The nucleolar RNA-binding protein B-36 is highly conserved among plants

The nucleolar protein B-36 is an RNA-associated protein which has a number of properties in common with pre-mRNA-binding proteins (hnRNP proteins). Like the hnRNP proteins, B-36 appears to be evolutionarily conserved among various eukaryotes (protists and several animal species). The conservation of B-36 throughout the plant kingdom has been investigated using a panel of nine monoclonal antibodies previously shown to recognize a minimum of four different epitopes in Physarum B-36, the protein used to generate the monoclonal antibodies. Two of the epitopes (I and III) are widely conserved in 34 kDa proteins (presumably B-36 homologues) from the various species tested (Chlamydomonas, moss, fern, oat, onion, carrot, and bean). Using immunofluorescence localization in moss and carrot protoplasts, the cross-reacting proteins were shown to be restricted to the nucleolus, further confirming their probable homology to B-36. Epitopes I and III are also unique to the B-36 homologues as demonstrated by the failure of any other bands to cross-react. Another epitope (IV) was specifically recognized in the plant B-36 homologues but exhibited greatly reduced affinity for the monoclonal antibody relative to Physarum B-36. The remaining epitope (II), unlike the others, exhibited variable conservation in the plant B-36 homologues and, in addition, was present in several other seemingly unrelated proteins. Finally, several of the plant species exhibited two cross-reacting variants at roughly the 34 kDa position and in at least one of these cases a single monoclonal antibody was able to distinguish between the two variants, a result indicating that the variants do have bona fide structural differences.(ABSTRACT TRUNCATED AT 250 WORDS)     

cDNA encoding a wheat (Triticum aestivum cv. Chinese Spring) glycine-rich RNA-binding protein

A wheat cDNA encoding a glycine-rich RNA-binding protein, whGRP-1, was isolated. WhGRP-1 contains two conserved domains, the RNA-binding motif (RNP motif) combined with a series of glycine-rich imperfect repeats, characteristic of a conserved family of plant RNA-binding proteins. Northern analysis revealed that whGRP-1 mRNA accumulates to high levels in roots and to lower levels in leaves of wheat seedlings. whGRP-1 mRNA accumulation is not enhanced by exogenous abscisic acid in seedlings and accumulates to very high levels during wheat embryo development, showing a pattern different from that of the ABA-inducible wheat Em gene.     

In vitro plantlet regeneration from cotyledon, hypocotyl and root explants of hybrid seed geranium

In vitro plantlet regeneration systems for the seed geranium (Pelargonium x hortorum Bailey) using cotyledon, hypocotyl and root explants were optimized by studying the influence of seedling age, growth regulators and excision orientation on organogenesis. Indole-3-acetic acid combined with zeatin yielded the highest rate of shoot production on cotyledon explants (0.2–2 shoots per explant). More shoots were produced on explants cut from the most basal region of cotyledons from 2 to 4-day-old seedlings than from older seedlings or more distal cut sites. Hypocotyl explants produced the highest number of shoots, up to 40 shoots per explant, on indole-3-acetic acid (2.8–5.6 mM) + zeatin (4.6 mM) or thidiazuron (4.5 mM). Maximum shoot formation (0.3–1.4 shoots per explant) on root explants occurred when they were cultured on medium containing zeatin. Regenerated shoots rooted best on a basal medium containing no growth regulators. There were substantial differences among cultivars in shoot formation from each of the explant systems.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA naphthaleneacetic acid - TDZ thidiazuron     

The mitochondrial genome of Moniliophthora roreri, the frosty pod rot pathogen of cacao

In this study, we report the sequence of the mitochondrial (mt) genome of the Basidiomycete fungus Moniliophthora roreri, which is the etiologic agent of frosty pod rot of cacao (Theobroma cacao L.). We also compare it to the mtDNA from the closely-related species Moniliophthora perniciosa, which causes witches' broom disease of cacao. The 94 Kb mtDNA genome of M. roreri has a circular topology and codes for the typical 14 mt genes involved in oxidative phosphorylation. It also codes for both rRNA genes, a ribosomal protein subunit, 13 intronic open reading frames (ORFs), and a full complement of 27 tRNA genes. The conserved genes of M. roreri mtDNA are completely syntenic with homologous genes of the 109 Kb mtDNA of M. perniciosa. As in M. perniciosa, M. roreri mtDNA contains a high number of hypothetical ORFs (28), a remarkable feature that make Moniliophthoras the largest reservoir of hypothetical ORFs among sequenced fungal mtDNA. Additionally, the mt genome of M. roreri has three free invertron-like linear mt plasmids, one of which is very similar to that previously described as integrated into the main M. perniciosa mtDNA molecule. Moniliophthora roreri mtDNA also has a region of suspected plasmid origin containing 15 hypothetical ORFs distributed in both strands. One of these ORFs is similar to an ORF in the mtDNA gene encoding DNA polymerase in Pleurotus ostreatus. The comparison to M. perniciosa showed that the 15 Kb difference in mtDNA sizes is mainly attributed to a lower abundance of repetitive regions in M. roreri (5.8 Kb vs 20.7 Kb). The most notable differences between M. roreri and M. perniciosa mtDNA are attributed to repeats and regions of plasmid origin. These elements might have contributed to the rapid evolution of mtDNA. Since M. roreri is the second species of the genus Moniliophthora whose mtDNA genome has been sequenced, the data presented here contribute valuable information for understanding the evolution of fungal mt genomes among closely-related species.     

