Intermediate Photosynthesis Research Articles

Transposable elements contribute to the establishment of the glycine shuttle in Brassicaceae species.

C3 -C4 intermediate photosynthesis has evolved at least five times convergently in the Brassicaceae, despite this family lacking bona fide C4 species. The establishment of this carbon concentrating mechanism is known to require a complex suite of ultrastructural modifications, as well as changes in spatial expression patterns, which are both thought to be underpinned by a reconfiguration of existing gene-regulatory networks. However, to date, the mechanisms which underpin the reconfiguration of these gene networks are largely unknown. In this study, we used a pan-genomic association approach to identify genomic features that could confer differential gene expression towards the C3 -C4 intermediate state by analysing eight C3 species and seven C3 -C4 species from five independent origins in the Brassicaceae. We found a strong correlation between transposable element (TE) insertions in cis-regulatory regions and C3 -C4 intermediacy. Specifically, our study revealed 113 gene models in which the presence of a TE within a gene correlates with C3 -C4 intermediate photosynthesis. In this set, genes involved in the photorespiratory glycine shuttle are enriched, including the glycine decarboxylase P-protein whose expression domain undergoes a spatial shift during the transition to C3 -C4 photosynthesis. When further interrogating this gene, we discovered independent TE insertions in its upstream region which we conclude to be responsible for causing the spatial shift in GLDP1 gene expression. Our findings hint at a pivotal role of TEs in the evolution of C3 -C4 intermediacy, especially in mediating differential spatial gene expression.

A genomic panel for studying C3-C4 intermediate photosynthesis in the Brassiceae tribe.

Research on C4 and C3-C4 photosynthesis has attracted significant attention because the understanding of the genetic underpinnings of these traits will support the introduction of its characteristics into commercially relevant crop species. We used a panel of 19 taxa of 18 Brassiceae species with different photosynthesis characteristics (C3 and C3-C4) with the following objectives: (i) create draft genome assemblies and annotations, (ii) quantify orthology levels using synteny maps between all pairs of taxa, (iii) ⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠describe the phylogenetic relatedness across all the species, and (iv) track the evolution of C3-C4 intermediate photosynthesis in the Brassiceae tribe. Our results indicate that the draft de novo genome assemblies are of high quality and cover at least 90% of the gene space. Therewith we more than doubled the sampling depth of genomes of the Brassiceae tribe that comprises commercially important as well as biologically interesting species. The gene annotation generated high-quality gene models, and for most genes extensive upstream sequences are available for all taxa, yielding potential to explore variants in regulatory sequences. The genome-based phylogenetic tree of the Brassiceae contained two main clades and indicated that the C3-C4 intermediate photosynthesis has evolved five times independently. Furthermore, our study provides the first genomic support of the hypothesis that Diplotaxis muralis is a natural hybrid of D. tenuifolia and D. viminea. Altogether, the de novo genome assemblies and the annotations reported in this study are a valuable resource for research on the evolution of C3-C4 intermediate photosynthesis.

De novo transcriptome assemblies of C3 and C4 non-model grass species reveal key differences in leaf development

