Pale Green Phenotype Research Articles

Large Scale Comparative Proteomics of a Chloroplast Clp Protease Mutant Reveals Folding Stress, Altered Protein Homeostasis, and Feedback Regulation of Metabolism

The clpr2-1 mutant is delayed in development due to reduction of the chloroplast ClpPR protease complex. To understand the role of Clp proteases in plastid biogenesis and homeostasis, leaf proteomes of young seedlings of clpr2-1 and wild type were compared using large scale mass spectrometry-based quantification using an LTQ-Orbitrap and spectral counting with significance determined by G-tests. Virtually only chloroplast-localized proteins were significantly affected, indicating that the molecular phenotype was confined to the chloroplast. A comparative chloroplast stromal proteome analysis of fully developed plants was used to complement the data set. Chloroplast unfoldase ClpB3 was strongly up-regulated in both young and mature leaves, suggesting widespread and persistent protein folding stress. The importance of ClpB3 in the clp2-1 mutant was demonstrated by the observation that a CLPR2 and CLPB3 double mutant was seedling-lethal. The observed up-regulation of chloroplast chaperones and protein sorting components further illustrated destabilization of protein homeostasis. Delayed rRNA processing and up-regulation of a chloroplast DEAD box RNA helicase and polynucleotide phosphorylase, but no significant change in accumulation of ribosomal subunits, suggested a bottleneck in ribosome assembly or RNA metabolism. Strong up-regulation of a chloroplast translational regulator TypA/BipA GTPase suggested a specific response in plastid gene expression to the distorted homeostasis. The stromal proteases PreP1,2 were up-regulated, likely constituting compensation for reduced Clp protease activity and possibly shared substrates between the ClpP and PreP protease systems. The thylakoid photosynthetic apparatus was decreased in the seedlings, whereas several structural thylakoid-associated plastoglobular proteins were strongly up-regulated. Two thylakoid-associated reductases involved in isoprenoid and chlorophyll synthesis were up-regulated reflecting feedback from rate-limiting photosynthetic electron transport. We discuss the quantitative proteomics data and the role of Clp proteolysis using a "systems view" of chloroplast homeostasis and metabolism and provide testable hypotheses and putative substrates to further determine the significance of Clp-driven proteolysis.

The Arabidopsis peroxisome division mutant pdd2 is defective in the DYNAMIN-RELATED PROTEIN3A (DRP3A) gene

In plants, the division of peroxisomes is mediated by several classes of proteins, including PEROXIN11 (PEX11), FISSION1 (FIS1), and DYNAMIN-RELATED PROTEIN3 (DRP3). DRP3A and DRP3B are two homologous dynamin-related proteins playing overlapping roles in the division of both peroxisomes and mitochondria, with DRP3A performing a stronger function than DRP3B in peroxisomal fission. Here, we report the identification and characterization of the peroxisome division defective 2 (pdd2) mutant, which was later proven to be another drp3A allele. The pdd2 mutant generates a truncated DRP3A protein and exhibits pale green and retarded growth phenotypes. Intriguingly, this mutant displays much stronger peroxisome division deficiency in root cells than in leaf mesophyll cells. Our data suggest that the partial GTPase effector domain retained in pdd2 may have contributed to the distinct mutant phenotype of this mutant.

Arabidopsis CHLI2 Can Substitute for CHLI1

The I subunit of magnesium-chelatase (CHLI) is encoded by two genes in Arabidopsis (Arabidopsis thaliana), CHLI1 and CHLI2. Conflicting results have been reported concerning the functions of the two proteins. We show here that the chli1/chli1 chli2/chli2 double knockout mutant was albino. Comparison with the pale-green phenotype of a chli1/chli1 single knockout mutant indicates that CHLI2 could support some chlorophyll biosynthesis in the complete absence of CHLI1. Real-time quantitative reverse transcription-polymerase chain reaction showed that CHLI2 was expressed at a much lower level than CHLI1. The chli1/chli1 chli2/chli2 double mutant could be fully rescued by expressing a transgene of CHLI2 driven by the CHLI1 promoter. These results suggest that differences between CHLI1 and CHLI2 lie mostly in their expression levels. Furthermore, both the chli1/chli1 and chli2/chli2 single knockout mutants had lower survival rates during de-etiolation than the wild type, suggesting that both genes are required for optimal growth during de-etiolation. In addition, we show that a semidominant chli1 mutant allele and the chli1/chli1 chli2/chli2 double mutant accumulated Lhcb1 transcripts when treated with the herbicide norflurazon, indicating that knocking out the CHLI activity causes the genome-uncoupled phenotype.

