SUMMARYMost vascular plants form a mutualistic association with arbuscular mycorrhizal (AM) fungi, known as AM symbiosis. The development of AM symbiosis is an asynchronous process, and mycorrhizal roots therefore typically contain several symbiotic structures and various cell types. Hence, the use of whole-plant organs for downstream analyses can mask cell-specific variations in gene expression. To obtain insight into cell-specific reprogramming during AM symbiosis, comparative analyses of various cell types were performed using laser capture microdissection combined with microarray hybridization. Remarkably, the most prominent transcriptome changes were observed in non-arbuscule-containing cells of mycorrhizal roots, indicating a drastic reprogramming of these cells during root colonization that may be related to subsequent fungal colonization. A high proportion of transcripts regulated in arbuscule-containing cells and non-arbuscule-containing cells encode proteins involved in transport processes, transcriptional regulation and lipid metabolism, indicating that reprogramming of these processes is of particular importance for AM symbiosis.

SummaryThe replacement of phospholipids by galacto-and sulfolipids in plant membranes represents an important adaptive process for growth on phosphate-limiting soils. Gene expression and lipid analyses revealed that the MYB transcription factor PHR1 that has been previously shown to regulate phosphate responses is not a major factor controlling membrane lipid changes. Candidate genes for phospholipid degradation were selected based on induction of expression during phosphate deprivation. Lipid measurements in the corresponding Arabidopsis mutants revealed that the non-specific phospholipase C5 (NPC5) is required for normal accumulation of digalactosyldiacylglycerol (DGDG) during phosphate limitation in leaves. The growth and DGDG content of a double mutant npc5 pho1 (between npc5 and the phosphate-deficient pho1 mutant) are reduced compared to parental lines. The amount of DGDG increases from approximately 15 mol% to 22 mol% in npc5, compared to 28 mol% in wild-type, indicating that NPC5 is responsible for approximately 50% of the DGDG synthesized during phosphate limitation in leaves. Expression in Escherichia coli revealed that NPC5 shows phospholipase C activity on phosphatidylcholine and phosphatidylethanolamine. A double mutant of npc5 and pldf2 (carrying a mutation in the phospholipase Df2 gene) was generated. Lipid measurements in npc5 pldf2 indicated that the contribution of PLDf2 to DGDG production in leaves is negligible. In contrast to the chloroplast envelope localization of galactolipid synthesis enzymes, NPC5 localizes to the cytosol, suggesting that, during phosphate limitation, soluble NPC5 associates with membranes where it contributes to the conversion of phospholipids to diacylglycerol, the substrate for galactolipid synthesis.

SummaryNitrogen is an essential nutrient for plants because it represents a major constituent of numerous cellular compounds, including proteins, amino acids, nucleic acids and lipids. While N deprivation is known to have severe consequences for primary carbon metabolism, the effect on chloroplast lipid metabolism has not been analysed in higher plants. Nitrogen limitation in Arabidopsis led to a decrease in the chloroplast galactolipid monogalactosyldiacylglycerol (MGDG) and a concomitant increase in digalactosyldiacylglycerol (DGDG), which correlated with an elevated expression of the DGDG synthase genes DGD1 and DGD2. The amounts of triacylglycerol and free fatty acids increased during N deprivation. Furthermore, phytyl esters accumulated containing medium-chain fatty acids (12:0, 14:0) and a large amount of hexadecatrienoic acid (16:3). Fatty acid phytyl esters were localized to chloroplasts, in particular to thylakoids and plastoglobules. Different polyunsaturated acyl groups were found in phytyl esters accumulating in Arabidopsis lipid mutants and in other plants, including 16:3 and 18:3 species. Therefore N deficiency in higher plants results in a co-ordinated breakdown of galactolipids and chlorophyll with deposition of specific fatty acid phytyl esters in thylakoids and plastoglobules of chloroplasts.

