Os03g0411500

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A nuclear gene coding caseinolyse positioned in plastid or mitochondrion regulating early morphogenesis. VYL is a gene in rice.Its RAP ID is Os03g0411500 and its MSU ID is LOC_Os03g29810. The mutant of this gene produces chlorotic leaves throughout the entire growth period.

Annotated Information

Function

The protein encoded by this gene belongs to the peptidase family S14 and hydrolyzes proteins into small peptides in the presence of ATP and magnesium. The protein is transported into mitochondrial matrix and is associated with the inner mitochondrial membrane[1], or plastid inner membrane in plants[2]. The plastidic caseinolytic protease (Clp) of higher plants is an evolutionarily conserved protein degradation apparatus composed of a proteolytic core complex (the P and R rings) and a set of accessory proteins (ClpT, ClpC, and ClpS).[2] Rice yellow leaf mutant vyl, the performance of the entire growth period, new leaves chlorotic phenotype, then gradually turn green from the top down.[2] VYL Arabidopsis Clp protease subunit ClpP6 homologous protein in rice, is one of the subunits of the chloroplast Clp protease, with the Clp protease subunit interactions OsClpP3 and OsClpP4 respectively, play an important role in the biosynthesis of rice chloroplast.[2] What's more, the gene D53 product shares predicted features with the class I Clp ATPase proteins and can form a complex with the a/b hydrolase protein DWARF 14 (D14) and the F-box protein DWARF 3 (D3), two previously identified signalling components potentially responsible for SL(Strigolactones) perception, which means, in a D14- and D3-dependentmanner, SLs induce D53 degradation by the proteasome and abrogate its activity in promoting axillary bud outgrowth.[3] The role and molecular composition of Clps in higher plants has just begun to be unraveled, mostly from studies with the model dicotyledonous plant Arabidopsis (Arabidopsis thaliana).Some researchers isolated a virescent yellow leaf (vyl) mutant in rice (Oryza sativa), which produces chlorotic leaves throughout the entire growth period.[2]

1.The vyl Mutant Displays Reduced Chlorophyll Accumulation

The vyl mutant was derived by transforming tissue cultures of the japonica rice variety Kita-ake. When grown under an alternating light/dark cycle (12 h of light at 30°C/12 h of darkness at 20°C) in a growth chamber, vyl mutant plants displayed a virescent yellow leaf pheno-type, and during development, leaves gradually turned green from their tips (more developed) to their bases (less developed; Fig. 1, A and B). At maturity, the vyl mutant plants also had reduced height and smaller seeds (Fig. 1, C and D). Mutant leaves also contained less chlorophyll than the wild type at various growth stages (Fig. 1E). Additionally, vyl mutants developed chlorotic leaves under different temperature conditions and light/dark cycles, suggesting that the virescent yellow phenotype of the vyl mutant was developmentally regulated but independent of external cues (such as temperature and light) Figure 1.png

2.The vyl Mutant Has Impaired Chloroplast Development

Next, researchers investigated whether the virescent yellow phenotype of the vyl mutant was associated with ultra-structural changes in the chloroplasts. Leaf samples of L3U (upper half of the third leaf), L3L (basal half of the third leaf), and L4 (fourth leaf above the shoot base) were collected from wild-type and vyl mutant seedlings and compared (Fig. 2A). Normally, when the third leaf has fully emerged from the shoot base of a rice plant, the shoot also contains the fourth to the seventh immature leaves. The leaf cells in the L3L and L3U samples contain mature chloroplasts, whereas those in the shoot base and L4 samples contain proplastids and early developing immature chloroplasts (Sugimoto et al., 2004). Similar to the wild type, the chloroplasts from the L3U green leaf sample (already turned green) of vyl mutant seedlings displayed well-developed lamellar structures and were equipped with normally stacked grana and thylakoid membranes (Fig. 2, B and C). By contrast, the chloroplasts from the L3L and L4 pale leaves (still wrapped in leaf sheath) of the vyl mutant had much reduced thylakoid membrane networks compared with wild-type plants (Fig. 2, D–G). This developmental defect is similar to the phenotype reported in the Arabidopsis CLPP6 antisense transgenic plants (Sjögren et al., 2006).

