Sixteen cytosolic glutamine synthetase genes identified in the Brassica napus L. genome are differentially regulated depending on nitrogen regimes and leaf senescence

Abstract

A total of 16 BnaGLN1 genes coding for cytosolic glutamine synthetase isoforms (EC 6.3.1.2.) were found in the Brassica napus genome. The total number of BnaGLN1 genes, their phylogenetic relationships, and genetic locations are in agreement with the evolutionary history of Brassica species. Two BnaGLN1.1, two BnaGLN1.2, six BnaGLN1.3, four BnaGLN1.4, and two BnaGLN1.5 genes were found and named according to the standardized nomenclature for the Brassica genus. Gene expression showed conserved responses to nitrogen availability and leaf senescence among the Brassiceae tribe. The BnaGLN1.1 and BnaGLN1.4 families are overexpressed during leaf senescence and in response to nitrogen limitation. The BnaGLN1.2 family is up-regulated under high nitrogen regimes. The members of the BnaGLN1.3 family are not affected by nitrogen availability and are more expressed in stems than in leaves. Expression of the two BnaGLN1.5 genes is almost undetectable in vegetative tissues. Regulations arising from plant interactions with their environment (such as nitrogen resources), final architecture, and therefore sink-source relations in planta, seem to be globally conserved between Arabidopsis and B. napus. Similarities of the coding sequence (CDS) and protein sequences, expression profiles, response to nitrogen availability, and ageing suggest that the roles of the different GLN1 families have been conserved among the Brassiceae tribe. These findings are encouraging the transfer of knowledge from the Arabidopsis model plant to the B. napus crop plant. They are of special interest when considering the role of glutamine synthetase in crop yield and grain quality in maize and wheat.

Keywords: Alloploidization; Brassica napus; Brassica oleracea; Brassica rapa; nitrogen metabolism; nitrogen remobilization; senescence..

Fig. 1.

Cytosolic glutamine synthetase (GS1) phylogenetic tree. DNA coding sequences (CDS) were aligned using Clustal. The distance matrix was computed using Dnadist with a Kimura 2 nucleotide substitution model (bootstrap analysis, 1000 iterations). A consensus unrooted tree was then generated using the Neighbor–Joining method from the Phylip 3.67 package. Black and grey dots indicate bootstrap values >90% and 50%, respectively. All programs are available at www.mobyle.pasteur.fr. ^aIncomplete CDS sequence when compared with the A. thaliana reference sequence.

Fig. 2.

Structure of BnaGLN1 genes. For each BnaGLN1 gene, the length of the 5′ and 3′ untranslated regions (UTRs) (white boxes), exons (black boxes), and introns (black lines) is represented by a number corresponding to base pairs.

Fig. 3.

Alignment of Brassica napus and Arabidopsis thaliana GS1 proteins. Protein sequences were deduced from DNA coding sequences and aligned using Clustal. Stars indicate residues involved in the ammonium/glutamate-binding pocket (Eisenberg et al., 2000). Boxes indicate residues involved in ammonium affinity properties (Ishiyama et al., 2006). Arrows indicate conserved domains (1) pfam 03951 Gln-synt_N glutamine synthetase bet-Gasp domain; and (2) pfam 00120 gln-synt_C catalytic domain. Residues are coloured according to their polarity properties (neutral non-polar as black, neutral polar as green, acidic as red, and basic as blue).

Fig. 4.

Leaf senescence markers on vegetative B. napus plants. F _v/F _m photosystem II capacity (A, B) and chlorophyll relative content (C, D) were monitored at the vegetative stage in all the leaf ranks of four B. napus plants grown under low (white bars) or high (black bars) nitrate conditions. The expression of the BnaA.GLN2.a and BnaC.GLN2.a marker genes was quantified (E, F) on selected leaf ranks (*) and confirmed differential senescence symptoms. Mean and standard deviation of four plant repeats are shown.

Fig. 5.

Expression of BnaGLN1 genes is modified depending on nitrate availability and leaf ageing. The relative expression level of BnaGLN1 genes was monitored in limbs and secondary veins of six leaf ranks harvested on vegetative plants grown under low (white bars) or high (black bars) nitrate conditions. Leaf ranks represented as number 1 (bottom and older leaf) to 6 (top and younger leaf) showed differential senescence symptoms. Mean and standard deviation of four plant repeats are shown.

Fig. 6.

Expression of BnaGLN1 genes is modified depending on nitrate availability in the taproot and crown of vegetative B. napus plants. The relative expression level of BnaGLN1 genes was monitored at the vegetative stage in the taproot and crown of four plants grown under low (white bars) or high (black bars) nitrate conditions. Mean and standard deviation of four plant repeats are shown.

Fig. 7.

BnaGLN1 genes are differentially expressed depending on flowering or seed filling stages and leaf age. The relative expression level of BnaGLN1 genes was monitored in young and old leaves of plants grown in the field and supplemented with nitrogen. Leaf limbs and stems were collected at flowering (white bars) and seed filling (black bars) stages. Mean and standard deviation of four plant repeats are shown. * indicates significant difference (Student’s t-test, P<0.05) between flowering and seed filling stages.