Cytochemical localization of ATPase activity in phloem tissues ofRicinus communis

Summary Standard lead precipitation procedures have been used to examine the localization of ATPase activity in phloem tissues ofRicinus communis. Reaction product was localized on the plasma membrane of the companion cells associated with sieve elements and of parenchyma cells in phloem tissues from the leaf, petiole, stem and root. ATPase activity was also present on the plasma membrane and dispersed P-protein of sieve elements in petiole, stem and root tissue, but was absent from the plasma membrane of these cells in the leaf minor veins. Substitution of-glycerophosphate for ATP produced no change in the localization of reaction product in leaf tissue. These findings are discussed in relation to current theories on the mechanism of sugar transport and phloem loading.     

Nucleolar organizing region in theApiaceae (Umbelliferae)

Data available for the nucleolus organizing region in the familyApiaceae are reviewed. An attempt has been made to establish the exact number of this region in various subfamilies and tribes through studies on the karyotype and nucleolus. Most of the taxa have a single nucleolar chromosome per haploid complement. The location of the nucleolar organizing region (NOR) on the chromosome varies. Members ofHydrocotyloideae differ drastically from those of subfam.Saniculoideae andApioideae, with respect to the location of NOR. Despite wide geographical distribution, varied ecological preferences and differences in morphology, anatomy and cytology, Umbellifers have attained stability in the number and location of NORs. Characters of NOR offer scope for utilization in understanding phylogenetic relationships at higher levels of taxonomic hierarchy.Studies on the Nucleolus and Nucleolar Chromosomes in Angiosperms XII.     

Relationship between secondary constrictions and nucleolus organizing regions inAllium sativum chromosomes

Summary Allium sativum L. (2 n=16) had three types of clones with regard to the number of chromosomes carrying well-defined secondary constrictions: the first type had two secondary constricted chromosomes (type I), the second had three (type II) and the third had four (type III). Silver staining was applied to these three types of cells to determine the number of nucleolus organizing regions (NORs) per cell and to study the relationship between the morphological appearance of the secondary constrictions and the ability of the chromosomes to form nucleoli. Ag-positive regions appeared on two chromosomes in type I, on three in type II and on four in type III. The comparison of Giemsa and Feulgen stained chromosomes with the silver stained ones clearly indicated that the positive reaction with silver occurred exclusively on the secondary constricted regions that responded negatively to both Giemsa and Feulgen staining, indicating that the size of the achromatic secondary constrictions directly reflects the volume of the Ag-positive materials. However, all three types of clones had a maximum of four nucleoli at interphase. Of the four nucleoli, either two or one was extremely small (less than 1 m in diameter) in types I and II respectively. The size variations of the other nucleoli seemed to be positively correlated with those of the Ag-positive regions. This and the observation that the maximum number of nucleoli per cell did not coincide with the number of Ag-positive regions on the metaphase chromosome complement suggest strongly that the NORs responsible for the minute nucleoli cannot be detected on the metaphase chromosomes. The present observations indicate that not all NORs are indicated by the morphological appearance of secondary constrictions.     

Phloem translocation of gibberellins in three species of higher plants

Phloem sap was collected from white lupin (Lupinus albus L.), cowpea (Vigna unguiculata L.) and castor bean (Ricinus communis L.) and analysed for gibberellins (GAs) using gas chromatography-mass spectrometry (GC-MS). A large number of GAs were found in the phloem exudate of all three species, particularly where the sap was collected from pods (white lupin and cowpea) and in these legumes GAs representing both the early C-13-hydroxylation and non-hydroxylation pathways of biosynthesis were identified. In the sap collected from the vegetative tissues of castor bean the number of GAs identified was fewer than that in the other species, representing mainly the non-hydroxylation pathway. Data from sap collected from the pedicel and stylar ends of pods and by making feeds of radiolabelled GAs to seeds in situ in white lupin indicate that the GAs present in the phloem are derived mainly from the vegetative tissues of the plant. No evidence for metabolism of GAs in the phloem could be found.     

EGTA和酒石酸对蓖麻Cd胁迫与积累的调控作用

通过盆栽试验研究了解毒剂酒石酸与螯合剂EGTA的单施与配施对强化蓖麻修复Cd污染土壤的作用,探讨重金属污染土壤植物修复中螯合剂与解毒剂配合使用的可行性。结果显示:(1)除酒石酸单施处理外,其余处理均可显著提高土壤中醋酸提取态Cd含量,增强土壤Cd的活性,并以酒石酸与EGTA配施的效果更显好,其土壤醋酸提取态Cd含量为对照的1.41~2.49倍。(2)EGTA能有效促进Cd从蓖麻根部向地上部的转移,但高剂量EGTA处理对蓖麻根系有明显的毒害作用;EGTA与酒石酸配合施能缓解Cd对植株的毒害作用,增大蓖麻生物量和Cd积累量,其地上部Cd积累量比对照增加4.56~8.32倍。(3)蓖麻叶片Cd含量、地上部积累总量以及土壤净化率随土壤醋酸提取态Cd含量的升高而增大,并且呈良好的线性递增关系。研究表明,酒石酸与EGTA配施可通过调控土壤Cd的植物可利用性和降低Cd的生理毒性来提高蓖麻对Cd的富集能力和对Cd污染土壤的修复效果。     

