Expression of Arabidopsis WEE1 in Tobacco Induces Unexpected Morphological and Developmental Changes

WEE1 regulates the cell cycle by inactivating cyclin dependent protein kinases (CDKs) via phosphorylation. In yeast and animal cells, CDC25 phosphatase dephosphorylates the CDK releasing cells into mitosis, but in plants, its role is less clear. Expression of fission yeast CDC25 (Spcdc25) in tobacco results in small cell size, premature flowering and increased shoot morphogenetic capacity in culture. When Arath;WEE1 is over-expressed in Arabidopsis, root apical meristem cell size increases, and morphogenetic capacity of cultured hypocotyls is reduced. However expression of Arath;WEE1 in tobacco plants resulted in precocious flowering and increased shoot morphogenesis of stem explants, and in BY2 cultures cell size was reduced. This phenotype is similar to expression of Spcdc25 and is consistent with a dominant negative effect on WEE1 action. Consistent with this putative mechanism, WEE1 protein levels fell and CDKB levels rose prematurely, coinciding with early mitosis. The phenotype is not due to sense-mediated silencing of WEE1, as overall levels of WEE1 transcript were not reduced in BY2 lines expressing Arath;WEE1. However the pattern of native WEE1 transcript accumulation through the cell cycle was altered by Arath;WEE1 expression, suggesting feedback inhibition of native WEE1 transcription

Epigenetic control of nuclear architecture

The cell nucleus is a highly structured compartment where nuclear components are thought to localize in non-random positions. Correct positioning of large chromatin domains may have a direct impact on the localization of other nuclear components, and can therefore influence the global functionality of the nuclear compartment. DNA methylation of cytosine residues in CpG dinucleotides is a prominent epigenetic modification of the chromatin fiber. DNA methylation, in conjunction with the biochemical modification pattern of histone tails, is known to lock chromatin in a close and transcriptionally inactive conformation. The relationship between DNA methylation and large-scale organization of nuclear architecture, however, is poorly understood. Here we briefly summarize present concepts of nuclear architecture and current data supporting a link between DNA methylation and the maintenance of large-scale nuclear organization

Characterization of a knotted1-like gene of Helianthus tuberosus

Several gene families cooperate in meristem signalling; among these Knotted-like Homeobox (KNOX) genes have been shown to play a crucial role. The homeodomain of KNOX genes contains a conserved secondary structure described as "helix-loop-helix-turn-helix" and conserved regions at the N-terminal: the amphipathic helix and the ELK domain. KNOX genes, belonging to the TALE (Three Amino acid Loop Extension) superclass homeobox genes, are further divided into two classes by amino acid sequence differences in the third helix of the homeodomain and distinctive expression patterns. The combined effects of class 1 KNOX genes are required for the prevention of improper stem cell differentiation in the peripheral zone of the SAM. The present work is aimed to characterize the expression pattern of a class 1 KNOX like gene isolated from Helianthus tuberosus (2n = 6x = 10 2) and its involvement on morphogenetic competence expressed by two somaclones (EMB-2 and EMB-9) isolated from the interspecific tetraploid (2n = 4x = 68) hybrid H. annuus x H. tuberosus. The reconstructed full-length cDNA sequence (HtKNOT1, 1398 bp), obtained from 3’ and 5’ RACE, contained a 1089 bp CDS, 54- nucleotides of 5’-untranslated region (UTR), and 255-nucleotides of 3’-UTR. The predicted protein (HtKNOT1) displayed 362 amino acids with a calculated molecular mass of 40.2 kDa. A BLAST search against the protein database of the National Center for Biotechnology Information (NCBI) indicated that the encoded protein shared high sequence identity with members of the class I KNOX subfamily. Sequence information from the HtKNOT1 cDNA was used to design specific primers and to isolate the full-length exons/introns region of the gene. The expression pattern of HtKNOT1 was examined in plants of H. tuberosus by both RT-PCR and in situ hybridisation. HtKNOT1 has been observed to be highly expressed in vegetative shoots and stems. Weak presence of transcripts was detected also in incipient leaf primordia and young leaves. In the last years, it has been demonstrated that the ectopic reactivation of KNOX genes may be sufficient to restore the meristematic potential in differentiated cells. Here, we report that misexpression of HtKNOT1 is intimately linked to the development of ectopic shoots and somatic embryos on leaves of the somaclone EMB-2. Misexpression of HtKNOT1 was also observed in somatic embryos developed from intact adventitious roots of in vitro-grown plantlets of the variant clone EMB-9. The effect of exogenous hormonal treatments on HtKNOT1 expression was evaluated. Notably, massive shoot/embryo regeneration along with a strong accumulation of HtKNOT1 transcripts was induced in EMB-2 non-epiphyllous leaves by in vitro zeatin treatment. However, epiphyllous structures were not formed in zeatin-treated control leaves, despite an enhancement of HtKNOT1 expression, suggesting that HtKNOT1 is not the unique factor, to trigger ectopic morphogenesis in EMB-2 leaves

Fruit volatilome profiling through GC × GC-ToF-MS and gene expression analyses reveal differences amongst peach cultivars in their response to cold storage

Peaches have a short shelf life and require chilling during storage and transport. Peach aroma is important for consumer preference and determined by underlying metabolic pathways and gene expression. Differences in aroma (profiles of volatile organic compounds, VOCs) have been widely reported across cultivars and in response to cold storage. However, few studies used intact peaches, or used equilibrium sampling methods subject to saturation. We analysed VOC profiles using TD-GC 7 GC-ToF-MS and expression of 12 key VOC pathway genes of intact fruit from six cultivars (three peaches, three nectarines) before and after storage at 1 \ub0C for 7 days including 36 h shelf life storage at 20 \ub0C. Two dimensional GC (GC 7 GC) significantly enhances discrimination of thermal desorption gas chromatography time-of-flight mass spectrometry (TD-GC-ToF-MS) and detected a total of 115 VOCs. A subset of 15 VOCs from analysis with Random Forest discriminated between cultivars. Another 16 VOCs correlated strongly with expression profiles of eleven key genes in the lipoxygenase pathway, and both expression profiles and VOCs discriminated amongst cultivars, peach versus nectarines and between treatments. The cultivar-specific response to cold storage underlines the need to understand more fully the genetic basis for VOC changes across cultivars

Distinct nuclear organization, DNA methylation pattern and cytokinin distribution mark juvenile, juvenile-like and adult vegetative apical meristems in peach (Prunus persica (L.) Batsch)

Chromatin organization, nuclear DNA methylation and endogenous zeatin localization were investigated in shoot apical meristems (SAM) during juvenile and adult phases of peach (Prunus persica (L.) Batsch). The aim was to examine the extent to which these parameters could discriminate the juvenile and adult SAMs. Seedlings (juvenile, cannot flower), basal shoots (called juvenile-like, because they exhibit juvenile macroscopic traits) and apical shoots (competent to form flowers) of adult plants were chosen. Nuclear chromatin exhibited chromocentres that were peripherally distributed in SAMs of juvenile and juvenile-like shoots, but were diffusely spread in those of adult shoots. These patterns coincided with a peripheral labelling of DNA methylation in juvenile and juvenile-like meristem nuclei versus a diffuse labelling pattern in adult meristem nuclei. During vegetative growth (from March to June), the level of nuclear DNA methylation was higher in adult meristems than in juvenile and juvenile-like ones. The immunolocalization of zeatin in juvenile SAM was in the subapical region, but adult meristems exhibited a widespread localization or a signal confined within the boundaries of the central zone. The extent to which the acquisition of a strongly zonated pattern of these parameters as markers of floral competence in adult SAMs is discussed