The RNA-binding proteins CSP41a and CSP41b may regulate transcription and translation of chloroplast-encoded RNAs in Arabidopsis

The chloroplast protein CSP41a both binds and cleaves RNA, particularly in stem-loops, and has been found associated with ribosomes. A related protein, CSP41b, co-purifies with CSP41a, ribosomes, and the plastid-encoded RNA polymerase. Here we show that Arabidopsis CSP41a and CSP41b interact in vivo, and that a csp41b null mutant becomes depleted of CSP41a in mature leaves, correlating with a pale green phenotype and reduced accumulation of the ATP synthase and cytochrome b ( 6 )/f complexes. RNA gel blot analyses revealed up to four-fold decreases in accumulation for some chloroplast RNAs, which run-on experiments suggested could tentatively be ascribed to decreased transcription. Depletion of both CSP41a and CSP41b triggered a promoter switch whereby atpBE became predominately transcribed from its nucleus-encoded polymerase promoter as opposed to its plastid-encoded polymerase promoter. Together with published proteomic data, this suggests that CSP41a and/or CSP41b enhances transcription by the plastid-encoded polymerase. Gradient analysis of rRNAs in the mutant suggest a defect in polysome assembly or stability, suggesting that CSP41a and/or CSP41b, which are not present in polysomal fractions, stabilize ribosome assembly intermediates. Although psbA and rbcL mRNAs are normally polysome-associated in the mutant, petD-containing RNAs have diminished association, perhaps accounting for reduced accumulation of its respective multimeric complex. In conclusion, our data suggest that CSP41a and CSP41b stimulate both transcription and translation in the chloroplast.

A Heterocomplex of Iron Superoxide Dismutases Defends Chloroplast Nucleoids against Oxidative Stress and Is Essential for Chloroplast Development inArabidopsis

There are three iron superoxide dismutases in Arabidopsis thaliana: FE SUPEROXIDE DISMUTASE1 (FSD1), FSD2, and FSD3. Their biological roles in chloroplast development are unknown. Here, we show that FSD2 and FSD3 play essential roles in early chloroplast development, whereas FSD1, which is found in the cytoplasm, does not. An fsd2-1 fsd3-1 double mutant had a severe albino phenotype on agar plates, whereas fsd2 and fsd3 single knockout mutants had pale green phenotypes. Chloroplast development was arrested in young seedlings of the double mutant. The mutant plants were highly sensitive to oxidative stress and developed increased levels of reactive oxygen species (ROS) during extended darkness. The FSD2 and FSD3 proteins formed a heteromeric protein complex in the chloroplast nucleoids. Furthermore, transgenic Arabidopsis plants overexpressing both the FSD2 and FSD3 genes showed greater tolerance to oxidative stress induced by methyl viologen than did the wild type or single FSD2- or FSD3-overexpressing lines. We propose that heteromeric FSD2 and FSD3 act as ROS scavengers in the maintenance of early chloroplast development by protecting the chloroplast nucleoids from ROS.

An Arabidopsis homolog of the bacterial peptidoglycan synthesis enzyme MurE has an essential role in chloroplast development

Enzymes encoded by bacterial MurE genes catalyze the ATP-dependent formation of uridine diphosphate-N-acetylmuramic acid-tripeptide in bacterial peptidoglycan biosynthesis. The Arabidopsis thaliana genome contains one gene with homology to the bacterial MurE:AtMurE. Under normal conditions AtMurE is expressed in leaves and flowers, but not in roots or stems. Sequence-based predictions and analyses of GFP fusions of the N terminus of AtMurE, as well as the full-length protein, suggest that AtMurE localizes to plastids. We identified three T-DNA-tagged and one Ds-tagged mutant alleles of AtMurE in A. thaliana. All four alleles show a white phenotype, and A. thaliana antisense AtMurE lines showed a pale-green phenotype. These results suggest that AtMurE is involved in chloroplast biogenesis. Cells of the mutants were inhibited in thylakoid membrane development. RT-PCR analysis of the mutant lines suggested that the expression of genes that depend on a multisubunit plastid-encoded RNA polymerase was decreased. To analyze the functional relationships between the MurE genes of cyanobacteria, the moss Physcomitrella patens and higher plants, a complementation assay was carried out with a P. patens (Pp) MurE knock-out line, which exhibits a small number of macrochloroplasts per cell. Although the Anabaena MurE, fused with the N-terminal region of PpMurE, complemented the macrochloroplast phenotype in P. patens, transformation with AtMurE did not complement this phenotype. These results suggest that AtMurE is functionally divergent from the bacterial and moss MurE proteins.