Use of mitochondrial DNA as a sensitive and specific marker for localization of mitochondria in fractionated plant extracts

Upon subfractionation of certain plant seed homogenates on sucrose density gradients, we encountered problems in defining the location and amount of mitochondria using marker enzymes. In order to overcome the inherent limitations of enzyme assays, we utilized a heterologous DNA probe specific foratp6 in maize orBrassica tournefortii to detect mitochondria. The samples were treated with SDS, proteinase K, and RNase A followed by agarose gel electrophoresis, and blotting. The immobilized DNA was detected with [³²P]-labelled probes, and quantified using a phosphor imager. The assay is specific, sensitive, and independent of species, cell type, and developmental stage, thus circumventing the need for expressed protein to assay enzyme activity.     

Ricin A Chain from <Emphasis Type="Italic">Ricinus sanguineus</Emphasis>: DNA Sequence,Structure and Toxicity

The gene coding for the ricin A-chain from Ricinus sanguineus (R_sTA) was isolated and sequenced (GB: DQ661048). Comparison of R_sTA with the ricin A-chain from Ricinus communis (RTA) revealed the presence of five differences in the gene sequence. At the protein level only two differences were noticed, the replacement of Asn136 by Ser (N136S) and Ile173 by Val (I174 V). From the R_sTA structure model (PMDB: PM0074652), the N136S mutation was predicted to have no effect on R_sTA structure. The I174V mutation is believed to have no effect on the R_sTA structure or on toxicity since this replacement was found in the Ricinus agglutinin’s A-chain and the latter has a comparable toxicity to RTA. The Ser221 of the putative lipase active site was found. Toxicity experiments showed that R_sTA and RTA have similar toxicities. This finding proves that the N136S and I174 V mutations have no effect on R_sTA toxicity.     

Satellite association of the nucleolar chromosomes in a plant

Summary A method for preparing chromosomes that included enzyme maceration and subsequent flame-drying allowed us to easily detect satellite association in the mitotic cells ofNothoscordum fragrans (2 n=19), which has six acrocentric nucleolar chromosomes in its chromosome complement. Of 593 metaphase plates examined, approximately 60% had satellite association. The number of chromosomes involved in the association varied from two to six, and the incidence decreased as the number of chromosomes involved in the association increased. Comparison of the same chromosomes stained with Giemsa and subsequently with silver demonstrated that the nucleolar organizing regions (NORs) that responded almost negatively to Giemsa and positively to silver was responsible for satellite association. The nucleoli may strongly correlate with satellite association since persistent nucleoli associated with a few metaphase chromosomes were sometimes found and the nucleoli had a strong tendency to fuse with each other at interphase. Four types of acrocentric chromosomes could be discriminated on the basis of the bands negatively staining with Hoechst. All four types were involved in satellite association and there were significant deviations from the expectation for random participation in the association.     

The plant nucleolar cycle under hypoxia

Summary Hypoxia disturbs the nucleolar cycle inAllium cepa L. meristems by diminishing the disorganizing stage and increasing the nucleologenesis time.Though nucleolar remnants persist in the enlarged metaphase recorded under hypoxia, prenucleolar bodies appear at the same time than in control meristems if related to the timing of nucleolar envelope breakage. Such appearance is apparently independent of the chromosome condensation cycle, since it took place in anaphase and not in midtelophase as in controls.Finally, the involvement of NORs in segregation is questioned since prenucleolar bodies are also segregated under hypoxia, both when integrated in the reforming nuclei or when dispersed in cytoplasm.     

Dynamic studies of phloem and xylein flow in fully differentiated plants by fast nuclear-magnetic-resonance microimaging

Summary A fast nuclear-magnetic-resonance imaging method was developed in order to measure simultaneously and quantitatively the water flow velocities in the xylem and the phloem of intact and transpiring plants. Due to technical improvements a temporal resolution of 7 min could be reached and flow measurements could be performed over a time course of 12–30 h. The novel method was applied to the hypocotyl of 35– to 40-day-old, leafy plants ofRicinus communis which were subjected to different light-dark regimes. The results showed that the xylem flow velocities and the xylem volume flow responded immediately to light on-off changes. Upon illumination the flow velocity and the volume flow increased as expected in respect to literature. In contrast, the phloem flow velocity did not change in response to the light-dark regimes. Interestingly, though, the volume flow in the phloem increased during darkness. These findings can be explained by assuming that the conducting area of the phloem becomes enlarged during the dark period due to opening of sieve pores.     

