The role of intercellular signals in plant development was investigated using phytochrome-induced formation of anthocyanin in cotyledons of white mustard as a model system. The problem was approached by irradiating different subregions of the cotyledon with a microbeam. This technique was combined with in situ hybridization of chalcone synthase mRNA after irradiation of the entire cotyledon. Individual cells that exhibited all-or-none responses with a resultant stochastic, patchy pattern were examined during early stages of anthocyanin synthesis. It was demonstrated that the responses of individual cells were subsequently integrated by long-range inhibitory signals. This process led to ordered and gradually developing patterns that could be detected when final stages were analyzed at the whole-organ level. The significance of these findings is discussed in terms of efforts toward a general understanding of photomorphogenesis in plants.
Genomic DNA from mustard was cloned in EMBL4 and screened for chalcone synthase (CHS) genes using a heterologous cDNA probe from parsley. Two clones which hybridized with the parsley cDNA probe were isolated. They showed different restriction patterns. One clone was sequenced and identified as a CHS gene by sequence comparison with published CHS sequences. The sequence of the coding region is 1188 bp, and encodes a protein of 43 kDa. The start-point of transcription was determined by primer extension. The sequence of 0.9 kbp at the 5' end of the transcription start and part of the noncoding 3' of this gene were also determined. The coding sequence is interrupted by a single intron of 523 bp. The coding and the noncoding 5' sequence of this gene was compared with CHS genes from other species. A very high homology was found with the Arabidopsis CHS coding region. A sequence motif (CACGTGT) which is present in most rbcS and all CHS upstream regions, and which specifically binds a protein factor from plant nuclear extracts, is also present in the upstream region of the mustard CHS gene. Measurements of CHS transcript levels show that phytochrome controls expression of this gene in cotyledons of mustard seedlings; however, blue/UV-light photoreceptors control expression in later stages of development.
SummaryPhotoregulation of chalcone synthase (CHS) mRNA accumulation was analysed in parsley (Petroselinum crispum) and mustard (Sinapis alba) plants at different developmental stages. In both species, CHS mRNA accumulation in young etiolated seedlings was primarily under phytochrome control. In leaves of adult reetiolated plants, a UV-B photoreceptor was predominantly involved in photocontrol. The reduced red light control in mature leaves was not due to the absence of immunoreactive phytochrome. The apparent dependence of photoreceptor usage on the developmental state of the cell or organism was in accordance with observations on the photoregulation of fusion constructs between CHS promoters from parsley or mustard and the P-glucuronidase reporter gene (GUS). When tested in the parsley protoplast transient expression system, both constructs yielded the same type of photoregulation as observed for the endogenous CHS gene.
I n cotyledons of etiolated mustard (Sinapis a b a L.) seedlings, phytochrome-far-red-absorbing forminduced flavonoid biosynthesis was found to be inhibited by short-term ultraviolet .(UV) irradiations. UV inhibition was shown for the synthesis of quercetin, anthocyanin, and also for the accumulation of the mRNA for chalcone synthase, the key enzyme of this pathway. The UV effect was more pronounced on flavonoid biosynthesis, a process that selectively occurs i n the epidermal layers, than on the synthesis of mRNA for chlorophyll a/b-binding protein localized in the mesophyll tissue. These UV inhibitory effects were accompanied by cyclobutane pyrimidine dimer (CPD) formation showing a linear fluence-response relationship. CPD formation and UV inhibition of flavonoid biosynthesis was found to be partially reversible by blue/UV-A light via DNA photolyase (PRE), allowing photoreactivation of the DNA by splitting of CPDs, which are the cause of the UV effect. Like flavonoid formation PRE was also induced by the far-red-absorbing form of phytochrome and induction was inhibited by UV. A potentia1 risk of inhibition, in response to solar UV-B irradiation, was shown for anthocyanin formation. This inhibition, however, occurred only if photoreactivation was experimentally reduced. The PRE activity present in the etiolated seedlings (further increasing about 5-fold during light acclimatization) appears to be sufficient to prevent the persistence of CPDs even under conditions of high solar irradiation.
Import of preproteins into mltochondria requires transport machineries in both mitochondrial membranes that have been characterized in Saccharomyces cerevisiae and Neurospora crassa. By cDNA analysis, we identified a human protein of 16 kDa with significant overall homology to the fungal mitochondrial import receptor Mom19, including the three typical characteristics: a hydrophobic N-terminal segment, a tetratrico peptide motif in the middle and a negatively charged C-terminus. The human Mom19 homolog is expressed in all tissues analyzed. When synthesized in vitro, the human Mom19 homolog is targeted to isolated yeast mitochondria and specifically associates with the outer membrane receptor complex, suggesting that indeed a mitochondrial import receptor was identified.
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