Front Plant Sci Mar 14

Case Study

Front Plant Sci. 2016 Mar 14 30

33 Study Background Cryptochromes (CRY) are blue-light photoreceptors that mediate various light responses in plants and animals. It is known that CRY1 and CRY2 regulate a variety of physiological processes, including seedling photomorphogenesis and photoperiodic flowering, respectively. Trends in Plant Science, Volume 17, Issue 10, p584 593, October 2012

34 About Cryptochromes 1 (CRY1) functional domains The C-termini of CRY1 (CCT1) is known to mediate CRY1 signaling. It has long been demonstrated that Arabidopsis CRY (CRY1 and CRY2) C-termini (CCT1 and CCT2) mediate light signaling through direct interaction with COP1. It is demonstrated that the N-termini of CRY1 (CNT1) are not only responsible for chromophores binding and blue light perception, but also mediation of their dimerization, which is essential for light activation of their photoreceptor activity.

35 Aimed Given that CNT1 and CCT1 are both involved in mediating CRY1 signaling, it is worth investigating how they are involved in regulating phytohormone-responsive gene expression at the transcriptomic level. In this study, we performed RNA-seq assay using transgenic plants expressing CCT1 fused to b-glucuronidase(gus) (GUS-CCT1, abbreviated as CCT1),and compared with the RNA-Seq results we obtained from cry1cry2 and CNT1 seedlings previously. Main findings CCT1 mediates CRY1 inhibition of GA promoted degradation of HY5 protein. The CCT1-mediated CRY1 regulation of GA response may be mediated through CCT1 regulation of COP1.

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37 Questions risen from transcriptome analytical study What are all the known associations with CRY1 in the plant database? CRY1 is related with cryptochrome, phototropin, auxin signaling pathways. CRY1 mediates light signaling through direct inhibition with COP1. In Cryptochrome signaling pathway, COP1 plays an important role to regulate plant photoperiodism, hypocotyl growth, photomorphogenesis, phototropism through inhibiting HY5, CO, HFR1, MYB18. Focus on specific interactions: How does CRY1 regulates the phytohormone-response? CRY1 may affects phytohormone response through regulating the activation of COP1 and the synthesis of Auxin and GA.

38 CRY1 in Pathway Studio CRY1 is involved in 6 existing pathways, 61 group, and highly associated with Cryptochrome, Phototropin, Auxin signaling pathways

40 Cryptochrome Signaling Pathway CRY1 mediates light signaling through direct inhibition with COP1 In Cryptochrome signaling pathway, COP1 plays an important role to regulate plant photoperiodism, hypocotyl growth, photomorphogenesis, phototropism through inhibiting HY5, CO, HFR1, MYB18.

ENTITY DEFINITIONS Cell Process * biological processes, most coincide with Gene Ontology Clinical Parameter measured parameters of the human body used in clinical practice Complex * several polypeptides that form a complex via physical interactions Disease health conditions and disease terms from MeSH Functional Class * most functional classes coincide with Gene Ontology Protein defined by Entrez Gene - represents both genes and the gene products, including proteins and mirnas. Small Molecule in Mammal, naturally occurring metabolites and small molecules found in mammalian cells; ChemEffect adds drugs and non-naturally occurring small molecules to the mammal database. In Plant, naturally occurring metabolites and small molecules and other plant related chemicals (ex. herbicides or research related chemicals). The category small molecule may contain some biologically active polypeptides and proteins that work as drugs, such as monoclonal antibodies. Treatment non-chemical treatments and environmental conditions, such as cold shock. * Container Entities these are valid entities but also can have proteins mapped to them. You can see the proteins for the container entities in the child concepts in the property records for the specific entity. 41

