Fruit Ripening – A D evelopmental E vent Driven by H ydration/ Dehydration

1. Fruit Ripening – A Developmental Event Driven by Hydration/ Dehydration Chaim Frenkel Rutgers – the State University of New Jersey Department Plant Biology and Pathology New Brunswick, NJ 08901 USA (848) 932-6236 frenkel@aesop.rutgers.edu

2. Fruit ripening, a form of senescence in plants, is a genetically programmed event manifesting abundant transcriptional and translational activity. Expressed sequence tag (EST) dataset in ripening tomato fruit assembled to different functional categories Fei Z et al (2004) The Plant Journal 40:47–59

3. Based on a ripening pattern fruit are classified into: 1. Climacteric fruit, displaying Climacteric rise in respiration and attended pattern in ethylene evolution during the ripening process. 2. Non climacteric fruit, which manifest continuous down drift in respiration and ethylene evolution. Ethylene production and action stimulate ripening in climacteric fruit but appears not to be important in the ripening of non climacteric fruit. Ethylene Climacteric and non climacteric respiration in ripening fruit Banana- a climacteric fruit Strawberry- non climacteric fruit Banana- a climacteric fruit Strawberry- a non climacteric fruit

4. Autonomous 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 ripening transcriptional program, ethylene action not withstanding. Ann Callahan and I [Frenkel and Callahan 1997] argue that fruit ripening is accompanied by and 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 response Distribution of gene products by different categories in ripening apricot fruit Distribution of ESTs in ripening tomato by different functional categories

5. But what type of stress?

6. Chemical Stress? ethylene 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-dependent transcriptional 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. H2O2 lipid hydroperoxide Ethylene evolution is relation to H2O2 and lipid hydroperoxide accumulation in ripening tomato fruit (Frenkel and Eskin 1977)

7. Hydration 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 status in ripening fruit and, furthermore, to asses whether hydration changes is a causation in the onset of fruit ripening .

8. We used azeotropic (toluene) distillation to extract and measure the water content in ripening climacteric and non climacteric fruit. fr Fruit tissue sample (around 25 g) Toluene Water, 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 1989 Fetzer WR (1951) Anal Chem 23:1062-1069

9. Changes in tissue moisture content in ripening climacteric fruit. Arrows indicate the Breaking (insipient color) ripening stage. A schematic chart showing stages in fruit background color during the ripening process Green White Breaking Pink Red

10. Changes in tissue moisture content in ripening non climacteric fruit. Arrows indicate the Breaking (insipient color) ripening stage.

11. Changes in moisture content in the pulp and peel of developing banana fruit. The data (from Offem and Thomas [1993]Food Res Intn26: 187) was presented in table form and was plotted and presented in graph form. Similar results (not shown) were observed in plantain fruit.

12. Other 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.

13. Ethylene might induce water deficit Ethylene was applied to potato tuber, an ethylene-responsive tissue, to examine whether the phytohormone might also lead to a decline in 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 and total weight, suggest expulsion onto or uptake of moisture from the ambient atmosphere in ethylene-treated potato tuber. Ethylene-induced changes in potato tuber: A. water content B. Total weight

14. Ripening-associated water deficit: Coincidental to or a cause of fruit ripening? It is not clear whether the ripening-related hydration/ dehydration is merely coincidental to or, 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 not produce 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 biosynthesis Oeller PW et al (1991) Science 254:437–9.

15. 60 hours at 12° C -------------- Cold Stress ----------------- I -----------------Stress Acclimation ------------ Changes in water content in ethylene-mutant tomato fruit, induced by cold stress and acclimation Fruit was held for 60 h at 12° C and next at 23° C for stress acclimation for up to 98 h. Tissue water content was measured periodically.

16. Effect of ethylene and comparable effect of cold stress and acclimation on ripening of ethylene-mutant tomato fruit. Ethylene-treated tomatoes were held at 23◦C and ventilate continuously for 5 days with air containing 10 μL ethylene/L air. Cold-treated fruit were held at 12◦ C for 60 h and nextfor 5 days at 23◦ C.

17. Ripening 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.

18. What is the cellular and metabolic origin of ripening-related water deficit? Cyclic changes in tissue water content might reflect changes in the hydration state of two major water compartments in plant cells: the central vacuole and the apoplast. Apoplast: Matter located between the plasma membrane of neighboring cells and harboring the cell wall Central vacuole

19. Cyclic changes in vacuolarwater 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 in detached tissues, off-the-vine ripened tomato or ethylene-treated potato tuber, for example. Ethylene-treated potato tuber

20. Cell 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) isolated from kiwifruit at different stages of ripening. CWM was stained with toluidine blue and allowed to settle. Redgwell RJ et al (1997) Planta203:162-173

21. Changes 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 tuber R2 = 0.9563 A high correlation coefficient (R2) values between tissue water content and swelling proficiency of cell walls, suggest that these variables are strongly interrelated. The results suggest, furthermore, that changes in cell wall swelling might reflect and actually account for corresponding changes in tissue water content. R2 = 0.6812 R2 = 0.9626

22. Low chemical potential High chemical potential Peroxidase, Oxidase H2O2, O2 Relaxed Tightened High osmotic pressure Low osmotic pressure A scheme showing the hydration state of relaxed and tightened plant cell wall Cell wall in a relaxed state, draw and hold water because molecular and segmental mobility of the wall constituents 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.

