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.     

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 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.     

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.     

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.     

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.     

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.     

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.     

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.     

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     

Growth of tree roots in hostile soil: A comparison of root growth pressures of tree seedlings with peas

Background and Aims

As part of a study on growth of tree roots in hostile soil, we envisaged that establishment and survival of trees on hard, dry soil may depend on their ability to exert axial root growth pressures of similar magnitude to those of the roots of agricultural plants (with significant root thickening when roots grow across an air gap or cracks and biopores). We selected tree species originating from a range of different soil and climatic conditions to evaluate whether their relative success on harsh soil (in an evolutionary sense) might be related to the magnitude of root growth pressures they could exert, or how they performed in the very early stages of growth after germination.

Methods

We measured the maximum axial root growth force (F_max) on single lateral root axes of 3- to 4- month old seedlings of 6 small-seeded eucalypts from 2 different habitats and 2 contrasting soil types. Root growth rate, root diameter and F_max were also measured on the primary root axes of a large-seeded acacia and a domesticated annual (Pisum sativum) seedling for up to 10 days following germination.

Results

The lateral roots of the 6 eucalypts and the primary roots of the acacia were considerably smaller than the primary roots of P. sativum and they exerted average forces of similar magnitude to one another (0.198 to 0.312 N). The maximum axial root growth pressures were all in the range 150 to 250 kPa but E. leucoxylon, E. loxophleba and A. salicina exerted the greatest pressures among the trees, and comparable pressures to those exerted by the primary roots of 2-day-old P. sativum (211-252 kPa). Although the primary roots of acacia seedlings exerted increasing axial root growth pressures over a 10-day period following germination, the pressures were still only slightly greater than those of the domesticated plant, P. sativum.

Conclusions

The lack of any very large differences in axial root growth pressures between trees and domesticated plants suggests that trees that grow well in harsh soil don’t do so by exerting higher root growth pressures alone but by also exploring the network of cracks and pores more effectively than do other plants that are less successful.     

Cholinesterases from Plant Tissues: III. Distribution and Subcellular Localization in Phaseolus aureus Roxb

The distribution and localization of cholinesterase in Phaseolus aureus, Glycine max, and Pisum sativum is described. The enzyme is present in roots, leaves, stems, root callus tissue, root cells suspension cultures, and root nodules. Cholinesterase in roots is found primarily in the cell wall. In cell fractionation experiments, at least 95% of the cholinesterase activity is associated with cell wall material. The enzyme can be solubilized by salt solutions, whereas Triton X-100 and sodium deoxycholate solubilize relatively small amounts of the enzyme. Cytochemical techniques have been employed to show the presence of cholinesterase activity at the cell surface and in the cell wall of certain cells of the root.     

The salt resistance of wild soybean (Glycine soja Sieb. et Zucc. ZYD 03262) under NaCl stress is mainly determined by Na+ distribution in the plant

Understanding the salt resistance mechanism of wild soybean is important in improving salt tolerance of cultivated soybean. Therefore, we comparatively analyzed effects of NaCl on photosynthesis, antioxidant enzyme activity, and ion distribution in a cultivated (Glycine. max) and a wild (Glycine soja) soybean to study the salt resistance mechanism of the G. soja. The results showed that more Na⁺ was accumulated in the G. soja roots than in the G. max roots, but the Na⁺ in the G. soja leaves was much less than that observed in the G. max leaves. The Na⁺ concentrations in the G. soja leaves were not high enough to affect the photosynthetic apparatus, which was demonstrated by less inhibition of photosynthetic activity, stomatal conductance, carboxylation efficiency in the G. soja leaves than in the G. max leaves after treated with different concentrations of NaCl. Meanwhile, there were no significant changes in intercellular CO₂ concentration, maximum PSII quantum yield, and relative water content in the G. soja leaves after NaCl treatment, while they significantly decreased in the G. max leaves. The non-photochemical quenching and the activities of superoxide dismutase (EC 1.15.1.1) and ascorbate peroxidase (EC 1.11.1.11) in the G. soja leaves increased with the increasing of NaCl concentrations, whereas only the activity of superoxide dismutase increased in G. max leaves. Based on these results, we suggested that the G. soja is able to accumulate higher levels of Na⁺ in its roots, and prevent the transportation of Na⁺ to leaves to protect photosynthetic apparatus from salt damage.     

Efficacy of Oxamyl Coated on Alfalfa Seed with a Polymer Sticker in Pratylenchus and Meloidogyne Infested Soils

A polymer sticker was used as a coating in which oxamyl was applied to seeds of alfalfa cultivar Saranac for the control of Pratylenchus penetrans and Meloidogyne hapla. The sticker, diluted 1:1 (sticker:water) to 1:5, delayed seedling emergence during the first 4 days after planting. By day 13, however, emergence from all sticker treatments was comparable to the control. Shoot growth of seedlings at day 21 was less than that of the control only from seeds coated with a 1:1 dilution; root growth and nodulation were not affected. Sticker-coated seeds absorbed 30-58% as much water in 3.5 hours as was absorbed by uncoated seeds. Oxamyl concentrations of 40-160 mg/ml in a 1:5 sticker : water mixture had no adverse affect on seedling emergence, growth, and nodulation over 3 weeks. Oxamyl at 160 mg/ml was more effective against P. penetrans than M. hapla. Growth of alfalfa in P. penetrans-infested soil was greater than that of the control in each sampling for 11 weeks. The reduction of number of P. penetrans in soil and roots moderated slowly over 11 weeks from 90% to 60%. Shoot and root growth of alfalfa from oxamyl-coated seed in M. hapla-infested soil were greater than those of the control for 7 and 11 weeks, respectively. The reduction in the number of M. hapla in the soil and roots changed from 80% at 7 weeks to 15% at 11 weeks.     

