Endogenous auxin accumulation/localization during zygotic and somatic embryogenesis of Capsicum chinense Jacq.

Zygotic and somatic embryogenesis in plants is a fascinating event that is finely regulated through the expression of a specific group of genes and dynamic levels of plant hormones whose concerted action determines the fate that specific cells follow towards zygotic or somatic embryo development. This work studied different stages of Capsicum chinense Jacq. zygotic and somatic embryogenesis. HPLC quantification determined that the levels of indole-3-acetic acid (IAA) increase as the zygotic or somatic embryogenesis progresses, being higher at maturity, thus supporting a positive correlation between embryo cell differentiation and IAA increase. A monoclonal anti-IAA-antibody was used to detect IAA levels. Findings revealed a dynamic pattern of auxin distribution along the different embryogenic embryonic stages. In the early stages of zygotic embryos, the IAA gradient was observed in the basal cells of the suspensor and the hypostases, suggesting that they are the initial source of the IAA hormone. As embryogenesis proceeds, the dynamic of the IAA gradient is displaced to the embryo and endosperm cells. In the case of induced somatic embryogenesis, the IAA gradient was detected in the dividing cells of the endodermis, from where pre-embryogenic cells emerge. However, the analysis of somatic embryos revealed that IAA was homogeneously distributed. This study shows evidence supporting a correlation between IAA levels during zygotic or somatic embryogenesis in Capsicum chinense species.

Genes involved in the deformations of the shoot apical meristem in somatic embryos of Capsicum chinense Jacq.

Somatic embryos (SE) of habanero pepper (Capsicum chinense Jacq.) represent persistent deformations in the shoot apical meristem (SAM), which inhibits their capacity to form organs and subsequently plants. In dicotyledonous plants, SAM is formed in the apex, between cotyledons and it plays a central role in postembryonic shoot organ formation. Based on the previous knowledge on the role of some families of gene in the formation, organization and maintenance of the SAM, the expression patterns of WUS, WOX2, NAM, STM, PIN1 and PIN7 genes were analysed, which would allow us to elucidate the possible implication of these genes in SAM deformations in the SE of C. chinense. The results show that the expression patterns of STM and PIN1 in the SE were completely opposite to the respective expression pattern obtained in zygotic embryos (ZE). Moreover, NAM and PIN7 showed an over accumulation of transcripts in SE, compared with ZE. This is the first time in the genus Capsicum that alterations in the expression pattern of key genes of the SE development are reported, as well as its possible implication in the persistent deformations of the SAM.

Determination of n-alkanes in C. annuum (bell pepper) fruit and seed using GC-MS: comparison of extraction methods and application to samples of different geographical origin.

An efficient extraction and analysis method was developed for the isolation and quantification of n-alkanes from bell peppers of different geographical locations. Five extraction techniques, i.e., accelerated solvent extraction (ASE), ball mill extraction, ultrasonication, rinsing, and shaking, were quantitatively compared using gas chromatography coupled to mass spectrometry (GC-MS). Rinsing of the surface wax layer of freeze-dried bell peppers with chloroform proved to be a relatively quick and easy method to efficiently extract the main n-alkanes C27, C29, C31, and C33. A combined cleanup and fractionation approach on Teflon-coated silica SPE columns resulted in clean chromatograms and gave reproducible results (recoveries 90-95 %). The GC-MS method was reproducible (R(2) = 0.994-0.997, peak area standard deviation = 2-5%) and sensitive (LODs, S/N = 3, 0.05-0.15 ng/µL). The total main n-alkane concentrations were in the range of 5-50 µg/g dry weight. Seed extractions resulted in much lower total amounts of extracted n-alkanes compared to flesh and surface extractions, demonstrating the need for further improvement of pre-concentration and cleanup. The method was applied to 131 pepper samples from four different countries, and by using the relative n-alkane concentration ratios, Dutch peppers could be discriminated from those of the other countries, with the exception of peppers from the same cultivar. Graphical Abstract Procedure for pepper origin determination.

The pepper late embryogenesis abundant protein CaLEA1 acts in regulating abscisic acid signaling, drought and salt stress response.

