Effects of hormonal priming on seed germination of pigeon pea under cadmium stress

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Germination of Cochlospermum regium Seeds: Influence of Seed Size, Vials, Vial Sealing In vitro, and Substrate In vivo

Aims: This work aimed to assess how seed size, vials, vial sealing (in vitro), and substrate (in vivo) affect C. regium germination and emergence. This study shall contribute to the viable production of C. regium seedlings. Study Design: The experimental design used in these experiments was randomized. Place and Duration of Study: Department of Plant Biotechnology, Universidade de RibeirãoPreto, between March 2010 and December 2010. Methodology: This work has evaluated how seed size, vials, vial sealing (in vitro), and substrate (in vivo) influence the germination and emergence of C. regium. Results: The results showed that cultivation of C. regium seedlings from seeds is viable, irrespective of seed size. Vial oxygenation is an important parameter to consider in vitro, to obtain a larger number of normal seedlings. As for in vivo conditions, germination should be conducted in sand, to ensure a greater amount of young seedlings. Conclusion: The results presented here attested that it is possible to produce C. regium seedlings from seeds of any size both in vivo and in vitro conditions. In vitro, it is important to consider vial oxygenation, in order to obtain a greater amount of normal seedlings. In vivo, germination should be conducted in sand, to ensure production of a large quantity of seedlings.

Isothermal seed germination of Adenanthera pavonina

(Isothermal seed germination of Adenanthera pavonina). This work reports aspects of seed germination at different temperatures of Adenanthera pavonina L., a woody Southeast Asian Leguminosae. Germination was studied by measuring the final percentages, the rate, the rate variance and the synchronisation of the individual seeds calculated by the minimal informational entropy of frequencies distribution of seed germination. Overlapping the germinability range with the range for the highest values of germination rates and the minimal informational entropy of frequencies distribution of seed germination, we found that the best temperature for the germination of A. pavonina seeds is 35 degrees C. The slope mu of the Arrhenius plot of the germination rates is positive for T < 35 degrees C and negative for T > 35 degrees C. The activation enthalpies, estimated from closely-spaced points, shows that vertical bar Delta H-vertical bar < 12 Cal mol(-1) occur for temperatures in the range between 25 degrees C and 40 degrees C. The ecological implication of these results are that this species may germinate very fast in tropical areas during the summer season. This may be an advantage to the establishment of this species under the climatic conditions in those areas.

Differential seed germination of a keystone palm (Euterpe edulis) dispersed by avian frugivores

FAPESP (BIOTA Program) [2007/03392-6, 2010/04927-3]

Isothermal seed germination of Adenanthera pavonina

This work reports aspects of seed germination at different temperatures of Adenanthera pavonina L., a woody Southeast Asian Leguminosae. Germination was studied by measuring the final percentages, the rate, the rate variance and the synchronisation of the individual seeds calculated by the minimal informational entropy of frequencies distribution of seed germination. Overlapping the germinability range with the range for the highest values of germination rates and the minimal informational entropy of frequencies distribution of seed germination, we found that the best temperature for the germination of A. pavonina seeds is 35 ºC. The slope µ of the Arrhenius plot of the germination rates is positive for T < 35 ºC and negative for T > 35 ºC. The activation enthalpies, estimated from closely-spaced points, shows that |ΔH-| < 12 Cal mol-1 occur for temperatures in the range between 25 ºC and 40 ºC. The ecological implication of these results are that this species may germinate very fast in tropical areas during the summer season. This may be an advantage to the establishment of this species under the climatic conditions in those areas.

Relationship between eggplant seed morphology and germination

Structural differences such as abnormalities, damage and free spaces in seeds may affect germination. The aim of this study was to study the relationship between eggplant seed morphology and seed germination. Ten seed lots of the eggplant cultivar Embu were evaluated by X-ray image analysis and the germination test. Seed image analysis was performed by Image Pro Plus® software and the whole seed area and free space between the embryo and endosperm were measured. The internal seed area filled by the embryo and endosperm was calculated from the difference between the whole seed and free space areas. Based on these results and visual seed analysis, seeds were classified into three categories and information on germination was obtained for each one. X-ray image analysis provides a perfect view of the internal seed parts and for seed morphology studies. An increase in seed area filled by the endosperm and embryo does not improve seed germination. Mechanical seed damage and deteriorated tissues can adversely affect seed germination.

Restoration of Cymodocea nodosa (Uchria) Ascherson seagrass prairies through seed propagation. Germination in vitro, plantlet acclimation and transplanting to natural meadows.

