Interactions between water and triacylglycerols may explain faster aging rates in stored germplasm at low temperatures

The purpose of this research is to explore the extent and significance of possible interacting factors on the viability of stored germplasm. Our work begins with characterizing the kinetics of TAG and water phase changes in peanut (Arachis hypogaea) and Papaya (Carica papaya) seeds equilibrated to different water contents and stored at temperatures between -5 and -80°C. Water and TAG phase was measured using a Perkin Elmer Differential Scanning Calorimeter. Cytoplasm ultra-structure was visualized without chemical fixatives using low temperature scanning electron microscopy (cryo-SEM) performed with a Zeiss DSN 960 scanning microscope equipped with a Cryotrans CT-1500 cold plate (Oxford, UK).

Ground cover and leaf area index relationship in a grass, legume and crucifer crop

Canopy characterization is essential for describing the interaction of a crop with its environment. The goal of this work was to determine the relationship between leaf area index (LAI) and ground cover (GC) in a grass, a legume and a crucifer crop, and to assess the feasibility of using these relationships as well as LAI-2000 readings to estimate LAI. Twelve plots were sown with either barley (Hordeum vulgare L.), vetch (Vicia sativa L.), or rape (Brassica napus L.). On 10 sampling dates the LAI (both direct and LAI-2000 estimations), fraction intercepted of photosynthetically active radiation (FIPAR) and GC were measured. Linear and quadratic models fitted to the relationship between the GC and LAI for all of the crops, but they reached a plateau in the grass when the LAI mayor que 4. Before reaching full cover, the slope of the linear relationship between both variables was within the range of 0.025 to 0.030. The LAI-2000 readings were linearly correlated with the LAI but they tended to overestimation. Corrections based on the clumping effect reduced the root mean square error of the estimated LAI from the LAI-2000 readings from 1.2 to less than 0.50 for the crucifer and the legume, but were not effective for barley.

Estudios termodinámicos de los procesos de vitrificación en la crioconservación de germoplasma vegetal=Thermodynamic studies of vitrification processes in plant germplasm cryopreservation

