Systematics and plastid genome evolution of the cryptically photosynthetic parasitic plant genus Cuscuta (Convolvulaceae).

BACKGROUND: The genus Cuscuta L. (Convolvulaceae), commonly known as dodders, are epiphytic vines that invade the stems of their host with haustorial feeding structures at the points of contact. Although they lack expanded leaves, some species are noticeably chlorophyllous, especially as seedlings and in maturing fruits. Some species are reported as crop pests of worldwide distribution, whereas others are extremely rare and have local distributions and apparent niche specificity. A strong phylogenetic framework for this large genus is essential to understand the interesting ecological, morphological and molecular phenomena that occur within these parasites in an evolutionary context. RESULTS: Here we present a well-supported phylogeny of Cuscuta using sequences of the nuclear ribosomal internal transcribed spacer and plastid rps2, rbcL and matK from representatives across most of the taxonomic diversity of the genus. We use the phylogeny to interpret morphological and plastid genome evolution within the genus. At least three currently recognized taxonomic sections are not monophyletic and subgenus Cuscuta is unequivocally paraphyletic. Plastid genes are extremely variable with regards to evolutionary constraint, with rbcL exhibiting even higher levels of purifying selection in Cuscuta than photosynthetic relatives. Nuclear genome size is highly variable within Cuscuta, particularly within subgenus Grammica, and in some cases may indicate the existence of cryptic species in this large clade of morphologically similar species. CONCLUSION: Some morphological characters traditionally used to define major taxonomic splits within Cuscuta are homoplastic and are of limited use in defining true evolutionary groups. Chloroplast genome evolution seems to have evolved in a punctuated fashion, with episodes of loss involving suites of genes or tRNAs followed by stabilization of gene content in major clades. Nearly all species of Cuscuta retain some photosynthetic ability, most likely for nutrient apportionment to their seeds, while complete loss of photosynthesis and possible loss of the entire chloroplast genome is limited to a single small clade of outcrossing species found primarily in western South America.

BVOCs: plant defense against climate warming?

Plants emit a substantial amount of biogenic volatile organic compounds (BVOCs) into the atmosphere. These BVOCs represent a large carbon loss and can be up to approximately 10% of that fixed by photosynthesis under stressful conditions and up to 100gCm(-2) per year in some tropical ecosystems. Among a variety of proven and unproven BVOC functions in plants and roles in atmospheric processes, recent data intriguingly link emission of these compounds to climate. Ongoing research demonstrates that BVOCs could protect plants against high temperatures. BVOC emissions are probably increasing with warming and with other factors associated to global change, including changes in land cover. These increases in BVOC emissions could contribute in a significant way (via negative and positive feedback) to the complex processes associated with global warming.

Identification of type 1 and type 2 light-harvesting chlorophyll a/b-binding proteins using monospecific antibodies.

The amino acid sequences of more than 40 apoproteins of the light-harvesting complex associated with Photosystem II (LHC II) of various plants have been deduced by sequencing their corresponding genes. These highly conserved sequences fall into two major categories, type 1 and type 2, that differ mainly in a small number of domains close to the N-terminus. We have made polyclonal, monospecific antibodies against synthetic peptides corresponding to the most unique sequence domains of the N-terminal regions of type 1 and type 2 LHC II apoproteins, using sequences derived from petunia genes. On Western blots our anti-type 1 and 2 antibodies crossreact with light-harvesting proteins of petunia, tomato, spinach and several other plants. By using a new gel-system based on ammediol (2-amino-2-methyl-1,3-propanediol), we are able to resolve up to eight LHC II apoproteins. On petunia, tomato and spinach blots the anti type 1 antibodies bind to two or more of the higher molecular weight LHC II polypeptides, whereas the anti type 2 antibodies recognize very specifically only one or two of the lower molecular weight LHC-proteins. In all plants studied, the type 1 LHC II apoproteins are more numerous and span a greater size range than the type 2 apoproteins. This is consistent with the smaller number of type 2 LHC II CAB genes that have been discovered to date.

A cytochrome b origin of photosynthetic reaction centers: an evolutionary link between respiration and photosynthesis.

