Cell- and Tissue-Specific Transcriptome Analyses of Medicago truncatula Root Nodules

Legumes have the unique ability to host nitrogen-fixing Rhizobium bacteria as symbiosomes inside root nodule cells. To get insight into this key process, which forms the heart of the endosymbiosis, we isolated specific cells/tissues at different stages of symbiosome formation from nodules of the model legume Medicago truncatula using laser-capture microdissection. Next, we determined their associated expression profiles using Affymetrix Medicago GeneChips. Cells were collected from the nodule infection zone divided into a distal (where symbiosome formation and division occur) and proximal region (where symbiosomes are mainly differentiating), as well as infected cells from the fixation zone containing mature nitrogen fixing symbiosomes. As non-infected cells/tissue we included nodule meristem cells and uninfected cells from the fixation zone. Here, we present a comprehensive gene expression map of an indeterminate Medicago nodule and selected genes that show specific enriched expression in the different cells or tissues. Validation of the obtained expression profiles, by comparison to published gene expression profiles and experimental verification, indicates that the data can be used as digital "in situ''. This digital "in situ'' offers a genome-wide insight into genes specifically associated with subsequent stages of symbiosome and nodule cell development, and can serve to guide future functional studies.

Rate of Belowground Carbon Allocation Differs with Successional Habit of Two Afromontane Trees

Background: Anthropogenic disturbance of old-growth tropical forests increases the abundance of early successional tree species at the cost of late successional ones. Quantifying differences in terms of carbon allocation and the proportion of recently fixed carbon in soil CO2 efflux is crucial for addressing the carbon footprint of creeping degradation. Methodology: We compared the carbon allocation pattern of the late successional gymnosperm Podocarpus falcatus (Thunb.) Mirb. and the early successional (gap filling) angiosperm Croton macrostachyus Hochst. es Del. in an Ethiopian Afromontane forest by whole tree (CO2)-C-13 pulse labeling. Over a one-year period we monitored the temporal resolution of the label in the foliage, the phloem sap, the arbuscular mycorrhiza, and in soil-derived CO2. Further, we quantified the overall losses of assimilated C-13 with soil CO2 efflux. Principal Findings: C-13 in leaves of C. macrostachyus declined more rapidly with a larger size of a fast pool (64% vs. 50% of the assimilated carbon), having a shorter mean residence time (14 h vs. 55 h) as in leaves of P. falcatus. Phloem sap velocity was about 4 times higher for C. macrostachyus. Likewise, the label appeared earlier in the arbuscular mycorrhiza of C. macrostachyus and in the soil CO2 efflux as in case of P. falcatus (24 h vs. 72 h). Within one year soil CO2 efflux amounted to a loss of 32% of assimilated carbon for the gap filling tree and to 15% for the late successional one. Conclusions: Our results showed clear differences in carbon allocation patterns between tree species, although we caution that this experiment was unreplicated. A shift in tree species composition of tropical montane forests (e. g., by degradation) accelerates carbon allocation belowground and increases respiratory carbon losses by the autotrophic community. If ongoing disturbance keeps early successional species in dominance, the larger allocation to fast cycling compartments may deplete soil organic carbon in the long run.

Composite Medicago truncatula plants harbouring Agrobacterium rhizogenes-transformed roots reveal normal mycorrhization by Glomus intraradices

Composite plants consisting of a wild-type shoot and a transgenic root are frequently used for functional genomics in legume research. Although transformation of roots using Agrobacterium rhizogenes leads to morphologically normal roots, the question arises as to whether such roots interact with arbuscular mycorrhizal (AM) fungi in the same way as wild-type roots. To address this question, roots transformed with a vector containing the fluorescence marker DsRed were used to analyse AM in terms of mycorrhization rate, morphology of fungal and plant subcellular structures, as well as transcript and secondary metabolite accumulations. Mycorrhization rate, appearance, and developmental stages of arbuscules were identical in both types of roots. Using Mt16kOLI1Plus microarrays, transcript profiling of mycorrhizal roots showed that 222 and 73 genes exhibited at least a 2-fold induction and less than half of the expression, respectively, most of them described as AM regulated in the same direction in wild-type roots. To verify this, typical AM marker genes were analysed by quantitative reverse transcription-PCR and revealed equal transcript accumulation in transgenic and wild-type roots. Regarding secondary metabolites, several isoflavonoids and apocarotenoids, all known to accumulate in mycorrhizal wild-type roots, have been found to be up-regulated in mycorrhizal in comparison with non-mycorrhizal transgenic roots. This set of data revealed a substantial similarity in mycorrhization of transgenic and wild-type roots of Medicago truncatula, validating the use of composite plants for studying AM-related effects.

Pre-announcement of symbiotic guests: transcriptional reprogramming by mycorrhizal lipochitooligosaccharides shows a strict co-dependency on the GRAS transcription factors NSP1 and RAM1

BACKGROUND: More than 80 % of all terrestrial plant species establish an arbuscular mycorrhiza (AM) symbiosis with Glomeromycota fungi. This plant-microbe interaction primarily improves phosphate uptake, but also supports nitrogen, mineral, and water aquisition. During the pre-contact stage, the AM symbiosis is controled by an exchange of diffusible factors from either partner. Amongst others, fungal signals were identified as a mix of sulfated and non-sulfated lipochitooligosaccharides (LCOs), being structurally related to rhizobial nodulation (Nod)-factor LCOs that in legumes induce the formation of nitrogen-fixing root nodules. LCO signals are transduced via a common symbiotic signaling pathway (CSSP) that activates a group of GRAS transcription factors (TFs). Using complex gene expression fingerprints as molecular phenotypes, this study primarily intended to shed light on the importance of the GRAS TFs NSP1 and RAM1 for LCO-activated gene expression during pre-symbiotic signaling. RESULTS: We investigated the genome-wide transcriptional responses in 5 days old primary roots of the Medicago truncatula wild type and four symbiotic mutants to a 6 h challenge with LCO signals supplied at 10(-7/-8) M. We were able to show that during the pre-symbiotic stage, sulfated Myc-, non-sulfated Myc-, and Nod-LCO-activated gene expression almost exclusively depends on the LysM receptor kinase NFP and is largely controled by the CSSP, although responses independent of this pathway exist. Our results show that downstream of the CSSP, gene expression activation by Myc-LCOs supplied at 10(-7/-8) M strictly required both the GRAS transcription factors RAM1 and NSP1, whereas those genes either co- or specifically activated by Nod-LCOs displayed a preferential NSP1-dependency. RAM1, a central regulator of root colonization by AM fungi, controled genes activated by non-sulfated Myc-LCOs during the pre-symbiotic stage that are also up-regulated in areas with early physical contact, e.g. hyphopodia and infecting hyphae; linking responses to externally applied LCOs with early root colonization. CONCLUSIONS: Since both RAM1 and NSP1 were essential for the pre-symbiotic transcriptional reprogramming by Myc-LCOs, we propose that downstream of the CSSP, these GRAS transcription factors act synergistically in the transduction of those diffusible signals that pre-announce the presence of symbiotic fungi.