Similarities between the Actions of Ethylene and Cyanide in Initiating the Climacteric and Ripening of Avocados

A continuous exposure of intact avocados (Persea americana) to 400 μl/l of cyanide results in a rapid increase in the rate of respiration, followed by a rise in ethylene production, and eventual ripening. The pattern of changes in the glycolytic intermediates glucose 6-phosphate, fructose diphosphate, 3-phosphoglyceric acid, and phosphoenolpyruvate during the rapid rise in respiration in both ethylene and cyanide-treated fruits is similar to that found in fruits made anaerobic where a 2.3- to 3-fold increase in the rate of glycolysis is observed. It is suggested that both during the climacteric and in response to cyanide, glycolysis is enhanced. It is proposed that cyanide implements the diversion of electrons to the cyanide-resistant electron path through structural alterations which are independent of the simultaneous inhibition of cytochrome oxidase.     

1-Phenyl-2-pentanone and methyl salicylate: new defense allomone components and their content shift during ontogenetic development of the millipede <Emphasis Type="Italic">Nedyopus tambanus mangaesinus</Emphasis> (Polydesmida: Paradoxosomatidae)

Twelve components were identified in hexane extracts of the polydesmid millipede Nedyopus tambanus mangaesinus (Attems, 1909) and their contents were examined at all stadia of ontogenetic development including two adult conditions (before and after hibernation). Two compounds, 1-phenyl-2-pentanone and methyl salicylate, were newly identified as components of the millipede together with six well-known polydesmoid compounds (benzaldehyde, benzyl alcohol, benzoic acid, benzoyl cyanide, mandelonitrile, and mandelonitrile benzoate) and four phenols (phenol, p-cresol, 2-methoxyphenol, and 2-methoxy-4-methylphenol). Benzaldehyde and benzoyl cyanide were distributed from nymphs at stadium I, and other components started to become detectable at more advanced stadia. The largest content (35.1%) of methyl salicylate was detected in nymphs at stadium I, together with benzaldehyde and benzoyl cyanide, implying the reinforcement of defensive functions during or after egg emergence. The content (18.8%) of 1-phenyl-2-pentanone reached a maximum in hibernated females among all stadia and conditions. Its female-biased distribution at the mating season (ca. 5.7-fold more than in males) implies its sexually related functions.     

Toxicity of Glucosinolates and Their Enzymatic Decomposition Products to Caenorhabditis elegans

An aquatic 24-hour lethality test using Caenorhabditis elegans was used to assess toxicity of glucosinolates and their enzymatic breakdown products. In the absence of the enzyme thioglucosidase (myrosinase), allyl glucosinolate (sinigrin) was found to be nontoxic at all concentrations tested, while a freeze-dried, dialyzed water extract of Crambe abyssinica containing 26% 2-hydroxyl 3-butenyl glucosinolate (epi-progoitrin) had a 50% lethal concentration (LC₅₀) of 18.5 g/liter. Addition of the enzyme increased the toxicity (LC₅₀ value) of sinigrin to 0.5 g/liter, but the enzyme had no effect on the toxicity of the C. abyssinica extract. Allyl isothiocyanate and allyl cyanide, two possible breakdown products of sinigrin, had an LC₅₀ value of 0.04 g/liter and approximately 3 g/liter, respectively. Liquid chromatographic studies showed that a portion of the sinigrin decomposed into allyl isothiocyanate. The results indicated that allyl isothiocyanate is nearly three orders of magnitude more toxic to C. elegans than the corresponding glncosinolate, suggesting isothiocyanate formation would improve nematode control from application of glucosinolates.     

拟南芥PMSR2对脂肪族芥子油苷侧链的修饰作用

芥子油苷是一类由氨基酸合成的次生代谢产物,脂肪族芥子油苷主要来源于甲硫氨酸,因侧链长度和结构的不同而拥有多样化的生物活性。根据拟南芥不同组织中芥子油苷组分和含量的特点及生物信息学分析,我们推断脂肪族芥子油苷的侧链修饰反应中可能存在由甲基亚磺酰基芥子油苷向甲硫基芥子油苷转化的还原反应,候选基因为甲硫氨酸硫还原酶2(Peptide Methionine Sulfoxide Reductase 2,PMSR2)。为了验证这一假设,我们构建了过量表达PMSR2基因的转基因拟南芥,对其芥子油苷组分及含量进行了测定,并与野生型和PMSR2基因缺失的突变体进行了对比分析,结果表明,PMSR2基因的过量表达并未使芥子油苷含量与组分发生明显变化,但PMSR2基因缺失的突变体与野生型相比,MS GSL/MT GSL的值显著提高,证明PMSR2参与了脂肪族芥子油苷侧链的修饰反应,可以将MS GSL中的硫还原生成MT GSL。该酶的鉴定进一步完善了对芥子油苷合成途径及其侧链修饰的认识,为深入研究脂肪族芥子油苷的生理功能奠定了理论基础。     

