Karen Klotz Fugate

Metabolic profile of wound-induced changes in primary carbon metabolism in sugarbeet root.

Injury to plant products by harvest and postharvest operations induces respiration rate and increases the demand for respiratory substrates. Alterations in primary carbon metabolism are likely to support the elevated demand for respiratory substrates, although the nature of these alterations is unknown. To gain insight into the metabolic changes that occur to provide substrates for wound-induced increases in respiration, changes in the concentrations of compounds that are substrates, intermediates or cofactors in the respiratory pathway were determined in sugarbeet (Beta vulgaris L.) roots in the 4days following injury. Both wounded and unwounded tissues of wounded roots were analyzed to provide information about localized and systemic changes that occur after wounding. In wounded tissue, respiration increased an average of 186%, fructose, glucose 6-phosphate, ADP and UDP concentrations increased, and fructose 1,6-bisphosphate, triose phosphate, citrate, isocitrate, succinate, ATP, UTP and NAD(+) concentrations decreased. In the non-wounded tissue of wounded roots, respiration rate increased an average of 21%, glucose 6-phosphate, fructose 6-phosphate, glucose 1-phosphate and ADP concentrations increased, and isocitrate, UTP, NAD(+), NADP(+), and NADPH concentrations declined. Changes in respiration rate and metabolite concentrations indicated that localized and systemic changes in primary carbon metabolism occurred in response to injury. In wounded tissue, metabolite concentration changes suggested that activities of the early glycolytic enzymes, fructokinase, phosphofructokinase, phosphoglucose isomerase, and phosphoglucomutase were limiting carbon flow through glycolysis. These restrictions in the respiratory pathway, however, were likely overcome by use of metabolic bypasses that allowed carbon compounds to enter the pathway at glycolytic and tricarboxylic acid (TCA) cycle downstream locations. In non-wounded tissue of wounded roots, metabolic concentration changes suggested that glycolysis and the TCA cycle were generally capable of supporting the small systemic elevation in respiration rate. Although the mechanism by which respiration is regulated in wounded sugarbeet roots is unknown, localized and systemic elevations in respiration were positively associated with one or more indicators of cellular redox status.

Generation and Characterization of a Sugarbeet Transcriptome and Transcript-Based SSR Markers

Sugarbeet is a major source of refined sucrose and increasingly grown for biofuel production. Demand for higher productivity for this crop requires greater knowledge of sugarbeet physiology, pathology, and genetics, which can be advanced by the development of new genomic resources. Towards this end, a sugarbeet transcriptome of expressed genes from leaf and root tissues at varying stages of development and production, and after elicitation with jasmonic acid (JA) or salicylic acid (SA), was constructed and used to generate simple sequence repeat (SSR) markers. The transcriptome was generated via paired‐end RNA sequencing and contains 82,404 unigenes. A total of 37,207 unigenes were annotated, of which 9480 were functionally classified using clusters of orthologous groups (COG) annotations, 17,191 were classified into biological process, molecular function, or cellular component using gene ontology (GO) terms, and 17,409 were assigned to 126 metabolic pathways using Kyoto Encyclopedia of Genes and Genomes (KEGG) identifiers. A SSR search of the transcriptome identified 7680 SSRs, including 6577 perfect SSRs, of which 3834 were located in unigenes with ungapped sequence. Primer‐pairs were designed for 288 SSR loci, and 72 of these primer‐pairs were tested for their ability to detect polymorphisms. Forty‐three primer‐pairs detected single polymorphic loci and effectively distinguished diversity among eight B. vulgaris genotypes. The transcriptome and SSR markers provide additional, public domain genomic resources for an important crop plant and can be used to increase understanding of the functional elements of the sugarbeet genome, aid in discovery of novel genes, facilitate RNA‐sequencing based expression research, and provide new tools for sugarbeet genetic research and selective breeding.

Glycolysis Is Dynamic and Relates Closely to Respiration Rate in Stored Sugarbeet Roots

Although respiration is the principal cause of the loss of sucrose in postharvest sugarbeet (Beta vulgaris L.), the internal mechanisms that control root respiration rate are unknown. Available evidence, however, indicates that respiration rate is likely to be controlled by the availability of respiratory substrates, and glycolysis has a central role in generating these substrates. To determine glycolytic changes that occur in sugarbeet roots after harvest and to elucidate relationships between glycolysis and respiration, sugarbeet roots were stored for up to 60 days, during which activities of glycolytic enzymes and concentrations of glycolytic substrates, intermediates, cofactors, and products were determined. Respiration rate was also determined, and relationships between respiration rate and glycolytic enzymes and metabolites were evaluated. Glycolysis was highly variable during storage, with 10 of 14 glycolytic activities and 14 of 17 glycolytic metabolites significantly altered during storage. Changes in glycolytic enzyme activities and metabolites occurred throughout the 60 day storage period, but were greatest in the first 4 days after harvest. Positive relationships between changes in glycolytic enzyme activities and root respiration rate were abundant, with 10 of 14 enzyme activities elevated when root respiration was elevated and 9 glycolytic activities static during periods of unchanging respiration rate. Major roles for pyruvate kinase and phosphofructokinase in the regulation of postharvest sugarbeet root glycolysis were indicated based on changes in enzymatic activities and concentrations of their substrates and products. Additionally, a strong positive relationship between respiration rate and pyruvate kinase activity was found indicating that downstream TCA cycle enzymes were unlikely to regulate or restrict root respiration in a major way. Overall, these results establish that glycolysis is not static during sugarbeet root storage and that changes in glycolysis are closely related to changes in sugarbeet root respiration.

