assay. This suggests that Ca2+-dependent binding of dephospho-RcCDPK1 to BTPC may trigger a structural change that leads to rapid autophosphorylation and subsequent substrate transphosphorylation. Tyr30 was identified as an autophosphorylation site via LC-MS/MS and immunoblotting with a phosphosite-specific antibody. Tyr30 occurs at the junction of RcCDPK1's N-terminal variable (NTVD) and catalytic domains and is widely conserved in plant and protist CDPKs. Interestingly, a reduced rate and extent of BTPC transphosphorylation occurred with a RcCDPK1Y30F mutant. Prior research demonstrated that RcCDPK1's NTVD is essential for its Ca2+-dependent autophosphorylation or BTPC transphosphorylation activities but plays no role in target recognition. We propose that Tyr30 autophosphorylation facilitates a Ca2+-dependent interaction between the NTVD and Ca2+-activation domain that primes RcCDPK1 for transphosphorylating BTPC at Ser451. Our results provide insights into links between the post-translational control of COS anaplerosis, Ca2+-dependent signaling and the biological significance of RcCDPK1 autophosphorylation.

Lupeol Accumulation Correlates with Auxin in the Epidermis of Castor.

Lupeol, a natural lupane-type pentacyclic triterpene, possesses various pharmacological properties, and its production attracts attention. Significant quantities of lupeol are deposited on the castor aerial organ surface and are easily extractable as a predominant wax constituent. Thus, castor might be considered as a potential bioreactor for the production of lupeol. The lupeol biosynthesis pathway is well known, but how it is regulated remains largely unknown. Among large numbers of castor cultivars, we targeted one accession line (337) with high levels of lupeol on its stem surface and low levels thereof on its hypocotyl surface, implicating that lupeol synthesis is differentially regulated in the two organs. To explore the underlying mechanisms, we did comparative transcriptome analysis of the first internode of 337 stem and the upper hypocotyl. Our results show that large amounts of auxin-related genes are differentially expressed in both parts, implying some possible interactions between auxin and lupeol production. We also found that several auxin-responsive cis-elements are present in promoter regions of HMGR and LUS genes encoding two key enzymes involved in lupeol production. Furthermore, auxin treatments apparently induced the expression levels of RcHMGR and RcLUS. Furthermore, we observed that auxin treatment significantly increased lupeol contents, whereas inhibiting auxin transport led to an opposite phenotype. Our study reveals some relationships between hormone activity and lupeol synthesis and might provide a promising way for improving lupeol yields in castor.

Green Synthesis, Characterization, Enzyme Inhibition, Antimicrobial Potential, and Cytotoxic Activity of Plant Mediated Silver Nanoparticle Using Ricinus communis Leaf and Root Extracts.

The need of non-toxic synthesis protocols for nanoparticles arises developing interest in biogenic approaches. The present project was focused on cost effective, environment congenial synthesis of Ag nanoparticles and their biological applications. Leaf and root extracts of Ricinus communis were used as a reducing and stabilizing agent in synthesis process. A Proposed mechanism in published literature suggested that Indole-3-acetic acid, l-valine, triethyl citrate, and quercetin-3-0-p-d-glucopyranoside phytoconstituents of Ricinus communis act as reducing and capping agents. The synthesized Ag NPs were characterized with a help X-ray diffractometer, Transmission electron microscopy, UV-Vis spectrophotometry and Fourier Transform Infrared Spectroscopy (FTIR). The XRD results inveterate the synthesis of pure nano size crystalline silver particles. The FTIR data revealed the possible functional groups of biomolecules involved in bio reduction and capping for efficient stabilization of silver nanoparticles. TEM analysis confirmed the almost spherical morphology of synthesized particles with mean size 29 and 38 nm for R-Ag-NPs (root) and L-Ag-NPs (leaf), respectively. The stability of synthesized nanoparticles was examined against heat and pH. It was observed that synthesized nanoparticles were stable up to 100 °C temperature and also showed stability in neutral, basic and slightly acidic medium (pH 05-06) for several months while below pH 5 were unstable. The synthesized silver nanoparticles had promising inhibition efficiency in multiple applications, including as bactericidal/fungicidal agents and Urease/Xanthine oxidase enzymes inhibitors. The cytotoxicity of synthesized nanoparticles shows that the concentration under 20 µg/mL were biologically compatible.

Phytoremediation of soil heavy metals (Cd and Zn) by castor seedlings: Tolerance, accumulation and subcellular distribution.

