Presentation on theme: "Chaim Frenkel Rutgers – the State University of New Jersey"— Presentation transcript:
1Fruit Ripening – A Developmental Event Driven by Hydration/ Dehydration Chaim FrenkelRutgers – the State University of New JerseyDepartment Plant Biology and PathologyNew Brunswick, NJ USA(848)
2Fruit ripening, a form of senescence in plants, is a genetically programmed event manifestingabundant transcriptional and translational activity.Expressed sequence tag (EST) dataset in ripening tomato fruitassembled to different functional categoriesFei Z et al (2004) The Plant Journal 40:47–59
3Banana- a climacteric fruit Strawberry- a non climacteric fruit Based on a ripening pattern fruit are classified into:1. Climacteric fruit, displaying Climacteric risein respiration and attended pattern in ethyleneevolution during the ripening process.2. Non climacteric fruit, which manifest continuousdown drift in respiration and ethylene evolution.Ethylene production and action stimulate ripeningin climacteric fruit but appears not to be importantin the ripening of non climacteric fruit.EthyleneClimacteric and non climacteric respiration in ripening fruitBanana- a climacteric fruit Strawberry- non climacteric fruitBanana- a climacteric fruit Strawberry- a non climacteric fruit
4Autonomous stress might trigger the onset of fruit ripening It is not clear, however, what might be a cellular or metabolic cue(s) that stimulate the ripeningtranscriptional program, ethylene action not withstanding.Ann Callahan and I [Frenkel and Callahan 1997] argue that fruit ripening is accompanied byand is driven by autonomous stress. Stress might be a major metabolic shift that, apparently,trigger the expression of ripening related genes and formation of corresponding gene products.Defense/stress responseDistribution of gene productsby different categories in ripening apricot fruitDistribution of ESTs in ripening tomatoby different functional categories
6Chemical Stress?An early study revealed that fruit ripening is accompanied by the accumulation of and is apparently driven by production of reactive oxygen species (ROS).However, accumulation and activity of ROS might jeopardize the ripening-dependenttranscriptional and translational programs.The molecular basis for ROS-driven fruit ripening wait clarification, but Stay tuned: oxidative stress might instigate water stress, as shown later.ethyleneH2O2lipidhydroperoxideEthylene evolution is relation to H2O2 and lipid hydroperoxideaccumulation in ripening tomato fruit (Frenkel and Eskin 1977)
7Hydration stress might be an alternative stress form, which initiate and drive the fruit ripening process.To examine this conjecture, we carried out measurement of the water statusin ripening fruit and, furthermore, to asses whether hydration changes is a causationin the onset of fruit ripening .
8fr We used azeotropic (toluene) distillation to extract and measure the water contentin ripening climacteric and non climacteric fruit.frFruit tissue sample(around 25 g)TolueneWater, an azeotrop, is removed from the fruit tissue sample by distillation with toluene.The moisture is collected in a water trap and its volume is measured.A.O.C.S official protocol 1989Fetzer WR (1951) Anal Chem 23:
9Changes in tissue moisture content in ripening climacteric fruit. Arrows indicate the Breaking (insipient color)ripening stage.A schematic chart showing stages in fruit backgroundcolor during the ripening processGreen White Breaking Pink Red
10Changes in tissue moisture content in ripening non climacteric fruit. Arrows indicate the Breaking (insipient color)ripening stage.
11Changes in moisture content in the pulp and peel of developing banana fruit. The data (from Offem and Thomas Food Res Intn26: 187) was presented in table form and wasplotted and presented in graph form. Similar results (not shown) were observed in plantain fruit.
12Other studies, in a different context noted occurrence of water deficit before the onset of ripening in both climacteric and non climacteric fruit.The present and other studies suggest that decline in tissue water content,preceding the onset of fruit ripening, might be a universal phenomenon.
13Ethylene might induce water deficit Ethylene was applied to potato tuber, an ethylene-responsive tissue,to examine whether the phytohormone might also lead to a declinein water content.The results suggest that ethylene-induced water deficit might account,in part, for ethylene action in fruit ripening.The results, showing comparable changes in water content andtotal weight, suggest expulsion onto or uptake of moisture from theambient atmosphere in ethylene-treated potato tuber.Ethylene-induced changes in potato tuber: A. water contentB. Total weight
14Ripening-associated water deficit: Coincidental to or a cause of fruit ripening? It is not clear whether the ripening-related hydration/ dehydration is merely coincidental toor, alternatively, ,might have a regulatory role in the onset of ripening.We examined this question with the use of an ethylene-mutant tomato*, which does notproduce ethylene but can be ripen by applied ethylene.We asked: Would artificially-induced water deficit lead to the initiation of ripening,even in the absence of ethylene ?* antisense to 1-aminocyclopropane carboxylate synthase, an intermediate in ethylene biosynthesisOeller PW et al (1991) Science 254:437–9.
