The identification of allergen proteins in sugar beet (Beta vulgaris) pollen causing occupational allergy in greenhouses

Background

During production of sugar beet (Beta vulgaris) seeds in greenhouses, workers frequently develop allergic symptoms. The aim of this study was to identify and characterize possible allergens in sugar beet pollen.

Methods

Sera from individuals at a local sugar beet seed producing company, having positive SPT and specific IgE to sugar beet pollen extract, were used for immunoblotting. Proteins in sugar beet pollen extracts were separated by 1- and 2-dimensional electrophoresis, and IgE-reactive proteins analyzed by liquid chromatography tandem mass spectrometry.

Results

A 14 kDa protein was identified as an allergen, since IgE-binding was inhibited by the well-characterized allergen Che a 2, profilin, from the related species Chenopodium album. The presence of 17 kDa and 14 kDa protein homologues to both the allergens Che a 1 and Che a 2 were detected in an extract from sugar beet pollen, and partial amino acid sequences were determined, using inclusion lists for tandem mass spectrometry based on homologous sequences.

Conclusion

Two occupational allergens were identified in sugar beet pollen showing sequence similarity with Chenopodium allergens. Sequence data were obtained by mass spectrometry (70 and 25%, respectively for Beta v 1 and Beta v 2), and can be used for cloning and recombinant expression of the allergens. As for treatment of Chenopodium pollinosis, immunotherapy with sugar beet pollen extracts may be feasible.     

Genetic analysis of bolting after winter in sugar beet (Beta vulgaris L.)

Key message

This study reveals for the first time a major QTL for post-winter bolting resistance in sugar beet ( Beta vulgaris L.). The knowledge of this QTL is a major contribution towards the development of a winter sugar beet with controlled bolting behavior.

Abstract

In cool temperate climates, sugar beets are currently grown as a spring crop. They are sown in spring and harvested in autumn. Growing sugar beet as a winter crop with an extended vegetation period fails due to bolting after winter. Bolting after winter might be controlled by accumulating genes for post-winter bolting resistance. Previously, we had observed in field experiments a low post-winter bolting rate of 0.5 for sugar beet accession BETA 1773. This accession was crossed with a biennial sugar beet with regular bolting behavior to develop a F₃ mapping population. The population was grown in the greenhouse, exposed to artificial cold treatment for 16 weeks and transplanted to the field. Bolting was recorded twice a week from May until October. Post-winter bolting behavior was assessed by two different factors, bolting delay (determined as days to bolt after cold treatment) and post-winter bolting resistance (bolting rate after winter). For days to bolt, means of F₃ families ranged from 25 to 164 days while for bolting rate F₃ families ranged from 0 to 1. For each factor one QTL explaining about 65 % of the phenotypic variation was mapped to the same region on linkage group 9 with a partially recessive allele increasing bolting delay and post-winter bolting resistance. The results are discussed in relation to the potential use of marker-assisted breeding of winter sugar beets with controlled bolting.     

An analysis of leaf growth in sugar beet.

The responses of leaf appearance and expansion to temperature in sugar beet were measured under controlled conditions, using ruler and auxanometers, to establish a basis for a subsequent analysis of leaf growth in field crops. The studies showed that leaf appearance rate responded linearly to temperature above 1°C, that leaf expansion rate responded likewise above 3°C and that both rates were zero below these base temperatures. Auxanometer measurements of leaf extension showed that daily rates of expansion of leaf area increased linearly with the daily integral of temperature. However, hourly rates of extension in length alternated with those in width during each 24 h cycle in patterns that were not clearly related to hourly changes of temperature or to the day/night sequence.     

Genetic diversity and linkage disequilibrium analysis in elite sugar beet breeding lines and wild beet accessions

Key message

Linkage disequilibrium decay in sugar beet is strongly affected by the breeding history, and varies extensively between and along chromosomes, allowing identification of known and unknown signatures of selection.

Abstract

Genetic diversity and linkage disequilibrium (LD) patterns were investigated in 233 elite sugar beet breeding lines and 91 wild beet accessions, using 454 single nucleotide polymorphisms (SNPs) and 418 SNPs, respectively. Principal coordinate analysis suggested the existence of three groups of germplasm, corresponding to the wild beets, the seed parent and the pollen parent breeding pool. LD was investigated in each of these groups, with and without correction for genetic relatedness. Without correction for genetic relatedness, in the pollen as well as the seed parent pool, LD persisted beyond 50 centiMorgan (cM) on four (2, 3, 4 and 5) and three chromosomes (2, 4 and 6), respectively; after correction for genetic relatedness, LD decayed after <6 cM on all chromosomes in both pools. In the wild beet accessions, there was a strong LD decay: on average LD disappeared after 1 cM when LD was calculated with a correction for genetic relatedness. Persistence of LD was not only observed between distant SNPs on the same chromosome, but also between SNPs on different chromosomes. Regions on chromosomes 3 and 4 that harbor disease resistance and monogermy loci showed strong genetic differentiation between the pollen and seed parent pools. Other regions, on chromosomes 8 and 9, for which no a priori information was available with respect to their contribution to the phenotype, still contributed to clustering of lines in the elite breeding material.     

