A role for salicylic acid and NPR1 in regulating cell growth in Arabidopsis

Salicylic acid (SA) plays a key role in activating defenses and cell death during plant-pathogen interactions. In response to some pathogens, SA also limits the extent of cell death, indicating that it acts positively or negatively depending on the host-pathogen interaction. In addition, we previously showed that SA affects cell growth in the Arabidopsis defense-related mutants accelerated cell death 6-1 (acd6-1) and aberrant growth and death 2 (agd2). Using acd6-1, agd2 and two other defense-related mutants, lesion simulating disease 6 (lsd6), suppressor of SA-insensitivity (ssi1), we show here in detail that SA regulates cell growth by specifically affecting cell enlargement, endoreduplication and/or cell division. We find that SA can act either positively or negatively to regulate cell growth depending on the context in which signaling occurs. Additionally, Nonexpressor of PR 1 (NPR1), a key SA signaling protein important for regulating defenses and cell death, also acts to promote cell division and/or suppress endoreduplication during leaf development. We propose that SA interacts with multiple receptors or signaling pathways to control cellular alterations during normal development, pathogen attack and/or stress situations. We suggest that SA and NPR1 play broader roles in cell fate control than has previously been understood.     

Evidence for a dual role for TC4 protein in regulating nuclear structure and cell cycle progression

TC4, a ras-like G protein, has been implicated in the feedback pathway linking the onset of mitosis to the completion of DNA replication. In this report we find distinct roles for TC4 in both nuclear assembly and cell cycle progression. Mutant and wild-type forms of TC4 were added to Xenopus egg extracts capable of assembling nuclei around chromatin templates in vitro. We found that a mutant TC4 protein defective in GTP binding (GDP-bound form) suppressed nuclear growth and prevented DNA replication. Nuclear transport under these conditions approximated normal levels. In a separate set of experiments using a cell-free extract of Xenopus eggs that cycles between S and M phases, the GDP- bound form of TC4 had dramatic effects, blocking entry into mitosis even in the complete absence of nuclei. The effect of this mutant TC4 protein on cell cycle progression is mediated by phosphorylation of p34cdc2 on tyrosine and threonine residues, negatively regulating cdc2 kinase activity. Therefore, we provide direct biochemical evidence for a role of TC4 in both maintaining nuclear structure and in the signaling pathways that regulate entry into mitosis.     

Evidence for a role of salicylic acid in the oxidative damage generated by NaCl and osmotic stress in Arabidopsis seedlings.

Previous studies have shown that salicylic acid (SA) is an essential component of the plant resistance to pathogens. We now show that SA plays a role in the plant response to adverse environmental conditions, such as salt and osmotic stresses. We have studied the responses of wild-type Arabidopsis and an SA-deficient transgenic line expressing a salicylate hydroxylase (NahG) gene to different abiotic stress conditions. Wild-type plants germinated under moderate light conditions in media supplemented with 100 mM NaCl or 270 mM mannitol showed extensive necrosis in the shoot. In contrast, NahG plants germinated under the same conditions remained green and developed true leaves. The lack of necrosis observed in NahG seedlings under the same conditions suggests that SA potentiates the generation of reactive oxygen species in photosynthetic tissues during salt and osmotic stresses. This hypothesis is supported by the following observations. First, the herbicide methyl viologen, a generator of superoxide radical during photosynthesis, produced a necrotic phenotype only in wild-type plants. Second, the presence of reactive oxygen-scavenging compounds in the germination media reversed the wild-type necrotic phenotype seen under salt and osmotic stress. Third, a greater increase in the oxidized state of the glutathione pool under NaCl stress was observed in wild-type seedlings compared with NahG seedlings. Fourth, greater oxidative damage occurred in wild-type seedlings compared with NahG seedlings under NaCl stress as measured by lipid peroxidation. Our data support a model for SA potentiating the stress response of the germinating Arabidopsis seedling.     

Evidence for a role for cardiac myosin in regulating the contractile response.

