Photosynthetic pathway variation among C4 grasses along a precipitation gradient in Argentina

Aim Based on the biochemical and physiological attributes of C₄ grasses, and on the close association between decarboxylation pathways and the taxa in which they evolved, the hypotheses tested were: (1) that C₄ grasses would become progressively more abundant as precipitation decreased, with grasses of the NADP‐me subtype more abundant in wetter sites and those of the NAD‐me subtype more common in arid regions; and (2) that the distribution of grass subfamilies would also be correlated with annual precipitation. Location The study was conducted along a precipitation gradient in central Argentina, from the eastern Pampas (>1000 mm year^?1) to the western deserts and semi‐deserts near the Andes (<100 mm year^?1). Methods Percentage of species and relative cover of C₃ and C₄ grasses (including C₄ subtypes) in local floras from 15 lowland sites of central Argentina were obtained from our own unpublished data and from recently published floristic surveys. Pearson correlation coefficients were obtained between grass distribution parameters and the available climatic data. Results The percentage of C₄ grasses increased towards the arid extreme and showed a strong negative correlation with annual rainfall (r = ?0.74, P < 0.01). Within the C₄ subtypes, the NADP‐me species showed a higher proportional representation at the wetter extreme, whereas the representation of NAD‐me species increased towards the more arid extreme. The relationship of PEP‐ck species with climatic parameters in central Argentina was less evident. The distributions of the Panicoideae and Chloridoideae subfamilies along the precipitation gradient were diametrically opposed, with the Panicoideae positively (r = 0.86, P < 0.001) and the Chloridoideae negatively (r = ?0.87, P < 0.001) correlated with annual precipitation. Main conclusions Our data are consistent with the broad observation that C₄ grasses tend to dominate in areas where the wet season falls in the warmer summer months. In agreement with previously reported results for Africa, Asia, Australia and North America, we describe here for the first time a significant relationship between annual precipitation and the prevalence of the NADP‐me and NAD‐me photosynthetic pathways along climatic gradients for the Neotropics. We also report for the first time that correlations between C₄ species and annual rainfall are stronger when the relative cover of grass species is considered. The association of grass subfamilies Panicoideae and Chloridoideae with rainfall is as strong as that recorded for the NADP‐me and NAD‐me variants, respectively, suggesting that characteristics other than decarboxylation type may be responsible for the geographic patterns described in this study.     

Phylogenetic niche conservatism in C4 grasses

Photosynthetic pathway is used widely to discriminate plant functional types in studies of global change. However, independent evolutionary lineages of C₄ grasses with different variants of C₄ photosynthesis show different biogeographical relationships with mean annual precipitation, suggesting phylogenetic niche conservatism (PNC). To investigate how phylogeny and photosynthetic type differentiate C₄ grasses, we compiled a dataset of morphological and habitat information of 185 genera belonging to two monophyletic subfamilies, Chloridoideae and Panicoideae, which together account for 90 % of the world’s C₄ grass species. We evaluated evolutionary variance and covariance of morphological and habitat traits. Strong phylogenetic signals were found in both morphological and habitat traits, arising mainly from the divergence of the two subfamilies. Genera in Chloridoideae had significantly smaller culm heights, leaf widths, 1,000-seed weights and stomata; they also appeared more in dry, open or saline habitats than those of Panicoideae. Controlling for phylogenetic structure showed significant covariation among morphological traits, supporting the hypothesis of phylogenetically independent scaling effects. However, associations between morphological and habitat traits showed limited phylogenetic covariance. Subfamily was a better explanation than photosynthetic type for the variance in most morphological traits. Morphology, habitat water availability, shading, and productivity are therefore all involved in the PNC of C₄ grass lineages. This study emphasized the importance of phylogenetic history in the ecology and biogeography of C₄ grasses, suggesting that divergent lineages need to be considered to fully understand the impacts of global change on plant distributions.     

