Suppression of a Prolyl 4 Hydroxylase Results in Delayed Abscission of Overripe Tomato Fruits

The tomato pedicel abscission zone (AZ) is considered a model system for flower and fruit abscission development, activation, and progression. O-glycosylated proteins such as the Arabidopsis IDA (INFLORESCENCE DEFICIENT IN ABSCISSION) peptide and Arabinogalactan proteins (AGPs) which undergo proline hydroxylation were demonstrated to participate in abscission regulation. Considering that the frequency of occurrence of proline hydroxylation might determine the structure as well the function of such proteins, the expression of a tomato prolyl 4 hydroxylase, SlP4H3 (Solanum lycopersicum Prolyl 4 Hydroxylase 3) was suppressed in order to investigate the physiological significance of this post-translational modification in tomato abscission. Silencing of SlP4H3 resulted in the delay of abscission progression in overripe tomato fruits 90 days after the breaker stage. The cause of this delay was attributed to the downregulation of the expression of cell wall hydrolases such as SlTAPGs (tomato abscission polygalacturonases) and cellulases as well as expansins. In addition, minor changes were observed in the mRNA levels of two SlAGPs and one extensin. Moreover, structural changes were observed in the silenced SlP4H3AZs. The fracture plane of the AZ was curved and not along a line as in wild type and there was a lack of lignin deposition in the AZs of overripe fruits 30 days after breaker. These results suggest that proline hydroxylation might play a role in the regulation of tomato pedicel abscission

AtHESPERIN: a novel regulator of circadian rhythms with poly(A)-degrading activity in plants

<p>We report the identification and characterization of a novel gene, <i>AtHesperin</i> (<i>AtHESP</i>) that codes for a deadenylase in <i>Arabidopsis thaliana</i>. The gene is under circadian clock-gene regulation and has similarity to the mammalian <i>Nocturnin</i>. AtHESP can efficiently degrade poly(A) substrates exhibiting allosteric kinetics. Size exclusion chromatography and native electrophoresis coupled with kinetic analysis support that the native enzyme is oligomeric with at least 3 binding sites. Knockdown and overexpression of <i>AtHESP</i> in plant lines affects the expression and rhythmicity of the clock core oscillator genes <i>TOC1</i> and <i>CCA1</i>. This study demonstrates an evolutionary conserved poly(A)-degrading activity in plants and suggests deadenylation as a mechanism involved in the regulation of the circadian clock. A role of <i>AtHESP</i> in stress response in plants is also depicted.</p

Role of lupeol synthase in Lotus japonicus nodule formation

P>Triterpenes are plant secondary metabolites, derived from the cyclization of 2,3-oxidosqualene by oxidosqualene cyclases (OSCs). Here, we investigated the role of lupeol synthase, encoded by OSC3, and its product, lupeol, in developing roots and nodules of the model legume Lotus japonicus. The expression patterns of OSC3 in different developmental stages of uninfected roots and in roots infected with Mesorhizobium loti were determined. The tissue specificity of OSC3 expression was analysed by in situ hybridization. Functional analysis, in which transgenic L. japonicus roots silenced for OSC3 were generated, was performed. The absence of lupeol in the silenced plant lines was determined by GC-MS. The expression of ENOD40, a marker gene for nodule primordia initiation, was increased significantly in the OSC3-silenced plant lines, suggesting that lupeol influences nodule formation. Silenced plants also showed a more rapid nodulation phenotype, consistent with this. Exogenous application of lupeol to M. loti-infected wild-type plants provided further evidence for a negative regulatory effect of lupeol on the expression of ENOD40. The synthesis of lupeol in L. japonicus roots and nodules can be solely attributed to OSC3. Taken together, our data suggest a role for lupeol biosynthesis in nodule formation through the regulation of ENOD40 gene expression

A metabolic gene cluster in Lotus japonicus discloses novel enzyme functions and products in triterpene biosynthesis

Genes for triterpene biosynthetic pathways exist as metabolic gene clusters in oat and Arabidopsis thaliana plants. We characterized the presence of an analogous gene cluster in the model legume Lotus japonicus. In the genomic regions flanking the oxidosqualene cyclase AMY2 gene, genes for two different classes of cytochrome P450 and a gene predicted to encode a reductase were identified. Functional characterization of the cluster genes was pursued by heterologous expression in Nicotiana benthamiana. The gene expression pattern was studied under different developmental and environmental conditions. The physiological role of the gene cluster in nodulation and plant development was studied in knockdown experiments. A novel triterpene structure, dihydrolupeol, was produced by AMY2. A new plant cytochrome P450, CYP71D353, which catalyses the formation of 20-hydroxybetulinic acid in a sequential three-step oxidation of 20-hydroxylupeol was characterized. The genes within the cluster are highly co-expressed during root and nodule development, in hormone-treated plants and under various environmental stresses. A transcriptional gene silencing mechanism that appears to be involved in the regulation of the cluster genes was also revealed. A tightly co-regulated cluster of functionally related genes is involved in legume triterpene biosynthesis, with a possible role in plant development