Time course and amplitude of DNA methylation in the shoot apical meristem are critical points for bolting induction in sugar beet and bolting tolerance between genotypes

An epigenetic control of vernalization has been demonstrated in annual plants such as Arabidopsis and cereals, but the situation remains unclear in biennial plants such as sugar beet that has an absolute requirement for vernalization. The role of DNA methylation in flowering induction and the identification of corresponding target loci also need to be clarified. In this context, sugar beet (Beta vulgaris altissima) genotypes differing in bolting tolerance were submitted to various bolting conditions such as different temperatures and/or methylating drugs. DNA hypomethylating treatment was not sufficient to induce bolting while DNA hypermethylation treatment inhibits and delays bolting. Vernalizing and devernalizing temperatures were shown to affect bolting as well as DNA methylation levels in the shoot apical meristem. In addition, a negative correlation was established between bolting and DNA methylation. Genotypes considered as resistant or sensitive to bolting could also be distinguished by their DNA methylation levels. Finally, sugar beet homologues of the Arabidopsis vernalization genes FLC and VIN3 exhibited distinct DNA methylation marks during vernalization independently to the variations of global DNA methylation. These vernalization genes also displayed differences in mRNA accumulation and methylation profiles between genotypes resistant or sensitive to bolting. Taken together, the data suggest that the time course and amplitude of DNA methylation variations are critical points for the induction of sugar beet bolting and represent an epigenetic component of the genotypic bolting tolerance, opening up new perspectives for sugar beet breeding.     

Fractionation by centrifugation of leaf proteins from varieties and cultivars of Helianthus annuus and Helianthus debilis as a function of pH and temperature

In order to select plant material for obtaining a high proportion of chlorophyll-free protein from Helianthus species by heat fractionation, leaf extracts from 11 field grown cultivars of H. annuus and H. debilis were investigated. In addition, press juices from H. annuus cv. Kinesisk were supplied with salts or urea before heat treatment in order to increase the proportion of chlorophyll-free protein during heat fractionation. The extracts were adjusted to pH 5.0, 6.0 and 8.0; then left for 20 min at 20°C, 50°C or 60°C and centrifuged at 2,500 g. The highest percentage chlorophyll-free protein of the total protein in the extract, more than 10%, was obtained for H. debilis cv. fl.pl. Sun Gold, H. annuus cv. Giganta, H. annuus cv. uniflorus and H. debilis cv. Stella, at pH 6.0 and 50°C. The low percentage chlorophyll-free protein obtained could be explained by the fact that a considerable part of the chlorophyll-free protein sedimented at original pH, low temperature and low centrifugation speed. This sedimentation at low temperature was probably due to interactions of phenolics. Besides, if the chlorophyll-associated membranes were highly fragmented during the disintegration of the plant material, the high temperature required to precipitate them completely led to further aggregation and thus to loss of chlorophyll-free proteins. Salts or urea present during heat treatment (pH 6.0/55°C for 20 min) did not considerably increase the proportion of chlorophyll-free proteins obtained.     

THE EFFECT OF MANURES ON THE BOLTING OF THE BEET PLANT

In sugar beet, the effect on bolting of additions of potash manures, of superphosphate, and of the mixture of these was not significant, whether applied at the time of sowing or previously. Dung applied in the previous autumn slightly increased the number of plants which went to seed, and when applied just before sowing it had a more marked effect. An amount of sulphate of ammonia which would have approximately the same stimulating effect as the dung gave a slightly larger amount of bolting.
On red beetroot, it was possible to compare larger and smaller dressings of various coarse organic manures, and of each of these with and without dressings of sulphate of ammonia. In all cases the sulphate of ammonia and each of the organic manures largely increased the amount of bolting, and a doubling of the organic manure dressing or the addition of sulphate of ammonia to it caused a further increase. Sewage sludge gave an abnormally large amount of bolting, but it is doubtful whether this was due to the larger amount of organic matter and nitrogen applied in this manure. In general, any manurial addition causing more vigorous growth leads to an increase in bolting. There is a very large variation in the amount of running to seed in the crop of red beetroot from different parts of the same field, but the effect of the manures on the proportion which bolted was not widely different.
The general question as to the reason why beet plants should bolt more in one season than in another is discussed, and it is suggested that a check to the plants in an early stage, whether caused by dryness or waterlogging, or by low temperature, followed by a vigorous growing period may have something to do with the matter. This would agree with the experiences recorded in the present paper.     

