Cardiopulmonary exercise testing excludes significant disease in patients recovering from COVID-19.

OBJECTIVE: Post-COVID-19 syndrome presents a health and economic challenge affecting ~10% of patients recovering from COVID-19. Accurate assessment of patients with post-COVID-19 syndrome is complicated by health anxiety and coincident symptomatic autonomic dysfunction. We sought to determine whether either symptoms or objective cardiopulmonary exercise testing could predict clinically significant findings. METHODS: 113 consecutive military patients were assessed in a comprehensive clinical pathway. This included symptom reporting, history, examination, spirometry, echocardiography and cardiopulmonary exercise testing (CPET) in all, with chest CT, dual-energy CT pulmonary angiography and cardiac MRI where indicated. Symptoms, CPET findings and presence/absence of significant pathology were reviewed. Data were analysed to identify diagnostic strategies that may be used to exclude significant disease. RESULTS: 7/113 (6%) patients had clinically significant disease adjudicated by cardiothoracic multidisciplinary team (MDT). These patients had reduced fitness (V̇O2 26.7 (±5.1) vs 34.6 (±7.0) mL/kg/min; p=0.002) and functional capacity (peak power 200 (±36) vs 247 (±55) W; p=0.026) compared with those without significant disease. Simple CPET criteria (oxygen uptake (V̇O2) >100% predicted and minute ventilation (VE)/carbon dioxide elimination (V̇CO2) slope <30.0 or VE/V̇CO2 slope <35.0 in isolation) excluded significant disease with sensitivity and specificity of 86% and 83%, respectively (area under the receiver operating characteristic curve (AUC) 0.89). The addition of capillary blood gases to estimate alveolar-arterial gradient improved diagnostic performance to 100% sensitivity and 78% specificity (AUC 0.92). Symptoms and spirometry did not discriminate significant disease. CONCLUSIONS: In a population recovering from SARS-CoV-2, there is reassuringly little organ pathology. CPET and functional capacity testing, but not reported symptoms, permit the exclusion of clinically significant disease.

In vitro susceptibilities of feline and canine Escherichia coli and Pseudomonas spp. isolates to ticarcillin and ticarcillin-clavulanic acid.

OBJECTIVES: To investigate in vitro susceptibilities of canine and feline Escherichia coli and canine Pseudomonas spp. isolates to ticarcillin and ticarcillin-clavulanic acid (T/C). DESIGN: In vitro susceptibility testing of bacterial isolates collected from infections. METHODS: We tested 148 (83 canine and 65 feline) E. coli and 61 canine Pseudomonas spp. isolates for susceptibility to T/C using both disc diffusion and Epsilometer tests (E-tests). Additionally, susceptibilities of 96 E. coli and 23 canine Pseudomonas spp. isolates were tested via disc diffusion to ticarcillin alone. RESULTS: Of the E. coli isolates obtained from canine and feline urine, 92% by disc diffusion and 91% by E-tests were susceptible to T/C. Of the canine Pseudomonas isolates, 90% by disc diffusion and 82% by E-tests were susceptible to T/C. Of the Pseudomonas spp. isolates from the canine ear canal or tympanic bullae, 12% of isolates tested via disc diffusion and 23% via E-tests were found to be resistant to T/C. The 50% minimum inhibitory concentration of T/C for all feline E. coli isolates was significantly lower than that for all canine E. coli isolates (P = 0.0031). The addition of clavulanic acid significantly increased the efficacy of ticarcillin against E. coli (P< 0.0001), but had negligible effect against canine Pseudomonas spp. isolates. CONCLUSION: Ticarcillin-clavulanic acid has reasonable in vitro efficacy against canine and feline E. coli, and canine Pseudomonas spp. isolates. However, decisions to use this drug therapeutically must be made on prudent considerations to minimise selection for bacterial resistance.

