M. Mercedes Lucas

Starch Granule Initiation in Arabidopsis Requires the Presence of Either Class IV or Class III Starch Synthases

The mechanisms underlying starch granule initiation remain unknown. We have recently reported that mutation of soluble starch synthase IV (SSIV) in Arabidopsis thaliana results in restriction of the number of starch granules to a single, large, particle per plastid, thereby defining an important component of the starch priming machinery. In this work, we provide further evidence for the function of SSIV in the priming process of starch granule formation and show that SSIV is necessary and sufficient to establish the correct number of starch granules observed in wild-type chloroplasts. The role of SSIV in granule seeding can be replaced, in part, by the phylogenetically related SSIII. Indeed, the simultaneous elimination of both proteins prevents Arabidopsis from synthesizing starch, thus demonstrating that other starch synthases cannot support starch synthesis despite remaining enzymatically active. Herein, we describe the substrate specificity and kinetic properties of SSIV and its subchloroplastic localization in specific regions associated with the edges of starch granules. The data presented in this work point to a complex mechanism for starch granule formation and to the different abilities of SSIV and SSIII to support this process in Arabidopsis leaves.

Characterization of Rhizobium spp. bean isolates from South- West Spain

Rhizobium spp. strains able to nodulate beans (Phaseolus vulgaris L.) were isolated from Andalusian (Southern Spain) soils with no record of recent bean cultivation (except soil 14) and no known history of bean inoculation in this area. The isolation methodology was devised to obtain an heterogeneous rhizobia population from each soil sample, by using three different bean cultivars as trap-host. No association was found between the presence of rhizobia nodulating bean and the chemical or textural properties of the soils. The isolates were grouped on the basis of their symbiotic effectiveness on bean cv. Canellini under greenhouse conditions, intrinsic antibiotic resistance (IAR), lipopolysaccharide (LPS) and protein profiles, melanin production, and by amplified rDNA restriction analysis (ARDRA). Most of the isolates were more effective than the reference strains Rhizobium leguminosarum bv. phaseoli TAL1121, R. etli type strain CFN42 and R. tropici type strain CIAT899. The symbiotic effectiveness of the isolates could not be related with other traits analyzed. Predominantly, a two bands-LPS profile was found amongst the isolates. Most of them have been assigned to R. etli by ARDRA and seem to be more competitive than R. gallicum or R. giardinii isolates. Additionally, a strong interaction between the bean cultivar and the native rhizobia populations was observed.

The Sinorhizobium fredii HH103 Lipopolysaccharide is not only relevant at early soybean nodulation stages but also for symbiosome stability in mature nodules

In this work we have characterised the Sinorhizobium fredii HH103 greA lpsB lpsCDE genetic region and analysed for the first time the symbiotic performance of Sinorhizobium fredii lps mutants on soybean. The organization of the S. fredii HH103 greA, lpsB, and lpsCDE genes was equal to that of Sinorhizobium meliloti 1021. S. fredii HH103 greA, lpsB, and lpsE mutant derivatives produced altered LPS profiles that were characteristic of the gene mutated. In addition, S. fredii HH103 greA mutants showed a reduction in bacterial mobility and an increase of auto-agglutination in liquid cultures. RT-PCR and qPCR experiments demonstrated that the HH103 greA gene has a positive effect on the transcription of lpsB. Soybean plants inoculated with HH103 greA, lpsB or lpsE mutants formed numerous ineffective pseudonodules and showed severe symptoms of nitrogen starvation. However, HH103 greA and lps mutants were also able to induce the formation of a reduced number of soybean nodules of normal external morphology, allowing the possibility of studying the importance of bacterial LPS in later stages of the S. fredii HH103-soybean symbiosis. The infected cells of these nodules showed signs of early termination of symbiosis and lytical clearance of bacteroids. These cells also had very thick walls and accumulation of phenolic-like compounds, pointing to induced defense reactions. Our results show the importance of bacterial LPS in later stages of the S. fredii HH103-soybean symbiosis and their role in preventing host cell defense reactions. S. fredii HH103 lpsB mutants also showed reduced nodulation with Vigna unguiculata, although the symbiotic impairment was less pronounced than in soybean.

