Glycerol Affects Root Development through Regulation of Multiple Pathways in Arabidopsis

Glycerol metabolism has been well studied biochemically. However, the means by which glycerol functions in plant development is not well understood. This study aimed to investigate the mechanism underlying the effects of glycerol on root development in Arabidopsis thaliana. Exogenous glycerol inhibited primary root growth and altered lateral root development in wild-type plants. These phenotypes appeared concurrently with increased endogenous glycerol-3-phosphate (G3P) and H₂O₂ contents in seedlings, and decreased phosphate levels in roots. Upon glycerol treatment, G3P level and root development did not change in glycerol kinase mutant gli1, but G3P level increased in gpdhc1 and fad-gpdh mutants, which resulted in more severely impaired root development. Overexpression of the FAD-GPDH gene attenuated the alterations in G3P, phosphate and H₂O₂ levels, leading to increased tolerance to exogenous glycerol, which suggested that FAD-GPDH plays an important role in modulating this response. Free indole-3-acetic acid (IAA) content increased by 46%, and DR5pro::GUS staining increased in the stele cells of the root meristem under glycerol treatment, suggesting that glycerol likely alters normal auxin distribution. Decreases in PIN1 and PIN7 expression, β-glucuronidase (GUS) staining in plants expressing PIN7pro::GUS and green fluorescent protein (GFP) fluorescence in plants expressing PIN7pro::PIN7-GFP were observed, indicating that polar auxin transport in the root was downregulated under glycerol treatment. Analyses with auxin-related mutants showed that TIR1 and ARF7 were involved in regulating root growth under glycerol treatment. Glycerol-treated plants showed significant reductions in root meristem size and cell number as revealed by CYCB1;1pro::GUS staining. Furthermore, the expression of CDKA and CYCB1 decreased significantly in treated plants compared with control plants, implying possible alterations in cell cycle progression. Our data demonstrated that glycerol treatment altered endogenous levels of G3P, phosphate and ROS, affected auxin distribution and cell division in the root meristem, and eventually resulted in modifications of root development.     

Chitin-supplemented Foliar Application of Serratia marcescens GPS 5 Improves Control of Late Leaf Spot Disease of Groundnut by Activating Defence-related Enzymes

Chitinolytic Serratia marcescens GPS 5 and non‐chitinolytic Pseudomonas aeruginosa GSE 18, with and without supplementation of chitin, were tested for their ability to activate defence‐related enzymes in groundnut leaves. Thirty‐day‐old groundnut (cv. TMV 2) plants pretreated with GPS 5 and GSE 18 (with and without supplementation of 1% colloidal chitin) were challenge inoculated after 24 h with Phaeoisariopsis personata, the causal agent of late leaf spot (LLS) disease of groundnut. GPS 5 and GSE 18, applied as a prophylactic spray, reduced the lesion frequency by 23% and 67%, respectively, compared with control. Chitin supplementation had no effect on the control of LLS by GSE 18, unlike GPS 5, which upon chitin supplementation reduced the lesion frequency by 64%, compared with chitin alone. In a time course study the activities of chitinase, β‐1,3‐glucanase, peroxidase and phenylalanine ammonia lyase were determined for the different treatments. There was an enhanced activity of the four defence‐related enzymes with all the bacterial treatments when compared with phosphate buffer and colloidal chitin‐treated controls. In correlation to disease severity in bacterial treatments, chitin‐supplemented GSE 18 was similar to GSE 18, whereas chitin‐supplemented GPS 5 was much more effective than GPS 5, in activation of the defence‐related enzymes. The high levels of enzyme activities following chitin‐supplemented GPS 5 application continued up to the measured 13 days after pathogen inoculation.     

