Search

Plants exploit phenotypic plasticity to adapt their growth and development to prevailing environmental conditions. Interpretation of light and temperature signals is aided by the circadian system, which provides a temporal context. Phenotypic plasticity provides a selective and competitive advantage in nature but is obstructive during large-scale, intensive agricultural practices since economically important traits (including vegetative growth and flowering time) can vary widely depending on local environmental conditions. This prevents accurate prediction of harvesting times and produces a variable crop. In this study, we sought to restrict phenotypic plasticity and circadian regulation by manipulating signaling systems that govern plants' responses to environmental signals. Mathematical modeling of plant growth and development predicted reduced plant responses to changing environments when circadian and light signaling pathways were manipulated. We tested this prediction by utilizing a constitutively active allele of the plant photoreceptor phytochrome B, along with disruption of the circadian system via mutation of EARLY FLOWERING3. We found that these manipulations produced plants that are less responsive to light and temperature cues and thus fail to anticipate dawn. These engineered plants have uniform vegetative growth and flowering time, demonstrating how phenotypic plasticity can be limited while maintaining plant productivity. This has significant implications for future agriculture in both open fields and controlled environments. Plants utilize phenotypic plasticity to adapt to prevailing environmental conditions. This work demonstrates that manipulation of phytochrome B- and EARLY FLOWERING3-regulated signaling pathways can limit phenotypic plasticity with respect to light and temperature signals. This has implications for future crop development by enabling consistent plant growth and development despite the effects of climate change. [ABSTRACT FROM AUTHOR]

The circadian clock system acts as an endogenous timing reference that coordinates many metabolic and physiological processes in plants. Previous studies have shown that the application of osmotic stress delays circadian rhythms via 3ʹ-phospho-adenosine 5ʹ-phosphate (PAP), a retrograde signalling metabolite that is produced in response to redox stress within organelles. PAP accumulation leads to the inhibition of exoribonucleases (XRNs), which are responsible for RNA degradation. Interestingly, we are now able to demonstrate that post-transcriptional processing is crucial for the circadian response to osmotic stress. Our data show that osmotic stress increases the stability of specific circadian RNAs, suggesting that RNA metabolism plays a vital role in circadian clock coordination during drought. Inactivation of XRN4 is sufficient to extend circadian rhythms as part of this response, with PRR7 and LWD1 identified as transcripts that are post-transcriptionally regulated to delay circadian progression. [ABSTRACT FROM AUTHOR]

Fish are an important source of the n−3 highly unsaturated fatty acids (HUFA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids that are crucial to the health of higher vertebrates. The synthesis of HUFA involves enzyme-mediated desaturation, and a Δ5 fatty acyl desaturase cDNA has been cloned from Atlantic salmon ( Salmo salar) and functionally characterization of a Δ6 fatty acyl desaturase of Atlantic salmon and describe its genomic structure, tissue expression, and nutritional regulation. A salmon genomic library was screened with a salmon Δ5 desaturase cDNA and positive recombinant phage isolated and subcloned. The full-length cDNA for the putative fatty acyl desaturase was shown to comprise 2106 bp containing an open reading frame of 1365 bp specifying a protein of 454 amino acids (GenBank accession no. AY458652). The protein sequence included three histidine boxes, two transmembrane regions, and an N-terminal cytochrome b5 domain containing the heme-binding motif HPGG, all of which are characteristic of microsomal fatty acid desaturases. Functional expression showed that this gene possessed predominantly Δ6 desaturase activity. Screening and sequence analysis of the genomic DNA of a single fish revealed that the Δ6 desaturase gene constituted 13 exons in 7965 bp of genomic DNA. Quantitative real-time PCR assay of gene expression in Atlantic salmon showed that both Δ6 and Δ5 fatty acyl desaturase genes, and a fatty acyl elongase gene, were highly expressed in intestine, liver, and brain, and less so in kidney, heart, gill, adipose tissue, muscle, and spleen. Furthermore, expression of both Δ6 and Δ5 fatty acyl desaturase genes in intestine, liver, red muscle, and adipose tissue was higher in salmon fed a diet containing vegetable oil than in fish fed a diet containing fish oil. [ABSTRACT FROM AUTHOR]

Fish are the only major dietary source for humans of α-3 highly unsaturated fatty acids (HUFAs) and with declining fisheries farmed fish such as Atlantic salmon (Salmo salar) constitute an increasing proportion of the fish in the human diet. However, the current high use of fish oils, derived from wild capture marine fisheries, in aquaculture feeds is not sustainable in the longer term and will constrain continuing growth of aquaculture activities. Greater understanding of how fish metabolize and biosynthesize HUFA may lead to more sustainable aquaculture diets. The study described here contributes to an effort to determine the molecular genetics of the HUFA biosynthetic pathway in salmon, with the overall aim being to determine mechanisms for optimizing the use of vegetable oils in Atlantic salmon culture. In this paper we describe the cloning and functional characterization of 2 genes from salmon involved in the biosynthesis of HUFA. A salmon desaturase complementary DNA, SalDes, was isolated that include an open reading frame of 1362 bp specifying a protein of 454 amino acids. The protein sequence includes all the characteristics of micomal fatty acid desaturases, including 3 histidine boxes, 2 transmembrane regions, and an N-terminal cytochrome b3 domain containing a heme-binding motif similar to that of other fatty acid desaturases. Functional expression in the yeast Saccharomyces cerevisiae showed SalDes is predominantly an α-3 Δ5 desaturase, a key enzyme in the synthesis of eicosapentaenoic acid (20:5n-3) from α-linolenic acid (18:3-3). The desaturase showed only low levels of Δ6 activity toward C18 polyunsaturated fatty acids. In addition, a fatty acid elongase cDNA, SalElo, was isolated that included an open reading frame of 888 bp, specifying a protein of 295 amino acids. The protein sequence of SalElo included characteristics of microsomal fatty acid elongases, including a histidine box and a transmembrane region. Upon expression in yeast SalElo showed broad substrate specificity for polyunsaturated fatty acids with a range of chain lengths, with the rank order being C18 > C20 > C22. Thus this one polypeptide product displays all fatty acid elongase activities required for the biosynthesis of docosahexaenoic acid (22:6n-3) from 18:3n-3. [ABSTRACT FROM AUTHOR]

