Establishing aqueous two-phase flotation coupled with preparative high performance liquid chromatography and its application for the purification of astragalin from Flaveria bidentis.

The strategy of aqueous two-phase flotation (ATPF) followed by preparative high performance liquid chromatography (prep-HPLC) was established and used for the separation of astragalin from Flaveria bidentis. In the ATPF, the effects of sublation solvent, solution pH, (NH4)2SO4 concentration in aqueous solution, cosolvent, N2 flow rate, flotation time and volumes of the PEG phase on the recovery of astragalin were investigated in detail, and the optimal conditions of ATPF were selected: 50 wt% PEG1000 ethanol solvent as the flotation solvent, pH 4, 350 g/L of (NH4)2SO4 concentration in 5 % ethanol aqueous phase, 40 mL/min of N2 flow rate, 30 min of flotation time, 10.0 mL of flotation solvent volume and twice. After ATPF enrichment, the flotation product was further purified by prep-HPLC. As determined by HPLC, the purity of astragalin was 98.8 %.

Increased α-ketoglutarate links the C3-C4 intermediate state to C4 photosynthesis in the genus Flaveria.

As a complex trait, C4 photosynthesis has multiple independent origins in evolution. Phylogenetic evidence and theoretical analysis suggest that C2 photosynthesis, which is driven by glycine decarboxylation in the bundle sheath cell, may function as a bridge from C3 to C4 photosynthesis. However, the exact molecular mechanism underlying the transition between C2 photosynthesis to C4 photosynthesis remains elusive. Here, we provide evidence suggesting a role of higher α-ketoglutarate (AKG) concentration during this transition. Metabolomic data of 12 Flaveria species, including multiple photosynthetic types, show that AKG concentration initially increased in the C3-C4 intermediate with a further increase in C4 species. Petiole feeding of AKG increases the concentrations of C4-related metabolites in C3-C4 and C4 species but not the activity of C4-related enzymes. Sequence analysis shows that glutamate synthase (Fd-GOGAT), which catalyzes the generation of glutamate using AKG, was under strong positive selection during the evolution of C4 photosynthesis. Simulations with a constraint-based model for C3-C4 intermediate further show that decreasing the activity of Fd-GOGAT facilitated the transition from a C2-dominant to a C4-dominant CO2 concentrating mechanism. All these results provide insight into the mechanistic switch from C3-C4 intermediate to C4 photosynthesis.

Evolution of gene regulatory network of C4 photosynthesis in the genus Flaveria reveals the evolutionary status of C3-C4 intermediate species.

C4 photosynthesis evolved from ancestral C3 photosynthesis by recruiting pre-existing genes to fulfill new functions. The enzymes and transporters required for the C4 metabolic pathway have been intensively studied and well documented; however, the transcription factors (TFs) that regulate these C4 metabolic genes are not yet well understood. In particular, how the TF regulatory network of C4 metabolic genes was rewired during the evolutionary process is unclear. Here, we constructed gene regulatory networks (GRNs) for four closely evolutionarily related species from the genus Flaveria, which represent four different evolutionary stages of C4 photosynthesis: C3 (F. robusta), type I C3-C4 (F. sonorensis), type II C3-C4 (F. ramosissima), and C4 (F. trinervia). Our results show that more than half of the co-regulatory relationships between TFs and core C4 metabolic genes are species specific. The counterparts of the C4 genes in C3 species were already co-regulated with photosynthesis-related genes, whereas the required TFs for C4 photosynthesis were recruited later. The TFs involved in C4 photosynthesis were widely recruited in the type I C3-C4 species; nevertheless, type II C3-C4 species showed a divergent GRN from C4 species. In line with these findings, a 13CO2 pulse-labeling experiment showed that the CO2 initially fixed into C4 acid was not directly released to the Calvin-Benson-Bassham cycle in the type II C3-C4 species. Therefore, our study uncovered dynamic changes in C4 genes and TF co-regulation during the evolutionary process; furthermore, we showed that the metabolic pathway of the type II C3-C4 species F. ramosissima represents an alternative evolutionary solution to the ammonia imbalance in C3-C4 intermediate species.

The Evolution of C4 Photosynthesis in Flaveria (Asteraceae): Insights from the Flaveria linearis Complex.

