Integrative transcriptomics reveals genotypic impact on sugar beet storability.

KEY MESSAGE: An integrative comparative transcriptomic approach on six sugar beet varieties showing different amount of sucrose loss during storage revealed genotype-specific main driver genes and pathways characterizing storability. Sugar beet is next to sugar cane one of the most important sugar crops accounting for about 15% of the sucrose produced worldwide. Since its processing is increasingly centralized, storage of beet roots over an extended time has become necessary. Sucrose loss during storage is a major concern for the sugar industry because the accumulation of invert sugar and byproducts severely affect sucrose manufacturing. This loss is mainly due to ongoing respiration, but changes in cell wall composition and pathogen infestation also contribute. While some varieties can cope better during storage, the underlying molecular mechanisms are currently undiscovered. We applied integrative transcriptomics on six varieties exhibiting different levels of sucrose loss during storage. Already prior to storage, well storable varieties were characterized by a higher number of parenchyma cells, a smaller cell area, and a thinner periderm. Supporting these findings, transcriptomics identified changes in genes involved in cell wall modifications. After 13 weeks of storage, over 900 differentially expressed genes were detected between well and badly storable varieties, mainly in the category of defense response but also in carbohydrate metabolism and the phenylpropanoid pathway. These findings were confirmed by gene co-expression network analysis where hub genes were identified as main drivers of invert sugar accumulation and sucrose loss. Our data provide insight into transcriptional changes in sugar beet roots during storage resulting in the characterization of key pathways and hub genes that might be further used as markers to improve pathogen resistance and storage properties.

Statistical shape analysis of tap roots: a methodological case study on laser scanned sugar beets.

BACKGROUND: The efficient and robust statistical analysis of the shape of plant organs of different cultivars is an important investigation issue in plant breeding and enables a robust cultivar description within the breeding progress. Laserscanning is a highly accurate and high resolution technique to acquire the 3D shape of plant surfaces. The computation of a shape based principal component analysis (PCA) built on concepts from continuum mechanics has proven to be an effective tool for a qualitative and quantitative shape examination. RESULTS: The shape based PCA was used for a statistical analysis of 140 sugar beet roots of different cultivars. The calculation of the mean sugar beet root shape and the description of the main variations was possible. Furthermore, unknown and individual tap roots could be attributed to their cultivar by means of a robust classification tool based on the PCA results. CONCLUSION: The method demonstrates that it is possible to identify principal modes of root shape variations automatically and to quantify associated variances out of laserscanned 3D sugar beet tap root models. The introduced approach is not limited to the 3D shape description by laser scanning. A transfer to 3D MRI or radar data is also conceivable.

Salt stress vs. salt shock - the case of sugar beet and its halophytic ancestor.

BACKGROUND: Sugar beet is a highly salt-tolerant crop. However, its ability to withstand high salinity is reduced compared to sea beet, a wild ancestor of all beet crops. The aim of this study was to investigate transcriptional patterns associated with physiological, cytological and biochemical mechanisms involved in salt response in these closely related subspecies. Salt acclimation strategies were assessed in plants subjected to either gradually increasing salt levels (salt-stress) or in excised leaves, exposed instantly to salinity (salt-shock). RESULT: The majority of DEGs was down-regulated under stress, which may lead to certain aspects of metabolism being reduced in this treatment, as exemplified by lowered transpiration and photosynthesis. This effect was more pronounced in sugar beet. Additionally, sugar beet, but not sea beet, growth was restricted. Silencing of genes encoding numerous transcription factors and signaling proteins was observed, concomitantly with the up-regulation of lipid transfer protein-encoding genes and those coding for NRTs. Bark storage protein genes were up-regulated in sugar beet to the level observed in unstressed sea beet. Osmotic adjustment, manifested by increased water and proline content, occurred in salt-shocked leaves of both genotypes, due to the concerted activation of genes encoding aquaporins, ion channels and osmoprotectants synthesizing enzymes. bHLH137 was the only TF-encoding gene induced by salt in a dose-dependent manner irrespective of the mode of salt treatment. Moreover, the incidence of bHLH-binding motives in promoter regions of salinity-regulated genes was significantly greater than in non-regulated ones. CONCLUSIONS: Maintaining homeostasis under salt stress requires deeper transcriptomic changes in the sugar beet than in the sea beet. In both genotypes salt shock elicits greater transcriptomic changes than stress and it results in greater number of up-regulated genes compared to the latter. NRTs and bark storage protein may play a yet undefined role in salt stress-acclimation in beet. bHLH is a putative regulator of salt response in beet leaves and a promising candidate for further studies.

