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Molecular aspects of plant biochemistry
Molecular aspects of plant biochemistry Nikolaus Amrhein and Jiirg Schmid Swiss Federal Institute of Technology,
The
integration
has
brought
topography
of molecular about
and function
of expression
cloning
encoding numerous
genetics,
significant
of the thylakoid
in appropriate
have been identified
and functionally biochemistry
the activities
of these enzymes
mutants
and carriers
Biochemistry relates molecular structures to biological functions and vice versa. Topics specifically related to the biochemistry of plants tend to be excluded not only &om the text books of biochemistry, but also from the minds of many biochemists. The exception is, of course, photosynthesis, a process unique to plants and certain microorganisms in which solar energy is converted to chemical energy, and in which biological matter is produced f?om carbon dioxide and inorganic salts. The use of recombinant DNA technology to integrate molecular genetics and protein chemistry, together with the determination of crystal structures of components of the thylakoid membrane, is revealing a clearer picture of the topography and function of this membrane, as discussed in the first section of this review. Many metabolic pathways in plants are still poorly understood, partly because it is often very difficult to extract and purify the respective enzymes from plant tissues, but partly also because it is tacitly assumed that many pathways operate in the same manner as in bacteria or yeast. Thus, little stimulus exists to address the same issues in plants all over again. It appears, however, that in spite of identical or similar reaction sequences, the mechanisms regulating such pathways may differ widely. By exploiting the frequently substantial homology between microbial and plant proteins, as well as the availability of mutants of bacteria, yeast, and algae defective in cellular and metabolic functions, numerous cDNA and genomic clones coding for proteins with important functions in plants have been identified. Some of the recent major advances in this area are outlined in the second section of this review.
Using
of
the
the technique cDNAs
membrane transporters, A new chapter in plant
been opened. in transgenic
Manipulation
of
plants is providing
and their regulatory mechanisms.
in Biotechnology
Introduction
and biophysics
understanding
of microorganisms,
characterized.
has thus
Switzerland
chemistry our
membrane.
increasing insight into whole plant functions
Current Opinion
in
and in particular
membrane
transport
protein
advances
plant enzymes,
Ziirich,
1995,
6:159-l
64
The complementation of appropriate yeast mutants has proven a particularly valuable strategy in the expression cloning of ion and metabolite transporters of the plasma membrane of plant cells; we describe some highlights in section three. The transfer of genes into plant cells and regeneration of intact and fertile plants from the transformed cells, is a technique no longer limited to a few model plant species; it is now applicable, within limits, to many crop plants previously considered recalcitrant, such as certain cultivars of legumes and cereals [l]. In the final section of this review, we explore the possibilities for the modulation of plant functions either by the suppression/over-expression of endogenous proteins or by the introduction of genes encoding foreign proteins. With the added possibility of directing the temporal and spatial expression of such transgenes, the function of their products may now be studied non-invasively in whole plants, thus opening avenues to the exciting fields of molecular plant physiology and physiological ecology.
Structure
and function
thylakoid
membrane
of components
of the
Electron transport in the photosynthesis of cyanobacteria, algae and plants (oxygenic photosynthesis) is mediated by three integral membrane protein complexes: photosystem (PS)-II (which is associated with its lightharvesting complex [LHC-II]), the cytochrome bsfcomplex, and PS-I. Although the atomic structure of PS-II has, as yet, not been determined, the known homology of its two reaction centre subunits (Dl and D2) with
Abbreviations ER-endoplasmic
reticulum; LHC(P)-light harvesting centre (apoprotein); MIP-major PS-photosystem; TIP--tonoplast intrinsic protein. 0 Current Biology Ltd ISSN 0958-1669
intrinsic protein;
159
160
Plantbiotechnology the L and M proteins of the purple bacteria reaction centre has greatly aided modelling of the PS-II reaction centre [2]. The three-dimensional structure of PS-I of the cyanobacterium Synechococcus sp. at 6 A resolution became available early in 1993 (see [2]) and revealed the positioning of the 4Fe-4S clusters (Fx, FA and FB), as well as of 28 helices. More recently, the LHC-II structure has been determined at 3.4A resolution by highresolution electron cryomicroscopy of two-dimensional crystals of detergent-solubilized and purified material [3°]. It revealed the positions of the three membranespanning et-helices, as well as a close spatial relationship between chlorophylls and carotenoids, which allows the latter to quench their chlorophyll triplets and thus to perform photoprotective function. LHC-II is organized in trimers. When the apoprotein of the protein-pigment complex (LHCP) is expressed in Escherichia coli and then purified, LHC-II monomers form upon the addition of chloroplast pigments. Trimerization occurs in the presence of a lipid fraction isolated from thylakoids, and the reconstituted trimers appear to be very similar to the native trimers [4°]. In particular, they form two-dimensional crystals as readily as the native trimers. Amino acid substitutions in LHCP can now be introduced by sitedirected mutagenesis, and their effect on the structure and function of LHC-II reconstituted in vitro from a homogeneous population of LHCP can then be analyzed. The cytochrome bffcomplex accepts electrons from PSII and donates them (via plastocyanin) to PS-I, at the same time contributing to the electrochemical proton gradient (proton motive force) that drives the synthesis of ATP (photophosphorylation). The complex consists of four polypeptides, of which cytochrome f is the largest. This c-type cytochrome is anchored in the thylakoid membrane by a single membrane-spanning a-helix which thus separates the large amino-terminal luminal segment of the protein (containing the heme) from a short segment on the stroma side of the membrane. The lumen-side polypeptide fragment from turnip (comprising 252 out of 285 amino acid residues in the protein) has recently been crystallized and its atomic structure determined at 2.3 A resolution [5"°]. Among other unique and unprecedented features of this cytochrome, the structure revealed that the sixth heme iron ligand is the 0t-amino group of the aminoterminal tyrosine residue (the other axial heme ligand is a 'conventional' histidine residue). As mentioned above, plastocyanin, a small copper-containing polypeptide, functions as a mobile shuttle of electrons between the cytochrome b ~ complex and PS-I. It has been well characterized at the structural level. Ill-nuclear magnetic resonance has now been used to determine the high-resolution solution structure of parsley plastocyanin, and the protein conformation explains how deletion of certain acidic residues in the consensus plastocyanin sequence are compensated by other acidic residues [6]. Site-directed mutagenesis of the spinach plastocyanin gene and expression of the mutant polypep--
tides in transgenic potato plants have been powerful tools in the analysis of the interaction of plastocyanin with P700 + as well as of the electron transfer [7]. At first glance, a report on the structure of F1-ATPase from bovine heart mitochondria [8°°] seems to be out of place in a review devoted to plant biochemistry. However, this enzyme (ATP synthase, FIF0-ATPase) is responsible for the energy conversion in mitochondria (inner membrane), chloroplasts (thylakoid membrane) and bacteria (plasma membrane), and ATPases from these widely divergent sources have similar structures. Thus, the solution structure of the F1-ATPase from bovine heart mitochondria is exciting news for plant biochemists too. F0 is a membrane-embedded hydrophobic complex which functions as a proton channel. The hydrophilic F 1 part (containing five subunits) has three catalytic sites for ATP synthesis. The crystal structure now available supports the binding-change mechanism of ATP synthesis. It has led Abrahams et al. [8°°] to propose a 'rotational catalysis' (comparable to a bacterial flagellar motor) during which a central asymmetric aggregate of subunits rotates, driven by the protons, relative to the stationary subunits containing the catalytic sites. It thus provides the binding changes required for catalysis. These findings and models will have great impact on our understanding of photophosphorylation at the thylakoid membrane.
