Regulation of Nitrate Reductase in Pea rrrbrb, rrRbRb, and RRrbrb Mutants during Nitrate Assimilation

Abstract

The principal laws of genetic inheritance were first discovered by Gregor Mendel in 1865 [1] in experiments with garden pea seeds differing in their shape, round or wrinkled. Later, it was demonstrated that mutations at r and rb loci control these morphological traits [2,3]. Both mutations reduce starch content and increase protein content in seeds [4]. Wrinkled seeds of r mutants are characterized by a higher content of amylose in starch, as compared to round seeds, whereas rb mutants contain less amylose in starch. These differences between the r and rb mutants with respect to starch content and composition are the consequences of mutations in the pathway of starch biosynthesis: the r mutation eliminates one of the isoforms of the starchbranching enzyme, which results in a decrease in starch content and the synthesis of its less branched form [5]; the rb mutation suppresses sharply the activity of glucosepyrophosphorylase, which also reduces starch content [6]. Thus, both the r and rb mutations with a wrinkled seed phenotype are characterized by a changed composition of storage compounds in seeds. In its turn, the changed composition of seed storage compounds can affect the total directionality and activity of metabolic processes in the developing plant, in particular of the assimilation of nitrate nitrogen, the main source of plant nitrogen nutrition. It turned out that these topics were not investigated. The objective of this work was to study the regulation of nitrate reductase (NR) in the rrrbrb, rrRbRb , and RRrbrb mutants and wild-type pea plants during nitrate assimilation at the developmental stages of heterotrophic and early autotrophic nutrition.Pea plants were grown in water culture in a controlled climatic chamber at a temperature of 22/18°ë (day/night) and a 16-h photoperiod. Experiments were performed on plants grown under a low illuminance (~3 klx) from luminescent lamps. In such a way, we limited the amount of photosynthates in plants, and this permitted the elucidation of glucose role in the control of NR activity. Glucose is a monomer of starch, and pea genotypes differed just in starch content. Plant nutrition is heterotrophic during the first 13 to 15 days of their growth; thereafter, nutrition becomes autotrophic. Plants were grown on modified Knop solution [7] containing 1.4, 7.1, and 14.2 mM nitrate in different treatments. NR activity was assayed by the in vivo method [8]. The actual NR activity was determined without adding nitrate to the incubation medium, whereas the potential NR activity was measured in the presence of nitrate in medium. NR activity was expressed in µ moles of nitrite produced by 1 g of fresh biomass for 1 h. Nitrate content in leaves and roots was measured by titration with salicylic acid [9]. It was expressed in µ moles per 1 g fr wt. Figure 1 shows that, at a low nitrate concentration (1.42 mM), wild-type plants differed substantially in NR activity from mutant plants. This is evident from the absolute values of actual and potential NR activities and from the patt...

Regulation of Glutamine Synthetase and Glutamate Dehydrogenase in Pea Mutants rrrbrb, rrRbRb, and RRrbrb during Nitrate Nitrogen Assimilation

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It was reported earlier that mutations at the r and rb pea genes, which lead to a decrease in the content of starch in seeds and change in their shape from round to wrinkled [1,2] cause changes in general metabolism due to intensification of basic processes, such as symbiotic fixation of molecular nitrogen and assimilation of nitrate nitrogen [3,4]. As a result, mutant plants and their seeds accumulate greater amounts of protein than the wild-type plants.

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