[EN] The seagrass Cymodocea nodosa (Ucria) Ascherson is the most abundant seagrass species in the Canary Islands (Spain), where it forms dense submerged, ecologically relevant communities as stable and protected habitats. As with other seagrasses, concern has arisen due to a decline in the number and extension of the communities as the result of adverse activities in coastal areas. Seed germination and planting are assumed as cost-effective method for restoration. In the frame of the restoration of natural populations of Cymodocea nodosa, pilot experiences not tested so far in the Canary Islands have been carried out to developed in vitro techniques to produce viable seedlings and its transference to the natural environment.

AtPTR1, a plasma membrane peptide transporter expressed during seed germination and in vascular tissue of Arabidopsis

For the efficient translocation of organic nitrogen, small peptides of two to three amino acids are posited as an important alternative to amino acids. A new transporter mediating the uptake of di- and tripeptides was isolated from Arabidopsis thaliana by heterologous complementation of a peptide transport-deficient Saccharomyces cerevisiae mutant. AtPTR1 mediated growth of S. cerevisiae cells on different di- and tripeptides and caused sensitivity to the phytotoxin phaseolotoxin. The spectrum of substrates recognized by AtPTR1 was determined in Xenopus laevis oocytes injected with AtPTR1 cRNA under voltage clamp conditions. AtPTR1 not only recognized a broad spectrum of di- and tripeptides, but also substrates lacking a peptide bond. However, amino acids, omega-amino fatty acids or peptides with more than three amino acid residues did not interact with AtPTR1. At pH 5.5 AtPTR1 had an apparent lower affinity (K-0.5 = 416 mum) for Ala-Asp compared with Ala-Ala (K-0.5 = 54 mum) and Ala-Lys (K-0.5 = 112 mum). Transient expression of AtPTR1/GFP fusion proteins in tobacco protoplasts showed that AtPTR1 is localized at the plasma membrane. In addition, transgenic plants expressing the beta-glucuronidase (uidA) gene under control of the AtPTR1 promoter demonstrated expression in the vascular tissue throughout the plant, indicative of a role in long-distance transport of di- and tripeptides.

Peptide and amino acid transporters are differentially regulated during seed development and germination in faba bean

Two peptide transporter (PTR) homologs have been isolated from developing seeds of faba bear, (Vicia faba). VfPTR1 was shown to be a functional peptide transporter through complementation of a yeast mutant. Expression patterns of VfPTR1 and VfPTR2 as well as of the amino acid permease VfAAP1 (Miranda et al., 2001) were compared throughout seed development and germination. In developing seeds, the highest levels of VfPTR1 transcripts were reached during midcotyledon development, whereas VfAAP1 transcripts were most abundant during early cotyledon development, before the appearance of storage protein gene transcripts, and were detectable until late cotyledon development. During early germination, VfPTR1 mRNA appeared first in cotyledons and later, during seedling growth, also in axes and roots. Expression of VfPTR2 and VfAAP1 was delayed compared with VfPTR1, and was restricted to the nascent organs of the seedlings. Localization of VfPTR1 transcripts showed that this FTR is temporally and spatially regulated during cotyledon development. In germinating seeds, VfPTR1 mRNA was localized in root hairs and root epidermal cells, suggesting a role in nutrient uptake from the soil. In seedling roots, VfPTR1 was repressed by a dipeptide and by an amino acid, whereas nitrate was without influence.

Flower and seed biomass, and germination rate of arctic tundra plants in response to long term experimental warming

We provide new information on changes in tundra plant sexual reproduction in response to long-term (12 years) experimental warming in the High Arctic. Open-top chambers (OTCs) were used to increase growing season temperatures by 1-2 °C across a range of vascular plant communities. The warming enhanced reproductive effort and success in most species; shrubs and graminoids appeared to be more responsive than forbs. We found that the measured effects of warming on sexual reproduction were more consistently positive and to a greater degree in polar oasis compared with polar semidesert vascular plant communities. Our findings support predictions that long-term warming in the High Arctic will likely enhance sexual reproduction in tundra plants, which could lead to an increase in plant cover. Greater abundance of vegetation has implications for primary consumers - via increased forage availability, and the global carbon budget - as a function of changes in permafrost and vegetation acting as a carbon sink. Enhanced sexual reproduction in Arctic vascular plants may lead to increased genetic variability of offspring, and consequently improved chances of survival in a changing environment. Our findings also indicate that with future warming, polar oases may play an important role as a seed source to the surrounding polar desert landscape.

