BioSentinel Project

Background of BioSentinel

What is a BioSentinel?

 It is a kind of 'biological instrument', an organism - typically a plant - that can help us understand constraints to our natural resource base, or use of it, and to act on these constraints.

Naturally occurring BioSentinels may include bacteria, honeybees, trees, native plants, or mosses and lichens. Bacteria are used to monitor toxic metals in soil (Trends in Biotech. 15: 500-506, 1997) whereas honey bees and their products, mosses and lichens are explored as potential BioSentinels of environmental pollution in Europe (Conti and Ceccetti, Environ Pollut. 114: 471-92, 2001; and Poikolainen et al., Sci Total Environ. 318: 171-85, 2004).  The classic example of a BioSentinel plant is the rose bush, which is planted in vineyards, to serve as BioSentinels for pest problems on grapes under certain environmental conditions (link).  Other examples include native plants species that are used to monitor airborne fluorides in Europe and USA (Weinstein and Davison, Environ Pollut. 125: 3-11, 2003).

BioSentinels can also be engineered.  These have been mainly designed to understand the role of nutrients in intracellular signaling processes in plants (Knight et al, 1991), measuring iron and phosphorous deficiencies (Eide et al., 1996; Vert et al, 2002; Hammond et al, 2003), assessing nuclear pollution caused by disastrous events (Kovalchuck et al, 1998), monitoring mineral toxicity and understanding physiological interactions within plants (Ezaki et al, 2000), and understanding ion dynamics in the cytoplasm and in the apoplast (Gao et al, 2004). 

Proof of concept BioSentinels include transgenic Arabidopsis whose leaves turn from green to red after 3-5 weeks exposure to nitrogen dioxide gas indicating presence of landmines (www.aresa.dk, 2004), transgenic Arabidopsis plants that are metal specific BioSentinels (Environ Toxic.and Chemis 22: 175-181, 2003), transgenic Arabidopsis plants that monitor their own phosphorus status (Plant Physiology 132: 578-596), and transgenic Arabidopsis that were explored as sensitive BioSentinels of nuclear pollution caused by the Chernobyl accident (Nature Biotech 16: 1054-9, 1998).

In our project, we propose

1. developing modules to engineer plants to become potential biological instruments

2. coordinating through collaboration with other inventors/institutes access to other complementary modules to construct a pool of enabled technologies

3. forming a community that will make these tools available under BiOS licenses.  

We believe this will provide new opportunities for empowering local choices about what to do with local crops, rather than bringing in crops and methods from elsewhere. The proposal integrates the use of novel methods of molecular biology with the basic understanding of a field problem¹. A biosentinel can be any species that can grow on the land of interest concurrently with the crop of interest, or at the best decision time. It receives a signal from the environment and translates it in a way that is readily observable by people who can then decide what to do about it. The signal could even come from the crop itself: for example, leaves beginning to wilt signals that fruit set will be hurt unless there is more water.  What is a modular approach and how it can be implemented?

1. Jefferson, R.A. (1993) 'Beyond model systems: New Strategies, Methods, and Mechanisms for Agricultural Research', Annals of the New York Academy of Sciences 700: 53-73

CAMBIA's Modular Approach

In a systems approach to get the engineered biosentinel concept working in a crop system to address real local constraints, we envision a biosentinel with at least five modular components:

• A suitable species and a universal transformation system:- Does the plant have to be in the food chain or the non-food chain? What are the consequences of either option?  Some of the desired characteristics for such a plant: a) highly relevant to the problem, b) easily transformable, c) can be rendered sterile, if need be, and d) from a regulatory perspective, preferably it contains the appropriate sensor or its homologue.  With the availability of CAMBIA's TransBacter project and the help from the Lemelson Foundation grant, the project focused on this first module and on building new universal gene transfer machinery to enable transformation of various crops.
• An identified or engineered sensor with a promoter: - preferably regulatory elements or genes that are specifically induced or regulated in response to the constraint that needs addressing.  CAMBIA developed a technology landscape on regulatory elements and in this project, we continue updating the list of publicly available promoters but we welcome your input by E-mail.
• Engineered sensor with a marker or a detectable signal:- this step may be critical in its design since it will depend on the type of the sensor used, the location in plant where the response is needed, the type of reporter used, and its regulation mode.  Regulation of the time and duration of the response is important (inducible system for short (hours) or long (days) or constitutive for indefinetely).  This step will depend on the purpose of the application needed and the previous steps.
• A detection system for humans (an inventive device, or one of the five senses), ideally nondestructive, inexpensive or costless to use, reliable under field conditions and requires no instrumentation. Translation of the signal into something that can be observed, such as wilting, an odor, or a visible or infra-red color change mediated by a GUS gene product, an anthocyanin gene or similar genes, expressed in a part of the plant that is observable. (Ideally, the signal should be quantifiable, expressed in proportion to the level of the physiological parameter being detected. In the best mode, two signals could be used, which can be compared against each other for high accuracy.
• Field applicability (How to measure the response? How does it perform in the field? Does it meet the intended specifications? How to account for microenvironment variability?  How to establish confidence levels in the measurement of the response?) and what are the ways to act on or share the information?