Figure 2 .png

3.The vyl Mutant Has Impaired Photosynthesis

Chloroplasts are the organelles in plant cells that perform photosynthesis; therefore, they play an essential role in plant growth. To test whether the photosynthetic apparatus was affected in vyl mutants, we compared some key parameters of PSI and PSII between vyl and wild-type plants. Distinct differences in photochemical efficiency of PSII (FPSII), electron transport rate (ETR), nonphotochemical quenching (NPQ), and photochemical quenching (Qp) were detected between vyl mutants and the wild type. In contrast, the maximal efficiency of PSII photochemistry (F v /F m ) values was almost comparable between vyl and wild-type plants (Table I). These observations indicate that vyl mutants absorbed much less light energy, as shown by the lower NPQ values. PSII photochemistry was reduced at both the donor and acceptor sites, as indicated by the greatly decreased Qp and ETR in vyl mutants. Notably, the PSII structure appeared intact in the mutant plants (indicated by the equivalent F v /F m values), but the actual FPSII was much lower in the mutants. These differences may underlie the defects in chloroplast biogenesis and retarded growth in the vyl mutant (Fig. 1, C and D) Table 1.png

4.Altered Expression of Genes Associated with Chloroplast Biogenesis and Photosynthesis in vyl Mutants

Chloroplast biogenesis and physiological changes are tightly regulated by the coordinated expression of plastid and nuclear genes during leaf development and facilitated by protein quality control (Kusumi et al., 2010; Clarke, 2012). To examine whether the expression of genes associated with chloroplast biogenesis and photosynthesis was altered in vyl mutants, quantitative real-time reverse transcription (qRT)-PCR was performed on total RNAs extracted from the L4 and L3U leaf sections of wild-type and vyl plants. Compared with the wild type, the transcript levels of Virescent1 (V1), V2, and V3 genes and some other genes encoding components of the plastid and nuclear transcription apparatus Sigma factor 2A (OsSig2A), Ribosomal Protein S15 (rps15) and the subunit RNA polymerase (RpoA and RpoTp) that are highly expressed in early stages of chloroplast development were significantly increased in vyl mutants. In contrast, several genes encoding components of the photosynthesis apparatus RuBisCO large subunit (RbcL), the subunit of photosystem I (PsaA), a core component of Photosystem II (PsbA), light-harvesting complex protein (Lhcp2) and Chlorophyll A/B binding protein1 (Cab1) that are highly expressed in later stages of chloroplast development were significantly reduced in vyl mutants (Fig. 3). These results suggested that VYL plays an important role in regulating chloroplast biogenesis.

Figure 3 .png

5.The vyl Locus Maps to a Putative Gene Encoding OsClpP6

Genetic analysis showed that the virescent yellow leaf phenotype in vyl mutants is controlled by a single recessive nuclear locus, VYL. Using a BC 1 F 2 mapping population of the vyl mutant and 93-11 (an indica variety), we mapped the vyl gene to a 137-kb genomic region on chromosome 3 between the insertion/deletion polymorphism (Indel) markers F17 and I53. Within this region, 17 open reading frames (ORFs) were predicted from published data (http:// www.gramene.org/; Fig. 4A). Genomic sequence analysis revealed that only the 13th ORF (LOC_Os03g29810) carries a single-nucleotide transition (G→T) at the position 1,509 bp from the ATG start codon (Fig. 4B). We obtained the full-length complementary DNA (cDNA) of LOC_Os03g29810 from both the wild type and the vyl mutant by reverse transcription (RT)-PCR. Sequence analysis showed that the full-length VYL cDNA is 780 bp long in the wild type but 842 bp in the mutant. Comparison of the genomic and cDNA sequences revealed that the single-nucleotide substitution in the vyl mutant genome created a new splicing site and the addition of a partial fourth intron sequence in the cDNA (Fig. 4, C and D). The mutant cDNA is predicted to encode a truncated vyl mutant protein (DVYL) lacking the classic Ser protease triad (Ser-His-Asp) in the active site and the polypeptide-binding site (Fig. 4, C and E). Phylogenetic analysis and protein sequence alignment showed that VYL is most closely related to the Arabidopsis ClpP6 protein with a conserved S14_ClpP_2 domain and more remotely related to several other Arabidopsis ClpP subunits, and it apparently represents a single-copy gene in the rice genome (Fig. 4F). To verify the identity of VYL, the plasmid pGVYL, containing a 6-kb genomic DNA fragment consisting of a 2.5-kb upstream sequence, the entire VYL coding region, including nine exons and eight introns, and a 0.6-kb downstream sequence, was constructed and introduced into the vyl mutant. All five transgenic lines containing pGVYL complemented the virescent phenotype of the vyl mutant (Fig. 5). To further confirm that disruption of the VYL gene was responsible for the vyl mutant phenotype, we generated RNA interference transgenic plants in the wild-type Kita-ake background and obtained more than eight independent transgenic lines. qRT-PCR analysis revealed that the expression of VYL was reduced in three tested transgenic lines compared with the wild-type plants. These VYL knockdown transgenic plants showed reduced accumulation of chlorophyll a and chlorophyll b. Together, these results confirmed that LOC_Os03g29810 indeed corresponds to the VYL gene. Figure 4 .png

