Biotechnology for Enhancing Micronutrients Acquisition and Uptake Efficiency

Author: Dr Suresh Kaushik

Iron (Fe), manganese (Mn), zinc (Zn), and copper (Cu) constitute a group of the micronutrient cations. Out of these, zinc has received attention nationally, and iron and manganese on a regional scale or under particular soil- and crop-situation specific conditions. Zinc deficiency plaques nearly 50% of the soils, particularly alkaline low organic matter ones under lowland rice. Although iron is the fourth most abundant element of earth crust, yet one hectare of plough-sole is not able to provide even one kg of iron to the plants under situations worsened by host of environment and soil factors. Use efficiency scenario of micronutrient fertilizers puts figures on their utilization by crops at less than 5%. Where does the rest of the amount of the added nutrients go? Are these utilized by the succeeding crops or do these ultimately deteriorate the environmental quality? The need of the hour is to make efforts to identify and/ or develop plant types which are capable of meeting their micronutrient requirements from the inaccessible pools without suffering yield decrements.

Micronutrient uptake and accumulation traits are inherited. Environmentally-safe strategy is to screen the existing plant types having genetically-inherent micronutrient acquisition and storage strategies because such plants can meet their micronutrient requirements from the relatively less labile pools. Sustenance of the productivity of the agricultural crops in the micronutrient-deficient soils without their accretion through natural/synthetic sources by way of their acquisition from the non-labile pools is the most challenging area of research. Progress made in recombinant DNA technology in recent years and the application of molecular techniques has advanced our understanding in unraveling the mechanisms of acquisition of micronutrients by the plants from less-labile of soil pools and role of genes involved in these processes.

Nutrient acquisition and uptake:
The molecular identity of a vast array of mineral-nutrient transporters has been determined in recent years. Transporter proteins can be grouped into three broad classes- pumps, carrier and channels. Pumps are active transporters that perform energy transduction; that is, they couple the chemical energy released by ATP hydrolysis to the electrochemical ion gradient by using the electrically favourable movement of one molecule to drive the unfavourable movement of another. Channels facilitate the unidirectional movement of ions down their electrochemical gradient and hence do not require an input of energy, other than the membrane potential. Genes involved in the acquisition of micronutrients such as iron, zinc, manganese and copper have been described in this section at molecular level.

