Salicylic acid has a role in regulating gene expression during leaf senescence

Leaf senescence is a complex process that is controlled by multiple developmental and environmental signals and is manifested by induced expression of a large number of different genes. In this paper we describe experiments that show, for the first time, that the salicylic acid (SA)-signalling pathway has a role in the control of gene expression during developmental senescence. Arabidopsis plants defective in the SA-signalling pathway (npr1 and pad4 mutants and NahG transgenic plants) were used to investigate senescence-enhanced gene expression, and a number of genes showed altered expression patterns. Senescence-induced expression of the cysteine protease gene SAG12, for example, was conditional on the presence of SA, together with another unidentified senescence-specific factor. Changes in gene expression patterns were accompanied by a delayed yellowing and reduced necrosis in the mutant plants defective in SA-signalling, suggesting a role for SA in the cell death that occurs at the final stage of senescence. We propose the presence of a minimum of three senescence-enhanced signalling factors in senescing leaves, one of which is SA. We also suggest that a combination of signalling factors is required for the optimum expression of many genes during senescence.     

Protease activity during rice leaf senescence

A protease activity was detected in rice (Oryza sativa L. cv. Ratna) leaves that hydrolysed hemoglobin more efficiently than bovine serum albumin. The activity was high when the enzyme was extracted and assayed with tris-maleate buffer [tris (hydroxymethyl) methyl amino-maleate] pH 7.0 rather than with water or with citrate-phosphate buffer pH 7.0. The enzyme had a strong dependence on sulfhydryl groups for its activity without which it was inaotive. The pH optimum was 7.0 and the temperature optimum was 40 °C. Protease activity expressed per unit leaf fresh weight (absolute activity) increased only little during senescence of detached rice leaves while the same activity expressed per unit soluble protein content (specific activity) increased by a greater factor (about 5 times) than absolute activity. Total and soluble protein content decreased during the senescence of detached leaves. Benzimidazole (10-3M) and kinetin (0.5xl0-5M) treatment arrested the increase of the protease activity and the deorease in the protein content during detached leaf senescence. It was indicative that protease protein was more stable than the bulk of other proteins in senescing leaves.     

A molecular model for diacylglycerol acyltransferase from Mortierella ramanniana var. angulispora

Acyl CoA diacylglycerol acyltransferase (DGAT, EC 2.3.120) is recognized as a key player of cellular diacylglycerol metabolism. It catalyzes the terminal, yet the committed step in triacylglycerol synthesis using diacylglycerol and fatty acyl CoA as substrates. The protein sequence of diacylglycerol acyltransferse (DGAT) Type 2B in Moretierella ramanniana var. angulispora (Protein_ID = {"type":"entrez-protein","attrs":{"text":"AAK84180.1","term_id":"15099961"}}AAK84180.1) was retrieved from GenBank. However, a structure is not yet available for this sequence. The 3D structure of DGAT Type 2B was modeled using a template structure (PDB ID: 1K30) obtained from Protein databank (PDB) identified by searching with position specific iterative BLAST (PSI-BLAST). The template (PDB ID: 1K30) describes the structure of DGAT from Cucurbita moschata. Modeling was performed using Modeller 9v2 and protein model is hence generated. The DGAT type 2B protein model was subsequently docked with six inhibitors (sphingosine; trifluoroperazine; phosphatidic acid; lysophospatidylserine; KCl; 1, 2-diolein) using AutoDock (a molecular docking program). The binding of inhibitors to the protein model of DGAT type 2B is discussed.     

Triacylglycerol synthesis in cultured rat hepatocytes. The rate-limiting role of diacylglycerol acyltransferase

The limiting role of diacylglycerol acyltransferase with respect to triacylglycerol synthesis in cultured rat hepatocytes was evaluated by following the inhibition of the overall synthetic flux by 2-bromooctanoate acting as an inhibitor of the diacylglycerol acyltransferase step. The flux-control coefficient of diacylglycerol acyltransferase in intact cultured hepatocytes amounted to 0.76 in the presence of saturating glycerol and either palmitate or oleate as the fatty acyl substrates. The flux-control coefficient of diacylglycerol acyltransferase in lysolecithin-permeabilized cultured hepatocytes amounted to 0.80 and 0.99 in the presence of saturating glycerol 3-phosphate and either palmitate or oleate as the fatty acyl substrate, respectively. Hence, triacylglycerol synthesis in liver cells under the experimental conditions employed is rate-limited by the diacylglycerol acyltransferase.     

