OsHemA gene, encoding glutamyl-tRNA reductase(GluTR) is essential for chlorophyll biosynthesis in rice(Oryza sativa)

Chlorophyll(Chl) biosynthesis is essential for photosynthesis and plant growth.Glutamyl-tRNA reductase(GluTR) catalyzes glutamyl-tRNA into glutamate-1-semialdehyde(GSA) and initiates the chlorophyll biosynthesis.Even though the main role of GluTR has been established,the effects caused by natural variations in its corresponding gene remain largely unknown.Here,we characterized a spontaneous mutant in paddy field with Chl biosynthesis deficiency,designated as cbd1.With intact thylakoid lamellar structure,the cbd1 plant showed light green leaves and reduced Chl and carotenoids(Cars) content significantly compared to the wild type.By map-based gene cloning,the mutation was restricted within a 57-kb region on chromosome 10,in which an mPingA miniature inverted-repeat transposable element(MITE) inserted in the promoter region of OsHemA gene.Both leaf color and the pigment contents in cbd1 were recovered in a complementation test,confirming OsHemA was responsible for the mutant phenotype.OsHemA was uniquely predicted to encode GluTR and its expression level was dramatically repressed in cbd1.Transient transformation in protoplasts demonstrated that GluTR localized in chloroplasts and a signal peptide exists in its N-terminus.A majority of Chl biosynthesis genes,except for POR and CHLG,were down-regulated synchronously by the repression of OsHemA,suggesting that an attenuation occurred in the Chl biosynthesis pathway.Interestingly,we found major agronomic traits involved in rice yield were statistically unaffected,except for the number of full grains per panicle was increased in cbd1.Collectively,OsHemA plays an essential role in Chl biosynthesis in rice and its weak allele can adjust leaf color and Chls content without compromise to rice yield.

Positive Regulation of Phytochrome B on Chlorophyll Biosynthesis and Chloroplast Development in Rice

Phytochromes in rice are encoded by a gene family composed of three members, PHYA, PHYB, and PHYC. Through characterizing the phytochrome mutants and wild type （WT） in terms of photomorphogenesis, roles of individual phytochromes have been preliminarily explored in regulating rice de-etiolation, flowering time and fertility. However, little information has been reported about whether or how phytochromes affect chlorophyll biosynthesis and chloroplast development in rice. In this study, we compared the chlorophyll contents of wild type and the phyA, phyB and phyAphyB mutants grown under either white light （WL） or red light （R）. The results suggest that phyB perceives R to positively regulate chlorophyll biosynthesis, while the role of phyA can be detected only in the phyB-deficient mutant. Analyses of the expression levels of genes involved in chlorophyll biosynthesis revealed that phytochromes affected the chlorophyll biosynthesis by regulating protochlorophyll oxidoreductase A （PORA） expression. The role of phyB in chloroplast development was also analyzed, and the results suggest that phyB perceives R to regulate chloroplast development by affecting the numbers of chloroplasts and grana, as well as the chloroplast membrane system.

Manganese toxicity-induced chlorosis in sugarcane seedlings involves inhibition of chlorophyll biosynthesis

Manganese(Mn)toxicity-induced leaf chlorosis limits crop production in acidic soils,but its underlying mechanisms remain unknown.The effects of excessive Mn on chlorophyll(Chl)biosynthesis in sugarcane(Saccharum officinarum L.)leaves were investigated.Under Mn treatment,Chl concentration decreased with Mn accumulation and chlorosis appeared in expanding leaves.Before that,levels of the initial Chl precursor 5-aminolevulinic acid(ALA)and its downstream intermediates decreased,whereas magnesium-protoporphyrin IX monomethyl ester(MgPME)accumulated.Overaccumulation of Mn in leaves downregulated the ALA biosynthetic gene GluTR(encoding glutamyl-tRNA reductase)and MgPME conversion gene MgPMEC(encoding MgPME cyclase),upregulated the ALA biosynthesis inhibitor FLU(encoding FLUORESCENT),but had no significant effect on the expression of other Chl biosynthetic genes.The above Mn-induced changes of Chl precursors and expression of corresponding genes commenced before the Chl decline and leaf chlorosis,and were reversed by ALA supplementation.Thus,excessive Mn-induced chlorosis in sugarcane is mediated by a Chl-biosynthesis disorder resulting from the inhibition of ALA synthesis and MgPME conversion.