Dickeya dadantii pectic enzymes necessary for virulence are also responsible for activation of the Arabidopsis thaliana innate immune system

Soft‐rot diseases of plants attributed to Dickeya dadantii result from lysis of the plant cell wall caused by pectic enzymes released by the bacterial cell by a type II secretion system (T2SS). Arabidopsis thaliana can express several lines of defence against this bacterium. We employed bacterial mutants with defective envelope structures or secreted proteins to examine early plant defence reactions. We focused on the production of AtrbohD‐dependent reactive oxygen species (ROS), callose deposition and cell death as indicators of these reactions. We observed a significant reduction in ROS and callose formation with a bacterial mutant in which genes encoding five pectate lyases (Pels) were disrupted. Treatment of plant leaves with bacterial culture filtrates containing Pels resulted in ROS and callose production, and both reactions were dependent on a functional AtrbohD gene. ROS and callose were produced in response to treatment with a cellular fraction of a T2SS‐negative mutant grown in a Pels‐inducing medium. Finally, ROS and callose were produced in leaves treated with purified Pels that had also been shown to induce the expression of jasmonic acid‐dependent defence genes. Pel catalytic activity is required for the induction of ROS accumulation. In contrast, cell death observed in leaves infected with the wild‐type strain appeared to be independent of a functional AtrbohD gene. It was also independent of the bacterial production of pectic enzymes and the type III secretion system (T3SS). In conclusion, the work presented here shows that D. dadantii is recognized by the A. thaliana innate immune system through the action of pectic enzymes secreted by bacteria at the site of infection. This recognition leads to AtrbohD‐dependent ROS and callose accumulation, but not cell death.     

Two thiadiazole compounds promote rice defence against Xanthomonas oryzae pv. oryzae by suppressing the bacterium's production of extracellular polysaccharides

Thiazole, isothiazole, thiadiazole, and their derivatives are used to control various human, animal and plant diseases. In addition to having direct anti‐microbial and anti‐fungal properties, these compounds are thought to induce host defences, but the mechanism of defence induction remains poorly understood. This article reports that the thiadiazoles of zinc thiazole and bismerthiazol induce H₂O₂ accumulation, up‐regulation of defence‐related genes, callose deposition and hypersensitive response‐like cell death in rice leaves infected with Xanthomonas oryaze pv. oryzae (Xoo) strain ZJ173, but not in non‐infected leaves. These defence responses in Xoo‐infected leaves were suppressed by the exogenous application of catalase, which reduces H₂O₂ accumulation. The application of extracellular polysaccharides (EPSs) extracted from strain ZJ173 significantly compromised rice defence against ZJ173 with or without thiadiazole treatment. The EPS‐deficient Xoo mutant ?gumH triggered a stronger defence than its parent strain ZJ173. The thiadiazole treatments reduced EPS production by strain ZJ173, but not by the thiadiazole‐resistant strain 2‐1‐1, which is thiadiazole resistant in vivo, but not in vitro; moreover, enhanced defence was not detected in thiadiazole‐treated rice inoculated with 2‐1‐1. Based on these data, we infer that zinc thiazole and bismerthiazol promote rice defence against Xoo by inhibiting the production of bacterial EPS.     

Drought-induced proline accumulation is uninvolved with increased nitric oxide,which alleviates drought stress by decreasing transpiration in rice

Accumulation of proline is trusted to be an adaptive response of plants against drought stress, and exogenous application of nitric oxide (NO) enhances proline accumulation in Cu-treated algae. In order to investigate whether NO works as a necessary signaling molecule in drought-induced proline accumulation in rice leaves, effects of drought stress on endogenous NO content and proline accumulation were studied in rice leaves, using sodium nitroprusside (SNP, a NO donor) and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO, a NO scavenger). The results showed that drought treatment increased both endogenous NO and proline contents in rice leaves, while foliar spray of various concentrations of SNP failed to induce proline accumulation in the leaves of well-watered rice and foliar spray of cPTIO failed to inhibit proline accumulation in the leaves of drought-stressed rice. These results indicate that increase of endogenous NO is dispensable for proline accumulation in the leaves of rice under drought stress. Further studies indicate that exogenous application of NO alleviates drought-induced water loss and ion leakage by decreasing transpiration rate of rice leaves.     