En la actualidad, las técnicas de crioconservación poseen una importancia creciente para el almacenamiento a largo plazo de germoplasma vegetal. En las dos últimas décadas, estos métodos experimentaron un gran desarrollo y se han elaborado protocolos adecuados a diferentes sistemas vegetales, utilizando diversas estrategias como la vitrificación, la encapsulación-desecación con cuentas de alginato y el método de “droplet”-vitrificación. La presente tesis doctoral tiene como objetivo aumentar el conocimiento sobre los procesos implicados en los distintos pasos de un protocolo de crioconservación, en relación con el estado del agua presente en los tejidos y sus cambios, abordado mediante diversas técnicas biofísicas, principalmente calorimetría diferencial de barrido (DSC) y microscopía electrónica de barrido a baja temperatura (crio-SEM). En un primer estudio sobre estos métodos de crioconservación, se describen las fases de enfriamiento hasta la temperatura del nitrógeno líquido y de calentamiento hasta temperatura ambiente, al final del periodo de almacenamiento, que son críticas para la supervivencia del material crioconservado. Tanto enfriamiento como calentamiento deben ser realizados lo más rápidamente posible pues, aunque los bajos contenidos en agua logrados en etapas previas de los protocolos reducen significativamente las probabilidades de formación de hielo, éstas no son del todo nulas. En ese contexto, se analiza también la influencia de las velocidades de enfriamiento y calentamiento de las soluciones de crioconservación de plantas en sus parámetros termofísicos referente a la vitrificación, en relación su composición y concentración de compuestos. Estas soluciones son empleadas en la mayor parte de los protocolos actualmente utilizados para la crioconservación de material vegetal. Además, se estudia la influencia de otros factores que pueden determinar la estabilidad del material vitrificado, tales como en envejecimiento del vidrio. Se ha llevado a cabo una investigación experimental en el empleo del crio-SEM como una herramienta para visualizar el estado vítreo de las células y tejidos sometidos a los procesos de crioconservación. Se ha comparado con la más conocida técnica de calorimetría diferencial de barrido, obteniéndose resultados muy concordantes y complementarios. Se exploró también por estas técnicas el efecto sobre tejidos vegetales de la adaptación a bajas temperaturas y de la deshidratación inducida por los diferentes tratamientos utilizados en los protocolos. Este estudio permite observar la evolución biofísica de los sistemas en el proceso de crioconservación. Por último, se estudió la aplicación de películas de quitosano en las cuentas de alginato utilizadas en el protocolo de encapsulación. No se observaron cambios significativos en su comportamiento frente a la deshidratación, en sus parámetros calorimétricos y en la superficie de las cuentas. Su aplicación puede conferir propiedades adicionales prometedoras. ABSTRACT Currently, cryopreservation techniques have a growing importance for long term plant germplasm storage. These methods have undergone great progress during the last two decades, and adequate protocols for different plant systems have been developed, making use of diverse strategies, such as vitrification, encapsulation-dehydration with alginate beads and the dropletvitrification method. This PhD thesis has the goal of increasing the knowledge on the processes underlying the different steps of cryopreservation protocols, in relation with the state of water on tissues and its changes, approached through diverse biophysical techniques, especially differential scanning calorimetry (DSC) and low-temperature scanning electron microscopy (cryo-SEM). The processes of cooling to liquid nitrogen temperature and warming to room temperature, at the end of the storage period, critical for the survival of the cryopreserved material, are described in a first study on these cryopreservation methods. Both cooling and warming must be carried out as quickly as possible because, although the low water content achieved during previous protocol steps significantly reduces ice formation probability, it does not completely disappear. Within this context, the influence of plant vitrification solutions cooling and warming rate on their vitrification related thermophysical parameters is also analyzed, in relation to its composition and component concentration. These solutions are used in most of the currently employed plant material cryopreservation protocols. Additionally, the influence of other factors determining the stability of vitrified material is studied, such as glass aging. An experimental research work has been carried out on the use of cryo-SEM as a tool for visualizing the glassy state in cells and tissues, submitted to cryopreservation processes. It has been compared with the better known differential scanning calorimetry technique, and results in good agreement and complementary have been obtained. The effect on plant tissues of adaptation to low temperature and of the dehydration induced by the different treatments used in the protocols was explored also by these techniques. This study allows observation of the system biophysical evolution in the cryopreservation process. Lastly, the potential use of an additional chitosan film over the alginate beads used in encapsulation protocols was examined. No significant changes could be observed in its dehydration and calorimetric behavior, as well as in its surface aspect; its application for conferring additional properties to gel beads is promising.

Influence of denitrifiers abundance on N2O emissions in long term tillage system under a rainfed legume crop

Current studies about nitrous oxide (N2O) emissions from legume crops have raised considerable doubt, observing a high variability between sites (0.03-7.09 kg N2O–N ha−1 y -1) [1]. This high variability has been associated to climate and soil conditions, legume species and soil management practices (e.g. conservation or conventional tillage). Conservation tillage (i.e. no tillage (NT) and minimum tillage (MT)) has spread during the last decades because promotes several positive effects (increase of soil organic content, reduction of soil erosion and enhancement of carbon (C) sequestration). However, these benefits could be partly counterbalanced by negative effects on the release of N2O emissions. Among processes responsible for N2O production and consumption in soils, denitrification plays an importantrole both in tilled and no-tilled ropping systems [2]. Recently, amplification of functional bacterial genes involved in denitrification is being used to examine denitrifiers abundance and evaluate their influence on N2O emissions. NirK and nirS are functional genes encoding the cytochrome cd1 and copper nitrite reductase, which is the key enzyme regulating the denitrification process.

Thermographic imaging: assessment of drought and heat tolerance in Spanish germplasm of Brachypodium distachyon.

