Microperforated Compostable Packaging Extends Shelf Life of Ethylene-Treated Banana Fruit.

Plastic packaging preserves the quality of ethylene-treated bananas by generating a beneficial modified atmosphere (MA). However, petroleum-based plastics cause environmental pollution, due to their slow decomposition. Biodegradable packaging may help resolve this controversy, provided it shows adequate preservation efficacy. In this study, we tested the compostable biodegradable polyester packaging of ethylene-treated bananas in comparison with commercially available petroleum-based plastic alternatives. When compostable packaging was used in a non-perforated form, it caused hypoxic fermentation, manifested as impaired ripening, off-flavor, and excessive softening. Micro-perforation prevented fermentation and allowed MA buildup. Furthermore, no water condensation was observed in the biodegradable packages, due to their somewhat higher water vapor permeability compared to conventional plastics. The fruit weight loss in biodegradable packaging was higher than in polypropylene, but 3-4-fold lower than in open containers. The control of senescence spotting was the major advantage of microperforated biodegradable packaging, combined with the preservation of acceptable fruit firmness and flavor, and low crown rot incidence. Optimal biodegradable packages extended the shelf life of bananas by four days compared with open containers, and by two days compared with the best commercial plastic package tested. Microperforated biodegradable packages combined the advantage of improved sustainability with superior fruit preservation.

Phenotypic Characterization of Postharvest Fruit Qualities in Astringent and Non-astringent Persimmon (Diospyros kaki) Cultivars.

Phenotypic characterization of postharvest traits is essential for the breeding of high-quality fruits. To compare postharvest traits of different genetic lines, it is essential to use a reference point during fruit development that will be common to all the lines. In this study, we employed a non-destructive parameter of chlorophyll levels to establish a similar physiological age and compared several postharvest traits of ten astringent and seven non-astringent persimmon cultivars. The fruit's traits examined were astringency, weight, total soluble solids (TSS), titratable acidity (TA), chlorophyll levels (I AD ), color (hue), firmness, color development and firmness loss during storage, crack development, and susceptibility to Alternaria infection. Although the chlorophyll (I AD ) index and color (hue) showed a high correlation among mature fruits of all cultivars, the chlorophyll parameter could detect higher variability in each cultivar, suggesting that I AD is a more rigorous parameter for detecting the developmental stage. The average weight, TSS, and TA were similar between astringent and non-astringent cultivars. Cracks appeared only on a few cultivars at harvest. Resistance to Alternaria infection and firmness were lower in astringent than in non-astringent cultivars. Only the astringent cultivar "32" was resistant to infection possibly due to the existence of an efficient peel barrier. It was concluded that a high correlation existed between astringency, susceptibility to Alternaria infection, and firmness. Cracks did not correlate with astringency or firmness. The phenotypic traits evaluated in this work can be used in future breeding programs for elite persimmon fruits.

Distinctive in-planta acclimation responses to basal growth and acute heat stress were induced in Arabidopsis by cattle manure biochar.

In-planta mechanisms of biochar (BC)-mediated improved growth were evaluated by examining oxidative stress, metabolic, and hormonal changes of Arabidopsis wild-type plants under basal or acute heat stress (-HS/ + HS) conditions with or without BC (+ BC/-BC). The oxidative stress was evaluated by using Arabidopsis expressing redox-sensitive green fluorescent protein in the plastids (pla-roGFP2). Fresh biomass and inflorescence height were greater in + BC(‒HS) plants than in the -BC(‒HS) plants, despite similar leaf nutrient levels, photosystem II (PSII) maximal efficiencies and similar oxidative poise. Endogenous levels of jasmonic and abscisic acids were higher in the + BC(‒HS) treatment, suggesting their role in growth improvement. HS in ‒BC plants caused reductions in inflorescence height and PSII maximum quantum yield, as well as significant oxidative stress symptoms manifested by increased lipid peroxidation, greater chloroplast redox poise (oxidized form of roGFP), increased expression of DNAJ heat shock proteins and Zn-finger genes, and reduced expression of glutathione-S-transferase gene in addition to higher abscisic acid and salicylic acid levels. Oxidative stress symptoms were significantly reduced by BC. Results suggest that growth improvements by BC occurring under basal and HS conditions are induced by acclimation mechanisms to 'microstresses' associated with basal growth and to oxidative stress of HS, respectively.

