Virescent Mutant Research Articles

Mutations in the chloroplast inner envelope protein TIC100 impair and repair chloroplast protein import and impact retrograde signaling.

Chloroplast biogenesis requires synthesis of proteins in the nucleocytoplasm and the chloroplast itself. Nucleus-encoded chloroplast proteins are imported via multiprotein translocons in the organelle’s envelope membranes. Controversy exists around whether a 1-MDa complex comprising TIC20, TIC100, and other proteins constitutes the inner membrane TIC translocon. The Arabidopsis thaliana cue8 virescent mutant is broadly defective in plastid development. We identify CUE8 as TIC100. The tic100cue8 mutant accumulates reduced levels of 1-MDa complex components and exhibits reduced import of two nucleus-encoded chloroplast proteins of different import profiles. A search for suppressors of tic100cue8 identified a second mutation within the same gene, tic100soh1, which rescues the visible, 1 MDa complex-subunit abundance, and chloroplast protein import phenotypes. tic100soh1 retains but rapidly exits virescence and rescues the synthetic lethality of tic100cue8 when retrograde signaling is impaired by a mutation in the GENOMES UNCOUPLED 1 gene. Alongside the strong virescence, changes in RNA editing and the presence of unimported precursor proteins show that a strong signaling response is triggered when TIC100 function is altered. Our results are consistent with a role for TIC100, and by extension the 1-MDa complex, in the chloroplast import of photosynthetic and nonphotosynthetic proteins, a process which initiates retrograde signaling.

Rice TSV2 encoding threonyl-tRNA synthetase is needed for early chloroplast development and seedling growth under cold stress.

Threonyl-tRNA synthetase (ThrRS), one of the aminoacyl-tRNA synthetases (AARSs), plays a crucial role in protein synthesis. However, the AARS functions on rice chloroplast development and growth were not fully appraised. In this study, a thermo-sensitive virescent mutant tsv2, which showed albino phenotype and lethal after the 4-leaf stage at 20°C but recovered to normal when the temperatures rose, was identified and characterized. Map-based cloning and complementation tests showed that TSV2 encoded a chloroplast-located ThrRS protein in rice. The Lys-to-Arg mutation in the anticodon-binding domain hampered chloroplast development under cold stress, while the loss of function of the ThrRS core domain in TSV2 fatally led to seedling death regardless of growing temperatures. In addition, TSV2 had a specific expression in early leaves. Its disruption obviously resulted in the downregulation of certain genes associated with chlorophyll biosynthesis, photosynthesis, and chloroplast development at cold conditions. Our observations revealed that rice nuclear-encoded TSV2 plays an important role in chloroplast development at the early leaf stage under cold stress.

Genome sequencing identified novel mechanisms underlying virescent mutation in upland cotton Gossypiuma hirsutum

BackgroundVirescent mutation broadly exists in plants and is an ideal experimental material to investigate regulatory mechanisms underlying chlorophyll synthesis, photosynthesis and plant growth. Up to date, the molecular mechanisms in two virescent mutations have been clarified in cottons (Gossypiuma hirsutum). A virescent mutation has been found in the cotton strain Sumian 22, and the underlying molecular mechanisms have been studied.MethodsThe virescent mutant and wild type (WT) of Sumian 22 were cross-bred, and the F1 population were self-pollinated to calculate the segregation ratio. Green and yellow leaves from F2 populations were subjected to genome sequencing and bulked-segregant analysis was performed to screen mutations. Real-time quantitative PCR (RT-qPCR) were performed to identify genes in relations to chlorophyll synthesis. Intermediate products for chlorophyll synthesis were determined to validate the RT-qPCR results.ResultsThe segregation ratio of green and virescent plants in F2 population complied with 3:1. Compared with WT, a 0.34 Mb highly mutated interval was identified on the chromosome D10 in mutant, which contained 31 genes. Among them, only ABCI1 displayed significantly lower levels in mutant than in WT. Meanwhile, the contents of Mg-protoporphyrin IX, protochlorophyllide, chlorophyll a and b were all significantly lower in mutant than in WT, which were consistent with the inhibited levels of ABCI1. In addition, a mutation from A to T at the -317 bp position from the start codon of ABCI1 was observed in the genome sequence of mutant.ConclusionsInhibited transcription of ABCI1 might be the mechanism causing virescent mutation in Sumian 22 cotton, which reduced the transportation of protoporphyrin IX to plastid, and then inhibited Mg-protoporphyrin IX, Protochlorophyllide and finally chlorophyll synthesis. These results provided novel insights into the molecular mechanisms underlying virescent mutation in cotton.