Preharvest application of ethephon and postharvest UV-B radiation improve quality traits of beetroot (Beta vulgaris L. ssp. vulgaris) as source of colourant.

BACKGROUND: Betanins have become excellent replacers for artificial red-purple food colourants. Red beet (Beta vulgaris L. spp. vulgaris) known as beetroot, is a rich source of betalains, which major forms are betanin (red to purple) and vulgaxanthin (yellow). Betalains and phenolic compounds are secondary metabolites, accumulation of which is often triggered by elicitors during plant stress responses. In the present study, pre-harvest applications of ethephon (an ethylene-releasing compound) and postharvest UV-B radiation were tested as elicitors of betalains and phenolic compounds in two beetroot cultivars. Their effects on quality parameters were investigated, and the expression of biosynthetic betalain genes in response to ethephon was determined. RESULTS: Ethephon was applied as foliar spray during the growth of beetroot, resulting in increased betanin (22.5%) and decreased soluble solids contents (9.4%), without detrimental effects on beetroot yield. The most rapid accumulation rate for betanin and soluble solids was observed between 3 and 6 weeks after sowing in both untreated and ethephon-treated beetroots. Overall, the expression of the betalain biosynthetic genes (CYP76AD1, CYP76AD5, CYP76AD6 and DODA1), determining the formation of both betanin and vulgaxanthin, increased in response to ethephon treatment, as did the expression of the betalain pathway activator BvMYB1. In the postharvest environment, the use of short-term UV-B radiation (1.23 kJ m- 2) followed by storages for 3 and 7 days at 15 °C resulted in increased betanin to vulgaxanthin ratio (51%) and phenolic content (15%). CONCLUSIONS: The results of this study provide novel strategies to improve key profitability traits in betalain production. High betanin concentration and high betanin to vulgaxanthin ratio increase the commercial value of the colourant product. In addition, lowering soluble solids levels facilitates higher concentration of beetroot colour during processing. Moreover, we show that enhanced betanin content in ethephon-treated beetroots is linked to increased expression of betalain biosynthetic genes.

DigR: a generic model and its open source simulation software to mimic three-dimensional root-system architecture diversity.

Background and Aims: Many studies exist in the literature dealing with mathematical representations of root systems, categorized, for example, as pure structure description, partial derivative equations or functional-structural plant models. However, in these studies, root architecture modelling has seldom been carried out at the organ level with the inclusion of environmental influences that can be integrated into a whole plant characterization. Methods: We have conducted a multidisciplinary study on root systems including field observations, architectural analysis, and formal and mathematical modelling. This integrative and coherent approach leads to a generic model (DigR) and its software simulator. Architecture analysis applied to root systems helps at root type classification and architectural unit design for each species. Roots belonging to a particular type share dynamic and morphological characteristics which consist of topological and geometric features. The DigR simulator is integrated into the Xplo environment, with a user interface to input parameter values and make output ready for dynamic 3-D visualization, statistical analysis and saving to standard formats. DigR is simulated in a quasi-parallel computing algorithm and may be used either as a standalone tool or integrated into other simulation platforms. The software is open-source and free to download at http://amapstudio.cirad.fr/soft/xplo/download. Key Results: DigR is based on three key points: (1) a root-system architectural analysis, (2) root type classification and modelling and (3) a restricted set of 23 root type parameters with flexible values indexed in terms of root position. Genericity and botanical accuracy of the model is demonstrated for growth, branching, mortality and reiteration processes, and for different root architectures. Plugin examples demonstrate the model's versatility at simulating plastic responses to environmental constraints. Outputs of the model include diverse root system structures such as tap-root, fasciculate, tuberous, nodulated and clustered root systems. Conclusions: DigR is based on plant architecture analysis which leads to specific root type classification and organization that are directly linked to field measurements. The open source simulator of the model has been included within a friendly user environment. DigR accuracy and versatility are demonstrated for growth simulations of complex root systems for both annual and perennial plants.

