Plants with Improved Quality of Nutrition

One of the important goals of biotechnology is the production of plants with improved quality of nutrition for humans as well as for domestic animals. Improved quality of seed storage proteins by tailoring particular amino acids have major health benefits for humans and economic benefits for poultry industries. In this contrast a major target has been increasing lysine and methionine content in particular group of plants.

Generally, human requires certain essential amino acids (atleast eight) that are supplied by the diet. Since these are present in proteins they must by supplied by different foods because one or another essential amino acids may be below the required level.

Most human diet however, consists of mixture of food and overcome amino acid deficiency problem. Similarly, poultry and other domestic animals are also required to supply essential amino acids. Most of the poultry industries have, however, costly practice of feeding synthetic amino acids along with poultry feeds.

Engineering of plant seed storage proteins for high value amino acids are highly desirable considering nutritional quality of poorly fed people from developing countries. This is because people from these regions rely on a single staple food such as corn or rice.

Seed Storage Proteins:

Plant seeds contain storage a protein varies from 10% in cereals to 40% in certain leg­umes and oil seeds of the dry weight. Vast majority of the individual proteins present in mature seeds have structural roles or metabolic in function. The same seeds may also contain one to many groups of proteins serve as huge amino acid reservoir for use in germination and the process of seedling growth.

These seed storage proteins reflect not only the total protein content of particular species but also quality of the protein, in turn can be assign­ing to nutritionally or low profile proteins. For example, the low content of lysine, thrionins and tryptophan in various cereals seeds and cysteine and methionine in legume seeds. These inturn limit the nutritional quality of the seeds in some purpose.

Some of the characteristic of seed storage proteins is that are specifically synthesised and accumulates in seeds. Secondly, their presences in mature seeds in discrete deposite are called protein bodies. One of the earliest and first isolated proteins is wheat gluten and Brazil nut globulin. Classification of proteins in to their groups is based on their solubility. Three protein groups have been categorised during classification.

They are:

(1) Albumin storage proteins (water soluble)

(2) Globulins (dilute salt soluble)

(3) Prolamins (alcohol/water mixture soluble) (Table 18.1).

One of the widely distributed proteins in dicot seed is the 25 albumin protein. The napin proteins are well known in oil seed rape plants. The 25 albumin and globulin storage proteins are widely distributed in flowering plants, the alcohol soluble prolamins are restricted to grass family that are present in major cereals. Exception of rice, nearly half of the prolamins are further classified into sulphur rich, sulphur poor and high molecular weight (HMW) prolamin based on their amino acid sequence.

The globulin storage proteins are widely distributed in dicots as well as in monocots. They are divided into two groups based on their sedimentation coefficient like 7S vicilin type, globulins and 11S legumin type globulins. They exhibit considerable nutritional significance in that they are deficient in cysteine and methionine.

The 11S globulins consist of six subunit pairs and each of these subunit pairs consist of acidic subunit (MW 40,000) and basic unit (MW 20,000) linked by a single disulfide bond. In contrast, 7S globulins are trimeric proteins of 150,000 to 190,000 that lack cysteine residue.

The α-Zein account for 75 to 80% of the total proteins in maize and are categorized into two groups. One is M 19,000 and second is 22,000. Both have similar structure, consisting of unique N and C-terminal domains flanking sequences. Major source of Zein proteins are cereals.

Engineering Plant Seed Storage Proteins:

Present strategy in the modification of seed storage protein is mainly focussed on in­creasing concentration of sulphur containing amino acids (S-rich amino acids) in legume seeds and lysine content in cereals.

Since animal system (including human) cannot synthesise certain essential amino acids, dietary supply can overcome this problem. Cereal grains containing lysine rich protein consumed as high profile energy source in the diet of humans and lives stock. Cereal seeds however, generally tend to be deficient in lysine, threonine and tryptophan.

In contrast, legumes seed is the richest source for proteins (upto 40%) but shows, deficient in methionine and cysteine content as well as tryptophan (sulphur containing amino acid) for example, pea seed protein containing around 0.8% methionine and 1.0% cysteine. This level is insufficient for growth and development.

Lives stock animals however, require 3.5% by weight of dietery protein. Besides, people who consumes only vegetarian food suffers health problem due to amino acid imbalance. Therefore, breeding and genetic engineering methods can drive increase in concentration of essential amino acid to overcome nutritional imbalance.

