Is Autophagy Involved in Pepper Fruit Ripening?

Autophagy is a universal self-degradation process involved in the removal and recycling of cellular constituents and organelles; however, little is known about its possible role in fruit ripening, in which the oxidation of lipids and proteins and changes in the metabolism of different cellular organelles occur. In this work, we analyzed several markers of autophagy in two critical maturation stages of pepper (Capsicum annuum L.) fruits where variations due to ripening become clearly visible. Using two commercial varieties that ripen to yellow and red fruits respectively, we studied changes in the gene expression and protein content of several autophagy (ATG) components, ATG4 activity, as well as the autophagy receptor NBR1 and the proteases LON1 and LON2. Additionally, the presence of intravacuolar vesicles was analyzed by electron microscopy. Altogether, our data reveal that autophagy plays a role in the metabolic changes which occur during ripening in the two studied varieties, suggesting that this process may be critical to acquiring final optimal quality of pepper fruits.

Holistic protocol for callus culture optimization using statistical modelling.

Plants endue a key role against illnesses caused by oxidative stress. These attributes are frequently associated with polyphenolic compounds. However, presence and concentration of secondary metabolites are affected by abiotic factors. The in vitro culture techniques can solve these drawbacks. Peppers can be a suitable alternative to obtain polyphenols. Aiming to optimise the callus culture stage from Capsicum baccatum to produce polyphenols, this work evaluated systemically the effects of the explant's origin (root, hypocotyl and cotyledon), growth hormone type (2,4-dichlorophenoxyacetic acid (2,4-D), benzylaminopurine (BAP) and a combination of 2,4-D/BAP at five-to-one ratio) and concentration (0.023-10.000 mg L-1) on callus culture efficiency parameters using a multilevel factorial design. The root explant in combination with BAP at 1.138 mg L-1 ensured the optimal values of the assessed responses; ​callus mass (225.03 mg), antioxidant activity (35.95%), total phenols (11.48 mg of GAE/g DE) and flavonoids (15.92 mg of RU/g DE) production.

Impedance Flow Cytometry: A Novel Technique in Pollen Analysis.

INTRODUCTION: An efficient and reliable method to estimate plant cell viability, especially of pollen, is important for plant breeding research and plant production processes. Pollen quality is determined by classical methods, like staining techniques or in vitro pollen germination, each having disadvantages with respect to reliability, analysis speed, and species dependency. Analysing single cells based on their dielectric properties by impedance flow cytometry (IFC) has developed into a common method for cellular characterisation in microbiology and medicine during the last decade. The aim of this study is to demonstrate the potential of IFC in plant cell analysis with the focus on pollen. METHOD: Developing and mature pollen grains were analysed during their passage through a microfluidic chip to which radio frequencies of 0.5 to 12 MHz were applied. The acquired data provided information about the developmental stage, viability, and germination capacity. The biological relevance of the acquired IFC data was confirmed by classical staining methods, inactivation controls, as well as pollen germination assays. RESULTS: Different stages of developing pollen, dead, viable and germinating pollen populations could be detected and quantified by IFC. Pollen viability analysis by classical FDA staining showed a high correlation with IFC data. In parallel, pollen with active germination potential could be discriminated from the dead and the viable but non-germinating population. CONCLUSION: The presented data demonstrate that IFC is an efficient, label-free, reliable and non-destructive technique to analyse pollen quality in a species-independent manner.

Novel genetic male sterility developed in (Capsicum annuum x C. chinense) x C. pubescens and induced by HNO2 showing Mendelian inheritance and aborted at telophase of microspore mother cell stage.

A novel genetic male sterile germplasm was developed by successively crossing of (C. annuum x C. chinense) x C. pubescens and by chemical mutagenesis in pepper. The sterile anthers showed morphological abnormalities, but pistils developed normally with fine pollination capability. We investigated fertility segregation through sib-crossing of the same strains and test crossing by male sterile plants with 6 advanced inbred lines. The results showed that male fertility in the pepper was dominant in the F1 generation and segregated at a rate of 3:1 in the F2 generation, suggesting that monogenic male sterility was recessive and conformed to Mendelian inheritance. Cyto-anatomy analysis revealed that microspore abortion of sterile anthers occurred during telophase in the microspore mother cell stage when tapetal cells showed excessive vacuolation, resulting in occupation of the loculi. The microspore mother cells self-destructed and autolyzed with the tapetum so that meiosis in pollen mother cells could not proceed past the tetrad stage.

Efficacy of bioconversion of paper mill bamboo sludge and lime waste by composting and vermiconversion technologies.

Paper mill bamboo sludge (PMBS) and Paper mill lime waste (PMLW) are extensively produced as solid wastes in paper mills. Untreated PMBS and PMLW contain substantial amount of heavy metals (Zn, Pb, Ni, Cd, Cr) in soluble forms. Efficiency of vermiconversion and aerobic composting with these wastes is reported here. Adopted bioconversion systems enhanced the availability of some essential nutrients (N, P, K and Zn) in various combinations of cow dung (CD) with PMBS and PMLW. Colonization of nitrogen fixing bacteria and phosphate solubilizing bacteria considerably intensified under the vermiconversion system. Moreover, significant metal detoxification occurred due to vermiconversion. Various combinations of bioconverted PMBS and PMLW were applied to tissue cultured bamboo (Bambusa tulda) and chilli (Capsicum annum). Accelerated nutrient uptake coupled with improved soil quality resulted in significant production of chilli. Furthermore, vermiconverted PMBS+CD (1:1) and PMLW+CD (1:3) confirmed as potential enriching substrate for tissue cultured bamboo.

Androgenesis in recalcitrant solanaceous crops.

