Publications

Macroautophagy (hereafter referred to as autophagy) is a conserved mechanism in eukaryotic cells that delivers unneeded cellular components for degradation in the lytic organelle. In plants, as in other eukaryotes, autophagy begins in the formation of cup-shaped double membranes that engulf cytosolic material. The double membrane closes to form autophagosomes that are then transported to the vacuole for degradation. Autophagy can function as a bulk nonselective process or as a selective process targeting specific proteins, protein aggregates, organelles, or other cellular components for degradation. The endoplasmic reticulum (ER) is linked to autophagy-related processes in multiple ways. The ER was suggested as a possible site for the nucleation of autophagosomes, and as a source for autophagosomal membranes. Furthermore, autophagy has an important role in ER homeostasis, and the ER is a target for a selective type of autophagy, ER-phagy, in response to ER stress. However, the detailed molecular mechanisms, especially in plants, are only now starting to be revealed. In this chapter, we describe the use of confocal imaging to follow the delivery of fluorescently tagged ER-associated proteins to the vacuole. We also describe the utilization of fluorescent protein fusions to look at the co-localization of a protein of interest with the autophagosome marker protein ATG8, a core autophagy machinery protein that is essential for selective autophagy processes.

Aspartate (Asp)-family pathway, via several metabolic branches, leads to four key essential amino acids: Lys, Met, Thr, and Ile. Among these, Lys and Met have received the most attention, as they are the most limiting amino acid in cereals and legumes crops, respectively. The metabolic pathways of these four essential amino acids and their interactions with regulatory networks have been well characterized. Using this knowledge, extensive efforts have been devoted to augmenting the levels of these amino acids in various plant organs, especially seeds, which serve as the main source of human food and livestock feed. Seeds store a number of storage proteins, which are utilized as nutrient and energy resources. Storage proteins are composed of amino acids, to guarantee the continuation of plant progeny. Thus, understanding the seed metabolism, especially with respect to the accumulation of aspartate-derived amino acids Lys and Met, is a crucial factor for sustainable agriculture. In this review, we summarized the Asp-family pathway, with some new examples of accumulated Asp-family amino acids, particularly Lys and Met, in plant seeds. We also discuss the recent advances in understanding the roles of Asp-family amino acids during seed development.

To feed the worlds growing population, increasing the yield of crops is not the only important factor, improving crop quality is also important, and it presents a significant challenge. Among the important crops, horticultural crops (particularly fruits and vegetables) provide numerous health compounds, such as vitamins, antioxidants, and amino acids. Essential amino acids are those that cannot be produced by the organism and, therefore, must be obtained from diet, particularly from meat, eggs, and milk, as well as a variety of plants. Extensive efforts have been devoted to increasing the levels of essential amino acids in plants. Yet, these efforts have been met with very little success due to the limited genetic resources for plant breeding and because high essential amino acid content is generally accompanied by limited plant growth. With a deep understanding of the biosynthetic pathways of essential amino acids and their interactions with the regulatory networks in plants, it should be possible to use genetic engineering to improve the essential amino acid content of horticultural plants, rendering these plants more nutritionally favorable crops. In the present report, we describe the recent advances in the enhancement of essential amino acids in horticultural plants and possible future directions towards their bio-fortification.

Lysine is considered an essential amino acid required at sufficient levels to prevent malnutrition and serious diseases, particularly in developing countries. It is mostly obtained from various plant foods. In the current issue (pages 42854296) Yang and colleagues report on successful stimulation of lysine biosynthesis and suppression of its catabolism in transgenic rice plants without changing the plant phenotype. This approach led to the production of high-lysine rice plants, rendering them as nutritionally favourable crops.

Although amino acids are critical for all forms of life, only proteogenic amino acids that humans and animals cannot synthesize de novo and therefore must acquire in their diets are classified as essential. Nine amino acids-lysine, methionine, threonine, phenylalanine, tryptophan, valine, isoleucine, leucine, and histidine-fit this definition. Despite their nutritional importance, several of these amino acids are present in limiting quantities in many of the world's major crops. In recent years, a combination of reverse genetic and biochemical approaches has been used to define the genes encoding the enzymes responsible for synthesizing, degrading, and regulating these amino acids. In this review, we describe recent advances in our understanding of the metabolism of the essential amino acids, discuss approaches for enhancing their levels in plants, and appraise efforts toward their biofortification in crop plants.

Environmental stresses such as high light intensity and temperature cause induction of the shikimate pathway, aromatic amino acids (AAA) pathways, and of pathways downstream from AAAs. The induction leads to production of specialized metabolites that protect the cells from oxidative damage. The regulation of the diverse AAA derived pathways is still not well understood. To gain insight on that regulation, we increased AAA production in red grape Vitis vinifera cv. Gamay Red cell suspension, without inducing external stress on the cells, and characterized the metabolic effect of this induction. Increased AAA production was achieved by expressing a feedback-insensitive bacterial form of 3-deoxy- D-arabino-heptulosonate 7-phosphate synthase enzyme (AroG*) of the shikimate pathway under a constitutive promoter. The presence of AroG* protein led to elevated levels of primary metabolites in the shikimate and AAA pathways including phenylalanine and tyrosine, and to a dramatic increase in phenylpropanoids. The AroG* transformed lines accumulated up to 20 and 150 fold higher levels of resveratrol and dihydroquercetin, respectively. Quercetin, formed from dihydroquercetin, and resveratrol, are health promoting metabolites that are induced due to environmental stresses. Testing the expression level of key genes along the stilbenoids, benzenoids, and phenylpropanoid pathways showed that transcription was not affected by AroG*. This suggests that concentrations of AAAs, and of phenylalanine in particular, are rate-limiting in production of these metabolites. In contrast, increased phenylalanine production did not lead to elevated concentrations of anthocyanins, even though they are also phenylpropanoid metabolites. This suggests a control mechanism of this pathway that is independent of AAA concentration. Interestingly, total anthocyanin concentrations were slightly lower in AroG* cells, and the relative frequencies of the different anthocyanins changed as well.

Degradation of chloroplasts is a hallmark of both natural and stress-induced plant senescence. Autophagy and senescence-associated vacuoles are two established cellular pathways for chloroplast degradation. Recently, a third independent pathway for chloroplast degradation was reported. Here we will discuss this new discovery in relation to the other known pathways.

Seeds are the major organs responsible for the evolutionary upkeep of angiosperm plants. Seeds accumulate significant amounts of storage compounds used as nutrients and energy reserves during the initial stages of seed germination. The accumulation of storage compounds requires significant amounts of energy, the generation of which can be limited due to reduced penetration of oxygen and light particularly into the inner parts of seeds. In this review, we discuss the adjustment of seed metabolism to limited energy production resulting from the suboptimal penetration of oxygen into the seed tissues. We also discuss the role of photosynthesis during seed development and its contribution to the energy status of developing seeds. Finally, we describe the contribution of amino acid metabolism to the seed energy status, focusing on the Asp-family pathway that leads to the synthesis and catabolism of Lys, Thr, Met, and Ile.