Respiratory Contribution of the Alternate Path during Various Stages of Ripening in Avocado and Banana Fruits

The respiration of fresh slices of preclimacteric avocado (Persea americana Mill. var. Hass) and banana (Musa cavendishii var. Valery) fruits is stimulated by cyanide and antimycin. The respiration is sensitive to m-chlorobenzhydroxamic acid in the presence of cyanide but much less so in the presence of antimycin. In the absence of cyanide the contribution of the cyanide-resistant pathway to the coupled preclimacteric respiration is zero. In uncoupled slices, by contrast, the alternate path is engaged and utilized fully in avocado, and extensively in banana. Midclimacteric and peak climacteric slices are also cyanide-resistant and, in the presence of cyanide, sensitive to m-chlorobenzhydroxamic acid. In the absence of uncoupler there is no contribution by the alternate path in either tissue. In uncoupled midclimacteric avocado slices the alternate path is fully engaged. Midclimacteric banana slices, however, do not respond to uncouplers, and the alternate path is not engaged. Avocado and banana slices at the climacteric peak neither respond to uncouplers nor utilize the alternate path in the presence or absence of uncoupler.

The maximal capacities of the cytochrome and alternate paths, V_cyt and V_alt, respectively, have been estimated in slices from preclimacteric and climacteric avocado fruit and found to remain unchanged. The total respiratory capacity in preclimacteric and climacteric slices exceeds the respiratory rise which attends fruit ripening. In banana V_alt decreases slightly with ripening.

The aging of thin preclimacteric avocado slices in moist air results in ripening with an accompanying climacteric rise. In this case the alternate path is fully engaged at the climacteric peak, and the respiration represents the total potential respiratory capacity present in preclimacteric tissue. The respiratory climacteric in intact avocado and banana fruits is cytochrome path-mediated, whereas the respiratory climacteric of ripened thin avocado slices comprises the alternate as well as the cytochrome path. The ripening of intact fruits is seemingly independent of the nature of the electron transport path.

Uncouplers are thought to stimulate glycolysis to the point where the glycolytic flux exceeds the oxidative capacity of the cytochrome path, with the result that the alternate path is engaged.

     

Malate Oxidation and Cyanide-Insensitive Respiration in Avocado Mitochondria during the Climacteric Cycle

After preparation on self-generated Percoll gradients, avocado (Persea americana Mill, var. Fuerte and Hass) mitochondria retain a high proportion of cyanide-insensitive respiration, especially with α-ketoglutarate and malate as substrates. Whereas α-ketoglutarate oxidation remains unchanged, the rate of malate oxidation increases as ripening advances through the climacteric. An enhancement of mitochondrial malic enzyme activity, measured by the accumulation of pyruvate, closely parallels the increase of malate oxidation. The capacity for cyanide-insensitive respiration is also considerably enhanced while respiratory control decreases (from 3.3 to 1.7), leading to high state 4 rates.

Both malate dehydrogenase and malic enzyme are functional in state 3, but malic enzyme appears to predominate before the addition of ADP and after its depletion. In the presence of cyanide, a membrane potential is generated when the alterntive pathway is operating. Cyanide-insensitive malate oxidation can be either coupled to the first phosphorylation site, sensitive to rotenone, or by-pass this site. In the absence of phosphate acceptor, malate oxidation is mainly carried out via malic enzyme and the alternative pathway. Experimental modification of the external mitochondrial environment in vitro (pH, NAD⁺, glutamade) results in changes in malate dehydrogenase and malic enzyme activities, which also modify cyanide resistance. It appears that a functional connection exists between malic enzyme and the alternative pathway via a rotenone-insensitive NADH dehydrogenase and that this pathway is responsible, in part, for nonphosphorylating respiratory activity during the climacteric.

     

Abscisic Acid levels and seed dormancy

Dormant seeds from Fraxinus species require cold-temperature after-ripening prior to germination. Earlier, we found that abscisic acid (ABA) will inhibit germination of excised nondormant embryos and that this can be reversed with a combination of gibberellic acid and kinetin. Using Milborrow's quantitative “racemate dilution” method the ABA concentration in 3 types of Fraxinus seed and pericarp were determined. While ABA was present in all tissues, the highest concentration was found in the seed and pericarp of dormant F. americana. During the chilling treatment of F. americana the ABA levels decreased 37% in the pericarp and 68% in the seed. The ABA concentration of the seed of the nondormant species, F. ornus, is as low as that found in F. americana seeds after cold treatment. Experiments with exogenously added ABA solutions indicate that it is unlikely that the ABA in the pericarp functions in the regulation of seed dormancy. However, the ABA in the seed does seem to have a regulatory role in germination.     

