Temperature-mediated shifts in chlorophyll biosynthesis in leaves of chlorophyll b-lacking rice (Oryza sativa L.)

Extreme temperatures have become a threat to crop yields. To maintain plant growth and yield, chlorophyll (Chl) biosynthesis plays a crucial role in adaptation to temperature stress. This study investigated the influence of temperature on the biosynthesis and characteristics of pigments (Chl a, Chl b, and carotenoids) in the leaves of Chl b-lacking mutant rice (Chlorina 1, ch1) and wild-type rice (Norin No.8, wt). The ch1 showed thinner stacked grana caused by a decrease in thylakoid membranes per granum at 15 °C, whereas the destacked grana were observed at 35 °C after 12 h incubation. However, the grana are stacked normally, along with the absence of Chl b, and a significantly decreased amount of Chl a in both wt and ch1 were observed after heat stress exposure, demonstrating that light-harvesting complex II proteins are involved in grana stacking. Ch1 was sensitive to 15 °C during the first 4 h of incubation but it subsequently adapted to the cold environment. In addition, there were no significant differences in the photosynthesis between wt and ch1 after 12 h incubation at 35 °C. Differentially expressed gene (DEGs) analysis revealed that GluRS expression decreased, which resulted in a decline in Chl biosynthesis in wt and ch1 at 35 °C. At 8 h and 12 h, there were no significant differences in the expression of DEGs involved in Chl biosynthesis and degradation between wt and ch1 at 15 °C. ALAD expression in wt and ch1 at 15 °C decreased until it was undetectable. These findings suggested that ch1 may adapt to temperatures ranging from 15 °C to 35 °C.

Transcription Profile Analysis of Chlorophyll Biosynthesis in Leaves of Wild-Type and Chlorophyll b-Deficient Rice (Oryza sativa L.)

Photosynthesis is an essential biological process and a key approach for raising crop yield. However, photosynthesis in rice is not fully investigated. This study reported the photosynthetic properties and transcriptomic profiles of chlorophyll (Chl) b-deficient mutant (ch11) and wild-type rice (Oryza sativa L.). Chl b-deficient rice revealed irregular chloroplast development (indistinct membranes, loss of starch granules, thinner grana, and numerous plastoglobuli). Next-generation sequencing approach application revealed that the differential expressed genes were related to photosynthesis machinery, Chl-biosynthesis, and degradation pathway in ch11. Two genes encoding PsbR (PSII core protein), FtsZ1, and PetH genes, were found to be down-regulated. The expression of the FtsZ1 and PetH genes resulted in disrupted chloroplast cell division and electron flow, respectively, consequently reducing Chl accumulation and the photosynthetic capacity of Chl b-deficient rice. Furthermore, this study found the up-regulated expression of the GluRS gene, whereas the POR gene was down-regulated in the Chl biosynthesis and degradation pathways. The results obtained from RT-qPCR analyses were generally consistent with those of transcription analysis, with the exception of the finding that MgCH genes were up-regulated which enhance the important intermediate products in the Mg branch of Chl biosynthesis. These results indicate a reduction in the accumulation of both Chl a and Chl b. This study suggested that a decline in Chl accumulation is caused by irregular chloroplast formation and down-regulation of POR genes; and Chl b might be degraded via the pheophorbide b pathway, which requires further elucidation.

AbhemC encoding porphobilinogen deaminase plays an important role in chlorophyll biosynthesis and function in albino Ananas comosus var. bracteatus leaves