RELATIONS DEFINITIONS Relation Types for the database: Binding direct physical interaction between two molecules. ChemicalReaction enzyme catalyzed reaction involving small molecules. DirectRegulation influences target activity by direct physical interaction (excluding promoter binding interactions). Expression regulator changes protein abundance by affecting levels of transcript or protein stability. mirnaeffect the inhibitory effect of a mirna on its mrna target. Regulation changes the activity of the target by an unknown mechanism (may be direct or indirect). This is a less specific relation type than others provided. MolSynthesis regulator changes the concentrations of the target (usually a small molecule target). MolTransport a regulator that changes the localization of the target (molecular translocation, export, import etc.) PromoterBinding a regulator that binds to the promoter of a gene. ProtModification a regulator that changes the modification of the target molecule, usually by a direct interaction such as phosphorylation, glycosylation, acetylation etc. 42

44 Questions risen from transcriptome analytical study What are all the known associations with CRY1 in the plant database? CRY1 is related with cryptochrome, phototropin, auxin signaling pathways. CRY1 mediates light signaling through direct interaction with COP1. In Cryptochrome signaling pathway, COP1 plays an important role to regulate plant photoperiodism, hypocotyl growth, photomorphogenesis, phototropism through inhibiting HY5, CO, HFR1, MYB18. Focus on specific interactions: How does CRY1 regulates the phytohormone-response? CRY1 may affects phytohormone response through regulating the activation of COP1 and the synthesis of Auxin and GA.

45 How does CRY1 regulates the phytohormone-response? CRY1 may affects phytohormone response through regulating the activation of COP1 and the synthesis of Auxin and Gibberellin (GA) 22 Proteins and Chemicals related to Phytohormone response

57 Questions risen from transcriptome analytical study What genes are regulated by CRY1, CCT1, CNT1, respectively? Genes with differential expression were identified with a p value < 0.01 and fold change (FC) > 2 2902 Genes are common regulated in group CRY1 and CCT1 1442 Genes are common regulated in group CRY1 and CNT1 1804 Genes are common regulated in group CCT1 and CNT1 1094 Genes are common regulated in 3 groups What functions/pathway are associated with CCT1, CNT1 regulated genes? Both CCT1 and CNT1 regulated Genes are associated with 9 GO functions such as defense response, response to UV-B, gibberellin biosynthetic process and 1 Cold-stress signaling pathway CCT1 regulated Genes are associated with photosynthesis, response to light CNT1 regulated Genes are associated protein ubiquitination, defense response, Gibberellin Signaling, Salicylic Acid Signaling

58 What genes are regulated by CRY1, CCT1, CNT1, respectively? Three RNA-seq datasets CRY1, CCT1, and CNT1 CCT1 3147 1808 710 1094 1023 348 1056 CRY1 CNT1 2902 Genes are common regulated in group CRY1 and CCT1 1442 Genes are common regulated in group CRY1 and CNT1 1804 Genes are common regulated in group CCT1 and CNT1 1094 Genes are common regulated in 3 groups

64 Questions risen from transcriptome analytical study What genes are regulated by CRY1, CCT1, CNT1, respectively? Genes with differential expression were identified with a p value < 0.01 and fold change (FC) > 2 2902 Genes are common regulated in group CRY1 and CCT1 1442 Genes are common regulated in group CRY1 and CNT1 1804 Genes are common regulated in group CCT1 and CNT1 1094 Genes are common regulated in 3 groups What functions/pathway are associated with CCT1, CNT1 regulated genes? Both CCT1 and CNT1 regulated Genes are associated with 9 GO functions such as defense response, response to UV-B, gibberellin biosynthetic process and 1 Cold-stress signaling pathway CCT1 regulated Genes are associated with photosynthesis, response to light CNT1 regulated Genes are associated protein ubiquitination, defense response, Gibberellin Signaling, Salicylic Acid Signaling