Interactions of the manganese hyperaccumulator <Emphasis Type="Italic">Phytolacca americana</Emphasis> L. with soil pH and phosphate

Hyperaccumulators are plants that store exceptionally high concentrations of heavy metals or metalloids in their leaves. Phytolacca americana is one of the few species known to hyperaccumulate manganese (Mn); however, it is a common weedy species and has no specific association with high-Mn soils. Neither the mechanism by which P. americana hyperaccumulates Mn nor the ecological significance of this trait are well understood. It has recently been suggested that P. americana secretes acids into the rhizosphere as a means of acquiring phosphate, which might coincidentally increase Mn uptake. To determine whether P. americana acidifies the surrounding soil, plants were grown in rhizoboxes providing access to living roots. A thin layer of agar containing bromocresol green pH indicator dye was placed on the roots to observe color changes indicating acidification. Comparative studies showed that P. americana acidifies the rhizosphere significantly more than the non-accumulating plant Acalypha rhomboidea. A second experiment studied whether adjustment of soil pH and phosphate affect foliar Mn concentrations of P. americana. Concentrations of Mn in leaves were highest when plants were grown in acidified soils but were significantly lower in soils that were alkaline and/or enriched with phosphate. These results suggest that Mn hyperaccumulation may be a side effect of rhizosphere acidification as a phosphorus-acquisition mechanism, rather than an adaptation in its own right. The findings provide fundamental information about hyperaccumulator physiology and evolution, and may be relevant to attempts to utilize P. americana for phytoremediation.     

Enhanced Benzaldehyde Tolerance in Zymomonas mobilis Biofilms and the Potential of Biofilm Applications in Fine-Chemical Production

Biotransformation plays an increasingly important role in the industrial production of fine chemicals due to its high product specificity and low energy requirement. One challenge in biotransformation is the toxicity of substrates and/or products to biocatalytic microorganisms and enzymes. Biofilms are known for their enhanced tolerance of hostile environments compared to planktonic free-living cells. Zymomonas mobilis was used in this study as a model organism to examine the potential of surface-associated biofilms for biotransformation of chemicals into value-added products. Z. mobilis formed a biofilm with a complex three-dimensional architecture comprised of microcolonies with an average thickness of 20 μm, interspersed with water channels. Microscopic analysis and metabolic activity studies revealed that Z. mobilis biofilm cells were more tolerant to the toxic substrate benzaldehyde than planktonic cells were. When exposed to 50 mM benzaldehyde for 1 h, biofilm cells exhibited an average of 45% residual metabolic activity, while planktonic cells were completely inactivated. Three hours of exposure to 30 mM benzaldehyde resulted in sixfold-higher residual metabolic activity in biofilm cells than in planktonic cells. Cells inactivated by benzaldehyde were evenly distributed throughout the biofilm, indicating that the resistance mechanism was different from mass transfer limitation. We also found that enhanced tolerance to benzaldehyde was not due to the conversion of benzaldehyde into less toxic compounds. In the presence of glucose, Z. mobilis biofilms in continuous cultures transformed 10 mM benzaldehyde into benzyl alcohol at a steady rate of 8.11 g (g dry weight)⁻¹ day⁻¹ with a 90% molar yield over a 45-h production period.     

Gene Duplication in the Diversification of Secondary Metabolism: Tandem 2-Oxoglutarate–Dependent Dioxygenases Control Glucosinolate Biosynthesis in Arabidopsis

