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BC

Vacúolo

Vacúolo de células vegetais

� Ocorrência� Origem� Funções� Alcalóides� Pigmentos� Inclusões minerais: Oxalato de cálcio

� Formação, Ocorrência, Dimensões e forma. Funções.

� Inclusões de Carbonato de cálcio.

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Vacúolo - Origem

� A Working Model for Transport Pathways in the Vacuolar Apparatus.

� Seven basic pathways are used for the biogenesis, maintenance, and supplying of vacuoles. Pathway 1: entry and transport in the early secretory pathway (from ER to late Golgi compartments). Pathway 2: sorting of vacuolarproteins in the trans-Golgi network (TGN) to a pre/provacuolar compartment (PVC) via an early biosynthetic vacuolar pathway. Pathway 3: transport from PVC to vacuole via the late biosynthetic vacuolar pathway. Pathway 4: transport from early secretory steps (ER to Golgi complex; pathway 1) to the vacuole via an alternative route with possible material accretion from Golgi (indicated by the asterisk). Pathway 5: endocytotic pathway from the cell surface to the vacuole via endosomes. Pathway 6: cytoplasm to vacuole through autophagy by degradative or biosynthetic pathways. Pathway 7: transport of ions and solutes across the tonoplast. AV, autophagic vacuole; E, early endosome; ER, endoplasmic reticulum; PVC, pre/provacuolar compartment; TGN, trans-Golgi network.

TonoplastoThe vacuole plays an important role in the homeostasis of the plant cell. It is involved in the control of cell volume and cell turgor; the regulation of cytoplasmic ions and pH; the storage of amino acids, sugars, and CO2; and the sequestration of toxic ions and xenobiotics. These activities are driven by specific proteins present in the tonoplast.

� Model of ABC Transporters, H1 Primary Pumps, H1-Coupled Transporters, and Channels in a Simplified Tonoplast.

� Glutathione S-conjugate (GS-X) and metabolite (M) transport is achieved by an ABC transporter(s). An electrogenicH1-ATPase (V-type) and an H1-PPase acidify the vacuole. The proton motive force provides energy for uptake and release of solutes (i.e., cations, anions, and organic solutes, denoted A, B, or C indiscriminately here) across the tonoplast through transporters and channels. Water channels (aquaporins) facilitate the passive exchange of water.

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Vacúolo

� São cavidades de forma e dimensões variadas delimitadas pelo tonoplasto.

� As células vegetais jovens apresentam vacúolos pequenos os quais se vão agregando com a idade até ocorrer um número reduzido, normalmente um, nas células velhas.

� Resultam da dilatação de túbulos do retículo endoplasmático e da fusão de vesículas do retículo e do complexo de Golgi.

� Contêm soluções de muitas substâncias:� gases atmosféricos,� sais inorgânicos,� sais de ácidos orgânicos, frequentemente na forma de cristais.� Também podem ser encontrados ácidos orgânicos, açucares,

proteínas solúveis em água, alcalóides e pigmentos.� Ao nível dos pigmentos estes compreendem os antociânicos (encarnados,

púrpura, azuis) presentes nos vacúolos da epiderme das pétalas de flores, flavonas (amarelos).

Funções� Os vacúolos das células de plantas desempenham funções diversas

sendo importantes no desenvolvimento da planta.� Desempenham funções na osmorregulação

� Juntamente com a parede desempenha função importante na manutenção da forma e no crescimento.

� Armazenam:� Produtos absorvidos

� Fosfatos, sulfatos, nitratos, cloretos, outros� Substâncias elaboradas pela célula e temporariamente armazenadas

� Malato, tartarato, cítrico, succínico, ...� Substâncias elaboradas pela célula de reserva

� Glúcidos, proteínas: Glucose, Frutose, Sacarose,Inulina, aleurona.� Produtos rejeitados (tóxicos)

� Oxalatos, taninos, alcalóides (papaverina, cafeína, nicotina, cocaína)� Algumas daquelas substâncias alelopáticas, depositadas em células

especializadas, servem de mediadores planta-planta, planta-microrganismos e interagem com herbívoros.

