Hyperhydricity of Prunus avium shoots cultured on gelrite: a controlled stress response.

Hyperhydricity is a physiological disorder frequently affecting shoots vegetatively propagated in vitro. Hyperhydric shoots are characterised by a translucent aspect due to a chlorophyll deficiency, a not very developed cell wall and a high water content. Hyperhydricity of Prunus avium shoots was expressed in vitro in one multiplication cycle by replacing the gelling agent agar (normal shoots: NS) by gelrite (hyperhydric shoots: HS). P. avium shoots evolving towards the hyperhydric state produced higher amounts of ethylene, polyamines (PAs) and proline, which are substances considered as stress markers. A higher activity of glutathione peroxidase (GPX; EC 1.11.1.9), involved in organic hydroperoxide elimination, suggested an increased production of these compounds in HS. The unchanged free fatty acid composition indicated no HS membrane damages compared to NS. The ploidy level of HS nuclei was not affected, but the bigger size and the lower percentage of nuclei during the S phase suggested a slowing down of the cell cycle. The results argued for a stress response of the HS, but no signs of oxidative damages of lipid membrane and nucleus were observed. The discussion points out paradoxical results in a classical analysis of stress and suggests an alternative way of defense mechanisms in HS, involving homeostatic regulation and controlled degradation processes to maintain integrity and vital functions of the cell.     

Predominance of bacteriophage SP82 over bacteriophage SP01 in mixed infections of Bacillus subtilis

In mixed infections with Bacillus subtilis phages SP82 and SP01, the SP82 genotype is predominant among the progeny. This predominance is determined by a specific region of the genome, the pos region, which apparently is located near genes 29 to 32 (by the SP01 numbering system). Recombination between SP82 and SP01 yields phage which have both the SP82 pos region and an SP01 mutation. This mutation then behaves in mixed infection as if it were part of an SP82 genome.     

Focus on Weed Control: Natural Compounds as Next-Generation Herbicides

Herbicides with new modes of action (MOAs) are badly needed due to the rapidly evolving resistance to commercial herbicides, but a new MOA has not been introduced in over 20 years. The greatest pest management challenge for organic agriculture is the lack of effective natural product herbicides. The structural diversity and evolved biological activity of natural phytotoxins offer opportunities for the development of both directly used natural compounds and synthetic herbicides with new target sites based on the structures of natural phytotoxins. Natural phytotoxins are also a source for the discovery of new herbicide target sites that can serve as the focus of traditional herbicide discovery efforts. There are many examples of strong natural phytotoxins with MOAs other than those used by commercial herbicides, which indicates that there are molecular targets of herbicides that can be added to the current repertoire of commercial herbicide MOAs.The evolutionary forces driving the survival of species include chemical interactions between organisms, which function in positive interactions such as mutualistic and symbiotic relationships and negative interactions such as competitive and parasitic relationships. These processes have led to the emergence of novel secondary metabolic pathways (often through gene duplication), producing a vast array of structurally diverse and biologically active molecules (Moore and Purugganan, 2005; Ober, 2005; Flagel and Wendel, 2009; Jiang et al., 2013). This evolutionary process is similar to a high-throughput screen. However, unlike conventional in vitro screens, which test many compounds on a single biochemical target over a very short period of time, this natural high-throughput process selects molecules based on their whole-organism activities, involving numerous chemical interactions between countless organisms and target sites over millions of years. To date, approximately 200,000 secondary metabolites have been identified (Tulp and Bohlin, 2005), with many more expected to be discovered. Few of these compounds have been examined for phytotoxicity, and the modes or mechanisms of action (MOAs) of even fewer known phytotoxins have been elucidated.The negative chemical interactions between organisms are often characterized using anthropomorphic language, such as chemical warfare, referring to the production of phytotoxins used by plant pathogens to invade their host plants (Maor and Shirasu, 2005), and the novel weapons hypothesis, which is associated with the chemical-based advantage of some invasive plant species over native plant populations (Callaway and Aschehoug, 2000; Callaway and Ridenour, 2004; Callaway and Maron, 2006; Cappuccino and Arnason, 2006; Callaway et al., 2008). While simplistic, this terminology illustrates how these toxin-based interactions exploit biochemical weaknesses between an organism and its host or enemy/competitor to enhance its own survival (Verhoeven et al., 2009). In fact, these interactions can even be multitrophic, such as when exotic plants enhance their invasiveness by promoting the growth of certain native soil pathogens noxious to native plants (Mangla et al., 2008; Barto et al., 2011).As humans evolved from a nomadic hunter-gatherer subsistence existence to an agricultural lifestyle, they learned to utilize certain biologically active secondary metabolites to manage agricultural pests. Indeed, the concept that nature is an excellent source of natural pesticides is captured in the following ancient Lithica poem (circa 400 B.C.): “All the pests that out of earth arise, the earth itself the antidote supplies” (Ibn et al., 1781). Less than a century later, Greek and Roman treatises described practices to control agricultural pests that include the use of essential oils. Similar documents are found in Chinese literature, such as a survey describing plant species used to control plant pests (Yang and Tang, 1988). The mid-20th century ushered in the use of synthetic pesticides, which have revolutionized agriculture. Like pharmaceuticals (Harvey, 1999, 2008; Newman and Cragg, 2012), many pesticides are based on natural compounds. However, natural products have not played a major role in herbicide discovery (Copping and Duke, 2007; Hüter, 2011).     