Cd and Zn pollution was observed to often occur simultaneously in soils. However, previous studies focused on single heavy metal instead of Cd and Zn combined pollution. Castor (Ricinus communis) is considered to have great potential for contaminated soil remediation. The resistance of castor seedlings to heavy metals and the mechanism behind it remain unknown. In this study, the tolerance and accumulation ability of castor seedlings to Cd and Zn were investigated, and the accumulation mechanism involving the subcellular distribution in different tissues was further explored. The results on biomass and chlorophyll revealed that castor seedlings have good tolerance to the pollution with 0-5 mg/kg Cd and 380 mg/kg Zn, while not to the heavy pollution with 25 mg/kg Cd and 380 mg/kg Zn. The maximum accumulation concentrations of Cd and Zn, 175.3 mg Cd/kg and 386.8 mg/kg Zn, appeared in castor seedling root instead of stem and leaf, indicating that root played a significant part in accumulating Zn and Cd. The relative low dosage of Cd (0-5 mg/kg) promoted the accumulation of Zn in the subcellular component, while high dosage (25 mg/kg) inhibited the accumulation of Zn. In subcellular accumulation and distribution of castor seedlings, Cd (27.1%-69.4%) and Zn (39.6%-66.6%) in the cell wall was the highest. With the increase of Cd addition, the accumulation of Cd increased in cell wall while decreased in organelle and soluble fraction. Hydroxyl, amino, amides and carboxyl functional groups on cell wall might provided the main binding sites for Cd and Zn.

Coupling phytoremediation efficiency and detoxification to assess the role of P in the Cu tolerant Ricinus communis L.

Phosphorous (P) fertilization is an important agronomic practice, but its role in enhancing phytoremediation efficacy and mediating detoxification has rarely been reported in environmental remediation studies. In this study, a pot experiment was undertaken to assess: firstly, the effect of P on phytoextraction of Cu by Ricinus communis L.; secondly, the potential mechanisms by differentiating the effects of the plant from that of P fertilizer (Ca(H2PO4)2); and thirdly, the role of P in physiological detoxification. Results showed that the application of P fertilizer significantly (p ≤ 0.05) increased the plant biomass as well as the Cu concentrations in plant tissues. This enhanced the phytoremediation efficiency represented by the total Cu extraction (up to 121.3 µg Cu plant-1). Phosphorous (P) fertilizer led to a negligible decline in soil pH (0.2 units) but significantly (p ≤ 0.05) reduced the concentrations of soil available in Cu and Fe, due to the formation of insoluble Cu/Fe-phosphate precipitates. Nevertheless, P fertilizer still improved the accumulation and extraction of Cu by R. communis, most likely attributable to the Fe-deficiency induced by applied P fertilizer. Moreover, the application of P fertilizer revealed a significant reduction in MDA, and a profound (p ≤ 0.05) elevation in the amount of photosynthetic pigments, GSH and AsA, along with the enhanced activities of antioxidative enzymes (SOD, POD, and CAT). In this way, Cu toxicity was alleviated. P fertilizers not only enhance the phytoremediation efficiency of Cu-contaminated soils by R. communis, but they also facilitate detoxification, which improves our understanding of the role of P in phytoremediation technologies.

Castor Stearoyl-ACP Desaturase Can Synthesize a Vicinal Diol by Dioxygenase Chemistry.

In previous work, we identified a triple mutant of the castor (Ricinus communis) stearoyl-Acyl Carrier Protein desaturase (T117R/G188L/D280K) that, in addition to introducing a double bond into stearate to produce oleate, performed an additional round of oxidation to convert oleate to a trans allylic alcohol acid. To determine the contributions of each mutation, in this work we generated individual castor desaturase mutants carrying residue changes corresponding to those in the triple mutant and investigated their catalytic activities. We observed that T117R, and to a lesser extent D280K, accumulated a novel product, namely erythro-9,10-dihydroxystearate, that we identified via its methyl ester through gas chromatography-mass spectrometry and comparison with authentic standards. The use of 18O2 labeling showed that the oxygens of both hydroxyl moieties originate from molecular oxygen rather than water. Incubation with an equimolar mixture of 18O2 and 16O2 demonstrated that both hydroxyl oxygens originate from a single molecule of O2, proving the product is the result of dioxygenase catalysis. Using prolonged incubation, we discovered that wild-type castor desaturase is also capable of forming erythro-9,10-dihydroxystearate, which presents a likely explanation for its accumulation to ∼0.7% in castor oil, the biosynthetic origin of which had remained enigmatic for decades. In summary, the findings presented here expand the documented constellation of di-iron enzyme catalysis to include a dioxygenase reactivity in which an unactivated alkene is converted to a vicinal diol.

