The use of auxins to improve the vase life of cut flowers is very limited. Previous studies demonstrated that a pulse treatment of Red Cestrum (Cestrum elegansSchlecht.) cut flowers with 2,4-dichlorophenoxyacetic acid (2,4-D) significantly reduced floret bud abscission, whereas 1-naphthaleneacetic acid (NAA) was ineffective. This difference resulted, at least in part, from the higher acropetal transport capability of 2,4-D compared to that of NAA. The present research focused on examining the factors affecting the acropetal transport, and hence the efficacy of the two auxins in reducing floret bud abscission of Red Cestrum cut flowers. We assumed that the differential acropetal transport capability of the two auxins results from the difference in their dissociation constants (pKa), with values of 2.75 and 4.23 for 2,4-D and NAA, respectively, which affects their pH-dependent physicochemical properties. Thus, increasing the pH of the pulsing solution above the pKa of both auxins might improve their acropetal movement. Indeed, the results of the present research show that raising the pH of the pulsing solution to pH 7.0 and above improved the efficacy of the two auxins in reducing floret bud abscission, with a higher effect on 2,4-D than that on NAA. Raising the pH of the pulsing solution decreased the adsorption and/or uptake of the two auxins by the cells adjacent to the xylem vessels, leading to an increase in their acropetal transport. The high pH of the pulsing solution increased the dissociation and hence decreased the lipophilicity of the auxin molecules, leading to improved acropetal movement. This effect was corroborated by the significant reduction in their 1-octanol/water partition coefficient (K-OW) values with the increase in the pH. A significant increase in theCeIAA1transcript level was obtained in response to 2,4-D pulsing at pH 7.0 and 8.25 and to NAA pulsing at pH 8.25, indicating that the acropetally transported auxins were taken up by the cells under these conditions. Our data suggest that raising the pH of the pulsing solution would significantly contribute to the increased efficacy of auxins in improving the vase life of cut flowers.

The present report describes the effect of 3-cyclopropyl-1-enyl-propanoic acid sodium salt (CPAS—WO 2010/0822930), a novel water soluble ethylene antagonist, on senescence and grain yield of several cultivars of common wheat (Triticum aestivum L.) and durum wheat (T. durum Desf.). CPAS abolished ethylene-induced leaf and ear organ senescence, as evidenced by the inhibition of chlorophyll degradation. CPAS application to wheat plants grown under controlled (120 mg L−1) or field conditions (110 mg L−1) significantly increased grain yield in the range of 9–13 % and 3–45 %, respectively, depending on treatment and variety. The CPAS-induced increase in grain yield was usually more significant under extreme weather conditions. In some cases spraying once at the medium milk stage was sufficient to obtain the maximum increase in grain yield, while in other cases spraying twice at the medium and late milk stages yielded the highest increase. The above data support the common view, that there is a positive causal relationship between the delay of leaf and ear organ senescence and grain yield. The results of the present study demonstrate that CPAS can effectively be used to increase wheat grain yield, and possibly the yield of other field crops, under field conditions.

The molecular responses of plants to anaerobic stress have been extensively studied in roots during and after flooding; far less has been done with ripening fruits, and very little is known about the behavior of citrus fruit during postharvest storage under restricted O-2 availability conditions. Using 'Star Ruby' grapefruit and 'Murcott' mandarin as plant materials and N-2 atmosphere treatment as the major experimental system, we studied the molecular responses of citrus fruits to anaerobiosis. The major response of citrus fruit to anaerobiosis is a metabolic shift towards ethanol fermentation, and the regulation of ethanol fermentation takes place at both transcriptional and posttranscriptional levels. Ethylene is not a major player in the responses to anaerobiosis. 'Murcott' mandarin is more sensitive than 'Star Ruby' grapefruit to anaerobiosis, peel (flavedo) and juice vesicles respond differently to anaerobiosis. Taken together, our data reveal both general and citrus fruit-specific anaerobic-response mechanisms.