An added value to the systems approach is that it can bring non-transgenic or post-transgenic modules to service. We envision an "Apollo Project" of biosentinel components that can be useful as part of a repertoire from which the appropriate modules can be chosen locally for empowering particular actions by farmers and breeders.  Such actions will more likely lead to adoption and further innovation of the technology as compared to the provision of a complete bioindicator prototype.

In this project, CAMBIA brings in more than 15 years of experience evaluating and thinking about Biosentinels, offers highly distinctive visual markers and  transactivation cassettes to help move this technology into a real working model.  

Realizing that there may be even better visual markers and promoters out there, we hope this provides impetus for talented groups that may identify and/or provide such useful markers for the bioindicators project as a public good. Do you know of any that might be really useful for this purpose?

Most of the previous research has been done on Arabidopsis using Agrobacterium methods encumbered by a variety of patents that prevent practical use outside companies that have closely held licenses ( view  ).

CAMBIA, via a BiOS license (info), is also offering the TransBacter technology to encourage participants to use the method to obviate these obstructive licensing practices for biosentinels for public good.

Modules developed by inovative individuals within this project may be patented, but the capability to use them for research and commercial purposes by all those interested to use and improve them for public good will be governed by a BiOS license (info).

Examples of BioSentinels

One significant aspect of the innovation will be identification of indicator species appropriate to the local cultivation system, sensitive to the condition of interest, and able to provide an observable signal.

In 1998, Kovalchuk et al., Bioindicator plants to detect nuclear pollution, engineered Arabidopsis plants to analyze the influence of chronic irradiation from the environment of the Chernobyl exclusion zone, on the stability of plant genomes. In this example, bioindicator plants were shown to be an effective indicator of change within the surrounding environment.

Another example encompasses research conducted by Aresa, a private company originally based at the University of Copenhagen, that is marketing bioindicator plants commercially. Aresa genetically engineered a weedy plant with a gene that produced a red-colored product when the gene's expression was induced by a receptor as a breakdown product of TNT. This allowed the plant to become a potential field biosentinel to alert about the presence of land mines. 

Proof of concept is thus established for Biosentinels.  For example, the underlying mechanism by which the color changes from green to red in the Arabidopsis plants, occurs is via an altered regulation of the natural pigment biosynthetic pathways in the plants.  The genetically engineered plants could then be modified in a way that allows these plants to turn red-purple if triggered by TNT in the soil. The field applicability of such a technology remains to be evaluated.

Shown below² at left is a photo of a soil tray planted with the engineered bioindicator seed in which the upper right quadrant of the soil has been drenched with liquid TNT. The photo at the right indicates the size of isolated plants.

Because CAMBIA's project goes beyond a model system approach,  we envision that the real field problems are going to be complex and variable and thus through the provision of various modules, the coordination of their accessability by an open source approach, and the evaluation of their potential field applicability, a more efficient and enabled innovation can be achieved, with the production of prototypes, by the end-users themselves. 

For example, scientists have identified gene products that might be applied to measuring iron and phosphate levels and heavy metals in soil³, and Aresa has announced a plan to commercialize biosentinels for bioremediation⁴.  Unfortunately, in the iron and phosphate plant sentinel prototype systems, the effects of the measurement are seen in the roots⁵ (not readily visible to the farmer) or as a very slow change in the shoots⁶ (timing that may not facilitate effective response measures), or like in the case of phosphate, we do not know yet the rate limiting step that determines level of phosphate in the plant.

New examples of detection methods include a color change on a de-greened background (Medford et al.), a shape change or a texture change, such as the presence or absence of trichomes. Such changes might appear in leaves or stem segments produced only in plastochrons immediately associated with the stress, and leaf or stem segment production would revert to normal in sectors produced after the stress passes.

If you have ideas about such changes and how to effect them and you are willing to contribute or participate in this project, please email us.

References

2. Images are used with permission, from Aresa's website, www.aresa.dk

3. Vert G, Grotz N, Dedaldechampa F, Gaymard F, Guerinot ML, Briat J-F, and Curie C (2002) 'IRT1, an Arabidopsis transporter essential for iron uptake from the soil and for plant growth'. Plant Cell 14: 1223-1233;

4. www.aresa.dk

5. Vert et al. ibid.

6. Hammond JP, Bennett MJ, Bowen HC, Martin R. Broadley MR, Eastwood DC, May ST, Rahn C, Swarup R, Woolaway KE, and White PJ (2003) 'Changes in gene expression in Arabidopsis shoots during phosphate starvation and the potential for developing smart plants'. Plant Physiology 132: 578-596.

Biosentinels for nutrient deficiency?