Phenylpropanoid Metabolism in Astringent and Nonastringent Persimmon (Diospyros kaki) Cultivars Determines Sensitivity to Alternaria Infection.

Fruits of nonastringent persimmon cultivars, as compared to astringent ones, were more resistant to Alternaria infection despite having lower polyphenol content. Metabolic analysis from the pulp of nonastringent "Shinshu", as compared to the astringent "Triumph", revealed a higher concentration of salicylic, coumaric, quinic, 5-o-feruloyl quinic, ferulic acids, ß-glucogallin, gallocatechin, catechin, and procyanidins. Selected compounds like salicylic, ferulic, and ρ-coumaric acids inhibited in vitro Alternaria growth, and higher activity was demonstrated for methyl ferulic and methyl ρ-coumaric acids. These compounds also reduced in vivo Alternaria growth and the black spot disease in stored fruits. On the other hand, methyl gallic acid was a predominant compound in the "Triumph" pulp, as compared to the "Shinshu" pulp, and it augmented Alternaria growth in vitro and in vivo. Our results might explain the high sensitivity of the cultivar "Triumph" to Alternaria. It also emphasizes that specific phenolic compounds, and not the total phenol, affect susceptibility to fungal infection.

Arabidopsis cysteine-rich trans-membrane module (CYSTM) small proteins play a protective role mainly against heat and UV stresses.

The genomes of Arabidopsis and other plants contain cysteine-rich small protein of unknown function, harbouring a transmembrane module (CYSTM proteins). In this work we show that the transcript of one gene (At1g05340) encoding a CYSTM protein is induced mainly by heat and to a lesser extent by UV, but less by NaCl or sorbitol. A functional analysis of At1g05340 and its paralog At2g32210 using T-DNA insertional mutants revealed a decrease in seedlings root length, and a lower PSII efficiency in mature plant, due to heat stress and to a lesser extent due to UV stress, in comparison to the effect on wild-type plants. The sensitivity of these mutants to salt or osmotic stresses did not differ from wild type response, indicating a specific function for these genes in heat and UV. Heat and UV increased reactive oxygen species levels in wild type; however, the levels were higher in the mutant line than in wild type due to heat treatment, but was similar in the mutant lines and wild type due to UV stress. Taken together, our results suggest that these small cysteine-rich proteins are necessary for thermotolerance and protection from UV exposure. The proteins encoded by these genes most likely, act in heat stress by reducing reactive oxygen species level by yet unknown mechanism.

MaMADS2 repression in banana fruits modifies hormone synthesis and signalling pathways prior to climacteric stage.

BACKGROUND: While the role of ethylene in fruit ripening has been widely studied, the contributions of additional plant hormones are less clear. Here we examined the interactions between the transcription factor MaMADS2-box which plays a major role in banana fruit ripening and hormonal regulation. Specifically, we used MaMADS2 repressed lines in transcriptome and hormonal analyses throughout ripening and assessed hormone and gene expression perturbations as compared to wild-type (WT) control fruit. RESULTS: Our analyses revealed major differences in hormones levels and in expression of hormone synthesis and signaling genes mediated by MaMADS2 especially in preclimacteric pulp. Genes encoding ethylene biosynthesis enzymes had lower expression in the pulp of the repressed lines, consistent with reduced ethylene production. Generally, the expression of other hormone (auxin, gibberellins, abscisic acid, jasmonic acid and salicylic acid) response pathway genes were down regulated in the WT pulp prior to ripening, but remained high in MaMADS2 repressed lines. Hormone levels of abscisic acid were also higher, however, active gibberellin levels were lower and auxin levels were similar with MaMADS2 repression as compared to WT. Although abscisic level was higher in MaMADS2 repression, exogenous abscisic acid shortened the time to ethylene production and increased MaMADS2 mRNA accumulation in WT. Exogenous ethylene did not influence abscisic acid level. CRE - a cytokinin receptor, increased its expression during maturation in WT and was lower especially at prebreaker in the repressed line and zeatin level was lower at mature green of the repressed line in comparison to WT. CONCLUSIONS: In addition to previously reported effects of MaMADS2 on ethylene, this transcription factor also influences other plant hormones, particularly at the pre-climacteric stage. The cytokinin pathway may play a previously unanticipated role via MaMADS2 in banana ripening. Finally, abscisic acid enhances MaMADS2 expression to promote ripening, but the transcription factor in turn auto inhibits ABA synthesis and signaling. Together, these results demonstrate a complex interaction of plant hormones and banana fruit ripening mediated by MaMADS2.