Function of C1A barley peptidases and their inhibitors (cystatins) in barley seed germination : Funcion de las peptidasas C1A de cebada y sus inhibidores (cistatinas) en la germinacion de la semilla

Plant proteolysis is a metabolic process where specific enzymes called peptidases degrade proteins. In plants, this complex process involves broad metabolic networks and different sub-cellular compartments. Several types of peptidases take part in the proteolytic process, mainly cysteine-, serine-, aspartyl- and metallo- peptidases. Among the cysteine-peptidases, the papain-like or C1A peptidases (family C1, clan CA) are extensively present in land plants and are classified into catepsins L-, B-, H- and Flike. The catalytic mechanism of these C1A peptidases is highly conserved and involves the three amino acids Cys, His and Asn in the catalytic triad, and a Gln residue which seems essential for maintaining an active enzyme conformation. These proteins are synthesized as inactive precursors, which comprise an N-terminal signal peptide, a propeptide, and the mature protein. In barley, we have identified 33 cysteine-peptidases from the papain-like family, classifying them into 8 different groups. Five of them corresponded to cathepsins L-like (5 subgroups), 1 cathepsin B-like group, 1 cathepsin F-like group and 1 cathepsin H-like group. Besides, C1A peptidases are the specific targets of the plant proteinaceous inhibitors known as phytocystatins (PhyCys). The cystatin inhibitory mechanism is produced by a tight and reversible interaction with their target enzymes. In barley, the cystatin gene family is comprised by 13 members. In this work we have tried to elucidate the role of the C1A cysteine-peptidases and their specific inhibitors (cystatins) in the germination process of the barley grain. Therefore, we selected a representative member of each group/subgroup of C1A peptidases (1 cathepsin B-like, 1 cathepsin F-like, 1 cathepsin H-like and 5 cathepsins L-like). The molecular characterization of the cysteine-peptidases was done and the peptidase-inhibitor interaction was analyzed in vitro and in vivo. A study in the structural basis for specificity of pro-peptide/enzyme interaction in barley C1A cysteine-peptidases has been also carried out by inhibitory assays and the modeling of the three-dimensional structures. The barley grain maturation produces the accumulation of storage proteins (prolamins) in the endosperm which are mobilized during germination to supply the required nutrients until the photosynthesis is fully established. In this work, we have demonstrated the participation of the cysteine-peptidases and their inhibitors in the degradation of the different storage protein fractions (hordeins, albumins and globulins) present in the barley grain. Besides, transgenic barley plants overexpressing or silencing cysteine-peptidases or cystatins were obtained by Agrobacterium-mediated transformation of barley immature embryos to analyze their physiological function in vivo. Preliminary assays were carried out with the T1 grains of several transgenic lines. Comparing the knock-out and the overexpressing lines with the WT, alterations in the germination process were detected and were correlated with their grain hordein content. These data will be validated with the homozygous grains that are being produced through the double haploid technique by microspore culture. Resumen La proteólisis es un proceso metabólico por el cual se lleva a cabo la degradación de las proteínas de un organismo a través de enzimas específicas llamadas proteasas. En plantas, este complejo proceso comprende un entramado de rutas metabólicas que implican, además, diferentes compartimentos subcelulares. En la proteólisis participan numerosas proteasas, principalmente cisteín-, serín-, aspartil-, y metalo-proteasas. Dentro de las cisteín-proteasas, las proteasas tipo papaína o C1A (familia C1, clan CA) están extensamente representadas en plantas terrestres, y se clasifican en catepsinas tipo L, B, H y F. El mecanismo catalítico de estas proteasas está altamente conservado y la triada catalítica formada por los aminoácidos Cys, His y Asn, y a un aminoácido Gln, que parece esencial para el mantenimiento de la conformación activa de la proteína. Las proteasas C1A se sintetizan como precursores inactivos y comprenden un péptido señal en el extremo N-terminal, un pro-péptido y la proteína madura. En cebada hemos identificado 33 cisteín-proteasas de tipo papaína y las hemos clasificado filogenéticamente en 8 grupos diferentes. Cinco de ellos pertenecen a las catepsinas tipo L (5 subgrupos), un grupo a las catepsinas tipo-B, otro a las catepsinas tipo-F y un último a las catepsinas tipo-H. Las proteasas C1A son además las dianas específicas de los inhibidores protéicos de plantas denominados fitocistatinas. El mecanismo de inhibición de las cistatinas está basado en una fuerte interacción reversible. En cebada, se conoce la familia génica completa de las cistatinas, que está formada por 13 miembros. En el presente trabajo se ha investigado el papel de las cisteín-proteasas de cebada y sus inhibidores específicos en el proceso de la germinación de la semilla. Para ello, se seleccionó una proteasa representante de cada grupo/subgrupo (1 catepsina tipo- B, 1 tipo-F, 1 tipo-H, y 5 tipo-L, una por cada subgrupo). Se ha llevado a cabo su caracterización molecular y se ha analizado la interacción enzima-inhibidor tanto in vivo como in vitro. También se han realizado estudios sobre las bases estructurales que demuestran la especificidad en la interacción enzima/propéptido en las proteasas C1A de cebada, mediante ensayos de inhibición y la predicción de modelos estructurales de la interacción. Finalmente, y dado que durante la maduración de la semilla se almacenan proteínas de reserva (prolaminas) en el endospermo que son movilizadas durante la germinación para suministrar los nutrientes necesarios hasta que la nueva planta pueda realizar la fotosíntesis, en este trabajo se ha demostrado la participación de las cisteínproteasas y sus inhibidores en la degradación de las diferentes tipos de proteínas de reserva (hordeinas, albúmins y globulinas) presentes en el grano de cebada. Además, se han obtenido plantas transgénicas de cebada que sobre-expresan o silencian cistatinas y cisteín-proteasas con el fin de analizar la función fisiológica in vivo. Se han realizado análisis preliminares en las semillas T1 de varias líneas tránsgenicas de cebada y al comparar las líneas knock-out y las líneas de sobre-expresión con las silvestres, se han detectado alteraciones en la germinación que están además correlacionadas con el contenido de hordeinas de las semillas. Estos datos serán validados en las semillas homocigotas que se están generando mediante la técnica de dobles haploides a partir del cultivo de microesporas.

