Identification of domains involved in tetramerization and malate inhibition of maize C4-NADP-malic enzyme. Drincovich, M. Raghavendra and R. Sage Dordrecht: Springer , — Edwards, G. Phosphoenolpyruvate carboxykinase in leaves of certain plants which fix CO 2 by the C4-dicarboxylic acid cycle of photosynthesis. Sage Dordrecht: Springer , 29— PubMed Abstract Google Scholar. Finnegan, P. Phosphoenolpyruvate carboxykinase in the C4 monocot Urochloa panicoides is encoded by four differentially expressed genes.
Furbank, R. Evolution of the C4 photosynthetic mechanism: are there really three C4 acid decarboxylation types? Ghannoum, O. Gutierrez, M. Biochemical and cytological relationships in C4 plants.
Planta , — Haberlandt, G. Physiologische Pflanzenanatomie , 2nd Edn. Leipzig: Wilhelm Engelman. Hatch, M. The C4-pathway of photosynthesis. Evidence for an intermediate pool of carbon dioxide and the identity of the donor C4-dicarboxylic acid.
C4 photosynthesis: a unique blend of modified biochemistry, anatomy and ultrastructure. Acta , 81— Subdivision of C4-pathway species based on differing C4 decarboxylating systems and ultrastructural features. Photosynthesis by sugar-cane leaves: a new carboxylation reaction and the pathway of sugar formation. Further studies on a new pathway of photosynthetic carbon dioxide fixation in sugar-cane and its occurrence in other plant species. Jenkins, C. Mechanism of C4 photosynthesis.
A model describing the inorganic carbon pool in bundle sheath cells. Kanai, R. Sage and R. Monson London: Academic Press , 49— Kortschak, H.
Carbon dioxide fixation in sugarcane leaves. Koteyeva, N. An assessment of the capacity for phosphoenolpyruvate carboxykinase to contribute to C4 photosynthesis. Kromdijk, J. Bundle-sheath leakiness in C4 photosynthesis: a careful balancing act between CO 2 concentration and assimilation. Lai, L. Leegood, R. The intercellular compartmentation of metabolites in leaves of Zea mays L. Identification of the regulatory steps in gluconeogenesis in cotyledons of Cucurbita pepo.
Acta , 1— Energetics of photosynthesis in Zea mays. Studies of the flash-induced electrochromic shift and fluorescence induction in bundle sheath cells.
Acta , — Organic acid metabolism in plants: from adaptive physiology to transgenic varieties for cultivation in extreme soils. Ludwig, M. Evolution of the C4 pathway: events at the cellular and molecular levels. Maier, A. Marshall, J. Plant Cell 9, — Implications for the evolution of C4 photosynthesis. Maurino, V. Non-photosynthetic malic enzyme from maize: a constitutively expressed enzyme that responds to plant defense inducers.
Meierhoff, K. Differential biogenesis of photosystem II in mesophyll and bundle-sheath cells of monocotyledonous NADP-malic enzyme-type C4 plants: the non-stoichiometric abundance of the subunits of photosystem II in the bundle-sheath chloroplasts and the translational activity of the plastome-encoded genes.
Planta , 23— Meister, M. The roles of malate and aspartate in C4 photosynthetic metabolism of Flaveria bidentis L. Muhaidat, R. Diversity of Kranz anatomy and biochemistry in C4 eudicots.
Penfield, S. Expression and manipulation of phosphoenolpyruvate carboxykinase 1 identifies a role for malate metabolism in stomatal closure. Plant J. Pick, T. Systems analysis of a maize leaf developmental gradient redefines the current C4 model and provides candidates for regulation. Plant Cell 23, — Sage, R. The C4 plant lineages of planet Earth. Saigo, M. Sharwood, R. Photosynthetic flexibility in maize exposed to salinity and shade.
Sommer, M. The dicotyledonous NAD malic enzyme C4 plant Cleome gynandra displays age-dependent plasticity of C4 decarboxylation biochemistry. Plant Biol. Stitt, M. Generation and maintenance of concentration gradients between mesophyll cell and bundle sheath in maize leaves.
The large pools of metabolites involved in intercellular metabolite shuttles in C4 photosynthesis provide enormous flexibility and robustness in a fluctuating light environment. Tausta, S. Maize C4 and non-C4-dependent malic enzymes are encoded by distinct genes derived from a plastid-localized ancestor. Moreover, PEP is a substrate of the citric acid cycle in mitochondria Krebs, Recent studies show that PEP is involved in nitrogen recycling from xylem Bailey and Leegood, and in nitrogen mobilization from aging leaves Taylor et al.
