Germination Sensu Stricto Research Articles

New insights into the rapid germination process of lentil and cowpea seeds: High thiamine and folate, and low α-galactoside content

During germination sensu-stricto in pulses, an increase in the content of thiamine (B1) and folate (B9) vitamins is expected, along with a reduction in α-galactoside levels. The aim of our study was to optimize germination to increase the nutritional quality of lentils and cowpeas. An experimental design was carried out at 12 h and 24 h of imbibition to analyze the effects of temperature, light, and water content on thiamine, folate, and α-galactoside content. Germination increased thiamine content by 152% in lentils, while in cowpeas, the increase was only 10%. Folate content in cowpea increased by 33%, while α-galactoside content decreased by 99% in cowpeas and by 48% in lentils. Germination sensu-stricto can be safely implemented by any food company worldwide as it is simple and involves less sanitary risk than sprouting. This opens up opportunities for enhancing food nutrient content and new ways of processing pulses.

Identification of Reference Genes for Precise Expression Analysis during Germination in Chenopodium quinoa Seeds under Salt Stress.

Chenopodium quinoa Willd. (quinoa), a member of the Amaranthaceae family, is an allotetraploid annual plant, endemic to South America. The plant of C. quinoa presents significant ecological plasticity with exceptional adaptability to several environmental stresses, including salinity. The resilience of quinoa to several abiotic stresses, as well as its nutritional attributes, have led to significant shifts in quinoa cultivation worldwide over the past century. This work first defines germination sensu stricto in quinoa where the breakage of the pericarp and the testa is followed by endosperm rupture (ER). Transcriptomic changes in early seed germination stages lead to unstable expression levels in commonly used reference genes that are typically stable in vegetative tissues. Noteworthy, no suitable reference genes have been previously identified specifically for quinoa seed germination under salt stress conditions. This work aims to identify these genes as a prerequisite step for normalizing qPCR data. To this end, germinating seeds from UDEC2 and UDEC4 accessions, with different tolerance to salt, have been analyzed under conditions of absence (0 mM NaCl) and in the presence (250 mM NaCl) of sodium chloride. Based on the relevant literature, six candidate reference genes, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Monensin sensitivity1 (MON1), Polypyrimidine tract-binding protein (PTB), Actin-7 (ACT7), Ubiquitin-conjugating enzyme (UBC), and 18S ribosomal RNA (18S), were selected and assessed for stability using the RefFinder Tool encompassing the statistical algorithms geNorm, NormFinder, BestKeeper, and ΔCt in the evaluation. The data presented support the suitability of CqACT7 and CqUBC as reference genes for normalizing gene expression during seed germination under salinity stress. These recommended reference genes can be valuable tools for consistent qPCR studies on quinoa seeds.

Transcripts Expressed during Germination Sensu Stricto Are Associated with Vigor in Soybean Seeds.

The rapid and uniform establishment of crop plants in the field underpins food security through uniform mechanical crop harvesting. In order to achieve this, seeds with greater vigor should be used. Vigor is a component of physiological quality related to seed resilience. Despite this importance, there is little knowledge of the association between events at the molecular level and seed vigor. In this study, we investigated the relationship between gene expression during germination and seed vigor in soybean. The expression level of twenty genes related to growth at the beginning of the germination process was correlated with vigor. In this paper, vigor was evaluated by different tests. Then we reported the identification of the genes Expansin-like A1, Xyloglucan endotransglucosylase/hydrolase 22, 65-kDa microtubule-associated protein, Xyloglucan endotransglucosylase/hydrolase 2, N-glycosylase/DNA lyase OGG1 and Cellulose synthase A catalytic subunit 2, which are expressed during germination, that correlated with several vigor tests commonly used in routine analysis of soybean seed quality. The identification of these transcripts provides tools to study vigor in soybean seeds at the molecular level.

Acceleration in Germination Sensu stricto Plays a Central Role on Seedling Vigor in Post-Germination.

