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)

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.
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?

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

Or improving internal P usage

Differential expression of PEPCase

The accumulation of anthocyanins in the aerial tissues is a characteristic response of P-deficient plants

Recycling and remobilizing of P

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).

root-mediated changes in the rhizophere chemistry and upregulation of Pi transporters
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.
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
Global regulator genes such as PHR1, which encodes an MYB transcription factor, are important in the signaling cascade initiated by P starvation
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
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?

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

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.

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.

Would expressing a bacterial gene in a plant root communicate the signal to bacteria in the soil?

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?

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.

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.

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.

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.

HAR1 encodes a serine/threonine kinase that is required for shoot controlled regulation of root growth, nodule formation and nitrate sensitivity of symbiotic development.

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)

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).