Components of C 4. Andreas P.M. Weber Institute of Plant Biochemistry Heinrich-Heine-University Düsseldorf

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1 Components of C 4 Andreas P.M. Weber Institute of Plant Biochemistry Heinrich-Heine-University Düsseldorf

2 C 4 photosynthesis is a complex trait Sage R F, Zhu X J. Exp. Bot. 2011;62:

3 Knowing the components is a prerequisite for understanding and reconstruction of the trait omics sys-bio syn-bio parts list blueprint engineered system

4 Identifying components by comparative (transcript)omics (within/between species) Calling differentially expressed genes Candidate genes for further analysis RNA isolation Assembly and annotation Read mapping RNA-Seq Preparation of sequencing libraries

5 NADP-ME C 4 photosynthesis in maize leaves

6 Onset of C 4 photosynthesis along a maize leaf Pick et al. (2011) Plant Cell

7 Leaf gradient chlorophyll and protein Pick et al. (2011) Plant Cell

8 Marker enzymes for NADP-ME C 4 malate malate OAA PEP P i P i OAA PEPC malate malate NADP-ME PEP CO 2 pyruvate pyruvate CO 2 pyruvate pyruvate H + H + 3-PGA 3-PGA 3-PGA 3-PGA TP TP TP TP

9 Activities of key C 4 photosynthetic enzymes increase from base to tip clear tip to base gradient for C 4 photosynthetic enzymes

10 Metabolite patterns along the gradient Pick et al. (2011) Plant Cell

11 The NADP-ME C 4 cycle (textbook ) CO 2 Mesophyll cell CO 2 Bundle Sheath cell C pyruvate P i PEP H + HCO 3 - H + pyruvate P i pyruvate pyruvate C Calvin cycle PEP OAA OAA HCO 3 - Malic acid CO 2 Malic acid Malic acid Malic acid Pick et al. (2011) Plant Cell

12 The NADP-ME C 4 cycle in maize current view CO 2 Mesophyll cell CO 2 Bundle Sheath cell C pyruvate P i PEP H + HCO 3 - H + pyruvate P i PEP alanine pyruvate alanine PEP pyruvate CO 2 AlaAT C Calvin cycle OAA OAA HCO 3 - OAA CO 2 Malic acid CO 2 aspartate aspartate Malic acid Malic acid Malic acid AspAT PEPCK Pick et al. (2011) Plant Cell

13 The C 4 cycle summary C HCO - Current model involves 3 AlaAT and AspAT PE P OA A Malic acid H + pyruvat e CO 2 H + pyruvat e alanine PE P Malic acid HCO 3 - pyruvat e aspartat e Malic acid pyruvate 2 2 OA PEPCK activity OA is required for N-balancing A CO 2 alanine Alanine is Pthe i main shuttle metabolite from Calvi P i BS to M aspartat e See also posters P30 and P57! AspA T PE P A CO CO 2 PEPC K AlaAT CO Malic acid C n cycle Pick et al. (2011) Plant Cell

14 Joe Berry, 1970, PhD thesis

15 Quantitative proteomics of maize leaf chloroplast envelope membranes isolate chloroplasts isolate envelope membranes and PLAMS Bräutigam et al., 2008

16 Top 20 most abundant proteins in envelope the Top20 account for 62.8% of the total envelope protein metabolite transporters, porins and import proteins are major constituents of the plastid envelope in maize classification annotation percentage of total protein metabolite transporter TPT 9.0 unknown membrane protein Mep2 7.3 porin OEP porin OEP unknown membrane protein Mep1 4.4 metabolite transporter PPT 4.3 import Tic unknown membrane protein Mep3 3.0 metabolite transporter DCT1 2.8 metabolite transporter DCT2/3 2.0 unknown membrane protein Mep3 2.0 import Tic porin OEP import? OEP unknown membrane protein Mep4 1.5 import? Tim22/ import Tic metabolite transporter OMT 1.4 enzyme membrane ascorbate peroxidase 1.3 unknown membrane protein ANTR2 1.3

17 MEP1 a highly abundant protein in maize chloroplast envelope membranes Highly abundant in maize leaf envelopes, low in pea envelopes Highly expressed in upper (photosynthetic) part of maize leaves, low in basal (sink) part Not abundant in NAD-ME C 4 plants Role in NADP-ME C 4 photosynthesis? Bräutigam et al., 2008