1560 hours at 12° CCold Stress I Stress AcclimationChanges in water content in ethylene-mutant tomato fruit, induced by cold stress and acclimationFruit was held for 60 h at 12° C and next at 23° C for stress acclimation for up to 98 h. Tissue water content wasmeasured periodically.
16of ethylene-mutant tomato fruit. Effect of ethylene and comparable effect of cold stress and acclimation on ripeningof ethylene-mutant tomato fruit.Ethylene-treated tomatoes were held at 23◦C and ventilate continuously for 5 dayswith air containing 10 μL ethylene/L air.Cold-treated fruit were held at 12◦ C for 60 h and next for 5 days at 23◦ C.
17Ripening of ethylene-mutant tomato induced by cold stress and acclimation Effect of cold stress and acclimation on ripening, in fruit held at 12° C for 60 h and next at 23° C for up to 8 d for stress acclimation.
18What is the cellular and metabolic origin of ripening-related water deficit? Cyclic changes in tissue water content might reflect changes in the hydration stateof two major water compartments in plant cells:the central vacuole andthe apoplast.Apoplast: Matter located between the plasma membrane ofneighboring cells and harboring the cell wallCentral vacuole
19Increase in water content would be predicated on solute uptake, Cyclic changes in vacuolar water content would necessitate comparable changes in the state of water:Increase in water content would be predicated on solute uptake,to decrease the chemical potential (increase the osmotic pressure).Decrease in water content would result from solute expulsion,to increase the chemical potential (decrease the osmotic pressure).This is not a likely scenario, particularly indetached tissues, off-the-vine ripened tomatoor ethylene-treated potato tuber, for example.Ethylene-treatedpotato tuber
20Cell wall might mediate cyclic changed in fruit water content. Cell walls might have an efficacy to mediate changes in the state of water content, based on changes in the physical confinement of the solvent, brought about by cell wall re-modeling:cell wall loosening, which increases the osmotic pressure leads to increased hydration.Cell wall tightening, resulting in lowering the osmotic pressure lead to water expulsion and dehydration.Cell wall swelling has been used to measure degree of cell wall hydration.Swelling of cell wall material (CWM) isolatedfrom kiwifruit at different stages of ripening.CWM was stained with toluidine blue andallowed to settle.Redgwell RJ et al (1997) Planta 203:
21Changes in tissue moisture content and comparable changes in swelling efficacy of cell walls:(A) ripening tomato fruit(B) ripening strawberry fruit(C) in ethylene-treated potato tuberR2 =A high correlation coefficient (R2) values betweentissue water content and swelling proficiency of cell walls,suggest that these variables are strongly interrelated.The results suggest, furthermore, that changes in cell wallswelling might reflect and actually account for correspondingchanges in tissue water content.R2 =R2 =
22Low chemical potential High chemical potentialPeroxidase, OxidaseH2O2, O2Relaxed TightenedHigh osmotic pressure Low osmotic pressureA scheme showing the hydration state of relaxed and tightened plant cell wallCell wall in a relaxed state, draw and hold water because molecular and segmental mobility of the wallconstituents lowers the chemical potential (increase the osmotic pressure) of water molecules. ( ).Cell wall tightening, resulting from oxidative cross-linking and consequent restriction on molecular mobility,leads to an increase in the wall water potential (decrease in osmotic pressure ( ).High-chemical potential water escapes the system resulting in cell wall dehydration.
