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

Over expression of Spcdc25 and of ATWEE1 in Arabidopsis alter the sensitivity of hypocotyl explants to auxin and cytokinin in culture

The cell cycle is regulated by plant growth regulators (PGRs), mainly auxins and cytokinins. In eukaryotes, two genes regulate entry into mitosis: cdc25 and wee1, although a confirmed homologue of cdc25 in plants has not yet been identified. Expressing the fission yeast cdc25 (Spcdc25) in tobacco BY2 cells allows them to bypass a cytokinin block at G2/M. In tobacco and Arabidopsis roots, Spcdc25 expression led to an increased frequency of lateral roots. Conversely, over-expression of AtWEE1 in Arabidopsis induces a slower rate of root elongation and a reduced frequency of lateral roots. Wild type Arabidopsis hypocotyl explants respond to increasing levels of cytokinin with rapid proliferation, greening and formation of shoots, but are unable to form shoots in the absence of auxin and cytokinin. We tested if hypocotyl explants from Arabidopsis lines over-expressing ATWEE1 and Spcdc25 respond like WT to auxin (NAA) and cytokinin (Kinetin). We used a system of two-way grids in which we increased concentration of NAA and kinetin in X- and Y-axes, respectively. There were clear differences in the response between the transgenic and WT. All concentrations of auxin and cytokinin suppressed growth of calli in the AtWEE1OE line compared to WT. Moreover, in the AtWEE1OE, both organogenesis and formation of root hair structures were suppressed. At higher PGR levels neither WT nor Spcdc25 lines produced shoots but both produced roots and hairs. A minimum level of NAA was required for callus induction in both WT and Spcdc25 lines. However at low NAA/Kinetin there was growth of calli and more roots in Spcdc25 lines compared with WT. In an inducible Spcdc25 line plus inducer, roots formed at concentrations which suppressed roots in the inducible Spcdc25 line without inducer These results are discussed in the context of plant growth regulators and regulatory genes of the plant cell cycle

Expression of Spcdc25 in Arabidopsis alters the response of hypocotyl explants to auxin & cytokinin in culture

The eukaryotic cell cycle comprises four distinct phases, mitosis (M), G1, DNA synthesis (S)-phase and G2 in which the key transitions are G1/S and G2/M. Progression of dividing cells through the cell cycle is controlled by key enzymes: cyclin-dependent kinases (CDKs), the products of cdc2-like genes. The regulation of CDK activity includes, amongst others, its activation by Cdc25 phosphatase at G2/M. This positive regulator is balanced by negative regulation by a protein kinase encoded by wee1. A plant homologue of the inactivating kinase, wee1, has already been cloned. However, a homologue of the activating phosphatase cdc25 has not been unequivocally identified in plants yet. The expression of cdc2-like genes can be influenced by many factors including plant growth regulators, mainly auxins and cytokinins. Expressing the fission yeast cdc25 (Spcdc25) in the tobacco BY2 cell line results in cells bypassing a cytokinin block, and in explants of tobacco expressing Spcdc25, shoot development is stimulated not only in a treatment that favours shoot formation (high cytokinin and low auxin) but also under root-stimulating conditions (high auxin and low cytokinin) and even without exogenous growth regulator treatment. Wild type Arabidopsis hypocotyls explants respond to increasing levels of cytokinin with rapid proliferation, greening and formation of shoots, but are unable to form shoots in the absence of auxin and cytokinin. We tested if hypocotyl explants from Arabidopsis transformed with constitutive and inducible lines Spcdc25 respond like wt to auxin (NAA) and cytokinin (Kinetin) in a system of grids in which we increase concentration of these exogenous plant growth regulators. We find that Wt and Spcdc25 hypocotiles segments are very sensible to higher liveles of ormons (1000 ngml-1 to 3000 ngml-1), calli grew with a lot of root hairs and the analysis of shots formation was impossible. Also is required a minimum level of NAA for calli induction in these strains: at 25 ngml-1 NAA/Kinetin we start to see grow of calli in Spcdc25 lines, plants in WT, not grow in not induced Spcdc25 line. Interesting results at 25, 50 ngml-1 NAA and 200ngml-1 Kinetin where in Spcdc25 there is grow of real roots, not visible in wt and not induced Spcdc25 line. At several concentrations of hormones Spcdc25 forms buds they will be show and discussed

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