Kranz Type C4 Research Articles

Methods of analysis of chloroplast genomes of C3, Kranz type C4 and Single Cell C4 photosynthetic members of Chenopodiaceae

BackgroundChloroplast genome information is critical to understanding forms of photosynthesis in the plant kingdom. During the evolutionary process, plants have developed different photosynthetic strategies that are accompanied by complementary biochemical and anatomical features. Members of family Chenopodiaceae have species with C3 photosynthesis, and variations of C4 photosynthesis in which photorespiration is reduced by concentrating CO2 around Rubisco through dual coordinated functioning of dimorphic chloroplasts. Among dicots, the family has the largest number of C4 species, and greatest structural and biochemical diversity in forms of C4 including the canonical dual-cell Kranz anatomy, and the recently identified single cell C4 with the presence of dimorphic chloroplasts separated by a vacuole. This is the first comparative analysis of chloroplast genomes in species representative of photosynthetic types in the family.ResultsMethodology with high throughput sequencing complemented with Sanger sequencing of selected loci provided high quality and complete chloroplast genomes of seven species in the family and one species in the closely related Amaranthaceae family, representing C3, Kranz type C4 and single cell C4 (SSC4) photosynthesis six of the eight chloroplast genomes are new, while two are improved versions of previously published genomes. The depth of coverage obtained using high-throughput sequencing complemented with targeted resequencing of certain loci enabled superior resolution of the border junctions, directionality and repeat region sequences. Comparison of the chloroplast genomes with previously sequenced plastid genomes revealed similar genome organization, gene order and content with a few revisions. High-quality complete chloroplast genome sequences resulted in correcting the orientation the LSC region of the published Bienertia sinuspersici chloroplast genome, identification of stop codons in the rpl23 gene in B. sinuspersici and B. cycloptera, and identifying an instance of IR expansion in the Haloxylon ammodendron inverted repeat sequence. The rare observation of a mitochondria-to-chloroplast inter-organellar gene transfer event was identified in family Chenopodiaceae.ConclusionsThis study reports complete chloroplast genomes from seven Chenopodiaceae and one Amaranthaceae species. The depth of coverage obtained using high-throughput sequencing complemented with targeted resequencing of certain loci enabled superior resolution of the border junctions, directionality, and repeat region sequences. Therefore, the use of high throughput and Sanger sequencing, in a hybrid method, reaffirms to be rapid, efficient, and reliable for chloroplast genome sequencing.

The Developmental Enhancement of a C4 System With Non-Typical C4 Physiological Characteristics in Salsola ferganica (Kranz Anatomy), an Annual Desert Halophyte.

Variations of photosynthetic structures in different tissues or cells are in coordination with changes in various aspects, e.g. physiology, biochemistry, gene expression, etc. Most C4 plant species undergo developmental enhancement of the photosynthetic system, which may present different modes of changes between anatomy and physiology/biochemistry. In the current study, we investigated a Kranz-type C4 species Salsola ferganica with the progressive development of photosynthetic (PS) structure, performance of PS physiology, induction of PS enzymes, and transcriptional and translational regulation of PS genes, results revealed that S. ferganica presented C3 type anatomy in cotyledons but C4 type in leaves (C3/L4), with the C4 system separation of initial carbon fixation in the palisade mesophyll (M) cells and the following incorporation into triosephosphates and sugars in the bundle sheath (BS) cells, respectively. The BS cells continuously surrounded the vascular bundles and water storage cells in leaf anatomic structure. Compared to the single-cell C4 species Suaeda aralocaspica, S. ferganica exhibited similar developmental enhancement of C4 syndrome temporally and spatially in anatomic structures, enzyme activities, and gene expression, which suggests that completion of differentiation of the photosynthetic system is necessary for a C4 assimilation pathway. Besides, S. ferganica also displayed some different characteristics compared to S. aralocaspica in photosynthetic physiology, e.g. a more flexible δ13C value, much lower phosphoenolpyruvate carboxylase (PEPC) activity, and an insensitive response to stimuli, etc., which were not typical C4 characteristics. We speculate that this may suggest a different status of these two species in the evolutionary process of the photosynthesis pathway. Our findings will contribute to further understanding of the diversity of photosynthesis systems in Kranz-type C4 species and the Salsola genus.

What is the fate of xylem-transported CO2 in Kranz-type C4 plants?