An obvious relationship between germination sensu stricto and seedling development during post-germination has been considered, but not explained concerning vigor. Taking this into account, we used measurements of water dynamics in germinating seeds and seedling development to clarify that relationship. The biological model was soybean seeds, since it is the most relevant ‘true seed’ produced around world. Our findings suggest that the way energy is used (acceleration) and not its input (velocity) is the main aspect relating seed germination and seedling development, especially when considering vigor. However, velocity and acceleration can be complementary in analyses of seed physiology. Other measurements proposed here also have potential uses for testing vigor in seed lots, such as seedling vigor index and biological activity in the lot. Therefore, water dynamics in germinating seeds can be an interesting way for testing seed lots, because it is an easier, faster and cheaper method in relation to other non-destructive procedures.

Elucidating ROS signaling networks and physiological changes involved in nanoscale zero valent iron primed rice seed germination sensu stricto

Reactive oxygen species (ROS) play pivotal roles during seed dormancy and germination. Metabolically active cells of seeds generate ROS and successful germination is governed by internal ROS contents, maintained within an optimum “oxidative window” by several ROS scavengers. Although ROS was previously considered hazardous, optimum ROS generation in seeds can mediate early seed germination by acting as messengers for cell signaling involved in endosperm weakening, stored food mobilization, etc. Recent reports suggest that nanopriming can expedite seed germination rates and enhance seed quality and crop performances. However, nanoparticle-driven signal cascades involved during seed germination are still unknown. The present study is aimed to explore molecular mechanisms for promoting germination in nanoprimed seeds and to investigate the plausible role of nanoparticle-mediated ROS generation in this process. Here rice seeds were primed with 20 mg L −1nanoscale zero valent iron (nZVI) for 72 h and several biochemical and physiological changes were monitored at different time points (5, 10, 20, 40, 60, and 80 h). To gain insight into roles of ROS in germination rate enhancement, intercellular ROS inhibitor, diphenyleneiodonium (DPI) was taken as another priming agent. Seed priming with DPI impaired seed germination percentage, hydrolytic enzyme activities due to ROS imbalance. On the contrary, seeds primed with both DPI and nZVI could recover from deleterious consequences of DPI treatment. Although DPI impaired intercellular ROS generation, nZVI can generate ROS independently which was confirmed from ROS localization assay. In both nZVI and the DPI and nZVI co-primed sets, significant up-regulation in genes like OsGA3Ox2, OsGAMYB were observed which are responsible for regulating the activity of several hydrolases and mediates efficient mobilization of storage food reserves of seeds. Thus, nZVI priming has potential to regulate intracellular ROS levels and orchestrate all the metabolic activities which eventually up-regulates seed germination rate and seed vigour.

Clues on an intraspecific communication system in seed‐seedling transition

How much interactivity is in a seed-seedling transition system? We hypothesize that seed-seed, seed-seedling, and seedling-seedling interactions can drive the early plant development in artificial growth systems directly due to mutual stimulation phenomena. To test the hypothesis, we performed seed germination measurements, gene expression in germination sensu stricto, water dynamics in germinating seeds, and information theory. For a biological model, we used Solanum lycocarpum A. St.-Hil. seeds. This is a neotropical species with high intraspecific variability in the seed sample. Our findings demonstrate that the dynamic and transient seed-seedling transition system is influenced by the number of individuals (seed or seedling) in the artificial system. In addition, we also discuss that: (1) the information entropy enables the quantification of system disturbance relative to individuals at the same physiological stage (seed-seed or seedling-seedling), which may be determinant for embryo growth during germination and (2) the intraspecific communication in seed-seedling transition systems formed by germinating seeds has the potential to alter the expression pattern of key genes for embryo development. Therefore, the phenomenon of mutual stimulation during the germination process can be an important aspect of seed-seedling transition, especially in laboratory conditions.