Breeding Strategy:

Several pain staking breeding experiments have evolved a novel approach in the im­provement of amino acid profiles in the seeds of several pulses and cereals. One of the earliest successes in breeding technique is the substantial increase in the concentration of lysine amino acid content in maize plant. Several mutation breeding experiments reveals that any increase in one particular amino acid is always followed by declining amino acid concentration. As a result of the above mutant work the S-amino acid content of total seed protein remains con­stant.

Improvement by Genetic Engineering:

Enhancement of Lysine Content in Transgenic Plant:

Many crop plants contain low levels of several essential amino acids. Cereals in particu­lar have limiting amount of lysine. Thus, genetic engineering of crop plants has been used to increase lysine content. Lysine is the most important and limited amino acid for the dietary requirements of many amino acid followed by tryptophan.

Soyabean meal, which is rich in lysine and tryptophan, is used to sup­plement corn in animal feed. Any modification process to increase the level of lysine amino acid with in the plant seed, would replace the supply of mixed or single grain feed with purified amino acids. Therefore, efforts have been made to modify natural seed storage proteins by addition or replacement of amino acids to increase the lysine content of the plant.

Some of the amino acids like lysine, methionine, threonine and isoluesine belong to aspartate family. Aspartate pathway is controlled by two enzymes such as aspartate kinase and dihydro picolinate synthase (DHPS). Plant isoenzymes of aspartate kinase are inactivated by feedback inhibition process by threonine or lysine.

DHPS is also feedback inhibition by lysine and inhibition ap­proaches 100% when lysine level less than 100 µm. The rate limiting step in the pathway for lysine synthesis is catalysed by DHPS, which is encoded by the Dap gene. Thus, in plant, the expression of lysine insensitive DHPS activities causes the accumulation of high levels of free lysine by deregulating the key-enzymes involved in the biosynthetic pathway of the lysine syn­thesis via aspartate.

A subtantial increase in free levels has been obtained in tobacco plants transferred with Escherichia coli dapA and E. coli lys C genes and re­cently, Cornebacterium dap A and E coli lys C genes driven by a seed specific promotives were expressed in soyabean plants. Transgenic experiments resulted in fivefold increase in the total lysine content of seeds.

Another approach is the insertion of lysine residue into storage protein in maize seeds. Modification of 19 kD α-Zein by inserting lysine residue possibilities with in a Zein molecule. Margaita (1997) have modified Zein genes by inserting synthetic oligonucleotides encoding lys. rich sequence. Transgenic plants expressing modified α-Zein genes in aleuron endosperm cells contain large quantities of lysine-rich Zein proteins.

Another synthetic gene design construct for lysine enhancement has been established in which designed gene for protein molecules are based on α-helical, coiled-coil structure (based on natural proteins like leusine zipper and tropomesin) because this coiled coil structure has a high surface to volume ratio which allows the incorporation of high percentage of charged resi­due such as lysine.

Genes encoding such designed proteins were tested initially in Escherichia coli and they expressed in tobacco. Transgenic tobacco seeds using seed specific phaseolin and soyabean β-conglycin promoter shows 31% lysine and 30% methionine accumulation and this is perhaps first report of a significant increase in seed lysine content.

In continuation with many more examples, the gene for lysine-rich protein from soyabean has been expressed in the rice endosperm. To accomplish optimum expression soyabean glycine protein was chosen which is 11S globulin protein are similar to rice glutenin. Transgenic rice in which both globulins were expressed and targeted efficiently.

Soyabean glycine protein has several nutrition values especially in reducing cholesterol. Therefore high expression of the soybean gene with high amount of lysine in transgenic rice endosperm can reduce cholesterol. This is an added advantage as rice is the main staple food among people of several developing countries.

Feedback inhibition strategies have been found to be successful in the process of lysine enhancement. In one of the classical example, genes encoding feedback insen­sitive forms of aspartate kinase and DHPS have been cloned and transferred to novel plants expressed under suitable promoters.

It was found that expression of feedback insensitive DHPS and aspartate kinase could increases substantiate level of lysine in transgenic plants. Three mutants of the soyabean Dap A genes were constructed in which one with single amino acid substitute at codon 104 and another single amino acid substitutes at codon 112, sufficient to become insensitive to lysine feedback inhibition.

Expression of these mutant genes using seed specific promoters resulted in 100% increase in lysine content in transgenic soyabean and canola seeds (Fig. 18.1). More precisely total seed lysine content was increased upto 25% in soyabean and 100% in canola seeds. The resulted transgenic technology allowed the increase of 6.8% of threonine and total methionine increased by 6.8%.

