Documente Academic
Documente Profesional
Documente Cultură
USA
Vol. 96, pp. 59735977, May 1999
Colloquium Paper
This paper was presented at the National Academy of Sciences colloquium Plants and Population: Is There Time?
held December 56, 1998, at the Arnold and Mabel Beckman Center in Irvine, CA.
ABSTRACT
Alternative agriculture, which expands the
uses of plants well beyond food and fiber, is beginning to
change plant biology. Two plant-based biotechnologies were
recently developed that take advantage of the ability of plant
roots to absorb or secrete various substances. They are (i)
phytoextraction, the use of plants to remove pollutants from
the environment and (ii) rhizosecretion, a subset of molecular
farming, designed to produce and secrete valuable natural
products and recombinant proteins from roots. Here we
discuss recent advances in these technologies and assess their
potential in soil remediation, drug discovery, and molecular
farming.
Biotechnology is transforming world agriculture, adding new
traits to crop plants at a greatly accelerated rate. Plants are
becoming more efficient producers of food, fiber, medicines,
and construction materials. In addition to these conventional
uses, biotechnology opens doors to unique uses of plants that
are gaining greater acceptance from the public and attention
from the scientific community. These so-called value-added
uses include phytoremediation, the use of plants to remove
pollutants from the environment or to render them harmless
(1), and molecular farming (phytomanufacturing), the use of
plants for the production of valuable organic molecules and
recombinant proteins (2, 3). Because of the growing number
of commercially successful applications and the lack of serious
environmental concerns, both technologies are gaining acceptance from the scientific community, the general public, and
regulators.
With the exception of root crops, plant roots are less utilized
and studied than shoots. However, this situation may be
changing because of the emerging biotechnologies described
below that exploit the ability of plants to transport valuable
molecules into and out of their roots. These root-based technologies include metal phytoextraction, a subset of phytoremediation, which uses plants to remove toxic heavy metals
from soil; and rhizosecretion, a subset of molecular farming,
which relies on the ability of plant roots to exude valuable
compounds. Both technologies exploit plants innate biological
mechanisms for human benefit.
Phytoextraction. Giant underground networks formed by
the roots of living plants function as solar-driven pumps that
extract and concentrate essential elements and compounds
from soil and water. Absorbed substances are used to support
reproductive function and carbon fixation within shoots. Metal
phytoextraction relies on metal-accumulating plants to transport and concentrate polluting metals, such as lead, uranium,
and cadmium, from the soil into the harvestable aboveground
shoots (1, 4, 5). Hydroponically grown plant roots can also
directly absorb, precipitate, and concentrate toxic metals from
polluted effluents in a process termed rhizofiltration (6).
D.G., N.V.B., L.G.B., R.K., A.P., and M.S. contributed equally to this
work.
aesop.rutgers.edu.
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FIG. 4. Rhizosecretion of jellyfish green fluorescent protein (GFP)(A), human placental alkaline phosphatase (SEAP)(B), and bacterial
(Clostridium thermocellum) xylanase (C) from the roots of transgenic Nicotiana tabacum L. (A) To direct GFP into the secretory pathway,
GFP-coding sequence was fused to the signal peptide derived from the resident ER protein calreticulin, and the resulting fusion placed in correct
orientation between the mannopine synthase (mas29) promoter (provided by Stanton Gelvin, Purdue University, West Lafayette, IN) and nos
terminator. GFP rhizosecretion from the hydroponically cultivated aseptic roots was visualized after illuminating the hydroponic medium contacting
roots with near-UV light. Media from nontransformed plants showed no fluorescence (data not shown). (B) Visualization of SEAP rhizosecretion
in the native gel. In transformed tobacco, coding sequence of SEAP with its own signal peptide was controlled by the cauliflower mosaic virus 35S
promoter (CaMV35S). Thirty micrograms of total protein concentrated from root exudates of transgenic and nontransformed plants was separated
on native PAGE, and SEAP activity was localized using the alkaline phosphatase isoenzymes procedure (Sigma). Lanes 1 and 2, transgenic tobacco
plants; lanes 3 and 4, nontransformed tobacco. (C) Rhizosecretion of bacterial xylanase from transgenic tobacco seedlings germinated on the
RBB-xylane-containing agar medium (dark blue), which becomes colorless when cleaved by xylanase (photographed upside down). Nontransformed
plants did not change the color of the medium (data not shown). Seeds of tobacco expressing a truncated C. thermocellum xylanase gene controlled
by the CaMV35S promoter and targeted to the apoplast by proteinase inhibitor II ER signal peptide were provided by Uwe Sonnewald.
protein (GFP) of the jellyfish Aequorea victoria, human placental secreted alkaline phosphatase (SEAP), and xylanase
from the thermophylic bacterium Clostridium thermocellum.
All three of these proteins were rhizosecreted from transgenic plants when their expression was controlled by a strong
root-expressed promoter and targeted by a secretory signal
peptide (Fig. 4). Daily rhizosecretion of GFP, released into
fresh medium unprotected from proteolysis, reached 2 mg/g
root dry weight, while SEAP rhizosecretion, quantified from
its activity, reached 20 mg/g root dry weight, a significant
amount considering that no attempts to optimize rhizosecretion had been made thus far. It is likely that methods for
increasing protein expression and secretion will be developed
along with plant varieties optimized for the rhizosecretion of
recombinant proteins.
Data suggest that plant roots can continuously produce and
secrete biologically active recombinant proteins of different
origins. The rhizosecretion system offers a simplified method
for the isolation of recombinant proteins from simple hydroponic medium rather than from complex plant extracts. As
with rhizosecretion of natural products, protein rhizosecretion
can be operated continuously without destroying the plant,
thus producing a higher total yield of the recombinant protein
over the life of the transgenic plant. In addition, recombinant
biopharmaceutical proteins purified from root exudates are
less likely to be contaminated with pathogenic viruses that may
be present in the milk or urine of transgenic animals. Rhizosecretion also borrows from many well developed and tested
methods of commercial hydroponic plant cultivation, and
therefore, will be relatively easy to scale up.
CONCLUSIONS
While the evolution of plant shoots followed primarily introverted paths by perfecting physical barriers between themselves and the environment, roots had to be more extroverted in their relationship with soil. This requirement created a
unique set of biological mechanisms, which until recently, were
understudied and underutilized. Phytoextraction and rhizosecretion are starting to change this, while allowing scientists to
take a radically new look at the darkest corners of plant
biology. These technologies also open the doors to the valueadded, nonagricultural uses of plants, which will continue to
expand in the new century.
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