Verena Cameron-Mills

Transcriptional and post-transcriptional regulation of gene expression in developing barley endosperm

SummaryThe expression of the genes encoding B-, C-, D- and γ-hordein, protein Z, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and histone H3 has been studied in developing barley endosperms. The levels of the mRNAs encoding hordein and protein Z increase 3- to 4-fold from 8 to 25 days after anthesis and thereafter decrease, whereas the GAPDH and histone H3 mRNAs are constant from 8 to 15 days and then decline. B- and C-hordein mRNAs are 7 to 100 times more abundant than any of the other mRNA species. Analysis of the transcription rate of the genes encoding B-, C- and D-hordein and protein Z in isolated endosperm nuclei revealed that the rate is correlated with their copy number in the genome. A comparison of the steady-state levels of endosperm-specific mRNA species with the in vitro transcription rates of their respective gene families suggests that the high levels of B- and C-hordein mRNAs are the result of post-transcriptional regulation. Mutanthor 2ca, defective in B-hordein synthesis, has a substantial increase in the level of mRNAs coding for C-hordein and GAPDH, whereas the levels of the other mRNAs are unaffected. In mutantlys 3a, which fails to synthesize the major storage polypeptides, the mRNA levels of B-, C- and γ-hordein and protein Z are dramatically reduced. The in vitro transcription activity of the genes encoding B- and C-hordein and protein Z is 50-fold lower inlys 3a nuclei than in the wild type, accounting for the reduced level of mRNAs and storage protein synthesis in this mutant.

Primary structure of A B1 hordein gene from barley

The 873 base pair coding region of a Hor-2 gene of barley and the adjacent 550 base pair upstream and 413 base pair downstream regions were sequenced. The gene is devoid of introns and encodes a 271 amino acid long B1 hordein polypeptide containing a putative 19 amino acid signal peptide. The remaining part of the coding sequence can be divided into three parts. In the 53 residue amino-terminal region there are 9 glutamine-proline blocks with a preferred core sequence PQQP, separated by one or two other residues giving a glutamine proline content of 78%. The second part encodes 164 amino acids, 41% of which are glutamine+proline organised in scattered blocks. Seven cysteine residues are coded for by this portion of the gene. The last part encodes the carboxyterminal 35 amino acids none of which is glutamine.In the 550 base pair upstream region the sequence TATAAATA is found at- 71 base pairs from the initiator methionine. In the 3′ non-coding region three putative polyadenylation signals, AATAAA, are present.Comparison of the gene with 3 partial cDNA clones indicates that the charge polymorphism in the B1 polypeptide group is due to point mutations in the part of the gene corresponding to the carboxy terminal half of the polypeptide. Comparison with the sequence of a second B hordein gene suggests that insertions or deletions of glutamine-proline blocks in the amino-terminal domain are a major source of size polymorphisms in the B hordein family.

Protein body formation in the developing barley endosperm

The ultrastructure of the pericarp, testa, aleurone and endosperm in a developing barley grain is presented in the form of a reconstructed section cut tangential to the dorsal surface of the grain and extending half way to the center of the endosperm.Protein body formation in the endosperm is examined in Carlsberg II and Bomi barley and two mutants defective in hordein synthesis. Protein bodies of complex morphology are deposited in large as well as small vacuoles. They comprise clusters of homogeneous components, embedded in a fibrillar matrix, associated with electron-dense spheres and numerous vesicles. The fibrillar matrix is interpreted to be a transient stage in the condensation of storage proteins into a homogeneous structure. The polypeptide composition of protein bodies determines their ultrastructure. The reduction in the synthesis of ‘B’ type hordein in mutant Risø 56 increases the proportion of the fibrillar matrix, while a more drastic alteration in storage protein condensation is observed in mutant Risø 1508, which is highly deficient in both ‘B’ and ‘C’ type hordein polypeptides.

Amber codon suppression: the in vivo and in vitro analysis of two C-hordein genes from barley

A 1420 bp genomic fragment (λ-horl-17) encompassing a Hor-1 gene encoding a C-hordein polypeptide is presented. The deduced amino acid sequence is 261 residues long. It comprises a 20 amino acid signal peptide, unique NH2- and COOH-terminal regions and a coding region comprised of pentapeptide (PQQPY) and octapeptide (PQQPFPQQ) repeat motifs. The 431 bp of 5′ non-coding region contains a ‘TATA box’ at −105, a ‘CACA box’ (−181 to −201) and a −300 prolamin element. In the 3′ noncoding region there are two putative polyadenylation signals located 88 and 142 bp downstream of the stop codon.The structure of λ-hor1-17 is compared with that of another gene (λ-hor1-14) encoding a C-hordein polypeptide, which contains an amber codon interrupting the ORF. A functional assay in which the 5′ non-coding regions of the two genes were fused to the β-glucuronidase (GUS) gene demonstrated that both genes were transcriptionally active and that circa 430 bp of the C-hordein promoters were sufficient to drive the expression of the GUS gene in developing barley endosperms. It also demonstrated that both promoters had transcriptional efficiencies comparable with that of the 35S CaMV promoter. The in vitro translation of the coding region of λ-hor1-14 in the wheat germ system showed that the premature stop codon could be partially suppressed. The suppression was also demonstrated in a transient expression assay in vivo using isolated barley endosperms.

