Transcript analysis in two alfalfa salt tolerance selected breeding populations relative to a non-tolerant population.

With the growing limitations on arable land, alfalfa (a widely cultivated, low-input forage) is now being selected to extend cultivation into saline lands for low-cost biofeedstock purposes. Here, minerals and transcriptome profiles were compared between two new salinity-tolerant North American alfalfa breeding populations and a more salinity-sensitive western Canadian alfalfa population grown under hydroponic saline conditions. All three populations accumulated two-fold higher sodium in roots than shoots as a function of increased electrical conductivity. At least 50% of differentially expressed genes (p < 0.05) were down-regulated in the salt-sensitive population growing under high salinity, while expression remained unchanged in the saline-tolerant populations. In particular, most reduction in transcript levels in the salt-sensitive population was observed in genes specifying cell wall structural components, lipids, secondary metabolism, auxin and ethylene hormones, development, transport, signalling, heat shock, proteolysis, pathogenesis-response, abiotic stress, RNA processing, and protein metabolism. Transcript diversity for transcription factors, protein modification, and protein degradation genes was also more strongly affected in salt-tolerant CW064027 than in salt-tolerant Bridgeview and salt-sensitive Rangelander, while both saline-tolerant populations showed more substantial up-regulation in redox-related genes and B-ZIP transcripts. The report highlights the first use of bulked genotypes as replicated samples to compare the transcriptomes of obligate out-cross breeding populations in alfalfa.

Modeling degradation ratios and nutrient availability of anthocyanidin-accumulating Lc-alfalfa populations in dairy cows.

Dairy cattle eating fresh or ensiled alfalfa as the main portion of their diet often have low protein efficiency because of the rapid initial rate of ruminal protein degradation of fresh and ensiled alfalfa. Ruminal protein degradation of alfalfa might be reduced by introducing a gene that stimulates the accumulation of mono- or polymeric anthocyanidins in alfalfa. The objectives of this study were to fractionate protein and carbohydrates by in situ and chemical approaches, to evaluate in situ ruminal degradation characteristics and synchronization ratios, to determine protein availability to dairy cattle using the 2007 digestible intestinal protein/rumen-degraded protein balance (DVE/OEB) protein system, and to determine net energy for lactation using the Dutch net energy for lactation (VEM) system for 3 newly developed transgenic winter hardy anthocyanidin-accumulating T(1)Lc-alfalfa populations. These T(1)Lc-alfalfa populations, called (T1)BeavLc1, (T1)RambLc3, and (T1)RangLc4, had an average anthocyanidin accumulation of 163.4 µg/g of DM, whereas AC Grazeland (selected for a low initial rate of degradation) did not accumulate anthocyanidin. The basic chemical composition of the original samples, soluble and potentially degradable fractions, and degradation characteristics of crude protein and carbohydrates were similar in T(1)Lc-alfalfa and AC Grazeland. The undegradable in situ crude protein and neutral detergent fiber fraction had 1.3% lower CP and 4.8% lower CHO, respectively, in T(1)Lc-alfalfa compared with the amounts in AC Grazeland. The T(1)Lc-alfalfa had a 0.34 MJ/kg of DM higher calculated net energy for lactation and 1.9% of CP higher buffer soluble protein compared with that in AC Grazeland. By the protein evaluation model, it was predicted that T(1)Lc-alfalfa tended to have 11.9, 6.9, and 8.4 g/kg of DM higher rumen degradable protein, OEB, and intestinal available protein, respectively, compared with the amounts in AC Grazeland. The hourly OEB included an initial and substantial peak (oversupply) of protein relative to energy, which was highest in (T1)RangLc4 and lowest in (T1)RambLc3. The hourly OEB between 4 and 24h was similar and more balanced for all 4 alfalfa populations. In conclusion, T(1)Lc-alfalfa accumulated anthocyanidin, tended to have higher predicted intestinal protein availability, and had higher predicted net energy of lactation availability for dairy cattle than did AC Grazeland.

Biodegradation Profiles of Proanthocyanidin-Accumulating Alfalfa Plants Coexpressing Lc- bHLH and C1-MYB Transcriptive Flavanoid Regulatory Genes.