DWARF and SMALL SEED1,a Novel Allele of OsDWARF,Controls Rice Plant Architecture,Seed Size,and Chlorophyll Biosynthesis

Plant architecture is a vital agronomic trait to control yield in rice(Oryza sativa L.).A dwarf and small seed 1(dss1)mutant were obtained from the ethyl methanesulfonate(EMS)mutagenized progeny of a Guizhou glutinous landrace cultivar,Lipingzabianhe.The dss1 mutant displayed phenotypes similar to those of brassinosteroid(BR)deficient mutants,such as dwarfing,dark green and rugose erect leaves,small seeds,and loner neck internode panicles with primary branching.In our previous study,the underlying DSS1 gene was isolated,a novel allele of OsDWARF(OsBR6ox)that encodes a cytochrome P450 protein involved in the BR biosynthetic pathway by MutMap technology.In this work,we confirmed that a Thr335Ile amino acid substitution residing in DSS1/OsDWARF was responsible for the dwarf,panicle architecture,and small seed phenotypes in the dss1 mutants by genetic transformation experiments.The overexpression of OsDWARF in the dss1 mutant background could not only recover dss1 to the normal plant height and panicle architecture but also rescued normal leaf angles,seed size,and leaf color.Thus,the specific mutation in DSS1/OsDWARF influenced plant architecture,seed size,and chlorophyll biosynthesis.

Growth response and toxic effects of three antibiotics on Selenastrum capricornutum evaluated by photosynthetic rate and chlorophyll biosynthesis

The effects of three types of antibiotics （erythromycin,ciprofloxacin and sulfamethoxazole） on the photosynthesis of freshwater algae,Selenastrum capricornutum Printz,were investigated by determining the growth rate,chloroplast pigments content,seven main precursors （including δ-aminolevulinic acid,porphobilinogen,uroporphyrinogen III,coproporphyrinogen III,protoporphyrin IX,Mg-proporphyrin IX and protochlorophyllide）,and photosynthetic rate during chlorophyll biosynthesis.The antibiotics significantly decreased the growth rate,chlorophyll content,and photosynthetic rate.Erythromycin induced a decreasing effect at a concentration of 0.06 mg/L,while ciprofloxacin and sulfamethoxazole achieved the same results at concentrations higher than 1.5 mg/L.Only erythromycin significantly inhibited chlorophyll biosynthesis,which indicated that it was considerably more toxic to S.capricornutum than ciprofloxacin and sulfamethoxazole,and may pose a high potential risk to aquatic ecosystems.

The SWi2/SNF2 Chromatin-Remodeling ATPase BRAHMA Regulates Chlorophyll Biosynthesis in Arabidopsis

Chlorophyll biosynthesis is critical for chloroplast development and photosynthesis in plants. Although reactions in the chlorophyll biosynthetic pathway have been largely known, little is known about the regu-latory mechanisms of this pathway. In this study, we found that the dark-grown knockout and knockdown mutants as well as RNA-interference transgenic seedlings of BRAHMA （BRM）, which encodes an SWI2/ SNF2 chromatin-remodeling ATPase, had iligher greening rates, accumulated less protochlorophyllide, and produced less reactive oxygen species than Arabidopsis wild-type plants did upon light exposure. The expression of NADPH：protochlorophyilide oxidoreductase A （PORA）, PORB, and PORC, which catalyze a key step in chlorophyll biosynthesis, was increased in the brm mutants. We found that BRM physically interacted with the bHLH transcription factor PHYTOCHRONIE-iNTERACTING FACTOR 1 （PIF1） through its N-terminal domains. Furthermore, we demonstrated that BRM was directly recruited to the cis-regula-tory regions of PORC, but not of PORA and PORB, at least partially in a PIF1-dependent manner and the level of histone H3 lysine 4 tri-methylation （H3K4me3） at PORC loci was increased in the brm mutant. Taken together, our data indicate that the chromatino-remodeling enzyme BRM modulates PORC expression through interacting with PIF1, providing a novel regulatory mechanism by which plants fine-tune chloro-phyll biosynthesis during the transition from heterotrophic to autotrophic growth.