Enzymes that increase the chain length of fatty acids are essential for biosynthesis of highly unsaturated fatty acids. The gLELO gene encodes a protein involved in the elongation of polyunsaturated fatty acids in the fungus Mortierella alpina. A search of the GenBank database identified several expressed sequence tag sequences, including one obtained from zebrafish (Danio rerio), with high similarity to gLELO. The full-length transcript ZfELO, encoding a polypeptide of 291 amino acid residues, was isolated from zebrafish liver cDNA. The predicted amino acid sequence of the open reading frame shared high similarity with the elongases of Caenorhabditis elegans and human. When expressed in Saccharomyces cerevisiae, the zebrafish open reading frame conferred the ability to lengthen the chain of a range of C18, C20, and C22 polyunsaturated fatty acids, indicating not only that biosynthesis of 22:6n-3 from 18:3n-3 via a 24-carbon intermediate is feasible, but also that one elongase enzyme can perform all three elongation steps required. The zebrafish enzyme was also able to elongate monounsaturated and saturated fatty acids, and thus demonstrates a greater level of promiscuity in terms of substrate use than any elongase enzyme described previously. [ABSTRACT FROM AUTHOR]

Galactomannan biosynthesis in legume seed endosperms involves two Golgi membrane-bound glycosyltransferases, mannan synthase and galactomannan galactosyltransferase (GMGT). GMGT specificity is an important factor regulating the distribution and amount of (1 → 6)-α-galactose (Gal) substitution of the (1 → 4)-β-1inked mannan backbone. The model legume Lotus japonicus is shown now to have endospermic seeds with endosperm cell walls that contain a high-Gal galactomannan (mannose [Man]/Gal = 1.2-1.3). Galactomannan biosynthesis in developing L. japonicus endosperms has been mapped, and a cDNA encoding a functional GMGT has been obtained from L. japonicus endosperms during galactomannan deposition. L. japonicus has been transformed with sense, antisense, and sense/antisense ("hairpin loop") constructs of the GMGT cDNA. Some of the sense, antisense, and sense/antisense transgenic lines exhibited galactomannans with altered (higher) Man/Gal values in their (T1 generation) seeds, at frequencies that were consistent with posttranscriptional silencing of GMGT. For T1 generation individuals, transgene inheritance was correlated with galactomannan composition and amount in the endosperm. All the azygous individuals had unchanged galactomannans, whereas those that had inherited a GMGT transgene exhibited a range of Man/Gal values, up to about 6 in some lines. For Man/Gal values up to 4, the results were consistent with lowered Gal substitution of a constant amount of mannan backbone. Further lowering of Gal substitution was accompanied by a slight decrease in the amount of mannan backbone. Microsomal membranes prepared from the developing T2 generation endosperms of transgenic lines showed reduced GMGT activity relative to mannan synthase. The results demonstrate structural modification of a plant cell wall polysaccharide by designed regulation of a Golgi-bound glycosyltransferase. [ABSTRACT FROM AUTHOR]

Examines the biosynthesis of galactomannans in the development of legume seeds. Interaction of two membrane-bound glycosyltransferases, mannan synthase and galactomannan galactosyltransferases; Elongation of mannan backbone; Analysis of the endosperm cell walls of tobacco.

Summary Galactomannan biosynthesis in vitro is catalysed by membrane preparations from developing fenugreek seed endosperms. Two enzymes interact: a GDP-mannose dependent (1→4)-β-d-mannan synthase and a UDP-galactose dependent (1→6)-α-d-galactosyltransferase. The statistical distribution of galactosyl substituents along the mannan backbone, and the degree of galactose substitution of the primary product of galactomannan biosynthesis appear to be regulated by the specificity of the galactosyltransferase. We now report the detergent solubilisation of the fenugreek galactosyltransferase with retention of activity, the identification on gels of a putative 51 kDa galactosyltransferase protein, and the isolation, cloning and sequencing of the corresponding cDNA. The solubilised galactosyltransferase has an absolute requirement for added acceptor substrates. Beta-(1→4)-linked d-manno-oligosaccharides with chain lengths greater than or equal to 5 acted as acceptors, as did galactomannans of low to medium galactose-substitution. The putative galactosyltransferase cDNA encodes a 51282 Da protein, with a single transmembrane alpha helix near the N terminus. We have also confirmed the identity of the galactosyltransferase by inserting the cDNA in frame into the genome of the methylotrophic yeast Pichia pastoris under the control of an AOX promoter and the yeast alpha secretion factor and observing the secretion of galactomannan α-galactosyltransferase activity. Particularly high activities were observed when a truncated sequence, lacking the membrane-spanning helix, was expressed. [ABSTRACT FROM AUTHOR]