Dinitrophenylated bovine cardiac myosin incorporates 1.3 mol of 1-fluoro-2,4-dinitro-benzene per 5 × 10⁵ g of protein. Concomitantly there was an activation of the Ca²⁺-ATPase activity and an inhibition of the K⁺(EDTA)-ATPase activity. The dinitrophenyl group is located in the smallest active proteolytic fragment, subfragment 1. Virtually all of the labeling occurs in the region containing the heavy chains of cardiac myosin as judged by dissociation experiments in sodium dodecyl sulfate. Dinitrophenylated myosin failed to form calcium-sensitive actomyosin when tested in an ATPase assay system containing actin, tropomyosin, troponin and ethylene glycol-bis(β-aminoethyl ether)N,N′-tetraacetic acid. Thiolysis of the dinitrophenyl group from myosin with 2-mercaptoethanol restored its ability to form a calcium-sensitive actomyosin. The Ca²⁺ and K⁺(EDTA)-ATPase activities were also restored to control values. These results indicate that cardiac myosin participates in the regulation of the interaction between the contractile proteins.     

高温强光对小麦叶绿体Deg1蛋白酶和D1蛋白的影响及水杨酸的调节作用

以小麦品种矮抗58为材料,采用0.3 mmol/L水杨酸(SA)溶液预处理灌浆期小麦叶片,以水预处理为对照,进行3种不同的光温处理:适宜温度中等光强(25℃,600 μmol m-2 s-1)2h、高温强光(38℃,1600μmol m-2 s-1)2h、高温强光2h后置于适宜温度中等光强下恢复3h.测定不同光温条件下,小麦叶绿体的Deg1蛋白酶、D1蛋白和PSⅡ功能的变化及SA的调节效应.结果表明,高温强光胁迫导致Deg1蛋白酶和D1蛋白降解,PSⅡ功能发生可逆损伤.与对照相比,水杨酸预处理不仅能够抑制高温强光下小麦叶绿体Deg1蛋白酶和D1蛋白的降解,维持较高的PSⅡ原初光化学效率(Fv/ Fm)、实际光化学效率(φPSⅡ)、电子传递速率和净光合速率(Pn),而且加快回到非逆境下PSⅡ功能的恢复.     

Evidence that the role of plant defensins in radish defense responses is independent of salicylic acid

Radish leaves contain two homologous 5-kDa plant defensins which accumulate systemically upon infection by fungal pathogens (F.R.G. Terras et al., 1995, Plant Cell 7: 573–588). Here we report on the molecular cloning of the cDNAs encoding the two pathogen-inducible plant defensin isoforms from radish (Raphanus sativus L.) leaves. Tissue-print and whole-leaf electroblot immunostaining showed that the plant defensin peptides not only accumulate at high levels at or immediately around the infection sites in leaves inoculated with Alternariabrassicicola, but also accumulate in healthy tissue further away from the infection sites and in non-infected leaves from infected plants. Gel blot analysis of RNA confirmed that expression of plant defensin genes is systemically triggered upon fungal infection whereas radish PR-1 gene expression is only activated locally. In contrast to the radish PR-1 gene(s), expression of the radish plant defensin genes was not induced by external application of salicylic acid. Activation of the plant defensin genes, but not that of PR-1 genes, occurred upon treatment with methyl jasmonate, ethylene and paraquat. Received: 3 December 1997 / Accepted: 3 March 1998     

Evidence for a role of phytochelatins in regulating arsenic accumulation in rice grain

Phytochelatins (PCs) play a crucial role in detoxifying cellular arsenic (As) through complexation of arsenite. Here, we investigated whether PCs influence As accumulation in rice grain by using six rice cultivars varying in grain As accumulation. The cultivars with low grain As had significantly higher PCs concentration in the shoots than the cultivars with high grain As, but lower glutathione concentration. Shoot PCs concentration correlated negatively with grain As accumulation. Foliar sprays with 0.5 mM l-buthionine-sulphoxime (BSO) on rice leaves at grain filling stage decreased GSH and PC accumulation in rice shoots by 40-63% and 20-55%, respectively, but did not significantly affect plant growth. Foliar sprays with BSO decreased shoot As concentration, while increased As concentrations in husk and brown rice significantly. These results suggest that PC complexation of arsenite in rice leaves reduces As translocation from leaves to grains, and implicate that manipulation of PC synthesis might mitigate As accumulation in rice grain.     

The role of salicylic acid and jasmonic acid in pathogen defence

Phytohormones are not only instrumental in regulating developmental processes in plants but also play important roles for the plant's responses to biotic and abiotic stresses. In particular, abscisic acid, ethylene, jasmonic acid, and salicylic acid have been shown to possess crucial functions in mediating or orchestrating stress responses in plants. Here, we review the role of salicylic acid and jasmonic acid in pathogen defence responses with special emphasis on their function in the solanaceous plant potato.     