Transformation of sugar beet cell suspension cultures

Summary A sugar beet transformation method was developed using particle bombardment of short-term suspension cultures of a breeding line FC607. Highly embryogenic suspension cultures derived from leaf callus were bombarded with the uidA (gusA) reporter gene under the control of either the osmotin or proteinase inhibitor II gene promoter, and the npt II selectable marker gene. Transient uidA expression was visualized as 500–4000 blue units per 200 mg of bombarded cells 2 d after bombardment. Stably-transformed calluses were recovered on both kanamycin and paromomycin media. The greatest number of GUS (+) calluses was obtained when 50 or 100 mgl⁻¹ of kanamycin was applied 2 d after transformation for 3–5 wk, followed by either no selection or reduced levels of the antibiotic. PCR analyses of the GUS (+) callus lines revealed the expected size fragment for uidA and npt II genes. Stable incorporation of the uidA gene into the genome was confirmed by Southern blot analyses. Several transformed embryos were detected by histochemical β-glucuronidase (GUS) staining.     

Translocation and the control of photosynthesis in sugar beet

Summary Evidence is presented that the size and activity of sinks for the products of photosynthesis exert a large measure of control over carbohydrate levels in the leaves of the sugar beet plant. Further data is presented to show that a correlation exists between the level carbohydrate in the leaf and the rate of photosynthesis. The rate of photosynthesis can thus be linked directly to the sinks of the plant by their ability to control rates of translocation from leaves.     

Role of free space in translocation in sugar beet

The involvement of the free space in phloem loading of sucrose was studied in sugar beet source leaves (Beta vulgaris, L.). Sucrose, supplied exogenously to the abraded upper surface of leaves at a concentration of 20 mm, was available for translocation at rates similar to those obtained with photosynthesis. The exogenous sucrose substituted as a source of translocate for assimilate derived from photosynthesis when the latter process was disrupted by plasmolysis of the leaf with 0.8 M mannitol. The mesophyll symplast was not completely disrupted by this treatment, however. Data from the sugar uptake experiments indicate that phloem loading can occur from the free space.     

Enhanced drought resistance in fructan-producing sugar beet

Fructans are soluble polymers of fructose that are produced by approximately 15 % of the flowering plant species. Production of bacterial fructans in tobacco has been shown previously to lead to improved biomass production under polyethylene glycol-mediated drought stress. Here, we used the same SacB gene from Bacillus subtilis to produce bacterial fructans in sugar beet (Beta vulgaris L.). The transgenic sugar beets accumulated fructans to low levels (max. 0.5 % of dry weight) in both roots and shoots. Two independent transgenic lines of fructan-producing sugar beets showed significantly better growth under drought stress than untransformed beets. Drought stressed fructan-producing plants attained higher total dry weights (+25–35 %) than wildtype sugar beet, due to higher biomass production of leaves (+30–33 %), storage roots (+16–33 %) and fibrous roots (+37–60 %). Under well-watered conditions, no significant differences were observed between the transgenic and wildtype beets. In conclusion, the introduction of fructan biosynthesis in transgenic plants is a promising approach to improve crop productivity under drought stress.     

Auxin-induced ethylene production as related to auxin metabolism in leaf discs of tobacco and sugar beet

Exogenously supplied indole-3-acetic acid (IAA) stimulated ethylene production in tobacco (Nicotiana glauca) leaf discs but not in those of sugar beet (Beta vulgaris L.). The stimulatory effect of IAA in tobacco was relatively small during the first 24 hours of incubation but became greater during the next 24 hours. It was found that leaf discs of these two species metabolized [1-¹⁴C]IAA quite differently. The rate of decarboxylation in sugar beet discs was much higher than in tobacco. The latter contained much less free IAA but a markedly higher level of IAA conjugates. The major conjugate in the sugar beet extracts was indole-3-acetylaspartic acid, whereas tobacco extracts contained mainly three polar IAA conjugates which were not found in the sugar beet extracts. The accumulation of the unidentified conjugates corresponded with the rise of ethylene production in the tobacco leaf discs. Reapplication of all the extracted IAA conjugates resulted in a great stimulation of ethylene production by tobacco leaf discs which was accompanied by decarboxylation of the IAA conjugates. The results suggest that in tobacco IAA-treated leaf discs the IAA conjugates could stimulate ethylene production by a slow release of free IAA. The inability of the exogenously supplied IAA to stimulate ethylene production in the sugar beet leaf discs was not due to a deficiency of free IAA within the tissue but rather to the lack of responsiveness of this tissue to IAA, probably because of an autoinhibitory mechanism existing in the sugar beet leaf discs.     

