Growth Substances in the Roots of Vicia faba II. THE EFFECTS OF AGEING AND EXCISION OF THE MAIN TAP-ROOT MERISTEM

A quantitative chromatographic study has been made of the changesin the activity and distribution of the main ether-soluble acidauxins of Vicia faba seedling root systems during developmentand resulting from excision of the main tap-root meristem. AnIAA-like auxin (AP (ii)) is apparently synthesized predominantlyin apical and at a lower rate in lateral meristems. Productionseems to stop when meristematic growth stops. Its concentrationin mature extended cells is much lower and may fall to zeroin old cells, suggesting active degradation by an auxin-oxidase.Excision of the main tap-root tip gradually results in a greatlyaugmented production of AP (ii) in lateral meristems, conceivablythe result of correlative growth promotion. A second auxin and root-growth inhibitor (AP (iii)) is presentat higher activity levels than AP (ii) in tap-root meristemsand at the same level in lateral meristems. In mature cellsits activity is much lower than that of AP (ii). In contrastto AP (ii) it accumulates in both tap-root meristems and maturetissue as the root system ages. It could also be produced duringmeristematic growth but is not subsequently degraded. As withAP (ii), excision of the tap-root tip brings about a great increasein its concentration in lateral tips. A third auxin and root-growth accelerator (AP (i)) (accelerator?) is present in lower concentrations in both meristem and maturetissue. Its concentration tends to decrease with ageing and,in lateral meristems, is not affected by tap-root tip excision. It is suggested that AP (ii) produced by the meristem is normallyat suboptimal levels in the extending cells and may be the principalhormone controlling extension growth. AP (iii) accumulationmay account for growth deceleration on ageing. The role of AP(i) remains obscure. It is unlikely that correlative effectsof the taproot tip on lateral root growth are exercised directlyvia these auxins.     

几种信号类物质对蚕豆气孔运动的效应

用表皮生物分析方法,研究H2O2、NO和多胺对蚕豆叶片气孔开放和关闭的单独及其综合效应.其结果表明:H2O2和NO明显促进气孔关闭,抑制气孔开放.腐胺(Put)效应相对较弱,与H2O2和NO未表现出明显叠加效应.     

Changes in the Chromosome Complements of Cells of Vicia faba Roots following Irradiation

1. Primary roots of Vicia faba were irradiate after one day'sgermination. The X-ray dose was 620 r 2. Cells with changed chromosome complements appeared in lateralroots. It is inferred that these cells are descended from cellsdamaged by irradiation; chromosome breakage was seen in thefirst mitoses in the irradiated roots but was not scored. 3. Six types of chromosomal rearrangements were found. One typehad been reported previously in primary roots of V. faba afterX-raying. 4. The new complements appear to be stable because they occurseveral cells of a lateral. Affected laterals could containup to five new complements. The primordium of such a lateralmust have consisted of five changed and at least one normalcell. It had previously been suggested that a root of Viciahad three or four initial cells. 5. The survival of the cells with changed complements is notdue solely to successful competition with, not to support by,normal cells, otherwise other types of changes should also havebeen found. 6. It is suggest that the changes found were perpetuated becausethey were induced in initial cells of the primary root, andthat cells in which they were present beca,e part of the primordialand continued as initial cells in apical meristems of lateralroots. 7. Apical cells appear to have a lower sensitivity than othermeristematic cells, so providing, after treatment, a reservoirof normal cells from which regeneration can occur.     

Distribution of Gibberellins and Abscisic Acid in Geotropically Stimulated Vicia faba Roots

The occurrence of gibberellins and abscisic acid (ABA) in extracts of roots of Vicia faba was demonstrated by gas-liquid chromatography (GLC) of the methylated eluates from the relevant zones of thin-layer chromatograms (TLC) of purified extracts. Quantitative determination of the hormone contents in extracts from upper and lower halves of roots which had been kept in the horizontal position for 30 min indicated a redistribution of the hormones during the geotropic stimulation. Gibberellins whose methyl esters appeared at the retention time of methylated gibberellic acid (GA₃), used as a standard, occurred in higher concentration in the upper than in the lower halves (ratio 2.08:1), whereas the concentration of ABA was highest in the lower halves (ratio 3.08:1). The ratio of the hormones in right and left halves of vertical roots was close to 1:1. Indoleacetic acid (IAA) and ABA were found to retard the elongation of roots of Vicia faba and Lepidium during the first 24 h. Additional experiments with Lepidium showed that this retardation occurs within the first hour after application. Low concentrations of GA₃, when applied to germinating seeds just after the radicles had broken the seed coat, stimulated root elongation in Vicia faba within 24 h and in Lepidium within 36 h. When applied to Lepidium seedlings with 20 mm long roots, GA₃ showed a stimulatory trend within the first 2 h, and distinct stimulation in the subsequent hours, particularly at the lowest concentrations, 0.01 and 0.001 mg/1. These results suggest the possibility of a participation of ABA and gibberellins (in addition to IAA) in the development of the positive geotropic curvature.     