Seed Germination and Bolting in Red Beet as Affected by Parent Plant Environment

Low temperature and long day regimes applied during seed development and maturation of red beet (Beta rulgaris var. rubra L.) increased the proportion of empty seed balls and reduced the germination ability of normally developed seeds. Low seed development temperatures also greatly increased the bolting susceptibility of seedlings grown at low temperatures or temperatures which are marginal for bolting, but were not able to bring about bolting under conditions which are not themselves inducive to flowering. In a field experiment a significant reduction of the root tuber yield was associated with these effects.     

Proteome analysis of sugar beet (Beta vulgaris L.) elucidates constitutive adaptation during the first phase of salt stress

Salinity is one of the major stress factors responsible for growth reduction of most of the higher plants. In this study, the effect of salt stress on protein pattern in shoots and roots of sugar beet (Beta vulgaris L.) was examined. Sugar beet plants were grown in hydroponics under control and 125 mM salt treatments. A significant growth reduction of shoots and roots was observed. The changes in protein expression, caused by salinity, were monitored using two-dimensional gel-electrophoresis. Most of the detected proteins in sugar beet showed stability under salt stress. The statistical analysis of detected proteins showed that the expression of only six proteins from shoots and three proteins from roots were significantly altered. At this stage, the significantly changed protein expressions we detected could not be attributed to sugar beet adaptation under salt stress. However, unchanged membrane bound proteins under salt stress did reveal the constitutive adaptation of sugar beet to salt stress at the plasma membrane level.     

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 approach to identify cold-induced low-abundant proteins in rice leaf

A proteomic approach has been adopted to investigate the low-abundant proteins in rice leaf in response to cold stress. Rice seedlings were exposed to different temperatures, such as 5 or 10 degrees C, and samples were collected after different time course. To eliminate the high-abundant proteins in leaf tissues such as ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), proteins were fractionated by polyethylene glycol (PEG). The elimination of Rubisco from the protein samples was confirmed by Western blot analysis. The PEG fractionated protein samples were separated by 2-DE and visualized by silver or CBB staining. A total 12 up-regulated protein spots were identified using the analysis of MALDI-TOF mass spectrometry or ESI MS/MS. We identified some novel proteins such as cysteine proteinase, thioredoxin peroxidase, a RING zinc finger protein-like, drought-inducible late embryogenesis abundant, and a fibrillin-like protein that had not yet been reported in the earlier reports on cold proteomic analysis. The identification of some novel low-abundant proteins in response to cold stress may provide a new homeostasis to develop enhanced cold tolerance transgenic plants. Thus, we propose that a PEG fractionation system can be used as an influential protein extraction method from the leaf samples, which can lead to knowledge of the expression pattern of low-abundant proteins in response to various biotic or abiotic stresses.     

Comparison of Physiology and Anatomy of Seedlings and Regenerants of Sugar Beet

Whole sugar beet (Beta vulgaris L. cv. Ras poly) plants were grown in the greenhouse from the same seed stock used for an in vitro shoot tip culture. In vitro produced sugar beet plants exhibited a high content of chlorophylls a and b, carotene, and total and soluble sugars. On the other hand, total protein content of in vivo plants was higher than that of in vitro plants. No differences were found by SDS-PAGE analysis in the nature and contents of soluble proteins of in vitro propagated plants and greenhouse-grown plants. Surfaces of epidermal cells were larger and palisade and spongy paranchyma tissues were thicker in leaves of regenerants than in leaves of seedlings. Vascular tissues in leaf petioles in regenerants were flat and more differentiated than in seedlings. Closed and undeveloped stomata were found on the abaxial leaf surface of regenerants, whereas in seedlings the stomata were open. This revised version was published online in July 2006 with corrections to the Cover Date.     

Testing the optimal defense theory and the growth-differentiation balance hypothesis in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Two prominent theories proposed to explain patterns of chemical defense expression in plants are the optimal defense theory (ODT) and the growth-differentiation balance hypothesis (GDBH). The ODT predicts that plant parts with high fitness value will be highly defended, and the GDBH predicts that slow growing plant parts will have more resources available for defense and thus will have higher defense levels than faster growing tissues. We examined growth rate, fitness value, and defense protein levels in leaves of a wild and lab ecotype of Arabidopsis thaliana to address whether patterns of defense protein expression in this plant conform to predictions of either the ODT or the GDBH. We divided leaves of A. thaliana into six leaf classes based on three developmental stages: vegetative, bolting, and flowering; with two leaf ages at each stage: young and old. We assessed the fitness value of leaves by determining the impact of the removal of each leaf class on total seed production and germination rates. Although A. thaliana was highly tolerant to defoliation, young leaves were more valuable than old in general, and young leaves on bolting plants were the most valuable leaf class in particular. Young leaves on vegetative plants grew fastest in both ecotypes, while old leaves on bolting and flowering plants grew slowest. Finally, defense levels were assessed in each leaf class by quantifying the constitutive and inducible expression of four defense-related proteins. Expression of guaiacol peroxidase and chitinase activity conformed largely to GDBH predictions. Expression of trypsin inhibitor and polyphenoloxidase activity varied by leaf class and treatment, but conformed to neither GDBH nor ODT predictions.     

