Integrace hormonálních signálů

Transkript

1 Integrace hormonálních signálů

2 SKOOG a MILLER auxin cytokinin

3 Příklady: Integrace regulací GA s dalšími fytohorm ony

4 IAA spouští zvýšenou produkci GAs. Narušení transportu IAA brzdí degradaci DELLA represorů působenou GAs.

5 BR a IAA Indukují řadu identických mrna.

6 Hladiny auxinu a cytokininů inverzně korelují. Zvýšení cytokininů stimuluje syntézu IAA, ale zvýšení IAA inhibuje hladinu cytokininů.

7

8

9 uxin stimuluje produkci etylenu stimulací genu pro syntézu ACC. tylen inhibuje polární transport auxinu.

10 Světlo a IAA Světlo zasahuje do auxinem regulovaných změn v dlouživém růstu buněk a diferenciace.

11 ABA interaguje antagonisticky s GAs i IAA. Přidání ABA může snižovat hladinu volné IAA a zvyšovat podíl neaktivních konjugátů; ABA interferuje se sig. drahami GAs.

12

13

14 Zvýšená hladina cytokinů a potlačení GAs je konstitutivním parametrem dělivého centra apikálního meristému. Naopak pro vývoj listových primordií je podmínkou zvýšená hladina GAs.

15 Interaguje prakticky každý s každým a to jak na úrovni přenosu signálů/regulace genové exprese, tak také na úrovni regulace biosyntézy - inhibice či aktivace akumulace partnerského "fytohormonu".

16

17 Plasmodesmata rostlina jako symplastická síť

18 Symplastická konektivita je dynamická, regulovaná! Plasmodesmy vznikají při buněčném dělení i de novo a mohou být regulovaně uzavřena.

19 Buněčně autonomní = molekula působí jen uvnitř buňky. Buněčně neautonomní = molekula působí mezi buňkami.

20

21 Systemické šíření RNA floemem

22 PD=plasmodesm NCAP = non cell autonom. prot. injekce FITC-MP bílkoviny;c nepoh. muta G NCAP pomáhá pohybu dextranu.

23 S transportem jsou spojeny regulované změny SEL (Size Exclusion Limit).

24 NCAP =non cell autonom. proteins Mechanismus mezibuněčného pohybu

25 Floemem se šíří květní signál bílkovina FT

26 Rostlinný organismus jako nadbuněčná integrovaná symplastická síť.

27 RNA viruses can block expression of a transgene if a copy of the transgene has been added Tobacco plant expressing GFP protein Infected with RNA virus with GFP gene Virus infection travels through veins GFP expression inhibited starting at veins.

28 Gene silencing and RNA viruses share potential to produce ds RNA Fire and Mello proved that ds RNA inhibits expression of endogenous genes homologous to that dsrna (Nobelova cena 2006). Ve skutečnosti první důkaz byl podán u rostlin (př. v laboratoři Davida Baulcomba JIC, Norwich)

29 a RNA interference: A type of gene regulation Involving small RNA molecules and induced by double stranded RNA

30 How does RNAi work? RNAi works postranscriptionally.. in key two steps!

31 Overview of RNAi Double-stranded RNA (dsrna) is processed by Dicer, an RNase III family member, to produce 21-23nt small interfering RNAs (sirnas) sirnas are manipulated by a multi-component nuclease called the RNA-induced silencing complex (RISC). RISC specifically cleaves mrnas that have perfect complementarity to an sirna strand

32 Components of the RNAi pathway Dicer u Arabidopsis 4x Dicer like = DCL Dicer belongs to the RNase III family of dsrna-specific ribonucleases. Contains dsrna-binding motifs and a PAZ domain believed to mediate protein-protein interactions. Argonaute homologs (Dicer/RISC associated) - u Arabidopsis 10x. argonaut byl popsán jako meristémový mutant Arabidopsis. Argonaute family members are highly basic proteins that contain PAZ and PIWI domains. RISC komplex rozmotává dvouřetězcové fragmenty RNA a ssrna RISC komplex pak působí po hybridizaci s cílem degradaci RNA, blok translace či metylaci DNA. RNA-dependent RNA polymerase (RdRP) Endogenous RdRPs influence RNAi in certain eukaryotes including fungi, plants, C. elegans. Function to amplify the dsrna signal.

33 Catalysis: RdRP copies mrna making more ds RNA. Dicer cuts that generating more sirna More RdRP is activated and more dsrna is made. Spread: dsrna transported to other cells

34 Dicer contains two RNAse III domains long dsrna sirnas

35 MicroRNAs (mirnas) mirnas are products of endogenous genes and function through the RNA interference (RNAi) pathway to posttranscriptionally regulate the expression of other genes.