Misunderstanding on germination sensu stricto leads us to a false positive germination‐dormancy balance in diaspores of Paepalanthus chiquitensis Herzog (Eriocaulaceae), a threatened everlasting flowering species

AbstractAlthough germination sensu stricto and germination–dormancy balance are essential to each other, few reports demonstrate the effects of their relationship on patterns of seed–seedling transition. We studied this relationship by using diaspores of Paepalanthus chiquitensis, a threatened everlasting flowering species. We assessed three aspects: (a) water dynamics in germinating diaspores, (b) thermal stimulation (35, 40, 50, 60 and 70°C for 30 min) and (c) imbibition (4 hr) in growth stimulators (100, 200 or 300 mg · L−1 GA3; 0.1% KNO3). Our findings demonstrate that there are no barriers in the diaspore for diffusion. Additionally, there is high variability in diaspore permeability, and the germination of the species has two steps divided into five stages. These stages were defined by inflections on velocity in water dynamics, which ranged from −0.05 to 0.05 g · hr−1, where only the last stage (Phase V) is associated with the second step of germination sensu stricto. The imbibition phase was the shortest germination phase (1.3 hr), whereas the predominantly biochemical phase was the longest (5 days after sowing [DAS]). Germinability was high (77.5–86.5%), with high daily embryo protrusion (2.46–3.03 diaspores · day−1). Protocorm emergence marked the end of the first step of germination sensu stricto (9 DAS), and first protocorm rupture (11 DAS) marked the end of the second step. The growth stimulus only concentrated the germination process, showing that the diaspores do not have primary dormancy. Knowing these diaspores’ peculiarities during germination sensu stricto avoids a false positive statement on the existence of diaspore dormancy.

In vivo and in vitro Activity of 1-aminocyclopropane-1-carboxylic Acid Oxidase in Germinating Seeds of China Aster (Callistephus chinensis Nees)

Abstract The activity of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO; EC 1.4.3.3) in germinating seeds of Callistephus chinensis was studied. For maximum recovery of ACO activity in vitro, the presence of 10% (w/v) insoluble polyvinylpolypyrrolidone (PVPP) and 30% of glycerol in the extraction medium was necessary. The optimum pH for this activity was 7.0. Ethylene production by whole achenes or enzymatic extract increased due to increasing 1-aminocyclopropane-1-carboxylic acid (ACC) concentrations. Saturation level of ACC for in vivo ACO activity was 10−1 M and Vmax was 10.89 nL C2H4·mg protein−1·h−1. For in vitro ACO activity, the saturation level of ACC was 10−3 M and Vmax was 2.299 nL C2H4·mg protein−1·h−1. Both, in vivo and in vitro ACO activities did not follow Michaelis-Menten kinetics. The Hill coefficients (h) were estimated on the basis of non-linear estimation. Their values were 0.63 for in vivo ACO activity and 1.73 for in vitro ACO activity. The experimental data show that ACO from C. chinensis seeds is an oligomeric enzyme with at least two active sites. During seed germination, in vitro ACO activity was detectable after 12 hours of imbibition, while in vivo ACC conversion to ethylene was observed after 24 h, i.e. – after radicle protrusion. The activity of ACO in C. chinensis seeds is associated with germination sensu stricto, and might be a good marker of this process.

The Endosperm-Derived Embryo Sheath Is an Anti-adhesive Structure that Facilitates Cotyledon Emergence during Germination in Arabidopsis.

