Genetics of complement C4. Two homoduplication haplotypes C4S C4S and C4F C4F in a family

Summary A family in which two homoduplicated C4 haplotypes (or supergenes) segregate is described. One haplotype C4F ^* 3 C4F ^*2.2 is composed of two C4F alleles and the other C4S ^* 5.1 C4S ^*1 of two C4S alleles. The C4F duplication haplotype is a partial inhibitor of the Rodgers antigen, and judged from our family and population material, it seems to be rather frequent and associated with HLAB ^*35, Bf ^* F, and HLAD/DR ^*1. The C4S duplication haplotype is Rg(a-) and is not identified in individuals without another S, Ch(a+) variant.This work was supported by grant No 12-1727 from the Danish Medical Research Council     

Human C4 haplotypes with duplicated C4A or C4B

In the course of study of families for the sixth chromosome markers HLA-A, C, B, D/DR, BF, and C2, the two loci for C4, C4A, and C4B, and glyoxalase I, we encountered five examples of probable duplication of one or the other of the two loci for C4. In one of these, both parents and one sib expressed two different structural genes for C4B, one sib expressed one, and one sib expressed none, suggesting that two C4B alleles were carried on a single haplotype: HLA-A2, B7, DR3, BFS1, C2C, C4A2, C4B1, C4B2, GLO1. In a second case, two siblings inherited C4B*1 and C4B*2 from one parent and C4B*Q0 from the other. This duplication appeared on the chromosome as HLA-AW33, B14, DR1, BFS, C2C, C4A2, C4B1, C4B2, GLO2. In a third, very large family with 3 generations, a duplication of the C4B locus occurred which was followed in 2 generations. In one individual, there were three C4B alleles and two C4A alleles. One of the C4B alleles had a hemolytically active product with electrophoretic mobility near C4B2 and was designated C4B*22. It segregated with C4B1 in the family studied. The complete haplotype was HLA-A11, CW1, BW56, DR5, BFS, C2C, C4A3, C4B22, C4B1, GLO2. In another family with 12 siblings, one parent and eight children expressed two C4A alleles on the haplotype HLA-AW30, BW38, DR1, BFF, C2C, C4A3, C4A2, C4BQ0, GLO1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Co-function of C3-and C4-photosynthetic pathways in C3, C4 and C3-C4 intermediate Flaveria species

The potential for C₄ photosynthesis was investigated in five C₃-C₄ intermediate species, one C₃ species, and one C₄ species in the genus Flaveria, using ¹⁴CO₂ pulse-¹²CO₂ chase techniques and quantum-yield measurements. All five intermediate species were capable of incorporating ¹⁴CO₂ into the C₄ acids malate and aspartate, following an 8-s pulse. The proportion of ¹⁴C label in these C₄ products ranged from 50–55% to 20–26% in the C₃-C₄ intermediates F. floridana Johnston and F. linearis Lag. respectively. All of the intermediate species incorporated as much, or more, ¹⁴CO₂ into aspartate as into malate. Generally, about 5–15% of the initial label in these species appeared as other organic acids. There was variation in the capacity for C₄ photosynthesis among the intermediate species based on the apparent rate of conversion of ¹⁴C label from the C₄ cycle to the C₃ cycle. In intermediate species such as F. pubescens Rydb., F. ramosissima Klatt., and F. floridana we observed a substantial decrease in label of C₄-cycle products and an increase in percentage label in C₃-cycle products during chase periods with ¹²CO₂, although the rate of change was slower than in the C₄ species, F. palmeri. In these C₃-C₄ intermediates both sucrose and fumarate were predominant products after a 20-min chase period. In the C₃-C₄ intermediates, F. anomala Robinson and f. linearis we observed no significant decrease in the label of C₄-cycle products during a 3-min chase period and a slow turnover during a 20-min chase, indicating a lower level of functional integration between the C₄ and C₃ cycles in these species, relative to the other intermediates. Although F. cronquistii Powell was previously identified as a C₃ species, 7–18% of the initial label was in malate+aspartate. However, only 40–50% of this label was in the C-4 position, indicating C₄-acid formation as secondary products of photosynthesis in F. cronquistii. In 21% O₂, the absorbed quantum yields for CO₂ uptake (in mol CO₂·[mol quanta]^-1) averaged 0.053 in F. cronquistii (C₃), 0.051 in F. trinervia (Spreng.) Mohr (C₄), 0.052 in F. ramosissima (C₃-C₄), 0.051 in F. anomala (C₃-C₄), 0.050 in F. linearis (C₃-C₄), 0.046 in F. floridana (C₃-C₄), and 0.044 in F. pubescens (C₃-C₄). In 2% O₂ an enhancement of the quantum yield was observed in all of the C₃-C₄ intermediate species, ranging from 21% in F. ramosissima to 43% in F. pubescens. In all intermediates the quantum yields in 2% O₂ were intermediate in value to the C₃ and C₄ species, indicating a co-function of the C₃ and C₄ cycles in CO₂ assimilation. The low quantum-yield values for F. pubescens and F. floridana in 21% O₂ presumably reflect an ineffcient transfer of carbon from the C₄ to the C₃ cycle. The response of the quantum yield to four increasing O₂ concentrations (2–35%) showed lower levels of O₂ inhibition in the C₃-C₄ intermediate F. ramosissima, relative to the C₃ species. This indicates that the co-function of the C₃ and C₄ cycles in this intermediate species leads to an increased CO₂ concentration at the site of ribulose-1,5-bisphosphate carboxylase/oxygenase and a concomitant decrease in the competitive inhibition by O₂.Abbreviations PEP phosphoenolpyruvate - PGA 3-phosphoglycerate - RuBP ribulose-1,5-bisphosphate     

