Functional Knockout of the Adenosine 5′-Phosphosulfate Reductase Gene in Physcomitrella patens Revives an Old Route of Sulfate Assimilation

Abstract

The reduction of adenosine 5-phosphosulfate (APS) to sulfite catalyzed by adenosine 5-phosphosulfate reductase is considered to be the key step of sulfate assimilation in higher plants. However, analogous to enteric bacteria, an alternative pathway of sulfate reduction via phosphoadenosine 5-phosphosulfate (PAPS) was proposed. To date, the presence of the corresponding enzyme, PAPS reductase, could be neither confirmed nor excluded in plants. To find possible alternative routes of sulfate assimilation we disrupted the adenosine 5-phosphosulfate reductase single copy gene in Physcomitrella patens by homologous recombination. This resulted in complete loss of the correct transcript and enzymatic activity. Surprisingly, the knockout plants grew on sulfate as the sole sulfur source, and the concentration of thiols in the knockouts did not differ from the wild type plants. However, when exposed to a sublethal concentration of cadmium, the knockouts were more sensitive than wild type plants. When fed [35 S]sulfate, the knockouts incorporated 35 S in thiols; the flux through sulfate reduction was ϳ50% lower than in the wild type plants. PAPS reductase activity could not be measured with thioredoxin as reductant, but a cDNA and a gene coding for this enzyme were detected in P. patens. The moss Physcomitrella patens is thus the first plant species wherein PAPS reductase was confirmed on the molecular level and also the first organism wherein both APS-and PAPS-dependent sulfate assimilation co-exist.

Identification and Functional Characterization of the Moss Physcomitrella patens Δ5-Desaturase Gene Involved in Arachidonic and Eicosapentaenoic Acid Biosynthesis

“…In contrast, D. discoideum ⌬ 5 -desaturase genes are not involved in long-chain PUFA metabolism, because they can add a double bond at the ⌬ 5 position of C16 and C18 monoenoic acids only (29,30). Recently a ⌬ 5 -desaturase from Phytophthora megasperma was described that specifically introduces an additional double bond only into C20 fatty acids degradation (46), photomorphology (47), and sulfate assimilation (48 S promoter in the targeted line confirmed the ⌬ 5 -desaturase activity specific for C20 fatty acids. However, we could not detect EPA (20:5 ⌬ 5,8,11,14,17 ) in protoplasts of the targeted strain, and so the absence of an -3 product in the overexpression transgenics is likely explained by the low level of substrate present in P. patens.…”

Targeted knock‐out of a gene encoding sulfite reductase in the moss Physcomitrella patens affects gametophytic and sporophytic development

“…Cadmium treatments were performed in two independent repetitions with three independent cultures each. The described DSiR1 mutants are deposited in the International Moss Stock Center with the accessions IMSC 40447 (1-6), IMSC 40450 (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17), IMSC 40454 (4-5) and IMSC 40455 (5-15).…”

“…Sensitivity to the toxic heavy metal cadmium is a typical consequence of disturbance of sulfur metabolism in P. patens [11,22].…”

“…patens has been widely used to address various questions of primary metabolism, such as fatty acid synthesis, N-glycosylation, or sulfur metabolism [10,11,[17][18][19][20]. Many such analyses revealed an unexpected variety of enzymes and pathways alternative to those in flowering plants.…”

Comparative genomics ofPhyscomitrella patensgametophytic transcriptome andArabidopsis thaliana: Implication for land plant evolution

“…A number of P. patens genes have been isolated and disrupted successfully (12)(13)(14)(15)(16). In addition to these individual gene analyses, EST projects are ongoing, and P. patens is the most suitable moss for genetic comparisons with the flowering plants.…”