The evolutionary origin of photosynthetic reaction centers has long remained elusive. Here, we use sequence and structural analysis to demonstrate an evolutionary link between the cytochrome b subunit of the cytochrome bc(1) complex and the core polypeptides of the photosynthetic bacterial reaction center. In particular, we have identified an area of significant sequence similarity between a three contiguous membrane-spanning domain of cytochrome b, which contains binding sites for two hemes, and a three contiguous membrane-spanning domain in the photosynthetic reaction center core subunits, which contains binding sites for cofactors such as (bacterio)chlorophylls, (bacterio)pheophytin and a non-heme iron. Three of the four heme ligands in cytochrome b are found to be conserved with the cofactor ligands in the reaction center polypeptides. Since cytochrome b and reaction center polypeptides both bind tetrapyrroles and quinones for electron transfer, the observed sequence, functional and structural similarities can best be explained with the assumption of a common evolutionary origin. Statistical analysis further supports a distant but significant homologous relationship. On the basis of previous evolutionary analyses that established a scenario that respiration evolved prior to photosynthesis, we consider it likely that cytochrome b is the evolutionary precursor for type II reaction center apoproteins. With a structural analysis confirming a common evolutionary origin of both type I and type II reaction centers, we further propose a novel "reaction center apoprotein early" hypothesis to account for the development of photosynthetic reaction center holoproteins.

Diffusive and metabolic limitations to photosynthesis under drought and salinity in C(3) plants.

Drought and salinity are two widespread environmental conditions leading to low water availability for plants. Low water availability is considered the main environmental factor limiting photosynthesis and, consequently, plant growth and yield worldwide. There has been a long-standing controversy as to whether drought and salt stresses mainly limit photosynthesis through diffusive resistances or by metabolic impairment. Reviewing in vitro and in vivo measurements, it is concluded that salt and drought stress predominantly affect diffusion of CO(2) in the leaves through a decrease of stomatal and mesophyll conductances, but not the biochemical capacity to assimilate CO(2), at mild to rather severe stress levels. The general failure of metabolism observed at more severe stress suggests the occurrence of secondary oxidative stresses, particularly under high-light conditions. Estimates of photosynthetic limitations based on the photosynthetic response to intercellular CO(2) may lead to artefactual conclusions, even if patchy stomatal closure and the relative increase of cuticular conductance are taken into account, as decreasing mesophyll conductance can cause the CO(2) concentration in chloroplasts of stressed leaves to be considerably lower than the intercellular CO(2) concentration. Measurements based on the photosynthetic response to chloroplast CO(2) often confirm that the photosynthetic capacity is preserved but photosynthesis is limited by diffusive resistances in drought and salt-stressed leaves.

Physiological responses of the CAM epiphyte Tillandsia usneoides L. (Bromeliaceae) to variations in light and water supply.

In an effort to understand the mechanisms that sustain rootless atmospheric plants, the modulation of Crassulacean acid metabolism (CAM) in response to variations in irradiance and water supply was investigated in the epiphyte Tillandsia usneoides. Plants were acclimated to three light regimes, i.e. high, intermediate and low, with integrated photon flux densities (PFD) of 14.40, 8.64 and 4.32 mol m-2 d-1 equivalent to an instantaneous PFD of 200, 100, and 50 mumol m-2 s-1, respectively. Daily watering was then withdrawn from half of the plants at each PFD for 7 d prior to sampling. In response to the three PFD treatments, chlorophyll content increased in plants acclimated to lower irradiances. Light response curves using non-invasive measurements of chlorophyll fluorescence demonstrated that photosystem II efficiency (phi PSII) was maintained in high PFD acclimated plants, as they exhibited a larger capacity for non-photochemical dissipation (NPQ) of excess light energy than low PFD acclimated plants. Net CO2 uptake increased in response to higher PFD, reflecting enhanced carboxylation capacity in terms of phosphoenolpyruvate carboxylase (PEPc) and ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) activities. After water was withdrawn, nocturnal net CO2 uptake and accumulated levels of acidity declined in all PFD treatments, concomitant with increased respiratory recycling of malate. Examining the strategies employed by epiphytes such as T. usneodies to tolerate extreme light and water regimes has demonstrated the importance of physiological mechanisms that allow flexible carboxylation capacity and continued carbon cycling to maintain photosynthetic integrity.

Evolution of C(4) phosphoenolpyruvate carboxylase in the genus Alternanthera: gene families and the enzymatic characteristics of the C(4) isozyme and its orthologues in C(3) and C(3)/C(4) Alternantheras.

Phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.3) is a key enzyme of C(4) photosynthesis. It has evolved from ancestral non-photosynthetic (C(3)) isoforms and thereby changed its kinetic and regulatory properties. We are interested in understanding the molecular changes, as the C(4) PEPCases were adapted to their new function in C(4) photosynthesis and have therefore analysed the PEPCase genes of various Alternanthera species. We isolated PEPCase cDNAs from the C(4) plant Alternanthera pungens H.B.K., the C(3)/C(4) intermediate plant A. tenella Colla, and the C(3) plant A. sessilis (L.) R.Br. and investigated the kinetic properties of the corresponding recombinant PEPCase proteins and their phylogenetic relationships. The three PEPCases are most likely derived from orthologous gene classes named ppcA. The affinity constant for the substrate phosphoenolpyruvate (K (0.5) PEP) and the degree of activation by glucose-6-phosphate classified the enzyme from A. pungens (C(4)) as a C(4) PEPCase isoform. In contrast, both the PEPCases from A. sessilis (C(3)) and A. tenella (C(3)/C(4)) were found to be typical C(3) PEPCase isozymes. The C(4) characteristics of the PEPCase of A. pungens were accompanied by the presence of the C(4)-invariant serine residue at position 775 reinforcing that a serine at this position is essential for being a C(4) PEPCase (Svensson et al. 2003). Genomic Southern blot experiments and sequence analysis of the 3' untranslated regions of these genes indicated the existence of PEPCase multigene family in all three plants which can be grouped into three classes named ppcA, ppcB and ppcC.

Night-time conductance in C3 and C4 species: do plants lose water at night?

Significant night-time stomatal conductance and transpiration were found for 11 out of 17 species with a range of life histories (herbaceous annual, perennial grass, shrub, tree), photosynthetic pathways (C(3), C(4)), and habitats in the western United States. Across species and habitats, higher night-time conductance and transpiration were associated with higher daytime values. The prevalence, mechanisms and ecological implications of substantial night-time water loss deserve further investigation.

Temperature response of mesophyll conductance. Implications for the determination of Rubisco enzyme kinetics and for limitations to photosynthesis in vivo.

CO(2) transfer conductance from the intercellular airspaces of the leaf into the chloroplast, defined as mesophyll conductance (g(m)), is finite. Therefore, it will limit photosynthesis when CO(2) is not saturating, as in C3 leaves in the present atmosphere. Little is known about the processes that determine the magnitude of g(m). The process dominating g(m) is uncertain, though carbonic anhydrase, aquaporins, and the diffusivity of CO(2) in water have all been suggested. The response of g(m) to temperature (10 degrees C-40 degrees C) in mature leaves of tobacco (Nicotiana tabacum L. cv W38) was determined using measurements of leaf carbon dioxide and water vapor exchange, coupled with modulated chlorophyll fluorescence. These measurements revealed a temperature coefficient (Q(10)) of approximately 2.2 for g(m), suggesting control by a protein-facilitated process because the Q(10) for diffusion of CO(2) in water is about 1.25. Further, g(m) values are maximal at 35 degrees C to 37.5 degrees C, again suggesting a protein-facilitated process, but with a lower energy of deactivation than Rubisco. Using the temperature response of g(m) to calculate CO(2) at Rubisco, the kinetic parameters of Rubisco were calculated in vivo from 10 degrees C to 40 degrees C. Using these parameters, we determined the limitation imposed on photosynthesis by g(m). Despite an exponential rise with temperature, g(m) does not keep pace with increased capacity for CO(2) uptake at the site of Rubisco. The fraction of the total limitations to CO(2) uptake within the leaf attributable to g(m) rose from 0.10 at 10 degrees C to 0.22 at 40 degrees C. This shows that transfer of CO(2) from the intercellular air space to Rubisco is a very substantial limitation on photosynthesis, especially at high temperature.

Genetic manipulation of glycine decarboxylation.

The glycine-serine interconversion, catalysed by glycine decarboxylase and serine hydroxymethyltransferase, is an important reaction of primary metabolism in all organisms including plants, by providing one-carbon units for many biosynthetic reactions. In plants, in addition, it is an integral part of the photorespiratory metabolic pathway and produces large amounts of photorespiratory CO(2) within mitochondria. Although controversial, there is significant evidence that this process, by the relocation of glycine decarboxylase within the leaves from the mesophyll to the bundle-sheath, contributed to the evolution of C(4) photosynthesis. In this review, some aspects of current knowledge about glycine decarboxylase and serine hydroxymethyltransferase and the role of these enzymes in metabolism, about the corresponding genes and their expression as well as about mutants and anti-sense plants related to these genes or processes will be summarized and discussed. From a comparison of the available information about the number and organization of GDC and SHMT genes in the genomes of Arabidopsis thaliana and Oryza sativa it appears that these and, possibly, other genes related to photorespiration, are similarly organized even in only very distantly related angiosperms.