Environmental factors determining the phylogenetic structure of C4 grass communities

Aim To determine how the distribution of species richness is associated with environmental factors for the four major C₄ grass lineages in South Africa, as a means to explore the mechanisms responsible. Location South Africa, Lesotho and Swaziland. Methods The geographical distributions of species richness for four major C₄ grass lineages (Aristidoideae, Chloridoideae, Andropogoneae and Paniceae) were sourced from a recently published flora that divided the study region into different vegetation types. Mean values of potential environmental correlates were calculated for each vegetation type, and the relative importances of these were determined using single‐ and multiple‐predictor generalized linear models, with and without control for spatial autocorrelation. Model selection of the multiple‐predictor generalized linear models was conducted using an Akaike’s information criterion–information theoretic approach. Association with wet, intermediate or dry, shady or open, and disturbed or undisturbed habitats was also determined for each C₄ grass clade using habitat data for all the grass species, and analysed using chi‐square tests of independence. Results Andropogoneae and Paniceae are most species‐rich in areas of high precipitation and in mesic habitats. Andropogoneae are associated with high fire frequencies. Species richness in Andropogoneae decreases and in Paniceae increases in relation to livestock density. Chloridoideae species richness is relatively constant across South Africa, but is highest where there are infrequent fires, high temperatures and basic soils, and in mesic and disturbed habitats. Aristidoideae are most species‐rich in arid regions and in habitats with high temperatures, and are associated with disturbed habitats. Main conclusions Environmental variables other than precipitation, including temperature, fire frequency and grazing pressure, are strongly associated with the contrasting distributions of species richness for the various C₄ grass clades in South Africa. Our results suggest that ecological sorting is an important determinant of phylogenetic patterns in the species richness of these C₄ grass lineages.     

Relationship between leaf nitrogen and photosynthetic rate for three NAD-ME and three NADP-ME C4 grasses

Theoretical considerations have suggested that there may be differences in photosynthetic nitrogen use efficiency (PNUE) among plants that use different biochemical variants of C(4) photosynthesis. To test this hypothesis we examined the leaf nitrogen content and photosynthetic rates of six grass species (three of C(4) subtype NAD-ME and three of C(4) subtype NADP-ME) grown over a wide range of nitrogen supply. While there were significant differences among the species in various traits, there were no consistent differences between the C(4) subtypes in either leaf nitrogen content at a given level of nitrogen supply or in the leaf nitrogen-photosynthesis relationship. We suggest that species-level variation in photosynthetic nitrogen use efficiency among C(4) species is large enough to mask any differences that may be due to C(4) subtype.     

C4 plants in the vegetation of Mongolia: their natural occurrence and geographical distribution in relation to climate

The natural geographical occurrence, carbon assimilation, and structural and biochemical diversity of species with C₄ photosynthesis in the vegetation of Mongolia was studied. The Mongolian flora was screened for C₄ plants by using ¹³C/¹²C isotope fractionation, determining the early products of ¹⁴CO₂ fixation, microscopy of leaf mesophyll cell anatomy, and from reported literature data. Eighty C₄ species were found among eight families: Amaranthaceae, Chenopodiaceae, Euphorbiaceae, Molluginaceae, Poaceae, Polygonaceae, Portulacaceae and Zygophyllaceae. Most of the C4 species were in three families: Chenopodiceae (41 species), Poaceae (25 species) and Polygonaceae, genus Calligonum (6 species). Some new C₄ species in Chenopodiaceae, Poaceae and Polygonaceae were detected. C₄ Chenopodiaceae species make up 45% of the total chenopods and are very important ecologically in saline areas and in cold arid deserts. C₄ grasses make up about 10% of the total Poaceae species and these species naturally concentrate in steppe zones. Naturalized grasses with Kranz anatomy,of genera such as Setaria, Echinochloa, Eragrostis, Panicum and Chloris, were found in almost all the botanical-geographical regions of Mongolia, where they commonly occur in annually disturbed areas and desert oases. We analyzed the relationships between the occurrence of C₄ plants in 16 natural botanical-geographical regions of Mongolia and their major climatic influences. The proportion of C₄ species increases with decreasing geographical latitude and along the north-to-south temperature gradient; however grasses and chenopods differ in their responses to climate. The abundance of Chenopodiaceae species was closely correlated with aridity, but the distribution of the C₄ grasses was more dependent on temperature. Also, we found a unique distribution of different C₄ Chenopodiaceae structural and biochemical subtypes along the aridity gradient. NADP-malic enzyme (NADP-ME) tree-like species with a salsoloid type of Kranz anatomy, such as Haloxylon ammodendron and Iljinia regelii, plus shrubby Salsola and Anabasis species, were the plants most resistant to ecological stress and conditions in highly arid Gobian deserts with less than 100 mm of annual precipitation. Most of the annual C₄ chenopod species were halophytes, succulent, and occurred in saline and arid environments in steppe and desert regions. The relative abundance of C₃ succulent chenopod species also increased along the aridity gradient. Native C₄ grasses were mainly annual and perennial species from the Cynodonteae tribe with NAD-ME and PEP-carboxykinase (PEP-CK) photosynthetic types. They occurred across much of Mongolia, but were most common in steppe zones where they are often dominant in grazing ecosystems. Received: 17 March 1999 / Accepted: 1 November 1999     