A novel water soluble inhibitor of ethylene action, 3-cyclopropyl-1-enyl-propanoic acid sodium salt [(CPAS) Patent Application number: PCT/IL2008/000995, US Application number 61/144758, International publication number: WO 2009/010981 AI] was synthesized in a highly purified form, and its effect to retard various exogenous or endogenous ethylene-mediated processes was tested. The inhibitor was applied by loading, dipping or spraying. CPAS retarded some ripening processes in avocado, banana, and peach fruit, delayed abscission of citrus leaf explants, inhibited leaf epinasty in tomato seedlings, and prolonged the vase-life of carnation and petunia flowers. The fact that CPAS is a solid, water soluble, non-phytotoxic, and odorless inhibitor of ethylene action renders it a promising candidate for pre- and post-harvest application in a wide rang of open growing environments.

The potency of 12 new volatile cyclopropenes to inhibit ethylene action synthesized at the laboratory of Edward C. Sisler was assessed. The inhibitory effect was evaluated in the following test systems: ethylene-induced ripening of climacteric fruits, ethylene-induced growth modifications in etiolated pea seedlings and abscission of citrus leaf explants. In some of the test systems, the new compounds were found to be more potent ethylene antagonists than 1-MCP, whereas in other systems they were less potent. A novel water soluble inhibitor of ethylene action 3-(cycloprop-1-enylpropanoic acid, sodium salt (CPAS, IL patent application 184729, WO 2009/010981 A1)), was synthesized by D-Pharm, Israel, from one of the new cyclopropene derivatives. CPAS was found to counteract ethylene-induced abscission of citrus leaf explants and of avocado fruit peduncles, to inhibit ethylene-induced leaf epinasty in tomato seedlings, and to prolong the vase-life of carnation and petunia cut flowers. It also considerably delayed fruit ripening processes, such as skin colour change in 'Hass' avocado and banana fruit and softening of peach fruit. Banana and avocado fruit softening were delayed by CPAS to a lesser extent, probably due to insufficient penetration.

Respiratory responses of citrus fruit to controlled atmospheres (CA) are of great importance, since CA treatments can be used in postharvest handling only as long as they do not cause any detrimental effects on fruit quality. In this study, we evaluated the physiological responses of 'Star Ruby' grapefruit (Citrus paradisi Macf., 'Star Ruby') and 'Murcott' mandarin (Citrus reticulata Blanco, 'Murcott') to different concentrations of O 2 (5, 10, 15, and 21%) or CO 2 (0, 5, 10, and 20%) at 20°C for a short-term period (1 week). Exposure to reduced O 2 atmospheres greatly reduced respiration rates and internal CO 2 levels, but 5% O 2 dramatically increased juice ethanol, and to a lesser extent, acetaldehyde (AA) levels. Exposure to 5% and 10% O 2 reduced ethylene evolution rates in grapefruit but increased their levels in mandarin. Exposure to 10% and 20% CO 2 markedly increased respiration rates and internal CO 2 levels, but reduced ethylene production. Exposure to 20% CO 2 substantially increased juice ethanol and AA levels as compared with air controls, but to a less extent as compared with exposure to 5% O 2 atmosphere. Mandarin exhibited stronger and more rapid responses to reduced O 2 than grapefruit, as revealed by the accumulation of juice ethanol and AA, two important indicators of anaerobic respiration. Mandarin also showed earlier and higher increases in respiration rates and internal CO 2 levels than grapefruit following exposure to elevated CO 2 . Results indicate that reduced O 2 and elevated CO 2 atmospheres affect citrus fruit' respiratory characteristics differently. The possible mechanisms involved are briefly discussed.

Compounds that can block the ethylene receptor and be applied either as a gas or as a salt by spray or dip have been prepared and tested. Cyclopropenes with a methyl group in the 1-position, on which was attached a substituted amine, were allowed to evaporate in the presence of bananas that were treated with the gas. The minimum amount of a given compound required to inhibit chlorophyll degradation in the banana peel (an indicator of protective effect of the compound against ethylene action) that was subsequently exposed to ethylene, varied considerably depending on the compound, but N,N-dipropyl-(1-cyclopropenylmethyl)amine and N,N-di-(1-cyclopropenylmethyl)amine were the most effective. The degree of response to the ethylene inhibitory effect was similar for all of the compounds tested (32–34d). The amount of cyclopropene compound required for inhibiting ethylene action following a 24h exposure of bananas to the salt followed by a 15h exposure to ethylene was higher than that required by the gas form used under the same conditions for the same effect. However, time of exposure could be much longer than 24h with the salt than with the gas. The bananas treated with the salt do not need to be in an air-tight container, but could be used in open spaces. Only the banana peel appeared to be protected against ethylene during the 24h interval when the salt was used. The pulp ripened upon exposure to ethylene.