23. H2O2, O2 A 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 state might 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 lignin D; 5-O-diferuloyl group (5-5linked dimer) E; 5-O-diferuloyl group(8-5dimer) F; 3-O-acetyl group G; arabinose-lignin Mathew S (2004) Critical Reviews in Biotechnology 24:59–83

24. Changes 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 Compound β–β, coniferyl alcohol/coniferylalcohol 8-8 ferulate/ferulate 518 8-β, ferulate/coniferyl alcohol Molecular Mass

25. Low chemical potential High chemical potential Peroxidase, Oxidase H2O2, O2 Relaxed Tightened High osmotic pressure Low osmotic pressure A scheme showing the hydration state of relaxed and tightened plant cell wall A similar reasoning might account to dehydration associated with human aging.

26. Age-related changes in body water in humans 20 years 65-80 years Decrease in Decrease in total body water extra-cellular water 17% 40% Shi S, Klotz U (2011) Current Drug Metabolism 12:603

27. 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

28. Beverly Rubik* presented data showing age-related decline in body water, suggesting that this phenomenon might arise from molecular aggregation. The present result reinforce this suggestion and emphasize that a major aggregation event Is 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 and other 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

29. Dehydration 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 stress and a metabolic shift that might trigger the ripening transcriptional program. Where to go next?

30. Hydration state (swelling efficacy) of cell wall from on-the-vine ripened tomato A literature search revealed that in the course of development, plant cell wall (CW) undergo cyclic changes in CW relaxation and tightening by cross-linking and accompanying hydration and dehydration, respectively. This scenario is relevant to CW changes occurring in ripening fruit, as shown for (on-the-vine) ripening tomato. H+ Acid-induced CW hydrolysis Oxidative Crosslinking and relaxation and CW tightening HYDRATIONDEHYDRATION Wolf S et al (2012) Annu Rev Plant Biol63:381–407

31. A 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 production Changes in cell wall composition and structure, notably oxidative cross-linking Production of ethylene Formation of callose (β-glucan, a β-1,3-linked glucose) These changes are also the hallmark of fruit ripening

32. We propose: perturbations in cell wall integrity (CWI), distortion in cellulose architecture brought about by turgor pressure for example, might launch signaling cascade that trigger the ripening transcriptional program. CWI sensing Turgor pressure Oxidative cross-linking Dehydration Signaling cascade Hydrated CW CW perturbation Ripening Transcriptional Program The scheme suggests that loosening and an accompanying hydration of fruit cell wall is a pre-requisite for sensing perturbation in cell wall and subsequent events leading to the onset of ripening. It also account for the universal expression of this developmental event in ripening fruit.

33. A preliminary study revealed that citral, which disrupt the cytoskeleton and consequent distortion in cellulose architecture, initiated ripening in ethylene-mutant tomato fruit, even in the absence of ethylene. Localization of the cellulose synthase complex (CSC) is microtubule (MT) dependent. Wightman R, Turner SR (2008) The Plant Journal 54:794–805 MT Citral CSC

34. Thank you for your attention. Questions are welcome.

37. These perturbation are perceived by ‘mechano-sensing’ apparatus, resulting in signaling cascade leading to transcriptional activation of the following: production of ethylene production other aging-relate phytohormones (salicylic acid or jasmonic acid) formation of callose (β-glucan, made of β-1,3-linked glucose) ROS production as well as changes in cell wall composition and structure These changes are a hall mark of fruit ripening as well as of stress responses, further attesting that the ripening process is expression of a stress response, evoked, apparently, by perturbation(s) in cell wall integrity.

38. 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 stress Turgor pressure When in a relaxed hydrated state, perturbations in cell wall integrity (CWI), for example distortion in cellulose architecture, may be brought about by turgor pressure.

39. Cell 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 Extensinand 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. Acidification, leading to hydrolysis of CW polysaccharides and consequent CW loosening and hydration Cross-linking and consequent CW tightening and dehydration H+ Cross-linking Wolf S et al (2012) Annu Rev Plant Biol 63:381–407

40. It 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 motions the 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 program Ethylene action not withstanding. Ann Callahan and I [Frenkel and Callahan 1997] argue that fruit ripening is accompanied by and is driven by autonomous stress. This metabolic shift triggers, apparently, the expression of ripening related genes. But what type of stress?

41. Brennan and Frenkel 1977 Frenkel and Eskin 1977

43. Model for regulation of climacteric ripening via coordinated signaling pathways. Transcription factors including LeMADS-RIN, LeNOR, likely additional MADS-box proteins, CNR and factors remaining to be discovered (?) represent the developmental signaling system that initiates ripening in climacteric fruit. Some components, such as those homologous to LeMADS-RIN can be used in non-climacteric species as well. The developmental signaling system regulates ethylene synthesis that is itself autocatalytic, in addition to non-ethylene-mediated ripening responses (represented by the red broken arrow). Light influences ripening, at least in tomato, only in relation to carotenoid accumulation and through activity 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

44. Functional classification of proteins expressed during citrus fruit ripening Distribution of 101 gene products during apricot fruit ripening D'Ambrosio C et al (2013) J Proteom78:39-57

45. CW 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 apoplasticreactive oxygen species (ROS), cross-linking, partial dehydration, and stiffening of the wall (Turgor-driven cell wall extension takes place, which in turn leads to the activation 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. 2009. Feeling green: mechanosensing in plants. Trends Cell Biol. 19:228–35