cDNAs and genes encoding enzymes of plant metabolism Several experimental approaches can be adopted for isolating cDNAs that encode plant enzymes. The most elegant and increasingly successful approach is expression cloning in an organism lacking the corresponding functional enzyme. Appropriate auxotrophs of yeast and E. coli, identified during several decades of research, are now available for functional complementation. For example, cDNA clones encoding imidazoleglycerolphosphate dehydratase, an enzyme of histidine biosynthesis which is still poorly understood in higher plants, have been obtained from Arabidopsis thatiana by complementation of an E. coli auxotroph [9]. The cDNAs encoding enzymes of the two-step C 5 pathway of 5-aminolevulinic acid biosynthesis, which operates in chloroplasts to provide the precursor for chlorophyll biosynthesis, have been isolated from Arabidopsis by a similar approach [10], as have been cDNAs encoding three purine biosynthetic enzymes from the same plant [11]. Yeast mutants are the organisms of choice, in particular when the cDNAs to be functionally expressed encode proteins whose occurrence is restricted to eukaryotic organisms. Recent examples are the expression of cDNAs encoding a GTP-binding protein from Arabidopsis likely to be involved in protein sorting [12], and an endoplasmic reticulum (EP,.) retention factor, which is responsible for the retention of soluble proteins in the lumen of the E R that
Molecular aspects of plant biochemistry Amrhein and Schmid
contain a carboxy-terminal recognition sequence [I 31. Two cDNA clones (StMetS-1 and StMet3-2) encoding ATP-sulphurylase from potato, the enzyme responsible for sulphate activation, have also been identified following transformation of the appropriate yeast mutant [14]. On the basis of an analysis of the amino-terminal amino acid sequences of the predicted proteins, it appears that the StMet3-2 clone encodes an ATPsulphurylase with a 48 amino acid chloroplast transit peptide, whereas the enzyme encoded by the StMet 3-1 clone does not appear to be targeted to the chloroplast. This work complements and extends the previous biochemical characterization of two ATP-sulfurylases from spinach [15]. Because enzymes of secondary plant metabolism, as a rule, are not produced by E. coli or yeast, the complementation approach cannot be used to isolate the corresponding cDNAs. Nevertheless, Corey et al. [16*] have successfully cloned a cycloartenol synthase cDNA by first functionally expressing an Arabidopsis cDNA in a yeast mutant lacking lanosterol synthases and then using a simple thin layer chromatographic assay to discover epoxysqualene mutase activity in homogenates of pools of transformants. A positive pool of transformants was then replated in order to detect single colonies, homogenates of which displayed the enzymic activity that cyclizes epoxysqualene to cycloartenol. It remains to be seen whether the successful cloning of a gene of plant secondary metabolism in yeast was, in this case, just fortuitous or whether this approach will be applicable to other genes of this type in the future. For obvious reasons, complementation in yeast is not possible for genes involved in functions related to the photoautotrophic growth of plants. It is, therefore, good news that gene isolation through genomic complementation of the green unicellular biflagellate alga Chlamydomonas reinhardtii has been achieved [17*,18] using a glass-bead transformation method [19]. A pooled library of C. reinhardtii DNA was used in the first study [17*]. Subsequently, an ordered (indexed) cosmid library has been employed [18]. In both cases, clones of the argininosuccinate lyase gene were successfully identified and isolated. Considering the wealth of C. reinhardtii mutants available, the road is now open for the isolation of genes involved in a multitude of functions using this genetically well characterized algal species.
The low-affinity inward-rectitjring potassium channels (KATl and AKTl) have recently been isolated from Arabidopsis by complementation of mutant yeast strains defective for K+ uptake [20,21], and one of them, KATl, has been functionally expressed in Xenopus oocytes [22]. Also using expression cloning, Schachtmann and Schroeder [23**] have now isolated a cDNA encoding a high-affinity K+ transporter (termed HKTl) from wheat roots. The K,, of Rb+ uptake of yeast cells expressing the transporter is 29@~l (i.e. within the range of apparent K,, values of Rb+ uptake by plant roots) and the selectivity sequence is K+>Cs+>Rb+>Na+>NH4+, as determined with HKTl-expressing Xenoptrs oocytes. K+/H+ co-uptake is the likely transport mechanism of high-affinity K+ uptake, and calculations show that K+ can thus be accumulated from very dilute external K+ solutions, against a steep concentration gradient. Energization is provided by the proton-extruding ATPase (H+-ATPase). Ultimately, the study of the K+ uptake transporter is certain to provide insight into important agronomical topics, such as K+ deficiency and alkali metal (Na+ and Cs+) toxicity. Analysis of whole cell current/voltage relationships in Arabidopsis root protoplasts by the patch clamp technique has also provided evidence that high-affinity K+ uptake transport occurs via a K+/H+-symport [24]. Using a biochemical approach, Zeilinger [25] has succeeded in isolating a Vicia faba mesophyll plasma membrane K+ channel by observing the functional reconstitution of the tetraethylammonium chloride-sensitive transport activity. This channel appears to be different from both AKTl and KATl (see above). Because of constraints of space, we can mention here only that the modulation of K+ channels by plant hormones (specifically, ion transport across the guard cell plasma membrane and its role in stomata1 opening and closing [26,27]) is currently under investigation.
Ion and metabolite transporters
Inorganic nitrogen is taken up by plants as nitrate or ammonium. Uptake studies usually reveal biphasic uptake kinetics, which indicate the presence of both low- and high-affinity uptake transporters. An Arabidopsis cDNA complementing a yeast mutant deficient in two NH4+ uptake systems has recently been shown to encode the high-affinity NH4+ transporter [28**]. The corresponding gene (MEP7) has also been isolated from yeast and has been shown to encode a protein highly related to the plant protein [29]. The plant transporter has a high affinity for methylamine (K, =65 @VI) and can thus conveniently be assayed using I%-labelled substrate. The Ki value for NH4+ is
The following section is devoted to the isolation of cDNAs and genes encoding proteins involved in the translocation of ions and metabolites across plant membranes, because unprecedented progress has been, and is rapidly being, made in this field as a result of both molecular and electrophysiological approaches.