� Em sementes, vacúolos armazenam proteínas utilizadas durante o desenvolvimento.

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ORIGEM DAS SUBSTÂNCIAS DO SUCO VACUOLAR :

Exterior

Acumulação temporária

Acumulação definitiva (ex: produtos de rejeição)

Metabolitos secundários: não estão directamente envolvidos no crescimento e reprodução, muitas vezes são específicos de cada espécie.

Vacúolo – ProteóliseA proteólise e reciclagem de aminoácidos, é uma das actividades mais importantes desempenhada pelos vacúolos.

� . Schematic overview of the functions of vacuolar proteolysis in cellular protein breakdown and amino acid recycling. Upper part: precursors of vacuolar proteases are synthesized at the ER and transferred in vesicles to the vacuole either via endomembrane progression through the Golgi and MVB (e.g. CCV) (for references see legend to Fig. 2) or bypassing the Golgi (PAC, precursor protease vesicles, ER bodies, riconosomes) (Chrispeels and Herman, 2000; Gietl and Schmidt, 2001; and references in the legend to Fig. 2). Lower part: in MVB and vacuoles (LV, PSV) precursors of processing enzymes (e.g. VPE) are activated auto-catalytically by limited proteolysis (precursor processing). Activated processing proteases process other precursor polypeptides and thereby activate enzymes and generate polypeptides involved in defence processes. Proteins to be degraded get into vacuoles as shown in Fig. 2. Only vacuolar protein transfer by variants of autophagy is indicated here because of its specific contribution to senescence and PCD. Limited and complete vacuolar proteolysis is a major process of cellular amino acid recycling after long-term storage of protein reserves, when selected proteins are broken down immediately after getting into the vacuole or during senescence. Mega-autophagy represents the final step in PCD when the release of proteases leads to cell corpse degradation.

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The components of Water Potential

� The components of Water Potential

� Pressure potential or pressure (Ψp or P)� Osmotic potential (solute potential) which is caused by the presence of solute

particles (Ψs or s)� In simple systems at constant temperatures, the water potential results from the

combined but opposite actions of the pressure and the osmotic potentials:

� Ψ = Ψp – Ψs or Ψ = P – s

� By convention P = 0 at atmospheric pressure.� An increase of pressure results in a positive pressure, and tension (suction or

pulling, the opposite of pressure) results in a negative pressure.

� Pressure is usually positive in living cells;� In dead xylem elements or soil is negative.�

� The components of water potential consist of solute and matric forces, which decreases the water potential, and pressure, which increases it.

� The osmotic potential of a solution is negative because the solvent water in the solution can do less work than pure water.

� Water flux from leaves to atmosphere (transpiration) is driven by vapor concentration gradient

� Flux = vapor pressure gradient * stomatal conductance

� The reduced Ψp in xylem causes liquid water to flow up the stem.

� Water flux through xylem is driven by ∆Ψp

� Flux = ∆Ψp * xylem conductance

� Transpiration establishes gradients of pressure (Ψp), or tension, along the continuous hydraulic pathway in transpiring plants, that is immediately transduced through the entire hydraulic pathway.

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Three ways that water (and other

materials) move in plants:

� Water movement� Difusion

� Diffusion is driven by a concentration gradient� Osmosis

� Osmosis is driven by a water potential difference across a membrane – ie, both pressure and concentration are important.

� Water moves across a semipermeable membrane to the side where the solute is most concentrated

� Mass Flux� Mass flow is driven by a pressure gradient� The mass flow (amount/time) of water movement is the

product of the potential difference (hidrostatic and osmotic), and conductance or conductivity of the material or space (depends on density and flow area) that the water is moving through (Flux = ∆Ψ * k).