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.     

A Differential Response to Colchicine of Meristems of Roots of Vicia faba

In whole root systems of Vicia faba, including primaries andlaterals, the meristems do not respond in a uniform manner totreatment with colchicine. The meristems of the primary andfully emerged lateral roots become mixoploid and these rootsshow a temporary inhibition of growth. Lateral roots that emergewithin 48 hours of treatment show no effects of colchicine;their growth is not inhibited and they contain few or no polyploidcells. The cells of the primordia that produce these lateralsappear to be insensitive to colchicine: at the insensitive stagethese primordia contain at least 1, 000 cells. Primordia withless than 800 cells are very sensitive to colchicine. They donot recover from treatment and appear to be completely inhibited.The stages of extreme sensitivity and resistance are transientphases in the morphogenesis of a lateral root. The change insensitivity to colchicine is accompanied by a fall in the mitoticindex; this is highest in young primordia, where it is abouttwice the value found in growing laterals.     

Axial Resistance to Water Movement in Broad Bean (Vicia faba) Roots

A new potometer method was devised to measure the rates of wateruptake by small portions of an intact root system. An analysisof the relative effects of radial resistance (calculated fromthe potometer data) and axial resistance (calculated from thexylem dimensions) snowed that axial resistance was not likelyto be important. Root water potentials were determined by findingthe osmotic potential of the solution in the potometer whichjust prevented water uptake by the root. Root-tip water potentialswere similar in long and short roots suggesting that any effectof axial resistance was similar in both types of root.     

Para-nitrophenyl Phosphate as a Substrate for Some Acid Phosphatases in Roots of Vicia faba

A cytochemical and biochemical study of acid phosphatases inroots of Vicia faba using p-nitrophenylphosphate(p-NPP) as substratehas shown the lack of specificity of the substrate which canbe acted upon by a K⁺-activated acyl phosphatase, nucleotidepyrophosphatase, glucox-6-phosphatase, 5' (3')-ribonucleotidephosphohydrolase, nucleotide phosph-transferase and those acidphosphatases demonstrable with ß-glycerophosphate.It is suggested that care should be taken in the interpretationof both biochemical and cytochemical studies employing sucha non-specific substrate as P-NPP. Vicia faba, broad bean, root, acid phosphatases     

Meristem Initial Cells in Irradiated Roots of Vicia faba: With one Figure in the Text

Beans (Vicia faba) were germinated for 24 hours and irradiatedwith X-rays. Primary roots were fixed after 9, 11, and 21 days.Aberrant chromosome complements, the result of chromosome changesinduced by irradiation, were present. They were used as cellmarkers to estimate the number of cell types present in themeristem initial cells of the growing root and the number ofprimordium initial cells from which root regeneration occurred.Up to 9 cell types occur as meristem initials. From the relativefrequencies of the different cell types, it is estimated thatthere are at least 32 actual meristem initial cells in regeneratingirradiated primary roots. This result is compatible with observationsmade on normal roots. The chimaerical nature of the regeneratingroot apparently does not interfere with the normal organizationof the meristem, but it serves to reveal what part of the organizationis.     

Nucleic Acid Metabolism of Vicia faba during Germination and Growth

During the growth of Vicia faba seedlings in the absence of an external nitrogen supply, the cotyledons decreased rapidly in dry weight and nucleic acid content. In the developing shoot the dry weight increased rapidly for four weeks and then very slowly over the next two weeks growth; the nucleic acid content of the shoot increased to a maximum after 4 weeks growth and decreased in amount during the next 2 weeks. On the other hand the roots increased in both dry weight and nucleic acid content throughout the growth period, although they only accounted for a small proportion of the total dry weight and nucleic acid content of the plant. These changes during germination and growth are discussed in relation to those occurring during these developmental stages in other plants.     

An Analysis of Cell Proliferation in the Apical Meristem of Lateral Roots of Vicia faba L.

The relative proportions of the various proliferating and quiescentcells, cell doubling time, mean cycle time and the durationof the mitotic cycle and its various phases as measured fromthe passage of labelled cells through mitosis have been determinedfor the initial cells of the cap, epidermis, cortex and stele,for the epidermis together with the cortex and for the steleat various distances basal to the cap-quiescent centre boundaryin 1-cm long lateral roots of Vicia faba. Cell doubling timegenerally increased basally along these tissues as a resultof a gradual decrease in the size of the proliferating populationof cells. Cycle time of the fast-dividing cell population, however,was less between 750 and 800 µm basal to the cap-quiescentcentre boundary than in the corresponding initial cells largelyas a result of a decrease in the duration of G1, although changesalso took place in the durations of the other phases of themitotic cycle as cells were displaced basally along the root.From data reported in this paper as well as other results inthe literature, it appears that the proportions of quiescentcells arrested in G1 and G2 vary in the different groups ofinitial cells. Moreover, the proportion arrested in G1 appearsto decrease basally along each tissue, while that in G2 increases.     

Proliferating and Quiescent Cells in the Apical Meristem of Elongating Lateral Roots of Vicia faba L.

