Effects of promoter,intron and enhancer elements on transient gene expression in sugar-cane and carrot protoplasts

Various chimaeric promoter regions coupled to the uidA -glucuronidase gene were evaluated for transient expression strength following electroporation into sugar-cane (monocot) and carrot (dicot) protoplasts. Multiple enhancer elements increased expression in sugar-cane, by up to 400-fold for the artificial Emu promoter relative to the CaMV 35S promoter. The relative expression strengths of promoters varied substantially between the species. Sugar-cane also differed in some respects from previously tested species in the family Poaceae. For example, in sugar-cane the nopaline synthase and CaMV 35S promoters were of equivalent strength, and insertion of Adh1 intron 1 into the 5 transcribed region decreased expression strength.     

Direct DNA transfer using electric discharge particle acceleration (ACCELL™ technology)

Direct DNA transfer methods based on particle bombardment have revolutionized plant genetic engineering. Major agronomic crops previously considered recalcitrant to gene transfer have been engineered using variations of this technology. In many cases variety-independent and efficient transformation methods have been developed enabling application of molecular biology techniques to crop improvement. The focus of this article is the development and performance of electric discharge particle bombardment (ACCELL™) technology. Unique advantages of this methodology compared to alternative propulsion technologies are discussed in terms of the range of species and genotypes that have been engineered, and the high transformation frequencies for major agronomic crops that enabled the technology to move from the R&D phase to commercialization. Creation of transgenic soybeans, cotton, and rice will be used as examples to illustrate the development of variety-independent and efficient gene transfer methods for most of the major agronomic crops. To our knowledge, no other gene transfer method based on particle bombardment has resulted in variety-independent and practical generation of large numbers of independently-derived crop plants. ACCELL™ technology is currently being utilized for the routine transfer of valuable genes into elite germplasm of soybean, cotton, bean, rice, corn, peanut and woody species.     

Characterization of a rice gene family encoding root-specific proteins

Two cDNA clones (RCc2 and RCc3) corresponding to mRNAs highly expressed only in root tissues of rice (Oryza sativa L.) seedlings were characterized. Respectively, they encode polypeptides of 146 (14.5 kDa) and 133 amino acids (13.4 kDa) that share high (<70%) sequence similarity with a polypeptide encoded by a cDNA (ZRP3) encoding an mRNA preferentially expressed in young maize roots. Genomic DNA blot analysis revealed that they are members of a small gene family and RCg2, the gene corresponding to RCc2, was isolated. A 1656 bp 5-upstream sequence of RCg2 was translationally fused to a -glucuronidase (GUS) reporter gene and stable introduction of the chimeric construct into rice was confirmed by PCR and genomic DNA blot analyses. Histochemical analysis of transgenic rice plants containing the full-length chimeric gene showed high levels of GUS activity in mature cells and the elongation and maturation zones of primary and secondary roots, and in the root caps, but no GUS activity was detected in root meristematic regions. Surprisingly, high GUS activity was also detected in leaves of the same plants. This raises the possibility that the RCg2 5-upstream element may not be sufficient for the proper spatial control of root specificity in transgenic rice.     

Thecab-m7 gene: a light-inducible,mesophyll-specific gene of maize

Southern blot analysis has revealed the existence in maize of perhaps 12 members of the nuclearcab multigene family encoding the chlorophylla- andb-binding proteins of the photosystem II light-harvesting complex. Hybridization with 3 probes derived from unsequenced cDNA clones showed that six members of this family differ from one another with respect to expression in mesophyll and/or bundle sheath cells and regulation by light. An additional member of this family, designatedcab-m7, that encodes a 28 kDa primary translation product has now been identified. It has been cloned from a maize genomic library and sequenced to begin to define the bases for differences in the expression of these genes. Thiscab gene is shown to be strongly preferentially expressed in the mesophyll (vs. bundle sheath) cells of maize. Furthermore, the gene is photo-responsive; although small amounts ofcab-m7 mRNA are present in etiolated leaves, the mRNA pool is 8-fold larger after six hours of illumination. DNA sequences upstream of thecab-m7 gene resemble those found in the 5-flanking regions of some other plant genes.     