Linkage of C4 and C4 deficiency to BF and GPLA

The C4, Bf, and GPLA phenotypes of homo- and heterozygous C4-deficient guinea pigs were studied. The electrophoretic patterns suggest that the deficiency in circulating C4 results from an impaired structural gene, allelic to the C4^F, C4^S, and C4^S1 alleles at the C4 locus. In family studies, support for linkage of C4 and Bf to theGPLA system was obtained. The defective gene appears to be the fourth allele, which is rare, in the polymorphism of the fourth component of guinea pig complement.Abbreviations used in this paper are as follows Bf locus for properdin factor B - MHC major histocompatibility complex - GPLA major histocompatibility complex of the guinea pig     

C3、C4和C3-C4中间型植物的进化

介绍了有关C3、C4和C3-C4中间型植物进化的形态学、生理学、分子生物学、遗传学等方面的证据;推断地球上首先出现C3植物,然后是C3-C4中间类型植物,最后出现C4植物.     

The complement regulator C4b-binding protein (C4BP) interacts with both the C4c and C4dg subfragments of the parent C4b ligand: evidence for synergy in C4BP subsite binding

C4b-binding protein (C4BP) is a multimeric serum protein that is a potent regulator of the classical and lectin complement pathways. The binding site for C4b has been localized to complement control protein (CCP) domains 1-3 of the C4BP alpha-chain and, in particular, to a cluster of positively charged amino acids predicted to be at the interface between CCP 1 and CCP 2. To determine the regions of C4b contributing to C4BP binding, we have examined via surface plasmon resonance technology the binding of the C4c and C4dg subfragments of C4b to C4BP. At half-physiologic ionic strength, specific and saturable binding was observed for both C4c and C4dg. C4c exhibited much greater ionic strength sensitivity in its binding than did C4dg. Analysis of the effect on binding of the subfragments to various C4b-binding-defective C4BP mutants, together with cross-competition experiments, suggests that the subsites in C4BP for C4c and C4dg are adjacent, but distinct. Additionally, we observed synergy in subsite filling such that the presence of C4dg enhanced the extent of C4c binding over its basal level, and vice versa. The enhanced binding of C4c in the presence of C4dg was not due to an increase in affinity but rather reflected a 2-3-fold increase in the number of sites capable of binding C4c. This suggests the existence of a conformational equilibrium between high- and low-affinity states in the C4c binding subsite within each C4BP subunit, an equilibrium which is shifted in favor of the high-affinity state by the filling of the C4dg subsite.     