In plants, inorganic nitrogen is fixed in leaves or roots into amino acids or other forms of reduced nitrogen, which are then distributed within the plant through the vascular bundles in both xylem and phloem. Functional complementation of yeast mutants defective in amino acid and peptide uptake has allowed the identification and isolation of cDNAs encoding several distinct amino
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Plant biotechnology
acid and peptide permeases [32,33*,34,35]. This increasing collection of amino acid/peptide permeases now provides the basis for molecular analysis and manipulation of nitrogen partitioning and utilization in plants, similar to that already achieved with carbohydrate partioning (see [36]).
transport (i.e. pores, channels, pumps, carriers, etc.) are emerging, and progress in this field, even within the single year covered by this report, is breathtaking.
The first cloning, characterization and functional analysis in transgenic plants of a sucrose-H+ co-transporter from higher plants have previously been reviewed in Current Opinion in Biotechnolqqy [36]. The ease of the preparation of vascular bundles from Planta~e major enabled the construction of a specific cDNA library which, in turn, has subsequently allowed the identification of a phloem-specific sucrose-H+ symporter from this plant, thus lending further support to the model of apoplastic phloem loading [37]. Two sucrose-H+ symporters (SUCl and SUC2) from Arubidopsis have been functionally analyzed by expression in yeast 138.1. In this study, a cDNA was constructed and expressed that encodes SUCl with a histidine tag at the carboxyl terminus. The histidine-tagged protein was found to be fully active and is now available for reconstitution and biochemical analysis.
Modulation
Although evidence for the existence of facilitated channel-mediated water movement through membranes from various sources, including plants, had been available for many years, such water channel proteins (aquaporins) have only recently been identified (see [39] for an update). Aquaporins are members of the major intrinsic protein (MIP) family, a superfamily of proteins with six membrane-spanning domains conserved throughout evolution. In plants, tonoplast intrinsic proteins (TIPS) were first identified by Chrispeels’ group (see [39] for references). The high expression of TIPS in cells of growing tissues indicates that they facilitate water flow that is required for expansion growth. Plasma membrane intrinsic proteins have now been identified in Arubidopsis, and their function as water channels has been established by expression in Xenoplrs oocytes [40**]. TIPS and MIPS will surely add a new dimension to the analysis of plantwater relations in the years to come. The term ‘porins’ has been reserved for voltage-dependent anion-selective channels in the outer membrane of Gram-negative bacteria and of mitochondria. They permit the free passage of molecules with molecular masses of up to 4-5 kDa. Until recently, plant porins had only been characterized at the physiological level. In the past year, however, cDNAs encoding porins born pea and maize plastids [41*] as well as from potato mitochondria 142.1 have been identified, and the corresponding proteins functionally analyzed in artificial lipid bilayers. Interestingly, the in vitro translated plastid porin is imported only into non-green plastids, not into chloroplasts [41’]. Within only a few years, the analysis of plant membrane transport has been revolutionized by the shrewd combination of molecular and biophysical techniques. The physical identities of the components of membrane
of plant biochemical
functions
by
transgenes In a recent review, Stitt [36] highlights the reverse genetics approach in the manipulation of carbohydrate partitioning. A substantial number of references cited as ‘in press’ in that review have since appeared in print and need not be discussed here again. Suffice it to say that transgenic plants over- or under-expressing enzymes of carbohydrate metabolism or sugar carriers continue to be invaluable tools in the determination of the roles of the respective proteins in the metabolism and allocation of assimilates. Many other pathways have recently been modulated in transgenic plants, but brevity dictates that we list only a few representative examples. Misawa et al. [43] have successfully introduced an Eriuinia uredovora phytoene desaturase gene into tobacco plants. This enzyme catalyzes a series of desaturation steps in the biosynthesis of carotenoids and is the target of many bleaching herbicides. The transformed plants are highly resistant to such herbicides and, in addition, have a spectrum of carotenoids that is different from the untransformed control plants. By introducing bacterial feedback-insensitive enzymes of lysine biosynthesis into tobacco plants, Shaul and Galili [44] were able to define the regulatory roles of the respective plant counterparts and, moreover, to consider the possibility of engineering plants that overproduce desirable levels of lysine and threonine. Suppression of enzyme levels by the expression of antisense constructs directed against the respective proteins is also a powerful approach for understanding their physiological functions. This strategy is particularly valuable when specific enzyme inhibitors are not available, or when organ specificity is to be analyzed. Transgenic tobacco plants expressing carbonic anhydrase at levels only 2% of those of the wild-type have been reported to show no morphological differrences from wild-type plants [45]. Thus, at least under the conditions employed, carbonic anhydrase activity does not produce a limitation on photosynthesis in the C3 plant tobacco. In general, repression of enzyme activity through antisense gene expression may allow elucidation of the h-action of an enzyme activity in a plant tissue required for normal plant development. For practical purposes, antisense inhibition of an enzyme can be considered an in viva model of herbicide action, and this strategy may thus be very useful in the identification of potential targets for herbicides. Two Hf-translocating enzymes are known to reside in the plant vacuolar membrane (tonoplast), one using ATP as an energized substrate, and the other pyrophosphate.
Molecular aspects of plant biochemistry Amrhein
Ellebracht et al. [46] have investigated whether pyrophosphate is required for the light-stimulated transport of protons into the vacuoles of leaf mesophyll cells. In leaves of transgenic tobacco plants expressing a soluble pyrophosphatase in the cytosol, light-dependent proton transport into vacuoles is not affected. This finding indicates that the activity of the tonoplast ATPase is sufficient for proton pumping. One can visualize many possibilities for specifically reducing the levels of metabolites by the introduction of such ‘pirate’ enzymes.
Conclusions:
future prospects
Plant biochemistry has recently seen many significant advances as a result of the application of molecular biological techniques. Proteins previously not accessible by biochemical means have been identified and functionally analyzed. This is particularly true for components of membrane transport systems. The possibility of manipulating the activity of such proteins in transgenic plants has advanced the new field of molecular plant physiology, which connects plant biochemistry with (whole) plant physiology in an unprecedented way. Old but pressing issues in plant physiology, such as the allocation of nutrients and assimilates, can now be addressed with a new repertoire of powerful techniques. It is gratifying to see that plant molecular biology, which initially was viewed with suspicion by many plant physiologists, has, in fact, become a strong supporting science in this field and has brought new life and fresh ideas. It is especially satisfying to see this happen at a time when the ever-increasing specialization of science is to be deplored.
