Overexpression of gamma-glutamyl cyclotransferase 2;1 (CsGGCT2;1) reduces arsenic toxicity and accumulation in Camelina sativa (L.).

KEY MESSAGE: Overexpressing CsGGCT2;1 in Camelina enhances arsenic tolerance, reducing arsenic accumulation by 40-60%. Genetically modified Camelina can potentially thrive on contaminated lands and help safeguard food quality and sustainable food and biofuel production. Environmental arsenic contamination is a serious global issue that adversely affects human health and diminishes the quality of harvested produce. Glutathione (GSH) is known to bind and detoxify arsenic and other toxic metals. A steady level of GSH is maintained within cells via the γ-glutamyl cycle. The γ-glutamyl cyclotransferases (GGCTs) have previously been shown to be involved in GSH degradation and increased tolerance to toxic metals in plants. In this study, we characterized the GGCT2;1 homolog from Camelina sativa for its role in arsenic tolerance and accumulation. Overexpression of CsGGCT2;1 in Camelina under CaMV35S constitutive promoter resulted in strong tolerance to arsenite (AsIII). The overexpression (OE) lines had 2.6-3.5-fold higher shoots and sevenfold to tenfold enhanced root biomass on media supplemented with AsIII, relative to wild-type plants. The CsGGCT2;1 OE lines accumulated 40-60% less arsenic in root and shoot tissues compared to wild-type plants. Further, the OE lines had ~ twofold higher chlorophyll content and 35% lesser levels of malondialdehyde (MDA), an indicator of membrane damage via lipid peroxidation. There was a slight but non-significant increase in 5-oxoproline (5-OP), a product of GSH degradation, in OE lines. However, the transcript levels of Oxoprolinase 1 (OXP1) were upregulated, indicating the accelerated conversion of 5-OP to glutamate, which is further utilized for the resynthesis of GSH to maintain GSH homeostasis. Overall, this research suggests that genetically modified Camelina may have the potential for cultivation on contaminated marginal lands to reduce As accumulation; thereby could help in addressing food safety issues as well as future food and biofuel needs.

Patterns of physical, chemical, and metabolic characteristics of sugar maple leaves with depth in the crown and in response to nitrogen and phosphorus addition.

Few previous studies have described the patterns of leaf characteristics in response to nutrient availability and depth in the crown. Sugar maple has been studied for both sensitivity to light, as a shade-tolerant species, and sensitivity to soil nutrient availability, as a species in decline due to acid rain. To explore leaf characteristics from the top to bottom of the canopy, we collected leaves along a vertical gradient within mature sugar maple crowns in a full-factorial nitrogen (N) by phosphorus (P) addition experiment in three forest stands in central New Hampshire, USA. Thirty-two of the 44 leaf characteristics had significant relationships with depth in the crown, with the effect of depth in the crown strongest for leaf area, photosynthetic pigments and polyamines. Nitrogen addition had a strong impact on the concentration of foliar N, chlorophyll, carotenoids, alanine and glutamate. For several other elements and amino acids, N addition changed patterns with depth in the crown. Phosphorus addition increased foliar P and boron (B); it also caused a steeper increase of P and B with depth in the crown. Since most of these leaf characteristics play a direct or indirect role in photosynthesis, metabolic regulation or cell division, studies that ignore the vertical gradient may not accurately represent whole-canopy performance.

Is foliar tissue drying and grinding required for reliable and reproducible extraction of total inorganic nutrients? A comparative study of three tissue preparation methods.

In response to abiotic and biotic stress or experimental treatment(s), foliar concentrations of inorganic nutrients and metabolites often change in concert to maintain a homeostatic balance within the cell's environment thus allowing normal functions to carry on. Therefore, whenever possible, changes in cellular chemistry, metabolism, and gene expressions should be simultaneously evaluated using a common pool of tissue. This will help advance the knowledge needed to fill the gaps in our understanding of how these variables function together to maintain cellular homeostasis. Currently, foliar samples of trees for total inorganic nutrients and metabolic analyses are often collected at different times and are stored and processed in different ways before analyses. The objective of the present study was to evaluate whether a pool of wet (previously frozen) intact tissue that is used for metabolic and molecular work would also be suitable for analyses of foliar total inorganic nutrients. We compared quantities of nutrients extracted from wet-intact, dried-intact, and dried-ground tissues taken from a common pool of previously frozen foliage of black oak (Quercus velutina L.), sugar maple (Acer saccharum Marshall), red spruce (Picea rubens Sarg.), and white pine (Pinus strobus L.). With a few exceptions in the case of hardwoods where concentrations of total Ca, Mg, K, and P extracted from wet-intact tissue were significantly higher than dry tissue, data pooled across all collection times suggest that the extracted nutrient concentrations were comparable among the three tissue preparation methods and all for species. Based on the data presented here, it may be concluded that drying and grinding of foliage may not be necessary for nutrient analyses thus making it possible to use the same pool of tissue for total inorganic nutrients and metabolic and/or genomic analyses. To our knowledge, this is the first report on such a comparison.