Functional analysis of endo-1,4-ß-glucanases in response to Botrytis cinerea and Pseudomonas syringae reveals their involvement in plant-pathogen interactions.

Plant cell wall modification is a critical component in stress responses. Endo-1,4-ß-glucanases (EGs) take part in cell wall editing processes, e.g. elongation, ripening and abscission. Here we studied the infection response of Solanum lycopersicum and Arabidopsis thaliana with impaired EGs. Transgenic TomCel1 and TomCel2 tomato antisense plants challenged with Pseudomonas syringae showed higher susceptibility, callose priming and increased jasmonic acid pathway marker gene expression. These two EGs could be resistance factors and may act as negative regulators of callose deposition, probably by interfering with the defence-signalling network. A study of a set of Arabidopsis EG T-DNA insertion mutants challenged with P. syringae and Botrytis cinerea revealed that the lack of other EGs interferes with infection phenotype, callose deposition, expression of signalling pathway marker genes and hormonal balance. We conclude that a lack of EGs could alter plant response to pathogens by modifying the properties of the cell wall and/or interfering with signalling pathways, contributing to generate the appropriate signalling outcomes. Analysis of microarray data demonstrates that EGs are differentially expressed upon many different plant-pathogen challenges, hormone treatments and many abiotic stresses. We found some Arabidopsis EG mutants with increased tolerance to osmotic and salt stress. Our results show that impairing EGs can alter plant-pathogen interactions and may contribute to appropriate signalling outcomes in many different biotic and abiotic plant stress responses.

Measuring natural pest suppression at different spatial scales affects the importance of local variables.

The role biodiversity plays in the provision of ecosystem services is widely recognized, yet few ecological studies have identified characteristics of natural systems that support and maintain ecosystem services. The purpose of this study was to identify landscape variables correlated with natural pest suppression carried out by arthropod natural enemies, predators and parasitoids. We conducted two field experiments, one observational and one experimental, where landscape variables at broad and local scales were measured and related to natural pest suppression. The first experiment measured natural pest suppression at 16 sites across an urban to rural landscape gradient in south central Wisconsin. We found natural enemy diversity positively affected natural pest suppression, whereas flower diversity negatively affected pest suppression. No relationship was found between natural pest suppression and broad scale variables, which measured the percentage of different land cover classes in the surrounding landscape. In the second experiment, we established small (2- by 3-m) replicated plots that experimentally varied flower diversity (0, 1, or 7 species) within a plot. We found no significant relationship between natural pest suppression and the different levels of flower diversity. The fact that we only found differences in natural pest suppression in our first experiment, which measured natural pest suppression at sites separated by larger distances than our second experiment, suggests the more appropriate scale for measuring ecosystem services performed by mobile organisms like insects, is across broad spatial scales where variation in natural enemies communities and the factors that affect them become more apparent.

The intersection between cell wall disassembly, ripening, and fruit susceptibility to Botrytis cinerea.

Fruit ripening is characterized by processes that modify texture and flavor but also by a dramatic increase in susceptibility to necrotrophic pathogens, such as Botrytis cinerea. Disassembly of the major structural polysaccharides of the cell wall (CW) is a significant process associated with ripening and contributes to fruit softening. In tomato, polygalacturonase (PG) and expansin (Exp) are among the CW proteins that cooperatively participate in ripening-associated CW disassembly. To determine whether endogenous CW disassembly influences the ripening-regulated increase in necrotropic pathogen susceptibility, B. cinerea susceptibility was assessed in transgenic fruit with suppressed polygalacturonase (LePG) and expansin (LeExp1) expression. Suppression of either LePG or LeExp1 alone did not reduce susceptibility but simultaneous suppression of both dramatically reduced the susceptibility of ripening fruit to B. cinerea, as measured by fungal biomass accumulation and by macerating lesion development. These results demonstrate that altering endogenous plant CW disassembly during ripening influences the course of infection by B. cinerea, perhaps by changing the structure or the accessibility of CW substrates to pathogen CW-degrading enzymes. Recognition of the role of ripening-associated CW metabolism in postharvest pathogen susceptibility may be useful in the design and development of strategies to limit pathogen losses during fruit storage, handling, and distribution.