A simple model to develop biosentinels for nutrients.

a.    Consider well studied examples such as genes that are upregulated under nutrient deficiency.
b.    Isolate their homologs from the crop of interest.
c.    Check their temporal gene expression profiles in their own host under various nutrient deficiency conditions (lab and/or greenhouse mainly).
d.    Introduce their promoters into expression cassettes.
e.    Transform the crop of interest and evaluate the biosentinel transgenic plants in the lab/ greenhouse.
f.    Assay their effectiveness in the field?
           i.   what sampling strategies to use?
           ii   how can the expression of the marker be  easily assessed? quantitative or qualitative assay?
           iii  how are the results interpreted and when action is recommended for farmers to adjust the level of nutrient in their field?
           iv  what controls are needed in the generation of plants and in the field (natural mutants? Non-transgenic?  Nutrient tolerant varieties?

What are some of the other complexities in designing a biosentinel plant for nutrients?

Below, I consider some nutrients and review what is known to enable a field-based design. 

First let us keep in mind some realities for nutrient deficiency:

a.    For MACROnutrients such as Nitrogen (N), Phosporous (P) and potassium (K), HOURS are needed to detect signals.
b.    For MICROnutrients such as Zinc (Zn), iron (Fe), Manganese (Mn), and Copper (Cu), DAYS are needed to detect signals.
c.    P, K, Zn, Mn, and Cu nutrients are limited in the rhizosphere and move by slow diffusion.
d.    The differential availability of soil nutrients to different genotypes is hard to measure.
e.    The rhizosphere rather than the plant is the most critical limiting step in improving nutrient efficiency.

Biosentinels for nutrient signaling cascades and ideas on when and where they may be developed/not developed-just some thoughts to share

  Phosphorous (Hammond et al, 2004)

Diagram of P cascade events in the plant/rhizosphere with the following overview

• Early signaling events (early genes specific and nonspecific)

1. Early responses to P deficiency include the expression of general stress related genes and low-specific P signaling cascades.  General stress response genes are differentially regulated in response to P deficiency, although the magnitude and temporal patterns of these changes may vary.
2. Initial response may be generic across stress and nutrient deficiency, however, specificity may be determined down the signaling cascade.

• Late genes response (morphology, metabolism, and physiology)

        are we after Improving P acquisition from soil?

1. Changes in root morphology and growth are proportional to the concentration of plant growth regulators, in particular auxins*, ethylene, and cytokinins
2. Role of Pi transporters?
3. Overproducing citrate improved Phosphate Use efficiency (PUE)
4. Transcription and activity of secreted acid phosphatases increased by P deficiency

    Or improving internal P usage

1. Differential expression of PEPCase
2. The accumulation of anthocyanins in the aerial tissues is a characteristic response of P-deficient plants
3. Recycling and remobilizing of P
4. Increased expression of phosphatases in P deficient plants may signal the start of a specific response

Mechanisms of phosphorous efficiency (Kochian et al, 2004; Schachtman, 1998; Hammond, 2003)

    Because Pi diffusion in the soil is slow, under P deficiency, there is evidence of increased root:shoot ratio and relocation of C resources to support the newly formed roots.  Proliferation may include root branching and increased root hairs. In addition, the association of roots with vesicular-arbuscular mycorrhizae was shown to enhance their contact with soil and thus expand Pi uptake (and Zn).

1. root-mediated changes in the rhizophere chemistry and upregulation of Pi transporters
2. root exudates and P mobilization from the soil:  P deficiency triggers malate and citrate production from the root.  These organic acid anions can desorb Pi from mineral surfaces, solubilize it from other associations (Al, Fe, Ca….), and cause the development of clusters or proteoid roots.
3. when the supply of P is limited, plants grow more roots, increase the rate of uptake by roots from the soil, and retranslocate Pi from older leaves.  Pi vacuolar pools are depleted whereas cytoplasmic P levels remain fairly constant
4. Global regulator genes such as PHR1, which encodes an MYB transcription factor, are important in the signaling cascade initiated by P starvation
5. root Pi uptake is mediated via a thermodynamically active H+-Pi co-transporter driven by plasma membrane H+-ATPase.  Several genes have been isolated and they are related to the H+-coupled co-transporters (Major facilitator Superfamily) that mediate sugar, aa, and inorganic anions uptake.  Several transporters for Pi have been identified across cellular membranes.  They are differentially expressed and have two affinity types.  The external supply of P regulates the transport
6. Rice P efficiency is due mainly to genotypic differences in root P uptake

Preliminary analysis of potential biosentinel systems for Phosphorous

• Evaluation of early and late P response elements that are upregulated under P starvation i.e. having biosentinels that give a temporal signal under field conditions to changes in P content.  Is this needed or useful?

1. Has little if any practical use in managing P in the field since P is applied only once before planting and if applied when deficiency is detected, the plant does not respond quickly enough
2. Breeding for phosphate tolerant variety is not a priority.  Most farmers use cheap rock phosphate fertilizers and their main issue is the bioavailability of Phosphate in soil rather than in the plant.

• Focus on early events of phosphate starvation in the rhizosphere and look for biosentinels that sense the organic acids produced or bacterial populations affected (engineer plant with promoter of AHL synthase fused to a reporter gene or the gene itself to check whether root colonizing bacterial populations are increasing under nutrient deficient conditions (Steidle et al., 2001)  where it may be needed?

