Tag: macetas de 30 litros

In the dwarf Arabidopsis mutant murus 1 L-galactose replaces L-fucose in several cell wall polysaccharides, including RGII, because the plant is unable to produce GDP-fucose in its shoots as it lacks GDP-D-mannose-4,6-dehydratase GMD1 . This has been shown to result in the incomplete formation of the A side-chain of RG-II, which in turn reduces the stability of the borate cross-linked dimer . Thus, the structural integrity of RG-II is probably important for its biological functions. Pectic and hemicellulosic polysaccharides are synthesized in the Golgi apparatus using activated donor substrates, typically in the form of nucleotide diphosphate-linked sugars . However, most NDPsugars are synthesized in the cytosol . Thus, NDP-sugar transporters are required to provide substrates for glycan synthesis . The Golgi-localized NST sub-family, which forms part of clade IIIa of the NST/triose phosphate transporter super family , comprises four members related to GONST1 , the first nucleotide sugar transporter described in Arabidopsis . The members of this family are the only Arabidopsis NSTs that contain a predicted GDP-binding motif . Arabidopsis is known to synthesize four GDP-linked sugars: GDP-L-fucose, GDP-L-galactose, GDP-D-glucose and GDP-D-mannose. GDP-mannose for the glycosylation of glycosylinositolphosphorylceramides is transported into the Golgi by GONST1 , whereas GDP-fucose is transported by GONST4, which has been renamed GDPfucose transporter1 . No Golgi-localized GDP-L-galactose transporters have been identified to date.Most GDP-L-galactose is then converted, via L-galactose, into L-ascorbate ,macetas 5 litros which is important for maintaining redox balance in the cell, particularly under abiotic or biotic stress .

However, some GDP-Lgalactose is required for cell wall polysaccharide synthesis since L-galactose is present in side chain A of RG-II, in the side-chains of xyloglucan from a limited number of plant species  and in corn bran glucuronoarabinoxylan . Here we provide evidence that GONST3 probably encodes a Golgi-localized GDP-L-galactose transporter, which we rename Golgi GDP-L-galactose transporter1 . We used RNA interference to suppress GGLT1 expression in Arabidopsis, since complete loss of GGLT1 is lethal. Plants with decreased GGLT1 expression have growth defects, which are rescued by increasing the amount of borate in their growth medium. Chemical analysis of the cell walls of GGLT1 knock-down plants revealed a substantial reduction in the L-galactose decoration of RG-II, which is correlated with a decrease in the proportion of RG-II dimer in the wall and a decrease in the stability of the crosslink. Our results underscore the importance of RG-II to plant survival, and highlight an unexpectedly critical role for L-galactose in borate crosslinking of this unusual pectic polysaccharide.Publicly available gene expression data reveal that GGLT1 is a ubiquitously expressed gene, with a level of expression that is slightly lower than GONST1 and GFT1 . In an earlier study, the subcellular localization of GGLT1 was not determined because tagged GGLT1 could not be expressed in vivo . To overcome this issue, the full-length GGLT1 coding sequence tagged with a fluorescent protein was introduced into onion epidermal cells by biolistic transformation. Confocal imaging revealed that the fluorescently tagged GGLT1 gave a punctate signal that co-localized with a Golgi marker .No Arabidopsis lines carrying a T-DNA insertion in the GGLT1 open reading frame have been reported. A single T-DNA line , with an insertion 841 bp upstream of the start of transcription was obtained, but we were unable to identify any plants homozygous for the TDNA insertion despite screening at least 30 different seedlings. Therefore, we took a targeted gene-knockdown approach and generated RNAi transgenic lines with a hairpin RNA construct, which specifically targeted GGLT1. Forty independent hpGGLT1 transformants were screened, and four were selected for characterization . These lines all had rosettes which were smaller than the empty vector control .

