Functional characterization of a melon alcohol acyl-transferase gene family involved in the biosynthesis of ester volatiles. Identification of the crucial role of a threonine residue for enzyme activity

Volatile esters, a major class of compounds contributing to the aroma of many fruit, are synthesized by alcohol acyl-transferases (AAT). We demonstrate here that, in Charentais melon (Cucumis melo var. cantalupensis), AAT are encoded by a gene family of at least four members with amino acid identity ranging from 84% (Cm-AAT1/Cm-AAT2) and 58% (Cm-AAT1/Cm-AAT3) to only 22% (Cm-AAT1/Cm-AAT4). All encoded proteins, except Cm-AAT2, were enzymatically active upon expression in yeast and show differential substrate preferences. Cm-AAT1 protein produces a wide range of short and long-chain acyl esters but has strong preference for the formation of E-2-hexenyl acetate and hexyl hexanoate. Cm-AAT3 also accepts a wide range of substrates but with very strong preference for producing benzyl acetate. Cm-AAT4 is almost exclusively devoted to the formation of acetates, with strong preference for cinnamoyl acetate. Site directed mutagenesis demonstrated that the failure of Cm-AAT2 to produce volatile esters is related to the presence of a 268-alanine residue instead of threonine as in all active AAT proteins. Mutating 268-A into 268-T of Cm-AAT2 restored enzyme activity, while mutating 268-T into 268-A abolished activity of Cm-AAT1. Activities of all three proteins measured with the prefered substrates sharply increase during fruit ripening. The expression of all Cm-AAT genes is up-regulated during ripening and inhibited in antisense ACC oxidase melons and in fruit treated with the ethylene antagonist 1-methylcyclopropene (1-MCP), indicating a positive regulation by ethylene. The data presented in this work suggest that the multiplicity of AAT genes accounts for the great diversity of esters formed in melon

Resistance of Malus domestica fruit to Botrytis cinerea depends on endogenous ethylene biosynthesis

The plant hormone ethylene regulates fruit ripening, other developmental processes, and a subset of defense responses. Here, we show that 1-aminocyclopropane-1-carboxylic acid synthase (ACS)-silenced apple (Malus domestica) fruit that express a sense construct of ACS were more susceptible to Botrytis cinerea than untransformed apple, demonstrating that ethylene strengthens fruit resistance to B. cinerea infection. Because ethylene response factors (ERFs) are known to contribute to resistance against B. cinerea via the ethylene-signaling pathway, we cloned four ERF cDNAs from fruit of M. domestica: MdERF3,-4,-5, and-6. Expression of all four MdERF mRNAs was ethylene dependent and induced by wounding or by B. cinerea infection. B. cinerea infection suppressed rapid induction of wound-related MdERF expression. MdERF3 was the only mRNA induced by wounding and B. cinerea infection in ACS-suppressed apple fruit, although its induction was reduced compared with wild-type apple. Promoter regions of all four MdERF genes were cloned and putative cis-elements were identified in each promoter. Transient expression of MdERF3 in tobacco increased expression of the GCC-box containing gene chitinase 48

Stress responses in citrus peel: Comparative analysis of host responses to Huanglongbing disease and puffing disorder