Mutational analysis of a role for salicylic acid in iron metabolism of Mycobacterium smegmatis

The role of salicylic acid in iron metabolism was examined in two wild-type strains (mc(2)155 and NCIMB 8548) and three mutant strains (mc(2)1292 [lacking exochelin], SM3 [lacking iron-dependent repressor protein IdeR] and S99 [a salicylate-requiring auxotroph derived in this study]) of Mycobacterium smegmatis. Synthesis of salicylate in SM3 was derepressed even in the presence of iron, as was synthesis of the siderophores exochelin, mycobactin, and carboxymycobactin. S99 was dependent on salicylate for growth and failed to grow with the three ferrisiderophores, suggesting that salicylate fulfills an additional function(s) other than being a precursor of mycobactin and carboxymycobactin. Salicylic acid at 100 microgram/ml repressed the formation of a 29-kDa cell envelope protein (putative exochelin receptor protein) in S99 grown both iron deficiently and iron sufficiently. In contrast, synthesis of this protein was affected only under iron-limited conditions in the parent strain, mc(2)155, and remained unaltered in SM3, suggesting an interaction between the IdeR protein and salicylate. Thus, salicylate may also function as a signal molecule for recognition of cellular iron status. Growth of all strains and mutants with p-aminosalicylate (PAS) at 100 microgram/ml increased salicylate accumulation between three- and eightfold under both iron-limited and iron-sufficient growth conditions and decreased mycobactin accumulation by 40 to 80% but increased carboxymycobactin accumulation by 50 to 55%. Thus, although PAS inhibited salicylate conversion to mycobactin, presumptively by blocking salicylate AMP kinase, PAS also interferes with the additional functions of salicylate, as its effect was heightened in S99 when the salicylate concentration was minimal.     

Evidence for a role of SNX16 in regulating traffic between the early and later endosomal compartments

Sorting nexins (SNXs) are a growing family of proteins characterized by the presence of a PX domain. The PX domain mediates membrane association by interaction with phosphoinositides. The SNXs are generally believed to participate in membrane trafficking, but information regarding the function of individual proteins is limited. In this report, we describe the major characteristics of one member, SNX16. SNX16 is a novel 343-amino acid protein consisting of a central PX domain followed by a potential coiled-coil domain and a C-terminal region. Like other sorting nexins, SNX16 associates with the membrane via the PX domain which interacts with the phospholipid phosphatidylinositol 3-phosphate. We show via biochemical and cellular studies that SNX16 is distributed in both early and late endosome/lysosome structures. The coiled-coil domain is necessary for localization to the later endosomal structures, as mutant SNX16 lacking this domain was found only in early endosomes. Trafficking of internalized epidermal growth factor was also delayed by this SNX16 mutant, as these cells showed a delay in the segregation of epidermal growth factor in the early endosome for its delivery to later compartments. In addition, the coiled-coil domain is shown here to be important for homo-oligomerization of SNX16. Taken together, these results suggest that SNX16 is a sorting nexin that may function in the trafficking of proteins between the early and late endosomal compartments.     

Evidence for a direct role for sialic acid in the attachment of encephalomyocarditis virus to human erythrocytes

Sialic acid residues are required in cellular receptors for many different mammalian viruses. Sialic acid could have a direct role, being an integral part of the virus binding site on the receptor. Alternatively, negatively charged sialic acid could have an indirect role, being responsible for holding the receptor in the required configuration for virus recognition, for instance, by interacting with positively charged amino acid residues found in the polypeptide chain of receptors. We have investigated the role of sialic acid in virus attachment by studying the interaction of the small RNA virus encephalomyocarditis (EMC) with glycophorin A, its receptor on human erythrocytes. In several experiments, influenza virus A was used for control purposes. Blocking positive charges on glycophorin either in lysine residues by acetylation or in arginine residues with butanedione did not affect its interaction with EMC virus. In contrast, blocking negatively charged carboxyl groups in sialic acid residues by amidation destroyed the ability of glycophorin to inhibit EMC virus attachment suggesting an important role for this part of sialic acid in EMC virus attachment. Removal of the polyhydroxy side chain in sialic acid residues of glycophorin by mild oxidation with periodate followed by reduction with borohydride had little effect on its interaction with EMC virus. Further, sialic acid species with either an acetyl or glycolyl group attached to the amino group on position 5 interacted equally well with EMC virus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for regulation of resistance in Arabidopsis to Egyptian cotton worm by salicylic and jasmonic acid signaling pathways