The utilization of the nutrient potential of alfalfa ( Medicago sativa L.) cannot be maximized because of its rapidly degradable protein content in the rumen, leading to waste and various digestive disorders. This might be alleviated if protein-binding proanthocyanidins are present in aerial parts of alfalfa forage in adequate amounts. The Lc (bHLH) and C1 (MYB) genes of maize are transcription factors known to be collectively involved in the regulation of anthocyanin biosynthetic pathways. The objective of this study was to investigate the effect of Lc and C1 gene transformations on the proanthocyanidin content, nutrient composition, and degradation characteristics of proteins and carbohydrates by comparing the transgenic alfalfa with its parental nontransgenic (NT) alfalfa and commercial AC-Grazeland cultivar. The DNA extracted from transgenic plants was tested for the presence of respective transgenes by amplification with specific primers of respective transgenes using PCR. Both Lc-single and LcC1-double transgenic alfalfa accumulated both monomeric and polymeric proanthocyanidins with total proanthocyanidins ranging from ca. 460 to 770 µg/g of DM. The C1-transgenic alfalfa did not accumulate proanthocyanidins similar to NT alfalfa. The C1 gene increased the NPN content significantly only in C1-single and Lc1C1-double transgenic alfalfa. The LcC1 combination seemed to have a synergic effect on reducing sugar in alfalfa. In contrast, the Lc gene appears to have a negative effect on starch content. The C1 gene tended to lower the PB3 content irrespective of the presence of the Lc gene. Although the cotransformation of Lc and C1 increased the total N/CHO ratio compared to Lc single gene transformation, the total N/CHO ratio of transgenic alfalfa was not significantly different from NT. In conclusion, Lc-bHLH single and LcC1 double gene transformation resulted in the accumulation of proanthocyanidins and affected the chemical profiles in alfalfa, which altered ruminal degradation patterns and impacted the nutrient availability of alfalfa in ruminant livestock systems.

A study of the interaction between progesterone and membrane lipids.

The ability of progesterone to associate with phospholipid was examined in a model membrane system. Molecular interaction was assessed by measuring the enthalpy of the phase transition of dipalmitoylphosphatidylcholine liposomes by differential scanning calorimetry. The response was compared to cholesterol, a constituent of cellular membranes. Unlike cholesterol, progesterone caused minimal disruption of the phospholipid bilayer phase properties at concentrations ranging from 5-33 mol %. However, it interacted with the phospholipid to a greater degree when cholesterol was included in the liposomes. These results indicate that progesterone can intercalate into phospholipid bilayers containing cholesterol, and raise the prospect that there may be some diffusion of the hormone across the plasma membrane.

Compositional and physical properties of microsomal membrane lipids from regressing rat corpora lutea.

Wide angle x-ray diffraction has revealed that during corpus luteum regression there is a liquid-crystalline to gel phase transition in the phospholipid molecules of the cellular membranes. In the present study we have examined the lipid composition of these membranes and looked for evidence of membrane protein involvement in this change. Lipid analysis of smooth microsomal membranes prepared from rat corpora lutea revealed no significant change in the cholesterol to phospholipid ratio or in the ratio of unsaturated to saturated fatty acids with advancing luteolysis. In addition, there was no clear trend for these changes in the relative proportions of the major fatty acids. Liposomes were prepared from smooth microsomal fractions of regressing rat corpora lutea, and examination of these lipid vesicles by x-ray diffraction revealed that the temperature of the liquid-crystalline to gel phase transition was much lower (approximately 25-30 C) than that for the corresponding microsomes. These observations are consistent with the view that membrane proteins contribute to the ordering of lipid that results in a mixture of liquid-crystalline and gel phases in membranes from regressed corpora lutea.

Development of DNA-mediated transformation systems for plants.

The genetic engineering of plants by DNA-mediated gene transfer requires that efficient transformation systems be developed. Considerable progress has been made in manipulating the Ti plasmid of Agrobacterium tumefaciens as a vehicle for delivery of foreign genes into protoplasts of dicotyle-donous plants. Part of the Ti plasmid, the T-DNA, can be incorporated into the genome of the host cell; the T-DNA can carry a foreign DNA sequence which co-integrates with it; under normal conditions, the tumorigenic-causing portion of the T-DNA can be inactivated so that transformed protoplasts can be regenerated and T-DNA with an inserted foreign gene can be stably maintained during regeneration, meiosis and gamete formation. A foreign gene has yet to be expressed in regenerated plants although a T-DNA gene for opine synthesis can function in regenerates. Developing a more ubiquitous transformation system for monocotyledons is further from fruition. Based on transformation systems for simple eukaryotic organisms, it is reasonable to expect that a DNA vector which is capable of amplifying a novel plant gene and which contains both a drug resistance marker to facilitate the selection of transformed plant protoplasts and a species-specific autonomously replicating sequence to ensure the stable maintenance of the input gene in the recipient cell can be constructed.

Responses of the crucifer flea beetle to Brassica volatiles in an olfactometer.

A suite of commercially available volatile compounds was tested in an olfactometer bioassay for responses by the crucifer flea beetle (Phyllotreta cruciferae). Flea beetles were inhibited by exposure to hexane, pentane, and ethanol. Allyl-isothiocyanate, a crucifer-specific volatile, was moderately attractive to spring and early fall flea beetles, but inhibitory to late fall flea beetles. Spring flea beetles were most attracted to (+)-sabinene and E-beta-ocimene, and 1-hexanol, 1-pentanol, and Z-3-hexen-1-ol were stronger attractants than allyl-isothiocyanate. Spring beetles were strongly inhibited by (-)-E-caryophyllene, beta-ionone, indole, (+/-)-linalool, (+)-limonene, E-geraniol, and (-)-beta-pinene and moderately inhibited by (-)-verbenene and hexenal. Our study showed that older leaves and flowers of Brassica napus variety AC Excel contained small amounts of beta-ionone, but seedlings did not. beta-Ionone has not been documented previously in B. napus.