Acknowledgement We
thank
Doris
Wewetzer
Papers of particular
interest, published review, have been highlighted as: . of special interest .. of outstanding interest
Hobe 5, Prytulla 5, Kuhlbrandt W, Paulsen H: Trimerization and crystallization of reconstituted light-harvesting chlorophyll a/b complex. EM60 I 1994, 13:3423-3429. LHC-II monomers, reconstituted from the heterologously expressed and purified apoprotein (LHCPJ and pigments, trimerize in vifro in the presence of a lipid fraction isolated from pea thylakoids. Structure and function of LHC-II can thus be studied in a highly defined system which can be modified by site-directed mutagenesis. 5. ..
Martinez SE, Huang D, Szczepaniak, A, Cramer WA, Smith JL: Crystal structure of chloroplast cytochrome f reveals a novel cytochrome fold and unexpected heme ligation. Structure 1994, 2:95-l 05. A landmark paper on the atomic structure of a subunit of the cytochrome bsfcomplex of the turnip thylakoid membrane, revealing several unique features of this cytochrome. The structure allows prediction of the docking site of plastocyanin and of the pathways of intra- and inter-protein electron transfer. 6.
Bagby 5, Driscoll PC, Harvey TS, Hill HAO: High-resolution solution structure of reduced parsley plastocyanin. Biochemistry 1994, 33:661 l-6622.
7.
Haehnel W, Jansen T, Cause K, Kloskgen RB, Stahl 8, Michl D, Huvermann 8, Karas M, Herrmann RG: Electron transfer from plastocyanin to photosystem I. EM60 / 1994, 13:102B-1038.
a.
Abrahams JP, Leslie ACW, Lutter R, Walker JE: Structure at 2.8A resolution of F,-ATPase from bovine heart mitochondria. Nature 1994, 370:621-628. The atomic structure of Ft-ATPase at 2.8/c resolution is described. A milestone paper of far-reaching impact for the understanding of the mechanism of the synthesis of ATP by oxidative and photo-phosphorylation. This is the largest asymmetric structure ever to be solved. ..
9.
Tada 5, Volrath 5, Cuyer D, Scheidegger A, Ryals J, Ohta D, Ward E: Isolation and characterization of cDNAs encoding imidazoleglycerolphosphate dehydratase from Arabidopsis thalima. P/ant Physiol 1994, 105:579-583.
10.
llag LL, Kumar AM, 5611 D: tight regulation of chlorophyll biosynthesis at the level of 5-aminolevulinate formation in Arabidopsis. Plant Cell 1994, 6:265-275.
11.
Schnorr KM, Nygaard P, Laloue M: Molecular characterization of Arabidopsis thaliana cDNAs encoding three purine biosynthetic enzymes. P/ant / 1994, 6:113-l 21.
12.
Bednarek SY, Reynolds TL, Schroeder M, Crabowski R, Hengst L, Gallwitz D, Raikhel NV: A small CTP-binding protein from Arabidopsis thaliana functionally complements the yeast YPT6 null mutant. P/ant Physiol 1994, 104:591-596.
13.
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14.
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15.
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Fischer K, Weber A, Brink S, Arbinger 6, Schiinemann D, Borchert S, Heldt HW, Popp B, Benz R, Link TA et a/.: Porins from plants. Molecular cloning and functional characterization of two new members of the porin family. / 6iol Chem 1994, 26925754-25760. Mitochondria and non-green plastids are shown to possess homologous porin proteins, whereas chloroplasts are characterized by a different type of porin. Heins L, Mentzel H, Scmid A, Benz R, Schmitz UK: Biochemical, molecular and functional characterization of porin isoforms from potato mitochondria. 1 Viol Chem 1994, 269:26402-26410. Presents a biochemical and molecular genetic analysis of two different porin polypeptides (M, 34 000 and M, 36 000) from the outer membrane of potato mitochondria. Neither of the two proteins is, however, able to complement the respiratory defect of a yeast por mutant.
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Shaul 0, Galili G: Concerted regulation of lysine and threonine synthesis in tobacco plants expressing bacterial feedback insensitive aspartate kinase and dihydrodipicolinate synthase. P/ant MO/ Biol 1993, 23:759-768.
45.
Price CD, Von Caemmerer S, Evans JR, Yu JW, Lloyd J, Oja V, Kell P, Harrison K, Gallagher A, Badger MR: Specific reduction of chloroplast carbonic anhydrase activity by antisense RNA in transgenic tobacco plants has a minor effect on photosynthesic CO2 assimilation. Planta 1994, 193:331-340.
46.
Ellebracht A, Heber U, Sonnewald U: Lightstimuhted proton transport into the vacuoles of leaf mesophyll cells does not require energization by the tonoplast pyrophosphatase. Planta 1994, 193:203-207.
N Amrhein and J Schmid, Institute of Plant Federal Institute of Technology, Universidtstrasse Switzerland.
Sciences,
Swiss
2, 8092 Ziirich,
The
integration
has
brought
topography
of molecular about
and function
of expression
cloning
encoding numerous
genetics,
significant
of the thylakoid
in appropriate
have been identified
and functionally biochemistry
the activities
of these enzymes
mutants
and carriers
Biochemistry relates molecular structures to biological functions and vice versa. Topics specifically related to the biochemistry of plants tend to be excluded not only &om the text books of biochemistry, but also from the minds of many biochemists. The exception is, of course, photosynthesis, a process unique to plants and certain microorganisms in which solar energy is converted to chemical energy, and in which biological matter is produced f?om carbon dioxide and inorganic salts. The use of recombinant DNA technology to integrate molecular genetics and protein chemistry, together with the determination of crystal structures of components of the thylakoid membrane, is revealing a clearer picture of the topography and function of this membrane, as discussed in the first section of this review. Many metabolic pathways in plants are still poorly understood, partly because it is often very difficult to extract and purify the respective enzymes from plant tissues, but partly also because it is tacitly assumed that many pathways operate in the same manner as in bacteria or yeast. Thus, little stimulus exists to address the same issues in plants all over again. It appears, however, that in spite of identical or similar reaction sequences, the mechanisms regulating such pathways may differ widely. By exploiting the frequently substantial homology between microbial and plant proteins, as well as the availability of mutants of bacteria, yeast, and algae defective in cellular and metabolic functions, numerous cDNA and genomic clones coding for proteins with important functions in plants have been identified. Some of the recent major advances in this area are outlined in the second section of this review.