Identification and Fine Mapping of a Gene Related to Pale Green Leaf Phenotype near the Centromere Region in Rice ( Oryza sativa)

A thermo-insensitive pale green leaf mutant ( pgl2) was isolated from T-DNA inserted transgenic lines of rice ( Oryza sativa L. subsp. japonica cv. Nipponbare). Genetic analysis indicated that the phenotype was caused by a recessive mutation in a single nuclear-encoded gene. To map the PGL2 gene, an F 2 population was constructed by crossing the mutant with Longtefu ( Oryza sativa L. subsp. indica). The PGL2 locus was roughly linked to SSR marker RM331 on chromosome 8. To finely map the gene, 14 new InDel markers were developed around the marker, and PGL2 was further mapped to a 2.37 Mb centromeric region. Analysis on chlorophyll contents of leaves showed that there was no obvious difference between the mutant and the wild type in total chlorophyll (Chl) content, while the ratio of Chl a/Chl b in the mutant was only about 1, which was distinctly lower than that in the wild type, suggesting that the PGL2 gene was related to the conversion between Chl a and Chl b. Moreover, the method of primer design around the centromeric region was discussed, which would provide insight into fine mapping of the functional genes in plant centromeres.

An Arabidopsis chloroplast‐targeted Hsp101 homologue, APG6, has an essential role in chloroplast development as well as heat‐stress response

Analysis of albino or pale-green (apg) mutants is important for identifying nuclear genes responsible for chloroplast development and pigment synthesis. We have identified 38 apg mutants by screening 11 000 Arabidopsis Ds-tagged lines. One mutant, apg6, contains a Ds insertion in a gene encoding APG6 (ClpB3), a homologue of the heat-shock protein Hsp101 (ClpB1). We isolated somatic revertants and identified two Ds-tagged and one T-DNA-tagged mutant alleles of apg6. All three alleles gave the same pale-green phenotype. These results suggest that APG6 is important for chloroplast development. The APG6 protein contains a transit peptide and is localized in chloroplasts. The plastids of apg6 pale-green cells were smaller than those of the wild type, and contained undeveloped thylakoid membranes. APG6 mRNA accumulated in response to heat shock in various organs, but not in response to other abiotic stresses. Under normal conditions, APG6 is constitutively expressed in the root tips, the organ boundary region, the reproductive tissues of mature plants where plastids exist as proplastids, and slightly in the stems and leaves. In addition, constitutive overexpression of APG6 in transgenic plants inhibited chloroplast development and resulted in a mild pale-green phenotype. The amounts of chloroplast proteins related to photosynthesis were markedly decreased in apg6 mutants. These results suggest that APG6 functions as a molecular chaperone involved in plastid differentiation mediating internal thylakoid membrane formation and conferring thermotolerance to chloroplasts during heat stress. The APG6 protein is not only involved in heat-stress response in chloroplasts, but is also essential for chloroplast development.

Downregulation of ClpR2 Leads to Reduced Accumulation of the ClpPRS Protease Complex and Defects in Chloroplast Biogenesis inArabidopsis