• How about phosphate reporter bacteria (lux-tagged) that sense level of P in the soil and around the roots (de Weger et al, 1994, 1997; kragelund et al.,1997)  The genes are available and known to be overexpressed in transgenic plants AHL signal molecules serve as universal communicator between different bacterial populations of the rhizosphere.

1. Alan Richardson’s work showed that transgenic plants overexpressing citrate are not more P efficient under soil condition, so there is no strong evidence that the produced organic acids lead to release more P from soil. 
   
2. Would expressing a bacterial gene in a plant root communicate the signal to bacteria in the soil?
3. Would bacterial population growth correlate with nutrient deficiency? This may require the use of a camera to trace bacteria in the soil and the bacteria may not compete with endogenous microbial populations present under different soil types.  

• The release of organic acids under P starvation leads to proteoid roots or lateral root clusters in white lupin. In Arabidopsis the elongation and density of root hairs are regulated by P availability in a dose-dependent manner (Ma et al., 2001).  Could we then develop a biosentinel for altered root morphology?  Can we develop a marker that predicts the elongation and density of root hairs?  Why it is important?

1. Architecture of the root system is complex (3 processes: cell division, lateral root formation and root-hair formation) and depends on soil structure, microbial populations, nutrient availability, and genetics of the plant. N, P and Fe alter root developmental processes.
2. PHR1, which is related to PSR1, thought to encode a MYB trascription factor that is involved in P signaling pathway (Rubio et al, 2001).  PHR1 has an effect on root/shoot ratio and thus may provide a visual biosentinel for P deficiency.
3. The role of auxin transporters in proteoid root formation was demonstrated in white lupin and Arabidopsis (lopez-bucio et al., 2002; 2003).  The expression of P transporters (auxin transporters) may influence lateral root growth so we may be able to develop a biosentinel for various lateral root types to provide breeders with standardized way of measuring nutrient effect on roots.
4. ANR1 gene encodes a NO3-  -inducible MADS-box transcription factor that Zhang and Forde (1998) showed its role in lateral root growth. Filleur et al., (2005) showed that ANR1 would require an external component of the regulatory pathway (external glutamate is suggested) to affect primary root growth.  It is worth studying this in detail.
5. HAR1 encodes a serine/threonine kinase that is required for shoot controlled regulation of root growth, nodule formation and nitrate sensitivity of symbiotic development.
6. How about the role of auxin-induced gene, OsRAA1, in rice under nutrient stress? Overexpression of OsRAA1 affected leaf, flower, and root development (lei at al., 2004) decline in leaf expansion due to withdrawal of N from the roots (root to shoot signal for Nutrient stress)
7. Although in response to P and Fe, plants have a similar root hair density, changes in the root-hair morphology in response to P and Fe were shown to be mediated by different signal pathways (Schmidt and Schikora, 2001).

Biosentinel for nitrogen defficiency

Mechanisms of Nitrogen efficiency (Good et al, 2004)

• Early signaling events:  cytokinins may well be the long-distance signals mediating the molecular response to changes in NO3- availability from roots to shoot (Forde, 2002)

Pathway- Nitrateammonium uptake overexpression of nitrate transporters did not show phenotypic effect on Nitrogen use efficiency (NUE) ammonium transporters effect on NUE is still unknown.  Two enzymatic steps are involved in reducing nitrate to ammonia: Nitrate reductase step (NR) and nitrite reductase step (NiR).  Overexpression of NR seems to reduce the level of nitrate in the tissue analyzed (inducible promoters).  Overexpression of NR or NiR in whole plants increased mRNA levels and generally affected N uptake without affecting yield though.  A couple of enzymes have a major role in this step (assimilating and recycling ammonium): glutamine synthetase (GS) and glutamine synthase (GSGAT).  Overexpression of GS1 gene in transgenics showed an enhanced root and grain yield and a higher N content.  So the overall nitrogen assimilation can be increased using GS1genes. 

Grain filling is still a limiting step to improve cereal crop yield.  How about overexpressing NADH-GOGAT in rice and its effect on nitrogen use and grain filling?

Can GS or GSGAT genes be used to enhance nitrogen use efficiency by various crops?

Translocation of Asparagine synthetase (AS) catalyses the formation of asparagines (Asn) and glutamate from glutamine (Gln) and aspartate and is encoded by a small gene family (ASN1, ASN2, and ASN3).  It is believed that when GS becomes limiting, AS becomes important in controlling the flux of nitrogen in the plant.  By overexpressing the AS genes, it was shown that it is possible to interfere with nitrogen metabolism and growth phenotype and thus this may be a way to improve nitrogen use efficiency in crop plants.

