IS TRIGONELLINE A PLANT HORMONE?

Experimental results are presented so that trigonelline may be evaluated as a plant hormone. Trigonelline promotes preferential cell arrest in G2 of the cell cycle for about 40% of the cell population in root meristems of Pisum sativum. Trigonelline is present in ungerminated seeds and is transported from cotyledons to other tissues during early seedling development. These experimental results show that trigonelline satisfies all six criteria that have been used to establish whether a substance is a hormone. As the seedlings age from day 3 to 10, the concentration of trigonelline in meristems decreases and so does the proportion of cells arrested in G2. Trigonelline may be isolated from excised cotyledons and can be added back to decotyledonized seedlings or excised root meristems to have the same effect as found in intact organisms. Predominant cell arrest in G2 occurs in roots of some plant species, although other species show preponderant cell arrest in G1. Many members of the Preiss-Handler metabolic pathway show some ability to promote cell arrest in G2 but only at concentrations 100 (10^-5 m ) or 1,000 (10^-4 m ) times the concentration of trigonelline (10^-7 m ) necessary for function. The proportion of cells arrested in G2 is highly correlated with the concentration of trigonelline within the root meristem.     

IS THE NUCLEAR DNA CONTENT OF MATURE ROOT CELLS PRESCRIBED IN THE ROOT MERISTEM?

Cells of the mature root exhibit arrest within the G1 and G2 periods of the mitotic cycle. The number of cells arrested with a 2C or 4C DNA amount in mature tissue was compared with that in meristems of excised primary root tips deprived of carbohydrate. Results from four plant species are described. Cells in mature tissue of seedling roots of Vicia and Pisum exhibited arrest predominately at the 4C while those of Triticum and Helianthus arrested preponderantly at the 2C DNA level. The proportion of cells arrested at the 2C and 4C levels in mature root tissue was specific for each species tested. In each species the cycle stage where most cells arrested was the same in carbohydrate-deficient root meristems as in mature root tissue; consequently, most meristematic cells are preconditioned or predetermined to arrest in a specific mitotic period. A test system was developed in Pisum in which the predominant period of arrest was altered by the removal of the cotyledons. The predominant arrest period changed from 4C to 2C in both mature root tissue and carbohydrate-deficient root meristems with cotyledon removal and indicated that mature root cells are preconditioned while meristematic as to where they will eventually arrest in the mitotic cycle.     

DEVELOPMENTAL ASPECTS OF ROOTS OF PISUM SATIVUM INFLUENCED BY THE G2 FACTOR FROM COTYLEDONS

A G2 Factor present in cotyledons of Pisum sativum influences several developmental events in roots. G2 Factor present in dry seeds (cotyledons and radicles) is transported to roots after germination and promotes cell arrest in G2 in about 35% of all root meristem cells. Present evidence suggests the G2 Factor promotes cell arrest in G2 only in cells that undergo normal cell differentiation (arrest) because the proportion of cells labeled with ³H-TdR after 16 hr does not differ among both seedlings or excised roots in the presence or absence of this substance. In this manner, trigonelline differs from chalones of animal tissues that usually suppress cell proliferation by cell arrest either in G1 or in G2. Experimental results suggest that cortex cells and not cells of vascular tissues in mature root tissues (20–22 mm from the meristem) are influenced by G2 Factor. Other recent publications indicate that the G2 Factor is trigonelline (N-methyl nicotinic acid) and concentrations of synthetic trigonelline from 10⁻⁵ to 10⁻⁷ m are effective in promoting cell arrest in G2 in one of the G2 Factor bioassays.     

IS THE PREDOMINANT PERIOD OF CELL ARREST AND PRESENCE OF ENDOREDUPLICATED CELLS COINCIDENT WITH PRODUCTION OF SECONDARY VASCULAR TISSUES IN INTACT AND CULTURED ROOTS OF RAPHANUS SATIVUS L.?