18 MEP1 is now called PLGG1 and it is required for photorespiration Pick, Bräutigam et al., 2013

19 plgg1 mutant phenotype in Arabidopsis Extensive cell death in plgg1 mutants; PLGG1 annotated as cell-death associated protein in bacteria Pick and Bräutigam et al. (2013) PNAS

20 plgg1 shows a photorespiratory phenotype shm1 WT plgg1 ambient air (0.038% CO 2 ) shm1 plgg1 WT Shift to ambient air (0.038% CO 2 ) elevated CO 2 (0.3% CO 2 ) plgg1 plgg1/ PLGG1 Pick and Bräutigam et al. (2013) PNAS

21 Metabolite profiling of plgg1 plants WT elevated CO 2 (0.3% CO 2 ) plgg1 Shift to ambient air (0.038% CO 2 ) plgg1 harvest after 2, 5 and 7 days

22 Metabolite accumulation in plgg1 plants glycolate WT mep1-1 plgg1 Pick and Bräutigam et al. (2013) PNAS

23 Metabolite accumulation in plgg1 plants glycolate WT plgg1 mep1-1 glycerate WT plgg1 mep1-1 Pick and Bräutigam et al. (2013) PNAS

24 PLGG1 a role in 2-PG/glycerate transport? Triose-P RuBP 3-PGA 2 O 2 Chloroplast RubisCO P-Glycolate (2x) ATP P i 2 Glycolate Glycerate

25 18 O 2 labeling of plgg1 Triose-P RuBP 2 O 2 RubisCO Chloroplast 3-PGA P-Glycolate (2x) ATP P i 2 Glycolate Glycerate

26 18 O 2 labeling of plgg1 Triose-P RuBP 3-PGA RubisCO 2 O 2 Chloroplast P-Glycolate (2x) ATP P i 2 Glycolate Glycerate plgg1

27 No export of glycolate in plgg1 plgg1 Triose-P RuBP 3-PGA RubisCO 2 O 2 Chloroplast P-Glycolate (2x) ATP P i 2 Glycolate X Glycerate plgg1 Pick and Bräutigam et al. (2013) PNAS

28 Glycerate-dependent oxygen evolution inhibited in plgg1 *** Pick and Bräutigam et al. (2013) PNAS

29 PLGG1 is the chloroplastidic glycolate/glycerate transporter RuBP 2 O 2 Chloroplast Triose-P RubisCO 3-PGA 2 2P-Glycolate P i Glycerate 2 Glycolate Glycerate NAD + 2 Glycolate Peroxisome O 2 NADH Hydroxypyruvate 2 Glyoxylate Glu Serine 2 Glycine 2-OG NH 4 + CO 2 Glycine Mitochondrion THF-C 1 Serine Glycine NADH NAD + ETC ATP O 2 H 2 O Figure by Marc Linka

30 Photorespiration is essential in maize A maize line with a non-functional GO, requires elevated CO 2 for growth when shifted from high CO 2 to ambient air Zelitch et al. (2008) Plant Physiol

31 Possible role of PLGG1 in maize? mesophyll cell glycerate glycerate GK 3-PGA NADPH GAPDH NADP + TP TP NH 4+, CO 2 bundle sheath cell glycerate glycolate partial photorespiratory pathway O 2 RubisCO PGP glycolate 2-PG

32 Maize runs an open Calvin-Benson cycle HCO PEP OAA malate malate pyruvate CO 2 + RubP NADPH PSI + PSII NADPH 2x 3PGA NADPH + ATP 3PGA DHAP TPT TPT 3PGA + DHAP DHAP mesophyll bundle sheath

33 PLGG1 might be priming the pump in maize Mesophyll Cell Bundle Sheath Cell DHAP NADP GAPDH NADPH 3-PGA ADP GK ATP Glycerate TPT Mep1 Glycerate Glycolate TPT Mep1 DHAP RuBP O 2 RubisCO P-Glycolate Glycolate P i Weber and Bräutigam, 2013

34 Kranz-Anatomy

35 C 3 Leaf BSC MC Evolutionary Progression towards C 4 Vascular Bundle MC BSC Vascular Bundle C 4 Leaf Denton et al., COPB 2013

36 C 4 photosynthesis in the Brassicales Cleome gynandra NAD-ME C4 photosynthesis Cleome hassleriana C3 photosynthesis Cleome are closely related to Arabidopsis thaliana (Order of the Brassicales)

37 Comparing leaf development in C 3 and C 4 ~ 2Days C. gynandra, C 4 T. hassleriana, C 3 See also poster P61! Canan Külahoglu