Using
of
the
the technique cDNAs
membrane transporters, A new chapter in plant
been opened. in transgenic
Manipulation
of
plants is providing
and their regulatory mechanisms.
in Biotechnology
Introduction
and biophysics
understanding
of microorganisms,
characterized.
has thus
Switzerland
chemistry our
membrane.
increasing insight into whole plant functions
Current Opinion
in
and in particular
membrane
transport
protein
advances
plant enzymes,
Ziirich,
1995,
6:159-l
64
The complementation of appropriate yeast mutants has proven a particularly valuable strategy in the expression cloning of ion and metabolite transporters of the plasma membrane of plant cells; we describe some highlights in section three. The transfer of genes into plant cells and regeneration of intact and fertile plants from the transformed cells, is a technique no longer limited to a few model plant species; it is now applicable, within limits, to many crop plants previously considered recalcitrant, such as certain cultivars of legumes and cereals [l]. In the final section of this review, we explore the possibilities for the modulation of plant functions either by the suppression/over-expression of endogenous proteins or by the introduction of genes encoding foreign proteins. With the added possibility of directing the temporal and spatial expression of such transgenes, the function of their products may now be studied non-invasively in whole plants, thus opening avenues to the exciting fields of molecular plant physiology and physiological ecology.
Structure
and function
thylakoid
membrane
of components
of the
Electron transport in the photosynthesis of cyanobacteria, algae and plants (oxygenic photosynthesis) is mediated by three integral membrane protein complexes: photosystem (PS)-II (which is associated with its lightharvesting complex [LHC-II]), the cytochrome bsfcomplex, and PS-I. Although the atomic structure of PS-II has, as yet, not been determined, the known homology of its two reaction centre subunits (Dl and D2) with
Abbreviations ER-endoplasmic
reticulum; LHC(P)-light harvesting centre (apoprotein); MIP-major PS-photosystem; TIP--tonoplast intrinsic protein. 0 Current Biology Ltd ISSN 0958-1669
intrinsic protein;
159
160
Plantbiotechnology the L and M proteins of the purple bacteria reaction centre has greatly aided modelling of the PS-II reaction centre [2]. The three-dimensional structure of PS-I of the cyanobacterium Synechococcus sp. at 6 A resolution became available early in 1993 (see [2]) and revealed the positioning of the 4Fe-4S clusters (Fx, FA and FB), as well as of 28 helices. More recently, the LHC-II structure has been determined at 3.4A resolution by highresolution electron cryomicroscopy of two-dimensional crystals of detergent-solubilized and purified material [3°]. It revealed the positions of the three membranespanning et-helices, as well as a close spatial relationship between chlorophylls and carotenoids, which allows the latter to quench their chlorophyll triplets and thus to perform photoprotective function. LHC-II is organized in trimers. When the apoprotein of the protein-pigment complex (LHCP) is expressed in Escherichia coli and then purified, LHC-II monomers form upon the addition of chloroplast pigments. Trimerization occurs in the presence of a lipid fraction isolated from thylakoids, and the reconstituted trimers appear to be very similar to the native trimers [4°]. In particular, they form two-dimensional crystals as readily as the native trimers. Amino acid substitutions in LHCP can now be introduced by sitedirected mutagenesis, and their effect on the structure and function of LHC-II reconstituted in vitro from a homogeneous population of LHCP can then be analyzed. The cytochrome bffcomplex accepts electrons from PSII and donates them (via plastocyanin) to PS-I, at the same time contributing to the electrochemical proton gradient (proton motive force) that drives the synthesis of ATP (photophosphorylation). The complex consists of four polypeptides, of which cytochrome f is the largest. This c-type cytochrome is anchored in the thylakoid membrane by a single membrane-spanning a-helix which thus separates the large amino-terminal luminal segment of the protein (containing the heme) from a short segment on the stroma side of the membrane. The lumen-side polypeptide fragment from turnip (comprising 252 out of 285 amino acid residues in the protein) has recently been crystallized and its atomic structure determined at 2.3 A resolution [5"°]. Among other unique and unprecedented features of this cytochrome, the structure revealed that the sixth heme iron ligand is the 0t-amino group of the aminoterminal tyrosine residue (the other axial heme ligand is a 'conventional' histidine residue). As mentioned above, plastocyanin, a small copper-containing polypeptide, functions as a mobile shuttle of electrons between the cytochrome b ~ complex and PS-I. It has been well characterized at the structural level. Ill-nuclear magnetic resonance has now been used to determine the high-resolution solution structure of parsley plastocyanin, and the protein conformation explains how deletion of certain acidic residues in the consensus plastocyanin sequence are compensated by other acidic residues [6]. Site-directed mutagenesis of the spinach plastocyanin gene and expression of the mutant polypep--
tides in transgenic potato plants have been powerful tools in the analysis of the interaction of plastocyanin with P700 + as well as of the electron transfer [7]. At first glance, a report on the structure of F1-ATPase from bovine heart mitochondria [8°°] seems to be out of place in a review devoted to plant biochemistry. However, this enzyme (ATP synthase, FIF0-ATPase) is responsible for the energy conversion in mitochondria (inner membrane), chloroplasts (thylakoid membrane) and bacteria (plasma membrane), and ATPases from these widely divergent sources have similar structures. Thus, the solution structure of the F1-ATPase from bovine heart mitochondria is exciting news for plant biochemists too. F0 is a membrane-embedded hydrophobic complex which functions as a proton channel. The hydrophilic F 1 part (containing five subunits) has three catalytic sites for ATP synthesis. The crystal structure now available supports the binding-change mechanism of ATP synthesis. It has led Abrahams et al. [8°°] to propose a 'rotational catalysis' (comparable to a bacterial flagellar motor) during which a central asymmetric aggregate of subunits rotates, driven by the protons, relative to the stationary subunits containing the catalytic sites. It thus provides the binding changes required for catalysis. These findings and models will have great impact on our understanding of photophosphorylation at the thylakoid membrane.