Ferric iron (III) is extremely insoluble at neutral or basic pHs and is the predominant form of iron in soil. Thus, before plants can take up iron for transport into the root, they must somehow solubilize these ferric iron. Plants solubilize and absorb Fe (III) using one of the following two strategies: Strategy-I: Dicotyledonous and non-graminaceous monocotyledonous plants use this strategy to acquire iron that is similar to the system used by S. cerevisiae. Strategy-I plants solubilize Fe (III) by releasing hydrogen ions and small organic acids to acidify the rhizosphere. These plants then reduce Fe (III) to Fe (II) through the action of enzymes such as Fe (III)-chelate reductase, and soluble Fe (II) is available for absorption by the roots with the help of Fe (II) transport activities. IRT1 gene from Arabidopsis is the first transporter gene to be isolated from plants and could be used for engineering plants to take up more iron. Two barley genes - ids2 and ids3 are also good candidate to increase the bioavailiability of iron in transgenic plants. Several other candidate genes (frohA, frohB, frohC, and frohD) that may encode Fe (III) reductase have been identified in Arabidopsis using degenerate polymerase chain reaction (PCR) with primers designed against motifs common to the yeast Fe (III) reductase proteins (Fre1p, Fre2p, and Frp1) and therefore may be potential candidate gene for developing transgenic plants with high iron uptake. The uptake of iron in transgenic tobacco was increased by constitutively expressing the yeast Fre2 gene encoding a ferric reductase. This was related to higher rate of Fe (III) reduction along the entire length of the roods and in the shoots. Transgenic plants were tolerant to iron deficiency and exhibited 50% higher Fe concentrations in younger leaves than nontransformed plants when cultivated in a iron-deficient medium. This suggests that the Fre2 gene may be used to improve iron uptake in crop plants. Strategy-II: Strategy-II plants, the grasses, release phytosiderphores, low molecular weight ferric-specific ligands, in response to iron deficiency. Phytosiderphores are a class of metal-chelating compounds produced by roots of graminaceous species to enhance the solubility and uptake of certain cationic micronutrients in the rhizosphere. These compounds have a high binding affinity for iron (III), and the intact phytosiderphores-ferric complex is directly transported across the root plasma membrane by specialized membrane transport. The first step in phytosiderphores synthesis is the combination of these molecules of S-adenosylmethionine to form nicotinamine (NA) via nicotinamine synthase (NAS). Nicotinamine is the main precursor compound for the biosynthesis of the phytosiderphores 2’-deoxymugienic acid and avenic acid. All three of these compounds have the ability to chelate iron (III) and are involved in the mobilization of iron for uptake into the plants. Several related genes including, NAS1, have been cloned from barley that encode nicotinamine synthase enzyme. Expression of the NAS genes is induced in iron-deficient roots, and repressed under iron-sufficient conditions. However, NAS genes are not expressed in shoots under either iron-deficient or-sufficient condition. Expression of NAS1 in E. coli was also found to be sufficient to allow E. coli to synthesize NA from exogenously supplied S-adenosymthionine. After formation of NA, NA aminotranferase (NAAT) transfer an amino group to produce an unstable intermediate that is rapidly reduced to form deoxymugienic acid. The gene for NAAT has also been cloned from barley and is induced by iron deficient. Two other barley genes - ids2 and ids3 - that may function in synthesis of mugineic acid have identified. Each encoded a protein with some similarity to 2-oxoglutartgate dioxygenases for the conversions of deoxymugineic acid and mugineic acid to epihyroxymugineic acid via hydroxylation. Use of genes encoding S-adenosymethionine synthase, nicotianamine synthase (NAS) and nicotianamine aminotrasferase (NAAT), and a through understanding of phytosiderophore (PS) biosynthesis and of the PS cation complex transport mechanism into the cytoplasm may allow the generation of dicot plants exhibiting strategies-II uptake and thus improved iron and zinc uptake. Additionally, high iron-containing transgenic plants have been produced by expression of cDNA coding for ferritin under the control of either constitutive (CaMV 35S) or seed specific promoters. A three-fold greater iron content in rice ( Oryza sativa) seeds was obtained.

Zinc is taken up from the soil solution as a divalent cation. Once taken up, Zn is neither oxidized nor reduced. Zn deficiency in soils has been recognized an important problem worldwide. Similarly, expression of Zn transporters may lead to increase zinc absorption in roots. Zinc transporters genes such as ZIP1, ZIP2, ZIP3, ZIP4, ZRT1, and ZAT could be good potential candidate genes to enhance zinc uptake and acquisition for the development of transgenic plants. This type of approach could also be evolved by increasing the level of methallothione for high zinc accumulation.

Manganese is taken into plants as the free Mn (II) ions. Concentrations of Mn can vary greatly in the soil, ranging from less than 0.1 mM in well-aerated alkaline soils to greater than 400 Micromolar in submerged soils. The Mn concentration required by plants also spans wide range, from 0.01 to 50 Micromolar. Nramp genes such as OsNramp1, OsNramp2, OsNramp3 (cloned from rice), AtNramp1, AtNramp2, AtNramp3 (cloned from Arabidopsis thaliana ) has significant potential for genetic enhancement of Mn extraction from soil.

Copper levels in soils range from 10-4 to 10-9 M, Most of the copper in the soil solution complexed to low molecular weight organic compounds. Plant require from 5 to 20 micromolar per g DW Cu, depending the species. Cu is a essential redox component required for a wide variety of processes such the photosynthesis (plastocyanin), the electron transfer reactions of respiration (cytochrome c oxidase), the detoxification of superoxide radicals (Cu-Zn superoxide dismutase) and lignification of plant cell wall (laccase). A COPT1 gene coding a putative copper transporter from Arabidopsis thaliana may be a potential candidate for copper uptake.