38 The rate of leaf expansion is similar in C 3 and C 4 Cleomaceae Canan Külahoglu

39 Similar outside, different inside C. gynandra, C 4 T. hassleriana, C 3 Canan Külahoglu

40 Visual comparison of leaf growth in C 3 and C 4 Cleomaceae ~ 2Days C. gynandra, C 4 T. hassleriana, C 3 Canan Külahoglu

41 Leaf developmental gradient C 4 leaf stays longer undifferentiated Leaf Age C 4 C 3 Canan Külahoglu

42 Leaf developmental gradient Leaf Age C 4 leaf stays longer undifferentiated C 4 C 3 Canan Külahoglu

43 Internal leaf development is differentially regulated between C 4 /C 3 leaves 240 transcriptional regulators are correlated between C 4 and C are not correlated, 30 are inversely correlated Correlated (<0.8) Not Correlated Inversely Correlated (>- 0.8) Canan Külahoglu

44 Many leaf developmental core elements between C 4 and C 3 are similar Similar development of leaf shape and size Correlated transcriptional regulators: 240 genes Canan Külahoglu

45 Genes involved in regulating leaf shape and architecture show similar patterns Leaf developmental Model STM PHV REV MP ET Many of the developmental transcriptional regulators involved in leaf shape have similar expression patterns in C 4 and C 3 Byrne, 2012 Canan Külahoglu

46 A small subset of regulators is inversely regulated between C 3 and C 4 leaves Canan Külahoglu

47 Several core cell cycle genes are inversely regulated C 4 up-regulated C 4 down-regulated Canan Külahoglu

48 Endoreduplication activators are upregulated in C 4 C 4 /C 3 (rpkms) 8 Endoreduplication activators Leaf0 Leaf1 Leaf2 Leaf3 Leaf4 Leaf5 E2Fb Dpa KRP-1/2 RBR CDKA CYC D3;1 CDC-6 CCS52 APC/C HBT SMR/LGO Change between leaf 4 and 5 Canan Külahoglu

49 Endoreduplictation repressors are downregulated in C 4 C 4 /C 3 (rpkms) 16 Endoreduplication repressors Leaf0 Leaf1 Leaf2 Leaf3 Leaf4 Leaf5 SPY KAK PYM CYCB/CDK2;2 GTL-1 HPY-2 ILP DEL Canan Külahoglu

50 Genes promoting in endoreduplication are upregulated in C 4? Increased nuclear content to maintain larger cells? Increased Ploidy : Enables cells to increase size Often found in highly metabolic active tissues Number of organelles correlates with cell size Canan Külahoglu

51 Anatomical comparison of C 4 and C 3 nuclei size C. gynandra T. hassleriana B M B M Canan Külahoglu

52 Anatomical comparison of C 4 and C 3 nuclei size C. gynandra T. hassleriana B M B M Canan Külahoglu

53 Flow cytometry of leaf developmental gradient C. gynandra ploidy during development ploidy frequency in % Leaf Age in Days C 4C 8C T. hassleriana ploidy during development ploidy frequency in % Leaf Age in Days C 4C 8C Canan Külahoglu

54 Flow cytometry of leaf developmental gradient T. hassleriana, C 3 stages 3-5 Canan Külahoglu

55 Flow cytometry of leaf developmental gradient C. gynandra, C 4 stages Canan Külahoglu

56 CAM and endocycling De Rocher et al. (1990) Science 250:

57 Conclusions Leaf cells stay undifferentiated for longer in Cleome C 4 Veins differentiate at a later stage Fewer cell divisions (inhibition of mitosis?) Increased endoreduplication in bundle sheath cells

58 Acknowledgements Heinrich-Heine-Universität Düsseldorf Thea Pick Alisandra Denton Simon Schliesky Canan Külahoglu Andrea Bräutigam Udo Gowik Peter Westhoff Michigan State University Robin Buell Max Planck Institute for Molecular Plant Physiology Toshiharu Obata Alisdair Fernie Cambridge Julian Hibberd David Heckmann Martin Lercher Posters 2, 16, 57, 61

59 Heckmann et al. (2013), Cell

60 Evolution of C 4 leaf anatomy C 3 BSC MC... MC... MC BSC Vein C 4 BSC MC MC BSC Vein C 4 has maximized vein density!

61 Anatomical comparison of C 4 and C 3 nuclei size C. gynandra T. hassleriana B S M M S B Canan Külahoglu

62 Lower cell number per leaf area in C 4 Cell Number per mm Cell division and expansion T. hassleriana C. gynandra Leaf Age in Days Canan Külahoglu