Osmosis

� Osmosis is a special case of diffusion� Osmosis involves the diffusion of water through a membrane� The membrane may be artificial and non-living e.g. Cellophane� In biology, the important membrane is the cell membrane� The osmotic pressure, ∆Π, is measured by the hidrostatic pressure observed at equilibrium

(∆Π = ∆P = ρg∆h)

� P = hydrostatic pressure (pascals);� ρ = water density (kg/m3);� g =gravitational acceleration (meter/s2);� h = height of fluid above (meter).�

� Pressure (Pascal)= Force/Area = gρh

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Vacúolo e osmorregulação

Os vacúolos são importantes estruturas celulares delimitadas por uma membrana unitária, o tonoplasto.

A existência do tonoplasto a separar o citoplasma do conteúdo vacuolar pode ser evidenciado se induzirmos a plasmólise celular.

Assim se colocarmos a célula numa solução hipertónica de NaCl ou sacarose vamos induzir a saída de água do interior do vacúolo com consequente diminuição de volume.

O vacúolo ao contrair-se provoca a deslocação do protoplasma que se destaca da parede celular e fica ligado à pasmodesmeos da parede por finas expansões. Se pelo contrário o meio for hipotónico (com concentração inferior à do meio intracelular) há entrada de água no vacúolo e consequente aumento do volume deste (turgecência).

CONCENTRAÇÃO DO SUCO VACUOLAR

PLASMÓLISE DESPLAMÓLISE ( TURGESCÊNCIA)

(meio exterior hipertónico) (meio hipotónico)

ORIGEM DAS SUBSTÂNCIAS DO SUCO VACUOLAR :

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Stomatal Mechanics and environmental effects

� Stomatal aperture changes in response to environmental variables .� Stomatal movements depend on changes in pressure potential ( Ψp) or turgor inside:

� the guard cells and� the subsidiary cells

� Changes in turgor results from a movement of water into/out of the guard cells.� Stomatal movements can be:

� (i) hydropassive due to changes in total water potential (Ψw of the leaf, or� (ii) hydroactive due to active changes in osmotic potential ( Ψπ) of the guard cells:

� Accumulation of solutes decreases Ψπ, thereby� Lowering Ψt and causing� Uptake of water until Ψp increases.

� Stomatal opening occurs when the Ψp in guard cells increases.

� Stomatal Opening� They open because of the presence of:

� Inelastic radially orientated cellulose microfibrils and� A thickened wall adjacent to the pore.

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Opening and closure - Opening

� When conditions are conducive to stomatal opening (e.g., high light intensity and high humidity), a proton pump drives H+ from the guard cells. The created negative potential opens potassium voltage - gated channels and so an uptake of K + occurs. To maintain this internal negative voltage so that entry of potassium ions does not stop, negative ions balance the influx of potassium. In some cases chloride ionsenter, while in other plants the organic ion malate is produced in guard cells. This in turn increases the osmotic inside the cell, drawing in water through osmosis. This increases the cell's volume and turgor pressure. Then, because of rings of cellulose microfibrils that prevent the width of the guard cells from swelling, and thus only allow the extra turgor pressure to elongate the guard cells, whose ends are held firmly in place by surrounding epidermal cells, the two guard cells lengthen by bowing apart from one another, creating an open pore through which gas can move.

ALCALÓIDES

São substâncias azotadas, básicas do grupo das aminas (derivam dos aminoácidos). Muitos são tóxicos, outros são também considerados princípios activos quando permitem o uso dessas plantas como plantas medicinais- ex: a sua actividade biológica pode ser anti-bacteriana).

Atropina (algumas Solanaceae: Datura stramonium, Atropa bella-donna)Morfina , papaverina (algumas Papaveraceae: Papaver somniferum)

Datura stramonium L.Papaver somniferum L.