Molecular analysis of endo-β-mannanase genes upon seed imbibition suggest a cross-talk between radicle and micropylar endosperm during germination of Arabidopsis thaliana.

The endo-β-mannanase (MAN) family is represented in the Arabidopsis genome by eight members, all with canonical signal peptides and only half of them being expressed in germinating seeds. The transcripts of these genes were localized in the radicle and micropylar endosperm (ME) before radicle protrusion and this expression disappears as soon as the endosperm is broken by the emerging radicle tip. However, only three of these MAN genes, AtMAN5, AtMAN7 and especially AtMAN6 influence the germination time (t50) as assessed by the analysis of the corresponding knock-out lines. The data suggest a possible interaction between embryo and ME regarding the role of MAN during the Arabidopsis germination process.

Modelling seed germination in forest tree species through survival analysis. The Pinus pinea L. case study

The direct application of existing models for seed germination may often be inadequate in the context of ecology and forestry germination experiments. This is because basic model assumptions are violated and variables available to forest managers are rarely used. In this paper, we present a method which addresses the aforementioned shortcomings. The approach is illustrated through a case study of Pinus pinea L. Our findings will also shed light on the role of germination in the general failure of natural regeneration in managed forests of this species. The presented technique consists of a mixed regression model based on survival analysis. Climate and stand covariates were tested. Data for fitting the model were gathered from a 5-year germination experiment in a mature, managed P. pinea stand in the Northern Plateau of Spain in which two different stand densities can be found. The model predictions proved to be unbiased and highly accurate when compared with the training data. Germination in P. pinea was controlled through thermal variables at stand level. At microsite level, low densities negatively affected the probability of germination. A time-lag in the response was also detected. Overall, the proposed technique provides a reliable alternative to germination modelling in ecology/forestry studies by using accessible/ suitable variables. The P. pinea case study highlights the importance of producing unbiased predictions. In this species, the occurrence and timing of germination suggest a very different regeneration strategy from that understood by forest managers until now, which may explain the high failure rate of natural regeneration in managed stands. In addition, these findings provide valuable information for the management of P. pinea under climate-change conditions.

Arabidopsis thaliana bZIP44: a transcription factor affecting seed germination and expression of the mannanase-encoding gene AtMAN7.

Endo-β-mannanases (MAN; EC. 3.2.1.78) catalyze the cleavage of β1[RIGHTWARDS ARROW]4 bonds in mannan polymers and have been associated with the process of weakening the tissues surrounding the embryo during seed germination. In germinating Arabidopsis thaliana seeds, the most highly expressed MAN gene is AtMAN7 and its transcripts are restricted to the micropylar endosperm and to the radicle tip just before radicle emergence. Mutants with a T-DNA insertion in AtMAN7 have a slower germination than the wild type. To gain insight into the transcriptional regulation of the AtMAN7 gene, a bioinformatic search for conserved non-coding cis-elements (phylogenetic shadowing) within the Brassicaceae MAN7 gene promoters has been done, and these conserved motifs have been used as bait to look for their interacting transcription factors (TFs), using as a prey an arrayed yeast library from A. thaliana. The basic-leucine zipper TF AtbZIP44, but not the closely related AtbZIP11, has thus been identified and its transcriptional activation upon AtMAN7 has been validated at the molecular level. In the knock-out lines of AtbZIP44, not only is the expression of the AtMAN7 gene drastically reduced, but these mutants have a significantly slower germination than the wild type, being affected in the two phases of the germination process, both in the rupture of the seed coat and in the breakage of the micropylar endosperm cell walls. In the over-expression lines the opposite phenotype is observed.