Germination sensu stricto in Arabidopsis involvesseed-coat and endosperm rupture by the emerging seedling root. Subsequently, the cotyledons emerge rapidly from the extra-embryonic tissues of the seed, allowing autotrophic seedling establishment [1, 2]. Seedling survival depends upon the presence of an intact seedling cuticle that prevents dehydration, which has hitherto been assumed to form the interface between the newly germinated seedling and its environment [3-5]. Here, we show that in Arabidopsis, this is not the case. The primary interface between the emerging seedling and its environment is formed by an extra-cuticular endosperm-derived glycoprotein-rich structure called the sheath, which is maintainedas a continuous layer at seedling surfaces during germination and becomes fragmented as cotyledons expand. Mutants lacking an endosperm-specific cysteine-rich peptide (KERBEROS [KRS]) show a complete loss of sheath production [6]. Although krs mutants have no defects in germinationsensu stricto, they show delayed cotyledonemergence, a defect not observed in seedlingswith defects in cuticle biosynthesis. Biophysical analyses reveal that the surfaces of wild-type cotyledons show minimal adhesion to silicabeads in an aqueous environment at cotyledon emergence but that adhesion increases as cotyledons expand. In contrast, krs mutant cotyledons show enhanced adhesion at germination. Mutantswith defects in cuticle biosynthesis, but no sheath defects, show a similar adhesion profile to wild-type seedlings at germination. We propose that the sheath reduces the adhesiveness of thecotyledon surface under the humid conditions necessary for seed germination and thus promotesseed-coat shedding and rapid seedling establishment.

A Possible Role of the Aleurone Expressed Gene HvMAN1 in the Hydrolysis of the Cell Wall Mannans of the Starchy Endosperm in Germinating Hordeum vulgare L. Seeds.

The barley endo-β-mannanase (MAN) gene family (HvMAN1-6) has been identified and the expression of its members analyzed throughout different plant organs, and upon grain development and germination. The HvMAN1 gene has been found to be highly expressed in developing and germinating grains. The MAN (EC 3.2.1.78) enzymatic activity gets a maximum in grains at 48 h of germination (post-germination event). Immunolocalization of mannan polymers in grains has revealed the presence of these polysaccharides in the endosperm cell walls (CWs). By mRNA in situ hybridization assays, the HvMAN1 transcripts have been localized to the aleurone layer, but not to the dead starchy endosperm cells. These data suggest that MAN1 is synthesized in the aleurone layer during early grain imbibition and moves potentially through the apoplast to the endosperm where the hydrolysis of the mannan polymers takes place after germination sensu stricto. Hence, mannans in the starchy endosperm CWs, besides their structural function, could be used as reserve compounds upon barley post-germination.

A molecular framework for the embryo growth in germinating seeds of Solanum lycocarpum A. St.‐Hil., a nurse plant species

AbstractWe are clarifying how the functional embryo growth occurs in germinating seeds of Solanum lycocarpum A. St.‐Hil., a nurse plant with a central role in forest dynamics in the Cerrado (a biodiversity hotspot). For that, we used classical seed germination measurements (germinability, mean germination time, mean germination rate, coefficient of variation of the germination time, synchronisation index and germination time range) and gene expression of mRNA codifying key proteins/enzymes for the success in the seed–seedling transition (Cyclin, Actin, Small Heat Shock Protein, Glutathione S‐transferase, Malate Dehydrogenase, Alcohol Dehydrogenase). Our findings demonstrate: (a) Although germination kinetics in S. lycocarpum seeds is slower than that in tomato seeds, the fold change of genes codifying key enzymes for the embryo development is similar in germinating seeds of both species. (b) The genes used here are useful, from a technical point of view, for classifying commercial seed samples of the species in relation to physiological quality. More notably, cyclin and malate dehydrogenase genes have a greater expression, both in germination sensu stricto and in immediate post‐germination. (c) A molecular framework for the embryo growth in germinating seeds of S. lycocarpum can be a functional explication for the species to be a nurse plant. Thus, the overlapping of classical and contemporary measurements is especially interesting to those species playing a central role in the environment, such as nurse plants, and may represent a new conservationist paradigm.

Germinative and Post-Germinative Behaviours of Brassica napus Seeds Are Impacted by the Severity of S Limitation Applied to the Parent Plants.