Whole root systems of Vicla faba were continuously exposed to³H-TdR for periods of up to 72h, following which LI was determinedin the cap initials, in the quiescent centre or in that partof the meristem in which a quiescent centre will develop, andin the stele and in the cortex-epidermis at intervals alongthe apical 800 µm basal to the junction between the capinitials and the rest of the meristem, in newly-emerged (NE),0.2 and 4.0 cm long lateral roots, after each exposure period.Cell doubling time (Td), mean cycling time (Tc) and the sizeof the growth fraction (GF) were then calculated for each partof the meristem investigated in each batch of roots, from thecurves recording increase in labelling index (LI) with increasein duration of the period of exposure to ³H-TdR and from therate of increase in LI over the initial l-12h labelling period.Since it is extremely difficult to eliminate all sources oferror in calculating GF from the values obtained for LI in continuouslabelling experiments, it is emphasized that the values of GFreported in the present paper may not be totally accurate. Thisis also true of the results obtained for Tc as Tc was derivedfrom the product of the corresponding values for Td and GF. Cell doubling time and mean cycling time were both longer inthe cells forming the quiescent centre in the 0.2 and 40 cmlong roots than in any other part of the apical meristem examined.The size of the GF was found to decrease basally along the steleand the cortex-epidermis from the most apical to the most basalsegment examined in the NE, while Td increased in duration.Similar changes took place along the stele of the 0.2 cm longlaterals, but not in the cortex-epidermis of these roots or,to any great extent, in any of the tissues examined in the 4.0cm secondary roots. No consistent trend was apparent in theduration of Tc basally along either tissue examined in theseroots. It was concluded from these results, and from supportingdata in the literature, that, as the laterals elongated fromNE to 4.0 cm, the apical meristem increased in length.     

Agrobacterium-mediated transformation of Vicia faba

Among the major grain legume crops, Vicia faba belongs to those where the production of transgenic plants has not yet convincingly been reported. We have produced stably transformed lines of faba bean with an Agrobacterium tumefaciens-mediated gene transfer system. Stem segments from aseptically germinated seedlings were inoculated with A. tumefaciens strains EHA101 or EHA105, carrying binary vectors conferring (1) uidA, (2) a mutant lysC gene, coding for a bacterial aspartate kinase insensitive to feedback control by threonine, and (3) the coding sequence for a methionine-rich sunflower 2S-albumin, each in combination with nptII as selectable marker. Kanamycin-resistant calluses were obtained on callus initiation medium at a frequency of 10–30%. Shoot regeneration was achieved on thidiazuron containing medium in a second culture step. A subsequent transfer of shoots to BA-containing medium was necessary for stem elongation and leaf development. Shoots were rooted or grafted onto young seedlings in vitro and mature plants were recovered. Molecular analysis confirmed the integration of the transgenes into the plant genome. Inheritance and expression of the foreign genes was demonstrated by Southern blot, PCR, western analysis and enzyme activity assays. Although at present the system is time-consuming and of relatively low efficiency, it represents a feasible approach for the production of genetically engineered faba beans.     

Nitrogen Metabolism of Vicia faba

Vicia faba plants were grown for four and six weeks without externally supplied nitrogen. Some nitrogen was transported to the plant axis from the cotyledons throughout this period, but the amount available was insufficient to support maximum shoot growth. During this period the protein content of the shoot declined whilst the free amino acids, especially aspartic acid, glutamic acid, histamine and the combined pool for threonine, serine, asparagine and glutamine and ammonia, increased in amount. In contrast to the shoot the protein content of the root increased as did their free amino acid content, but the increase in the latter was less than in the shoot and only the combined value for threonine, serine, asparagines and glutamine increased significantly. During tbe last two weeks growth, some soluble non-amino acid compound appeared to donate nitrogen to the pool of free amino acids in the root and shoot.     

Effect of Exogenous DNA on Acid Deoxyribonuclease Activity in Intact Roots of Vicia faba L.

Exogenous DNA has been administered to roots of Vicia faba inwhich this DNA stimulates an increase in acid deoxyribonucleaseactivity. This enzyme activity is associated with vesicles whichfollow whatappears to be the cytological pathway of foreignDNA in host tissues.     

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.     

Glutamine synthetase in the chloroplasts of Vicia faba

Summary A glutamine synthetase has been localised in the chloroplasts of Vicia faba. The enzyme has requirements for Mg²⁺ and ATP in the biosynthetic reaction and in addition will catalyse a -glutamyl transferase reaction in the presence of Mn²⁺ and arsenate. The enzyme is inhibited by AMP, CTP, glycine and alanine. These results are discussed in relation to the possible chloroplastic synthesis of nucleotide bases. Estimations of glutamine amide-2-oxoglutarate amino transferase (oxido-reductase) have demonstrated only low levels of activity in the chloroplast extracts. This enzyme is generally active in organisms where GS has an assimilary role. It is coneluded that glutamine synthetase has a biosynthetic and not an assimilatory role in the chloroplast.