63 Flow cytometry of leaf developmental gradient C. gynandra ploidy during development ploidy frequency in % C 4C 8C T. hassleriana ploidy during development Higher nuclear content is correlated with larger bundle sheath 40 size Leaf Age in Days ploidy frequency in % Leaf Age in Days C 4C 8C

64 C 3 to C 4 transitions in the Brassicales Mya Cleomaceae NAD-ME C 4 C. gynandra C 3 < 10 My C. hassleriana C 3 only Brassicaceae Capparaceae

65 C 4 vs. C 3 leaf ontogeny in Cleome C. gynandra C 4 C. hassleriana C 3

66 C 4 vs. C 3 leaf ontogeny in Cleome C. gynandra C 4 C. hassleriana C 3

67 Comparative RNA-seq of C 3 and C 4 Calling differentially expressed genes Candidate genes for further analysis RNA isolation Assembly and annotation Read mapping RNA-Seq Preparation of sequencing libraries

68 Cell cycle genes are differentially expressed between C 3 and C 4 leaves C 4 up-regulated C 4 down-regulated

69 C.gyn/C.hassl rpkms Endoreduplication activators are upregulated in C 4 leaves 32 Endoreduplication Activators Leaf0 Leaf1 Leaf2 Leaf3 Leaf4 Leaf5 E2Fa Dpa KRP-1/2 RBR CDKA CYC D3;1 CDC-6 CCS52 APC/C HBT

70 C.gyn/C.hassl rpkms Endoreduplication repressors are downregulated in C 4 leaves Endoreduplication Repressors 2 SPY 1 Leaf0 Leaf1 Leaf2 Leaf3 Leaf4 Leaf5 KAK PYM CYCA 0.5 CYCB/CDK2;1 CYCB/CDK2;2 HPY-2 CYCA2, ILP DP-E2F-like

71 Endoreduplication is upregulated in C 4 Breuer et al., 2010

72 Anatomical comparison of C 4 and C 3 nuclei size C. gynandra C. hassleriana B S M M S B

73 Anatomical comparison of C 4 and C 3 nuclei size B C. gynandra T. hassleriana M B M

74 Anatomical comparison of C 4 and C 3 nuclei size F. trinervia F. pringlei B B M S M

75 Anatomical Comparison of C 4 and C 3 nuclei size P. maximum B B P. clandestinum M S M B S

76 Conclusions Increasing plant productivity requires reduction of photorespiration Photorespiration is the basis of C 2 photosynthesis C 2 photosynthesis provides the bridge to C 4 photosynthesis Mechanistic understanding of the evolutionary progression from C 3 to C 4 provides the blueprint for engineering approaches

77 CO 2 assimilation and biomass production are strictly correlated Heckmann et al. (2013), Cell

78 Quantum requirements for CO 2 assimilation (no free lunch ) 13 photons per CO photons per CO 2 Break even for C 4 at approx. 23 C Amthor, New Phytol, 2010

79 Photosynthetic performance of the bou-2 mutant CO 2 fixation rate ** *** ns *** CO 2 compensation point * ** ** ns Eisenhut and Planchais et al. (2013), Plant Journal

80 The split Calvin-cycle problem HCO PEP OAA malate malate pyruvate CO 2 + RubP NADPH 2x 3PGA PSI + PSII NADPH NADPH + ATP 3PGA DHAP TPT TPT 3PGA + DHAP DHAP mesophyll bundle sheath

81 RubisCO evolved under conditions of low O 2 Xiong & Bauer, Annu Rev Plant Biol, 2002

82 Oxygenic photosynthesis changed the composition of the atmosphere Beerling and Franks, Nature, 2010

83 C 3 plants one transport step per 3 molecules of CO 2 assimilated Ru 1,5bP + CO 2 Triosephosphate Intra-chloroplastic metabolism Export to cytosol

84 C 4 plants: > 10 transport steps per 1 molecule of CO 2 assimilated Transporters catalyzing these high fluxes must be highly abundant proteins in C 4 envelopes!