Cell-Wall-Degrading Enzymes-Related Genes Originating from Rhizoctonia solani Increase Sugar Beet Root Damage in the Presence of Leuconostoc mesenteroides.

Sugar beet crown and root rot caused by Rhizoctonia solani is a major yield constraint. Root rot is highly increased when R. solani and Leuconostoc mesenteroides co-infect roots. We hypothesized that the absence of plant cell-wall-degrading enzymes in L. mesenteroides and their supply by R. solani during close contact, causes increased damage. In planta root inoculation with or without cell-wall-degrading enzymes showed greater rot when L. mesenteroides was combined with cellulase (22 mm rot), polygalacturonase (47 mm), and pectin lyase (57 mm) versus these enzymes (0-26 mm), R. solani (20 mm), and L. mesenteroides (13 mm) individually. Carbohydrate analysis revealed increased simpler carbohydrates (namely glucose + galactose, and fructose) in the infected roots versus mock control, possibly due to the degradation of complex cell wall carbohydrates. Expression of R. solani cellulase, polygalacturonase, and pectin lyase genes during root infection corroborated well with the enzyme data. Global mRNAseq analysis identified candidate genes and highly co-expressed gene modules in all three organisms that might be critical in host plant defense and pathogenesis. Targeting R. solani cell-wall-degrading enzymes in the future could be an effective strategy to mitigate root damage during its interaction with L. mesenteroides.

Regulatory Roles of Small Non-coding RNAs in Sugar Beet Resistance Against Beet curly top virus.

Beet curly top virus (BCTV) mediated yield loss in sugar beets is a major problem worldwide. The circular single-stranded DNA virus is transmitted by the beet leafhopper. Genetic sources of BCTV resistance in sugar beet are limited and commercial cultivars rely on chemical treatments versus durable genetic resistance. Phenotypic selection and double haploid production have resulted in sugar beet germplasm (KDH13; 13 and KDH4-9; 4) that are highly resistant to BCTV. The molecular mechanism of resistance to the virus is unknown, especially the role of small non-coding RNAs (sncRNAs) during early plant-viral interaction. Using the resistant lines along with a susceptible line (KDH19-17; 19), we demonstrate the role of sugar beet microRNAs (miRNAs) in BCTV resistance during early infection stages when symptoms are not yet visible. The differentially expressed miRNAs altered the expression of their corresponding target genes such as pyruvate dehydrogenase (EL10Ac1g02046), carboxylesterase (EL10Ac1g01087), serine/threonine protein phosphatase (EL10Ac1g01374), and leucine-rich repeats (LRR) receptor-like (EL10Ac7g17778), that were highly expressed in the resistant lines versus susceptible lines. Pathway enrichment analysis of the miRNA target genes showed an enrichment of genes involved in glycolysis/gluconeogenesis, galactose metabolism, starch, and sucrose metabolism to name a few. Carbohydrate analysis revealed altered glucose, galactose, fructose, and sucrose concentrations in the infected leaves of resistant versus susceptible lines. We also demonstrate differential regulation of BCTV derived sncRNAs in the resistant versus susceptible lines that target sugar beet genes such as LRR (EL10Ac1g01206), 7-deoxyloganetic acid glucosyltransferase (EL10Ac5g12605), and transmembrane emp24 domain containing (EL10Ac6g14074) and altered their expression. In response to viral infection, we found that plant derived miRNAs targeted BCTV capsid protein/replication related genes and showed differences in expression among resistant and susceptible lines. The data presented here demonstrate the contribution of miRNA mediated regulation of metabolic pathways and cross-kingdom RNA interference (RNAi) in sugar beet BCTV resistance.

Red maple (Acer rubrum L.) trees demonstrate acclimation to urban conditions in deciduous forests embedded in cities.