Sequence variation in the Trichuris trichiura beta-tubulin locus: implications for the development of benzimidazole resistance.

Benzimidazole resistance has evolved in a variety of organisms and typically results from mutations in the beta-tubulin locus at specific amino acid sites. Despite widespread treatment of human intestinal nematodes with benzimidazole drugs, there have been no unambiguous reports of resistance. However, since beta-tubulin mutations conferring resistance are generally recessive, frequencies of resistance alleles less than 30% would be difficult to detect on the basis of drug treatment failures. Here we investigate sequence variation in a 1079 bp segment of the beta-tubulin locus in the human whipworm Trichuris trichiura from 72 individual nematodes from seven countries. We did not observe any alleles with amino acid mutations indicative of resistance, and of 40 point mutations there were only four non-synonymous mutations all of which were singletons. Estimated effective population sizes are an order of magnitude lower than those from another nematode species in which benzimidazole resistance has developed (Haemonchus contortus). Both the lower diversity and reduced population sizes suggest that benzimidazole resistance is likely to evolve less rapidly in Trichuris than in trichostrongyle parasites of livestock. We observed moderate levels of population subdivision (Phi(ST)=0.26) comparable with that previously observed in Ascaris lumbricoides, and identical alleles were frequently found in parasites from different continents, suggestive of recent admixture. A particularly interesting feature of the data is the high nucleotide diversities observed in nematodes from the Caribbean. This genetic complexity may be a direct result of extensive admixture and complex history of human populations in this region of the world. These data should encourage (but not make complacent) those involved in large-scale benzimidazole treatment of human intestinal nematodes.

Characterization of a tomato xyloglucan endotransglycosylase gene that is down-regulated by auxin in etiolated hypocotyls.

The reorganization of the cellulose-xyloglucan matrix is proposed to serve as an important mechanism in the control of strength and extensibility of the plant primary cell wall. One of the key enzymes associated with xyloglucan metabolism is xyloglucan endotransglycosylase (XET), which catalyzes the endocleavage and religation of xyloglucan molecules. As with other plant species, XETs are encoded by a gene family in tomato (Lycopersicon esculentum cv T5). In a previous study, we demonstrated that the tomato XET gene LeEXT was abundantly expressed in the rapidly expanding region of the etiolated hypocotyl and was induced to higher levels by auxin. Here, we report the identification of a new tomato XET gene, LeXET2, that shows a different spatial expression and diametrically opposite pattern of auxin regulation from LeEXT. LeXET2 was expressed more abundantly in the mature nonelongating regions of the hypocotyl, and its mRNA abundance decreased dramatically following auxin treatment of etiolated hypocotyl segments. Analysis of the effect of several plant hormones on LeXET2 expression revealed that the inhibition of LeXET2 mRNA accumulation also occurred with cytokinin treatment. LeXET2 mRNA levels increased significantly in hypocotyl segments treated with gibberellin, but this increase could be prevented by adding auxin or cytokinin to the incubation media. Recombinant LeXET2 protein obtained by heterologous expression in Pichia pastoris exhibited greater XET activity against xyloglucan from tomato than that from three other species. The opposite patterns of expression and differential auxin regulation of LeXET2 and LeEXT suggest that they encode XETs with distinct roles during plant growth and development.

MIP genes are down-regulated under drought stress in Nicotiana glauca.