Effect of Cuscuta campestris parasitism on the physiological and anatomical changes in untreated and herbicide-treated sugar beet.

The effects of field dodder on physiological and anatomical processes in untreated sugar beet plants and the effects of propyzamide on field dodder were examined under controlled conditions. The experiment included the following variants: N-noninfested sugar beet plants (control); I - infested sugar beet plants (untreated), and infested plants treated with propyzamide (1500 g a.i. ha-1 (T1) and 2000 g a.i. ha-1(T2)). The following parameters were checked: physiological-pigment contents (chlorophyll a, chlorophyll b, total carotenoids); anatomical -leaf parameters: thickness of epidermis, parenchyma and spongy tissue, mesophyll and underside leaf epidermis, and diameter of bundle sheath cells; petiole parameters: diameter of tracheid, petiole hydraulic conductance, xylem surface, phloem cell diameter and phloem area in sugar beet plants. A conventional paraffin wax method was used to prepare the samples for microscopy. Pigment contents were measured spectrophotometrically after methanol extraction. All parameters were measured: prior to herbicide application (0 assessment), then 7, 14, 21, 28 and 35 days after application (DAA). Field dodder was found to affect the pigment contents in untreated sugar beet plants, causing significant reductions. Conversely, reduction in the treated plants decreased 27% to 4% for chlorophyll a, from 21% to 5% for chlorophyll b, and from 28% to 5% for carotenoids (T1). Also, in treatment T2, reduction decreased in infested and treated plants from 19% to 2% for chlorophyll a, from 21% to 2% for chlorophyll b, from 23% to 3% for carotenoids and stimulation of 1% and 2% was observed 28 and 35 DAA, respectively. Plants infested (untreated) by field dodder had lower values of most anatomical parameters, compared to noninfested plants. The measured anatomical parameters of sugar beet leaves and petiole had significantly higher values in noninfested plants and plants treated with propyzamide than in untreated plants. Also, the results showed that propyzamide is an adequate herbicide for control of field dodder at the stage of early infestation.

Growing larger with domestication: a matter of physiology, morphology or allocation?

Domestication might affect plant size. We investigated whether herbaceous crops are larger than their wild progenitors, and the traits that influence size variation. We grew six crop plants and their wild progenitors under common garden conditions. We measured the aboveground biomass gain by individual plants during the vegetative stage. We then tested whether photosynthesis rate, biomass allocation to leaves, leaf size and specific leaf area (SLA) accounted for variations in whole-plant photosynthesis, and ultimately in aboveground biomass. Despite variations among crops, domestication generally increased the aboveground biomass (average effect +1.38, Cohen's d effect size). Domesticated plants invested less in leaves and more in stems than their wild progenitors. Photosynthesis rates remained similar after domestication. Variations in whole-plant C gains could not be explained by changes in leaf photosynthesis. Leaves were larger after domestication, which provided the main contribution to increases in leaf area per plant and plant-level C gain, and ultimately to larger aboveground biomass. In general, cultivated plants have become larger since domestication. In our six crops, this occurred despite lower investment in leaves, comparable leaf-level photosynthesis and similar biomass costs of leaf area (i.e. SLA) than their wild progenitors. Increased leaf size was the main driver of increases in aboveground size. Thus, we suggest that large seeds, which are also typical of crops, might produce individuals with larger organs (i.e. leaves) via cascading effects throughout ontogeny. Larger leaves would then scale into larger whole plants, which might partly explain the increases in size that accompanied domestication.