High concentrations of dissolved inorganic carbon in stems of herbaceous and woody C3 plants exit leaves in the dark. In the light, C3 species use a small portion of xylem-transported CO2 for leaf photosynthesis. However, it is not known if xylem-transported CO2 will exit leaves in the dark or be used for photosynthesis in the light in Kranz-type C4 plants. Cut leaves of Amaranthus hypochondriacus were placed in one of three solutions of [NaH13 CO3 ] dissolved in KCl water to measure the efflux of xylem-transported CO2 exiting the leaf in the dark or rates of assimilation of xylem-transported CO2 * in the light, in real-time, using a tunable diode laser absorption spectroscope. In the dark, the efflux of xylem-transported CO2 increased with increasing rates of transpiration and [13 CO2 *]; however, rates of 13 Cefflux in A.hypochondriacus were lower compared to C3 species. In the light, A.hypochondriacus fixed nearly 75% of the xylem-transported CO2 supplied to the leaf. Kranz anatomy and biochemistry likely influence the efflux of xylem-transported CO2 out of cut leaves of A.hypochondriacus in the dark, as well as the use of xylem-transported CO2 * for photosynthesis in the light. Thus increasing the carbon use efficiency of Kranz-type C4 species over C3 species.

Exploring mechanisms linked to differentiation and function of dimorphic chloroplasts in the single cell C4 species Bienertia sinuspersici.

BackgroundIn the model single-cell C4 plant Bienertia sinuspersici, chloroplast- and nuclear-encoded photosynthetic enzymes, characteristically confined to either bundle sheath or mesophyll cells in Kranz-type C4 leaves, all occur together within individual leaf chlorenchyma cells. Intracellular separation of dimorphic chloroplasts and key enzymes within central and peripheral compartments allow for C4 carbon fixation analogous to NAD-malic enzyme (NAD-ME) Kranz type species. Several methods were used to investigate dimorphic chloroplast differentiation in B. sinuspersici.ResultsConfocal analysis revealed that Rubisco-containing chloroplasts in the central compartment chloroplasts (CCC) contained more photosystem II proteins than the peripheral compartment chloroplasts (PCC) which contain pyruvate,Pi dikinase (PPDK), a pattern analogous to the cell type-specific chloroplasts of many Kranz type NAD-ME species. Transient expression analysis using GFP fusion constructs containing various lengths of a B. sinuspersici Rubisco small subunit (RbcS) gene and the transit peptide of PPDK revealed that their import was not specific to either chloroplast type. Immunolocalization showed the rbcL-specific mRNA binding protein RLSB to be selectively localized to the CCC in B. sinuspersici, and to Rubisco-containing BS chloroplasts in the closely related Kranz species Suaeda taxifolia. Comparative fluorescence analyses were made using redox-sensitive and insensitive GFP forms, as well comparative staining using the peroxidase indicator 3,3-diaminobenzidine (DAB), which demonstrated differences in stromal redox potential, with the CCC having a more negative potential than the PCC.ConclusionsBoth CCC RLSB localization and the differential chloroplast redox state are suggested to have a role in post-transcriptional rbcL expression.

Using evolution as a guide to engineer kranz-type c4 photosynthesis.

Kranz-type C4 photosynthesis has independently and rapidly evolved over 60 times to dramatically increase radiation use efficiency in both monocots and eudicots. Indeed, it is one of the most exceptional examples of convergent evolution in the history of life. The repeated and rapid evolution of Kranz-type C4 suggests that it may be a derivative of a conserved developmental pathway that is present in all angiosperms. Here, I argue that the Kranz-type C4 photosynthetic system is an extension of the endodermis/starch sheath, that is normally only found in the roots and stems, into photosynthetic structures such as leaves. Support for this hypothesis was recently provided by a study that showed that the same genetic pathway that gives rise to the endodermis in roots, the SCARECROW/SHORT-ROOT radial patterning system, also regulates the development of Kranz anatomy and C4 physiology in leaves. This new hypothesis for the evolution of Kranz-type C4 photosynthesis has opened new opportunities to explore the underlying genetic networks that regulate the development and physiology of C4 and provides new potential avenues for the engineering of the mechanism into C3 crops.

How do single cell C4 species form dimorphic chloroplasts?