The impacts of urbanization, such as urban heat island (UHI) and nutrient loads, can influence tree function through altered physiology and metabolism and stress response, which has implications for urban forest health in cities across the world. Our goal was to compare growth-stimulating and stress-mitigating acclimation patterns of red maple (Acer rubrum) trees in deciduous forests embedded in a small (Newark, DE, US) and a large (Philadelphia, PA, US) city. The study was conducted in a long-term urban forest network on seventy-nine mature red maple trees spanning ten forests across Newark and Philadelphia. We hypothesized that red maples in Philadelphia forests compared to Newark forests will be healthier and more acclimated to warmer temperatures, elevated CO2 concentrations and reactive nitrogen (Nr) deposition, and higher nutrient/heavy metal loads. Therefore, these red maples will have higher foliar pigments, nutrients, and stress-indicating elements, enriched δ15N isotopes and increased free polyamines and amino acids to support a growth-stimulating and stress-induced response to urbanization. Our results indicate red maples are potentially growth-stimulated and stress-acclimated in Philadelphia forests experiencing a greater magnitude of urban intensity. Red maples in Philadelphia forests contained higher concentrations of foliar chlorophyll, %N, δ15N, and nutrients than those in Newark forests. Similarly, lower foliar magnesium and manganese, and higher foliar zinc, cadmium, lead, and aluminum reflected the difference in soil biogeochemistry in Philadelphia forests. Accumulation patterns of foliar free amino acids, polyamines, phosphorous, and potassium ions in red maples in Philadelphia forests shows a reallocation in cellular metabolism and nutrient uptake pathways responsible for physiological acclimation. Our results suggest the approach used here can serve as a model for investigating 'plant physiology' and the use of urban trees as a biomonitor of the impacts of 'urban pollution' on urban forests. The results suggest that cellular oxidative stress in trees caused by pollutant uptake is mitigated by the accumulation of free amino acids, polyamines, and nutrients in a larger city. Our study provides a framework for determining whether trees respond to complex urban environments through stress memory and/or acclimation.

Contribution of Maize Polyamine and Amino Acid Metabolism Toward Resistance Against Aspergillus flavus Infection and Aflatoxin Production.

Polyamines (PAs) are ubiquitous polycations found in plants and other organisms that are essential for growth, development, and resistance against abiotic and biotic stresses. The role of PAs in plant disease resistance depends on the relative abundance of higher PAs [spermidine (Spd), spermine (Spm)] vs. the diamine putrescine (Put) and PA catabolism. With respect to the pathogen, PAs are required to achieve successful pathogenesis of the host. Maize is an important food and feed crop, which is highly susceptible to Aspergillus flavus infection. Upon infection, the fungus produces carcinogenic aflatoxins and numerous other toxic secondary metabolites that adversely affect human health and crop value worldwide. To evaluate the role of PAs in aflatoxin resistance in maize, in vitro kernel infection assays were performed using maize lines that are susceptible (SC212) or resistant (TZAR102, MI82) to aflatoxin production. Results indicated significant induction of both PA biosynthetic and catabolic genes upon A. flavus infection. As compared to the susceptible line, the resistant maize lines showed higher basal expression of PA metabolism genes in mock-inoculated kernels that increased upon fungal infection. In general, increased biosynthesis and conversion of Put to Spd and Spm along with their increased catabolism was evident in the resistant lines vs. the susceptible line SC212. There were higher concentrations of amino acids such as glutamate (Glu), glutamine (Gln) and γ-aminobutyric acid (GABA) in SC212. The resistant lines were significantly lower in fungal load and aflatoxin production as compared to the susceptible line. The data presented here demonstrate an important role of PA metabolism in the resistance of maize to A. flavus colonization and aflatoxin contamination. These results provide future direction for the manipulation of PA metabolism in susceptible maize genotypes to improve aflatoxin resistance and overall stress tolerance.

The Aspergillus flavus Spermidine Synthase (spds) Gene, Is Required for Normal Development, Aflatoxin Production, and Pathogenesis During Infection of Maize Kernels.