Retrograde signalling in a virescent mutant triggers an anterograde delay of chloroplast biogenesis that requires GUN1 and is essential for survival.

Defects in chloroplast development are ‘retrograde-signalled’ to the nucleus, reducing synthesis of photosynthetic or related proteins. The Arabidopsis cue8 mutant manifests virescence, a slow-greening phenotype, and is defective at an early stage in plastid development. Greening cotyledons or early leaf cells of cue8 exhibit immature chloroplasts which fail to fill the available cellular space. Such chloroplasts show reduced expression of genes of photosynthetic function, dependent on the plastid-encoded polymerase (PEP), while the expression of genes of housekeeping function driven by the nucleus-encoded polymerase (NEP) is elevated, a phenotype shared with mutants in plastid genetic functions. We attribute this phenotype to reduced expression of specific PEP-controlling sigma factors, elevated expression of RPOT (NEP) genes and maintained replication of plastid genomes (resulting in densely coalesced nucleoids in the mutant), i.e. it is due to an anterograde nucleus-to-chloroplast correction, analogous to retention of a juvenile plastid state. Mutants in plastid protein import components, particularly those involved in housekeeping protein import, also show this ‘retro-anterograde’ correction. Loss of CUE8 also causes changes in mRNA editing. The overall response has strong fitness value: loss of GUN1, an integrator of retrograde signalling, abolishes elements of it (albeit not others, including editing changes), causing bleaching and eventual seedling lethality upon cue8 gun1. This highlights the adaptive significance of virescence and retrograde signalling.This article is part of the theme issue ‘Retrograde signalling from endosymbiotic organelles’.

The tomato WV gene encoding a thioredoxin protein is essential for chloroplast development at low temperature and high light intensity

BackgroundChloroplast biogenesis, a complex process in higher plants, is the key to photoautotrophic growth in plants. White virescent (wv) mutants have been used to unfold the molecular mechanisms underlying the regulation of chloroplast development and chloroplast gene expression in plants. However, most of genes controlling white virescent phenotype still remain unknown.ResultsIn this study, we identified a temperature- and light intensity-sensitive mutant, named as wv. The content of chlorophyll was dramatically decreased in the immature leaves of wv mutant under the conditions of low temperature and high-light intensity. TEM observation showed that the chloroplasts in the young leaves of wv mutant lacked an organized thylakoid membrane, whereas crescent-shaped chloroplasts with well-developed stromal and stacked grana thylakoids in the mature leaves were developed. Immunoblot analyses suggested that proteins of photosynthetic complexes were decreased substantially in wv mutants. Based on map-based cloning and transgenic analysis, we determined that the wv phenotype was caused by single base mutation in the first intron of WV gene, which encoded a thioredoxin protein with 365 amino acids. qRT-PCR analysis revealed that the expression of WV gene was significantly down-regulated in wv mutant. In addition, knockdown of WV gene through RNAi also resulted in white virescent young leaves, suggesting that the mutation possibly blocks the differentiation of chloroplasts through inhibiting the expression of WV gene. Furthermore, the expression of WV peaked in apical buds and gradually decreased along with the developmental stage, which was consistent with the wv mutant phenotype. Expression analysis of chloroplast-encoded genes by qRT-PCR showed that the wv mutation affected the expression pattern of chloroplast-encoded PEP dependent genes.ConclusionOur results suggested that wv mutant was sensitive to low temperature and light intensity. WV gene was essential for chloroplast differentiation. A single base mutation in the first intron resulted in down-regulation of WV gene expression, which inhibited the expression of chloroplast-encoded genes, thereby blocking chloroplast formation and chlorophyll synthesis.

