Protein and Nitrate Content of Lemna Sp. as a Function of Developmental Stage and Incubation Temperature

Abstract

Lemna protein per frond and per root increases with developmental stage until plants are at least two generations old. Protein per frond, per root, and per unit dry weight is greater in plants grown at 23.9 C than at 18.3 C. More protein is found in fronds than in roots, and more nitrate occurs in roots than in fronds. Nitrate per root increases with developmental stage and is higher (per root) in plants grown at 23.9 C than in those grown at 18.3 C. The distribution of generations within a growing population is constant for at least eight doubling times. Whether populations multiply slowly at 15.6 C or more rapidly at 23.9 C, fronds which have not yet produced progeny form 62% of the population; fronds which are one generation old form 24% of the population; and fronds which are two generations old form 9% of the population. These experiments were undertaken to evaluate the possibility of using duckweeds to remove nitrate from irrigation return water. Protein and nitrate were measured in fronds and roots at different developmental stages. The distribution of developmental stages in the growing population was also monitored. Results allow preliminary estimates of nitrate removal as a function of time after frond inoculation, but the great difference between protein content (per plant and per unit dry weight) of plants grown at 18.3 C and at 23.9 C implies that reliable models will require a large data base. MATERIALS AND METHODS Culturing of Plants. Lemna (species unknown)2 was obtained from local ponds, sterilized by hypochlorite treatment (10), and grown in sterile Hoagland solution modified to contain 20 mg 1-1 nitrogen (nitrate-N). This nitrate concentration was chosen because it is typical of agricultural return water in the area (8). The modification did not affect the pH of the incubation medium. All plant material used for the experiments was derived from a single progenitor frond and was presumably genetically identical. Culture vessels were Kimax crystallizing dishes containing 250 ml sterile medium and attached by parafilm to sterile, disposable Plexiglas covers. Dishes were incubated in growth chambers at 15.6 C, 18.3 C, or 23.9 C, under continuous irradiation from 14 metal halide lamps plus 12 self-ballasted mercury lamps with ' maintained in the laboratory of Dr. Allen W. Knight. deluxe white phosphor which, together, provided 350 ,uE m-2 s-' on the surface of the dish lids. Nutrient medium was changed daily by gently lifting plants into new dishes containing freshly sterilized medium. 'Frond' is used in this paper in the sense of Ashby et al. (1) and refers to a single leaf from which root and younger (or older) attached appendages have been dissected away. 'Plant' is a frond with its root and pocket containing developing fronds. 'Colony' is used in the sense of Datko et al. (4) and refers to a collection of plants which are physically bound as a group. Protein, Nitrate, and Dry Weight Determinations. For protein assays, five replicate samples of 20 plants were harvested from each generation. A razor blade was used to separate fronds from roots. Upon harvesting, tissue was chilled, rinsed with deionized H20, and ground in a homogenizer, and the extract was frozen. The frozen samples were accumulated gradually over a period of several months. Accumulated extracts were washed with 5% tri-chloroacetic acid and assayed for soluble protein by the Lowry method (17). For nitrate analysis, five replicate samples of nine plants were harvested from each generation. After separation of fronds from roots, tissue was chilled, rinsed with deionized water, ground in a homogenizer, and filtered through a Millipore HA filter (0.45-,um pore size), and the filtrate was frozen. Nitrate concentration was measured by the high pressure liquid chromatography method of Thayer and Huffaker (20). Dry weight was determined on five replicate tissue samples, containing between 9 and 20 fronds or roots, after 24 h of incubation at 41 C. Generation Study. Frond lineage was followed by marking fronds with small dots of colored ink (which appeared not to harm fronds or change growth rates) and periodically placing siblings into separate, marked culture vessels. The development is continuous ; but, for convenience when a new frond emerged from a side pocket, it was called the 0 (daughter) stage, and it simultaneously caused its progenitor frond to reach the 1 (parent) stage. The numbers of fronds at each generational stage were recorded daily for approximately 2 weeks at 15.6 C, 18.3 C, and 23.9 C. Numerical Methods. In analyzing the growth data (Figs. 9, 10) for each temperature, the conventional relationship, (In total frond number) = A x time + B, was assumed. The regression coefficient A and the y-intercept B were calculated using a linear regression program. Among the different experiments, A varied with temperature and B with initial population size. For each point in Figures 9 and 10, the number of doubling times was then computed as (time x A)/ln 2, and log2 (frond number) was computed as (frond number-B)/ln 2. This transformation had the effect of normalizing each time course to an initial value of one frond at time zero. In actual experiments, the initial total frond number was between 8 and 29, with the final population growing to well over 500 to 600 fronds. A two-way analysis of variance was used to determine whether the differences between the two incubation temperatures were significant. To test for monotonic increase of 127