Hijacking the Factory Management—Using Viral Vectors for Protein Expression

There are, of course, alternative ways of exploiting the protein synthesis machinery of plants for the production of foreign proteins. The earliest proponents of this process, however, were not plant molecular biologists, but rather the plant viruses that hijack plant cells and use them for self-reproduction. These viruses provide a direct method for the transient production of recombinant proteins in crop plants, and this is the subject of the chapter written by Grill (see Chapter 40). By inserting target genes into the viral genomes, the hijacker can be turned into a molecular farmer.

In discussing how plant expression can be achieved using stably transformed plants or transient viral expression, Grill uses as examples two plant-produced proteins that have already entered clinical trials in the United States. The protein made in stably transformed plants was a humanised version of the Avicidin antibody for the treatment of prostate cancer, while the transient viral expression approach was used for the production of personalised vaccines produced from the cancerous B cells of non-Hodgkins lymphoma patients. What these examples illustrate is the flexibility of the viral vector system. Essentially, using this approach, personalised plant-based production of treatments for individual patients becomes possible, which is not feasible using stably transformed plants.

Grill then gives a detailed description of how viral vectors work in practice. Most plant viruses have single-stranded positive sense RNA genomes, and those that are assembled into rod-like particles can accommodate large inserted genes because it is possible to increase the length of the virion. Using recombinant viruses to infect plants and then produce the chosen recombinant proteins is rapid, compared to standard transgenic approaches, and has been tested in the field since the early 1990s. Grill argues that the rapid high-level expression of proteins is what sets viral vectors apart from the classical methods. Scaling up the methods for plant inoculation with viral vectors from the laboratory to the field has involved innovative solutions. An example, shown in the review figures, illustrates plants being inoculated using high-pressure sprays containing an abrasive and the recombinant viruses. A further interesting aspect of viral vectors is that there is no requirement for the cultivation of transgenic plant lines in the field. However, proteins transiently produced by viral infection have to be harvested from green leafy tissue and cannot be stored as easily as the seeds produced by transgenic plants.

Grill puts the case, and we agree, that while viral vectors compare well with transgenic plants overall, their real advantage is the ability to produce proteins rapidly. Indeed, the use of viral vectors to produce patient-personalised vaccines against non-Hodgkins lymphoma is such a compelling prospect that we feel viral vectors must remain an important tool for farming molecular medicines in the future.

A Perennial Production System

In their contribution, D'Aoust et al. describe the use of perennial plants for the production of recombinant proteins. Perennial plants are interesting as production systems because, with several harvests possible from the same crop, the plants can be used for repeated extraction of the protein.

The authors argue the case for forage legumes and, in particular, discuss the merits of using alfalfa. Because of the difficulties that had to be overcome to enable protein expression and plant transformation, forage legumes are relatively new to molecular farming. The authors describe the advances in promoter technology that have led to the construction of efficient inducible or strong leaf-specific expression cassettes. The features of the available transformation methods are discussed for alfalfa, which was first transformed more than 15 years ago, with D'Aoust et al. stating that reliable and efficient Agrobacterium-mediated transformation has been made possible by the optimal choice of genetic background and the use of standardised transformation protocols.

Because alfalfa is a fodder crop for ruminants, high-protein-yield varieties have been obtained and large-scale protein extraction techniques have been established by the animal feed industry. The authors describe how they are adapting these techniques for the production of plant-derived vaccines and, through the description of a case study, illustrate successful antibody expression in alfalfa.

Harvesting Recombinant Proteins from Food Crops

Many recombinant proteins have been produced in those plants that are ideally suited for laboratory experiments, such as tobacco or Arabidopsis thaliana, but these are not suited to long-term or large-scale production. The global agricultural industry has focused on high-yield crops that have been optimised over generations to be cost-effective for the large-scale production of food, animal fodder and other products, such as fibres for clothing and pulp for paper-making. Therefore, research on molecular farming has moved towards evaluating these commercial species for the production of pharmaceuticals and other recombinant proteins.

In their contribution, Stoger et al. first discuss the nature of the recombinant proteins expressed in plants before progressing to a detailed discussion of the issues that are related to choosing the most suitable species for protein expression. These include a meticulous review of the decisions that need to be taken during protein-expression projects and an indication of where the potential advantages lie with each crop species. Their contribution provides a solid framework and entry point for researchers new to molecular farming.

Industrial ‘Plants’ of the Future

In her chapter, Hood describes the exploitation of transgenic plants as a production system for proteins that are of industrial importance. These include recombinant enzymes, the use of which would benefit industrial production processes that are currently based on synthetic chemistry. The high specificity and activity of enzymes is what makes them attractive to industry, and the argument presented in the contribution is that plants, with their high biomass and large-scale production potential, are ideal for mass production of these proteins. The central thesis is that using plants will reduce the cost of these proteins and make them available for a wide number of applications in industries where they were previously unaffordable.

The use of plants to produce industrially relevant enzymes has been shown to be practical in a number of species. For example, phytase and αα-amylase have been produced in tobacco. However, the author argues that the expression of proteins in leaf tissue is not ideally suited to molecular farming. This is primarily because of the expense and the difficulties involved in extracting the proteins from leaves. She discusses, therefore, using other plant species, such as alfalfa or oilseed crops, to produce industrially important proteins. The author makes the case for maize (Zea) as a production system, her well-reasoned argument being based on a comparison of the production costs of various crops. Although alfalfa has the greatest potential for the production of recombinant protein per hectare, this is offset by the need to extract the protein from leaf material. Of the crops surveyed, soybean has the lowest cost for protein production, but the methods required for transformation are labour intensive and expensive. The cereal crops, such as rice and wheat, are shown to have positive advantages for expression but, for pragmatic reasons, maize was their selected production platform. First, maize is the largest crop produced in North America and its two major advantages are the low cost and ease of large-scale production. The rationale for the use of maize rests on the sophisticated existing production and harvesting infrastructure and on the advantages of using a seed-based production system.

One contentious and difficult issue covered by Hood is that of containment, which is of serious concern given the large amounts of maize produced within the United States for both human and animal consumption. The author presents a series of measures that can be used to control inadvertent mixing of corn destined for food or fodder with the transgenic variety. It can be argued that this is where industry has to be at its most vigilant, because the consequences of transgenic crops entering the food chain are potentially very large. Overall, however, with close control over its use, maize could become an acceptable and profitable species for use as an ‘industrial plant’ of the future.

An Introduction to Industrial and Pharmaceutical Protein Production in Plants

Advances in plant molecular biotechnology do not merely mean that farmers and research scientists alike can now contemplate a more than adequate harvest from newly sown crops. During the last decade, transgenic plants have been shown to provide a practical and feasible method of producing recombinant proteins. This technology has now progressed to the point where plants can be used as a platform for the expression of proteins intended for use in the treatment, or diagnosis, of a number of diseases. Such proteins include recombinant antibodies, cytokines and blood substitutes.

This research area—the combination of molecular biotechnology and agriculture, which is referred to as ‘molecular farming’—focuses on producing valuable proteins in plants, and forms the subject of the following contributions to this handbook. Drawing on expertise from both industry and academia, we present reviews of both the plant species and strategies that are being used to transfer molecular farming from the research laboratory to the field.

It is widely predicted that the world capacity for recombinant protein production will soon be exceeded by the demand and that this demand will continue to increase. Yet, the justification for the use of plants for recombinant protein production may not be immediately obvious. While transgenic cell lines, animals and microbes will continue to have significant roles to play as expression hosts for recombinant pharmaceuticals, the future role of plants should not be underestimated. One reason for this is that plants constitute a mass-production platform that can be used for the economical, large-scale production of proteins for industrial use in processes that were previously unaffordable. This makes them particularly relevant for the production of the recombinant proteins that will be required to treat the diseases we shall be challenged by in the 21st century.

Molecular farming is a fast-developing research area where fundamental research into protein expression and purification is coupled to the practicalities of plant growth and harvesting. This is reflected in the following contributions of how to express proteins, where to express them and how to choose the most appropriate host plant for protein expression. At present, there is no consensus on either the ideal expression method or choice of species, which has, therefore, to be determined empirically in individual cases. It should be emphasised that crops producing recombinant proteins have been in commercial production since 1997 (see Chapter 41) and crops producing recombinant therapeutics have already entered clinical trials (see Chapter 40). The development of edible vaccines using plants, as described by Mor and Mason (see Chapter 39), will have a great impact on world health and protection from disease. It is certain that the impact of molecular farming will increase as the technique develops both scientifically and commercially.

