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Characterization and Complexity of Wheat Developing Endosperm mRNAs
1Laboratoire d'Etude des Protéines, Département de Physiologie et Biochimie végétales, Centre I.N.R.A., route de St Cyr, 78000 Versailles, France.
Abstract:
Free and membrane-bound (MB) polysomes and the corresponding polyadenylated RNAs (polyA(+) RNAs) have been isolated from developing wheat endosperm (Triticum aestivum L.) Free and MB poly(A)(+) RNAs, analyzed on isokinetic sucrose gradient with [(3)H]polyuridylic acid [poly(U)] hybridization detection, appear to be 11S to 12S in size with a 7% poly(A) tail for MB RNAs. cDNAs synthesized using both of these mRNA populations in presence of a potent RNase inhibitor (RNasin), have been used for hybridization kinetics experiments. The mean square fitting analysis of the hybridization kinetics between MB cDNA and its template reveals the presence of two abundance classes representing roughly (2/3) and (1/3) of the MB poly(A)(+) RNAs and containing the information for approximately 75 superabundant species (21,000 copies per cell) and 750 intermediate species (530 copies per cell), respectively. The mRNA population extracted from free polysomes is divided into three abundance classes. The first one is composed of superabundant sequences which would correspond to the MB superabundant mRNAs. The free mRNAs consist of about 11,000 diverse sequences, most of them being rare sequences. Heterologous hybridizations of MB cDNAs to free mRNAs have shown that some mRNAs are common to both populations. This could be explained either by a partial contamination or by free polysomes en route to their membrane destination. Contrary to the low number of diverse mRNAs corresponding to the legume seed storage proteins, the wheat endosperm superabundant mRNAs consist of about 75 different sequences which would encode most of the seed storage proteins, especially gliadins.
Insights
Wheat endosperm studies reveal distinct mRNA populations in free and membrane-bound polysomes. Membrane-bound RNAs encode abundant seed storage proteins, primarily gliadins, while free polysomes contain diverse, mostly rare, sequences.
Area of Science:
- Molecular Biology
- Plant Biochemistry
- Gene Expression
Background:
- Developing wheat endosperm utilizes distinct polysome populations for protein synthesis.
- Polyadenylated RNAs (polyA(+) RNAs) are crucial for translation regulation.
Purpose of the Study:
- To characterize and compare mRNA populations associated with free and membrane-bound (MB) polysomes in wheat endosperm.
- To determine the abundance classes and diversity of mRNAs in these fractions.
Main Methods:
- Isolation of free and MB polysomes and their associated polyA(+) RNAs from developing wheat endosperm (Triticum aestivum L.).
- Analysis of RNA size and poly(A) tail length using sucrose gradient centrifugation and hybridization.
- Synthesis of complementary DNAs (cDNAs) and hybridization kinetics experiments to quantify mRNA abundance classes.
Main Results:
- MB poly(A)(+) RNAs (11S-12S) possess a 7% poly(A) tail and comprise two abundance classes: superabundant (75 species) and intermediate (750 species).
- Free polysomal mRNA is divided into three classes, including superabundant sequences overlapping with MB mRNAs and a large population of rare sequences (~11,000 diverse sequences).
- Some shared mRNAs between free and MB populations suggest potential contamination or polysome trafficking.
Conclusions:
- Wheat endosperm superabundant mRNAs, primarily encoding seed storage proteins like gliadins, are concentrated in MB polysomes.
- Free polysomes contain a greater diversity of mRNAs, predominantly rare sequences, with some overlap with MB populations.
- The findings provide insights into the differential distribution and function of polysomal mRNA populations during wheat seed development.
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