Fine mapping and molecular characterization of the virescent gene vsp in Upland cotton (Gossypium hirsutum).

The vsp gene was fine mapped to a 353.7-kb region, and a 201-bp deletion that affected chloroplast development and chlorophyll biosynthesis was found in the candidate gene GhPUR4. Virescent mutations can be used as marker traits in heterosis breeding and can also be used to research chloroplast development, chlorophyll biosynthesis and photosynthesis mechanisms. Here, we obtained a light-sensitive virescent mutant, vsp, that has reduced chlorophyll (Chl) content and abnormal chloroplast development. Then, the virescent space (vsp) gene in the vsp mutant was preliminarily mapped to a 38.32-Mb region of chromosome D04 using a high-density SNP genetic map with a total length of 5384.33cM and 4472 bin markers. Furthermore, the vsp gene was narrowed down to a 353.7-kb region that contains 15 candidate genes using 484 virescent individuals from an F2 population. Sequence analysis of genes in this region showed that a 201-bp deletion was present in the Gh_D04G1108 (GhPUR4) gene in the vsp mutant. The 201-bp deletion of Gh_D04G1108 caused the deletion of 67 AAs in the GhPUR4 protein. Virus-induced gene silencing (VIGS) of GhPUR4 in normal plants caused reduced GhPUR4 gene expression levels, reduced Chl content, abnormal chloroplast development and virescent true leaves. This study could help us unravel the function of GhPUR4 in chloroplast development and Chl biosynthesis at the early developmental stages of the true leaves in cotton, which could promote the research and application of virescent mutations in cotton heterosis breeding.

A Single Nucleotide Mutation of the IspE Gene Participating in the MEP Pathway for Isoprenoid Biosynthesis Causes a Green-Revertible Yellow Leaf Phenotype in Rice.

Plant isoprenoids are dependent on two independent pathways, the cytosolic mevalonate (MVA) pathway and the plastidic methylerythritol phosphate (MEP) pathway. IspE is one of seven known enzymes in the MEP pathway. Currently, no IspE gene has been identified in rice. In addition, no virescent mutants have been reported to result from a gene mutation affecting the MEP pathway. In this study, we isolated a green-revertible yellow leaf mutant gry340 in rice. The mutant exhibited a reduced level of photosynthetic pigments, and an arrested development of chloroplasts and mitochondria in its yellow leaves. Map-based cloning revealed a missense mutation in OsIspE (LOC_Os01g58790) in gry340 mutant plants. OsIspE is constitutively expressed in all tissues, and its encoded protein is targeted to the chloroplast. Further, the mutant phenotype of gry340 was rescued by introduction of the wild-type gene. Therefore, we have successfully identified an IspE gene in monocotyledons via map-based cloning, and confirmed that the green-revertible yellow leaf phenotype of gry340 does result from a single nucleotide mutation in the IspE gene. In addition, the ispE ispF double mutant displayed an etiolation lethal phenotype, indicating that the isoprenoid precursors from the cytosol cannot efficiently compensate for the deficiency of the MEP pathway in rice chloroplasts. Furthermore, real-time quantitative reverse transcription-PCR suggested that this functional defect in OsIspE affected the expression of not only other MEP pathway genes but also that of MVA pathway genes, photosynthetic genes and mitochondrial genes.

Rice TSV3 Encoding Obg-Like GTPase Protein Is Essential for Chloroplast Development During the Early Leaf Stage Under Cold Stress.