Macroautophagy (hereafter referred to as autophagy) is a cellular mechanism dedicated to the degradation and recycling of unnecessary cytosolic components by their removal to the lytic compartment of the cell (the vacuole in plants). Autophagy is generally induced by stresses causing energy deprivation and its operation occurs by special vesicles, termed autophagosomes. Autophagy also operates in a selective manner, recycling specific components, such as organelles, protein aggregates or even specific proteins, and selective autophagy is implicated in both cellular housekeeping and response to stresses. In plants, selective autophagy has recently been shown to degrade mitochondria, plastids and peroxisomes, or organelle components such as the endoplasmic-reticulum (ER) membrane and chloroplast-derived proteins such as Rubisco. This ability places selective-autophagy as a major factor in cellular steady-state maintenance, both under stress and favorable environmental conditions. Here we review the recent advances documented in plants for this cellular process and further discuss its impact on plant physiology.

The exocyst complex is a multi-subunits evolutionary conserved complex, which was originally shown to be primarily associated with vesicular transport to the plasma membrane. A recent report (Kulich et al., 2013 Traffic; In Press) revealed that AtEXO70B1, one of the multiple subunits of the exocyst complex of Arabidopsis thaliana plants, is co-transported with the autophagy-associated Atg8f protein to the vacuole. This pathway does not involve the Golgi apparatus. The co-localization of AtEXO70B1 and Atg8f suggests either that both of these proteins are co-transported together to the vacuole or, alternatively, that Atg8 binds to a putative Atg8 interacting motif (AIM) located within the AtEXO70B1 polypeptide, apparently forming a tethering complex for an autophagic complex that is transported to the vacuole. In the present addendum, by tooling a bioinformatics approach, we show that AtEXO70B1 as well as the additional 20 paralogs of Arabidopsis EXO70 exocyst subunits each possess one or more AIMs whose consensus sequence implies their high fidelity binding to Atg8. This indicates that the autophagy machinery is strongly involved in the assembly, transport, and apparently also the function of AtEXO70B1 as well as the exocyst sub complex.

The aim of this work was to investigate the effect of decreased cytosolic phosphoenolpyruvate carboxykinase (PEPCK) and plastidic NADP-dependent malic enzyme (ME) on tomato (Solanum lycopersicum) ripening. Transgenic tomato plants with strongly reduced levels of PEPCK and plastidic NADP-ME were generated by RNA interference gene silencing under the control of a ripening-specific E8 promoter. While these genetic modifications had relatively little effect on the total fruit yield and size, they had strong effects on fruit metabolism. Both transformants were characterized by lower levels of starch at breaker stage. Analysis of the activation state of ADP-glucose pyrophosphorylase correlated with the decrease of starch in both transformants, which suggests that it is due to an altered cellular redox status. Moreover, metabolic profiling and feeding experiments involving positionally labeled glucoses of fruits lacking in plastidic NADP-ME and cytosolic PEPCK activities revealed differential changes in overall respiration rates and tricarboxylic acid (TCA) cycle flux. Inactivation of cytosolic PEPCK affected the respiration rate, which suggests that an excess of oxaloacetate is converted to aspartate and reintroduced in the TCA cycle via 2-oxoglutarate/glutamate. On the other hand, the plastidic NADP-ME antisense lines were characterized by no changes in respiration rates and TCA cycle flux, which together with increases of pyruvate kinase and phosphoenolpyruvate carboxylase activities indicate that pyruvate is supplied through these enzymes to the TCA cycle. These results are discussed in the context of current models of the importance of malate during tomato fruit ripening.

Plants need to continuously adjust their transcriptome in response to various stresses that lead to inhibition of photosynthesis and the deprivation of cellular energy. This adjustment is triggered in part by a coordinated re-programming of the energy-associated transcriptome to slow down photosynthesis and activate other energy-promoting gene networks. Therefore, understanding the stress-related transcriptional networks of genes belonging to energy-associated pathways is of major importance for engineering stress tolerance. In a bioinformatics approach developed by our group, termed 'gene coordination', we previously divided genes encoding for enzymes and transcription factors in Arabidopsis thaliana into three clusters, displaying altered coordinated transcriptional behaviors in response to multiple biotic and abiotic stresses (Plant Cell, 23, 2011, 1264). Enrichment analysis indicated further that genes controlling energy-associated metabolism operate as a compound network in response to stress. In the present paper, we describe in detail the network association of genes belonging to six central energy-associated pathways in each of these three clusters described in our previous paper. Our results expose extensive stress-associated intra- and inter-pathway interactions between genes from these pathways, indicating that genes encoding proteins involved in energy-associated metabolism are expressed in a highly coordinated manner. We also provide examples showing that this approach can be further utilized to elucidate candidate genes for stress tolerance and functions of isozymes.

Autophagy is a mechanism used for the transport of macromolecules to the vacuole for degradation. It can be either non-selective or selective, resulting from the specific binding of target proteins to Atg8, an essential autophagyrelated protein. Nine Atg8 homologs exist in the model plant Arabidopsis thaliana, suggesting possible different roles for different homologs. In a previous report published in the Plant Cell, our group identified two plant-specific proteins, termed ATI1 and ATI2, which bind Atg8f, as a representative of the nine Atg8 homologs. The proteins were shown to associate with novel starvationinduced bodies that move on the ER network and reach the lytic vacuole. Altered expression level of the proteins was also shown to affect the ability of seeds to germinate in the presence of the germination inhibiting hormone ABA. In the present addendum article, we demonstrate that, in addition to Atg8f, ATI1 binds Atg8h, an Atg8 homolog from a different sub-family, indicating that ATI1 is not a specific target of Atg8f.

Autophagy is an evolutionary conserved process of bulk degradation and nutrient sequestration that occurs in all eukaryotic cells. Yet, in recent years, autophagy has also been shown to play a role in the specific degradation of individual proteins or protein aggregates as well as of damaged organelles. The process was initially discovered in yeast and has also been very well studied in mammals and, to a lesser extent, in plants. In this review, we summarize what is known regarding the various functions of autopahgy in plants but also attempt to address some specific issues concerning plant autophagy, such as the insufficient knowledge regarding autophagy in various plant species other than Arabidopsis, the fact that some genes belonging to the core autophagy machinery in various organisms are still missing in plants, the existence of autophagy multigene families in plants and the possible operation of selective autophagy in plants, a study that is still in its infancy. In addition, we point to plant-specific autophagy processes, such as the participation of autophagy during development and germination of the seed, a unique plant organ. Throughout this review, we demonstrate that the use of innovative bioinformatic resources, together with recent biological discoveries (such as the ATG8-interacting motif), should pave the way to a more comprehensive understanding of the multiple functions of plant autophagy.