cDNAs and genes encoding enzymes of plant metabolism Several experimental approaches can be adopted for isolating cDNAs that encode plant enzymes. The most elegant and increasingly successful approach is expression cloning in an organism lacking the corresponding functional enzyme. Appropriate auxotrophs of yeast and E. coli, identified during several decades of research, are now available for functional complementation. For example, cDNA clones encoding imidazoleglycerolphosphate dehydratase, an enzyme of histidine biosynthesis which is still poorly understood in higher plants, have been obtained from Arabidopsis thatiana by complementation of an E. coli auxotroph [9]. The cDNAs encoding enzymes of the two-step C 5 pathway of 5-aminolevulinic acid biosynthesis, which operates in chloroplasts to provide the precursor for chlorophyll biosynthesis, have been isolated from Arabidopsis by a similar approach [10], as have been cDNAs encoding three purine biosynthetic enzymes from the same plant [11]. Yeast mutants are the organisms of choice, in particular when the cDNAs to be functionally expressed encode proteins whose occurrence is restricted to eukaryotic organisms. Recent examples are the expression of cDNAs encoding a GTP-binding protein from Arabidopsis likely to be involved in protein sorting [12], and an endoplasmic reticulum (EP,.) retention factor, which is responsible for the retention of soluble proteins in the lumen of the E R that
Molecular aspects of plant biochemistry Amrhein and Schmid
contain a carboxy-terminal recognition sequence [I 31. Two cDNA clones (StMetS-1 and StMet3-2) encoding ATP-sulphurylase from potato, the enzyme responsible for sulphate activation, have also been identified following transformation of the appropriate yeast mutant [14]. On the basis of an analysis of the amino-terminal amino acid sequences of the predicted proteins, it appears that the StMet3-2 clone encodes an ATPsulphurylase with a 48 amino acid chloroplast transit peptide, whereas the enzyme encoded by the StMet 3-1 clone does not appear to be targeted to the chloroplast. This work complements and extends the previous biochemical characterization of two ATP-sulfurylases from spinach [15]. Because enzymes of secondary plant metabolism, as a rule, are not produced by E. coli or yeast, the complementation approach cannot be used to isolate the corresponding cDNAs. Nevertheless, Corey et al. [16*] have successfully cloned a cycloartenol synthase cDNA by first functionally expressing an Arabidopsis cDNA in a yeast mutant lacking lanosterol synthases and then using a simple thin layer chromatographic assay to discover epoxysqualene mutase activity in homogenates of pools of transformants. A positive pool of transformants was then replated in order to detect single colonies, homogenates of which displayed the enzymic activity that cyclizes epoxysqualene to cycloartenol. It remains to be seen whether the successful cloning of a gene of plant secondary metabolism in yeast was, in this case, just fortuitous or whether this approach will be applicable to other genes of this type in the future. For obvious reasons, complementation in yeast is not possible for genes involved in functions related to the photoautotrophic growth of plants. It is, therefore, good news that gene isolation through genomic complementation of the green unicellular biflagellate alga Chlamydomonas reinhardtii has been achieved [17*,18] using a glass-bead transformation method [19]. A pooled library of C. reinhardtii DNA was used in the first study [17*]. Subsequently, an ordered (indexed) cosmid library has been employed [18]. In both cases, clones of the argininosuccinate lyase gene were successfully identified and isolated. Considering the wealth of C. reinhardtii mutants available, the road is now open for the isolation of genes involved in a multitude of functions using this genetically well characterized algal species.
The low-affinity inward-rectitjring potassium channels (KATl and AKTl) have recently been isolated from Arabidopsis by complementation of mutant yeast strains defective for K+ uptake [20,21], and one of them, KATl, has been functionally expressed in Xenopus oocytes [22]. Also using expression cloning, Schachtmann and Schroeder [23**] have now isolated a cDNA encoding a high-affinity K+ transporter (termed HKTl) from wheat roots. The K,, of Rb+ uptake of yeast cells expressing the transporter is 29@~l (i.e. within the range of apparent K,, values of Rb+ uptake by plant roots) and the selectivity sequence is K+>Cs+>Rb+>Na+>NH4+, as determined with HKTl-expressing Xenoptrs oocytes. K+/H+ co-uptake is the likely transport mechanism of high-affinity K+ uptake, and calculations show that K+ can thus be accumulated from very dilute external K+ solutions, against a steep concentration gradient. Energization is provided by the proton-extruding ATPase (H+-ATPase). Ultimately, the study of the K+ uptake transporter is certain to provide insight into important agronomical topics, such as K+ deficiency and alkali metal (Na+ and Cs+) toxicity. Analysis of whole cell current/voltage relationships in Arabidopsis root protoplasts by the patch clamp technique has also provided evidence that high-affinity K+ uptake transport occurs via a K+/H+-symport [24]. Using a biochemical approach, Zeilinger [25] has succeeded in isolating a Vicia faba mesophyll plasma membrane K+ channel by observing the functional reconstitution of the tetraethylammonium chloride-sensitive transport activity. This channel appears to be different from both AKTl and KATl (see above). Because of constraints of space, we can mention here only that the modulation of K+ channels by plant hormones (specifically, ion transport across the guard cell plasma membrane and its role in stomata1 opening and closing [26,27]) is currently under investigation.
Ion and metabolite transporters
Inorganic nitrogen is taken up by plants as nitrate or ammonium. Uptake studies usually reveal biphasic uptake kinetics, which indicate the presence of both low- and high-affinity uptake transporters. An Arabidopsis cDNA complementing a yeast mutant deficient in two NH4+ uptake systems has recently been shown to encode the high-affinity NH4+ transporter [28**]. The corresponding gene (MEP7) has also been isolated from yeast and has been shown to encode a protein highly related to the plant protein [29]. The plant transporter has a high affinity for methylamine (K, =65 @VI) and can thus conveniently be assayed using I%-labelled substrate. The Ki value for NH4+ is
The following section is devoted to the isolation of cDNAs and genes encoding proteins involved in the translocation of ions and metabolites across plant membranes, because unprecedented progress has been, and is rapidly being, made in this field as a result of both molecular and electrophysiological approaches.
In plants, inorganic nitrogen is fixed in leaves or roots into amino acids or other forms of reduced nitrogen, which are then distributed within the plant through the vascular bundles in both xylem and phloem. Functional complementation of yeast mutants defective in amino acid and peptide uptake has allowed the identification and isolation of cDNAs encoding several distinct amino
161
162
Plant biotechnology
acid and peptide permeases [32,33*,34,35]. This increasing collection of amino acid/peptide permeases now provides the basis for molecular analysis and manipulation of nitrogen partitioning and utilization in plants, similar to that already achieved with carbohydrate partioning (see [36]).
transport (i.e. pores, channels, pumps, carriers, etc.) are emerging, and progress in this field, even within the single year covered by this report, is breathtaking.
The first cloning, characterization and functional analysis in transgenic plants of a sucrose-H+ co-transporter from higher plants have previously been reviewed in Current Opinion in Biotechnolqqy [36]. The ease of the preparation of vascular bundles from Planta~e major enabled the construction of a specific cDNA library which, in turn, has subsequently allowed the identification of a phloem-specific sucrose-H+ symporter from this plant, thus lending further support to the model of apoplastic phloem loading [37]. Two sucrose-H+ symporters (SUCl and SUC2) from Arubidopsis have been functionally analyzed by expression in yeast 138.1. In this study, a cDNA was constructed and expressed that encodes SUCl with a histidine tag at the carboxyl terminus. The histidine-tagged protein was found to be fully active and is now available for reconstitution and biochemical analysis.