Below, we briefly introduce the contributions to this section and focus on what we regard as the most interesting issues covered in each review.

Branched-Chain Amino Acids: Valine and Leucine

Pyruvate (2-oxopropionate) and acetyl CoA provide the carbon backbone for the two ‘branched-chain’ amino acids, valine and leucine. Isoleucine is also a branched chain amino acid, similar in structure to leucine, but the carbon backbone is derived from aspartate. These three amino acids are often grouped together, because they share several common enzymes in their respective biosynthetic pathways (Bryan, 1980; Bryan, 1990). The same enzymes that convert 2-oxobutyrate to isoleucine also convert pyruvate to valine in a parallel but distinct pathway, with no sharing of intermediates. Synthesis of both amino acids, valine and leucine, begins with the formation of acetolactate from two molecules of pyruvate. Acetolactate synthase (ALS) also often denominated acetohydroxyacid synthase (AHAS), catalysing this condensation reaction, contains thiamine pyrophosphate as its prosthetic group. Acetolactate is subsequently reduced, rearranged and the release of water yields 2-oxoketoisovalerate. Finally, a transamination reaction by glutamate produces valine. The branch point in this pathway is 2-oxoketoisovalerate. In a methylation reaction from acetyl coA, isopropylmalate is formed. Isomerisation and decarboxylation produces 2-oxoisocaproate, which is then transaminated to leucine.

These pathways appear to be in the chloroplast since isolated chloroplasts can synthesise valine from 14CO2, and several enzymes of the pathway have been found in isolated chloroplasts. The synthesis of branched chain amino acids is also subject to feedback control by the end products. Isopropylmalate synthase is inhibited by leucine. The first enzyme, ALS, is inhibited by valine and leucine. The sulfonyl urea (e.g. chlorsulphurone) and imidazoline herbicides (e.g. imazethapyr) are very strong inhibitors of ALS, where they bind to the pyruvate-, respectively, 2-oxobutyrate-binding site and antisense mediated repression of ALS also proved ALS to be essential for plant growth and survival (Höfgen et al., 1995). However, upon overexpression of the enzyme there was no increase in the soluble level of valine, leucine and isoleucine indicating that the enzyme is not rate-limiting the biosynthetic pathway (Smith et al., 1988).

Aspartate-Derived Amino Acid Biosynthesis: Lysine, Threonine, Isoleucine, and Methionine

The aspartate pathway is a highly branched pathway, leading to the synthesis of the amino acids lysine, threonine, methionine and isoleucine (Figure 27.5). This pathway is therefore subject to a complex control by enzyme feedback inhibition loops as well as transcriptional and post-transcriptional regulation of expression of genes encoding pathway enzymes (see, Galili, 1995; Saito, 2000; Galili, 2002). Aspartate is formed either through a specific transamination catalysed by glutamate-oxalacetate transaminase or by a deamination of asparagine catalysed by asparaginase. The first two reactions of the aspartate pathway are common to all of its end-product amino acids and include the synthesis of aspartic semialdehyde from aspartate, catalysed by the enzymes aspartate kinase and aspartate semialdehyde dehydrogenase. Aspartate semialdehyde is at an important branch point, since it can either be reduced to homoserine or condensed with pyruvate to give dihydrodipicolinic acid, which subsequently undergoes a series of six enzymatic reactions to yield lysine (see section on ‘Lysine Biosynthesis and Degradation’). The other branch starts with homoserine, an amino acid not found in proteins and usually not present in appreciable concentrations in plants, with the exception of peas, where it can constitute 70% of the soluble nitrogen in 1-week old seedlings. In most plants homoserine is quickly phosphorylated to O-phosphohomoserine, which represents the next metabolic branch point, since it can be converted in a three step mechanism to methionine (see section on ‘Lysine Biosynthesis and Degradation’) or in a single step to threonine by the enzyme threonine synthase. Threonine can either be used for protein synthesis or is further metabolised to isoleucine, the synthesis of which begins with the deamination and dehydratation of threonine to 2-oxobutyrate catalysed by threonine deaminase (TD). All branched chain amino acids share a number of common enzymes converting either 2-oxobutyrate to isoleucine or pyruvate (2-oxopropionate) to valine and leucine. The steps leading to isoleucine are catalysed by acetohydroxyacid synthase (ALS or AHAS) resulting in 2-acetohydroxybutyrate, acetohydroxyacid isomerase (AHAI) yielding 2,3-dihydroxy-3-methylvalerate, which is oxidised to 2-keto-3-methylvalerate through the activity of dihydroxyacid dehydratase (DHAD). Here the pathway to leucine branches out with four further steps while isoleucine is subsequently formed by transamination through a branched chain amino acid specific aminotransferase (KAAT), specific for 2-keto-3-methylvalerate leading to isoleucine and for 2-ketoisovalerate leading to valine, respectively.

As the biosynthesis of the amino acids lysine and methionine are currently the main focus of plant biotechnology a substantial body of knowledge has accumulated recently. Therefore, we devote a separate section to each of these two pathways.

Aromatic Amino Acids: Phenylalanine, Tyrosine and Tryptophan

The shikimate pathway provides the basic building blocks for the synthesis of the three aromatic amino acids as well as an array of other aromatic compounds required for functions as different as UV protection, electron transport, signalling, communication, plant defence, structural components and the wound response (Schmid and Amrhein, 1995; Radwanski and Last, 1995; Herrmann and Weaver, 1999). The pathway is firmly rooted in primary metabolism and forms a major link between primary and secondary metabolism in higher plants. From just this brief survey of products derived from the shikimate pathway it is not surprising to find that up to 35% of the ultimate plant mass in dry weight has its metabolic origins in this pathway as for example lignins are derived from the shikimate pathway. The first seven reactions of the pathway lead from erythrose 4-phosphate and PEP via shikimate to chorismate and are also referred to as the main trunk of the shikimate pathway, or the pre-chorismate pathway.

The shikimate pathway is present only in bacteria, fungi and plants. The absence of the pathway in all other genera has rendered the enzymes catalysing these reactions potentially useful targets for the development of new antibiotics and herbicides (Siehl, 1992).

Some of these reactions are unique in nature: for example, 5-enolpyruvylshikimate 3-phosphate synthase (EPSP-synthase), the sixth enzyme of the pre-chorismate pathway, catalyses the transfer of the intact enolpyruvate to shikimate 3-phosphate.

The first step of the synthesis of these three amino acids is the condensation of erythrose 4′-phosphate (derived from the oxidative pentose phosphate pathway or the Calvin cycle) with phosphoenolpyruvate (from glycolysis) to produce 3′-deoxy D-arabino heptulosonic acid 7′-phosphate (DAHP). This undergoes a series of reactions, including loss of a phosphate, ring closure and a reduction to give shikimic acid, which is then phosphorylated by shikimate kinase. Shikimate phosphate is combined with a further molecule PEP to give 3-enolpyruvylshikimate 5-phosphate (EPSP). The enzyme EPSP synthase, which has received considerable attention because it is inhibited by the herbicide, glyphosate, catalyses this latter reaction. EPSP is converted to chorismic acid, which is at a branch point in this pathway, and can undergo two different reactions, one leading to tryptophan, and the other to phenylalanine and tyrosine.

Anthranilate synthase (AS) catalyses the first reaction in the multi-step tryptophan biosynthesis branch by converting chorismate to anthranilate. AS is feedback inhibited by the end product tryptophan, which binds to an allosteric site on the AS catalytic αα-subunit. The fact that AS is the control point in the tryptophan branch in plant cells is indicated by pathway intermediate feeding and many other studies. But conversion of chorismate to tryptophan has significance beyond amino acid biosynthesis. This is the branch point from which the essential aromatic amino acids as well as many important secondary plant metabolites are derived. Plants use this pathway to produce precursors for numerous secondary metabolites, including the hormone auxine (e.g. indoleacetic acid), indole alkaloids, phytoalexins, cyclic hydroxamic acids, indole glucosinolates, acridone alkaloids, tetrahydrofolate, ubiquinone and vitamine K. These metabolites serve as growth regulators, defence agents and signals for insect pollinators and herbivores. Some of these alkaloids have great pharmacological value, including the anticancer drugs vinblastine and vincristine.