Experimental results show that predominant cell arrest in G2 and the presence of endoreduplicated cells are coincident with presence of secondary vascular tissues while preponderant cell arrest in G1 and absence of polyploid cells are coincident with an absence of secondary vascular tissues in mature root tissues of intact and cultured roots of Raphanus sativus L. In mature tissues of intact seedling roots, most cells arrest in G2, and both polyploid cells and secondary vascular tissues are present. If excised roots are grown on simple medium, most mature cells arrest in G1, none undergo endoreduplication, and only primary vascular tissues are present. When bases of these excised roots are later placed in a medium with auxin, cytokinin, and myo-inositol that produces secondary vascular tissues in vitro, preponderant cell arrest occurred in G2 with some polyploid cells. The general relationship of predominant period of cell arrest, presence of polyploid cells, and presence of secondary vascular tissues in mature roots among plants of various taxa is surveyed.     

The age-distribution of cell cycle populations in plant root meristems : Complex tissues

The durations of the mitotic cycle periods, the proportions of cells in each cycle period of the proliferative and nonproliferative populations, and the rates of cell progression from G1-S and from G2-M were used to characterize the cytokinetics in root meristems of four plant species. The observed age-distribution of cells in the cycle of each meristem was not comparable to either a theoretical exponential or uniform age-distribution. A more exact fit of the observed age-distributions with theoretical distributions was hindered by proliferative cells which halted temporarily in G1 and/or in G2 in relative proportions similar to the nonproliferative cells in the meristem. Moreover, the preponderant cycle period in which cells halted temporarily differed among the species observed: it was G1 in Helianthus and Triticum and G2 in Pisum and Vicia. The cell populations of these complex tissues can be subdivided kinetically into three types: (1) rapidly proliferating cells; (2) slowly proliferating cells that halt temporarily in G1 and/or in G2; and (3) nonproliferating cells that halt in G1 and/or in G2.     

CELL CYCLE KINETICS OF ENDOREDUPLICATION IN GAMMA-IRRADIATED ROOT MERISTEMS OF PISUM SATIVUM

Label and mitotic indices and microspectrophotometry of unlabeled interphase cells were used to measure the proportion of root meristem cells of Pisum sativum in each cell cycle stage after exposure to protracted gamma irradiation. Three seedling types were investigated: 1) intact seedlings, 2) seedlings with cotyledons detached and treated with lanolin paste applied to the area of cotyledon excision, and 3) seedlings with detached cotyledons and treated with a G2 Factor applied to the area of cotyledon excision in lanolin paste. In intact seedling meristems, predominant cell arrest occurred with a 4C amount of DNA while 0.30 of the cells underwent endoreduplication to arrest with an 8C amount of DNA. Only 0.07 cells arrested with a 2C amount of DNA. Polyploid cells were produced several days after the start of irradiation and were derived from a diploid cell population. In seedlings exposed to lanolin only, without cotyledons, most cells arrested with a 2C amount of DNA with no polyploid cells. In seedlings exposed to a G2 Factor in lanolin after cotyledon excision, most cells arrested with a 4C amount of DNA but no cells underwent endoreduplication. These experimental results suggest that the G2 Factor derived from cotyledons of Pisum sativum was necessary for predominant cell arrest in G2 but alone was not sufficient for the polyploidization step.     

PROMOTION OF CELL ARREST IN G2 DURING CELL MATURATION IN ROOTS OF PISUM SATIVUM BY EXOGENOUSLY APPLIED CYCLIC AMP

Cyclic AMP and dibutyryl cyclic AMP promoted cell arrest in G2 in roots during tissue maturation in seedlings of Pisum sativum when applied in lanolin after cotyledon excision. 5' AMP did not promote arrest in G2 in a similar manner. Results demonstrate that cyclic AMP did not inhibit cell proliferation in root meristems when applied exogenously.     