36 mirnas in development mirnas are differentially expressed among various tissue types and at various stages in cellular differentiation. e.g. there exist stem cell and neuronal cell specific mirnas. Disruption of RNAi pathway results in early embryonic lethality due to depletion of stem cells. Certain mirnas are known to control developmental timing and fate specification in C. elegans, leaf morphogenesis in plants, and hematopoetic lineage differentiation in mice.

37 mirna hrají např. klíčovou roli v iniciaci ad- vs. ab-axiální vývojové dráhy při tvorbě listů z primordia.

38 o mirna hovoříme tam, kde vznikají z nedokonalé vlásenky kodované v genomu. Cílem jejich regulace/inhibice jsou transkripční faktory a jsou tak důležité pro regulaci vývoje rostlin. sirna vzniká z celé délky dokonale homologní dvoušroubovicové RNA (produkt RDR nebo příp. endog. inv. opakování). Jsou produkty i intermediáty obrany proti invasivním/aberantním/abundantním RNA jako jsou vir, transgeny a transposony.

39 A Dvojí shrnující schema.

40 B RdDM = RNA dependent DNA methylation. RDR = RNA dependent RNA polymerázy

41

42

43 Stress Responses & Gene Expression plants must adapt to stresses because of their sedentary lifestyle

44 Regulace iontové homeostasy během reakce na zasolení.

45 Reakce na osmotický stress a ABA.

46 Transkripční regulace v odpovědi na chlad +TF -E3

47 Heat Shock Proteins (hsp) ~100, ~90, ~70, and ~60 kda Low molecular weight (LMW) hsp: ~27, ~20-22, ~15-18 kda all induced within 30 min. more LMW hsp in plants 2-Dimensional gel electrophoresis and molecular cloning indicates most hsps are families of related proteins, particularly hsp70 and the LMW hsps

48 HSP70, a chaperonin Essential gene Homologues found in cytoplasm, ER lumen, mitochondria, and chloroplasts function in protein targeting and assembly in normal (non-stressed) cells, hydrolyze ATP Constitutive & heat-induced (cytoplasmic) forms the heat-induced form first appears in the nucleolus, then goes to cytoplasm (may protect pre-ribosomes from heat stress?) Also, some hsp70s are light-induced; chloroplast hsp70 helps protect PSII from light/heat damage in Chlamy

49 LMW HSPs highly heat-induced 4 nuclear gene families: 1. Class I cytoplasmic 2. Class II cytoplasmic 3. Chloroplast localized 4. Endomembrane localized (ER) found in organelles only in plants function mostly unknown aggregate in vivo into "heat shock granules"

50 HSP regulation most work on LMW hsp in plants induction is mainly transcriptional but also translational control (hsp mrnas preferentially translated) genes induced coordinately, but not equally in all tissues light can also induce some LMW hsps

51 Cis-acting transcriptional regulatory elements HSE (heat shock elements) in the 5' regions: ~10-15 bp partial palindromes multiple copies required also found in other HS genes (e.g., hsp 70) similar to HSEs in animals

52 Heat-shock transcription factor (HSF) studied mostly in animals and yeast Binds to HSEs Contains leucine zipper motifs Binds DNA as a trimer Activity is induced by heat, and phosphorylation Activity Repressed by HSP70

53 Fig , Buchanan et al.

54 ig , Buchanan et al.

55 HSFs u Arabidopsis Sequencing of the Arabidopsis genome revealed a unique complexity of the plant heat stress transcription factor (Hsf) family. By structural characteristics and phylogenetic comparison, the 21 representatives are assigned to 3 classes and 14 groups. Particularly striking is the finding of a new class of Hsfs (AtHsfC1) closely related to Hsf1 from rice and to Hsfs identified from frequently found expressed sequence tags of tomato, potato, barley, and soybean. Evidently, this new type of Hsf is well expressed in different plant tissues. Besides the DNA binding and oligomerization domains (HR-A/B region), we identified other functional modules of Arabidopsis Hsfs by sequence comparison with the wellcharacterized tomato Hsfs. These are putative motifs for nuclear import and export and transcriptional activation (AHA motifs). There is intriguing flexibility of size and sequence in certain parts of the otherwise strongly conserved N-terminal half of these Hsfs. We have speculated about possible exon-intron borders in this region in the ancient precursor gene of plant Hsfs, similar to the exon-intron structure of the present mammalian Hsf-encoding genes.

56

57 PCD rostlinné buňky a vakuola

58 Geneticky a vývojově řízená sebevražda buňky.

59 Programmed Cell Death = PCD programovaná buněč ěčná smrt Concept developed in animal systems Each cell has intrinsic genetic program for ordered cell death Cell suicide pathway encoded by genome of dying cell Self-execution execution and dismantling in the context of the organism.

60 Cleavage at DNA linker sites between nucleosomes,, resulting in DNA fragments multimers of 180 bp Results in DNA laddering

61

62

63 TUNEL assay- (Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling) uses terminal deoxynucleotidyl transferase (TdT) to transfer biotin-dutp to these strand breaks of cleaved DNA. The biotin-labeled cleavage sites are then detected by reaction with HRP conjugated streptavidin and visualized by DAB showing brown color.

