Cloning, characterization and expression analysis of tonoplast intrinsic proteins and glutamine synthetase in ryegrass (Lolium perenne L.).
Perennial ryegrass (Lolium perenne L.) is the most important turf and forage grass species of the temperate regions. It requires substantial input of nitrogen fertilizer for optimum yield. Improved nitrogen use efficiency (NUE) is therefore one of the main breeding targets. However, limited knowledge is currently available on the genes controlling NUE in perennial ryegrass. The aim of the present study was to isolate genes involved in ammonium transport and assimilation. In silico screening of a Lolium EST-library using known sequences of tonoplast intrinsic proteins (TIPs) and cytosolic glutamine synthetase (GS1) revealed a number of homologous sequences. Using these sequences, primers were designed to obtain the full-length sequences by RACE-PCR. Three TIP genes (LpTIP1;1, LpTIP1;2 and LpTIP2;1) and two GS genes (LpGS1a and LpGS1b) were isolated. Characterization in S. cerevisiae confirmed a function in ammonium transport for LpTIP1;1 and LpTIP2;1 and in synthesis of glutamine for LpGS1a and LpGS1b. Cytoimmunochemical studies showed that GS protein was present in the chloroplasts and cytosol of leaf cells, while TIP1 proteins localized to the tonoplast. At the expression level, Lolium GS1 genes responded to N starvation and re-supply in a manner consistent with functions in primary N assimilation and N remobilization. Similarly, the expression of LpTIPs complied with a role in vacuolar ammonium storage. Together, the reported results provide new understanding of the genetic basis for N assimilation and storage in ryegrass.
Nord-Larsen
Pia H
PH
Plant and Soil Science Laboratory, Department of Agriculture and Ecology, Faculty of Life Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg, Denmark.
Kichey
Thomas
T
Jahn
Thomas P
TP
Jensen
Christian S
CS
Nielsen
Klaus K
KK
Hegelund
Josefine N
JN
Schjoerring
Jan K
JK
D003001:Cloning, Molecular; D020224:Expressed Sequence Tags; D018506:Gene Expression Regulation, Plant; D005974:Glutamate-Ammonia Ligase / Q000235:genetics / Q000378:metabolism*; D008129:Lolium / Q000201:enzymology / Q000235:genetics*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009584:Nitrogen / Q000378:metabolism; D018515:Plant Leaves / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D018517:Plant Roots / Q000378:metabolism; D020033:Protein Isoforms; D018749:RNA, Plant / Q000235:genetics
D008565:Membrane Proteins; D010940:Plant Proteins; D020033:Protein Isoforms; D018749:RNA, Plant; D005974:Glutamate-Ammonia Ligase; D009584:Nitrogen