Trigonelline and promotion of cell arrest in G2 of various legumes

Trigonelline, present in dry seeds of Pisum sativum, is transported to enlarging roots and shoots during early seedling ontogeny and promotes cell arrest in G2 in 40% of all root cells. In the absence of trigonelline, this cell population arrests in G1. Results presented herein show that trigonelline also promotes cell arrest in G2 in roots of Glycine max and Phaseolus vulgaris and that the percentage of cells that arrest in G2 in roots of G. max decreases during seedling ontogeny, as it does in P. sativum. During development, trigonelline is synthesized in leaves and is translocated to pods and eventually to seeds during fruit maturation in P. sativum and G. max. Seeds of most legumes have high concentrations of trigonelline and those of some non-legumes have low concentrations.     

Nicotinic acid and nicotinamide metabolism and promotion of cell arrest in G2 in Pisum sativum

Nicotinic acid and nicotinamide are immediate precursors of trigonelline, a hormone present in cotyledons of Pisum sativum L. which promotes cell arrest in G2 during cell maturation in roots and shoots. All three compounds are members of the pyridine nucleotide pathway for the synthesis of NAD and NADP. Concentrations of nicotinic acid and nicotinamide in excised roots grown for 3 days in White's medium with sucrose were determined by HPLC. Results suggest that nicotinamide is rapidly converted first to nicotinic acid and then trigonelline. High nicotinic acid concentrations may occur in excised roots. Conversion of trigonelline to nicotinic acid in excised roots did not occur in these experiments. The concentrations of either nicotinamide or nicotinic acid in roots are not related to the proportions of cells arrested in G2. Trigonelline promotes cell arrest in G2, and nicotinic acid and nicotinamide are active only because they are converted to trigonelline.     

The response of apical meristems of primary roots of Vicia faba L. to colchicine treatments

The effects of 0.5% and 0.025% solutions of colchicine on the passage of cells through the mitotic cycle in apical meristems of primary roots of Vicia faba have been examined. Both treatments affected cell progression through the mitotic cycle in the same way: S and G1 were shorter, and G2 and mitosis longer, than the corresponding control values. The duration of the various phases of the mitotic cycle were similar to those reported previously for apical meristems of lateral roots though cycle time itself was longer. Recovery of root proliferating tissues from colchicine-induced inhibition of growth is correlated with the presence of quiescent cells. Meristems which have no quiescent cells do not recover from eolchicine treatment, while meristems which contain many quiescent cells recover faster than those which contain few. The growth fraction and the proportion of proliferating cells with a short cycle time are linearly related to the duration of the S period in root meristems.     

NUCLEAR DNA CONTENT OF EMBRYONIC RADICLES AND CULTURED STATIONARY PHASE ROOT TIPS OF PEA (PISUM SATIVUM L.)

Cells in mature embryos and stationary phase (SP) root meristems of pea arrest in G1 and G2 of the cell cycle. The patterns of distribution of G2 nuclei in radicles and SP meristems, with and without G2 factor, were compared by using cytophotometric analysis of the relative amount of DNA/nucleus in sectioned material. Radicles and SP meristems were each divided into 5 zones and the ratio of G1 to G2 nuclei was determined for each zone. The G2 population in the radicle is restricted mainly to the embryonic cortex. A small part of the G2 population was located in the central cylinder and the root cap. In SP meristems without G2 factor, the pattern of distribution of G2 cells was similar to that in radicles. SP meristems with G2 factor contained G2 arrested nuclei in all regions of the root tip. In each region the percentage of G2 nuclei was higher than that in the same region of SP meristems without G2 factor. This indicates that the population of cells that responds to G2 factor is distributed throughout the root tip.     