A γ-hordein gene

The 1614 bp nucleotide sequence of a barley gene encoding a γ-hordein endosperm storage polypeptide is presented. The deduced amino acid sequence is 305 amino acids long. It comprises a 19 amino acid signal peptide, an N-terminal half composed of proline-glutamine blocks organized in repeating units and a C-terminal half where the repeats are dispersed and less conserved. The deduced amino acid sequence shows strong homology to a γ-gliadin polypeptide from wheat and a γ-secalin polypeptide from rye and less homology to a B1 hordein polypeptide from barley. The 378 bp 5′ non-coding region contains a TATA box at-85, an AGGA sequence at-105 and a-300 element typical of prolamin storage protein genes. The transcript start is 56 bp upstream of the ATG codon and 30 bp downstream of the TATA box. The 318 bp 3′ non-coding region contains 2 putative polyadenylation signals, 76 and 132 bp downstream of the stop codon. γ-Hordein polypeptides are encoded by a small multigene family. The γ-hordein gene family is not part of the deleted chromosome 5 region, containing the Hor 2 locus, in the B hordein-deficient mutant hor 2ca. Two mRNA size classes of 1350 and 1450 nt are detectable in wild-type endosperms from 8 to 26 days after anthesis. The mutant hor 2ca contains as much γ-hordein mRNA as the wild type, whereas the B and C hordein-deficient mutant lys 3a contains barely detectable amounts.

Transfer of in vitro synthesized barley endosperm proteins into the lumen of the endoplasmic reticulum

Microsomes were prepared from 20 day old Bomi barley endosperm by sucrose density gradient centrifugation. Electron microscopy revealed the presence of ribosome-studded vesicles in the material banding at the 1.75–2.26m sucrose interface. The isolated microsomes were active in the wheat-germ cell-free protein synthesizing system. Hordeins were present among the in vitro synthesized products and were identified by their solubility in 55% isopropanol and by their co-migration with native hordeins on SDS-polyacrylamide gels. The microsome-and polysome-directed, in vitro synthesized hordeins were analysed before and after chymotrypsin treatment by SDS-polyacrylamide gel electrophoresis. The hordeins were degraded when polysomes were used as a template. However, hordeins synthesized on microsomes showed significant protection from proteolysis. This protection could be abolished by treatment with membrane-solubilizing detergents. The reported experiments show that hordeins synthesized on microsomes were discharged vectorially into the lumen of the microsomes.

In vitro synthesis and transport of barley endosperm proteins: Reconstitution of functional rough microsomes from polyribosomes and stripped microsomes

Synthesis of hordein, a hydrophobic storage protein of developing barley endosperms, and its transport into the lumen of the endoplasmic reticulum has been studied in vitro. Microsomes of the rough endoplasmic reticulum have been prepared from 20 day old Bomi barley endosperms and their ability to synthesize all the major hordein polypeptides during in vitro cell-free protein synthesis demonstrated. Some 50% of these hordein polypeptides are discharged into the lumen of the microsome and are consequently inaccessible to chymotrypsin digestion.Stripped microsomes, derived from rough microsomes by EDTA-treatment, have been combined with detached polysomes from Bomi endosperm during cell-free protein synthesis. An in vitro reconstitution of rough microsomes ensued, with a proportion of all the synthesized hordein polypeptides being discharged vectorially into the microsomal lumen. Both co-and post-translational transport of processed hordein polypeptides has been demonstrated with reconstituted Bomi rough microsomes.For mutant no. 1508, which is deficient in hordein synthesis, in vitro synthesis of certain hordein polypeptides has only been demonstrated on detached polysomes isolated from mutant endosperms. Not only are mutant rough microsomes unable to synthesize and transport hordein polypeptides, but attempts to reconstitute active hybrid rough microsomes from Bomi polysomes and mutant stripped microsomes or vice versa were unsuccessful.

The structure and composition of protein bodies purified from barley endosperm by silica sol density gradients

A Percoll density gradient has been devised for the separation of protein bodies from cytoplasmic membrane components of barley endosperm. Protein bodies band isopycnically at a density of 1.13 g.ml−1, whereas stripped and smooth microsomes band at a density of 1.05 g.ml−1 in a sigmoidal Percoll density gradient generated in situ. Sucrose density gradients are shown to be unsuitable for the isolation of immature barley protein bodies owing to the isopycnic banding of smooth microsomes together with immature protein bodies at a density of 1.18 g.ml−1.The complex ultrastructure of protein bodies observed in situ in barley endosperm is preserved in the isolated protein bodies prepared from Bomi and Carlsberg II barley and their high-lysine mutants Risø 1508 and Risø 56, respectively. The changes in protein body structure observed in these high-lysine mutants are thought to reflect the qualitative and quantitative changes in hordein biosynthesis caused by these gene mutations.Electrophoretic analysis of the polypeptide composition of the purified protein bodies reveals that all the major hordein polypeptides synthesized in barley endosperm are stored in protein bodies. However, several additional polypeptides, which are not hordein, are specifically associated with protein bodies of each barley genotype.

The signal peptide cleavage site of a B1 hordein determined by radiosequencing of the in vitro synthesized and processed polypeptide

The major storage proteins which accumulate in the endosperm of the developing barley grain are the hordein polypeptides. They are synthesized on membrane-bound polysomes and co-translationally transported into the lumen of the ER and are ultimately deposited in the vacuole. The processing step, which accompanies transport across the ER membrane, was studied in vitro with the B1 hordein gene, λhor2-4 in the expression plasmid pDS6. Microsomes were prepared from barley endosperm which transported and processed the B1 hordein polypeptide to a mature form with a high degree of efficiency. Because the B1 hordein was previously found to be NH2-terminally blocked, the site of processing was unknown. The in vitro synthesized precursor and processed B1 hordein polypeptides were subjected to N-chlorosuccinimide peptide mapping and NH2-terminal radiosequencing. The signal peptide cleavage site, predicted from the deduced amino acid sequence, was confirmed.