Citokininas y desarrollo vegetal

Presentación del tema: "Citokininas y desarrollo vegetal"— Transcripción de la presentación:

1 Citokininas y desarrollo vegetal
• Estimula división celular • Promueve morfogénesis in vitro • Induce tallos laterales • Regula desarrollo de cloroplastos In 1913, Gottlieb Haberlandt discovered that a compound found in phloem had the ability to stimulate cell division (Haberlandt, 1913). In 1941, Johannes van Overbeek discovered that the milky endosperm from coconut also had this ability. He also showed that various other plant species had compounds which stimulated cell division (van Overbeek, 1941). In 1954, Jablonski and Skoog extended the work of Haberlandt showing that vascular tissues contained compounds which promote cell division (Jablonski and Skoog, 1954). The first cytokinin was isolated from herring sperm in 1955 by Miller and his associates (Miller et al., 1955). This compound was named kinetin because of its ability to promote cytokinesis. Hall and deRopp reported that kinetin could be formed from DNA degradation products in 1955 (Hall and deRopp, 1955). The first naturally occurring cytokinin was isolated from corn in 1961 by Miller (Miller, 1961). It was later called zeatin. # Stimulates cell division. # Stimulates morphogenesis (shoot initiation/bud formation) in tissue culture. # Stimulates the growth of lateral buds-release of apical dominance. # Stimulates leaf expansion resulting from cell enlargement. # May enhance stomatal opening in some species. # Promotes the conversion of etioplasts into chloroplasts via stimulation of chlorophyll synthesis. • Retrasa la senescencia Zeatina

2 Estructura química Moléculas basadas en la conjugación de la posición N6 de la adenina Citokininas naturales Derivadas de adenina con un resto isoprenoide isopenteniladenina (i6ade) trans-zeatina Citoquininas sintéticas Derivadas de la adenina y de otros compuestos kinetina benciladenina (BA)

3 Citokininas y división celular
Growth of tobacco cells on control medium containing auxin (left) and on the same medium with addition of the silver nitrate precipitable material. This image is from the original work of Miller in 1953. Tabaco + auxinas Tabaco + auxinas + “kinetina” Miller (1953)

4 Inducción del crecimiento de callos por citokininas
Tobacco tissues depend on cytokinin for their growth in culture. Cultured tobacco callus was transferred to fresh medium that contained an auxin and either zeatin or kinetin at the indicated concentrations. The tissues were weighed after growing for 1 month on the various media. The results show that zeatin is more effective than kinetin in supporting tobacco tissue growth. The maximum response was obtained with 5 × 10–8 M zeatin, and higher zeatin concentrations either did not stimulate additional growth or were slightly inhibitory. The kinetin concentration had to be at least tenfold higher to achieve the same growth stimulation. (From Leonard et al )

5 Citokininas y fotomorfogénesis
1 semana Plastos en la oscuridad + CKs 2 semanas When wild-type Arabidopsis seedlings are germinated in darkness in the presence of cytokinin, they develop many of the characteristics of the det mutants: Hypocotyls shorten, cotyledons expand, the apical meristem initiates leaves, and there is partial development of the chloroplasts, including the synthesis of some photosynthetic enzymes. As Web Figure 21.7.A shows, this effect is proportional to the cytokinin dosage given to the seedlings (Chory et al. 1994). Cytokinin is said to produce a phenocopy of the det mutations. As the cytokinin concentration was increased, the inhibition of hypocotyl elongation became more pronounced, while the cotyledons expanded somewhat and leaves were initiated from the shoot apical meristem. At the higher cytokinin concentrations the seedlings were phenocopies of det mutants. Cytokinin treatment also resulted in thylakoid formation in the plastids of dark-grown seedlings (E) as compared to the development of plastids as etioplasts in the untreated, dark-grown wild-type control (D) 3 semanas

6 Citokininas y senescencia
Tabaco (58 días de edad) The control tobacco plant (left) is a 58-day-old cutting. The same type of tobacco plant (right), engineered in the experiment to express the Sho gene, is also 58 days old. Courtesy of Elena Zubko and Peter Meyer/ University of Leeds. By influencing the decline in proteins involved in photosynthetic metabolism. silvestre superproductor de CKs