Structure and growth of infection threads in the legume symbiosis with Rhizobium leguminosarum

Specific antibodies and enzyme–gold probes were used to study the structure and development of infection threads in nodules induced by Rhizobium leguminosarum on the roots of Vicia, Pisum and Phaseolus. In Pisum nodules, the tubular infection thread wall contains polysaccharides antigenically similar to those of the cell wall, including cellulose, xyloglucan, methyl-esterified pectin and non-esterified pectin, but none of these wall components is present around the infection droplet structures from which bacteria are internalized by plant plasma membrane. As reported previously for pea nodules, the luminal matrix of infection threads and infection droplets contains a plant glycoprotein; this glycoprotein is also secreted by infected and uninfected cortical cells of a Vicia root at the earliest stages of nodule initiation. Synthesis of a transcellular infection thread apparently involves reorganized deposition of components normally targeted to the cell wall, and infection thread growth is orientated anticlinally through the outer cortex in the same plane observed for the deposition of new cell walls following mitosis. Both the development of infection threads in the outer cortex and the initiation of cell division in the inner cortex are preceded by a similar process of cell reactivation involving centralization of nuclei and the development of anticlinal transvacuolar strands. It is therefore suggested that the two Rhizobium-induced processes of infection thread growth and cortical cell division may both be consequences of a similar plant cell response in the inner and outer root cortex, respectively. Phaseolus nodules contained only short intracellular infection structures which terminated within individual cells and contained no luminal matrix material. The differences in infection thread structure between Pisum and Phaseolus nodules may reflect differences in ontogeny between “indeterminate” and “determinate” nodule meristems.     

Cell division and DNA topisomerase I activity in root meristems of pea seedlings during water stress

ABSTRACT

Low water potential, generated by PEG addition to the liquid medium of hydroponically grown pea seedlings, induces a fall in moisture content in the roots, followed by the arrest of elongation. This water stress reduces the mitotic index of root meristems during the treatment and induces the appearance of a peak of mitosis at 12 hours from the beginning of recovery. This peak suggests that during water stress the cell cycle is blocked in G2 or late S phase. In a first attempt to understand the biochemical events leading to cell cycle arrest, we tested the in vitro activity of DNA topoisomerase I extracted from stressed or control root meristems. The activity of this enzyme in extracts from stressed seedlings was lower than in controls, whereas it was higher in extracts from seedlings which had recovered from water stress for a few hours. The highest specific activity was observed with seedlings at 24 hours from the start of recovery. The fact that during stress treatments and recovery there was no variation in the synthesis of a 45 kDa protein, indicated as DNA topoisomerase I, suggested that the activity of this enzyme could be posttranslationally regulated. The hypothesis that variations in the concentration of unknown endogenous regulators of the activity of this enzyme may take place during water loss or uptake in the cytosol of meristematic cells is discussed.     

IS POLYPLOIDY NECESSARY FOR TISSUE DIFFERENTIATION IN HIGHER PLANTS?

Measurements of relative DNA per nucleus of cells from various tissues show that cell differentiation can occur in the absence of polyploidy in higher plants. In Pisum polyploidy was present in roots, sepals, pods, pistils, and stamens but not in petals or leaves. In Triticum cells of leaves exhibited some polyploidy, but no polyploid cells were present in mature roots. No polyploid cells were found in any tissue of Helianthus examined (roots, cotyledons, stems, leaves, sepals, petals, pistils, and stamens). Therefore, as a general rule, polyploidy should not be considered essential in tissue or organ differentiation of higher plants. In Helianthus polyploidy is unnecessary for the completion of the life cycle.     

A natural substance that regulates the cell cycle in complex plant tissues

A natural substance which regulates the cell cycle of seedling roots of Pisum sativum has been isolated and identified as 1-(3-(4,5-dihydro-2-furanone)-5-(hydroxymethyl)pyrrole-2-carboxyaldehyde. This compound interacts with trigonelline to determine the percentages of cells in G1 and in G2 in pea root meristems. Both purified natural and synthetic compounds are active at concentrations of 5 × 10⁻⁶ M.     

Relationship Between Root Morphogenesis and Period of Predominant Cell Arrest in Intact and Aseptically-cultured Roots of Helianthus annuus L.