BackgroundC4 photosynthesis is a mechanism that plants have evolved to reduce the rate of photorespiration during the carbon fixation process. The C4 pathway allows plants to adapt to high temperatures and light while more efficiently using resources, such as water and nitrogen. Despite decades of studies, the evolution of the C4 pathway from a C3 ancestor remains a biological enigma. Interestingly, species with C3-C4 intermediates photosynthesis are usually found closely related to the C4 lineages. Indeed, current models indicate that the assembly of C4 photosynthesis was a gradual process that included the relocalization of photorespiratory enzymes, and the establishment of intermediate photosynthesis subtypes. More than a third of the C4 origins occurred within the grass family (Poaceae). In particular, the Otachyriinae subtribe (Paspaleae tribe) includes 35 American species from C3, C4, and intermediates taxa making it an interesting lineage to answer questions about the evolution of photosynthesis.ResultsTo explore the molecular mechanisms that underpin the evolution of C4 photosynthesis, the transcriptomic dynamics along four different leaf segments, that capture different stages of development, were compared among Otachyriinae non-model species. For this, leaf transcriptomes were sequenced, de novo assembled, and annotated. Gene expression patterns of key pathways along the leaf segments showed distinct differences between photosynthetic subtypes. In addition, genes associated with photorespiration and the C4 cycle were differentially expressed between C4 and C3 species, but their expression patterns were well preserved throughout leaf development.ConclusionsNew, high-confidence, protein-coding leaf transcriptomes were generated using high-throughput short-read sequencing. These transcriptomes expand what is currently known about gene expression in leaves of non-model grass species. We found conserved expression patterns of C4 cycle and photorespiratory genes among C3, intermediate, and C4 species, suggesting a prerequisite for the evolution of C4 photosynthesis. This dataset represents a valuable contribution to the existing genomic resources and provides new tools for future investigation of photosynthesis evolution.

Nuclear phylogeny and hypothesized allopolyploidization events in the Subtribe Otachyriinae (Paspaleae, Poaceae)

Otachyriinae Butzin, one of the three subtribes within the Paspaleae tribe of grasses, includes seven genera and approximately 35 species. Interestingly, this subtribe comprises species with C3, C3-C4 intermediate, and C4 photosynthesis. The circumscription of Otachyriinae has changed through time, and still varies among treatments in current use. The monophyly of this subtribe has been recovered in previous studies based on plastid markers. A phylogenetic study taking into account polyploidy and a low-copy nuclear gene (LCNG) is still lacking in Paspaleae. A phylogeny including data from a LCNG is paramount to uncover reticulated evolution within the group. The purpose of the present study was to reconstruct the evolutionary history of Otachyriinae, based on both chloroplast and LCNG DNA with a focus on polyploidization. Several incongruences between gene trees allowed us to explore relationships between diploid and polyploid taxa. Our study identified several promising topics for future studies: genetic allopolyploidy and autopolyploidy was here documented using the characteristic pattern of double-labelled gene trees. The molecular evidence indicates that at least 40% of species of Otachyriinae show phylogenetic signature of polyploidy (16 taxa appear double-labelled in the nuclear gene trees); furthermore, the results support an allopolyploid origin of at least nine taxa in the subtribe: Rugoloa polygonata, a species with unknown photosynthetic pathway, the proto-kranz species Steinchisma laxum and Rugoloa hylaeica, and the C3 species Hymenachne felliana, H. grumosa, H. hemitomon, H. donacifolia, H. pernambucense, and P. grande. Also, our results confirm that the C4 genus Anthaenantia is unambiguously monophyletic and show that Anthaenantia lanata is an autopolyploid. We recognized six genera within subtribe Otachyriinae: Anthaenantia, Hymenachne, Otachyrium, Plagiantha, Rugoloa, and Steinchisma. Finally, Panicum species with historically ambiguous placements, i.e. P. condensatum, P. grande, P. harleyi, P. leptachne, P. longum, and P. stagnatile were transferred to the genus Hymenachne.

Promotion of cyclic electron transport around photosystem I during the evolution of NADP–malic enzyme‐type C4 photosynthesis in the genus Flaveria

C4 plants display higher cyclic electron transport activity than C3 plants. This activity is suggested to be important for the production of ATPs required for C4 metabolism. To understand the process by which photosystem I (PSI) cyclic electron transport was promoted during C4 evolution, we conducted comparative analyses of the functionality of PSI cyclic electron transport among members of the genus Flaveria, which contains several C3, C3-C4 intermediate, C4-like and C4 species. The abundance of NDH-H, a subunit of NADH dehydrogenase-like complex, increased markedly in bundle sheath cells with the activity of the C4 cycle. By contrast, PROTON GRADIENT REGULATION5 (PGR5) and PGR5-LIKE1 increased in both mesophyll and bundle sheath cells in C4-like Flaveria palmeri and C4 species. Grana stacks were drastically reduced in bundle sheath chloroplasts of C4-like F. palmeri and C4 species; these species showed a marked increase in PSI cyclic electron transport activity. These results suggest that both the expression of proteins involved in PSI cyclic electron transport and changes in thylakoid structure contribute to the high activity of cyclic electron flow in NADP-malic enzyme-type C4 photosynthesis. We propose that these changes were important for the establishment of C4 photosynthesis from C3-C4 intermediate photosynthesis in Flaveria.

