Mapping of unmethylated sites in rDNA repeats in barley NOR deletion line.

Extensive cytosine methylation is characteristic of plant rDNA. Evidence exists, however, that the active rRNA genes are less methylated. In this work we report on the mapping of unmethylated CCGG sites in Hordeum vulgare rDNA repeats by digestion with methylation sensitive restriction enzyme HpaII and indirect end-labeling of the generated fragments. For mapping we used genomic DNA from barley deletion line with a single NOR on chromosome 5H. This NOR is more active in order to compensate for the missing NOR 6H. The enhanced NOR 5H activity in the deletion mutant is not due to higher multiplicity of the rRNA genes or, as sequencing showed, to changes in the subunit structure of the intergenic spacer. The HpaII sites in barley rDNA are heavily methylated. Nevertheless, a fraction of the rDNA repeats is hypomethylated with unmethylated CCGG sites at various positions. One unmethylated CCGG sequence is close to the transcription start site, downstream of the 135bp subrepeats. Unmethylated sites are also present in the external transcribed spacer and in the genes coding mature rRNAs. The patterns of unmethylated sites in the barley deletion line and in lines with two NORs were compared. It is hypothesized that the occurrence of unmethylated sites on a fixed subset of rDNA repeats correlates with their transcriptional activity.

Darkness affects differentially the expression of plastid-encoded genes and delays the senescence-induced down-regulation of chloroplast transcription in cotyledons of Cucurbita pepo L. (Zucchini).

In contrast to differentiated leaves, the regulatory mechanisms of chloroplast gene expression in darkened cotyledons have not been elucidated. Although some results have been reported indicating accelerated senescence in Arabidopsis upon reillumination, the capacity of cotyledons to recover after dark stress remains unclear. We analysed the effect of two-days dark stress, applied locally or at the whole-plant level, on plastid gene expression in zucchini cotyledons. Our results showed that in the dark the overall chloroplast transcription rate was much more inhibited than the nuclear run-on transcription. While the activities of the plastid-encoded RNA polymerase (PEP) and nuclear RNA polymerase II were strongly reduced, the activities of the nuclear-encoded plastid RNA polymerase (NEP) and nuclear RNA polymerase I were less affected. During recovery upon reillumination, chloroplast transcription in the cotyledons was strongly stimulated (3-fold) compared with the naturally senescing controls, suggesting delayed senescence. Northern blot and dot blot analyses of the expression of key chloroplast-encoded photosynthetic genes showed that in contrast to psbA, which remained almost unaffected, both the transcription rate and mRNA content of psaB and rbcL were substantially decreased.

Senescence progression in a single darkened cotyledon depends on the light status of the other cotyledon in Cucurbita pepo (zucchini) seedlings: potential involvement of cytokinins and cytokinin oxidase/dehydrogenase activity.

Darkness mediates different senescence-related responses depending on the targeting of dark treatment (whole plants or individual leaves) and on the organs that perceive the signal (leaves or cotyledons). As no data are available on the potential role of darkness to promote senescence when applied to individual cotyledons, we have investigated how darkness affects the progression of senescence in either a single or both individually darkened cotyledons of young 10-day-old Cucurbita pepo (zucchini) seedlings. Strong acceleration of senescence was observed when both cotyledons were darkened as judged by the damage in their anatomical structure, deterioration of chloroplast ultrastructure in parallel with decreased photosynthetic rate and photochemical quantum efficiency of PSII. In addition, the endogenous levels of cytokinins (CKs) and IAA were strongly reduced. In a single individually darkened cotyledon, the structure and function of the photosynthetic apparatus as well as the contents of endogenous CKs and IAA were much less affected by darkness, thus suggesting inhibitory effect of the illuminated cotyledon on the senescence of the darkened one. Apparently, the effect of darkness to accelerate/delay senescence in a single darkened cotyledon depends on the light status of the other cotyledon from the pair. The close positive correlation between CK content and the activity of CK oxidase/dehydrogenase (CKX; EC 1.4.3.18/1.5.99.12) suggested that CKX was essentially involved in the mechanisms of downregulation of endogenous CK levels. Our results indicated that CKX-regulated CK signaling could be a possible regulatory mechanism controlling senescence in individually darkened cotyledons.

Methyl jasmonate is a more effective senescence-promoting factor in Cucurbita pepo (zucchini) cotyledons when compared with darkness at the early stage of senescence.

The effects of short-term darkening and methyl jasmonate (MeJA) on cotyledon senescence were studied 24h after transfer of intact 7-day-old Cucurbita pepo (zucchini) seedlings to darkness or spraying with 100 microM MeJA. The jasmonate inhibitory effect on chlorophyll content and chloroplast transcriptional activity was stronger compared with darkness. Further, MeJA reduced the photosynthetic rate whereas darkness did not affect photosynthesis. Neither stress factor affected the photochemical quantum efficiency of photosystem II (PSII) estimated by the variable fluorescence (F(v))/maximal fluorescence (F(m)) ratio, suggesting the existence of mechanisms protecting the functional activity of PSII at earlier stages of senescence, thus making this parameter more stable compared to others used to quantify senescence. Both stress factors caused a decrease in the content of physiologically active cytokinins, especially trans-zeatin (Z), with the jasmonate effect being much more pronounced when compared to darkness. Our results indicate that MeJA is a more potent inducer of senescence in zucchini cotyledons, at least within the relatively short period of the 24h treatment. This is likely due to its stronger down-regulatory effect on the levels of physiologically active cytokinins.