Evolutionary transition from C3 to C4 photosynthesis and the route to C4 rice

Compared with C₃ plants, C₄ plants possess a mechanism to concentrate CO₂ around the ribulose-1,5-bisphosphate carboxylase/oxygenase in chloroplasts of bundle sheath cells so that the carboxylation reaction work at a much more efficient rate, thereby substantially eliminate the oxygenation reaction and the resulting photorespiration. It is observed that C₄ photosynthesis is more efficient than C₃ photosynthesis under conditions of low atmospheric CO₂, heat, drought and salinity, suggesting that these factors are the important drivers to promote C₄ evolution. Although C₄ evolution took over 66 times independently, it is hypothesized that it shared the following evolutionary trajectory: 1) gene duplication followed by neofunctionalization; 2) anatomical and ultrastructral changes of leaf architecture to improve the hydraulic systems; 3) establishment of two-celled photorespiratory pump; 4) addition of transport system; 5) co-option of the duplicated genes into C₄ pathway and adaptive changes of C₄ enzymes. Based on our current understanding on C₄ evolution, several strategies for engineering C₄ rice have been proposed to increase both photosynthetic efficiency and yield significantly in order to avoid international food crisis in the future, especially in the developing countries. Here we summarize the latest progresses on the studies of C₄ evolution and discuss the strategies to introduce two-celled C₄ pathway into rice.     

Effects of irradiance and temperature on photosynthesis in C3, C4 and C3/C4 Panicum species

Species in the Laxa and Grandia groups of the genus Panicum are adapted to low, wet areas of tropical and subtropical America. Panicum milioides is a species with C₃ photosynthesis and low apparent photorespiration and has been classified as a C₃/C₄ intermediate. Other species in the Laxa group are C₃ with normal photorespiration. Panicum prionitis is a C₄ species in the Grandia group. Since P. milioides has some leaf characteristics intermediate to C₃ and C₄ species, its photosynthetic response to irradiance and temperature was compared to the closely related C₃ species, P. laxum and P. boliviense and to P. prionitis. The response of apparent photosynthesis to irradiance and temperature was similar to that of P. laxum and P. boliviense, with saturation at a photosynthetic photo flux density of about 1 mmol m^-2 s^-1 at 30°C and temperature optimum near 30°C. In contrast, P. prionitis showed no light saturation up to 2 mmol m^-2 s^-1 and an optimum temperature near 40°C. P. milioides exhibited low CO₂ loss into CO₂-free air in the light and this loss was nearly insensitive to temperature. Loss of CO₂ in the light in the C₃ species, P. laxum and P. boliviense, was several-fold higher than in P. milioides and increased 2- to 5-fold with increases in temperature from 10 to 40°C. The level of dark respiration and its response to temperature were similar in all four Panicum species examined. It is concluded that the low apparent photorespiration in P. milioides does not influence its response of apparent photosynthesis to irradiance and temperature in comparison to closely related C₃ Panicum species.Abbreviations AP apparent photosynthesis - I CO₂ compensation point - gl leaf conductance; gm, mesophyll conductance - PPFD photosynthetic photon flux density - PR apparent photorespiration rate - RuBPC sibulose bisphosphate carboxylase     

Isotyping of human C4 complement using differences in the functional activity of C4A and C4B isotypes

The difference in the functional activity of the isotypes A and B of component C4 of human complement was used to determine their ratio and to detect the inherited deficiency of the isotypes. ELISA methods were developed for the quantitative assay of component C4 (conventional sandwich method) and its functional activity. When determining the functional activity, the classic pathway of the complement and therefore of component C4 was activated by activators sorbed on ELISA microplates (immunoglobulin IgG3 or liposaccharide of the Shigella sonnei cell walls, which activates the complement by binding component C1). The nascent fragment C4b is covalently bound to the target activator; C4Ab binds better to the target protein (immunoglobulin), and C4Bb to the target carbohydrate (liposaccharide). Therefore, when immunoglobulin is a target activator, isotype C4A is bound and determined; and when the complement is activated by liposaccharide, isotype C4B is determined. The ratio of the activities determined by the two methods indicates a deficiency in the individual isotypes of component C4 or its absence. The rabbit polyclonal monospecific antibodies against the human component C4 and the conjugates of these antibodies with horseradish peroxidase were used in the methods described.     

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.     