Magnetic resonance imaging of sugar beet taproots in soil reveals growth reduction and morphological changes during foliar Cercospora beticola infestation.

Cercospora leaf spot (CLS) infection can cause severe yield loss in sugar beet. Introduction of Cercospora-resistant varieties in breeding programmes is important for plant protection to reduce both fungicide applications and the risk of the development of fungal resistance. However, in vivo monitoring of the sugar-containing taproots at early stages of foliar symptoms and the characterization of the temporal development of disease progression has proven difficult. Non-invasive magnetic resonance imaging (MRI) measurements were conducted to quantify taproot development of genotypes with high (HS) and low (LS) levels of susceptibility after foliar Cercospora inoculation. Fourteen days post-inoculation (dpi) the ratio of infected leaf area was still low (~7%) in both the HS and LS genotypes. However, during this period, the volumetric growth of the taproot had already started to decrease. Additionally, inoculated plants showed a reduction of the increase in width of inner cambial rings while the width of outer rings increased slightly compared with non-inoculated plants. This response partly compensated for the reduced development of inner rings that had a vascular connection with Cercospora-inoculated leaves. Hence, alterations in taproot anatomical features such as volume and cambial ring development can be non-invasively detected already at 14 dpi, providing information on the early impact of the infection on whole-plant performance. All these findings show that MRI is a suitable tool to identify promising candidate parent lines with improved resistance to Cercospora, for example with comparatively lower taproot growth reduction at early stages of canopy infection, for future introduction into breeing programmes.

Assessment of the physiological responses to drought in different sugar beet genotypes in connection with their genetic distance.

Drought affects many physiological processes, which influences plant productivity. The aim of this study was to evaluate the degree of genotypic diversity in drought tolerance of sugar beet genotypes (Beta vulgaris L.) in connection with their genetic distance. Three hybrid genotypes produced by crossing double haploid genotype (P-pollinator) with cytoplasmic male-sterile female part (MS), as well as with two parent lines, were examined. Drought conditions were imposed by the cessation of watering at the 3-4 leaf stage for about three months, after which irrigation was resumed. Control plants were optimally irrigated throughout the entire vegetation period. Long-term drought significantly increased the wilting of leaves (Wilt.), specific leaf weight (SLW), the succulence index (Suc.I), leaf senescence and membrane damage (El-l). Simultaneously, the osmotic potential (ψs), leaf area index (LAI), absorption of photosynthetic active radiation (PAR) and the efficiency of the photosynthetic apparatus (Φ PSII) declined under water deficit conditions. The examined genotypes demonstrated a clear diversity in their physiological response to drought. Based on these findings, we suggest that traits that are strongly correlated with root and sugar yield, e.g. Φ PSII, LAI, PAR absorption and ψs, could be used as potential selection criteria in physiological-associated breeding strategies to improve drought tolerance in sugar beet. There was not a significant correlation between the genetic distance separating different sugar beet genotypes and the observed heterotic effect of root or sugar yields, with the exception of heterosis of root yield under optimal conditions, where the correlation was negative.

[Fruit set variation associated with apozygotic reproduction in sugar beet (Beta vulgaris l.)].

Fruit set upon apozygotic reproduction was studied for four years in the msSOAN-5 pollen-sterile inbred sugar beet line. The progenies obtained from pollen-sterile plants by apozygotic reproduction had both fruits with normal seeds and parthenocarpic fruits without seeds, which was not an occasional event. Growth conditions proved to strongly affect the fruit set and seed quality. For instance, water deficiency during early plant development increased the proportion ofparthenocarpic seeds. Waiter deficiency combined with a lower temperature during flowering additionally caused a substantial decrease in the total number of fruits. Under the same growth conditions, related accessions did not differ in seed productivity, but varied in the proportion of normal (with seeds) and parthenocarpic (without seeds) fruits.

Floral and inflorescence morphology and ontogeny in Beta vulgaris, with special emphasis on the ovary position.