Modulation
Although evidence for the existence of facilitated channel-mediated water movement through membranes from various sources, including plants, had been available for many years, such water channel proteins (aquaporins) have only recently been identified (see [39] for an update). Aquaporins are members of the major intrinsic protein (MIP) family, a superfamily of proteins with six membrane-spanning domains conserved throughout evolution. In plants, tonoplast intrinsic proteins (TIPS) were first identified by Chrispeels’ group (see [39] for references). The high expression of TIPS in cells of growing tissues indicates that they facilitate water flow that is required for expansion growth. Plasma membrane intrinsic proteins have now been identified in Arubidopsis, and their function as water channels has been established by expression in Xenoplrs oocytes [40**]. TIPS and MIPS will surely add a new dimension to the analysis of plantwater relations in the years to come. The term ‘porins’ has been reserved for voltage-dependent anion-selective channels in the outer membrane of Gram-negative bacteria and of mitochondria. They permit the free passage of molecules with molecular masses of up to 4-5 kDa. Until recently, plant porins had only been characterized at the physiological level. In the past year, however, cDNAs encoding porins born pea and maize plastids [41*] as well as from potato mitochondria 142.1 have been identified, and the corresponding proteins functionally analyzed in artificial lipid bilayers. Interestingly, the in vitro translated plastid porin is imported only into non-green plastids, not into chloroplasts [41’]. Within only a few years, the analysis of plant membrane transport has been revolutionized by the shrewd combination of molecular and biophysical techniques. The physical identities of the components of membrane
of plant biochemical
functions
by
transgenes In a recent review, Stitt [36] highlights the reverse genetics approach in the manipulation of carbohydrate partitioning. A substantial number of references cited as ‘in press’ in that review have since appeared in print and need not be discussed here again. Suffice it to say that transgenic plants over- or under-expressing enzymes of carbohydrate metabolism or sugar carriers continue to be invaluable tools in the determination of the roles of the respective proteins in the metabolism and allocation of assimilates. Many other pathways have recently been modulated in transgenic plants, but brevity dictates that we list only a few representative examples. Misawa et al. [43] have successfully introduced an Eriuinia uredovora phytoene desaturase gene into tobacco plants. This enzyme catalyzes a series of desaturation steps in the biosynthesis of carotenoids and is the target of many bleaching herbicides. The transformed plants are highly resistant to such herbicides and, in addition, have a spectrum of carotenoids that is different from the untransformed control plants. By introducing bacterial feedback-insensitive enzymes of lysine biosynthesis into tobacco plants, Shaul and Galili [44] were able to define the regulatory roles of the respective plant counterparts and, moreover, to consider the possibility of engineering plants that overproduce desirable levels of lysine and threonine. Suppression of enzyme levels by the expression of antisense constructs directed against the respective proteins is also a powerful approach for understanding their physiological functions. This strategy is particularly valuable when specific enzyme inhibitors are not available, or when organ specificity is to be analyzed. Transgenic tobacco plants expressing carbonic anhydrase at levels only 2% of those of the wild-type have been reported to show no morphological differrences from wild-type plants [45]. Thus, at least under the conditions employed, carbonic anhydrase activity does not produce a limitation on photosynthesis in the C3 plant tobacco. In general, repression of enzyme activity through antisense gene expression may allow elucidation of the h-action of an enzyme activity in a plant tissue required for normal plant development. For practical purposes, antisense inhibition of an enzyme can be considered an in viva model of herbicide action, and this strategy may thus be very useful in the identification of potential targets for herbicides. Two Hf-translocating enzymes are known to reside in the plant vacuolar membrane (tonoplast), one using ATP as an energized substrate, and the other pyrophosphate.
Molecular aspects of plant biochemistry Amrhein
Ellebracht et al. [46] have investigated whether pyrophosphate is required for the light-stimulated transport of protons into the vacuoles of leaf mesophyll cells. In leaves of transgenic tobacco plants expressing a soluble pyrophosphatase in the cytosol, light-dependent proton transport into vacuoles is not affected. This finding indicates that the activity of the tonoplast ATPase is sufficient for proton pumping. One can visualize many possibilities for specifically reducing the levels of metabolites by the introduction of such ‘pirate’ enzymes.
Conclusions:
future prospects
Plant biochemistry has recently seen many significant advances as a result of the application of molecular biological techniques. Proteins previously not accessible by biochemical means have been identified and functionally analyzed. This is particularly true for components of membrane transport systems. The possibility of manipulating the activity of such proteins in transgenic plants has advanced the new field of molecular plant physiology, which connects plant biochemistry with (whole) plant physiology in an unprecedented way. Old but pressing issues in plant physiology, such as the allocation of nutrients and assimilates, can now be addressed with a new repertoire of powerful techniques. It is gratifying to see that plant molecular biology, which initially was viewed with suspicion by many plant physiologists, has, in fact, become a strong supporting science in this field and has brought new life and fresh ideas. It is especially satisfying to see this happen at a time when the ever-increasing specialization of science is to be deplored.
Acknowledgement We
thank
Doris
Wewetzer
Papers of particular
interest, published review, have been highlighted as: . of special interest .. of outstanding interest
Hobe 5, Prytulla 5, Kuhlbrandt W, Paulsen H: Trimerization and crystallization of reconstituted light-harvesting chlorophyll a/b complex. EM60 I 1994, 13:3423-3429. LHC-II monomers, reconstituted from the heterologously expressed and purified apoprotein (LHCPJ and pigments, trimerize in vifro in the presence of a lipid fraction isolated from pea thylakoids. Structure and function of LHC-II can thus be studied in a highly defined system which can be modified by site-directed mutagenesis. 5. ..
Martinez SE, Huang D, Szczepaniak, A, Cramer WA, Smith JL: Crystal structure of chloroplast cytochrome f reveals a novel cytochrome fold and unexpected heme ligation. Structure 1994, 2:95-l 05. A landmark paper on the atomic structure of a subunit of the cytochrome bsfcomplex of the turnip thylakoid membrane, revealing several unique features of this cytochrome. The structure allows prediction of the docking site of plastocyanin and of the pathways of intra- and inter-protein electron transfer. 6.
Bagby 5, Driscoll PC, Harvey TS, Hill HAO: High-resolution solution structure of reduced parsley plastocyanin. Biochemistry 1994, 33:661 l-6622.
7.
Haehnel W, Jansen T, Cause K, Kloskgen RB, Stahl 8, Michl D, Huvermann 8, Karas M, Herrmann RG: Electron transfer from plastocyanin to photosystem I. EM60 / 1994, 13:102B-1038.
a.