As sessile organisms, plants are constantly challenged by environmental stresses, including drought and high salinity. Among the various abiotic stresses, osmotic stress is one of the most important factors for growth and significantly reduces crop productivity in agriculture. Here, we report a function of the CaLEA1 protein in the defense responses of plants to osmotic stress. Our analyses showed that the CaLEA1 gene was strongly induced in pepper leaves exposed to drought and increased salinity. Furthermore, we determined that the CaLEA1 protein has a late embryogenesis abundant (LEA)_3 homolog domain highly conserved among other known group 5 LEA proteins and is localized in the processing body. We generated CaLEA1-silenced peppers and CaLEA1-overexpressing (OX) transgenic Arabidopsis plants to evaluate their responses to dehydration and high salinity. Virus-induced gene silencing of CaLEA1 in pepper plants conferred enhanced sensitivity to drought and salt stresses, which was accompanied by high levels of lipid peroxidation in dehydrated and NaCl-treated leaves. CaLEA1-OX plants exhibited enhanced sensitivity to abscisic acid (ABA) during seed germination and in the seedling stage; furthermore, these plants were more tolerant to drought and salt stress than the wild-type plants because of enhanced stomatal closure and increased expression of stress-responsive genes. Collectively, our data suggest that CaLEA1 positively regulates drought and salinity tolerance through ABA-mediated cell signaling.

Microspore reprogramming to embryogenesis induces changes in cell wall and starch accumulation dynamics associated with proliferation and differentiation events.

Plant cell wall polymers are regulated during development, but the specific roles of their different molecular components and the functional meaning of cell wall changes in different cell types and cell processes are still unclear. In the present work the presence and distribution of different cell wall components in Capsicum annuum L. pollen have been analyzed in situ in order to monitor how they change during two developmental programs. These programs are: pollen development, which is a differentiation process, and stress-induced pollen reprogramming to embryogenesis, which involves proliferation followed later by differentiation processes. Specific antibodies recognizing the major cell wall polymers, the major hemicellulose, xyloglucan (XG), the rhamnogalacturonan II (RGII) pectin domain and high- and low-methyl-esterified pectins were used for both dot-blot and immunolocalization assays at light and electron microscopy levels during defined developmental stages. For comparison purposes, a similar approach was also used in zygotic embryogenesis and root apical tip growth. Results showed differences in the distribution pattern of these molecular complexes, in the proportion of esterified and de-esterified pectins in the two pollen developmental pathways, and defined wall changes during microspore reprogramming. These changes were associated with proliferation and differentiation events where highly esterified pectins were characteristic of proliferation, while de-esterified pectins, XG and RGII were abundant in walls of differentiating cells. Starch deposits were also studied and the results revealed changes in starch synthesis dynamics after switching the pollen embryogenic developmental program. These changes occurred together with modifications in the distribution patterns of cell wall polymers, starch accumulation being associated with cell differentiation. As in the case of proliferating cells, esterified pectins were also abundant in the apertures of developing microspores, regions of new cell wall formation. The different distribution patterns of cell wall polymers were common for proliferating cells and differentiating cells in all the plant systems analyzed, including zygotic embryos and root tip cells, suggesting that these patterns are markers of proliferation and differentiation events as well as markers of pollen reprogramming to embryogenesis.

Cell wall components and pectin esterification levels as markers of proliferation and differentiation events during pollen development and pollen embryogenesis in Capsicum annuum L.