Production of Raphanus sativus (C3)-Moricandia arvensis (C3-C4 intermediate) Monosomic and Disomic Addition Lines with Each Parentl Cytoplasmic Background and their Photorespiratory Characteristics

We are maintaining five Moricandia arvensis monosomic addition lines of Raphanus sativus carrying R. sativus cytoplasm (autoplasmic MALs) and twelve M. arvensis MALs of R. sativus carrying M. arvensis cytoplasm (alloplasmic MALs) from BC6 to BC8 generation, and newly produced five M. arvensis disomic addition lines of R. sativus (autoplasmic DALs) and seven M. arvensis DALs of R. sativus carrying M. arvensis cytoplasm (alloplasmic DALs) from selfing and sib-crossing of the MALs and DALs in S3BC5 and S2BC6 generations. The structural, biochemical and physiological characteristics related to photorespiration of these MALs and DALs were compared to study the genetic mechanisms of the C3-C4 intermediate photosynthesis in the individual chromosomes of M. arvensis. The CO2 compensation point of the autoplasmic and alloplasmic DALs (RMa-b and MaR-b DALs) with one pair of M. arvensis ‘b’ chromosome were 29.4 and 30.1 μmol mol-1, respectively, which were significantly lower than that of other DALs and MALs as well as R. sativus (34.5 𰂼mol mol-1). An immunogold electron microscopic study of the P-protein of glycine decarboxylase (GDC) in photosynthetic cells of the RMa-b DAL revealed that the bundle sheath cell (BSC) mitochondria were more intensively labeled for the protein than the mesophyll cell (MC) mitochondria. The ratio of the labeling density of the BSC mitochondria to that of the MC mitochondria was 1.13, which lies between values in M. arvensis (2.66) and R. sativus (0.76). These data suggest that the ‘b’ chromosome of M. arvensis genome controls the expression of C3-C4 intermediate characteristics.

C3 and C4 pathways of photosynthetic carbon assimilation in marine diatoms are under genetic, not environmental, control.

Marine diatoms are responsible for up to 20% of global CO(2) fixation. Their photosynthetic efficiency is enhanced by concentrating CO(2) around Rubisco, diminishing photorespiration, but the mechanism is yet to be resolved. Diatoms have been regarded as C(3) photosynthesizers, but recent metabolic labeling and genome sequencing data suggest that they perform C(4) photosynthesis. We studied the pathways of photosynthetic carbon assimilation in two diatoms by short-term metabolic (14)C labeling. In Thalassiosira weissflogii, both C3 (glycerate-P and triose-P) and C4 (mainly malate) compounds were major initial (2-5 s) products, whereas Thalassiosira pseudonana produced mainly C3 and C6 (hexose-P) compounds. The data provide evidence of C(3)-C(4) intermediate photosynthesis in T. weissflogii, but exclusively C(3) photosynthesis in T. pseudonana. The labeling patterns were the same for cells grown at near-ambient (380 microL L(-1)) and low (100 microL L(-1)) CO(2) concentrations. The lack of environmental modulation of carbon assimilatory pathways was supported in T. pseudonana by measurements of gene transcript and protein abundances of C(4)-metabolic enzymes (phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase) and Rubisco. This study suggests that the photosynthetic pathways of diatoms are diverse, and may involve combined CO(2)-concentrating mechanisms. Furthermore, it emphasizes the requirement for metabolic and functional genetic and enzymic analyses before accepting the presence of C(4)-metabolic enzymes as evidence for C(4) photosynthesis.

