In vitro gas production and rumen fermentation profile of fresh and ensiled genetically modified high–metabolizable energy ryegrass

We previously generated a high–metabolizable energy (HME) perennial ryegrass (Lolium perenne) by genetically modifying the plant to increase the leaf lipid content. Although substantial progress has been made toward characterizing physiological changes of HME ryegrass, very limited information exists for feeding value and its suitability for adoption into the pastoral system. In this study, independent HME ryegrass lines with a range of elevated leaf lipid concentrations were analyzed for changes in fatty acids and possible associated changes in the broader nutritional profile, including the gross energy, which was found to increase by 6.8%. Because ryegrass is often ensiled and fermentation in the rumen leads to biohydrogenation of fatty acids as well as enteric methane production, we sought to investigate these effects on HME ryegrass. This was achieved by performing mini-scale silos and using an automated gas measurement system to incubate the material in rumen fluid in vitro for 24 h. Our study included treatments comprising 3 independent HME ryegrass genotypes and wild-type control materials prepared fresh and as silage, employing in total 5 incubation studies, using rumen fluids collected from 4 nonlactating Jersey × Holstein cows. At intervals during the incubation, the production of gases, volatile fatty acids, and the degree of biohydrogenation were measured. Statistical data analysis indicated that differences in the nutritional compositions of the ensiled materials largely reflected those of their fresh counterparts. Incubation of both fresh and ensiled HME ryegrass in rumen fluid resulted in: (1) a greater percentage of valuable unsaturated fatty acids compared with the control; (2) a significant reduction of butyrate; and (3) a 10 to 15% decrease in the methane proportion of the total gas production. We conclude that ensiling could be a convenient option for preserving HME as a locally produced high-value supplementary feed; however, large-scale application needs to be investigated. In this paper we discuss the potential use of HME ryegrass to enhancing forage feeding value and the potential environmental benefits to the pastoral agriculture industry

Single domain antibodies: promising experimental and therapeutic tools in infection and immunity

Antibodies are important tools for experimental research and medical applications. Most antibodies are composed of two heavy and two light chains. Both chains contribute to the antigen-binding site which is usually flat or concave. In addition to these conventional antibodies, llamas, other camelids, and sharks also produce antibodies composed only of heavy chains. The antigen-binding site of these unusual heavy chain antibodies (hcAbs) is formed only by a single domain, designated VHH in camelid hcAbs and VNAR in shark hcAbs. VHH and VNAR are easily produced as recombinant proteins, designated single domain antibodies (sdAbs) or nanobodies. The CDR3 region of these sdAbs possesses the extraordinary capacity to form long fingerlike extensions that can extend into cavities on antigens, e.g., the active site crevice of enzymes. Other advantageous features of nanobodies include their small size, high solubility, thermal stability, refolding capacity, and good tissue penetration in vivo. Here we review the results of several recent proof-of-principle studies that open the exciting perspective of using sdAbs for modulating immune functions and for targeting toxins and microbes

Analysis of the asparagus (Asparagus officinalis) asparagines synthetase gene promoter identifies evolutionarily conserved cis-regulatory elements that mediate Suc-repression

In asparagus (Asparagus officinalis L.), increased levels of asparagine (Asn) and Asn synthetase (AS) transcript are detected during foliar senescence and in harvested spears, possibly triggered by signals from a reduced supply of carbohydrate. To identify cis-elements mediating this regulation, the asparagus AS gene promoter was isolated and analysed by DNA sequencing, followed by expression of AS::GUS (beta-glucuronidase) reporter-gene constructs in transgenic tissue, and electrophoretic mobility shift assays (EMSA). The 1958-base pair ( bp) region of the AS promoter upstream of the translation initiation ATG(-1958 bp region) was sufficient to confer sucrose (Suc)-regulated expression on the GUS reporter gene in asparagus callus and protoplasts, which were transformed by particle bombardment and electroporation, respectively. Removal of Suc from callus or protoplast media resulted in the induction of GUS activity. Deletion analysis of this 1958-bp fragment identified elements in the -640 to -266 bp region as important for both high GUS levels and mediating the Suc response. This was supported by EMSA results, which showed the formation of three nuclear protein-DNA complexes with the -558 to -284 bp fragment of the promoter. A 20-bp oligonucleotide, designed to match the sequence from -423 to -404 bp, was able to out-compete formation of one of these protein-DNA complexes, suggesting a specific interaction with this sequence. This region of the promoter, overlapping with the 20-bp oligonucleotide sequence, contains a 10-bp stretch identical to a sequence previously shown to mediate low Suc induction of an Oryza sativa (rice) alpha-amylase gene, and may thus represent a conserved Suc-responsive element