The synthesis of tryptophan from chorismate begins with the reaction of chorismate with the amide group of glutamine to produce anthranilic acid, which subsequently condenses with phosphoribosyl pyrophosphate (derived from ribose 5′-phosphate) to give phosphoribosyl anthranilate. This molecule undergoes a further series of reactions to produce indole glycerol phosphate, which then reacts with serine to produce tryptophan (catalysed by tryptophan synthase).

The synthesis of phenylalanine and tyrosine starts with the rearrangement of chorismate by chorismate mutase to prephenic acid, whose further metabolism has been subject to some debate. For some time the synthesis of phenylalanine and tyrosine from prephenate in plants was assumed to be the same as in bacteria, where the prephenate is either dehydrated to phenylpyruvate (prephenate dehydratase) or oxidatively decarboxylated to hydroxyphenylpyruvate (prephenate dehydratase). Both of these keto acids are subsequently aminated by tranaminases, the former to phenylalanine and the latter to tyrosine. In addition, although phenylalanine, tyrosine and tryptophan are necessary for protein biosynthesis, phenylanine is also a substrate for the phenylpropanoid pathway that produces numerous secondary plant products, such as anthocyanins, lignin, growth promoters, growth inhibitors and phenolics.

Although some of the enzymes involved in this route have been found in plants, there is a growing body of evidence which suggests that another route is either also, or in some plants solely, in operation. Formerly called the ‘pretyrosine’ pathway, it is now generally referred to as the ‘arogenate pathway’, and involves the transamination of prephenate to arogenate, which is then directly converted to either phenylalanine (arogenate dehydratase) or tyrosine (arogenate dehydratase). Arogenate dehydratase has been purified from sorghum and is activity shown to be inhibited by phenylalanine and stimulated by tyrosine, as might be expected from its position in the pathway.

Many of the enzymes of tryptophan synthesis have been found in the chloroplast, and labelling studies with ¹⁴CO₂ have shown that chloroplasts contain the complete pathways for the synthesis of the aromatic amino acids. It is believed that these pathways also exist in the cytosol and perhaps other subcellular compartments. As might be expected, feedback inhibition by tryptophan affects the synthesis of anthranilate from chorismate. Phenylalanine and tyrosine also inhibit their own synthesis, but it is not clear how this occurs. Two isoforms of chorismate mutase exist in a variety of plants, one being sensitive to inhibition by phenylalanine, tyrosine and tryptophan, whereas the other is not. The inhibition, however, is very much dependent on assay conditions and is not well defined.

The Metabolic Role of Amino Acids in Plants

Various transaminases transfer the reduced amino group to 2-oxo organic acids to form amino acids. Besides 20 proteinogenic amino acids which are used as building blocks of proteins, a vast number of non-proteinogenic amino acids are formed, either as intermediates of amino acid biosynthetic pathways, e.g. homoserine or homocysteine, or as products of certain pathways where these amino acids serve special functions in metabolism, such as ornithine and citrulline as part of the urea cycle or canavanine A as insect antifeedants.

Furthermore, a huge body of the amino acids are precursors for metabolic pathways (see, Buchanan et al., 2000). Glutamine is the precursor of heme groups and chlorophylls and methionine, respectively its direct activated product, S-adenosylmethionine (SAM), is precursor of the vitamin biotin, or is used directly as a major methyl group donor of numerous reactions of the C1 metabolism in cells, to synthesise the ‘aging’ hormone ethylene responsible for fruit ripening as well as the precursor of polyamines which are involved in stabilising and regulating DNA. The aromatic amino acids are beside other compounds precursors of pigments, phytoalexins, structural compounds as lignins, (here methionine is involved again in methylation reactions) as well as precursor of the plant hormone auxin and an almost innumerable number of secondary metabolites plants being able to synthesise. Furthermore, amino acids are involved in stress responses such as scavenging of active oxygen species, either directly or through regeneration of ascorbic acid by glutathione, a tripeptide (γγ-glutamyl-cysteinyl-glycine) or metal detoxification by phytochelatins (poly-glutathione). Thioredoxins are involved in controlling the redox status of a cell; they control photosynthesis by transmitting the dark-to-light switching signal. These examples are just a glimpse of the role of amino acids and peptides; several more are likely to emerge in the coming years.

Nutrient Uptake and Assimilation

Among the various minerals essential for plant growth the most limiting macronutrients are nitrogen, phosphorus and sulphur. A limitation of any of these minerals limits biomass formation in natural ecosystems and, in particular, plant production in agriculture.

Nitrogen Assimilation and Reduction

Nitrogen represents the mineral nutrient required in the largest quantities by plants and is most limiting where maximal biomass production is desired (Stitt, 1999; Tischner, 2000). The uptake and metabolism of nitrate and ammonia have been extensively investigated and analysed (Figure 27.1). It is now established that all steps of primary nitrogen assimilation are targets of several signal transduction cascades that integrate external stimuli and internal conditions of the plant. Within this regulatory network, nitrate reductase (NR) catalyses one of the most controlled reactions in plants, receiving input from light, photosynthesis, CO2, oxygen availability and nutrient status at the transcriptional and post-translational level. Nitrate uptake systems across membranes exist as high- and low-affinity forms. High-affinity nitrate transporters are either encoded by nitrate inducible or constitutive genes and have Michaelis-Menten constants for nitrate of 6–100 μμM. They are mostly expressed in the outer layers of roots to mediate increased uptake when the external nitrate supply is low. A constitutive low-affinity uptake system operates at nitrate concentrations above ~0.25 mM. Whereas the role of high-affinity transport systems is evident, the function of low-affinity transport systems is less clear. Optimal plant growth is achieved with balanced ratios of nitrate and ammonia but not with nitrogen source alone. The relative contributions of nitrate and ammonia to total nitrogen uptake differ considerably between plant species and ambient availability in soil. Ammonia uptake is mediated by families of active membrane transporters (Km <0.5–40 μμM), which exhibit differential expression in response to light, tissue and nutrient status.
The conversion of nitrate to nitrite is performed by NR, and of nitrite to ammonia by nitrite reductase (NiR). NR is cytosolic, mainly located in root epidermis and cortical cells and leaf mesophyll cells. NiR is chloroplast localised and encoded in the nuclear genome. In photosynthetic tissues reducing equivalents for the reduction of nitrate to ammonia are derived directly from photosynthetic electron transport. Reduced ferredoxin (Fd) is the electron donor that fuels the catalytic activity of NiR. In non-photosynthetic tissue, NADPH derived from the oxidative pentose phosphate pathway can be used to generate ammonia from nitrate. Experimental supplementation with nitrate induces genes of nitrate assimilation and carbon metabolism, mainly to provide carbon skeletons as acceptors for reduced nitrogen. Cross talk between assimilation pathways is further indicated by the finding that low sugar levels are able to repress NR expression, even in the presence of otherwise inductive nitrate concentrations and a down-regulation of NR expression is even observed under conditions of sulphate limitation. However, neither the chemical nature of the signal compounds nor the sensors responsible for these reactions are precisely known. Nitrate and ammonia could be sensed as free ions by extra cellular or intracellular receptors, but downstream products or the carbon could also exert regulation as well as the nitrogen to carbon ratio.

Though the picture is far from being resolved a number of nitrogen sensing and regulatory systems have been described such as a MADS-box like transcription factor responding to local nitrate supply, a transcription factor of the myb structural family and a putative protein kinase gene up regulated by nitrogen deficiency. Furthermore, an extra cellular nitrate reduction system which might act as a nitrate sensor via the release of nitric oxide (NO), GLB1 which is a structural PII homologue of the bacterial glutamine synthetase regulator and up regulated by light and sucrose, respectively repressed by glutamine and glutamate, and 14-3-3 protein family members that reversibly bind to phosphopeptide motifs in diverse target proteins in plant, fungi and animals, resulting in altered activities of enzymes and regulatory proteins.

