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

PREVENTION OF CARCINOGENESIS AND CANCER CELL PROLIFERATION BY AN INHIBITOR OF POLYAMINE BIOSYNTHESIS (DFMO, HELA, MAMMARY, TUMOR)

The induction of mammary cancer in female Sprague-Dawley rats by a single of 1-methyl-1-nitrosourea was used as an experimental model system for identifying an effective agent in the prevention of breast cancer. The polyamine concentrations in whole mammary gland or mammary epithelial cells remained unchanged in the post-initiation stages of mammary carcinogenesis prior to the appearance of detectable tumors. However, mammary tumors had much higher concentrations of polyamines as compared to normal mammary gland. The development of chemically-induced carcinomas in rats was dramatically suppressed by the provision of a 1% solution of D,L-(alpha)-difluoromethyl-ornithine (DFMO), an inhibitor of polyamine biosynthesis, in drinking water for 6 months. This treatment significantly reduced cancer incidence and the average number of cancers per rat and prolonged cancer-free time. The average weight of mammary gland tumors induced in animals receiving DFMO was reduced ten-fold in comparison to tumors occurring in untreated rats. The concentrations of polyamines and ornithine decarboxylase activity in the tumors of rats receiving 0.5% DFMO were significantly reduced in comparison with data obtained in tumors of control animals. DFMO did not affect polyamine concentrations in the mammary gland of saline-treated animals. DFMO appeared to block some aspect of promotional stage(s) of mammary carcinogenesis dependent upon polyamines. To study the molecular mechanism by which DFMO acts as an antiproliferative agent in carcinogenesis, HeLa cells were used. HeLa cells were synchronized by the double thymidine block procedure and depleted of polyamines by the use of 1 mM DFMO. These polyamine-depleted cells exhibited a severe deficiency in S phase DNA synthesis and cell proliferation. This inhibitory effect of DFMO could be partially reversed by the addition of polyamines to the cell cultures at the beginning of S phase. Polyamine-deficient cells did not lack any essential cytosol factor(s) for DNA synthesis. Furthermore, salt extracts of nuclei containing DNA polymerase activity did not restore DNA synthesis in nuclei of polyamine-deficient cells. Polyamines may be required for the synthesis of molecules or for their interaction in control mechanisms preceding S phase DNA synthesis

Soil bacterial communities of a calcium-supplemented and a reference watershed at the Hubbard Brook Experimental Forest (HBEF), New Hampshire, USA

Soil Ca depletion because of acidic deposition-related soil chemistry changes has led to the decline of forest productivity and carbon sequestration in the northeastern USA. In 1999, acidic watershed (WS) 1 at the Hubbard Brook Experimental Forest (HBEF), NH, USA was amended with Ca silicate to restore soil Ca pools. In 2006, soil samples were collected from the Ca-amended (WS1) and reference watershed (WS3) for comparison of bacterial community composition between the two watersheds. The sites were about 125 m apart and were known to have similar stream chemistry and tree populations before Ca amendment. Ca-amended soil had higher Ca and P, and lower Al and acidity as compared with the reference soils. Analysis of bacterial populations by PhyloChip revealed that the bacterial community structure in the Ca-amended and the reference soils was significantly different and that the differences were more pronounced in the mineral soils. Overall, the relative abundance of 300 taxa was significantly affected. Numbers of detectable taxa in families such as Acidobacteriaceae, Comamonadaceae, and Pseudomonadaceae were lower in the Ca-amended soils, while Flavobacteriaceae and Geobacteraceae were higher. The other functionally important groups, e.g. ammonia-oxidizing Nitrosomonadaceae, had lower numbers of taxa in the Ca-amended organic soil but higher in the mineral soil

Seasonal changes in foliar calcium oxalate concentrations in conifer and hardwood trees: a potentially bioavailable source of cellular calcium and/or oxalate under stress