BACKGROUND AND AIMS: In spite of recent phylogenetic analyses for the Chenopodiaceae-Amaranthaceae complex, some morphological characters are not unambiguously interpreted, which raises homology questions. Therefore, ontogenetic investigations, emphasizing on 'bracteoles' in Atripliceae and flowers in Chenopodioideae, were conducted. This first paper presents original ontogenetic observations in Beta vulgaris, which was chosen as a reference species for further comparative investigation because of its unclarified phylogenetic position and its flowers with a (semi-)inferior ovary, whereas all other Chenopodiaceae-Amaranthaceae have hypogynous flowers. METHODS: Inflorescences and flowers were examined using scanning electron microscopy and light microscopy. KEY RESULTS: Floral development starts from an inflorescence unit primordium subtended by a lateral bract. This primordium develops into a determinate axis on which two opposite lateral flowers originate, each subtended by a bracteole. On a flower primordium, first five tepal primordia appear, followed by five opposite stamen primordia. Simultaneously, a convex floral apex appears, which differentiates into an annular ovary primordium with three stigma primordia, surrounding a central, single ovule. A floral tube, which raises the outer floral whorls, envelops the ovary, resulting in a semi-inferior ovary at mature stage. Similarly, a stamen tube is formed, raising the insertion points of the stamens, and forming a staminal ring, which does not contain stomata. During floral development, the calyces of the terminal flower and of one of the lateral flowers often fuse, forming a compound fruit structure. CONCLUSIONS: In Beta vulgaris, the inflorescence is compound, consisting of an indeterminate main axis with many elementary dichasia as inflorescence units, of which the terminal flower and one lateral flower fuse at a later stage. Floral parts develop starting from the outer whorl towards the gynoecium. Because of the formation of an epigynous hypanthium, the ovary becomes semi-inferior in the course of floral development.

Association mapping in multiple segregating populations of sugar beet (Beta vulgaris L.).

Association mapping in multiple segregating populations (AMMSP) combines high power to detect QTL in genome-wide approaches of linkage mapping with high mapping resolution of association mapping. The main objectives of this study were to (1) examine the applicability of AMMSP in a plant breeding context based on segregating populations of various size of sugar beet (Beta vulgaris L.), (2) compare different biometric approaches for AMMSP, and (3) detect markers with significant main effect across locations for nine traits in sugar beet. We used 768 F(n) (n = 2, 3, 4) sugar beet genotypes which were randomly derived from 19 crosses among diploid elite sugar beet clones. For all nine traits, the genotypic and genotype x location interaction variances were highly significant (P < 0.01). Using a one-step AMMSP approach, the total number of significant (P < 0.05) marker-phenotype associations was 44. The identification of genome regions associated with the traits under consideration indicated that not only segregating populations derived from crosses of parental genotypes in a systematic manner could be used for AMMSP but also populations routinely derived in plant breeding programs from multiple, related crosses. Furthermore, our results suggest that data sets, whose size does not permit analysis by the one-step AMMSP approach, might be analyzed using the two-step approach based on adjusted entry means for each location without losing too much power for detection of marker-phenotype associations.

[Effect of the epimutagen 5-azacytidine on the structure of floral-stalk metameres in sugar beet (Beta vulgaris L)].

This study is dedicated to the effect of the epimutagen 5-azacytidine on the structure of floral-stalk metameres in sugar beet Beta vulgaris L. Simple phytomeres consist of separate flowers (unianthous plants; UA phenotype), while complex metameres occur in synanthous plants (SA phenotype). Treatment of the synanthous line mcSOAN-5 dramatically reduced the number of flowers on phytomeres as early as in generation zero (A0Az0). For the first time, plants with simple phytomeres were found in this line. The proportion of plants with the SA phenotype in generation A0Az0 was 7.7%. In generation A1Az1, the tendency for reduction of the number of flowers on phytomeres persisted. The proportion of plants with simple phytomeres reached 77%; i.e., the frequency of SA phenotypes in the progeny (A1Az1) increased tenfold in comparison with the parents A0Az0. The high frequency of morphogenetic changes in the floral stalk structure under the influence of the epimutagen suggests that the variability of the UA--SA character in beet populations is of epigenetic rather than mutational nature.

