Impaired Chloroplast Development Research Articles

Loss-function-of a UMP kinase leads to impaired chloroplast development and photosynthesis efficiency in cucumber

Loss-function-of a UMP kinase leads to impaired chloroplast development and photosynthesis efficiency in cucumber

Integrated Cytological, Physiological, and Transcriptome Analyses Provide Insight into the Albino Phenotype of Chinese Plum (Prunus salicina).

Albino seedlings that arise during seed reproduction can have a significant impact on plant growth and breeding. In this research, we present the first report of albino occurrences in the seed reproduction process of Prunus salicina and describe the cytological, physiological, and transcriptomic changes observed in albino seedlings. The albino seedlings which were observed in several plum cultivars exhibited abnormal chloroplast ultrastructure and perturbed stomatal structure. Compared to normal seedlings, the photosynthetic pigment contents in albino seedlings decreased by more than 90%, accompanied by significant reductions in several chlorophyll fluorescence parameters. Furthermore, substantially changed photosynthetic parameters indicated that the photosynthetic capacity and stomatal function were impaired in albino seedlings. Additionally, the activities of the antioxidant enzyme were drastically altered against the background of higher proline and lower ascorbic acid in leaves of albino seedlings. A total of 4048 differentially expressed genes (DEGs) were identified through transcriptomic sequencing, and the downregulated DEGs in albino seedlings were greatly enriched in the pathways for photosynthetic antenna proteins and flavonoid biosynthesis. GLK1 and Ftsz were identified as candidate genes responsible for the impaired chloroplast development and division in albino seedlings. Additionally, the substantial decline in the expression levels of examined photosystem-related chloroplast genes was validated in albino seedlings. Our findings shed light on the intricate physiological and molecular mechanisms driving albino plum seedling manifestation, which will contribute to improving the reproductive and breeding efforts of plums.

PWL1, a G-type lectin receptor-like kinase, positively regulates leaf senescence and heat tolerance but negatively regulates resistance to Xanthomonas oryzae in rice.

Plant leaf senescence, caused by multiple internal and environmental factors, has an important impact on agricultural production. The lectin receptor-like kinase (LecRLK) family members participate in plant development and responses to biotic and abiotic stresses, but their roles in regulating leaf senescence remain elusive. Here, we identify and characterize a rice premature withered leaf 1 (pwl1) mutant, which exhibits premature leaf senescence throughout the plant life cycle. The pwl1 mutant displayed withered and whitish leaf tips, decreased chlorophyll content, and accelerated chloroplast degradation. Map-based cloning revealed an amino acid substitution (Gly412Arg) in LOC_Os03g62180 (PWL1) was responsible for the phenotypes of pwl1. The expression of PWL1 was detected in all tissues, but predominantly in tillering and mature leaves. PWL1 encodes a G-type LecRLK with active kinase and autophosphorylation activities. PWL1 is localized to the plasma membrane and can self-associate, mainly mediated by the plasminogen-apple-nematode (PAN) domain. Substitution of the PAN domain significantly diminished the self-interaction of PWL1. Moreover, the pwl1 mutant showed enhanced reactive oxygen species (ROS) accumulation, cell death, and severe DNA fragmentation. RNA sequencing analysis revealed that PWL1 was involved in the regulation of multiple biological processes, like carbon metabolism, ribosome, and peroxisome pathways. Meanwhile, interfering of biological processes induced by the PWL1 mutation also enhanced heat sensitivity and resistance to bacterial blight and bacterial leaf streak with excessive accumulation of ROS and impaired chloroplast development in rice. Natural variation analysis indicated more variations in indica varieties, and the vast majority of japonica varieties harbour the PWL1Hap1 allele. Together, our results suggest that PWL1, a member of LecRLKs, exerts multiple roles in regulating plant growth and development, heat-tolerance, and resistance to bacterial pathogens.

Genetic mapping and molecular characterization of the delayed green gene dg in watermelon (Citrullus lanatus).