"HAIRY CANOLA"--Arabidopsis GL3 induces a dense covering of trichomes on Brassica napus seedlings.

Transformation with the Arabidopsis bHLH gene 35S:GLABRA3 (GL3) produced novel B. napus plants with an extremely dense coverage of trichomes on seedling tissues (stems and young leaves). In contrast, trichomes were strongly induced in seedling stems and moderately induced in leaves of a hairy, purple phenotype transformed with a 2.2 kb allele of the maize anthocyanin regulator LEAF COLOUR (Lc), but only weakly induced by BOOSTER (B-Peru), the maize Lc 2.4 kb allele, or the Arabidopsis trichome MYB gene GLABRA1 (GL1). B. napus plants containing only the GL3 transgene had a greater proportion of trichomes on the adaxial leaf surface, whereas all other plant types had a greater proportion on the abaxial surface. Progeny of crosses between GL3+ and GL1+ plants resulted in trichome densities intermediate between a single-insertion GL3+ plant and a double-insertion GL3+ plant. None of the transformations stimulated trichomes on Brassica cotyledons or on non-seedling tissues. A small portion of bHLH gene-induced trichomes had a swollen terminal structure. The results suggest that trichome development in B. napus may be regulated differently from Arabidopsis. They also imply that insertion of GL3 into Brassica species under a tissue-specific promoter has strong potential for developing insect-resistant crop plants.

Cloned manganese superoxide dismutase reduces oxidative stress in Escherichia coli and Anacystis nidulans.

The Mn superoxide dismutase gene of Escherichia coli was subcloned into the E. coli-Anacystis nidulans shuttle vector pSG111 to make the plasmid pMYG1. Transformation of E. coli HB101 with pMYG1 resulted in a 6-fold increase in superoxide dismutase activity. There was also induction of Mn superoxide dismutase in the transformants upon exposure to paraquat, as evidenced by dramatically increased levels of the Mn superoxide dismutase polypeptide in cytoplasmic extracts and a 16-fold further increase in superoxide dismutase activity. As well, the E. coli transformants showed resistance to paraquat-mediated inhibition of growth. Anacystis nidulans, a cyanobacterium that has no detectable Mn superoxide dismutase and is, consequently, very sensitive to oxidative stress, was also transformed with pMYG1. The transformants had detectable levels of Mn superoxide dismutase protein and showed resistance to paraquat-mediated inhibition of growth and photobleaching of pigments. Paraquat is known to promote formation of the superoxide radical anion, O2-., and thus the data have been interpreted as indicating that the cloned Mn superoxide dismutase provides protection in both E. coli and A. nidulans against damage attributable to O2-..

Improved yield of plasma membrane from mammalian cells through modifications of the two-phase polymer isolation procedure.

Modifications to the two-phase polymer gradient procedure for isolating plasma membrane from mammalian cells have resulted in greatly increased yields of purified plasma membrane. First, the cells were not treated with a membrane stabilizer (ZnCl2) prior to homogenization. This reduced the severity of homogenization required for disruption and allowed a greater proportion of the surface membrane to form large, flattened sheets that are more easily purified than the smaller fragments formed during more severe homogenization. Second, three crude fractions obtained from the homogenate (600g, 2000g, and 12,000g pellets), rather than a single, low-speed pellet (600g) containing only large sheets of membrane, were subjected to gradient centrifugation to obtain plasma membrane. This modification allowed purification of small as well as large fragments of plasmalemma and greatly increased the yield of purified membrane. Mg+2-dependent, Na+-K+-stimulated ATPase, a marker enzyme for plasma membrane, was enriched in the purified fraction by congruent to 17-fold relative to homogenate on a specific activity basis, and the yield of isolated plasma membrane averaged 70%, and was occasionally as high as 90%.

The effect of prolactin pretreatment on prostaglandin F2 alpha-associated structural changes in membranes from rat corpora lutea.

The ability of prolactin treatment to antagonize the luteolytic effect of prostaglandin F2 alpha (PGF2 alpha) was examined in the rat. Animals were superovulated, treated with PGF2 alpha and various doses of prolactin. Plasma progesterone concentrations were measured to assess luteal function. Microsomes were prepared from ovarian homogenates and examined by wide-angle x-ray diffraction for evidence of structural changes in the cellular membranes during luteolysis. In addition, the concentrations of various lipids were analyzed for alterations in membrane lipid composition. In preparations from control animals, all of the membrane lipid was in the liquid-crystalline phase at body temperature. However, in samples from PGF2 alpha-treated rats, portions of the bilayer underwent a structural alteration from liquid-crystalline to gel phase. This phase transition was not accompanied by significant changes in the relative concentrations of various lipids. Prolactin treatment was effective in inhibiting this membrane breakdown in a dose-dependent manner. These results suggest that PGF2 alpha and prolactin may control luteal function by affecting membrane structure.