Infection Biology of Moniliophthora perniciosa on Theobroma cacao and Alternate Solanaceous Hosts

The C-biotype of Moniliophthora perniciosa is the causal agent of witches’ broom disease of Theobroma cacao L. While this disease is of major economic importance, the pathogenicity mechanisms and plant responses underlying the disease are difficult to study given the cacao tree’s long life cycle and the limited availability of genetic and genomic resources for this system. The S-biotype of M. perniciosa infects solanaceous hosts, particularly pepper (Capsicum annuum) and tomato (Solanum lycopersicum). These species are much more amenable for performing studies of mechanisms underpinning host-pathogen interactions as compared to cacao. A phylogenetic analysis performed in this study demonstrated that S-biotype strains clustered with C-biotype strains, indicating that these biotypes are not genetically distinct. A comparative analysis demonstrated that disease progression in tomato infected with the S- biotype is similar to that described for cacao infected with the C- biotype. The major symptoms observed in both systems are swelling of the infected shoots and activation and proliferation of axillary meristems. Cellular changes observed in infected tissues correspond to an increase in cell size and numbers of xylem vessels and phloem parenchyma along the infected stem. Observations revealed that fungal colonization is biotrophic during the first phase of infection, with appearance of calcium oxalate crystals in close association with hyphal growth. In summary, despite different host specificity, both biotypes of M. perniciosa exhibit similar disease-related characteristics, indicating a degree of conservation of pathogenicity mechanisms between the two biotypes.     

Starch-branching enzyme IIa is required for proper diurnal cycling of starch in leaves of maize

Starch-branching enzyme (SBE), a glucosyl transferase, is required for the highly regular pattern of α-1,6 bonds in the amylopectin component of starch. In the absence of SBEIIa, as shown previously in the sbe2a mutant of maize (Zea mays), leaf starch has drastically reduced branching and the leaves exhibit a severe senescence-like phenotype. Detailed characterization of the maize sbe2a mutant revealed that SBEIIa is the primary active branching enzyme in the leaf and that in its absence plant growth is affected. Both seedling and mature sbe2a mutant leaves do not properly degrade starch during the night, resulting in hyperaccumulation. In mature sbe2a leaves, starch hyperaccumulation is greatest in visibly senescing regions but also observed in green tissue and is correlated to a drastic reduction in photosynthesis within the leaf. Starch granules from sbe2a leaves observed via scanning electron microscopy and transmission electron microscopy analyses are larger, irregular, and amorphous as compared with the highly regular, discoid starch granules observed in wild-type leaves. This appears to trigger premature senescence, as shown by an increased expression of genes encoding proteins known to be involved in senescence and programmed cell death processes. Together, these results indicate that SBEIIa is required for the proper diurnal cycling of transitory starch within the leaf and suggest that SBEIIa is necessary in producing an amylopectin structure amenable to degradation by starch metabolism enzymes.     

Rapid,efficient production of homozygous transgenic tobacco plants withagrobacterium tumefaciens: A seed-to-seed protocol

An optimized complete protocol was developed forAgrobacterium tumefaciens-mediated transformation of tobacco (Nicotiana tabacum L. cultivar SR1), producing T₁ flowering plants homozygous for the inserted T-DNA as verified by kanamycin resistance in T₂ seedlings in 6 to 7 months from the time of cocultivation withAgrobacterium. Previous protocols require up to 9 to 12 months to obtain similar results. Procedures unique and important to this protocol include; a modified “whole-leaf” transformation coupled with a long duration of cocultivation, resulting in high rates of transformation, high levels of kanamycin in selection media resulting in few escapes, and extensive rooting of regenerants prior to a greenhouse hardening procedure. Once in the greenhouse, primary regenerants were maintained in small containers with long day photoperiod and high light levels, greatly shortening the time to seed set. Flowers from primary transformants were bagged to allow self pollination, and seed capsules harvested and dried prior to normal maturation on the plant. T₁ and T₂ seeds were plated and selected on kanamycin media by an improved seed plating technique which eliminates the need for the placement of individual seeds, saving time and improving selection homogeneity. Using this protocol, over 130 independent tobacco lines from six separate gene constructs have been generated in a very short time period. Of these 130, nearly 60 percent segregated 3∶1 for kanamycin resistance: susceptibility, indicating single transgene insertion events.