Signaling cross-talk between wound- and pathogen-response pathways influences resistance of plants to insects and disease. To elucidate potential interactions between salicylic acid (SA) and jasmonic acid (JA) defense pathways, we exploited the availability of characterized mutants of Arabidopsis thaliana (L.) Heynh. and monitored resistance to Egyptian cotton worm (Spodoptera littoralis Boisd.; Lepidoptera: Noctuidae). This generalist herbivore is sensitive to induced plant defense pathways and is thus a useful model for a mechanistic analysis of insect resistance. As expected, treatment of wild-type Arabidopsis with JA enhanced resistance to Egyptian cotton worm. Conversely, the coil mutant, with a deficiency in the JA response pathway, was more susceptible to Egyptian cotton worm than wild-type Arabidopsis. By contrast, the nprl mutant, with defects in systemic disease resistance, exhibited enhanced resistance to Egyptian cotton worm. Pretreatment with SA significantly reduced this enhanced resistance of nprl plants but had no influence on the resistance of wild-type plants. However, exogenous SA reduced the amount of JA that Egyptian cotton worm induced in both npr1 mutant and wild-type plants. Thus, this generalist herbivore engages two different induced defense pathways that interact to mediate resistance in Arabidopsis.     

Effect of diabetes and protein malnutrition on the rate of muscle protein breakdown in rats

Male streptocozin diabetic rats were fed ad libitum in two diets, one a control, adequate in protein and energy, and another, depleted in protein, but adequate in energy. Within each one of these dietary groups, three hormone-treated groups were made as follows: rats receiving vehicle, or 0.25 or 0.50 I.U. insulin/100 g body weight/day i.p. for 21 days. A fourth group of intact rats, receiving vehicle injection, was included as a control. Every day urine excretion was collected for urea-N and 3-methylhistidine (3-Mehis) determination. Body weight and food intake were recorded daily. At the end of the experiment, all animals were sacrificed, and a sample of blood was taken for plasma insulin assay. Liver, as well as gastrocnemius, soleus and extensor digitorum longus muscles were excised and weighed. Results showed that diabetic animals had a reduced body weight gain, although the food intake was elevated in all groups, as compared to the intact rats. Gastrocnemius and soleus muscle weights were, respectively, reduced and increased in the diabetic animals fed the low-protein diet. Urea-N output was elevated in all groups fed the control diet, but a marked reduction was observed in the protein depleted rats. A reduction in 3-Mehis output was displayed by the diabetic animals, specially those fed the low-protein diet. The results of this experiment showed that in streptocozin diabetic rats there was a reduction in the rate of myofibrillar protein breakdown, specially marked when fed a protein depleted diet.     

A role for retinoic acid in regulating the regeneration of deer antlers

Deer antlers are the only mammalian organs that can be repeatedly regenerated; each year, these complex structures are shed and then regrow to be used for display and fighting. To date, the molecular mechanisms controlling antler regeneration are not well understood. Vitamin A and its derivatives, retinoic acids, play important roles in embryonic skeletal development. Here, we provide several lines of evidence consistent with retinoids playing a functional role in controlling cellular differentiation during bone formation in the regenerating antler. Three receptors (alpha, beta, gamma) for both the retinoic acid receptor (RAR) and retinoid X receptor (RXR) families show distinct patterns of expression in the growing antler tip, the site of endochondral ossification. RAR alpha and RXR beta are expressed in skin ("velvet") and the underlying perichondrium. In cartilage, which is vascularised, RXR beta is specifically expressed in chondrocytes, which express type II collagen, and RAR alpha in perivascular cells, which also express type I collagen, a marker of the osteoblast phenotype. High-performance liquid chromatography analysis shows significant amounts of Vitamin A (retinol) in antler tissues at all stages of differentiation. The metabolites all-trans-RA and 4-oxo-RA are found in skin, perichondrium, cartilage, bone, and periosteum. The RXR ligand, 9-cis-RA, is found in perichondrium, mineralised cartilage, and bone. To further define sites of RA synthesis in antler, we immunolocalised retinaldehyde dehydrogenase type 2 (RALDH-2), a major retinoic acid-generating enzyme. RALDH-2 is expressed in the skin and perichondrium and in perivascular cells in cartilage, although chondroprogenitors and chondrocytes express very low levels. At sites of bone formation, differentiated osteoblasts which express the bone-specific protein osteocalcin express high levels of RALDH2. The effect of RA on antler cell differentiation was studied in vitro; all-trans-RA inhibits expression of the chondrocyte phenotype, an effect that is blocked by addition of the RAR antagonist Ro41-5253. In monolayer cultures of mesenchymal progenitor cells, all-trans-RA increases the expression of alkaline phosphatase, a marker of the osteoblastic phenotype. In summary, this study has shown that antler tissues contain endogenous retinoids, including 9-cis RA, and the enzyme RALDH2 that generates RA. Sites of RA synthesis in antler correspond closely with the localisation of cells which express receptors for these ligands and which respond to the effects of RA.     

