Moving On Up: Internode Growth in Cereals. Sarah McKim Division of Plant Sciences

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1 Moving On Up: Internode Growth in Cereals Sarah McKim Division of Plant Sciences

2 Phytomer Growth node lateral organ shoot meristem lateral organ primordium internode node axillary bud Phytomer

3 Phytomers vary depending on growth phase vegetative reproductive

4 Phase change modifies existing phytomers Internode elongation vegetative reproductive

5 Many plants elongate stems after flowering Grain crops show this growth habit Lodging

6 Norman Borlaug The Green Revolution variation in internode elongation

7 Internode growth in barley flag leaf Sreenivasulu and Schnurbusch, 2012

8 Intercalary meristems fuel internode growth Dicots Ruonala et al, 2008 peduncle Intercalary Meristem Unique to grasses, hornworts and horsetail ferns

9 growth Model of internode zonation Maturation Zone Testing the Model: Expansion Zone Precision phenotyping Hormone networks Genome-wide analyses Mutant genetics Division Zone Intercalary meristem

10 Epidermal Cell Length (μm) Mitotic Index Development of internode zonation A kinematic analysis Cell length 4 3 Mitotic Index cm bin 1mm bins V. Patil et al, in prep

11 Mitotic Index Proliferation and growth of Epidermal layer Tissue section Histone4 in situ p fl p fl p 600μm d17 d24 Days post-germination

12 Mitotic Index Epidermal Cell Length (μm) Coupled and restricted to base col col lacuna mm bins 0 1 1cm bin node node 1.5cm

13 Epidermal Cell Length (μm) 1 cm 2cm 3cm 4cm 5cm Consistent elongation in the 300 5cm Lignification profile cm bins

14 spatial Proliferation Dominant Three Phase Model PHASE 1 PHASE 2 PHASE 3 Proliferation + Expansion Expansion dominant temporal

15 spatial Internode growth dynamics similar to leaves PHASE 1 PHASE 2 PHASE 3 temporal Nelissen et al (2016)

16 1 cm 2cm 3cm 4cm 5cm Dynamic Internode Transcriptome Bw.1cm Bw.2cm Bw.3cm +log2 -log2 Bw.4cm Bw.5cm 28 CLUSTERS Cyclins, histones Proliferation TFs Cell wall synthesis Cell wall extension Differentiation Carbohydrate metab. Lignification Photosynthesis

17 1 cm 2cm 3cm 4cm 5cm Regulatory Modules Suppress expansion Tb1 CUC2/ BOP2 Suppress proliferation ULT1 MND/PLA1 Differentiation AS2 BP, BLH6, KNAT7 Slows differentiation rate Suppress stem cells BLH4 AG, KAN4 WOX3 WOX2 Founder recruitment Proliferation GRF5,4,2 AN3 Proliferation duration ANT/AIL ERF TCP

18 Zeo alleles cause semi-dwarfism and dense spikes Bw Zeo1.b ^ Bw ^ peduncle Zeo1.b Bw Zeo1.b Houston and McKim et al (2013) PNAS

19 Culm Length (cm) HvAP2 delays spike differentiation and limits internode growth Bw Zeo1.b Bowman 40 Zeo1.b dpg mir172 HvAP2 mir172 HvAP2 slow Houston and McKim et al (2013) PNAS

20 mir172 regulation of HvAP2 promotes internode phase transitions Bw mir172 down-regulation of HvAP2 promotes cell transfer rate to the Zeo1.b HvAP2 does not control size or density

21 The Dynamic Internode Transcriptome S2 Bw S3 5cm 4cm Bw 3cm 2cm 2cm 1cm 1cm Zeo1.b Zeo1.b 2cm 2cm 1cm 1cm

22 Robust DETs between Bw and Zeo1.b S2 - Bw and Zeo1.b S3 Bw and Zeo1.b Overlap of S2-S3 Bw (S2-S3 Bw) Overlap of S2-S3 Zeo1.b (S2-S3 Zeo)