Leaf color mutants are common in higher plants that can be used as markers in crop breeding and are important tools in understanding regulatory mechanisms of chlorophyll biosynthesis and chloroplast development. Genetic analysis was performed by evaluating F1, F2 and BC1 populations derived from two parental lines (Charleston gray with green leaf color and Houlv with delayed green leaf color), suggesting that a single recessive gene controls the delayed green leaf color. In this study, the delayed green mutant showed a conditional pale green leaf color at the early leaf development but turned to green as the leaf development progressed. Delayed green leaf plants showed reduced pigment content, photosynthetic, chlorophyll fluorescence parameters, and impaired chloroplast development compared with green leaf plants. The delayed green (dg) locus was mapped to 7.48 Mb on chromosome 3 through bulk segregant analysis approach, and the gene controlling delayed green leaf color was narrowed to 53.54 kb between SNP130 and SNP135 markers containing three candidate genes. Sequence alignment of the three genes indicated that there was a single SNP mutation (G/A) in the coding region of ClCG03G010030 in the Houlv parent, which causes an amino acid change from Arginine to Lysine. The ClCG03G010030 gene encoded FtsH extracellular protease protein family is involved in early delayed green leaf development. The expression level of ClCG03G010030 was significantly reduced in delayed green leaf plants than in green leaf plants. These results indicated that the ClCG03G010030 might control watermelon green leaf color and the single SNP variation in ClCG03G010030 may result in early delayed green leaf color development during evolutionary process.

The DnaJ domain-containing heat-shock protein NAL11 determines plant architecture by mediating gibberellin homeostasis in rice (Oryza sativa).

Ideal Plant Architecture 1 (IPA1) is a key regulator of plant architecture. However, knowledge of downstream genes applicable for improving rice plant architecture is very limited. We identified the plant architecture regulatory gene NARROW LEAF 11 (NAL11), which encodes a heat-shock protein (HSP) containing a DnaJ domain. A promising rare allele of NAL11 (NAL11-923del-1552 ) positively selected in Aus cultivars was identified; this allele exhibited increased expression and generated relatively few tillers, thick stems, and large panicles, components of the ideal plant architecture (IPA). NAL11 is involved in regulating the cell cycle and cell proliferation. NAL11 loss-of-function mutants present impaired chloroplast development and gibberellin (GA) defects. Biochemical analyses show that IPA1 directly binds to elements in the missing fragment of the NAL11-923del-1552 promoter and negatively regulates NAL11 expression. Genetic analyses support the hypothesis that NAL11 acts downstream of IPA1 to regulate IPA by modulating GA homeostasis, and NAL11 may be an essential complement for IPA1. Our work revealed that avoidance of the inhibition of NAL11-923del-1552 caused by IPA1 represents a positive strategy for rescuing GA defects accompanied by the upregulation of IPA1 in breeding high-yield rice.

SlERF.J2 reduces chlorophyll accumulation and inhibits chloroplast biogenesis and development in tomato leaves

Chlorophyll metabolism and chloroplast biogenesis in tomato (Solanum lycopersicum) leaves contribute to photosynthesis; however, their molecular mechanisms are poorly understood. In this study, we found that overexpression of SlERF.J2 (ethylene transcription factor) resulted in a decrease in leaf chlorophyll content and reduced accumulation of starch and soluble sugar. The slerf.j2 knockout mutant showed no apparent change. Further observation of tissue sections and transmission electron microscopy (TEM) showed that SlERF.J2 was involved in chlorophyll accumulation and chloroplast formation. RNA-seq of mature SlERF.J2-OE leaves showed that many genes involved in chlorophyll biosynthesis and chloroplast formation were significantly downregulated compared with those in WT leaves. Genome global scanning of the ERF TF binding site combined with RNA-seq differential gene expression and qRT-PCR detection analysis showed that COP1 was a potential target gene of SlERF.J2. Tobacco transient expression technology, a dual-luciferase reporter system and Y1H technology were employed to verify that SlERF.J2 could bind to the COP1 promoter. Notably, overexpression of SlERF.J2 in Nr mutants resulted in impaired chloroplast biogenesis and development. Taken together, our findings demonstrated that SlERF.J2 plays an essential role in chlorophyll accumulation and chloroplast formation, laying a foundation for enhancing plant photosynthesis.