Sulphur Assimilation and Reduction

Sulphate uptake and assimilation are carried out in plants by a unique pathway that is distinct from that in bacteria and fungi (Anderson, 1990; Leustek and Saito, 1999; Hawkesford, 2000; Hawkesford and Wray, 2000; Leustek et al., 2000; Saito, 2000; Grossman and Takahashi, 2001; Hawkesford, 2002) (Figure 27.2). Beside the most regulatory step, the sulphate uptake by roots which enables the plant to achieve the inner-cellular homeostasis, significant regulatory steps of sulphur incorporation into organic compounds are catalysed by adenosine 5′-phosphosulphate reductase (APR). APR controls the flux of intermediates to yield sufficient reduced sulphur (Suter et al., 2000; Tsakraklides et al., 2002). A further step of control is exerted by the cysteine synthase complex providing the carbon/nitrogen backbone for cysteine formation (Blaszczyk et al., 1999; Harms et al., 2000). Alterations to any of these three processes can have profound effects on cysteine biosynthesis and on the capacity of plants to grow in soils in which nutrient resources are limiting. Essentially most of the reduced sulphur is channelled from cysteine into methionine, Fe/S clusters, vitamin cofactors and proteins required to carry out crucial structural, catalytic and regulatory functions in the cell. Despite this importance for plant biochemistry, plant sulphur metabolism has been much less thoroughly investigated than that of nitrogen. It has, however, gained much more attention in recent years after the unexpected observation of sulphur limitation in agricultural production at least in Europe due to reduced aerial pollution.
Although the uptake and transport of sulphate probably take the same combined apoplastic/symplastic route as, for example, nitrate and phosphate, the sulphate ion seems to be much less mobile after deposition in vacuoles of the source tissue. Sulphate transporters have been cloned and functionally characterised from several species. They can be grouped into high (Km 0.1–1 μμM) and low (Km 1–10 μμM) affinity proton/co-transporters. Expression analysis of sulphate transporters demonstrated that they are present in root hairs and epidermis for sulphate acquisition and in vascular bundles of root and leaf for the allocation of sulphate. Several sulphate transporter genes are induced within hours of sulphate deficiency and are rapidly repressed upon renewed supply of sulphate. Once the sulphate enters the cell it is activated by ATP sulphurylase to form adenosine 5′-phosphosulphate (APS). ATP sulphurylase (ATP-S) isoforms in plants are located either in plastids or in the cytosol. The cDNAs for these isoforms were first isolated from potato. In Arabidopsis there appear to be at least three plastidic and one putatively cytosolic ATP-S. The APS generated by ATP-S can serve as a substrate for sulphate reduction or can be phosphorylated by APS kinase to yield 3′-phosphoadenosine 5′-phosphosulphate (PAPS). PAPS is the substrate of various sulphotransferases to catalyse the sulphatation of a range of metabolites including flavanols, choline, and glucosides.

The sulphate of APS is reduced to sulphite by the plastid-localised APS sulphotransferase, also termed APS reductase (APR). The reductant used by the enzyme is probably reduced glutathione; a domain of the enzyme resembles a glutathione-dependent reductase. APR transcript accumulates during S-starvation, suggesting that a key juncture for controlling assimilatory processes occurs at the point at which APS interacts with either APS kinase or APR. The sulphite generated in the APR catalysed reaction is reduced to sulphide by plastidial sulphite reductase (SiR), the gene of which has been recently identified. Electrons used for sulphite reduction are donated by reduced ferredoxin. Sulphide is finally transferred to activated serine, O-acetyl serine, yielding cysteine through the activity of the enzyme O-acetyl serine-(thiol) lyase (OASTL). Cysteine is the first organic compound carrying reduced sulphur and the precursor of all following metabolic steps carrying a sulphur or thiol moiety.

The allocation of reduced sulphur proceeds via the phloem. Glutathione (GSH) and S-methylmethionine (SMM) appear to have a role in transport as well as being an interorgan signal for the sulphur status from the shoot to the root. In plants such as wheat, substantial amounts of reduced sulphur are transported as SMM from source leaves to sink tissues. Similar to nitrate assimilation, either sulphate or its downstream metabolites are suspected to trigger changes in the mRNA levels of the sulphate transporter and APR genes. An activating effect on uptake and APR activity has been demonstrated for O-acetylserine (OAS), an intermediate in cysteine biosynthesis (for review see Buchanan et al., 2000). An investigation of changes in OAS levels may suggest a link between sulphur, nitrogen and carbon metabolism, because external supplies of these macronutrients mutually affect at least single steps within the assimilatory activities of each pathway and photosynthesis.

As a number of amino acid related topics will be covered through other sections of the handbook, this chapter will highlight examples in which combined molecular, biochemical and genetic approaches have helped to define the pathways and uncover regulatory mechanisms of amino acid biosynthesis in plants. Especially, we will focus on biotechnology driven research and its implications for both basic and applied research. Rational engineering of amino acid biosynthesis is exploited for as diverse aspects as herbicide design and quality improvement of crop plants. A comprehensive review of the general biochemistry of amino acid synthesis can be found elsewhere (Miflin and Lea, 1990; Singh et al., 1992; Singh, 1999; Buchanan et al., 2000).

Amino Acids in Plants

In plants, amino acids fulfil a wide variety of functions. Their common role is to serve as building blocks of proteins, which exert manifold functions in plant metabolism, and as metabolites and precursors they are involved in plant defence, vitamin, nucleotide and hormone biosynthesis, and as precursors of a huge variety of secondary compounds. One way or the other, as active catalysts or as precursors, amino acids are essentially involved in all metabolic, regulatory, and physiological aspects of plant metabolism (for comprehensive reviews see, Miflin and Lea, 1990; Singh et al., 1992; Singh, 1999; Buchanan et al., 2000).

Uncovering plant specific aspects of amino acid biosynthesis contributed both to fundamental and applied research. The goals are to understand the biosynthetic pathways and their regulation and to understand the regulation of genes controlling growth-limiting processes (e.g., the assimilation of inorganic nitrogen into amino acids). A second huge field of investigations are the pathways and factors regulating the synthesis of secondary plant compounds derived from amino acid precursors. However, these areas are mainly covered in other chapters of this handbook. In addition to their obvious role in protein synthesis, amino acids perform essential functions in both primary and secondary plant metabolism. Some amino acids as glutamine and asparagine serve to assimilate and transport fixed nitrogen from sources to sinks. The aromatic amino acids serve as precursors to secondary products such as hormones and compounds involved in plant defence. Thus, the synthesis of amino acids controls directly or indirectly various aspects of plant growth and development. Such studies should also provide a framework for manipulating amino acid biosynthesis pathways in transgenic plants. For example, enzymes in several pathways have been identified as targets for herbicides. In some cases, the genes encoding these enzymes have been used for engineering herbicide resistance in plant of the first generation of transgenic plants. Eventually, future progress in amino acid biosynthesis research may provide enhanced crop resistance to osmotic stress and improved food protein composition. Therefore, the structural and regulatory genes controlling amino acid biosynthesis in plants are of interest not only to biochemists but also to agricultural biotechnologists, breeders and agrochemical industry to develop new transgenic plants with benefit to the consumer, sometimes befitted as the ‘second generation’ of genetically modified organisms (GMOs).

Beside the proteinogenic amino acids, many plants channel large amounts of nitrogen into amino acids that are not usually constituents of proteins (Herrmann, 1995; DellaPenna, 1999; Grusak and DellaPenna, 1999). These non-protein amino acids comprise a diverse and often complex group of compounds: several hundreds of them have been found in plants, most often in seeds (especially those of legumes), where they can accumulate to high levels. Non-protein amino acids are found in all plant tissues as intermediates in the synthesis of protein amino acids (e.g., homoserine, diaminopimelic acid, ornithine, citrulline), and in a more restricted range of plants as metabolic ‘end products’. The function of the latter one is often unclear, but they are frequently toxic to animals and found in seeds where they appear to serve both as a storage reserve of reduced nitrogen and as a feeding-deterrent to herbivores. The mode of toxicity varies, but is usually based on interference with regulation, transport or protein synthesis. One amino acid fairly common in legumes is canavanine A, which can account for up to 6&percnt; of the fresh weight of seeds of the Jack bean. It is very similar in structure to arginine and is thus able to interfere with arginine metabolism in animals that ingest those seeds. When metabolised, canavanine A causes a variety of toxic effects, including pupal malformation in insects and immune dysfunction in vertebrates. Other toxic arginine analogues include homoarginine and indospicine, also found in legumes.

Carbon and nitrogen are the principal constituents of amino acids. The carbon backbones are derived at different branching points from primary carbon metabolism while reduced nitrogen is transferred to these 2-oxo acids to form the common amino acid head group. The unique feature of this amino acid head group is the ability to form peptide bonds and thus polymerise to proteins. The side groups determine the chemical properties of the respective amino acids and later the derived protein structures (Table 27.1). These side groups are either directly derived from the carbon moiety or modified, e.g. through binding of reduced sulphur (cysteine and methionine), or through cyclisation (proline and aromatic amino acids). Actually amino acids are the entry port of the macronutrients N and S into plant metabolism. Nitrogen and sulphur assimilation first results in amino acids, i.e. glutamine and cysteine, respectively. These two amino acids serve as precursors of all further organic molecules containing any of these macronutrients.