The response of plants to environmental cues, particularly stresses, involves the coordinated induction or repression of gene expression. In a previous study, we developed a bioinformatics approach to analyze the mutual expression pattern of genes encoding transcription factors and metabolic enzymes upon exposure of Arabidopsis plants to abiotic and biotic stresses. The analysis resulted in three gene clusters, each displaying a unique expression pattern. In the present addendum, we address the composition of each of these three clusters in regard to the functional identity of their encoded proteins as enzymes or transcription factors.

The expression pattern of any pair of genes may be negatively correlated, positively correlated, or not correlated at all in response to different stresses and even different progression stages of the stress. This makes it difficult to identify such relationships by classical statistical tools such as the Pearson correlation coefficient. Hence, dedicated bioinformatics approaches that are able to identify groups of cues in which there is a positive or negative expression correlation between pairs or groups of genes are called for. We herein introduce and discuss a bioinformatics approach, termed Gene Coordination, that is devoted to the identification of specific or multiple cues in which there is a positive or negative coordination between pairs of genes and can further incorporate additional coordinated genes to form large coordinated gene networks. We demonstrate the utility of this approach by providing a case study in which we were able to discover distinct expression behavior of the energy-associated gene network in response to distinct biotic and abiotic stresses. This bioinformatics approach is suitable to a broad range of studies that compare treatments versus controls, such as effects of various cues, or expression changes between a mutant and the control wild-type genotype.

Amino acid metabolism is among the most important and best recognized networks within biological systems. In plants, amino acids serve multiple functions associated with growth. Besides their function in protein synthesis, the amino acids are also catabolized into energyassociated metabolites as well we into numerous secondary metabolites, which are essential for plant growth and response to various stresses. Despite the central importance of amino acids in plants growth, elucidation of the regulation of amino acid metabolism within the context of the entire system, particularly transcriptional regulation, is still in its infancy. The different amino acids are synthesized by a number of distinct metabolic networks, which are expected to possess regulatory cross interactions between them for proper coordination of their interactive functions, such as incorporation into proteins. Yet, individual amino acid metabolic networks are also expected to differentially cross interact with various genome-wide gene expression programs and metabolic networks, in respect to their functions as precursors for various metabolites with distinct functions. In the present review, we discuss our recent genomics, metabolic and bioinformatics studies, which were aimed at addressing these questions, focusing mainly on the Asp-family metabolic network as the main example and also comparing it to the aromatic amino acids metabolic network as a second example (Angelovici et al. in Plant Physiol 151:2058-2072, 2009; Less and Galili in BMC Syst Biol 3:14, 2009; Tzin et al. in Plant J 60:156- 167, 2009). Our focus on these two networks is because of the followings: (i) both networks are central to plant metabolism and growth and are also precursors for a wide range of primary and secondary metabolites that are indispensable to plant growth; (ii) the amino acids produced by these two networks are also essential to the nutrition and health of human and farm animals; and (iii) both networks contain branched pathways requiring extensive regulation of fluxes between the different branches. Additional views on the biochemistry, regulation and functional significance of the Asp-family and aromatic amino acid networks and some of their associated metabolites that are discussed in the present report, as well as the nutritional importance of Lys and Trp to human and farm animals, and attempts to improve Lys level in crop plants, can be obtained from the following reviews as examples (Radwanski and Last in Plant Cell 7:921-934, 1995; Halkier and Gershenzon in Annu Rev Plant Biol 57:303-333, 2006; Ufaz and Galili in Plant Physiol 147:954-961, 2008; Jander and Joshi in Mol Plant 3:54-65, 2010).

The creation of a water-stress environment usually starts with a reduction in air relative humidity (RH) while soil water potential still reflects favorable conditions. Arabidopsis plants subjected to low RH (17%) exhibited a significant increase in leaf specific hydraulic conductance, reducing the water potential. However, no detectable effects on stomatal performance or osmotic leaf adjustment were noted relative to plants exposed to high RH. In the present study, we profiled gene expression in roots 2.5 and 5 h after shoot exposure to low RH. Multiple genes with various putative biological roles were identified as differentially expressed under these conditions; among them were aquaporins, some of whose expression was induced under low RH. Transcription of two aquaporin was localized to the roots, especially to cells around the vascular system and to cells of the differentiation zone, and to leaf trichomes. Our results suggest that plant roots perceive the low RH stimulus from shoots through a sensing mechanism(s), leading to distinct plant transcriptional responses, potentially reflecting activation of various biological processes. This activation might be a prelude to the expected forthcoming drought conditions, providing the plant with enhanced resistance to future water-stress situations.

Keywords: Biochemistry & Molecular Biology; Genetics & Heredity; Medicine, Research & Experimental

Keywords: Biochemistry & Molecular Biology; Genetics & Heredity; Medicine, Research & Experimental

Gaucher's disease, a lysosomal storage disorder caused by mutations in the gene encoding glucocerebrosidase (GCD), is currently treated by enzyme replacement therapy using recombinant GCD (Cerezyme®) expressed in Chinese hamster ovary (CHO) cells. As complex glycans in mammalian cells do not terminate in mannose residues, which are essential for the biological uptake of GCD via macrophage mannose receptors in human patients with Gaucher's disease, an in vitro glycan modification is required in order to expose the mannose residues on the glycans of Cerezyme®. In this report, the production of a recombinant human GCD in a carrot cell suspension culture is described. The recombinant plant-derived GCD (prGCD) is targeted to the storage vacuoles, using a plant-specific C-terminal sorting signal. Notably, the recombinant human GCD expressed in the carrot cells naturally contains terminal mannose residues on its complex glycans, apparently as a result of the activity of a special vacuolar enzyme that modifies complex glycans. Hence, the plant-produced recombinant human GCD does not require exposure of mannose residues in vitro, which is a requirement for the production of Cerezyme®. prGCD also displays a level of biological activity similar to that of Cerezyme® produced in CHO cells, as well as a highly homologous high-resolution three-dimensional structure, determined by X-ray crystallography. A single-dose toxicity study with prGCD in mice demonstrated the absence of treatment-related adverse reactions or clinical findings, indicating the potential safety of prGCD. prGCD is currently undergoing clinical studies, and may offer a new and alternative therapeutic option for Gaucher's disease.