Corrigendum: Different Preclimacteric Events in Apple Cultivars with Modified Ripening Physiology.

[This corrects the article on p. 1502 in vol. 8, PMID: 28928755.].

Different Preclimacteric Events in Apple Cultivars with Modified Ripening Physiology.

"Anna" is an early season apple cultivar exhibiting a fast softening and juiciness loss during storage, in comparison to two mid-late season cultivars "Galaxy" and "GD." The poor storage capacity of "Anna" was correlated with high lipid oxidation-related autoluminescence, high respiration and ethylene production rates, associated with high expression of MdACO1, 2, 4, 7, and MdACS1. All cultivars at harvest responded to exogenous ethylene by enhancing ethylene production, typical of system-II. The contribution of pre-climacteric events to the poor storage capacity of "Anna" was examined by comparing respiration and ethylene production rates, response to exogenous ethylene, expression of genes responsible for ethylene biosynthesis and response, and developmental regulators in the three cultivars throughout fruit development. In contrast to the "Galaxy" and "GD," "Anna" showed higher ethylene production and respiration rates during fruit development, and exhibited auto-stimulatory (system II-like) effect in response to exogenous ethylene. The higher ethylene production rate in "Anna" was correlated with higher expression of ethylene biosynthesis genes, MdACS3a MdACO2, 4, and 7 during early fruit development. The expression of negative regulators of ripening (AP2/ERF) and ethylene response pathway, (MdETR1,2 and MdCTR1) was lower in "Anna" in comparison to the other two cultivars throughout development and ripening. Similar pattern of gene expression was found for SQUAMOSA promoter binding protein (SBP)-box genes, including MdCNR and for MdFUL. Taken together, this study provides new understanding on pre-climacteric events in "Anna" that might affect its ripening behavior and physiology following storage.

Banana MaMADS Transcription Factors Are Necessary for Fruit Ripening and Molecular Tools to Promote Shelf-Life and Food Security.

Genetic solutions to postharvest crop loss can reduce cost and energy inputs while increasing food security, especially for banana (Musa acuminata), which is a significant component of worldwide food commerce. We have functionally characterized two banana E class (SEPALLATA3 [SEP3]) MADS box genes, MaMADS1 and MaMADS2, homologous to the tomato (Solanum lycopersicum) RIN-MADS ripening gene. Transgenic banana plants repressing either gene (via antisense or RNA interference [RNAi]) were created and exhibited specific ripening delay and extended shelf-life phenotypes, including delayed color development and softening. The delay in fruit ripening is associated with a delay in climacteric respiration and reduced synthesis of the ripening hormone ethylene; in the most severe repressed lines, no ethylene was produced and ripening was most delayed. Unlike tomato rin mutants, banana fruits of all transgenic repression lines responded to exogenous ethylene by ripening normally, likely due to incomplete transgene repression and/or compensation by other MADS box genes. Our results show that, although MADS box ripening gene necessity is conserved across diverse taxa (monocots to dicots), unlike tomato, banana ripening requires at least two necessary members of the SEPALLATA MADS box gene group, and either can serve as a target for ripening control. The utility of such genes as tools for ripening control is especially relevant in important parthenocarpic crops such as the vegetatively propagated and widely consumed Cavendish banana, where breeding options for trait improvement are severely limited.

Flowering shoots of ornamental crops as a model to study cellular and molecular aspects of plant gravitropism.