Aspergillus flavus is a soil-borne saprophyte and an opportunistic pathogen of both humans and plants. This fungus not only causes disease in important food and feed crops such as maize, peanut, cottonseed, and tree nuts but also produces the toxic and carcinogenic secondary metabolites (SMs) known as aflatoxins. Polyamines (PAs) are ubiquitous polycations that influence normal growth, development, and stress responses in living organisms and have been shown to play a significant role in fungal pathogenesis. Biosynthesis of spermidine (Spd) is critical for cell growth as it is required for hypusination-mediated activation of eukaryotic translation initiation factor 5A (eIF5A), and other biochemical functions. The tri-amine Spd is synthesized from the diamine putrescine (Put) by the enzyme spermidine synthase (Spds). Inactivation of spds resulted in a total loss of growth and sporulation in vitro which could be partially restored by addition of exogenous Spd. Complementation of the Δspds mutant with a wild type (WT) A. flavus spds gene restored the WT phenotype. In WT A. flavus, exogenous supply of Spd (in vitro) significantly increased the production of sclerotia and SMs. Infection of maize kernels with the Δspds mutant resulted in a significant reduction in fungal growth, sporulation, and aflatoxin production compared to controls. Quantitative PCR of Δspds mutant infected seeds showed down-regulation of aflatoxin biosynthetic genes in the mutant compared to WT A. flavus infected seeds. Expression analyses of PA metabolism/transport genes during A. flavus-maize interaction showed significant increase in the expression of arginine decarboxylase (Adc) and S-adenosylmethionine decarboxylase (Samdc) genes in the maize host and PA uptake transporters in the fungus. The results presented here demonstrate that Spd biosynthesis is critical for normal development and pathogenesis of A. flavus and pre-treatment of a Δspds mutant with Spd or Spd uptake from the host plant, are insufficient to restore WT levels of pathogenesis and aflatoxin production during seed infection. The data presented here suggest that future studies targeting spermidine biosynthesis in A. flavus, using RNA interference-based host-induced gene silencing approaches, may be an effective strategy to reduce aflatoxin contamination in maize and possibly in other susceptible crops.

Polyamine- and Amino Acid-Related Metabolism: The Roles of Arginine and Ornithine are Associated with the Embryogenic Potential.

The mechanisms that control polyamine (PA) metabolism in plant cell lines with different embryogenic potential are not well understood. This study involved the use of two Araucaria angustifolia cell lines, one of which was defined as being blocked, in that the cells were incapable of developing somatic embryos, and the other as being responsive, as the cells could generate somatic embryos. Cellular PA metabolism was modulated by using 5 mM arginine (Arg) or ornithine (Orn) at two time points during cell growth. Two days after subculturing with Arg, an increase in citrulline (Cit) content was observed, followed by a higher expression of genes related to PA catabolism in the responsive cell line; whereas, in the blocked cell line, we only observed an accumulation of PAs. After 14 d, metabolism was directed towards putrescine accumulation in both cell lines. Exogenous Arg and Orn not only caused a change in cellular contents of PAs, but also altered the abundance of a broader spectrum of amino acids. Specifically, Cit was the predominant amino acid. We also noted changes in the expression of genes related to PA biosynthesis and catabolism. These results indicate that Arg and Orn act as regulators of both biosynthetic and catabolic PA metabolites; however, we suggest that they have distinct roles associated with embryogenic potential of the cells.

Polyamines in the Context of Metabolic Networks.

Polyamines (PAs) are essential biomolecules that are known to be involved in the regulation of many plant developmental and growth processes as well as their response to different environmental stimuli. Maintaining the cellular pools of PAs or their metabolic precursors and by-products is critical to accomplish their normal functions. Therefore, the titre of PAs in the cells must be under tight regulation to enable cellular PA homeostasis. Polyamine homeostasis is hence achieved by the regulation of their input into the cellular PA pool, their conversion into secondary metabolites, their transport to other issues/organs, and their catabolism or turnover. The major contributors of input to the PA pools are their in vivo biosynthesis, interconversion between different PAs, and transport from other tissues/organs; while the output or turnover of PAs is facilitated by transport, conjugation and catabolism. Polyamine metabolic pathways including the biosynthesis, catabolism/turnover and conjugation with various organic molecules have been widely studied in all kingdoms. Discoveries on the molecular transporters facilitating the intracellular and intercellular translocation of PAs have also been reported. Numerous recent studies using transgenic approaches and mutagenesis have shown that plants can tolerate quite large concentrations of PAs in the cells; even though, at times, high cellular accumulation of PAs is quite detrimental, and so is high rate of catabolism. The mechanism by which plants tolerate such large quantities of PAs is still unclear. Interestingly, enhanced PA biosynthesis via manipulation of the PA metabolic networks has been suggested to contribute directly to increased growth and improvements in plant abiotic and biotic stress responses; hence greater biomass and productivity. Genetic manipulation of the PA metabolic networks has also been shown to improve plant nitrogen assimilation capacity, which may in turn lead to enhanced carbon assimilation. These potential benefits on top of the widely accepted role of PAs in improving plants' tolerance to biotic and abiotic stressors are invaluable tools for future plant improvement strategies.

