RESUMO
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.
Assuntos
Flaveria/metabolismo , Glicolatos/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Aminoácidos/metabolismo , Dióxido de Carbono/metabolismo , Fotossíntese , Plantas Geneticamente ModificadasRESUMO
The bundle sheath provides a conduit linking veins and mesophyll cells. In the C3 plant Arabidopsis thaliana, it also plays important roles in oxidative stress and sulphur metabolism. However, the mechanisms responsible for the patterns of gene expression that underpin these metabolic specializations are poorly understood. Here, we used the Arabidopsis SULTR2;2 gene as a model to better understand mechanisms that restrict expression to the bundle sheath. Deletion analysis indicated that the SULTR2;2 promoter contains a short region necessary for expression in the bundle sheath and veins. This sequence acts as a positive regulator and is tolerant to multiple consecutive deletions indicating considerable redundancy in the cis-elements involved. It is highly conserved in SULTR2;2 genes of the Brassicaceae and is functional in the distantly related C4 species Flaveria bidentis that belongs to the Asteraceae. We conclude that expression of SULTR2;2 in the bundle sheath and veins is underpinned by a highly redundant sequence that likely represents an ancient and conserved mechanism found in families as diverse as the Asteraceae and Brassicaceae.
Assuntos
Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Folhas de Planta/metabolismo , Feixe Vascular de Plantas/metabolismo , Arabidopsis/metabolismo , Sequência de Bases , Alinhamento de SequênciaRESUMO
C4 photosynthesis is nature's most efficient answer to the dual activity of ribulose-1,5-bisphosphate carboxylase/oxygenase and the resulting loss of CO(2) by photorespiration. Gly decarboxylase (GDC) is the key component of photorespiratory CO(2) release in plants and is active in all photosynthetic tissues of C(3) plants, but only in the bundle sheath cells of C(4) plants. The restriction of GDC to the bundle sheath is assumed to be an essential and early step in the evolution of C(4) photosynthesis, leading to a photorespiratory CO(2) concentrating mechanism. In this study, we analyzed how the P-protein of GDC (GLDP) became restricted to the bundle sheath during the transition from C(3) to C(4) photosynthesis in the genus Flaveria. We found that C(3) Flaveria species already contain a bundle sheath-expressed GLDP gene in addition to a ubiquitously expressed second gene, which became a pseudogene in C(4) Flaveria species. Analyses of C(3)-C(4) intermediate Flaveria species revealed that the photorespiratory CO(2) pump was not established in one single step, but gradually. The knowledge gained by this study sheds light on the early steps in C(4) evolution.
Assuntos
Flaveria/metabolismo , Glicina Desidrogenase (Descarboxilante)/metabolismo , Fotossíntese , Proteínas de Plantas/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismo , Dióxido de Carbono/metabolismo , Evolução Molecular , Flaveria/classificação , Flaveria/genética , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Glicina Desidrogenase (Descarboxilante)/classificação , Glicina Desidrogenase (Descarboxilante)/genética , Isoenzimas/classificação , Isoenzimas/genética , Isoenzimas/metabolismo , Dados de Sequência Molecular , Consumo de Oxigênio/efeitos da radiação , Filogenia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/efeitos da radiação , Proteínas de Plantas/genética , Splicing de RNA , Ribulose-Bifosfato Carboxilase/genética , Especificidade da EspécieRESUMO
The mitochondrial Gly decarboxylase complex (GDC) is a key component of the photorespiratory pathway that occurs in all photosynthetically active tissues of C(3) plants but is restricted to bundle sheath cells in C(4) species. GDC is also required for general cellular C(1) metabolism. In the Asteracean C(4) species Flaveria trinervia, a single functional GLDP gene, GLDPA, encodes the P-subunit of GDC, a decarboxylating Gly dehydrogenase. GLDPA promoter reporter gene fusion studies revealed that this promoter is active in bundle sheath cells and the vasculature of transgenic Flaveria bidentis (C(4)) and the Brassicacean C(3) species Arabidopsis thaliana, suggesting the existence of an evolutionarily conserved gene regulatory system in the bundle sheath. Here, we demonstrate that GLDPA gene regulation is achieved by an intricate interplay of transcriptional and posttranscriptional mechanisms. The GLDPA promoter is composed of two tandem promoters, P(R2) and P(R7), that together ensure a strong bundle sheath expression. While the proximal promoter (P(R7)) is active in the bundle sheath and vasculature, the distal promoter (P(R2)) drives uniform expression in all leaf chlorenchyma cells and the vasculature. An intron in the 5' untranslated leader of P(R2)-derived transcripts is inefficiently spliced and apparently suppresses the output of P(R2) by eliciting RNA decay.
Assuntos
Flaveria/metabolismo , Proteínas de Plantas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Flaveria/genética , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Dados de Sequência Molecular , Proteínas de Plantas/genética , Regiões Promotoras Genéticas/genéticaRESUMO
BACKGROUND: The key enzymes of photosynthetic carbon assimilation in C4 plants have evolved independently several times from C3 isoforms that were present in the C3 ancestral species. The C4 isoform of phosphoenolpyruvate carboxylase (PEPC), the primary CO2-fixing enzyme of the C4 cycle, is specifically expressed at high levels in mesophyll cells of the leaves of C4 species. We are interested in understanding the molecular changes that are responsible for the evolution of this C4-characteristic PEPC expression pattern, and we are using the genus Flaveria (Asteraceae) as a model system. It is known that cis-regulatory sequences for mesophyll-specific expression of the ppcA1 gene of F. trinervia (C4) are located within a distal promoter region (DR). RESULTS: In this study we focus on the proximal region (PR) of the ppcA1 promoter of F. trinervia and present an analysis of its function in establishing a C4-specific expression pattern. We demonstrate that the PR harbours cis-regulatory determinants which account for high levels of PEPC expression in the leaf. Our results further suggest that an intron in the 5' untranslated leader region of the PR is not essential for the control of ppcA1 gene expression. CONCLUSION: The allocation of cis-regulatory elements for enhanced expression levels to the proximal region of the ppcA1 promoter provides further insight into the regulation of PEPC expression in C4 leaves.