Novel receptor‐like kinases in cacao contain PR‐1 extracellular domains

Members of the pathogenesis‐related protein 1 (PR‐1) family are well‐known markers of plant defence responses, forming part of the arsenal of the secreted proteins produced on pathogen recognition. Here, we report the identification of two cacao (Theobroma cacao L.) PR‐1s that are fused to transmembrane regions and serine/threonine kinase domains, in a manner characteristic of receptor‐like kinases (RLKs). These proteins (TcPR‐1f and TcPR‐1g) were named PR‐1 receptor kinases (PR‐1RKs). Phylogenetic analysis of RLKs and PR‐1 proteins from cacao indicated that PR‐1RKs originated from a fusion between sequences encoding PR‐1 and the kinase domain of a LecRLK (Lectin Receptor‐Like Kinase). Retrotransposition marks surround TcPR‐1f, suggesting that retrotransposition was involved in the origin of PR‐1RKs. Genes with a similar domain architecture to cacao PR‐1RKs were found in rice (Oryza sativa), barrel medic (Medicago truncatula) and a nonphototrophic bacterium (Herpetosiphon aurantiacus). However, their kinase domains differed from those found in LecRLKs, indicating the occurrence of convergent evolution. TcPR‐1g expression was up‐regulated in the biotrophic stage of witches' broom disease, suggesting a role for PR‐1RKs during cacao defence responses. We hypothesize that PR‐1RKs transduce a defence signal by interacting with a PR‐1 ligand.     

Determinants of substrate specificity and biochemical properties of the sn‐glycerol‐3‐phosphate ATP binding cassette transporter (UgpB–AEC2) of Escherichia coli

Under phosphate starvation conditions, Escherichia coli can utilize sn‐glycerol‐3‐phosphate (G3P) and G3P diesters as phosphate source when transported by an ATP binding cassette importer composed of the periplasmic binding protein, UgpB, the transmembrane subunits, UgpA and UgpE, and a homodimer of the nucleotide binding subunit, UgpC. The current knowledge on the Ugp transporter is solely based on genetic evidence and transport assays using intact cells. Thus, we set out to characterize its properties at the level of purified protein components. UgpB was demonstrated to bind G3P and glycerophosphocholine with dissociation constants of 0.68 ± 0.02 μM and 5.1 ± 0.3 μM, respectively, while glycerol‐2‐phosphate (G2P) is not a substrate. The crystal structure of UgpB in complex with G3P was solved at 1.8 Å resolution and revealed the interaction with two tryptophan residues as key to the preferential binding of linear G3P in contrast to the branched G2P. Mutational analysis validated the crucial role of Trp‐169 for G3P binding. The purified UgpAEC₂ complex displayed UgpB/G3P‐stimulated ATPase activity in proteoliposomes that was neither inhibited by phosphate nor by the signal transducing protein PhoU or the phosphodiesterase UgpQ. Furthermore, a hybrid transporter composed of MalFG–UgpC could be functionally reconstituted while a UgpAE–MalK complex was unstable.     

Development of a Saccharomyces cerevisiae strain for increasing the accumulation of triacylglycerol as a microbial oil feedstock for biodiesel production using glycerol as a substrate