In oilseed rape (Brassica napus L.), sulphur (S) limitation leads to a reduction of seed yield and nutritional quality, but also to a reduction of seed viability and vigour. S metabolism is known to be involved in the control of germination sensu stricto and seedling establishment. Nevertheless, how the germination and the first steps of plant growth are impacted in seeds produced by plants subjected to various sulphate limitations remains largely unknown. Therefore, this study aimed at determining the impact of various S-limited conditions applied to the mother plants on the germination indexes and the rate of viable seedlings in a spring oilseed rape cultivar (cv. Yudal). Using a 34S-sulphate pulse method, the sulphate uptake capacity during the seedling development was also investigated. The rate of viable seedlings was significantly reduced for seeds produced under the strongest S-limited conditions. This is related to a reduction of germination vigour and to perturbations of post-germinative events. Compared to green seedlings obtained from seeds produced by well-S-supplied plants, the viable seedlings coming from seeds harvested on plants subjected to severe S-limitation treatment showed nonetheless a higher dry biomass and were able to enhance the sulphate uptake by roots and the S translocation to shoots.

Integrating proteomics and enzymatic profiling to decipher seed metabolism affected by temperature in seed dormancy and germination

Temperature is an important environmental factor affecting seed dormancy and germination. The mechanism by which temperature induces germination in dormant seeds is however still unclear. Proteomic study has been performed in dormant sunflower seeds during imbibition at permissive and non-permissive temperatures for germination, 20 and 10 °C, respectively. Proteome analysis showed an increase of proteins belonging to metabolism and energy from the first hours of imbibition followed by a decrease of proteins involved in protein metabolism and seed storage in germinating compared to non-germinating seeds. Proteomic study was completed by polysome and proteasome activity assessment and enzymatic profiling on several altered proteins involved in metabolism and energy. Results showed that 20 °C treatment induced the activation of both protein synthesis and degradation processes, the latter being related to proteasome activity during the germination sensu stricto, and to other degradation processes such as proteases during the post-germination. Interestingly, enzymatic profiles showed that TCA cycle and glycolysis were more active in non-germinating seeds in the phase I of the germination sensu stricto. This result suggests the regulation of central metabolism activity in germinating seeds. The control of energy production during imbibition seems to be involved in molecular networks controlling seed dormancy and germination.

Nitric oxide-polyamines cross-talk during dormancy release and germination of apple embryos

Nitric oxide (NO) and polyamines (PAs) belong to plant growth and development regulators. These compounds play a key role in numerous physiological processes e.g. seed germination. Based on the suggestion of overlapping of NO and PAs biosynthetic pathways, we demonstrated a cross-talk of NO and PAs in regulation of embryonic dormancy release. The aim of the work was to investigate an impact of PAs (Put, Spd and Spm) or NO short-term fumigation on nitrite, urea, Arg and ornithine (Orn) content, NO synthase-like (NOS-like) and arginase activity in axes of apple (Malus domestica Borkh.) embryos during dormancy alleviation and at the stage of termination of germination sensu stricto. NO, Put/Spd induced dormancy breakage and germination of apple embryos corresponded to stimulation of urea cycle and high free Arg pool in seedlings roots. After two days of the culture Put and Spd stimulated Arg dependent NO formation, inhibition of which was observed after Spm application. Put or Spd application as well as NO short-term pretreatment of apple embryos influenced level of ubiquitin-conjugated proteins. Higher abundance of such modified proteins correlated well to the declined content of nitrated proteins, suggesting their important role in regulation of embryo germination. NO led to stimulation of embryos germination by increasing level of free PAs (mostly Put). While transcriptomic approach showed down regulation of Spm synthesis and up-regulation of Spm degradation by NO, confirming negative role of Spm over-accumulation in embryo dormancy removal. Our data clearly indicate positive relationship of NO-Put/Spd acting as dormancy removing factors.

Modification of the endogenous NO level influences apple embryos dormancy by alterations of nitrated and biotinylated protein patterns.