Extensive phenotypic variation in early flowering mutants of Arabidopsis

Flowering time, the major regulatory transition of plant sequential development, is modulated by multiple endogenous and environmental factors. By phenotypic profiling of 80 early flowering mutants of Arabidopsis, we examine how mutational reduction of floral repression is associated with changes in phenotypic plasticity and stability. Flowering time measurements in mutants reveal deviations from the linear relationship between the number of leaves and number of days to bolting described for natural accessions and late flowering mutants. The deviations correspond to relative early bolting and relative late bolting phenotypes. Only a minority of mutants presents no detectable phenotypic variation. Mutants are characterized by a broad release of morphological pleiotropy under short days, with leaf characters being most variable. They also exhibit changes in phenotypic plasticity across environments for florigenic-related responses, including the reaction to light and dark, photoperiodic behavior, and Suc sensitivity. Morphological pleiotropy and plasticity modifications are differentially distributed among mutants, resulting in a large diversity of multiple phenotypic changes. The pleiotropic effects observed may indicate that floral repression defects are linked to global developmental perturbations. This first, to our knowledge, extensive characterization of phenotypic variation in early flowering mutants correlates with the reports that most factors recruited in floral repression at the molecular genetic level correspond to ubiquitous regulators. We discuss the importance of functional ubiquity for floral repression with respect to robustness and flexibility of network biological systems.     

Novel Bioassay for the Discovery of Inhibitors of the 2-C-Methyl-D-erythritol 4-Phosphate (MEP) and Terpenoid Pathways Leading to Carotenoid Biosynthesis

The 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway leads to the synthesis of isopentenyl diphosphate in plastids. It is a major branch point providing precursors for the synthesis of carotenoids, tocopherols, plastoquinone and the phytyl chain of chlorophylls, as well as the hormones abscisic acid and gibberellins. Consequently, disruption of this pathway is harmful to plants. We developed an in vivo bioassay that can measure the carbon flow through the carotenoid pathway. Leaf cuttings are incubated in the presence of a phytoene desaturase inhibitor to induce phytoene accumulation. Any compound reducing the level of phytoene accumulation is likely to interfere with either one of the steps in the MEP pathway or the synthesis of geranylgeranyl diphosphate. This concept was tested with known inhibitors of steps of the MEP pathway. The specificity of this in vivo bioassay was also verified by testing representative herbicides known to target processes outside of the MEP and carotenoid pathways. This assay enables the rapid screen of new inhibitors of enzymes preceding the synthesis of phytoene, though there are some limitations related to the non-specific effect of some inhibitors on this assay.     

Intracellular maturation and transport of tumor necrosis factor alpha converting enzyme

The tumor necrosis factor alpha converting enzyme (TACE) activity is required for the shedding of a variety of biologically active membrane bound precursors. The activation of TACE necessitates the proteolytic cleavage of its prodomain, a process that was suggested to be catalyzed by the proprotein convertase furin. However, the involvement of furin in this activation process has never been experimentally demonstrated. We have shown that the furinlike cleavage site (R-V-K-R(214)) localized between the prodomain and the metalloprotease domain of TACE is the sole site that can be in vitro cleaved by furin. In Cos7 cells, the release of TACE-processed substrates was reduced by the overexpression of the furin-specific proprotein convertase inhibitor Portland alpha1-antitrypsin inhibitor, but the release of TACE-processed substrates was increased by overexpression of furin in LoVo cells (deficient in furin activity) in which a mature form of TACE was identified. The immature form of TACE was detected at the surface of LoVo cells and at the surface of Cos7 and HT29 cells upon proprotein convertase inhibition. These results suggest that furin is the major proprotein convertase involved in the maturation/activation of TACE which is not a prerequisite for its cell-surface expression.     

