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.     

Promotion of cell arrest in G2 in root and shoot meristems in Pisum by a factor from the cotyledons

Characterization of a factor within the cotyledons of Pisum that promotes cell arrest in G2 in mature root tissue and stationary phase root and shoot meristems is presented. Diffusion of the G2 factor into aseptic liquid and solid agar media enabled us to perform experiments focused on its cellular effect. The factor promotes cell arrest in G2 in shoots and roots of Pisum and roots of Vicia indicating a lack of species and organ specificity. In seedling roots of Pisum the factor promotes arrest in G2 in a large portion of the cell population. However, because pea cotyledons have a limited supply which is depleted 8–10 days after seed germination, cells previously responsive to the G2 factor in Pisum root meristems eventually assume preponderant arrest in G1. Once these cells arrest in G1 they are no longer influenced to arrest in G2. The G2 factor doss not promote arrest in G2 in meristem root cells of Helianthus and Triticum which normally show preponderant arrest in G1.     

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.     

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.     

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.     

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.     

Induction of cell arrest in G2: structural specificity of trigonelline

Synthetic analogues of N-methyl nicotinic acid, trigonelline, were prepared to test the structural features necessary for the induction of cellular arrest in G2 in Pisum sativum. Analogues that (1) were regioisomers of trigonelline, (2) possessed different 1,3-substituents, and (3) contained additional substituents on the pyridine ring were tested for their ability to induce cell arrest in G2 and to antagonize trigonelline induced arrest in G2. Only N-methyi-3-quinoline-carboxylic acid and 1-methyl nicotinamide induced cell arrest in G2, and 1-methyl-4-pyridine carboxylic acid and 1-methyl-2-pyridine carboxylic acid were effective trigonelline antagonists. These data further support a specific role for trigonelline in the induction of cell arrest in G2.     

Cell arrest in G2 in root meristems: A control factor from the cotyledons

A substance promoting cell cycle arrest in G 2 in the root meristem is demonstrated. This substance is produced in the cotyledons and is transported to the root.     

INFLUENCE OF THE TONIC EFFECT OF GRAVITATION AND AUXIN ON CELL ELONGATION AND POLARITY IN ROOTS

The influence of a longitudinal (tonic) gravitational force and of auxin on the pattern of growth and cell polarity has been studied on intact roots of wheat seedlings. A klinostat technique was used for controlling gravitation. Growth in length was evaluated as cell division activity, rate of cell elongation (μ/h) and duration of elongation (h). Exogenous auxin (1-NAA) increases the rate of cell elongation in all concentrations tested (10⁻⁸ — 3 × 10⁻⁷m ) and shortens the time of elongation with increasing concentration. It promotes rate of cell elongation in roots as it does in shoots. It also accentuates the polar insertion of root hairs and their growth. The tonic effect of gravitation resembles that of an increase in auxin both in light and darkness. The results are discussed in relation to plagiotropic growth of roots, root growth promotions by auxin, and the difference between root and shoot growth.     

Relationship between trigonelline concentration and promotion of cell arrest in G2 in cultured roots of Pisum sativum

Trigonelline, G2 Factor, present in cotyledons of Pisum sativum is transported to roots and shoots after germination. This hormone promotes prefere     

Resumption of DNA Synthesis and Cell Division in Wheat Roots as Related to Lateral Root Initiation

