The genetic and molecular analysis of trichome development in Arabidopsis thaliana has generated a detailed knowledge about the underlying regulatory genes and networks. However, how rapidly these mechanisms diverge during evolution is unknown. To address this problem, we used an unbiased forward genetic approach to identify most genes involved in trichome development in the related crucifer species Arabis alpina. In general, we found most trichome mutant classes known in A. thaliana. We identified orthologous genes of the relevant A. thaliana genes by sequence similarity and synteny and sequenced candidate genes in the A. alpina mutants. While in most cases we found a highly similar genephenotype relationship as known from Arabidopsis, there were also striking differences in the regulation of trichome patterning, differentiation, and morphogenesis. Our analysis of trichome patterning suggests that the formation of two classes of trichomes is regulated differentially by the homeodomain transcription factor AaGL2. Moreover, we show that overexpression of the GL3 basic helix-loop-helix transcription factor in A. alpina leads to the opposite phenotype as described in A. thaliana. Mathematical modeling helps to explain how this nonintuitive behavior can be explained by different ratios of GL3 and GL1 in the two species.Arabis alpina | trichomes | genetic analysis

SummaryContractile vacuoles (CVs) are essential for osmoregulation in many protists. To investigate the mechanism of CV function in Chlamydomonas, we isolated novel osmoregulatory mutants. Four of the isolated mutant cell lines carried the same 33,641 base deletion, rendering the cell lines unable to grow under strong hypotonic conditions. One mutant cell line (Osmo75) was analyzed in detail. The CV morphology was variable in mutant cells, and most cells had multiple small CVs. In addition, one or two enlarged CVs or no visible CVs at all, were observed by light microscopy. These findings suggest that the mutant is impaired in homotypic vacuolar and exocytotic membrane fusion. Furthermore the mutants had long flagella. One of the affected genes is the only SEC6 homologue in Chlamydomonas (CreSEC6). The SEC6 protein is a component of the exocyst complex that is required for efficient exocytosis. Transformation of the Osmo75 mutant with a CreSEC6-GFP construct rescued the mutant completely (osmoregulation and flagellar length). Rescued strains overexpressed CreSEC6 (as a GFP-tagged protein) and displayed a modified CV activity. CVs were larger, whereas the CV contraction interval remained unchanged, leading to increased water efflux rates. Electron microscopy analysis of Osmo75 cells showed that the mutant is able to form the close contact zones between the plasma membrane and the CV membrane observed during late diastole and systole. These results indicate that CreSEC6 is essential for CV function and required for homotypic vesicle fusion during diastole and water expulsion during systole. In addition, CreSEC6 is not only necessary for CV function, but possibly influences the CV cycle in an indirect manner and flagellar length in Chlamydomonas.

Arabis alpina is a perennial arctic-alpine plant and an upcoming model organism for genetics and molecular biology for the Brassicaceae family. One essential method for most molecular approaches is the analysis of gene expression by reverse-transcription quantitative Real-Time PCR (RT-qPCR). For the normalisation of expression data in RT-qPCR experiments, it is essential to use reliable reference genes that are not affected under a wide range of conditions. In this study we establish a set of 15 A . alpina reference genes that were tested under different conditions including cold, drought, heat, salt and gibberellic acid treatments. Data analyses with geNORM, BestKeeper and NormFinder revealed the most stable reference genes for the tested conditions: RAN3 , HCF and PSB33 are most suitable for cold treatments; UBQ10 and TUA5 for drought; RAN3 , PSB33 and EIF4a for heat; CAC , TUA5 , ACTIN 2 and PSB33 for salt and PSB33 and TUA5 for gibberellic acid treatments. CAC and ACTIN 2 showed the least variation over all tested samples. In addition, we show that two reference genes are sufficient to normalize RT-qPCR data under our treatment conditions. In future studies, these reference genes can be used for an adequate normalisation and thus help to generate high quality RT-qPCR data in A . alpina .

Beige and Chediak Higashi (BEACH) domain proteins mediate membrane-dependent processes in eukaryotic cells. The plant BEACH domain protein SPIRRIG in A. thaliana (AtSPI) was shown to display a similar molecular behavior as its yeast and animal homologs, along with a range of cell morphological defects. In addition, AtSPI was shown to interact with the P-body component DCP1, to differentially effect RNA levels and to be involved in the regulation of RNA stability in the context of salt stress responses. To determine, whether the dual function of SPI in apparently unrelated molecular pathways and traits is evolutionary conserved, we analyzed three Aaspi alleles in Arabis alpina. We show that the molecular behavior of the SPI protein and the role in cell morphogenesis and salt stress response are similar in the two species, though we observed distinct deviations in the phenotypic spectrum.

13Arabis alpina is a perennial arctic-alpine plant and an upcoming model organism for genetics and 14 molecular biology for the Brassicaceae family. One essential method for most molecular approaches 15 is the analysis of gene expression by quantitative Real-Time PCR (qPCR). For the normalisation of 16 expression data in qPCR experiments, it is essential to use reliable reference genes that are not 17 affected under a wide range of conditions. In this study we establish a set of 15 A. alpina reference 18 genes that were tested under different conditions including cold, drought, heat, salt and gibberellic 19 acid treatments. Data analyses with geNORM, BestKeeper and NormFinder revealed the most stable 20 reference genes for the tested conditions: RAN3, HCF and PSB33 are most suitable for cold 21 treatments; UBQ10 and TUA5 for drought; RAN3, PSB33 and EIF4a for heat; CAC, TUA5, ACTIN 2 and 22 PSB33 for salt and PSB33 and TUA5 for gibberellic acid treatments. CAC and ACTIN 2 showed the 23 least variation over all tested samples. In addition, we show that two reference genes are sufficient 24 to normalize qPCR data under our treatment conditions. In future studies, these reference genes can 25 be used for an adequate normalisation and thus help to generate high quality qPCR data in A. alpina. 2 26 130 131Normalisation of the results was carried out with one or two of the most stable reference genes 132 determined in this study (Fig 3). For cold, normalisation was carried out with RAN3 and/or HCF. 133 Drought samples were normalised to UBQ10 and/or TUA5. GA and salt responsive genes were 134 normalised with PSB33 and/or TUA5. Finally, the heat samples were normalised with RAN3 and/or 135 PSB33. The results clearly show that all treatments were successful, leading to an increased (cold, 136 drought, heat, salt) or decreased (GA) expression of the responsive genes. Individual normalisation 137with each reference gene led to differences in the calculated fold changes of 28.9 % (cold), 7.5 %