The essential amino acid methionine is a substrate for the synthesis of S-adenosyl-methionine (SAM), that donates its methyl group to numerous methylation reactions, and from which polyamines and ethylene are generated. To study the regulatory role of methionine synthesis in tomato fruit ripening, which requires a sharp increase in ethylene production, we cloned a cDNA encoding cystathionine γ-synthase (CGS) from tomato and analysed its mRNA and protein levels during tomato fruit ripening. CGS mRNA and protein levels peaked at the "turning" stage and declined as the fruit ripened. Notably, the tomato CGS mRNA level in both leaves and fruit was negatively affected by methionine feeding, a regulation that Arabidopsis, but not potato CGS mRNA is subject to. A positive correlation was found between elevated ethylene production and increased CGS mRNA levels during the ethylene burst of the climacteric ripening of tomato fruit. In addition, wounding of pericarp from tomato fruit at the mature green stage stimulated both ethylene production and CGS mRNA level. Application of exogenous methionine to pericarp of mature green fruit increased ethylene evolution, suggesting that soluble methionine may be a rate limiting metabolite for ethylene synthesis. Moreover, treatment of mature green tomato fruit with the ethylene-releasing reagent Ethephon caused an induction of CGS mRNA level, indicating that CGS gene expression is regulated by ethylene. Taken together, these results imply that in addition to recycling of the methionine moieties via the Yang pathway, operating during synthesis of ethylene, de novo synthesis of methionine may be required when high rates of ethylene production are induced.

Lysine catabolism in plants is initiated by a bifunctional LKR/SDH (lysine-ketoglutarate reductase/saccharopine dehydrogenase) enzyme encoded by a single LKR/SDH gene. Yet, the AtLKR/SDH gene of Arabidopsis also encodes a second gene product, namely a monofunctional SDH. To elucidate the regulation of lysine catabolism in Arabidopsis through these two gene products of the AtLKR/SDH gene, an analysis was carried out on the effects of the hormones, abscisic acid and jasmonate, as well as various metabolic and stress signals, including lysine itself, on their mRNA and protein levels. The response of the two gene products to the various treatments was only partially co-ordinated, but the levels of the monofunctional SDH mRNA and protein were always in excess over their bifunctional LKR/SDH counterparts. These results suggest that lysine catabolism is regulated primarily by the first enzyme LKR, while the excess level of SDH enables efficient flux of lysine catabolism following the LKR step. Analysis of transgenic plants expressing β-glucoronidase fusion constructs with the AtLKR/SDH and monofunctional AtSDH promoters demonstrated that transcriptional regulation contributes to the modulation of expression of the bifunctional LKR/SDH and monofunctional SDH gene products in response to hormonal and metabolic signals. To test whether the enhanced expression of the LKR/SDH gene under various hormonal and metabolic signals is correlated with enhanced lysine catabolism, wild-type Arabidopsis and a knockout mutant lacking lysine catabolism were exposed to abscisic acid and sugar starvation. Free lysine accumulated to significantly higher levels in this knockout mutant than in the wild-type plants.

RNA interference (RNAi) is an ancient mechanism of gene suppression, whose machinery and biological functions are only partially understood. Intensive studies have focused on developing RNAi technologies for treating human diseases and for improving plant traits. Yet application of RNAi to improving the nutritional value of plants for human and animal nutrition, and development of the related RNAi technologies are still in their infancy. Here we discuss current knowledge of plant RNAi function, as well as concepts and strategies for the improvement of plant nutritional value through the development of plant RNAi technologies.

Arbuscular mycorrhizae (AM) represent an ancient symbiosis between mycorrhizal fungi and plant roots which co-evolved to exhibit a finely tuned, multistage interaction that assists plant growth. Direct screening efforts for Myc^- plant mutants resulted in the identification of a tomato (Lycopersicon esculentum L. cv. Micro-Tom) mutant, M20, which was impaired in its ability to support the premycorrhizal infection (pmi) stages. The Myc^- phenotype of the M20 mutant was a single Mendelian recessive trait, stable for nine generations, and nonallelic to a previously identified M161 pmi mutant. The M20 mutant was resistant to infection by isolated AM spores and colonized roots. Formation of Glomus intraradices appressoria on M20 roots was normal, as on wild-type (WT) plants, but in significantly reduced numbers. A significant reduction in spore germination was observed in vitro in the presence of M20 exudates relative to WT. Our results indicate that this new mutant shares similar physiological characteristics with the M161 pmi mutant, but has a more suppressive Myc^- phenotype response.

To elucidate the relative significance of Lys synthesis and catabolism in determining Lys level in plant seeds, we expressed a bacterial feedback-insensitive dihydrodipicolinate synthase (DHPS) in a seed-specific manner in wild-type Arabidopsis as well as in an Arabidopsis knockout mutant in the Lys catabolism pathway. Transgenic plants expressing the bacterial DHPS, or the knockout mutant, contained ∼12-fold or ∼5-fold higher levels, respectively, of seed free Lys than wild-type plants. However, the combination of these two traits caused a synergistic ∼80-fold increase in seed free Lys level. The dramatic increase in free Lys in the knockout mutant expressing the bacterial DHPS was associated with a significant reduction in the levels of Glu and Asp but also with an unexpected increase in the levels of Gln and Asn. This finding suggested a special regulatory interaction between Lys metabolism and amide amino acid metabolism in seeds. Notably, the level of free Met, which competes with Lys for Asp and Glu as precursors, was increased unexpectedly by up to ∼38-fold in the various transgenic and knockout plants. Together, our results show that Lys catabolism plays a major regulatory role in Lys accumulation in Arabidopsis seeds and reveal novel regulatory networks of seed amino acid metabolism.

The 3 cleavage and polyadenylation of mRNAs has been studied in detail in animals and yeast, but not in plants. Aimed at elucidating the regulation of mRNA 3 end formation in plants, three Arabidopsis cDNAs encoding homologues of the animal proteins CstF-64, CstF-77 and CstF-50 that form the cleavage stimulating factor of the polyadenylation machinery have been cloned. It is shown experimentally that the N-terminal domain of the Arabidopsis CstF-64 homologue binds the mRNA 3 non-coding region in an analogous manner to the animal protein. It is also shown that the Arabidopsis CstF-64 and CstF-77 homologues strongly interact with each other in a similar way to their animal counterparts. These results imply that these Arabidopsis homologues belong to the polyadenylation machinery of nuclear mRNAs.

The sulfur-containing amino acid methionine is a nutritionally important essential amino acid and is the precursor of several metabolites that regulate plant growth and responses to the environment. Methionine production is largely regulated by cystathionine γ-synthase, the first specific enzyme for its synthesis. This enzyme competes in a complex manner with threonine synthase, the last enzyme in threonine biosynthesis, for their common substrate O-phosphohomoserine. New genetic and molecular data suggest that methionine synthesis and catabolism are coordinately regulated by novel post-transcriptional and post-translational mechanisms that are associated with a regulatory part within the N-terminal part of cystathionine γ-synthase.