Flaveria is a leading model for C4 plant evolution due to the presence of a dozen C3-C4 intermediate species, many of which are associated with a phylogenetic complex centered around Flaveria linearis. To investigate C4 evolution in Flaveria, we updated the Flaveria phylogeny and evaluated gas exchange, starch δ13C, and activity of C4 cycle enzymes in 19 Flaveria species and 28 populations within the F. linearis complex. A principal component analysis identified six functional clusters: (1) C3, (2) sub-C2, (3) full C2, (4) enriched C2, (5) sub-C4, and (6) fully C4 species. The sub-C2 species lacked a functional C4 cycle, while a gradient was present in the C2 clusters from little to modest C4 cycle activity as indicated by δ13C and enzyme activities. Three Yucatan populations of F. linearis had photosynthetic CO2 compensation points equivalent to C4 plants but showed little evidence for an enhanced C4 cycle, indicating they have an optimized C2 pathway that recaptures all photorespired CO2 in the bundle sheath (BS) tissue. All C2 species had enhanced aspartate aminotransferase activity relative to C3 species and most had enhanced alanine aminotransferase activity. These aminotransferases form aspartate and alanine from glutamate and in doing so could help return photorespiratory nitrogen (N) from BS to mesophyll cells, preventing glutamate feedback onto photorespiratory N assimilation. Their use requires upregulation of parts of the C4 metabolic cycle to generate carbon skeletons to sustain N return to the mesophyll, and thus could facilitate the evolution of the full C4 photosynthetic pathway.

Effects of water extracts of Flaveria bidentis on the seed germination and seedling growth of three plants.

To further explore the mechanism behind the allelopathic effects of Flaveria bidentis, we investigated the allelopathic effects of water extracts from Flaveria bidentis leaves on three plants, Shanghai green, barnyard grass and wheat. The results showed that the water extracts inhibited the germination potential, germination rate, seedling height, root length, chlorophyll content, fresh weight and dry weight of the three plants, and increasing the extract concentration further increased the inhibitory effect. The allelopathic effects of the water extracts from Flaveria bidentis leaves on the three receptor plants differed in strength from strong to weak as follows: Shanghai green > barnyard grass > wheat. Thus, wheat had strong resistance to the allelopathic effects of Flaveria bidentis and could be planted in area where Flaveria bidentis occurs. The effect of the water extract from Flaveria bidentis leaves on the seed germination and seedling growth of barnyard grass was obvious; thus, this extract could be used for the biological control of barnyard grass.

The evolution of stomatal traits along the trajectory toward C4 photosynthesis.

C4 photosynthesis optimizes plant carbon and water relations, allowing high photosynthetic rates with low stomatal conductance. Stomata have long been considered a part of the C4 syndrome. However, it remains unclear how stomatal traits evolved along the path from C3 to C4. Here, we examined stomata in the Flaveria genus, a model used for C4 evolutionary study. Comparative, transgenic, and semi-in vitro experiments were performed to study the molecular basis that underlies the changes of stomatal traits in C4 evolution. The evolution from C3 to C4 species is accompanied by a gradual rather than an abrupt change in stomatal traits. The initial change appears near the Type I intermediate stage. Co-evolution of the photosynthetic pathway and stomatal traits is supported. On the road to C4, stomata tend to be fewer in number but larger in size and stomatal density dominates changes in anatomical maximum stomatal conductance (gsmax). Reduction of FSTOMAGEN expression underlies decreased gsmax in Flaveria and likely occurs in other C4 lineages. Decreased gsmax contributes to the increase in intrinsic water-use efficiency in C4 evolution. This work highlights the stomatal traits in the current C4 evolutionary model. Our study provides insights into the pattern, mechanism, and role of stomatal evolution along the road toward C4.

Overexpression of cytoplasmic C4 Flaveria bidentis carbonic anhydrase in C3 Arabidopsis thaliana increases amino acids, photosynthetic potential, and biomass.

An important method to improve photosynthesis in C3 crops, such as rice and wheat, is to transfer efficient C4 characters to them. Here, cytosolic carbonic anhydrase (CA: ßCA3) of the C4 Flaveria bidentis (Fb) was overexpressed under the control of 35 S promoter in Arabidopsis thaliana, a C3 plant, to enhance its photosynthetic efficiency. Overexpression of CA resulted in a better supply of the substrate HCO3- for the endogenous phosphoenolpyruvate carboxylase in the cytosol of the overexpressers, and increased its activity for generating malate that feeds into the tricarboxylic acid cycle. This provided additional carbon skeleton for increased synthesis of amino acids aspartate, asparagine, glutamate, and glutamine. Increased amino acids contributed to higher protein content in the transgenics. Furthermore, expression of FbßCA3 in Arabidopsis led to a better growth due to expression of several genes leading to higher chlorophyll content, electron transport, and photosynthetic carbon assimilation in the transformants. Enhanced CO2 assimilation resulted in increased sugar and starch content, and plant dry weight. In addition, transgenic plants had lower stomatal conductance, reduced transpiration rate, and higher water-use efficiency. These results, taken together, show that expression of C4 CA in the cytosol of a C3 plant can indeed improve its photosynthetic capacity with enhanced water-use efficiency.