Water flux across cell membranes has been shown to occur not only through the lipid bilayer, but also through aquaporins, which are members of the major intrinsic protein (MIP) super-family of channel proteins. Aquaporins greatly increase the membrane permeability for water, but may also be regulated, allowing cellular control over the rate of water influx/efflux. Water flux is crucial for stomatal opening and closing, but little is known about the role that aquaporins play in stomatal physiology. Our initial goal was to isolate and characterize the MIP genes expressed in guard cells of the model plant, Nicotiana glauca. Degenerate oligonucleotides corresponding to amino acid sequences conserved in tonoplast intrinsic proteins (TIPs) or plasma membrane intrinsic proteins (PIPs) were used to amplify portions of MIP genes by RT-PCR. These PCR products were used as probes in screening a N. glauca guard cell cDNA library. We isolated three clones (NgMIP1, NgMIP2 and NgMIP3) homologous to TIPs and two clones (NgMIP4 and NgMIP5) homologous to PIPs. All of the MIP genes we characterized displayed highest levels of mRNA accumulation in roots or stems, with lower levels of expression in mesophyll cells and whole leaves, and lowest transcript accumulation in guard cell RNA. Interestingly, the accumulation of transcripts arising from NgMIP2, NgMIP3 and NgMIP4 diminished dramatically in drought-stressed plants. This down-regulation of MIP gene expression may result in reduced membrane water permeability and may encourage cellular water conservation during periods of dehydration stress.

Transgenic expression of pear PGIP in tomato limits fungal colonization.

Transgenic tomato plants expressing the pear fruit polygalacturonase inhibitor protein (pPGIP) were used to demonstrate that this inhibitor of fungal pathogen endopolygalacturonases (endo-PGs) influences disease development. Transgenic expression of pPGIP resulted in abundant accumulation of the heterologous protein in all tissues and did not alter the expression of an endogenous tomato fruit PGIP (tPGIP). The pPGIP protein was detected, as expected, in the cell wall protein fraction in all transgenic tissues. Despite differential glycosylation in vegetative and fruit tissues, the expressed pPGIP was active in both tissues as an inhibitor of endo-PGs from Botrytis cinerea. The growth of B. cinerea on ripe tomato fruit expressing pPGIP was reduced, and tissue breakdown was diminished by as much as 15%, compared with nontransgenic fruit In transgenic leaves, the expression of pPGIP reduced lesions of macerated tissue approximately 25%, a reduction of symptoms of fungal growth similar to that observed with a B. cinerea strain in which a single endo-PG gene, Bcpg1, had been deleted (A. ten Have, W. Mulder, J. Visser, and J. A. L. van Kan, Mol. Plant-Microbe Interact. 11:1009-1016, 1998). Heterologous expression of pPGIP has demonstrated that PGIP inhibition of fungal PGs slows the expansion of disease lesions and the associated tissue maceration.

Detection of expansin proteins and activity during tomato fruit ontogeny.

Expansins are plant proteins that have the capacity to induce extension in isolated cell walls and are thought to mediate pH-dependent cell expansion. J.K.C. Rose, H.H. Lee, and A.B. Bennett ([1997] Proc Natl Acad Sci USA 94: 5955-5960) reported the identification of an expansin gene (LeExp1) that is specifically expressed in ripening tomato (Lycopersicon esculentum) fruit where cell wall disassembly, but not cell expansion, is prominent. Expansin expression during fruit ontogeny was examined using antibodies raised to recombinant LeExp1 or a cell elongation-related expansin from cucumber (CsExp1). The LeExp1 antiserum detected expansins in extracts from ripe, but not preripe tomato fruit, in agreement with the pattern of LeExp1 mRNA accumulation. In contrast, antibodies to CsExp1 cross-reacted with expansins in early fruit development and the onset of ripening, but not at a later ripening stage. These data suggest that ripening-related and expansion-related expansin proteins have distinct antigenic epitopes despite overall high sequence identity. Expansin proteins were detected in a range of fruit species and showed considerable variation in abundance; however, appreciable levels of expansin were not present in fruit of the rin or Nr tomato mutants that exhibit delayed and reduced softening. LeExp1 protein accumulation was ethylene-regulated and matched the previously described expression of mRNA, suggesting that expression is not regulated at the level of translation. We report the first detection of expansin activity in several stages of fruit development and while characteristic creep activity was detected in young and developing tomato fruit and in ripe pear, avocado, and pepper, creep activity in ripe tomato showed qualitative differences, suggesting both hydrolytic and expansin activities.