Secondary metabolites are a diverse set of plant compounds believed to have numerous functions in plant–environment interactions. The large chemical diversity of secondary metabolites undoubtedly arises from an equally diverse set of enzymes responsible for their biosynthesis. However, little is known about the evolution of enzymes involved in secondary metabolism. We are studying the biosynthesis of glucosinolates, a large group of secondary metabolites, in Arabidopsis to investigate the evolution of enzymes involved in secondary metabolism. Arabidopsis contains natural variations in the presence of methylsulfinylalkyl, alkenyl, and hydroxyalkyl glucosinolates. In this article, we report the identification of genes encoding two 2-oxoglutarate–dependent dioxygenases that are responsible for this variation. These genes, AOP2 and AOP3, which map to the same position on chromosome IV, result from an apparent gene duplication and control the conversion of methylsulfinylalkyl glucosinolate to either the alkenyl or the hydroxyalkyl form. By heterologous expression in Escherichia and the correlation of gene expression patterns to the glucosinolate phenotype, we show that AOP2 catalyzes the conversion of methylsulfinylalkyl glucosinolates to alkenyl glucosinolates. Conversely, AOP3 directs the formation of hydroxyalkyl glucosinolates from methylsulfinylalkyl glucosinolates. No ecotype coexpressed both genes. Furthermore, the absence of functional AOP2 and AOP3 leads to the accumulation of the precursor methylsulfinylalkyl glucosinolates. A third member of this gene family, AOP1, is present in at least two forms and found in all ecotypes examined. However, its catalytic role is still uncertain.     

Physiological pattern and further characterization of the haemolymph violet carotenoprotein of the fly Rhynchosciara americana

• 1.1. An electrophoretic purification procedure for the haemolymph violet carotenoprotein of R. americana was described. The purified protein was used for obtaining a specific antiserum.
• 2.2. This carotenoprotein contains: (1) a high weight percentage of glutamic acid, threonine and proline and a low weight percentage of histidine; (2) mannose and/or glucose as suggested by the interaction with concanavalin A; (3) phosphoryl groups.
• 3.3. The concentration of the violet carotenoprotein in the haemolymph is approximately constant during all the life cycle of R. americana.
• 4.4. The haemolymph of four species of Rhynchosciara genus shows the presence of proteins immunologically related with the R. americana violet carotenoprotein.

     

Integration of Biosynthesis and Long-Distance Transport Establish Organ-Specific Glucosinolate Profiles in Vegetative Arabidopsis

Although it is essential for plant survival to synthesize and transport defense compounds, little is known about the coordination of these processes. Here, we investigate the above- and belowground source-sink relationship of the defense compounds glucosinolates in vegetative Arabidopsis thaliana. In vivo feeding experiments demonstrate that the glucosinolate transporters1 and 2 (GTR1 and GTR2), which are essential for accumulation of glucosinolates in seeds, are likely to also be involved in bidirectional distribution of glucosinolates between the roots and rosettes, indicating phloem and xylem as their transport pathways. Grafting of wild-type, biosynthetic, and transport mutants show that both the rosette and roots are able to synthesize aliphatic and indole glucosinolates. While rosettes constitute the major source and storage site for short-chained aliphatic glucosinolates, long-chained aliphatic glucosinolates are synthesized both in roots and rosettes with roots as the major storage site. Our grafting experiments thus indicate that in vegetative Arabidopsis, GTR1 and GTR2 are involved in bidirectional long-distance transport of aliphatic but not indole glucosinolates. Our data further suggest that the distinct rosette and root glucosinolate profiles in Arabidopsis are shaped by long-distance transport and spatially separated biosynthesis, suggesting that integration of these processes is critical for plant fitness in complex natural environments.     

Photosynthetic parameters of two invasive tree species of the Brazilian Pantanal in response to seasonal flooding

The Pantanal is the largest wetland in the world with extremely high plant and animal diversity, but large areas have been invaded by Vochysia divergens Pohl (Vochysiaceae), a tree that is native to the Amazon Basin, and Curatella americana L. (Dilleniaceae), a tree that is native to the Brazilian savanna (cerrado). V. divergens is reportedly floodadapted, thus its ability to invade the Pantanal may not be surprising, but the invasion of C. americana is counterintuitive, because this species is adapted to the well-drained soils of the cerrado. Thus, we were interested in comparing the photosynthetic capacity, in terms of CO₂ conductance, carboxylation, and electron transport of these species over a seasonal flooding cycle. Given that V. divergens is reportedly flood-adapted, we predicted that this species would have a higher photosynthetic capacity than C. americana, especially under flooding. To test this hypothesis we measured the photosynthetic CO₂ response (P _N/C _c) of V. divergens and C. americana within 1 year to determine, if photosynthetic capacity varied systematically over time and between species. Contrary to our hypothesis, V. divergens did not always have a higher photosynthetic capacity than C. americana. Rather, species differences were influenced by temporal variations in flooding and the leaf age. Leaf CO₂ assimilation and photosynthetic capacity of both species were lower during the flood period, but the differences were not statistically significant. The physiological performance of both species was strongly related to leaf N and P concentrations, but P limitation appeared to be more important than N limitation for these species and ecosystem. Photosynthetic capacity was higher and more stable for V. divergens, but such an advantage did not result in a statistically significant increase in P _N. Our results suggest that both species are tolerant to flooding even though they are adapted to very different hydrological conditions. Such physiological plasticity, especially for C. americana, might be a key feature for the ability to survive and persist in the seasonally flooded Pantanal.     