A role for diacylglycerol in antibacterial autophagy

Antibacterial autophagy is understood to be a key cellular immune response to invading microbes. However, the mechanism(s) by which bacteria are selected as targets of autophagy remain unclear. We recently identified diacylglycerol as a novel signaling molecule that targets bacteria to the autophagy pathway, and show that it acts via protein kinase C activation. We also found that Pkc1 is required for autophagy in yeast, indicating that this kinase plays a conserved role in autophagy regulation.Key words: bacteria, Salmonella, innate immunity, adaptor, lipid second messenger, diacylglycerol, ubiquitin, NDP52, p62, SQSTM1The mechanism by which bacteria and other subcellular targets are identified and degraded by the autophagy pathway is an area of intense research. Ubiquitin has been recently found to act as an essential signal required for the autophagy of bacteria and proteins. We have previously observed ubiquitin on autophagy-targeted Salmonella enterica serovar Typhimurium (S. Typhimurium) but were surprised to see that only 50% of these bacteria were positive for ubiquitin. This indicated the possibility that an alternate signal was required for efficient autophagic targeting of the nonubiquitinated population of these bacteria.We initially performed a screen quantifying the colocalization of different lipid second messengers (diacylglycerol (DAG), PtdIns(3)P, PtdIns(4,5)P₂, PtdIns(3,4) P₂, and PtdIns(3,4,5)P₃) with autophagytargeted (i.e., LC3⁺) S. Typhimurium. We observed that DAG preferentially localizes with LC3⁺ bacteria. A kinetic analysis revealed that maximal DAG colocalization with bacteria (45 min post-infection) precedes maximal autophagy of the bacteria (60 min post-infection). Using pharmacological agents, siRNA and dominant negative constructs we were able to determine that DAG localization to the bacteria requires the action of phospholipase D (PLD; phosphatidylcholine to phosphatidic acid conversion) and phosphatidic acid phosphatase (PAP; phosphatidic acid to DAG conversion). We observed that inhibition of these pathways significantly reduces DAG localization to bacteria as well as concomitant autophagy of the bacteria, indicating a role for this lipid second messenger in the regulation of this process.Having determined that DAG is necessary for autophagy of bacteria we subsequently wanted to identify the effector through which it was signaling. Conventional and novel isoforms of the protein kinase C (PKC) family contain DAG-binding C1 domains. Accordingly, we targeted PKC isoforms using pharmacological agents, siRNA and knockout cell lines and were able to determine that DAG is signaling through the δ isoform of PKC. Inhibition of this serine/threonine kinase results in significant inhibition of antibacterial autophagy. Furthermore, bacterial replication in PKCδ knockout mouse embryonic fibroblasts is significantly higher compared to control fibroblasts, consistent with previous observations demonstrating that autophagy impairs intracellular replication of S. Typhimurium (Birmingham et al. 2006).We addressed the possibility that DAG and ubiquitin are functioning in a cooperative manner to target Salmonella for degradation by autophagy. We simultaneously inhibited both pathways using siRNA or pharmacological agents and observed additive inhibitory effects on autophagy of the bacteria. While this is indicative of two independent pathways, we cannot discount the possibility that there is still cooperation between the two pathways, especially as we did observe a small population of bacteria that were positive for both DAG and ubiquitin (Fig. 1). There are also a number of technical limitations in the methods we used, such as detection levels of the probes and antibodies that warrant caution in concluding that the two pathways are completely independent. Nonetheless, our studies clearly demonstrate a role for both DAG (Shahnazari et al. 2010) and ubiquitin (Zheng et al. 2009) in autophagy of S. Typhimurium. Future studies are required to further examine how these signals contribute to regulation of antibacterial autophagy.Open in a separate windowFigure 1Autophagic targeting of Salmonella Typhimurium. Invading S. Typhimurium can be targeted to the autophagy pathway by two independent signaling mechanisms. The first requires ubiquitin and the autophagy adaptors p62 and NDP52. The second requires DAG generation and PKCδ function. DAG generation on the SCV may occur through interaction of the SCV with DAG-positive endocytic vesicles (pathway 1) or through direct DAG production on the SCV (pathway 2). SCV, Salmonella-containing vacuole; PA, phosphatidic acid; DAG, diacylglycerol; PAP, phosphatidic acid phosphatase; PKCδ, protein kinase C delta; Ub, ubiquitin.Having characterized this pathway in antibacterial autophagy we were interested in determining whether these components were required for general autophagy. We therefore tested whether DAG localizes with rapamycin-induced autophagosomes. We observed DAG on these compartments and also found a requirement for PAP and PKCδ in this process. Other PKC isoforms are involved in alternate types of autophagy including ER stress-induced autophagy (Sakaki et al. 2008) as well as hypoxia-induced autophagy (Chen et al. 2009). As a result, we were interested in determining whether PKC function in autophagy was evolutionarily conserved. We therefore tested a role for the yeast ortholog, Pkc1, in this process and observed that it is required for starvation-induced autophagy in Saccharomyces cerevisiae.Having identified and characterized a novel signal and effector for antibacterial autophagy, further work still remains to be done in order to obtain a complete picture of this process. This includes additional study of the mechanism by which DAG is generated and the subcellular localization of PLD and PAP during this process. It is possible that DAG⁺ endocytic vesicles fuse with the Salmonella-containing vacuole (SCV) coating this compartment with DAG (pathway 1, see Fig. 1). It is also possible that both PLD and PAP function directly on the SCV, converting phosphatidylcholine to DAG via the phosphatidic acid intermediate (pathway 2, Fig. 1).More work also needs to be done to dissect DAG and ubiquitin signaling contributions to this pathway. Questions to be answered include the identification of the ubiquitinated protein(s) on the SCV, which may be host or bacterial proteins. Additionally, while we know that DAG is present on the SCV we do not yet know the signal that induces its generation. One intriguing possibility is that DAG generation occurs in response to bacterial-induced damage to the SCV during invasion. To date, PKC has been implicated in at least three different types of autophagy, and the possibility exists that other PKC isoforms (DAG responsive or not) are also involved in this process.     