Triacylglycerol (TAG) is a microbial oil feedstock for biodiesel production that uses an inexpensive substrate, such as glycerol. Here, we demonstrated the overproduction of TAG from glycerol in engineered Saccharomyces cerevisiae via the glycerol‐3‐phosphate (G3P) pathway by overexpressing the major TAG synthesis. The G3P accumulation was increased 2.4‐fold with the increased glycerol utilization gained by the overexpression of glycerol kinase (GUT1). By overexpressing diacylglycerol acyltransferase (DGA1) and phospholipid diacylglycerol acyltransferase (LRO1), the engineered YPH499 (pGutDgaLro1) strain produced 23.0 mg/L lipids, whereas the YPH499 (pESC‐TRP) strain produced 6.2 mg/L total lipids and showed a lipid content that was increased 1.4‐fold compared with 3.6% for the wild‐type strain after 96 h of cultivation. After 96 h of cultivation using glycerol, the overall content of TAG in the engineered strain, YPH499 (pGutDgaLro1), yielded 8.2% TAG, representing a 2.3‐fold improvement, compared with 3.6% for the wild‐type strain. The results should allow a reduction of costs and a more sustainable production of biodiesel. Biotechnol. Bioeng. 2013; 110: 343–347. © 2012 Wiley Periodicals, Inc.     

A role for β‐sitosterol to stigmasterol conversion in plant–pathogen interactions

Upon inoculation with pathogenic microbes, plants induce an array of metabolic changes that potentially contribute to induced resistance or even enhance susceptibility. When analysing leaf lipid composition during the Arabidopsis thaliana–Pseudomonas syringae interaction, we found that accumulation of the phytosterol stigmasterol is a significant plant metabolic process that occurs upon bacterial leaf infection. Stigmasterol is synthesized from β‐sitosterol by the cytochrome P450 CYP710A1 via C22 desaturation. Arabidopsis cyp710A1 mutant lines impaired in pathogen‐inducible expression of the C22 desaturase and concomitant stigmasterol accumulation are more resistant to both avirulent and virulent P. syringae strains than wild‐type plants, and exogenous application of stigmasterol attenuates this resistance phenotype. These data indicate that induced sterol desaturation in wild‐type plants favours pathogen multiplication and plant susceptibility. Stigmasterol formation is triggered through perception of pathogen‐associated molecular patterns such as flagellin and lipopolysaccharides, and through production of reactive oxygen species, but does not depend on the salicylic acid, jasmonic acid or ethylene defence pathways. Isolated microsomal and plasma membrane preparations exhibited a similar increase in the stigmasterol/β‐sitosterol ratio as whole‐leaf extracts after leaf inoculation with P. syringae, indicating that the stigmasterol produced is incorporated into plant membranes. The increased contents of stigmasterol in leaves after pathogen attack do not influence salicylic acid‐mediated defence signalling but attenuate pathogen‐induced expression of the defence regulator flavin‐dependent monooxygenase 1. P. syringae thus promotes plant disease susceptibility through stimulation of sterol C22 desaturation in leaves, which increases the stigmasterol to β‐sitosterol ratio in plant membranes.     

Role of salicylic acid and fatty acid desaturation pathways in ssi2-mediated signaling

Stearoyl-acyl carrier protein desaturase-mediated conversion of stearic acid to oleic acid (18:1) is the key step that regulates the levels of unsaturated fatty acids (FAs) in cells. Our previous work with the Arabidopsis (Arabidopsis thaliana) ssi2/fab2 mutant and its suppressors demonstrated that a balance between glycerol-3-phosphate (G3P) and 18:1 levels is critical for the regulation of salicylic acid (SA)- and jasmonic acid-mediated defense signaling in the plant. In this study, we have evaluated the role of various genes that have an impact on SA, resistance gene-mediated, or FA desaturation (FAD) pathways on ssi2-mediated signaling. We show that ssi2-triggered resistance is dependent on EDS1, PAD4, EDS5, SID2, and FAD7 FAD8 genes. However, ssi2-triggered defects in the jasmonic acid pathway, morphology, and cell death phenotypes are independent of the EDS1, EDS5, PAD4, NDR1, SID2, FAD3, FAD4, FAD5, DGD1, FAD7, and FAD7 FAD8 genes. Furthermore, the act1-mediated rescue of ssi2 phenotypes is also independent of the FAD2, FAD3, FAD4, FAD5, FAD7, and DGD1 genes. Since exogenous application of glycerol converts wild-type plants into ssi2 mimics, we also studied the effect of exogenous application of glycerol on mutants impaired in resistance-gene signaling, SA, or fad pathways. Glycerol increased SA levels and induced pathogenesis-related gene expression in all but sid2, nahG, fad7, and fad7 fad8 plants. Furthermore, glycerol-induced phenotypes in various mutant lines correlate with a concomitant reduction in 18:1 levels. Inability to convert glycerol into G3P due to a mutation in the nho1-encoded glycerol kinase renders plants tolerant to glycerol and unable to induce the SA-dependent pathway. A reduction in the NHO1-derived G3P pool also results in a partial age-dependent rescue of the ssi2 morphological and cell death phenotypes in the ssi2 nho1 plants. The glycerol-mediated induction of defense was not associated with any major changes in the lipid profile and/or levels of phosphatidic acid. Taken together, our results suggest that glycerol application and the ssi2 mutation in various mutant backgrounds produce similar effects and that restoration of ssi2 phenotypes is not associated with the further desaturation of 18:1 to linoleic or linolenic acids in plastidal or extraplastidal lipids.     

