William A. Tramontano

Trigonelline accumulation in salt-stressed legumes and the role of other osmoregulators as cell cycle control agents

Abstract Plants, when exposed to excess salt, accumulate osmoregulators such as glycine betaine, proline and trigonelline to prevent water loss. Alfalfa (Medicago sativa) shows a five-fold proline increase and a two-fold trigonelline increase after salt-stress. Trigonelline also has a second function as a cell cycle regulator during the early growth of many legume root meristems. In this study, greenhouse grown soybeans (Glycine max) were exposed to salt-stress and levels (μg g−1, dry wt) of trigonelline were determined in leaf samples until senescence. Significant trigonelline increases occurred, beginning several weeks after salt exposure. Further experiments determined if other known osmoregulators (proline, glycine betaine) could affect the cell cycle parameters in cultured root meristems of peas (Pisum sativum), in a manner similar to trigonelline. Mitotic values remained near 4% of controls. At concentrations of 10−4–10−7 M trigonelline, an accumulation of G2 nuclei occurred, whereas proline was ineffective and glycine betaine only slightly effective in promoting G2 nuclei accumulation. These results demonstrate that the cell cycle effects of the trigonelline molecule are indeed specific and that its role as an osmoregulator in salt-stressed legumes is strengthened.

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

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

Trigonelline and promotion of cell arrest in G2 of various legumes

Abstract 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.

A survey of trigonelline concentrations in dry seeds of the dicotyledoneae

Abstract Ungerminated seeds of representatives of 21 of 24 superorders of the Dicotyledoneae were examined by high-pressure liquid chromatography for amounts of trigonelline (TRG), to determine if phylogenetic relationships are reflected in the relative amounts of TRG. With the exception of the Fabiflorae, the levels of TRG were quite variable within several superorders, whereas other superorders such as the Violiflorae, Primuliflorae, Rosiflorae, Rutiflorae, Araliiflorae and Gentianiflorae showed values that were consistently low. Overall, the amount of TRG present was relatively low in all of the superorders when compared with the relatively high TRG values of the Fabiflorae, the legumes. Many legumes have been shown to respond to TRG by predominant cell arrest in G2 and these possessed high concentrations of TRG. However, there are many non-legume nitrogen fixors, as well as several legumes that do not have significant TRG concentrations. From the above it appears that the concentrations of TRG in dry seeds are not well correlated with nitrogen fixation.

Distribution of α-tocopherol in early foliage samples in several forage crops

Abstract Concentrations of α-tocopherol in foliage samples of the forages annual rye, winter rye, red fescue, Kentucky blue-grass and white clover were determined. Concentrations of α-tocopherol increased with age in all four of the grasses tested during the six-week experimental period to values of 70.9, 80.9, 94.0 and 146.7 μg g−1 dry wt in annual rye, winter rye, red fescue and Kentucky blue-grass, respectively. White clover, the only legume forage tested, did not increase α-tocopherol concentrations with age, remaining at about 20 μg g−1 dry wt for each time sample. These results support the hypothesis that concentrations of α-tocopherol may act as indicators of biological age, although exceptions to this concept are likely to exist.

Age dependent α-tocopherol concentrations in leaves of soybean and pinto beans

Abstract The concentrations of α-tocopherol in foliage samples of soybeans ( Glycine max ) and pinto beans ( Phaseolus vulgaris ) were investigated. In both species, the α-tocopherol concentrations increased with age. Four-week-old soybeans had α-tocopherol concentrations of 135.1, 64.2 and 48.9 μg g −1 dry wt in unifoliates, first trifoliates and second trifoliates, respectively, while four-week-old pinto beans had α-tocopherol concentrations of 288.7, 123.3 and 40.6 μg g −1 dry wt in unifoliates, first trifoliates and second trifoliates. These results strengthen the hypothesis that increasing levels of α-tocopherol may act as an indicator of biological age in various plant species.

Histone deacetylase inhibitors and cell proliferation in pea root meristems

The histone deacetylase (HDA) inhibitors, trichostatin A (TSA) and HC toxin halt mitosis in cultured root meristems of Pisnum sativum, while the anti-protozoan HDA inhibitor apicidin is ineffective. Two-dimensional PAGE of proteins from root meristems exposed to TSA and HC toxin did not show significant differences compared to controls, although a previously tested HDA inhibitor, butyrate, exhibited dramatic variations in its protein profile. Northern analysis of butyrate- and TSA-treated root meristems indicated that non-proliferating cells are expressing significant amounts of transcripts of the known cell proliferation associated genes: histone H2A, MAP kinase, cycA2:1 and cdc2. Western analysis reveals the presence of hyperacetylated nuclear proteins in HDA-inhibitor treated cells. These results suggest that the HDA inhibitors, butyrate and TSA, halt mitosis without down-regulating genes that typically have low or nonexistent expression levels in non-dividing cells.

Trigonelline, nicotinic acid and nicotinamide in seedlings of Pisum sativum

Abstract Levels of trigonelline, nicotinic acid and nicotinamide have been examined in pea plants from the ungerminated seed to 10 days of age. The total quantity of trigonelline ranged between 70 and 81 μg in seeds to 7-day-old plants. At 10 days, the quantity of trigonelline increased to 101 μg. During the first 10 days, the concentration of trigonelline decreased in all regions of the seedling. Quantities of nicotinic acid within the seedling fell immediately after germination from 11.4 to 4.4 μg at 3 days but increased to 18.3 μg at 10 days. Unlike the decreasing trigonelline concentrations, nicotinic acid concentration throughout the seedling appeared relatively stable. Quantities of nicotinamide were less than 1% that of trigonelline, and never reached 1 μg total. The only noticeable increase in the concentration of nicotinamide occurred in leaves of 10-day-old plants. When [14C]trigonelline was injected at the cotyledonary node, it was transported to all regions of the plant and the majority (65%) went to epicotyl tissues.

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

Abstract 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.

Effects of cytokinins on promotion of cell arrest in G2 by trigonelline and trigonelline concentrations in cultured roots of Pisum sativum and Glycine max

Abstract Effects of cytokinins on trigonellines promotion of cell arrest in G2 were determined. Previous results demonstrate that trigonelline, (N-methyl nicotinic acid), a hormone present in cotyledons of young seedlings of Pisum sativum L. and Glycine max Merrill, is transported to roots and shoots where it stops cell progression in the G2 stage of the cell cycle during normal cell maturation. In P. sativum, two natural cytokinins, isopentenyl adenine and zeatin, and both their ribosides (all at 10−7 M) were antagonistic to trigonellines function when placed individually in aseptic culture with trigonelline at 10−6 M. Elevated trigonelline concentrations (10−4 M) negated the antagonistic effect on these cytokinins. Kinetin and benzyl adenine (synthetic cytokinins) were not antagonistic to trigonelline at any concentration. In G. max, two natural cytokinins, isopentenyl adenine and zeatin, at 10−7 and 10−8 M, promoted cell arrest in G2 in the absence of trigonelline. Moreover, the proportion of cells arrested in G2 was higher in roots cultured with isopentenyl adenine in combination with trigonelline, than with either hormone alone. In both legumes, these natural cytokinins did not influence trigonelline concentrations in roots. These results demonstrate an interaction between natural cytokinins and trigonelline in control of the cell cycle in peas and soybeans. This is the first report of an interaction of two naturally occurring hormones that affect cell arrest in either G1 or G2 in tissues of either plants or animals.