Background The chimeric leaves of Ananas comosus var. bracteatus are composed of normal green parts (Grs) and albino white parts (Whs). Although the underlying mechanism of albinism in A. comosus var. bracteatus leaves is not fully understood, it is likely associated with the chlorophyll (Chl) biosynthesis. In this biosynthetic process, porphobilinogen deaminase (PBGD) plays a crucial role by catalyzing the conversion of porphobilinogen (PBG) to uroporphyrinogen III (Urogen III). Therefore, its encoding gene AbhemC was investigated here in association with Chl biosynthesis and albinism in chimeric A. comosus var. bracteatus leaves. Methods The Chl content, main Chl biosynthesis precursor content, and main enzyme activity were determined and compared between the Whs and Grs of A. comosus var. bracteatus leaves. In addition, AbhemC was cloned and its transcriptional expression and prokaryotic protein expression were analyzed. Furthermore, RNAi-mediated silencing of AbhemC was produced and assessed in tobacco plants. Results The concentration of Chl a and Chl b in the Grs was significantly higher than that in the Whs, respectively. Additionally, the content of the Chl biosynthesis precursor Urogen III decreased significantly in the Whs compared with the Grs. Thus, the transition of PBG to Urogen III may be the first rate-limiting step leading to albinism in the chimeric leaves of A. comosus var. bracteatus. The gene AbhemC comprised 1,135 bp and was encoded into a protein with 371 amino acids; phylogenetically, AbhemC was most closely related to hemC of pineapple. Prokaryotic expression and in vitro enzyme activity analysis showed that the cloned mRNA sequence of AbhemC was successfully integrated and had PBGD activity. Compared with control plants, transgenic tobacco leaves with pFGC5941-AbhemC-RNAi vector were substantially less green with significantly reduced hemC expression and Chl content, as well as reduced PBGD enzyme activity and significantly decreased content of Chl biosynthesis precursors from Urogen III onwards. Our results suggest that the absence of hemC expression reduces the enzyme activity of PBGD, which blocks the transition of PBG to Urogen III, and in turn suppresses Chl synthesis leading to the pale-green leaf color. Therefore, we suggest that AbhemC plays an important role in Chl synthesis and may be an important factor in the albinism of A. comosus var. bracteatus leaves.

Photocatalytic plant LPOR forms helical lattices that shape membranes for chlorophyll synthesis

Chlorophyll (Chl) biosynthesis, crucial to life on Earth, is tightly regulated because its precursors are phototoxic1. In flowering plants, the enzyme Light-dependent Protochlorophyllide OxidoReductase (LPOR) captures photons to catalyze the penultimate reaction: the reduction of a double-bond within protochlorophyllide (Pchlide) to generate chlorophyllide (Chlide)2,3. In darkness, LPOR oligomerizes to facilitate photon energy transfer and catalysis4,5. However, the complete 3D structure of LPOR, the higher-order architecture of LPOR oligomers, and the implications of these self-assembled states for catalysis, including how LPOR positions Pchlide and the cofactor NADPH, remain unknown. Here we report the atomic structure of LPOR assemblies by electron cryo-microscopy (cryoEM). LPOR polymerizes with its substrates into helical filaments around constricted lipid bilayer tubes. Portions of LPOR and Pchlide insert into the outer membrane leaflet, targeting the product, Chlide, to the membrane for the final reaction site of chlorophyll biosynthesis. In addition to its crucial photocatalytic role, we show that in darkness LPOR filaments directly shape membranes into high-curvature tubules with the spectral properties of the prolammelar body, whose light-triggered disassembly provides lipids for thylakoid assembly. Our structure of the catalytic site, moreover, challenges previously proposed reaction mechanisms6. Together, our results reveal a new and unexpected synergy between photosynthetic membrane biogenesis and chlorophyll synthesis in plants orchestrated by LPOR.

Physiological and Transcriptome Analysis of a Yellow-Green Leaf Mutant in Birch (Betula platyphylla × B. Pendula)

Chlorophyll (Chl)-deficient mutants are ideal materials for the study of Chl biosynthesis, chloroplast development, and photosynthesis. Although the genes encoding key enzymes related to Chl biosynthesis have been well-characterized in herbaceous plants, rice (Oryza sativa L.), Arabidopsis (Arabidopsis thaliana), and maize (Zea mays L.), yellow-green leaf mutants have not yet been fully studied in tree species. In this work, we explored the molecular mechanism of the leaf color formation in a yellow-green leaf mutant (yl). We investigated the differentially expressed genes (DEGs) between yl and control plants (wild type birch (WT) and BpCCR1 overexpression line 11, (C11)) by transcriptome sequencing. Approximately 1163 genes (874 down-regulated and 289 up-regulated) and 930 genes (755 down-regulated and 175 up-regulated) were found to be differentially expressed in yl compared with WT and C11, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis for DEGs revealed that photosynthesis antenna proteins represent the most significant enriched pathway. The expressions of photosynthesis antenna proteins are crucial to the leaf color formation in yl. We also found that Chl accumulate, leaf anatomical structure, photosynthesis, and growth were affected in yl. Taken together, our results not only provide the difference of phenomenal, physiological, and gene expression characteristics in leaves between yl mutant and control plants, but also provide a new insight into the mutation underlying the chlorotic leaf phenotype in birch.

