Lateral root formation in Vicia faba L.

MI was found to decrease in LP with increase in cell number, and reached minimal values just before the emergence of LP to form lateral roots. These changes in MI have been correlated with the accumulation of cells in G1, 24 hours before a lateral root is formed. The durations of C and the various phases of the mitotic cycle were also investigated in LP, and compared with those in small primordia. As lateral root primordia increase in cell number, the durations of C, S and G2 become longer, while G1 becomes shorter. Also MI and GF decrease while the proportion of quiescent cells increases. Thus, there is a gradual decrease in the rate of cell proliferation as primordia increase in size. The changes which take place in these parameters during lateral root primordium development have been compared with the events which occur in seed proliferating tissues at the onset of dormancy.     

Tissue specification and intracellular distribution of actin isoforms inVicia faba L.

Summary Two dimensional gel electrophoresis of total cell protein extracts from not expanded, and primary leaves, petioles, and roots ofVicia faba resulted in four actin isoforms at 43 kDa with pI values from 5.9 to 6.05. In contrast to root extracts, in all leaf extracts an additional immunoreactive polypeptide with a molecular mass of 51 kDa and pI 5.75 was detected. This polypeptide was present in high amounts in protein extracts of purified chloroplasts, whereas no actin isoform at 43 kDa could be demonstrated. Compared to the tissue extracts, two actin isoforms at 43 kDa with pI values of 5.9 and 6.0 were enriched, when purified plasma membranes and the membranous fraction of vacuoles were analysed. In contrast, the soluble protein fraction of the plasma membrane preparation contained only two isoactins with pI values of 5.95 and 6.05 and a molecular mass of 43 kDa. These results indicate, that the four actin isoforms at 43 kDa detected in all examined tissues ofV. faba fulfill different functions at specific intracellular compartments, for example, the anchorage of actin microfilaments to membranes.Abbreviations BSA bovine serum albumin - BCIP 5-bromo-4-chloro-3-indolyl phosphate - DDM n-decyl -D-maltopyranoside - EDTA ethylenediamine-tetraacetic acid - HG n-hexyl -D-glucopyranoside - IEF isoelectrical focusing - MES morpholinoethanesulfonic acid - 2-ME 2 mercaptoethanol - NBT nitro blue tetrazolium - pCMB p-chloromercuribenzoic acid - PVP polyvinylpyrrolidone - Tris tris (hydroxymethyl) aminomethane     

Stereological analysis of cotyledon tissue inVicia faba L. var. aquadulce during germination

Summary Stereological analysis was carried out on cotyledon tissue at three different stages of germination. The tissue was selected by reference to adjacent sections which were assayed histochemically for protease activity. Day six tissue had no activity while eight and 14-day tissue did. Results show that during germination both tissue and cellular components undergo changes. The cells increase in size, the intercellular spaces increase, the cytoplasm increases in volume, the protein bodies swell and fuse, and small starch grains appear while the large starch grains do not undergo any major changes.     

Ethylene-enhanced ethylene oxidation inVicia faba

The alkene oxygenase (AO) of fababean (Vicia faba L.) converts ethylene to ethylene oxide. Treatment of fababeans with 10μl/liter ethylene increases the activity of this enzyme within 2 hours of ethylene treatment. Though other alkenes were taken up by fababean seedlings, ethylene was the most active in inducing AO activity. The ability of ethylene to increase AO was blocked 60% by cycloheximide, an inhibitor of protein synthesis, and 35% by AgNO₃, an inhibitor of ethylene action.     

Embryo rescue inVicia faba andVicia narbonensis

Embryo rescue in twoVicia faba L. cultivars (‘Polycarpe’ and ‘A-107’) and oneV. narbonensis L. population (A-202) was studied under a 22 ± 2°C/ 16 ± 1° day/night temperature regime. Very young ovules (1.0–1.8 mm long) cultured, in-ovule, on five liquid media remained green for a longer period of time on modified B5, modified Murashige and Skoog and modified Beasley and Ting media than on modified Phillips and Collins and modified Bourgin and Nitsch media. However, no embryo growth or embryo germination was observed. In-ovule culture of older ovules, 6 and 8 days forV. narbonensis and 10 and 14 days forV. faba, on modified B5 liquid medium allowed 6-day-oldV. narbonensis and 14-day-oldV. faba embryos to be rescued. Finally, culture of whole pods of the two species resulted in the rescue of even younger embryos. Thus, plantlets were obtained from as young as 4-day-oldV. narbonensis pods and 11-day-oldV. faba pods.     