The annual grass Brachypodium distachyon has been recently recognized as the model plant for functional genomics of temperate grasses, including cereals of economic relevance like wheat and barley. Sixty-two lines of B. distachyon were assessed for response to drought stress and heat tolerance. All these lines, except the reference genotype BD21, derive from specimens collected in 32 distinct locations of the Iberian Peninsula, covering a wide range of geo- climatic conditions. Sixteen lines of Brachypodium hybridum, an allotetraploid closely related to B. distachyon were used as reference of abiotic-stress well-adapted genotypes. Drought tolerance was assessed in a green-house trial. At the rosette-stage, no irrigation was applied to treated plants whereas their replicates at the control were maintained well watered during all the experiment. Thermographic images of treated and control plants were taken after 2 and 3 weeks of drought treatment, when stressed plants showed medium and extreme wilting symptoms. The mean leaf temperature of stressed (LTs) and control (LTc) plants was estimated based upon thermographic records from selected pixels (183 per image) that strictly correspond to leaf tissue. The response to drought was based on the analysis of two parameters: LTs and the thermal difference (TD) between stressed and control plants (LTs – LTc). The response to heat stress was based on LTc. Comparison of the mean values of these parameters showed that: 1) Genotypes better adapted to drought (B. hybridum lines) presented a higher LTs and TD than B. distachyon lines. 2) Under high temperature conditions, watered plants of B. hybridum lines maintained lower LTc than those of B. distachyon. Those results suggest that in these species adaptation to drought is linked to a more efficient stomata regulation: under water stress stomata are closed, increasing foliar temperature but also water use efficiency by reducing transpiration. With high temperature and water availability the results are less definite, but still seems that opening stomata allow plants to increase transpiration and therefore to diminish foliar temperature.

Metal Transport in the Rhizobium-Legume Symbiosis

Metal Transport in the Rhizobium-legume Symbiosis

Nutritive value for ruminants of winter oats-legume intercrops in organic cultivation

Winter oats were grown according to European organic farming regulations in monoculture (oats) and in intercropping with bard vetch (BAV), bitter vetch (BIV) or both legumes (MIX) to evaluate the effects of intercropping on forage yield and nutritive value for ruminants. The experiment was carried out as a randomised complete block design with four replications, and whole forage samples were obtained at two harvest dates (June and July). For both harvest times, all intercrops increased (P < 0.05) forage yield compared with oats, but forage crude protein content was only increased (P < 0.05) for BAV and MIX. Compared with oats, intercropping with BAV increased (P < 0.05) in vitro rate of gas production and total volatile fatty acid production, indicating a higher rate and extent of rumen degradation of BAV forage. In contrast, BIV forage harvested in June had lower (P < 0.05) rate of gas production and total volatile fatty acid production than June oats, but in general no differences in the in vitro rumen fermentation were detected between oats and BIV samples harvested in July. The results indicate that forage yield and quality can be enhanced by intercropping oats with BAV; however, intercropping with BIV increased yield but decreased nutritive value of the forage.

Populations genomics analysis of legume host preference for specific rhizobial genotypes in the Rhizobium leguminosarum bv. viciae symbioses

Rhizobium leguminosarum bv. viciae establishes root nodule symbioses with several legume genera. Although most isolates are equally effective in establishing symbioses with all host genera, previous evidence suggests that hosts select specific rhizobial genotypes among those present in the soil. We have used population genomics to further investigate this observation. P. sativum, L. culinaris, V. sativa, and V. faba plants were used to trap rhizobia from a well-characterized soil, and pooled genomic DNAs from one-hundred isolates from each plant were sequenced. Sequence reads were aligned to the R. leguminosarum bv. viciae 3841 reference genome. High overall conservation of sequences was observed in all subpopulations, although several multigenic regions were absent from the soil population. A large fraction (16-22%) of sequence reads could not be recruited to the reference genome, suggesting that they represent sequences specific to that particular soil population. Although highly conserved, the 16S-23S rRNA gene region presented single nucleotide polymorphisms (SNPs) regarding the reference genome, but no striking differences could be found among plant-selected subpopulations. Plant-specific SNP patterns were, however, clearly observed within the nod gene cluster, supporting the existence of a plant preference for specific rhizobial genotypes. This was also shown after genome-wide analysis of SNP patterns.

