Role In Chlorophyll Degradation Research Articles

Genome-Wide Identification and Characterization of Tea SGR Family Members Reveal Their Potential Roles in Chlorophyll Degradation and Stress Tolerance

Photosynthesis plays vital roles in plant growth and development. Stay-green (SGR) proteins are responsible for chlorophyll degradation and photosynthetic metabolism. To identify SGR family members and determine their potential functions in tea plants, we identified and cloned three SGR genes. Phylogenetic analysis revealed that the tea SGR homologs were classified into the SGR subfamily (named CsSGR1 and CsSGR2) and the SGRL subfamily (named CsSGRL). Cis-element analysis indicated that the promoters of CsSGR1, CsSGR2 and CsSGRL contained light-, phytohormone- and stress-related elements. Subcellular localization confirmed that CsSGR1 was localized in the chloroplast, while CsSGR2 and CsSGRL were localized in the chloroplast, membrane and nucleus. The RT-qPCR results showed that the three genes in the matures of albino tea cultivars were expressed higher than in the green tea cultivar. However, only CsSGR2-overexpressing tobacco leaves exhibited a yellowish phenotype and significantly lower Fv/Fm values. CsSGR1 and CsSGR2 exhibited similar expression patterns in different tissues after infection with the pathogen Colletotrichum camelliae, which was opposite to the pattern observed for CsSGRL. In addition, CsSGR1 was significantly induced in response to cold stress, SA, JA and ABA in C. camelliae. These findings identified valuable candidate genes for elucidating the mechanism of leaf albinism, stress response and phytohormone signaling in tea plants.

Functional Characterization of the Pheophytinase Gene, ZjPPH, From Zoysia japonica in Regulating Chlorophyll Degradation and Photosynthesis.

Pheophytinase (PPH), the phytol hydrolase, plays important roles in chlorophyll degradation. Nevertheless, little attention has been paid to the PPHs in warm-season grass species; neither its detailed function in photosynthesis has been systematically explored to date. In this study, we isolated ZjPPH from Zoysia japonica, an excellent warm-season turfgrass species. Quantitative real-time PCR analysis and promoter activity characterization revealed that the expression of ZjPPH could be induced by senescence, ABA, and dark induction. Subcellular localization observation proved that ZjPPH was localized in the chloroplasts. Overexpression of ZjPPH accelerated the chlorophyll degradation and rescued the stay-green phenotype of the Arabidopsis pph mutant. Moreover, ZjPPH promoted senescence with the accumulation of ABA and soluble sugar contents, as well as the increased transcriptional level of SAG12 and SAG14. Transmission electron microscopy investigation revealed that ZjPPH caused the decomposition of chloroplasts ultrastructure in stable transformed Arabidopsis. Furthermore, chlorophyll a fluorescence transient measurement analysis suggested that ZjPPH suppressed photosynthesis efficiency by mainly suppressing both photosystem II (PSII) and photosystem I (PSI). In conclusion, ZjPPH plays an important role in chlorophyll degradation and senescence. It could be a valuable target for genetic editing to cultivate new germplasms with stay-green performance and improved photosynthetic efficiency.

Characterization of a novel allele encoding pheophorbide a oxygenase in rice

ABSTRACT We identified a rapid cell death 2 (rcd2) mutant from an indica cultivar Zhongjian100 mutant bank. The red-brown lesions appeared firstly on young seedling leaves, then gradually merged and the leaves completely withered at the late tillering stage. rcd2 displayed apparent cell death at/around the lesions, accumulation of superoxide anion (O2 −) and disturbed ROS scavenging system, impaired photosynthetic capacity with significantly reduced chlorophyll content. The lesion formation was controlled by a single recessive nuclear gene and induced by natural light as well as mechanical wounding. A single base mutation (A1726T) at the 6th exon of OsMH_03G0040800 resulted in I576F substitution in the encoding protein, pheophorbide a oxygenase (PAO). Functional complementation could rescue the mutant phenotype and PAO-knockout lines exhibited the similar phenotype to rcd2. The activity of PAO decreased significantly while the content of PAO substrate, pheophorbide a, increased apparently in rcd2. The expression of chlorophyll synthesis/degradation-related genes and the contents of metabolic intermediates were largely changed. Furthermore, the level of chlorophyllide a, the product of chlorophyllase, increased significantly, indicating chlorophyllase might play a role in chlorophyll degradation in rice. Our results suggested that the I576F substitution disrupted PAO function, leading to O2 − accumulation and chlorophyll degradation breakdown in rice.