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Vacúolo - Pigmentos

O vacúolo contém além de água, substâncias diversas, minerais e orgânicas susceptíveis de formar com a água soluções ou pseudosoluções. Estas substâncias têm três origens possíveis: vindas do exterior; elaboradas pela célula e acumuladas no vacúolo; elaboradas pela célula mas rejeitadas do citoplasma por acumulação no vacúolo onde persistem como dejectos celulares. Os produtos de rejeição mais frequentes nos vacúolos são os pigmentos antociânicos e flavonas.

As antocianinas são substâncias tóxicas que conferem aos vacúolos uma coloração que varia em função do pH:vermelho a pH ácido; violeta a pH neutro; azul a pH básico. Estes pigmentos são responsáveis pela coloração de frutos, algumas raízes e caules, mas sobretudo pela coloração de flores.

As flavonas são heterósidos amarelos ou brancos. Vacúolos há que não apresentam coloração.

PIGMENTOS ANTOCIÂNICOS

pH básico → azul

pH neutro → violeta

pH ácido → vermelho

Observação de vacúolos com pigmentos antociânicos:

Procedimento experimental:1-Retirar, com a ajuda de uma pinça, película epidérmica, da

porção mais corada, da página superior de uma corola de Ipomoea acuminata (Convolvulaceae).

2-Montar em água entre lâmina e lamela (preparação extemporânea).

3-Provocar(*): mudança de cor adicionando à preparação HCl0,1 Na plasmólise pela adição de NaCl 8%

(*) juntar algumas gotas do reagente no bordo da lamela e absorver no bordo oposto com papel de filtro

Metabolitos secundários

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Oxalato de cálcio

� Morfologia dos cristais� Distribuição� Função

� Regulação de Cálcio� Protecção da Planta� Complexação com Al e outros metais� Biossíntese

Roots model of absorption

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How does water move from soil-leaves-atmosphere

through plants?

A simplified model of CaOx formation in crystals idioblasts. Calcium entering an organ, such as a leaf, with the xylem stream is distributed among cells via the apoplast (wall). Most cells regulate Ca levels by pumping it back out or by compartmentation, but crystal idioblastsaccumulate large amounts of Ca via the activity of channels or pumps. Ca in the cytosol is taken up by the extensive endoplasmic reticulum and buffered by the activity of calreticulin. Oxalic acid can be synthesized in noncrystal cells; however, transport of this strong acid and chelator of Ca in the cytoplasm and via plasmodesmata is not likely, at least at levels that can support rapid growth of crystals. Oxalate can be generated from ascorbate in crystal idioblasts and transferred to the vacuole along with Ca. In this raphide crystal model, Ca and oxalate are transferred across the crystal chamber membrane and added along all the surfaces of young crystals, but as the crystal grows, they are added primarily to the ends. Mature crystals no longer add Ca and oxalate, although the cells are still living. Matrix proteins in the growing parts of the crystals may regulate precipitation or shape. Evidence for Ca channels, ER Ca accumulation, oxalate synthesis, matrix proteins, and the growth dynamics in crystal idioblasts has been published. Mechanisms for transfer of Ca and oxalate to the vacuole and into the crystals have not been identified.

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Morphology of CaOx crystals. All are scanning electron micrographs. Insets are light micrographs. (A) Prismatic crystals in bean seed coat. Arrows indicate kinked twin crystals. (B) Crystal sand in sugar beet leaf cells. (C) Raphide crystals from a ruptured Pistia raphideidioblast. Note barbs on one end of the crystals (arrows). Inset shows large raphideidioblast in the leaf relative to much smaller adjacent mesophyll cells. (D) Developing druse crystals from Pistia. Only a few facets can be seen (arrows). (E) Isolated Peperomia druse crystal. Note the many facets radiating from a central core.