Isolation of genes predominantly expressed in guard cells and epidermal cells of Nicotiana glauca.

Guard cells are specialized and metabolically active cells which arise during the differentiation of the epidermis. Using Nicotiana glauca epidermal peels as a source of purified guard cells, we have constructed a cDNA library from guard cell RNA. In order to isolate genes that are predominantly expressed in guard cells, we performed a differential screen of this library, comparing the hybridization of a radiolabeled cDNA probe synthesized from guard cell RNA to that from a mesophyll cell cDNA probe. Sixteen clones were isolated based on their greater level of hybridization with the guard cell probe. Of these, eight had high homology to lipid transfer protein (LTP), two were similar to glycine-rich protein (GRP), and one displayed high homology to proline-rich proteins from Arabidopsis thaliana (AtPRP2, AtPRP4) and from potato guard cells (GPP). Northern analysis confirmed that one or more NgLTP genes, NgGRP1, and NgGPP1 are all differentially expressed, with highest levels in guard cells, and low or undetectable levels in mesophyll cells and in roots. In addition, all are induced to some degree in drought-stressed guard cells. NgLTP and NgGRP1 expression was localized by in situ hybridization to the guard cells and pavement cells in the epidermis. NgGRP1 expression was also detected in cells of the vasculature. Genomic Southern analysis indicated that LTP is encoded by a family of highly similar genes in N. glauca. This work has identified members of a subset of epidermis- and guard cell-predominant genes, whose protein products are likely to contribute to the unique properties acquired by guard cells and pavement cells during differentiation.

Auxin-regulated genes encoding cell wall-modifying proteins are expressed during early tomato fruit growth.

An expansin gene, LeExp2, was isolated from auxin-treated, etiolated tomato (Lycopersicon esculentum cv T5) hypocotyls. LeExp2 mRNA expression was restricted to the growing regions of the tomato hypocotyl and was up-regulated during incubation of hypocotyl segments with auxin. The pattern of expression of LeExp2 was also studied during tomato fruit growth, a developmental process involving rapid cell enlargement. The expression of genes encoding a xyloglucan endotransglycosylase (LeEXT1) and an endo-1, 4-beta-glucanase (Cel7), which, like LeExp2, are auxin-regulated in etiolated hypocotyls (C. Catalá, J.K.C. Rose, A.B. Bennett [1997] Plant J 12: 417-426), was also studied to examine the potential for synergistic action with expansins. LeExp2 and LeEXT1 genes were coordinately regulated, with their mRNA accumulation peaking during the stages of highest growth, while Cel7 mRNA abundance increased and remained constant during later stages of fruit growth. The expression of LeExp2, LeEXT1, and Cel7 was undetectable or negligible at the onset of and during fruit ripening, which is consistent with a specific role of these genes in regulating cell wall loosening during fruit growth, not in ripening-associated cell wall disassembly.

Characterization of ripening-regulated cDNAs and their expression in ethylene-suppressed charentais melon fruit.