The Spo0B-associated GTP-binding (Obg) proteins are essential for the viability of nearly all bacteria. However, the detailed roles of Obg proteins in higher plants have not yet been elucidated. In this study, we identified a novel rice (Oryza sativa L.) thermo-sensitive virescent mutant (tsv3) that displayed an albino phenotype at 20° before the three-leaf stage while being a normal green at 32° or even at 20° after the four-leaf stage. The mutant phenotype was consistent with altered chlorophyll content and chloroplast structure in leaves. Map-based cloning and complementation experiments showed that TSV3 encoded a small GTP-binding protein. Subcellular localization studies revealed that TSV3 was localized to the chloroplasts. Expression of TSV3 was high in leaves and weak or undetectable in other tissues, suggesting a tissue-specific expression of TSV3. In the tsv3 mutant, expression levels of genes associated with the biogenesis of the chloroplast ribosome 50S subunit were severely decreased at the three-leaf stage under cold stress (20°), but could be recovered to normal levels at a higher temperature (32°). These observations suggest that the rice nuclear-encoded TSV3 plays important roles in chloroplast development at the early leaf stage under cold stress.

Fine mapping and candidate gene analysis of the virescent gene v 1 in Upland cotton (Gossypium hirsutum).

The young leaves of virescent mutants are yellowish and gradually turn green as the plants reach maturity. Understanding the genetic basis of virescent mutants can aid research of the regulatory mechanisms underlying chloroplast development and chlorophyll biosynthesis, as well as contribute to the application of virescent traits in crop breeding. In this study, fine mapping was employed, and a recessive gene (v 1) from a virescent mutant of Upland cotton was narrowed to an 84.1-Kb region containing ten candidate genes. The GhChlI gene encodes the cotton Mg-chelatase I subunit (CHLI) and was identified as the candidate gene for the virescent mutation using gene annotation. BLAST analysis showed that the GhChlI gene has two copies, Gh_A10G0282 and Gh_D10G0283. Sequence analysis indicated that the coding region (CDS) of GhChlI is 1269bp in length, with three predicted exons and one non-synonymous nucleotide mutation (G1082A) in the third exon of Gh_D10G0283, with an amino acid (AA) substitution of arginine (R) to lysine (K). GhChlI-silenced TM-1 plants exhibited a lower GhChlI expression level, a lower chlorophyll content, and the virescent phenotype. Analysis of upstream regulatory elements and expression levels of GhChlI showed that the expression quantity of GhChlI may be normal, and with the development of the true leaf, the increase in the Gh_A10G0282 dosage may partially make up for the deficiency of Gh_D10G0283 in the v 1 mutant. Phylogenetic analysis and sequence alignment revealed that the protein sequence encoded by the third exon of GhChlI is highly conserved across diverse plant species, in which AA substitutions among the completely conserved residues frequently result in changes in leaf color in various species. These results suggest that the mutation (G1082A) within the GhChlI gene may cause a functional defect of the GhCHLI subunit and thus the virescent phenotype in the v1 mutant. The GhChlI mutation not only provides a tool for understanding the associations of CHLI protein function and the chlorophyll biosynthesis pathway but also has implications for cotton breeding.

TSV, a putative plastidic oxidoreductase, protects rice chloroplasts from cold stress during development by interacting with plastidic thioredoxin Z.

Rice is vulnerable to cold stress. Seedlings are very sensitive to cold stress and this harms global rice production. The effects of cold on chloroplast development are well known, but little is known about the underlying molecular mechanisms. Here, we isolated a temperature-sensitive virescent (tsv) mutant that is extremely sensitive to cold stress. It displayed defective chloroplasts, decreased chlorophyll and zero survivorship under cold stress. We isolated and identified TSV by map-based cloning and rescue experiments, combined with genetic, cytological and molecular biological analyses. We found that TSV, a putative plastidic oxidoreductase, is a new type of virescent protein. A mutation in tsv causes premature termination of the gene product. The activity of plastid-encoded RNA polymerase (PEP) and the expression of genes participating in chlorophyll synthesis were severely reduced in the tsv mutant under cold stress, but not at normal temperatures. TSV expression was induced by low temperatures. Strikingly, TSV interacted with OsTrxZ (a subunit of PEP in chloroplasts) and enhanced OsTrxZ stability under low temperatures. We demonstrated that TSV protects rice chloroplasts from cold stress by interacting with OsTrxZ. These results provide novel insights into ways in which rice chloroplast development and chlorophyll synthesis are protected by TSV under cold stress.