Genetic manipulation of putrescine biosynthesis reprograms the cellular transcriptome and the metabolome.

BACKGROUND: With the increasing interest in metabolic engineering of plants using genetic manipulation and gene editing technologies to enhance growth, nutritional value and environmental adaptation, a major concern is the potential of undesirable broad and distant effects of manipulating the target gene or metabolic step in the resulting plant. A comprehensive transcriptomic and metabolomic analysis of the product may shed some useful light in this regard. The present study used these two techniques with plant cell cultures to analyze the effects of genetic manipulation of a single step in the biosynthesis of polyamines because of their well-known roles in plant growth, development and stress responses. RESULTS: The transcriptomes and metabolomes of a control and a high putrescine (HP) producing cell line of poplar (Populus nigra x maximowiczii) were compared using microarrays and GC/MS. The HP cells expressed an ornithine decarboxylase transgene and accumulated several-fold higher concentrations of putrescine, with only small changes in spermidine and spermine. The results show that up-regulation of a single step in the polyamine biosynthetic pathway (i.e. ornithine → putrescine) altered the expression of a broad spectrum of genes; many of which were involved in transcription, translation, membrane transport, osmoregulation, shock/stress/wounding, and cell wall metabolism. More than half of the 200 detected metabolites were significantly altered (p ≤ 0.05) in the HP cells irrespective of sampling date. The most noteworthy differences were in organic acids, carbohydrates and nitrogen-containing metabolites. CONCLUSIONS: The results provide valuable information about the role of polyamines in regulating nitrogen and carbon use pathways in cell cultures of high putrescine producing transgenic cells of poplar vs. their low putrescine counterparts. The results underscore the complexity of cellular responses to genetic perturbation of a single metabolic step related to nitrogen metabolism in plants. Combined with recent studies from our lab, where we showed that higher putrescine production caused an increased flux of glutamate into ornithine concurrent with enhancement in glutamate production via additional nitrogen and carbon assimilation, the results from this study provide guidance in designing transgenic plants with increased nitrogen use efficiency, especially in plants intended for non-food/feed applications (e.g. increased biomass production for biofuels).

Glutamate, Ornithine, Arginine, Proline, and Polyamine Metabolic Interactions: The Pathway Is Regulated at the Post-Transcriptional Level.

The metabolism of glutamate into ornithine, arginine, proline, and polyamines is a major network of nitrogen-metabolizing pathways in plants, which also produces intermediates like nitric oxide, and γ-aminobutyric acid (GABA) that play critical roles in plant development and stress. While the accumulations of intermediates and the products of this network depend primarily on nitrogen assimilation, the overall regulation of the interacting sub-pathways is not well understood. We tested the hypothesis that diversion of ornithine into polyamine biosynthesis (by transgenic approach) not only plays a role in regulating its own biosynthesis from glutamate but also affects arginine and proline biosynthesis. Using two high putrescine producing lines of Arabidopsis thaliana (containing a transgenic mouse ornithine decarboxylase gene), we studied the: (1) effects of exogenous supply of carbon and nitrogen on polyamines and pools of soluble amino acids; and, (2) expression of genes encoding key enzymes in the interactive pathways of arginine, proline and GABA biosynthesis as well as the catabolism of polyamines. Our findings suggest that: (1) the overall conversion of glutamate to arginine and polyamines is enhanced by increased utilization of ornithine for polyamine biosynthesis by the transgene product; (2) proline and arginine biosynthesis are regulated independently of polyamines and GABA biosynthesis; (3) the expression of most genes (28 that were studied) that encode enzymes of the interacting sub-pathways of arginine and GABA biosynthesis does not change even though overall biosynthesis of Orn from glutamate is increased several fold; and (4) increased polyamine biosynthesis results in increased assimilation of both nitrogen and carbon by the cells.

Reduced Silver Nanoparticle Phytotoxicity in Crambe abyssinica with Enhanced Glutathione Production by Overexpressing Bacterial γ-Glutamylcysteine Synthase.

