Kanamycin resistance as a selectable marker for plastid transformation in tobacco

We report on a novel chimeric gene that confers kanamycin resistance on tobacco plastids. The kan gene from the bacterial transposon Tn5, encoding neomycin phosphotransferase (NPTII), was placed under control of plastid expression signals and cloned between rbcL and ORF512 plastid gene sequences to target the insertion of the chimeric gene into the plastid genome. Transforming plasmid pTNH32 DNA was introduced into tobacco leaves by the biolistic procedure, and plastid transformants were selected by their resistance to 50 g/ml of kanamycin monosulfate. The regenerated plants uniformly transmitted the transplastome to the maternal progeny. Resistant clones resulting from incorporation of the chimeric gene into the nuclear genome were also obtained. However, most of these could be eliminated by screening for resistance to high levels of kanamycin (500 g/ml). Incorporation of kan into the plastid genome led to its amplification to a high copy number, about 10000 per leaf cell, and accumulation of NPTII to about 1% of total cellular protein.     

Diversity and symbiotic effectiveness of beta-rhizobia isolated from sub-tropical legumes of a Brazilian Araucaria Forest

While the occurrence of Betaproteobacteria occupying the nodules of tropical legumes has been shown, little is known about subtropical areas. Araucaria Forest is a subtropical endangered ecosystem, and a better understanding of the legume-rhizobial symbionts may allow their use in land reclamation. The 16S rRNA gene of bacteria isolated from nine leguminous species was sequenced and their nodulation tested in Mimosa scabrella and Phaseolus vulgaris. 196 isolates were identified as eight genotypes: Pantoea, Pseudomonas, Bradyrhizobium sp1-2, Rhizobium, and Burkholderia sp1-3. The majority of the isolates from native plants (87 %) were taxonomically related to β-rhizobia, namely Burkholderia, however the legumes Galactia crassifolia and Collea speciosa were nodulated by both α and β-rhizobia, and Acacia dealbata, an exotic plant, only by α-rhizobia. The nifH genes of some isolates were sequenced and N-fixing potential shown by the acetylene reduction test. Most of the isolates nodulated the test plants, some were effective in M. scabrella, but all presented low efficiency in the exotic promiscuous legume P. vulgaris. Pantoea and Pseudomonas did not nodulate and probably are endophytic bacteria. The presented data shows diversity of α, β and γ-Proteobacteria in nodules of subtropical legumes, and suggests host specificity with β-rhizobia. Potential isolates were found for M. scabrella, indicating that a high N-fixing strain may be further inoculated in plants for use in reforestation.     

Expression of Arabidopsis Bax Inhibitor‐1 in transgenic sugarcane confers drought tolerance

The sustainability of global crop production is critically dependent on improving tolerance of crop plants to various types of environmental stress. Thus, identification of genes that confer stress tolerance in crops has become a top priority especially in view of expected changes in global climatic patterns. Drought stress is one of the abiotic stresses that can result in dramatic loss of crop productivity. In this work, we show that transgenic expression of a highly conserved cell death suppressor, Bax Inhibitor‐1 from Arabidopsis thaliana (AtBI‐1), can confer increased tolerance of sugarcane plants to long‐term (>20 days) water stress conditions. This robust trait is correlated with an increased tolerance of the transgenic sugarcane plants, especially in the roots, to induction of endoplasmic reticulum (ER) stress by the protein glycosylation inhibitor tunicamycin. Our findings suggest that suppression of ER stress in C₄ grasses, which include important crops such as sorghum and maize, can be an effective means of conferring improved tolerance to long‐term water deficit. This result could potentially lead to improved resilience and yield of major crops in the world.     

The genome sequence of the gram-positive sugarcane pathogen Leifsonia xyli subsp. xyli

The genome sequence of Leifsonia xyli subsp. xyli, which causes ratoon stunting disease and affects sugarcane worldwide, was determined. The single circular chromosome of Leifsonia xyli subsp. xyli CTCB07 was 2.6 Mb in length with a GC content of 68% and 2,044 predicted open reading frames. The analysis also revealed 307 predicted pseudogenes, which is more than any bacterial plant pathogen sequenced to date. Many of these pseudogenes, if functional, would likely be involved in the degradation of plant heteropolysaccharides, uptake of free sugars, and synthesis of amino acids. Although L. xyli subsp. xyli has only been identified colonizing the xylem vessels of sugarcane, the numbers of predicted regulatory genes and sugar transporters are similar to those in free-living organisms. Some of the predicted pathogenicity genes appear to have been acquired by lateral transfer and include genes for cellulase, pectinase, wilt-inducing protein, lysozyme, and desaturase. The presence of the latter may contribute to stunting, since it is likely involved in the synthesis of abscisic acid, a hormone that arrests growth. Our findings are consistent with the nutritionally fastidious behavior exhibited by L. xyli subsp. xyli and suggest an ongoing adaptation to the restricted ecological niche it inhabits.