Dof1 (maize transcription factor, a member of a family that is unique to plants) is an activator for multiple genes associated with organic acid metabolism.  Expression of this gene in arabidopsis induced the upregulation of genes involved in carbon skeleton production and resulted in a marked increase in aa content in the transgenic plants.  More importantly, the Dof1 transgenic exhibited improved growth under low nitrogen conditions.  Remobilization grain yield may be based not only on nitrate uptake before flowering but also on the remobilization of leaf N during seed maturation.  Several genes have been identified that are specifically activated during the remobilization of nitrogen, carbon, and minerals during leaf senescence.

Biosentinels for micronutrient defficiency

➢    Zinc

Mechanisms of Zinc efficiency (Huang et al, 2000; Rengel, 2001; Hacisalihoglu, 2003)

1. Increased root surface and a longer length of fine roots mainly in early stages of growth (micorrhizae help increasing the length of roots but it is present at low level in OZ)
2. Release of phytosiderophores (increased number of fluorescent Pseudomonas)
3. Increased zinc uptake in the grain which would eventually lead to increased seed Zn content.
4. Increased activity of carbonic anhydrase**in efficient genotypes (expression of Zn-requiring enzymes: Cu/SOD and CA was studied.  Results showed that expression of SOD1.1 was upregulated in the Zn-efficient wheat but there were no apparent increase in the expression of CA.  There seems to be a positive correlation between Zn efficiency and Cu/SOD activity and CA activity in Zn-efficient wheat genotypes.  This work led to the following hypothesis: the genotypic variation in the expression and activity of Zn-requiring enzymes is closely related to Zn efficiency), decreased level of superoxide dismutase , and production of specific plasma membrane polypeptides
5. Differential Fe transport to shoots

➢    Iron

Mechanisms of Fe efficiency ( Eide et al, 1996; Rengel, 2001; Graham and Stangoulis, 2004)

Iron is 10,000 times greater in the soil than in the vegetation grown in it, yet iron deficiency is common in crop plants.  Plants exhibit two distinct strategies coping with Fe deficiency:

Strategy I:  dicotyledons and non graminaceous monocots (see picture in graham and stangoulis, 2004)

Upregulation the ferric reductase and the proton-extrusion pump
Excretion of iron-binding ligands and reductants (phenols)
All these processes occur in the apical zones of the roots
Differential expression of dgl gene in the shoots
Differential production of nicotianamine?

Strategy II: grasses

Grasses are insensitive to bicarbonate. 
Instead of upregulating ferric reductase, they release phytosiderophores.   Wheat, rice, and corn release DMA whereas rye releases HMA.
Have a constitutively a highly specific transporter protein (IRT1 family?).  This protein (not found in strategy I plants) recognizes and transports its ferric chelates across the membrane.  In the cytoplasm, the ligand is separated from the metal and stored or transported further in the plant with ferrous specific ligands such as nicotianamine.
Increase Imax?

IRT1 gene has a role in iron uptake from the rhizophere across the plasma membrane in the root epidermal cell layer.

➢    Manganese

Mechanisms of Mn efficiency

1.    better internal compartmentalization and remobilization of Mn
2.    excretion by roots of high amounts of protons, reductants, Mn-binding ligands, microbial stimulants
3.    seed Mn content
4.    increased number of fluorescent Pseudomonas

Biosentinels for biotic or abiotic stresses?

Signals for biotic stresses

    Soil-borne pathogens of food, fiber, and ornamental crops such as take-all disease, potato scab decline, Pythium, Rhizoctonia root rot, phytophtera, and rice leaf blast

1.    does the best approach include engineering  local bacterial populations to produce higher levels of PCA or 2,4-DAPG antibiotics known to suppress the disease?
2.    sentinel plants that sense the level of antibiotic produced and produces a signal?
3.    sentinel plant that recognizes the  pathogen and induces the production of antibiotic(s)?
4.    markers to distinguish the various pseudomonas spp that are producers of PCA or 2,4 DAPG?
5.    differentiate between suppressive soils and conducive ones?

Signals for abiotic stresses

    Regulation of invertase (s) expression in cereal anthers and their potential use as biosensors of water deficit in high water-use-efficient cereal crops breeding programs in rainfed ecosystems

In a real cropping system, yield and water do not have a linear relationship because yield may also be constrained by several other factors such as weeds, diseases, frost, inadequate nutrients, acid soil, etc..  If and when water is limiting, yield then becomes a function of 1) the amount of water used by the crop; 2) how efficiently the crop uses this water for biomass growth (above ground); and 3) the harvest index (ratio of grain yield /aboveground biomass) (Passioura, 1977).  These three components are relatively independent and thus understanding and manipulating anyone of these would translate into a yield increase.

The amount of water used by the crop depends on the level of initial underground water, rainfall or irrigation schedules, temperature, and the efficiency of the water uptake, use, and distribution by the plant.   Since attempting genetic increases in yield under rainfed conditions is complicated by the challenging genotype x season x location interactions, a focus on maximizing water use efficiency within a plant is important. 