A nuclear-localized cysteine desulfhydrase plays a role in fruit ripening in tomato

Hydrogen sulfide (H2S) is a gaseous signaling molecule that plays multiple roles in plant development. However, whether endogenous H2S plays a role in fruit ripening in tomato is still unknown. In this study, we show that the H2S-producing enzyme l-cysteine desulfhydrase SlLCD1 localizes to the nucleus. By constructing mutated forms of SlLCD1, we show that the amino acid residue K24 of SlLCD1 is the key amino acid that determines nuclear localization. Silencing of SlLCD1 by TRV-SlLCD1 accelerated fruit ripening and reduced H2S production compared with the control. A SlLCD1 gene-edited mutant obtained through CRISPR/Cas9 modification displayed a slightly dwarfed phenotype and accelerated fruit ripening. This mutant also showed increased cysteine content and produced less H2S, suggesting a role of SlLCD1 in H2S generation. Chlorophyll degradation and carotenoid accumulation were enhanced in the SlLCD1 mutant. Other ripening-related genes that play roles in chlorophyll degradation, carotenoid biosynthesis, cell wall degradation, ethylene biosynthesis, and the ethylene signaling pathway were enhanced at the transcriptional level in the lcd1 mutant. Total RNA was sequenced from unripe tomato fruit treated with exogenous H2S, and transcriptome analysis showed that ripening-related gene expression was suppressed. Based on the results for a SlLCD1 gene-edited mutant and exogenous H2S application, we propose that the nuclear-localized cysteine desulfhydrase SlLCD1 is required for endogenous H2S generation and participates in the regulation of tomato fruit ripening.

Transcriptomic and physiological analysis of OsCAO1 knockout lines using the CRISPR/Cas9 system in rice.

The altered rice leaf color based on the knockout of CAO1 gene generated using CRISPR/Cas9 technology plays important roles in chlorophyll degradation and ROS scavenging to regulate both natural and induced senescence in rice. Rice chlorophyllide a oxygenase (OsCAO1), identified as the chlorophyll b synthesis under light condition, plays a critical role in regulating rice plant photosynthesis. In this study, the development of edited lines with pale green leaves by knockout of OsCAO1 gene known as a chlorophyll synthesis process is reported. Eighty-one genetically edited lines out of 181 T0 plants were generated through CRISPR/Cas9 system. The edited lines have short narrow flag leaves and pale green leaves compared with wild-type 'Dongjin' plants (WT). Additionally, edited lines have lower chlorophyll b and carotenoid contents both at seedling and mature stages. A transcriptome analysis identified 580 up-regulated and 206 downregulated genes in the edited lines. The differentially expressed genes (DEGs) involved in chlorophyll biosynthesis, magnesium chelatase subunit (CHLH), and glutamate-1-semialdehyde2, 1-aminomutase (GSA) metabolism decreased significantly. Meanwhile, the gel consistency (GC) levels of rice grains, chalkiness ratios and chalkiness degrees (CD) decreased in the edited lines. Thus, knockout of OsCAO1 influenced growth period, leaf development and grain quality characters of rice. Overall, the result suggests that OsCAO1 also plays important roles in chlorophyll degradation and ROS scavenging to regulate both natural and induced rice senescence.

