A mutant in Arabidopsis Lacking a Chloroplast Specific Lipid. Lewis Kurschner and Karen Thulasi Masters in Botany

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1 A mutant in Arabidopsis Lacking a Chloroplast Specific Lipid Lewis Kurschner and Karen Thulasi Masters in Botany

2 Fatty acid nomenclature Fatty acyl composition Chain length Degree of unsaturation and position of double bond(s) Configuration of double bond

3 Chloroplasts Chloroplasts are semi autonomous plant cellular organelles whose main function is in synthesizing carbohydrates by photosynthesis. These chloroplasts are thought to be the result of a endosymbiosis event. The chloroplasts contain the light harvesting structures called the thylakoids. The membranes of these thylakoids have a unique lipid composition compared to the membranes in the rest of the cell. The role that these lipids play in the photosynthetic process is not well known. Studies have discovered mutant plants which are deficient in chloroplast lipids which help us understand their role in biological processes.

4 Parallels to Cyanobacteria

5 Chloroplast glycerolipid composition is more similar to Cyanobacteria than it is to eukaryotic plasma membrane glycerolipid composition Kobayashi and Wada, 2016

6 Background and previous hypotheses Functional significance of unsaturation was largely a mystery at the time of publication for both animal and plants. The hypothesis at the time was that there was a causal relationship between membrane saturation and chilling sensitivity. Another hypothesis stated that the unusual fatty acid composition in membranes of chloroplasts had to do with photosynthetic ability. To obtain clarity on such questions this study was carried out.

7 Forward genetics

8 Generation of mutants using EMS Mutagenesis using EMS is based on the principle that Ethyl methanesulfonate converts Guanine to 6-Ethylguanine. 6-Ethylguanine base pairs with Thymine and not cytosine during DNA replication. This results in point mutations (transition mutation G-C to A-T) WT seeds were mutagenised using 0.3% aqueous EMS and these M1 seeds were grown and M2 seeds were harvested. The M2 plants were taken for analysis. Klug and Cummings, 1997

9 Esterification of the Lipids Lipids from 2000 M2 plants leaves were analysed using GC. The lipids were first esterified The esterified lipids were then analysed using gas-liquid chromatography.

10 Gas chromatography with flame ionization detection

11 Generation and propagation of mutants 89/2000 putative mutants with differing FA profile from WT 89 M2 were self fertilized to generate M3 seeds M3 plants were propagated using single-seed descent 7 of the original 89 lines had a stable mutation causing abnormal fatty acid composition Classified 7 mutations into 5 categories (How?) 89 M2 putative mutants

12 Total Fatty Acid Composition of WT and JB60 Doesn t contain Δ3-trans 16:1 Subsequent increase in 16:0 (Why?)

13 Δ3-trans-hexadecenoic acid Mutation in one line (JB60) made the plant unable to produce Δ3-trans 16:1 Increase in 16:0 fatty acyl as it was not being converted to Δ3-trans 16:1 16:1 is already uncommon in plants but the trans configuration even more so Carboxyl end Methyl end Δ3

14 Testing the genetic basis of the mutation Cross JB60 (homozygous mutant) to WT F1 progeny had 50% less Δ3-trans 16:1 than WT (What does this indicate about the nature of the mutation?) F2 progeny had 13/57 (44:13) mutants fitting the 3:1 hypothesis for Mendelian inheritance What kind of mutation is found in JB60? P1 x P2 F1 x F1 X M M WT WT/M WT/M WT WT/M WT/M X M WT M M/M WT/M WT WT/M WT/WT

15 Phosphatidylglycerol (PG) Glycerolipid that has a glycerol-phosphate backbone on sn-3 of the glycerol backbone Fatty acyl side chains on sn-1 and sn-2 Δ3-trans 16:1 is frequently found in this molecule attached only to sn-2

16 PG synthesis Phosphatidic acid gets converted to phosphatidylglycerol 16:0 gets desaturated to Δ3-trans 16:1 X

17 PG and Thylakoids PG is most abundant in the thylakoid membranes of the chloroplast Thylakoid membranes stack together to form the granum in higher plants This is where photosynthesis occurs

18 Thin Layer Chromatography to separate lipids Why use TLC Trans 16:1 lipid is specifically present in the Phosphatidylglycerol fraction of the total plant lipids. Hence using TLC they separated the PG fraction of the total lipid present in JB60 and WT leaves.

19 Iodine staining to visualize lipid containing zones The lipids present in the Wild Type and Mutant Arabidopsis plants were separated by running the chloroform-methanol leaf extracts on TLC plates. The lipid spots were visualized using iodine vapors. The PG spot was scraped off and the fatty acids were measured in a similar way using GC.

20 Composition of PG in WT vs. JB60 WT Peak absent in JB60 at 4 minutes which is present in WT (compared to standard) Large increase in 16:0 attached to PG in JB60 that is representative excess 16:0 JB60 confirmed to lack a desaturase enzyme that converts 16:0 to Δ3-trans 16:1 JB60

21 Light harvesting chlorophyll a/b protein complex (LHCP) Accumulates in thylakoid membranes during normal chloroplast development to increase efficiency of light capture in low light Possible role in the formation of appressed membranes of the grana Δ3-trans 16:1 may be associated with LHCP and the formation of grana

22 Transmission electron microscopy No obvious differences were found in the size or extent of grana development or other features in the chloroplast What does this mean? WT JB60

23 LHCP form complexes with integrated Δ3-trans 16:1-PG LHCP is found as a trimer of 3 identical subunits 1 Δ3-trans 16:1-PG molecule is found per LHCP monomer LHCP devoid of Δ3-trans 16:1-PG were analyzed in order to determine if JB60 had a reduction in light capture efficiency 3 subunits each bound with Δ3-trans 16:1-PG HLCP - white part is embedded in the thylakoid membrane

24 Photosystem I and II LHCP and other proteins make up PSI and PSII Light strikes PSII and an electron travels between various proteins through an electron transport chain What would we expect if Δ3-trans 16:1 is involved with the LHCP complex

25 Photosynthesis-irradiance (PI) curve A graphical representation of the relationship between solar irradiance and photosynthesis to approximate photosynthetic capability JB60 would show a decrease in the low light intensity sections if it had major involvement with light capture efficiency

26 Photosynthetic capability of WT vs. JB60 Measured rate of O2 production in PSII Inhibited PSII and measured rate of O2 removal in PSI Both the WT and JB60 are similar in photosynthetic capabilities at low irradiance Experiments with PSII at differing temperature and salt concentrations yielded no differences (data not shown) B = WT W = JB60 PSII PSI

27 Conclusions PG-Δ3-trans-16:1 plays no structural or functional role related to light reactions of photosynthesis in Arabidopsis. Further research has shown that the lipid trans-16:1 is an essential component of the antenna complex in Chlamydomonas reinhardtii (Gamier et al. 1987, Giroud et al. 1988). This shows that though PG-Δ3-trans-16:1 is not essential in Arabidopsis, it may have a necessary role in other species.

28 Importance of the paper First paper that discovered fatty acid mutants in Arabidopsis. First use of GC analysis to screen for FA mutants that didn t have a noticeable phenotype.