No abstract

Salinity is one of the major factors that limits geographical distribution of plants and adversely affects crop productivity and quality. We report here high-level expression of betaine aldehyde dehydrogenase (BADH) in cultured cells, roots, and leaves of carrot (Daucus carota) via plastid genetic engineering. Homoplasmic transgenic plants exhibiting high levels of salt tolerance were regenerated from bombarded cell cultures via somatic embryogenesis. Transformation efficiency of carrot somatic embryos was very high, with one transgenic event per approximately seven bombarded plates under optimal conditions. In vitro transgenic carrot cells transformed with the badh transgene were visually green in color when compared to untransformed carrot cells, and this offered a visual selection for transgenic lines. BADH enzyme activity was enhanced 8-fold in transgenic carrot cell cultures, grew 7-fold more, and accumulated 50-to 54-fold more betaine (93-101 mmol g 21 dry weight of b-Ala betaine and Gly betaine) than untransformed cells grown in liquid medium containing 100 mM NaCl. Transgenic carrot plants expressing BADH grew in the presence of high concentrations of NaCl (up to 400 mM), the highest level of salt tolerance reported so far among genetically modified crop plants. BADH expression was 74.8% in non-green edible parts (carrots) containing chromoplasts, and 53% in proplastids of cultured cells when compared to chloroplasts (100%) in leaves. Demonstration of plastid transformation via somatic embryogenesis utilizing non-green tissues as recipients of foreign DNA for the first time overcomes two of the major obstacles in extending this technology to important crop plants.Salt stress is a major abiotic stress in plant agriculture. The problem of soil salinity has been compounded by irrigation and excessive use of fertilizers. About 20% of the world's irrigated lands are affected by salinity (Zhu, 2001). Currently, high salinity limits crop production in 30% of the irrigated land in the United States and 20 million hectares globally. High salinity causes ion imbalance, toxic levels of cytoplasmic sodium, and drought stress (Ward et al., 2003). Plants utilize a number of protective mechanisms to maintain normal cellular metabolism and prevent damage to cellular components (Wood et al., 1996). One of the metabolic adaptations to salt stress is the accumulation of osmoprotectants. Gly betaine and b-Ala betaine are quaternary ammonium compounds that accumulate in many plant species in response to salt stress Rhodes and Hanson, 1993;Rathinasabapathi et al., 2001). Gly betaine protects the cell from salt stress by maintaining an osmotic balance with the environment (Robinson and Jones, 1986) and by stabilizing the quaternary structure of complex proteins (Papageorgiou and Murata, 1995). This substance occurs naturally in some crops, like sugar beet and cotton, as well as in many highly salt-or drought-tolerant wild plants, including halophytes (Rhodes and Hanson, 1993;Nishimura et al., 2001). However, many stress-suscep...

Cyclic electron flow (CEFI) has been proposed to balance the chloroplast energy budget, but the pathway, mechanism, and physiological role remain unclear. We isolated a new class of mutant in Arabidopsis thaliana, hcef for high CEF1, which shows constitutively elevated CEF1. The first of these, hcef1, was mapped to chloroplast fructose-1,6-bisphosphatase. Crossing hcef1 with pgr5, which is deficient in the antimycin A-sensitive pathway for plastoquinone reduction, resulted in a double mutant that maintained the high CEF1 phenotype, implying that the PGR5-dependent pathway is not involved. By contrast, crossing hcef1 with crr2-2, deficient in thylakoid NADPH dehydrogenase (NDH) complex, results in a double mutant that is highly light sensitive and lacks elevated CEF1, suggesting that NDH plays a direct role in catalyzing or regulating CEF1. Additionally, the NdhI component of the NDH complex was highly expressed in hcef1, whereas other photosynthetic complexes, as well as PGR5, decreased. We propose that (1) NDH is specifically upregulated in hcef1, allowing for increased CEF1; (2) the hcef1 mutation imposes an elevated ATP demand that may trigger CEF1; and (3) alternative mechanisms for augmenting ATP cannot compensate for the loss of CEF1 through NDH.

