Leon Dure

Common amino acid sequence domains among the LEA proteins of higher plants

LEA proteins are late embryogenesis abundant in the seeds of many higher plants and are probably universal in occurrence in plant seeds. LEA mRNAs and proteins can be induced to appear at other stages in the plants life by desiccation stress and/or treatment with the plant hormone abscisic acid (ABA). A role in protecting plant structures during water loss is likely for these proteins, with ABA functioning in the stress transduction process. Presented here are conserved tracts of amino acid sequence among LEA proteins from several species that may represent domains functionally important in desiccation protection. Curiously, an 11 amino acid sequence motif is found tandemly repeated in a group of LEA proteins of vastly different sizes. Analysis of this motif suggests that it exists as an amphiphilic α helix which may serve as the basis for higher order structure.

Developmental biochemistry of cotton seed embryogenesis and germination. VII. Characterization of the cotton genome.

Abstract The DNA of cotton, Gossypium hirsutum, has been characterized as to spectral characteristics, buoyant density in CsCl, base composition, and genetic complexity. The haploid genome size is found to bo 0.795 pg DNA/cell. However, the amount of DNA per cell in the cotyledons increases during embryogenesis to an average ploidy level of 12N in the mature seed cotyledons. Reassociation kinetics indicate that this increase is due to endoreduplication of the entire genome. Non-repetitive deoxynucleotide sequences account for approximately 60.5% of the cotton genome ( C 0 t 1 2 pure ¶ = 437); highly repetitive sequences (> 10,000 repetition frequency) constitute about 7.7% of the genome. ( C 0 t 1 2 pure = 4.6 × 10 −4 ) and intermediately repetitive sequences constitute the remaining 27% of the genome ( C 0 t 1 2 pure = 1.46 ). Hybridization of 125I-labeled cytoplasmic ribosomal RNA to whole-cell DNA on filters and in solution indicate approximately 300 to 350 copies of the rRNA cistrons per haploid genome. The interspersion of repetitive sequences that reassociate between C0t values of 0.1 and 50 with non-repetitive sequences of the cotton genome has been examined by determining the reassociation kinetics of DNA of varying fragment lengths and by the electron microscopy of reassociated molecules. About 60% of the genome consists of non-repetitive regions that average 1800 base-pairs interpersed with repetitive sequences that average 1250 base-pairs. Approximately 20% of the genome may be involved in a longer period interspersion pattern containing non-repetitive sequences of approximately 4000 base-pairs between repetitive sequences. Most of the individual sequences of the interspersed repetitive component are much smaller than the mass average size, containing between 200 and 800 base-pairs. Sequence divergence is evident among the members of this component. Highly repetitive sequence elements that are reassociated by a C0t value of 0.1 average 2500 base-pairs in length, appear to have highly divergent regions and do not appear to be highly clustered. A portion of this highly repetitive component reassociates by C0t = 10−4, zero-time binding DNA, and accounts for less than 3% of the genome. At least a third of these sequences appear by electron microscopy to be intramolecular duplexes (palindromes) of 150 to 200 base-pairs and to occur in clusters.

LONG-LIVED MESSENGER RNA: EVIDENCE FROM COTTON SEED GERMINATION.

In germinating cotton embryos the partial inhibition of RNA synthesis by actinomycin D does not inhibit the incorporation of leucine-14C into soluble protein nor cause a loss of polyribosomes during the first 16 hours of germination. This suggests that the protein synthesis observed during this period is directed by messenger RNA which exists in the mature seed and which is not rapidly degraded and resynthesized.

The globulin seed storage proteins of flowering plants are derived from two ancestral genes

SummaryThe cDNA and/or genomic DNA sequences of 13 globulin storage proteins from flowering plants (angiosperms) are now known. They represent 8 genera, 5 families and 5 orders of plants and include one monocotyledonous species. Here, the coding nucleotide and amino acid sequences of these proteins are compared by dot matrix analysis and gross protein domains visualized by hydropathy analyses. The vestigial homologies visualized by these means indicate that all of the globulin storage proteins of flowering plants have emanated from 2 genes that existed at the beginning of angiosperm evolution.A curious polypeptide domain of 150–200 amino acids located near the N terminus is found in a globulin subgroup of 2 genera widely separated phylogenetically. The domain appears to have resulted from an ancient insertion that has been deleted in most of its descendant genes.

Ribonucleic acid synthesis in germinating cotton seeds.