Recent advances in gene isolation, plant transformation, and genetic engineering are being used extensively to alter metabolic pathways in plants by tailormade modifications to single or multiple genes. Many of these modifications are directed toward increasing the nutritional value of plant-derived foods and feeds. These approaches are based on rapidly growing basic knowledge, understanding, and predictions of metabolic fluxes and networks. Some of the predictions appear to be accurate, while others are not, reflecting the fact that plant metabolism is more complex than we presently understand. Tailor-made modifications of plant metabolism has so far been directed into improving the levels of primary metabolites that are essential for growth and development of humans and their livestock. Yet, the list of improved metabolites is expected to grow tremendously after new discoveries in nutritional, medical, and health sciences. Despite our extensive knowledge of metabolic networks, many of the genes encoding enzymes, particularly those involved in secondary metabolism, are still unknown. These genes are being discovered at an accelerated rate by recent advances in genetic and genomics approaches. In the present review, we discuss examples in which the nutritional and health values of plant-derived foods and feeds were improved by metabolic engineering. These include modifications of the levels of several essential amino acids, lipids, fatty acids, minerals, nutraceuticals, antinutritional compounds, and aromas.

Lysine is among the most important essential amino acids in the diet of human and livestock because it is presented in severely limiting amounts in cereals and other important crops. Attempts to increase lysine levels in plants were made by reducing the sensitivity of the key enzyme in lysine biosynthesis, namely dihydrodipicolinate synthase, to feedback inhibition by lysine. However, these studies showed that in plant seeds, lysine accumulation is determined not only by the rate of its synthesis, but also by the rate of its catabolism via the α-amino adipic acid pathway. Our laboratory is currently studying the regulation of lysine catabolism in plants in order to explore potentials of reducing lysine catabolic fluxes in transgenic plants. In plants, like animals, lysine is catabolized via saccharopine by two consecutive enzymes, lysine-ketoglutarate reductase (LKR) and saccharopine dehydrogenase (SDH), which are linked on a single bifunctional polypeptide. Yet SDH activity of the LKR/SDH polypeptide may be limiting in vivo because of its non-physiological pH optimum of activity. In some plants, like Arabidopsis and canola, this is overcome by the presence of an additional monofunctional SDH enzyme, which is encoded by the same locus that encodes the bifunctional LKR/SDH enzyme. Results from our, and other laboratories imply that attempts to generate high-lysine crop plants should take into account a seed-specific reduction of lysine catabolism.

Both plants and animals catabolise lysine via saccharopine by two consecutive enzymes, lysine-ketoglutarate reductase (LKR) and saccharopine dehydrogenase (SDH), which are linked on a single polypeptide. We recently demonstrated that Arabidopsis plants possess not only a bifunctional LKR/SDH but in addition a monofunctional SDH enzyme. We also speculated that these two enzymes may be controlled by a single gane (G. Tang et al., Plant Cell, 1997, 9, 1305-1318). By expressing several epitope-tagged and GUS reporter constructs, we demonstrate in the present study that the Arabidopsis monofunctional SDH is encoded by a distinct gene, which is, however, nested entirely within the coding and 3' non-coding regions of the larger bifunctional LKR/SDH gene. The entire open reading frame of the monofunctional SDH gene, as well as some components of its promoter, are also parts of the translated coding sequence of the bifunctional LKR/SDH gene. These special structural characteristics, combined with the fact that the two genes encode simultaneously two metabolically related but distinct enzymes, render the LKR/SDH locus a novel type of a composite locus. Not all plant species possess an active monofunctional SDH gene and the production of this enzyme is correlated with an increased flux of lysine catabolism. Taken together, our results suggest that the composite LKR/SDH locus serves to control an efficient, highly regulated flux of lysine catabolism.

Arabidopsis plants possess a composite AtLKR/SDH locus encoding two different polypeptides involved in lysine catabolism: A bifunctional lysine-ketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH) enzyme and a monofunctional SDH enzyme. To unravel the physiological significance of these two enzymes, we analyzed their subcellular localization and detailed biochemical properties. Sucrose gradient analysis showed that the two enzymes are localized in the cytosol and therefore may operate at relatively neutral pH values in vivo. Yet while the physiological pH may provide an optimum environment for LKR activity, the pH optima for the activities of both the linked and non-linked SDH enzymes were above pH 9, suggesting that these two enzymes may operate under suboptimal conditions in vivo. The basic biochemical properties of the monofunctional SDH, including its pH optimum as well as the apparent Michaelis constant (K(m)) values for its substrates saccharopine and nicotinamide adenine dinucleotide at neutral and basic pH values, were similar to those of its SDH counterpart that is linked to LKR. Taken together, our results suggest that production of the monofunctional SDH provides Arabidopsis plants with enhanced levels of SDH activity (maximum initial velocity), rather than with an SDH isozyme with significantly altered kinetic parameters. Excess levels of this enzyme might enable efficient flux of lysine catabolism via the SDH reaction in the unfavorable physiological pH of the cytosol.

Seeds of many crop plants are considered as a poor nutritional value food source, due to very low levels of the essential amino acid lysine. Attempts to increase lysine content in seeds are hampered by the presence of an active catabolic process that degrades lysine via saccharopine into other metabolites. The first enzyme in this catabolic process is lysine ketoglutarate reductase (LKR). We have recently obtained evidence that strongly suggest that lysine finely controls its own level in seeds by maintaining LKR activity balanced using two separate complementary mechanisms. I) LKR activity is post-translationally stimulated by lysine via a Ca+2 dependent intracellular signaling cascade that ends in the phosphorylation of the enzyme, thus, preventing lysine to accumulate to high levels. LI) subsequent binding of lysine to the active site of the enzyme may expose phosphate residues on the surface of LKR rendering them more accessible to dephosphorylation, thus allowing lysine to accumulate to a sufficient level needed for protein synthesis.

Although the control of carbon fixation and nitrogen assimilation has been studied in detail, relatively little is known about the regulation of carbon and nitrogen flow into amino acids. In this paper we report our study of the metabolic regulation of expression of an Arabidopsis aspartate kinase/homoserine dehydrogenase (AK/ HSD) gene, which encodes two linked key enzymes in the biosynthetic pathway of aspartate family amino acids. Northern blot analyses, as well as expression of chimeric AK/HSD-β-glucuronidase constructs, have shown that the expression of this gene is regulated by the photosynthesis-related metabolites sucrose and phosphate but not by nitrogenous compounds. In addition, analysis of AK/HSD promoter deletions suggested that a CTTGACTCTA sequence, resembling the binding site for the yeast GCN4 transcription factor, is likely to play a functional role in the expression of this gene. Nevertheless, longer promoter fragments, lacking the GCN4-like element, were still able to confer sugar inducibility, implying that the metabolic regulation of this gene is apparently obtained by multiple and redundant promoter sequences. The present and previous studies suggest that the conversion of aspartate into either the storage amino acid asparagine or aspartate family amino acids is subject to a coordinated, reciprocal metabolic control, and this biochemical branch point is a part of a larger, coordinated regulatory mechanism of nitrogen and carbon storage and utilization.