Role of light and jasmonic acid signaling in regulating foliar phloem cell wall ingrowth development

Phloem cells adjacent to sieve elements can possess wall invaginations. The role of light and jasmonic acid signaling in wall ingrowth development was examined in pea companion cells (CCs), Arabidopsis thaliana phloem parenchyma cells (PCs), and in Senecio vulgaris (with ingrowths in both cell types). Features characterized included wall ingrowths (from electron microscopic images), foliar vein density and photosynthetic capacity. In Arabidopsis, wall ingrowths were bulky compared with finger-like invaginations in pea and S. vulgaris. Relative to low light (LL), wall invagination in both CCs and PCs was greater in high light (HL). Treatment with methyl jasmonate in LL had no effect on CCs, but increased PC wall ingrowths. LL-to-HL transfer resulted in significantly less wall ingrowth in the fad7-1 fad8-1 (jasmonate-deficient) Arabidopsis mutant relative to the wild type. These results suggest that chloroplast oxidative status, via chloroplast-derived jasmonates, may modulate phloem structure and function. While CC wall ingrowths facilitate phloem loading by expanding the membrane area available for active uptake, one can speculate that phloem PC ingrowths may have two potential roles: to increase the efflux of sugars and/or protons into the apoplast to augment phloem loading; and/or to protect the phloem against pathogens and/or insects.     

The Arabidopsis NPR1 protein is a receptor for the plant defense hormone salicylic acid

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Highlights? NPR1 is a salicylic acid (SA) receptor, binding specifically to SA via Cys^521/529 ? NPR1 binds copper in vivo via Cys^521/529, and metals are required for SA binding ? SA directly regulates the conformation of NPR1 by deoligomerizing NPR1 into a dimer ? The NPR1 BTB/POZ domain autoinhibits the function of the NPR1 transactivation domain     

Questioning the role of salicylic acid and cytosolic acidification in mitogen-activated protein kinase activation induced by cryptogein in tobacco cells

Elicitors of plant defence reactions, oligogalacturonides and cryptogein, an elicitin produced by Phytophthora cryptogea, were previously shown to induce a rapid and transient activation of two mitogen-activated protein kinases (MAPKs) in cells of tobacco [ Nicotiana tabacum L. cv. Xanthi; A. Lebrun-Garcia et al. (1998) Plant J 15:773-781]. We verified that these two MAPKs correspond to the salicylic acid-induced protein kinase (SIPK) and the wound-induced protein kinase (WIPK). The involvement of salicylic acid (SA) in cryptogein-induced MAPK activation was investigated using transgenic NahG tobacco cells expressing the salicylate hydroxylase gene and thus unable to accumulate SA. The large and sustained activation of both MAPKs by cryptogein was maintained in transgenic cells compared with non-transgenic tobacco cells. Moreover, weak acids, namely SA, 4-hydroxybenzoic acid, an ineffective analogue of SA in plant resistance, and butyric acid acidified the cytosol, a physiological event also induced by cryptogein, but activated both MAPKs only slightly and transiently in tobacco cells. These results indicate that MAPK activation by cryptogein is not mediated by SA, that cytosolic acidification can be transduced by MAPKs, and that in cryptogein-treated cells, cytosolic acidification should contribute poorly to MAPK activation.