A scheme for C4 evolution derived from a comparative analysis of the closely related C3, C3-C4 intermediate, C4-like, and C4 species in the genus Flaveria.

KEY MESSAGE: A comparative analysis of the genus Flaveria showed a C4 evolutionary process in which the anatomical and metabolic features of C4 photosynthesis were gradually acquired through C3-C4 intermediate stages. C4 photosynthesis has been acquired in multiple lineages of angiosperms during evolution to suppress photorespiration. Crops that perform C4 photosynthesis exhibit high rates of CO2 assimilation and high grain production even under high-temperature in semiarid environments; therefore, engineering C4 photosynthesis in C3 plants is of great importance in the application field. The genus Flaveria contains a large number of C3, C3-C4 intermediate, C4-like, and C4 species, making it a good model genus to study the evolution of C4 photosynthesis, and these studies indicate the direction for C4 engineering. C4 photosynthesis was acquired gradually through the C3-C4 intermediate stage. First, a two-celled C2 cycle called C2 photosynthesis was acquired by localizing glycine decarboxylase activity in the mitochondria of bundle sheath cells. With the development of two-cell metabolism, anatomical features also changed. Next, the replacement of the two-celled C2 cycle by the two-celled C4 cycle was induced by the acquisition of cell-selective expression in addition to the upregulation of enzymes in the C4 cycle during the C3-C4 intermediate stage. This was supported by an increase in cyclic electron transport activity in response to an increase in the ATP/NADPH demand for metabolism. Suppression of the C3 cycle in mesophyll cells was induced after the functional establishment of the C4 cycle, and optimization of electron transport by suppressing the activity of photosystem II also occurred during the final phase of C4 evolution.

Metabolic profiles in C3, C3-C4 intermediate, C4-like, and C4 species in the genus Flaveria.

C4 photosynthesis concentrates CO2 around Rubisco in the bundle sheath, favouring carboxylation over oxygenation and decreasing photorespiration. This complex trait evolved independently in >60 angiosperm lineages. Its evolution can be investigated in genera such as Flaveria (Asteraceae) that contain species representing intermediate stages between C3 and C4 photosynthesis. Previous studies have indicated that the first major change in metabolism probably involved relocation of glycine decarboxylase and photorespiratory CO2 release to the bundle sheath and establishment of intercellular shuttles to maintain nitrogen stoichiometry. This was followed by selection for a CO2-concentrating cycle between phosphoenolpyruvate carboxylase in the mesophyll and decarboxylases in the bundle sheath, and relocation of Rubisco to the latter. We have profiled 52 metabolites in nine Flaveria species and analysed 13CO2 labelling patterns for four species. Our results point to operation of multiple shuttles, including movement of aspartate in C3-C4 intermediates and a switch towards a malate/pyruvate shuttle in C4-like species. The malate/pyruvate shuttle increases from C4-like to complete C4 species, accompanied by a rise in ancillary organic acid pools. Our findings support current models and uncover further modifications of metabolism along the evolutionary path to C4 photosynthesis in the genus Flaveria.

The limiting factors and regulatory processes that control the environmental responses of C3, C3-C4 intermediate, and C4 photosynthesis.

Here, we describe a model of C3, C3-C4 intermediate, and C4 photosynthesis that is designed to facilitate quantitative analysis of physiological measurements. The model relates the factors limiting electron transport and carbon metabolism, the regulatory processes that coordinate these metabolic domains, and the responses to light, carbon dioxide, and temperature. It has three unique features. First, mechanistic expressions describe how the cytochrome b6f complex controls electron transport in mesophyll and bundle sheath chloroplasts. Second, the coupling between the mesophyll and bundle sheath expressions represents how feedback regulation of Cyt b6f coordinates electron transport and carbon metabolism. Third, the temperature sensitivity of Cyt b6f is differentiated from that of the coupling between NADPH, Fd, and ATP production. Using this model, we present simulations demonstrating that the light dependence of the carbon dioxide compensation point in C3-C4 leaves can be explained by co-occurrence of light saturation in the mesophyll and light limitation in the bundle sheath. We also present inversions demonstrating that population-level variation in the carbon dioxide compensation point in a Type I C3-C4 plant, Flaveria chloraefolia, can be explained by variable allocation of photosynthetic capacity to the bundle sheath. These results suggest that Type I C3-C4 intermediate plants adjust pigment and protein distributions to optimize the glycine shuttle under different light and temperature regimes, and that the malate and aspartate shuttles may have originally functioned to smooth out the energy supply and demand associated with the glycine shuttle. This model has a wide range of potential applications to physiological, ecological, and evolutionary questions.

