Ovule Gene Expression Analysis in Sexual and Aposporous Apomictic Hypericum perforatum L. (Hypericaceae) Accessions.

Hypericum perforatum L. (2n = 4x = 32) is an attractive model system for the study of aposporous apomixis. The earliest phenotypic features of aposporous apomixis in this species are the mitotic formation of unreduced embryo sacs from a somatic cell of the ovule nucellus and the avoidance of meiosis. In this research we addressed gene expression variation in sexual and apomictic plants, by focusing on the ovule nucellus, which is the cellular domain primarily involved into the differentiation of meiocyte precursors and aposporous embryo sacs, at a pre-meiotic developmental stage. Gene expression analyses performed by RNAseq identified 396 differentially expressed genes and 1834 transcripts displaying phenotype-specific expression. Furthermore, the sequencing and assembly of the genome from a diploid sexual accession allowed the annotation of a 50 kb sequence portion located upstream the HAPPY locus and to address the extent to which single transcripts were assembled in multiple variants and their co-expression levels. About one third of identified DEGs and phenotype-specific transcripts were associated to transcript variants with alternative expression patterns. Additionally, considering DEGs and phenotype-specific transcript, the co-expression level was estimated in about two transcripts per locus. Our gene expression study shows massive differences in the expression of several genes encoding for transposable elements. Transcriptional differences in the ovule nucellus and pistil terminal developmental stages were also found for subset of genes encoding for potentially interacting proteins involved in pre-mRNA splicing. Furthermore, the sexual and aposporous ovule transcriptomes were characterized by differential expression in genes operating in RNA silencing, RNA-mediated DNA methylation (RdDM) and histone and chromatin modifications. These findings are consistent with a role of these processes in regulating cell fate determination in the ovule, as indicated by forward genetic studies in sexual model species. The association between aposporous apomixis, pre-mRNA splicing and DNA methylation mediated by sRNAs, which is supported by expression data and by the enrichment in GO terms related to these processes, is consistent with the massive differential expression of multiple transposon-related sequences observed in ovules collected from both sexual and aposporous apomictic accessions. Overall, our data suggest that phenotypic expression of aposporous apomixis is concomitant with the modulation of key genes involved in the two interconnected processes: RNA splicing and RNA-directed DNA methylation.

An apomixis-linked ORC3-like pseudogene is associated with silencing of its functional homolog in apomictic Paspalum simplex.

Apomixis in plants consists of asexual reproduction by seeds. Here we characterized at structural and functional levels an apomixis-linked sequence of Paspalum simplex homologous to subunit 3 of the ORIGIN RECOGNITION COMPLEX (ORC3). ORC is a multiprotein complex which controls DNA replication and cell differentiation in eukaryotes. Three PsORC3 copies were identified, each one characterized by a specific expression profile. Of these, PsORC3a, specific for apomictic genotypes, is a pseudogene that was poorly and constitutively expressed in all developmental stages of apomictic flowers, whereas PsORC3b, the putative functional gene in sexual flowers, showed a precise time-related regulation. Sense transcripts of PsORC3 were expressed in the female cell lineage of both apomictic and sexual reproductive phenotypes, and in aposporous initials. Although strong expression was detected in sexual early endosperm, no expression was present in the apomictic endosperm. Antisense PsORC3 transcripts were revealed exclusively in apomictic germ cell lineages. Defective orc3 mutants of rice and Arabidopsis showed normal female gametophytes although the embryo and endosperm were arrested at early phases of development. We hypothesize that PsORC3a is associated with the down-regulation of its functional homolog and with the development of apomictic endosperm which deviates from the canonical 2(maternal):1(paternal) genome ratio.

Overexpression of the olive acyl carrier protein gene (OeACP1) produces alterations in fatty acid composition of tobacco leaves.

Taking into account that fatty acid (FA) biosynthesis plays a crucial role in lipid accumulation in olive (Olea europaea L.) mesocarp, we investigated the effect of olive acyl carrier protein (ACP) on FA composition by overexpressing an olive ACP cDNA in tobacco plants. The OeACP1.1A cDNA was inserted in the nucleus or in the chloroplast DNA of different tobacco plants, resulting in extensive transcription of the transgenes. The transplastomic plants accumulated lower olive ACP levels in comparison to nuclear-transformed plants. Moreover, the phenotype of the former plants was characterized by pale green/white cotyledons with abnormal chloroplasts, delayed germination and reduced growth. We suggest that the transplastomic phenotype was likely caused by inefficient olive ACP mRNA translation in chloroplast stroma. Conversely, total lipids from leaves of nuclear transformants expressing high olive ACP levels showed a significant increase in oleic acid (18:1) and linolenic acid (18:3), and a concomitant significant reduction of hexadecadienoic acid (16:2) and hexadecatrienoic acid (16:3). This implies that in leaves of tobacco transformants, as likely in the mesocarp of olive fruit, olive ACP not only plays a general role in FA synthesis, but seems to be specifically involved in chain length regulation forwarding the elongation to C18 FAs and the subsequent desaturation to 18:1 and 18:3.