A pair of light signaling factors FHY3 and FAR1 regulates plant immunity by modulating chlorophyll biosynthesis

Light and chloroplast function is known to affect the plant immune response; however, the underlying mechanism remains elusive. We previously demonstrated that two light signaling factors, FAR-RED ELONGATED HYPOCOTYL 3（FHY3）and FAR-RED IMPAIRED RESPONSE 1（FAR1）, regulate chlorophyll biosynthesis and seedling growth via controlling HEMB1 expression in Arabidopsis thaliana. In this study, we reveal that FHY3 and FAR1 are involved in modulating plant immunity. We showed that the fhy3 far1 double null mutant displayed high levels of reactive oxygen species and salicylic acid（SA） and increased resistance to Pseudomonas syringae pathogen infection. Microarray analysis revealed that a large proportion of pathogen-related genes, particularly genes encoding nucleotide-binding and leucine-rich repeat domain resistant proteins, are highly induced in fhy3 far1. Genetic studies indicated that the defects of fhy3 far1 can be largely rescued by reducing SA signaling or blocking SA accumulation, and by overexpression of HEMB1, which encodes a 5-aminolevulinic acid dehydratase in the chlorophyll biosynthetic pathway.Furthermore, we found that transgenic plants with reduced expression of HEMB1 exhibit a phenotype similar to fhy3 far1.Taken together, this study demonstrates an important role of FHY3 and FAR1 in regulating plant immunity, through integrating chlorophyll biosynthesis and the SA signaling pathway.

Arabidopsis transcription factor TCP4 represses chlorophyll biosynthesis to prevent petal greening

Green petals pose a challenge for pollinators to distinguish flowers from leaves,but they are valuable as a specialty flower trait.However,little is understood about the molecular mechanisms that underlie the development of green petals.Here,we report that CINCINNATA(CIN)-like TEOSINTE BRANCHED 1/CYCLOIDEA/PCF(TCP)proteins play key roles in the control of petal color.The septuple tcp2/3/4/5/10/13/17 mutant produced flowers with green petals due to chlorophyll accumulation.Expression of TCP4 complemented the petal phenotype of tcp2/3/4/5/10/13/17.We found that chloroplasts were converted into leucoplasts in the distal parts of wild-type petals but not in the proximal parts during flower development,whereas plastid conversion was compromised in the distal parts of tcp2/3/4/5/10/13/17 petals.TCP4 and most CIN-like TCPs were predominantly expressed in distal petal regions,consistent with the green–white pattern in wild-type petals and the petal greening observed in the distal parts of tcp2/3/4/5/10/13/17 petals.RNA-sequencing data revealed that most chlorophyll biosynthesis genes were downregulated in the white distal parts of wild-type petals,but these genes had elevated expression in the distal green parts of tcp2/3/4/5/10/13/17 petals and the green proximal parts of wild-type petals.We revealed that TCP4 repressed chlorophyll biosynthesis by directly binding to the promoters of PROTOCHLOROPHYLLIDE REDUCTASE(PORB),DIVINYL REDUCTASE(DVR),and SUPPRESSOR OF OVEREXPRESSION OF CO 1(SOC1),which are known to promote petal greening.We found that the conversion of chloroplasts to leucoplasts and the green coloration in the proximal parts of petals appeared to be conserved among plant species.Our findings uncover a major molecular mechanism that underpins the formation of petal color patterns and provide a foundation for the breeding of plants with green flowers.

Contributions of DPOR at Low Light Intensity to Chlorophyll Biosynthesis and Growth in the Synechocystis sp. PCC 6803

The chlL gene encoding one component of light-independent (dark) protochlorophyllide oxido reductase (DPOR) was deleted in cyanobacterium Synechocystis sp. PCC 6803 (S.6803). The resulting chlL- mutant lost DPOR activity. No significant differences of chlorophyll (Chl) content and growth rate were observed between the wild and the mutant strains grown at 50 mE·m2·s1 light intensity for photomixtrophic and photoautotrophic growth. However, differences were observed at 1 mE·m2·s1 light intensity. For photomixtrophic growth, the mutant Chl content was 50% of the wild content with continuous light and 35.7% of the wild content with a 10 h light/ 14 h dark cycle. For photoautotriphic growth, the mutant Chl level was 76.3% of the wild content with continuous light and 63.2% with a 10 h light/ 14 h dark cycle. The results indicate that DPOR contributes to Chl synthesis and increases the growth rate in cyanobacteria phototrophically cultured at 1mE·m2·s1 light intensity. In contrast, the photosynthetic capacity on a per-cell basis of the mutant is 5% higher than that of the wild strain with continuous light and 27% higher than that of the wild strain with a 10 h light/14 h dark cycle at 1 mE·m2·s1 light intensity for photoautotrophic growth. With the low Chl content, the cyanobacteria have the ability to improve their photosynthetic capacity by decreasing the ratio of PSI to PSII by unknown morphological or physiological means.