Methyl jasmonate changes the levels of rubisco and other leaf proteins in Ricinus communis

The effect of methyl jasmonate (MJ) on the water-soluble protein pattern of Ricinus communis leaves was analyzed. Several dynamic changes occurred after a period of 24 and 48h including six proteins (M_r 13,000, 15,000, 16,000, 27,000, 29,000 and 60,000) whose levels increased by 48h and seven others (M_r 11,000, 18,000, 20,000 30,000, 37,000, 40,000 and 58,000) whose levels decreased. Four proteins (M_r 24,000, 34,000, 64,000 and 66,000) were induced after 24h of treatment, but returned to control levels by 48h. On the other hand, the levels of three proteins (M_r 74,000, 84,000 and 88,000) decreased after 24h, but returned to control levels after 48h. One of the proteins that accumulated after 48 h had the 13 first residues sequenced. This polypeptide named MJRC-15, was identical to the C-terminal sequence of Rubisco-large chain polypeptide (position 337–350) from tobacco chloroplast. Western-blot analysis using polyclonal antibodies against Rubisco supports the hypothesis that MJRC-15 is a degradation product of Rubisco.     

Behavior of silver stainable material during mitosis inVicia faba

To reveal the behavior of silver stainable material localized mainly in the nucleoli and nucleolar organizing regions (NORs), the somatic cells ofVicia faba were investigated by silver staining throughout the mitotic cell cycle. Nucleoli of interphase and early prophase nuclei were darkly stained. From late prophase to anaphase the secondary constrictions were discriminated as silver stained NORs and many silver grains appeared throughout the cytoplasm. At late prophase the NOR condensed at the same rate as the chromosome arm. Small spherical bodies and two new nucleoli appeared in telophase nuclei and at the same time the cytoplasmic grains disappeared. On the basis of the above observations on the silver stainable material during each mitotic phase, the behavior of silver stainable material is interpreted.     

Monosomic and double monosomic substitutions of Hordeum bulbosum L. chromosomes into H. vulgare L.

Summary One of the aims of the interspecific crossing programme between Hordeum vulgare L. and H. bulbosum L. has been to introgress desirable genes into barley from the wild species. However, despite their close taxonomic relationship there have been few reports of achieving this objective using amphidiploid hybrids. In order to broaden the range of available hybrids, partially fertile triploids between H. vulgare (2n = 2x = 14) and H. bulbosum (2n = 4x = 28) were developed and crossed with H. vulgare as female parent. From 580 progeny which were screened, eight putative single monosomic chromosome substitution lines and one double monosomic substitution were identified by cytological analysis. These involved the substitution of H. vulgare chromosome 1 (two lines), 6 (four lines), 6L (one line), 7 (one line) and 1 + 4 (one line) by their respective H. bulbosum homoeologues. The H. bulbosum chromosome was frequently eliminated during plant development, but it was observed regularly in pollen mother cells of two lines. However, pairing between the H. bulbosum chromosome and its H. vulgare homoeologue was low. Several of the lines were more resistant than their H. vulgare parents to powdery mildew (Erysiphe graminis DC. f.sp. hordei Em. Marchai), net blotch (Drechslera teres Sacc.) and scald (Rhynchosporium secalis (Oudem.) Davis). Apart from their use in studying genome relationships, their value to plant breeders will depend on the ease of inducing translocations between the parental chromosomes.     

The fine structure of chickpea (Cicer arietinum L.) chromosomes as revealed by pachytene analysis

A standard pachytene karyotype of chickpea (Cicer arietinum L.) is presented for the first time. Individual pachytene chromosomes were identified and described in detail. An idiogram was prepared on the basis of chromosome length, arm ratio, and distribution of heterochromatin and euchromatin. Chickpea pachytene chromosomes belong to the differentiated type with darker staining heterochromatin proximal to and lighter staining euchromatin distal to the centromeres. Chromosomes were numbered from 1 to 8 following a descending order of length. The total length of the chromosome complement at pachytene was 335.33 , and chromosome size ranged from 58.05 to 30.53 .     