Assuntos
Evolução Molecular , Flaveria/genética , Regulação da Expressão Gênica de Plantas , Fosfoenolpiruvato Carboxilase/genética , Regiões Promotoras Genéticas , Regiões 5' não Traduzidas , Sequência de Bases , Flaveria/enzimologia , Íntrons , Dados de Sequência Molecular , Fosfoenolpiruvato Carboxilase/metabolismo , Folhas de Planta/enzimologia , Plantas Geneticamente Modificadas/enzimologia , Deleção de Sequência , Homologia de Sequência do Ácido NucleicoRESUMO
Glycine decarboxylase (GDC) plays an important role in the photorespiratory metabolism of plants. GDC is composed of four subunits (P, H, L, and T) with the P-subunit (GLDP) serving as the actual decarboxylating unit. In C(3) plants, GDC can be found in all photosynthetic cells, whereas in leaves of C(3)-C(4) intermediate and C(4) species its occurrence is restricted to bundle-sheath cells. The specific expression of GLDP in bundle-sheath cells might have constituted a biochemical starting point for the evolution of C(4) photosynthesis. To understand the molecular mechanisms responsible for restricting GLDP expression to bundle-sheath cells, we performed a functional analysis of the GLDPA promoter from the C(4) species Flaveria trinervia. Expression of a promoter-reporter gene fusion in transgenic plants of the transformable C(4) species Flaveria bidentis (C(4)) showed that 1,571 bp of the GLDPA 5' flanking region contain all the necessary information for the specific expression in bundle-sheath cells and vascular bundles. Interestingly, we found that the GLDPA promoter of F. trinervia exhibits a C(4)-like spatial activity also in the C(3) plant Arabidopsis (Arabidopsis thaliana), indicating that a mechanism for bundle-sheath-specific expression is also present in this C(3) species. Using transgenic Arabidopsis, promoter deletion studies identified two regions in the GLDPA promoter, one conferring repression of gene expression in mesophyll cells and one functioning as a general transcriptional enhancer. Subsequent analyses in transgenic F. bidentis confirmed that these two segments fulfill the same function also in the C(4) context.
Assuntos
Arabidopsis/genética , Flaveria/enzimologia , Glicina Desidrogenase (Descarboxilante)/genética , Transcrição Gênica , Sequência de Bases , Primers do DNA , Flaveria/genética , Deleção de Genes , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Hibridização In Situ , Plantas Geneticamente Modificadas , Regiões Promotoras GenéticasRESUMO
C(4) photosynthesis presents a sophisticated integration of two complementary cell types, mesophyll and bundle sheath cells. It relies on the differential expression of the genes encoding the component enzymes and transporters of this pathway. The entry enzyme of C(4) photosynthesis, phosphoenolpyruvate carboxylase (PEPC), is found exclusively in mesophyll cells, and the expression of the corresponding gene is regulated at the transcriptional level. In the C(4) dicot Flaveria trinervia, the mesophyll-specific expression of the C(4) PEPC gene (ppcA) depends on a 41-bp segment in the distal promoter region referred to as MEM1 (for mesophyll expression module1). Here, we show that a MEM1 sequence found in the orthologous ppcA gene from the C(3) species Flaveria pringlei is not able to direct mesophyll-specific gene expression. The two orthologous MEM1 sequences of F. pringlei and F. trinervia differ at two positions, a G-to-A exchange and the insertion of the tetranucleotide CACT. Changes at these two positions in the C(3) MEM1 sequence were necessary and sufficient to create a mesophyll-specificity element during C(4) evolution. The MEM1 of F. trinervia enhances mesophyll expression and concomitantly represses expression in bundle sheath cells and vascular bundles.
Assuntos
Carbono/metabolismo , Evolução Molecular , Flaveria/genética , Regulação da Expressão Gênica de Plantas , Sequências Reguladoras de Ácido Nucleico/genética , Arabidopsis/enzimologia , Arabidopsis/genética , Sequência de Bases , Flaveria/citologia , Genes Reporter , Glucuronidase/metabolismo , Dados de Sequência Molecular , Mutagênese Insercional , Especificidade de Órgãos , Folhas de Planta/citologia , Folhas de Planta/enzimologia , Folhas de Planta/genética , Plantas Geneticamente Modificadas , Polimorfismo Genético , Regiões Promotoras Genéticas , Transcrição GênicaRESUMO
C(4) photosynthesis depends on the strict compartmentalization of CO(2) assimilatory enzymes. cis-regulatory mechanisms are described that ensure mesophyll-specific expression of the gene encoding the C(4) isoform of phosphoenolpyruvate carboxylase (ppcA1) of the C(4) dicot Flaveria trinervia. To elucidate and understand the anatomy of the C(4) ppcA1 promoter, detailed promoter/reporter gene studies were performed in the closely related C(4) species F. bidentis, revealing that the C(4) promoter contains two regions, a proximal segment up to -570 and a distal part from -1566 to -2141, which are necessary but also sufficient for high mesophyll-specific expression of the beta-glucuronidase reporter gene. The distal region behaves as an enhancer-like expression module that can direct mesophyll-specific expression when inserted into the ppcA1 promoter of the C(3) plant F. pringlei. Mesophyll expression determinants were restricted to a 41-bp segment, referred to as mesophyll expression module 1 (Mem1). Evolutionary and functional studies identified the tetranucleotide sequence CACT as a key component of Mem1.