A Putative Chloroplast Thylakoid Metalloprotease VIRESCENT3 Regulates Chloroplast Development in Arabidopsis thaliana

The chloroplast is the site of photosynthesis and many other essential plant metabolic processes, and chloroplast development is an integral part of plant growth and development. Mutants defective in chloroplast development can display various color phenotypes including the intriguing virescence phenotype, which shows yellow/white coloration at the leaf base and greening toward the leaf tip. Through large scale genetic screens, we identified a series of new virescent mutants including virescent3-1 (vir3-1), vir4-1, and vir5-1 in Arabidopsis thaliana. We showed that VIR3 encodes a putative chloroplast metalloprotease by map-based cloning. Through site-directed mutagenesis, we showed that the conserved histidine 235 residue in the zinc binding motif HEAGH of VIR3 is indispensable for VIR3 accumulation in the chloroplast. The chloroplast localization of VIR3 was confirmed by the transient expression of VIR3-GFP in leaf protoplasts. Furthermore, taking advantage of transgenic lines expressing VIR3-FLAG, we demonstrated that VIR3 is an intrinsic thylakoid membrane protein that mainly resides in the stromal lamellae. Moreover, topology analysis using transgenic lines expressing a dual epitope-tagged VIR3 indicated that both the N and C termini of VIR3 are located in the stroma, and the catalytic domain of VIR3 is probably facing the stroma. Blue native gel analysis indicated that VIR3 is likely present as a monomer or part of a small complex in the thylakoid membrane. This work not only implicates VIR3 as a new factor involved in early chloroplast development but also provides more insight into the roles of chloroplast proteases in chloroplast biogenesis.

Assimilatory pigments, photosynthetic activity and ultrastructure of chloroplasts of the variegated-leaf chimera of Acer platanoides L.

The assimilatory pigment composition, photosynthetic activity and ultrastructure of chloroplasts were studied in the chlorophyll - deficient chimera of <i>Acer platanoides</i> L. Part of the crown of this chimera is a virescent mutant with variegated leaves. It was found that there exists no qualitative difference in the pigment composition between normal and variegated leaves. The accumulation of chlorophyll in the mutated part is more delayed that the accumulation of carotenoids. The photosynthetic rate on a chlorophyll basis is much higher in variegated than in green leaves. This difference gradually falls off with development. In the early spring, chloroplasts from the yellow spots of leaf blade have no lamellar system but only many vesicles are dispersed in the stroma. Occasionally also a single granum consisting of a few thylakoids occurs in the stroma. At the end of summer chloroplasts from yellow spots of variegated leaves possess a poorly developed lamellar system.

The rice OsV4 encoding a novel pentatricopeptide repeat protein is required for chloroplast development during the early leaf stage under cold stress

Pentatricopeptide repeat (PPR) proteins, characterized by tandem arrays of a 35 amino acid motif, have been suggested to play central and broad roles in modulating the expression of organelle genes in plants. However, the molecular mechanisms of most rice PPR genes remains unclear. In this paper, we isolated and characterized a temperature-conditional virescent mutant, OsV4, in rice (Oryza sativa cultivar Jiahua1 (WT, japonica rice variety)). The mutant displays albino phenotype and abnormal chloroplasts at the three leaf stage, which gradually turns green after the four leaf stage at a low temperature (20 °C). But the mutant always develops green leaves and well-developed chloroplasts at a high temperature (32 °C). Genetic and molecular analyses uncovered that OsV4 encodes a novel chloroplast-targeted PPR protein including four PPR motifs. Further investigations show that the mutant phenotype is associated with changes in chlorophyll content and chloroplast development. The OsV4 transcripts only accumulate to high levels in young leaves, indicating that its expression is tissue-specific. In addition, transcript levels of some ribosomal components and plastid-encoded polymerase-dependent genes are dramatically reduced in the albino mutants grown at 20 °C. These findings suggest that OsV4 plays an important role during early chloroplast development under cold stress in rice.