Wheat high molecular weight glutenin subunits (HMW-GS) are the most important determinants of its superiority for making leavened bread. Following synthesis, these proteins are sequestered into the endoplasmic reticulum and assemble into extremely large elastic polymers, linked by noncovalent and intermolecular disulfide bonds. To study the structural requirements for the assembly of HMW-GS, we have expressed in transgenic wheat a recombinant protein between two cognate x- and y-type subunits. In contrast to the natural polymerized x- and y-type HMW-GS, a significant amount of the recombinant subunit remained monomeric. Nonreducing SDS- polyacrylamide gel electrophoresis, coupled with limited proteolysis, showed that the monomeric form of the recombinant subunit contained an unusual intramolecular disulfide bond, linking an N-terminal cysteine to the single C-terminal cysteine residue. In addition, sucrose gradient analysis revealed that this intramolecular disulfide bond impeded the ability of the recombinant subunit to assemble into polymers. Despite of its altered assembly, a notable amount of the overexpressed recombinant subunit was also present in glutenin polymers. Moreover, its presence significantly altered the subunit composition of the polymer. Our results show that it is possible to modify gluten assembly and properties by expressing recombinant HMW-GS in transgenic wheat, and have a major implication for the improvement of wheat breadmaking quality.

Although the regulation of amino acid synthesis has been studied extensively at the biochemical level, it is still not known how genes encoding amino acid biosynthesis enzymes ate regulated during plant development. In the present report, we have used the β-glucuronidase (GUS) reporter gene to study the regulation of expression of an Arabidopsis thaliana aspartate kinase-homoserine dehydrogenase (AK/HSD) gene in transgenic tobacco plants. The polypeptide encoded by the AK/HSD gene comprises two linked key enzymes in the biosynthesis of aspartate-family amino acids. AK/HSD-GUS gene expression was highly stimulated in apical and lateral meristems, lateral buds, young leaves, trichomes, vascular and cortical tissues of growing stems, tapetum and other tissues of anthers, pollen grains, various parts of the developing gynoecium, developing seeds, and, in some transgenic plants, also in stem and leaf epidermal trichomes. AK/HSD-GUS gene expression gradually diminished upon maturation of leaves, stems, floral tissues, and embryos. GUS expression was relatively low in roots. During seed development, expression of the AK/HSD gene in the embryo was coordinated with the initiation and onset of storage protein synthesis, whereas in the endosperm it was coordinated with the onset of seed desiccation. Upon germination, AK/HSD-GUS gene expression in the hypocotyl and the cotyledons was significantly affected by light. The expression pattern of the A. thaliana AK/HSD-GUS reporter gene positively correlated with the levels of aspartate-family amino acids and was also very similar to the expression pattern of the endogenous tobacco AK/HSD mRNA as determined by in situ hybridization.

Plants generally contain low proportions of several essential amino acids, particularly lysine. The potential of increasing lysine content in vegetative tissues and storage organs of plants was studied by expression in transgenic tobacco plants of a chimeric gene encoding a bacterial dihydrodipicolinate synthase (DHPS), a key enzyme in lysine biosynthesis. The bacterial enzyme is much less sensitive to feedback inhibition by lysine than its plant counterparts. Constitutive expression of the bacterial enzyme caused a dramatic elevation of free lysine in vegetative tissues, which in some cases amounted up to 40 mol % of total free amino acids. In addition, constitutive lysine overproduction caused an alteration in plant phenotype, including loss of apical dominance, reduced plant height, bushy appearance, altered leaf structure and delayed flowering. In order to study the potential of the bacterial DHPS to increase lysine content in storage organs, we have expressed this bacterial enzyme in a seed-specific manner in transgenic tobacco plants. Although lysine synthesis was enhanced during seed development in the transgenic plants, this amino acid failed to over accumulate in mature seeds. The low lysine levels in mature seeds of the transgenic plants was correlated with a lysine-dependent stimulation of another enzyme, lysine-ketoglutarate reductase, which catabolizes lysine into saccharopine. This stimulation was shown to operate via an intracellular signaling cascade, mediated by Ca2+ and protein phosphorylation. The potential of the bacterial DHPS, either alone or in combination with additional genes, to improve the nutritional quality of forage, grain and tuber crops is discussed.

Wheat storage proteins are co-translationally inserted into the endoplasmic reticulum (ER) and then accumulate in protein bodies inside vacuoles. It appears that a significant amount of these storage proteins assemble into protein bodies within the ER and that these protein bodies are subsequently internalized into vacuoles by a process that is analogous to autophagy and does not utilize the Golgi complex. Folding and assembly of the storage proteins within the ER is not a spontaneous process, but is apparently assisted by ER-resident proteins. These include molecular chaperones as well as enzymes that catalyze the formation, isomerization and perhaps also dissociation of disulfide bonds.

Wheat (Triticum aestivum) storage proteins fold and assemble into complexes that are linked by intra- and intermolecular disulfide bonds, but it is not yet clear whether these processes are spontaneous or require the assistance of endoplasmic reticulum (ER)-resident enzymes and molecular chaperones. Aiming to unravel these processes, we have purified and characterized the enzyme protein disulfide isomerase (PDI) from wheat endosperm, as well as studied its developmental expression and intracellular localization. This ER-resident enzyme was previously shown to be involved in the formation of disulfide bonds in secretory proteins. Wheat PDI appears as a 60-kD glycoprotein and is among the most abundant proteins within the ER of developing grains. PDI is notably upregulated in developing endosperm in comparison to embryos, leaves, and roots. In addition, the increase in PDI expression in grains appears at relatively early stages of development, preceding the onset of storage protein accumulation by several days. Subcellular localization analysis and immunogold labeling of electron micrographs showed that PDI is not only present in the lumen of the ER but is also co-localized with the storage proteins in the dense protein bodies. These observations are consistent with the hypothesis that PDI is involved in the assembly of wheat storage proteins within the ER.