Gene transfer:

Advancement made in recent years in the area of recombinant DNA technology have provided an altogether new dimension to agricultural research. New, it is possible to harness genes of economic importance form sexually incompatible wide and weedy relatives of crop plants and from related and unrelated species and phyla. This has created a situation where the whole biological world is now being considered as a 'single gene pool'. The expectation are that in near future, the availability of expanded genetic base will provide new and novel genes or gene combinations for accelerating the speed and quantum of all rounds growth in agriculture through the use of modern tools of biotechnology. 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.

Transgenic technology :

Plants containing a gene or genes which have been artificially inserted instead of the plant acquiring them through pollination is known as transgenic plants or genetically modified or GM crops. Transgenic crops are now being cultivated in as many as 18 countries including 8 developing nations. During the first generation of the transgenic technology, emphasis was laid on the agronomic advantages like resistance to pests and disease, and tolerance against herbicides, but in the second generation of transgenic technology, emphasis is on improvising the quality of plant produce such as the improvement of carbohydrates, proteins, oil quality, enrichment of crucial vitamins and minerals (Fe, Zn) composition of stable foods. The inserted gene sequence (known as the transgene) may come from another unrelated plant or from a completely different species. Hence, genetic engineering is a specific process in which gene from a species are modified or genes from unrelated species can be introduced into the crop species by transformation methods, followed by regeneration, which is the subsequent selection in tissue culture of transformed cell, under conditions where each cell will express its totipotency and finally, form a new viable plant. There are many methods for genetic transformation such as Agrobacterium-mediated, particle bombardment and, protoplast fusion. Plant transformation is generally accomplished using techniques developed from a naturally occurring bacterial disease called crown gall. The casual agent, Agrobacterium tumefaciens, transfers some of its own DNA into a plant cell nucleus. Subsequent expression of the bacterial genes by the 'transformed' plant cell causes it to proliferate into a gall structure and produces a food source for the bacteria. For genetic engineering purposes, the A. tumefaciens DNA has been modified so that the genes that cause gall formation have been deleted and in their place genes have added to enable selection of plant tissue that contains the transferred DNA (T-DNA) along with desired trait gene or genes. Once a desired gene has been isolated, the functional region identified and, if necessary, modification made to ensure expression of the gene in the new host, the genetic engineering process will result in transgenic plants that contain the desired trait. Although broad-host range A. tumefaciens strains exist, many of the major crop plant including most cereals and legumes have only successfully been transformed by alternative methods. Genetically engineering technology has several advantages over conventional breeding methods for crop improvements such as the broadening of the germplasm base from which new character can be transferred, the ability to repeatedly transfer new genes directly into existing cultivars without many generations of additional crosses, the ability to transfer discrete gene without many unknown closely linked genes, and the ability to alter gene formulations that will produce new plant characteristics.

Conclusion :

There are now convincing evidences that the genes coding for several transporters and other proteins for nutrient acquisition opens up many options to improve nutrient uptake from the soils. Genetic engineering and molecular biological techniques have advance our understanding of different transport processes in plants and provide adequate insight in the key steps in nutrients uptake and accumulation. Genes encoding diverse transporters have been identifies and isolated from a number of organisms. The functional significance species of high and low affinity transporters for nutrient have been cloned and characterized by studying the mutants and over-expression study. Most of the genes encoding transporters expressed in roots. But, several intracellular transporters have also been cloned and characterized. Improvement of micronutrient acquisition in area where micronutrients deficiency in soils limits crop productivity is probably the most challenging and rewarding areas of research to achieve the sustainable productivity of agricultural crops.

About Author / Additional Info:
A biotechnology professional with Ph.D. in Molecular Biology and Biotechnology from India.