The coordination of major events in C4 photosynthesis evolution in the genus Flaveria.

C4 photosynthesis is a remarkable complex trait, elucidations of the evolutionary trajectory of C4 photosynthesis from its ancestral C3 pathway can help us better understand the generic principles of the evolution of complex traits and guide the engineering of C3 crops for higher yields. Here, we used the genus Flaveria that contains C3, C3-C4, C4-like and C4 species as a system to study the evolution of C4 photosynthesis. We first mapped transcript abundance, protein sequence and morphological features onto the phylogenetic tree of the genus Flaveria, and calculated the evolutionary correlation of different features; we then predicted the relative changes of ancestral nodes of those features to illustrate the major events during the evolution of C4 photosynthesis. We found that gene expression and protein sequence showed consistent modification patterns in the phylogenetic tree. High correlation coefficients ranging from 0.46 to 0.9 among gene expression, protein sequence and morphology were observed. The greatest modification of those different features consistently occurred at the transition between C3-C4 species and C4-like species. Our results show highly coordinated changes in gene expression, protein sequence and morphological features, which support evolutionary major events during the evolution of C4 metabolism.

Change in expression levels of NAD kinase-encoding genes in Flaveria species.

Nicotinamide adenine dinucleotides (NAD(H)) and NAD phosphates (NADP(H)) are electron carriers involved in redox reactions and metabolic processes in all organisms. NAD kinase (NADK) is the only enzyme that phosphorylates NAD+ into NADP+, using ATP as a phosphate donor. In NADP-dependent malic enzyme (NADP-ME)-type C4 photosynthesis, NADP(H) are required for dehydrogenation by NADP-dependent malate dehydrogenase (NADP-MDH) in mesophyll cells, and decarboxylation by NADP-ME in bundle sheath cells. In this study, we identified five NADK genes (FbNADK1a, 1b, 2a, 2b, and 3) from the C4 model species Flaveria bidentis. RNA-Seq database analysis revealed higher transcript abundance in one of the chloroplast-type NADK2 genes of C4F. bidentis (FbNADK2a). Comparative analysis of NADK activity in leaves of C3, C3-C4, and C4Flaveria showed that C4Flaveria (F. bidentis and F. trinervia) had higher NADK activity than the other photosynthetic-types of Flaveria. Taken together, our results suggest that chloroplastic NAD kinase appeared to increase in importance as C3 plants evolved into C4 plants in the genus Flaveria.

Dynamic changes of genome sizes and gradual gain of cell-specific distribution of C4 enzymes during C4 evolution in genus Flaveria.

C4 plants are believed to have evolved from C3 plants through various C3 -C4 intermediate stages in which a photorespiration-dependent CO2 concentration system known as C2 photosynthesis operates. Genes involved in the C4 cycle were thought to be recruited from orthologs present in C3 species and developed cell-specific expression during C4 evolution. To understand the process of establishing C4 photosynthesis, we performed whole-genome sequencing and investigated expression and mesophyll- or bundle-sheath-cell-specific localization of phosphoenolpyruvate carboxylase (PEPC), NADP-malic enzyme (NADP-ME), pyruvate, orthophosphate dikinase (PPDK) in C3 , C3 -C4 intermediate, C4 -like, and C4 Flaveria species. While genome sizes vary greatly, the number of predicted protein-coding genes was similar among C3 , C3 -C4 intermediate, C4 -like, and C4 Flaveria species. Cell-specific localization of the PEPC, NADP-ME, and PPDK transcripts was insignificant or weak in C3 -C4 intermediate species, whereas these transcripts were expressed cell-type specific in C4 -like species. These results showed that elevation of gene expression and cell-specific control of pre-existing C4 cycle genes in C3 species was involved in C4 evolution. Gene expression was gradually enhanced during C4 evolution, whereas cell-specific control was gained independently of quantitative transcriptional activation during evolution from C3 -C4 intermediate to C4 photosynthesis in genus Flaveria.