Quantitative real-time PCR showed that in the rosette leaves of the hpRNAi lines 1–3 the levels of GGLT1 silencing were similar . These data, together with the lack of T-DNA lines, suggest that stronger suppression of GGLT1 or null mutants will produce plants that are not viable. The expression of GFT1, the closest homolog of GGLT1, was not affected in lines 3 and 4 but was decreased by up to 50% in lines 1 and 2 . The monosaccharide compositions of the walls, including fucose, were not significantly altered in any of the hpRNAi lines , indicating that their phenotypes do not result from altered fucosylation of cell wall glycans and are thus a consequence of GGLT1 silencing. Moreover, the shortened petiole phenotype that is characteristic of silenced GFT1 plants as well as mur1 plants, which also have cell walls with reduced fucose , was not observed in our hpGGLT1 knock-down lines .L-Galactose replaces L-fucose in the xyloglucan formed by mur1 and GFT1-silenced plants where GDP-fucose synthesis or transport is perturbed . Since GGLT1 and GFT1 are closely related NSTs, we first determined if xyloglucan fucosylation is altered in hpGGLT1 . No differences were discernible in the matrix-assisted laser desorption–ionization time-of-flight mass spectra of the oligosaccharides generated by enzymatic fragmentation of the xyloglucan from hpGGLT1 and EV control lines . The presence of fucosylated side chains, together with no substantial increase in the abundance of galactosylated side-chains in the hpGGLT1 lines, supports our assertion that GDP-fucose transport is unaffected in the silenced plants. GGLT1 is in the same NST subclade as GONST1, which provides GDP-mannose specifically for GIPC glycosylation, as opposed to polysaccharide biosynthesis . Although glycosylation of GIPCs is still poorly understood, it is possible that other GDP-sugars, inaddition to GDP-mannose are required. Therefore, we used thin layer chromatography and LC-MS to determine the GIPC glycan composition of hpGGLT1. No major differences were discernible between hpGGLT1 and EV GIPCs. The overall sphingolipidomic composition was also unchanged . Together, the combined results of these studies show that GGLT1 does not encode a Golgi-localized protein involved in the transport of GDP-L-fucose or GDP-D-mannose. Thus, we next investigated if the L-galactose content of the wall was altered in the GGLT1 suppressed lines.

No significant differences were detected in the wall monosaccharide compositions of leaves from soil-grown EV and hpGGLT1 lines . This is not surprising since in primary cell walls D-galactose is far more abundant than L-galactose . Moreover, D-galactose and L-galactose are not separated when the monosaccharide composition of the cell wall is determined by high-performance anion exchange chromatography with pulsed amperometric detection . Rhamnogalacturonan-II is the only known L-galactosecontaining polysaccharide present in wild-type Arabidopsis cell walls, so we next determined whether the structure of RG-II differed in hpGGLT1 and EV plants. Material enriched in pectic polysaccharides, including RG-II, was obtained by extracting hpGGLT1 and EV leaf alcohol-insoluble residue with ammonium oxalate, a calcium chelator. This material was then treated with endopolygalacturonase and the products separated by size-exclusion chromatography . This separates RG-II from RG-I and oligogalacturonides, and also separates the RG-II monomer and dimer. In EV control plants the dimer accounts for 77% of the total RG-II isolated from the wall. Somewhat unexpectedly, the dimer accounts for only 49% of the hpGGLT1 RG-II,macetas de 30 litros and makes up only 6% of the RG-II in mur1-1 . This led us to suspect that the ability of hpGGLT1 RG-II to form dimers or the stability of those dimers had been altered. The latter notion is supported by our SEC data for the RG-II released by EPG treatment of the AIR . Under these conditions, in the absence of a chelating agent, the dimer accounted for 97% of the RG-II in the EV control plants , 87% of the RG-II in the hpGGLT1 lines and 70% of the mur1-1 . These results, together with data showing that calcium chelators partially convert the RG-II dimer to the monomer , strongly suggest that both the extent of formation and the stability of the borate cross-link in RG-II are affected in the hpGGLT1 lines. The differences in dimer abundance in the EPG and oxalate fractions were most pronounced with mur1-1 plants. This mutant produces RG-II that lacks L-galactose because its A side-chain is truncated , which led us to suspect that the L-galactose content of side-chain A of the RG-II from the hpGGLT1 lines may also be reduced. To determine if RG-II structure is indeed altered in the hpGGLT1 lines we isolated the total RG-II from the silenced and EV plants. Glycosyl residue composition analyses showed that D/L-galactose was reduced by about 35% in the most strongly affected hpGGLT1 lines . We then treated the RG-II with warm trifluoroacetic acid  to release side chains A and B. The MALDI-TOF MS analysis showed that a substantial portion of side-chain A from hpGGLT1 RG-II existed as a heptasaccharide whereas virtually all the A chain from the EV control was present as an octasaccharide . The A side-chains produced by hpGGLT1 and EV plants differ in mass by 162 Da, corresponding to a hexose residue, which we consider likely to be L-galactose. The side-chain B of RG-II contains a D-galactose residue . However, no differences were discernible in the structures of this side-chain from RG-II of hpGGLT1 and EV plants . Our structural data provide compelling evidence that the abundance of terminal L-galactose present on the A side-chain of RG-II is specifically affected in hpGGLT1 plants. To confirm the identity of the missing hexose in sidechain A, the RG-II monomers generated from the hpGGLT1 and EV plants were treated with a recently identified a-Lgalactosidase 95 from Bacteroides thetaiotaomicron that specifically removes the terminal L-galactose from side-chain A of RGII .