The Prospects of Developing Medicinal Plants (Present & Future)

Medicinal plants are the local heritage with global importance, World is endowed with a rich wealth of medicinal plants. Herbs have always been the principal form of medicine in India and presently they are becoming popular throughout the developed world, as people strive to stay healthy in the face of chronic stress and pollution, and to treat illness with medicines that work in concert with the body's own defense. People in Europe, North America and Australia are consulting trained herbal professionals and are using the plant medicines. Medicinal plants also play an important role in the lives of rural people, particularly in remote parts of developing countries with few health facilities.

The variety and sheer number of plants with therapeutic properties is quite astonishing. It is estimated that around 70,000 plant species, from lichens to towering trees, have been used at one time or another for medicinal purposes. The herbs provide the starting material for the isolation or synthesis of conventional drugs.

In Ayurveda about 2,000 plant species are considered to have medicinal value, while the Chinese Pharmacopoeia lists over 5,700 traditional medicines, most of which are of plant origin. About 500 herbs are still employed within conventional medicine, although whole plants are rarely used.

In India, medicinal plants have made a good contribution to the development of ancient Indian Material Medica. One of the earliest treatises on Indian medicine, the Charak Samhita (1000 B.C.), records the use of over 340 drugs of vegetable origin. Most of these continue to be gathered from wild plants to meet the demand of the medical profession. Thus, despite the rich heritage of knowledge on the use of plant drugs, little attention had been paid to grow them as field crops in the country till the latter part of the nineteenth century.

Medicinal plants help in alleviating human suffering. These plants "are being integrated to the field of foods as additives, beverages and cosmetics. They are widely used as sweeteners, as biters, as spices, as natural colouring agent and as insecticides. Mass selection recurrent selection, hybridization, clonal selection mutation and biotechnology are some of major techniques at their use for many proven medicinal plants. There are still several constraints.

During the past one century there has been a rapid extension of the allopathic system of medical treatment in India. It generated commercial demand for pharmacopoeial drugs and their products in India. Efforts have been made to introduce many of these drug plants to farmers. Several research institutes have undertaken studies on the cultivation practices of medicinal plants, which were found suitable and remunerative for commercial cultivation. The agronomic practices for growing poppy, isabgol, senna, cinchona, ipecac, belladonna, ergot and few others have been developed and there is now localized cultivation of these medicinal plants commercially.

Medicinal plants have curative properties due to the presence of various complex chemical substances of different composition, which are found as secondary plant metabolites in one or more parts of these plants. These plant metabolites, according to their composition, are grouped as alkaloids, glycosides, corticosteroids, essential oils, etc. The alkaloids form the largest group, which includes morphine and codeine (Poppy), strychnine and brucine (Nux vomica), quinine (Cinchona), ergotamine (Ergot), hyocyamine (Belladonna) scolapomine (Datura), emetine (Ipecac), cocaine (Coco), ephedrine (Ephedra), reserpine (Rauwolfia), caffeine (Tea dust), aconitine (Aconite), vascine (Vasaca) santonin (Artemisia), lobelin (Lobelia) and a large number of others. Glycosides form another important group represented by digoxin (Foxglove), stropanthin (Strophanthus), glycyrrhizin (Liquorice), barbolin (Aloe), sannocides (Senna), etc. Corticosteroids have come into sannocides (Senna), etc. Corticosteroids have come into prominence recently and diosgenin (Dioscorea), solasodin (Solanum sp.), etc. now command a large world demand. Some essential oils such as those of valerian kutch and peppermint also possess medicating properties and are used in the pharmaceutical industry. However, it should be stated in all fairness that our knowledge of the genetic and physiological make-up of most of the medicinal plants is poor and we know still less about the biosynthetic pathways leading to the formation of active constituents for which these crops are valued.

Medicinal and aromatic plants are found in forest areas throughout South Asia, from the plains to the high Himalayas, with the greatest concentration in the tropical and subtropical belts and arid region of Thar desert. India recognizes more than 2,500 plant species as having medicinal value, Sri Lanka about 1,400, and Nepal around 700. Some of these, found at high altitudes in particularly stressful environments, grow very slowly and cannot live elsewhere. Others are more broadly distributed and adapt more easily to different ecological conditions.

During the past decade, a dramatic increase in exports of medicinal plants attests to worldwide interest in these products as well as in traditional health systems. In the last 10 years, for example, India's exports of medicinal plants have trebled. But with most of these plants being taken from the wild, hundreds of species are now threatened with extinction because of over-harvesting, destructive collection techniques, and conversion of habitats to crop-based agriculture. For instance, the small coniferous Himalayan yew (Taxus baccata) has recently become a heavily traded species. Similarly, senna is being grown extensively in arid region of India.

The pharmaceutical industries have made massive investment on pharmacological, clinical and chemical researches all over the world in past five decades. Efforts have been made to discover still more potent plant drugs. In fact, a few new drug plants have successfully been passed the tests of commercial screening. The benefits of these efforts would reach to the masses in future if farmers initiate commercial cultivation of medicinal plants. In fact, agricultural studies on medicinal plants, by its very nature, demand an equally large investment and higher priority. India, in particular, has a big scope for the development of pharmaceutical and phytochemical industry.

The subcontinent, India is blessed with varieties of aromatic and medicinal plants. The agroclimatic conditions and rainfall favouring this bio-availability. More than 7,500 species of medicinal plants are grown in India. Owing to this India is considered as the botanical garden of the world and treasure house of the biodiversity. Ayurveda, our indigenous system of health care is accepted everywhere especially abroad. Vedas and other ancient scriptures give cleanout evidences of using herbs and medicinal plants. Ayurveda alone describes about 2000 species of plants, which constitutes more than 10,000 formulations.

Over the past 10 years there has been a considerable interest in the use of herbal medicines in the world. Regarding the export of medicinal plants India's contribution to the international market is comparatively very low. Utilizing our biodiversity and proper planning, Indian products can very well enter the overseas markets. This can be achieved only through proper development of medicinal plants, standardization of the extracts and keeping the quality. WHO has recognized the effectiveness of traditional system of medicine and its safety.

The Indian Pharmacopoeia (1966) recognized eighty five drug plants whose ingredients are used in various pharmaceutical preparations. The text is however; confine to a few important commercially grown medicinal plants whose cultivation deserves priority in out national economy.

According to R. B. S. Rawat and R. C. Uniyal, National Medicinal Plants Board Department of ISM&H (Agrobios News Letter, Vol. 1, No. 8, January 2003) the use of medicinal plants is as old as human civilization. India has a glorious tradition of health care system based on plants, which dates back to Vedic era. In Rig Veda which is the oldest known repository of human knowledge and wisdom (4500-2500 B.C.) mentions about hundred medicinal plants used by the Aryans while in Atharva Veda (2500-2000 B.C.) elaborate description of medicinal plants are given. Later in Samhita period the science of medicine systematically organized with clear concept and theories based on the treatises the Charak Samhita - 2000 B. C, Sushruta Samhita - 1000-800 B.C. Besides this there are other works on Ayurveda and medicinal plants by Nagarjun, Chakradatta, Sharangadhar and Bangasen - 1000-500 B.C. Vaghabhatta Junior - 800 A. D. complied most of the books on Ayurveda and wrote Ashtanga Hariday Samhita.

They further indicated that India is bestowed with unique diversity in culture and natural vegetation exhibiting rich plant diversity. It has all known types of agro-climatic, ecologic and edaphic conditions. It also have unique biogeographical positions having all known types of eco­systems ranging from coldest place, the dry cold desert of Ladakh (Nubra Valley with - 57°C), to temperate, alpine and sub-tropical regions of north-west and trans-Himalayas; rain forests with high rainfall; wet evergreen humid tropics of western ghats and arid and semi-arid regions of peninsular India; dry desert conditions of Rajasthan and Gujarat to the tidal mangroves of Sunderban. It harbors 17500 flowering plants out of which 2000 plants are used in various classical system of medicine like Ayurveda, Siddha and Unani. The tribal and other communities use about 8000 species of wild plants as traditional medicine. The drugs used in ISM are 90% based on plant material and are considered to be safe, cost effective and with minimal or no side effects when genuine ingredients are used.