6.Interactions between VYL, OsClpP3, OsClpP4, OsClpP5, and OsClpT

To gain an insight into the role of VYL in the rice Clp complex assembly, researchers sought to identify the proteins that directly interact with VYL. We first used a mass spectrometry-based tandem affinity purification proteomics approach. We generated a VYL-HBH construct in which the C terminus of VYL was fused with a Hisbiotin tag (Tagwerker et al., 2006), and the fusion gene was driven by the Ubiquitin promoter. The VYL-HBH fusion construct was introduced into the vyl mutant via Agrobacterium tumefaciens-mediated transformation. The VYL-HBH fusion protein transgene recovered the chlorotic phenotype of the vyl mutant, indicating that the fusion protein is biologically functiona SDS-PAGE and immunoblot analyses showed that the VYL-HBH fusion protein and several additional proteins could be effectively purified from the pUbi:: VYL-HBH transgenic plants using Ni 2+ -Sepharose and streptavidin beads (Fig. 8A). The copurified protein bands were excised from the SDS-PAGE gel and analyzed by matrix-assisted laser-desorption ionization time of flight (MALDI-TOF). Three bands were identified with confidence (P , 0.05), including two putative components of the Clp complex (LOC_Os03g29810/ VYL/OsClpP6 and LOC_Os10g43050/OsClpP4) and a vacuolar ATP synthase subunit E (Loc_Oso1g46980/ v-ATP-E; Fig. 8B; Table II). This result suggested that VYL is a bona fide component of the OsClp complex in vivo. To further investigate the role of VYL in the OsClp core proteolytic complex assembly, we cloned the rice homologs of Arabidopsis ClpR1, ClpP3, ClpP5, and ClpT and named them OsClpR1, OsClpP3, OsClpP5, and OsClpT, respectively. It is noticeable that there is only one copy of the OsClpT gene in the rice genome. Using yeast two-hybrid assays, we found that VYL protein directly interacted with OsClpP3 and OsClpP4 but not with vacuolar ATP synthase subunit E (v-ATP-E; Fig. 8C). Furthermore, we found that the yeast strain (Gold Saccharomyces cerevisiae) carrying BD-VYL+AD-OsClpP4 grew and developed the blue color faster than the yeast strain (Gold Saccharomyces cerevisiae) carrying BD-DVYL+AD-OsClpP4 or BD-VYL+AD-OsClpP3 on yeast growth medium containing 5-Bromo-4-chloro-3- indoylla-galactoside (X-a-Gal). Furthermore, the yeast strain(GoldSaccharomycescerevisiae)carryingBD-DVYL+AD-OsClpP3 did not grow even after incubation for 5 d in a growth chamber (Fig. 8D). This result suggests that the C-terminal polypeptide-binding site of VYL likely plays a role in mediating the interaction between VYL with OsClpP3 and OsClpP4. In addition, they also found that OsClpP3 interacted with OsClpT, OsClpP4 interacted with OsClpP5 and OsClpT, and that both OsClpP4 and OsClpT interacted with themselves but OsClpP5 and OsClpP3 did not (Fig. 8E). Due to the self-activation activity of VYL-AD, we were not able to test whether VYL can homodimerize.