Association of NADP- and NAD-linked malic enzyme acitivities in Zea mays: relation to C4 pathway photosynthesis.

Two of the three metabolic subtypes of species utilizing C₄-pathway photosynthesis are defined by high activities of either NADP malic enzyme (NADP malic enzyme type) or a coenzyme A (CoA)- and acetyl-CoA-activated NAD malic enzyme (NAD malic enzyme type). These enzymes function to decarboxylate malate as an integral part of the photosynthetic process. Leaves of NADP malic enzyme-type species also contain significant NAD-dependent malic enzyme activity. The purpose of the present study was to examine the nature and photosynthetic role of this activity. With Zea mays, this NAD-dependent activity was found to vary widely in fresh leaf extracts. Incubating extracts at 25 °C resulted in a disproportionate increase in NAD activity so that the final ratio of NADP to NAD activity was always about 5. Strong evidence was provided that the NADP and NAD malic enzyme activities in Z. mays extracts were catalyzed by the same enzyme. These activities remained associated during purification and were coincident after polyacrylamide gel electrophoresis. The pH optimum for NAD-dependent activity was about 7.1, compared with 8.3 for NADP malic enzyme activity. Other properties of the NAD-dependent activity are described, a particularly notable feature being the inhibition of this activity by less than 1 μm NADP and NADPH. Evidence is provided that the NADP malic enzyme of several other NADP malic enzyme-type C₄ species also has associated activity toward NAD. We concluded that the NAD-dependent malic enzyme activity would have no significant function in photosynthesis.     

Evolutionarily conserved and functionally important residues in the I-CeuI homing endonuclease.

Two approaches were used to discern critical amino acid residues for the function of the I- Ceu I homing endonuclease: sequence comparison of subfamilies of homologous proteins and genetic selection. The first approach revealed residues potentially involved in catalysis and DNA recognition. Because I- Ceu I is lethal in Escherichia coli , enzyme variants not perturbing cell viability were readily selected from an expression library. A collection of 49 variants with single amino acid substitutions at 37 positions was assembled. Most of these positions are clustered within or around the LAGLI-DADG dodecapeptide and the TQH sequence, two motifs found in all protein subfamilies examined. The Km and kcat values of the wild-type and nine variant enzymes synthesized in vitro were determined. Three variants, including one showing a substitution of the glutamine residue in the TQH motif, revealed no detectable endonuclease activity; five others showed reduced activity compared to the wild-type enzyme; whereas the remaining variant cleaved the top strand about three times more efficiently than the wild-type. Our results not only confirm recent reports indicating that amino acids in the LAGLI-DADG dodecapeptide are functionally critical, but they also suggest that some residues outside this motif directly participate in catalysis.     

The influence of climatic factors on the distribution of C4 species in Europe

A checklist of the currently-known C4 species which occur in Europe was compiled, and the number of these found in European territories was ascertained. Their contribution to the local floras range from 4.35% in the Azores to 0% in Svalbard. Subsequently, a stepwise multiple regression analysis was used to correlate the relative abundances of four subdivisions of C4 species with climatic variables derived for each territory from meteorological tables. The four subdivisions were: (a) total number of C4 species; (b) number of native C4 species; (c) C4 monocots and (d) C4 dicots. These values were expressed as percentages of either the total flora, (a), or the native flora ((b), (c) and (d)) for the regressions. The abundance of each C4 subdivision was found to be strongly correlated with temperature, and to a lesser extent, negatively with precipitation. Habitat information about the European C4 species was also analysed and it was concluded that their apparent preference for maritime and ruderal habitats indicates some form of competitive advantage of the pathway under saline or disturbed conditions. A small number of European C4 species was identified whose distribution appears to be anomalous with respect to temperature and/or soil moisture content.Nomenclature follows Tutin et al. Flora Europaea.     