23 Bw Robust DETs between Bw and Zeo1.b Bw Proliferation WOX2 GRF5 AGL14 ERF1/10 AGLs ARF19 Cell cycle/ division -ve regulators or repressed by Jasmonate (JA) JAZ WRKY70/ WRKY27/WRKY 41 Jasmonate antagonises Gibberellin, proliferation and promotes lignification Differentiation/ Growth suppression +ve by JA Zeo1.b BLH1, BLH6 KNAT7 LSH4 BOP Expansins, MYB87 Thionin PDF2 OMT, LOX2

24 Conserved mirna-target network regulates phase change vegetative JA reproductive APETALA2-like mir172 mir156 SPLs The phase change network, specifically AP2, controls developmental transitions through modulation of JA

25 HvAP2 underlies GWAS peak associated with internode length HvAP2 Ahmad Alqudah Thorsten Schnurbusch Patil et al in prep

26 Acknowledgments McKim Lab Vrushali Patil Hannah McDermott Monika Zwirek Jennifer Shoesmith Nikoleta Tzioutziou Trisha McAllister James Hutton Institute Robbie Waugh Kelly Houston Pete Hedley Leibniz Institute of Plant Genetics and Crop Plant Research Ahmad Alqudah Thorsten Schnurbusch

27 * Semidwarfism associated with disruption of mir172-regulation of HvAPETALA2 mir172 IIIIIIIII mir172 IIIIIIIII SNP HvAP2 mrna HvAP2 mrna mir172 HvAP2 mir172 HvAP2 Bw HvAP2 Zeo1.b HvAP2

28 Other Phase Change Factors in Barley? Identify and characterise HvAP2-likes and HvSPLs 3 other HvAP2-likes 8 HvSPLs regulated by mir172 OVEREXPRESS (OX) HvAP2-like or HvSPL GFP Mutated mirna-binding site KNOCK-OUT (KO) HvAP2-like and HvSPL CRISPR-Cas Transgenic plant phenotype

29 mir172 controls HvAP2 levels specifically Zeo1.b/ BW relative HvAP2 gen expression after the reproductive transition HvAP2 qpcr Days postgermination 7 (veg) * 14 aerial tissue * spike tissue Bw vegetative Zeo1.b mir172 HvAP2 reproductive mir172 HvAP2

30 % total rachis growth rachis length (mm) mir172 promotes spike phase progression Bw G H I d d26 BW Zeo1.b BW Zeo1.b mir172 HvAP2 Zeo1.b mir initiation d21 spikelet differentiation internode elongation d26 Days post-germination development paused Days post-germination HvAP2 Prolonged spikelet initiation late differentiation reduced internode elongation

31 Epidermal Cell Length (μm) Mitotic Index Development of internode zonation A kinematic analysis Cell length Mitotic Index Section from base (1 mm)

32 Mitotic Index Phase 1 Proliferation and genesis of Epidermal layer Tissue section Histone4 in situ col col col p p fl fl p 600μm d17 d24 Days post-germination

33 Mitotic Index Epidermal Cell length (um) Phase 2- Coupled and Zonation restricts to base col col lacuna fl mm bins from base node node

34 Mitotic Index Phase 2: proximo-distal size is constant 10cm internode 1.5cm internode Bins from base (1 mm)

35 Epidermal Cell Length (μm) Phase 2: Consistent elongation in the 300 5cm Lignification profile

36 Phase 2: Consistent elongation in the 300 5cm cm 15cm

37 Mitotic Index Phase 3: shrinks and eliminated 1.5cm 10cm internode 15cm Bins from base (1 mm)

38 Altered phase timing effects plant architecture WT 35S::miR156 Prolonged vegetative phase MicroRNAs control phase change timing in plants and animals

39 Conserved mirna-target network regulates phase change vegetative leaves, shoots APETALA2-like reproductive flowers, seeds mir172 mir156 SPLs mrna mirna mirna target mrna

40 Rice Ideal Plant Architecture Traditional IPA Ideal Plant Architecture OsSPL14 IIIIIIIII mir156 OsSPL14 X * mir156 Miura et al., 2010 Jiao et al, 2010