Although plant proteins enriched in lysine content such as the poplar bark storage pro­teins and histone proteins have been identified, no natural seed storage proteins enriched in lysine relative to average lysine content of plant proteins have been identified.

Increase in Methionine Content:

Presently, genetic engineering technique has been used to express number of high sul­phur proteins in plants. Apart from producing nutritive value proteins. High protein diet for poultry animals have been produced by many company using plant gene technology.

They transfer genes that encode methionine rich protein into soyabean. The reason that the feed industry can replace supply of synthetic amino acids if transgenic soyabeans are loaded with adequate methionine in their proteins. These are rich in Brazil nut and sunflower. Nearly 20% of their proteins contain methionine.

One of the novel strategies has been the isolation of gene for naturally occurring, sulphur poor seed protein and modify its nucleotide sequence for enriched sulphur amino acids. This technique has been extended to protein like phaseolin from phaseolus vulgaris. This was modi­fied by the transfer of methionine rich 45 bp nucleotide sequences from Maize Zein seed storage proteins into β-phaseolin gene.

The modification has increased number of methionine content marginally. In order to raise content of the total seed proteins to greater than 1.6% by weight, engineered proteins would have to be expressed at high levels. Alternatively; a novel idea is the construction of entirely a synthetic gene sequence encoding artificial protein with a high level of methionine content.

Synthetic genes encoding novel proteins that are essentially a key com­ponent of amino acids like lysine, tryptophan and methionine. These gene sequences are cloned and expressed in E. coli for stability. The same gene when expressed in the seeds of transgenic tobacco shows protein contains of 31% lysine and 22% methionine.

Similarly, one such synthetic protein containing 13% methionine expressed in sweet potato. Several naturally occurring pro­teins with high levels of methionine content have been noticed among several plants. Some of the most promising methionine rich proteins are 21-kD Zein protein contains 28% methionine, 10 kD Zein containing 23% methionine, residue from maize and 10 kD prolamine containing 20% methionine synthesised in seeds.

In addition, dicotyledon 2S seed albumin from Brazil nut (Bertholletia exelsa) and its protein is commonly known as Brazil nut albumin (BNA) contain­ing 18% methionine. Similarly, 2S albumin from sunflower seed (sunflower seed albumin SSA) containing 16% methionine.

Generally, methionine protein from monocots are transferred and expressed in transgenic monocot seeds as proteins from dicots are transferred and expressed in transgenic dicot seeds.

Increased Methionine Content in Monocot:

In view of enhancing methionine content in monocot seeds, gene encoding methionine rich 10 kDa Zein has been transferred to maize. The transferred lines shows higher methionine content and seed methionine content reached upto 30%. These results highlighted that the expression of introduced protein compete with endogeneous sulphur, rich proteins for limited sulphur reserve.

Increased Methionine Content in Dicot:

Brazil nut proteins (BN 2S) has been an attractive candidate for modifications and en­hancing methionine content, inturn increased the nutritive value of plants, especially legumes and tuber crops. Which are generally contains less sulphur amino acids. Some of the novel approaches have increased high level of methionine content in dicot seeds.

In a classic case, the 2S albumin of Brazil nut (Bertholletia excessa) contains 18% methionine and this level is raised upto 30% in transgenic tobacco seeds after fusion to the (3-phaseolin promoter. This encouraged results and its outcome has been extended to other plants like Arabidopsis 2S albumin. These proteins were selected as a system for modification of storage proteins.

It has been shown that A. thaliana 2S albumins expressed in transgenic tobacco seeds are correctly processed and targetted to the protein bodies. Transgenic expression of chimeric Brazil nut 2S albumin genes has since shown in several plant system including Arabidopsis and tobacco (Table 18.2).

In addition, expression of the Brazil nut methionine rich 2S albumin has also been reported in transgenic potato. The feasibility of increasing the net content of transgenic BN 2S protein through sequence modification and gene driven by 35S promoter has been clearly established. It was also shown that 2S protein is a large subunit can potentially tolerate significant sequence size changes without affecting either its processing or stability.

The construction of chemeric gene by fusing phaseolin gene promoter to Brazil nut albumin BNA was also transferred to canola. Like maize, canola was selected basically because of its potential use in feed mixes. Canola lines expressing BNA at levels upto 4% of total seed protein. These results supported the use of these proteins to enhance nutritional quality of plants.

