Changing plastid dynamics within early root and shoot apical meristem-derived tissue of A. thaliana

Whilst plastids are fundamental to many aspects of plant biology and the production of enhanced crop cultivars, research into the dynamics of non-green plastids has remained somewhat disregarded by the scientific community compared to chloroplasts. They are equally pivotal to normal plant development however, and are now increasingly becoming the focus of research made possible by genetic manipulation and reporter gene constructs. The total plastid content of all plant cells originates from small, undifferentiated plastids termed proplastids found within the meristematic regions of both root and shoot tissue. The cellular regulatory mechanisms controlling the development of plastids in young tissues are poorly understood, especially in the case of non-green plastids in roots. This investigation consequently aimed to elucidate the differences in plastid content, morphology and subcellular localization within epidermal cells derived from the root and shoot apical meristems (RAM and SAM respectively) of Arabidopsis thaliana. Quantification of non-green plastids was facilitated via the use of confocal laser scanning microscopy in conjunction with the expression of plastid-targeted green fluorescent protein driven by a constitutive promoter. Characterization of early seedling development and tissue diversification was also achieved by assessing epidermal cell size relative to developmental progression, ultimately facilitating comparative analyses of plastid dynamics on both a temporal and tissue-varietal basis. The number of plastids in epidermal cells within RAM-derived tissue was shown to increase across regions of cell division before being regulated throughout subsequent zones of elongation and maturing root tissue. In contrast, epidermal cells of the hypocotyl exhibit a more generalized increase in plastid number and less strict maintenance of cell plan area coverage during tissue expansion. The findings presented here suggest the functioning of distinct mechanisms regulating plastid division and growth in relation to cell size within shoot and root apical meristem-derived tissues

Redox regulation of cell proliferation: Bioinformatics and redox proteomics approaches to identify redox-sensitive cell cycle regulators

Plant stem cells are the foundation of plant growth and development. The balance of quiescence and division is highly regulated, while ensuring that proliferating cells are protected from the adverse effects of environment fluctuations that may damage the genome. Redox regulation is important in both the activation of proliferation and arrest of the cell cycle upon perception of environmental stress. Within this context, reactive oxygen species serve as ‘pro-life’ signals with positive roles in the regulation of the cell cycle and survival. However, very little is known about the metabolic mechanisms and redox-sensitive proteins that influence cell cycle progression. We have identified cysteine residues on known cell cycle regulators in Arabidopsis that are potentially accessible, and could play a role in redox regulation, based on secondary structure and solvent accessibility likelihoods for each protein. We propose that redox regulation may function alongside other known posttranslational modifications to control the functions of core cell cycle regulators such as the retinoblastoma protein. Since our current understanding of how redox regulation is involved in cell cycle control is hindered by a lack of knowledge regarding both which residues are important and how modification of those residues alters protein function, we discuss how critical redox modifications can be mapped at the molecular level

Reverse breeding: a novel breeding approach based on engineered meiosis

Reverse breeding (RB) is a novel plant breeding technique designed to directly produce parental lines for any heterozygous plant, one of the most sought after goals in plant breeding. RB generates perfectly complementing homozygous parental lines through engineered meiosis. The method is based on reducing genetic recombination in the selected heterozygote by eliminating meiotic crossing over. Male or female spores obtained from such plants contain combinations of non-recombinant parental chromosomes which can be cultured in vitro to generate homozygous doubled haploid plants (DHs). From these DHs, complementary parents can be selected and used to reconstitute the heterozygote in perpetuity. Since the fixation of unknown heterozygous genotypes is impossible in traditional plant breeding, RB could fundamentally change future plant breeding. In this review, we discuss various other applications of RB, including breeding per chromosome

Human papillomavirus L1 protein expressed in tobacco chloroplasts self-assembles into virus-like particles that are highly immunogenic