Over-expression of calcium-dependent protein kinase 13 and calreticulin interacting protein 1 confers cold tolerance on rice plants

Calcium is a ubiquitous signaling molecule and changes in cytosolic calcium concentration are involved in plant responses to various stimuli. The rice calcium-dependent protein kinase 13 (CDPK13) and calreticulin interacting protein 1 (CRTintP1) have previously been reported to be involved in cold stress response in rice. In this study, rice lines transformed with sense CDPK13 or CRTintP1 constructs were produced and used to investigate the function of these proteins. When the plants were incubated at 5°C for 3 days, leaf blades of both the sense transgenic and vector control rice plants became wilted and curled. When the plants were transferred back to non-stress conditions after cold treatment, the leaf blades died, but the sheaths remained green in the sense transgenic rice plants. Expression of CDPK13 or CRTintP1 was further examined in several rice varieties including cold-tolerant rice varieties. Accumulation of these proteins in the cold-tolerant rice variety was higher than that in rice varieties that are intermediate in their cold tolerance. To examine whether over-expression of CDPK13 and CRTintP1 would have any effect on the proteins or not, sense transgenic rice plants were analyzed using proteomics. The 2D-PAGE profiles of proteins from the vector control were compared with those of the sense transgenic rice plants. Two of the proteins that differed between these lines were calreticulins. The results suggest that CDPK13, calreticulin and CRTintP1 might be important signaling components for response to cold stress in rice.     

Fast-Growing, Aerobic, Heterotrophic Bacteria from the Rhizosphere of Young Sugar Beet Plants

Fast-growing, aerobic, heterotrophic bacteria from the root surface of young sugar beet plants were inventoried. Isolation of the most abundant bacteria from the root surface of each of 1,100 plants between the second and tenth leaf stage yielded 5,600 isolates. These plants originated from different fields in Belgium and Spain. All isolates were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total cellular proteins. Comparison of protein fingerprints allowed us to inventory the bacteria of individual plants of different fields or leaf stages and to analyze the composition and variability of the rhizobacterial population of young sugar beet plants. Each field harbored a specific population of bacteria which showed a highly hierarchic structure. A small number of bacteria occurring frequently at high densities dominated in each field. The major bacteria were identified as Pseudomonas fluorescens, Xanthomonas maltophilia, Pseudomonas paucimobilis, and Phyllobacterium sp. The former three species showed a high genetic variability as they were represented by different protein fingerprint types on the same or different fields or leaf stages. Twinspan analysis and relative abundance plots showed that the structure and composition of the bacterial populations varied strongly over time. Pseudomonads were typically early colonizers which were later replaced by X. maltophilia or Phyllobacterium sp.     

超表达AVP1基因提高甜菜的耐低磷和耐盐抗旱性

为探讨H⁺-焦磷酸酶编码基因对甜菜磷吸收和抗性的影响,实现优良基因在甜菜基因工程中的利用,研究在甜菜中超表达拟南芥液泡膜H⁺-焦磷酸酶编码基因AVP1,对转基因甜菜分析其耐低磷、耐盐性和抗旱性。结果显示,AVP1基因在甜菜植株的叶片和块根中表达,且在逆境胁迫下增强表达量响应胁迫;低磷处理条件下,转基因甜菜与野生型甜菜相比具有更高的含磷量,可提高甜菜对磷的吸收利用效率;干旱、盐胁迫处理条件下,AVP1基因在转基因甜菜中显著上升,在盐胁迫或干旱处理条件下,转基因植株的生长受抑程度相对较轻。随着盐和干旱胁迫的加剧,转基因植株体内MDA含量与野生型植株相比较低而脯氨酸含量显著增加,AVP1基因可通过减轻逆境对甜菜细胞膜的损伤及提高甜菜细胞的渗透调节能力,进而增强甜菜对高盐和干旱胁迫的抗性。     