Plastids contain tetradecameric Clp protease core complexes, with five ClpP Ser-type proteases, four nonproteolytic ClpR, and two associated ClpS proteins. Accumulation of total ClpPRS complex decreased twofold to threefold in an Arabidopsis thaliana T-DNA insertion mutant in CLPR2 designated clpr2-1. Differential stable isotope labeling of the ClpPRS complex with iTRAQ revealed a fivefold reduction in assembled ClpR2 accumulation and twofold to fivefold reductions in the other subunits. A ClpR2:(his)(6) fusion protein that incorporated into the chloroplast ClpPRS complex fully complemented clpr2-1. The reduced accumulation of the ClpPRS protease complex led to a pale-green phenotype with delayed shoot development, smaller chloroplasts, decreased thylakoid accumulation, and increased plastoglobule accumulation. Stromal ClpC1 and 2 were both recruited to the thylakoid surface in clpr2-1. The thylakoid membrane of clpr2-1 showed increased carotenoid content, partial inactivation of photosystem II, and upregulated thylakoid proteases and stromal chaperones, suggesting an imbalance in chloroplast protein homeostasis and a well-coordinated network of proteolysis and chaperone activities. Interestingly, a subpopulation of PsaF and several light-harvesting complex II proteins accumulated in the thylakoid with unprocessed chloroplast transit peptides. We conclude that ClpR2 cannot be functionally replaced by other ClpP/R homologues and that the ClpPRS complex is central to chloroplast biogenesis, thylakoid protein homeostasis, and plant development.

A nuclear‐encoded sigma factor, Arabidopsis SIG6, recognizes sigma‐70 type chloroplast promoters and regulates early chloroplast development in cotyledons

Eubacterial-type multi-subunit plastid RNA polymerase (PEP) is responsible for the principal transcription activity in chloroplasts. PEP is composed of plastid-encoded core subunits and one of multiple nuclear-encoded sigma factors that confer promoter specificity on PEP. Thus, the replacement of sigma factors associated with PEP has been assumed to be a major mechanism for the switching of transcription patterns during chloroplast development. The null mutant (sig6-1) of plastid sigma factor gene AtSIG6 exhibited a cotyledon-specific pale green phenotype. Light-dependent chloroplast development was significantly delayed in the sig6-1 mutant. Genetic complementation of the mutant phenotype by the AtSIG6 cDNA demonstrated that AtSIG6 plays a key role in light-dependent chloroplast development. Northern and array-based global analyses for plastid transcripts revealed that the transcript levels of most PEP-dependent genes were greatly reduced in the sig6-1 mutant, but that the accumulation of nuclear-encoded RNA polymerase (NEP)-dependent transcripts generally increased. As the PEP alpha subunit and PEP-dependent trnV accumulated at normal levels in the sig6-1 mutant, the AtSIG6 knockout mutant probably retained functional PEP, and the transcriptional defects are likely to have been directly caused by AtSIG6 deficiency. Most of the AtSIG6-dependent genes are preceded by sigma70-type promoters comprised of conserved -35/-10 elements. Thus, AtSIG6 may act as a major general sigma factor in chloroplasts during early plant development. On the other hand, the mutant phenotype was restored in older seedlings. Arabidopsis probably contains another late general sigma factor, the promoter specificity of which widely overlaps with that of AtSIG6.

T7 RNA Polymerase-Directed Expression of an Antibody Fragment Transgene in Plastids Causes a Semi-Lethal Pale-Green Seedling Phenotype

A T7 promoter-controlled transgene, AbL, encoding a camel single-domain antibody fragment that binds to the model antigen chicken egg-white lysozyme was introduced into the plastid genome of tobacco. AbL expression was activated in the transplastomic line by introducing a nuclear transgene, ST7, encoding a light-regulated plastid-targeted T7RNAP by cross-pollination. The resulting AbL x ST7 progeny seedlings developed a pale-green phenotype and ceased growth soon after germination. High levels of AbL transcripts accumulated in AbL x ST7 seedlings and expression of functional AbL antibody was detected by ELISA. Transplastomic AbL plants were also crossed with nuclear-transformed tobacco plants containing a salicylic acid-inducible transgene encoding a plastid-targeted T7RNAP (PR-T7 transgene). The resulting AbL x PR-T7 progeny were wild-type in appearance but were slow growing and prone to wilting even when provided with adequate water. Although AbL transcription was inducible by treating AbL x PR-T7 leaves with salicylic acid, high levels of T7RNAP-dependent AbL transcripts also accumulated in the absence of induction. However, AbL antibody did not accumulate at levels detectable by immunoblotting or ELISA in AbL x PR-T7 plants despite the fact that total leaf RNA containing AbL transcripts was capable of directing AbL antibody synthesis in an E. coli-derived in vitro translation system.

