Sequence Characteristics and Transcripts of rbcS Genes from Brassica napus: Temporal and Spatial Expression During Crucifer Seedling Morphogenesis*

Abstract

The small subunit of ribulose‐1,5‐bisphosphate carboxylase/oxygenase is encoded by a nuclear multigene family (rbcS genes). We have cloned and characterized three rbcS cDNA sequences from Brassica napus. These cDNA clones all appear to encode the same protein, but they differ in their nucleotide sequence, which has been exploited in the construction of clone‐specific oligonucleotides as selective hybridization probes. By using a nonselective RNA probe, the temporal expression of the entire rbcS gene family was analyzed during seedling development of Brassica napus and of Sinapis alba. In both crucifer species, rbcS transcripts show transient peak levels and then decrease, although to a different degree. Only a moderate (twofold) difference in transcript pool sizes is observed in light‐grown versus dark‐grown seedlings at the time of peak levels, while a much higher light/dark ratio is found in late‐stage seedlings. The oligonucleotide probes reveal three subsets of transcripts which differ in their accumulation kinetics and light/dark ratio. Assessment of the spatial distribution by using in situ hybridization indicates that rbcS transcripts are uniformly localized in cross‐sections of cotyledons from either light‐grown or dark‐grown seedlings, whereas they are undetectable in root sections.

Temporal and spatial pattern of plastid gene expression during crucifer seedling development and embryogenesis

“…3 F, H), both in the case of rbcS and psbA transcripts. This reflects the higher transcript levels in light-grown as compared to dark-grown seedlings determined by Northern hybridization (Hughes et al 1987;Fiebig et al 1990). A similar distribution of rbcS and psbA transcripts across sections of cotyledons has been observed in the case of two other crucifer species, Brassica napus and Arabidopsis thaliana (data not shown).…”

“…standard for quantifi- to 30 h after sowing, (ii) a major accumulation of transcripts up to a maximum around 60 h, (iii) a decrease in rbcS transcript levels during later stages of seedling development, and (iv) an approximately twofold higher accumulation at the 60-h peak levels in light-grown than in dark-grown seedlings, but a much higher light/dark ratio at the later stages. Analogous hybridization experiments using RNA from Sinapis alba cotyledons have shown that the temporal expression characteristics of the rbcS gene family during seedling development are very similar in the two closely-related crucifer species (Fiebig et al 1990). …”

“…0.8-kb transcript by using these cDNA sequences as probes (Fiebig et al 1990). To determine relative rbcS transcript levels during seedling development in Brassica napus, a Northern-blot analysis was carried out with an antisense RNA probe that represents cloned rbcS sequences.…”

“…The expression of rbcS genes in Brassica napus thus resembles the temporal expression pattern of light-modulated chloroplast genes in Sinapis alba (Dietrich etal. 1987;Hughes et al 1987) and also that of the rbcS gene family in Sinapis alba (Fiebig et al 1990). It appears that the expression of this nuclear gene family is likewise characterized by a light-independent 'competence' phase (Mohr 1984) as a prerequisite for further modulation of transcript levels by light (Oelmfiller et al 1986).…”

Sequence and expression characteristics of three g-Box-binding factor cdnas from brassica napus

“…Riboprobes, complementary to rbcS and GBF mRNAs (i.e. antisense RNAs), were labelled by incorporation of [c(3~p]UTP during in vitro transcription oflinearized plasmids pBnPTrc 1 [6,10] and pBnGBFlb and 2a with T7 RNA polymerase. After 6 h of prehybridization at 65 °C in a solution containing 50~o formamide, 5 x SSC, Complete removal of the probe was checked by long-term exposure to X-ray films.…”