Search

Glutamine synthetase (E.C. 6.3.1.2) is a key enzyme of plant nitrogen metabolism that assimilates ammonia into glutamine. The Arabidopsis thaliana genome encodes one chloroplastic (GLN2) and five cytosolic (GLN1;1 – GLN1;5) isoforms with different expression patterns, kinetic properties, regulation and functions. Physiological roles of different isoforms have been elucidated mainly by studying knockout mutants. However, the role of GLN1;5, which is expressed in dry seeds, remains unknown. To clarifty the function of GLN1;5, we studied a GLN1;5 knockout line (GLN1;5KO) homozygous for T-DNA insertion within the GLN1;5. GLN1;5 deficiency results in a phenotype with slightly delayed bolting and fewer siliques. The dry weight of GLN1;5KO seeds was 73.3% of wild-type (WT) seed weight, with seed length 90.9% of WT seeds. Finally, only 18.33% of the mutant seeds germinated in water within 10 days in comparison to 34.67% of WT seeds. KNO3 strongly stimulated germination of both GLN1;5KO and WT seeds, while germination in the presence of increasing NH4Cl concentrations potentiated the differences between the two genotypes. It can be concluded that GLN1;5 activity supports silique development and grain filling and that it has a role in ammonium reassimilation in the seed, as well as assimilation and/or detoxification of ammonium from the environment. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. ON173024 and Grant no. ON173015]

Over 20% of all protein domains are currently annotated as “domains of unknown function” or DUFs. In a recently identified Centaurium erythraea arabinogalactan peptide, CeAGP3 (AGN92423), a conserved DUF1070 domain was found. Since identifying functions for DUFs is important in systems biology, we have analyzed the distribution and structure of DUF1070 domain (pfam06376) using a set of bioinformatics tools. There are 271 publically available DUF1070 members from 25 diverse families of vascular plants, and most are short sequences (50-100 aa). The N-terminal signal peptide (Nsp) was found in almost all complete sequences. In 233 sequences, at least two noncontiguous prolines were found as clustered dipeptides predicted to be hydroxylated and glycosylated with type II arabino-3,6-galactans, thus representing AG-II glycomodules. In addition, 35 sequences contained a region rich in basic residues (basic linker, BL). The N-terminal part of the DUF1070 domain is comprised of (part of) AG-II and/or BL, while the highly conserved C-terminus is a region of 26 aa, termed SH26. In 212 sequences, SH26 was a typical glycosylphosphatidylinositol lipid anchor signal peptide (GPIsp), but in 83 cases GPIsp was not predicted due to software constraints. In sequences where both Nsp and GPIsp were predicted, the length of mature peptides could be calculated, and it was 10-16 aa. Our analysis suggests that DUF1070 members are arabinogalactan (AG) peptides, of which the majority are GPI-anchored. DUF1070 is the only conserved domain found in classical arabinogalactan proteins and AG peptides. The SH26 region can be used for mining and annotation of AG peptides. [Projekat Ministarstva nauke Republike Srbije, br. ТR31019]

Primary and secondary ammonium assimilation is catalyzed by the glutamine synthetase-glutamate synthase (GS-GOGAT) pathway in plants. The Arabidopsis genome contains five cytosolic GS1 genes (GLN1;1 - GLN1;5), one nuclear gene for chloroplastic GS2 isoform (GLN2), two Fd-GOGAT genes (GLU1 and GLU2) and a GLT1 gene coding for NADH-GOGAT. Even though the regulation of GS and GOGAT isoforms has been extensively studied in response to various environmental and metabolic cues in many plant species, little is known about the effects of phytohormones on their regulation. The objective of this study was to investigate the impact of representative plant growth regulators, kinetin (KIN), abscisic acid (ABA), gibberellic acid (GA3) and 2,4-dichlorophenoxyacetic acid (2,4-D), on the expression of A. thaliana GS and GOGAT genes. The obtained results indicate that GS and GOGAT genes are differentially regulated by growth regulators in shoots and roots. KIN and 2,4-D repressed GS and GOGAT expression in roots, with little effect on transcript levels in shoots. KIN affected all tested genes; 2,4-D was apparently more selective and less potent. ABA induced the expression of GLN1;1 and GLU2 in whole seedlings, while GA3 enhanced the expression of all tested genes in shoots, except GLU2. The observed expression patterns are discussed in relation to physiological roles of investigated plant growth regulators and N-assimilating enzymes. [Projekat Ministarstva nauke Republike Srbije, br. ON173024]

Glutamine synthetase (GS) is a key nitrogen-assimilating enzyme in plants and a target for the broad-spectrum herbicide glufosinate. Understanding its kinetic and structural properties is of major agricultural importance. Spinach (Spinacia oleracea) is classified as a plant expressing only chloroplastic GS activity. We have analyzed soluble proteins in the spinach by coupling native polyacrylamide gel electrophoresis (PAGE)-activity detection, based on phosphate precipitation, with SDS-PAGE/immunoblotting. One cytosolic (GS1) isoform from the roots and two chloroplastic (GS2) isoforms expressed in leaves were resolved by native PAGE. The identity of the obtained bands was established by the application of GS-specific inhibitors, L-methionine sulfoximine and glufosinate. Examination by sodium dodecyl sulfate (SDS)-PAGE/ Western analysis with anti-GS antibodies, confirmed the identity of the active bands and revealed that both chloroplastic isoforms are composed of 44 kDa subunits, while the cytosolic isoform consists of 40 kDa subunits. The presence of more GS2 isozymes than encoded in the spinach genome is discussed in terms of posttranslational modifications.

To determine the manganese requirement necessary for optimal development of lesser centaury [Centaurium pulchellum (Sw.) Druce] in vitro, we investigated the effect of exogenously applied Mn on different developmental processes such as growth, flowering, fruiting, and seed germination. The application of Mn had no effect on stem length, except at the highest concentration of 10-2 M, which was inhibitory. In addition, C. pulchellum plants were capable of in vitro flowering and fruiting even on media without added Mn. However, Mn content in the media affected seed dimensions, since both length and width of the seeds increased with increasing Mn concentration. Moreover, both excess and absence of Mn in the media caused appearance of necrotic plants. Exogenously applied Mn had no effect on seed germination percentage, except at concentrations greater than 3x10-3 M.