Medicinal plants are living and irreparable resource, which is exhaustible if over used and sustainable if used with care and wisdom. The importance of medicinal plants has been overlooked in the past. However, at present medicinal plants are looked upon not only as a source of affordable health care but also as a source of income. According to WHO report, over 80% of the world population relies on traditional medicine largely plant based for their primary healthcare needs.

The forest areas have been the traditional source of medicinal plants and herbs. The position cannot be sustained much further because on the one hand the areas under forests have been steadily shrinking and on the other the requirement of medicinal plants and herbs has increased steeply. This has resulted in unscientific and over exploitation of medicinal plants in the forests. One indication of the scarcity of some medicinal plants is their steep prices. The Ministry of Environment and Forests have already banned 29 endangered species of medicinal plans from their natural habitat. While medicinal plants are being utilized in the preparation of a number of modern drugs, there is a new trend worldwide of using natural products. Besides medicinal values, Pharmaceuticals, herbal food supplements, toiletries and cosmetics are growing in consumption in the international market.

The Role of Roots in Plant Growth

Root (botany), organ of higher plants, usually subterranean and having several functions, including the absorption and conduction of water and dissolved minerals, food storage, and anchorage of the plant in the soil. The root is distinguished from the stem by its structure, by the manner in which it is formed, and by the lack of such appendages as buds and leaves. The first root of the plant, known as the radicle, elongates during germination of the seed and forms the primary root. Roots that branch from the primary root are called secondary roots. In many plants the primary root is known as a taproot because it is much larger than secondary roots and penetrates deeper into the soil. Beets and carrots are examples of plants with very large taproots. Some plants having taproots cannot be transplanted easily, for breaking the taproot may result in the loss of most of the root system and cause the death of the plant.

Roots arising from the stem are known as adventitious roots. Such roots may be seen near the base of a corn stem. Adventitious roots formed high up on a stem are termed aerial roots or prop roots. Such roots aid in supporting the stem, as in the banyan, the mangrove, and certain orchids.

COMPOSITION

The root is composed of three types of tissue: the epidermis, or surface layer; the ground tissue, or cortex; and the vascular core, situated at the center of the root. Certain cells of the epidermis are modified for an absorptive function. Long, tubelike projections, called root hairs, grow from these cells into the absorptive surface of the root and anchor the root to soil particles. Water absorbed by root hairs is transferred across the cortex, the region of water and food storage, and into the vascular core, which carries it up into the stem. Organization of the vascular core in a root is markedly different from that in a stem. In the stem the vascular tissues xylem and phloem are grouped together in vascular bundles. In the root a central core of xylem has radial bands that extend outward toward the cortex, and between these bands are strands of phloem. In aerial roots the xylem core, which is usually solid in subterranean roots, often has a central zone of pith.

GROWTH

Under normal conditions the growth of roots is influenced chiefly by gravity and by the presence of water. Roots tend to grow downward into soil, unless water is more readily available at the surface. In addition to the primary growth in length occurring at the apex of the root, a secondary growth occurs that adds xylem, or wood, to the inside of the root and phloem toward the outside. Phloem produced in this manner becomes involved in the formation of bark, which covers old roots as well as old stems. Old roots often are virtually identical therefore with old stems.

Because in many plants roots can be formed from a cut end of a stem, cuttings may be used for plant propagation. Some plants, such as the willow or geranium, root quite easily, whereas others, such as the conifer, rarely root without special treatment. Root formation can be stimulated on cuttings of many plants by the application of the so-called root hormones, substances found naturally in the plant when new roots are formed. Most commercial preparations of root hormones contain indoleacetic acid, one of the most common root-stimulating substances. Occasionally roots may be formed from leaves, as in the African violet, which may be propagated by rooting the cut end of a leaf base in water. In some plants roots may give rise to shoots. For example, the stems that are formed at various distances from the base of a Lombardy poplar arise from roots.

USE

Roots of many plants are edible and contain considerable quantities of food materials, particularly starch. Root crops important in agriculture include the sweet potato, beet, turnip, carrot, parsnip, and cassava. The wild forms of these plants have much smaller roots than the cultivated forms because continued development by agricultural peoples has improved the size, texture, food value, and flavor of the roots in cultivated varieties.

Phytochrome Functions in Natural and Crop Environments

In nature, plants are exposed to broad-band irradiation for long periods of time. This radiation is periodic on a daily cycle, and superimposed on the daily cycle is an annual seasonal cycle. Daylight covers the whole spectrum over which photochemical reactions are possible (i.e. ca. 350–1200 nm) but peaks in photon units around 600 nm. Solar radiation is filtered in the atmosphere by various elements and molecules which absorb narrow bands to a lesser or greater effect. The result is a radiation spectrum for photochemical action that effectively ranges from ca. 400 nm to ca. 1200 nm, with a number of apparent peaks and hollows.

To relate this spectrum to phytochrome action we can estimate the relative amounts of radiation incident upon a plant at the absorption maxima of Pr and Pfr (i.e., the R:FR ratio); in open daylight this value is close to 1.15, and hardly changes with cloud cover or weather conditions (see Smith, 1983). Solar radiation with such a R:FR ratio will establish, at photoequilibrium, about 55% of the total phytochrome as Pfr; that is, Pfr/P = 0.55. Such a concentration of Pfr is sufficient to saturate most LFRs, such as seed germination, the inhibition of hypocotyl extension and the stimulation of leaf development during seedling establishment. Also, of course, all VLFRs will be saturated at such Pfr concentrations. In effect, therefore, daylight may be regarded as red light as far as most photomorphogenic phenomena are concerned.

When daylight interacts with vegetation the spectrum of the radiation transmitted or reflected from that vegetation is massively altered. The photosynthetic pigments absorb most of the blue and red photons and also much of the green, but radiation beyond ca. 700 nm is unaffected. Thus, transmitted or reflected radiation is low in R and relatively high in FR. Indeed, it is possible to use a suitable leaf, such as a bean leaf, as a FR filter in simple laboratory experiments. When plants grow in dense stands, as in vigorous herbaceous communities, forests or, indeed, in crop plantations, the radiation transmitted and reflected from leaves represents a major component of radiation present within the stands. Thus, the R:FR ratio above a crop may be 1.15, whereas within the crop it can be as low as 0.1. At such a R:FR ratio the phytochrome photoequilibrium will be less than 0.2, that is, less than 20% of the total phytochrome will be present in the active Pfr form.

Many LFR responses would still be saturated by such Pfr concentrations, but in mature plants, as opposed to seeds or seedlings, the low Pfr level causes a number of responses, known collectively as the shade avoidance syndrome, which has a marked effect on growth, architecture, and reproduction. These R:FR ratio responses, briefly mentioned above, allow plants to respond sensitively to the competitive threat posed by neighbours. The most striking response is a strong stimulation of stem elongation. When this is successful it elevates the leaves to positions within the canopy where unfiltered radiation is available; it is essentially a strategy to enhance the capture of radiation for photosynthesis. In natural communities this response, mediated by phyB principally but with subsidiary action by phyD and phyE, is a potent competitive strategy. In very dense canopies, such as forests, competitive strategies are obviously useless for relatively small herbs, and in these conditions another component of shade avoidance improves the chances of survival of the germ line. Deep shade, via the associated low R:FR ratio, accelerates flowering, sometimes in a quite spectacular fashion. In some ecotypes of Arabidopsis, for example, flowering may take 50–60 days in white light but in a low R:FR simulating deep shade it happens within 10 days (Botto and Smith, 2002).

What does shade avoidance mean for crop plants? Most crops are grown in dense plantations, for example, in the UK it is common practice for farmers to drill up to 400 seeds per m². Not all of these survive till maturity, but the resulting stands are extremely dense leading to very low R:FR ratios even early in the growing season. Shade avoidance in these stands causes such extreme stem elongation that the proportion of dry matter allocated to the seeds may be quite strongly reduced compared with the ideal. Furthermore, and in practice more important, such long-stemmed plants are susceptible to lodging, which can result in big losses in yield. The take-home message for this chapter is that modifications to shade avoidance resulting from the transgenic manipulation of the phytochromes could potentially have considerable benefits in the field.