Male flowers in Tulipa pumila Moench (Liliaceae) potentially originate from gender diphasy

We found functionally male individuals in an otherwise hermaphroditic population of Tulipa pumila (Liliaceae) located in Tuscany (central Italy). We investigated the sex ratio of this population, followed by morphometric analyses of the scape, leaves and flowers, and tests on pollen germinability and seed number and mass, in order to infer which sexual strategy produced the observed co-occurrence of male and hermaphrodite individuals. We found that sex ratio deviated from 1:1, and functionally male individuals showed a smaller plant size and a reduced pollen fitness (germinability and siring ability) compared to hermaphrodites. These findings point to a resource-dependent sexual allocation strategy, probably associated with gender diphasy.     

Diversification of the RAB Guanosine Triphosphatase Family in Dicots and Monocots

RAB guanosine triphosphatases （GTPases） are key regulators of vesicle trafficking and are essential to the growth and development of all eukaryotic cells. During evolution, the RAB family has expanded in different patterns to facilitate distinct cellular, developmental and physiological adaptations. Yeast has only 11 family members, whereas mammalian RABs have expanded to 18 RAB subfamilies. Plant RABs have diversified primarily by duplicating members within a single subfamily. Plant RABs are divided into eight subfamilies, corresponding to mammalian RAB1, RAB2, RAB5, RAB6, RAB7, RAB8, RAB11 and RAB18. Functional diversification of these is exemplified by the RAB1 ls, orthologs of which are partitioned into unique cell compartments in plants where they function to transport vesicles during localized tip growth. Similarly, the RAB2 family in grasses is likely involved in vesicle secretion associated with wall expansion, as determined by analysis of over-expression mutants. We propose that dicots and monocots have also diverged in their RAB profiles to accommodate unique cellular functions between the two groups. Here we present a bioinformatics analysis comparing the RAB sub-families of rice, maize and Arabidopsis. These results will guide future functional studies to test for the role of diversification of subfamilies unique to monocots compared to dicots.     

Isolation and characterization of cDNAs encoding mitochondrial phosphate transporters in soybean,maize, rice,and Arabidopsis

cDNA clones encoding mitochondrial phosphate transporters were isolated from four herbaceous plants. The cDNAs for the soybean, maize and rice transporters contained entire coding regions, whereas the Arabidopsis cDNA lacked the 5 portion. The hydropathy profiles of the deduced amino acid sequences predicted the existence of six membrane-spanning domains which are highly conserved in the mitochondrial transporter family. In soybeans, the mRNA level for the transporter was high in tissues containing dividing cells. It was suggested that there are multiple copies of transporter genes in both dicots and monocots. The soybean transporter was expressed as inclusion bodies in Escherichia coli, solubilized with detergents, and then reconstituted into liposomes. The resulting proteoliposomes exhibited high phosphate transport activity. The activity was inhibited by N-ethylmaleimide, like those of mammalian phosphate transporters.     

Transformation of the monocot Alstroemeria by Agrobacterium rhizogenes

An efficient procedure is described for transformation of calli of the monocotyledonous plant Alstroemeria by Agrobacterium rhizogenes. Calli were co-cultivated with A. rhizogenes strain A13 that harbored both a wild-type Ri-plasmid and the binary vector plasmid pIG121Hm, which included a gene for neomycin phosphotransferase II (NPTII) under the control of the nopaline synthase (NOS) promoter, a gene for hygromycin phosphotransferase (HPT) under the control of the cauliflower mosaic virus (CaMV) 35S promoter, and a gene for -glucuronidase (GUS) with an intron fused to the CaMV 35S promoter. Inoculated calli were plated on medium that contained cefotaxime to eliminate bacteria. Four weeks later, transformed cells were selected on medium that contained 20 mg L^–1 hygromycin. A histochemical assay for GUS activity revealed that selection by hygromycin was complete after eight weeks. The integration of the T-DNA of the Ri-plasmid and pIG121Hm into the plant genome was confirmed by PCR. Plants derived from transformed calli were produced on half-strength MS medium supplemented with 0.1 mg L^–1 GA₃ after about 5 months of culture. The presence of the gusA, nptII, and rol genes in the genomic DNA of regenerated plants was detected by PCR and Southern hybridization, and the expression of these transgenes was verified by RT-PCR.