In the life of a cereal crop, seed germination and reproductive development are the most water-stress-sensitive phases.  Within the reproductive phase, the sensitivity of male organs increases dramatically from the start of meiosis to the break-up of tetrad, events that last 24 h in a single anther (Koonjul et al., 2005).  By contrast, the female tissue remains insensitive to water stress during the same period (Saini and Aspinall, 1981).  Water deficit during meiosis induces pollen sterility and lead to a failure in fertilization and hence grain set (Saini and westgate, 2000).  Such an impact on cereal yield under rainfed conditions can be detrimental to farming communities.

In wheat, pollen development fails when a brief episode of moderately severe water deficit coincides with meiosis (Saini, 1997).  The failure appears to be due to some cellular lesion that is triggered by unidentified signal from the vegetatitve organs (Koonjul et al., 2005).  During their final stages of development, normal pollen grains of wheat and other cereals accumulate large quantities of starch that is used later to support pollen germination and growth of pollen tube.  Water-stressed-affected wheat pollen grains do not accumulate starch (Saini et al., 1984).  Upon studying the mechanisms that regulate this deficiency, Dorion et al. (1996) found and later Koonjul et al., ascertained  that water stress during meiosis irreversibly impairs invertase activity in anthers.  This effect precedes any visible developmental lesion and it is specific because other enzymes in the starch biosynthesis pathway are not affected.  The decline in invertase activity is followed by an accumulation of sucrose, a change in the profile of other sugars, and some spatial redistribution of starch within the anther (Dorion et al., 1996; Lalonde et al., 1997a).  A similar pattern of events was also reported in rice (Sheoran and Saini, 1996).  

Since invertase is the dominant sucrolytic enzyme in wheat anthers, these results suggest that the inhibition of invertase-mediated sucrose utilization in anthers may be the signal for pollen development failure under stress.  So, Koonjul et al. (2005) selected three invertase cDNAs cloned from a wheat anther cDNA library and showed that the effect of water stress is at the transcriptional level and is highly gene and cell specific.

We can propose developing biosentinels for the differential expression of these invertases in the anther and check their utility for breeding water efficient rice, wheat, or barley varieties under rainfed conditions.  By the way, one homologue invertase from rice was characterized and it shares 57% aa identity with that in wheat.  No reports yet for anther invertases in barley.

All these are just thoughts based on my readings, if you have any suggestions or you would like to add/modify the information, please email me (osmat@cambia.org).

Field case-Australia

Field-case: Phosphorous and Nitrogen use in Australia

Australia is the second driest continent after Antartica.  Such a dry climate impacts on salt leaching and bedrocking and leads to having shallow soils and fragile hydrologic conditions.

  Fig. 1. Land use in Australia

Three broad zones can be visualized in Australia:

1. conserved or protected uncultivated areas
2. livestock grazing areas with occassional wheat or other grain production depending on market prices and
3. dryland or irrigated agricultural areas (Fig. 1).  

To encourage economic and agricultural development, Australia subsidized irrigation, nitrogen and phosphate fertilizers between 1966 and 1984 (Hyberg, 1991).  Whereas these subsidies facilitated intensive agricultural systems, the overuse of fertilizers lead to increased salinity problems, soil acidity, and contaminated fresh and ground water in the 1980s.   By the 1990s and with reduced pasture areas, the introduction of break crops (ex. Canola) in between cereal croppings, and the adoption of semi-dwarf cultivars, Australia has been experiencing a selective increase in nitrogen applications with a fluctuating upward trend for phosphorous applications (Fig. 2).

Although fertilisers have boosted Australia’s agricultural production, there is a general concern about the impacts of these fertilisers on the environment and aquatic ecosystems.   For example, during the manufacturing of phosphorous, a by-product, known as phosphogypsum which contains radium, is produced.  In addition, due to the persistence of trace metal contaminants at higher concentrations, phosphorous application affects off-site water quality.  By far, the most important environmental concern reported is the export of 5% of the applied phosphorous into surface waters and to a lesser extent groundwater (Nash 2004).  For nitrogen what are the concerns?

 Fig. 2. Tonnes of Nitrogen (N), Phosphorous (P), and Potassium (K) provided as fertilizers in Australia between 1983 and 2005.

The water is considered degraded if the phosphorous level is 0.05mg/L.  On many farms, the phosphorous concentration in soil water can exceed 1mg/L.
How phosphorous export potential is currently measured?
The Olsen and Colwell P tests are the two most common agronomic soil tests available to measure soil phosphorous.  Both use Bicarbonate solutions to extract phosphorous, however, Olsen P test is shorter (30 min) and measures both quantity and concentration of phosphorous in soil whereas colwell P test measures only the quantity and is longer (16 hours).  Unfortunately, none of these tests measure the flow or rate of dissolved phosphorous by water.

Is there any potential for a biosentinel plant here?

Frequently Asked Questions

• What are biosentinels?

• What is unique about this particular project?

• What is the modular strategy for the BioForge bioindicators project?

• How will the community access and help in the innovation of plant bioindicators?

• References

What are Biosentinels?