85 The textbook view is too simple MDH malate PEPC NADP ME PPDK pyruvate

86 Reality is complex PEPC MDH malate PPDK pyruvate NADP ME different intercellular gradients required for C4 additional layers of regulation different redox balance calculations PEPC AspAT MDH PPDK additional transporters needed metabolite homoeostasis altered aspartate malate PEP pyruvate AspAT NADP ME PEP CK AlaAT alanine AlaAT Pick et al. Plant Cell (2011)

87 Pumping CO 2 pump - another solution to the RubisCO problem

88 C 4 Photosynthesis is a CO 2 Pump ATP -> ADP + Pi [CO 2 ] = 130 µl/l [CO 2 ] = 2,400 µl/l

89 Advantages of C 4 plants Corn Sugar cane Miscanthus High water use efficiency High nitrogen use efficiency High photosynthetic rate

90 The long term vision - converting C3 to C4 Rice (C3) Wheat (C3)

91 CO 2 Calvin cycle 3-PGA RubisCO Glycerate O 2 Glycolate Hydroxypyruvate Serine Glyoxylate Glycine CO2 + NH 3 Refixation

92 Coexpressed clusters in maize leaves

93 CO 2 Calvin cycle 3-PGA RubisCO Glycerate O 2 Glycolate Hydroxypyruvate Serine Glyoxylate Glycine CO2 + NH 3 Refixation

94 otorespiration, became restricted to the bundle sheath cells (Monson et al., 1984; Sag C 2 photosynthesis a bridge to C 4? 04; Bauwe, 2011; Sage et al., 2012) (Figure 4). gure 4. The photorespiratory glycine shuttle - a CO 2 pump. ycine produced in the mesophyll cells has to be shuttled in the bundle sheath cells where the glyc

95 Modeling the evolutionary trajectory from C 3 to C 4 Heckmann et al. (2013), Cell

96 Fitness changes along a greedy path through the fitness landscape Heckmann et al. (2013), Cell

97 Projections of trajectories through the sixdimensional fitness landscape Heckmann et al. (2013), Cell

98 CO 2, O 2 mesophyll cell chloro RubisCO CO 2 O 2 chloro RubisCO CO 2 3PGA mito C 1 -THF 3PGA 2PG 2PG perox biomass GDC biomass Gly bundle sheath cell Gly C 1 -THF GDC Gly mito CO 2 loss of biomass

99 C 2 photosynthesis provides CO 2 to BSCs GDC/SHMT CO 2 2x Gly 2x Gly NH 3 CH 3 -THF Ser Mesophyll Cell Bundle Sheath Cell

100 C 2 photosynthesis creates N-imbalance GDC/SHMT CO 2 2x Gly 2x Gly NH 3 CH 3 -THF 2 N out, 1 N back Ser Ser Mesophyll Cell Bundle Sheath Cell

101 Anaplerotic pathway supporting C 2 photosynthesis NH 3 Malate MDH OAA Asp Malate MDH OAA NH 3 GS1/GOGAT Glu Asp + 2-OG Mesophyll Cell Bundle Sheath Cell

102 From C 2 to C 4 photosynthesis MDH OAA Malate Malate NH 3 GS1/GOGAT CO 2 PEP ME CO 2 pyruvate Glu pyruvate Ala Ala + 2-OG NH 3 Mesophyll Cell Bundle Sheath Cell

103 PEPC Asp-AT NAD-ME NADP-MDH PEPCK mesophyll cell bundle sheath cell malate malate Asp As p Asp-AT OAA mito OAA OAA chloro CO 2 malate NAD + NADH VB HCO 3 - Calvin cycle ATP pyruvate PEP PPDK PEP PEP pyruvate Ala Ala-AT Ala Ala-AT pyruvate

104 Candidate genes: C 4 leaf anatomy KAC2 (Kinesin actin based chloroplast movement 2) CHUP1 (Chloroplast Unusual Positioning 1) beta 1,3 glucanase plasmodesmata associated protein Defectively organized tributaries 3 Reveille1&2 SAUR type protein GTL Signaling peptides such as root meristem growth factor CLE43 LRR & cysteine-rich kinases

105 Which regulators lead to the differences in leaf architecture C 4 /C 3? Phase shift in expression patterns based on leaf differentiation 105

106 Which regulators lead to the differences in leaf architecture C 4 /C 3? Our hypothesis: Development has a phase shift in differentiation Does this also hold true for the transcriptional regulators? Correlated (<0.8) Not Correlated Inversely Correlated (>-0.8) 755 transcriptional regulators are not correlated between C 4 and C 3 Canan Külahoglu

107 What are the inversely regulated transcription factors? Our hypothesis: - General leaf developmental transcriptional regulators have a similar expression pattern - Internal leaf development is differentially regulated between C4 and C3 Correlated (<0.8) Not Correlated 30 transcriptional regulators are inversely correlated between C 4 and C 3 Inversely Correlated (>-0.8)