Sarah Koo, Grant Lee, Tania Lucinian & Jennifer Tak

1. The lys5 Mutations of Barley Reveal the Nature and Importance of Plastidial ADP-Glc Transporters for Starch Synthesis in Cereal EndospermPatron et. al, 2004 Sarah Koo, Grant Lee, Tania Lucinian & Jennifer Tak

2. Low starch mutants SCREEN 3 mutants discovered Lys5 locus mutations Plastids from these mutants synthesize starch at normal rates from Glucose-1-Phosphate but not from ADP-Glc… hmmm?

3. ADP-Glc • Necessary for starch synthesis • Glucose donor in reaction by starch synthase • Starch is synthesized in pathways involving both the cytoplasm and the plastids (Hannah, et. al, 1997)

4. Starch Synthesis

5. Graminaceous endosperms • Starch synthesis in cereals: the location of the enzyme ADP glucose pyrophosphorylase (AGPase) in the cells of the endosperm is different from that in all other plant organs. • In cereal endosperms, AGPase occurs in both the plastids and the cytosol whereas in the other organs of cereals and in non-cereals, all of the AGPase activity is plastidial. The cytosolic AGPase in cereals plays an important role in supplying ADPG for starch synthesis. Mutants that lack cytosolic AGPase activity but have normal plastidial AGPase activity have reduced starch contents • What does this reveal? PLASTIDIAL AGPASE ALONE IS NOT SUFFICIENT TO SUPPORT NORMAL RATES OF STARCH SYNTHESIS… NEED SYNTHESIS OF ADP-GLC IN CYTOSOL FOR NORMAL RATES OF STARCH SYNTHESIS

6. WHAT DO GRAMINACEOUS ENDOSPERMS NEED? ADP-Glc transporter! a.k.a. Nucleotide-Sugar Transporter (NST) Its f(x)? -to import ADP-Glc synthesized in cytosol to site of starch synthesis in the plastid stroma

7. Maize • Brittle1 (BT1) locus encodes a plastidial envelope that transports ADP-Glc in exchange for AMP or ADP • Without BT1, the ADP-Glc synthesize by cytosolic isoform of AGPase accumulates in cytosol rate of starch synthesis = 80% ↓ (Tobias et al., 1992)

8. This study… • AIM: discover nature and importance of ADP-Glc transporter in barley endosperm • How? Study mutants that are unable to transport ADP-Glc into the plastid • Method: measure ADP-Glc contents of developing endosperms from barley mutant lines with low starch content and compare to WT endosperms • Hypothesis: mutants with reduced capacity to transport ADP-Glc across the plastid envelope would have elevated levels of ADP-Glc in the endosperm

9. Identification of ADP-Glc Accumulating Mutants

10. Mutants with lesions in Lys5 locus

11. Starch Granule Morphology Wild-Type Phenotype Low-starch mutant phenotype Also, RisØ 16 showed similar phenotype to the RisØ 13. Possibly due to similar reductions in amount of starch accumulation in the mutants. Outward grooves and sunken cheeks

12. Table III: Starch Synthesis by Isolated Plastids Objective: To discover if the ADP-Glc transport is affected by lys5 mutants Method: To isolate the plastids from the WT Bomi and a lys5 mutant, Riso 13. Table III.Starch synthesis by isolated plastids Cultivar/Line Incorporation of Glc into Starch ADP-Glc Glc-1-P nmol min–1 per unit alkaline pyrophosphatase activity Bomi 36.02 ± 3.47 2.96 ± 0.44 Risø 13 10.48 ± 2.39 4.04 ± 0.87 What are they looking for? To see ability to take up metabolites and make starch NOTE: Major difference in rate of starch synthesis

13. Results of Table III: -Intact plastids of WT: - able to synthesize starch from ADP-Glc at rates comparable to the in vivo rate. - Also can synthesize starch from Glc-1P - However, at significantly lower rate than ADP-Glc THUS, this indicates that ADP-Glc is more abundant in plastids than Glc-1P b/c ADP-Glc is what is being transported into the plastid. -Intact plastids of mutant: - able to synthesize starch from Glc-1P similar to rate of the WT -BUT…rate of starch from ADP-Glc was <30% of WT rate. THUS, this suggests that R13 has a reduced ability of ADP-Glc transport into the endosperm plastid.

14. Hypothesis If Lys5 mutations disable the ADP-Glc transport into endosperm plastids, then the mutation in Riso 16 should be epistatic to Lys5 mutations. and the double mutant plants should NOT have increased levels of ADP-Glc.

15. Facts behind the Hypothesis -Why cross R16 with R13? - Both are high-Lys mutants -Riso 16 lacks cytosolic AGPase activity reduces [ADP-Glc] reduces starch content -Riso 13 contains Lys5 mutation  increases [ADP-Glc]  reduces starch content - Thus…. - both mutants provide 2 distinguishable phenotypes - double mutants can provide information of ordering of pathway - Now it brings us to the meaning of epistasis…..

16. Background of Epistasis Epistasis= an interaction when an allele of one gene dominates the expression of alleles of another gene, and expresses its own phenotype. A double mutant of Riso 13 x Riso 16 will help to identify the sequence of the pathway of starch biosynthesis. Ordered pathway further explained after data results are shown.