Storing carbon in leaf lipid sinks enhances perennial ryegrass carbon capture especially under high N and elevated CO₂

By modifying two genes involved in lipid biosynthesis and storage [cysteine oleosin (cys-OLE)/diacylglycerol O-acyltransferase (DGAT)], the accumulation of stable lipid droplets in perennial ryegrass (Lolium perenne) leaves was achieved. Growth, biomass allocation, leaf structure, gas exchange parameters, fatty acids, and water-soluble carbohydrates were quantified for a high-expressing cys-OLE/DGAT ryegrass transformant (HL) and a wild-type (WT) control grown under controlled conditions with 1–10 mM nitrogen (N) supply at ambient and elevated atmospheric CO₂. A dramatic shift in leaf carbon (C) storage occurred in HL leaves, away from readily mobilizable carbohydrates and towards stable lipid droplets. HL exhibited an increased growth rate, mainly in non-photosynthetic organs, leading to a decreased leaf mass fraction. HL leaves, however, displayed an increased specific leaf area and photosynthetic rate per unit leaf area, delivering greater overall C capture and leaf growth at high N supply. HL also exhibited a greater photosynthesis response to elevated atmospheric CO₂. We speculate that by behaving as uniquely stable microsinks for C, cys-OLE-encapsulated lipid droplets can reduce feedback inhibition of photosynthesis and drive greater C capture. Manipulation of many genes and gene combinations has been used to increase non-seed lipid content. However, the cys-OLE/DGAT technology remains the only reported case that increases plant biomass. We contrast cys-OLE/DGAT with other lipid accumulation strategies and discuss the implications of introducing lipid sinks into non-seed organs for plant energy homeostasis and growth

Delivery of grasses with high levels of unsaturated, protected fatty acids

Our goal is to increase the metabolisable energy of forage species (such as Lolium perenne, perennial ryegrass) via accumulation of lipids in the leaves, and further improve upon this by the delivery of polyunsaturated fatty acids whilst maintaining a low input agricultural system. Keywords: lipid protection, triacylglyceride, DGAT, oleosin, biohydrogenation</jats:p

Elevation of oil body integrity and emulsion stability by polyoleosins, multiple oleosin units joined in tandem head-to-tail fusions

We have successfully created polyoleosins by joining multiple oleosin units in tandem head-to-tail fusions. Constructs encoding recombinant proteins of 1, 3 and 6 oleosin repeats were purposely expressed both in planta and in Escherichia coli. Recombinant polyoleosins accumulated in the seed oil bodies of transgenic plants and in the inclusion bodies of E. coli. Although polyoleosin was estimated to only accumulate to < 2% of the total oil body protein in planta, their presence increased the freezing tolerance of imbibed seeds as well as emulsion stability and structural integrity of purified oil bodies; these increases were greater with increasing oleosin repeat number. Interestingly, the hexameric form of polyoleosin also led to an observable delay in germination which could be overcome with the addition of external sucrose. Prokaryotically produced polyoleosin was purified and used to generate artificial oil bodies and the increase in structural integrity of artificial oil bodies-containing polyoleosin was found to mimic those produced in planta. We describe here the construction of polyoleosins, their purification from E. coli, and properties imparted on seeds as well as native and artificial oil bodies. A putative mechanism to account for these properties is also proposed