The Amidohydrolases IAR3 and ILL6 Contribute to Jasmonoyl-Isoleucine Hormone Turnover and Generate 12-Hydroxyjasmonic Acid Upon Wounding in Arabidopsis Leaves

Jasmonates (JAs) are a class of signaling compounds that mediate complex developmental and adaptative responses in plants. JAs derive from jasmonic acid (JA) through various enzymatic modifications, including conjugation to amino acids or oxidation, yielding an array of derivatives. The main hormonal signal, jasmonoyl-l-isoleucine (JA-Ile), has been found recently to undergo catabolic inactivation by cytochrome P450-mediated oxidation. We characterize here two amidohydrolases, IAR3 and ILL6, that define a second pathway for JA-Ile turnover during the wound response in Arabidopsis leaves. Biochemical and genetic evidence indicates that these two enzymes cleave the JA-Ile signal, but act also on the 12OH-JA-Ile conjugate. We also show that unexpectedly, the abundant accumulation of tuberonic acid (12OH-JA) after wounding originates partly through a sequential pathway involving (i) conjugation of JA to Ile, (ii) oxidation of the JA-Ile conjugate, and (iii) cleavage under the action of the amidohydrolases. The coordinated actions of oxidative and hydrolytic branches in the jasmonate pathway highlight novel mechanisms of JA-Ile hormone turnover and redefine the dynamic metabolic grid of jasmonate conversion in the wound response.     

Homothorax switches function of Drosophila photoreceptors from color to polarized light sensors

Different classes of photoreceptors (PRs) allow animals to perceive various types of visual information. In the Drosophila eye, the outer PRs of each ommatidium are involved in motion detection while the inner PRs mediate color vision. In addition, flies use a specialized class of inner PRs in the "dorsal rim area" of the eye (DRA) to detect the e-vector of polarized light, allowing them to exploit skylight polarization for orientation. We show that homothorax is both necessary and sufficient for inner PRs to adopt the polarization-sensitive DRA fate instead of the color-sensitive default state. Homothorax increases rhabdomere size and uncouples R7-R8 communication to allow both cells to express the same opsin rather than different ones as required for color vision. Homothorax expression is induced by the iroquois complex and the wingless (wg) pathway. However, crucial wg pathway components are not required, suggesting that additional signals are involved.     

CYP704B1 Is a Long-Chain Fatty Acid ω-Hydroxylase Essential for Sporopollenin Synthesis in Pollen of Arabidopsis