Occurrence of (+)-7-iso-jasmonic acid inVicia faba L. and its biological activity

The plant growth regulator (–)-jasmonic acid (JA) and its stereoisomer (+)-7-iso-jasmonic acid (7-iso-JA) have been isolated from young fruits ofVicia faba L. and identified by TLC, GC, GC-MS, and chemical transformation. Different isolation procedures gave different ratios of JA/7-iso-JA because of partial isomerization of (+)-7-iso-JA to (–)-JA. Optimal conditions, excluding artificial isomerization, were checked by the addition of [U-¹⁴C]-7-iso-JA. The naturally occurring isomer ratio was determined to be 65% (–)-JA: 35% (+)-7-iso-JA in immature broad bean fruits. The biological activities of both isomers of JA have been studied using several bioassays. Growth of wheat and GA₃-stimulated dwarf rice seedlings is inhibited more effectively by (+)-7-iso-JA than by (–)-JA. In growing seedlings of barley and oat, both isomers caused a senescence-like bleaching effect characterized by chlorophyll and carotenoid decrease. The free acids are more active than their methyl esters in intact barley plants in contrast to results obtained in leaf segment tests. Highest activity was obtained with (+)-7-iso-JA.     

Position and extent of the elongation zone in the root tip of the broad beanVicia faba L.

It was found by measuring the length of the cortex cells of the root tips of the broad beanVicia faba L. that the beginning of the elongation zone lies at about 1–2 mm from the initials and its end at about 7–8 mm from the initials. Shrinkage of the object during microtechnical treatment was negligible. The autonomy of the individual tissues of the root tip was taken into account.     

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.     

A comparative study of ethylene oxidation inVicia faba andMycobacterium paraffinicum

Vicia faba L. ‘Herz Freya’ (fababean) cotyledons andMycobacterium paraffinicum Bardane strain (MPB) cells were studied to describe and compare physiological and biochemical factors regulating ethylene oxidation. Both organisms demonstrated a linear rate of ethylene uptake as a function of concentration from 1 ppm to 1,000 ppm. CO₂ did not influence ethylene oxidation by either organism. Zero degree temperatures and CO inhibited ethylene oxidation by fababeans but not by MPB. An N₂ gas phase blocked ethylene consumption by fababeans. In contrast, MPB continued to consume ethylene at a reduced rate under anaerobic conditions. Hydrocarbon oxidation was limited to alkenes. Alkanes were not oxidized by either organism. Both organisms were sensitive to diethyldithiocarbamic acid, o-phenanthroline, carbonyl cyanidem-chlorophenyl hydrazone, and CS₂. The possibility that CS₂ acted as a suicide substrate is discussed. Evidence is presented that hydrocarbon gas oxidation by fababeans is not a part of, or reflection of, the way ethylene acts as a hormone.     

The influence of incorporated bromodeoxyuridine on mutagenicity testing by sister chromatid exchange induction inVicia faba root tip cells

The induction of sister chromatid exchanges (SCEs) inVicia faba root-tip cells after short-term (2 h) and long-term (24 h) treatments with alkylating agents (N-methyl-N-nitrosourea, ethyl methanesulphonate) and maleic hydrazide was studied. The primary roots were treated with mutagens before or after 5-bromodeoxyuridine (BrdU) incorporation into DNA and the influence of mutagen application on SCE induction in the cells with non- and BrdU-substituted chromosomal DNA. On the contrary, application of maleic hydrazide after the incorporation of BrdU into DNA strongly increased the rate of SCEs. The lowest limit concentrations of mutagens capable of significantly increasing SCE frequency in the cells with non-substituted DNA after the long-term treatment were estimated.     

Cell Expansion during the Elongation of Lateral Roots of Vicia faba L.