Glycine betaine involvement in freezing tolerance and water stress in Arabidopsis thaliana

Levels of endogenous glycine betaine in the leaves were measured in response to cold acclimation, water stress and exogenous ABA application in Arabidopsis thaliana. The endogenous glycine betaine level in the leaves increased sharply during cold acclimation treatment as plants gained freezing tolerance. When glycine betaine (10 mM) was applied exogenously to the plants as a foliar spray, the freezing tolerance increased from -3.1 to -4.5 degrees C. In addition, when ABA (1 mM) was applied exogenously, the endogenous glycine betaine level and the freezing tolerance in the leaves increased. However, the increase in the leaf glycine betaine level induced by ABA was only about half of that by the cold acclimation treatment. Furthermore, when plants were subjected to water stress (leaf water potential of approximately -1.6 MPa), the endogenous leaf glycine betaine level increased by about 18-fold over that in the control plants. Water stress lead to significant increase in the freezing tolerance, which was slightly less than that induced by the cold acclimation treatment. The results suggest that glycine betaine is involved in the induction of freezing tolerance in response to cold acclimation, ABA, and water stress in Arabidopsis plants.     

Ecological significance of light quality in optimizing plant defence

Plants balance the allocation of resources between growth and defence to optimize fitness in a competitive environment. Perception of neighbour‐detection cues, such as a low ratio of red to far‐red (R:FR) radiation, activates a suite of shade‐avoidance responses that include stem elongation and upward leaf movement, whilst simultaneously downregulating defence. This downregulation is hypothesized to benefit the plant either by mediating the growth‐defence balance in favour of growth in high plant densities or, alternatively, by mediating defence of individual leaves such that those most photosynthetically productive are best protected. To test these hypotheses, we used a 3D functional–structural plant model of Brassica nigra that mechanistically simulates the interactions between plant architecture, herbivory, and the light environment. Our results show that plant‐level defence expression is a strong determinant of plant fitness and that leaf‐level defence mediation by R:FR can provide a fitness benefit in high densities. However, optimal plant‐level defence expression does not decrease monotonically with plant density, indicating that R:FR mediation of defence alone is not enough to optimize defence between densities. Therefore, assessing the ecological significance of R:FR‐mediated defence is paramount to better understand the evolution of this physiological linkage and its implications for crop breeding.     

Carbon source-dependent variation of acquired mutagen resistance of Moniliophthora perniciosa: similarities in natural and artificial systems

The basidiomycete Moniliophthora perniciosa causes Witches' Broom disease in Theobroma cacao. We studied the influence of carbon source on conditioning hyphae to oxidative stress agents (H(2)O(2), paraquat, 4NQO) and to UVC, toward the goal of assessing the ability of this pathogen to avoid plant defenses involving ROS. Cells exhibited increased resistance to H(2)O(2) when shifted from glucose to glycerol and from glycerol to glycerol. When exposed to paraquat, cells grown in fresh medium were always more resistant. Apparently glycerol and/or fresh media, but not old glucose media, up-regulate oxidative stress defenses in this fungus. For the mutagens UVC and 4NQO, whose prime action on DNA is not via ROS, change of carbon source did not elicit a clear change in sensitivity/resistance. These results correlate with expression of fungal genes that protect against ROS and with biochemical changes observed in infected cacao tissues, where glycerol and high amounts of ROS have been detected in green brooms.     