7 Biosíntesis de citokininas
IPT Proposed biosynthetic and metabolic pathway for cytokinins. Left, The proposed biosynthesis of zeatin tri-/diphosphate in Arabidopsis. Both ADP and ATP are likely substrates for the plant IPT enzyme, and these and their di- and triphosphate derivatives are indicted together (e.g. ATP/ADP). The biosynthesis of cytokinins in bacteria (e.g. A. tumefaciens) is compared next to it. Right, Several possible modifications and the degradation of zeatin. The diagram only depicts reactions that are described in the text; cytokinin metabolism is more complex than the pathways shown (see Mok and Mok, 2001). Cytokinins (CKs) are a group of phytohormones that play a crucial role in the regulation of plant growth and development. Identification of the enzymes and the corresponding genes that are involved in CK metabolism allowed us to understand how plants synthesize CKs and adjust CK activity to optimal levels. A major accomplishment toward these goals was the identification of genes for the first enzyme in the CK biosynthetic pathway, adenosine phosphate-isopentenyltransferase (IPT). In Arabidopsis thaliana and Agrobacterium tumefaciens, detailed analyses of IPTs were conducted through not only enzymatic characterization but also molecular structural approaches. These studies revealed the molecular basis for the Agrobacterium-origin of IPT used for the efficient biosynthesis of trans-zeatin that promotes tumorigenesis in host plants. Another landmark in CK research was the identification of CYP735A as an enzyme that converts iP-nucleotide to tZ-nucleotide. Furthermore, the identification of a CK-activating enzyme, LOG, which catalyzes a novel activation pathway, is a remarkable recent achievement in CK research. Collectively, these advances have revealed the complexity of the entire metabolic scheme for CK biosynthesis.

8 Citokininas y plásmido Ti
Tabaco infectado con Agrobacterium tumefaciens The Ti plasmid (tumor-inducing plasmid) of Agrobacterium tumefaciens has been developed as a vehicle for introducing foreign genes into plants (Web Figure 21.5.A). When Agrobacterium infects plants, a region of the Ti plasmid called the T-DNA is taken up by the plant cell and incorporated into one of its chromosomes. The genes in the T-DNA are referred to as phyto-oncogenes because they induce neoplastic, or tumor-producing, growth (Matzke and Chilton 1981). To use the Ti plasmid as a vector for introducing new genes into plants, it is necessary to disarm the plasmid so that it does not cause tumors. Researchers accomplished this task by deleting the genes in the T-DNA that encode the enzymes controlling auxin and cytokinin synthesis. In addition, it is necessary to introduce a gene into the T-DNA that will enable the investigator to select the transformed cells. Genes for antibiotic resistance are normally used for this purpose.

9 Biosíntesis de citokininas
IPT LOG CYP735A Proposed biosynthetic and metabolic pathway for cytokinins. Left, The proposed biosynthesis of zeatin tri-/diphosphate in Arabidopsis. Both ADP and ATP are likely substrates for the plant IPT enzyme, and these and their di- and triphosphate derivatives are indicted together (e.g. ATP/ADP). The biosynthesis of cytokinins in bacteria (e.g. A. tumefaciens) is compared next to it. Right, Several possible modifications and the degradation of zeatin. The diagram only depicts reactions that are described in the text; cytokinin metabolism is more complex than the pathways shown (see Mok and Mok, 2001). Cytokinins (CKs) are a group of phytohormones that play a crucial role in the regulation of plant growth and development. Identification of the enzymes and the corresponding genes that are involved in CK metabolism allowed us to understand how plants synthesize CKs and adjust CK activity to optimal levels. A major accomplishment toward these goals was the identification of genes for the first enzyme in the CK biosynthetic pathway, adenosine phosphate-isopentenyltransferase (IPT). In Arabidopsis thaliana and Agrobacterium tumefaciens, detailed analyses of IPTs were conducted through not only enzymatic characterization but also molecular structural approaches. These studies revealed the molecular basis for the Agrobacterium-origin of IPT used for the efficient biosynthesis of trans-zeatin that promotes tumorigenesis in host plants. Another landmark in CK research was the identification of CYP735A as an enzyme that converts iP-nucleotide to tZ-nucleotide. Furthermore, the identification of a CK-activating enzyme, LOG, which catalyzes a novel activation pathway, is a remarkable recent achievement in CK research. Collectively, these advances have revealed the complexity of the entire metabolic scheme for CK biosynthesis.

10 LONELY GUY en arroz SAM Conclusion:
Cytokinins in the shoot meristem function as a LOCAL PARACRINE* SIGNAL rather than as a LONG-DISTANCE SIGNAL transported from other organs.