Short- and long-term changes in sugarbeet (Beta vulgaris L.) gene expression due to postharvest jasmonic acid treatment - Data

Jasmonic acid is a natural plant hormone that induces native defense responses in plants. Sugarbeet (Beta vulgaris L.) root unigenes that were differentially expressed 2 and 60 days after a postharvest jasmonic acid treatment are presented. Data include changes in unigene expression relative to water-treated controls, unigene annotations against nonredundant (Nr), Swiss-Prot, Clusters of Orthologous Groups (COG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) protein databases, and unigene annotations with Gene Ontology (GO) terms. Putative defense unigenes are compiled and annotated against the sugarbeet genome. Differential gene expression data were generated by RNA sequencing. Interpretation of the data is available in the research article, “Jasmonic acid causes short- and long-term alterations to the transcriptome and the expression of defense genes in sugarbeet roots” (K.K. Fugate, L.S. Oliveira, J.P. Ferrareze, M.D. Bolton, E.L. Deckard, F.L. Finger, 2017) [1]. Public dissemination of this dataset will allow further analyses of the data.

USE OF JASMONIC ACID AND SALICYLIC ACID TO INHIBIT GROWTH OF SUGARBEET STORAGE ROT PATHOGENS

Jasmonic acid (JA) and salicylic acid (SA) are endogenous plant hormones that induce native plant defense responses and provide protection against a wide range of diseases. Previously, JA, applied after harvest, was shown to protect sugarbeet roots against the storage pathogens, Botrytis cinerea, Penicillium claviforme, and Phoma betae by reducing the severity of rot symptoms due to these pathogens by 51, 44, and 71%, respectively (Fugate et al., 2012, Postharvest Biol. Technol., 65:1-4). Research was conducted to determine the ability of SA to protect sugarbeet roots from these storage rot pathogens and to investigate the use of preharvest treatments of JA or methyl jasmonate (MeJA), a low cost derivative of JA, to reduce storage rot due to B. cinerea, P. claviforme, or P. betae. The effect of water stress on severity of rot symptoms due to B. cinerea, P. claviforme, and P. betae was also investigated. SA, applied after harvest at concentrations of 0.01, 0.1, 1.0 or 10 mM, had no effect on the severity of storage rot symptoms in roots obtained from healthy, unstressed plants after inoculation with B. cinerea, P. claviforme, and P. betae. However, when roots were obtained from water-stressed plants, 0.01 to 10 mM SA reduced the severity of rot symptoms due to B. cinerea by 49—58%, P. claviforme by 30—53%, and P. betae by 47—74%. All concentrations of SA provided statistically similar reductions in the weight of rotted tissue for each of the three pathogens, and on average, postharvest SA treatment reduced the weight of rotted tissue due to B. cinerea, P. claviforme, and P. betae by 54, 45, and 58% respectively. SA reduced the weight of rotted tissue in roots from water-stressed plants by reducing lesion size, but had no effect on the incidence of infection. The ability of SA to reduce rot severity in water-stressed roots but not in roots harvested from plants that received sufficient water prior to harvest suggests that SA mitigated the negative effects of water stress, but did not directly protect roots against storage pathogens. Results from SA experiments described above suggested that drought stress increased root rot severity due to B. cinerea, P. claviforme, and P. betae. To verify this, water was withheld from greenhouse-grown plants, roots were harvested two days after plants were severely wilted, and the harvested roots were inoculated with B. cinerea, P. claviforme, or P. betae. Relative to roots obtained from well-watered plants, roots from water-stressed plants had 2.3-fold more rot due to B. cinerea, 1.4-fold more rot due to P. claviforme, and 2.4-fold more rot due to P. betae. Field-grown roots from water-stressed plants also exhibited increases in rot severity due to B. cinerea and P. betae, but not P. claviforme. The ability of preharvest JA treatments to reduce storage rot was determined by application of 0.01 or 10 μM JA or MeJA to foliage 3, 7, 14, or 30 days prior to harvest. Although this research is not complete, preliminary results suggest that preharvest JA or MeJA treatments reduce the severity of storage rot due to B. cinerea, P. claviforme, and P. betae. In general, 0.01 μM JA was more effective in reducing storage rot than 10 μM JA. As was observed previously for postharvest JA treatments, preharvest JA treatments were most effective against P. betae and least effective against P. claviforme.