23monomer undergoing oxidative coupling to a wall-bound dimer. H2O2, O2A scheme showing cell wall-bound phenyl propanoid (ferulic acid)monomer undergoing oxidative coupling to a wall-bound dimer.The scheme suggests that cell wall remodeling is predicated on onset of oxidative metabolism.Oxidant-induced cell wall remodeling and consequent changes in wall and tissue hydration statemight be a cellular and metabolic context for regulation of fruit ripening by oxidative metabolism.Cell wall constituents are denoted by A through G:A; β-(1-4) linked xylan backbone B; Xylose-arabinose linkage C; 5-O-feruloyl ligninD; 5-O-diferuloyl group (5-5linked dimer) E; 5-O-diferuloyl group(8-5dimer)F; 3-O-acetyl group G; arabinose-ligninMathew S (2004) Critical Reviews in Biotechnology 24:59–83
24Changes in the relative abundance of cell wall-bound and cross-linked phenolic acid residues measured in on-the-vine ripened tomato fruit at various stages of ripening including (A) Green, (B) Breaker, (C) Pink, and (D) Red.The results show a progressive increase in the abundance of mostly ferulic acid dimers as fruit become ripe.These results suggest that progressive cross-linking and tightening of cell wall structure might be a molecular basis for water expulsion from cell walls and corresponding decrease in tissue water content.Some of the dimers were tentatively identified:Molecular mass Compoundβ–β, coniferyl alcohol/coniferyl alcohol8-8 ferulate/ferulateβ, ferulate/coniferyl alcoholMolecular Mass
25Low chemical potential High chemical potentialPeroxidase, OxidaseH2O2, O2Relaxed TightenedHigh osmotic pressure Low osmotic pressureA scheme showing the hydration state of relaxed and tightened plant cell wallA similar reasoning might account to dehydration associated with human aging.
26Age-related changes in body water in humans 20 years 65-80 years Decrease in Decrease intotal body water extra-cellular water17% %Shi S, Klotz U (2011) Current Drug Metabolism 12:603
27particularly in females. Age-related decline in extracellular water is more pronounced than intracellular water,particularly in females.Decline in extracellular water might reflect age-related cross-linking in the extracellular matrix,perhaps collagen or elastin.Steen B (1988) Nutrition Reviews 46:45-51
28this phenomenon might arise from molecular aggregation. Beverly Rubik* presented data showing age-related decline in body water, suggesting thatthis phenomenon might arise from molecular aggregation.The present result reinforce this suggestion and emphasize that a major aggregation eventIs cross-linking of the extracellular matrix.This event leads to an increase in water potential (decrease in osmotic pressure)and consequent escape of water from the extracellular space.** An analogy is also the dehydration of insect larval cuticle during hardening (tanning),resulting from the cuticle protein aggregation, through the formation of hydrogen andother bonds or induced artificially by cross-linking agents.* Beverly Rubik. Studies & Observations on a “Functional” Water, Ionized Alkaline Water.Seventh Annual Conference on the Physics, Chemistry and Biology of Water 2012** Julian Vincent. If it's sclerotised it must be dry: Phenolic tanning controls hydration.Fifth Annual Conference on the Physics, Chemistry and Biology of Water 2010
29Dehydration of the extra-cellular matrix might be common to aging plant and animal tissues. In fruit, dehydration of the extra-cellular matrix is apparently an origin of water stressand a metabolic shift that might trigger the ripening transcriptional program.Where to go next?
30H+ A literature search revealed that in the course of development, Hydration state (swelling efficacy) ofcell wall from on-the-vine ripened tomatoA literature search revealed that in the course of development,plant cell wall (CW) undergo cyclic changes in CW relaxationand tightening by cross-linking and accompanying hydrationand dehydration, respectively.This scenario is relevant to CW changes occurring in ripeningfruit, as shown for (on-the-vine) ripening tomato.H+Acid-induced CW hydrolysis Oxidative Crosslinkingand relaxation and CW tighteningHYDRATION DEHYDRATIONWolf S et al (2012) Annu Rev Plant Biol 63:381–407
31A dedicated plant cell wall integrity (CWI) maintenance mechanism monitors and maintains functional integrity of the cell wall.When in a relaxed hydrated state, perturbations in cell wall integrity lead to:ROS productionChanges in cell wall composition and structure, notably oxidative cross-linkingProduction of ethyleneFormation of callose (β-glucan, a β-1,3-linked glucose)These changes are also the hallmark of fruit ripening
32architecture brought about by turgor pressure for example, might We propose: perturbations in cell wall integrity (CWI), distortion in cellulosearchitecture brought about by turgor pressure for example, mightlaunch signaling cascade that trigger the ripening transcriptional program.Turgor pressureCWI sensingOxidative cross-linkingDehydrationSignalingcascadeHydrated CW CW perturbationRipening Transcriptional ProgramThe scheme suggests that loosening and an accompanying hydration offruit cell wall is a pre-requisite for sensing perturbation in cell wall andsubsequent events leading to the onset of ripening.It also account for the universal expression of this developmental eventin ripening fruit.