Flowering shoots offer a very convenient and excellent model system for in-depth study of shoot gravitropism in regular stems rather than in special aboveground organs, showing how plants cope with the force of gravity on Earth and change in orientation. Regarding the emerging notion that roots and shoots execute their gravitropic bending by different mechanisms, the use of flowering shoots offers additional confirmation for the suggested shoot-sensing mechanisms initially found in Arabidopsis. As a part of confirming this mechanism, studying this unique model system also enabled elucidation of the sequence of events operating in gravity signalling in shoots. Hence, using the system of flowering shoots provided an additional dimension to our understanding of shoot gravitropism and its hormonal regulation, which has been less advanced than root gravitropism. This is particularly important since the term "shoots" includes various aboveground organs. Hence, unlike other aboveground organs such as pulvini, the asymmetric growth in response to change in shoot orientation is accompanied in cut ornamental spikes by a continuous growth process. This chapter provides an overview of the basic methods, specifically developed or adapted from other graviresponding systems, for determining the main components which play a key role in gravistimulation signalling in flowering shoots.

Pre-symptomatic transcriptome changes during cold storage of chilling sensitive and resistant peach cultivars to elucidate chilling injury mechanisms.

BACKGROUND: Cold storage induces chilling injury (CI) disorders in peach fruit (woolliness/mealiness, flesh browning and reddening/bleeding) manifested when ripened at shelf life. To gain insight into the mechanisms underlying CI, we analyzed the transcriptome of 'Oded' (high tolerant) and 'Hermoza' (relatively tolerant to woolliness, but sensitive to browning and bleeding) peach cultivars at pre-symptomatic stages. The expression profiles were compared and validated with two previously analyzed pools (high and low sensitive to woolliness) from the Pop-DG population. The four fruit types cover a wide range of sensitivity to CI. The four fruit types were also investigated with the ROSMETER that provides information on the specificity of the transcriptomic response to oxidative stress. RESULTS: We identified quantitative differences in a subset of core cold responsive genes that correlated with sensitivity or tolerance to CI at harvest and during cold storage, and also subsets of genes correlating specifically with high sensitivity to woolliness and browning. Functional analysis indicated that elevated levels, at harvest and during cold storage, of genes related to antioxidant systems and the biosynthesis of metabolites with antioxidant activity correlates with tolerance. Consistent with these results, ROSMETER analysis revealed oxidative stress in 'Hermoza' and the progeny pools, but not in the cold resistant 'Oded'. By contrast, cold storage induced, in sensitivity to woolliness dependant manner, a gene expression program involving the biosynthesis of secondary cell wall and pectins. Furthermore, our results indicated that while ethylene is related to CI tolerance, differential auxin subcellular accumulation and signaling may play a role in determining chilling sensitivity/tolerance. In addition, sugar partitioning and demand during cold storage may also play a role in the tolerance/sensitive mechanism. The analysis also indicates that vesicle trafficking, membrane dynamics and cytoskeleton organization could have a role in the tolerance/sensitive mechanism. In the case of browning, our results suggest that elevated acetaldehyde related genes together with the core cold responses may increase sensitivity to browning in shelf life. CONCLUSIONS: Our data suggest that in sensitive fruit a cold response program is activated and regulated by auxin distribution and ethylene and these hormones have a role in sensitivity to CI even before fruit are cold stored.

Kinetics of retrograde signalling initiation in the high light response of Arabidopsis thaliana.

High light acclimation depends on retrograde control of nuclear gene expression. Retrograde regulation uses multiple signalling pathways and thus exploits signal patterns. To maximally challenge the acclimation system, Arabidopsis thaliana plants were either adapted to 8 (low light (L-light)) or 80 µmol quanta m(-2) s(-1) (normal light (N-light)) and subsequently exposed to a 100- and 10-fold light intensity increase, respectively, to high light (H-light, 800 µmol quanta m(-2) s(-1)), for up to 6 h. Both L → H- and N → H-light plants efficiently regulated CO2 assimilation to a constant level without apparent damage and inhibition. This experimental set-up was scrutinized for time-dependent regulation and efficiency of adjustment. Transcriptome profiles revealed that N-light and L-light plants differentially accumulated 2119 transcripts. After 6 h in H-light, only 205 remained differently regulated between the L → H- and N → H-light plants, indicating efficient regulation allowing the plants to reach a similar transcriptome state. Time-dependent analysis of transcripts as markers for signalling pathways, and of metabolites and hormones as possibly involved transmitters, suggests that oxylipins such as oxophytodienoic acid and jasmonic acid, metabolites and redox cues predominantly control the acclimation response, whereas abscisic acid, salicylic acid and auxins play an insignificant or minor role.