Physiological and transcriptomic analysis of yellow leaf coloration inPopulus deltoidesMarsh

As important deciduous tree,Populus deltoidesMarsh possesses a high ornamental value for its leaves remaining yellow during the non-dormant period. However, little is known about the regulatory mechanism of leaf coloration inPopulus deltoidesMarsh. Thus, we analyzed physiological and transcriptional differences of yellow leaves (mutant) and green leaves (wild-type) ofPopulus deltoidesMarsh. Physiological experiments showed that the contents of chlorophyll (Chl) and carotenoid are lower in mutant, the flavonoid content is not differed significantly between mutant and wild-type. Transcriptomic sequencing was further used to identify 153 differentially expressed genes (DEGs). Functional classifications based on Gene Ontology enrichment and Genomes enrichment analysis indicated that the DEGs were involved in Chl biosynthesis and flavonoid biosynthesis pathway. Among these, geranylgeranyl diphosphate (CHLP) genes associated with Chl biosynthesis showed down-regulation, while chlorophyllase (CLH) genes associated with Chl degradation were up-regulated in yellow leaves. The expression levels of these genes were further confirmed using quantitative real-time PCR (RT-qPCR). Furthermore, the measurement of the main precursors of Chl confirmed that CHLP is vital enzymes for the yellow leaf color phenotype. Consequently, the formation of yellow leaf color is due to disruption of Chl synthesis and catabolism rather than flavonoid content. These results contribute to our understanding of mechanisms and regulation of leaf color variation in poplar at the transcriptional level.

Chloroplast SRP43 acts as a chaperone for glutamyl-tRNA reductase, the rate-limiting enzyme in tetrapyrrole biosynthesis

Assembly of light-harvesting complexes requires synchronization of chlorophyll (Chl) biosynthesis with biogenesis of light-harvesting Chl a/b-binding proteins (LHCPs). The chloroplast signal recognition particle (cpSRP) pathway is responsible for transport of nucleus-encoded LHCPs in the stroma of the plastid and their integration into the thylakoid membranes. Correct folding and assembly of LHCPs require the incorporation of Chls, whose biosynthesis must therefore be precisely coordinated with membrane insertion of LHCPs. How the spatiotemporal coordination between the cpSRP machinery and Chl biosynthesis is achieved is poorly understood. In this work, we demonstrate a direct interaction between cpSRP43, the chaperone that mediates LHCP targeting and insertion, and glutamyl-tRNA reductase (GluTR), a rate-limiting enzyme in tetrapyrrole biosynthesis. Concurrent deficiency for cpSRP43 and the GluTR-binding protein (GBP) additively reduces GluTR levels, indicating that cpSRP43 and GBP act nonredundantly to stabilize GluTR. The substrate-binding domain of cpSRP43 binds to the N-terminal region of GluTR, which harbors aggregation-prone motifs, and the chaperone activity of cpSRP43 efficiently prevents aggregation of these regions. Our work thus reveals a function of cpSRP43 in Chl biosynthesis and suggests a striking mechanism for posttranslational coordination of LHCP insertion with Chl biosynthesis.

A Novel “Oxygen-induced” Greening Process in a Cyanobacterial Mutant Lacking the Transcriptional Activator ChlR Involved in Low-oxygen Adaptation of Tetrapyrrole Biosynthesis

ChlR activates the transcription of the chlAII-ho2-hemN operon in response to low-oxygen conditions in the cyanobacterium Synechocystis sp. PCC 6803. Three genes in the operon encode low-oxygen-type enzymes to bypass three oxygen-dependent reactions in tetrapyrrole biosynthesis. A chlR-lacking mutant, ΔchlR, shows poor photoautotrophic growth due to low chlorophyll (Chl) content under low-oxygen conditions, which is caused by no induction of the operon. Here, we characterized the processes of etiolation of ΔchlR cells in low-oxygen conditions and the subsequent regreening of the etiolated cells upon exposure to oxygen, by HPLC, Western blotting, and low-temperature fluorescence spectra. The Chl content of the etiolated ΔchlR cells incubated under low-oxygen conditions for 7 days was only 10% of that of the wild-type with accumulation of almost all intermediates of the magnesium branch of Chl biosynthesis. Both photosystem I (PSI) and photosystem II (PSII) were significantly decreased, accompanied by a preferential decrease of antenna Chl in PSI. Upon exposure to oxygen, the etiolated ΔchlR cells resumed to produce Chl after a short lag (∼2 h), and the level at 72 h was 80% of that of the wild-type. During this novel “oxygen-induced” greening process, the PSI and PSII contents were largely increased in parallel with the increase in Chl contents. After 72 h, the PSI content reached ∼50% of the wild-type level in contrast to the full recovery of PSII. ΔchlR provides a promising alternative system to investigate the biogenesis of PSI and PSII.