NO donors and Arg remove dormancy of apple embryos and stimulate germination. Compounds lowering NO level (cPTIO, L -NAME, CAN) strengthen dormancy. Embryo transition from dormancy state to germination is linked to increased nitric oxide synthase (NOS)-like activity. Germination of embryos is associated with declined level of biotin containing proteins and nitrated proteins in soluble protein fraction of root axis. Pattern of nitrated proteins suggest that storage proteins are putative targets of nitration. Nitric oxide (NO) acts as a key regulatory factor in removal of seed dormancy and is a signal necessary for seed transition from dormant state into germination. Modulation of NO concentration in apple (Malus domestica Borkh.) embryos by NO fumigation, treatment with NO donor (S-nitroso-N-acetyl-D,L-penicillamine, SNAP), application of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), N ω-nitro-L-arginine methyl ester (L-NAME), canavanine (CAN) or arginine (Arg) allowed us to investigate the NO impact on seed dormancy status. Arg analogs and NO scavenger strengthened embryo dormancy by lowering reactive nitrogen species level in embryonic axes. This effect was accompanied by strong inhibition of NOS-like activity, without significant influence on tissue NO2 (-) concentration. Germination sensu stricto of apple embryos initiated by dormancy breakage via short term NO treatment or Arg supplementation were linked to a reduced level of biotinylated proteins in root axis. Decrease of total soluble nitrated proteins was observed at the termination of germination sensu stricto. Also modulation of NO tissue status leads to modification in nitrated protein pattern. Among protein bands that correspond to molecular mass of approximately 95kDa, storage proteins (legumin A-like and seed biotin-containing protein) were identified, and can be considered as good markers for seed dormancy status. Moreover, pattern of nitrated proteins suggest that biotin containing proteins are also targets of nitration.

Shotgun proteomic analysis of soybean embryonic axes during germination under salt stress.

Seed imbibition and radicle emergence are generally less affected by salinity in soybean than in other crop plants. In order to unveil the mechanisms underlying this remarkable salt tolerance of soybean at seed germination, a comparative label-free shotgun proteomic analysis of embryonic axes exposed to salinity during germination sensu stricto (GSS) was conducted. The results revealed that the application of 100 and 200 mmol/L NaCl stress was accompanied by significant changes (>2-fold, P<0.05) of 97 and 75 proteins, respectively. Most of these salt-responsive proteins (70%) were classified into three major functional categories: disease/defense response, protein destination and storage and primary metabolism. The involvement of these proteins in salt tolerance of soybean was discussed, and some of them were suggested to be potential salt-tolerant proteins. Furthermore, our results suggest that the cross-protection against aldehydes, oxidative as well as osmotic stress, is the major adaptive response to salinity in soybean.

A review: what is the spermosphere and how can it be studied?

The spermosphere is the zone surrounding seeds where interactions between the soil, microbial communities and germinating seeds take place. The concept of the spermosphere is usually only applied during germination sensu stricto. Despite the transient nature of this very small zone of soil around the germinating seed, the microbial activities which occur there may have long-lasting impacts on plants. The spermosphere is indirectly characterized by either (i) seed exudates, which could be inhibitors or stimulators of micro-organism growth or (ii) the composition of the microbiome on and around the germinating seeds. The microbial communities present in the spermosphere directly reflect that of the germination medium or are host-dependent and influenced quantitatively and qualitatively by host exudates. Despite its strong impact on the future development of plants, the spermosphere remains little studied. This can be explained by the technical difficulties related to characterizing this concept due to its short duration, small size and biomass, and the number and complexity of the interactions that take place. However, recent technical methods, such as metabolite profiling, combining phenotypic methods with DNA- and RNA-based methods, could be used to investigate seed exudates, microbial communities and their interactions with the soil environment.