The peribacteroid membrane (PBM) surrounding nitrogen fixing rhizobia in the nodules of legumes is crucial for the exchange of ammonium and nutrients between the bacteria and the host cell. Digalactosyldiacylglycerol (DGDG), a galactolipid abundant in chloroplasts, was detected in the PBM of soybean (Glycine max) and Lotus japonicus. Analyses of membrane marker proteins and of fatty acid composition confirmed that DGDG represents an authentic PBM lipid of plant origin and is not derived from the bacteria or from plastid contamination. In Arabidopsis, DGDG is known to accumulate in extraplastidic membranes during phosphate deprivation. However, the presence of DGDG in soybean PBM was not restricted to phosphate limiting conditions. Complementary DNA sequences corresponding to the two DGDG synthases, DGD1 and DGD2 from Arabidopsis, were isolated from soybean and Lotus. The two genes were expressed during later stages of nodule development in infected cells and in cortical tissue. Because nodule development depends on the presence of high amounts of phosphate in the growth medium, the accumulation of the non-phosphorus galactolipid DGDG in the PBM might be important to save phosphate for other essential processes, i.e. nucleic acid synthesis in bacteroids and host cells.

BackgroundLegumes have the unique capability to undergo root nodule and arbuscular mycorrhizal symbiosis. Both types of root endosymbiosis are regulated by NSP2, which is a target of microRNA171h (miR171h). Although, recent data implies that miR171h specifically restricts arbuscular mycorrhizal symbiosis in the root elongation zone of Medicago truncatula roots, there is limited knowledge available about the spatio-temporal regulation of miR171h expression at different physiological and symbiotic conditions.ResultsWe show that miR171h is functionally expressed from an unusual long primary transcript, previously predicted to encode two identical miR171h strands. Both miR171h and NSP2 transcripts display a complex regulation pattern, which involves the symbiotic status and the fertilization regime of the plant. Quantitative Real-time PCR revealed that miR171h and NSP2 transcript levels show a clear anti-correlation in all tested conditions except in mycorrhizal roots, where NSP2 transcript levels were induced despite of an increased miR171h expression. This was also supported by a clear correlation of transcript levels of NSP2 and MtPt4, a phosphate transporter specifically expressed in a functional AM symbiosis. MiR171h is strongly induced in plants growing in sufficient phosphate conditions, which we demonstrate to be independent of the CRE1 signaling pathway and which is also not required for transcriptional induction of NSP2 in mycorrhizal roots. In situ hybridization and promoter activity analysis of both genes confirmed the complex regulation involving the symbiotic status, P and N nutrition, where both genes show a mainly mutual exclusive expression pattern. Overexpression of miR171h in M. truncatula roots led to a reduction in mycorrhizal colonization and to a reduced nodulation by Sinorhizobium meliloti.ConclusionThe spatio-temporal expression of miR171h and NSP2 is tightly linked to the nutritional status of the plant and, together with the results from the overexpression analysis, points to an important function of miR171h to integrate the nutrient homeostasis in order to safeguard the expression domain of NSP2 during both, arbuscular mycorrhizal and root nodule symbiosis.