Leaf vascular systems in C(3) and C(4) grasses: a two-dimensional analysis

BACKGROUND AND AIMS: It is well documented that C(4) grasses have a shorter distance between longitudinal veins in the leaves than C(3) grasses. In grass leaves, however, veins with different structures and functions are differentiated: large longitudinal veins, small longitudinal veins and transverse veins. Thus, the densities of the three types of vein in leaves of C(3) and C(4) grasses were investigated from a two-dimensional perspective. METHODS: Vein densities in cleared leaves of 15 C(3) and 26 C(4) grasses representing different taxonomic groups and photosynthetic subtypes were analysed. KEY RESULTS: The C(4) grasses had denser transverse veins and denser small longitudinal veins than the C(3) grasses (1.9 and 2.1 times in interveinal distance), but there was no significant difference in large longitudinal veins. The total length of the three vein types per unit area in the C(4) grasses was 2.1 times that in the C(3) grasses. The ratio of transverse vein length to total vein length was 14.3 % in C(3) grasses and 9.9 % in C(4) grasses. The C(3) grasses generally had greater species variation in the vascular distances than the C(4) grasses. The bambusoid and panicoid C(3) grasses tended to have a denser vascular system than the festucoid C(3) grasses. There were no significant differences in the interveinal distances of the three vein types between C(4) subtypes, although the NADP-malic enzyme grasses tended to have a shorter distance between small longitudinal veins than the NAD-malic enzyme and phosphoenolpyruvate carboxykinase grasses. CONCLUSIONS: It seems that C(4) grasses have structurally a superior photosynthate translocation and water distribution system by developing denser networks of small longitudinal and transverse veins, while keeping a constant density of large longitudinal veins. The bambusoid and panicoid C(3) grasses have a vascular system that is more similar to that in C(4) grasses than to that in the festucoid C(3) grasses.     

Km (CO2) values of ribulose-1,5-bisphosphate carboxylase in grasses of different C4 type

Statistical analysis of K_m (CO₂) values of ribulose-1,5-bisphosphate (RuBP) carboxylase from 35 C₄ grass species shows that the mean value for PEP-carboxykinase (PCK) type C₄ species (41.4±s.e. 2.2 μM CO₂) is significantly different from that of NAD-malic enzyme (NAD-ME) type species (55.3±3.1 μM CO₂) or NADP-malic enzyme (NADP-ME type species (52.5±s.e. 2.0μM CO₂). These C₄ type differences remain detectable within both the eu-panicoid and chloridoid grass subfamilies. By contrast, no between-subfamily differences were found within C₄ types. Variation in K_m (CO₂) values of RuBP carboxylase may be related to in vivo differences in CO₂ concentration at the enzyme site, mediated perhaps by differences in CO₂-leakiness of C₄ leaf ‘photosynthetic carbon reduction’ (PCR or ‘Kranz’) tissue.     

