Quantitative expression analysis of selected transcription factors in pavement, basal and trichome cells of mature leaves from Arabidopsis thaliana.

Gene expression levels of several transcription factors from Arabidopsis thaliana that were described previously to be involved in leaf development and trichome formation were analysed in trichome, basal and pavement cells of mature leaves. Single cell samples of these three cells types were collected by glass micro-capillaries. Real-time reverse transcription (RT)-PCR was used to analyse expression patterns of the following transcription factors: MYB23, MYB55, AtHB1, FILAMENTOUS FLOWER (FIL)/YABBY1 (YAB1), TRIPTYCHON (TRY) and CAPRICE (CPC). A difference in the expression patterns of TRY and CPC was revealed. Contrary to the CPC expression pattern, no transcripts of TRY could be detected in pavement cells. FIL/YAB1 was exclusively expressed in trichome cells. AtHB1 was highly expressed throughout all three cell types. MYB55 was higher expressed in basal cells than in trichome and pavement cells. MYB23 showed a pattern of low expression in pavement cells, medium in basal cells and high expression in trichomes. Expression patterns obtained by single cell sampling and real-time RT-PCR were compared to promoter GUS fusions of the selected transcription factors. Therefore, we regenerated two transgenic Arabidopsis lines that expressed the GUS reporter gene under control of the promoters of MYB55 and YAB1. In conclusion, despite their function in leaf morphogenesis, all six transcription factors were detected in mature leaves. Furthermore, single cell sampling and promoter GUS staining patterns demonstrated the predominant presence of MYB55 in basal cells as compared to pavement cells and trichomes.

Tissue-specific down-regulation of LjAMT1;1 compromises nodule function and enhances nodulation in Lotus japonicus.

Plant ammonium transporters of the AMT1 family are involved in N-uptake from the soil and ammonium transport, and recycling within the plant. Although AMT1 genes are known to be expressed in nitrogen-fixing nodules of legumes, their precise roles in this specialized organ remain unknown. We have taken a reverse-genetic approach to decipher the physiological role of LjAMT1;1 in Lotus japonicus nodules. LjAMT1;1 is normally expressed in both the infected zone and the vascular tissue of Lotus nodules. Inhibition of LjAMT1;1 gene expression, using an antisense gene construct driven by a leghemoglobin promoter resulted in a substantial reduction of LjAMT1;1 transcript in the infected tissue but not the vascular bundles of transgenic plants. As a result, the nitrogen-fixing activity of nodules was partially impaired and nodule number increased compared to control plants. Expression of LjAMT1;1-GFP fusion protein in plant cells indicated a plasma-membrane location for the LjAMT1;1 protein. Taken together, the results are consistent with a role of LjAMT1;1 in retaining ammonium derived from symbiotic nitrogen fixation in plant cells prior to its assimilation.

Gene expression profiling of single epidermal, basal and trichome cells of Arabidopsis thaliana.

Samples of single epidermal, basal and trichome cells were collected by glass microcapillaries from 7-week-old Arabidopsis thaliana leaves. Transcript amplification of these single-cell samples was performed by RT PCR. For gene expression profiling, we hybridized the amplified transcriptome of each individual cell type to nylon membranes spotted with 16,000 Arabidopsis expressed sequence tags (ESTs). Initial analysis of the array filter data enabled us to functionally categorize transcripts that were present in each individual cell type. In order to confirm the filter array data, we used RT PCR. Results of this RT PCR approach confirmed the presence of 12 selected candidate genes in agreement with array filter hybridization data. Further, transcripts involved in detoxification and sulfur metabolism could be identified in epidermal cell extracts. Together, the results of our study provide the localization of approximately 1000 expressed genes to either pavement, basal or trichome cells. To cluster transcripts with similar expression levels, we developed a novel mathematical algorithm. Based on the mean and standard deviation, ratios of expression levels of a transcript were defined for pairs of the three cell types. This numerical analysis enabled subdivision into 67 categories of genes differentially expressed in epidermal, basal and trichome cells. Transcripts in each category displayed similar ratios of expression levels in the three cell types. Examples of these clusters are presented and discussed in Appendix A.

Cell-specific protein profiling in Arabidopsis thaliana trichomes: identification of trichome-located proteins involved in sulfur metabolism and detoxification.

Metabolite, protein, and transcript analysis at the cellular level gives unparalleled insight into the complex roles tissues play in the plant system. However, while capillary electrophoresis and PCR amplification strategies make the profiling of metabolites and transcripts in specific cell types possible, the profiling of proteins in small samples represents a bottleneck. Here for the first time protein profiling has been achieved in a specific plant cell type: The application of specific cell sampling and shotgun peptide sequencing (nano LC/MS/MS) resulted in the identification of 63 unique proteins from pooled Arabidopsis trichome cells. A complete S-adenosylmethionine pathway cluster, two S-adenosylmethionine synthase isoforms, a glutathione S-conjugate translocator and other proteins involved in sulfur metabolism and detoxification are shown to be present in these cells, in agreement with previous work done at the level of trichome transcript analysis. The technology described here brings the simultaneous identification and localization of physiologically relevant cellular proteins within reach.

Characterization of three functional high-affinity ammonium transporters in Lotus japonicus with differential transcriptional regulation and spatial expression.

Ammonium is a primary source of nitrogen for plants. In legume plants ammonium can also be obtained by symbiotic nitrogen fixation, and NH(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(4)(+). The K(i) for ammonium was 1.7, 3, and 15 microm 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-beta-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.

Molecular and cellular characterisation of LjAMT2;1, an ammonium transporter from the model legume Lotus japonicus.

Two related families of ammonium transporters have been identified and partially characterised in plants in the past; the AMT1 and AMT2 families. Most attention has focused on the larger of the two families, the AMT1 family, which contains members that are likely to fulfil different, possibly overlapping physiological roles in plants, including uptake of ammonium from the soil. The possible physiological functions of AMT2 proteins are less clear. Lack of data on cellular and tissue location of gene expression, and the intracellular location of proteins limit our understanding of the physiological role of all AMT proteins. We have cloned the first AMT2 family member from a legume, LjAMT2;1 of Lotus japonicus, and demonstrated that it functions as an ammonium transporter by complementing a yeast mutant defective in ammonium uptake. However, like AtAMT2 from Arabidopsis, and unlike AMT1 transporters from several plant species, LjAMT2;1 was unable to transport methylammonium. The LjAMT2;1 gene was found to be expressed constitutively throughout Lotus plants. In situ RNA hybridisation revealed LjAMT2;1 expression in all major tissues of nodules. Transient expression of LjAMT2;1-GFP fusion protein in plant cells indicated that the transporter is located on the plasma membrane. In view of the fact that nodules derive ammonium internally, rather than from the soil, the results implicate LjAMT2;1 in the recovery of ammonium lost from nodule cells by efflux. A similar role may be fulfilled in other organs, especially leaves, which liberate ammonium during normal metabolism.