WRINKLED1 homologs highly and functionally express in oil-rich endosperms of oat and castor.

Oat (Avena sativa) and castor (Ricinus communis) accumulate a large amount of lipids in their endosperms, however the molecular mechanism remains unknown. In this study, differences in oil regulators between oat and wheat (Triticum aestivum) as well as common features between oat and castor were tested by analyzing their transcriptomes with further q-PCR analysis. Results indicated that WRINKLED1 (WRI1) homologs and their target genes highly expressed in the endosperms of oat and castor, but not in the starchy endosperms of wheat. Expression pattern of WRI1s was in agreement with that of oil accumulation. Three AsWRI1s (AsWRI1a, AsWRI1b and AsWRI1c) and one RcWRI1 were identified in the endosperms of oat and castor, respectively. AsWRI1c lacks VYL motif, which is different from the other three WRI1s. Expressions of these four WRI1s all complemented the phenotypes of Arabidopsis wri1-1 mutant. Overexpression of these WRI1s in Arabidopsis and tobacco BY2 cells increased oil contents of seeds and total fatty acids of the cells, respectively. Moreover, this overexpression also resulted in up-regulations of WRI1 target genes, such as PKp-ß1. Taken together, our results suggest that high and functional expression of WRI1 play a key role in the oil-rich endosperms and the VYL motif is dispensable for WRI1 function.

Tissue-specific differences in metabolites and transcripts contribute to the heterogeneity of ricinoleic acid accumulation in Ricinus communis L. (castor) seeds.

INTRODUCTION: Castor (Ricinus communis L.) seeds are valued for their production of oils which can comprise up to 90% hydroxy-fatty acids (ricinoleic acid). Castor oil contains mono-, di- and tri- ricinoleic acid containing triacylglycerols (TAGs). Although the enzymatic synthesis of ricinoleic acid is well described, the differential compartmentalization of these TAG molecular species has remained undefined. OBJECTIVES: To examine the distribution of hydroxy fatty acid accumulation within the endosperm and embryo tissues of castor seeds. METHODS: Matrix assisted laser desorption/ionization mass spectrometry imaging was used to map the distribution of triacylglycerols in tissue sections of castor seeds. In addition, the endosperm and embryo (cotyledons and embryonic axis) tissues were dissected and extracted for quantitative lipidomics analysis and Illumina-based RNA deep sequencing. RESULTS: This study revealed an unexpected heterogeneous tissue distribution of mono-, di- and tri- hydroxy-triacylglycerols in the embryo and endosperm tissues of castor seeds. Pathway analysis based on transcript abundance suggested that distinct embryo- and endosperm-specific mechanisms may exist for the shuttling of ricinoleic acid away from phosphatidylcholine (PC) and into hydroxy TAG production. The embryo-biased mechanism appears to favor removal of ricinoleic acid from PC through phophatidylcholine: diacylglycerol acyltransferase while the endosperm pathway appears to remove ricinoleic acid from the PC pool by preferences of phospholipase A (PLA2α) and/or phosphatidylcholine: diacylglycerol cholinephosphotransferase. CONCLUSIONS: Collectively, a combination of lipidomics and transcriptomics analyses revealed previously undefined spatial aspects of hydroxy fatty acid metabolism in castor seeds. These studies underscore a need for tissue-specific studies as a means to better understand the regulation of triacylglycerol accumulation in oilseeds.

Synthesis and characterization of Zinc oxide nanoparticles utilizing seed source of Ricinus communis and study of its antioxidant, antifungal and anticancer activity.