Silver nanoparticles (Ag NPs) are widely used in consumer products, and their release has raised serious concerns about the risk of their exposure to the environment and to human health. However, biochemical mechanisms by which plants counteract NP toxicity are largely unknown. We have previously engineered Crambe abyssinica plants expressing the bacterial γ-glutamylecysteine synthase (γ-ECS) for enhancing glutathione (GSH) levels. In this study, we investigated if enhanced levels of GSH and its derivatives can protect plants from Ag NPs and AgNO3 (Ag(+) ions). Our results showed that transgenic lines, when exposed to Ag NPs and Ag(+) ions, were significantly more tolerant, attaining a 28%-46% higher biomass and 34-49% more chlorophyll content, as well as maintaining 35-46% higher transpiration rates as compared to those of wild type (WT) plants. Transgenic γ-ECS lines showed 2-6-fold Ag accumulation in shoot tissue and slightly lower or no difference in root tissue relative to levels in WT plants. The levels of malondialdehyde (MDA) in γ-ECS lines were also 27.3-32.5% lower than those in WT Crambe. These results indicate that GSH and related peptides protect plants from Ag nanotoxicity. To our knowledge, this is the first direct report of Ag NP detoxification by GSH in transgenic plants, and these results will be highly useful in developing strategies to counteract the phytotoxicty of metal-based nanoparticles in crop plants.

Long-term trends of changes in pine and oak foliar nitrogen metabolism in response to chronic nitrogen amendments at Harvard Forest, MA.

We evaluated the long-term (1995-2008) trends in foliar and sapwood metabolism, soil solution chemistry and tree mortality rates in response to chronic nitrogen (N) additions to pine and hardwood stands at the Harvard Forest Long Term Ecological Research (LTER) site. Common stress-related metabolites like polyamines (PAs), free amino acids (AAs) and inorganic elements were analyzed for control, low N (LN, 50 kg NH4NO3 ha(-1) year(-1)) and high N (HN, 150 kg NH4NO3 ha(-1) year(-1)) treatments. In the pine stands, partitioning of excess N into foliar PAs and AAs increased with both N treatments until 2002. By 2005, several of these effects on N metabolites disappeared for HN, and by 2008 they were mostly observed for LN plot. A significant decline in foliar Ca and P was observed mostly with HN for a few years until 2005. However, sapwood data actually showed an increase in Ca, Mg and Mn and no change in PAs in the HN plot for 2008, while AAs data revealed trends that were generally similar to foliage for 2008. Concomitant with these changes, mortality data revealed a large number of dead trees in HN pine plots by 2002; the mortality rate started to decline by 2005. Oak trees in the hardwood plot did not exhibit any major changes in PAs, AAs, nutrients and mortality rate with LN treatment, indicating that oak trees were able to tolerate the yearly doses of 50 kg NH4NO3 ha(-1) year(-1). However, HN trees suffered from physiological and nutritional stress along with increased mortality in 2008. In this case also, foliar data were supported by the sapwood data. Overall, both low and high N applications resulted in greater physiological stress to the pine trees than the oaks. In general, the time course of changes in metabolic data are in agreement with the published reports on changes in soil chemistry and microbial community structure, rates of soil carbon sequestration and production of woody biomass for this chronic N study. This correspondence of selected metabolites with other measures of forest functions suggests that the metabolite analyses are useful for long-term monitoring of the health of forest trees.

Extraction and estimation of the quantity of calcium oxalate crystals in the foliage of conifer and hardwood trees.

The main goal of this study was to develop a method for the extraction and indirect estimation of the quantity of calcium oxalate (CaOx) in the foliage of trees. Foliar tissue was collected from a single tree of each species (five conifers and five hardwoods) for comparison of extractions in different solvents using 10 replicates per species from the same pool of tissue. For each species, calcium (Ca) and oxalate were extracted sequentially in double deionized water and 2N acetic acid, and finally, five replicate samples were extracted in 5% (0.83N) perchloric acid (PCA) and the other five in 2N hydrochloric acid (HCl); three cycles of freezing and thawing were used for each solvent. Total ions were extracted by microwave digestion. Calcium was quantified with an inductively coupled plasma emission spectrophotometer method and oxalate was eluted and quantified using a high performance liquid chromatography method. This experiment was repeated again with two conifer and two hardwood species using four trees per species, and two analytical replicates for each tree. We report here that, regardless of age of individual trees within a species, time of collection or species type, the third extraction in PCA or HCl resulted in near equimolar quantities of Ca and oxalate (r(2) ≥ 0.99). This method provides an easy estimate of the quantity of CaOx crystals using a small sample of foliar tissue. An additional benefit of PCA is that it precipitates the nucleic acids and proteins, allowing the quantification of several free/soluble metabolites such as amino acids, polyamines, organic acids and inorganic elements all from a single sample extract.