Galactose was the only monosaccharide detected by HPAEC-PAD following hydrolysis of EV control RG-II with the a-L-galactosidase . Less galactose was released from the RG-II of the hpGGLT1- silenced lines relative to the control . The MALDI-TOF MS analysis of side-chain A, released by mild TFA hydrolysis following a-L-galactosidase treatment of RG-II monomer, revealed that the predominant oligosaccharides in the EV control plants correspond to side-chain A lacking L-galactose . The L-galactose was almost completely removed as only low-intensity signals corresponding to L-galactosylated A side-chains were discernible . The mass spectra of side-chain A from both hpGGLT1 silenced lines are similar to that of the EV control , demonstrating that the mass difference of 162 Da between the EV control and hpGGLT1 lines in Figure 3 is due to the specific loss of L-galactose. It has been proposed that pectin domains may be linked covalently to each other or to other cell wall components . To investigate whether the altered RG-II structure in the hpGGLT1 silenced lines had affected other pectic domains, the oxalate cell wall fraction was used to perform immune dotblots with a panel of antibodies raided against different pectin epitopes . However, no difference was observed between the EV control and the silenced lines. In combination with the monosaccharide composition data and the xyloglucan data we conclude that the reduction in GGLT1 expression does not affect non-RG-II polymers. These data provide strong evidence that silencing of GGLT1 leads to a reduction in the abundance of L-galactose on side-chain A of RG-II, and provides additional evidence that the absence of this sugar leads to a decrease in the ability of the RG-II monomer to self-assemble into a borate cross-linked dimer. Moreover, this L-galactosedepleted dimer is less stable in the presence of calcium chelators than its wild-type counterpart, a result consistent with the notion that interactions of borate and calcium with RG-II are important for plant growth .Several growth phenotypes, including the dwarf phenotype of mur1, that have been attributed to defects in RG-II structure and cross-linking have been reported to be rescued by supplementing the growth medium with additional borate . To further explore the observed growth phenotypes of the hpGGLT1 lines , plants were grown hydroponically to control the availability of all macro- and micro-nutrients, including borate. In low-borate media the hpGGLT1 lines are severely stressed, and their rosette diameter is about 70% smaller than that of EV control plants . However, this phenotype is not observed when the silenced plants are grown in high-borate media . The amount of borate in the growth medium did not affect GGLT1 expression, thereby excluding a potential effect of borate deficiency or supplementation on transgene expression and silencing strength . Therefore, we conclude that partial loss of the RG-II L-galactose decoration in hpGGLT1 reduces the rate of RG-II borate-dependent dimerization, directly affecting plant development.

Volatiles that signal the presence of flush may be important components in scent lures. Flushing shoots are essential for ACP reproduction. Signature odors might include cuticular hydrocarbons, ammonia, methanol, and carbon dioxide, all of which are emitted during leaf expansion. These compounds may synergize ACP response to terpenes emitted by foliar scent glands. Sesquiterpenes may be important signal compounds. These terpenes are prevalent in the aromas of ‘super hosts’ but most are commercially unobtainable; little is known about their effect on psyllid attraction. A better understanding of these factors could lead to the development of scent lures that are consistently effective in a variety of application situations. Better monitoring tools would especially be useful where ACP population densities are low or for tracking psyllid movement patterns. Citrus groves receiving nutritional sprays to manage HLB were compared with groves in the same areas receiving conventional fertilization treatments.In 2009–2012, HLB fruit were sorted into symptomatic and asymptomatic , while later , no distinction was made between symptomatic and asymptomatic HLB fruit. Fruit were washed and juiced using JBT extractors and finishers, then pasteurized following industry standards. Sensory tests were performed with trained and untrained panelists. In 2009–2010, differences between juice from healthy and HLBa fruit were minimal, and only perceived by trained panelists. Juice from HLBs was off-flavored, bitter, sour, with some metallic, tingling, and umami taste. These descriptors were mostly used for early season Hamlin,macetas 5 litros while the effect of HLB on Valencia juice flavor was minimal. Nutritional treatments had no consistent effects on juice flavor over the 5-year duration of the study.