Examples of CaOx crystals as static or active defense structures in plants. (A) Cross section through a leaf of Claoxylon sandwisence, showing sections along two large styloid crystal idioblasts(brackets), which can be viewed as static defenses. Both almost span the entire width of the leaf and the enclosed crystals (C) can be envisioned as potentially wounding the mouth and soft tissues of a grazing animal. The smaller styloid crystal is still developing. (B) An example of another type of static crystal defense. These developing raspberry fruits have a distinct layer or sheath of prismatic crystals in the developing seed coat (arrows) that would provide a tough physical barrier. The crystals appear bright in this image taken with crossed polarizing filters. (C) Vascular bundles in the stem of a Piper sp., seen in cross section. The phloem (P) of the two bundles is surrounded by a sheath of druse crystals (arrows). The phloem bundle is also protected by a fiber cap at the top and the tough xylem (X) at the bottom. (D) A living raphide idioblast in the stem of Pistia. Note the tapered tip of the cell, which is much thinner than the general cell wall. (E) When the idioblast is subjected to mechanical pressure, the tip is ruptured and the crystals are forcibly expelled. (F) Scanning electron micrograph of the Pistia raphide crystals showing sharply pointed tips and grooves (arrows) along the edges that help channel toxins into wounds created by the crystals.

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Inclusões minerais (acumulação temporária ou definitiva)

OXALATO DE CÁLCIO

A planta sintetiza ácido oxálico que é tóxico para a célula. Sais de cálcio são absorvidos pelas raízes e os iões cálcio combinam-se com o ácido oxálico dando origem a oxalato de cálcio, que cristaliza sob diversas formas, consoante o grau de hidratação do vacúolo.

→RÁFIDES (cristalização no sistema monoclínico): oxalato de cálcio mono-hidratado

→MACLAS ou CRISTAIS CÚBICOS (cristalização no sistema cúbico): oxalato de cálcio di-hidratado

Observação de feixes de ráfides no mesófilo da folha de Agave sp. e de maclas no pecíolo de Begonia sp.

1- Efectue um corte longitudinal (paralelo às fibras de celulose) no mesófilo da folha de Agave; efectue um corte transversal no pecíolo de Begonia.

2- Monte o corte entre lâmina e lamela, utilizando a água como meio de montagem.

3- Observe ao microscópio.

CARBONATO DE CÁLCIO

Ocorre sob a forma de cistólitos em células especializadas, os litocistos.

Encontram-se nas folhas (ex: epiderme), raízes e caules, em todos os tecidos excepto nos condutores.

CISTÓLITOS

Pedicelo - de natureza celulósico-péctica

Corpo - trama celulósico-péctica sobre a qual se deposita, em camadas concêntricas, carbonato de cálcio associado a silicatos

SEM; Ornamentação do cistólito de Parietaria sp. (f: folhado; p: protuberâncias)

SEM; Cistólito globoso de Parietaria punctata

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Bibliografia

� Becker, W.M., Reece, J.B. & Poenie, M.F.. 1996. The World of the Cell. 3ª Edição. The Benjamin/Cummings Publishing Company. Menlo Park, California.

� Cooper, G.M.. 1997. The Cell, A Molecular Approach. ASM Press. Washington, DC.� Franceschi, V.R. & Nakata, P.A.. 2005. Calcium Oxalate in Plants: Formation and

Function. Annu. Rev. Plant Biol., 56 : 41–71.� Marty, F.. 1999. Plant Vacuoles. The Plant Cell, 11: 587–599.� Mol, J., Cornish, E., Mason, J. & Koes, R.. 1999. Novel coloured flowers. Current

Opinion in Biotechnology, 10:198–201.� Müntz, K.. 2007. Protein dynamics and proteolysis in plant vacuoles. Journal of

Experimental Botany, 58, (10): 2391–2407.� Purves, W.K., Sadava, D., Orians, G.H. & Heller, H. C.. 2004. Life: The Science of

Biology. 7º ed.. Ed. Sinauer Associates Inc. Sunderland, MA.� Salisbury, F.B. & Ross, C.W.. 1992. Plant Physiology. Wadsworth Publishing

Company, Belmonte.