The galactolipid digalactosyldiacylglycerol (DGD) is an abundant thylakoid lipid in chloroplasts. The introduction of the bacterial lipid glucosylgalactosyldiacylglycerol (GGD) from Chloroflexus aurantiacus into the DGD-deficient Arabidopsis (Arabidopsis thaliana) dgd1 mutant was previously shown to result in complementation of growth, but photosynthetic efficiency was only partially restored. Here, we demonstrate that GGD accumulation in the double mutant dgd1dgd2, which is totally devoid of DGD, also complements growth at normal and high-light conditions, but photosynthetic efficiency in the GGD-containing dgd1dgd2 line remains decreased. This is attributable to an increased susceptibility of photosystem II to photodamage, resulting in reduced photosystem II accumulation already at normal light intensities. The chloroplasts of dgd1 and dgd1dgd2 show alterations in thylakoid ultrastructure, a phenotype that is restored in the GGD-containing lines. These data suggest that the strong growth retardation of the DGD-deficient lines dgd1 and dgd1dgd2 can be primarily attributed to a decreased capacity for chloroplast membrane assembly and proliferation and, to a smaller extent, to photosynthetic deficiency. During phosphate limitation, GGD increases in plastidial and extraplastidial membranes of the transgenic lines to an extent similar to that of DGD in the wild type, indicating that synthesis and transport of the bacterial lipid (GGD) and of the authentic plant lipid (DGD) are subject to the same mechanisms of regulation.Higher plants contain two galactolipids, monogalactosyldiacylglycerol (MGD) and digalactosyldiacylglycerol (DGD), that constitute about 75 mol % of the thylakoid lipids in chloroplasts (Benson, 1971;Joyard et al., 1998; Dörmann and Benning, 2002). MGD and DGD are synthesized from UDP-Gal and diacylglycerol by MGD and DGD synthases in the chloroplast envelope membranes. While MGD is mostly produced from diacylglycerol originating from the plastid (''prokaryotic lipid''), DGD is largely derived from endoplasmic reticulum (ER) lipid precursors (''eukaryotic lipid'';Heinz and Roughan, 1983;Browse et al., 1986b). The lipid transport from the ER to the plastid is not well understood. However, recent results suggest that an ATP-binding cassette-type transport complex is involved in the transfer of eukaryotic lipids from the ER to the chloroplast (Xu et al., 2003). Galactolipids do not only establish the lipid bilayer into which the photosynthetic complexes are embedded. Structural analysis of crystallized protein complexes revealed that galactolipids are also found within the structures of PSI and PSII, light-harvesting complex II (LHCII), and cytochrome b 6 /f (Jordan et al., 2001;Stroebel et al., 2003;Liu et al., 2004;Loll et al., 2005;Jones, 2007). MGD and DGD play crucial roles in photosynthetic light reactions, and this is in agreement with results obtained from the analysis of galactolipid-deficient Arabidopsis (Arabidopsis thaliana) mutants (Härtel et al., 1997;Reifarth et al., 1997;Guo et al., 2...

BackgroundLegumes have the unique capacity to undergo two important root endosymbioses: the root nodule symbiosis and the arbuscular mycorrhizal symbiosis. Medicago truncatula is widely used to unravel the functions of genes during these root symbioses. Here we describe the development of an artificial microRNA (amiR)-mediated gene silencing system for M. truncatula roots.ResultsThe endogenous microRNA (miR) mtr-miR159b was selected as a backbone molecule for driving amiR expression. Heterologous expression of mtr-miR159b-amiR constructs in tobacco showed that the backbone is functional and mediates an efficient gene silencing. amiR-mediated silencing of a visible marker was also effective after root transformation of M. truncatula constitutively expressing the visible marker. Most importantly, we applied the novel amiR system to shed light on the function of a putative transcription factor, MtErf1, which was strongly induced in arbuscule-containing cells during mycorrhizal symbiosis. MtPt4 promoter driven amiR-silencing led to strongly decreased transcript levels and deformed, non-fully truncated arbuscules indicating that MtErf1 is required for arbuscule development.ConclusionsThe endogenous amiR system demonstrated here presents a novel and highly efficient tool to unravel gene functions during root endosymbioses.

Keywords: arbuscular mycorrhizal symbiosis (AM symbiosis), laser capture microdissection (LCM), Medicago truncatula, proteomics, liquid chromatography-tandem mass spectrometry (LC/MS/MS)The development of an arbuscular mycorrhizal (AM) symbiosis is a non-synchronous process with typical mycorrhizal root containing different symbiotic stages at one time. Methods providing cell type-specific resolution are therefore required to separate these stages and analyze each particular structure independently from each other. We established an experimental system for analyzing specific proteomic changes in arbuscule-containing cells of Glomus intraradices colonized Medicago truncatula roots. The combination of laser capture microdissection (LCM) and liquid chromatography-tandem mass chromatography (LC-MS/MS) allowed the identification of proteins with specific or increased expression in arbuscule-containing cells. Consistent with previous transcriptome data, the proteome of arbuscule-containing cells showed an increased number of proteins involved in lipid metabolism, most likely related to the synthesis of the periarbuscular membrane. In addition, transcriptome data of non-colonized cells of mycorrhizal roots suggest mobilization of carbon resources and their symplastic transport toward arbuscule-containing cells for the synthesis of periarbuscular membranes. This highlights the periarbuscular membrane as important carbon sink in the mycorrhizal symbiosis.