ZnO nanoparticles have been synthesized using solution combustion technique and its antioxidant, antifungal, anticancer activity was studied. Ricinus communis plant seed extract used as fuel in synthesis by the solution combustion technique. Powder X-ray diffraction (PXRD) demonstrates the arrangement of a crystalline hexagonal stage (ICDD card number 89-1397) with space aggregate P63mc (186) and cell parameters a = b = 3.253, c = 5.213 Å. The normal crystallite measure is 20 nm which is ascertained by Debye - Scherer's formula. The Purity of the sample and metal to oxygen bond development was affirmed by utilizing Fourier transformation infrared (FTIR) spectroscopy and the particle size and shape was confirmed by HRTEM. Antifungal action of ZnO NPs was studied against Aspergillus and Penicillium by well dispersion strategy. The antifungal activity shows that ZnO NPs constitute as an effective fungicidal agent against both Aspergillus (4 ±â€¯0.5 mm) and Penicillium (3 mm ±â€¯0.4 mm) at 30 µg/mL fixation. ZnO nanoparticles were subjected to antioxidant activity. The objective of the study was to analyze the anticancer property of ZnO NPs on MDA-MB 231 cancer cells. To check the efficacy of the synthesized drug ZnO NPs MTT assay was performed, that determines % viability and/or cytotoxicity. IC50 of ZnO NPs in case of MDA-MB-231 breast cancer was 7.103 µg/mL. Anticancer outcome demonstrates that ZnO NPs is active against in MDA-MB-231 cells.

In-Depth Proteome Analysis of Ricinus communis Pollens.

Pollen grains are tiny structures vital for sexual reproduction and consequently seed and fruit production in angiosperms, and a source of many allergenic components responsible for deleterious implications for health worldwide. Current pollen research is mainly focused on unraveling the molecular mechanisms underlying the pollen germination and tube formation passing from the quiescent stage. In this context, an in-depth proteome analysis of the pollens from Ricinus communis at three different stages-that is, mature, hydrated, and in vitro germinated-is performed. This analysis results in the identification of 1950 proteins, including 1773, 1313, and 858, from mature, hydrated, and germinated pollens, respectively. Based on label-free quantification, 164 proteins are found to be significantly differentially abundant from mature to hydrated pollens, 40 proteins from hydrated to germinated, and 57 proteins from mature to germinated pollens, respectively. Most of the differentially abundant proteins are related to protein, carbohydrate, and energy metabolism and signaling. Besides other functional classes, a reasonable number of the proteins are predicted to be allergenic proteins, previously undiscovered. This is the first in-deep proteome analysis of the R. communis pollens and, to the best of our knowledge, one of the most complete proteome dataset identified from the pollens of any plant species, thus providing a reference proteome for researchers interested in pollen biology.

Chromium Accumulation in Medicinal Plants Growing Naturally on Tannery Contaminated and Non-contaminated Soils.

Herbal preparations used to treat human ailments globally can be contaminated with various heavy metals (HMs) originating from the raw materials or from the manufacturing processes. Therefore, we assessed 22 medicinal plants growing naturally on tannery pollutant contaminated (Site-C) and non-contaminated (Site-NC) sites for their ability to accumulate chromium (Cr). The Cr contents in soil and various plant parts were estimated using an atomic absorption spectrophotometer. Translocation and bioconcentration factors were calculated. The soil at Site-C had 27-fold higher concentration of total Cr than at Site-NC. Chromium accumulation is reported for the first time in 50 % of the medicinal plants examined and varied significantly among the sites. Shoots of Ricinus communis and Amaranthus viridis had maximum concentrations of Cr at Site-C, whereas in Site-NC, none of the plants had Cr accumulation >30 ppm. Ricinus communis, Amaranthus viridis, and Amaranthus spinosus had translocation factor (TF) greater than the one in the Site-C and Lantana camara had TF >1 in Site-NC. The bioconcentration factor (BCF) was >1 only for Ricinus communis at both the sites. The majority of the medicinal plants at Site-NC had Cr content exceeding the permissible limit of 2 ppm suggested for herbal raw material. The results of the study clearly emphasize the need for screening plants of therapeutic value for the presence of HMs even when collected from non-contaminated soils. Moreover, proportional allocation of Cr in different plant parts provided an insight on the safety of these parts when specifically used in herbal preparations.

High light exposure on seed coat increases lipid accumulation in seeds of castor bean (Ricinus communis L.), a nongreen oilseed crop.

Little was known on how sunlight affects the seed metabolism in nongreen seeds. Castor bean (Ricinus communis L.) is a typical nongreen oilseed crop and its seed oil is an important feedstock in industry. In this study, photosynthetic activity of seed coat tissues of castor bean in natural conditions was evaluated in comparison to shaded conditions. Our results indicate that exposure to high light enhances photosynthetic activity in seed coats and consequently increases oil accumulation. Consistent results were also reached using cultured seeds. High-throughput RNA-Seq analyses further revealed that genes involved in photosynthesis and carbon conversion in both the Calvin-Benson cycle and malate transport were differentially expressed between seeds cultured under light and dark conditions, implying several venues potentially contributing to light-enhanced lipid accumulation such as increased reducing power and CO2 refixation which underlie the overall lipid biosynthesis. This study demonstrated the effects of light exposure on oil accumulation in nongreen oilseeds and greatly expands our understanding of the physiological roles that light may play during seed development in nongreen oilseeds. Essentially, our studies suggest that potential exists to enhance castor oil yield through increasing exposure of the inflorescences to sunlight either by genetically changing the plant architecture (smart canopy) or its growing environment.