ROSMETER: a bioinformatic tool for the identification of transcriptomic imprints related to reactive oxygen species type and origin provides new insights into stress responses.

The chemical identity of the reactive oxygen species (ROS) and its subcellular origin will leave a specific imprint on the transcriptome response. In order to facilitate the appreciation of ROS signaling, we developed a tool that is tuned to qualify this imprint. Transcriptome data from experiments in Arabidopsis (Arabidopsis thaliana) for which the ROS type and organelle origin are known were compiled into indices and made accessible by a Web-based interface called ROSMETER. The ROSMETER algorithm uses a vector-based algorithm to portray the ROS signature for a given transcriptome. The ROSMETER platform was applied to identify the ROS signatures profiles in transcriptomes of senescing plants and of those exposed to abiotic and biotic stresses. An unexpected highly significant ROS transcriptome signature of mitochondrial stress was detected during the early presymptomatic stages of leaf senescence, which was accompanied by the specific oxidation of mitochondria-targeted redox-sensitive green fluorescent protein probe. The ROSMETER analysis of diverse stresses revealed both commonalties and prominent differences between various abiotic stress conditions, such as salt, cold, ultraviolet light, drought, heat, and pathogens. Interestingly, early responses to the various abiotic stresses clustered together, independent of later responses, and exhibited negative correlations to several ROS indices. In general, the ROS transcriptome signature of abiotic stresses showed limited correlation to a few indices, while biotic stresses showed broad correlation with multiple indices. The ROSMETER platform can assist in formulating hypotheses to delineate the role of ROS in plant acclimation to environmental stress conditions and to elucidate the molecular mechanisms of the oxidative stress response in plants.

Effect of seed on ripening control components during avocado fruit development.

Seedless avocado fruit are produced alongside seeded fruit in the cultivar Arad, and both reach maturity at the same time. Using this system, it was possible to show that avocado seed inhibits the ripening process: seedless fruits exhibited higher response to exogenous ethylene already at the fruitlet stage, and also at the immature and mature fruit stages. They produced higher CO2 levels, and the ethylene peak was apparent at the fruitlet stage of seedless fruit, but not of seeded ones. The expression levels of PaETR, PaERS1 and PaCTR1 on the day of harvest at all developmental stages were very similar between seeded and seedless fruit, except that PaCTR1 was higher in seedless fruit only at very early stages. This expression pattern suggests that the seed does not have an effect on components of the ethylene response pathway when fruits are just picked. The expression of MADS-box genes, PaAG1 and PaAGL9, preceded the increase in ethylene production of mature seeded fruit, but not at earlier stages. However, only PaAGL9 was induced in seedless fruit at early stages of development. Taken together, these data suggest that these genes are perhaps involved in climacteric response in seeded fruit, and the seed is responsible for their induction at normal fruit ripening.

Actomyosin mediates gravisensing and early transduction events in reoriented cut snapdragon spikes.

We investigated the involvement of the actomyosin network in the early events of the gravitropic response of cut snapdragon (Antirrhinum majus L.) spikes. The effects of the actin-modulating drug, cytochalasin D (CD) and/or the myosin inhibitor, 2,3-butanedione-2-monoxime (BDM) on amyloplast displacement, lateral auxin transport and consequently on stem bending were examined. The inhibitory effect on cytoskeleton integrity was studied by using indirect immunofluorescence double-labeling of actin and myosin. Our results demonstrate that no organizational changes in actin filaments occurred in cortical and endodermal cells of the stem bending zone during reorientation. These results suggest that actin depolymerization is not required for amyloplast sedimentation. Unlike the chloroplasts in the cortex, the amyloplasts in the endodermis were surrounded by actin and myosin, indicating that amyloplasts may be attached to the actin filaments via the motor protein, myosin. This suggests the involvement of myosin as part of the actomyosin complex in amyloplast movement in vertical as well as in reoriented stems. This suggestion was supported by the findings showing that: (a) BDM or CD disrupted the normal organization of actin either by altering characteristic distribution patterns of myosin-like protein in the cortex (BDM), or by causing actin fragmentation (CD); (b) both compounds inhibited the gravity-induced amyloplast displacement in the endodermis. Additionally, these compounds also inhibited lateral auxin transport across the stem and stem gravitropic bending. Our study suggests that during stem reorientation amyloplasts possibly remain attached to the actin filaments, using myosin as a motor protein. Thus, gravisensing and early transduction events in the gravitropic response of snapdragon spikes, manifested by amyloplast displacement and lateral auxin transport, are mediated by the actomyosin complex.