Identification and molecular characterization of a novel Chlamydomonas reinhardtii mutant defective in chlorophyll biosynthesis

The green micro-alga Chlamydomonas reinhardtii is an elegant model organism to study all aspects of oxygenic photosynthesis. Chlorophyll (Chl) and heme are major tetrapyrroles that play an essential role in energy metabolism in photosynthetic organisms and are synthesized via a common branched tetrapyrrole biosynthetic pathway. One of the enzymes in the pathway is Mg chelatase (MgChel) which inserts Mg2+ into protoporphyrin IX (PPIX, proto) to form magnesium-protoporphyrin IX (MgPPIX, Mgproto), the first biosynthetic intermediate in the Chl branch. MgChel is a multimeric enzyme that consists of three subunits designated CHLD, CHLI and CHLH. Plants have two isozymes of CHLI (CHLI1 and CHLI2) which are 70%-81% identical in protein sequences. Although the functional role of CHLI1 is well characterized, that of CHLI2 is not. We have isolated a non-photosynthetic light sensitive mutant 5A7 by random DNA insertional mutagenesis that is devoid of any detectable Chl. PCR based analyses show that 5A7 is missing the CHLI1 gene and at least eight additional functionally uncharacterized genes. 5A7 has an intact CHLI2 gene. Complementation with a functional copy of the CHLI1 gene restored Chl biosynthesis, photo-autotrophic growth and light tolerance in 5A7. We have identified the first chli1 (chli1-1) mutant of Chlamydomonas reinhardtii and in green algae. Our results show that in the wild type Chlamydomonas CHLI2 protein amount is lower than that of CHLI1 and the chli1-1 mutant has a drastic reduction in CHLI2 protein levels although it possesses the CHLI2 gene. Our chli1-1 mutant opens up new avenues to explore the functional roles of CHLI1 and CHLI2 in Chl biosynthesis in Chlamydomonas, which has never been studied before.

Identification and molecular characterization of a novel Chlamydomonas reinhardtii mutant defective in chlorophyll biosynthesis

The green micro-alga Chlamydomonas reinhardtii is an elegant model organism to study all aspects of oxygenic photosynthesis. Chlorophyll (Chl) and heme are major tetrapyrroles that play an essential role in energy metabolism in photosynthetic organisms and are synthesized via a common branched tetrapyrrole biosynthetic pathway. One of the enzymes in the pathway is Mg chelatase (MgChel) which inserts Mg2+ into protoporphyrin IX (PPIX, proto) to form magnesium-protoporphyrin IX (MgPPIX, Mgproto), the first biosynthetic intermediate in the Chl branch. MgChel is a multimeric enzyme that consists of three subunits designated CHLD, CHLI and CHLH. Plants have two isozymes of CHLI (CHLI1 and CHLI2) which are 70%-81% identical in protein sequences. Although the functional role of CHLI1 is well characterized, that of CHLI2 is not. We have isolated a non-photosynthetic light sensitive mutant 5A7 by random DNA insertional mutagenesis that is devoid of any detectable Chl. PCR based analyses show that 5A7 is missing the CHLI1 gene and at least eight additional functionally uncharacterized genes. 5A7 has an intact CHLI2 gene. Complementation with a functional copy of the CHLI1 gene restored Chl biosynthesis, photo-autotrophic growth and light tolerance in 5A7. We have identified the first chli1 mutant of Chlamydomonas reinhardtii and in green algae. Our results show that in the wild type Chlamydomonas CHLI2 protein amount is lower than that of CHLI1 and the chli1 mutant has a drastic reduction in CHLI2 protein levels although it possesses the CHLI2 gene. Our chli1 mutant opens up new avenues to explore the functional roles of CHLI1 and CHLI2 in Chl biosynthesis and chloroplast to nucleus retrograde signaling in Chlamydomonas, which has never been studied before.