It is also a substrate of the shikimate pathway SP in chloroplasts, which is expected to exist in both MC and BSC purple lines ; moreover, phospho enol pyruvate transporter PEP is a substrate for the citrate pathway in mitochondria red lines and glycolysis in cytosol black lines. PEP is also involved in nitrogen recycling from xylem orange lines. Considering that PEP functions in multiple metabolic pathways, it is safe to infer that the PEP transporting process is crucial in plants.
Here, we conducted a systematic comparison of different properties of PPT between C 3 and C 4 plants. Specifically, we first constructed a phylogeny of PPT in Viridiplantae, which includes 23 species spanning chlorophytes to angiosperms to infer the orthologous relationships and copy number of PPT.
Then, we compared a number of properties of PPT between C 3 and C 4 species, including PPT gene expression, amino acid sequences, and physiological functions. Our results showed that the paralog with relatively low transcript abundance in leaf of C 3 species was constantly recruited for C 4 photosynthesis in multiple C 4 lineages. Comparing PPT1 between C 4 and C 3 species showed that PPT1 has dramatic modifications in the coding region, however, its metabolic function remained the same.
The evolutionary changes of PPT suggest that high transcript abundance in the proper location is the key feature of transporters for C 4 photosynthesis. These included representative species along the phylogeny of Viridiplantae, spanning from basal species belonging to chlorophytes Micromonas pusilla and Chlamydomonas reinhardtii , embryophytes Marchantia polymorpha , tracheophytes Selaginella moellendorffii , and to higher angiosperm plants Amborella trichopoda.
Among these species, ten are eudicots and eight are monocots Figure 2. The tree was inferred from the alignment of protein sequences of PPT using the maximum likelihood method. Numbers beside each node are the bootstrap scores from 1, simulated trees; bootstrap scores lower than 60 in the major branch are shown full bootstrap scores are in Figure S1. PPT1 has one or two copies in eudicot species and two or three copies in monocot species. Red circles represent PPT1 and blue circles represent PPT2, large circles stand for original copies, and small circles for duplicated copies after the division of monocots and dicots.
The phylogenetic relationship of species is inferred from the Phytozome website. The genome-wide protein sequences of these 23 species were downloaded from Phytozome. We used OrthoFinder V2. The robustness of the tree topology was evaluated by bootstrap scores, which were calculated from 1, independently constructed gene trees. High transcript abundance is suggested as a major feature of genes recruited to support C 4 functions Moreno-Villena et al.
Specifically, we compared the transcript abundance of PPT1 and PPT2 in leaf among species with different photosynthetic types; we also compared the expression patterns of PPT1 and PPT2 in different tissues and cell types.
RNA isolation, quality control, library preparation, and sequencing procedures are summarized in Johnson et al. Briefly, RNA-Seq data were generated using Illumina with a paired-end sequencing strategy with a read length of 90 bp.
Transcripts were assembled using Trinity version 2. Transcript abundance was analyzed by mapping short reads to assembled contigs of corresponding species and then normalizing the transcript abundance to the Fragments Per Kilobase of transcript per Million mapped reads FPKM using the RSEM package version 1.
Functional annotations of transcripts from dicot species, namely, Heliotropium , Mollugo , and Flaveria , were determined by searching for the best hit in the protein dataset of A. We annotated Neurachne transcripts by searching for the best hit in the protein dataset of Zea mays Table S6. The protein sequences of Z. When comparing transcript abundance of PPTs in roots and leaves from C 3 and C 4 species, we surveyed processed RNA-seq data and identified species that have RNA-Seq data from both roots and leaves, which include two Flaveria species e.
The photosynthetic type and abbreviations of species are listed in Table S1. Given that the genus Flaveria includes species at different evolutionary stages of C 4 photosynthesis, we further used this genus as a model to examine how the transcript abundance of PPT1 and PPT2 evolved along with the evolution of C 4 photosynthesis.
Specifically, we studied this in five species representing four different photosynthetic types, i. Rowan F. Sage University of Toronto. The Flaveria plants were watered twice a week and fertilized weekly. To study the gene expression differences of PPT1 and PPT2 in response to illumination, 1-month old plants were put into darkness at 6 pm. The dark-adapted plants were illuminated at am the next day.
Fully expanded leaves, usually the 2 nd or 3 rd leaf pair counted from the top, were cut after the leaves were illuminated for different time periods, i. For each gene, three technical and three biological replicates were performed. The primers used here are listed in Table S2. RNA-seq was performed with the Illumina NovaSeq platform in the paired-end mode with a read length of bp. The amino acid sequences of PPT1 and PPT2 of different Flaveria species were predicted based on de novo assembled transcripts as described in Lyu et al.