The arrest of DNA synthesis and termination of cell division in basal meristematic cells as well as the resumption of these processes as related to the initiation of lateral root primordia (LRP) were studied in tissues of Triticum aestivumroots incubated with ³H-thymidine. All cells of the stelar parenchyma and cortex as well as most endodermal and pericycle cells left the mitotic cycle and ceased proliferative activity at the basal end of the meristem and at the beginning of the elongation zone. Some endodermal and pericycle cells started DNA synthesis in the basal part of the meristem and completed it later on during their elongation, but they did not divide. In the cells of these tissues, DNA synthesis resumed above the elongation zone, the cells being located much closer to the root tip than the first newly dividing cells. Thus, the initiation of LRP started much closer to the root tip than it was previously believed judging from the distance of the first dividing pericycle cells from the root tip. DNA synthesizing and dividing cells first appeared in the stelar parenchyma, then, in the pericycle, and later, in the endodermis and cortex. It seems likely that a release from the inhibition of DNA synthesis allows the cells that completed mitotic cycle in the basal part of meristem in the G₁phase to cease the proliferative arrest above the elongation zone and to continue their cycling. The location of the first DNA synthesizing and dividing cells in the stelar parenchyma and pericycle did not strictly correspond to the LRP initiation sites and proximity to the xylem or phloem poles. This indicates that LRP initiation results from the resumption of DNA synthesis in all pericycle and stelar parenchyma cells that retained the ability to synthesize DNA and occurs only in the pericycle sector situated between the two tracheal protoxylem strands, all cells of which terminated their mitotic cycles in the G₁phase.     

REGULATION OF GROWTH AND CELLULAR DIFFERENTIATION IN DEVELOPING AVENA INTERNODES BY GIBBERELLIC ACID AND INDOLE-3-ACETIC ACID

Auxin (IAA) at physiological concentrations causes significant reduction of GA₃-promoted growth in excised Avena stem segments. IAA is thus considered to be a gibberellin antagonist in this system. It was found to act non-competitively in repressing GA₃-augmented growth in these segments. In intercalary meristem cells at the base of the elongating internode, GA₃ blocks cell division activity and causes a marked increase in cell lengthening. IAA substantially promotes lateral expansion in comparable intercalary meristem cells, particularly in the vicinity of vascular bundles underlying the epidermis. It also alters the plane of cell division in differentiating stomata. IAA at high concentrations (10⁻³, 10⁻⁴ m ), in combination with GA₃, overrides the effects of GA₃ on cell lengthening, while with low concentrations of IAA (10⁻⁹, 10⁻¹⁰m ), the effects of GA₃ are clearly dominant. At intermediate concentrations of IAA (10⁻⁶, 10⁻⁷m ), in the presence of GA₃, the effects of this treatment on cell differentiation closely parallel the pattern of differentiation in untreated tissue. It is postulated that a lateral gradient of auxin and gibberellin could control cell expansion in long epidermal cells during intercalary growth of the internode.     

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.     

Restoration of cell growth and proliferation in the wheat roots after their inhibition by nickel sulfate

The dynamics of cell growth and proliferation restoration in different tissues and quiescent center (QC) in the wheat (Triticum aestivum L.) seedling roots and also the differentiation of rhizodermal cells and lateral root initiation after 48-h treatment with 100 μM NiSO₄ were studied. Within 24 h after nickel removal from medium, root growth was resumed due to the increase in the rate of cell growth in the meristem and the region where cell elongation started in control roots. Stimulation of cell proliferation was restored in the main part of the meristem and later in the initial cells of the files and QC. Cell proliferation was not observed in the QC. The time of cell proliferation resumption in the roots and in tested tissues depended on the degree of their injury by nickel treatment. In most tested roots, DNA synthesis and cell division were restored in 32 h. In the cells leaving the meristem due to the resumption of their growth and proliferation, growth of root hairs started. In 48 h, the number of roots with perished cells in the rhizodermis in the meristem was sharply increased and the regeneration of the damaged region by the cells of outer cortex was observed. Only after the appearance of root hairs, the cells coming from the meristem started to elongate. In most roots, the formation of the new elongation zone occurred in 56 h. During its formation, the initiation of lateral root primordia was shifted in the basipetal direction. It was concluded that the cessation of cell growth and proliferation under the influence of high concentration of heavy metal (HM) ions is not lethal for the root. At the action of toxic HM concentrations, the plant strategy is the maintenance of meristematic cell capacity for cell growth and proliferation resumption. The cellular mechanism of this capacity maintenance is the transition of meristematic cells from G₁ phase to dormancy due to growth inhibition and the inhibition of the transition to DNA synthesis.     