Exposure of citrus fruit to anaerobic conditions results in induction of anaerobic respiration and accumulation of the off flavor volatiles ethanol and acetaldehyde. In this study, we evaluated the effects of anaerobic stress (exposure to N2 atmospheres for 24h) on the proteome of mandarins and grapefruit. With two-dimension polyacrylamide gel electrophoresis (2D-PAGE), we detected more than 400 protein spots in the flavedo tissue and 300 spots in the juice vesicle tissue, that were reproducibly stained in mandarins and grapefruit. Exposure to the anaerobic treatment significantly affected the abundances of 33 different proteins by a factor of at least 1.5. Identification of the citrus anaerobic proteins (ANPs) by mass spectrometry (MS) and annotation according to the Munich Information Center for Protein Sequence (MIPS) revealed tissue- and cultivar-specific differences in the anaerobic response of citrus fruit. In the peel tissue, 64% of the detected ANPs were stress-related proteins involved in cell rescue, defense and virulence and only 6% in energy production, whereas 38% of the ANPs in juice vesicle tissue were involved in energy and 31% in either cell rescue and defense or in cell cycle and protein fate. Furthermore, exposure to N2 for 24h had only minor affects on protein abundance in grapefruit juice vesicle tissue (suppression of 5 proteins) but remarkably affected protein accumulation in mandarins, including induction of glycolytic enzymes, a 10-fold increase in the abundance of alcohol dehydrogenase (ADH), and induction of stress proteins, such as heat shock proteins and ascorbate peroxidase. Overall, the present study provides the first 2D-PAGE proteome analysis of fruit tissue responses to anaerobic stress, and the observed data reflect both common as well as citrus-specific anaerobic-response mechanisms.

A study was undertaken to assess the potency of 1-methylcyclopropene (1-MCP) analogues to block the ethylene receptor and thereby inhibit ethylene action. Eight structural analogues of 1-MCP with substitution in the 1-position and a side chain containing 2–10 carbons were synthesized and their potency to inhibit ethylene-induced plant processes was tested on climacteric fruit like avocado, and tomato, on ethylene-induced growth modification in etiolated pea seedlings and on abscission in citrus leaf explants. High concentrations of ethylene were used under conditions which hasten ethylene-induced processes. The results showed differences in the responses of the various tissues tested as related to the concentrations of the inhibitors. Some required much higher concentration to exert the same effect, while some, when applied at the same concentration, blocked the receptor for a longer period of time than the others. Fruits responded differently than other plant organs to the same inhibitor, indicating possible differences in characteristics and availability of the ethylene receptors in the various tissues. The potency of the inhibitors was greatly affected by their molecular structure and size. The highest potency of a given inhibitor was obtained when the treatment was applied before the onset of ethylene action. The relationship between ethylene and the inhibitors was found to be of an apparent non-competitive nature. All the fruits treated with the various inhibitors resumed normal ripening after recovery from the inhibition which is crucial when considering the putative inhibitors for practical use.