A comparison between transcriptomic responses to puffing disorder and Huanglongbing disease was conducted to decipher differences and similarities in gene and pathway regulation induced by abiotic (puffing) and biotic stresses (Huanglongbing) in citrus peel tissues. We functionally analyzed two previously published datasets: the first obtained for the study of puffing disorder using an Affymetrix citrus microarray and the second consisting of a deep sequencing analysis of symptomatic responses to Huanglongbing disease. Transcriptomic data were mined using bioinformatic tools to highlight genes and pathways playing a key role in modulating responses to different types of stress in citrus fruit. Puffing disorder was linked to altered expression of genes involved in abiotic stress, vesicle transport, and protein targeting while Huanglongbing disease induced biotic stress responses and transport pathways. Sucrose and starch metabolism were the most significantly regulated pathways in both the two stresses. Huanglongbing disease significantly affected secondary metabolism (phenylpropanoid, flavonoid, and terpenoid pathways) while puffing disorder was more linked to primary metabolism (fatty acid, pentose phosphate, and glycerolipid pathways). Key genes were analyzed by qRT-PCR to define possible host biomarkers specific to each stress or which could act as general indicators of stress. Ethylene-related genes in the fruit peel were more affected by Huanglongbing than puffing. Gibberellin signaling genes (GASA1 and GASA5) were repressed under both stresses. Huanglongbing upregulated key genes involved in biotroph responses such as methylsalicylate and WRKY70. A protein\u2013protein network revealed that heat shock proteins were major, transcriptionally regulated hubs under stress conditions as shown by the repression of HSP82

Somatic embryogenesis and plant regeneration in carob ( Ceratonia siliqua L.)

Summary Somatic embryos of carob (Ceratonia siliqua L.) were induced from cotyledonary segments excised from immature seeds when cultured on Murashige and Skoog media supplemented with several combinations of 6-benzylaminopurine (BA) and indole-3-butyric acid (IBA). The best frequencies of induction (33.8%) were obtained when 4.4 µM BA and 0.5 µM IBA were used. Shoots were also sporadically formed in the same media. When IBA was replaced by other auxins in the induction media, only a-naphthaleneacetic acid (NAA) and indole-3-acetic acid (IAA) could induce somatic embryogenesis, although at lower rates than IBA. 2,4-Dichlorophenoxyacetic acid and 4-amino-3,5,6-trichloropicolinic acid were completely ineffective. Besides culture media composition, the developmental stage of the explants at the time of culture showed a strong influence on somatic embryogenesis induction, with cotyledons from stage II pods providing the highest levels of induction. By contrast, the genotype of the explant did not determine a significant role in the induction process. Attempts to achieve somatic embryo germination were mostly unsuccessful, since only shoot development was observed; the highest frequencies of development occurred on media containing only gibberellic acid (3.0 µM). For plant regeneration, the developed shoots were further rooted on IBA-supplemented media, and the plantlets obtained were transferred to soil, where c. 88% of them survived. Histological observations showed the presence of morphologically normal and abnormal somatic embryos, the latter displaying an abnormal pattern of vascular bundles. Ultrastructural analysis showed that the cells of the globular embryos had a dense cytoplasm, whereas those not involved in somatic embryo formation showed signs of senescence. Histological studies were also used to distinguish between somatic embryos and shoots originated in the same media

Spall strength of glass fiber reinforced polymer composites

AbstractIn the present paper results of a series of plate impact experiments designed to study spall strength in glass–fiber reinforced polymer composites (GRP) are presented. Two GRP architectures are investigated—S2 glass woven roving in Cycom 4102 polyester resin matrix and a balanced 5-harness satin weave E-glass in a Ciba epoxy (LY564) matrix. The GRP specimens were shock loaded using an 82.5mm bore single-stage gas-gun. A velocity interferometer was used to measure the particle velocity profile at the rear (free) surface of the target plate. The spall strength of the GRP was obtained as a function of the normal component of the impact stress and the applied shear-strain by subjecting the GRP specimens to normal shock compression and combined shock compression and shear loading, respectively. The spall strengths of the two GRP composites were observed to decrease with increasing levels of normal shock compression. Moreover, superposition of shear-strain on the normal shock compression was found to be highly detrimental to the spall strength. The E-glass reinforced GRP composite was found to have a much higher level of spall strength under both normal shock compression and combined compression and shear loading when compared to the S2-glass GRP composite. The maximum spall strength of the E-glass GRP composite was found to be 119.5MPa, while the maximum spall strength for the S2 glass GRP composite was only 53.7MPa. These relatively low spall strength levels of the S2-glass and the E-glass fiber reinforced composites have important implications to the design and development of GRP-based light-weight integral armor