Contribution of C3 carboxylation to the circadian rhythm of carbon dioxide uptake in a Crassulacean acid metabolism plant Kalanchoë daigremontiana.

During the endogenous circadian rhythm of carbon dioxide uptake in continuous light by a Crassula cean acid metabolism plant, Kalanchoë daigremontiana, the two carboxylating enzymes, phosphoenolpyruvate carboxylase (PEPC) and ribulose 1,5 bisphosphate carboxylase/oxygenase (Rubisco), are active simultaneously, although, until now, only the role of PEPC in generating the rhythm has been acknowledged. According to the established model, the rhythm is primarily regulated at the PEPC activity level, modulated by periodic compartmentation of its inhibitor, malate, in the vacuole and controlled by tension/relaxation of the tonoplast. However, the circadian accumulation of malic acid (the main indicator of PEPC activity) dampened significantly within the first few periods without affecting the rhythm's amplitude. Moreover, the amount of malate accumulated during a free-running oscillation was several-fold lower than the amount expected if PEPC were the key carboxylating enzyme, based on a 1:1 stoichiometry of CO(2) and malate. Together with the observation that rates of CO(2) uptake under continuous light were higher than in darkness, the evidence shows that C(3) carboxylation greatly contributes to the generation of rhythmic CO(2) uptake in continuous light in this 'obligate' CAM plant. Because the shift from predominantly CAM to predominantly C(3) carboxylation is smooth and does not distort the trajectory of the rhythm, its control probably arises from a robust network of oscillators, perhaps also involving stomata.

Characterization of a cDNA encoding a PSII-associated chlorophyll a/b-binding protein (CAB) from Chlamydomonas moewusii fitting into neither type I nor type II.

A full-length 1010-bp cDNA clone from Chlamydomonas moewusii coding for the precursor of a chlorophyll a/b-binding protein (CAB) was characterized. Northern analysis shows hybridization to a single 1150-base light-stimulated mRNA. Complementary hybrid-selected mRNAs were translated in vitro; SDS-PAGE indicates the synthesis of three polypeptides of 25, 27 and 28 kDa. Comparison of the deduced polypeptide sequence with other published CABs reveals greater similarity with PSII-associated proteins but, as with other algal CABs, our sequence does not meet established criteria for inclusion into either type I or type II, so branching of CABs into two types seems to have occurred after the divergence between algae and land plants.

Blue-light photoreceptors in higher plants.

In the past few years great progress has been made in identifying and characterizing plant photoreceptors active in the blue/UV-A regions of the spectrum. These photoreceptors include cryptochrome 1 and cryptochrome 2, which are similar in structure and chromophore composition to the prokaryotic DNA photolyases. However, they have a C-terminal extension that is not present in photolyases and lack photolyase activity. They are involved in regulation of cell elongation and in many other processes, including interfacing with circadian rhythms and activating gene transcription. Animal cryptochromes that play a photoreceptor role in circadian rhythms have also been characterized. Phototropin, the protein product of the NPH1 gene in Arabidopsis, likely serves as the photoreceptor for phototropism and appears to have no other role. A plasma membrane protein, it serves as photoreceptor, kinase, and substrate for light-activated phosphorylation. The carotenoid zeaxanthin may serve as the chromophore for a photoreceptor involved in blue-light-activated stomatal opening. The properties of these photoreceptors and some of the downstream events they are known to activate are discussed.

Evolution of c4 phosphoenolpyruvate carboxylase. Genes and proteins: a case study with the genus Flaveria.

C4 photosynthesis is characterized by a division of labour between two different photosynthetic cell types, mesophyll and bundle-sheath cells. Relying on phosphoenolpyruvate carboxylase (PEPC) as the primary carboxylase in the mesophyll cells a CO2 pump is established in C4 plants that concentrates CO2 at the site of ribulose 1,5-bisphosphate carboxylase/oxygenase in the bundle-sheath cells. The C4 photosynthetic pathway evolved polyphyletically implying that the genes encoding the C4 PEPC originated from non-photosynthetic PEPC progenitor genes that were already present in the C3 ancestral species. The dicot genus Flaveria (Asteraceae) is a unique system in which to investigate the molcular changes that had to occur in order to adapt a C3 ancestral PEPC gene to the special conditions of C4 photosynthesis. Flaveria contains not only C3 and C4 species but also a large number of C3-C4 intermediates which vary to the degree in which C4 photosynthetic traits are expressed. The C4 PEPC gene of Flaveria trinervia, which is encoded by the ppcA gene class, is highly expressed but only in mesophyll cells. The encoded PEPC protein possesses the typical kinetic and regulatory features of a C4-type PEPC. The orthologous ppcA gene of the C3 species Flaveria pringlei encodes a typical non-photosynthetic, C3-type PEPC and is weakly expressed with no apparent cell or organ specificity. PEPCs of the ppcA type have been detected also in C3-C4 intermediate Flaveria species. These orthologous PEPCs have been used to determine the molecular basis for C4 enzyme characteristics and to understand their evolution. Comparative and functional analyses of the ppcA promoters from F. trinervia and F. pringlei make it possible to identity the cis-regulatory sequences for mesophyll-specific gene expression and to search for the corresponding trans-regulatory factors.