Antifreeze protein accumulation in freezing-tolerant cereals

Freezing-tolerant plants withstand extracellular ice formation at subzero temperatures. Previous studies have shown that winter rye ( Secale cereale L.) accumulates proteins in the leaf apoplast during cold acclimation that have antifreeze properties and are similar to pathogenesis-related proteins. To determine whether the accumulation of these antifreeze proteins is common among herbaceous plants, we assayed antifreeze activity and total protein content in leaf apoplastic extracts from a number of species grown at low temperature, including both monocotyledons (winter and spring rye, winter and spring wheat, winter barley, spring oats, maize) and dicotyledons (spinach, winter and spring oilseed rape [canola], kale, tobacco). Apoplastic polypeptides were also separated by SDS-PAGE and immunoblotted to determine whether plants generally respond to low temperature by accumulating pathogenesis-related proteins. Our results showed that significant levels of antifreeze activity were present only in the apoplast of freezing-tolerant monocotyledons after cold acclimation at 5/2⁰C. Moreover, only a closely related group of plants, rye, wheat and barley, accumulated antifreeze proteins similar to pathogenesis-related proteins during cold acclimation. The results indicate that the accumulation of antifreeze proteins is a specific response that may be important in the freezing tolerance of some plants, rather than a general response of all plants to low temperature stress.     

Evaluation of chloroform/methanol extraction to facilitate the study of membrane proteins of non-model plants

Membrane proteins are of great interest to plant physiologists because of their important function in many physiological processes. However, their study is hampered by their low abundance and poor solubility in aqueous buffers. Proteomics studies of non-model plants are generally restricted to gel-based methods. Unfortunately, all gel-based techniques for membrane proteomics lack resolving power. Therefore, a very stringent enrichment method is needed before protein separation. In this study, protein extraction in a mixture of chloroform and methanol in combination with gel electrophoresis is evaluated as a method to study membrane proteins in non-model plants. Benefits as well as disadvantages of the method are discussed. To demonstrate the pitfalls of working with non-model plants and to give a proof of principle, the method was first applied to whole leaves of the model plant Arabidopsis. Subsequently, a comparison with proteins extracted from leaves of the non-model plant, banana, was made. To estimate the tissue and organelle specificity of the method, it was also applied on banana meristems. Abundant membrane or lipid-associated proteins could be identified in both tissues, with the leaf extract yielding a higher number of membrane proteins.     

Effects of low temperature, high salinity and exogenous ABA on the synthesis of two chloroplastic drought-induced proteins in Solanum tuberosum

Two chloroplastic proteins of 32 and 34 kDa were previously shown to be substantially synthesized in response to a progressive water deficit in whole Solanum tuberosum plants (G. Pruvot, S. Cuiné, N. Gault, G. Peltier and P. Rey, unpublished data; G. Pruvot, S. Cuiné, G. Peltier and P. Rey. 1996. Planta 198: 471–479). These chloroplastic drought-induced stress proteins, named CDSP 32 and CDSP 34, accumulated in the stroma and in the thylakoids, respectively. In this study, we investigated the effects of low temperature and high salinity on the synthesis of the CDSP proteins. Whereas the CDSP 32 synthesis was not modified in response to a cold treatment, an increased synthesis of CDSP 32 was observed in salt-stressed plants, resulting in accumulation of the protein. The thylakoid CDSP 34 protein exhibited enhanced synthesis and substantial accumulation in response to cold and high salinity. A significant increase in the leaf abscisic acid content (at least 2.5-fold) was measured in plants subjected to water deficit, high salinity or low temperature. The contribution of ABA to the synthesis of the two proteins was investigated by spraying well-watered plants with a 100 μ M / ABA solution for 15 days. This treatment resulted in a 15-fold increase in the leaf ABA content. Whereas synthesis of the CDSP 32 protein was not affected by exogenous ABA, synthesis of the CDSP 34 protein was substantially enhanced. Based on these results, we conclude that ABA likely mediates the increased synthesis of CDSP 34 upon drought, low temperature and high salinity and suggest that another signal, likely related to high osmolarity, is involved in the induction of CDSP 32 synthesis.     

Proteome analysis of sugar beet leaves under drought stress

Drought is one of the major factors limiting the yield of sugar beet (Beta vulgaris L.). The identification of candidate genes for marker-assisted selection (MAS) could greatly improve the efficiency of breeding for increased drought tolerance. Drought-induced changes in the proteome could highlight important genes. Two genotypes of sugar beet (7112 and 7219-P.69) differing in genetic background were cultivated in the field. A line-source sprinkler irrigation system was used to apply irrigated and water deficit treatments beginning at the four-leaf stage. At 157 days after sowing, leaf samples were collected from well-watered and drought-stressed plants for protein extraction and to measure shoot biomass and leaf relative water content. Changes induced in leaf proteins were studied by two-dimensional gel electrophoresis and quantitatively analyzed using image analysis software. Out of more than 500 protein spots reproducibly detected and analyzed, 79 spots showed significant changes under drought. Some proteins showed genotype-specific patterns of up- or downregulation in response to drought. Twenty protein spots were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), leading to identification of Rubisco and 11 other proteins involved in redox regulation, oxidative stress, signal transduction, and chaperone activities. Some of these proteins could contribute a physiological advantage under drought, making them potential targets for MAS.     