PoolSeq analysis of the selection of the Rhizobium genotypes by the legume host plant

Rhizobium leguminosarum establishes highly specific nitrogen-fixing symbioses. We have applied a Pool-Seq approach to study plant host selection of genotypes. Our results confirm, at the genomic level, previous observations regarding plant selection of specific genotypes

Metagenomic Anlaysis of microsymbiont selection by the legume plant host

Rhizobium leguminosarum bv.viciae is able to establish nitrogen-fixing symbioses with legumes of the genera Pisum, Lens, Lathyrus and Vicia. Classic studies using trap plants (Laguerre et al., Young et al.) provided evidence that different plant hosts are able to select different rhizobial genotypes among those available in a given soil. However, these studies were necessarily limited by the paucity of relevant biodiversity markers. We have now reappraised this problem with the help of genomic tools. A well-characterized agricultural soil (INRA Bretennieres) was used as source of rhizobia. Plants of Pisum sativum, Lens culinaris, Vicia sativa and V. faba were used as traps. Isolates from 100 nodules were pooled, and DNA from each pool was sequenced (BGI-Hong Kong; Illumina Hiseq 2000, 500 bp PE libraries, 100 bp reads, 12 Mreads). Reads were quality filtered (FastQC, Trimmomatic), mapped against reference R. leguminosarum genomes (Bowtie2, Samtools), and visualized (IGV). An important fraction of the filtered reads were not recruited by reference genomes, suggesting that plant isolates contain genes that are not present in the reference genomes. For this study, we focused on three conserved genomic regions: 16S-23S rDNA, atpD and nodDABC, and a Single Nucleotide Polymorphism (SNP) analysis was carried out with meta / multigenomes from each plant. Although the level of polymorphism varied (lowest in the rRNA region), polymorphic sites could be identified that define the specific soil population vs. reference genomes. More importantly, a plant-specific SNP distribution was observed. This could be confirmed with many other regions extracted from the reference genomes (data not shown). Our results confirm at the genomic level previous observations regarding plant selection of specific genotypes. We expect that further, ongoing comparative studies on differential meta / multigenomic sequences will identify specific gene components of the plant-selected genotypes

Genomics of host specificity in the Rhizobium-legume simbiosis

Most Rhizobium leguminosarum bv. viciae isolates are able to specifically nodulate plants of any of four different legume genera: Pisum, Lens, Vicia, and Lathyrus. However, previous evidence suggests that some genotypes are more adapted to a given plant host than others, and that the plant host can select specific genotypes among those present in a given soil population. We have used a population genomics approach to confirm that this is indeed the case, and to analyze the specific genotypic characteristics that each plant host selects

Effect of the Legume Plant Host on Rhizobial Soil Population Dynamics

Rhizobium leguminosarum bv viciae (Rlv) is a soil bacterium able to establish specific root-nodule symbioses with legumes of four different genera: Pisum, Vicia, Lens and Lathyrus. Rlv isolates from nodules of any of these legumes can nodulate any of them; however, it has been shown that plants select specific rhizobial genotypes from those present in the soil (1,2). We have previously shown this at the genomic level by following a population genomics approach. Pool genomic sequences from 100 isolates from each of four plant species: P. sativum, L. culinaris, V. faba and V. sativa, show different, specific profiles at the single nucleotide polymorphism (SNP) level for relevant genes. In this work, the extent of Rlv selection from a well-characterized soil population by different legume plant hosts: P. sativum, L. culinaris, V. faba and V. sativa, after a medium-term mesocosm study is described. Direct soil isolates from each of these mesocosm studies have been tested for specific rhizobial genes (glnII and fnrN) and symbiotic genes (nodC and nifH). Different populations were characterized further by Sanger sequencing of both the rpoB phylogenetic marker gene and the symbiotic genes nodC and nifH. The distribution and size of the rhizobial population for each legume host showed changes during the medium-term mesocosm study. Particularly, a non-symbiotic group of rhizobia was enriched by all four hosts, in contrast to the symbiotic rhizobia profile, which was specific for each legume plant host.