Water status and water diffusion transport in lupine roots exposed to lead

Water status and diffusion transport were studied in the roots of yellow lupine (Lupinus luteus L., cv. Juno) treated for 48 h with two selected concentrations of Pb(NO₃)₂: 150 mg l⁻¹, which inhibited root growth by about 50% (medium stress intensity), as well as 350 mg l⁻¹, which almost entirely suppressed root elongation (severe stress intensity). Relative water content (RWC), which characterizes the degree of root water saturation, slightly increased at the lower lead concentration and remained unchanged at the higher lead dose. Ultrastructure analyses under a transmission electron microscope revealed that plasmolysis was not evoked by lead in the apical part of the meristem. Moreover, direct observation of meristem cells using Nomarsky optics indicated enhanced vacuolization in the presence of both lead concentrations. These data suggest that the water status of the roots was not affected by the metal. Due to the fact that proline is involved in the maintenance of turgor in the cells, the metabolism of this amino acid was investigated. In the roots, the activity of enzymes involved in proline synthesis, such as pyrroline-5-carboxylate synthetase (P5CS) and ornithine aminotransferase (OAT), increased at 150 mg l⁻¹ Pb²⁺; nevertheless, proline content was diminished at the lower lead concentration. This effect is likely the result of proline degradation by proline dehydrogenase (PDH), since the activity of this enzyme increased at the lower lead dose. On the other hand, in the presence of 350 mg l⁻¹ Pb²⁺, a low level of proline was correlated with a decrease in the activity of P5CS and OAT, as well as unchanged PDH activity in lupine roots. These data may imply that enzymatic synthesis of proline was strongly damaged by the metal ions. The low level of proline in both experimental variants suggests that proline accumulation is inessential to maintaining the osmotic uptake of water into root cells. NMR spectroscopy showed that exposition of lupine seedlings to lead caused a deceleration in water transport in the roots due to a reduction in the water transfer rate across the membranes (transmembrane transfer) and vacuoles continuum, as well as water diffusion along the root apoplast. Fluorescence staining and immunogold labeling showed the presence of callose strands in cell walls and/or in the vicinity of them. In lead-treated lupine roots, callose was mainly localized in the parenchyma cortex placed lengthwise to the vascular cylinder. Callose deposits in the cell walls may reduce vacuolar transport, as well as increase cell wall resistance to water flow. Deceleration of diffusional water movement to the vascular system, may in turn, influence the rate of long-distance water transport to aerial parts of the plant.     

Extra DNA synthesis in the dedifferentiating cells ofVicia faba roots

Summary Cell dedifferentiation was induced inVicia faba root tissues by removing the whole root meristem (decapitation) and the behaviour of the nuclear DNA in the dedifferentiating cells was studied by means of cytophotometric and autoradiographic analyses. Cytophotometric determination after Feulgen-staining showed that: 1. the vast majority of nuclei in differentiated cells were in the DNA postsynthetic phase, but their Feulgen absorption was lower than that of DNA postsynthetic nuclei (G₂, 4 C) in the meristem; 2. such a Feulgen absorption was detected in certain nuclei after root decapitation; 3. all the mitoses in the dedifferentiating tissues were diploid, fully matching the Feulgen absorption of mitoses in the meristem.After³H-thymidine (³H-T) feeding of the decapitated roots and autoradiography, the following results were obtained: 1. two populations of labeled nuclei, characterized by two different levels of scattered labeling occurred in dedifferentiating tissues, slightly labeled nuclei being much more numerous than heavily labeled nuclei; 2. the percentage of labeled nuclei was much greater than that of DNA presynthetic nuclei in the root tissues; 3. almost all the mitoses were labeled after a 16-hour³H-T feeding; 4. the percentage of slightly labeled nuclei paralleled that of dedifferentiating cells; 5. the duration of the DNA synthesis phase and that of the gap between completion of DNA synthesis and mitosis differed in heavily and slightly labeled nuclei; 6. all nuclei which entered DNA synthesis also entered mitosis.These results are interpreted to mean that: 1. after decapitation, two different DNA syntheses occur in the dedifferentiating root tissues ofV. faba: DNA reduplication in cells which dedifferentiate starting from a DNA presynthetic nuclear condition (heavily labeled nuclei) and extra DNA synthesis in cells which dedifferentiate starting from a DNA postsynthetic nuclear condition (slightly labeled nuclei); 2. extra DNA synthesis is required in these dedifferentiating cells for entry into mitosis.     