The potency to inhibit ethylene action of twelve new water-insoluble volatile cyclopropenes was assessed. This inhibitory effect was tested on the following test systems: ethylene-induced ripening of climacteric fruits, ethylene-induced growth modifications in etiolated pea seedlings and abscission of citrus leaf explants. In some of the test systems, the new putative inhibitors were found to be more potent ethylene antagonists than 1-MCP, whereas in other systems they were less potent. A novel water soluble, non-volatile inhibitor of ethylene action (WS-CPD, patent pending), was synthesized from one of the new cyclopropene derivatives. It was found to counteract ethylene-induced abscission of citrus leaf explants and peduncles of avocado fruits. It also inhibited ethylene-induced leaf epinasty in tomato seedlings, and prolonged the vase-life of carnation and petunia cut flowers. It considerably delayed fruit ripening processes, like skin color changes in ‘Hass’ avocado and banana fruit, as well as softening of peach fruit. WS-CPD delayed to a lesser extent banana and avocado fruit softening, probably due to insufficient penetration. INTRODUCTION Ethylene-induced accelerated ripening and senescence account for most of the preand postharvest losses of perishable agricultural commodities. Ethylene is a natural product of plant metabolism and plants respond to both endogenous and exogenous ethylene (Abeles et al., 1992). Antagonists of ethylene action are considered to be very beneficial for agricultural use, since they protect the tissues from both endogenous and exogenous ethylene. Ethylene antagonists inhibit the action of ethylene at the molecular level by blocking its receptor site (Sisler and Wood, 1988; Sisler. and Serek 1997). Thus, application of effective inhibitors of ethylene action may allow extending the harvest season of crops, and improving the keeping quality and prolonging the storability and shelf life of fruits, herbs, leafy vegetables, and flowers. Antagonists of ethylene action have been described in the past. Of the inhibitors of ethylene action developed so far, 1-methylcyclopropene (1-MCP) is considered to be the most promising antagonist for commercial use (Serek et al., 1995; Sisler et al., 2000). However, its practical use is limited, mainly due to the following reasons: (a) very low solubility in water; (b) it is a gas at temperatures above 0C and must be applied as such in sealed systems, (c) as a gas it cannot be used for dip loading of cut flowers or applied as a spray in the field (d) attempts to use 1-MCP as a spray revealed that very high concentrations are required to render it effective, which pose a threat to other plants and crops in the environment. In addition, being volatile, the compound dissipates rapidly depending on the wind velocity, and therefore its effectiveness is totally unpredictable. Consequently, it is obvious that a potent, water-soluble, broad-spectrum ethylene antagonist is required by the agriculture industry. MATERIALS AND METHODS The studies were conducted following the procedures described in Goldschmidt et al. (2007). The cyclopropenes tested are listed in Table 1. Table 1. List and structure of 1-MCP and twelve 1-substituted cyclopropene (cp) analogous newly synthesized and tested for potency as antagonists of ethylene action. RESULTS AND DISCUSSION In this communication we present results of two lines of research. The first stage was aimed to broaden the spectrum of volatile water-insoluble antagonists of ethylene action that can protect plants and fruits from ethylene for various lengths of time. The second stage of the research was aimed to develop a water soluble non-volatile ethylene antagonist. 3-Methylamine-1-cp-2-carboxylic acid N-(1-Methyl-cp)-ethyl-amine 3-(1-cp)-Propanoic acid-isopropyl amine salt 3-(1-cp)-Propanoic acid 1-(3-Hydroxypropyl)-cp 2 Structure Inhibitor No. Name -CH2N(CH2)3CH3 -COOH 3

Physiological and molecular evidence supported the autocatalytic ethylene production in young “Star Ruby” grapefruit and “Murcott” mandarins, and the autoinhibitory ethylene production in mature fruit. Ethylene upregulated expression of pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) in both fruitlets and mature “Star Ruby”, and increased acetaldehyde (AA) and ethanol production, while ethylene action inhibitors counteracted the effect. N2 not only changed the expression of genes for ethylene biosynthesis but also genes for ethylene perception in both young and mature fruits.