41 Agronomic traits are regulated by phase change Crop Gene Trait Maize Rice TS6 TGA LG GL15 SID1 IDS1 OsSPL16 OsSPL14 OsLG SHAT1 SNB IDS1 Male fertility Kernel accessibility Leaf shape, branch angle Leaf wax Kernel number Kernel number Grain size IPA Panicle angle Grain retention Panicle branching Panicle branching Wheat Q AP2 Height, grain retention Barley UNKNOWN UNKNOWN

42 Development of Plant Architecture Phase Changes in Barley What happens in barley??? vegetative leaves, shoots APETALA2-like reproductive flowers, seeds mir172 mir156 SPLs

43 Research Journey McKim Lab Architectural Roles of Phase Change Network in Barley Internode Elongation

44 Internode Patterning barley rice maize Briggs, 1978 Kende et al, 1998 Scanlon, 1996; 2000

45 Concentration (ng/g DW) Concentration (ng/g DW) Concentration (ng/g DW) Concentration (ng/g DW) Internode Hormone Profile at 5cm Bw 1 Gibberellins Bw 2 Bw 3 Bw 4 Bw 5 GA53 GA51 GA44 GA34 GA19 GA8 GA Bw 1 Bw 2 Cytokinins Bw 3 Bw 4 Bw 5 c-zog t-zog t-zr c-zr ipr t-z Auxins IAA IAA-Asp ACC ACC Bw 1 Bw 2 Bw 3 Bw 4 Bw 5 Bw 1 Bw 2 Bw 3 Bw 4 Bw 5

46 1 cm 2cm 3cm 4cm 5cm Integrated Model of Internode Growth Differentiation CK GA IAA ACC Suppress expansion Slow differentiation rate Expansion CK GA IAA ACC Suppress proliferation Suppress stem cell identity Proliferation Founder recruitment CK GA IAA ACC Patil et al in prep

47 1 cm 2cm 3cm 4cm 5cm Dynamic Internode Transcriptome Patil et al in prep

48 Post-embryonic body plan development Wheat Tulip Coconut Corn Teosinte

49 Epidermal Cell Length (μm) 1 cm 2cm 3cm 4cm 5cm Epidermal Cell Length (μm) Consistent elongation in the 300 5cm cm 15cm M Z cm bins

50 Mitotic Index Expansion dominant shrinks and eliminated 15cm 1.5cm 10cm internode Bins from base (1 mm)

51 Epidermal Cell Length (μm) Expansion dominant cm Bin

52 Epidermal cell length (μm) HvAP2 represses both proliferation and expansion Bw Zeo1.b bottom top peduncle (1cm bins)

53 Zeo1.b is sensitive to Gibberellin (GA) but does not phenocopy a GA-deficient dwarf, sdw1.a

54 * Semidwarfism caused by disruption of mir172- regulation of HvAPETALA2 (HvAP2) mir172 IIIIIIIII mir172 IIIIIIIII SNP HvAP2 mrna HvAP2 mrna mir172 HvAP2 mir172 HvAP2 Bw HvAP2 Zeo1.b HvAP2 Houston and McKim et al (2013) PNAS

55 Ancient regulatory network? hornworts

56 Research Journey Testing the model Three Phase Model Novel Regulators why? Dynamic transcriptome Recapitulation of lateral organ programme Elevated HvAP2 = proliferative TFs, differentiation TFs, cell wall enzymes JA mir172 regulation of HvAP2: promotes cell proliferation rate and transfer from to links lignification with zonation may promote GA signalling

57 Basis of Semi-dwarfism in Zeo1.b Wild type Short internode Fewer cells Smaller cells Combination of both Proliferation Expansion

58 spatial Loss of mir172 regulation of HvAP2 uncouples lignification from stem zonation Bw Zeo1.b - - temporal

59 Comparative Transcriptome of Peduncle Initials S1 Bw Zeo1.b Bw Zeo1.b 1mm Bw 68 Zeo 193 Differentially Expressed Transcripts (DETs) 2485 Zeo1.b/ 261 Bw

60 DETs show Gene Ontology (GO) Enrichment for meristem function Stem cell differentiation Maintenance of meristem identity Negative regulation of cell differentiation WUSCHEL-like homeoboxes = WOXs AGL TFs All downregulated in Zeo1.b