Inheritance of C3―C4 Intermediate Photosynthesis in Reciprocal Hybrids Between Moricandia Arvensis (C3―C4) and Brassica Oleracea (C3) That Differ in Their Genome Constitution

To Elucidate The Genetic Mechanisms Underlying C3―C4 intermediate Photosynthesis, We investigated The Structural and Photosynthetic Characteristics of Leaves of Reciprocal Hybrids Between The C3―C4 intermediate Species Moricandia Arvensis (L.) Dc. (Mama) and The C3 Species Brassica Oleracea L. (Cabbage; Cc), Which Differ in Genome Constitution. Moricandia Arvensis Bundle Sheath (Bs) Cells included Many Centripetally Located Chloroplasts and Mitochondria, Whereas Those of Cabbage Had Few Organelles. Hybrid Leaves Were Structurally intermediate Between Those of The Parents and Showed Stronger intermediate C3―C4 Features As The Proportion of The Ma Genome increased. The P-Protein of Glycine Decarboxylase (Gdc) Was Confined Mainly To Bs Mitochondria in M. Arvensis, But Accumulated More in The Mesophyll (M) of Cabbage. in The Hybrids, The Accumulation of Gdc in Bs Cells increased With An increasing Ma:C Ratio. Hybrids Exhibited Gradients in Structural and Biochemical Features, Even in Reciprocal Crosses. The Co2 Compensation Point of Reciprocal Hybrids With High Ma:C Ratios Was Lower Than That of Cabbage But Higher Than That of M. Arvensis. Thus, The Structural and Biochemical Features in Hybrid Leaves Reduced Photorespiration. Moricandia Arvensis Had A Higher Photosynthetic Rate Than Cabbage, But The Photosynthetic Rates of Hybrids Were intermediate Between Those of The Parents Or Comparable To That of M. Arvensis. Our Results Demonstrate That The C3―C4 intermediate Characteristics Are inherited Based On The Ratio of The Parent Genomes, and That There Is No Evidence of Cytoplasmic inheritance in These Characteristics.

Leaf anatomy, gas exchange and photosynthetic enzyme activity in Flaveria kochiana.

Flaveria (Asteraceae) is one of the few genera known to contain both C3 and C4 species, in addition to numerous biochemically-intermediate species. C3-C4 and C4-like intermediate photosynthesis have arisen more than once in different phylogenetic clades of Flaveria. Here, we characterise for the first time the photosynthetic pathway of the recently described species Flaveria kochiana B.L. Turner. We examined leaf anatomy, activity and localisation of key photosynthetic enzymes, and gas exchange characteristics and compared these trait values with those from related C4 and C4-like Flaveria species. F. kochiana has Kranz anatomy that is typical of other C4 Flaveria species. As in the other C4 lineages within the Flaveria genus, the primary decarboxylating enzyme is NADP-malic enzyme. Immunolocalisation of the major C4 cycle enzymes, PEP carboxylase and pyruvate, orthophosphate dikinase, were restricted to the mesophyll, while Rubisco was largely localised to the bundle sheath. Gas exchange analysis demonstrated that F. kochiana operates a fully functional C4 pathway with little sensitivity to ambient oxygen levels. The CO2 compensation point (2.2 µbar) was typical for C4 species, and the O2-response of the CO2 compensation point was the same as the C4 species F. trinervia. Notably, F. vaginata (B.L. Robinson & Greenman), a putative C4-like species that is the nearest relative of F. kochiana, had an identical response of the CO2 compensation point to O2. Furthermore, F. vaginata, exhibited a carbon isotope ratio (-15.4‰) similar to C4 species including F. australasica Hooker, F. trinervia Spreng. C. Mohr and the newly characterised F. kochiana. F. vaginata could be considered a C4 species, but additional studies are necessary to confirm this hypothesis. In addition, our results show that F. kochiana uses an efficient C4 cycle, with the highest initial slope of the A/Ci curve of any C4 Flaveria species.