A light and electron microscopy analysis of the events leading to male sterility in Ogu-INRA CMS of rapeseed (Brassica napus)

Ogura cytoplasmic male sterility (CMS) occurs naturally in radish and has been introduced into rapeseed (Brassica napus) by protoplast fusion. As with all CMS systems, it involves a constitutively expressed mitochondrial gene which induces male sterility to otherwise hermaphroditic plants (so they become females) and a nuclear gene named restorer of fertility that restores pollen production in plants carrying a sterility-inducing cytoplasm. A correlative approach using light and electron microscopy was applied to define what stages throughout development were affected and the subcellular events leading to the abortion of the developing pollen grains upon the expression of the mitochondrial protein. Three central stages of development (tetrad, mid-microspore and vacuolate microspore) were compared between fertile, restored, and sterile plants. At each stage observed, the pollen in fertile and restored plants had similar cellular structures and organization. The deleterious effect of the sterility protein expression started as early as the tetrad stage. No typical mitochondria were identified in the tapetum at any developmental stage and in the vacuolate microspores of the sterile plants. In addition, some striking ultrastructural alterations of the cells organization were also observed compared with the normal pattern of development. The results showed that Ogu-INRA CMS was due to premature cell death events of the tapetal cells, presumably by an autolysis process rather than a normal PCD, which impairs pollen development at the vacuolate microspore stage, in the absence of functional mitochondria.

Immunolocalization of ferritin in determinate and indeterminate legume root nodules

SummaryIn eukaryotic organisms ferritin is a protein involved in the storage of iron. The occurrence of ferritin and its relationship to the effectiveness of the nitrogen-fixing activity have been previously studied during the early stages of the nodule development by biochemical methods. We have used immunocytochemistry techniques to determine the precise location of ferritin and the behavior of this protein along the nodule development. The major localization was found in plastids and amyloplasts of infected and uninfected cells of the three legume nodules studied. A decrease of the immunolabelling was observed in infected cells of lupin and soybean senescing nodules and in the senescent zone of indeterminate alfalfa nodules. In the cortex of soybean and lupin nodules, ferritin increased during nodule ageing and the immunogold particles were mainly located in crystalline structures. The putative role of ferritin and plastids during nodule development is discussed.

Overexpression of Flavodoxin in Bacteroids Induces Changes in Antioxidant Metabolism Leading to Delayed Senescence and Starch Accumulation in Alfalfa Root Nodules

Sinorhizobium meliloti cells were engineered to overexpress Anabaena variabilis flavodoxin, a protein that is involved in the response to oxidative stress. Nodule natural senescence was characterized in alfalfa (Medicago sativa) plants nodulated by the flavodoxin-overexpressing rhizobia or the corresponding control bacteria. The decline of nitrogenase activity and the nodule structural and ultrastructural alterations that are associated with nodule senescence were significantly delayed in flavodoxin-expressing nodules. Substantial changes in nodule antioxidant metabolism, involving antioxidant enzymes and ascorbate-glutathione cycle enzymes and metabolites, were detected in flavodoxin-containing nodules. Lipid peroxidation was also significantly lower in flavodoxin-expressing nodules than in control nodules. The observed amelioration of the oxidative balance suggests that the delay in nodule senescence was most likely due to a role of the protein in reactive oxygen species detoxification. Flavodoxin overexpression also led to high starch accumulation in nodules, without reduction of the nitrogen-fixing activity.

The future of lupin as a protein crop in Europe

Europe has become heavily dependent on soya bean imports, entailing trade agreements and quality standards that do not satisfy the European citizen’s expectations. White, yellow, and narrow-leafed lupins are native European legumes that can become true alternatives to soya bean, given their elevated and high-quality protein content, potential health benefits, suitability for sustainable production, and acceptability to consumers. Nevertheless, lupin cultivation in Europe remains largely insufficient to guarantee a steady supply to the food industry, which in turn must innovate to produce attractive lupin-based protein-rich foods. Here, we address different aspects of the food supply chain that should be considered for lupin exploitation as a high-value protein source. Advanced breeding techniques are needed to provide new lupin varieties for socio-economically and environmentally sustainable cultivation. Novel processes should be optimized to obtain high-quality, safe lupin protein ingredients, and marketable foods need to be developed and offered to consumers. With such an integrated strategy, lupins can be established as an alternative protein crop, capable of promoting socio-economic growth and environmental benefits in Europe.