Biosentinels also known as bioindicators, ambiosensors, Sentinel plants, bio-reporters, biomarkers, “Smart” plants, and biosensors, are in vivo reporter systems applied to resolve a specific field problem, are live organisms that their sole purpose is to mark an event at a specific time and in a particular microenvironment.  Biological indicators include natural organisms that are used to monitor the atmosphere or soil. Some examples include

• mosses, which are used as bioindicators for heavy metal deposition in the atmosphere in Finland,
  
• native plants species that are used to monitor airborne fluorides in Europe and USA,
  
• lichens, which are used as bioindicators of air pollution in Italy,
  
• honeybees and their products, which are explored as potential bioindicators of environmental pollution also in Italy, and
  
• bacteria that were used to monitor toxic metals in soil.

Engineered bioindicators consist mainly of transgenic plants that are designed to monitor levels of specific contaminants in natural resources such as soil, water or sediments. Specific examples include transgenic plants whose leaves turn from green to red after 3-5 weeks exposure to nitrogen dioxide gas, making the plants useful bioindicators of landmines (www.aresa.dk), transgenic Arabidopsis plants that are metal specific bioindicators5, transgenic Arabidopsis plants that monitor their own phosphorus status6, and transgenic Arabidopsis that were explored as sensitive bioindicators of nuclear pollution caused by the Chernobyl accident.7

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What is unique about this particular project? 

This project goes beyond the model system approach. It seeks to develop specifications for biosentinel plants (rather than prototypes) and modules (component parts), if you will, that can be accessed fairly, assembled or disaasembled and reassembled again by various end users based on their own needs.  CAMBIA's intention not to claim ownership for this stratigic project, instead provides some modules and expertise to facilitate project development by various collaborators and institutes.  Our ultimate aim is to enable informed decision-making and maximization of limited resources by farmers themselves (for more details, see Jefferson, R.A. (1993) 'Beyond model systems: New Strategies, Methods, and Mechanisms for Agricultural Research', Annals of the New York Academy of Sciences 700: 53-73).

The community approach to this project, as opposed to a single lab for a single crop, may bring a deliverable mechanism to lab outputs and link them to address field challenges in heterogeneous environments and unpredictable agricultural ecosystems. 

Our approach to develop modular parts that can be combined in various ways is also supported by a delivery mechanism that is royalty-free and under a BiOS license (info), to ensure open access and fair use by local groups wishing to develop their own biosentinel systems.  The project began by voluntary contributions but now is supported through funding by the Lemelson Foundation, and we hope that your direct and indirect contributions will enrich it and sustain it as a public good.

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What is the modular strategy for the BioForge bioindicators project? 

To engineer and develop a bioindicator plant, our approach envisions that various modules can be recombined for different crops and settings:

1. Identification of the potential bioindicator plant: what plants will grow well in the context of the field problem that you are trying to solve? Does the proposed indicator plant enter the food chain or not? What are the consequences of either option?

   Some of the desired characteristics for an indicator plant: 

   • relevance to the problem and crop-growing context;

   • easily transformable;

   • can be sterile, if need be; and

   • ideally, it already contains the appropriate sensor or a homologue (which is helpful from a regulatory perspective).

2. Sensor identification: preferably one or more regulatory element(s) or gene(s) that are specifically induced or up-regulated in response to the constraint the bioindicator is desired to address, such as soil available concentration of a nutrient. Many such sensors are found in the published literature and more may be found via interactions with the BioForge user community.

3. Engineering the sensor with a known reporter system, such as GUS or GUSPlus, GFP, etc. The choice will depend on interactions with the type of sensor used, the position in the plant where the response must be visualised, and its mode of regulation.

4. Regulation of the time and duration of the response: inducibility, how long the system is "turned on" (i.e. the signal persists through to plant senescence or it is turned on and off based on the initiation for perhaps a few days to provide the needed response).

5. Detection: this can be as simple as an induced change in the color of the leaf/stem or productive organs, so that the response will be obvious and easily detected by inexperienced persons. Alternatively, it can be designed to induce a strong unusual smell to serve the same purpose. Such sensory systems are available, but they need to be tuned to the purpose of this project.

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How will the community access and help in the innovation of plant bioindicators? 

The scientific and IP teams at CAMBIA have developed some initial modules (such as the TransBacter and GUSPlus technologies) and will continue to further develop much needed modules. But also we would welcome contributions, suggestions, new ideas or tools that you would like to share under an open source business models.  All the available technologies will be available under a BiOS license (info).

We also expect, with the assistance of the community, to develop for each module one or more technology landscapes or "Freedom to co-operate" analyses for those components of modules that are not in CAMBIA's "protected commons" yet.

Communities interested in customizing the modules to develop a preferred bioindicator can then acquire the parts cost-free, assemble and develop the product and test it under local conditions. Within the community of BiOS licensees, shared successes and failures will be discussed and reported, and improved versions of the modules/products will continue to be widely available as public goods.