Cell width, breadth, length, cross-sectional area, volume andthe ratio of the volume of the nucleus to that of the cell havebeen determined in the epidermis, cortex and stele over theapical mm of lateral roots of Vicia faba as they elongated fromjust-emerged to 4 cm in length. Cell volume increased basallyalong the root in each tissue, but this was not a result ofcell expansion taking place in all dimensions in the epidermis,cortex and stele. Thus, while increase in cell length, was themajor factor involved in cell volume increase basally alongthe root in the stele, the corresponding dimensions in the epidermisand cortex were cell width and cell breadth respectively. Cellvolume was greater in the cortex than in the other tissues andusually greater in the epidermis than in the stele. Cell lengthwas greater in the stele than in the other tissues, while cellbreadth was maximal in the cortex and cell width in the epidermis.Changes took place in the various measurements made as the rootselongated. These are discussed with respect to the onset oflateral root growth and to changes in the rate of growth ofthese roots as they elongate.     

Cap Formation during the Elongation of Lateral Roots of Vicia faba L.

Cell proliferation was examined in the cap initials of newly-emerged0·2 and 4·0 cm long lateral roots of Vicia fabafrom an investigation of the passage of labelled cells throughmitosis following a 1-h pulse with tritiated thymidine. Celldoubling time was found to increase in this group of initialcells as the secondary roots elongated, this increase beinga result of a gradual lengthening in the duration of the mitoticcycle of both the fast and slow cycling meristematic cells andof a decrease in the size of both the growth fraction and thoseproliferating cells with a short cycle time. The rate of cellproduction by the cap initials was maximal in the newly emergedsecondary roots and showed a gradual decline with subsequentlateral elongation. This change in the rate of cell proliferationin the cap initials has been shown to be related to the initiationof the quiescent centre following lateral root emergence fromthe tissues of the primary. The number of cells making up the cap of 0·2–4·0cm long secondary roots is known to be constant and thus therate at which cells are sloughed off of the cap into the soilmust be the same as the rate of cap cell formation in theseroots, all of the cap cells being replaced every 6–9 days.The rate of cap cell formation in 0·2–4·0cm long secondary roots was used to calculate that one 11-dayold Vicia plant will have contributed between 56 000 and 85000 cap cells to the rhizosphere from its root system sincegermination.     

Interaction of chromatid lesions induced by N-nitroso-N-methylurea and N-nitroso-N-ethylurea inVicia faba

After treatment of primary roots ofVicia faba with N-nitroso-N-methylurea (NMU) and afterwards with N-nitroso-N-ethylurea (NEU) only an additive effect has been found. When NEU was used for the first treatment and NMU was applied afterwards an interaction of induced lesions was observed in the production of interchanges. These results might be interpreted as follows: NMU induces primary chromatid lesions in a later stage of interphase than NEU, so that these lesions cannot be induced in the same cell, if NMU is applied first. Because of the complexity of events leading to aberrations, a lot of which are unknown, our interpretation should be accepted with due reserve and cannot be more than a working hypothesis for further experimental tests.     

Electrophoretic investigation of proteins in different root zones ofvicia faba L.

The differentiation of tissues is closely connected with the proteosynthesis. One can therfore assume that tissues with different types of cell growth (meristematic or elongation growth) and with different degrees of differentiation are different in their protein composition. In order to compare the protein composition of different plant organs, the method of disc electrophoresis on a polyacrylamide gel has been used by some authors. As compared with other methods used up to now, e. g. isolating proteins on DEAE cellulose or in Sephadex, this method does not need so much material and its resolution ability seems to be higher. It is also quicker and enables the study of several samples simultaneously. Its disadvantage is that proteins can be identified mainly by means of Rf and their quantity, measured from the intensity of staining of individual fractions in the gel, which may be misleading due to different sorption capacity of different proteins (Fri?Fri?ová 1967). None the less, it is good for comparison of protein composition of individual parts of the plant body. Different methods have been used to compare protein composition of individual growth zones in roots.Barsky,Ivanov andPushakova (1965) used luminiscence microscopy and found that in maize roots it is not possible to find substantial differences by this method.Morgan andReith (1954) arrived at similar conclusions. On the other hand,Steward et al. (1965) andMorris (1966) found qualitative differences in protein composition of different parts of pea roots using acrylamide electrophoresis. The results of the last named authors show considerable discrepancies in details, due perhaps to a different method of extraction (buffer, pH, purifying method). We have used acrylamide gel electrophoresis for investigating proteins in precisely defined growth zones of theVicia faba root.     

Tritiated-thymidine Labelled Nuclei in Primordia and Newly-emerged Lateral Roots of Vicia faba L.