Difference-from-control tests showed improvements due to nutritional treatments on Hamlin in January 2012, December 2012, and January 2013, i.e., panelists could not detect differences between juice from healthy and HLB fruit. Trained panelists found more differences between juice from healthy and HLB fruit than between juice from different nutritional treatments, except for one Hamlin nutritional juice in December 2012. Blends made with 75:25, 50:50, and 25:75 ratio of healthy and HLB Hamlin juice were also tested. Trained panelists classified the 75:25 blend with healthy juice, and 25:75 with HLB juice, confirming previous blending studies, while the flavor of the 50:50 blend varied with harvest. In conclusion, no management practice in the field was able to alleviate the effect of HLB on juice flavor. However, much of the off-flavor depended on the season and levels of sugars, acids, and other components in the juice. The Asian citrus psyllid was detected in California in October 2008 and has invaded most of the citrus grown in southern California. Huanglongbing was detected in Los Angeles County in 2012 and poses a significant threat to California’s ~$2 billion citrus industry. Establishment of ACP and HLB can cause huge economic loss and it is important to slow the spread of the HLB pathogen. At present, this can best be achieved by controlling the size and spread of psyllid population through integrated management strategies. A series of lab trials were conducted to evaluate the residual efficacy of various conventional and organic chemicals for ACP control and for selection of chemicals that were least toxic to natural enemies like Tamarixia radiata. The most persistent organic insecticide tested was Entrust + oil and to a lesser extent Trilogy. Among four pyrethroids, Danitol was most effective and persistent, while Mustang was weaker when tested with adult ACP. However, when toxicity was tested with adult Tamarixia, both the organic and conventional pesticides were not as toxic as they were to ACP adults. In a separate study, a field trial was conducted to test the efficacy of organic insecticides against ACP nymphs. Among the materials tested, Entrust + oil provided better control in comparison to two other products.

Future plans include further trials under lab and field conditions to evaluate products for ACP control that are least toxic to natural enemies.The Asian citrus psyllid vectors pathogens that cause Huanglongbing or citrus greening disease. Management of ACP is critical in all habitats including organic citrus for area-wide suppression of HLB. Mature citrus trees in Florida go through dormancy in winter producing little or no new growth required for ACP to develop and reproduce. Targeting overwintering ACP adults with sprays of broad-spectrum insecticides has been shown to provide significant reduction into growing season and opportunity for biological control and selective insecticides. This tactic is adopted area-wide in Florida and Texas and also used in California. However, organic farmers struggle with management of ACP and HLB because they cannot use synthetic insecticides. Consequently, we have initiated development of ACP control programs testing use of organic insecticides and release of Tamarixia radiata also suitable for conventional citrus and urban habitats. We evaluated dormant winter sprays of an organic insecticide, Pyganic applied in November, December, and January compared to a single spray of the synthetic pyrethroid Danitol in January against ACP in Valencia and Hamlin oranges. Pyganic was applied either alone, with horticultural mineral oil , or soybean oil at 2% volume by volume. Significant effects of Pyganic plus either oil lasted for 1–2 weeks after November and December applications. Pyganic + HMO and Danitol maintained ACP at or below 0.1 adults per tap sample for 4–5 weeks, and significant suppression lasted 2 months after the January application. Monthly applications of Pyganic with HMO appear to be a reasonable option for organic growers to suppress overwintering ACP. Organic programs evaluating promising insecticides alone or altered with oils or other surfactants are being evaluated parallel to a conventional program. Tamarixia radiata are also being released for integration in these programs. Findings and implications for ACP management will be discussed. Tolerance, or resistance, to citrus Huanglongbing will be important as a long-term solution for this disease. In a field trial conducted with over 1000 plants belonging to different genera in the sub-family Aurantioideae, we observed field tolerance in many Australian citrus relatives. To confirm these results in a controlled greenhouse setting, we have exposed 140 plants to Candidatus Liberibacter asiaticus via psyllid feeding under no-choice conditions for a period of 2 weeks.