Cytokinin Content during Early Stages of Legume-Rhizobial symbiosis and effect of hypothermia

The effects of pea (Pisum sativum L.) seedling inoculation with nodule bacteria (Rhizobium leguminosarum bv. viceae) and hypothermia on the content of cytokinins (Ck) in seedling roots during 8 days after external factor action were studied. As soon as a day after inoculation with Rh. leguninosarum, the content of Ck in the seedling roots increased, especially in the root zone most susceptible to infection (0?C20 mm from the tip); this not only affected growth of the whole roots but also determined the initiation of nodule primordia. Hypothermia (8°C) reduced the content of CK as soon as in a day. After 5 and 8 days of hypothermia, the content of Ck in inoculated plants was approximately twice higher than in non-inoculated plant roots.     

Gibberellin Metabolism and Transport During Germination and Young Seedling Growth of Pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.)

The role of gibberellins (GAs) during germination and early seedling growth is examined by following the metabolism and transport of radiolabeled GAs in cotyledon, shoot, and root tissues of pea (Pisum sativum L.) using an aseptic culture system. Mature pea seeds have significant endogenous GA₂₀ levels that fall during germination and early seedling growth, a period when the seedling develops the capacity to transport GA₂₀ from the cotyledon to the shoot and root of the seedling. Even though cotyledons at 0–2 days after imbibition have appreciable amounts of GA₂₀, the cotyledons retain the ability to metabolize labeled GA₁₉ to GA₂₀ and express significant levels of PsGA20ox2 message (which encodes a GA biosynthesis enzyme, GA 20-oxidase). The large pool of cotyledonary GA₂₀ likely provides substrate for GA₁ synthesis in the cotyledons during germination, as well as for shoots and roots during early seedling growth. The shoots and roots express GA metabolism genes (PsGA3ox genes which encode GA 3-oxidases for synthesis of bioactive GA₁, and PsGA2ox genes which encode GA 2-oxidases for deactivation of GAs to GA₂₉ and GA₈), and they develop the capacity to metabolize GAs as necessary for seedling establishment. Auxins also show an interesting pattern during early seedling growth, with higher levels of 4-chloro-indole-3-acetic acid (4-Cl-IAA) in mature seeds and higher levels of indole-3-acetic acid (IAA) in young root and shoot tissues. This suggests a changing role for auxins during early seedling development.     

Coating Soybean Seed with Oxamyl for Control of Heterodera glycines

Oxamyl coated on soybean (Glycine max (L.) Merr. cv. Elgin) seeds in solutions of 20, 40, 80, and 160 mg/ml had no serious deleterious effects on seedling emergence and growth when planted in sterile soil. Seedling emergence on day 3 was less than that of the uncoated control, but by day 7 emergence was equal to, or greater than, the control. Shoot and root growth from seed coated with oxamyl in 40 and 80 mg/ml solutions was greater than that of the control. In soil infested with soybean cyst nematode, Heterodera glycines, shoot weight of soybean plants from seeds coated with oxamyl in 80 mg/ml solution was 11 and 9% greater at weeks 3 and 7, respectively, than from uncoated seeds. Numbers of juveniles (J3 and J4) and adults of H. glycines observed on the roots of plants from oxamyl-coated seeds were 83, 42, and 49% less at weeks 3, 5, and 7, respectively, than numbers on the roots of the untreated control. Numbers of J2 extracted from the roots of plants from oxamyl-coated seeds were 75% less at weeks 5 and 7 than those extracted from roots of uncoated seeds. The numbers of J2 extracted from the soil planted to oxamyl-coated seeds were 51 and 33% less at weeks 5 and 7, respectively, than from soil planted to uncoated seed.     

Maximum axial and radial growth pressures of plant roots

Summary The axial root growth force exerted by seedlings of pea (Pisum sativum cv. Greenfeast), cotton (Gossypium hirsutum cv. Sicot 3) and sunflower (Helianthus annuus cv. Hysun) was measured. Effects of different seedling age and different batches of seeds on axial root growth pressure were investigated. Mean values of the maximum axial root growth pressure (P_a) estimated from the maximum axial root growth force (F_max) and root diameter were 497, 289, and 238 kPa respectively for pea, cotton and sunflower seedlings of same size. P_a and F_max were significantly influenced by seedling age and for pea seedlings of same age they varied with the seed batch. A new technique was developed for estimating radial root growth pressure and was tested on pea seedlings. Each pea root was confined both in the axial and radial directions in a cylindrical chalk sample at a constant water potential. The roots exerted radial stress which caused tensile failure in a proportion of the chalks. The measurement of tensile strength of duplicate chalks enabled estimation of the maximum radial pressures exerted by the roots. The maximum axial and radial root growth pressures were of comparable magnitude.     

Effect of triethanolamine and silatranes on thermotolerance and accumulation of stress proteins in pea seedlings

A capability of triethanolamine (TEA) and its organosilicon derivatives methyl-, chloromethyl-, and ethoxy silatrane (MS, CMS, and ES, respectively) in low and ultra-low concentrations (from 10^?3 to 10^?13 M) to increase pea (Pisum sativum L.) thermotolerance and the relation of this process with heat-shock protein (HSP) accumulation in the roots were studied. Low and ultra-low concentrations of CMS and MS improved seedling survival under conditions of the heat shock (45°C). This process was not accompanied by stress protein accumulation. ES and CMS affected seedling survival and HSP accumulation differently in dependence on the temperature and preparation concentration.