The role of sugars in integrating environmental signals during the regulation of leaf senescence

Although leaf senescence results in a loss of photosynthetic carbon fixation, the senescence-dependent release of nutrients, especially of nitrogen, is important for the growth of young leaves and for reproduction. Environmental regulation of senescence is therefore a vital factor in the carbon and nitrogen economy of plants. Leaf senescence is a highly plastic trait that is affected by a range of different environmental factors including light, nutrient supply, CO2 concentration, and abiotic and biotic stress. In this review, the focus is on the impact of environmental conditions on sugar accumulation and sugar signalling during senescence. By signalling a high availability of carbon relative to nitrogen in the old leaves, sugar accumulation can trigger leaf senescence. Sugar-induced senescence is therefore particularly important under low nitrogen availability and may also play a role in light signalling. Whether or not sugars are involved in regulating the senescence response of plants to elevated CO2 remains unresolved. Senescence can be delayed or accelerated in elevated CO2 and no clear relationship between sugar accumulation and senescence has been found. Plasticity in the response to environmental factors, such as daylength and sugar accumulation, varies between different Arabidopsis accessions. This natural variation can be exploited to analyse the genetic basis of the regulation of senescence and the consequences for growth and fecundity. Different evolutionary strategies, i.e. early senescence combined with a high reproductive effort or late senescence combined with a low reproductive effort, may be an important adaptation of Arabidopsis accessions to their natural habitat.     

High-content assays for evaluating cellular and hepatic diacylglycerol acyltransferase activity

Acyl-CoA:diacylglycerol acyltransferase (DGAT) catalyzes the terminal step in triglyceride (TG) synthesis using diacylglycerol (DAG) and fatty acyl-CoA as substrates. In the liver, the production of VLDL permits the delivery of hydrophobic TG from the liver to peripheral tissues for energy metabolism. We describe here a novel high-content, high-throughput LC/MS/MS-based cellular assay for determining DGAT activity. We treated endogenous DGAT-expressing cells with stable isotope-labeled [¹³C₁₈]oleic acid. The [¹³C₁₈]oleoyl-incorporated TG and DAG lipid species were profiled. The TG synthesis pathway assay was optimized to a one-step extraction, followed by LC/MS/MS quantification. Further, we report a novel LC/MS/MS method for tracing hepatic TG synthesis and VLDL-TG secretion in vivo by administering [¹³C₁₈]oleic acid to rats. The [¹³C₁₈]oleic acid-incorporated VLDL-TG was detected after one-step extraction without conventional separation of TG and recovery by derivatizing [¹³C₁₈]oleic acid for detection. Using potent and selective DGAT1 inhibitors as pharmacological tools, we measured changes in [¹³C₁₈]oleoyl-incorporated TG and DAG and demonstrated that DGAT1 inhibition significantly reduced [¹³C₁₈]oleoyl-incorporated VLDL-TG. This DGAT1-selective assay will enable researchers to discern differences between the roles of DGAT1 and DGAT2 in TG synthesis in vitro and in vivo.     