Molecular aspects of ethanol fermentation in citrus fruit were investigated in immature and mature ‘Star Ruby’ grapefruit ( Citrus paradisi Macf.) and ‘Murcott’ mandarin ( Citrus reticulata Blanco). Transcript levels of pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH), which play a central role in ethanol fermentation, were detectable in all stages of fruit development, but accumulation of acetaldehyde (AA) and ethanol was evident only as fruit approached maturation or after several weeks of storage. Treatment of mature fruit with ethylene enhanced ethanol fermentation in grapefruit but not in mandarin. Immature fruit of both cultivars, on the other hand, responded to ethylene by prominent, although transient, enhancement of ethanol fermentation. Exposure of mature or immature fruit to anaerobic conditions (N 2 atmosphere) upregulated the expression of PDC and ADH, and increased the levels of AA and ethanol. Exposure of mature fruit to anaerobic conditions also increased the enzymatic activities of PDC and ADH. The data indicate that the potential for ethanol fermentation exists in citrus fruit throughout development, even under aerobic conditions, but AA and ethanol are detected mainly toward maturation or under prolonged storage. However, prominent, long-term molecular induction of ethanol fermentation occurs only under anaerobic conditions imposed by N 2 atmosphere.

During postharvest storage or after exposure to anaerobic atmospheres, mandarins develop off-flavors much more rapidly than other citrus varieties, and the occurrence of these off-flavors is associated with increases in juice ethanol and acetaldehyde (AA) levels. However, the reasons why mandarins accumulate more off-flavor volatiles than other varieties are not yet understood. We studied the roles of various molecular, biochemical and anatomical factors in governing ethanolic fermentation in ‘Murcott’ mandarins and ‘Star Ruby’ grapefruit under aerobic and anaerobic conditions, and found that exposure to N2 atmospheres for 24h increased accumulation of ethanol and AA in the juice and peel of mandarins much more than in grapefruit. However, exposure to anaerobic atmospheres increased pyruvate kinase (PK), pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) transcript levels in both cultivars by similar amounts, suggesting that post-transcriptional and perhaps other regulatory mechanisms must be involved in governing ethanol fermentation rates. Exposure to anaerobic atmospheres increased PDC enzyme activity in both mandarins and grapefruit but had only minor effects on ADH activity. Nevertheless, mandarins had much higher (3.8-fold) levels of ADH enzyme activity in their juice vesicles than grapefruit. Anatomical observations revealed that although the total thickness of the peel (comprising the albedo, the white inner layer and the flavedo, the colored outer layer) was greater in grapefruit, the dense flavedo layer was considerably thicker in mandarins. In mandarins, the flavedo also contained more oil glands than that of grapefruit, and the albedo was thinner but more condensed. Accordingly, gas diffusion tests indicated that the peel of mandarins was less permeable to gases, and especially to ethanol vapors, than that of grapefruit. Overall, we conclude that mandarins accumulate larger amounts of AA and ethanol after harvest than grapefruit do because of higher ADH enzyme activity levels in the juice, and because their peel is less permeable to gases. The latter characteristic prevents the release of the produced off-flavor volatiles from mandarins, which results in buildup of ethanol and AA in the internal atmosphere of the fruit and the consequent perception of off-flavors.

BACKGROUND AND AIMS:A previous study showed that the relative effectiveness of 2,4-dichlorophenoxyacetic acid (2,4-D) compared with that of 1-naphthaleneacetic acid (NAA) in reducing floret bud abscission in cestrum (Cestrum elegans) cut flowers was due to its acropetal transport. The aim of the present study was to examine if the differential effect of these auxins on floret abscission is reflected in the expression of Aux/IAA genes in the floret abscission zone (AZ).METHODS:cDNAs were isolated by PCR-based cloning from the floret AZ of auxin-treated cut flowers. The expression patterns of the cDNAs in various tissues and the effect of indole-3-acetic acid (IAA), applied with or without cycloheximide, on their expression in the floret AZ were examined by northern blot analysis. The regulation of transcript accumulation in the floret AZ in response to NAA or 2,4-D was measured by real-time PCR during auxin pulsing of cut flowers and vase life, concomitantly with floret abscission.KEY RESULTS:Six isolated cDNAs were identified to represent Aux/IAA homologous genes, designated as Cestrum elegans (Ce)-IAA1 to Ce-IAA6. Four Ce-IAA genes were characterized as early auxin-responsive genes (ARGs), and two (Ce-IAA1 and Ce-IAA5) as late ARGs. Only Ce-IAA5 was AZ-specific in floret buds. A temporal regulation of Ce-IAA transcript levels in the floret AZ was found, with 2,4-D inducing higher expression levels than NAA in floret buds. These Ce-IAA expression levels were negatively correlated with floret abscission.CONCLUSIONS:The differential transport characteristics of NAA and 2,4-D in cestrum cut flowers were reflected in differential activation of the Ce-IAA genes identified in the floret AZ. Therefore, Aux/IAA genes can be used as molecular markers to measure auxin activity, which reflects free auxin level in the AZ. Two of the identified genes, Ce-IAA1 and Ce-IAA5, may also have a regulatory role in abscission.