Optimizing harvest of corn stover fractions based on overall sugar yields following ammonia fiber expansion pretreatment and enzymatic hydrolysis

Background  

Corn stover composition changes considerably throughout the growing season and also varies between the various fractions of the plant. These differences can impact optimal pretreatment conditions, enzymatic digestibility and maximum achievable sugar yields in the process of converting lignocellulosics to ethanol. The goal of this project was to determine which combination of corn stover fractions provides the most benefit to the biorefinery in terms of sugar yields and to determine the preferential order in which fractions should be harvested. Ammonia fiber expansion (AFEX) pretreatment, followed by enzymatic hydrolysis, was performed on early and late harvest corn stover fractions (stem, leaf, husk and cob). Sugar yields were used to optimize scenarios for the selective harvest of corn stover assuming 70% or 30% collection of the total available stover.     

Ethylene is differentially regulated during sugar beet germination and affects early root growth in a dose-dependent manner

Main conclusion

By integrating molecular, biochemical, and physiological data, ethylene biosynthesis in sugar beet was shown to be differentially regulated, affecting root elongation in a concentration-dependent manner. There is a close relation between ethylene production and seedling growth of sugar beet (Beta vulgaris L.), yet the exact function of ethylene during this early developmental stage is still unclear. While ethylene is mostly considered to be a root growth inhibitor, we found that external 1-aminocyclopropane-1-carboxylic acid (ACC) regulates root growth in sugar beet in a concentration-dependent manner: low concentrations stimulate root growth while high concentrations inhibit root growth. These results reveal that ethylene action during root elongation is strongly concentration dependent. Furthermore our detailed study of ethylene biosynthesis kinetics revealed a very strict gene regulation pattern of ACC synthase (ACS) and ACC oxidase (ACO), in which ACS is the rate liming step during sugar beet seedling development.     

Colonization study of gfp-tagged Achromobacter marplatensis strain in sugar beet

This study details the introduction of a gfp marker into an endophytic bacterial strain (Achromobacter marplatensis strain 17, isolated from sugar beet) to monitor its colonization of sugar beet (Beta. vulgaris L.). Stability of the plasmid encoding the gfp was confirmed in vitro for at least 72 h of bacterial growth and after the colonization of tissues, under nonselective conditions. The colonization was observed using fluorescence microscopy and enumeration of culturable endophytes in inoculated sugar beet plants that grew for 10 or 20 days. gfp-Expressing strains were re-isolated from the inner tissues of surface-sterilized roots and stems of inoculated plants, and the survival of the Achromobacter marplatensis 17:gfp strain in plants 20 days after inoculation, even in the absence of selective pressure, suggests that it is good colonizer. These results also suggest that this strain could be a useful tool for the delivery of enzymes or other proteins into plants. In addition, the study highlights that sugar beet plants can be used effectively for detailed in vitro studies on the interactions between A. marplatensis strain 17 and its host, particularly if a gfp-tagged strain of the pathogen is used.     

Potassium nutrition and translocation in sugar beet

The effect of increased net foliar K⁺ accumulation on translocation of carbon was studied in sugar beet (Beta vulgaris, L. var. Klein E and US H20) plants. Net accumulation of recently absorbed K⁺ was studied by observing arrival of ⁴²K⁺ per unit area of leaf. Labeled K⁺ was added to give an initial concentration at 2 or 10 millimolar K⁺ in mineral nutrient solution. Because the newly arrived K⁺ constitutes a small part of the total leaf K⁺ in plants raised in 10 millimolar K⁺, export of ⁴²K⁺ by phloem was negligible over the 2- to 3-day period; consequently, accumulation is a measure of arrival in the xylem. In leaves from plants in 2 millimolar K⁺, export by the phloem was estimated to be of the same order as import by the xylem; K⁺ per area was observed to remain at a steady-state level. Increasing the supply of K⁺ to 10 millimolar caused arrival in the xylem to increase 2- to 3-fold; K⁺ per area increased gradually in the mature leaves. Neither net carbon exchange nor translocation of sugar increased in response to a faster rate of arrival of K⁺ over a 6- to 8-hour period. In the absence of short-term effects, it is suggested that K⁺-promoted increase in synthetic metabolism may be the basis of the increased carbon assimilation and translocation in plants supplied with an above-minimal level of K⁺.     

