Applications of Cell Biology in Plant Biotechnology

Another research line of our group involves the application of the cell biology techniques available in our Laboratory, to specific problems related to plant biology and biotechnology. In particular, our group has years of experience in the application of techniques such as in vitro culture, flow cytometry, light and fluorescence microscopy, transmission and scanning electron microscopy to the study of plant material. We are also experts in analytic techniques based on microscopy, such as immunolocalization (immunohistochemistry and immunocytochemistry), fluorescence (FISH) and ultrastructural (electron microscopy) in situ hybridization. We also handle image analysis, quantification and three-dimensional reconstruction techniques based on microscopy, such as stereology, three-dimensional reconstruction and modeling of serial sections, or high-resolution electron tomography.

In our laboratory we have established a number of scientific collaborations with research groups within and outside Spain, centered on the use of these techniques on the following topics:


Following this philosophy, we are open to any proposal for collaboration with other groups, beneficial in scientific terms to all participants.

 

IN SITU IDENTIFICATION OF THERAPEUTIC BIOMOLECULES IN BIOFACTORY PLANTS

From 2007 we have been collaborating with various groups in the identification of various proteins of therapeutic interest in transgenic plant tissues and cells, using molecular in situ localization techniques. These proteins are produced by genetically modified plants, engineered for the production of these biomolecules. Our job includes their in situ detection in those tissues, cells or organelles responsible for their production. For example, in 2008 one of these works in collaboration with the Institute of Agrobiotechnology of the Public University of Navarra (Fernandez-San Millan et al., 2008) was published in a leading journal in the field of Plant Biotechnology (Plant Biotechnology Journal). This study demonstrated the immunogenicity of L1 proteins produced in tobacco plants. The L1 protein is a component of the capsid of human papilloma virus, responsible for women’s cervical cancer. In this work, transgenic tobacco plants expressing large amounts of L1 in their chloroplasts were obtained. Our collaboration was the identification, using specific antibodies, of this protein in the chloroplasts of transgenic plants (Figure 1).

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 Figure 1. Ultrastructural localization of L1 proteins in chloroplasts of transgenic tobacco leaves.

 

A similar methodological approach has recently allowed us to identify other recombinant proteins of therapeutic interest such as human serum albumin and cardiotrophin, or endogenous thioredoxins in transgenic tobacco chloroplasts. In addition, we have recently started another collaboration in this same topic with the ITQB of the Universidade Nova de Lisboa, Portugal.

 

     IN SITU LOCALIZATIONS OF PROTEINS INVOLVED ON PLANT PHYSIOLOGICAL OR PATHOLOGICAL PROCESSES

 

In situ localization of arginine decarboxylase (ADC) during organogenic nodule formation in hop (Humulus lupulus L.)

In this collaboration with researchers from the Plant Systems Biology Lab, Center for Biodiversity, Functional & Integrative Genomics (BioFIG), ICAT, University of Lisbon in Portugal, we identified and quantified the presence and abundance of the ADC enzyme in hop organogenic nodules at different stages of development, once induced in vitro (Figure 2). The results have been published in Plant Signaling & Behavior (Fortes et al., 2011).

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Figure 2. Structure of organogenic nodules (A), ultrastructural localization (B) of ADC by ADC immunolabeling with antibodies, and quantification of the signal (C) in different cellular compartments and organelles of nodular cells.

 

Ultrastructural characterization of infection and in situ localization of virus movement proteins that affect crops of agronomic interest: Melon Necrotic Spot Virus (MNSV)

Through this collaboration with the group of Molecular Plant Virology from the Institute of Molecular and Cellular Biology of Plants (IBMCP), we aim to shed light on the cellular and vascular transport of an important virus in agronomic terms, in order to gain knowledge to design future antiviral strategies based on inhibiting the movement of these viruses. Specifically, this collaborative study focuses on the effects of infection in melon plants with MNSV at the ultrastructural level (Figure 3), and the subcellular localization of movement (MPs) and capsid proteins of these viruses.

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Figure 3. Ultrastructural characterization of infection and in situ localization of the Melon Necrotic Spot Virus (MNSV).

 

HISTOLOGICAL AND ULTRASTRUCTURAL STUDIES IN DIFFERENT PLANT CELL TYPES AND SPECIES

We also collaborate with other groups in the histological and ultrastructural characterization of different plant cells, tissues and species, obtained under both in vitro and ex vitro conditions, in order to help to solve particular biotechnological problems. For instante, we collaborate with the Group of Plant Physiology of the ETSIAMN (Universitat Politècnica de València) in the characterization of saffron calli obtained through in vitro culture of corms.

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Figure 4. Histological organization of calli obtained through in Vitro culture of saffron corms.

 

 

References

 

Fernandez-San Millan, A., Ortigosa, S.M., Hervas-Stubbs, S., Corral-Martinez, P., Seguí-Simarro, J.M., Gaetan, J., Coursaget, P. y Veramendi, J. (2008). Human papillomavirus L1 protein expressed in tobacco chloroplasts self-assembles into virus-like particles that are highly immunogenic. Plant Biotechnol. J. 6, 427-441.

 

Fortes, A.M., Costa, J., Santos, F., Seguí-Simarro, J.M., Cordeiro, A., Palme, K., Altabella, T., Tiburcio, A.F. y Pais, M.S. (2011). Arginine Decarboxylase expression, polyamines biosynthesis and reactive oxygen species during organogenic nodule formation in hop. Plant Signaling and Behavior In press.

 

Sanz-Barrio, Ruth, Alicia Fernández-San Millán, Patricia Corral-Martínez, José M. Seguí-Simarro and Inmaculada Farran. (2011). Tobacco plastidial thioredoxins as modulators of recombinant protein production in transgenic chloroplasts. Plant Biotechnology Journal, 9 (6): 639-650.

 

Ruth Sanz-Barrio, Alicia Fernández-San Millán, Jon Carballeda, Patricia Corral-Martínez, José M. Seguí-Simarro and Inmaculada Farran (2011). Chaperone-like properties of tobacco plastid thioredoxins f and m. Journal of Experimental Botany, En prensa. doi: 10.1093/jxb/err282.

 

 

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Group of cell biology

  • e-mail: seguisim@btc.upv.es
  • Phone: (+34)963879047
                 (+34)963877000 ext 88472(Lab.)
  • Location: Building 8E - Access I -Floor 1 - Cell biology lab
  • CPI - Universidad Politécnica de Valencia