SCL, Encoding a Chloroplast Signal Recognition Particle Receptor, Affects Chlorophyll Synthesis and Chloroplast Development in Rice

Crop yield is largely determined by the solar energy utilization efficiency of photosynthesis; plants with long stay-green periods have greater total photosynthetic production levels and crop yields. Here, a novel seedling chlorosis and lethality (scl) mutant exhibiting a yellow leaf and seedling-lethal phenotype was identified in rice (Oryza sativa L.). The mutant had deformed chloroplasts and almost no protein complexes in thylakoid membranes. The expression levels of photosynthesis-associated genes were significantly down-regulated in scl compared with the wild-type (WT). Positive transgenic lines generated by Agrobacterium tumefaciens-mediated transformation of the scl mutant with a complementation vector harboring SCL cDNA exhibited the normal green leaf phenotype, whereas the scl seedling harboring the empty vector displayed the yellow leaf phenotype, indicating that SCL is LOC_Os01g72800. A fusion protein expressing SCL with green fluorescent protein revealed the fluorescence signal localized to chloroplasts. The expression patterns of chloroplast development and chlorophyll biosynthesis and degradation-related genes were disordered in scl mutant, possibly resulting in the yellow leaf phenotype. These results indicated that the SCL loss of function impaired chloroplast development, chlorophyll biosynthesis, and light-harvesting chlorophyll-binding protein transportation in rice.

A rice XANTHINE DEHYDROGENASE gene regulates leaf senescence and response to abiotic stresses

Xanthine dehydrogenase, a member of the molybdenum enzyme family, participates in purine metabolism and catalyzes the generation of ureides from xanthine and hypoxanthine. However, the mechanisms by which xanthine dehydrogenase affects rice growth and development are poorly understood. In the present study, we identified a mutant with early leaf senescence and reduced tillering that we named early senescence and less-tillering 1 (esl1). Map-based cloning revealed that ESL1 encodes a xanthine dehydrogenase, and it was expressed in all tissues. Chlorophyll content was reduced and chloroplast maldevelopment was severe in the esl1 mutant. Mutation of ESL1 led to decreases in allantoin, allantoate, and ABA contents. Further analysis revealed that the accumulation of reactive oxygen species in esl1 resulted in decreased photosynthesis and impaired chloroplast development, along with increased sensitivity to abscisic acid and abiotic stresses. Ttranscriptome analysis showed that the ESL1 mutation altered the expression of genes involved in the photosynthesis process and reactive oxygen species metabolism. Our results suggest that ESL1 is involved in purine metabolism and the induction of leaf senescence. These findings reveal novel molecular mechanisms of ESL1 gene-mediated plant growth and leaf senescence.

Roles for the chloroplast-localized pentatricopeptide repeat protein 30 and the 'mitochondrial' transcription termination factor 9 in chloroplast quality control.

Chloroplasts constantly experience photo-oxidative stress while performing photosynthesis. This is particularly true under abiotic stresses that lead to the accumulation of reactive oxygen species (ROS) which oxidize DNA, proteins and lipids. Reactive oxygen species can also act as signals to induce acclimation through chloroplast degradation, cell death and nuclear gene expression. To better understand the mechanisms behind ROS signaling from chloroplasts, we have used the Arabidopsis thaliana mutant plastid ferrochelatase two (fc2) that conditionally accumulates the ROS singlet oxygen (1 O2 ) leading to chloroplast degradation and eventually cell death. Here we have mapped mutations that suppress chloroplast degradation in the fc2 mutant and demonstrate that they affect two independent loci (PPR30 and mTERF9) encoding chloroplast proteins predicted to be involved in post-transcriptional gene expression. These mutants exhibited broadly reduced chloroplast gene expression, impaired chloroplast development and reduced chloroplast stress signaling. Levels of 1 O2 , however, could be uncoupled from chloroplast degradation, suggesting that PPR30 and mTERF9 are involved in ROS signaling pathways. In the wild-type background, ppr30 and mTERF9 mutants were also observed to be less susceptible to cell death induced by excess light stress. While broad inhibition of plastid transcription with rifampicin was also able to suppress cell death in fc2 mutants, specific reductions in plastid gene expression using other mutations was not always sufficient. Together these results suggest that plastid gene expression, or the expression of specific plastid genes by PPR30 and mTERF0, is a necessary prerequisite for chloroplasts to activate the 1 O2 signaling pathways to induce chloroplast quality control pathways and/or cell death.