17. Fig 1: Comparison of Mutants and Wild-Type What exactly are we comparing? -Wild-Type: - Bomi, Carlsberg II: parental cultivars -Single Lys5 mutants: -Riso 13, Riso 29, Riso 86 -Double Lys5 mutants: -R16/R13 (1) and (2) What does this show? -The relative amounts of ADP-Glc and UDP-Glc in each line

18. Fig 1: Metabolite Contents of WT and Mutant Endosperm Results: -[UDP-Glc] do not differ significantly from one another -Significant increase in [ADP-Glc] due to Lys5 mutation [ADP-Glc] of double mutants do not vary much from the WT WT parents Lys5 mutants Double mutants

19. Ordering of Pathway Transporter Commited step Glucose-1P ADP-Glc ADP-Glc Starch and ATP starch synthase AGPase cytosol plastid Riso 13 mutation Riso 16 mutation So why is this specific enzyme regulated?? Note: AGPase= ADP-Glucose Pyrophosphorylase Possible Pathways: MutationAccumulateConclusion R16 Glc-1P, ATP R16 has lesion in AGPasehence, inactive R13 ADP-Glc R13 with lys5 mutation affects step in starch biosynthesis AFTER ADP-Glc production R16/R13 Glc-1P, ATP R16 is epistatic to lys5 mutation (R13)

20. Conclusions: • Phenotype of Lys5 locus mutations: High [ADP-Glc] • Phenotype of double mutant: normal WT [ADP-Glc] • Since Riso16 mutation is epistatic to Riso 13 lys5 mutation, then the lys5 mutation must cause a defect in ADP-Glc transport for starch synthesis. • Hence, the hypothesis stated earlier proves true. • Inconclusive Data: Avg Wt. of Double mutants < Avg. Wt. of Single mutants or WT Thus…there is a relation between the Riso13 and Riso16 in terms of grain weight HOWEVER… “cannot be explained at present” (Patron et al.)

21. Table IV Other enzymes involved in the biosynthesis of starch most likely did not affect the inhibition of starch synthesis.

22. Question: Do the plastid envelopes in barley have a protein similar to maize BT1? Method: Isolate plastid envelope proteins of wild-type and mutant barley and perform SDS-PAGE.

23. SDS-PAGE Rupture of cells High Speed Centrifugation

24. Figure 3 Could the major protein of the plastid envelope be the protein similar to maize BT1?

25. Q-TOF Mass Spectroscopy http://www.chem.vt.edu/chem-ed/ms/quadrupo.html

26. Barley NST1, the most abundant protein in the plastid envelope, is similar to maize BT1. Black indicates identical amino acids Gray indicates similar amino acids Figure 4

27. Other Results • NST1 is 71.5% identical, 75% similar to maize BT1 • A maize BT1 antiserum failed to recognize the barley NST1 transporter • The Hv.Nst1 gene is found on the same chromosome as the lys5 locus

28. Hv.Nst1 Sequence Differences Comparison between the two WT cultivars, Bomi and Carlsberg II • Hv.Nst1 sequence differed by one base • Bomi : C-548, Carlsberg II : G-548 GGA GGT GGG GGC GCA GCT GCG GCC Glycine-184 Alanine-184

29. Hv.Nst1 Sequence Differences Comparison with the three mutants: Risø 13, Risø 29, and Risø 86 Nucleotide sequence at DNA position 548 Carlsberg II Bomi Risø 13 Risø 29 Risø 86 ? Possible that some or none of these lines are pure-breeding. Conclusion:

30. Bomi Risø 13 Hv.Nst1 Sequence Differences Looking at (point) mutations elsewhere in Hv.Nst1 gene Carlsberg II Risø 29 Risø 86 818: T  G 682: C  T GTA GTG GAA GAG CCA CCT CCG CCC TCA TCT TCG TCC Pro  Ser at 228 Val  Glu at 273

31. Bomi Risø 13 Hv.Nst1 Sequence Differences Hv.Nst1 mutations found in all three lys5 mutants = Hv.Nst1 located on Lys5 locus Carlsberg II Risø 29 Risø 86 818: T  G 682: C  T Pro  Ser at 228 Val  Glu at 273

32. Are there any homologs to Hv.NST1 and BT1 proteins? Found most similarities when compared with the transporters of the mitochondrial carrier family (MCF). MCF proteins are integral membrane proteins which transport metabolites and cofactors; used in TCA cycle, malate shuttle system, and β-oxidation. MCF, with ~2000 members, do not have any nucleotide-sugar transporting members.

33. Using bovine ATP/ADP translocator as a reference Proteins of the MCF have, in their sequences, a triplet of tandem repeats in close proximity of each other. For each tandem repeat there was a conserved Pro residue (6 aa) on one helix. D = Asp E = Glu K = Lys R = Arg [P] X [D or E] X X [K or R] The Pro residue creates a kink on that helix, such that the kink is on every other helix.

34. Proteomic Analysis of Hv.NST1 Intermembrane space Inner membrane Stroma Fig. 5a , 5b

35. From Pebay-Peyroula et al.(2003)

36. Starch biosynthesis in amyloplasts ADP? ADP? ADP-Glc From Dr Merchant lecture, 1/31/05 ; project adaptation

37. Proteomic Analysis of Hv.NST1 WT cultivars difference Val  Glu @ 273 Pro  Ser @ 228

38. Single aa substitutions explain loss of ADP-Glc transport Kinked helices are essential for transport functionality 228: Pro  Ser in Risø 29 and Risø 86 would prevent kink formation 273: Val  Glu in Risø 13 would change aa property from hydrophobic to hydrophilic The Ala/Gly discrepancy between the Bomi and Carlsberg II WT cultivars did not have a statistically significant effect on ADP-Glc transport or starch synthesis

39. Paper Conclusions • Hv.NST1 is a plastidial ADP-Glc transporter for barley endosperm • Hv.Nst1 lies in the Lys5 locus • Hv.NST1 is first BT1 homolog studied extensively • BT1 homologs possibly exist for all graminaceous plants