Despite the prior use of approximately 9000 bp, deep-level relationships within the angiosperm clade, Saxifragales remain enigmatic, due to an ancient, rapid radiation (89.5 to 110 Ma based on the fossil record). To resolve these deep relationships, we constructed several new data sets: (1) 16 genes representing the three genomic compartments within plant cells (2 nuclear, 10 plastid, 4 mitochondrial; aligned, analyzed length = 21,460 bp) for 28 taxa; (2) the entire plastid inverted repeat (IR; 26,625 bp) for 17 taxa; (3) "total evidence" (50,845 bp) for both 17 and 28 taxa (the latter missing the IR). Bayesian and ML methods yielded identical topologies across partitions with most clades receiving high posterior probability (pp = 1.0) and bootstrap (95% to 100%) values, suggesting that with sufficient data, rapid radiations can be resolved. In contrast, parsimony analyses of different partitions yielded conflicting topologies, particularly with respect to the placement of Paeoniaceae, a clade characterized by a long branch. In agreement with published simulations, the addition of characters increased bootstrap support for the putatively erroneous placement of Paeoniaceae. Although having far fewer parsimony-informative sites, slowly evolving plastid genes provided higher resolution and support for deep-level relationships than rapidly evolving plastid genes, yielding a topology close to the Bayesian and ML total evidence tree. The plastid IR region may be an ideal source of slowly evolving genes for resolution of deep-level angiosperm divergences that date to 90 My or more. Rapidly evolving genes provided support for tip relationships not recovered with slowly evolving genes, indicating some complementarity. Age estimates using penalized likelihood with and without age constraints for the 28-taxon, total evidence data set are comparable to fossil dates, whereas estimates based on the 17-taxon data are much older than implied by the fossil record. Hence, sufficient taxon density, and not simply numerous base pairs, is important in reliably estimating ages. Age estimates indicate that the early diversification of Saxifragales occurred rapidly, over a time span as short as 6 million years. Between 25,000 and 50,000 bp were needed to resolve this radiation with high support values. Extrapolating from Saxifragales, a similar number of base pairs may be needed to resolve the many other deep-level radiations of comparable age in angiosperms.

dRhizoctonia bare patch and root rot disease of wheat, caused by Rhizoctonia solani AG-8, develops as distinct patches of stunted plants and limits the yield of direct-seeded (no-till) wheat in the Pacific Northwest of the United States. At the site of a long-term cropping systems study near Ritzville, WA, a decline in Rhizoctonia patch disease was observed over an 11-year period. Bacterial communities from bulk and rhizosphere soil of plants from inside the patches, outside the patches, and recovered patches were analyzed by using pyrosequencing with primers designed for 16S rRNA. Taxa in the class Acidobacteria and the genus Gemmatimonas were found at higher frequencies in the rhizosphere of healthy plants outside the patches than in that of diseased plants from inside the patches. Dyella and Acidobacteria subgroup Gp7 were found at higher frequencies in recovered patches. Chitinophaga, Pedobacter, Oxalobacteriaceae (Duganella and Massilia), and Chyseobacterium were found at higher frequencies in the rhizosphere of diseased plants from inside the patches. For selected taxa, trends were validated by quantitative PCR (qPCR), and observed shifts of frequencies in the rhizosphere over time were duplicated in cycling experiments in the greenhouse that involved successive plantings of wheat in Rhizoctonia-inoculated soil. Chryseobacterium soldanellicola was isolated from the rhizosphere inside the patches and exhibited significant antagonism against R. solani AG-8 in vitro and in greenhouse tests. In conclusion, we identified novel bacterial taxa that respond to conditions affecting bare patch disease symptoms and that may be involved in suppression of Rhizoctonia root rot and bare batch disease.

JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact support@jstor.org.. Molecular phylogenetics has revolutionized our understanding of the Caryophyllales, and yet many relationships have remained uncertain, particularly at deeper levels. We have performed parsimony and maximum likelihood analyses on separate and combined data sets comprising nine plastid genes (;12,000 bp), two nuclear genes (;5000 bp), and the plastid inverted repeat (;24,000 bp), giving a combined analyzed length of 42,006 bp for 36 species of Caryophyllales and four outgroups. We have recovered strong support for deep-level relationships across the order. Two major subclades are well supported, the noncore and core Caryophyllales; Rhabdodendron followed by Simmondsia are sisters to the core Caryophyllales, Limeum and Stegnosperma are successive sisters to the ''globular inclusion'' clade, Gisekia is a distinct lineage well separated from Rivina within the ''raphide'' clade, and Rivina and Phytolaccaceae are disparate lineages, with Rivina sister to Nyctaginaceae. The placement of Sarcobatus and relationships within the portulacaceous cohort remain problematic. Within the latter, Halophytum is sister to Basellaceae and Didiereaceae, and the clade comprising Portulaca, Talinum, and Cactaceae is well supported. Classical hypotheses argued that the early Caryophyllales had evolved in open, dry, marginal environments at a time when pollinators were scarce, and, as such, the ancestral caryophyllid flower was wind pollinated with an undifferentiated perianth. We reevaluated these hypotheses in light of our phylogeny and find little support for anemophily as the ancestral condition; however, the early caryophyllid flower is suggested to have possessed an undifferentiated perianth. A subsequent minimum of nine origins of differentiated perianth is inferred. We discuss the evidence for independent origins of differentiated perianth and highlight the research opportunities that this pattern offers to the field of evolutionary developmental genetics.

Recent plastid phylogenomic studies have helped clarify the backbone phylogeny of angiosperms. However, the relatively limited taxon sampling in these studies has precluded strongly supported resolution of some regions of angiosperm phylogeny. Other recent work has suggested that the 25,000-bp plastid inverted repeat (IR) region may be a valuable source of characters for resolving these remaining problematic nodes. Consequently, we aligned all available angiosperm IR sequences to produce a matrix of 24,702 aligned bases for 246 accessions, including 36 new accessions. Maximum likelihood analyses of the complete data set yielded a generally well-supported topology that is highly congruent with those of recent plastid phylogenomic analyses. However, reducing taxon sampling to match a recent 83-gene plastid analysis resulted in significant changes in bootstrap support at some nodes. Notably, IR analyses resolved Pentapetalae into three wellsupported clades: (1) superasterids (comprising Santalales, Caryophyllales, Berberidopsidales, and Asteridae), (2) superrosids (comprising Vitaceae, Saxifragales, and Rosidae), and (3) Dilleniaceae. These results provide important new evidence for a stable, well-supported phylogenetic framework for angiosperms and demonstrate the utility of IR data for resolving the deeper levels of angiosperm phylogeny. They also reiterate the importance of carefully considering taxon sampling in phylogenomic studies.

Ribulose-1,5-bisphosphate carboxylase͞oxygenase (Rubisco) is a key enzyme that converts atmospheric carbon to food and supports life on this planet. Its low catalytic activity and specificity for oxygen leads to photorespiration, severely limiting photosynthesis and crop productivity. Consequently, Rubisco is a primary target for genetic engineering. Separate localization of the genes in the nuclear and chloroplast genomes and a complex assembly process resulting in a very low catalytic activity of hybrid Rubisco enzymes have rendered several earlier attempts of Rubisco engineering unsuccessful. Here we demonstrate that the RbcS gene, when integrated at a transcriptionally active spacer region of the chloroplast genome, in a nuclear RbcS antisense line and expressed under the regulation of heterologous (gene 10) or native (psbA) UTRs, results in the assembly of a functional holoenzyme and normal plant growth under ambient CO2 conditions, fully shortcircuiting nuclear control of gene regulation. There was Ϸ150-fold more RbcS transcript in chloroplast transgenic lines when compared with the nuclear RbcS antisense line, whereas the wild type has 7-fold more transcript. The small subunit protein levels in the gene 10͞RbcS and psbA͞RbcS plants were 60% and 106%, respectively, of the wild type. Photosynthesis of gene 10͞RbcS plants was approximately double that of the antisense plants, whereas that of psbA͞RbcS plants was restored almost completely to the wild-type rates. These results have opened an avenue for using chloroplast engineering for the evaluation of foreign Rubisco genes in planta that eventually can result in achieving efficient photosynthesis and increased crop productivity.