The molecular basis of complete complement C4A and C4B deficiencies in a systemic lupus erythematosus patient with homozygous C4A and C4B mutant genes

The disease course of a complete C4-deficient patient in the U.S. was followed for 18 years. The patient experienced multiple episodes of infection, and he was diagnosed with systemic lupus erythematosus at age 9 years. The disease progressed to WHO class III mild lupus nephritis and to fatal CNS vasculitis at age 23 years. Immunochemical experiments showed that the patient and his sibling had complete absence of C4A and C4B proteins and were negative for the Rodgers and Chido blood group Ags. Segregation and definitive RFLP analyses demonstrated that the patient and his sibling inherited two identical haplotypes, HLA A2 B12 DR6, each of which carries a defective long C4A gene and a defective short C4B gene. PCR and DNA sequencing revealed that the mutant C4A contained a 2-bp insertion in exon 29 at the sequence for codon 1213. The identical mutation was absent in the mutant C4B. The C4B mutant gene was selectively amplified by long range PCR, and its 41 exons were completely sequenced. The C4B mutant had a novel single C nucleotide deletion at the sequence for codon 522 in exon 13, leading to frame-shift mutation and premature termination. Thus, a multiplex PCR is designed by which known mutations in C4A and C4B can be elucidated conveniently. Among the 28 individuals reported with complete C4 deficiency, 75-96% of the subjects (dependent on the inclusion criteria) were afflicted with autoimmune or immune complex disorders. Hence, complete C4 deficiency is one of the most penetrant genetic risk factors for human systemic lupus erythematosus.     

Phosphorus responses of C3 and C4 species

An hypothesis was formulated that phosphorus (P) partitioningin tissues of C₄ leaves would permit C₄ plants to resist P deficiencybetter than C₃ plants. To test this hypothesis, 12 C₃, C₄, andC₃–C₄ intermediate species were grown at adequate, deficient,and severely deficient P supply in a solid-phase-buffered sandculture system to characterize photosynthetic and growth responses.Species differed considerably in response to P stress. The growthof C₃ species was more sensitive to P supply than C₄ species,but C₃ and C₄ species had similar photosynthetic P use efficiency,and C₄ species did not have low leaf P content, contrary toour hypothesis. In fact, leaf photosynthetic rates were notcorrelated with growth responses. Moncots had lower leaf P contentand better maintenance of leaf production under P stress thandicots, because of greater inhibition of branching (dicots)than of tillering (monocots). The most P efficient species inthis survey was Brachiaria, a C₄ monocot that increased rootbiomass allocation under stress while maintaining P allocationto the shoot. It is concluded that C₄ species are not inherentlymore P efficient than C₃ species, but that monocots are moreP efficient than dicots, because of contrasting P and biomassallocation under stress. Key words: Phosphorus deficiency, C₃ plants, C₄ plants, growth response     

Insect herbivory on C3 and C4 grasses

Summary This study tested the hypothesis that grasses with the C₄ photosynthetic pathway are avoided as a food source by insect herbivores in natural communities. Insects were sampled from ten pairs of C₃–C₄ grasses and their distributions analyzed by paired comparisons tests. Results showed no statistically significant differences in herbivore utilization of C₃–C₄ species. However, there was a trend towards heavier utilization of C₃ species when means for both plant groups were compared. In particular, Homoptera and Diptera showed heavier usage of C₃ plants. Significant correlations between insect abundances and plant protein levels suggest that herbivores respond to the higher protein content of C₃ grasses. ¹³C values for six of the most common grasshopper species in the study area indicated that three species fed on C₃ plants, two species fed on C₄ plants, and one species consumed a mixture of C₃ and C₄ tissue.Welder Wildlife Refuge Contribution Number 213     

Deficiency of human complement protein C4 due to identical frameshift mutations in the C4A and C4B genes

The complement protein C4, encoded by two genes (C4A and C4B) on chromosome 6p, is the most polymorphic among the MHC III gene products. We investigated the molecular basis of C4 deficiency in a Finnish woman with systemic lupus erythematosus. C4-specific mRNA was present at low concentrations in C4-deficient (C4D) patient fibroblasts, but no pro-C4 protein was detected. This defect in C4 expression was specific in that synthesis of two other complement proteins was normal. Analysis of genomic DNA showed that the proposita had both deleted and nonexpressed C4 genes. Each of her nonexpressed genes, a C4A null gene inherited from the mother, a C4A null gene, and a C4B null gene inherited from the father, all contained an identical 2-bp insertion (TC) after nucleotide 5880 in exon 29, providing the first confirmatory proof of the C4B pseudogene. This mutation has been previously found only in C4A null genes. Although the exon 29/30 junction is spliced accurately, this frameshift mutation generates a premature stop at codon 3 in exon 30. These truncated C4A and C4B gene products were confirmed through RT-PCR and sequence analysis. Among the possible genetic mechanisms that produce identical mutations is both genes, the most likely is a mutation in C4A followed by a gene conversion to generate the mutated C4B allele.     

