A lambda recombinant phage, carrying a radish rDNA fragment spanning the complete external spacer and its borders, has been isolated and characterized by sequencing. The fragment is 2911 bp long and includes 486 bp of the 3' end of the 25s rRNA sequence, 2349 bp of spacer and the first 76 bp of the 5' end of the 18s rRNA sequence. The spacer can be divided into three regions: two unique domains flanking a 830-bp region of repeated sequences. Seven repeats ranging from 80 to 103 bp can be recognized. They are separated by short arrays of 12 -21 adenylic residues. Each repeat slightly differs from the others by single-nucleotide changes or short deletions. Examination of single-nucleotide changes common to two units suggests that a duplication arose during the evolution of this sequence. The repeated region was subcloned and used as a probe to demonstrate that it is highly species-specific: in stringent conditions it does not cross-hybridize with the spacer of ribosomal genes from closely related species such as Brassica. Transcription products, starting or finishing within the spacer sequence, were mapped by northern blotting, primer extension and S1 mapping. Two major precursors were identified starting respectively at positions 2095 and 2280. The region surrounding the start at 2095 presents extensive homology with an analogous region in maize, rye, mung bean, Xenopus and tse-tse fly. However, longer transcripts can be detected. Several 3' ends downstream of the 25s terminus were also observed. Taken together these results indicate that rDNA transcription and pre-rRNA processing in plants are more complex than anticipated from previous studies.Higher plant genomes contain multiple copies of ribosomal RNA nuclear genes [I, 21. The general organization of these genes is similar to that of animals [3]. They are organized in tandem repeats, the coding sequences being separated by a small internal transcribed spacer and a large external spacer, which is considered to be only partially transcribed (ENTS : external non-transcribed spacer and ETS : external transcribed spacer).Several plant rDNAs have been characterized by molecular cloning and restriction mapping. In many species these studies revealed an important heterogeneity between the numerous genes of a single individual. Several types of genes were reported (reviews [4, 51). Some species, such as flax or soybean, were shown to have fairly homogeneous genes; wheat, barley, rice, broad bean, mung bean, cucumber and pea present length heterogeneity; onion and radish present sequence heterogeneity. Finally other species present both length and sequence heterogeneity. Variability is .also evident at the species level and it is very often possible to distinguish between different cultivars or different wild-type populations from a given species by their restriction pattern [5 -111. Therefore, this variability seems to be very useful for plant breeding and evolutionary studies.The molecular basis for length heterogeneity has been recently elucidated. Variations in th...

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.

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Natural senescence of Cucurbita pepo (zucchini) cotyledons was accompanied by a gradual degradation of reserve proteins (globulins) and an intensive decrease in the content of both large subunit (LSU) and small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The net photosynthetic rate, the primary photochemical activity of PSII, estimated by the variable fluorescence (F v )/maximal fluorescence (F m ) ratio (F v /F m ) and the actual quantum yield of PSII electron transport in the light-adapted state (UPSII) also progressively decreased during natural senescence. In contrast, the fraction of the absorbed light energy, which is not used for photochemistry (LNU) increased with progression of senescence. The decline in the photosynthetic rate started earlier in ontogenesis compared with the down-regulation of the functional activity of PSII, thus suggesting the existence of protective mechanisms which maintain higher efficiency of the photochemical electron transport reactions of photosynthesis compared with the dark reactions of the Calvin cycle during earlier stages of natural senescence. Decapitation of the epicotyl above the senescing cotyledons resulted in full recovery of the polypeptide profile in the rejuvenated cotyledons. In addition, the photosynthetic rate increased reaching values that exceeded those measured in juvenile cotyledons. The photochemical efficiency of PSII also gradually recovered, although it did not reach the maximum values measured in the presenescent cotyledons. KeywordsCotyledons of Cucurbita pepo L. (zucchini) Á Natural senescence Á Photosynthesis Á Polypeptide pattern Á Rejuvenation Abbreviations PSIIPhotosystem II UPSIIThe actual quantum yield of PSII electron transport in the light-adapted state LSU