Regulation of Glycerol Catabolism in Klebsiella aerogenes

The utilization of glycerol as a carbon source for growth by Klebsiella aerogenes, strain 2103, involves separate aerobic (sn-glycerol-3-phosphate or G3P) and anaerobic (dihydroxyacetone or DHA) pathways of catabolism. Enzyme and transport activities of the aerobic pathway are elevated in cells grown under oxygenated conditions on glycerol or G3P. Anaerobic growth on G3P as carbon source requires the presence of an exogenous hydrogen acceptor such as fumarate; cells thus grown also are highly induced in the G3P pathway. Anaerobic growth on glycerol requires no exogenous hydrogen acceptors; cells thus grown are highly induced in the DHA pathway but almost uninduced in the G3P pathway and the addition of fumarate electron acceptors has no effect on the relative levels of the two pathways. When both glycerol and G3P are provided anaerobically with fumarate, the DHA pathway is still preferentially induced, which probably accounts for the exclusive utilization of glycerol until its exhaustion. These observations suggest the presence of a regulatory control of G3P pathway imposed by the operation of the DHA pathway.     

Auxin modification of the incompatibility response in Theobroma cacao

The time course and control of floral abscission and fruit set in Theobroma cacao were studied after spray application of growth regulators. 1-Naphthaleneacetic acid (NAA) prevented flower abscission in a concentration dependent manner and induced the early stages of fruit development. The cytokinin benzylaminopurine (BAP) counteracted NAA but resulted in longer fruit retention. Measurements of endogenous levels of indole-3-acetic acid showed an inverse correlation between the number of flowers per plant and auxin content. The results suggest that the genetic control of self-incompatibility in T. cacao may be modulated by the hormonal content of the flower.     

Early Detection of Bacterium-induced Basal Resistance in Tobacco Leaves with Diaminobenzidine and Dichlorofluorescein Diacetate

The present study demonstrate that in tobacco leaves the diaminobenzidine (DAB) and 2′,7′‐dichlorofluorescein diacetate (DCFH‐DA) staining is a useful indicator of the basal (also known as general or innate) defence‐associated reactions, especially of the early developing form of basal resistance (EBR). DAB and DCFH‐DA, in the presence of H₂O₂ and peroxidase converts to a brown polymer and fluorescent DCF respectively. In the present study, the hypersensitive response (HR)‐inducing avirulent Pseudomonas syringae pv. syringae 61, its HR‐negative hrp/hrc mutants and even non‐pathogenic bacteria such as P. fluorescens and Escherichia coli caused DAB and DCFH‐DA staining, if the dyes were injected 3–4 h after bacterial inoculation into tobacco leaves. The conditions that enable the staining of plant leaves infiltrated with HR‐negative bacteria were persisted for 1 to several days depending on the physiological state of the plant, and plant activity was required to the development of the staining. The live virulent P. syringae pv. tabaci was able to suppress the development of the staining reaction. Bacteria that induced more intensive staining reaction triggered stronger local resistance response, which was verified by its ability to inhibit the HR by challenging avirulent bacteria and by expression analysis of genes that are activated during the basal defence response. The peroxidase enzyme activity increased in bacterially treated tobacco tissue, and inhibition of peroxidase activity blocked the development of the staining. The results showed that in tobacco leaves the staining reactions were associated with the general recognition and basal defence reaction of tobacco plant and can be used as markers in tobacco leaves for testing the occurrence of this type of defence.