We further identified consistent amino acid modifications between C 3 and C 4 species, which were defined as sites that showed differences between C 3 and C 4 species, but that were conserved within C 3 species and also conserved within C 4 species. These consistently identified modifications were mapped to the phylogenetic tree of Flaveria Lyu et al.
Phylogeny of the Flaveria species was inferred from our previous work Lyu et al. Considering that the phylogeny of Flaveria contains two clades, we conducted the positive selection in two independent ways: either including species of both clade A and clade B, or excluding species from clade B which lacks a true C 4 species.
A threshold p -value of 0. The primers are listed in Table S2. A CDS with the amino acid 52 aa insertion deleted, i. All primers are listed in Table S2. The promoter used was a CaMV 35S promoter. The final plasmids were verified by Sanger-sequencing Sangon Biotech, Shanghai. The autofluorescence signal from chlorophyll was used as a marker for chloroplast thylakoids, with an excitation wavelength of nm and an emission wavelenth of nm.
The A. Agrobacterium cells were pelleted and re-suspended in transformation buffer 50 g sucrose, 2. The floral dipping process was repeated 1 week later. The positive T 1 transformants were transferred to soil. The T 2 lines were used to examine morphological phenotypes. These species were selected to capture the major events in Viridiplantae evolution with one species representing a major evolutionary stage of the Viridiplantae phylogeny. Furthermore, we included 10 eudicot species with six species from the Brassicaceae family and eight monocot species with seven species from the grass family Figure 2.
These two families contain the most sequenced genomes; hence they can be used to study how PPTs evolved within families. The gene tree showed that PPTs were present in all selected species. PPT had one copy in species that evolved before angiosperms, including the two chlorophyte species, M. The angiosperm species A. In contrast, there were two copies in other angiosperms with one being the ortholog of A.
PPTs from lower species showed higher similarity with A. Furthermore the single copy of PPT in A. The physiological significance and underlying mechanisms behind these different evolutionary speeds are unknown. First, we examined the transcript abundances of PPT1 and PPT2 in a few sets of species that are evolutionarily closely related but have different photosynthetic types. These species are from four genera with each representing an independent C 4 lineage.
Among these four genera, three are dicots, i. In the analysis, data from mature leaves were used. Photosynthetic types are marked with different colors, red: C 3 ; green: C 3 —C 4 ; purple: C 4 -like; blue: C 4. All the data are from published RNA-seq data; the data source is detailed in the Materials and Methods section. Species abbreviations are listed in Table S1. Therefore, PPT1, the copy recruited to support C 4 photosynthesis, did not have higher expression levels than PPT2 in the leaf tissue of C 3 plants; however, the gene had higher transcript abundance than PPT2 in root.
During the evolution of C 4 photosynthesis, the transcript abundance of PPT1 was decreased in root and increased in leaf, implying a major shift in tissue specificity. Considering that C 4 photosynthesis occurs in two cell types, which is a major evolutionary innovation, we further examined the changes in cellular specificity of PPT expression during C 4 evolution.
RNA-seq data from transcript residency on ribosomes Aubry et al. We investigated the expression with a focus on developmental scale, in which the average was calculated from samples at the same development stage regardless of tissue type and cell type, and on the scale of cell type.
The dominant role of PPT1 was more obvious in root tip in C 3 species. The mechanism by which PPT1 gained new expression patterns to support C 4 photosynthesis, e. We quantified the transcript abundance using qRT-PCR in five Flaveria species, representing different photosynthetic types, i. Our results demonstrated a gradual increase in the speed of changes of PPT1 transcript abundance to light from C 3 to C 3 —C 4 intermediate to C 4 species.
In the C 4 species F. Therefore, during C 4 evolution, PPT1 acquired new mechanisms enabling it to be rapidly up-regulated upon illumination. We further examined the patterns of increase in transcript abundance of PPT upon illumination change along the evolution from C 3 to C 4 species.
Specifically, in the C 3 F. An up-regulated expression level of PPT2 in F. Nevertheless, in both C 3 —C 4 species and the C 4 species F. Although PPT2 was induced at 0. Therefore, during evolution PPT1 gained not only higher transcript abundance in leaf, in particular in the MC, but also a more rapid and long-lasting response to light illumination, while PPT2 gradually lost its light responsiveness. Changes in transcriptional responses to external stimuli can be driven by changes in gene regulatory mechanisms.