Overproduction of PGR5 enhances the electron sink downstream of photosystem I in a C4 plant, Flaveria bidentis.

C4 plants can fix CO2 efficiently using CO2 -concentrating mechanisms (CCMs), but they require additional ATP. To supply the additional ATP, C4 plants operate at higher rates of cyclic electron transport around photosystem I (PSI), in which electrons are transferred from ferredoxin to plastoquinone. Recently, it has been reported that the NAD(P)H dehydrogenase-like complex (NDH) accumulated in the thylakoid membrane in leaves of C4 plants, making it a candidate for the additional synthesis of ATP used in the CCM. In addition, C4 plants have higher levels of PROTON GRADIENT REGULATION 5 (PGR5) expression, but it has been unknown how PGR5 functions in C4 photosynthesis. In this study, PGR5 was overexpressed in a C4 dicot, Flaveria bidentis. In PGR5-overproducing (OP) lines, PGR5 levels were 2.3- to 3.0-fold greater compared with wild-type plants. PGR5-like PHOTOSYNTHETIC PHENOTYPE 1 (PGRL1), which cooperates with PGR5, increased with PGR5. A spectroscopic analysis indicated that in the PGR5-OP lines, the acceptor side limitation of PSI was reduced in response to a rapid increase in photon flux density. Although it did not affect CO2 assimilation, the overproduction of PGR5 contributed to an enhanced electron sink downstream of PSI.

Targeted misexpression of NAC052, acting in H3K4 demethylation, alters leaf morphological and anatomical traits in Arabidopsis thaliana.

In an effort to identify genetic regulators for the cell ontogeny around the veins in Arabidopsis thaliana leaves, an activation-tagged mutant line with altered leaf morphology and altered bundle sheath anatomy was characterized. This mutant had a small rosette area with wrinkled leaves and chlorotic leaf edges, as well as enhanced chloroplast numbers in the (pre-)bundle sheath tissue. It had a bundle-specific promoter from the gene GLYCINE DECARBOXYLASE SUBUNIT-T from the C4 species Flaveria trinervia (GLDTFt promoter) inserted in the coding region of the transcriptional repressor NAC052, functioning in H3K4 demethylation, in front of an alternative start codon in-frame with the natural start codon. Reconstruction of the mutation event of our activation-tagged line by creating a line expressing an N-terminally truncated sequence of NAC052 under control of the GLDTFt promoter confirmed the involvement of NAC052 in leaf development. Our study not only reveals leaf anatomic and transcriptomic effects of an N-terminally truncated NAC052 under control of the GLDTFt promoter, but also identifies NAC052 as a novel genetic regulator of leaf development.

Efficient 2-phosphoglycolate degradation is required to maintain carbon assimilation and allocation in the C4 plant Flaveria bidentis.

Photorespiration is indispensable for oxygenic photosynthesis since it detoxifies and recycles 2-phosphoglycolate (2PG), which is the primary oxygenation product of Rubisco. However, C4 plant species typically display very low rates of photorespiration due to their efficient biochemical carbon-concentrating mechanism. Thus, the broader relevance of photorespiration in these organisms remains unclear. In this study, we assessed the importance of a functional photorespiratory pathway in the C4 plant Flaveria bidentis using knockdown of the first enzymatic step, namely 2PG phosphatase (PGLP). The isolated RNAi lines showed strongly reduced amounts of PGLP protein, but distinct signs of the photorespiratory phenotype only emerged below 5% residual PGLP protein. Lines with this characteristic were stunted in growth, had strongly increased 2PG content, exhibited accelerated leaf senescence, and accumulated high amounts of branched-chain and aromatic amino acids, which are both characteristics of incipient carbon starvation. Oxygen-dependent gas-exchange measurements consistently suggested the cumulative impairment of ribulose-1,5-bisphosphate regeneration with increased photorespiratory pressure. Our results indicate that photorespiration is essential for maintaining high rates of C4 photosynthesis by preventing the 2PG-mediated inhibition of carbon utilization efficiency. However, considerably higher 2PG accumulation can be tolerated compared to equivalent lines of C3 plants due to the differential distribution of specific enzymatic steps between the mesophyll and bundle sheath cells.