Cytological and genetic analysis of a virescent mutant in upland cotton (Gossypium hirsutum L.)

It is well known that genetic mutation could be generated by physical treatment (for example, γ-irradiation) and chemical treatment (for example methylnitrosourea and ethyl methanesulfonate). Here we reported identification of a virescent mutation (vsp) after exposing the upland cotton (Gossypium hirsutum L.) CCRI58 seeds in space environments. vsp mutant was characterized at the morphological, agronomic, cellular and genetic levels. vsp mutant showed an earlier virescence and specific only to true leaves. Agronomic traits of vsp mutant, such as plant height, number of bolls, boll weight, yield and fiber quality were significantly lower than those of CCRI 58. Chlorophyll level, carotenoid level and photochemical efficiency of vsp mutant true leaves were significantly lower compared to CCRI 58 at young leave stage. Anatomical studies of chloroplasts showed that vsp mutant lacked grana in the thylakoids of the mesophyll cells at young leave stage, while CCRI 58 showed normal grana in the thylakoids of the mesophyll cells at young leave stage. This indicated that chlorophyll and carotenoid levels were related with chloroplast structure. Genetic analysis indicated that vsp was controlled by one recessive gene in nucleus. Allelic tests showed that vsp was nonallelic to 12 virescent genes currently available at Anyang, China. In summary, we identified a vsp mutant after exposing the upland cotton (Gossypium hirsutum L.) seeds in space environments. vsp could be a newly identified virescent gene. vsp may also be used as a marker in cotton breeding programs. Exposing seeds in space environments could cause new spectrum of genetic mutations and could be used for breeding programs.

Photosynthesis and Growth of Cotton (Gossypium hirsutum L.) Lines Deficient in Chlorophyll Accumulation

Three virescent mutants and two wild-type lines of upland cotton were evaluated for chlorophyll (Chl), carotenoids (Car), carbon exchange ratio (CER), water use efficiency (WUE), chlorophyll fluorescence, and growth. Lines 30 and 174 were allelic for the virescent locus and had reduced ratios of ChlTot, Chla, Chlb, CER, and WUE compared to DP90ne (in both 1995 and 1996). Line 174 had a higher CER than line 30 in both 1995 and 1996. The CER values of line 174 were similar to those of line 31. The only differences in fluorescent measurements were found in initial fluorescence (F0) values, reflecting the concentrations of Chl. Of the three plant growth indicators, only plant height showed consistent statistical differences during growth. Reduced plant height was associated with lower chlorophyll concentrations. The number of main-stem nodes was slightly affected, with statistical differences occurring on only two dates—41 and 82 days after planting. The data indicate that these virescence alleles negatively influence most measurements of photosynthesis and cotton growth, but not all. Also, as previously reported in other crops, cotton leaf CER is not totally dependent on ChlTot levels.

Large Scale Comparative Proteomics of a Chloroplast Clp Protease Mutant Reveals Folding Stress, Altered Protein Homeostasis, and Feedback Regulation of Metabolism

The clpr2-1 mutant is delayed in development due to reduction of the chloroplast ClpPR protease complex. To understand the role of Clp proteases in plastid biogenesis and homeostasis, leaf proteomes of young seedlings of clpr2-1 and wild type were compared using large scale mass spectrometry-based quantification using an LTQ-Orbitrap and spectral counting with significance determined by G-tests. Virtually only chloroplast-localized proteins were significantly affected, indicating that the molecular phenotype was confined to the chloroplast. A comparative chloroplast stromal proteome analysis of fully developed plants was used to complement the data set. Chloroplast unfoldase ClpB3 was strongly up-regulated in both young and mature leaves, suggesting widespread and persistent protein folding stress. The importance of ClpB3 in the clp2-1 mutant was demonstrated by the observation that a CLPR2 and CLPB3 double mutant was seedling-lethal. The observed up-regulation of chloroplast chaperones and protein sorting components further illustrated destabilization of protein homeostasis. Delayed rRNA processing and up-regulation of a chloroplast DEAD box RNA helicase and polynucleotide phosphorylase, but no significant change in accumulation of ribosomal subunits, suggested a bottleneck in ribosome assembly or RNA metabolism. Strong up-regulation of a chloroplast translational regulator TypA/BipA GTPase suggested a specific response in plastid gene expression to the distorted homeostasis. The stromal proteases PreP1,2 were up-regulated, likely constituting compensation for reduced Clp protease activity and possibly shared substrates between the ClpP and PreP protease systems. The thylakoid photosynthetic apparatus was decreased in the seedlings, whereas several structural thylakoid-associated plastoglobular proteins were strongly up-regulated. Two thylakoid-associated reductases involved in isoprenoid and chlorophyll synthesis were up-regulated reflecting feedback from rate-limiting photosynthetic electron transport. We discuss the quantitative proteomics data and the role of Clp proteolysis using a "systems view" of chloroplast homeostasis and metabolism and provide testable hypotheses and putative substrates to further determine the significance of Clp-driven proteolysis.