C4GEM, a genome-scale metabolic model to study C4 plant metabolism

Leaves of C(4) grasses (such as maize [Zea mays], sugarcane [Saccharum officinarum], and sorghum [Sorghum bicolor]) form a classical Kranz leaf anatomy. Unlike C(3) plants, where photosynthetic CO(2) fixation proceeds in the mesophyll (M), the fixation process in C(4) plants is distributed between two cell types, the M cell and the bundle sheath (BS) cell. Here, we develop a C(4) genome-scale model (C4GEM) for the investigation of flux distribution in M and BS cells during C(4) photosynthesis. C4GEM, to our knowledge, is the first large-scale metabolic model that encapsulates metabolic interactions between two different cell types. C4GEM is based on the Arabidopsis (Arabidopsis thaliana) model (AraGEM) but has been extended by adding reactions and transporters responsible to represent three different C(4) subtypes (NADP-ME [for malic enzyme], NAD-ME, and phosphoenolpyruvate carboxykinase). C4GEM has been validated for its ability to synthesize 47 biomass components and consists of 1,588 unique reactions, 1,755 metabolites, 83 interorganelle transporters, and 29 external transporters (including transport through plasmodesmata). Reactions in the common C(4) model have been associated with well-annotated C(4) species (NADP-ME subtypes): 3,557 genes in sorghum, 11,623 genes in maize, and 3,881 genes in sugarcane. The number of essential reactions not assigned to genes is 131, 135, and 156 in sorghum, maize, and sugarcane, respectively. Flux balance analysis was used to assess the metabolic activity in M and BS cells during C(4) photosynthesis. Our simulations were consistent with chloroplast proteomic studies, and C4GEM predicted the classical C(4) photosynthesis pathway and its major effect in organelle function in M and BS. The model also highlights differences in metabolic activities around photosystem I and photosystem II for three different C(4) subtypes. Effects of CO(2) leakage were also explored. C4GEM is a viable framework for in silico analysis of cell cooperation between M and BS cells during photosynthesis and can be used to explore C(4) plant metabolism.     

Comparative analysis of thylakoid protein complexes in the mesophyll and bundle sheath cells from C3, C4 and C3–C4 Paniceae grasses

To better understand the coordination between dark and light reactions during the transition from C₃ to C₄ photosynthesis, we optimized a method for separating thylakoids from mesophyll (MC) and bundle sheath cells (BSCs) across different plant species. We grew six Paniceae grasses including representatives from the C₃, C₃–C₄ and C₄ photosynthetic types and all three C₄ biochemical subtypes [nicotinamide adenine dinucleotide phosphate‐dependent malic enzyme (NADP‐ME), nicotinamide adenine dinucleotide‐dependent malic enzyme (NAD‐ME) and phosphoenolpyruvate carboxykinase (PEPCK)] in addition to Zea mays under control conditions (1000 μmol quanta m^?2 s^?1 and 400 ppm of CO₂). Proteomics analysis of thylakoids under native conditions, using blue native polyacrylamide gel electrophoresis followed by liquid chromatography‐mass spectrometry (LC‐MS), demonstrated the presence of subunits of all light‐reaction‐related complexes in all species and cell types. C₄ NADP‐ME species showed a higher photosystems I/II ratio and a clear accumulation of the NADH dehydrogenase‐like complexes in BSCs, while Cytb₆f was more abundant in BSCs of C₄ NAD‐ME species. The C₄ PEPCK species showed no clear differences between cell types. Our study presents, for the first time, a good separation between BSC and MC for a C₃–C₄ intermediate grass which did not show noticeable differences in the distribution of the thylakoid complexes. For the NADP‐ME species Panicum antidotale, growth at glacial CO₂ (180 ppm of CO₂) had no effect on the distribution of the light‐reaction complexes, while growth at low light (200 μmol quanta m^?2 s^?1) promoted the accumulation of light‐harvesting proteins in both cell types. These results add to our understanding of thylakoid distribution across photosynthetic types and subtypes, and introduce thylakoid distribution between the MC and BSC of a C₃–C₄ intermediate species.     

Malate decarboxylases: evolution and roles of NAD(P)-ME isoforms in species performing C(4) and C(3) photosynthesis

In the C(4) pathway of photosynthesis two types of malate decarboxylases release CO(2) in bundle sheath cells, NADP- and NAD-dependent malic enzyme (NADP-ME and NAD-ME), located in the chloroplasts and the mitochondria of these cells, respectively. The C(4) decarboxylases involved in C(4) photosynthesis did not evolve de novo; they were recruited from existing housekeeping isoforms. NADP-ME housekeeping isoforms would function in the control of malate levels during hypoxia, pathogen defence responses, and microspore separation, while NAD-ME participates in the respiration of malate in the tricarboxylic acid cycle. Recently, the existence of three enzymatic NAD-ME entities in Arabidopsis, occurring by alternative association of two subunits, was described as a novel mechanism to regulate NAD-ME activity under changing metabolic environments. The C(4) NADP-ME is thought to have evolved from a C(3) chloroplastic ancestor, which in turn would have evolved from an ancient cytosolic enzyme. In this way, the C(4) NADP-ME would have emerged through gene duplication, acquisition of a new promoter, and neo-functionalization. In contrast, there would exist a unique NAD-ME in C(4) plants, which would have been adapted to perform a dual function through changes in the kinetic and regulatory properties of the C(3) ancestors. In addition to this, for the evolution of C(4) NAD-ME, insertion of promoters or enhancers into the single-copy genes of the C(3) ancestors would have changed the expression without gene duplication.     