Abrahams JP, Leslie ACW, Lutter R, Walker JE: Structure at 2.8A resolution of F,-ATPase from bovine heart mitochondria. Nature 1994, 370:621-628. The atomic structure of Ft-ATPase at 2.8/c resolution is described. A milestone paper of far-reaching impact for the understanding of the mechanism of the synthesis of ATP by oxidative and photo-phosphorylation. This is the largest asymmetric structure ever to be solved. ..
9.
Tada 5, Volrath 5, Cuyer D, Scheidegger A, Ryals J, Ohta D, Ward E: Isolation and characterization of cDNAs encoding imidazoleglycerolphosphate dehydratase from Arabidopsis thalima. P/ant Physiol 1994, 105:579-583.
10.
llag LL, Kumar AM, 5611 D: tight regulation of chlorophyll biosynthesis at the level of 5-aminolevulinate formation in Arabidopsis. Plant Cell 1994, 6:265-275.
11.
Schnorr KM, Nygaard P, Laloue M: Molecular characterization of Arabidopsis thaliana cDNAs encoding three purine biosynthetic enzymes. P/ant / 1994, 6:113-l 21.
12.
Bednarek SY, Reynolds TL, Schroeder M, Crabowski R, Hengst L, Gallwitz D, Raikhel NV: A small CTP-binding protein from Arabidopsis thaliana functionally complements the yeast YPT6 null mutant. P/ant Physiol 1994, 104:591-596.
13.
Lee H, Gal 5, Newman TC, Raikhel NV: The Arabidopsis endoplasmic reticulum retention receptor functions in yeast. Proc Nat/ Acad Sci USA 1994, 90:11433-11437.
14.
Klonus D, Hofgen R, Willmitzer L, Riesmeier JW: Isolation and characterization of two cDNA clones encoding ATP-sulfurylases from potato by complementation of a yeast mutant. P/ant ) 1994, 6:105-112.
15.
Renesto F, Pate1 HC, Martin RL, Thomassian C, Zimmermann G, Segel IH: ATP sulfurylase from higher plants: kinetic and structural characterization of the chloroplast and cytosol enzymes from spinach leaves. Arch Biochem Biophys 1993, 3071272-285.
reading within
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Barber J, Andersson B: Revealing the blueprint thesis. Nature 1994, 370:3 l-34.
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Kiihlbrandt W, Wang DN, Fujiyoshi Y: Atomic model of plant light-harvesting complex by electron crystallography. Name 1994, 367~614421. An important contribution that provides the basis for the understanding of energy transfer in an antenna. Two of the three membrane-spanning a-helices of LHC-II are held together by ion pairs formed by charged residues that also serve as chlorophyll ligands. In the centre of the complex, chlorophylls a and b are in close contact with each other as well as with two carotenoids. 3. .
4. .
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Ninnemann 0, Jauniaux JC, Frommer WB: Identification of a high affinity NHq+ transporter from plants. EMEO / 1994, 13:3464-3471. The first identification of an NH4+ transporter in a plant at the molecular level. This study opens the possibility of manipulating the nitrogen uptake characteristics in transgenic plants. 28. ..
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Kwart M, Hirner 8, Hummel S, Frommer WB: Differential expression of two related amino acid transporters with differing substrate specificity in Aralddopsis thaliana. P/ant I 1993, 4:993-1002. Two new amino acid transporter genes are identified by complementation of citrulline and histidine uptake deficiencies in yeast mutants. The encoded transporters differ in their substrate specificity as well as in their organ-specific expression pattern. 34.
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L, Naider F, Becker JM, Stacey transporter is a member of a
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Sauer N, Stolz J: SUCl and SUCZ: two sucrose transporters from Arabidopsis thaliana; expression and characterization in baker’s yeast and identification of the histidine-tagged protein. P/ant I 1994, 6:67-77. The first sucrose transporter to be cloned was from spinach (see 136)). This paper reports the cloning of two such transporters from Arabidopsis. More importantly, a cDNA encoding a histidine tag at the SUCl carboxyl terminus is expressed in yeast and the tagged protein found to be fully active. 39.
Chrispeels MJ, Maurel C: Aquaporins: the molecular basis of facilitated water movement through living plant cells? P/ant Physiol 1994, 105:9-l 3.
40. ..
Kammerloher W, Fischer V, Piechottka GP, Schaffner AR: Water channels in the plant plasma membrane cloned by immunoselection from a mammalian expression system. P/ant J 1994, 6:187-l 99. An important paper demonstrating for the first time the presence of water channels in the plant plasma membrane. 41. .
Fischer K, Weber A, Brink S, Arbinger 6, Schiinemann D, Borchert S, Heldt HW, Popp B, Benz R, Link TA et a/.: Porins from plants. Molecular cloning and functional characterization of two new members of the porin family. / 6iol Chem 1994, 26925754-25760. Mitochondria and non-green plastids are shown to possess homologous porin proteins, whereas chloroplasts are characterized by a different type of porin. Heins L, Mentzel H, Scmid A, Benz R, Schmitz UK: Biochemical, molecular and functional characterization of porin isoforms from potato mitochondria. 1 Viol Chem 1994, 269:26402-26410. Presents a biochemical and molecular genetic analysis of two different porin polypeptides (M, 34 000 and M, 36 000) from the outer membrane of potato mitochondria. Neither of the two proteins is, however, able to complement the respiratory defect of a yeast por mutant.
42.
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43.
Misawa N, Nasamoto K, Hori T, Ohtani T, BOger P, Sandmann G: Expression of an Frwinia phytoene desaturase gene not only confers multiple resistance to herbicides interfering with carotenoid biosynthesis, but also alters xanthophyll metabolism in transgenic plants. P/ant / 1994, 6:481-489.
44.
Shaul 0, Galili G: Concerted regulation of lysine and threonine synthesis in tobacco plants expressing bacterial feedback insensitive aspartate kinase and dihydrodipicolinate synthase. P/ant MO/ Biol 1993, 23:759-768.
45.
Price CD, Von Caemmerer S, Evans JR, Yu JW, Lloyd J, Oja V, Kell P, Harrison K, Gallagher A, Badger MR: Specific reduction of chloroplast carbonic anhydrase activity by antisense RNA in transgenic tobacco plants has a minor effect on photosynthesic CO2 assimilation. Planta 1994, 193:331-340.
46.
Ellebracht A, Heber U, Sonnewald U: Lightstimuhted proton transport into the vacuoles of leaf mesophyll cells does not require energization by the tonoplast pyrophosphatase. Planta 1994, 193:203-207.
N Amrhein and J Schmid, Institute of Plant Federal Institute of Technology, Universidtstrasse Switzerland.
Sciences,
Swiss
2, 8092 Ziirich,