Chloroplast SRP54s are Essential for Chloroplast Development in Rice

BackgroundThe chloroplast signal recognition particle 54 (cpSRP54) is known for targeting the light-harvesting complex proteins to thylakoids and plays a critical role for chloroplast development in Arabidopsis, but little is known in rice. Here, we reported two homologous cpSRP54s that affect chloroplast development and plant survival in rice.ResultsTwo rice cpSRP54 homologues, OscpSRP54a and OscpSRP54b, were identified in present study. The defective OscpSRP54a (LOC_Os11g05552) was responsible for the pale green leaf phenotype of the viable pale green leaf 14 (pgl14) mutant. A single nucleotide substitution from G to A at the position 278, the first intron splicing site, was detected in LOC_Os11g05552 in pgl14. The wild type allele could rescue the mutant phenotype. Knockout lines of OscpSRP54b (LOC_Os11g05556) exhibited similar pale green phenotype to pgl14 with reduced chlorophyll contents and impaired chloroplast development, but showed apparently arrested-growth and died within 3 weeks. Both OscpSRP54a and OscpSRP54b were constitutively expressed mainly in shoots and leaves at the vegetative growth stage. Subcellular location indicated that both OscpSRP54a and OscpSRP54b were chloroplast-localized. Both OscpSRP54a and OscpSRP54b were able to interact with OscpSRP43, respectively. The transcript level of OscpSRP43 was significantly reduced while the transcript level of OscpSRP54b was apparently increased in pgl14. In contrast, the transcript levels of OscpSRP54a, OscpSRP43 and OscpSRP54b were all significantly decreased in OscpSRP54b knockout lines.ConclusionOur study demonstrated that both OscpSRP54a and OscpSRP54b were essential for normal chloroplast development by interacting with OscpSRP43 in rice. OscpSRP54a and OscpSRP54b might play distinct roles in transporting different chloroplast proteins into thylakoids through cpSRP-mediated pathway.

Mutation of DELAYED GREENING1 impairs chloroplast RNA editing at elevated ambient temperature in Arabidopsis

Chloroplasts are important for plant growth and development. RNA editing in chloroplast converts cytidines (Cs) to uridines (Us) at specific transcript positions and provides a correction mechanism to restore conserved codons or creates start or stop codons. However, the underlined molecular mechanism is not yet fully understood. In the present study, we identified a thermo-sensitive mutant in leaf color 1 (tsl1) and found that TSL1 is allelic to DELAYED GREENING 1 (DG1). The missense mutation of DG1 in tsl1 mutant confers a high temperature sensitivity and impaired chloroplast development at an elevated ambient temperature in Arabidopsis. Subsequent analysis showed that chloroplast RNA editing at several sites including accD-1568, ndhD-2, and petL-5 is impaired in tsl1 mutant plants grown at an elevated temperature. DG1 interacts with MORF2 and other proteins such as DYW1 and DYW2 involved in chloroplast RNA editing. In vitro RNA electrophoretic mobility shift assay demonstrated that DG1 binds to RNA targets such as accD, ndhD, and petL. Thus, our results revealed that DG1 is important for maintaining chloroplast mRNA editing in Arabidopsis.

Plastid-targeted forms of restriction endonucleases enhance the plastid genome rearrangement rate and trigger the reorganization of its genomic architecture.