11 Biosíntesis de citokininas
IPT LOG CYP735A Proposed biosynthetic and metabolic pathway for cytokinins. Left, The proposed biosynthesis of zeatin tri-/diphosphate in Arabidopsis. Both ADP and ATP are likely substrates for the plant IPT enzyme, and these and their di- and triphosphate derivatives are indicted together (e.g. ATP/ADP). The biosynthesis of cytokinins in bacteria (e.g. A. tumefaciens) is compared next to it. Right, Several possible modifications and the degradation of zeatin. The diagram only depicts reactions that are described in the text; cytokinin metabolism is more complex than the pathways shown (see Mok and Mok, 2001). Cytokinins (CKs) are a group of phytohormones that play a crucial role in the regulation of plant growth and development. Identification of the enzymes and the corresponding genes that are involved in CK metabolism allowed us to understand how plants synthesize CKs and adjust CK activity to optimal levels. A major accomplishment toward these goals was the identification of genes for the first enzyme in the CK biosynthetic pathway, adenosine phosphate-isopentenyltransferase (IPT). In Arabidopsis thaliana and Agrobacterium tumefaciens, detailed analyses of IPTs were conducted through not only enzymatic characterization but also molecular structural approaches. These studies revealed the molecular basis for the Agrobacterium-origin of IPT used for the efficient biosynthesis of trans-zeatin that promotes tumorigenesis in host plants. Another landmark in CK research was the identification of CYP735A as an enzyme that converts iP-nucleotide to tZ-nucleotide. Furthermore, the identification of a CK-activating enzyme, LOG, which catalyzes a novel activation pathway, is a remarkable recent achievement in CK research. Collectively, these advances have revealed the complexity of the entire metabolic scheme for CK biosynthesis.

12 Señalización de citokininas: CRE1
silvestre cre1

13 CRE1: una HisK del “sistema de dos componentes”
A, Evidence that CRE1 is a cytokinin receptor. The left-most pathway depicts the osmosensing pathway in wild-type yeast: the His kinase SLN1, which suppresses the activity of SSK2 (a mitogen-activated protein kinase kinase kinase) activity via a phosphorelay consisting of YPD1 (an Hpt) and SSK1 (a response regulator). A deletion mutant of SLN1 is lethal due to overactivation of SSK2. CRE1 can suppress the growth defect in an SLN1 deletion only in the presence of cytokinins (two right pathways). B, Model of cytokinin signaling in Arabidopsis. Cytokinin binds to the N-terminal domain of CRE1 (and likely other similar sensor kinase) and activates its His kinase activity. CRE1 phosphorylate the AHPs, which in turn transfer the phosphate to the receiver domain of ARR1 (and presumably to other type-B ARRs), thus activating their output (transcriptional activator) domain. Type-A ARRs (and possibly other primary target genes) are transcriptionally induced by the activated type-B ARRs. The type-A ARRs also interact with the AHPs, and are also likely phosphorylated. The activated type-A ARRs, perhaps in parallel and/or in combination with the activated type-B proteins, interact with various effectors to alter cellular function, including the a feedback inhibition of their own expression. The curved arrows indicate phosphotransfer.

14 CRE1: complementación en levadura

15 Mutantes de receptores de citokininas
Receptores HisK: AHK2, AHK3, AHK4/CRE1 Arabidopsis, 5 semanas Mutational Phenotypes of the ahk Multiple Mutants in the Reproductive Growth Phase. (A) Morphology of 5-week-old plants. From left to right are Col, Ws, ahk2-1 ahk4-1, ahk3-1 ahk4-1, ahk2-1 ahk3-1, and ahk2-1 ahk3-1 ahk4-1 plants. Bar = 10 cm. (B) Close-up view of the 7-week-old ahk2-1 ahk3-1 ahk4-1 triple mutant. Bar = 1 cm. (C) to (E) Close-up views of Col (C), Ws (D), and ahk2-1 ahk3-1 ahk4-1 (E) flowers. Bars = 1 mm. Plant Cell June; 16(6): 1365–1377. silvestres ahk2 ahk4 ahk3 ahk4 ahk2 ahk3 ahk2 ahk3 ahk4

16 Expresión de receptores de citokininas
AHK2 AHK3 AHK4/CRE1 Expression of AHK:GUS Fusion Genes. GUS staining of AHK2:GUS (left column), AHK3:GUS (middle column), and AHK4:GUS (right column) is shown. Plants were grown on MS gellan gum plates or on soil with 16-h-light/8-h-dark fluorescent illumination at 22°C. (A), (B), and (C) Five-day-old seedlings and close-up views of SAM and young leaf primordia (insets). (D), (E), and (F) Growing lateral root primordia of 5-d-old seedlings. (G), (H), and (I) Root tips of 5-d-old seedlings. (J), (K), and (L) Close-up views of the surface of first mature leaves. Ten-day-old rosette plants were stained. (M), (N), and (O) Cross-sections of influorescences of 4-week-old plants. (P), (Q), and (R) Floral tissues of 4-week-old plants. Plant Cell June; 16(6): 1365–1377.