转高粱C4型NADP-ME基因水稻植株的光合生理特征

NADP-苹果酸酶(NADP-ME)是C4型植物C4光合途径的一个分离得到了编码高梁(Sorghum vuklgare L.)C4型NADP-ME的全长cDNA.该cDNA全长为2 139 bp,其开放可读框为1 911bp,共编码636个氨基酸和一个终止密码子(GenBank登录号为AY274836).利用农杆菌介导的转化系统将其转入水稻品种"农垦58".经Southern杂交、Northern杂交和酶活性检测表明,高粱C4型NADP-ME可以在水稻中有效表达,酶活性可被提高1～7倍.对转基因水稻进行光合生理检测表明,转NADP-ME基因水稻CO2交换特征没有明显改变,但是在中午强光条件下光抑制加剧.     

Development of molecular approaches to estimating germinal mutation rates. I. Detection of insertion/deletion/rearrangement variants in the human genome

DNA from 130 individuals was studied with up to 18 (primarily cDNA) probes for the frequency of variants in this initial experiment to determine the feasibility of this approach to screening for germinal gene mutations. This approach, a modification of the usual restriction enzyme mapping strategy, focuses on the detection of insertion/deletion/rearrangement (I/D/R) variants, because the DNA is digested with only two restriction enzymes before transfer to membranes and hybridization with an extensive series of unrelated probes. Some 4000 noncontiguous, independent DNA fragments ("loci"), functional loci, pseudogenes or anonymous fragments, (a total of approximately 77,400 kb) were screened. 19 different classes and 31 copies of presumably I/D/R variants were detected while 4 different classes and 24 individuals exhibiting base substitution variants were observed. 18 of the 19 I/D/R classes were rare variants, that is, each were observed at a frequency, within this population, of less than 0.01; 3 of the base substitution classes existed at polymorphic frequencies and only 1 was a rare variant. 10 of the I/D/R classes, occurring in a total of 18 individuals, were detected with probes which are not known to be associated with repetitive elements. This is a variant frequency for I/D/R variants without known repetitive elements of 0.15 classes and 0.23 copies for each 1000 kb screened; this would extrapolate to 1600 such variant sites in the genome of each individual. Within the context of a mutation screening program, the rare variants, either with or without repetitive elements, would have a higher probability of being de novo mutations than would polymorphic variants; this former group would be the focus of family studies to test for the heritability of the allele (fragment pattern). Sufficient DNA probes are available to screen a significant portion of the human genome for genetic variation and de novo mutations of this type.     

C4 urernic variant: An acquired C4 allotype

The major histocompatibility complex (MHC)-linked complotype region includes alleles for B, C2, and C4 loci. These loci are closely linked to each other and to HLA-DR on chromosome 6. The duplicated C4 loci,, C4A and B, are especially polymorphic. In seven patients with renal insufficiency, we observed a C4 variant with electrophoretic mobility between C4B2 and C4B3. Four of these patients were detected during a study of MHC markers in mernbranoproliferative glomerulonephritis. Complete complotype and HLA data from families of five of the seven patients demonstrated that the variant was not inherited. The pattern was revealed by immunofixation electrophoresis and also by C4-specific hemolytic overlay. In serial plasma specimens taken from one patient, the C4 variant appeared only after the patient became uremic. However, the variant could not be produced in normal plasma after incubation with C4-depleted uremic plasma. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions of immunoprecipitated C4 from these patients showed C4A and C4B chains of normal molecular mass; incompletely processed forms of C4 were not observed. We believe that this variant is probably acquired in the presence of uremia and may represent the C4B2.9 allele found by Wank and co-workers in many patients with glomerulonephritis. Family studies are mandatory to distinguish genetic variants from acquired alterations in the C4 phenotype.