Plant cells have acquired chloroplasts (plastids) with a unique genome (ptDNA), which developed during the evolution of endosymbiosis. The gene content and genome structure of ptDNAs in land plants are considerably stable, although those of algal ptDNAs are highly varied. Plant cells seem, therefore, to be intolerant of any structural or organizational changes in the ptDNA. Genome rearrangement functions as a driver of genomic evolutionary divergence. Here, we aimed to create various types of rearrangements in the ptDNA of Arabidopsis genomes using plastid-targeted forms of restriction endonucleases (pREs). Arabidopsis plants expressing each of the three specific pREs, i.e., pTaqI, pHinP1I, and pMseI, were generated; they showed the leaf variegation phenotypes associated with impaired chloroplast development. We confirmed that these pREs caused double-stranded breaks (DSB) at their recognition sites in ptDNAs. Genome-wide analysis of ptDNAs revealed that the transgenic lines exhibited a large number of rearrangements such as inversions and deletions/duplications, which were dominantly repaired by microhomology-mediated recombination and microhomology-mediated end-joining, and less by non-homologous end-joining. Notably, pHinP1I, which recognized a small number of sites in ptDNA, induced drastic structural changes, including regional copy number variations throughout ptDNAs. In contrast, the transient expression of either pTaqI or pMseI, whose recognition site numbers were relatively larger, resulted in small-scale changes at the whole genome level. These results indicated that DSB frequencies and their distribution are major determinants in shaping ptDNAs.

Protective Role of Leaf Variegation in Pittosporum tobira under Low Temperature: Insights into the Physio-Biochemical and Molecular Mechanisms.

Leaf variegation has been demonstrated to have adaptive functions such as cold tolerance. Pittosporum tobira is an ornamental plant with natural leaf variegated cultivars grown in temperate regions. Herein, we investigated the role of leaf variegation in low temperature responses by comparing variegated “Variegatum” and non-variegated “Green Pittosporum” cultivars. We found that leaf variegation is associated with impaired chloroplast development in the yellow sector, reduced chlorophyll content, strong accumulation of carotenoids and high levels of ROS. However, the photosynthetic efficiency was not obviously impaired in the variegated leaves. Also, leaf variegation plays low temperature protective function since “Variegatum” displayed strong and efficient ROS-scavenging enzymatic systems to buffer cold (10 °C)-induced damages. Transcriptome analysis under cold conditions revealed 309 differentially expressed genes between both cultivars. Distinctly, the strong cold response observed in “Variegatum” was essentially attributed to the up-regulation of HSP70/90 genes involved in cellular homeostasis; up-regulation of POD genes responsible for cell detoxification and up-regulation of FAD2 genes and subsequent down-regulation of GDSL genes leading to high accumulation of polyunsaturated fatty acids for cell membrane fluidity. Overall, our results indicated that leaf variegation is associated with changes in physiological, biochemical and molecular components playing low temperature protective function in P. tobira.

Leaf Variegation and Impaired Chloroplast Development Caused by a Truncated CCT Domain Gene in albostrians Barley.

Chloroplasts fuel plant development and growth by converting solar energy into chemical energy. They mature from proplastids through the concerted action of genes in both the organellar and the nuclear genome. Defects in such genes impair chloroplast development and may lead to pigment-deficient seedlings or seedlings with variegated leaves. Such mutants are instrumental as tools for dissecting genetic factors underlying the mechanisms involved in chloroplast biogenesis. Characterization of the green-white variegated albostrians mutant of barley (Hordeum vulgare) has greatly broadened the field of chloroplast biology, including the discovery of retrograde signaling. Here, we report identification of the ALBOSTRIANS gene HvAST (also known as Hordeum vulgare CCT Motif Family gene 7, HvCMF7) by positional cloning as well as its functional validation based on independently induced mutants by Targeting Induced Local Lesions in Genomes (TILLING) and RNA-guided clustered regularly interspaced short palindromic repeats-associated protein 9 endonuclease-mediated gene editing. The phenotypes of the independent HvAST mutants imply residual activity of HvCMF7 in the original albostrians allele conferring an imperfect penetrance of the variegated phenotype even at homozygous state of the mutation. HvCMF7 is a homolog of the Arabidopsis (Arabidopsis thaliana) CONSTANS, CO-like, and TOC1 (CCT) Motif transcription factor gene CHLOROPLAST IMPORT APPARATUS2, which was reported to be involved in the expression of nuclear genes essential for chloroplast biogenesis. Notably, in barley we localized HvCMF7 to the chloroplast, without any clear evidence for nuclear localization.