Predictive capability of a leaf optical meter for determining leaf pigment status during senescence

We conducted an experiment to assess the predictive capability of a leaf optical meter for determining leaf pigment status of Acer mono Maxim., A. ginnala Maxim., Quercus mongolica Fisch., and Cornus alba displaying a range of visually different leaf colors during senescence. Concentrations of chlorophyll (Chl) a, Chl b, and total Chl [i.e., Chl (a+b)] decreased while the concentration of carotenoids (Car) remained relatively static for all species as leaf development continued from maturity to senescence. C. alba exhibited the lowest average concentration of Chl (a+b), Chl a, and Car, but the highest relative anthocyanin concentration, while Q. mongolica exhibited the highest Chl (a+b), Chl b, and the lowest relative anthocyanin concentration. A. mono exhibited the highest Chl a and Car concentrations. The relationships between leaf pigments and the values measured by the optical meter generally followed an exponential function. The strongest relationships between leaf pigments and optical measurements were for A. mono, A. ginnala, and Q. mongolica (R ² ranged from 0.64 to 0.95), and the weakest relationships were for C. alba (R ² ranged from 0.13 to 0.67). Moreover, optical measurements were more strongly related to Chl a than to Chl b or Chl (a+b). Optical measurements were not related to Car or relative anthocyanin concentrations. We predicted that weak relationships between leaf pigments and optical measurements would occur under very low Chl concentrations or under very high anthocyanin concentrations; however, these factors could not explain the weak relationship between Chl and optical measurements observed in C. alba. Overall, our results indicated that an optical meter can accurately estimate leaf pigment concentrations during leaf senescence — a time when pigment concentrations are dynamically changing — but that the accuracy of the estimate varies across species. Future research should investigate how species-specific leaf traits may influence the accuracy of pigment estimates derived from optical meters.     

Protein degradation and nitrogen remobilization during leaf senescence

Leaf senescence, a type of programmed cell death, is a complex and highly regulated process that involves the degradation of macromolecules, including proteins, nucleic acids, and lipids. Nutrients, especially nitrogen, are re-mobilized from senescing leaves to newly developing tissues or reserve organs. Our review focuses on three pathways for protein breakdown and the resorption of N during this process: the ubiquitin/proteosome system, the chloroplast degradation pathway, and the vacuolar and autophagic pathway. We propose that two relative biochemical cycles exist for amino acid recycling and N-export — the GS/GOGAT cycle and the PPDK-GS/GOGAT cycle.     

Activation of latent phenolase during spinach leaf senescence

The observed increase of phenolase activity and of its rate of activation during spinach leaf senescence is due to reduced binding of latent phenolase to the thylakoid membranes and not to de novo synthesis. The same amount of phenolase which is active in isolated thylakoid membranes from senescent leaves can be found in the membranes of non-senescent leaves after activation of latent enzyme. Tracer experiments give evidence that one multiple form which is responsible for the bulk activity in senescent leaves, is synthesized before, but not after the onset of senescence, indicating that pre-existing latent phenolase is converted to easily activating forms.     

Measurement of diacylglycerol acyltransferase activity in isolated hepatocytes

An assay procedure for diacylglycerol acyltransferase that allows rapid measurement of the activity of this enzyme in isolated hepatocytes is described. The one-step procedure involves permeabilization of the plasma membrane with digitonin and simultaneous measurement of diacylglycerol acyltransferase activity. Digitonin at a concentration of 64 microg/mg of cellular protein was found to be optimal for exposing microsomal diacylglycerol acyltransferase to the components of the assay. The enzyme assay is linear with time up to 4 min and with protein concentrations in the range 0.25-2.4 mg of cellular protein/assay. It is shown that there is a good correlation of cellular enzyme activity as determined in digitonin-permeabilized hepatocytes with the rate of triacylglycerol synthesis in intact hepatocytes.     