A dominant gdcP‐specific marker derived from Moricandia nitens used for introducing the C3‐C4 character from M. nitens into Brassica crops

AbstractDifferential localization of decarboxylation and carboxylation in association with Kranz anatomy distinguish species with C4 or C3‐C4 intermediate photosynthesis from species with C3 photosynthesis. Moricandia nitens has a C3‐C4 intermediate photosynthetic mechanism and yet is closely related to C3Brassica species. In order to introduce the C3‐C4 character from M. nitens into Brassica crops, sesquitetraploids (MACC) were synthesized by crossing Brassica napus (AACC) and a somatic hybrid (M. nitens+B. oleracea, MMCC). Homologous fragments (2966 and 3254 bp) of P‐protein gene of glycine decarboxylase (gdcP) were isolated from M. nitens and B. napus, respectively, to develop a PCR‐based marker for assisted selection. Reverse transcriptase‐polymerase chain reaction (RT‐PCR) of the partial encoding regions of both gdcPs indicated that the mRNAs were expressed in the expanded leaf. The characterization of high divergence, but with large conserved island in the regulatory region of the two C3 species, implies that they may play an important role in maintaining the C3 photosynthetic pathway. Thereafter, a dominant gene‐specific marker was developed within the highly divergent region for subsequent introduction of C3‐C4 photosynthesis from Moricandia nitens into Brassica crops.

Environmental regulation of photosynthetic metabolism in the amphibious sedge Eleocharis baldwinii and comparisons with related species

ABSTRACTThe amphibious leafless sedge, Eleocharis baldwinii, expresses C4 characteristics in the terrestrial form and intermediate characteristics between C3 and C4 photosynthesis in the submerged form. This study examined the immunocytochemical localization of C3 and C4 enzymes in culms of the two forms to elucidate the regulatory mechanism of photosynthetic metabolism and compared the activities and amounts of C3 and C4 enzymes with those in other Eleocharis species, E. vivipara and E. retroflexa, which show C4 characteristics on land but C3 and C4 characteristics under water. The terrestrial form of E. baldwinii exhibited a C4‐like pattern of enzyme localization. The submerged form exhibited a modified anatomy with well‐developed mesophyll cells and small Kranz cells. The C4 enzyme levels declined conspicuously in outer mesophyll cells adjacent to the epidermis, whereas Rubisco levels increased throughout the mesophyll in the submerged form. These results suggest that intermediate photosynthesis between C3 and C4 photosynthesis in the submerged form results from the predominant operation of the C3 pathway in the outer mesophyll cells and the C4 pathway in both the inner mesophyll and Kranz cells. Differences in the degree of C4 expression in terrestrial forms of Eleocharis species may cause the differences in the expression of photosynthetic modes under water.

Development of a strategy for transgenic studies and monitoring of transgene expression in two closely related Moricandia species possessing a C3 or C3–C4 intermediate photosynthetic phenotype

In order to establish a model system for comparative studies of C3 and C3–C4 intermediate photosynthesis, the development of efficient transformation systems and the monitoring of transgene behaviour and stability were carried out in two closely related Moricandia species (Brassicaceae): the C3–C4 photosynthetic intermediate species M. arvensis and the C3 species M. moricandioides. In this study the green fluorescent protein (gfp) reporter gene was used as a vital marker gene while the use of the β‐glucuronidase (gusA) gene was based on the highly sensitive detection of its activity. For Agrobacterium‐mediated transformation of leaf explants, a cauliflower mosaic virus 35S promoter‐driven, modified version of gfp, the mgfp5‐ER gene and the gusA gene, respectively, were introduced into the new dual binary transformation vector system pGreen/pSoup (Hellens et al. 2000, Plant Mol Bio 42: 819–832). GFP5 produced bright‐green fluorescence in transformed tissues that was distinctly detected 5–12 days following transformation in developing calli of the two species. Visual screening, combined with antibiotic selection, enabled early and easy identification of transformation events and contributed to improvements in the transformation strategies. Transgene integration studies demonstrated that mgfp5‐ER was inserted with low copy number in the M. arvensis plant lines and the transgene was transmitted in a Mendelian fashion to T1 and T2 progenies. GFP5 expression levels in a population of 100 independent primary transformed M. arvensis plant lines (T0) showed great variation between transformation events (coefficient of variation of 108%). The mgfp5‐ER or gusA reporter genes were expressed in 90–95% of the kanamycin‐resistant M. arvensis plant lines and in up to 98% of the independent M. moricandioides plant lines.