ABA‐stimulated SoDOG1 expression is after‐ripening inhibited during early imbibition of germinating Sisymbrium officinale seeds

DELAY OF GERMINATION 1 (AtDOG1) was the first gene identified as dormancy-associated, but its physiological role in germination is far from being understood. Here, an orthologue of AtDOG1 in Sisymbrium officinale (SoDOG1; KM009050) is being reported. Phylogenetically, the SoDOG1 gene is included into the dicotyledonous group together with DOG1 from Arabidopsis thaliana (EF028470), Brassica rapa (AC189537), Lepidium papillosum (JX512183, JX512185) and Lepidium sativum (GQ411192). The SoDOG1 expression peaked at the onset of the silique maturation stage and there was presence of SoDOG1-mRNA in the freshly collected viable dry seed (i.e. AR0). The SoDOG1 transcripts were also found in other organs, such as open and closed flowers and to a lesser degree in roots and stems. We have previously reported in S. officinale seeds in which sensu stricto germination is positively affected by nitrate and both testa and micropylar endosperm ruptures are temporally separated. In dry viable seeds, the SoDOG1-mRNA level in three different after-ripening (AR) status was AR0 ≈ AR7 (optimal AR) < AR27 (optimal AR was almost lost). The presence of nitrate in the AR0 seed imbibition medium markedly decreased the SoDOG1 expression during sensu stricto germination. However, the nitrate stimulated the SoDOG1 expression during imbibition of AR7 compared to AR0. At the early AR0 seed imbibition (3-6 h), exogenous ABA provoked a very strong stimulation of the SoDOG1 expression. AR inhibits ABA-induced SoDOG1 expression during early germination and gibberellins (GA) can partially mimic this AR effect. A view on the integration of all found results in the sensu stricto germination of S. officinale was conducted.

Fate of nickel and calcium in seedlings of the hyperaccumulator Berkheya coddii during germination

Little is known about Ni storage in seeds of hyperaccumulating plants and its possible role in the first stages of plant development. The aim of this study was to determine Ni distribution in seeds and seedlings during germination and to test its role during germination with and without an external Ni supply. Field-harvested seeds from the South African Ni-hyperaccumulator Berkheya coddii Roessler were germinated either in Ni-free deionised water or in ultramafic soil. Sections of seeds and seedlings were analyzed using micro-proton induced X-ray emission (micro-PIXE) in order to localise Ni and other elements. Results show that high amounts of Ni were stored within the seeds. In germinating seeds, Ni was located in different parts: the lower epidermis, margins of cotyledons, and the pericarp in the micropylar area. The Ni and Ca were not mobilised during germination sensu stricto. Emergence of the first leaf seemed to trigger the translocation of Ni and Ca within the seedling. Besides, no effect of Ni supply from soil on its redistribution could be established for the germination stage.

Mannans and endo-β-mannanases (MAN) in Brachypodium distachyon: expression profiling and possible role of the BdMAN genes during coleorhiza-limited seed germination.

Immunolocalization of mannans in the seeds of Brachypodium distachyon reveals the presence of these polysaccharides in the root embryo and in the coleorhiza in the early stages of germination (12h), decreasing thereafter to the point of being hardly detected at 27h. Concurrently, the activity of endo-β-mannanases (MANs; EC 3.2.1.78) that catalyse the hydrolysis of β-1,4 bonds in mannan polymers, increases as germination progresses. The MAN gene family is represented by six members in the Brachypodium genome, and their expression has been explored in different organs and especially in germinating seeds. Transcripts of BdMAN2, BdMAN4 and BdMAN6 accumulate in embryos, with a maximum at 24-30h, and are detected in the coleorhiza and in the root by in situ hybridization analyses, before root protrusion (germination sensu stricto). BdMAN4 is not only present in the embryo root and coleorhiza, but is abundant in the de-embryonated (endosperm) imbibed seeds, while BdMAN2 and BdMAN6 are faintly expressed in endosperm during post-germination (36-42h). BdMAN4 and BdMAN6 transcripts are detected in the aleurone layer. These data indicate that BdMAN2, BdMAN4 and BdMAN6 are important for germination sensu stricto and that BdMAN4 and BdMAN6 may also influence reserve mobilization. Whether the coleorhiza in monocots and the micropylar endosperm in eudicots have similar functions, is discussed.