Cervical cancer is the second most prevalent cancer in women worldwide. It is linked to infection with human papillomavirus (HPV). As the virus cannot be propagated in culture, vaccines based on virus‐like particles have been developed and recently marketed. However, their high costs constitute an important drawback for widespread use in developing countries, where the incidence of cervical cancer is highest. In a search for alternative production systems, the major structural protein of the HPV‐16 capsid, L1, was expressed in tobacco chloroplasts. A very high yield of production was achieved in mature plants (~3 mg L1/g fresh weight; equivalent to 24% of total soluble protein). This is the highest expression level of HPV L1 protein reported in plants. A single mature plant synthesized ~240 mg of L1. The chloroplast‐derived L1 protein displayed conformation‐specific epitopes and assembled into virus‐like particles, visible by transmission electron microscopy. Furthermore, leaf protein extracts from L1 transgenic plants were highly immunogenic in mice after intraperitoneal injection, and neutralizing antibodies were detected. Taken together, these results predict a promising future for the development of a plant‐based vaccine against HPV.This work was supported by Grants BIO2005‐00155 and CPE03‐022‐C5‐2 from the Ministerio de Educación y Ciencia and INIA (Spain)

Tobacco plastidial thioredoxins as modulators of recombinant protein production in transgenic chloroplasts

Thioredoxins (Trxs) are small ubiquitous disulphide proteins widely known to enhance expression and solubility of recombinant proteins in microbial expression systems. Given the common evolutionary heritage of chloroplasts and bacteria, we attempted to analyse whether plastid Trxs could also act as modulators of recombinant protein expression in transgenic chloroplasts. For that purpose, two tobacco Trxs (m and f) with different phylogenetic origins were assessed. Using plastid transformation, we assayed two strategies: the fusion and the co-expression of Trxs with human serum albumin (HSA), which was previously observed to form large protein bodies in tobacco chloroplasts. Our results indicate that both Trxs behave similarly as regards HSA accumulation, although they act differently when fused or co-expressed with HSA. Trxs-HSA fusions markedly increased the final yield of HSA (up to 26% of total protein) when compared with control lines that only expressed HSA; this increase was mainly caused by higher HSA stability of the fused proteins. However, the fusion strategy failed to prevent the formation of protein bodies within chloroplasts. On the other hand, the co-expression constructs gave rise to an absence of large protein bodies although no more soluble HSA was accumulated. In these plants, electron micrographs showed HSA and Trxs co-localization in small protein bodies with fibrillar texture, suggesting a possible influence of Trxs on HSA solubilization. Moreover, the in vitro chaperone activity of Trx m and f was demonstrated, which supports the hypothesis of a direct relationship between Trx presence and HSA aggregates solubilization in plants co-expressing both proteins. © 2011 The Authors. Plant Biotechnology Journal © 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.Authors wish to thank MJ Villafranca for excellent plant care and cultivation. This work was supported by grants Res. 17/2004 and IIM10865.RI1 (Proyecto EUROINNOVA) from Gobierno de Navarra (Spain). RSB and PCM were supported by predoctoral fellowships from CSIC and Generalitat Valenciana, respectively. AFS was supported by a postdoctoral fellowship from Public University of Navarra.Sanz-Barrio, R.; Fernandez-San Millan, A.; Corral Martínez, P.; Seguí-Simarro, JM.; Farran, I. (2011). Tobacco plastidial thioredoxins as modulators of recombinant protein production in transgenic chloroplasts. Plant Biotechnology Journal. 9(6):639-650. https://doi.org/10.1111/j.1467-7652.2011.00608.xS6396509

Arabidopsis R-SNARE Proteins VAMP721 and VAMP722 Are Required for Cell Plate Formation