Structural and biochemical dissection of photorespiration in hybrids differing in genome constitution between Diplotaxis tenuifolia (C3-C4) and radish (C3).

We compared the structural, biochemical, and physiological characteristics involved in photorespiration of intergeneric hybrids differing in genome constitution (DtDtR, DtDtRR, and DtRR) between the C(3)-C(4) intermediate species Diplotaxis tenuifolia (DtDt) and the C(3) species radish (Raphanus sativus; RR). The bundle sheath (BS) cells in D. tenuifolia included many centripetally located chloroplasts and mitochondria, but those of radish had only a few chloroplasts and mitochondria. In the hybrids, the numbers of chloroplasts and mitochondria, the ratio of centripetally located organelles to total organelles, and the mitochondrial size in the BS cells increased with an increase in the constitution ratio of the Dt:R genome. The P-protein of glycine decarboxylase (GDC) was confined to the BS mitochondria in D. tenuifolia, whereas in radish, it accumulated more densely in the mesophyll than in the BS mitochondria. In the hybrids, more intense accumulation of GDC in the BS relative to the mesophyll mitochondria occurred with an increase in the Dt:R ratio. These structural and biochemical features in the hybrids were reflected in the gas exchange characteristics of leaves, such as the CO(2) compensation point. Our data indicate that the leaf structure, the intercellular pattern of GDC expression, and the gas exchange characteristics of C(3)-C(4) intermediate photosynthesis are inherited in the hybrids depending on the constitution ratio of the parent genomes. Our findings also demonstrate that the apparent reduced photorespiration in C(3)-C(4) intermediate plants is mainly due to the structural differentiation of mitochondria and chloroplasts in the BS cells combined with the BS-dominant expression of GDC.

Photosynthesis in Salsola Species (Chenopodiaceae) from Southern Africa Relative to their C4 Syndrome Origin and their African‐Asian Arid Zone Migration Pathways

Abstract: Over 60 Salsola species of Chenopodiaceae from South Africa were studied for their photosynthesis type, using δ13C analysis and light microscopy of leaf anatomy. These species cover about 70 % of the total list of Southern African Salsola species and grow naturally in South and Southwest African desert regions. All species are shrubby forms and belong to the single subsection Caroxylon. Only C4 photosynthesis was found in the Salsola species determined with 13C/12C carbon isotope discrimination values that ranged from ‐ 11.04 to ‐ 14.03 % (PDB), plus the presence of a Kranz type assimilation tissue anatomy. The apparent absence of C3 in Salsola in South and Southwest Africa and the known presence of C3 and C3 ‐ C4 intermediate photosynthesis in Caroxylon, Salsola species in Asia strongly indicate that the genus Salsola originated in Asia and later migrated to South Africa.