The multigene family of lysophosphatidate acyltransferase (LPAT)-related enzymes in Ricinus communis: cloning and molecular characterization of two LPAT genes that are expressed in castor seeds.

The multigene family encoding proteins related to lysophosphatidyl-acyltransferases (LPATs) has been analyzed in the castor plant Ricinus communis. Among them, two genes designated RcLPAT2 and RcLPATB, encoding proteins with LPAT activity and expressed in the developing seed, have been cloned and characterized in some detail. RcLPAT2 groups with well characterized members of the so-called A-class LPATs and it shows a generalized expression pattern in the plant and along seed development. Enzymatic assays of RcLPAT2 indicate a preference for ricinoleoyl-CoA over other fatty acid thioesters when ricinoleoyl-LPA is used as the acyl acceptor, while oleoyl-CoA is the preferred substrate when oleoyl-LPA is employed. RcLPATB groups with B-class LPAT enzymes described as seed specific and selective for unusual fatty acids. However, RcLPATB exhibit a broad specificity on the acyl-CoAs, with saturated fatty acids (12:0-16:0) being the preferred substrates. RcLPATB is upregulated coinciding with seed triacylglycerol accumulation, but its expression is not restricted to the seed. These results are discussed in the light of a possible role for LPAT isoenzymes in the channelling of ricinoleic acid into castor bean triacylglycerol.

Cadmium tolerance and its phytoremediation by two oil yielding plants Ricinus communis (L.) and Brassica juncea (L.) from the contaminated soil.

The effect of increasing level of cadmium in soil was investigated on biomass production, antioxidants, Cd bioaccumulation and translocation in Ricinus communis vis-à-vis a commonly studied oil crop Brassica juncea. The plants were exposed to 25, 50, 75, 100, and 150 mg Cd/Kg soil for up to 60 days. It was found that R. communis produced higher biomass at all the contamination levels than that of B. juncea. Proline and malondialdehyde in the leaves increased with increase in Cd level in both the species, whereas soluble protein decreased. The bioaccumulation of Cd was higher in B. juncea on the basis of the per unit biomass, total metal accumulation per plant was higher in R. communis. The translocation of Cdfrom roots to shoot was also higher in B. juncea at all Cd concentrations. R. communis appeared more tolerant and capable to clean Cd contaminated soil for longer period in one sowing than B. juncea and the former can grow in wasteland soil also in which later cannot be cultivated.

Inoculation of Ni-resistant plant growth promoting bacterium Psychrobacter sp. strain SRS8 for the improvement of nickel phytoextraction by energy crops.

This study was conducted to elucidate effects of inoculating plant growth-promoting bacterium Psychrobacter sp. SRS8 on the growth and phytoextraction potential of energy crops Ricinus communis and Helianthus annuus in artificially Ni contaminated soils. The toxicity symptom in plants under Ni stress expressed as chlorophyll, protein content, growth inhibition, and Fe, P concentrations were studied, and the possible relationship among them were also discussed. The PGPB SRS8 was found capable of stimulating plant growth and Ni accumulation in both plant species. Further, the stimulation effect on plant biomass, chlorophyll, and protein content was concomitant with increased Fe and P assimilation from soil to plants. Further, the induction of catalase and peroxidase activities was also involved in the ability of SRS8 to increase the tolerance in both plant species under Ni stress. The findings suggest that strain SRS8 play an important role in promoting the growth and phytoextraction efficiency of R. communis and H. annuus, which may be used for remediation of metal contaminated sites.

[Microbiological characteristics of phytoremediation plant root-soil interface for petroleum contaminated soil].

With the petroleum-contaminated soil under Ricinus communis L. phytoremediation in Shenfu irrigation area of Shenyang as test material, this paper studied the quantitative variations of bacteria and fungi, physiological and biochemical characteristics of dominant microbial species, and nutritional types of bacteria in root zone, rhizosphere, root plane and root inside. The results showed that the quantity of bacteria decreased in the order of rhizosphere > root plane > root inside, while that of fungi was in the sequence of root inside > root plane > rhizosphere. The dominant species of bacteria and fungi were most abundant in root plane and root inside, respectively, and the dominant bacterial species in root plane and root inside had a stronger capability in degrading macro-molecular substances. The nutritional demand of bacteria in root plane was of amino acid type.