Plant cell walls and their polymers are regulated during plant development, but the specific roles of their molecular components are still unclear, as well as the functional meaning of wall changes in different cell types and processes. In this work the in situ analysis of the distribution of different cell wall components was performed during two developmental programmes, gametophytic pollen development, which is a differentiation process, and stress-induced pollen embryogenesis, which involves proliferation followed by differentiation processes. The changes in cell wall polymers were compared with a system of plant cell proliferation and differentiation, the root apical meristem. The analysis was also carried out during the first stages of zygotic embryogenesis. Specific antibodies recognizing the major cell wall polymers, xyloglucan (XG) and the rhamnogalacturonan II (RGII) pectin domain, and antibodies against high- and low-methyl-esterified pectins were used for both dot-blot and immunolocalization with light and electron microscopy. The results showed differences in the distribution pattern of these molecular complexes, as well as in the proportion of esterified and non-esterified pectins in the two pollen developmental pathways. Highly esterified pectins were characteristics of proliferation, whereas high levels of the non-esterified pectins, XG and RGII were abundant in walls of differentiating cells. Distribution patterns similar to those of pollen embryos were found in zygotic embryos. The wall changes reported are characteristic of proliferation and differentiation events as markers of these processes that take place during pollen development and embryogenesis.

[Histological and cytological study on the ontogeny of embryoid in pepper anther culture].

The characteristics of histology and cytology of embryogenesis in pepper anther culture were examined with fluorescence microscopy, scanning microscopy, and electron microscopy. Pepper was characterized by a strong asynchrony of pollen development within a single anther. With the change of culture period, the proportion of dead pollen increased drastically from 2 day after culture. Microspores that were cultured at the late-uninucleate stage followed one of two developmental pathways. In the more common route, the first sporophytic division was asymmetric and produced what appeared to be typical bicellular pollen. Embryogenic pollen was formed by repeated divisions of the vegetative nuclei. An exine with its specific pattern had already been formed, when microspores were released from tetrads. During subsequent pollen development, microspores increased in size and continued to strengthen the exine. After 24 h in culture, the microspores had increased in size. Thereafter, embryogenesis was indicated in some microspores by two different morphological changes. One featured an expansion in volume of the cell cluster around the germination aperture, the other showed cell cluster volume expansion over the entire microspore surface. Morphogenesis of microspore-derived embryos has been analyzed, at both light and electron microscopical levels. The changes in cell organization after embryogenesis induction, and the characterization of the time sequence of a set of structural events, had been also explained. These changes mainly affected the plastids, the vacuolar compartment, the cell wall and the nucleus. Further differentiation processes mimicked that of the zygotic development.

Embryogenesis and plant regeneration of hot pepper (Capsicum annuum L.) through isolated microspore culture.

We report high frequencies of embryo production and plant regeneration through isolated microspore culture of hot pepper (Capsicum annuum L.). Microspores cultured in modified NLN medium (NLNS) divided and developed to embryos. Globular and heart-shaped embryos were observed from 3 weeks after the beginning of culture, and many embryos reached the cotyledonary stage after 4 weeks of culture. These cotyledonary embryos developed to plantlets after transfer to solid B5 basal medium. We also optimized conditions for embryo production by varying the pretreatment media, the carbon sources, and culture densities. Heat shock treatment in sucrose-starvation medium was more effective than in B5 medium. Direct comparisons of sucrose and maltose as carbon sources clearly demonstrated the superiority of sucrose compared to maltose, with the highest frequency of embryo production being obtained in 9% (w/v) sucrose. Microspore plating density was critical for efficient embryonic induction and development, with an optimal plating density of 8 x 10(4)-10 x 10(4)/ml. Under our optimized culture conditions, we obtained over 54 embryos, and an average of 5.5 cotyledonary embryos when 10 x 10(4) microspores were grown on an individual plate.

[Embryogenesis of microspore derived multicells in Capsicum annuum L].

Microspores and derived multicells were isolated and cultured in modified liquid CP medium after a 15d's preculture of anthers on solidified medium. Thirty days later in suspension culture, at 28 degrees C dark condition embryoids with different developmental stages were formed. Up to 22 embryoids could be formed from the cell suspension of 12 anthers, and about 23% of the embryoids were at the cotyledonary stage. Fluorescence and light microscope observations revealed that these embryoids derived from microspores. After several symmetrical division of the nuclei of uninucleated microspores, multi-nuclei cells or multi-cells were formed, and developed further into embryoids. There were white hairs on the surface of pepper embryoids, and some embryoids showed low vigor while others showed normal by TTC staining. Plants could be formed from torpedo and cotyledonary stage embryoids on solidified medium. Embryoids could be induced by 7 degrees C, 32 degrees C or 35 degrees C stress treatment on anthers, Higher embryogenesis frequencies were got at 7 degrees C and 35 degrees C condition in anther culture while 35 degrees C and 32 degrees C treatment showed a higher embryogenesis in isolated multicell culture. The reason of this result was discussed. There were obvious differences in embryogenesis frequency among different genotypes and different temperature stress conditions. Flow cytometric analysis revealed that there were haploidy, doubled haploidy and haploid-diploid chimera in the regenerated plants.

