Commentary: Extracellular peptidase hunting for improvement of protein production in plant cells and roots

Agroinfiltration is used to treat plants with modified strains of Agrobacterium tumefaciens for the purpose of transient in planta expression of genes transferred from the bacterium. These genes encode valuable recombinant proteins for therapeutic or industrial applications. Treatment of large quantities of plants for industrial-scale protein production exposes bacteria (harboring genes of interest) to agroinfiltration medium that is devoid of nutrients and carbon sources for prolonged periods of time (possibly upwards of 24 h). Such conditions may negatively influence bacterial viability, infectivity of plant cells, and target protein production. Here, we explored the role of timing in bacterial culture preparation for agroinfiltration using mass spectrometry-based proteomics to define changes in cellular processes. We observed distinct profiles associated with bacterial treatment conditions and exposure timing, including significant changes in proteins involved in pathogenesis, motility, and nutrient acquisition systems as the bacteria adapt to the new environment. These data suggest a progression towards increased cellular remodelling over time. In addition, we described changes in growth- and environment-specific processes over time, underscoring the interconnectivity of pathogenesis and chemotaxis-associated proteins with transport and metabolism. Overall, our results have important implications for the production of transiently expressed target protein products, as prolonged exposure to agroinfiltration medium suggests remodelling of the bacterial proteins towards enhanced infection of plant cells.

Download Full-text

Cellular engineering of plant cells for improved therapeutic protein production

Plant Cell Reports ◽

10.1007/s00299-021-02693-6 ◽

2021 ◽

Author(s):

Uddhab Karki ◽

Hong Fang ◽

Wenzheng Guo ◽

Carmela Unnold-Cofre ◽

Jianfeng Xu

Keyword(s):

Protein Production ◽

Plant Cells ◽

Therapeutic Protein ◽

Cellular Engineering

Download Full-text

Foreign protein production using plant cells: Opportunities and challenges

Biotechnology Advances ◽

10.1016/j.biotechadv.2011.08.002 ◽

2012 ◽

Vol 30 (2) ◽

pp. 385-386 ◽

Cited By ~ 2

Author(s):

Amanda M. Walmsley ◽

Pauline M. Doran

Keyword(s):

Protein Production ◽

Plant Cells ◽

Foreign Protein

Download Full-text

Gene Delivery into Plant Cells for Recombinant Protein Production

BioMed Research International ◽

10.1155/2015/932161 ◽

2015 ◽

Vol 2015 ◽

pp. 1-10 ◽

Cited By ~ 17

Author(s):

Qiang Chen ◽

Huafang Lai

Keyword(s):

Gene Delivery ◽

Recombinant Protein ◽

Recombinant Proteins ◽

Recombinant Protein Production ◽

Large Scale ◽

Protein Production ◽

Plant Cells ◽

High Volume ◽

Extensive Discussion ◽

Transgene Delivery

Recombinant proteins are primarily produced from cultures of mammalian, insect, and bacteria cells. In recent years, the development of deconstructed virus-based vectors has allowed plants to become a viable platform for recombinant protein production, with advantages in versatility, speed, cost, scalability, and safety over the current production paradigms. In this paper, we review the recent progress in the methodology of agroinfiltration, a solution to overcome the challenge of transgene delivery into plant cells for large-scale manufacturing of recombinant proteins. General gene delivery methodologies in plants are first summarized, followed by extensive discussion on the application and scalability of each agroinfiltration method. New development of a spray-based agroinfiltration and its application on field-grown plants is highlighted. The discussion of agroinfiltration vectors focuses on their applications for producing complex and heteromultimeric proteins and is updated with the development of bridge vectors. Progress on agroinfiltration inNicotianaand non-Nicotianaplant hosts is subsequently showcased in context of their applications for producing high-value human biologics and low-cost and high-volume industrial enzymes. These new advancements in agroinfiltration greatly enhance the robustness and scalability of transgene delivery in plants, facilitating the adoption of plant transient expression systems for manufacturing recombinant proteins with a broad range of applications.

