No abstract

Despite 14‐3‐3 proteins being implicated in the control of the eukaryotic cell cycle, metabolism, cell signalling and survival, little is known about the global regulation or functions of the phosphorylation‐dependent binding of 14‐3‐3s to diverse target proteins. We identified Arabidopsis cytosolic proteins that bound 14‐3‐3s in competition with a 14‐3‐3‐binding phosphopeptide, including nitrate reductase, glyceraldehyde‐ 3‐phosphate dehydrogenase, a calcium‐dependent protein kinase, sucrose‐phosphate synthase (SPS) and glutamyl‐tRNA synthetase. Remarkably, in cells starved of sugars or fed with non‐metabolizable glucose analogues, all 14‐3‐3 binding was lost and the target proteins were selectively cleaved into proteolytic fragments. 14‐3‐3 binding reappeared after several hours of re‐feeding with sugars. Starvation‐induced degradation was blocked by 5‐amino imidazole‐4‐carboxamide riboside (which is converted to an AMP‐mimetic) or the protease inhibitor MG132 (Cbz‐leu‐leu‐leucinal). Extracts of sugar‐starved (but not sugar‐fed) Arabidopsis cells contained an ATP‐independent, MG132‐sensitive, neutral protease that cleaved Arabidopsis SPS, and the mammalian 14‐3‐3‐regulated transcription factor, FKHR. Cleavage of SPS and phosphorylated FKHR in vitro was blocked by binding to 14‐3‐3s. The finding that 14‐3‐3s participate in a nutrient‐sensing pathway controlling cleavage of many targets may underlie the effects of these proteins on plant development.

Nitric oxide (NO) is a diffusible, very reactive gas that is involved in the regulation of many processes in plants. Several enzymatic sources of NO production have been identified in recent years. Nitrate reductase (NR) is one of them and it has been shown that this well-known plant protein, apart from its role in nitrate reduction and assimilation, can also catalyse the reduction of nitrite to NO. This reaction can produce large amounts of NO, or at least more than is needed for signalling, as some escape of NO to the outside medium can be detected after NR activation. A role for NO and NR in stomata functioning in response to abscisic acid has also been proposed. The question that remains is whether this NR-derived NO is a signalling molecule or the mere product of an enzymatic side reaction like the products generated by the oxygenase activity of RuBisCO.

Health diets that contain immunostimulants and other functional ingredients can strengthen the immune response in Atlantic salmon, Salmo salar, and thereby reduce sea lice, Lepeophtheirus salmonis, infection levels. Such diets can be used to supplement other treatments and will potentially reduce the need for delousing and medication. A sea lice infection trial was conducted on fish with an average weight of 215 g. One control diet and four experimental diets containing functional ingredients were produced. The diets were fed to salmon for 4 weeks before infection with sea lice copepodids. When lice had developed to chalimus III/IV, 88 fish per diet were examined for lice loads. Mucus samples from fish fed the different diets were taken before and after lice infection. Mass spectrometry-based proteomics was used to characterize the protein composition in the epidermal mucus of Atlantic salmon and to identify quantitative alterations in protein expression. Multivariate analysis of the generated data sets was performed to identify protein biomarkers. Putative biomarkers associated with functional feed intake and with sea lice infection have been identified and can form the basis for strategic validation experiments with selected functional feeds.

In wild-type Nicotiana plumbaginifolia Viv. and other higher plants, nitrate reductase (NR) is regulated at the post-translational level and is rapidly inactivated in response to, for example, a light-to-dark transition. This inactivation is caused by phosphorylation of a conserved regulatory serine residue, Ser 521 in tobacco, and interaction with divalent cations or polyamines, and 14-3-3 proteins. The physiological importance of the post-translational NR modulation is presently under investigation using a transgenic N. plumbaginifolia line. This line expresses a mutated tobacco NR where Ser 521 has been changed into aspartic acid (Asp) by site-directed mutagenesis, resulting in a permanently active NR enzyme. When cut leaves or roots of this line (S(521)) were placed in darkness in a buffer containing 50 mM KNO(3), nitrite was excreted from the tissue at rates of 0.08-0.2 micromol (g FW)(-1) h(-1) for at least 5 h. For the control transgenic plant (C1), which had the regulatory serine of NR intact, nitrite excretion was low and halted completely after 1-3 h. Without nitrate in the buffer in which the tissue was immersed, nitrite excretion was also low for S(521), although 20-40 micromol (g FW)(-1) nitrate was present inside the tissue. Apparently, stored nitrate was not readily available for reduction in darkness. Leaf tissue and root segments of S(521) also emitted much more nitric oxide (NO) than the control. Importantly, NO emission from leaf tissue of S(521) was higher in the dark than in the light, opposite to what was usually observed when post-translational NR modulation was operating.