Stable genetic transformation of castor (<Emphasis Type="Italic">Ricinus communis</Emphasis> L.) via particle gun-mediated gene transfer using embryo axes from mature seeds

The first successful attempt to produce stably transformed castor plants through direct gene transfer using particle gun (BioRad) is described. Decotyledonated embryos from mature seeds were germinated and the embryonic axis was induced to proliferate on Murashige and Skoog (MS) medium supplemented with 0.5 mg l(-1) thidiazuron (TDZ) and subjected to bombardment after 5-7 days of pre-incubation. The physical parameters for transient transformation were optimized using the UidA gene encoding beta-glucuronidase (GUS) as the reporter gene and with hygromycin-phosphotransferase (hptII) gene as selectable marker. Statistical analysis revealed that helium pressure, target distance, osmoticum, microcarrier type and size, DNA quantity, explant type and number of bombardments had significant influence on transformation efficiency, while the effect of genotype was non-significant. Of the different variables evaluated, embryonic axes from mature seeds, a target distance of 6.0 cm, helium pressure of 1,100 psi, 0.6 mum gold microcarriers, single time bombardment and with both pre- and post-osmoticum were found ideal. Selection of putative transformants was done on MS medium supplemented with 0.5 mg l(-1) BA and hygromycin (20, 40 and 60 mg l(-1)) for 3 cycles. The stable integration of the incorporated gene into castor genome was confirmed with PCR and Southern analysis of T(0) and T(1) plants. Transformation frequency in terms of plants grown to maturity and showing the presence of the introduced genes was 1.4%. The present results demonstrate the possibility of transformation of embryonic meristematic tissues of castor through particle delivery system.     

EGTA对Cd胁迫下蓖麻Cd积累和营养元素吸收的影响

以‘淄蓖麻5号’蓖麻品种为材料,通过盆栽试验研究了重度Cd土壤污染（100 mg·kg^-1）条件下,不同浓度（0、0.5、1.0、2.0 mmol·kg^-1）外源螯合剂——乙二醇双(2-氨基乙基醚)四乙酸(EGTA)对蓖麻植株生长、Cd积累和营养元素吸收的影响,探讨外源螯合剂调控Cd污染土壤上植物生长和修复效应。结果显示：（1）在Cd胁迫下,土壤中外源添加0.5~2.0 mmol·kg^-1EGTA使蓖麻根系鲜、干重比不添加EGTA对照不同程度降低,但植株总干重没有受到显著影响。（2）外源EGTA能有效促进Cd从蓖麻根部向地上部的转移,2.0 mmol·kg^-1的EGTA处理使蓖麻叶片Cd 含量显著增加了41.34倍;与不添加EGTA对照相比,外源EGTA处理蓖麻叶片中Cd积累量随添加EGTA的浓度增加而显著大幅度增加14.0~45.6倍,占相应植株总积累量的36.89%~58.63%,而茎中Cd积累量增加幅度较小,根中Cd积累量则显著降低。（3）Cd胁迫条件下,外源EGTA对蓖麻各器官矿质元素含量的影响不一,EGTA促进K向蓖麻地上部的转运,同时抑制Mg向植株地上部转运;随土壤添加的EGTA浓度提高,蓖麻植株对Ca吸收表现为低促高抑,叶片Zn含量和植株Cu含量逐渐增加,叶片和根系Fe含量及植株各器官Mn含量显著增加。与无Cd胁迫对照相比,EGTA在提高植株Cd积累的同时,降低了根系对K的吸收。研究表明,Cd胁迫显著抑制了蓖麻植株的生长,适宜浓度的外源EGTA对Cd的这种抑制有显著的缓解作用;外源EGTA改变了Cd在蓖麻根、茎、叶中的积累分布情况,提高了Cd从根系向地上部,尤其是向叶片的转移能力,从而强化了蓖麻对Cd污染土壤的修复效率;在采用EGTA强化植物修复Cd污染土壤时,应适量增施K肥以保证植株的正常生理代谢。     

The phytoremediation potential of bioenergy crop Ricinus communis for DDTs and cadmium co-contaminated soil

Cadmium (Cd) and dichlorodiphenyltrichloroethane (DDT) or its metabolite residues are frequently detected in agricultural soils and food, posing a threat to human health. The objective of this study was to compare the ability of 23 genotypes of Ricinus communis in mobilizing and uptake of Cd and DDTs (p,p′-DDT, o,p′-DDT, p,p′-DDD and p,p′-DDE) in the co-contaminated soil. The plant genotypes varied largely in the uptake and accumulation of DDTs and Cd, with mean concentrations of 0.37, 0.43 and 70.51 for DDTs, and 1.22, 2.27 and 37.63 mg kg⁻¹ dw for Cd in leaf, stem and root, respectively. The total uptake of DDTs and Cd varied from 83.1 to 267.8 and 66.0 to 155.1 μg per pot, respectively. These results indicate that R. communis has great potential for removing DDTs and Cd from contaminated soils attributed to its fast growth, high biomass, strong absorption and accumulation for both DDTs and Cd.