Screening of the Rice Viviparous Mutants Generated by Endogenous Retrotransposon Tos17 Insertion. Tagging of a Zeaxanthin Epoxidase Gene and a Novel OsTATCGene

The rice (Oryza sativa) retrotransposon Tos17 is one of a few active retrotransposons in plants and its transposition is activated by tissue culture. Here, we present the characterization of viviparous mutants of rice induced by tissue culture to demonstrate the feasibility of the use of retrotransposon Tos17 as an endogenous insertional mutagen and cloning of the tagged gene for forward genetics in unraveling the gene function. Two mutants were shown to be caused by the insertion of Tos17. Osaba1, a strong viviparous mutant with wilty phenotype, displayed low abscisic acid level and almost no further increase in its levels upon drought. The mutant is shown to be impaired in the epoxidation of zeaxanthin. On the other hand, Ostatc, a mutant with weak phenotype, exhibited the pale green phenotype and slight increase in abscisic acid levels upon drought. Deduced amino acids of the causative genes of Osaba1 and Ostatc manifested a significantly high homology with zeaxanthin epoxidase isolated from other plant species and with bacterial Sec-independent translocase TATC protein, respectively. This is the first example of transposon tagging in rice.

A library of Arabidopsis 35S-cDNA lines for identifying novel mutants.

We have developed a system to over-express or co-suppress random cDNAs in Arabidopsis thaliana upon Agrobacterium tumefaciens-mediated transformation. We constructed a binary vector containing a novel Arabidopsis cDNA library driven by the cauliflower mosaic virus (CaMV) 35S promoter. The vector, 35SpBARN, offers in terra selection with glufosinate ammonium (BASTA) and the ability to identify the cDNA insert using PCR with flanking primers. We introduced this overexpression library into Arabidopsis and selected over 30,000 transformants. A random sample of 50 T1 plants was analyzed to determine the quality of the cDNA library in planta. About 90% of T1 plants in the collection have inserts, the average insert size is ca. 1.1 kb, and ca. 43% of these inserts appear to encode full-length proteins. T1 plants were screened for visible abnormalities, and one mutant, V5, was chosen for further study. This mutant displays a pale green phenotype, and its transgene contains a partial petH cDNA encoding chloroplast ferredoxin-NADP+ reductase (EC 1.18.1.2). This construct co-suppresses the endogenous petH transcript. We recapitulated the mutant phenotype by expressing either the full-length or truncated petH cDNA from the CaMV 35S promoter in wild-type Arabidopsis. Our results indicate that co-suppressing endogenous genes can cause dominant phenotypes as expected. As we have also used the 35SpBARN vector to successfully over-express other transcripts in planta, we predict that this system will be generally useful for identifying genes that yield phenotypes upon over-expression as well.

Chloroplast development in Arabidopsis thaliana requires the nuclear-encoded transcription factor Sigma B

Development of plastids into chloroplasts, the organelles of photosynthesis, is triggered by light. However, little is known of the factors involved in the complex coordination of light-induced plastid gene expression, which must be directed by both nuclear and plastid genomes. We have isolated an Arabidopsis mutant, abc1, with impaired chloroplast development, which results in a pale green leaf phenotype. The mutated nuclear gene encodes a sigma factor, SigB, presumably for the eubacterial-like plastid RNA polymerase. Our results provide direct evidence that a nuclear-derived prokaryotic-like SigB protein, plays a critical role in the coordination of the two genomes for chloroplast development.

Site-selected transposon mutagenesis at the hcf106 locus in maize.

The High chlorophyll fluorescence106 (Hcf106) gene in maize is required for chloroplast membrane biogenesis, and the hcf106-mum1 allele is caused by the insertion of a Robertson's Mutator Mu1 element into the promoter of the gene. Seedlings homozygous for hcf106-mum1 are pale green and die 3 weeks after germination, but only in the presence of Mutator activity conferred by active, autonomous Mu regulatory transposons elsewhere in the genome. When Mutator activity is lost, the mutant phenotype is suppressed, and homozygous plants have an almost wild-type phenotype. To isolate derivative alleles at the hcf106 locus that no longer require Mutator activity for phenotypic expression, we have developed a method for site-selected transposon mutagenesis in maize. This procedure, first described for Caenorhabditis elegans and Drosophila, involves using polymerase chain reaction (PCR) to screen pools of individuals for insertions and deletions in genes of known sequence. Pools of seedlings segregating for the progenitor allele hcf106-mum1 were screened by PCR for insertions and deletions associated with Robertson's Mutator. In a 360-bp target region, two new insertions and one deletion were identified in only 700 Mu-active gametes screened. One of the insertions was in the progenitor hcf106-mum1 allele and the other was in the wild-type allele, but all three new alleles were found to have break-points at the same nucleotide in the first intron. Unlike the hcf-106-mum1 progenitor allele, the deletion and one of the insertions conferred pale green seedling lethal phenotypes in the absence of mutator activity. However, the second insertion had a weak, viable phenotype under these conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Inactivation of maize transposon Mu suppresses a mutant phenotype by activating an outward-reading promoter near the end of Mu1.