Download Full-text

SEM study of the morphological effects of kinetin and zeatin on the growth and development of the apical meristem in wheat

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100141263 ◽

1995 ◽

Vol 53 ◽

pp. 978-979

Author(s):

G. M. Hutchins ◽

J. S. Gardner

Keyword(s):

Growth And Development ◽

Furfuryl Alcohol ◽

Apical Meristem ◽

Plant Cells ◽

Triticum Aestivum L ◽

Morphological Development ◽

Naturally Occurring ◽

Chinese Spring Wheat ◽

Sem Study ◽

Moist Environment

Cytokinins are plant hormones that play a large and incompletely understood role in the life-cycle of plants. The goal of this study was to determine what roles cytokinins play in the morphological development of wheat. To achieve any real success in altering the development and growth of wheat, the cytokinins must be applied directly to the apical meristem, or spike of the plant. It is in this region that the plant cells are actively undergoing mitosis. Kinetin and Zeatin were the two cytokinins chosen for this experiment. Kinetin is an artificial hormone that was originally extracted from old or heated DNA. Kinetin is easily made from the reaction of adenine and furfuryl alcohol. Zeatin is a naturally occurring hormone found in corn, wheat, and many other plants.Chinese Spring Wheat (Triticum aestivum L.) was used for this experiment. Prior to planting, the seeds were germinated in a moist environment for 72 hours.

Download Full-text

Confocal microscopy of the cytoskeleton in living plant cells following microinjection of fluorescent probes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100146497 ◽

1993 ◽

Vol 51 ◽

pp. 130-131

Author(s):

Ann Cleary

Keyword(s):

Cell Division ◽

Fluorescent Probes ◽

Actin Filaments ◽

Intracellular Transport ◽

Cell Structure ◽

Plant Cells ◽

Cell Plate ◽

Cell Cortex ◽

Living Plant ◽

Rhodamine Phalloidin

Microinjection of fluorescent probes into living plant cells reveals new aspects of cell structure and function. Microtubules and actin filaments are dynamic components of the cytoskeleton and are involved in cell growth, division and intracellular transport. To date, cytoskeletal probes used in microinjection studies have included rhodamine-phalloidin for labelling actin filaments and fluorescently labelled animal tubulin for incorporation into microtubules. From a recent study of Tradescantia stamen hair cells it appears that actin may have a role in defining the plane of cell division. Unlike microtubules, actin is present in the cell cortex and delimits the division site throughout mitosis. Herein, I shall describe actin, its arrangement and putative role in cell plate placement, in another material, living cells of Tradescantia leaf epidermis.The epidermis is peeled from the abaxial surface of young leaves usually without disruption to cytoplasmic streaming or cell division. The peel is stuck to the base of a well slide using 0.1% polyethylenimine and bathed in a solution of 1% mannitol +/− 1 mM probenecid.

Download Full-text

Scanning electron microscopy of plant cells using a variable-pressure SEM and cryogenic techniques

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100147144 ◽

1993 ◽

Vol 51 ◽

pp. 260-261

Author(s):

M. Yamada ◽

K. Ueda ◽

K. Kuboki ◽

H. Matsushima ◽

S. Joens

Keyword(s):

Electron Microscopy ◽

Scanning Electron Microscopy ◽

Saturated Vapor ◽

Plant Cells ◽

Variable Pressure ◽

Specimen Preparation ◽

Operating Pressure ◽

Vapor Pressures ◽

Low Temperature Microscopy ◽

Scanning Electron

Use of variable Pressure SEMs is spreading among electron microscopists The variable Pressure SEM does not necessarily require specimen Preparation such as fixation, dehydration, coating, etc which have been required for conventional scanning electron microscopy. The variable Pressure SEM allows operating Pressure of 1˜270 Pa in specimen chamber It does not allow microscopy of water-containing specimens under a saturated vapor Pressure of water. Therefore, it may cause shrink or deformation of water-containing soft specimens such as plant cells due to evaporation of water. A solution to this Problem is to lower the specimen temperature and maintain saturated vapor Pressures of water at low as shown in Fig. 1 On this technique, there is a Published report of experiment to have sufficient signal to noise ratio for scondary electron imaging at a relatively long working distance using an environmental SEM. We report here a new low temperature microscopy of soft Plant cells using a variable Pressure SEM (Hitachi S-225ON).

Download Full-text