The LOV domain family: photoresponsive signaling modules coupled to diverse output domains.

For single-cell and multicellular systems to survive, they must accurately sense and respond to their cellular and extracellular environment. Light is a nearly ubiquitous environmental factor, and many species have evolved the capability to respond to this extracellular stimulus. Numerous photoreceptors underlie the activation of light-sensitive signal transduction cascades controlling these responses. Here, we review the properties of the light, oxygen, or voltage (LOV) family of blue-light photoreceptor domains, a subset of the Per-ARNT-Sim (PAS) superfamily. These flavin-binding domains, first identified in the higher-plant phototropins, are now shown to be present in plants, fungi, and bacteria. Notably, LOV domains are coupled to a wide array of other domains, including kinases, phosphodiesterases, F-box domains, STAS domains, and zinc fingers, which suggests that the absorption of blue light by LOV domains regulates the activity of these structurally and functionally diverse domains. LOV domains contain a conserved molecular volume extending from the flavin cofactor, which is the locus for light-driven structural change, to the molecular surface. We discuss the role of this conserved volume of structure in LOV-regulated processes.

Dramatic difference in the responses of phosphoenolpyruvate carboxylase to temperature in leaves of C3 and C4 plants.

Temperature caused phenomenal modulation of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) in leaf discs of Amaranthus hypochondriacus (NAD-ME type C(4) species), compared to the pattern in Pisum sativum (a C(3) plant). The optimal incubation temperature for PEPC in A. hypochondriacus (C(4)) was 45 degrees C compared to 30 degrees C in P. sativum (C(3)). A. hypochondriacus (C(4)) lost nearly 70% of PEPC activity on exposure to a low temperature of 15 degrees C, compared to only about a 35% loss in the case of P. sativum (C(3)). Thus, the C(4) enzyme was less sensitive to supra-optimal temperature and more sensitive to sub-optimal temperature than that of the C(3) species. As the temperature was raised from 15 degrees C to 50 degrees C, there was a sharp decrease in malate sensitivity of PEPC. The extent of such a decrease in C(4) plants (45%) was more than that in C(3) species (30%). The maintenance of high enzyme activity at warm temperatures, together with a sharp decrease in the malate sensitivity of PEPC was also noticed in other C(4) plants. The temperature-induced changes in PEPC of both A. hypochondriacus (C(4)) and P. sativum (C(3)) were reversible to a large extent. There was no difference in the extent of phosphorylation of PEPC in leaves of A. hypochondriacus on exposure to varying temperatures, unlike the marked increase in the phosphorylation of enzyme on illumination of the leaves. These results demonstrate that (i) there are marked differences in the temperature sensitivity of PEPC in C(3) and C(4) plants, (ii) the temperature induced changes are reversible, and (iii) these changes are not related to the phosphorylation state of the enzyme. The inclusion of PEG-6000, during the assay, dampened the modulation by temperature of malate sensitivity of PEPC in A. hypochondriacus. It is suggested that the variation in temperature may cause significant conformational changes in C(4)-PEPC.

Origin and early evolution of photosynthesis.

Photosynthesis was well-established on the earth at least 3.5 thousand million years ago, and it is widely believed that these ancient organisms had similar metabolic capabilities to modern cyanobacteria. This requires that development of two photosystems and the oxygen evolution capability occurred very early in the earth's history, and that a presumed phase of evolution involving non-oxygen evolving photosynthetic organisms took place even earlier. The evolutionary relationships of the reaction center complexes found in all the classes of currently existing organisms have been analyzed using sequence analysis and biophysical measurements. The results indicate that all reaction centers fall into two basic groups, those with pheophytin and a pair of quinones as early acceptors, and those with iron sulfur clusters as early acceptors. No simple linear branching evolutionary scheme can account for the distribution patterns of reaction centers in existing photosynthetic organisms, and lateral transfer of genetic information is considered as a likely possibility. Possible scenarios for the development of primitive reaction centers into the heterodimeric protein structures found in existing reaction centers and for the development of organisms with two linked photosystems are presented.