Oligotyping reveals stronger relationship of organic soil bacterial community structure with N-amendments and soil chemistry in comparison to that of mineral soil at Harvard Forest, MA, USA.

The impact of chronic nitrogen amendments on bacterial communities was evaluated at Harvard Forest, Petersham, MA, USA. Thirty soil samples (3 treatments × 2 soil horizons × 5 subplots) were collected in 2009 from untreated (control), low nitrogen-amended (LN; 50 kg NH4NO3 ha(-1) yr(-1)) and high nitrogen-amended (HN; 150 kg NH4NO3 ha(-1) yr(-1)) plots. PCR-amplified partial 16S rRNA gene sequences made from soil DNA were subjected to pyrosequencing (Turlapati et al., 2013) and analyses using oligotyping. The parameters M (the minimum count of the most abundant unique sequence in an oligotype) and s (the minimum number of samples in which an oligotype is expected to be present) had to be optimized for forest soils because of high diversity and the presence of rare organisms. Comparative analyses of the pyrosequencing data by oligotyping and operational taxonomic unit clustering tools indicated that the former yields more refined units of taxonomy with sequence similarity of ≥99.5%. Sequences affiliated with four new phyla and 73 genera were identified in the present study as compared to 27 genera reported earlier from the same data (Turlapati et al., 2013). Significant rearrangements in the bacterial community structure were observed with N-amendments revealing the presence of additional genera in N-amended plots with the absence of some that were present in the control plots. Permutational MANOVA analyses indicated significant variation associated with soil horizon and N treatment for a majority of the phyla. In most cases soil horizon partitioned more variation relative to treatment and treatment effects were more evident for the organic (Org) horizon. Mantel test results for Org soil showed significant positive correlations between bacterial communities and most soil parameters including NH4 and NO3. In mineral soil, correlations were seen only with pH, NH4, and NO3. Regardless of the pipeline used, a major hindrance for such a study remains to be the lack of reference databases for forest soils.

Polyamines and abiotic stress in plants: a complex relationship.

The physiological relationship between abiotic stress in plants and polyamines was reported more than 40 years ago. Ever since there has been a debate as to whether increased polyamines protect plants against abiotic stress (e.g., due to their ability to deal with oxidative radicals) or cause damage to them (perhaps due to hydrogen peroxide produced by their catabolism). The observation that cellular polyamines are typically elevated in plants under both short-term as well as long-term abiotic stress conditions is consistent with the possibility of their dual effects, i.e., being protectors from as well as perpetrators of stress damage to the cells. The observed increase in tolerance of plants to abiotic stress when their cellular contents are elevated by either exogenous treatment with polyamines or through genetic engineering with genes encoding polyamine biosynthetic enzymes is indicative of a protective role for them. However, through their catabolic production of hydrogen peroxide and acrolein, both strong oxidizers, they can potentially be the cause of cellular harm during stress. In fact, somewhat enigmatic but strong positive relationship between abiotic stress and foliar polyamines has been proposed as a potential biochemical marker of persistent environmental stress in forest trees in which phenotypic symptoms of stress are not yet visible. Such markers may help forewarn forest managers to undertake amelioration strategies before the appearance of visual symptoms of stress and damage at which stage it is often too late for implementing strategies for stress remediation and reversal of damage. This review provides a comprehensive and critical evaluation of the published literature on interactions between abiotic stress and polyamines in plants, and examines the experimental strategies used to understand the functional significance of this relationship with the aim of improving plant productivity, especially under conditions of abiotic stress.

Putrescine overproduction does not affect the catabolism of spermidine and spermine in poplar and Arabidopsis.