Origin and evolution of C 4 photosynthesis in the tribe Salsoleae (Chenopodiaceae) based on anatomical and biochemical types in leaves and cotyledons

The Chenopodiaceae genus Salsola contains a large number of species with C4 photosynthesis. Along with derivative genera they have a prominent position among the desert vegetation of Asia and Africa. About 130 species from Asia and Africa were investigated to determine the occurrence of C3 versus C4 syndrome in leaves and cotyledons, and to study specific anatomical and biochemical features of photosynthesis in both photosynthetic organs. The species studied belong to all six previously identified sections of the tribe Salsoleae based on morphological characters. Types of photosynthesis were identified using carbon 13C/12C isotope fractionation. The representatives of all systematic groups were investigated for mesophyll anatomy and biochemical subtypes by determination of enzyme activity (RUBPC, PEPC, NAD- and NADP-ME and AAT) and primary photosynthetic products. Two photosynthetic types (C3 and C4) and two biochemical subtypes (NAD- and NADP-ME) were identified in both leaves and cotyledons. Both Kranz and non-Kranz type anatomy were found in leaves and cotyledons, but cotyledons had more diversity in anatomical structure. Strong relationships between anatomical types and biochemical subtypes in leaves and cotyledons were shown. We found convincing evidence for a similar pattern of structural and biochemical features of photosynthesis in leaves and cotyledons within systematic groups, and evaluated their relevance at the evolutionary level. We identified six groups in tribe Salsoleae with respect to photosynthetic types and mesophyll structure in leaves and cotyledons. Two separate lineages of biochemical and anatomical evolution within Salsoleae were demonstrated based on studies of leaves and cotyledons. The sections Caroxylon, Malpighipila, Cardiandra and Belanthera have no C3 species and only the NAD-ME C4 subtype has been found in leaves. We suggest the C4 species in the NADP-ME lineage evolved in Coccosalsola and Salsola sections, and originated in the subsection Arbuscula. Coccosalsola contains many species with C3 and/or C3-C4 intermediate photosynthesis. Within these main evolutionary lineages, species of different taxonomic groups (sections and subsections) had differences in anatomical or/and biochemical features in leaves and cotyledons. We conclude that structural and biochemical changes in the photosynthetic apparatus in species of the tribe Salsoleae were a key factor in their evolution and broad distribution in extreme desert environments.

Molecular phylogeny of Flaveria as deduced from the analysis of nucleotide sequences encoding the H‐protein of the glycine cleavage system

ABSTRACTSequence comparisons have shown that nucleotide sequences of the H‐protein, a component of the glycine cleavage system, are only moderately conserved and can be used as molecular markers for intrageneric phylogenetic studies. We have analysed the respective cDNA sequences from 12 species of Flaveria, and a more limited set of gdcsH upstream regions. These data are discussed with respect to a phylogenetic reconstruction of Flaveria, a small genus which includes species of different photo‐synthetic types, namely C3, C3‐C4, C4‐like and C4. Our analysis essentially supports an earlier hypothesis, based on morphological and eco‐geographical data, of the evolution of Flaveria (Powell 1978). This close agreement shows the usefulness of H‐protein nucleotide sequences at a low taxonomic level. Our analysis independently confirms that C4 photosynthesis has evolved two times in different lineages of Flaveria. Most remarkably, the C4 taxa of Flaveria appear as derived relative to the C3‐C4 intermediate taxa, i.e. they probably have common direct predecessors. This is the first direct evidence for a phylo‐genetically intermediate position of C3‐C4 intermediate photosynthesis. Our data also confirm the antiquity of C3 photosynthesis in Flaveria but suggest that the collection of F.pringlei used in our experiments, although clearly of C3 photosynthetic metabolism, possibly originated from hybridization with a more advanced taxon.

Novel characteristics of cassava, Manihot esculenta Crantz, a reputed C3-C4 intermediate photosynthesis species