The synthesis of RNA has been mapped during the initial (dark) phase of germination in cotton seed. During the first 16 hours whole embryos synthesize new ribosomes and protein. Treatment of whole embryos with cycloheximide during this period inhibits protein synthesis and stops further development. Actinomycin D inhibits the synthesis of new ribosomes. This inhibition does not decrease the total ribosomal population, inhibit the synthesis of protein, or retard normal development during this time. These observations suggest that the protein synthesis observed during this period is directed by stable messenger RNA and ribosomes which exist preformed in the mature seed. The synthesis of ribosomes ceases after about 40 hours in the cotyledons and 48 hours in the roots and stems. After this time the synthesis of ribosomal RNA continues but it does not become incorporated into ribosomes. Rather, this RNA is incorporated into a particle, detectable only by radioactivity, which sediments more slowly than monomeric ribosomes on a sucrose density-gradient. On prolonged incubation, the radioactivity associated with the particle moves into mature ribosomes. The particle appears to be an intermediate in the synthesis of ribosomes and has properties similar to those reported for ribosomal precursor particles in bacteria. Its synthesis is completely suppressed by light. During the period of precursor particle synthesis in the roots and stems, protein and RNA synthesis is necessary for further development as determined by inhibition studies with cycloheximide and actinomycin D. The required RNA synthesis is possibly messenger RNA. In cotyledons, during this period, protein synthesis appears to be required for normal development. Whether this protein synthesis is dependent upon concurrent RNA synthesis cannot be evaluated by the use of actinomycin D, probably due to an inability to get the antibiotic into the cells during this period.

Hormonal regulation of translation inhibition requiring RNA synthesis

Abstract The control of the translation inhibition of germination mRNAs during cotton embryogenesis has been studied by determining the events necessary to remove the translation inhibition during precocious germination. The results indicate that the plant hormone abscisic acid may control translation inhibition by a mechanism involving RNA synthesis.

Developmental biochemistry of cottonseed embryogenesis and germination XVIII cDNA and amino acid sequences of members of the storage protein families

SummaryWe have sequenced cDNA clones representing each of the three distinct groups of storage proteins of the cotton seed. Characteristics of their mRNAs and derived proteins are given. Dot matrix analysis of the nucleotide and amino acid sequences shows that 2 of these groups of proteins have a great deal of vestigial homology at low stringency and should be considered subfamilies of a single storage protein gene family. The remaining group is quite distinct and should be considered a separate multigene family. It also can be divived into 2 subfamilies based on the presence or absence of glycosyl residues and other sequence differences.These proteins are processed to smaller species during embryogenesis, and all of the mature storage proteins of cotton can be traced back to these 2 gene families.In view of these relationships we propose that these 2 families be called the α and β globulins of cotton storage proteins, each comprised of an A and B subfamily.

Action of 3′d adenosine (cordycepin) and 3′d cytidine on the translation of the stored mRNA of cotton cotyledons

Abstract Cordycepin (3′dAdo), an inhibitor of RNA synthesis and poly(A) addition to pre-mRNA, inhibits protein synthesis and the appearance of enzyme activity thought to come from stored mRNA in germinating cotton cotyledons. 3′dCyd and Actinomycin D, other RNA synthesis inhibitors, have no effect on early germination. The period of sensitivity to 3′dAdo is short, comprising the 6th–30th hour of germination. This is consistent with the idea that the stored mRNA of cotton cotyledons is not processed until germination.

Developmental biochemistry of cottonseed embryogenesis and germination : XVI. Analysis of the principal cotton storage protein gene family with cloned cDNA probes.

DNAs complementary to the mRNAs coding for the major cotton 48 kD and 52 kD storage proteins have been cloned and used to characterize the principal cotton storage protein gene family. The principal storage proteins are found to emanate from three subsets of genes that share some homology, as shown by common antigenic determinants shared by the proteins themselves, but that are distinguishable by nucleic acid hybridization. A single sequence subfamily of 2.26 kb mRNAs codes for the 69 kD preproteins (precursors to the mature 48 kD proteins) and two sequence subfamilies of 1.96 kb mRNAs each code for 60 kD preproproteins (precursors to the mature 52 kD proteins). Hybrid arrested translation shows that cloned members of these three subfamilies hybridize only with the mRNAs of a single subfamily at moderate criterion. These three subfamilies comprise all of the principal storage protein mRNAs detectable byin vitro translation. With hybridization at low criterion, some homology has been detected between the two 1.96 kb mRNA families, although no homology has yet been detected between the 2.26 kb mRNA family and either of the two 1.96 kb mRNA families.

Developmental biochemistry of cottonseed embryogenesis and germination. XIX. Sequences and genomic organization of the α globulin (vicilin) genes of cottonseed

The α globulin storage protein genes of cotton are found to exist as gene tandems that contain a gene from each of the 2 α globulin subfamilies separated by a spacer region of about 2700 or 3400 base pairs. Three different tandems have been identified by restriction endonuclease mapping of genomic DNA. A cDNA that is different from the genes of the tandems in map sites and/or in nucleotide sequence indicates that a fourth tandem probably exists in the cotton genome. Since the species of cotton used here (Gossypium hirsutum) is an amphidiploid, it is likely that two of the tandems are contributed from each genome.Considerable divergence in nucleotide sequence (18%) and in derived amino acid sequence (28%) is found when the 2 genes of a sequenced tandem are compared. The sequence of the cDNA closely resembles one of the genes in the tandem showing only a 4% divergence in nucleotides and a 4.2% divergence in amino acids. Thus the 2 genes of each tandem represent a relatively ancient gene duplication that has given rise to the two α globulin subfamilies of cotton. Only one subfamily has a glycosylation site and the glycosylation of its derived proteins gives rise to the 2 molecular weight sets of α globulins seen on gel electrophoresis.Other basic features of these genes and their derived proteins are presented.