Organelles contribute differentially to reactive oxygen species-related events during extended darkness.

Treatment of Arabidopsis (Arabidopsis thaliana) leaves by extended darkness generates a genetically activated senescence program that culminates in cell death. The transcriptome of leaves subjected to extended darkness was found to contain a variety of reactive oxygen species (ROS)-specific signatures. The levels of transcripts constituting the transcriptome footprints of chloroplasts and cytoplasm ROS stresses decreased in leaves, as early as the second day of darkness. In contrast, an increase was detected in transcripts associated with mitochondrial and peroxisomal ROS stresses. The sequential changes in the redox state of the organelles during darkness were examined by redox-sensitive green fluorescent protein probes (roGFP) that were targeted to specific organelles. In plastids, roGFP showed a decreased level of oxidation as early as the first day of darkness, followed by a gradual increase to starting levels. However, in mitochondria, the level of oxidation of roGFP rapidly increased as early as the first day of darkness, followed by an increase in the peroxisomal level of oxidation of roGFP on the second day. No changes in the probe oxidation were observed in the cytoplasm until the third day. The increase in mitochondrial roGFP degree of oxidation was abolished by sucrose treatment, implying that oxidation is caused by energy deprivation. The dynamic redox state visualized by roGFP probes and the analysis of microarray results are consistent with a scenario in which ROS stresses emanating from the mitochondria and peroxisomes occur early during darkness at a presymptomatic stage and jointly contribute to the senescence program.

The regulation of MADS-box gene expression during ripening of banana and their regulatory interaction with ethylene.

Six MaMADS-box genes have been cloned from the banana fruit cultivar Grand Nain. The similarity of these genes to tomato LeRIN is low and neither MaMADS2 nor MaMADS1 complement the tomato rin mutation. Nevertheless, the expression patterns, specifically in fruit and the induction during ripening and in response to ethylene and 1-MCP, suggest that some of these genes may participate in ripening. MaMADS1, 2, and 3, are highly expressed in fruit only, while the others are expressed in fruit as well as in other organs. Moreover, the suites of MaMADS-box genes and their temporal expression differ in peel and pulp during ripening. In the pulp, the increase in MaMADS2, 3, 4, and 5 expression preceded an increase in ethylene production, but coincides with the CO(2) peak. However, MaMADS1 expression in pulp coincided with ethylene production, but a massive increase in its expression occurred late during ripening, together with a second wave in the expression of MaMADS2, 3, and 4. In the peel, on the other hand, an increase in expression of MaMADS1, 3, and to a lesser degree also of MaMADS4 and 2 coincided with an increase in ethylene production. Except MaMADS3, which was induced by ethylene in pulp and peel, only MaMADS4, and 5 in pulp and MaMADS1 in peel were induced by ethylene. 1-MCP applied at the onset of the increase in ethylene production, increased the levels of MaMADS4 and MaMADS1 in pulp, while it decreased MaMADS1, 3, 4, and 5 in peel, suggesting that MaMADS4 and MaMADS1 are negatively controlled by ethylene at the onset of ethylene production only in pulp. Only MaMADS2 is neither induced by ethylene nor by 1-MCP, and it is expressed mainly in pulp. Our results suggest that two independent ripening programs are employed in pulp and peel which involve the activation of mainly MaMADS2, 4, and 5 and later on also MaMADS1 in pulp, and mainly MaMADS1, and 3 in peel. Hence, our results are consistent with MaMADS2, a SEP3 homologue, acting in the pulp upstream of the increase in ethylene production similarly to LeMADS-RIN.

A fluorometer-based method for monitoring oxidation of redox-sensitive GFP (roGFP) during development and extended dark stress.