Figure 5 .pngFigure 6 .png

Expression

Expression is constitutive in most tissues examined (roots, stems, leaves, leaf sheath, panicle)but most abundant in leaf sections containing chloroplasts in early stages of development,which can be light-mediated.[2] The young chlorotic leaves turn green in later developmental stages, accompanied by alterations in chlorophyll accumulation, chloroplast ultrastructure, and the expression of chloroplast development- and photosynthesis-related genes. Positional cloning revealed that the VYL gene encodes a protein homologous to the Arabidopsis ClpP6 subunit and that it is targeted to the chloroplast. VYL expression is constitutive in most tissues examined but most abundant in leaf sections containing chloroplasts in early stages of development. The mutation in vyl causes premature termination of the predicted gene product and loss of the conserved catalytic triad (serine-histidine-aspartate) and the polypeptide-binding site of VYL. Using a tandem affinity purification approach and mass spectrometry analysis, we identified OsClpP4 as a VYL-associated protein in vivo. In addition, yeast two-hybrid assays demonstrated that VYL directly interacts with OsClpP3 and OsClpP4. Furthermore, we found that OsClpP3 directly interacts with OsClpT, that OsClpP4 directly interacts with OsClpP5 and OsClpT, and that both OsClpP4 and OsClpT can homodimerize. Together, our data provide new insights into the function, assembly, and regulation of Clps in higher plants.[2] To investigate the expression patterns of VYL during chloroplast and leaf development, researchers analyzed VYL expression in different sections of leaves at various leaf developmental stages by qRT-PCR (Fig. 2A). They found that in wild-type plants, VYL was most highly expressed in the L4 section at the early chloroplast and leaf development stage (Fig. 6A). Furthermore, qRT-PCR analysis and histochemical staining of the pVYL::GUS reporter gene transgenic plants showed that VYL was constitutively expressed in young buds, young roots, stems, leaves, leaf shoots, and panicles (Fig. 6, B and C). To test whether VYL expression is regulated by light, They analyzed VYL expression during greening of etiolated seedlings. Wild-type rice plants were grown in continuous darkness for 10 d and subsequently exposed to light for 3, 6, 9, 12, 15, 18, 21, or 24 h. The expression of VYL was highly induced after 3 h of illumination and peaked after 6 h of illumination, then its expression gradually decreased over time, and by 15 h after illumination, its expression returned to the preillumination basal level (Fig. 6D). These observations suggested that VYL likely plays a role in the light regulation of chloroplast development. We next conducted qRT-PCR analysis to examine a possible effect of the vyl mutation on the expression of other genes encoding various components of the rice Clp. We found that OsClpPs, OsClpT, and OsClpR4 all had similar expression patterns to VYL, with a peak accumulation at the early stage of chloroplast development, but the expression levels of these genes were higher in the vyl mutants compared with the wild type (Fig. 7). This observation suggested that there may be a compensatory mechanism to increase the expression of OsClpPs, OsClpT, and OsClpR4 in vyl mutants.

Figure 7 .pngFigure 8 .png

Evolution

ATP-dependent Clp protease proteolytic subunit is an enzyme that in humans is encoded by the CLPP gene.[4][1] It is found in mitochondria and is widely distributed in bacterial species. In several bacteria, such as E. coli, proteins tagged with the SsrA peptide (ANDENYALAA) encoded by tmRNA are digested by Clp proteases.[5]

612px-Protein CLPP PDB 1tg6.png1867.png

Labs working on this gene

National Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University, Nanjing 210095, People’s Republic of China

References

  1. 1.0 1.1 a b "Entrez Gene: CLPP ClpP caseinolytic peptidase, ATP-dependent, proteolytic subunit homolog (E. coli)".
  2. 2.0 2.1 2.2 2.3 2.4 2.5 2.6 Hui Dong et al. A Rice Virescent-Yellow Leaf Mutant Reveals New Insights into the Role and Assembly of Plastid Caseinolytic Protease in Higher Plants. Plant Physiology, 2013, 162(4): 1867-1880
  3. Zhou Feng et al.D14–SCFD3-dependent degradation of D53 regulates strigolactone signaling. Nature,2013. doi:10.1038/nature12878
  4. Bross P, Andresen BS, Knudsen I, Kruse TA, Gregersen N (Feb 1996). "Human ClpP protease: cDNA sequence, tissue-specific expression and chromosomal assignment of the gene". FEBS Lett 377 (2): 249–52. doi:10.1016/0014-5793(95)01353-9. PMID 8543061.
  5. Gottesman S, Roche E, Zhou Y, Sauer RT (1998). "The ClpXP and ClpAP proteases degrade proteins with carboxy-terminal peptide tails added by the SsrA-tagging system". Genes Dev 12 (9): 1338–47. doi:10.1101/gad.12.9.1338. PMC 316764. PMID 9573050

Structured Information