A Thylakoid Membrane Protein Functions Synergistically with GUN5 in Chlorophyll Biosynthesis

Chlorophyll(Chl)is essential for photosynthetic reactions and chloroplast development.While the enzymatic pathway for Chl biosynthesis is well established,the regulatory mechanism underlying the homeostasis of Chl levels remains largely unknown.In this study,we identified CBD1(Chlorophyll Biosynthetic Defect1),which functions in the regulation of chlorophyll biosynthesis.The CBD1 gene was expressed specifically in green tissues and its protein product was embedded in the thylakoid membrane.Furthermore,CBD1 was precisely co-expressed and functionally correlated with GUN5(Genome Uncoupled 5).Analysis of chlorophyll metabolic intermediates indicated that cbd1 and cbd1gun5 mutants over-accumulatedmagnesium protoporphyrin Ⅸ(Mg-Proto Ⅸ).In addition,the cbd1 mutant thylakoid contained less Mg than the wild type not only as a result of lower Chl content,but also implicating CBD1 in Mg transport.This was supported by the finding that CBD1 complemented a Mg^(2+)uptake-deficient Salmonella strain under low Mg conditions.Taken together,these results indicate that CBD1 functions synergistically with CHLH/GUN5 in Mg-Proto IX processing,and may serve as a Mg-transport protein to maintain Mg homeostasis in the chloroplast.

Bn.YCO affects chloroplast development in Brassica napus L.

Mature chloroplasts,as the main sites of photosynthesis,are essential for seedling growth in higher plants.Loss of function of genes involved in chloroplast development changes plant phenotype.We obtained a YELLOW COTYLEDON (YCO) mutant in rapeseed (Brassica napus L.) using CRISPR-Cas9.Bn.YCO,a gene of unknown function,has two homologous copies (Bna A01.YCO and Bna C01.YCO) in B.napus.Homozygous mutation of these two homologs resulted in yellow cotyledons and chlorotic true leaves.Transmission electron microscopy revealed that the formation of thylakoid membranes was inhibited in yellow cotyledons.Sequence similarity search revealed that YCO was conserved in different species,and a subcellular location assay verified that Bn.YCO was located in the chloroplast.Bn.YCO was expressed in multiple tissues,most highly in cotyledons.Knockout of Bn.YCO blocked the transcription of plastid genes,especially those of photosystem genes transcribed by plastid-encoded polymerase.Transcriptome sequencing showed that the majority of genes involved in ribosome assembly and photosynthesis were down-regulated in Bn.yco mutants.These results suggested that loss of function of Bn.YCO affected plastid gene transcription,which influenced chloroplast biogenesis in rapeseed seedlings.

Identification and Characterization of a Novel Yellow Leaf Mutant yl1 in Rice

Leaf-color mutants play an important role in the study of chlorophyll metabolism,chloroplast development,and photosynthesis system.In this study,the yellow leaf 1(yl1)rice mutant was identified from the ethyl methane sulfonate-treated mutant progeny of Lailong,a glutinous japonica rice landrace cultivated in Guizhou Province,China.Results showed that yl1 exhibited yellow leaves with decreased chlorophyll content throughout the growth period.Chloroplast development in the yl1 mutant was disrupted,and the grana lamellae was loosely packed and disordered.RNA sequencing and real-time quantitative polymerase chain reaction(qRT-PCR)analysis revealed that the chlorophyll synthesis-related genes OsCHLH,OsCHLM,OsCHLG,PORB,and YGL8,as well as the chloroplast development-related genes FtsZ,OsRpoTp,and RbcL,were down-regulated in the yl1 mutant.Genetic analysis revealed that the yellow leaf phenotype of yl1 was controlled by recessive nuclear gene.By employing the MutMap method,the mutation responsible for the phenotype was mapped to a 6.17 Mb region between 17.34 and 23.51 Mb on chromosome 3.Two non-synonymous single-nucleotide polymorphisms(SNPs)located in the gene locus LOC_Os03g31210 and LOC_Os03g36760 were detected in this region.The two SNPs were further confirmed by PCR and Sanger sequencing.The expression patterns of the two candidate genes indicated that LOC_Os03g36760 showed greater potential for functional verification.Subcellular protein localization revealed that the encoded product of LOC_Os03g36760 was localized in the nucleus,cytoplasm,and plasma membrane.These results will be useful for further characterization and cloning of the yl1 gene,and for research on the molecular mechanisms controlling biogenesis and chloroplast biochemical processes.