A frameshift mutation of the chloroplast matK coding region is associated with chlorophyll deficiency in the Cryptomeria japonica virescent mutant Wogon-Sugi

Wogon-Sugi has been reported as a cytoplasmically inherited virescent mutant selected from a horticultural variety of Cryptomeria japonica. Although previous studies of plastid structure and inheritance indicated that at least some mutations are encoded by the chloroplast genome, the causative gene responsible for the primary chlorophyll deficiency in Wogon-Sugi, has not been identified. In this study, we identified this gene by genomic sequencing of chloroplast DNA and genetic analysis. Chloroplast DNA sequencing of 16 wild-type and 16 Wogon-Sugi plants showed a 19-bp insertional sequence in the matK coding region in the Wogon-Sugi. This insertion disrupted the matK reading frame. Although an indel mutation in the ycf1 and ycf2 coding region was detected in Wogon-Sugi, sequence variations similar to that of Wogon-Sugi were also detected in several wild-type lines, and they maintained the reading frame. Genetic analysis of the 19 bp insertional mutation in the matK coding region showed that it was found only in the chlorophyll-deficient sector of 125 full-sibling seedlings. Therefore, the 19-bp insertion in the matK coding region is the most likely candidate at present for a mutation underlying the Wogon-Sugi phenotype.Electronic supplementary materialThe online version of this article (doi:10.1007/s00294-009-0247-9) contains supplementary material, which is available to authorized users.

An Arabidopsis thaliana virescent mutant reveals a role for ClpR1 in plastid development

The ATP-dependent Clp protease has been well-characterized in Escherichia coli, but knowledge of its function in higher plants is limited. In bacteria, this two-component protease consists of a Ser-type endopeptidase ClpP, which relies on the ATP-dependent unfolding activity from an Hsp100 molecular chaperone to initiate protein degradation. In the chloroplasts of higher plants, multiple isoforms of the proteolytic subunit exist, with Arabidopsis having five ClpPs and four ClpP-like proteins termed ClpR predicted in its genome. In this work we characterized an Arabidopsis mutant impaired in one subunit of the chloroplast-localized Clp protease core, ClpR1. clpR1-1, a virescent mutant, carries a pre-mature stop codon in the clpR1 gene, resulting in no detectable ClpR1 protein. The accumulation of several chloroplast proteins, as well as most of the chloroplast-localized Clp protease subunits, is inhibited in clpR1-1. Unexpectedly, some plastid-encoded proteins do not accumulate, although their transcripts accumulate to wild-type levels. Maturation of 23S and 4.5S chloroplast ribosomal RNA (cp-rRNA) is delayed in clpR1-1, and both RNAs accumulate as higher molecular weight precursors. Also, chloroplasts in clpR1-1 are smaller than in wild type and have fewer thylakoid membranes with smaller grana stacks. We propose that a ClpR1-containing activity is required for chloroplast development and differentiation and in its absence both are delayed.