Phloem metabolism and function have to cope with low internal oxygen.

We have investigated the consequences of endogenous limitations in oxygen delivery for phloem transport in Ricinus communis. In situ oxygen profiles were measured directly across stems of plants growing in air (21% [v/v] oxygen), using a microsensor with a tip diameter of approximately 30 microm. Oxygen levels decreased from 21% (v/v) at the surface to 7% (v/v) in the vascular region and increased again to 15% (v/v) toward the hollow center of the stem. Phloem sap exuding from small incisions in the bark of the stem was hypoxic, and the ATP to ADP ratio (4.1) and energy charge (0.78) were also low. When 5-cm stem segments of intact plants were exposed to zero external oxygen for 90 min, oxygen levels within the phloem decreased to approximately 2% (v/v), and ATP to ADP ratio and adenylate energy charge dropped further to 1.92 and 0.68, respectively. This was accompanied by a marked decrease in the phloem sucrose (Suc) concentration and Suc transport rate, which is likely to be explained by the inhibition of retrieval processes in the phloem. Germinating seedlings were used to analyze the effect of a stepwise decrease in oxygen tension on phloem transport and energy metabolism in more detail. Within the endosperm embedding the cotyledons-next to the phloem loading sites-oxygen decreased from approximately 14% (v/v) in 6-d-old seedlings down to approximately 6% (v/v) in 10-d-old seedlings. This was paralleled by a similar decrease of oxygen inside the hypocotyl. When the endosperm was removed and cotyledons incubated in a 100 mM Suc solution with 21%, 6%, 3%, or 0.5% (v/v) oxygen for 3 h before phloem sap was analyzed, decreasing oxygen tensions led to a progressive decrease in phloem energy state, indicating a partial inhibition of respiration. The estimated ratio of NADH to NAD(+) in the phloem exudate remained low (approximately 0.0014) when oxygen was decreased to 6% and 3% (v/v) but increased markedly (to approximately 0.008) at 0.5% (v/v) oxygen, paralleled by an increase in lactate and ethanol. Suc concentration and translocation decreased when oxygen was decreased to 3% and 0.5% (v/v). Falling oxygen led to a progressive increase in amino acids, especially of alanine, gamma-aminobutyrat, methionine, and isoleucine, a progressive decrease in the C to N ratio, and an increase in the succinate to malate ratio in the phloem. These results show that oxygen concentration is low inside the transport phloem in planta and that this results in adaptive changes in phloem metabolism and function.

Evidence for high activity of xylem parenchyma and ray cells in the interface of host stem and Agrobacterium tumefaciens-induced tumours of Ricinus communis.

Rapidly developing tumours at hypocotyls of Ricinus communis, induced by Agrobacterium tumefaciens strain C58, were characterized by strong differentiation of vascular bundles and their functional connection to the host bundles. The stem/tumour interface showed increased xylem, with numerous vessels accompanied by multiseriate unlignified rays. To know how nutrients efficiently accumulate in the tumour sink tissue, cell electropotentials (E(m)) in cross-sections were mapped. The measured cells were identified by injected Lucifer Yellow. Xylem and phloem parenchyma cells and stem/tumour-located rays hyperpolarized to E(m) values of about -170 mV, which suggest high plasma membrane proton pump activities. Rapidly dividing cells of cambia or small tumour parenchyma cells had low E(m). The tumour aerenchyma and the stem cortex cells displayed values close to the energy-independent diffusion potential. The lowest values were recorded in stem pith cells. Cell K(+) concentrations largely matched the respective E(m). The pattern of individual cell electropotentials was supplemented by whole organ voltage measurements. The voltage differences between the tumour surface and the xylem perfusion solution in stems attached to the tumours, the trans-tumour electropotentials (TTP), confirm the findings of respiration-dependent and phytohormone-stimulated high plasma membrane proton pump activity in intact tumours, mainly in the xylem and phloem parenchyma and ray cells. TTPs were inhibited by addition of NaN(3), CN(-) plus SHAM or N(2) gas in the xylem perfusion solution and by external N(2) flushing. The data provide functional evidence for the structural basis of priority over the host shoot in nutrient flow from the stem to the tumour.