Analysis of genetic variability in a sample of the durum wheat (Triticum durum Desf.) Spanish collection based on gliadin markers

In this work gliadin proteins were used to analyse the genetic variability in a sample of the durum wheat Spanish collection conserved at the CRF-INIA. In total 38 different alleles were identified at the loci Gli-A1, Gli-A3, Gli-B5, Gli-B1, Gli-A2 and Gli-B2. All the gliadin loci were polymorphic, possessed large genetic diversity and small and large differentiation within and between varieties, respectively. The Gli-A2 and Gli-B2 loci were the most polymorphic, the most fixed within varieties and the most useful to distinguish among varieties. Alternatively, Gli-B1 locus presented the least genetic variability out of the four main loci Gli-A1, Gli-B1, Gli-A2 and Gli-B2. The Gli-B1 alleles coding for the gliadin γ-45, associated with good quality, had an accumulated frequency of 69.7%, showing that the Spanish germplasm could be a good source for breeding quality. The Spanish landraces studied showed new gliadin alleles not catalogued so far. These new alleles might be associated with specific Spanish environment factors. The large number of new alleles identified also indicates that durum wheat Spanish germplasm is rather unique.

Growth response to ozone of annual species from Mediterranean pastures

Ozone (O3) phytotoxicity has been reported on a wide range of plant species. However, scarce information has been provided regarding the sensitivity of semi-natural grassland species, especially those from dehesa Mediterranean grasslands, in spite of their great biological diversity and the high O3 levels recorded in the region. A screening study was carried out in open-top chambers (OTCs) to assess the O3-sensitivity of representative therophytes of these ecosystems based on the response of selected growth-related parameters. Three O3 treatments and 3 OTCs per treatment were used. Legume species were very sensitive to O3, because 78% of the tested species showed detrimental effects on their total biomass relative growth rate (RGR) following their exposure to O3. The Trifolium genus was particularly sensitive showing O3-induced adverse effects on most of the assessed parameters. Gramineae plants were less sensitive than Leguminosae species because detrimental effects on total biomass RGR were only observed in 14% of the assessed species. No relationship was found between relative growth rates when growing in clean air and O3 susceptibility. The implications of these effects on the performance of dehesa acidic grasslands and on the definition of ozone critical levels for the protection of semi-natural vegetation are discussed.

Endosymbiotic bacteria nodulating a new endemic lupine Lupinus mariae-josephi from alkaline soils in Eastern Spain represent a new lineage within the Bradyrhizobium genus

Lupinus mariae-josephi is a recently described endemic Lupinus species from a small area in Eastern Spain where it thrives in soils with active lime and high pH. The L. mariae-josephi root symbionts were shown to be very slow-growing bacteria with different phenotypic and symbiotic characteristics from those of Bradyrhizobium strains nodulating other Lupinus. Their phylogenetic status was examined by multilocus sequence analyses of four housekeeping genes (16S rRNA, glnII, recA, and atpD) and showed the existence of a distinct evolutionary lineage for L. mariae-josephi that also included Bradyrhizobium jicamae. Within this lineage, the tested isolates clustered in three different sub-groups that might correspond to novel sister Bradyrhizobium species. These core gene analyses consistently showed that all the endosymbiotic bacteria isolated from other Lupinus species of the Iberian Peninsula were related to strains of the B. canariense or B. japonicum lineages and were separate from the L. mariae-josephi isolates. Phylogenetic analysis based on nodC symbiotic gene sequences showed that L. mariae-josephi bacteria also constituted a new symbiotic lineage distant from those previously defined in the genus Bradyrhizobium. In contrast, the nodC genes of isolates from other Lupinus spp. from the Iberian Peninsula were again clearly related to the B. canariense and B. japonicum bv. genistearum lineages. Speciation of L. mariae-josephi bradyrhizobia may result from the colonization of a singular habitat by their unique legume host.