Sporopollenin is the major component of the outer pollen wall (exine). Fatty acid derivatives and phenolics are thought to be its monomeric building blocks, but the precise structure, biosynthetic route, and genetics of sporopollenin are poorly understood. Based on a phenotypic mutant screen in Arabidopsis (Arabidopsis thaliana), we identified a cytochrome P450, designated CYP704B1, as being essential for exine development. CYP704B1 is expressed in the developing anthers. Mutations in CYP704B1 result in impaired pollen walls that lack a normal exine layer and exhibit a characteristic striped surface, termed zebra phenotype. Heterologous expression of CYP704B1 in yeast cells demonstrated that it catalyzes ω-hydroxylation of long-chain fatty acids, implicating these molecules in sporopollenin synthesis. Recently, an anther-specific cytochrome P450, denoted CYP703A2, that catalyzes in-chain hydroxylation of lauric acid was also shown to be involved in sporopollenin synthesis. This shows that different classes of hydroxylated fatty acids serve as essential compounds for sporopollenin formation. The genetic relationships between CYP704B1, CYP703A2, and another exine gene, MALE STERILITY2, which encodes a fatty acyl reductase, were explored. Mutations in all three genes resulted in pollen with remarkably similar zebra phenotypes, distinct from those of other known exine mutants. The double and triple mutant combinations did not result in the appearance of novel phenotypes or enhancement of single mutant phenotypes. This implies that each of the three genes is required to provide an indispensable subset of fatty acid-derived components within the sporopollenin biosynthesis framework.The biopolymer sporopollenin is the major component of the outer walls in pollen and spores (exines). It is highly resistant to nonoxidative physical, chemical, and biological treatments and is insoluble in both aqueous and organic solvents. While the stability and resistance of sporopollenin account for the preservation of ancient pollen grains for millions of years with nearly full retention of morphology (Doyle and Hickey, 1976; Friis et al., 2001), these same qualities make it extremely difficult to study the chemical structure of sporopollenin. Thus, although the first studies on the composition of sporopollenin were reported in 1928 (Zetzsche and Huggler, 1928), the exact structure of sporopollenin remains unresolved. At present, it is thought that sporopollenin is a complex polymer primarily made of a mixture of fatty acids and phenolic compounds (Guilford et al., 1988; Wiermann et al., 2001).Fatty acids were first implicated as sporopollenin components when ozonolysis of Lycopodium clavatum and Pinus sylvestris exine yielded significant amounts of straight- and branched-chain monocarboxylic acids, characteristic fatty acid breakdown products (Shaw and Yeadon, 1966). More recently, improved purification and degradation techniques coupled with analytical methods, such as solid-state ¹³C-NMR spectroscopy, Fourier transform infrared spectroscopy, and ¹H-NMR, have shown that sporopollenin is made up of polyhydroxylated unbranched aliphatic units and also contains small amounts of oxygenated aromatic rings and phenylpropanoids (Guilford et al., 1988; Ahlers et al., 1999; Domínguez et al., 1999; Bubert et al., 2002). Biochemical studies using thiocarbamate herbicide inhibition of the chain-elongating steps in the synthesis of long-chain fatty acids and radioactive tracer experiments provided further evidence that lipid metabolism is involved in the biosynthesis of sporopollenin (Wilwesmeier and Wiermann, 1995; Meuter-Gerhards et al., 1999).Relatively little is known about the genetic network that determines sporopollenin synthesis. However, several Arabidopsis (Arabidopsis thaliana) genes implicated in exine biosynthesis encode proteins with sequence homology to enzymes that are involved in fatty acid metabolism. Mutations in MALE STERILITY2 (MS2) eliminate exine and affect a protein with sequence similarity to fatty acyl reductases; the predicted inability of ms2 plants to reduce pollen wall fatty acids to the corresponding alcohols suggests that this reaction is a key step in sporopollenin synthesis (Aarts et al., 1997). The FACELESS POLLEN1 (FLP1) gene, whose loss causes the flp1 exine defect, encodes a protein similar to those involved in wax synthesis (Ariizumi et al., 2003). The no exine formation1 (nef1) mutant accumulates reduced levels of lipids, and the NEF1 protein was suggested to be involved in either lipid transport or the maintenance of plastid membrane integrity, including those plastids in the secretory tapetum of anthers, where many of the sporopollenin components are synthesized (Ariizumi et al., 2004). The dex2 mutant has mutations in the evolutionarily conserved anther-specific cytochrome P450, CYP703A2 (Morant et al., 2007), which catalyzes in-chain hydroxylation of saturated medium-chain fatty acids, with lauric acid (C12:0) as a preferred substrate (Morant et al., 2007). A recently described gene, ACOS5, encodes a fatty acyl-CoA synthetase that has in vitro preference for medium-chain fatty acids (de Azevedo Souza et al., 2009). Mutations in all of these genes compromise exine formation.Here, we describe an evolutionarily conserved cytochrome P450, CYP704B1, and demonstrate that this gene is essential for exine biosynthesis and plays a role different from that of CYP703A2. Heterologously expressed CYP704B1 catalyzed ω-hydroxylation of several saturated and unsaturated C14-C18 fatty acids. These results suggest the possibility that ω-hydroxylated fatty acids produced by CYP704B1, together with in-chain hydroxylated lauric acids provided by the action of CYP703A2, may serve as key monomeric aliphatic building blocks in sporopollenin formation. Analyses of the genetic relationships between CYP704B1, MS2, and CYP703A2 suggest that all three genes are involved in the same pathway within the sporopollenin biosynthesis framework.