Transcriptomic analysis of Litchi chinensis pericarp during maturation with a focus on chlorophyll degradation and flavonoid biosynthesis.

BackgroundThe fruit of litchi (Litchi chinensis) comprises a white translucent edible aril surrounded by a pericarp. The pericarp of litchi has been the focus of studies associated with fruit size, coloration, cracking and shelf life. However, research at the molecular level has been limited by the lack of genomic and transcriptomic information. In this study, an analysis of the transcriptome of litchi pericarp was performed to obtain information regarding the molecular mechanisms underlying the physiological changes in the pericarp, including those leading to fruit surface coloration.ResultsCoincident with the rapid break down of chlorophyll, but substantial increase of anthocyanins in litchi pericarp as fruit developed, two major physiological changes, degreening and pigmentation were visually apparent. In this study, a cDNA library of litchi pericarp with three different coloration stages was constructed. A total of 4.7 Gb of raw RNA-Seq data was generated and this was then de novo assembled into 51,089 unigenes with a mean length of 737 bp. Approximately 70% of the unigenes (34,705) could be annotated based on public protein databases and, of these, 3,649 genes were significantly differentially expressed between any two coloration stages, while 156 genes were differentially expressed among all three stages. Genes encoding enzymes involved in chlorophyll degradation and flavonoid biosynthesis were identified in the transcriptome dataset. The transcript expression patterns of the Stay Green (SGR) protein suggested a key role in chlorophyll degradation in the litchi pericarp, and this conclusion was supported by the result of an assay over-expressing LcSGR protein in tobacco leaves. We also found that the expression levels of most genes especially late anthocyanin biosynthesis genes were co-ordinated up-regulated coincident with the accumulation of anthocyanins, and that candidate MYB transcription factors that likely regulate flavonoid biosynthesis were identified.ConclusionsThis study provides a large collection of transcripts and expression profiles associated with litchi fruit maturation processes, including coloration. Since most of the unigenes were annotated, they provide a platform for litchi functional genomic research within this species.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1433-4) contains supplementary material, which is available to authorized users.

Spatio-temporal regulation of the OsHFP gene promoter establishes the involvement of this protein in rice anther development

Anther development leading to pollen maturation, anther dehiscence and pollen dispersal depends upon the precise timing of programmed cell death (PCD) in specified anther tissues. The PCD necessitates a properly tuned transcriptional regulation of some crucial genes. However, the detailed genetic regulation of this PCD in rice anther is yet to be deciphered. Recently, we have established that the OsHFP, a structurally novel hemopexin fold protein of rice is a flower-specific heme binding protein, and plays a role in chlorophyll degradation. Here, we report the spatio-temporal transcriptional regulation of the OsHFP gene, which is proposed to be involved in anther PCD. The OsHFP was immunodetected in rice anthers, and OsHFP-related proteins were also found to be present in anthers of other monocot (lily) and dicot (tobacco) plant species. Unique cis-acting elements, possibly involved in the activation and anther-specificity of the OsHFP promoter were identified based upon in silico prediction and in planta expression profiling of the reporter gene driven by the OsHFP promoter (2051bp) and its two deleted versions (1057bp and 437bp). The temporal regulation of the OsHFP promoter in different developmental stages of tobacco anther implies the physiological function of this protein in anther PCD.

A structurally novel hemopexin fold protein of rice plays role in chlorophyll degradation

Proteins containing hemopexin fold domain are suggested to have diverse functions in various living organisms. In order to investigate the structure and function of this type of protein in rice plant (Oryza sativa), the gene encoding a hemopexin fold protein (OsHFP) was cloned, analyzed in silico and characterized. Molecular modeling revealed that the OsHFP is closely related to other hemopexin fold proteins, but is unique with a cylindrical central tunnel as well as extended N- and C-terminal domains. The recombinant OsHFP was found to bind hemin, the oxidized form of heme in vitro. The expression of the single copy OsHFP gene was detected in rice flower buds. Heterologous expression of OsHFP in green leaf tissues resulted in chlorophyll degradation; however, stable expression of OsHFP was observed in transgenic hairy roots, a non-green tissue. The possible role of OsHFP in regulating programmed cell death in anther green tissues of rice is proposed.