Phytochrome Response Modes

Decades of physiological investigation have resulted in the identification of several distinct ‘response modes’, based on photobiological criteria (for review see Smith, 1995). The classical phytochrome-mediated response, first demonstrated by the pioneering studies of Sterling Hendricks, Harry Borthwick and their colleagues in the 1950s (Borthwick et al., 1952) is the low fluence response (LFR). LFRs (such as the stimulation of seed germination, the inhibition of seedling elongation and the control of flowering by night breaks) are saturated by low fluences of R and reversed by similarly low fluences of FR.

This R/FR reversibility became, and remains, the classical criterion of phytochrome action. Decades later, a ‘very low fluence response’ (VLFR) was recognised, in which minute amounts of light saturate response by establishing very low concentrations of Pfr. Because Pr has a tail of absorption stretching up to 730 nm, even FR radiation will establish low levels of Pfr and therefore the VLFR is not FR-reversible. The most important VLFR in nature is the stimulation of germination of small seeds buried beneath the ground and briefly exposed to daylight by disturbance. Both the LFR and the VLFR require only brief periods of irradiation, and when the light is kept on for several hours a different mode of action becomes apparent. This ‘high irradiance reaction’ (HIR) requires continued irradiation for several hours and the response is usually a diminishing logarithmic function of irradiance (or fluence rate). The HIR can be observed in etiolated seedlings grown under continuous irradiation. It is characterised by dependence on fluence rate, usually with an action maximum in the FR, and by non-conformation to the reciprocity law (for review see Mancinelli, 1994).

All three of these response modes (LFR, VLFR, HIR) can, and often are, involved in the control of germination and de-etiolation. Also, the role of the phytochromes in photoperiodism involves interaction with the circadian rhythms, and can take the form of either an LFR or HIR. Finally, phytochromes regulate growth and flowering in mature plants in the natural environment via a R:FR ratio response. A wide range of phenomena, including elongation growth and the rate of flowering (separately from the induction of flowering), exhibit a direct linear relationship to the proportion of Pfr established by the incident radiation (Smith, 1983, 1995, 2000). These R:FR ratio responses, which are the basis of proximity perception and shade avoidance, may indicate a quite separate response mode, or may represent a sub-set of LFR responses conditioned by acting within the environment of light-grown tissues.

Relating the different response modes to the individual phytochromes became possible with the generation of null mutants that lack functional phytochromes (for review, see Whitelam and Devlin, 1997). The conclusion from many mutant studies is that the VLFR and the FR–HIR are both mediated by phyA, whereas the LFR is mediated predominantly by phyB. Furthermore, the R:FR ratio responses are mediated predominantly by phyB, with supplementary action by phyD and phyE. These discoveries opened the way to exploiting our knowledge of the individual functions of the members of the phytochrome family by transgenic over-expression of the PHY genes. Over-expression has been important for fundamental objectives, to analyse the molecular actions of the phytochromes and to help elucidate signal transduction pathways, but increasingly transgenic methods are being applied towards the improvement of crop plant performance.

The Phytochromes and Their Functions

The Phytochrome Family of Photoreceptors

The phytochromes are a family of photoreceptors that absorb radiation across the 600–800 nm waveband. The 600–700 nm band is conventionally referred to as red light (R) while the 700–800 nm band as far-red (FR). Each phytochrome can exist in two photoconvertible conformers: Pr has an absorption maximum at ca. 665 nm and is converted to Pfr, which absorbs maximally at ca. 730 nm, being thereby converted back to Pr. The overall scheme, first worked out by Sterling Hendricks and Harry Borthwick, with their colleagues at Beltsville in the 1940s and 1950s on the basis of supremely elegant physiological experiments, is as follows:

Both Pr and Pfr have relatively broad absorption bands and these overlap below ca. 700 nm; this means that in broad-band irradiation, such as daylight, Pr and Pfr are continually being inter-converted resulting in a dynamic equilibrium, known as the phytochrome photoequilibrium, which is a quantitative function of the relative amounts of R and FR incident upon the plant. The photoequilibrium is conventionally expressed numerically as a proportion of the total phytochrome present as Pfr, or Pfr/P. The roles and mechanisms of Pr and Pfr have been investigated almost entirely by reductionist approaches, growing plants in darkness and exposing them to brief or prolonged irradiation from narrow-beam sources. For over fifty years this research has been and continues to be a tour de force of modern biology, and the knowledge accrued can now be applied to the infinitely more complex situations where plants growing in a natural environment are subject to continuous and often rapid fluctuations in light signals over a wide wavelength range.

The phytochromes are encoded by a small multi-gene family comprising five genes in Arabidopsis but possibly only three in the cereals (Mathews et al., 1996). The five Arabidopsis phytochromes are known as phytochrome A (phyA) to phytochrome E (phyE). Prior to molecular characterisation, phytochromes were classified into two main pools, based on their biochemical and kinetic characteristics as determined from in vivo and in vitro spectrophotometry. The so-called type I phytochrome is the predominant phytochrome present in etiolated seedlings, the first to be identified and still the most completely characterised. It is described as being ‘light-labile’, although it is only the Pfr form which is labile; thus type I phytochromes are also labile in the dark as long as Pfr has been previously generated.

Degradation half-lives of Pfr are typically around 20–45 minutes, and thus Pfr is essentially absent from plants exposed to more than ca. 24 hours of white light. Phytochrome A (phyA) is the only known representative of the type I phytochrome pool. In etiolated seedlings and dark-grown tissues phyA accumulates to relatively high levels in the Pr form. Exposure to light causes rapid loss of phyA, not only through degradation of Pfr_A, but also because phyA mRNA is highly unstable and because transcription of the PHYA gene is under feedback down-regulation by Pfr. Thus, in light-grown plants phyA is present at barely detectable levels. Although this may appear a technical detail, it becomes crucial to the role of transgenic phyA in the field, as will be seen.

In contrast, type II phytochromes are present in low steady-state concentrations in both dark-grown and light-grown plants because the Pfr forms are relatively stable (Furuya and Schäfer, 1996). All other members of the phytochrome family, that is, phyB to phyE, have these characteristics and are thus regarded as type II phytochromes. PhyB to phyE are synthesised slowly and appear not to be under such strict transcriptional regulation as phyA. In most cases studied, phyB is the predominant phytochrome in light-grown plants. As phytochrome action is a function of the concentration of Pfr, regulation of the rates of synthesis and degradation of the individual phytochromes is clearly crucial in determining the physiological response. It follows that attempts to regulate development by the transgenic expression of phytochrome genes must take account of the rates of synthesis and degradation of the transgene products.

Genetics and Phytoremediation Strategies

Genetic Engineering Possibilities

To determine possible mechanisms for genetically engineering a plant to increase its remediation ability, it is best to begin with the mechanism for arsenic uptake and storage. It is believed that arsenate is taken up from the soil by the phosphate transporter. This has been suggested by exclusion experiments in which inorganic arsenate competed with inorganic phosphate for transport by a phosphate transporter. (Csanaky and Gregus 2001) It is believed that the high affinity phosphate uptake system is involved, because it has a lower selectivity than the low affinity system, and suppression of the high affinity system results in smaller arsenic uptake and accumulation. (Fitz and Wenzel 2002) One mechanism that could be useful would be to overexpress the phosphate ion transporter gene in plant roots. This would allow for more arsenic uptake by the plant, and could therefore lead to greater arsenic sensitivity, due to increased concentration of arsenate in the plant, and accumulation.

Once inside the plant, the arsenate travels with the transpiration flow through the xylem and into the stems and leaves. (Tu et.al 2002) Here it is reduced, possibly by an arsenate reductase enzyme (ArsC) into its more toxic form, arsenite. (Dhankher et.al 2002) Another possible mechanism would be to overexpress the arsenate reductase gene in plant shoots. Studies have coupled the arsenate reductase gene to a soybean ribulose bisphosphate carboxylase small-subunit gene promoter, which will give it strong light-induced expression, insuring that the arsenate is reduced in the shoots, not roots. (Dhankher et.al 2002) Since arsenite is more toxic than arsenate, all the transgenic plants were more sensitive to arsenic in the growing medium. (Dhankher et.al 2002)

Arsenite has a great affinity for thiol groups and consequently binds to peptide-thiol molecules, also known as phytochelatins. (Dhankher et.al 2002) Phytochelatin production appears to be activated by the presence of heavy metals and arsenic. (Sauge-Merle et.al 2003) One enzyme critical in the pathway for the formation of these peptide-thiol molecules is y-glutamylcysteine synthetase (y-ECS). (Dhankher et.al 2002) This enzyme produces the first, and possibly limiting enzyme in the phytochelatin producing pathway. (Dhankher et.al 2002) Studies have coupled this gene to a constitutively expressed actin promoter, giving a constant high level of phytochelatins in the plant and providing a sink for reduced arsenite within the cells. (Dhankher et.al 2002) Plants transformed with both ArsC and y-ECS produced four times more biomass than the control when grown four weeks on a 200 micromolar arsenate medium, showing greater tolerance as well as accumulation. (Dhankher et.al 2002)

This complex is then transported via a specific transport mechanism into a vacuole or into the cell wall. (Pickering et.al 2000) Another possible genetic approach would be to overexpress the gene for the transport protein into the vacuole or cell wall. This would remove the arsenite more quickly from the cells, to reduce the toxicity to the cell, and allow for more arsenic to be taken up.