The host specificity of Moniliformis dubius (Acanthocephala), a parasite of cockroaches

Moniliformis dubius apparently evades the cellular defence reaction of the normal intermediate host, Periplaneta americana, by the production of a non-cellular envelope. Larval development will occasionally occur in other species of cockroaches: though some of these species reject tissue transplants from P. americana, they do not respond to the envelope of M. dubius when the parasite is transplanted from P. americana. This contrasts with the response elicited by gregarines transplanted from P. americana into other cockroach species, and it is suggested that the envelope surface of M. dubius mimics, but is not identical to, the surfaces of tissues from P. americana.     

The Effects of Glucosinolates and Their Breakdown Products on Necrotrophic Fungi

Glucosinolates are a diverse class of S- and N-containing secondary metabolites that play a variety of roles in plant defense. In this study, we used Arabidopsis thaliana mutants that contain different amounts of glucosinolates and glucosinolate-breakdown products to study the effects of these phytochemicals on phytopathogenic fungi. We compared the fungus Botrytis cinerea, which infects a variety of hosts, with the Brassicaceae-specific fungus Alternaria brassicicola. B. cinerea isolates showed variable composition-dependent sensitivity to glucosinolates and their hydrolysis products, while A. brassicicola was more strongly affected by aliphatic glucosinolates and isothiocyanates as decomposition products. We also found that B. cinerea stimulates the accumulation of glucosinolates to a greater extent than A. brassicicola. In our work with A. brassicicola, we found that the type of glucosinolate-breakdown product is more important than the type of glucosinolate from which that product was derived, as demonstrated by the sensitivity of the Ler background and the sensitivity gained in Col-0 plants expressing epithiospecifier protein both of which accumulate simple nitrile and epithionitriles, but not isothiocyanates. Furthermore, in vivo, hydrolysis products of indole glucosinolates were found to be involved in defense against B. cinerea, but not in the host response to A. brassicicola. We suggest that the Brassicaceae-specialist A. brassicicola has adapted to the presence of indolic glucosinolates and can cope with their hydrolysis products. In contrast, some isolates of the generalist B. cinerea are more sensitive to these phytochemicals.     

Tissue localization of aggregation pheromones in the American cockroach, <Emphasis Type="Italic">Periplaneta americana</Emphasis> (Blattodea: Blattidae)

The American cockroach, Periplaneta americana, excretes feces containing aggregation pheromones to attract conspecific individuals. It is thought that these pheromones play an important role in establishing and maintaining colonies. However, despite previous extensive efforts, the aggregation pheromones of P. americana have not been isolated. It is also unclear whether the aggregation pheromones of P. americana are truly biosynthesized by the insect, as most previous experiments extracted chemicals from feces. Here, we investigate the tissue localization of aggregation agents in P. americana. To reduce the effects of diet-derived odorants, we developed a new diet for P. americana consisting of only agar and sugars, and tested the attractiveness of fecal extracts from animals reared on this new diet. Our results show that the aggregation agents of fecal extracts are insect-derived, and the extracts from the midgut evoke stronger behavioral responses than those from other parts of the alimentary tract. This suggests that the midgut may be the production site or the storage organ of the aggregation agents. Thus, our data will facilitate the future identification of the aggregation pheromones of P. americana.     

Distribution and chemotaxonomic significance of glucosinolates in certain Middle-Eastern cruciferae

Eighty-four collections of 51 species of Middle-eastern Cruciferae were analysed by paper and gas-liquid chromatography for seed glucosinolates. Thirty-two species were investigated for the first time. Thirty compounds were identified and three unknowns of limited occurrence were found, p-Hydroxybenzylglucosinolate was found in six species of various tribes. 4-Methylsulfinyl-3-butenyl, 3-methoxycarbonylpropyl, and p-rhamnopyranosyloxybenzyl glucosinolates, which are rare in the Cruciferae, were found in Sinapis aucheri two species of Erysimum and Thlaspi perfoliatum, respectively. The biosynthetic precursors of all but one of the 30 glucosinolates are protein amino acids and at least 20 glucosinolates are derived from methionine. The distribution of glucosinolates is very useful at the species and genus levels but may provide only minimal support to the tribal alliance of certain genera. It supports the removal of Sinapis aucheri from the genus, the recognition of Fibigia clypeata and F. macrocarpa as distinct species, and the alliance of Ochthodium with Euclidium and Arabis with Drabopsis.