Effect of nickel on growth,proliferation, and differentiation of root cells in <Emphasis Type="Italic">Triticum aestivum</Emphasis> seedlings

The effect of 10^–6 and 10^–4 M NiSO₄ on cell growth, proliferation, and differentiation was studied over 48 h in seminal and lateral roots of five-day-old Triticum aestivum seedlings. 10^–6 M NiSO₄ did not significantly affect the root system, whereas 10^–4 M NiSO₄ inhibited its development. However, 10^–6 M NiSO₄ disturbed the contacts between the groups of closely related cells of the rhizodermis in the meristem. In the exodermis, an additional layer of cells was formed. At the nickel concentration of 10^–4 M, cell divisions in the outer layers of the root cells and metaxylem ceased earlier than in other root tissues positioned both centripetally and acropetally. Differentiation of protophloem sieve elements was completed in the meristem but at a greater distance from the root tip. Cell elongation started at the same distance from the root tip as in control plants. The rate of elongation decreased, and acropetally it stopped. Therefore, the cells of the xylem and metaphloem started to differentiate, and primordia of lateral roots were initiated and formed closer to the root tip. At a lethal concentration (10^–4 M), nickel induced necroses of elongating cells of the endodermis and pericycle. Nickel is supposed to enter the tissues of the central cylinder predominantly via the protoxylem and rapidly translocate along the xylem. As a result, the incubation of the roots at this concentration for 48 h almost did not affect the development of the phloem and probably sugar unloading, that makes possible to maintain the growth of meristematic cells and the cell division of the most important tissues for longer time.Translated from Fiziologiya Rastenii, Vol. 52, No. 2, 2005, pp. 250–258.Original Russian Text Copyright © 2005 by N. Demchenko, Kalimova, K. Demchenko.This revised version was published online in April 2005 with a corrected cover date.     

Analysis of Cell Division and Elongation Underlying the Developmental Acceleration of Root Growth in Arabidopsis thaliana

To investigate the relation between cell division and expansion in the regulation of organ growth rate, we used Arabidopsis thaliana primary roots grown vertically at 20°C with an elongation rate that increased steadily during the first 14 d after germination. We measured spatial profiles of longitudinal velocity and cell length and calculated parameters of cell expansion and division, including rates of local cell production (cells mm⁻¹ h⁻¹) and cell division (cells cell⁻¹ h⁻¹). Data were obtained for the root cortex and also for the two types of epidermal cell, trichoblasts and atrichoblasts. Accelerating root elongation was caused by an increasingly longer growth zone, while maximal strain rates remained unchanged. The enlargement of the growth zone and, hence, the accelerating root elongation rate, were accompanied by a nearly proportionally increased cell production. This increased production was caused by increasingly numerous dividing cells, whereas their rates of division remained approximately constant. Additionally, the spatial profile of cell division rate was essentially constant. The meristem was longer than generally assumed, extending well into the region where cells elongated rapidly. In the two epidermal cell types, meristem length and cell division rate were both very similar to that of cortical cells, and differences in cell length between the two epidermal cell types originated at the apex of the meristem. These results highlight the importance of controlling the number of dividing cells, both to generate tissues with different cell lengths and to regulate the rate of organ enlargement.     

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