Arsenic has been shown to have deleterious effects on seed germination, as well as root length and mass, which can detract from the effectiveness of a phytoremediation treatment. (Nie et.al 2002) Some bacteria, such as Enterobacter cloacae promote plant growth and minimize arsenic stress on the roots. (Nie et.al 2002) The Enterobacter cloacae UW4 1-aminocyclopropane-1-carboxylate deaminase gene in plants has been shown to increase the interaction between plants and these growth promoting bacteria. (Nie et.al 2002) In addition, transformed plants accumulated four times as much arsenic as non-transformed plants. (Nie et.al 2002) Overexpression of this gene is another possible mechanism to increase plant growth and phytoremediation of arsenic-contaminated soils.

A combination of these transformations and others could create a plant that was capable of high levels of arsenic accumulation and tolerance, when used to transform a species with high biomass that is adapted to living in the environment in which the arsenic-contaminated site exists. A tree such as cottonwood would be ideal for this purpose, and researchers are concentrating their efforts on performing this transformation. (www.edie.net) Further studies must be performed on the unique hyperaccumulating species, such as Pteris vittata and Pityrogramma calomelanos, to determine if they possess a novel mechanism or just an abnormally high expression of a given enzyme in the accumulation pathway. These hyperaccumulators or the transgenic accumulators can then be used with various phytoremediation strategies.

Phytoremediation Strategies

Phytostabilization is a possible strategy for control of arsenic, where arsenic tolerant plants, such as Spergularia grandis and Agrostis catellana are used to keep Arsenic contaminated soil in place to prevent erosion and leaching of the contaminant. (Fitz and Wenzel 2002)

Phytovolatilization is the use of plants to turn a pollutant into a gas to remove it from a site. Although this does occur to some extent in nature, volatile arsenic is still toxic, so phytovolatilization is not really a feasible option. (Fitz and Wenzel 2002)

One positive phytoremediation option is rhizofiltration, the removal of pollutants from water using a hydroponic system. Arsenic contaminated runoff could be run through a set-up with Lepidium sativum, which has been shown to remove arsenic from anaerobic flooded environments. (Robinson et.al 2003) This can also be used in conjunction with bioleaching, which would produce a heavily arsenic-contaminated runoff from soil with initially low arsenic bioavailability. (Seidel et.al 2002)

One positive phytoremediation option is rhizofiltration, the removal of pollutants from water using a hydroponic system. Arsenic contaminated runoff could be run through a set-up with Lepidium sativum, which has been shown to remove arsenic from anaerobic flooded environments. (Robinson et.al 2003) This can also be used in conjunction with bioleaching, which would produce a heavily arsenic-contaminated runoff from soil with initially low arsenic bioavailability. (Seidel et.al 2002)

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Phytoremediation: Cleaning Up With Plants

Have you ever heard of locoweed? As a child, I watched a lot of Bgrade movie Westerns and one thing I learned was that, if you are a rancher, you don’t want your cattle grazing on locoweed! Locoweed is the common name given to several species of Astragalus, a genus in the Fabaceae or pea family. Also known as milk vetch or poison vetch, many species of Astragalus take up unusually large quantities of selenium from the alkaline soils of the western plains.

The high selenium content contributes to a disease known as alkali poisoning or “blind staggers” in cattle unfortunate enough to graze on this plant—the cattle literally behave as though they are crazy. There are many regions where natural geochemical processes have produced soils that are rich in metals such as nickel, chromium, gold, cadmium, selenium, and arsenic. Normally high levels of heavy metals would be toxic to plants, just as they are to humans, yet many plants actually thrive on soils rich in such metals. For some plants, the metals are not a problem simply because the cell membranes surrounding the root cells prevent the metals from entering the root. Other plants actually take up the metals and accumulate them to levels that would be toxic to most other plants.

In Astragalus, for example, selenium may account for as much as 10% of the dry weight of the seeds. In soils that are rich in nickel, some plants may contain 200,000 times more nickel than plants growing in normal soils. Many years ago, such plants were known as “indicator species,” and prospectors would take the presence of such plants as an early indication that the soils may have contained a mineral of interest, such as gold. This was called phytoprospecting. We now call these plants accumulator species, which are not injured by high concentrations of heavy metals because they sequester (isolate) the metals with small proteins called phytochelatins.

The sequestered metals are then stored in the large central vacuole of the plant cell, where they cannot interfere with the cell’s metabolism. There has recently been a renewed interest in accumulator species because these plants may have the potential to assist in cleaning up soils contaminated with heavy metals as a result of twentieth-century industrial activities. Using plants to clean up soils is called phytoremediation (phyto meaning “plant” and remediation meaning “to correct a fault”).

The idea is to grow accumulator species on mine tailings and wastes from paper mills, for example, where they would extract the heavy metals. Plants will naturally take longer to do the job, but plants are much more cost-effective and would not create even more ecological problems as engineering-based technologies often do.

Phytoremediation would also help to stabilize contaminated sites because the plants help to control erosion. An additional benefit of accumulator species is that they begin the revegetation of barren industrial sites and assist in the recovery of useful metals. Phytomining, as it is called, has proven effective in the recovery of both nickel and thallium in demonstrations. Inother trials, various species of willows (Salix) have shown promise for extraction of heavy metals from soils treated with sewage sludge. The advantage of using plants is that they can be harvested and burned. The heavy metals remain concentrated in the ash, which makes their disposal much easier.

Green Super Rice Have Been Planted

Rice bred to perform well in the toughest conditions where the poorest farmers grow rice is a step away from reaching farmers thanks to a major project led by the Chinese Academy of Agricultural Sciences and the International Rice Research Institute (IRRI).

Green Super Rice is actually a mix of more than 250 different potential rice varieties and hybrids variously adapted to difficult growing conditions such as drought and low inputs, including no pesticide and less fertilizer, and with rapid establishment rates to out-compete weeds, thus reducing the need for herbicides. More types of Green Super Rice that combine a number of of these traits are in the pipeline.

As published in the latest issue of Rice Today, Green Super Rice is already in the hands of national agricultural agencies in key rice-growing countries for testing and development.

Green Super Rice is an example of what is needed as part of a "Greener Revolution," which is called for by rice researchers around the world and is one of the driving concepts behind the Global Rice Science Partnership (GRiSP) - a plan to improve international partnerships in rice research, its delivery, and impact that would also ensure that rice is grown in an environmentally sustainable way.

With the theme Rice for Future Generations, the 3rd International Rice Congress held in November last year was the perfect venue for the launch of GRiSP. Incredible sharing of rice research and ideas occurred, which Rice Today features in a suite of stories outlining some of the highlights and activities of the event that was attended by more than 1,900 people.

Our Grain of Truth article links Latin America in with GRiSP, highlighting the benefits of sharing expertise and experiences, while in Africa we learn about how improving the quality of rice is critical to reducing the continent's rice imports.

In our mapping section, we see how much yield and yield stability have improved since the 1960s - and also notice how much room for improvement remains.

IRRI's rodent experts, headed by Dr. Grant Singleton, take us on a journey to the northern Philippines to discover both "good" and "bad" rat species. And, we see how they are working with a local community to adopt practices to help reduce rat damage in rice crops - in 2010, rats destroyed between 30% and 50% of the rice crop there.

India is our country profile this issue and we take a look at some rice awareness-raising activities in Singapore. Meanwhile, IRRI's senior economist Dr. Samarendu Mohanty observes the recent fluctuations of rice prices and suggests that freeing up the market and creating a strategic rice reserve would help keep rice prices stable in the long term.

Finally, it is a pleasant surprise to see that nine World Food Prize laureates have had a connection with IRRI - a reminder that rice science is having an impact where it really matters.

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