Aldehyde oxidase (AO) in the root nodules of Lupinus albus and Medicago truncatula: identification of AO in meristematic and infection zones

Phytohormones are involved in the organogenesis of legume root nodules. The source of the auxin indole-3-acetic acid (IAA) in nodules has not been clearly determined. We studied the enzyme aldehyde oxidase (AO; EC 1.2.3.1), that catalyzes the last step of IAA biosynthesis in plants, in the nodules of Lupinus albus and Medicago truncatula. Primordia and young lupin nodules and mature M. truncatula nodules showed AO activity bands after native polyacrylamide gel electrophoresis. Gel activity analyses using indole-3-aldehyde as substrate indicated that the nodules of white lupin and M. truncatula have the capability to synthesize IAA via the indole-3-pyruvic acid pathway. Immunolocalization and in situ hybridization experiments revealed that AO is preferentially expressed in the meristematic and the invasion zones in Medicago nodules and in the lateral meristematic zone of Lupinus nodules. High IAA immunolabeling was also detected in the meristematic and invasion zones. Low expression levels and no AO activity were detected in lupin Fix- nodules that displayed restricted growth and early senescence. We propose that local synthesis of IAA in the root nodule meristem and modulation of AO expression and activity are involved in regulation of nodule development.

Cytochemical Study of Catalase and Peroxidase in the Mesophyll of Lolium rigidum Plants Treated with Isoproturon

Summary A study was made of the number of peroxisomes appearing in the mesophyll of Lolium rigidum in control and isoproturon-treated plants and also, using the diamino benzidine (DAB) technique, the cytochemical localization of catalase and peroxidase enzymes in the same tissues. The number of peroxisomes increased when growing in the presence of a 3.4x10-5 M isoproturon concentration. Isoproturon also somewhat changed the normal position of the peroxisomes. With regard to the cytochemical results, DAB precipitate (osmium black reaction product) was located mainly in the cell wall, vacuole membrane, and in the chloroplasts of the isoproturon-treated mesophyll. However, there was a decrease in DAB in the microbodies of these treated plants. In this study, microbody-like structures associated with a kind of network were observed, and we believe these to be microbodies in development, the Golgi vesicles contributing to their formation. They also presented DAB precipitate in their small internal compartments, probably corresponding to catalase activity. The results are discussed in relation to the senescence of the tissues studied.

Nuclear DNA Endoreduplication and Expression of the Mitotic Inhibitor Ccs52 Associated to Determinate and Lupinoid Nodule Organogenesis

Lotus japonicus determinate nodules differ greatly from indeterminate nodules in their organogenesis and morphological characteristics, whereas Lupinus albus lupinoid nodules share features of determinate and indeterminate nodules. The mitotic inhibitor Ccs52A is essential for endoreduplication and ploidy-dependent cell enlargement during symbiotic cell differentiation in Medicago truncatula indeterminate nodules. ccs52A homolog genes were isolated from lupin and lotus nodules; the deduced Ccs52A proteins showed high sequence similarity with other Cdh-1-type activators of the anaphase-promoting complex and were grouped with A-type Ccs52 proteins from different plants. In lupin, ccs52A expression was restricted to the earlier stages of nodule development, whereas ccs52A transcripts accumulated in lotus nodule primordia and, to a lesser extent, in mature nodules. Nodule development in Lupinus albus involved a progressive increase in nuclear and cellular size and ploidy level; similarly, Lotus japonicus nodules contained polyploid nuclei and enlarged cells in the infected zone. Nevertheless, in situ hybridization experiments showed the highest ccs52A expression in the inner cortex cells of the lupin nodule primordium, probably associated to the increased size of these cells in mature nodules. In view of our results, Ccs52A-mediated endoreduplication appears to be a universal mechanism required for nodule cell differentiation during the establishment of nitrogen-fixing symbioses.