65 What functions/pathway are associated with CCT1, CNT1 regulated genes? Common associated functions/pathway Name Cold stress signaling # of Measured p-value in # of Measured p-value in # of Entities terpenoid biosynthetic process Entities in CCT1 CCT1 Entities in CNT1 CNT1 Biological Process anthocyanin-containing compound response to UV-B 49 34 7.41E-09 16 0.025688 biosynthetic process plant-type cell wall modification cellular response to hypoxia 25 9 0.003766 11 0.001605 defense response lipid storage 527 200 0.008775 97 CNT1 0.002897 defense response, incompatible interaction 87 40 6.35E-05 44 CCT1 0.009149 gibberellin biosynthetic process gibberellin biosynthetic process 57 25 0.006767 16 0.003992 # of Entities lipid storage defense response, incompatible interaction 104 29 0.005838 14 0.008075 plant-type cell wall modification 171 33 0.005678 26 0.001966 response to UV-B defense response 99 50 4.73E-10 16 0.045407 terpenoid biosynthetic cellular process response to hypoxia 53 20 0.00301 10 0.033438 Pathway anthocyanin-containing Cold-Stress Signaling compound biosynthetic process 31 12 0.000528 12 0.008012 0 100 200 300 400 500 600 Both CCT1 and CNT1 regulated Genes are associated with 9 GO functions such as defense response, response to UV-B, gibberellin biosynthetic process and 1 Cold-stress signaling pathway

66 What functions/pathway are associated with CCT1, CNT1 regulated genes? Unique associated functions/pathway with CCT1 regulated genes Name # of Entities # of Measured Entities p-value cell differentiation 211 85 0.005717 cell fate specification 38 9 0.006571 cellulose catabolic process 25 13 0.00181 cysteine biosynthetic process 211 99 0.006987 detection of biotic stimulus 102 38 0.005445 divalent metal ion transport 82 38 0.00403 epidermal cell differentiation 16 5 0.002335 flavonoid biosynthetic process 174 79 0.000365 glucosinolate biosynthetic process 167 87 0.001369 leaf morphogenesis 186 78 0.001724 leaf senescence 72 16 0.003968 myo-inositol hexakisphosphate biosynthetic process 65 41 1.68E-05 oxidation-reduction process 1367 489 2.83E-05 pentose-phosphate shunt 193 139 2.83E-08 photosynthesis 152 109 5.95E-08 photosynthesis, light harvesting 24 21 0.000208 photosynthesis, light reaction 111 88 1E-06 photosynthetic electron transport in photosystem I 46 43 1.86E-05 photosystem II assembly 154 117 3.32E-06 plastid organization 83 71 4.26E-07 protein-chromophore linkage 38 28 2.75E-05 reductive pentose-phosphate cycle 19 13 0.003655 regulation of multi-organism process 91 35 0.002589 response to blue light 106 76 7.16E-06 response to far red light 96 73 1.2E-07 response to karrikin 128 54 0.003681 response to red light 98 76 1.91E-07 response to sucrose 204 100 4.21E-08 rrna processing 247 148 0.004022 secondary metabolite biosynthetic process 138 65 0.000142 starch CCT1 biosynthetic regulated process Genes are associated 199 129 with 0.007877 trichoblast differentiation 45 38 5.24E-06 unsaturated fatty acid biosynthetic process 78 46 0.000985 photosynthesis, response to light 33 unique associated function from Sub Network enrichment Analysis (SNEA) with GO biological categories