64

65 Důležitou roli hrají mitochondrie Leakage of cytochrome C from mitochondria into cytoplasm precedes death a výtok cytochromu C z nich.

66 Formation of apoptotic bodies- blebbing of DNA into membrane-bound bound bodies and engulfment by phagocytosis to prevent an inflammatory response

67 Živočišná vs. rostlinná buňka = autofágie.

68 Iniciace PCD je regulována preteázami - kaspázami Regulated by caspases- cysteine proteases Very specific proteases known- usually no more than 1 or 2 breaks substrate Orchestrates cell death- eg.. Caspase-3 3 an executioner caspase that starts a cascade

69 Účast PCD na vývoji rostlin příklady Aleurone cells- aleurone in monocots form a secretory tissue that releases hydrolases to digest the endosperm and nourish the embryo. Die after germination is complete.

70 Root cap cells -protect the root apical meristem during seed germination and growth. Root cap are continually displaced to the root periphery by new cells. Happens when growing in water, not a caused by abrasion. TUNEL positive

71 Tracheid elements- part of xylem Functional cells are dead After they elongate, deposit cell wall components, including lignin, then undergo autolysis. Actinomycin D or cycloheximide block cell death

72 Leaf senescence- induced by ethylene- a way of recapturing cellular material for use in other organs- i.e. to the roots. Membrane integrity and cellular compartmentalization are maintained until late into the process, so there is little leakage

73 Hypoxia- results in aerenchyma formation- empty cells with internal air spaces to allow transfer of O 2 from aerial organs to waterlogged stem bases and roots. Can be blocked by mutants in ETR (ethylene responsive gene)

74

75 Hypersensitivní Reakce hypersens.. resp. (HR) A hallmark of resistance- non-host, host specific the rapid death of plant cells in association with the restriction of pathogen growth Effective against biotrophic fungi, bacteria, and viruses je nespecifická a širokospektrá

76 JE HR FORMOU PCD? Do plant cells show PCD or apoptotic characteristics during infection? Ryerson and Heath showed TUNEL and DNA laddering in resistant reaction with soybean rust. A N O

77 Evidence pro PCD v HR Lesion mimicks of Arabidopsis- Single-gene gene mutations in regulatory pathway results in phenotype similar to HR mimick the effect of infection in the absence of the pathogen. Thus, plants contain an intrinsic genetic program that initiates and carries out cell death sentences on infected cells.

78 Lsd mutants- Lsd 1-5 (lesion simulating disease) Acd mutants (accelerated( cell death) Lls mutants (lethal( leaf spot) in maize

79 Lsd1 mutant

80 Funkční analýza LSD1 LSD1- gene has been cloned. A zinc-finger protein needed to restrict lesion size during HR. May be a transcription factor that down regulates or dampens cell death-a a negative regulator of PCD This pro-death pathway requires ROS LSD1 = Negativní regulátor HR lezí.

81

82 Rop GTPázy všude i v PCD

83

84 Účastní se kaspázy PCDrostlin? No homologue to animal caspases have been found- looked through genome of Arabidopsis.

85 ale Chichkova et al identified a metacaspase in tobacco Used a substrate of human caspase-3 3 (VirD2 from Agrobacterium) ) as a target, and found an enzyme that specifically cleaved this in HR-induced with TMV CLPs

86 Caspase inhibitors, effective in animal systems, also block PCD in plants Lam and del Pozo,, 2000, Del Pozo and Lam, used p35 from baculovirus,, to inhibit caspase and HR when expressed in tobacco. Disrupted N-mediated N resistance- TMV became systemic Mutated versions of p35, impaired in caspase inhibition, were not effective

87 VPE - vakuolární kaspázy (Casp.LikeProt.) u rostlin Vacuolar Processing Enzyme (VPE) je vakuolární cysteinová proteáza, která štěpí substráty specifické pro kaspázy.

88 Rostliny s potlačenou expresí VPE mají částečně potlačen také kolaps vakuoly při HR. Ale PCD je celkem normální u kombinovaných mutantů bez VPE proteáz

89

90 Hlavní nástroj genetické transformace rostlin je derivátem biotické interakce mezi rostlinami a Agrobakteriemi.

91 Genetic engineering of plants with Agrobacterium tumefaciens A. tumefaciens: used extensively for genetic engineering of plants. Contains T-DNA (bacterial plasmid) Genes colud be integrated into the plant chromosomes when the T-DNA is transferred. Tumor induced by A. tumefaciens

92

93

94 U Arabidopsis je možná vysoce účinná transformace ponořením mladých květenství do suspenze Agrobacteria. Dochází posléze k transformaci zárodečných vaků a vzniku heterozygotních transformantů po opylení. Účinnost je kol. 2%.