The cassava plant, Manihot esculenta, grows exceptionally well in low fertility and drought prone environments, but the mechanisms that allow this growth are unknown. Earlier, and sometimes contradictory, work speculated about the presence of a C4-type photosynthesis in cassava leaves. In the present work we found no evidence for a C4 metabolism in mature attached cassava leaves as indicated i) by the low, 2 to 8%, incorporation of (14)CO2 into C4 organic acids in short time periods, 10 s, and the lack of (14)C transfer from C4 acids to other compounds in (12)CO2, ii) by the lack of C4 enzyme activity changes during leaf development and the inability to detect C4 acid decarboxylases, and iii) by leaf CO2 compensation values between 49 and 65 μl of CO2 1(-1) and by other infrared gas exchange photosynthetic measurements. It is concluded that the leaf biochemistry of cassava follows the C3 pathway of photosynthesis with no indication of a C3-C4 mechanism.However, cassava leaves exhibit several novel characteristics. Attached leaves have the ability to effectively partition carbon into sucrose with nearly 45% of the label in sucrose in about one min of (14)CO2 photosynthesis, contrasting with 34% in soybean (C3) and 25% in pigweed (C4). Cassava leaves displayed a strong preference for the synthesis of sucrose versus starch. Field grown cassava leaves exhibited high rates of photosynthesis and curvilinear responses to increasing sunlight irradiances with a tendency to saturate only at high irradiances, above 1500 μmol m(-2) s(-1). Morphologically, the cassava leaf has papillose epidermal cells on its lower mesophyll surface that form 'fence-like' arrangements encircling guard cells. It is proposed that the active synthesis of sugars has osmotic functions in the cassava plant and that the papillose epidermal cells function to maintain a healthy leaf water status in various environments.

Plant regeneration from mesophyll protoplasts of Moricandia arvensis

Moricandia arvensis is of interest as it is a dicotyledonous species which has C3 - C4 intermediate photosynthesis, a mechanism which results in enhanced recapture of photorespired CO2. Leaves from cultured shoot tips were used as a source for mesophyll cell protoplasts. Approximately 1% of the protoplasts which survived the first few days of culture produced calli. On a suitable regeneration medium, 30-60% of the calli regenerated one or more shoots. From among the regenerating shoots eight were selected, transferred to soil and grown to flowering in the glasshouse; all were fertile. The development of a protoplast regeneration system provides the opportunity to use transformation and somaclonal variation as tools in the genetic analysis of the C3-C4 character in this species.

C3–C4 intermediate photosynthesis: linking physiology to gene expression

C₃–C₄ intermediate photosynthesis: linking physiology to gene expression

PHOTOSYNTHETIC CHARACTERISTICS OF C3‐C4 INTERMEDIATE FLAVERIA FLORIDANA (ASTERACEAE) IN NATURAL HABITATS: EVIDENCE OF ADVANTAGES TO C3‐C4 PHOTOSYNTHESIS AT HIGH LEAF TEMPERATURES

Measurements of leaf gas exchange were conducted in situ for the C3‐C4 intermediate plant Flaveria floridana. Leaves exhibited measurable CO2 assimilation at atmospheric CO2 concentrations as low as 20 μmol/mol. This result demonstrates that the low CO2 compensation points observed in past studies of greenhouse‐grown C3‐C4 intermediate plants also exist in plants growing in their natural habitat. Photosynthesis rates in F. floridana were near their maximum at intercellular CO2 concentrations as low as 112 μmol/mol. The existence of near‐maximum photosynthesis rates at such low intercellular CO2 concentrations is interpreted as evidence for the existence of a CO2‐concentrating mechanism in F. floridana. Such a mechanism would also explain the observed lack of response in photosynthesis rates to reductions in stomatal conductance and intercellular CO2 concentration as the leaf‐to‐air water vapor concentration gradient is increased. Photosynthetic rates were relatively high at leaf temperatures between 35 and 40 C, compared to most C3 plants. At midday during May, when leaf temperatures were between 35 and 42 C, F. floridana leaves exhibited photosynthesis rates that were four times higher than a sympatric C3 species (Eustoma exaltatum) of similar growth form and ecological habit. The high photosynthesis rates at high leaf temperatures in F. floridana were not due to higher leaf nitrogen contents, but rather to its reduced rate of photorespiration. These results confirm that C3‐C4 intermediate photosynthesis can provide plants with an advantage at high leaf temperatures, compared to C3 plants.