黄土高原甜菜叶片生长特性及其对块根产量、含糖量的影响

通过对旱地甜菜叶片生长特性及摘除不同叶组对块根产量,含糖量,显微结构的影响研究,结果表明：甜菜第10-20片叶的叶龄最长,积温最高,是甜菜的主要功能叶;甜菜从第20片叶期起进入块根,糖份增长期,从第55叶期起进入糖份积累期;摘除不同叶组的叶片对甜菜块根产量,含糖量及显微结构均有不同程度降低作用,摘除前期叶组对甜菜块根产量,产糖量,根径减幅较大,摘除后期叶组对块根含糖量,维管束环数,维管束环密度减幅较大;摘除第1-30片叶对甜菜影响最大。     

Evidence for active Phloem loading in the minor veins of sugar beet

Phloem loading in source leaves of sugar beet (Beta vulgaris, L.) was studied to determine the extent of dependence on energy metabolism and the involvement of a carrier system. Dinitrophenol at a concentration of 4 mm uncoupled respiration, lowered source leaf ATP to approximately 40% of the level in the control leaf and inhibited translocation of exogenously supplied ¹⁴C-sucrose to approximately 20% of the control. Dinitrophenol at a concentration of 8 mm inhibited rather than promoted CO₂ production, indicating a mechanism of inhibition other than uncoupling of respiration. The 8 mm dinitrophenol also reduced ATP to approximately 40% of the level in the control source leaf and reduced translocation of exogenous sucrose to approximately 10% of the control. Application of 4 mm ATP to an untreated source leaf promoted the translocation rate by approximately 80% over the control, while in leaves treated with 4 mm dinitrophenol, 4 mm ATP restored translocation to the control level. No recovery of translocation was observed when ATP was applied to leaves treated with 8 mm dinitrophenol. The results indicate an energy-requiring process for both phloem loading and translocation in the source leaf.     

Localization of QTLs for tolerance to Cercospora beticola on sugar beet linkage groups

We present a new linkage map for sugar beet (Beta vulgaris) which has been developed using a population segregating for genetic factors that confer tolerance to the leaf spot fungus (Cercospora beticola), the causal factor of leaf spot disease in sugar beet). In the F₂ population studied, a subset of 36 RFLP probes, mapping on eight out of the nine linkage groups of sugar beet, provided the anchor markers to assign chromosomes. A total of 224 markers, including RFLPs, AFLPs, SCARs and microsatellites, were mapped. Estimates of leaf damage in F₂ and test-cross families were repeated at different stages of plant development. Each set of data was analysed as such. An average estimate was also considered. QTLs with highly significant LOD scores revealed both by the F₂ and test-cross analyses were localized on linkage groups 2, 6 and 9. Linkage groups 4 and 5 gave a clear indication of the presence of a QTL only when F₂ data were considered. One highly significant QTL with a LOD of 16.0 was revealed only by the data obtained under conditions of artificial inoculation. This QTL maps at position 90 on chromosome 3. Received: 3 February 1999 / Accepted: 20 February 1999     

Effect of water deficit on respiration of conducting bundles in leaf petioles of sugar beet

Isolated fibrovascular bundles from source leaf petioles of sugar beet (Beta vulgaris L.) and hog-weed (Heracleum sosnovskyi L.) were used to study the influence of long-term drought on the oxygen uptake rate and activities of mitochondrial oxidases, i.e., cytochrome oxidase and salicylhydroxamic acid-sensitive alternative oxidase (AO). Under normal soil moisture content (70% of full water-retaining capacity, WRC), the oxygen uptake by sugar beet conducting bundles was characterized by a high rate (> 700 μl O₂/(g fr wt h)) and by distinct cytochrome oxidase-dependent manner of terminal oxidation (up to 80% inhibition of respiration in the presence of 0.5 mM KCN). After long-term water deficit (40% of WRC), the bundle respiration proceeded at nearly the same rate but featured an elevated resistance to cyanide. At early drought stage (10 days), a decrease in the activity of cytochrome-mediated oxidation pathway was largely counterbalanced by activation of mitochondrial AO, whereas long-term dehydration of plants was accompanied by activation of additional oxidative systems insensitive to both KCN and SHAM. Similar but even more pronounced changes in activities of terminal oxidases were discovered in conducting bundles of wild-grown hogweed plants exposed to long-term natural drought. It is supposed that the suppression of cytochrome-mediated oxidation coupled with ATP synthesis in the cells of sugar beet source leaves impedes the translocation of assimilates and their accumulation in the taproot, which represents an important factor of drastic decrease in the yield of this agricultural crop under conditions of water deficit.