Long time exposure to cold stress and continuous light shows enhanced chlorophyll

Anna Beike tested cold stress conditions on sfd plate cultures (Beike 2013), but under the tested conditions (4 °C, 4 days, 16 h light/8 h dark cycle) no difference was detected between wild type and sfd. Exposure of moss cultures to cold stress for longer time periods was not analyzed in that study. Therefore, P. patens wild type and 3 sfd KO-lines were grown for a prolonged time (3 months) under continuous light and low temperature (6 °C) for phenotype analysis. As a control, the same lines were grown similarly, but at normal temperature (24 °C). Additionally, 2 lines of the FA-elongase KO-mutant elo (PSE1, (Zank, Zähringer et al. 2002)), which is necessary for the synthesis of the FA 20:4, were analyzed as well to verify if the presence of the FA 20:4 has an impact on growth in P. patens.

Chemotypes of all KO-lines were also verified via LC-MS analysis (data not shown).

At normal temperature, plate cultures of wild type and sfd grown at gametophore inducing conditions (without tartrate) look similar for all lines. Gametophores develop normally, tissue remains green and no signs of chlorotic or dried out tissue was detected (see Appendix 8).

When grown at medium containing tartrate (causing P. patens cultures to grow predominantly as protonema), wild type and sfd moss cultures show browning of tissues after 3 months of growth (see Figures 3.18. & 3.19.). The moss colonies form sponge-like structures which seem to dry out, especially when compared to gametophore-induced cultures (Appendix 9 & Appendix 10). At normal growth conditions, tissue of sfd lines appears to dry out stronger compared to wild type. The mutant lines have overall browner colonies and some cultures show a straw-like coloration of protonema tissue. The intensity of this phenotype is however not consistent for all plate cultures, some sfd colonies appear to be less dehydrated than others, even when from the same line. Lines sfd10 and sfd11 appear to show the most drying of the PpSFD KO-lines at normal temperature (see Figure 3.18.).

Figure 3.18. Moss plate cultures at normal temperature (24 °C) and protonema-inducing growth conditions (with tartrate). P. patens strains used are wild type (WT) and PpSFD KO-lines sfd3, sfd10 and sfd11. Agar plate cultures were grown for 3 months at continuous light. Pictures were taken from two independent plate cultures for each strain. Scale bar

= 0.5 cm

Cold-stressed (6 °C) wild type plate cultures with tartrate look overall more healthy than those grown at room temperature, generally being greener and developing larger gametophytes (see Figure 3.19.). The sfd KO-lines are again drier and show stronger

browning of tissue and straw-like colorations compared to wild type. Lines sfd3 and sfd11 appear to be stronger affected by the growth conditions than sfd10.

Figure 3.19. Moss plate cultures at cold stress temperature (6°C) and protonema-inducing growth conditions (with tartrate). P. patens strains used are wild type (WT) and PpSFD KO-lines sfd3, sfd10 and sfd11. Agar plate cultures were grown for 3 months at continuous light. Pictures were taken from two independent plate cultures for each strain. Scale bar = 0.5 cm

In gametophore-inducing growth conditions (without tartrate) moss cultures look overall healthier than those with tartrate. All lines form colonies with large gametophores and no visible sponge-like tissue. At normal temperatures, wild type and sfd KO-lines are visually indistinguishable (see Appendix 9). At cold stress, some sfd show a slight brown coloration, but overall tissue formation seems to be similar to wild type (see Appendix 10).

KO lines of PpELO look mostly similar to wild type, be it without tartrate (Appendix 11) or with tartrate (Appendix 12). The line elo35 shows slightly more browning of tissue when grown at cold stress without tartrate compared to wild type. Both elo35 and elo131 produced a lot more protonema on medium with tartrate compared to wild type, forming dome-like colonies.

To verify the observed phenotypes, moss tissue from tartrate-containing plate cultures was furthermore scraped off the plate cultures, lyophilized and analyzed for its chlorophyll content. Chlorophyll was extracted using an acetic acetate/acetone mixture and the light absorption was measured at 646 and 664 nm (see Figure 3.20.).

Figure 3.20. Chlorophyll content of normal grown (24°C) and cold stressed (6°C) of P. patens wild type and KO-lines from protonema-inducing growth conditions (with tartrate). P. patens strains used are wild type (WT), PpSFD KO-lines sfd3, sfd10, sfd11, and PpELO KO-lines elo35 & elo131. Agar plate cultures were grown for 3 months at continuous light. Chlorophyll was extracted from lyophilized moss tissue with acetic acetate/acetone. Absorption of wavelengths 646 & 664 nm was measured with a photometer. Data represent average and standard deviations of 2 extractions except for normally grown wild type (1 extraction) for a single experiment.

At normal temperatures, protonema-induced plants of wild type P. patens have a much higher chlorophyll content compared to sfd and elo KO-lines. At cold stress conditions wild type contains less chlorophyll than at normal temperatures. On average, both sfd and elo lines have still less chlorophyll content than wild type, even though not by much. Cold stress in general does not have a strong impact on the chlorophyll content of sfd and elo lines, which remains equally low for both conditions. Wild type, however, reduces its chlorophyll content at cold stress.

For gametophore-induced plate cultures, it was not possible to harvest sufficient amounts of plant material for chlorophyll analysis.

3.4. Complementation of A. thaliana ads2.1 KO-mutant