Recovery from stolbur disease in grapevine involves changes in sugar transport and metabolism.

Grapevine can be severely affected by phytoplasmas, which are phytopathogenic Mollicutes invading the sieve elements of the host plant. The biochemical and molecular relationships between phytoplasmas and their hosts remain largely unexplored. Equally unknown is an interesting aspect of the pathogen-plant interaction called "recovery," which is a spontaneous remission of symptoms in previously symptomatic plants. Recovered plants develop resistance mechanisms correlated with ultrastructural and biochemical changes in the sieve elements. Callose as well as sugars are involved in several plant defense processes and signaling. In the present work we have examined the possible involvement of callose, as well as callose synthase, sugar transporter, and cell wall invertase genes, during the infection and after "recovery" of grapevine from bois noir (BN). Ultrastructural investigation of leaf tissue showed that callose accumulated in the sieve elements of diseased grapevine; moreover, two genes encoding for callose synthase were up-regulated in the infected leaves. Regarding sucrose, expression analysis showed that sucrose transport and cleavage were severely affected by BN phytoplasma, which induced the establishment of a carbohydrate sink in the source leaf, and was analogous to other obligate biotrophs that acquire most of their nutrients from the host plant. Interestingly, whereas in recovered plants the transcript level of sucrose synthase was similar to healthy plants, sucrose transporters as well as cell wall invertase were expressed to a greater degree in recovered leaves than in healthy ones. Recovered plants seem to acquire structural and molecular changes leading to increases in sucrose transport ability and defense signaling.

Laser microdissection of grapevine leaf phloem infected by stolbur reveals site-specific gene responses associated to sucrose transport and metabolism.

Bois Noir is an emergent disease of grapevine that has been associated to a phytoplasma belonging to the XII-A stolbur group. In plants, phytoplasmas have been found mainly in phloem sieve elements, from where they spread moving through the pores of plates, accumulating especially in source leaves. To examine the expression of grapevine genes involved in sucrose transport and metabolism, phloem tissue, including sieve element/companion cell complexes and some parenchyma cells, was isolated from healthy and infected leaves by means of laser microdissection pressure catapulting (LMPC). Site-specific expression analysis dramatically increased sensitivity, allowing us to identify specific process components almost completely masked in whole-leaf analysis. Our findings showed decreased phloem loading through inhibition of sucrose transport and increased sucrose cleavage activity, which are metabolic changes strongly suggesting the establishment of a phytoplasma-induced switch from carbohydrate source to sink. The analysis focused at the infection site also showed a differential regulation and specificity of two pathogenesis-related thaumatin-like genes (TL4 and TL5) of the PR-5 family.

A polyphasic approach for the characterization of endophytic Alternaria strains isolated from grapevines.

A polyphasic approach was set up and applied to characterize 20 fungal endophytes belonging to the genus Alternaria, recovered from grapevine in different Italian regions. Morphological, microscopical, molecular and chemical investigations were performed and the obtained results were combined in a pooled cluster analysis. Following morphological analyses, all strains were grouped according to their three-dimensional sporulation pattern on PCA and to the colony characteristics on different substrates. After DNA extraction, all strains were analyzed by RAPD-PCR and the resulting profiles were subjected to cluster analysis. The metabolites extracted from the 20 Alternaria endophytes were analyzed by a HPLC and the resulting metabolite profiles were subjected to multivariate statistic analyses. In comparison with reference 'small-spored' Alternaria species, the 20 strains were segregated into two morphological groups: one belonging to the A. arborescens species-group and a second to the A. tenuissima species-group. RAPD analysis also showed that grapevine endophytes belonged to either the A. arborescens or the A. tenuissima species-group and that they were molecularly distinct from strains belonging to A. alternata. Chemotaxonomy gave the same grouping: the grapevine endophytic strains belong to A. arborescens or A. tenuissima species-groups producing known metabolites typical of these species-groups. Interestingly, the 20 grapevine endophytes were able to produce also a number of unknown metabolites, whose characterization could be useful for a more precise segregation of the two species-groups. The results show how complementary morphological, molecular and chemical data can clarify relationships among endophyte species-groups of low morphological divergence.