Isolation and characterization of a chlorophyll degradation regulatory gene from tall fescue

The non-yellowing gene (NYE1), initially identified from Arabidopsis, is a key regulatory gene responsible for chlorophyll degradation during senescence. Here, FaNYE1, an orthologue of AtNYE1, was further identified from a major type of cool-season turf grass, tall fescue (Festuca arundinacea Schreb.), by RACE-PCR. It consists of 1,441bp, with an open reading frame of 834bp, encoding a predicted polypeptide of 278 amino acids. Sequence similarity as well as exon and intron characteristics clearly suggested that FaNYE1 encoded an AtNYE1-like chloroplast protein. FaNYE1 could be strongly induced by dark treatment and natural senescence. FaNYE1, driven by a 1.5kb upstream fragment of AtNYE1, could rescue the stay-green phenotype of nye1-1. Constitutive overexpression of FaNYE1 in Arabidopsis resulted in the whole spectrum of leaf yellowing phenotypes, the severity of which correlates with its transcript level. These results collectively indicate that FaNYE1 might play an important regulatory role in chlorophyll degradation during senescence in tall fescue, and therefore is a valuable gene for improving the green period or lawn color of turf grasses by genetic engineering.

Reverse Genetic Identification of CRN1 and its Distinctive Role in Chlorophyll Degradation in Arabidopsis

Recent identification of NYE1/SGR1 brought up a new era for the exploration of the regulatory mechanism of Chlorophyll (Chl) degradation. Cluster analysis of senescence associated genes with putative chloroplast targeting sequences revealed several genes sharing a similar expression pattern with NYE1. Further characterization of available T-DNA insertion lines led to the discovery of a novel stay-green gene CRN1 (Co-regulated with NYE1). Chl breakdown was significantly restrained in crn1-1 under diversified senescence scenarios, which is comparable with that in acd1-20, but much more severe than that in nye1-1. Notably, various Chl binding proteins, especially trimeric LHCP II, were markedly retained in crn1-1 four days after dark-treatment, possibly due to a lesion in disassociation of protein-pigment complex. Nevertheless, the photochemical efficiency of PSII in crn1-1 declined, even more rapidly, two days after dark-treatment, compared to those in Col-0 and nye1-1. Our results suggest that CRN1 plays a crucial role in Chl degradation, and that loss of its function produces various side-effects, including those on the breakdown of Ch-protein complex and the maintenance of the residual photosynthetic capability during leaf senescence.

Hormonal Regulation, and Intracellular Localization of a 33-kD Cationic Peroxidase in Excised Cucumber Cotyledons

Ethylene enhanced chlorosis and levels of 33-kilodalton cationic peroxidase (33-CPO) in excised cucumber (Cucumis sativus L. cv ;Poinsett 76') cotyledons. Compared to other hormones, such as kinetin, indoleacetic acid, gibberellic acid, and abscisic acid, ethylene was the only effective promoter of 33-CPO synthesis. The hypothesis that peroxidase plays a role in chlorophyll degradation was tested by comparing levels of 33-CPO in cotyledons treated with compounds thought to either retard (kinetin, indoleacetic acid and gibberellic acid), or promote (abscisic acid and methyl jasmonate [MJ]) senescence. It was concluded that 33-CPO did not play a role in senescence since no direct correlation between chlorophyll content and 33-CPO was observed. MJ was as effective as ethylene in inducing senescence. However, ethylene did not appear to be involved in the action of MJ. Using immunocytochemistry, 33-CPO was found to be located primarily around starch grains and near the plasmalemma. High levels of 33-CPO were also found in cells destined to be vascular tissue.