Bienertia sinuspersici is one of only three higher land plant species known to perform C4 photosynthesis without Kranz anatomy through partitioning of photosynthetic functions between dimorphic chloroplasts in a single photosynthetic cell. We recently reported the successful separation of the two chloroplast types, and biochemical and functional analyses revealed differences in protein composition and specialization of photosynthetic functions. In Kranz type C4 species, spatial (or cell-specific) control of transcription of nuclear genes contributes to development of dimorphic chloroplasts, but obviously this cannot be involved in formation of dimorphic chloroplasts within individual photosynthetic cells. Therefore, we address here the question of how nuclear encoded proteins could be selectively targeted to plastids within a cell to form two types of chloroplasts. We discuss current knowledge of chloroplast differentiation in single cell C4 species and present three hypothetical mechanisms for how this could occur.

Photosynthetic features of non-Kranz type C4 versus Kranz type C4 and C3 species in subfamily Suaedoideae (Chenopodiaceae).

The objective of this study was to characterise photosynthesis in terrestrial non-Kranz (NK) C4 species, Bienertia sinuspersici Akhani and Suaeda aralocaspica (Bunge) Freitag & Schütze (formerly Borszczowia aralocaspica), compared with closely related Kranz type C4 Suaeda eltonica Iljin and Suaeda taxifolia Standley, and C3 species Suaeda heterophylla Bunge and Suaeda maritima Dumort in subfamily Suaedoideae (Chenopodiaceae). Traditional Kranz type C4 photosynthesis has several advantages over C3 photosynthesis under certain environmental conditions by suppressing photorespiration. The different photosynthetic types were evaluated under varying levels of CO2 and light at 25°C. Both NK and Kranz type species had C4 type CO2 compensation points (corrected for dark-type respiration) and half maximum saturation of photosynthesis at similar levels of atmospheric CO2 (average of 145 µbar for the C4 species v. 330 µbar CO2 for C3 species) characteristic of C4 photosynthesis. CO2 saturated rates of photosynthesis per unit chlorophyll was higher in the C3 (at ~2.5 current ambient CO2 levels) than the C4 species, which is likely related to their higher Rubisco content. The amount of Rubisco as a percentage of total protein was similar in NK and Kranz type species (mean 10.2%), but much lower than in the C3 species (35%). Light saturated rates of CO2 fixation per unit leaf area at 25°C and 340 µbar CO2 were higher in the Kranz species and the NK C4 S. aralocaspica than in the C3 species. In response to light at 340 µbar CO2, there was a difference in rates of photosynthesis per unit Rubisco with NK > Kranz > C3 species. There were no significant differences between the three photosynthetic types in maximum quantum yields, convexity of light response curves, and light compensation points at 25°C. The water use efficiency (CO2 fixed per water transpired) at 340 µbar CO2, 25°C and 1000 µmol quanta m-2 s-1 was on average 3-fold higher in the C4 (NK and Kranz) compared with the C3 species. The results show that the NK species have several C4 traits like the Kranz type species in subfamily Suaedoideae.

Species having C4 single-cell-type photosynthesis in the Chenopodiaceae family evolved a photosynthetic phosphoenolpyruvate carboxylase like that of Kranz-type C4 species.

Spatial and temporal regulation of phosphoenolpyruvate carboxylase (PEPC) is critical to the function of C(4) photosynthesis. The photosynthetic isoform of PEPC in the cytosol of mesophyll cells in Kranz-type C(4) photosynthesis has distinctive kinetic and regulatory properties. Some species in the Chenopodiaceae family perform C(4) photosynthesis without Kranz anatomy by spatial separation of initial fixation of atmospheric CO(2) via PEPC from C(4) acid decarboxylation and CO(2) donation to Rubisco within individual chlorenchyma cells. We studied molecular and functional features of PEPC in two single-cell functioning C(4) species (Bienertia sinuspersici, Suaeda aralocaspica) as compared to Kranz type (Haloxylon persicum, Salsola richteri, Suaeda eltonica) and C(3) (Suaeda linifolia) chenopods. It was found that PEPC from both types of C(4) chenopods displays higher specific activity than that of the C(3) species and shows kinetic and regulatory characteristics similar to those of C(4) species in other families in that they are subject to light/dark regulation by phosphorylation and display differential malate sensitivity. Also, the deduced amino acid sequence from leaf cDNA indicates that the single-cell functioning C(4) species possesses a Kranz-type C(4) isoform with a Ser in the amino terminal. A phylogeny of PEPC shows that isoforms in the two single-cell functioning C(4) species are in a clade with the C(3) and Kranz C(4) Suaeda spp. with high sequence homology. Overall, this study indicates that B. sinuspersici and S. aralocaspica have a C(4)-type PEPC similar to that in Kranz C(4) plants, which likely is required for effective function of C(4) photosynthesis.