Apparent Inhibition of Chloroplast Protein Import by Cold Temperatures Is Due to Energetic Considerations Not Membrane Fluidity

The transport of proteins across virtually all types of biological membranes has been reported to be inhibited by low temperatures. Paradoxically, plants are able to acclimate to growth at temperatures below which protein import into chloroplasts is known to be blocked. In examining this incongruity, we made a number of unexpected observations. First, chloroplasts isolated from plants grown at 7/1[deg]C in light/dark and from plants grown at 25[deg]C were able to import proteins with the same efficiency over a temperature range from 5 to 21[deg]C, indicating that no functional adaptation had taken place in the protein import machinery of chloroplasts in these cold-grown plants. Second, chloroplasts from warm-grown plants were able to take up proteins at temperatures as low as 4[deg]C provided that they were illuminated. We determined that light mediates the import process at 5[deg]C by driving ATP synthesis in the stroma, the site of its utilization during protein transport. Direct measurement of the envelope phase transition temperature as well as the activity of the ATP/ADP translocator in the inner envelope membrane at 5 and 25[deg]C demonstrated that the cold block of protein import into chloroplasts observed in vitro is due primarily to energetic considerations and not to decreased membrane fluidity.     

Fractionation of leaf proteins by differential centrifugation and gel filtration

The quantity and quality of fractionated leaf proteins from different plant species were investigated. Leaf extracts (pH range 7.0–8.3) were prepared on a laboratory scale from greenhouse cultivated plants. The proteins were fractionated by differential centrifugation followed by gel filtration. Amaranthus caudatus and Chenopodium quinoa seem favourable for production of non-green leaf protein concentrates, since the water-soluble protein comprised nearly 50% of the extracted protein. The chlorophyll-associated protein in fresh extracts from these species was, however, difficult to sediment. A practical separation of chlorophyll-associated protein from the chlorophyll-free proteins will require some kind of pre-aggregation of the chlorophyll-associated proteins. In extracts from Dactylis glomerata, Lolium perenne and Vicia sativa a considerable proportion of the extracted protein sedimented rapidly, but at least for the grasses some of the chlorophyll-associated protein remained in the supernatants even after centrifugation at 30 000 g for 180 min. Practical separation of all the chlorophyll-associated protein from the extract requires some kind of pre-aggregation of the chlorophyll-associated proteins even for these species. The situation was similar for extracts from Helianthus annuus, but a very high percentage of the total chlorophyll-associated proteins could be sedimented at low centrifugation speed. Brassica oleracea was the most suitable species for the removal of the chlorophyll-associated proteins by centrifugation alone. Moreover, the proportion of chlorophyll-free protein in the extracts was relatively high, more than 40%. The in vitro digestibility of membrane-bound protein fractions was high for species with co-aggregation of water-soluble protein and membrane-bound protein. Causes underlying the different distributions of the chlorophyll-associated and chlorophyll-free proteins and the reason for the different size of the chlorophyll-containing particles are discussed.     

The Response of Leaf Water Potential and Crassulacean Acid Metabolism to Prolonged Drought in Sedum rubrotinctum

Plants of Sedum rubrotinctum R. T. Clausen were studied in a green-house over a 2-year period without watering. Only the apical leaves survived and were turgid at the end of the experiment. The midday leaf water potential of these apical leaves was −1.20 megapascals, while the leaf water potential of comparable leaves on well-watered control plants was −0.20 megapascals. The unwatered plants appear to have maintained turgor by means of an osmotic adjustment. After 2 years without water the plants no longer exhibited a nocturnal accumulation of titratable acidity. However, the daytime levels of titratable acidity of the unwatered plants were more than 2-fold greater than the levels in well-watered control plants. Well-watered plants of S. rubrotinctum exhibited seasonal shifts in biomass stble carbon isotope ratios, indicating a greater proportion of day versus night CO₂ uptake in the winter than in the summer. The imposition of water stress prevented the expression of this seasonal rhythm and restricted the plants to dark CO₂ uptake.