We described previously a mutation in maize, hcf106, caused by the insertion of a Mu1 transposon. When the Mu transposon system is in an active phase, hcf106 conditions a nonphotosynthetic, pale green phenotype. However, when the Mu system is inactive (a state correlated with hypermethylation of Mu elements), the plant adopts a normal phenotype despite the continued presence of the transposon within the gene. The molecular mechanisms that mediate this suppression of the mutant phenotype have now been investigated. We show here that the Mu element responsible for the hcf106 lesion lies within sequences encoding the 5'-untranslated leader of the Hcf106 mRNA. When the Mu transposon system is active, this insertion interferes with the accumulation of mRNA from the hcf106 allele. However, when Mu is inactive, mRNA similar in size and abundance to that transcribed from the normal allele accumulates. These transcripts initiate at many sites throughout a 70-base-pair region, within and immediately downstream of the Mu1 insertion. Thus, an unusual promoter spanning the downstream junction between Mu1 and Hcf106 substitutes for the normal Hcf106 promoter but only when Mu is inactive. The pattern of mRNA accumulation in different organs and in response to light suggests that the activity of this promoter is conditional not only upon the phase of Mu activity, but also upon signals that regulate the normal Hcf106 promoter.

Light Intensity During Leaf Growth Affects Chlorophyll Concentration and CO2 Assimilation of a Soybean Chlorophyll Mutant1

An evaluation was made of leaf diffusive resistance and chlorophyll concentrations of dark green (normal) and pale green (chlorophyll deficient) phenotypes of a soybean [Glycine max (L.) Merrill] mutant when grown 10‐ and 30‐klux light intensities. When grown at the 10‐klux intensity the two leaf types had similar mesophyll and total CO2 diffusive resistances and similar net photosynthetic rates per unit leaf area at normal ambient CO2 concentrations (e.g., 300 µl/liter). Chloropyhll concentration of the pale green leaf was one‐fourth that of the dark green leaf. On a chlorophyll basis, rate of CO2 fixation was four times faster in the pale green type than in the dark green type. The similarity in net photosynthetic rates per unit leaf area of the two phenotypes when grown at the 10‐klux light intensity was the result of (1) similar CO2 diffusive resistances and (2) the ability of the photosynthetic apparatus of the pale green leaf to fix CO2 as rapidly as that of the dark green leaf, in spite of a much lower amount of chlorophyll.When grown at the 30‐klux light intensity, chlorophyll concentration of the pale green phenotype progressively declined, to one‐tenth the amount at the 10‐klux intensity. Chlorophyll concentration of the dark green phenotype was not significantly affected by light intensity. Photosynthesis of the pale green leaf grown at the 30‐klux intensity became CO2 saturated at concentrations well below 300 µl/liter. Therefore, light intensity during leaf growth strongly influenced the relative CO2 assimilation rates of the dark green and pale green phenotypes.

Continuity of the Chloroplast Membrane Systems in Zea mays L.

Ultrastructural studies of the chloroplasts of the normal, yellow-green, and pale green phenotypes of Zea mays L. indicate that the internal membrane system is continuous with the plastid envelop. The intramembraneous spaces, loculi, and fret channels are also continuous with inner component of the plastid envelop. High energy compounds or other photosynthates, formed in the grana or frets are thus separated from both stroma and cytoplasm by a single membrane, either the fret membrane or the outer component of the plastid envelop. Since this type of plastid ultrastructure is apparently found only in plants exhibiting the Hatch and Slack pathways of photosynthesis there may be a relation between plastid ultrastructure and the pathways of photosynthetic carbon fixation.