Engineering Crops for Efficient NPK Capture from Soils
By: S.C. Kaushik

The global population is expected to reach ten billion by the year 2070. Feeding this many people will require more efficient use of agricultural land. Creating designer crops with enhanced nutrient uptake could help in getting this goal by reducing the need for fertilizer application. Molecular genetic tools have given the researcher the possibility to identify key regulatory steps in the acquisition of nutrients by plants. There are now convincing evidences that the genes coding for several target traits, mainly the transporters and other mechanisms for nutrients acquisition opens up several options to improve nutrient uptake and utilization in soils with low fertility. The transfer of the corresponding genes to agriculturally important crops might therefore allow to increase their nutrient uptake capacity. Genetic engineering and biotechnology hold great potential for plant breeding as it promises to expedite the time taken to produce crop varieties with desirable characters. With the use of molecular techniques it would now be possible to hasten the transfer of desirable genes among varieties and to introgress novel genes from related or unrelated species.

Uptake of Nutrients

A plant's ability to absorb nutrients is often limited by the availability by nutrients at the surface of the root. Plants increase the solubility of nutrients by extruding enzymes, organic acids and other compounds that after the chemistry of the rhizosphere. The secretion of root exudates varies according to the species, the soil conditions and the nutritional status of the plant.

Many nutrients are concentrated within plant tissue to values far in excess of their levels in the soil through the action of plasma-membrane transport proteins. A vast array of mineral-nutrient transporters has been identified at molecular level in recent years such as transporter of nitrate, ammonium transporter, potassium transporter, phosphate transporter, iron transporter, zinc transporter, manganese transporter, copper transporter etc. In a broad sense, transporter proteins can be classified into three groups - pumps, carrier and channels. Pumps are active transporters that perform energy transduction. They couple the chemical energy released by ATP hydrolysis to the transport of ions or small molecules across the membrane. The major pump activity in the plasma-membrane of plant is the H+ ATPase , which expels protons from the cell and thus establishes a pH gradient and membrane potential, leading the movement of nutrients via carriers and channels. Carriers use the energy in an electrochemical ion gradient such as a H+ gradient by using the energetically favorable movement of one molecule to drive the unfavorable movement of another. Channel facilitate the unidirectional movement of ions down their electrochemical gradient and so do not require an input of energy other than the membrane potential.

The concentration dependence of the uptake of most nutrients by intact plant roots is multiphasic i.e. multiple uptake system, thus leading to models describing separate high-affinity and low-affinity uptake systems. These terms apply to the concentration range in which a particular transporter operates.

This article discuss the various strategies plants use to solubilize and absorb nutrients such as nitrate, phosphorous and potassium, and the benefits to be gained from enhancing their uptake by the application of genetic engineering in plants.

Nitrogen

The major source of inorganic nitrogen in a typical agricultural soil is a mixture of NO3- and NH4+, with NO3- being the predominant form. Plants can absorb NO3- over a wide concentration range, with its uptake showing complex kinetics. Plant must absorb large amounts of nitrogen (as NO3- or NH4+) from the soil, because it can be limiting for growth. It is estimated that one third of the applied nitrogen is not retained by crops, as NO3- is highly soluble and not retained by the negatively charged soil matrix. Thus, excess NO3- may leach into the water supply, and it is a contaminant of drinking water in some agricultural areas that rely on ground water for human consumption.

Two families of genes encoding NO3- uptake proteins have been identified in plants - NRT1 and NRT2 families. The NRT1 family includes genes that are induced by NO3- and others that are constitutively expressed. The NRT2 genes, which show no sequence similarity to the RT1 family, encode high-affinity transporters that are inducibly expressed in response to NO3-. Both the NRT1 and NRT2 families of genes are believed to encode carrier that drive NO3- uptake by cotranporting at least two protons into the cell for every one NO3- ion. The over-expression of NO3- transporters may have an impact on the plant's ability to absorb NO3-, because of these genes have shown to be cell-specific and highly regulated.

The Arabidopsis AtNRT1 (CHL1) gene encodes an inducible component of low-affinity nitrate uptake. It provides a primary mechanism for nitrate uptake and is expected to localize to the epidermis and cortex of the mature root, where the bulk of nitrate uptake occurs. A nitrate gene, OsNRT1, was cloned from rice (Oryza sativa) and is a new member of a growing transporter family PTR. OsNRT1 is constitutively expressed in the most external layer of the root, epidermis and root hair.

A gene, AtNR1, has been identified that can alter the proliferation of lateral roots in response to NO3- levels in the soil. This gene encodes a member of the MADS-box family of transcription factors. It may be possible to enhance uptake by genetically altering genes in the ANR1 pathway as a way to coax plants into building more-expensive root system.

So it is now possible to create plants that are better able to absorb NO3-, thus leading to reduce NO3- contamination of ground water and lesser consumption of nitrogenous chemical fertilizers.

Phosphorus

Phosphate (Pi) is one of the least available plant nutrient found in the soil. Plant absorbs Pi from the soil as inorganic orthophosphate ions. In most soils, 30-70% (w/w) of the total phosphorus present is complexed with carbon (organic phosphorus or Porg). Phosphorous is the most limiting nutrient for growth and Pi levels in soil can vary considerably, from 1M in unfertilized soil to 1 mM or more in fertilized soils. The uptake of Pi is also multiphasic, active and career mediated. Pi binds strongly to soil surfaces and is highly insoluble causing the absorption of Pi to be strongly limited by its delivery to the root surface.