A missense mutation of plastid RPS4 is associated with chlorophyll deficiency in Chinese cabbage (Brassica campestris ssp. pekinensis)

BackgroundPlastome mutants are ideal resources for elucidating the functions of plastid genes. Numerous studies have been conducted for the function of plastid genes in barley and tobacco; however, related information is limited in Chinese cabbage.ResultsA chlorophyll-deficient mutant of Chinese cabbage that was derived by ethyl methanesulfonate treatment on isolated microspores showed uniformly pale green inner leaves and slow growth compared with that shown by the wild type “Fukuda 50′ (‘FT’). Genetic analysis revealed that cdm was cytoplasmically inherited. Physiological and ultrastructural analyses of cdm showed impaired photosynthesis and abnormal chloroplast development. Utilizing next generation sequencing, the complete plastomes of cdm and ‘FT’ were respectively re-mapped to the reference genome of Chinese cabbage, and an A-to-C base substitution with a mutation ratio higher than 99% was detected. The missense mutation of plastid ribosomal protein S4 led to valine substitution for glycine at residue 193. The expression level of rps4 was analyzed using quantitative real-time PCR and found lower in than in ‘FT’. RNA gel-blot assays showed that the abundance of mature 23S rRNA, 16S rRNA, 5S rRNA, and 4.5S rRNA significantly decreased and that the processing of 23S, 16S rRNA, and 4.5S rRNA was seriously impaired, affecting the ribosomal function in cdm.ConclusionsThese findings indicated that cdm was a plastome mutant and that chlorophyll deficiency might be due to an A-to-C base substitution of the plastome-encoded rps4 that impaired the rRNA processing and affected the ribosomal function.

PTAC10, an S1-domain-containing component of the transcriptionally active chromosome complex, is essential for plastid gene expression in Arabidopsis thaliana and is phosphorylated by chloroplast-targeted casein kinase II.

In higher plant chloroplasts, the plastid-encoded RNA polymerase (PEP) consists of four catalytic subunits and numerous nuclear-encoded accessory proteins, including pTAC10, an S1-domain-containing protein. In this study, pTAC10 knockout lines were characterized. Two ptac10 mutants had an albino phenotype and severely impaired chloroplast development. The pTAC10 genomic sequence fused to a four-tandem MYC tag driven by its own promoter functionally complemented the ptac10-1 mutant phenotype. pTAC10 was present in both the chloroplast stroma and thylakoids. Two-dimensional blue native polyacrylamide gel electrophoresis (BN-PAGE), and immunoblotting assays showed that pTAC10:MYC co-migrates with one of the PEP core subunits, RpoB. A comprehensive investigation of the plastid gene expression profiles by quantitative RT-PCR revealed that, compared with wild-type plants, the abundance of PEP-dependent plastid transcripts is severely decreased in the ptac10-1 mutant, while the amount of plastid transcripts exclusively transcribed by NEP either barely changes or even increases. RNA blot analysis confirmed that PEP-dependent chloroplast transcripts, including psaB, psbA and rbcL, substantially decrease in the ptac10-1 mutant. Immunoblotting showed reduced accumulation of most chloroplast proteins in the ptac10 mutants. These data indicate the essential role of pTAC10 in plastid gene expression and plastid development. pTAC10 interacts with chloroplast-targeted casein kinase 2 (cpCK2) in vitro and in vivo and can be phosphorylated by Arabidopsis cpCK2 in vitro at sites Ser95, Ser396 and Ser434. RNA-EMSA assays showed that pTAC10 is able to bind to the psbA, atpE and accD transcripts, suggesting a non-specific RNA-binding activity of pTAC10. The RNA affinity of pTAC10 was enhanced by phosphorylation and decreased by the amino acid substitution Ser434-Ala of pTAC10. These data show that pTAC10 is essential for plastid gene expression in Arabidopsis and that it can be phosphorylated by cpCK2.

EMB2738, which encodes a putative plastid-targeted GTP-binding protein, is essential for embryogenesis and chloroplast development in higher plants.