Charentais melons (Cucumis melo cv Reticulatus) are climacteric and undergo extremely rapid ripening. Sixteen cDNAs corresponding to mRNAs whose abundance is ripening regulated were isolated to characterize the changes in gene expression that accompany this very rapid ripening process. Sequence comparisons indicated that eight of these cDNA clones encoded proteins that have been previously characterized, with one corresponding to ACC (1-aminocyclopropane-1-carboxylic acid) oxidase, three to proteins associated with pathogen responses, two to proteins involved in sulfur amino acid biosynthesis, and two having significant homology to a seed storage protein or a yeast secretory protein. The remaining eight cDNA sequences did not reveal significant sequence similarities to previously characterized proteins. The majority of the 16 ripening-regulated cDNAs corresponded to mRNAs that were fruit specific, although three were expressed at low levels in vegetative tissues. When examined in transgenic antisense ACC oxidase melon fruit, three distinct patterns of mRNA accumulation were observed. One group of cDNAs corresponded to mRNAs whose abundance was reduced in transgenic fruit but inducible by ethylene treatment, indicating that these genes are directly regulated by ethylene. A second group of mRNAs was not significantly altered in the transgenic fruit and was unaffected by treatment with ethylene, indicating that these genes are regulated by ethylene-independent developmental cues. The third and largest group of cDNAs showed an unexpected pattern of expression, with levels of mRNA reduced in transgenic fruit and remaining low after exposure to ethylene. Regulation of this third group of genes thus appears to ethylene independent, but may be regulated by developmental cues that require ethylene at a certain stage in fruit development. The results confirm that both ethylene-dependent and ethylene-independent pathways of gene regulation coexist in climacteric fruit.

An expansin gene expressed in ripening strawberry fruit.

Tissue softening accompanies the ripening of many fruit and initiates the processes of irreversible deterioration. Expansins are plant cell wall proteins proposed to disrupt hydrogen bonds within the cell wall polymer matrix. Expression of specific expansin genes has been observed in tomato (Lycopersicon esculentum) meristems, expanding tissues, and ripening fruit. It has been proposed that a tomato ripening-regulated expansin might contribute to cell wall polymer disassembly and fruit softening by increasing the accessibility of specific cell wall polymers to hydrolase action. To assess whether ripening-regulated expansins are present in all ripening fruit, we examined expansin gene expression in strawberry (Fragaria x ananassa Duch.). Strawberry differs significantly from tomato in that the fruit is derived from receptacle rather than ovary tissue and strawberry is non-climacteric. A full-length cDNA encoding a ripening-regulated expansin, FaExp2, was isolated from strawberry fruit. The deduced amino acid sequence of FaExp2 is most closely related to an expansin expressed in early tomato development and to expansins expressed in apricot fruit rather than the previously identified tomato ripening-regulated expansin, LeExp1. Nearly all previously identified ripening-regulated genes in strawberry are negatively regulated by auxin. Surprisingly, FaExp2 expression was largely unaffected by auxin. Overall, our results suggest that expansins are a common component of ripening and that non-climacteric signals other than auxin may coordinate the onset of ripening in strawberry.

Expression of a polygalacturonase associated with tomato seed germination.

Radicle protrusion from tomato (Lycopersicon esculentum Mill.) seeds to complete germination requires weakening of the endosperm tissue opposite the radicle tip. In common with other cell wall disassembly processes in plants, polygalacturonases (PGs) may be involved. Only calcium-dependent exo-PG activity was detected in tomato seed protein extracts. Chromatographic profiles of a partially acid-hydrolyzed fraction of polygalacturonic acid further digested with seed extract were consistent with the presence of only calcium-dependent exo-PG activity. In addition, a transcript encoding a previously unknown PG was detected prior to the completion of germination. The mRNA, produced from a gene (LeXPG1) estimated by Southern analysis to be represented once in the genome, was also present in flowers (anthers) and in lower amounts in roots and stems. LeXPG1 mRNA abundance was low during seed development, increased during imbibition, and was even greater in seeds that had completed germination. Expression of LeXPG1 during germination predominates in the endosperm cap and radicle tip, and in the radicle appears as a distinct band possibly associated with vascular tissue differentiation. We suggest that PG is involved in cell wall loosening of the endosperm necessary for radicle protrusion from tomato seeds and in subsequent embryo and seedling growth.

Antisense suppression of tomato endo-1,4-beta-glucanase Cel2 mRNA accumulation increases the force required to break fruit abscission zones but does not affect fruit softening.