A persistent low level of available phosphorous in the soil solution has led to numerous morphological, physiological biochemical and molecular adaptations by plants to survive in the nature. The root to shoot ratio of plants increases under phosphate (Pi) stress and the root diameter decreases, while the amount of absorptive surface is relative to root volume increases. Enhanced root growth under Pi starvation results in increased root surface area available for Pi acquisition. Root hairs play an important role in Pi acquisition under nutrient deficiency conditions. A highly branched, actively growing root system of some of the bean genotypes is positively correlated with phosphorous efficiency. Proteoid root development in white lupins is a classical example of plant adaptation to Pi deficiency.

Phosphate deficiency results in distinct changes in gene expression. Some of these altered gene products may serve as molecular determinants of plant adaptation to Pi deficiency. The coding for proteins such as phosphate transporters, phosphatse, phytase, RNases have a distinct role in Pi nutrition of plants. The number of genes known to be expressed under Pi deficiency is increasing rapidly.

Plant roots have a high-affinity uptake system that is specifically depressed by phosphate deprivation. The cloning and analysis of plant phosphate transporter genes were possible by the availability of fungal and yeast tranporter mutants and clones. The plant genes show extensive similarity among themselves, and they show regulation in response to phosphate starvation.

Eukaryotic high-affinity phosphate tranporter genes were initially isolated from Saccharomyces cerevisiae (PHO84), Neurospora crassa (PHO5), and the arbuscular mycorrhizal fungus Glomus veriforme (GvPT). cDNA clones with related sequences were then identified in the Arabidopsis (AtPT1 and PHT1), tomato (LePT1), potato (StPT1) and Medicago truncatula (MePT1). All of the plant genes respond to phosphate deprivation with a dramatic increase in transcript levels, the most to the root epidermis. The findings also indicate that the cloned plant genes encode high-affinity phosphate transporters.

In response to persistent Pi deficiency plant have developed many adaptive mechanisms to enhance the availability and increase the uptake of Pi. One such adaptive mechanism is production and secretion of phosphatase to release Pi from organic forms. The activity of these phosphatases is induced by the depletion of Porg and varies with species and soil type. Genes encoding secreted phophatases have been cloned from Neurospora crassa. A phosphate starvation induced acid phosphatase (AtACP5) has been cloned that could involve in phosphate mobilization. Recently, a novel tomato (Lycopersicon esculentum) phosphate starvation-induced gene (LePS2) representing an acid phosphatase has been isolated and characterized.

Many plants secrete organic acids such as citric acids to solubilize Pi. Proteoid roots, an adaptation to Pi starvation in white lupin, are highly efficient in synthesis and secretion of organic acids to the rhizosphere. Secretion of organic acids enhances the release of Pi from Ca, Fe and Al phosphate complexes in the rhizosphere Increase secretion of organic may involves activation or synthesis of enzymes and anion channels to enhance the secretory processes.

Some legume crop like Cajanus cajan, pigeon pea, grow well in low P soils due to their special capacity to acquire Pi. Piscidic acid (p-hydroxybenzyl tartaric acid), a phenolic compund known to release Pi from iron complexes, is secreted in case of pigeon pea. The extraction of P from soils is one of the most promising areas for genetic manipulation. Plants like tobacco, papaya engineered to overexpress a bacterial citrate-synthase gene excreted large amount of citric acid. Being a chelator of Pi, organic acids could also improve plants extraction of Pi. By identifying gene involved in the biosynthesis of specialized organic acids, it may be possible to introduce these genes into other species to enable these species to extract Pi from adverse soils.

Some other genes induced under Pi deficiency are Ca2+ATPase, PEPcase, vegetative storage protein, enolase and pyruvate formate-lyase, b-glucosidase and novel gene such as TPS1 and Mt4. Identification of genes involved in phosphate uptake is a major first step towards the eventual development of plants, which can absorb phosphorus from soil in an efficient manner.

Potassium

Plants roots can absorb K+ over more than a 1000-fold concentration range. K+ uptake by roots has complex kinetics, indicating the presence of multiple uptake systems. Genes families encoding K+-channels have been identified in several gene is predominantly expressed in roots and it mediates the uptake of K+ in both the iM and mM ranges. Active transporters also participate in K+ uptake. Several candidate gene encoding energized transporters have been identified. The HKT1 transporter from wheat can cause K+ accumulation in yeast. It may function as a sodium-coupled transporter and is up-regulated in response to low K+ levels. KT or KUP genes are homologous to K+ transporter from E. coli and their products functions as K+ transporters when expressed in yeast. Some of the KT genes are expressed in roots and induced by K+ starvation.

Potassium is readily accessible to the root by simple diffusion in the soil solutions. The levels in fertilized soils are generally sufficient for optimal growth. Consequently, simple overexpressing K+-uptake genes may not produce any appreciable increase in crop yield. In fact, potassium uptake is limited by the presence of Na+, which compete for the uptake with K+. Potassium-uptake systems are highly selective for K+ when K+ and Na+ are present at comparable levels, but they are blocked by or transport Na+ when Na+ levels are high. Mutation study in K+ channels and the HKT1 transporter has been identified that decrease the inhibition of K+ uptake by Na+. SOS, a mutant gene has been cloned. Such mutations provide possible avenues for improving the K+ selectivity of roots in salt-contaminated soils and may prove to be useful for creating salt-tolerant crops.

Conclusion

Strategies to absorb nutrients from soil in an efficient manner, may play an increasingly role in the future to deal with the problems of poor soil fertility and to reduce the dependency on fertilizer application. This would be particularly welcome by resource-poor farmers in developing countries such as India. Molecular identification of more genes involved in nutrient solubilization and uptake has significant potential for the genetic enhancement of nutrient extraction from soil.

About Author / Additional Info:
A Biotechnogical Professional from India
Dr. Suresh Kaushik
drsckaushik@gmail.com
http://in.linkedin.com/in/sckaushik