Pepper seed yield and quality in relation to fruit position on the mother plant.

Variation in seed yield and quality in relation to fruit position were determined in hot pepper cultivar during 2004 and 2005. Fruits were harvested from different layers (first, second and third) of the plant. In each layer, fruits were harvested 65 days after anthesis to obtain similar seed maturation stage. Fruit weight, seed yield, seed moisture content, germination at 15 and 25 degrees C, germination after controlled deterioration and mean germination time were determined. Fruit weight and seed yield gradually decreased throughout fructification, from the first to the third layer. Seed moisture content did not change between layers. Moisture remained at values of about 48-50%. The seeds extracted from fruits of the first layer had higher germination and vigour, lower mean germination time than those from the other layers. Seeds from the third layer caused decline in seed quality especially vigour with 69.3%.

A differentially expressed proteomic analysis in placental tissues in relation to pungency during the pepper fruit development.

Using proteomic analysis including 2-DE, image analysis, and protein identification with LC-MS/MS, an investigation aimed at a better understanding of the differentially expressed proteins and/or gene products was carried out with total cell extracts from placental tissues in nonpungent (Capsicum annuum cv. Saeng-Ryeog #213) and pungent peppers (C. annuum cv. Saeng-Ryeog #211). Mobilization of the most abundant proteins, which were on the gels of pH ranges of 4-7, 4.5-5.5, 5.5-6.7, and 6-9, and showed very similar profiles in the two tissues, revealing approximately 2600 protein spots consisting of 1200 on pH 4-7, 600 on 4.5-5.5, 550 on 5.5-6.7, 250 on 6-9. Of these, 37 protein spots, which appeared in only pungent tissues but not in nonpungent tissues or markedly increased in their staining intensities on the gels from pungent tissue, were selected, excised, in-gel trypsin digested, and analyzed by LC-ESI-MS/MS. Peptide MS/MS data were searched against publicly available protein and EST databases, and 22 proteins were identified. Based on this result, we tested and compared the differential expression during fruit development on the 2-DE gels with total cell extracts from placental tissues of pungent and nonpungent peppers at an interval of 10 days from 10 to 40 days after flowering. In addition, this differential protein expression was further confirmed for some subsets of candidates by Northern-blot analysis with RNA samples from placental tissues harvested from each pepper fruit at the same sampling intervals. In this study, the physiological implications, revealed from the experimental data in the levels of proteome and transcripts, are discussed in the context of a complex biosynthesis network of capsaicinoids in pepper cells responsive to pungency.

Successful development of a shed-microspore culture protocol for doubled haploid production in Indonesian hot pepper (Capsicum annuum L.).

Various systems of anther and microspore cultures were studied to establish an efficient doubled haploid production method for Indonesian hot pepper (Capsicum annuum L.). A shed-microspore culture protocol was developed which outperformed all the previously reported methods of haploid production in pepper. The critical factors of the protocol are: selection of flower buds with more than 50% late unicellular microspores, a 1 day 4 degrees C pretreatment of the buds, followed by culture of the anthers in double-layer medium system for 1 week at 9 degrees C and thereafter at 28 degrees C in continuous darkness. The medium contained Nitsch components and 2% maltose, with 1% activated charcoal in the solid under layer and 2.5 muM zeatin and 5 muM indole-3-acetic acid in the liquid upper layer. All the ten genotypes of hot pepper tested, responded to this protocol. The best genotypes produced four to seven plants per original flower bud. This protocol can be used as a potential tool for producing doubled haploid plants for hot pepper breeding.