33in ethylene-mutant tomato fruit, even in the absence of ethylene. A preliminary study revealed that citral, which disrupt the cytoskeletonand consequent distortion in cellulose architecture, initiated ripeningin ethylene-mutant tomato fruit, even in the absence of ethylene.MTLocalization of the cellulose synthase complex (CSC) is microtubule(MT) dependent.Wightman R, Turner SR (2008) The Plant Journal 54:794–805CitralCSC
34Thank you for your attention. Questions are welcome.
37These perturbation are perceived by ‘mechano-sensing’ apparatus, resulting in signaling cascade leading to transcriptional activation of the following:production of ethyleneproduction other aging-relate phytohormones (salicylic acid or jasmonic acid)formation of callose (β-glucan, made of β-1,3-linked glucose)ROS productionas well as changes in cell wall composition and structureThese changes are a hall mark of fruit ripening as well as of stress responses, further attestingthat the ripening process is expression of a stress response, evoked, apparently,by perturbation(s) in cell wall integrity.
38Abiotic, biotic stress Turgor pressure Changes in CW structure reflect the existence of a dedicated plant cell wall integrity (CWI) maintenance mechanism, which monitors and maintains functional integrity of the cell wall.Perturbations in CWI, for example distortion in cellulose architecture of CW in a relaxed hydrated state, may be brought about by turgor pressure or external forces.Abiotic, biotic stressTurgor pressureWhen in a relaxed hydrated state, perturbations in cell wall integrity (CWI), for example distortion in cellulose architecture, may be brought about by turgor pressure.
39Cell walls and growth control Plant growth is predicated on cell wall (CW) loosening, instigated through acidity-induced hydrolysis of CW matrix polysaccharides. The loosened CW structure swells by taking up water.Growth is inhibited through cross-linking of the wall protein Extensin and matrix polysaccharides. The tightening of the CW results in water expulsion.Cell wall remodeling is regulated by signaling cascade induced by phytohormones and/ or biotic and abiotic cues.H+Acidification, leading to hydrolysisof CW polysaccharides and consequentCW loosening and hydrationCross-linking and consequentCW tightening and dehydrationCross-linkingWolf S et al (2012) Annu Rev Plant Biol 63:381–407
40It is generally accepted that ethylene is a ripening hormone, because the gas strongly stimulate various ripening process,and, accordingly, stimulate the expression of ripening-related genes.However, other phytohormones (Brassinosteroids, for example) also control the ripening process.Ethylene activity might not be important in the ripening of non climacteric fruit.Importantly, abiotic and biotic stresses also stimulate fruit ripening.A fundamental question is:What are the metabolic and cellular events that might trigger hormonal or other signaling cues, which set in motionsthe biosynthesis and action of ethylene and perhaps other signaling mechanism?It is not clear, however, what might be a cellular or metabolic cue(s) that stimulate the ripening transcriptional programEthylene action not withstanding.Ann Callahan and I [Frenkel and Callahan 1997] argue that fruit ripening is accompanied by and is driven byautonomous stress. This metabolic shift triggers, apparently, the expression of ripening related genes.But what type of stress?
43Model for regulation of climacteric ripening via coordinated signaling pathways. Transcription factors including LeMADS-RIN, LeNOR, likely additionalMADS-box proteins, CNR and factors remaining to be discovered (?) representthe developmental signaling system that initiates ripening in climacteric fruit.Some components, such as those homologous to LeMADS-RIN can be used innon-climacteric species as well. The developmental signaling system regulatesethylene synthesis that is itself autocatalytic, in addition to non-ethylene-mediatedripening responses (represented by the red broken arrow). Light influences ripening,at least in tomato, only in relation to carotenoid accumulation and throughactivity of the DET1 (hp2) and DDB1 (hp1) gene products.Adams-Phillips L et al (2004) TRENDS in Plant Science Vol.9 No.7 July 2004
44Distribution of 101 gene products during apricot fruit ripening Functional classification of proteins expressed during citrus fruit ripeningDistribution of 101 gene products during apricot fruit ripeningD'Ambrosio C et al (2013) J Proteom78:39-57
45CW expansion:In principle, one can distinguish the following events:(a) hydration of newly deposited cell wall material and wall relaxation (Figure 2a)(b) turgor-driven deformation of the cell wall(c) mechanosensing, followed by the release of apoplastic reactive oxygen species (ROS), cross-linking, partialdehydration, and stiffening of the wall (Turgor-driven cell wall extension takes place, which in turn leads to theactivation of the mechanosensing feedback loop, cell wall cross-linking, and dehydration)(d ) the secretion of new wall material. In what follows, these events are discussed separately.Monshausen GB, Gilroy S Feeling green: mechanosensing in plants. Trends Cell Biol. 19:228–35