Plants of tomato (Lycopersicon esculentum Mill. cv. T5) were transformed with an antisense endo-1,4-beta-glucanase (cellulase, EC 3.2.1.4) Cel2 transgene under the control of the constitutive cauliflower mosaic virus 35S promoter in order to suppress mRNA accumulation of Cel2. In two independent transgenic lines, Cel2 mRNA abundance was reduced by >95% in ripe fruit pericarp and ca. 80% in fruit abscission zones relative to non-transgenic controls. In both transgenic lines the softening of antisense Cel2 fruit pericarp measured using stress-relaxation analysis was indistinguishable from control fruit. No differences in ethylene evolution were observed between fruit of control and antisense Cel2 genotypes. However, in fruit abscission zones the suppression of Cel2 mRNA accumulation caused a significant (P<0.001) increase in the force required to cause breakage of the abscission zone at 4 days post breaker, an increase of 27% in one transgenic line and of 46% in the other transgenic line. Thus the Cel2 gene product contributes to cell wall disassembly occurring in cell separation during fruit abscission, but its role, if any, in softening or textural changes occurring in fruit pericarp during ripening was not revealed by suppression of Cel2 gene expression.

Alternative transcription initiation sites generate two LCA1 Ca2+-ATPase mRNA transcripts in tomato roots.

The tomato LCA1 gene encodes a Ca2+-ATPase and gives rise to two major mRNA transcripts and two distinct protein products of different size in tomato roots. The basis of the transcript size difference was investigated to assess whether the mRNA transcripts encoded distinct protein products. Primer extension and S1 nuclease analysis identified two transcription initiation sites at -72 and -1392 from the start of translation. RNA gel blot analysis of poly(A)+ RNA isolated from phosphate-starved tomato roots using probes designed to domains of the 5'-untranslated region (UTR) or the full-length LCA1 cDNA identified mRNAs of 4.7 and 3.6 kb, corresponding to mRNA originating from transcription initiation sites -1392 and -72, respectively. Screening of a cDNA library derived from phosphate-starved tomato roots yielded three cDNA clones, LCA1A, LCA1B and LCA1C (3.6, 4.5 and 5.1 kb respectively). These cDNAs contain full-length LCA1 mRNA sequence derived from each transcription initiation site, with LCA1C additionally containing an intron of 0.6 kb. Sequence analysis indicated 100% identity between the three size classes of cDNA clones except for the differential 5'-UTR and the unspliced intron. Overall, the results indicate that the two major LCA1 mRNA transcripts are derived by differential transcription initiation and that two of the mRNAs may encode identical protein products, while a third mRNA may correspond to a non-functional truncated protein.

A gel diffusion assay for quantification of pectin methylesterase activity.

Increased binding of ruthenium red to pectin as the number of methyl esters attached to the pectin decreases was used as the basis for a gel diffusion assay for pectin methylesterase (PME, EC 3.1.1.11) activity. The stained zone diameters resulting from the hydrolysis of 0.1% (w/v) 90% esterified pectin in an agarose gel by diffused, commercial PME were log-linear over 4 orders of magnitude, with a minimum detection limit of 3.6 pkatals. Pectin deesterification as the cause for a stained zone after PME incubation was confirmed when only 1 N NaOH, which will chemically deesterify the pectin, and not methanol or acid, the two products formed when PME acts on a methyl ester, resulted in the characteristic stained zone. The stained zone diameters decreased with increasing percentage of substrate esterification, were independent of pH, and were insensitive to simultaneous incubation with two forms of pectin lyase (EC 4.2.2.10), polygalacturonase (EC 3.2.1.15), or all combinations. PME extracted from tomato seeds, cotton fibers, and melon fruit showed pH optima of 6, 6, and 8, respectively. Using individual tomato seed parts, the assay was adapted to quantify diffusate activity and to localize activity in tissue prints. The sensitivity, specificity, and simplicity of this PME assay are superior to all others.