Photosynthetic performance of cold‐sensitive mutants of maize at low temperature

The virescent character is a genetic variant in pigmentation characterized by a delay in greening. Seedlings of the virescent mutants v1, v2, v3, v4, v13, v16, v18, v19 and v26 of maize exhibit chlorosis when grown at low temperature. Chlorotic leaves contain plastids that appear to have been arrested at an early stage of development. The results indicated that V16, V2, V3 and V4 loci control early stages of chloroplast development while V1, V13 and V19 may play a role at the end of development. The mutations in the V18 and V26 loci may control an intermediate step. At the pigment level, the virescent mutants of maize differ widely from analogous mutations existing in other plants. The mutations were characterized by a reduced amount of chlorophyll a and b (up to 100 times in v16) and chlorophyll a/b ratio above normal (up to 13.7 in v16). Lutein content was reduced in all mutants (less than 3% in v16 compared to wild type) but v13, while pigments of the xanthophyll cycle were found at higher levels in v1 and v13 (more than 10 and 90%, respectively). The v2, v3, v4, v16 and v18 mutants that are most depleted in β‐carotene (36 times less in average than wild type) are also deprived in D1 and D2 polypeptides. Moreover, the v2, v3, v4, v16 and v18 mutants characterized by a lower accumulation in lutein are most depleted of light‐harvesting complex II. All mutants possess a functioning, fully reversible, non‐photochemical quenching mechanism. This is most developed in the v13 and v19 mutants (φn = 0.48 and 0.44, respectively). These two mutants also have a relatively high primary photochemical yield for photosystem II and a functioning photosystem I (φp = 0.23 and 0.39, respectively). The most interesting mutant is v13 that shows severe chlorosis and possesses the most effective non‐photochemical quenching mechanism(s), which is thought to provide protection against excess photon absorption by photosystem II.

Molecular and cytological characterization of a cytoplasmic-specific mutant in pima cotton (Gossypium barbadense L.)

A cytoplasmic mutant of Gossypium barbadense L., cyt-V was characterized at the morphological, cellular, genetic and molecular levels using comparison analysis with v 7 v 7, a nuclear virescent mutant to identify molecular effects of the cyt-V mutation. The yellow phenotype was specific only to leaves in the cyt-V mutant (CM-1-90) but the same phenotype was present in both leaves and cotyledons of v 7 v 7, a nuclear virescent mutant, suggesting that cyt-V and v 7 v 7, had different organ-specific gene actions. Chlorophyll and carotenoid levels of CM-1-90, CM-1-90 × PS-7 and CM-1-90 × v 7 v 7 true leaves were significantly lower than in the true leaves of PS-7 × CM-1-90, v 7 v 7 × CM-1-90 and PS-7. Anatomical studies of chloroplast showed that CM-1-90, CM-1-90 × PS-7 and CM-1-90 × v 7 v 7 lacked grana in the thylakoids of the mesophyll cells. This indicated that chlorophyll and carotenoid levels correlated with chloroplast structure. SDS-PAGE analysis of thylakoid preparations revealed decreases of several granalocalized PSII proteins in CM-1-90, CM-1-90 × PS-7 and CM-1-90 × v 7 v 7. cDNA-AFLP differential display studies identified several differentially expressed transcripts in the leaves of reciprocal crosses (PS-7 × CM-1-90, v 7 v 7 × CM-1-90 and CM-1-90 × PS-7 and CM-1-90 × v 7 v 7), including one possessing a high sequence homology to a psbA gene. Western blot analysis further confirmed the absence of D1 protein encoded by psbA in CM-1-90 × PS-7′ CM-1-90 × v 7 v 7 and CM-1-90 true leaves. Overall, we studied cyt-V and v 7 v 7 that both are developmental mutants, as all the virescents of cotton mutants, and as such it was difficult to separate cause and effect in the observation; however, we verified that the source of cyt-V mutation was in chloroplast and elucidated that its gene action was different from v 7 v 7. Results indicated that cyt-V is inherited as a single gene but it affects several chloroplast and nucleus-encoded genes. We identified several transcripts that associated with the cyt-V mutation. This study also suggested that chloroplast-encoded gene products might affect the expression of nuclear genes, possibly at the transcriptional stage.