The present study compared seasonal changes in the concentrations of calcium oxalate (CaOx) crystals and total calcium (Ca) in the foliage of red spruce (Picea rubens Sarg.), white pine (Pinus strobus L.), black oak (Quercus velutina L.), and sugar maple (Acer saccharum Marshall) trees. Samples were collected from the same four replicate trees of each species starting in June 2014 through September 2015 for a total of six times for conifers and four times for hardwoods. Calcium oxalate was extracted from tissues using a method developed in our laboratory in 2015. The purity of the extracted CaOx was indicated by an r2 of 0.98 between Ca and oxalate (Ox) for the data pooled across all species and all sampling times. As expected, the concentrations of CaOx varied between species. We hypothesized that the only role of CaOx crystals is to bind excess Ca, so based on this hypothesis the concentrations of CaOx would increase over the growing season both in conifer and hardwood trees, and in conifers, its quantities would be higher in the older relative to the younger needles. However, we found, that for most species, CaOx concentrations were not significantly different from each other for all collection times. In addition, relative to total Ca, the percent of Ca that existed in the form of CaOx varied widely with species, time of collection within a species, and needle age. Thus, no specific trend was observed for CaOx accumulations with changes in seasons. Concentrations of CaOx were indeed higher in older spruce and pine needles. Based on the available literature on this topic and our data, this could mean that CaOx amounts are dynamic and are continuously being adjusted according to the metabolic needs of cells for either Ca or Ox while still performing the function of shedding off excess Ca

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

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

Climate change influences foliar nutrition and metabolism of red maple (Acer rubrum) trees in a northern hardwood forest

© The Author(s), 2022. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Blagden, M., Harrison, J. L., Minocha, R., Sanders-DeMott, R., Long, S., & Templer, P. H. Climate change influences foliar nutrition and metabolism of red maple (Acer rubrum) trees in a northern hardwood forest. Ecosphere, 13(2), (2022): e03859. https://doi.org/10.1002/ecs2.3859.Mean annual air temperatures are projected to increase, while the winter snowpack is expected to shrink in depth and duration for many mid- and high-latitude temperate forest ecosystems over the next several decades. Together, these changes will lead to warmer growing season soil temperatures and an increased frequency of soil freeze–thaw cycles (FTCs) in winter. We took advantage of the Climate Change Across Seasons Experiment (CCASE) at the Hubbard Brook Experimental Forest in the White Mountains of New Hampshire, USA, to determine how these changes in soil temperature affect foliar nitrogen (N) and carbon metabolism of red maple (Acer rubrum) trees in 2015 and 2017. Earlier work from this study revealed a similar increase in foliar N concentrations with growing season soil warming, with or without the occurrence of soil FTCs in winter. However, these changes in soil warming could differentially affect the availability of cellular nutrients, concentrations of primary and secondary metabolites, and the rates of photosynthesis that are all responsive to climate change. We found that foliar concentrations of phosphorus (P), potassium (K), N, spermine (a polyamine), amino acids (alanine, histidine, and phenylalanine), chlorophyll, carotenoids, sucrose, and rates of photosynthesis increased with growing season soil warming. Despite similar concentrations of foliar N with soil warming with and without soil FTCs in winter, winter soil FTCs affected other foliar metabolic responses. The combination of growing season soil warming and winter soil FTCs led to increased concentrations of two polyamines (putrescine and spermine) and amino acids (alanine, proline, aspartic acid, γ-aminobutyric acid, valine, leucine, and isoleucine). Treatment-specific metabolic changes indicated that while responses to growing season warming were more connected to their role as growth modulators, soil warming + FTC treatment-related effects revealed their dual role in growth and stress tolerance. Together, the results of this study demonstrate that growing season soil warming has multiple positive effects on foliar N and cellular metabolism in trees and that some of these foliar responses are further modified by the addition of stress from winter soil FTCs.This research was supported by an NSF Long Term Ecological Research (LTER) Grant to Hubbard Brook (NSF 1114804 and 1637685) and an NSF CAREER grant to PHT (NSF DEB1149929). RSD was supported by NSF DGE0947950, a Boston University (BU) Dean's Fellowship, and the BU Program in Biogeoscience. Jamie Harrison was supported by a BU Dean's Fellowship. Megan Blagden was supported by a BU Undergraduate Research Opportunity Program fellowship. This manuscript is a contribution to the Hubbard Brook Ecosystem Study. Hubbard Brook is part of the LTER network, which is supported by the NSF

Cell wall degrading enzymes 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 (49 mm rot), polygalacturonase (48 mm), and pectin lyase (35 mm) versus these enzymes (0–11 mm), R. solani (13 mm), and L. mesenteroides (22 mm) individually. Carbohydrate analysis revealed increase in 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 clusters (in all 3 organisms) that might be critical in host plant defense and pathogenesis. Future targeting of R. solani cell wall degrading enzymes could be an effective strategy to mitigate root damage during interaction with L. mesenteroides

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

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 noncoding 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 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 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 concentration 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 RNAi in sugar beet BCTV resistance