Isotyping of component C4 of human complement using differences in the functional activity of isotypes C4A and C4B

The difference in the functional activity of the isotypes A and B of component C4 of human complement was used to determine their ratio and to detect the inherited deficiency of the isotypes. ELISA methods were developed for the quantitative assay of component C4 (conventional sandwich method) and its functional activity. When determining the functional activity, the classic pathway of the complement and therefore of component C4 was activated on activators sorbed on ELISA microplates: immunoglobulin IgG3 or liposaccharide of theShigella sonnei cell walls, which activates the complement by binding component C1. The nascent fragment C4b is covalently bound to the target activator; C4Ab binds better to the target protein (immunoglobulin), and C4Bb to the target carbohydrate (liposaccharide). Therefore, when immunoglobulin is a target activator, isotype C4A is bound and determined; and when the complement is activated with liposaccharide, isotype C4B is determined. The radio of the activities determined by the two methods indicates the deficiency in the individual isotypes of component C4 or its absence. The rabbit polyclonal monospecific antibodies against the human component C4 and the conjugates of these antibodies with horseradish peroxidase were used in the methods described.     

C4 photosynthesis and water stress

Background

In contrast to C₃ photosynthesis, the response of C₄ photosynthesis to water stress has been less-well studied in spite of the significant contribution of C₄ plants to the global carbon budget and food security. The key feature of C₄ photosynthesis is the operation of a CO₂-concentrating mechanism in the leaves, which serves to saturate photosynthesis and suppress photorespiration in normal air. This article reviews the current state of understanding about the response of C₄ photosynthesis to water stress, including the interaction with elevated CO₂ concentration. Major gaps in our knowledge in this area are identified and further required research is suggested.

Scope

Evidence indicates that C₄ photosynthesis is highly sensitive to water stress. With declining leaf water status, CO₂ assimilation rate and stomatal conductance decrease rapidly and photosynthesis goes through three successive phases. The initial, mainly stomatal phase, may or may not be detected as a decline in assimilation rates depending on environmental conditions. This is because the CO₂-concentrating mechanism is capable of saturating C₄ photosynthesis under relatively low intercellular CO₂ concentrations. In addition, photorespired CO₂ is likely to be refixed before escaping the bundle sheath. This is followed by a mixed stomatal and non-stomatal phase and, finally, a mainly non-stomatal phase. The main non-stomatal factors include reduced activity of photosynthetic enzymes; inhibition of nitrate assimilation, induction of early senescence, and changes to the leaf anatomy and ultrastructure. Results from the literature about CO₂ enrichment indicate that when C₄ plants experience drought in their natural environment, elevated CO₂ concentration alleviates the effect of water stress on plant productivity indirectly via improved soil moisture and plant water status as a result of decreased stomatal conductance and reduced leaf transpiration.

Conclusions

It is suggested that there is a limited capacity for photorespiration or the Mehler reaction to act as significant alternative electron sinks under water stress in C₄ photosynthesis. This may explain why C₄ photosynthesis is equally or even more sensitive to water stress than its C₃ counterpart in spite of the greater capacity and water use efficiency of the C₄ photosynthetic pathway.Key words: C₃ and C₄ photosynthesis, stomatal and non-stomatal limitation, high CO₂, water stress     

Evolution of C4 photosynthetic genes and overexpression of maize C4 genes in rice

C3 plants including many agronomically important crops exhibit a lower photosynthetic efficiency due to inhibition of photosynthesis by O₂ and the associated photorespiration. C4 plants had evolved the C4 pathway to overcome low CO₂ and photorespiration. This review first focuses on the generation of a system for high level expression of the C4-specific gene for pyruvate, orthophosphate dikinase (Pdk), one of the key enzyme in C4 photosynthesis. Based on the results with transgenic rice plants, we have demonstrated that the regulatory system controlling thePdk expression in maize is not unique to C4 plants but rice (C3 plant) posses a similar system. Second, we discussed the possibility of the high level expression of maize C4-specific genes in transgenic rice plants. Introduction of the maize intact phosphoenolpyruvate carboxylase gene (Ppc) caused 30–100 fold higher PEPC activities than non-transgenic rice. These results demonstrated that intact C4-type genes are available for high level expression of C4 enzymes in rice plants. The extended abstract of a paper presented at the 13th International Symposium in Conjugation with Award of the International Prize for Biology “Frontier of Plant Biology”