ABSTRACT Floret abscission in Red Cestrum ( Cestrum elegans Schlecht) cut flower shoots was significantly delayed by a pulse treatment of 2,4-dichlorophenoxyacetic acid (2,4-D) while 1-naphthaleneacetic acid (NAA) was less effective. This phenomenon is attributed to the findings showing that significant amount of 2,4-D moved acropetally and accumulated in florets, leaves, and upper parts of the stem, while NAA remained in the lower parts of the stem. In addition, a significant amount of the accumulated 2,4-D remained in the active free form for a relatively longer period of time during vase life, while NAA was quickly metabolized. 2,4-D induced higher rates of ethylene evolution and increased expression levels of Aux/IAA homologous genes, cloned from the floret abscission zone, compared to those observed in response to NAA. This suggests that ethylene evolution and expression of Aux/IAA homologous genes may serve as markers for the activity of these two synthetic auxins. INTRODUCTION It is well accepted that both the natural auxin, indole-3-acetic acid (IAA), and the synthetic auxins exhibit polar transport (Lomax et al., 1995). This is also true for the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D), which has transport characteristics similar to those of IAA, except for a slower transport rate (McCready, 1963; McCready and Jacobs, 1963). When 2,4-D was combined with silver thiosulfate (STS) for pulsing ‘Red Cestrum’ (

Arabidopsis thaliana CEL1 protein was detected in young expanding tissues. Immunostaining revealed that CEL1 accumulated mostly in xylem cells. The primary, as well as the secondary xylem showed considerable CEL1 staining. CEL1 was also observed in young epidermal cells, in which the thicker lateral and tangential walls stained more intensely than the inner walls. In newly formed cell walls, the lateral tangential walls were labeled more intensively than the inner walls. Cellulase activity was found to be significantly higher in growing tissue compared to mature parts of the plant. Cel1 expression concurrently with cellulase activity could be restored in detached matured leaves by sucrose treatment after 48 h in the culture medium.

Red Cestrum (Cestrum elegans Schlecht.) cut flowers are considered as a new export crop from Israel. However, the cut flwers exhibit both bud and floret abscission during shipment. Pulsing cut flowers for 4 h at 20°C and 16 h at 4°C with 2,4-dichlorophenoxyacetie acid (2,4-D) combined with sliver thiosulfate (STS) significantly reduced abscission, while a similar treatment with 1-naphthaleneacetic acid (NAA) failed to do so. Contrary to its inhibitory effect on floret abscission, 2,4-D induced a higher level of ethylene evolution in the inflorescences compared to NAA. The data suggest that 2,4-D moved acropetally in a significant amount, sufficient to reduce floret abscission, while NAA did not. These unexpected results led us to study the mode of transport of 2,4-D and NAA in stem sections and cut flowers. In stem sections, transport of NAA was polar with a velocity of 5.9 mm per h. Although 2,4-D exhibited a polar transport, it was also transported non-polarly showing a fast transport during the first 15 minutes both in the acropetal and basipetal directions, followed by progressively increased basipetal polar transport and decreased acropetal transport. During the transport period of 3 to 11 h, 2,4-D moved at a rate of 1.9 mm per h in the basipetal direction. Studies with cut flowers also showed a significant acropetal transport and accumulation of 2,4-D in the upper parts of the stem, leaves and florets, while NAA mainly accumulated in the lower parts of the stem. In addition, NAA was metabolized faster in florets and leaves compared with 2,4-D. This suggests that like in other abscission systems, the level of free auxin in the tissues controls abscission of buds and florets in cut flowers.