The fucoxanthin-chlorophyll proteins from a chromophyte alga are part of a large multigene family: structural and evolutionary relationships to other light harvesting antennae.

A fucoxanthin-chlorophyll protein (FCP) cDNA from the raphidophyte Heterosigma carterae encodes a 210-amino acid polypeptide that has similarity to other FCPs and to the chlorophyll a/b-binding proteins (CABs) of terrestrial plants and green algae. The putative transit sequence has characteristics that resemble a signal sequence. The Heterosigma fcp genes are part of a large multigene family which includes members encoding at least two significantly different polypeptides (Fcp1, Fcp2). Comparison of the FCP sequences to the recently determined three-dimensional structure of the pea LHC II complex indicates that many of the key amino acids thought to participate in the binding of chlorophyll and the formation of complex-stabilizing ionic interactions are well conserved. Phylogenetic analyses of sequences of light-harvesting proteins shows that the FCPs of several chromophyte phyla form a natural group separate from the intrinisic peridinin-chlorophyll proteins (iPCPs) of the dinoflagellates: Although the FCP and CAB genes shared a common ancestor, these lineages diverged from each other prior to the separation of the CAB LHC I and LHC II sequences in the green algae and terrestrial plants.

Water-soluble chlorophyll protein in Brassicaceae plants is a stress-induced chlorophyll-binding protein.

Two kinds of water-soluble chlorophyll (Chl) proteins (WSCPs) have been found, e.g., a WSCP from Chenopodium, Atriplex, Polygonum, and Amaranthus species (class I) and that from Brassica, Raphanus, and Lepidium species (class II). Classes I and II WSCPs differ mainly in their photoconvertiblity. Class I WSCPs show a light-induced absorption change, whereas Class II WSCPs do not. The molecular and functional properties of Class I WSCP are largely uncertain. On the other hand, recent studies on the adaptation of plants to osmotic stress revealed the participation of drought-stress induced proteins with molecular masses of 20-22 kDa possessing a sequence similarity with class II WSCPs. This mini review focuses on the molecular signature of class II WSCPs. The physiological function of class II WSCPs has not been clarified either, but, their water-solubility, low Chl content, and stress-inducibility suggested little contribution to photosynthesis. Several molecular properties predicting its physiological role are as follows. The WSCP tetramer, may have only one or no Chl molecules in each subunit. All WSCPs possess a motif for Künitz-type proteinase inhibitor family in their sequence. WSCP is induced by drought- and heat-stresses suggesting its protective role during stress conditions. Monomeric recombinant apo-WSCP is able to remove Chls from the thylakoid membrane in aqueous solution and form into a tetramer. Brassica-WSCP contains a signal sequence targeted to endoplasmic reticulum. The highly conserved, C-terminal region is missing in the mature WSCP. Possible functions of class II WSCPs in plant tissues are discussed.

Photosynthetic enzyme accumulation during leaf development of Arundinella hirta, a C4 grass having Kranz cells not associated with veins.

The leaf of the NADP-malic enzyme type C(4) grass, Arundinella hirta, has not only mesophyll cells (MCs) and bundle sheath cells (BSCs, usual Kranz cells) but also another type of Kranz cells (distinctive cells; DCs) that are not associated with vascular bundles. We investigated photosynthetic enzyme accumulation along the base-to-tip maturation gradient of developing leaves by immunogold electron microscopy. In mature leaves, phosphoenolpyruvate carboxylase (PEPC) and ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) were detected in the MC cytosol and in the BSC and DC chloroplasts, respectively. Pyruvate, P(i) dikinase (PPDK) was present in the chloroplasts of all photosynthetic cells but with higher levels in the MCs. Rubisco was first detected in the basal region of emerging leaf blades where the BSCs and DCs became discernable. Subsequently, the accumulation of PEPC and PPDK was initiated in the region where the granal proliferation in the chloroplasts was conspicuous; and, suberized lamellae were formed in the cell walls of the Kranz cells. There was no difference in the patterns of cellular development and enzyme accumulation between the BSCs and DCs or between the MCs adjacent to each type of Kranz cells. These results demonstrate that, although the DCs are not associated with veins, they behaved like BSCs with respect to enzyme induction and cellular differentiation.