This chapter discusses the synthesis of plant proteins in heterologous systems. The recent advent in recombinant DNA and gene transfer technologieswhich enables the construction of mutant genes and their subsequent expression in plant and animal cellshas provided an impetus for a wide array of studies on the synthesis, assembly, and transport of plant proteins. An important outcome of these studies is that many processes, though not all, are independent of the eukaryotic cell type in which the protein of interest is synthesized. It appears, for instance, that plant secretory proteins can fold, assemble, and acquire transport competence when expressed in animal cells. The major advantages of such heterologous systems are the ease of analysis and fast results, compared to the use of transgenic plants. Moreover, in specific cases, heterologous systems can be used for specific experiments that are either difficult or impossible to perform in plant cells.

Following sequestration into the endoplasmic reticulum, wheat high molecular weight glutenin subunits (HMW-GS) assemble into polymers through intermolecular disulfide bond formation. These polymers, which also include low molecular weight glutenin subunits (LMW-GS), have a broad distribution of molecular mass reaching up to several million daltons. To study the mechanism of assembly of the HMW-GS, we have expressed x- and y-type HMW-GS in transgenic tobacco plants. Both types, when expressed individually or in combination, were incorporated into polymers. Partial reduction of polymers formed by different subunits resulted in different patterns of release of homodimers, heterodimers, and monomers. This suggested different arrangements of intermolecular disulfide bonds or different peptide conformations in the vicinity of the disulfide bonds linking x-x, y-y, and x-y type HMW-GS. A mutant of the x-type subunit, lacking a conserved cysteine in the C-terminal domain, assembled into oligomers linked by intermolecular disulfide bonds, but not into large polymers. This mutant was deposited, however, in dense protein bodies, similar to those formed by the native HMW-GS, suggesting that polymer formation and packaging into protein bodies may be the result of different types of interactions. Pulse-chase labeling of proteins in wheat endosperm showed that the assembly of the HMW-GS into insoluble polymers occurs by a slow process which apparently continues after the initiation of protein body formation.

Following sequestration into the endoplasmic reticulum, wheat gliadin storage proteins may either be retained and packaged into protein bodies inside the organelle or be transported via the Golgi apparatus to vacuoles and condense into protein bodies at a post-endoplasmic reticulum location. To unravel the mechanism of this complex process of deposition, we expressed wild-type and mutant forms of two closely related γ and aggregated gliadins in Xenopus oocytes. Although a considerable amount of the γ-gliadin was secreted to the medium, its closely related aggregated gliadin was entirely retained within the oocytes. This differential secretion was largely due to structural variations in the C-terminal regions of the proteins. Retention of the wild-type aggregated and γ-gliadins within the endoplasmic reticulum could not be explained by rapid assembly into insoluble deposits inasmuch as both proteins could diffuse rather efficiently within the organelle for several hours. To address more closely the role of the C-terminal region in the transport and assembly of the γ-gliadin within the endoplasmic reticulum, 3 cysteine codons in this region were mutated, one at a time, to serine codons. The cysteine-replacement mutants improperly aggregated within the endoplasmic reticulum forming denser deposits compared with the wild- type protein.

Plant seeds store nitrogen by accumulating storage proteins in protein bodies within various compartments of the endomembrane system. The prolamin storage proteins of some cereal species are normally retained and assembled into protein bodies within the ER. Yet, these proteins lack a C-terminal KDEL/HDEL signal, suggesting that their retention is regulated by novel mechanisms. Furthermore, in other cereal species, such protein bodies formed within the ER may be subsequently internalized into vacuoles by a special route that does not utilize the Golgi complex. Thus, studies of the routing of seed storage proteins are revealing novel mechanisms of protein assembly and transport in the endomembrane system.

The 3 AU-rich region of human beta-1 interferon (hu-IFNβ) mRNA was found to act as a translational inhibitory element. The translational regulation of this 3 AU-rich sequence and the effect of its association with the poly(A) tail were studied in cell-free rabbit reticulocyte lysate. A poly(A)-rich hu-IFNβ mRNA (110 A residues) served as an inefficient template for protein synthesis. However, translational efficiency was considerably improved when the poly(A) tract was shortened (11 A residues) or when the 3 AU-rich sequence was deleted, indicating that interaction between these two regions was responsible for the reduced translation of the poly(A)-rich hu-IFNβ mRNA. Differences in translational efficiency of the various hu-IFNβ mRNAs correlated well with their polysomal distribution. The poly(A)-rich hu-IFNβ mRNA failed to form large polysemes, while its counterpart bearing a short poly(A) tail was recruited more efficiently into large polysemes. The AU-rich sequence-binding activity was reduced when the RNA probe contained both the 3 AU-rich sequence and long poly(A) tail, supporting a physical association between these two regions. Further evidence for this interaction was achieved by RNase H protection assay. We suggest that the 3 AU-rich sequence may regulate the translation of hu-IFNβ mRNA by interacting with the poly(A) tail.

Following their sequestration into the endoplasmic reticulum (ER), wheat storage proteins may either be retained and packaged into protein bodies within this organelle or transported via the Colgi to vacuoles. We attempted to study the processes of transport and packaging of wheat storage proteins using the heterologous expression system of yeast. A wild-type wheat γ-gliadin, expressed in the yeast cells, accumulated mostly within the ER and was deposited in protein bodies with similar density to natural protein bodies from wheat endosperm. This suggested that wheat storage proteins contain sufficient information to initiate the formation of protein bodies in the ER of a heterologous system. Only a small amount of the γ-gliadin was transported to the yeast vacuoles. When a deletion mutant of the γ-gliadin, lacking the entire N-terminal repetitive region, was expressed in the yeast cells, the mutant was unable to initiate the formation of protein bodies within the ER and was completely transported to the yeast vacuole. This strongly indicated that the information for packaging into dense protein bodies within the ER resides in the N-terminal repetitive region of the γ-gliadin. The advantage of using yeast to identify the signals and mechanisms controlling the transport of wheat storage proteins and their deposition in protein bodies is discussed.

Genetic transformation of cereals by direct DNA delivery via microprojectile bombardment has become an established procedure in recent years. But the derivation of functional transgenic plants, especially in wheat, is still problematic, mainly due to low efficiency of DNA delivery and the reduced regeneration capability of microprojectile-bombarded tissue. We focussed on these two aspects and found that the regeneration of scutellar calli of wheat can be rendered highly efficient and considerably accelerated by a liquid culture phase in screen rafts. We also found that the expression of a reporter gene following DNA delivery by microprojectile can be improved by maintaining the scutellar calli in 0.25 M mannitol before and after bombardment, by bombardment in the presence of silver thiosulfate and Ca(NO₃)₂ (rather than CaCl₂) and by the elimination of spermidine from the DNA/microprojectile mixture. A protocol that includes all these features leads to several-fold higher transient expression of the reporter gene than have previously published procedures.