Root morphogenesis and cell cycle kinetics of intact and aseptically-grownexcised roots of Helianthus annuus L. were studied. Intact rootsshow predominant cell arrest in G1 with an absence of polyploidcells coincident with secondary vascularization. Exposure ofthe cut ends of aseptically grown excised roots to known concentrationsof indol-3-yl acetic acid, benzyladenine, and myo-inositol for8 weeks initiated the production of secondary vascular tissuesand predominant cell arrest in G2 concommitant with poiyploidization.Excised roots grown in the absence of these substances producedroots with only primary vascularization and predominant cellarrest in G1 coincident with an absence of polyploidization.These results indicate that (a) root cells of H. annuus havethe ability to undergo polyploidization that may be inducedby exogeneously applied chemicals, (b) a general relationshipbetween predominant cell arrest in G1 coincident with the absenceof secondary vascularization does not hold true and (c) althoughsecondary vascularization occurs in cultured roots exposed toall three additives similar to secondary vascularization inintact roots, the two roots should not be considered identicalin all respects. Helianthus annus L., sunflower, root, morphogenesis, cell cycle kinetics, polyploidy, cell differentiation, vascularization     

SHORT-ROOT 1 is critical to cell division and tracheary element development in rice roots

The exocyst is a key factor in vesicle transport and is involved in cell secretion, cell growth, cell division and other cytological processes in eukaryotes. EXO70 is the key exocyst subunit. We obtained a gene, SHORT-ROOT 1 (SR1), through map-based cloning and genetic complementation. SR1 is a conserved protein with an EXO70 domain in plants. SR1 mutation affected the whole root-development process: producing shorter radicles, adventitious roots and lateral roots, and demonstrating abnormal xylem development, resulting in dwarfing and reduced water potential and moisture content. SR1 was largely expressed in the roots, but only in developing root meristems and tracheary elements. The shortness of the sr1 mutant roots was caused by the presence of fewer meristem cells. The in situ histone H4 expression patterns confirmed that cell proliferation during root development was impaired. Tracheary element dysplasia was caused by marked decreases in the inner diameters of and distances between the perforations of adjacent tracheary elements. The membrane transport of sr1 mutants was blocked, affecting cell division in the root apical region and the development of root tracheary elements. The study of SR1 will deepen our understanding of the function of EXO70 genes in Oryza sativa (rice) and guide future studies on the molecular mechanisms involved in plant root development.     

Lateral Root Anlage Development in Excised Roots of Vicia faba L., Pisum sativum L., Zea mays L. and Phaseolus vulgaris L.

The development of lateral root primordia has been investigatedin excised roots of Vicia faba, Pisum sativum, Zea mays andPhaseolus vulgaris cultured in White's medium supplemented with2 per cent sucrose and compared with previously published dataon such development in primaries of the corresponding intactplants (control roots). Primordia were produced in each batchof excised roots over the 6 day culture period but at a lowerrate (number day^–1) than in the controls. Such primordia in cultured roots of Zea and Phaseolus completedtheir development and grew out as lateral roots over a periodsimilar in length to that found in the controls, but with acell number of only about 33 per cent of that attained at thetime of secondary emergence in the primaries of the latter roots.These lower cell numbers were at least partly a reflection ofincreases in mean cell doubling time over the period of anlagedevelopment investigated in the excised roots relative to thecorresponding values found in the controls. Primordia initiated in excised roots of Pisum and Vicia didnot complete their development in culture, i.e. no lateral rootsemerged and arrest took place with cell numbers of only 37 (Pisum)and 17 (Vicia) per cent of the numbers determined at the timeof secondary root emergence in the controls. Such arrested primordiahad few nuclei in S and none in mitosis. Moreover, at leastin Pisum, the frequency distribution of the relative DNA contentof the nuclei in the latter primordia approximated that foundin the apical meristem of primary roots following the establishmentof the stationary phase under conditions of carbohydrate starvation. It has also been demonstrated in the course of these investigationsthat lateral root primordium development in all four speciesis at least biphasic and possibly triphasic. Vicia faba L., broad bean, Pisum sativum L., garden pea, Zea mays L., maize, Phaseolus vulgaris L., dwarf bean, root primordia, anlage, cell doubling time, lateral root emergence