We have maintained these seedlings under greenhouse conditions and conducted detailed analysis on the presence of the pathogen and level of tolerance observed in various plants after exposure to the pathogen. We have analyzed the samples using qPCR and droplet digital PCR to estimate the bacterial titer in accessions that were either susceptible, tolerant, or resistant in field studies. To confirm the presence or absence of the pathogen in plants with questionable qPCR results, ddPCR analysis was conducted targeting multiple genomic regions of Candidatus Liberibacter asiaticus. Since these experiments are conducted using open pollinated seeds where the pollen parent is not known, we have also conducted about 2000 pollinations using Microcitrus, Eremocitrus, pummelo, and mandarin as both seed and pollen parents. The targeted crosses were also conducted with known HLB tolerant cultivars of mandarins like Temple tangor and Fallglo mandarin. Citrus greening, also known as Huanglongbing , is a devastating disease on Citrus plants and is caused by the yet unculturable bacterial Candidatus liberibacter spp. This gram-negative bacterium is limited to vascular tissue and mainly accumulates in the symplasm of autotrophic organs. In addition, the accurate quantification of viable bacteria is necessary to assess the status of potentially-infected trees as well as to test any possible control strategies. A number of methods have been broadly described to quantify total bacteria content; which includes the detection of both live and dead bacteria as well as the different bacterial pathovars. In the present work,macetas de 30 litros an improved detection method, based on PCR is described, which employs new designed primers. Real-time PCR was also employed to test the sensitivity of the method, which was able to detect 10 copies per genome in tree-infected trees. In addition, Droplet Digital PCR was also developed to detect two copies per plant genome, which is the highest sensitivity reached in current protocols. The accuracy of the tested techniques will allow assessing the feasibility of possible controlling methods of this devastating disease. Researchers have been exploring more effective ways to control the Asian Citrus Psyllid , the only known insect vector of the phloem-limited bacterium “Candidatus Liberibacter sp.” responsible for Citrus Greening or Huanglongbing disease. Research results are reported on the use of a crowd sourcing challenge-based strategy to identify gene targets in the ACP that can function as interdiction points to control the insect using an oral uptake RNA interference strategy. This challenge was sponsored and developed by the Florida citrus industry and posted on the web through Innocentive, Inc. Interested participants were allowed to submit suggested targets with justifications for orally administered dsRNAs that would be hypothesized to induce mortality by RNAi. Of the sequences submitted, a committee of experts selected 43 for an oral uptake bio-assay performed using an artificial diet screen to identify those that induced mortality. Subsequently, a citrus phloem-limited virus vector was used to engineer citrus to produce candidate dsRNAs in the phloem. When ACP fed on these plants, there was much greater ACP mortality than was observed by artificial diet delivery, in some cases producing 100% adult ACP mortality in detached-leaf bioassays.

RNA Seq analysis shows that resulting ACP transcriptome changes were attributable to genes functionally linked to the gene targeted by RNAi. New methods are needed to control the spread of the citrus greening pathogen, Candidatus Liberibacter asiaticus , by the Asian Citrus Psyllid . This collaborative project funded by the California Citrus Research Board is focused on discovery of protein-protein interactions involved in Huanglongbing transmission. During transmission of CLas within the ACP, interactions between the vector and host regulate how the pathogen moves through the insect, replicates, and is transmitted into a plant. In this study, Protein Interaction Reporter technology has been used to characterize protein interactions between CLas and ACP. The PIR strategy was developed by the Bruce Lab and uses novel crosslinker molecules which covalently link interacting proteins. Mass spectrometric analysis of PIR cross-linked peptides provides information on the relative proximity and orientation of surface exposed lysine residues in cross-linked protein complexes. The Bruce and Cilia labs have previously successfully applied PIR technology to study interactions with an aphid-transmitted virus. Percoll density gradient centrifugation has been applied to purify enriched populations of CLas cells from infected ACP. Using this method, intact CLas cells retaining interactions with ACP tissues are recovered. Infected psyllids were homogenized in a non-denaturing buffer, and the concentrated lysate was applied to a Percoll gradient. Centrifugation resulted in two distinct bands in the Percoll gradient—these fractions were recovered, the cells were washed, and qPCR was used to quantitate the amount of CLas cells present. The PIR cross-linker was used to capture protein interactions in the enriched CLas sample, and the cross-linked peptides were subjected to mass spectrometry analysis. Given the challenges of working with this non-culturable pathogen, this Percoll method for CLas enrichment has a range of potential applications, including the use of purified CLas cells as immunogen for antibody production.Huanglongbing is the most serious recent threat to the US citrus industry. Several studies have indicated that Poncirus is resistant to HLB, but the Poncirus genes for HLB resistance are to be identified and characterized. The Ctv locus in Poncirus confers durable broad-spectrum resistance to citrus tristeza virus , a virus that is phloem-limited like CLas, has been identified and sequenced. We compared the genomic organization of the Ctv region to the corresponding regions from three susceptible citrus species and analyzed the genes, particularly the resistance genes within the locus, in an attempt to gain perspective of the to-be-identified HLB resistance gene in Poncirus. Copy number and localization of R-genes at the Ctv and ctv regions vary considerably from species to species; R-genes have been on faster paces of sequence change than surrounding genes. Dramatic structural rearrangements have occurred, resulting in an expansion of ~60 kb at the Ctv region over the ctv region. While the center of the Ctv region is packed with a rich and diverse group of retrotransposable elements , the ctv regions are free of REs. Pondering these phenomena at Ctv, it is intriguing to speculate the likely structure and evolution of the Poncirus genomic regions harboring HLB resistance genes.Huanglongbing is the most devastating disease of citrus.