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References 

1. Poikolainen J, Kubin E, Piispanen J, Karhu J (2004) Atmospheric heavy metal deposition in Finland during 1985-2000 using mosses as bioindicators?. The Science of the Total Environment 318 (1-3): 171-185.
2. Weinstein LH, Davison AW (2003) Native plant species suitable as bioindicators and biomonitors for airborne fluoride, Environmental Pollution 125 (1): 3-11.
3. Conti ME, Cecchetti G (2001) Biological monitoring: lichens as bioindicators of air pollution assessment: a review, Environmental Pollution 114 (3): 471-492.
4. Ramanathan S, Ensor M, Daunert S (1997) Bacterial biosensors for monitoring toxic metals. Trends in Biotechnology 15 (12): 500-506.
5. Krizek BA, Prost V, Joshi RM, Stoming T, and Glenn TC (2003) 'Developing transgenic Arabidopsis plants to be metal-specific bioindicators', Environmental Toxicology and Chemistry  22 (1): 175-181
6. Hammond JP, Bennett MJ, Bowen HC, Martin R. Broadley MR, Eastwood DC, May ST, Rahn C, Swarup R, Woolaway KE, and White PJ (2003) ' Changes in gene expression in Arabidopsis shoots during phosphate starvation and the potential for developing smart plants'. Plant Physiology 132: 578-596.
7. Kovalchuk I, Kovalchuk O, Arkhipov A, Hohn B (1998) Transgenic plants are sensitive bioindicators of nuclear pollution caused by the Chernobyl accident. Nature Biotechnology 16: 1054-1059.

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BioSentinel Resources

If you are interested in a preliminary overview on biosentinel technologies, please check  Bioindicator technology landscape on Patent Lens website.

Written References

• Gene transfer to plants by diverse species of bacteria', Nature 43: 629-633, and patents published as US 2005/0289672 and US 2005/0289667, and as PCT Publication WO 2006/004914. See also Commentary to the Nature paper by Mary Dell Chilton, from Nature Biotechnology23(5) March 2005.
• M.B. Connett Porceddu and R.A. Jefferson (2007) BiOSentinel Plants:  Enabling Farmer Choice.  Hainan, China.  See this presentation.
• Conti ME, Cecchetti G (2001) Biological monitoring: lichens as bioindicators of air pollution assessment: a review, Environmental Pollution 114 (3): 471-492.
• Hammond JP, Bennett MJ, Bowen HC, Martin R. Broadley MR, Eastwood DC, May ST, Rahn C, Swarup R, Woolaway KE, and White PJ (2003) ' Changes in gene expression in Arabidopsis shoots during phosphate starvation and the potential for developing smart plants'. Plant Physiology132: 578-596.
• Jefferson, R.A. (1993) 'Beyond model systems: New Strategies, Methods, and Mechanisms for Agricultural Research', Annals of the New York Academy of Sciences700: 53-73
• Jefferson, R.A. (1992) 'The GUS Fusion System: Summary and Future Perspectives' in GUS Protocols: Using the GUS Gene as a Reporter of Gene Expression. ed. S. Gallagher. Academic Press, New York.
• Kovalchuk I, Kovalchuk O, Arkhipov A, Hohn B (1998) Transgenic plants are sensitive bioindicators of nuclear pollution caused by the Chernobyl accident. Nature Biotechnology 16: 1054-1059.
• Krizek BA, Prost V, Joshi RM, Stoming T, and Glenn TC (2003) 'Developing transgenic Arabidopsis plants to be metal-specific bioindicators', Environmental Toxicology and Chemistry 22 (1): 175-181
• Poikolainen J, Kubin E, Piispanen J, Karhu J (2004) Atmospheric heavy metal deposition in Finland during 1985-2000 using mosses as bioindicators. The Science of the Total Environment 318 (1-3): 171-185.
• Ramanathan S, Ensor M, Daunert S (1997) Bacterial biosensors for monitoring toxic metals. Trends in Biotechnology 15 (12): 500-506.
• Roa-Rodriguez, C. & Nottenburg, C. (2003) 'Agrobacterium-mediated transformation of plants', CAMBIA technology landscape paper (view online)
• Thach Tranh et al.. manuscripts in prep. from Ph.D. thesis at CAMBIA (see also related patent application, published as WO 01/21781).
• Tuan Nguyen, manuscripts in prep from Ph.D thesis at CAMBIA. See also GUSPlus BioForge project.
• Vert G, Grotz N, Dedaldechampa F, Gaymard F, Guerinot ML, Briat J-F, and Curie C (2002) 'IRT1, an Arabidopsis transporter essential for iron uptake from the soil and for plant growth'. Plant Cell 14: 1223-1233.
• Weinstein LH, Davison AW (2003) Native plant species suitable as bioindicators and biomonitors for airborne fluoride, Environmental Pollution 125 (1): 3-11.

Web References

• Some images are used by permission from the Aresa website, www.aresa.dk

The information contained in this page was believed to be correct at the time it was collated. New patents and patent applications, altered status of patents, and case law may have resulted in changes in the landscape. CAMBIA makes no warranty that it is correct or up to date at this time and accepts no liability for any use that might be made of it. Corrections or updates to the information are welcome, please send an email to info@bios.net.