The utilization of ³H-TdR in DNA synthesis and the distributionof labelled nuclei have been determined in large primordia (LP)and newly emerged lateral roots (JE) of Vicia faba followingdifferent exposure periods. This nucleoside appears to reachLP and the more basal, unemerged parts of JE by way of the vasculartissue of the primary root, while those parts of JE which projectout from the epidermis of the primary obtain ³H-TdR directlyfrom the surrounding medium. The utilization of ³H-TdR in thesynthesis of DNA in basal segments of JE is also complicatedby the temporary quiescence of nuclei in this part of thesemeristems as LP elongate to form lateral roots.     

The Emergence and Early Growth of the Lateral Root in Vicia faba L.

The duration of the mitotic cycle, as well as the proportionof cells with long and short cycle times and quiescent cells,have been investigated in the apical meristems of young lateralroots of Vicia faba. No changes took place in the duration ofC or in the phases of the mitotic cycle as the lateral rootemerged from the primary root, though the proportion of proliferatingcells increased and the quiescent fraction of cells decreased.It is suggested that the low frequency with which newly emergedlateral roots label with ³H-TdR is a result of the formationof a large endogenous pool of TdR in the meristems during theperiod they are temporarily quiescent. The changes which tookplace in the parameters of cell proliferation during the earlygrowth of the lateral root have been correlated with those inroot apical meristems following the onset of seed germination.     

The Development of a Quiescent Centre in Lateral Roots of Vicia faba L.

During lateral root elongation there was a reduction in thenumber of cells present in the apical mm of the root, in thewidth of the root and in the number of rows of cells acrossthe root. At the same time, a quiescent centre, which was absentfrom large primordia and from lateral roots which had just emergedfrom the tissues of the primary root, was established. The quiescentcentre increased in size and cell number as the lateral rootelongated from 0·2 to 4 cm, the most rapid increase takingplace in roots between 0·2 and 0·5 cm in length.There was no correlation between root width and size of thequiescent centre in these roots. The cells in the quiescentcentre were smaller in size and had a lower labelling indexthan cells in other parts of the meristem. The distributionof labelled nuclei within the quiescent centrewas determinedand is discussed with respect to the site of root initial cells.     

Cytological localization of fast renaturing and satellite DNA sequences inVicia faba

Summary DNA sequences reassociating within a Cot value of 1.8×10^–1 and those producing a light satellite in a CsCl density gradient were isolated fromVicia faba DNA and hybridizedin situ on squashes of roots of the same species. Silver grains were seen to be scattered over both the interphase nuclei and the metaphase chromosomes after hybridization with fast renaturing DNA sequences, indicating these are fairly regularly interspersed in theV. faba genome. Clustered labeling occurred after hybridization with satellite DNA sequences, indicating these are clustered in the genome. The localization of satellite DNA in chromosomes appeared to correspond closely to the position of the bright bands detectable after staining with quinacrine mustard. After hybridization with both DNA probes, labeling intensity over the nuclei of meristematic cells was higher than that over the nuclei of differentiating and/or differentiated cells. These results are discussed in relation to the structure of the cell nucleus, the mechanism of quinacrine banding and to previous data suggesting underrepresentation of nuclear repeated DNA sequences in differentiatingV. faba root cells.     

Lateral root emission in woody taproots of Fraxinus ornus L.

Abstract

We applied environmental stresses, namely dehydration, pruning and bending, to woody taproots of Fraxinus ornus L. in order to: (i) identify a method that could be applied in routine studies of lateral root development from a secondary structure; and (ii) carry out anatomical investigations to identify the tissue involved in the recruitment of lateral root mother cells (LRMC). We found that all three methods induce the formation of new lateral roots from a woody parental root. However, bending stress considerably reduced the zone of the woody parental root (the curvature) for analysis when studying the process of emission of a new lateral root. The trace left by a new lateral root in the taproot secondary xylem forms a V-shaped insertion zone that starts in contact with a growth ring and enlarges toward the periphery. This type of insertion zone suggests that the vascular cambium is the tissue-source of initials that produce the root primordium of a new lateral root. In the case of root bending, the emission of a new lateral root occurs also in the convex side of the curvature and is preceded by the formation, at the same site, of a small amount of reaction wood. Thus, reaction wood and lateral root emission are two aspects of the same response mechanism to bending. Consequently, anatomical and cytological studies of lateral root formation should focus on this part of the woody taproot. By peeling off the bark at this site, one has direct access to the underlying living tissues and can thus investigate lateral root formation also at a biochemical and molecular level.