Tomato, eggplant, and pepper are three solanaceous crops of outstanding importance worldwide. For hybrid seed production in these species, a fast and cheap method to obtain pure (homozygous) lines is a priority. Traditionally, pure lines are produced by classical inbreeding and selection techniques, which are time consuming (several years) and costly. Alternatively, it has become possible to accelerate the production of homozygous lines through a biotechnological approach: the induction of androgenesis to generate doubled haploid (homozygous) plants. This biotechnological in vitro tool reduces the process to only one generation, which implies important time and costs savings. These facts make androgenic doubled haploids the choice in a number of important crops where the methodology is well set up. Unfortunately, recalcitrant solanaceous crops such as tomato, eggplant, and pepper are still far from an efficient and reliable technology to be applied on a routine basis to different genotypes in breeding programs. In eggplant and pepper, only anther cultures are known to work relatively well. Unfortunately, a more efficient and promising technique, the culture of isolated microspores, is not sufficiently developed yet. In tomato, none of these methods is available nowadays. However, recent advances in the knowledge of embryo development are filling the gaps and opening new ways to achieve the final goal of an efficient protocol in these three recalcitrant species. In this review, we outline the state of the art on androgenic induction in tomato, eggplant, and pepper, and postulate new experimental ways in order to overcome current limitations.

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.

A novel pepper (Capsicum annuum) receptor-like kinase functions as a negative regulator of plant cell death via accumulation of superoxide anions.

Plant receptor-like kinases belong to a large gene family. The Capsicum annuum receptor-like kinase 1 (CaRLK1) gene encodes a transmembrane protein with a cytoplasmic kinase domain and an extracellular domain. The CaRLK1 extracellular domain (ECD)-green fluorescent protein (GFP) fusion protein was targeted to the plasma membrane, and the kinase domain of the CaRLK1 protein exhibited autophosphorylation activity. CaRLK1 transcripts were more strongly induced in treatment with Xag8ra than in treatment with Xag8-13. Furthermore, infection with incompatible Xanthomonas campestris pv. vesicatoria race 3 induced expression of CaRLK1 more strongly than in the compatible interaction. Cell death caused by both a disease-forming and an HR-inducing pathogen was delayed in the CaRLK1-transgenic plants. Ectopic expression of CaRLK1 also induced transcripts of the lesion stimulating disease (LSD) gene, a negative regulator of cell death. Respiratory burst oxidase homolog (RBOH) genes were up-regulated in the transgenic plants compared with the wild type, as the concentration of the superoxide anion was increased. In contrast, the concentration of H(2)O(2) did not differ between the transgenic and wild-type plants. These results support the theory that the suppression of plant cell death by CaRLK1 is associated with consistent production of the superoxide anion and induction of the RBOH genes and the LSD gene, but not with the concentration of H(2)O(2). Thus, CaRLK1 may be a receptor of an as yet unidentified pathogen molecular pattern and may function as a negative regulator of plant cell death.

[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.

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.

Biotransformation of protocatechuic aldehyde and caffeic acid to vanillin and capsaicin in freely suspended and immobilized cell cultures of Capsicum frutescens.

Freely suspended cells and immobilized cell cultures of Capsicum frutescens Mill. were treated with phenylpropanoid intermediates--protocatechuic aldehyde and caffeic acid to study their biotransformation ability. It was found that externally fed protocatechuic aldehyde and caffeic acids were biotransformed to vanillin and capsaicin. It was noted that this culture biotransformed externally fed protocatechuic aldehyde to vanillin more than its conversion to capsaicin, whereas, caffeic acid-treated cultures accumulated more capsaicin than vanillin. The maximum accumulation of vanillin (5.63 mg l(-1)) and capsaicin (3.83 mg l(-1)) was recorded on the 6th and 15th day, respectively in immobilized C. frutescens cell cultures treated with protocatechuic aldehyde, which was 1.8 and 1.4 times higher than in protocatechuic aldehyde-treated freely suspended cell cultures. Caffeic acid-treated immobilized C. frutescens cell cultures accumulated maximum vanillin and capsaicin at 2.68 and 3.03 mg l(-1) culture, respectively, on the 9th and 12th day, which was 1.65 and 1.33 times over freely suspended cultures treated with caffeic acid. The addition of S-adenosyl-L-methionine, a methyl donor, to protocatechuic aldehyde-treated immobilized C. frutescens cell cultures, resulted in accumulation of vanillin (14.08 mg l(-1)) on the 4th day, which was 2.5-fold higher than that in cultures treated with protocatechuic aldehyde alone, suggesting the influence of S-adenosyl-L-methionine on O-methylation of protocatechuic aldehyde, resulting in more vanillin accumulation. The increase in vanillin accumulation was well correlated with an increase in specific activity of caffeic acid O-methyltransferase in protocatechuic aldehyde and S-adenosyl-L-methionine-treated immobilized C. frutescens cell cultures. This study also provides an example for an alternative route to formation of vanillin by C. frutescens cell cultures.

Computer-aided modeling and optimization for capsaicinoid production by immobilized Capsicum frutescens cells.

Production of capsaicinoids by immobilized cell cultures of Capsicum entrapped in calcium alginate matrix is influenced by several factors related to immobilization techniques, including media composition and the biosynthetic capability of cells. Central composite rotatable design (CCRD), one of the response surface methodologies, was used to arrive at optimal conditions for maximizing the yields of capsaicinoids in only 53 experiments, instead of 15,625. Immobilization of cells in 2.5% sodium alginate with 15 min contact time in 60 mM calcium chloride and culture in Murashige and Skoog medium supplemented with 3 mg l-1 2,4-dichlorophenoxyacetic acid and 0.2% curdlan gave the highest yield of 220 micrograms g-1 in 3 days.