Biotechnological mechanism for improving plant remobilization of phosphorus during leaf senescence

Phosphorus enrichment of aquatic ecosystems through diffuse source pollution is an ongoing issue worldwide. A potential solution lies in the use of fast‐growing, multipurpose feedstocks, such as trees, to limit the flow of phosphorus into riparian areas through luxury consumption. However, the perennial nature of trees and their use of leaves as storage organs for excess phosphorus may reduce the effectiveness of contaminant removal during periods of leaf abscission. In an attempt to improve phosphorus remobilization during autumnal senescence, transgenic hybrid poplar P39 (Populus alba × Populus grandidentata) and Arabidopsis thaliana harbouring a constitutively expressed low‐affinity potato phosphate transporter (35S::StPht1‐1) were generated using Agrobacterium‐mediated transformation. For both species, the highest expressing 35S::StPht1‐1 lines were grown alongside wild‐type plants and subjected to increasing phosphate applications. StPht1‐1 expression in A. thaliana led to a reduction in biomass when grown under high‐phosphate conditions and had no effect on phosphate remobilization during senescence. In contrast, StPht1‐1 constitutive expression in P39 resulted in increased leaf phosphate content in the highest expressing transgenic line and minimal to no effect on P resorption efficiency. Surprisingly, sulphate resorption showed the greatest improvement in all three transgenic poplar lines, displaying a 31%–37% increase in resorption efficiency. These results highlight the complexity of nutrient resorption mechanisms in plants.     

Evidence that Plasmodium falciparum diacylglycerol acyltransferase is essential for intraerythrocytic proliferation

In triacylglycerol (TAG)-accumulating organisms, the physiological roles of diacylglycerol acyltransferase (DGAT), a principal enzyme in the major biosynthetic pathway for TAG, appear to be diverse. Apicomplexan parasite, Plasmodium falciparum, shows unique features in TAG metabolism and trafficking during intraerythrocytic development, and unlike most eukaryotes, only one open reading frame (ORF) encoding a candidate DGAT could be found in its genome. However, whether this candidate ORF encodes P. falciparum DGAT and its physiological relevance have not been assessed. Here, we demonstrate that the ORF is transcribed as a approximately 3.6 kb single mRNA throughout intraerythrocytic development, markedly elevated at trophozoite, schizont, and segmented schizont, and indeed encodes a protein exhibiting DGAT activity. Further, we provide evidence that the parasite in which the ORF was disrupted via double crossover recombination cannot be enriched, implying a fundamental role of PfDGAT in intraerythrocytic proliferation.     

The TAG1 locus of Arabidopsis encodes for a diacylglycerol acyltransferase

Diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) is a membrane enzyme that drives the final step in the formation of oils using diacylglycerol (DAG) and acyl-CoA to yield triacylglycerol (TAG). We identified a putative plant DGAT gene (TRIACYLGLYCEROL1: TAG1) and demonstrated its function by the cloning of two mutated alleles, designated AS11 (tag1-1) and ABX45 (tag1-2). One allele, AS11, has been previously characterised at the biochemical level. Mutant seeds contained less oil with a modified fatty acid profile and have reduced germination rates compared to wild-type controls. The TAG1 cDNA encodes for a 520-aa protein that possesses multiple putative transmembrane domains and shows 70 % similarity to a human DGAT cDNA.     

Functional size of acyl coenzyme A:diacylglycerol acyltransferase by radiation inactivation

Rat liver acyl coenzyme A:diacylglycerol acyltransferase, an intrinsic membrane activity associated with the endoplasmic reticulum, catalyzes the terminal and rate-limiting step in triglyceride synthesis. This enzyme has never been purified nor has its gene been isolated. Inactivation by ionizing radiation and target analysis were used to determine its functional size in situ. Monoexponential radiation inactivation curves were obtained which indicated that a single-sized unit of 72 +/- 4 kDa is required for expression of activity. The size corresponds only to the protein portion of the target and may represent one or several polypeptides.     

Accumulation of pathogenesis-related proteins in barley leaf intercellular spaces during leaf senescence

Accumulation of the pathogenesis-related (PR) proteins localised in intercellular spaces of barley primary leaves, chlorophyll content, structure of chloroplasts, and photosynthesis were examined during natural and in vitro induced leaf senescence (cultivation of whole plants in the dark or detached leaves under nutrient deficiency). Some of PR proteins accumulated during natural senescence, but their accumulation pattern was different from those of pathogen-induced as well as during in vitro-induced senescence, which indicate different molecular bases of these processes. Photosynthetic rate and chlorophyll content indicate that natural senescence of barley primary leaves began from 15th day after sowing. In 35-d-old first leaves, the chloroplasts showed typical characteristics of senescence as significant decrease of size, greater grana, and prominent plastoglobuli. The chloroplasts contained more grana under in vitro induced senescence and they had reduced length in the dark. Correspondingly, accumulation of PR proteins was detectable on about the 15th day but the content of some PR proteins increased in later stages of senescence. This revised version was published online in July 2006 with corrections to the Cover Date.