We tested whether C 4 PPT1 might have acquired new cis -elements that are responsible for the altered expression patterns.
Based on the draft genome sequences of four Flaveria species, we found that there are two copies of PPT1 in F. The promoter sequences 3 kbp upstream of the start codon of two F.
Moreover, the promoter sequences of the two PPT1s from F. For PPT2, all species have one copy, which also show comparable transcript abundance in the four species Figure 5A.
Numbers on each node show bootstrap scores from 1, independent simulated trees. Abbreviations: Ftri, F. Further examination of the promoter structure shows that there is a highly conserved region between the proximal region of PPT1 promoter from F. The conserved region was divided into two parts by an insertion in F. Moreover, the two conserved parts were also observed in the promoters of PPT1 from F.
Usually the functional changes of a protein are underlined by changes in the amino acid sequence. Because of a lack of genome reference for some Flaveria species, the protein sequences of PPT1 and PPT2 were predicted based on de novo assembled transcripts for those species, and the genes that showed the highest sequence similarity with PPT1 and PPT2 from F.
We specifically examined the number of consistent amino acid modifications, which were defined as sites that have the same amino acid sequences in C 4 species but differ with those in C 3 species.
Specifically, the amino acid sequence of PPT1 had 19 consistent amino acid modifications between C 3 and C 4 species; in contrast, PPT2 exhibited eight consistent amino acid modifications Figure 6. To test whether these modifications were specific adaptations gained during evolution of C 4 photosynthesis, we performed a positive selection test in protein coding sequences of C 4 species against that of C 3 species in the genus Flaveria.
While horsepower figures are a major portion of performance, horsepower is not the only element in the equation, especially if you are limited by fuel-mileage requirements. The engineers and designers knew this and began with a completely new mindset toward technology, utilizing new materials not available in prior generations of Corvettes. Before the C4, no new Corvette program had been approved by GM in over 20 years. The time had come. While and hp levels were not going to happen overnight, it set a standard to target.
On the other side of the performance coin, the new car had to handle. The goal was 1 g of lateral acceleration, a lofty number usually achieved by only the most serious racing engineers. This necessitated a much wider, high-cornering-stiffness tire.
Dave explains that steering changes of just one degree produce over pounds of cornering force per tire, half again the cornering stiffness of a normal passenger-car tire.
Therefore, keeping the tire squarely planted to the road was important, and the C4 was designed to be stiff by using stiff springs and heavy rollbars fore and aft. While the early C4s have been criticized for having a harsh ride, most enthusiast drivers know why. Dave and his crew were to meet their intended goal of 1 g, and, most of all, they did it in a production car.
Due to the extensive design and performance targets, the introduction of the C4, which was intended as an '83 model, was delayed a year, with no official Corvettes produced that year.
All the Corvettes produced in were certified as '84s regardless of the actual delivery date. From there, it was a year parade that was to become the fourth-generation "C4" Corvette. Let's take a look at some contributions and significant changes that happened to Corvette through the C4 years, and the effect they had on Corvettes to follow. While you can forget about ever owning one, you can see a real '83 Corvette at the National Corvette Museum www.
This is the only '83 known to exist today. It is the foundation that all other C4s were built upon. GM realized that performance and fuel economy meant having fuel control. Cross-Fire was an early attempt at controlling the V-8's fuel diet and, while it helped, it still didn't give enough control since there were two injectors for eight cylinders.
What You Don't See: In an attempt to provide fuel mileage that met government standards and performance that met buyers' standards, GM also carried over the R4 automatic from the '82 Corvette. Calling it a manual transmission is a bit of a misnomer because the front portion is basically a four-speed transmission with an overdrive unit mounted behind it that operated on the top three gears.
GM was on the verge of losing its production source for transmissions, so joining with Doug Nash on these overdriven standard transmissions bridged the gap until something better was designed. These transmissions can be found in any standard-equipped Corvette from '' Having individual injectors for each cylinder gave much more control over the fuel trim of the engine; the tuned-length runners helped boost torque to 40 lb-ft, and horsepower increased from to This shaved almost a second off the previous year's quarter-mile times at the track.
Also, the air was regulated by a single throttle body no more equalizing throttle bodies and the amount of air fed into the engine was measured by a Mass Air Flow meter MAF. This helped increase fuel mileage because a What You Don't See: Along with added control of the fuel, the '85 Corvette's computer was upgraded to better control engine performance.
The suspension harshness issue was addressed, netting a better ride. Corvette was pacing the Indy , so what kind of pace car would it be if it weren't a convertible? Also, with the introduction of the convertible came aluminum heads for Corvette.
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