Microspore-derived embryogenesis in pepper (Capsicum annuum L.): subcellular rearrangements through development.

Background information. In vitro-cultured microspores, after an appropriate stress treatment, can switch towards an embryogenic pathway. This process, known as microspore embryogenesis, is an important tool in plant breeding. Basic studies on this process in economically interesting crops, especially in recalcitrant plants, are very limited and the sequence of events is poorly understood. In situ studies are very convenient for an appropriate dissection of microspore embryogenesis, a process in which a mixture of different cell populations (induced and non-induced) develop asynchronically.Results. In the present study, the occurrence of defined subcellular rearrangements has been investigated during early microspore embryogenesis in pepper, an horticultural crop of agronomic interest, in relation to proliferation and differentiation events. Haploid plants of Capsicum annuum L. (var. Yolo Wonder B) have been regenerated from in vitro anther cultures by a heat treatment at 35 degrees C for 8 days. Morphogenesis of microspore-derived embryos has been analysed, at both light and electron microscopy levels, using low-temperature-processed, well-preserved specimens. The comparison with the normal gametophytic development revealed changes in cell organization after embryogenesis induction, and permitted the characterization of the time sequence of a set of structural events, not previously defined in pepper, related to the activation of proliferative activity and differentiation. These changes mainly affected the plastids, the vacuolar compartment, the cell wall and the nucleus. Further differentiation processes mimicked that of the zygotic development.Conclusions. The reported changes can be considered as markers of the microspore embryogenesis. They have increased the understanding of the mechanisms controlling the switch and progression of the microspore embryogenesis, which could help to improve its efficiency and to direct strategies, especially in agronomically interesting crops.

New in situ approaches to study the induction of pollen embryogenesis in Capsicum annuum L.

The induction of pollen embryogenesis in Capsicum annuum L. has been studied at the cellular level using various in situ approaches with several molecular probes for DNA, RNA and proteins. The late vacuolated microspore and the young bicellular pollen grain are stages of gametophytic development in which embryogenesis can be induced. Our results show that the late vacuolated microspore stage is most responsive to embryogenesis induction. The proliferating cell nuclear antigen (PCNA) has been immunolocalized at the electron microscopy level, in order to map replication sites in relation to the fine structure of chromatin. It shows different patterns of labelling at both developmental stages studied, revealing that the late vacuolated microspore is in a period of replication. Other in situ studies have been performed to characterize the state of nuclear activity at the specific developmental stages in which the embryogenic induction can occur. The modern in situ terminal-deoxy-nucleotidyl transferase (TdT) reaction for DNA, the immunolocalization of various nuclear antigens (as snRNPs, fibrillarin, RNA) and the ultrastructural in situ hybridization using 18S and 25S ribosomal probes provided valuable data bout the specific features displayed by the functional nuclear compartments of the microspore, and the young vegetative and generative cells. They are related not only to the state of gene activity but also with probably the ability to switch to the sporophytic pathway at specific developmental times of their gametophytic program.

The immunolocalization of nuclear antigens during the pollen developmental program and the induction of pollen embryogenesis.

The immunolocalization of nuclear antigens, combined with cytochemical procedures as well as in situ hybridization and recent in situ molecular methods, has been applied at different steps of pollen development to characterize the functional organization of the nucleus during the formation of the male gametophyte in an agronomically interesting plant, Capsicum annuum L. Pollen embryogenesis has been induced in pepper and the first stages of the process have been studied at the cellular level. Low temperature processing methods including cryosections and Lowicryl sections were very convenient for performing the various in situ techniques used in the pollen grains. Different molecular probes for localizing DNA, RNA, snRNPs, specific nucleolar proteins, various rRNA species, and DNA/RNA hybrids provided positive results in the pollen nuclei. The data obtained, and the changes observed in the organization of the nuclear compartments during pollen development, are related to the variations in gene activity undergone by the male gametophyte. The methodology used is proposed as a very convenient approach to localize molecules and events involved in the nuclear function in both gametophytic and sporophytic pollen development.