Publications - Cell and Metabolic Biology

Genetic resources for the model plant Arabidopsis comprise mutant lines defective in almost any single gene in reference accession Columbia. However, gene redundancy and/or close linkage often render it extremely laborious or even impossible to isolate a desired line lacking a specific function or set of genes from segregating populations. Therefore, we here evaluated strategies and efficiencies for the inactivation of multiple genes by Cas9-based nucleases and multiplexing. In first attempts, we succeeded in isolating a mutant line carrying a 70 kb deletion, which occurred at a frequency of ~ 1.6% in the T2 generation, through PCR-based screening of numerous individuals. However, we failed to isolate a line lacking Lhcb1 genes, which are present in five copies organized at two loci in the Arabidopsis genome. To improve efficiency of our Cas9-based nuclease system, regulatory sequences controlling Cas9 expression levels and timing were systematically compared. Indeed, use of DD45 and RPS5a promoters improved efficiency of our genome editing system by approximately 25–30-fold in comparison to the previous ubiquitin promoter. Using an optimized genome editing system with RPS5a promoter-driven Cas9, putatively quintuple mutant lines lacking detectable amounts of Lhcb1 protein represented approximately 30% of T1 transformants. These results show how improved genome editing systems facilitate the isolation of complex mutant alleles, previously considered impossible to generate, at high frequency even in a single (T1) generation.

Expression takes place for most of the jasmonic acid (JA)-induced genes in a COI1-dependent manner via perception of its conjugate JA-Ile in the SCFCOI1-JAZ co-receptor complex. There are, however, numerous genes and processes, which are preferentially induced COI1-independently by the precursor of JA, 12-oxo-phytodienoic acid (OPDA). After recent identification of the Ile-conjugate of OPDA, OPDA-Ile, biological activity of this compound could be unequivocally proven in terms of gene expression. Any interference of OPDA, JA, or JA-Ile in OPDA-Ile-induced gene expression could be excluded by using different genetic background. The data suggest individual signaling properties of OPDA-Ile. Future studies for analysis of an SCFCOI1-JAZ co-receptor-independent route of signaling are proposed.

Jasmonates (JAs) are ubiquitously occurring signaling compounds in plants formed in response to biotic and abiotic stress as well as in development. (+)-7-iso-jasmonoyl isoleucine, the bioactive JA, is involved in most JA-dependent processes mediated by the F-box protein COI1 in a proteasome-dependent manner. However, there is an increasing number of examples, where the precursor of JA biosynthesis, cis-(+)-12-oxophytodienoic acid (OPDA) is active in a JA/COI1-independent manner. Here, we discuss those OPDA-dependent processes, thereby giving emphasis on tomato embryo development. Recent data on seed coat-generated OPDA and its role in embryo development is discussed based on biochemical and genetic evidences.

Oxidative tailoring of C40 carotenoids by double bond-specific cleavage enzymes (carotenoid cleavage dioxygenases, CCDs) gives rise to various apocarotenoids. AtCCD1 generating C13 and C14 apocarotenoids and orthologous enzymes in other plants are the only CCDs acting in the cytosol, while the hitherto presumed C40 substrate is localized in the plastid. A new model for CCD1 action arising from a RNAi-mediated CCD1 gene silencing study in mycorrhizal hairy roots of Medicago truncatula may solve this contradiction. This approach unexpectedly resulted in the accumulation of C27 apocarotenoids but not C40 carotenoids suggesting C27 as the main substrates for CCD1 in planta. It further implies a consecutive two-step cleavage process, in which another CCD performs the primary cleavage of C40 to C27 in the plastid followed by C27 export and further cleavage by CCD1 in the cytosol. We compare the specificities and subcellular locations of the various CCDs and propose the plastidial CCD7 to be the first player in mycorrhizal apocarotenoid biogenesis.

The mutualistic interaction of plants with arbuscular mycorrhizal (AM) fungi is characterized by an exchange of nutrients. The plant provides sugars in the form of hexoses to the heterotrophic fungus in return for phosphate as well as nitrogen, water, and micronutrients. Plant sucrose-cleaving enzymes are predicted to play a crucial role in hexose mobilization as these enzymes appear to be absent in the fungal partner. Here, recent findings concerning the function of plant apoplastic invertases in the AM symbiosis are discussed. Plants with modulated enzyme activity in roots and leaves provide additional insight on the complexity of the regulation of the AM interaction by apoplastic invertases as mycorrhization could be reduced or stimulated depending on the level of invertase activity and its tissue-specific expression.