Emergence of gynodioecy in wild beet (Beta vulgaris ssp. maritima L.): a genealogical approach using chloroplastic nucleotide sequences.

Gynodioecy is a breeding system where both hermaphroditic and female individuals coexist within plant populations. This dimorphism is the result of a genomic interaction between maternally inherited cytoplasmic male sterility (CMS) genes and bi-parentally inherited nuclear male fertility restorers. As opposed to other gynodioecious species, where every cytoplasm seems to be associated with male sterility, wild beet Beta vulgaris ssp. maritima exhibits a minority of sterilizing cytoplasms among numerous non-sterilizing ones. Many studies on population genetics have explored the molecular diversity of different CMS cytoplasms, but questions remain concerning their evolutionary dynamics. In this paper we report one of the first investigations on phylogenetic relationships between CMS and non-CMS lineages. We investigated the phylogenetic relationships between 35 individuals exhibiting different mitochondrial haplotypes. Relying on the high linkage disequilibrium between chloroplastic and mitochondrial genomes, we chose to analyse the nucleotide sequence diversity of three chloroplastic fragments (trnK intron, trnD-trnT and trnL-trnF intergenic spacers). Nucleotide diversity appeared to be low, suggesting a recent bottleneck during the evolutionary history of B. vulgaris ssp. maritima. Statistical parsimony analyses revealed a star-like genealogy and showed that sterilizing haplotypes all belong to different lineages derived from an ancestral non-sterilizing cytoplasm. These results suggest a rapid evolution of male sterility in this taxon. The emergence of gynodioecy in wild beet is confronted with theoretical expectations, describing either gynodioecy dynamics as the maintenance of CMS factors through balancing selection or as a constant turnover of new CMSs.

Taproot promoters cause tissue specific gene expression within the storage root of sugar beet.

The storage root (taproot) of sugar beet (Beta vulgaris L.) originates from hypocotyl and primary root and contains many different tissues such as central xylem, primary and secondary cambium, secondary xylem and phloem, and parenchyma. It was the aim of this work to characterize the promoters of three taproot-expressed genes with respect to their tissue specificity. To investigate this, promoters for the genes Tlp, His1-r, and Mll were cloned from sugar beet, linked to reporter genes and transformed into sugar beet and tobacco. Reporter gene expression analysis in transgenic sugar beet plants revealed that all three promoters are active in the storage root. Expression in storage root tissues is either restricted to the vascular zone (Tlp, His1-r) or is observed in the whole organ (Mll). The Mll gene is highly organ specific throughout different developmental stages of the sugar beet. In tobacco, the Tlp and Mll promoters drive reporter gene expression preferentially in hypocotyl and roots. The properties of the Mll promoter may be advantageous for the modification of sucrose metabolism in storage roots.

[Specific density of leaf as a characteristic of the photosynthetic apparatus]. / Udel'naia plotnost' lista kak kharakteristika fotosinteticheskogo apparata.

At early stages of ontogeny (up to 50-60% of the maximum leaf area) of wheat (Triticum aestivum L.), meadow fescue (Festuca pratensis Huds.), reed fescue (F. arindinacea Schreb.), and sugar beet (Beta vulgaris L. var. saccharifera (Alef) Krass), there is correlation between changes in the specific leaf density (SLD), rate of photosynthetic CO2 assimilation; activity of the key photosynthetic enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39); and concentration of chlorophyll (Chl) a, Chl b, carotenoids, and soluble leaf proteins. However, there is no correlation of SLD with the activity of phospho(enol)pyruvate carboxylase (EC 4.1.1.31). Senescence of leaves was accompanied by a decrease in the SLD value. Treatment with cytokininomimetics (6-benzylaminopurine and Metribuzin) caused an increase in the SLD value. The specific leaf density is suggested to be a structural and functional characteristic of the photosynthetic apparatus of agricultural plants.