Designing a Biosentinel plant

A simple model to develop biosentinels for nutrients.

  • Consider well studied examples such as genes that are upregulated under nutrient deficiency.
  • Isolate their homologs from the crop of interest.
  • Check their temporal gene expression profiles in their own host under various nutrient deficiency conditions (lab and/or greenhouse mainly).
  • Introduce their promoters into expression cassettes.
  • Transform the crop of interest and evaluate the biosentinel transgenic plants in the lab/ greenhouse.
  • Assay their effectiveness in the field?
    • what sampling strategies to use?
    • how can the expression of the marker be  easily assessed? quantitative or qualitative assay?
    • how are the results interpreted and when action is recommended for farmers to adjust the level of nutrient in their field?
    • 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:

  1. For MACROnutrients such as Nitrogen (N), Phosporous (P) and potassium (K), HOURS are needed to detect signals.
  2. For MICROnutrients such as Zinc (Zn), iron (Fe), Manganese (Mn), and Copper (Cu), DAYS are needed to detect signals.
  3. P, K, Zn, Mn, and Cu nutrients are limited in the rhizosphere and move by slow diffusion.
  4. The differential availability of soil nutrients to different genotypes is hard to measure.
  5. 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).