17 Modelo hipotético de señalización

18 Histidine Phosphotransfer Proteins (AHPs)

19 ARRs en Arabidopsis Phylogenetic tree of Arabidopsis response regulators. The top part of the figure shows a phylogenetic tree that represents the degree of relatedness of the receiver domains present in the Arabidopsis genome. The closer two proteins are on the tree, the more similar are their amino acid sequences. Note that these proteins fall into two distinct groups, or clades, called the type-A ARRs (blue) and the type-B ARRs (red). These differences in sequence are also reflected in a distinct domain structure, as depicted below the tree. The type-A ARRs consist solely of a receiver domain, but the type-A proteins also contain a fused output domain at the carboxy-terminus.

20 Localización de ARRs en Arabidopsis
Expression of ARR5. The pattern of ARR5 expression was examined by fusion of the promoter to a GUS reporter gene (A) or by whole-mount in situ hybridization (B and C). For the latter, the tissue is hybridized with labeled single-stranded ARR5 RNA in either the sense orientation (B) or the antisense (C). The sense RNA is a negative control and reveals background, nonspecific staining. The antisense probe specifically hybridizes with the ARR5 mRNA present in the tissue, thereby revealing its spatial distribution. With both methods, ARR5 expression is observed primarily in the apical meristems. (From D’Agostino et al )

21 Modelo hipotético de señalización

22 Dianas de ARRs

23 Bifurcación en la señalización de CKs
The plant hormone cytokinin regulates numerous growth and developmental processes. A signal transduction pathway for cytokinin has been elucidated that is similar to bacterial two-component phosphorelays. In Arabidopsis, this pathway is comprised of receptors that are similar to sensor histidine kinases, histidine-containing phosphotransfer proteins, and response regulators (ARRs). There are two classes of response regulators, the type-A ARRs, which act as negative regulators of cytokinin responses, and the type-B ARRs, which are transcription factors that play a positive role in mediating cytokinin-regulated gene expression. Here we show that several closely related members of the Arabidopsis AP2 gene family of unknown function are transcriptionally up-regulated by cytokinin through this pathway, and we have designated these AP2 genes CYTOKININ RESPONSE FACTORS (CRFs). In addition to their transcriptional regulation by cytokinin, the CRF proteins rapidly accumulate in the nucleus in response to cytokinin, and this relocalization depends on the histidine kinases and the downstream histidine-containing phosphotransfer proteins, but is independent of the ARRs. Analysis of loss-of-function mutations reveals that the CRFs function redundantly to regulate the development of embryos, cotyledons, and leaves. Furthermore, the CRFs mediate a large fraction of the transcriptional response to cytokinin, affecting a set of cytokinin-responsive genes that largely overlaps with type-B ARR targets. These results indicate that the CRF proteins function in tandem with the type-B ARRs to mediate the initial cytokinin response. Thus, the evolutionarily ancient two-component system that is used by cytokinin branches to incorporate a unique family of plant-specific transcription factors.

24 Señalización: CRE1 Cytokinin is perceived by the AHK plasma membrane receptors. Cytokinin signal is further amplified by phosphorelay events starting from AHKs, which lead to the activation and subsequent nuclear translocation of AHP proteins. AHP proteins transfer the phosphoryl group to type A or type B ARR proteins. The former act as repressors of cytokinin signaling, whereas the latter act as positive transcriptional regulators of cytokinin-induced genes, including those encoding type A ARRs. CRF proteins are also activated by cytokinin, and after translocation to the nucleus they act as activators of cytokinin-regulated genes.

25 Autoinhibición de la senescencia con pSAG12-IPT
Silvestre (8) pSAG12-IPT (13) Flores producidas 178 ± ± 46 Semillas (g/planta) 20 ± ± 4 Biomasa (g/planta) 107 ± ± 20 Altura (cm) 176 ± ± 8 Hojas en tallo principal 33 ± ± 1.4

26 Autoinhibición de la senescencia con pSAG12-IPT
Capacidad fotosintética tr wt tr wt tr wt 3.5w 6w wt tr Regulación del promotor pSAG12 tr wt 12w 20w

27 Genes IPTs Arabidopsis contains nine different IPT genes, several of which form a distinct clade with other plant sequences.