Redox-sensitive GFP (roGFP) localized to different compartments has been shown to be suitable for determination of redox potentials in plants via imaging. Long-term measurements bring out the need for analyzing a large number of samples which are averaged over a large population of cells. Because this goal is too tedious to be achieved by confocal imaging, we have examined the possibility of using a fluorometer to monitor changes in roGFP localized to different subcellular compartments during development and dark-induced senescence. The degree of oxidations determined by a fluorometer for different probes was similar to values obtained by confocal image analysis. Comparison of young and old leaves indicated that in younger cells higher levels of H(2)O(2) were required to achieve full roGFP oxidation, a parameter which is necessary for calculation of the degree of oxidation of the probe and the actual redox potential. Therefore, it is necessary to carefully determine the H(2)O(2) concentration required to achieve full oxidation of the probe. In addition, there is an increase in autofluorescence during development and extended dark stress, which might interfere with the ability to detect changes in oxidation-reduction dependent fluorescence of roGFP. Nevertheless, it was possible to determine the full dynamic range between the oxidized and the reduced forms of the different probes in the various organelles until the third day of darkness and during plant development, thereby enabling further analysis of probe oxidation. Hence, fluorometer measurements of roGFP can be used for extended measurements enabling the processing of multiple samples. It is envisaged that this technology may be applicable to the analysis of redox changes in response to other stresses or to various mutants.

Induction of ethylene in avocado fruit in response to chilling stress on tree.

Chilling of avocado fruit (Persea americana cv. Arad) in the orchard caused a dramatic induction of fruit ripening and a parallel increase in ethylene biosynthesis and receptor genes' expression during shelf life. In-orchard chilling stress stimulated ethylene and CO(2) production already in fruit attached to the tree, and these reduced thereafter during 20 degrees C storage. In non-chilled control fruit, ethylene and CO(2) production started after 3d at 20 degrees C and exhibited a climacteric peak. In-orchard chilling stress also led to membrane destruction expressed as higher electrical conductivity (EC) in chilling stressed (CS) fruit and accelerated softening compared with control fruit. The increase in ethylene production on the day of harvest in CS fruit was accompanied by high expression of two 1-aminocyclopropane-1-carboxylic aCSd (ACC) synthase genes: PaACS1 and PaACS2, and ACC oxidase PaACO. The initial gene expressions of PaACS1, PaACS2, and PaACO in the CS fruit at the day of harvest was similar to the levels reached by the control fruit after 4d at 20 degrees C. The expression levels of both PaETR and PaERS1 in CS fruit on tree were 25 times higher than the control. In control fruit, expression of ethylene receptor genes was very low at harvest and increased in parallel to the onset of the climacteric ethylene peak. PaCTR1 transcript levels were less affected by chilling stress, and small changes (less than 3-fold) were observed in CS fruit on the day of harvest. Together, our results suggest that ethylene biosynthesis and ethylene response-pathway genes are involved in regulation of ethylene responsiveness in response to in-orchard chilling stress and during ripening.

The role of the embryo and ethylene in avocado fruit mesocarp discoloration.

Chilling injury (CI) symptoms in avocado (Persea americana Mill.) fruit, expressed as mesocarp discoloration, were found to be associated with embryo growth and ethylene production during cold storage. In cvs Ettinger and Arad most mesocarp discoloration was located close to the base of the seed and was induced by ethylene treatment in seeded avocado fruit. However, ethylene did not increase mesocarp discoloration in seedless fruit stored at 5 degrees C. Application of ethylene to whole fruit induced embryo development inside the seed. It also induced seedling elongation when seeds were imbibed separately. Persea americana ethylene receptor (PaETR) gene expression and polyphenol oxidase activity were highest close to the base of the seed and decreased gradually toward the blossom end. By contrast, expressions of PaETR transcript and polyphenol oxidase activity in seedless avocado fruit were similar throughout the pulp at the base of the fruit. Application of the ethylene inhibitor, 1-methylcyclopropene, decreased mesocarp browning, embryo development, seedling growth, and ion leakage, and down-regulated polyphenol oxidase activity. The results demonstrate that ethylene-mediated embryo growth in whole fruit is involved in the mesocarp response to ethylene perception and the development of CI disorders.