67 Name What functions/pathway are associated with CCT1, CNT1 regulated genes? Unique associated functions/pathway with CNT1 regulated genes # of Entities # of Measured Entities p-value abscisic acid-activated signaling pathway 269 45 0.028491 actin filament-based movement 82 10 0.031992 copper ion transport 22 7 0.005606 defense response by callose deposition 47 13 0.028161 defense response to fungus 597 109 5.44E-06 defense response to insect 33 9 0.014358 endoplasmic reticulum unfolded protein response 186 29 0.041374 ethylene biosynthetic process 116 46 1.43E-08 ethylene-activated signaling pathway 237 54 1.1E-05 hyperosmotic salinity response 161 38 0.034673 intracellular signal transduction 277 71 0.003915 jasmonic acid mediated signaling pathway 279 81 0.00327 killing of cells of other organism 279 15 0.003388 MAPK cascade 217 67 0.004274 meristem development 44 6 0.018965 meristem structural organization 95 8 0.041725 negative regulation of defense response 269 70 0.007327 negative regulation of programmed cell death 166 35 0.000883 photomorphogenesis 210 11 0.007134 pollen germination 49 5 0.016876 pollen tube growth 252 32 0.005164 protein ubiquitination 589 51 0.031199 purine nucleobase transport 123 14 0.034503 regulation of flower development 291 16 0.018152 regulation of hydrogen peroxide metabolic process 184 61 0.015335 Name # of Entitie # of Measure p-value s d Entities regulation of immune response 16 5 0.017839 regulation of innate immune response 45 20 3.27E-05 regulation of timing of transition from vegetative to reproductive phase 32 5 0.002259 respiratory burst involved in defense response 122 51 0.000146 response to bacterium 178 37 0.033654 response to chitin 421 155 4.2E-09 response to endoplasmic reticulum stress 187 23 0.015222 response to ethylene 263 58 6.33E-05 response to freezing 95 14 0.004964 response to fungus 115 31 0.019961 response to hypoxia 84 12 0.012746 response to jasmonic acid 277 72 0.005894 response to mechanical stimulus 55 26 4.29E-06 response to molecule of bacterial origin 99 18 0.014205 response to ozone 37 10 0.020965 response to red or far red light 51 6 0.017232 response to salicylic acid 164 38 0.011671 response to water deprivation 337 82 0.018476 response to wounding 334 97 9.03E-06 salicylic acid mediated signaling pathway 163 40 0.000162 sexual reproduction 27 8 0.033244 signal transduction 540 74 0.024208 stamen development 89 7 0.049327 stamen filament development 8 5 0.042189 systemic acquired resistance 251 52 0.032952 systemic acquired resistance, salicylic acid mediated signaling pathway 252 66 0.006884 vasculature development 26 9 0.004422 CRY1 and CRY2 interact with COP1 s E3 ubiquitin ligase enhancer, SPA1 (SUPPRESSOR OFPHYTOCHROME A 1), through their C and N termini, respectively (Lian et al., 2011; Liu et al., 2011; Zuo et al., 2011). COP1 has E3 ubiquitin ligase activity, and physically interacts with various substrates, such as HY5/HYH, HFR1, LAF1, and CONSTANS, to promote their ubiquitination and Defense response and immune response degradation to regulate photomorphogenesis and flowering (Holm et al., 2002; Seo et al., 2003; Yang et al., 2005; Liu et al., 2008).

68 What functions/pathway are associated with CCT1, CNT1 regulated genes? Unique associated functions/pathway with CNT1 regulated genes Gibberellin Signaling P-value: 0.00288 # of entities: 51 # of measured entities: 8 Gibberellins (GAs) are plant hormones that regulate growth and influence various developmental processes, including stem elongation, germination, dormancy, flowering, sex expression, enzyme induction, and leaf and fruit senescence

73 Conclusion What are all the known associations with CRY1 in the plant database? CRY1 is related with cryptochrome, phototropin, auxin signaling pathways. CRY1 mediates light signaling through direct inhibition with COP1. In Cryptochrome signaling pathway, COP1 plays an important role to regulate plant photoperiodism, hypocotyl growth, photomorphogenesis, phototropism through inhibiting HY5, CO, HFR1, MYB18. Focus on specific interactions: How does CRY1 regulates the phytohormone-response? CRY1 may affects phytohormone response through regulating the activation of COP1 and the synthesis of Auxin and GA.

74 Conclusion What genes are regulated by CRY1, CCT1, CNT1, respectively? Genes with differential expression were identified with a p value < 0.01 and fold change (FC) > 2 2902 Genes are common regulated in group CRY1 and CCT1 1442 Genes are common regulated in group CRY1 and CNT1 1804 Genes are common regulated in group CCT1 and CNT1 1094 Genes are common regulated in 3 groups What functions/pathway are associated with CCT1, CNT1 regulated genes? Both CCT1 and CNT1 regulated Genes are associated with 9 GO functions such as defense response, response to UV-B, gibberellin biosynthetic process and 1 Cold-stress signaling pathway CCT1 regulated Genes are associated with photosynthesis, response to light CNT1 regulated Genes are associated protein ubiquitination, defense response, Gibberellin Signaling, Salicylic Acid Signaling

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