After feasable success in tobacco a chimeric gene containing BNA under phaseolin pro­moter has been transferred to soyabean. Transgenic soyabean expressed BNA at more than 10% of total seed protein resulting in nearly 50% increase in seed methionine content. In the resulting transgenic success, the total methionine content would be 1.8%. Which is within the range of methionine concentrations required by animals for optimal growth?

The BNA protein gene has been transferred to norbon bean. Expression of this gene under the controlled of the promoter LeB₄ legumin gene from Vicia faba resulted in accumulation of BNA upto 4.8% and methionine enhancement fulfills range required for optimal animal nutrition. In another alternative strategy for Brazil nut albumin protein is the transfer of sunflower albumin (SSA) into the leaf of lupin (lupinus angustifolius).

Lupinus pulses are used as poultry feeds for both ruminant and non-ruminant animals. Transgenic lupinus with high protein and fibre accumulate approximately 5% of salt extractable seed protein and is almost double when compared with untransferred plant. The same chimeric encoding SSA gene has also been transferred to chick pea and pea associated with considerable increase in methionine as well as total proteins in the extractable (2.5%) seed protein.

Improvement of 2S albumin protein of Brazil nut which is sulphur rich but lacks the essential amino acid tryptophan has been carried out by inserting tryptophan residues without compromising the structure. Three constructs, one with 5 consecutive tryptophan codons and the other two modified genes encoding proteins carrying single tryptophan residues under 35S CaMV promoter were used in transgenic work.

Improvement of Wheat and Corn Proteins:

The high-molecular weight gluten subunits (HMW-9S) is a one of the families of seed storage proteins synthesised in wheat endosperm at developing stages are important determinants of the processing characteristics of wheat flor. The total levels of HMW-98 are between 5% to 10% of total seed and play a central role in determining the elasticity of wheat dough.

Thus, this protein is important candidate for genetic engineering. Bleach and Anderson (1996) produced transgenic wheat by changing the composition and level of HMW-GS protein after addition of gene copies to the wheat genome. The transgenic wheat contains unique coding region created by the fusion of two native genes able to distinguish the product if the transgene for native endosperm protein.

In the improvement of nutritional quality of the corn, Zein genes have been modified by introducing lysine codon through site-directed mutagenesis. The modified Zein genes have been cloned along with the strong promoters like CaMV 35S and reintroduced into the corn. These transgenic corns produce the lysine rich zeins in the seeds.

Improvement of Crucifer Storage Proteins:

The napin or 2S protein is one of the attractive candidates for modifications interm of nutritional quality and also for molecular pharming. There have been several promising at­tempts for the genetic engineering of 2S albumins. The 5′-upstream sequences of Brassica napus and Brassica juncea 2S protein confer seed specificity of expression of an introduced gene.

The modified 2S protein is correctly expressed and targeted in a heterologous host. BNA protein gene and a methionine enriched engineered napin genes were introduced into B. napus and were expressed to improvise methionine level of total seed protein. Highest expression level of BNA in transgenic B. napus ranging from 1.7% to 4% of total seed protein.

This 4% methionine rich protein contributed about 33% more methionine in the total seed protein. Certain legume lectin promoters were used in these studies. In another novel strategy, the antisense gene for cruciferin was introduced into B. napus which consequently suppresses cruciferin and increases in the relative proportion of napin as a more balanced protein.

Enhancement of Leaf Protein Quality for Ruminant Animals:

Ruminant animals have commercial significance due to their wool production. Animal wool is basically made up of protein, rich in sulphur-amino acids, significantly greater demand for methionine and cysteine. The performance of ruminant animals can be improvised by supplementing the diet with methionine.

Sunflower seed albumin is a novel protein, directly targeted their S-amino acid into intestive without undergoing degradation in the rumen. Thus, vegetative parts (leaf) containing SSA is would be more appropriate to supply S.amino acids for increased wool production. Earlier attempts to express pea vicillin protein in the leaves of transgenic plants exhibit limited success until the protein coding sequence was modified to include signal peptide for endoplasmic reticulum.

A modified SSA chimeric gene with signal peptide was transferred to subterranean clover (Trifolium subterranean) expressing high level of methionine. This work demonstrated the feasability of the approach for modifying the S-amino acid content of vegetative plant material for the benefit of grazing animals.