The essential amino acid lysine is synthesized in higher plants by a complex pathway that is predominantly regulated by feedback inhibition of two enzymes, namely aspartate kinase (AK) and dihydrodipicolinate synthase (DHPS). Although DHPS is thought to play a major role in this regulation, the relative importance of AK is not known. In order to study this regulation, we have expressed in the chloroplasts of transgenic potato plants a DHPS derived from Escherichia coli at a level 50-fold above the endogenous DHPS. The bacterial enzyme is much less sensitive to lysine inhibition than its potato counterpart. DHPS activity in leaves, roots and tubers of the transgenic plants was considerably higher and more resistant to lysine inhibition than in control untransformed plants. Furthermore, this activity was accompanied by a significant increase in level of free lysine in all three tissues. Yet, the extent of lysine overproduction in potato leaves was significantly lower than that previously reported in leaves of transgenic plants expressing the same bacterial enzyme, suggesting that in potato, AK may also play a major regulatory role in lysine biosynthesis. Indeed, the elevated level of free lysine in the transgenic potato plants was shown to inhibit the lysine-sensitive AK activity in vivo. Our results support previous reports showing that DHPS is the major rate-limiting enzyme for lysine synthesis in higher plants, but they suggest that additional plant-specific regulatory factors are also involved.

The high molecular weight glutenin subunits are considered one of the most important components of wheat (Triticum aestivum) gluten, but their structure and interactions with other gluten proteins are still unknown. Understanding the role of these proteins in gluten formation may be aided by analyses of the conformation and interactions of individual wild-type and modified subunits expressed in heterologous systems. In the present report, the bacterium Escherichia coli was used to synthesize four naturally occurring X- and Y-type wheat high molecular weight glutenin subunits of the Glu-1D locus, as well as four bipartite chimeras of these proteins. Naturally occurring subunits synthesized in the bacteria exhibited sodium dodecyl sulfate-polyacrylamide gel electrophoresis migration properties identical to those of high molecular weight glutenin subunits extracted from wheat grains. Wild-type and chimeric subunits migrated in sodium dodecyl sulfate gels differently than expected based on their molecular weights due to conformational properties of their N- and C-terminal regions. Results from cycles of reductive cleavage and oxidative reformation were consistent with the formation of both inter- and intramolecular disulfide bonds in patterns and proportions that differed among specific high molecular weight glutenin species. Comparison of the chimeric and wild-type proteins indicated that the two C-terminal cysteines of the Y-type subunits are linked by intramolecular disulfide bonds, suggesting that the role of these cysteines in glutenin polymerization may be limited.

Variation in the electrophoretic pattern of the high-molecular-weight (HMW) glutenin subunits was studied in the Ammiad population of wild tetraploid wheat, Triticum turgidum var. dicoccoides (genome AABB), during a 5-year period (1983-4 to 1987-8). These storage proteins were analysed following one-dimensional sodium-dodecyl-sulphate polyacrylamide-gel electrophoresis (SDS PAGE), using seeds collected annually from individual plants at 249 defined sampling sites distributed in 11 habitats. Since plants did mt grow at all the sampling points each year, 1108 accessions were analysed altogether. The population was found to be highly polymorphic: the HMW glutenin loci of genome A, Glu-Al-1 and Glu-Al-2, had four and two alleles. respectively, and those of genome B. Glu-Bl-1 and Glu-Bl-2, had five and seven alleles, respectively. The A-genome alleles appeared in 4 combinations, and the B-genome alleles appeared in 12 combinations. There were 18 intergenomic combinations (A and B genotypes), some of which were very rare while others were abundant and distributed along transects in clusters. The spatial distribution of these genotypes was nonrandom, with each of the 11 habitats characterized by different genotype frequencies. Yearly changes in genotypes, mostly occurring in the last 2 years of the study, had little effect on the total frequencies of die various genotypes. A high affinity was found between specific HMW glutenin genotypes and certain habitats. This affinity may have resulted from a random fixation of specific genotypes in different habitats (founder effect) or, alternatively, from natural selection, thus indicating either linkage between HMW glutenin alleles and adaptive genes (hitchhiking effect) or fitness of some of these allele combinations to specific micro-environments.

Zeins, the storage proteins of maize, are totally lacking in the essential amino acids lysine and tryptophan. Lysine codons and lysine- and typtophan-encoding oligonucleotides were introduced at several positions into a 19-kilodalton zein complementary DNA by oligonucleotide-mediated mutagenesis. A 450-base pair open reading frame from a simian virus 40 (SV40) coat protein was also engineered into the zein coding region. Messenger RNAs for the modified zeins were synthesized in vitro with an SP6 RNA polymerase system and injected into Xenopus laevis oocytes. The modifications did not affect the translation, signal peptide cleavage, or stability of the zeins. The ability of the modified zeins to assemble into structures similar to maize protein bodies was assayed by two criteria: assembly into membrane-bound vesicles resistant to exogenously added protease, and ability to self-aggregate into dense structures. All of the modified zeins were membrane-bound; only the one containing a 17-kilodalton SV40 protein fragment was unable to aggregate. These findings suggest that it may be possible to create high-lysine corn by genetic engineering.

Total endosperm proteins extracted from both several common wheat cultivars and some intervarietal substitution lines derived from them were fractionated according to their molecular weight in a high resolution one-dimensional gel electrophoresis. The four donor cultivars and the recipient one - 'Chinese Spring', possessed differentially migrating protein bands in the fractions of high molecular weight (HMW) glutenins and gliadins. Several of these bands were identified for the first time in this study. By utilizing intervarietal substitution lines the control of the HMW glutenins and gliadins by chromosomes of homoeologous group 1 was either reaffirmed or, for the new bands, established. Several HMW gliadin subunits showed a considerable variation in their staining intensity in the intervarietal substitution lines indicating that their expression was dependent on the genetic background.

Endosperm protein subunits of 109 primitive and modern lines of hexaploid wheat, Triticum aestivum L. em. Thell., were fractionated by one-dimensional, high resolution, sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (PAGE). A wide range of both qualitative and quantitative variation was observed in the fractions of the high molecular weight (HMW) glutenin and gliadin subunits of the different lines. The qualitative variation was expressed in the number of subunits per fraction and in their molecular weight, as determined by the differential rate of migration. The quantitative variation was expressed in the differential staining intensity of several subunits. The widest variation was detected in the HMW glutenin and gliadin subunits controlled by chromosome 1B while a much smaller variation was observed in those subunits controlled by chromosome 1A and further smaller variation in the subunits controlled by 1D. Only a small number of subunits in both fractions was found to be controlled by chromosome 1A indicating that diploidization of endosperm protein genes in common wheat has been non-random. The genetic and evolutionary implications of these findings are discussed.