The effect of up-regulation of putrescine (Put) production by genetic manipulation on the turnover of spermidine (Spd) and spermine (Spm) was investigated in transgenic cells of poplar (Populus nigra × maximowiczii) and seedlings of Arabidopsis thaliana. Several-fold increase in Put production was achieved by expressing a mouse ornithine decarboxylase cDNA either under the control of a constitutive (in poplar) or an inducible (in Arabidopsis) promoter. The transgenic poplar cells produced and accumulated 8-10 times higher amounts of Put than the non-transgenic cells, whereas the Arabidopsis seedlings accumulated up to 40-fold higher amounts of Put; however, in neither case the cellular Spd or Spm increased consistently. The rate of Spd and Spm catabolism and the half-life of cellular Spd and Spm were measured by pulse-chase experiments using [(14)C]Spd or [(14)C]Spm. Spermidine half-life was calculated to be about 22-32 h in poplar and 52-56 h in Arabidopsis. The half-life of cellular Spm was calculated to be approximately 24 h in Arabidopsis and 36-48 h in poplar. Both species were able to convert Spd to Spm and Put, and Spm to Spd and Put. The rates of Spd and Spm catabolism in both species were several-fold slower than those of Put, and the overproduction of Put had only a small effect on the overall rates of turnover of Spd or Spm. There was little effect on the rates of Spd to Spm conversion as well as the conversion of Spm into lower polyamines. While Spm was mainly converted back to Spd and not terminally degraded, Spd was removed from the cells largely through terminal catabolism in both species.

Enhanced flux of substrates into polyamine biosynthesis but not ethylene in tomato fruit engineered with yeast S-adenosylmethionine decarboxylase gene.

S-adenosylmethionine (SAM), a major substrate in 1-C metabolism is a common precursor in the biosynthetic pathways of polyamines and ethylene, two important plant growth regulators, which exhibit opposing developmental effects, especially during fruit ripening. However, the flux of various substrates including SAM into the two competing pathways in plants has not yet been characterized. We used radiolabeled (14)C-Arg, (14)C-Orn, L-[U-(14)C]Met, (14)C-SAM and (14)C-Put to quantify flux through these pathways in tomato fruit and evaluate the effects of perturbing these pathways via transgenic expression of a yeast SAM decarboxylase (ySAMDC) gene using the fruit ripening-specific promoter E8. We show that polyamines in tomato fruit are synthesized both from Arg and Orn; however, the relative contribution of Orn pathway declines in the later stages of ripening. Expression of ySAMDC reversed the ripening associated decline in spermidine (Spd) and spermine (Spm) levels observed in the azygous control fruit. About 2- to 3-fold higher levels of labeled-Spd in transgenic fruit (556HO and 579HO lines) expressing ySAMDC confirmed the enzymatic function of the introduced gene. The incorporation of L-[U-(14)C]Met into Spd, Spm, ethylene and 1-aminocyclopropane-1-carboxylic acid (ACC) was used to determine Met-flux into these metabolites. The incorporation of (14)C-Met into Spd/Spm declined during ripening of the control azygous fruit but this was reversed in fruits expressing ySAMDC. However, incorporation of (14)C-Met into ethylene or ACC during ripening was not altered by the expression of ySAMDC in the fruit. Taken together these results show that: (1) There is an inverse relationship between the production of higher polyamines and ethylene during fruit ripening, (2) the inverse relationship between higher polyamines and ethylene is modulated by ySAMDC expression in that the decline in Spd/Spm during fruit ripening can be reversed without significantly altering ethylene biosynthesis, and (3) cellular flux of SAM in plants is homeostatically regulated based on its demand for competing pathways.

Ornithine: the overlooked molecule in the regulation of polyamine metabolism.

We overexpressed a mouse ornithine decarboxylase gene under the control of a constitutive and an estradiol-inducible promoter in Arabidopsis thaliana to increase our understanding of the regulation of polyamine metabolism. Of particular interest was the role of the substrate ornithine not only in the regulation of polyamine biosynthesis, but also in the accumulation of related amino acids in response to short-term induction of this enzyme. We hypothesized that the inducible expression of the transgene would mimic the natural responses of plants to changing conditions, e.g. under stress conditions and during rapid growth. Our results reveal that ornithine, even though present in relatively small quantities (compared with other amino acids of the glutamate-arginine-proline pathway), may not only be the key regulator of polyamine biosynthesis in Arabidopsis, but it may also regulate the entire subset of pathways for glutamate to arginine and to proline. Indirectly, it could also regulate putrescine catabolism, therefore contributing to the γ-aminobutyric acid content of the cells. Furthermore, the induction of mouse ornithine decarboxylase resulted in up- and down-regulation of several amino acids in the transgenic plants. It was learned that the turnover of putrescine in both the wild type and the transgenic plants occurs rapidly, with a half-life of 6-8 h.