Effects of high CO_2 treatment on green-ripening and peel senescence in banana and plantain fruits

Banana fruit（Musa,AAA group,cv.Brazil） peel fails to fully degreen but the pulp ripens normally at temperatures above24°C.This abnormal ripening,known as green-ripening,does not occur in plantains（Musa,ABB group,cv.Dajiao）.Based on the fact that un-completely yellowing was also observed for bananas in poorly ventilated atmospheres,in the present study,the effect of high CO2 with regular O2（21%） on banana ripening was investigated along with that on plantains at20℃.The results showed that high CO2 conferred different effects on the color changing of bananas and plantains.After6 d ripening in 20%CO2,plantains fully yellowed,while bananas retained high chlorophyll content and stayed green.In contrast to the differentiated color changing patterns,the patterns of the softening,starch degradation and soluble sugar accumulation in the pulp of 20%CO2 treated bananas and plantains displayed similarly as the patterns in the fruits ripening in regular air,indicating that the pulp ripening was not inhibited by 20%CO2,and the abnormal ripening of bananas in 20%CO2 can be considered as green ripening.Similar expression levels of chlorophyll degradation related genes,SGR,NYC and PaO,were detected in the peel of the control and treated fruits,indicating that the repressed degreening in 20%CO2treated bananas was not due to the down-regulation of the chlorophyll degradation related genes.Compared to the effect on plantains,20%CO2 treatment delayed the decline in the chlorophyll florescence（F√F_m values and in the mRNA levels of a gene coding small subunit of Rubisco（SSU）,and postponed the disruption of the ultrastructure of chloroplast in the peel tissue of bananas,indicating that the senescence of the green cells in the exocarp layer was delayed by 20%CO2,to more extent in bananas than in plantains.High CO2 reduced the ethylene production and the expression of the related biosynthesis gene,ACS,but elevated the respiration rates in both cultivars.The up-regulation of the expression of anaerobic respiration pathway genes,ADH and PDC,might be responsible for the subtle effect of high CO2 on the pulp ripening.Taken together,the atmosphere of high CO2 and regular O2,delayed the senescence of the green cells in the exocarp layer of the banana peel,but conferred no obvious inhibition on the pulp ripening,leading to a distinct green-ripening that was different from the phenomenon induced by high temperatures.

Chloroplast Proteomics and the Compartmentation of Plastidial Isoprenoid Biosynthetic Pathways

Recent advances in the proteomic field have allowed high-throughput experiments to be conducted on chloroplast samples. Many proteomic investigations have focused on either whole chloroplast or sub-plastidial fractions. To date, the Plant Protein Database （PPDB, Sun et al., 2009） presents the most exhaustive chloroplast proteome available online. However, the accurate localization of many proteins that were identified in different sub-plastidial compartments remains hypothetical. Ferro et al. （2009） went a step further into the knowledge of Arabidopsis thaliana chloroplast proteins with regards to their accurate localization within the chloroplast by using a semi-quantitative proteomic approach known as spectral counting. Their proteomic strategy was based on the accurate mass and time tags （AMT） database approach and they built up AT_CHLORO, a comprehensive chloroplast proteome database with sub-plastidial localization and curated information on envelope proteins. Comparing these two extensive databases, we focus here on about 100 enzymes involved in the synthesis of chloroplast-specific isoprenoids. Well known pathways （i.e. compartmentation of the methyl erythritol phosphate biosynthetic pathway, of tetrapyrroles and chlorophyll biosynthesis and breakdown within chloroplasts） validate the spectral counting-based strategy. The same strategy was then used to identify the precise localization of the biosynthesis of carotenoids and prenylquinones within chloroplasts （i.e. in envelope membranes, stroma, and/or thylakoids） that remains unclear until now.