A single serine to alanine substitution decreases bicarbonate affinity of phosphoenolpyruvate carboxylase in C4Flaveria trinervia.

Phosphoenolpyruvate (PEP) carboxylase (PEPc) catalyzes the first committed step of C4 photosynthesis generating oxaloacetate from bicarbonate (HCO3-) and PEP. It is hypothesized that PEPc affinity for HCO3- has undergone selective pressure for a lower KHCO3 (Km for HCO3-) to increase the carbon flux entering the C4 cycle, particularly during conditions that limit CO2 availability. However, the decrease in KHCO3 has been hypothesized to cause an unavoidable increase in KPEP (Km for PEP). Therefore, the amino acid residue S774 in the C4 enzyme, which has been shown to increase KPEP, should lead to a decrease in KHCO3. Several studies reported the effect S774 has on KPEP; however, the influence of this amino acid substitution on KHCO3 has not been tested. To test these hypotheses, membrane-inlet mass spectrometry (MIMS) was used to measure the KHCO3 of the photosynthetic PEPc from the C4Flaveria trinervia and the non-photosynthetic PEPc from the C3F. pringlei. The cDNAs for these enzymes were overexpressed and purified from the PEPc-less PCR1 Escherichia coli strain. Our work in comparison with previous reports suggests that KHCO3 and KPEP are linked by specific amino acids, such as S774; however, these kinetic parameters respond differently to the tested allosteric regulators, malate and glucose-6-phosphate.

Sulfate Metabolism in C4 Flaveria Species Is Controlled by the Root and Connected to Serine Biosynthesis.

The evolution of C4 photosynthesis led to an increase in carbon assimilation rates and plant growth compared to C3 photosynthetic plants. This enhanced plant growth, in turn, affects the requirement for soil-derived mineral nutrients. However, mineral plant nutrition has scarcely been considered in connection with C4 photosynthesis. Sulfur is crucial for plant growth and development, and preliminary studies in the genus Flaveria suggested metabolic differences in sulfate assimilation along the C4 evolutionary trajectory. Here, we show that in controlled conditions, foliar accumulation of the reduced sulfur compounds Cys and glutathione (GSH) increased with progressing establishment of the C4 photosynthetic cycle in different Flaveria species. An enhanced demand for reduced sulfur in C4 Flaveria species is reflected in high rates of [35S]sulfate incorporation into GSH upon sulfate deprivation and increased GSH turnover as a reaction to the inhibition of GSH synthesis. Expression analyses indicate that the γ-glutamyl cycle is crucial for the recycling of GSH in C4 species. Sulfate reduction and GSH synthesis seems to be preferentially localized in the roots of C4 species, which might be linked to its colocalization with the phosphorylated pathway of Ser biosynthesis. Interspecies grafting experiments of F. robusta (C3) and F. bidentis (C4) revealed that the root system primarily controls sulfate acquisition, GSH synthesis, and sulfate and metabolite allocation in C3 and C4 plants. This study thus shows that evolution of C4 photosynthesis resulted in a wide range of adaptations of sulfur metabolism and points out the need for broader studies on importance of mineral nutrition for C4 plants.

An rbcL mRNA-binding protein is associated with C3 to C4 evolution and light-induced production of Rubisco in Flaveria.

Nuclear-encoded RLSB protein binds chloroplastic rbcL mRNA encoding the Rubisco large subunit. RLSB is highly conserved across all groups of land plants and is associated with positive post-transcriptional regulation of rbcL expression. In C3 leaves, RLSB and Rubisco occur in all chlorenchyma cell chloroplasts, while in C4 leaves these accumulate only within bundle sheath (BS) chloroplasts. RLSB's role in rbcL expression makes modification of its localization a likely prerequisite for the evolutionary restriction of Rubisco to BS cells. Taking advantage of evolutionarily conserved RLSB orthologs in several C3, C3-C4, C4-like, and C4 photosynthetic types within the genus Flaveria, we show that low level RLSB sequence divergence and modification to BS specificity coincided with ontogeny of Rubisco specificity and Kranz anatomy during C3 to C4 evolution. In both C3 and C4 species, Rubisco production reflected RLSB production in all cell types, tissues, and conditions examined. Co-localization occurred only in photosynthetic tissues, and both proteins were co-ordinately induced by light at post-transcriptional levels. RLSB is currently the only mRNA-binding protein to be associated with rbcL gene regulation in any plant, with variations in sequence and acquisition of cell type specificity reflecting the progression of C4 evolution within the genus Flaveria.