Background: Cell plate formation during plant cytokinesis is facilitated by SNARE complex-mediated vesicle fusion at the cell-division plane. However, our knowledge regarding R-SNARE components of membrane fusion machinery for cell plate formation remains quite limited. Methodology/Principal Findings: We report the in vivo function of Arabidopsis VAMP721 and VAMP722, two closely sequence-related R-SNAREs, in cell plate formation. Double homozygous vamp721vamp722 mutant seedlings showed lethal dwarf phenotypes and were characterized by rudimentary roots, cotyledons and hypocotyls. Furthermore, cell wall stubs and incomplete cytokinesis were frequently observed in vamp721vamp722 seedlings. Confocal images revealed that green fluorescent protein-tagged VAMP721 and VAMP722 were preferentially localized to the expanding cell plates in dividing cells. Drug treatments and co-localization analyses demonstrated that punctuate organelles labeled with VAMP721 and VAMP722 represented early endosomes overlapped with VHA-a1-labeled TGN, which were distinct from Golgi stacks and prevacuolar compartments. In addition, protein traffic to the plasma membrane, but not to the vacuole, was severely disrupted in vamp721vamp722 seedlings by subcellular localization of marker proteins. Conclusion/Significance: These observations suggest that VAMP721 and VAMP722 are involved in secretory trafficking to the plasma membrane via TGN/early endosomal compartment, which contributes substantially to cell plate formatio

Nuclear translocation of haeme oxygenase-1 is associated to prostate cancer

The role of oxidative stress in prostate cancer has been increasingly recognised. Acute and chronic inflammations generate reactive oxygen species that result in damage to cellular structures. Haeme oxygenase-1 (HO-1) has cytoprotective effects against oxidative damage. We hypothesise that modulation of HO-1 expression may be involved in the process of prostate carcinogenesis and prostate cancer progression. We thus studied HO-1 expression and localisation in 85 samples of organ-confined primary prostate cancer obtained via radical prostatectomy (Gleason grades 4–9) and in 39 specimens of benign prostatic hyperplasia (BPH). We assessed HO-1 expression by immunohistochemical staining. No significant difference was observed in the cytoplasmic positive reactivity among tumours (84%), non-neoplastic surrounding parenchyma (89%), or BPH samples (87%) (P=0.53). Haeme oxygenase-1 immunostaining was detected in the nuclei of prostate cancer cells in 55 of 85 (65%) patients but less often in non-neoplastic surrounding parenchyma (30 of 85, 35%) or in BPH (9 of 39, 23%) (P<0.0001). Immunocytochemical and western blot analysis showed HO-1 only in the cytoplasmic compartment of PC3 and LNCaP prostate cancer cell lines. Treatment with hemin, a well-known specific inducer of HO-1, led to clear nuclear localisation of HO-1 in both cell lines and highly induced HO-1 expression in both cellular compartments. These findings have demonstrated, for the first time, that HO-1 expression and nuclear localisation can define a new subgroup of prostate cancer primary tumours and that the modulation of HO-1 expression and its nuclear translocation could represent new avenues for therapy

Doubled haploidy in model and recalcitrant species

Doubled haploids (DHs) are powerful tools to reduce the time and costs needed to produce pure lines to be used in breeding programs. DHs are also useful for genetic mapping of complex qualitative traits, to avoid transgenic hemizygotes, for studies of linkage and estimation of recombination fractions, for screening of recessive mutants. These are just some of the advantages that make DH technology one of the most exciting fields of present and future plant biotechnology. All of the DH methods have model species where these technologies have been developed, or that respond every efficiently to their corresponding induction treatment. However, not all the species of economical/agronomical interest respond to these methodologies as they should be in order to obtain DHs on a routine basis. Indeed, many of them are still considered as low-responding or recalcitrant to these treatments, including many of the most important crops worldwide. Although many groups are making significant progresses in the understanding of these intriguing experimental pathways, little is known about the origin, causes and ways to overcome recalcitrancy. It would be very important to shed light on the particularities of recalcitrant species and the special conditions they need to be induced. In parallel, the knowledge gained from the study of basic aspects in model species could also be beneficial to overcome recalcitrancy. In this e-book, we present a compilation of different approaches leading to the generation of DHs in model and in recalcitrant species, and different studies on new and relevant aspects of this process, useful to extract common traits and features, to know better these processes, and eventually, to elucidate how to make DH technology more efficient