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Summary• The role of plastidic glutamine synthetase (GS2) in proline biosynthesis and drought stress responses in Lotus japonicus was investigated using the GS2 mutant, Ljgln2-2.• Wild-type (WT) and mutant plants were submitted to different lengths of time of water and nutrient solution deprivation. Several biochemical markers were measured and the transcriptional response to drought was determined by both quantitative real-time polymerase chain reaction and transcriptomics.• The Ljgln2-2 mutant exhibited normal sensitivity to mild water deprivation, but physiological, biochemical and massive transcriptional differences were detected in the mutant, which compromised recovery (rehydration) following re-watering after severe drought stress. Proline accumulation during drought was substantially lower in mutant than in WT plants, and significant differences in the pattern of expression of the genes involved in proline metabolism were observed. Transcriptomic analysis revealed that about three times as many genes were regulated in response to drought in Ljgln2-2 plants compared with WT.• The transcriptomic and accompanying biochemical data indicate that the Ljgln2-2 mutant is subject to more intense cellular stress than WT during drought. The results presented here implicate plastidic GS2 in proline production during stress and provide interesting insights into the function of proline in response to drought.

Recycling of the 2-phosphoglycolate generated by the oxygenase reaction of Rubisco requires a complex and energy-consuming set of reactions collectively known as the photorespiratory cycle. Several approaches aimed at reducing the rates of photorespiratory energy or carbon loss have been proposed, based either on screening for natural variation or by means of genetic engineering. Recent work indicates that plant yield can be substantially improved by the alteration of photorespiratory fluxes or by engineering artificial bypasses to photorespiration. However, there is also evidence indicating that, under certain environmental and/or nutritional conditions, reduced photorespiratory capacity may be detrimental to plant performance. Here we summarize recent advances obtained in photorespiratory engineering and discuss prospects for these advances to be transferred to major crops to help address the globally increasing demand for food and biomass production.

Sandal et al. MPMI 4 INTRODUCTIONGenetic analysis and application of genetic approaches in the model legume Lotus japonicus (Handberg and Stougaard 1992) has progressed rapidly. Several key genes important for symbiosis with mycorrhizal fungi, root nodule development and other developmental processes have been identified using molecular genetics. The developmental regulators Nin (Schauser et al. 1999) and Pfo (Zhang et al. 2002) were isolated by transposon tagging while map-based cloning led to the molecular characterisation of Har1, SymRK, Nfr1, Nfr5, Castor and Pollux involved in autoregulation, Nod-factor signal perception or signal transduction (Schauser et al. 1999, Krusell et al. 2002 Nishimura et al. 2002a;Stracke et al. 2002;Radutoiu et al. 2003;Madsen et al. 2003; Imaizumi-Anraku et al. 2005). Genetic loci required for the early stages of endosymbiosis have attracted particular interest. Diallelic crosses together with phenotypical studies defined seven loci, SymRK, Nup133, Castor, Pollux, Sym6, Sym15,Sym24, in the common pathway required for both rhizobial and mycorrhizal symbiosis (Kistner et al. unpublished data) and map-based cloning of these loci has been accomplished or is advancing rapidly. A similar interest and effort is now emerging for genetic dissection of nodule organogenesis and function using the Fix -mutants arrested at various stages of nodule development or impaired in nodule function. Cloning of the Sst1 sulfate transporter required in functional root nodules is a first example (Krusell et al. 2005).Continuous isolation of new plant mutant lines is important for completing the genetic dissection of symbiosis and so far six independent mutant populations have been obtained by chemical (EMS) mutagenesis (Perry et al. 2003;Szczyglowski et al. 1998; Webb et al. unpublished data; Gresshoff et al. unpublished data), four populations after T-DNA or transposon insertion mutagenesis (Thykjaer et al. 1995;Schauser et al. 1998;Webb et al. 2000; Gresshoff et al. unpublished data), one population made with fast neutrons (Gresshoff et al. unpublished Umehara and Kouchi (unpublished data). All in all more than 400 symbiotic Lotus mutant lines were identified by screening in these populations and more are likely to follow. Assignment to complementation groups is next logical step in order to determine the number of loci involved, identify all alleles that contribute to phenotypic characterisation of mutants and genotyping of loci. However, diallelic crossing is a relatively slow process where progress is determined by generation time and slowed by a continuously increasing number of individual crosses necessary to keep up with mutant isolation programs. Given the number of symbiotic mutant lines already available and considering the time used to define seven complementation groups with a total of 26 alleles constituting the common pathway (Kistner et al. unpublished data), this approach is unlikely to encompass all alleles in near future. Detection of alleles in already cloned genes ...

(M.B., A.M.) Ammonium is a primary source of nitrogen for plants. In legume plants ammonium can also be obtained by symbiotic nitrogen fixation, and NH 1 4 is also a regulator of early and late symbiotic interaction steps. Ammonium transporters are likely to play important roles in the control of nodule formation as well as in nitrogen assimilation. Two new genes, LjAMT1;2 and LjAMT1;3, were cloned from Lotus japonicus. Both were able to complement the growth defect of a yeast (Saccharomyces cerevisiae) ammonium transport mutant. Measurement of [ 14 C]methylammonium uptake rates and competition experiments revealed that each transporter had a high affinity for NH 1 4 . The K i for ammonium was 1.7, 3, and 15 mM for LjAMT1;1, 1;2, and 1;3, respectively. Real-time PCR revealed higher expression of LjAMT1;1, 1;2, and 1;3 genes in leaves than in roots and nodule, with expression levels decreasing in the order LjAMT1;1 [ 1;2 [ 1;3 except in flowers, in which LjAMT1;3 was expressed at higher level than in leaves, and LjAMT1;1 showed the lowest level of expression. Expression of LjAMT1;1 and 1;2 in roots was induced by nitrogen deprivation. Expression of LjAMT1;1 was repressed in leaves exposed to elevated CO 2 concentrations, which also suppress photorespiration. Tissue and cellular localization of LjAMT1 genes expression, using promoter-b-glucuronidase and in situ RNA hybridization approaches, revealed distinct cellular spatial localization in different organs, including nodules, suggesting differential roles in the nitrogen metabolism of these organs.