In higher plants, chloroplasts carry out many important functions, and normal chloroplast development is required for embryogenesis. Numerous chloroplast-targeted proteins involved in embryogenesis have been identified. Nevertheless, their functions remain unclear. In this study, a chloroplast-localized protein, EMB2738, was reported to be involved in Arabidopsis embryogenesis. EMB2738 knockout led to defective embryos, and the embryo development in emb2738 was interrupted after the globular stage. Complementation experiments identified the AT3G12080 locus as EMB2738. Cellular observation indicated that severely impaired chloroplast development was observed in these aborted embryos. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis showed that chloroplast-encoded photosynthetic genes, which are transcribed by plastid-encoded RNA polymerase (PEP), are predominantly decreased in defective embryogenesis, compared with those in the wild-type. In contrast, genes encoding PEP core subunits, which are transcribed by nucleus-encoded RNA polymerase (NEP), were increased. These results suggested that the knockout of EMB2738 strongly blocked chloroplast-encoded photosynthesis gene expression in embryos. Silencing of the EMB2738 orthologue in tobacco through a virus-induced genome silencing technique resulted in an albinism phenotype, vacuolated chloroplasts and decreased PEP-dependent plastid transcription. These results suggested that NtEMB2738 might be involved in plastid gene expression. Nevertheless, genetic analysis showed that the NtEMB2738 coding sequence could not fully rescue the defective embryogenesis of the emb2738 mutant, which suggested functional divergence between NtEMB2738 and EMB2738 in embryogenesis. Taken together, these results indicated that both EMB2738 and NtEMB2738 are involved in the expression of plastid genes in higher plants, and there is a functional divergence between NtEMB2738 and EMB2738 in embryogenesis.

A Young Seedling Stripe2 phenotype in rice is caused by mutation of a chloroplast-localized nucleoside diphosphate kinase 2 required for chloroplast biogenesis.

Chloroplast development and chlorophyll (Chl) biosynthesis in plants are regulated by many genes, but the underlying molecular mechanisms remain largely elusive. We isolated a rice mutant named yss2 (young seedling stripe2) with a striated seedling phenotype beginning from leaf 2 of delayed plant growth. The mutant developed normal green leaves from leaf 5, but reduced tillering and chlorotic leaves and panicles appeared later. Chlorotic yss2 seedlings have decreased pigment contents and impaired chloroplast development. Genetic analysis showed that the mutant phenotype was due to a single recessive gene. Positional cloning and sequence analysis identified a single nucleotide substitution in YSS2 gene causing an amino acid change from Gly to Asp. The YSS2 allele encodes a NDPK2 (nucleoside diphosphate kinase 2) protein showing high similarity to other types of NDPKs. Real-time RT-PCR analysis demonstrated that YSS2 transcripts accumulated highly in L4 sections at the early leaf development stage. Expression levels of genes associated with Chl biosynthesis and photosynthesis in yss2 were mostly decreased, but genes involved in chloroplast biogenesis were up-regulated compared to the wild type. The YSS2 protein was associated with punctate structures in the chloroplasts of rice protoplasts. Our overall data suggest that YSS2 has important roles in chloroplast biogenesis.

Physiological properties and chlorophyll biosynthesis in a Pak-choi (Brassica rapa L. ssp. chinensis) yellow leaf mutant, pylm

The chlorophyll (Chl)-deficient mutant pylm is a double haploid (DH) line of Pak-choi (Brassica rapa L. ssp. chinensis) that was obtained from Pak-choi variety ‘Huaguan’ by isolated microspore culture. In this study, the aim was to examine the physiological properties and Chl biosynthesis in the pylm mutant. Compared to the control line ‘CK-51’, pylm is slender, with an earlier flowering phenotype and obviously elongated hypocotyls. Normally grown pylm seedlings had yellow leaves with reduced total Chl content but an increased Chl a/b ratio. Ultrastructural analysis showed that the pylm mutant has a loose grana lamellae structure and few thylakoid stacks, resulting in impaired chloroplast development. Measurement of photosynthetic gas exchange and Chl fluorescence parameters indicated that the pylm mutant has lower photosynthetic activity and photochemical conversion efficiency than ‘CK-51’ plants. We investigated the biochemical mechanism of Chl deficiency in pylm by examining Chl biosynthesis precursors in this mutant, showing that Chl biosynthesis is blocked in the mutant at the Chl a production step. In addition, the expression of a number of genes related with Chl biosynthesis is down-regulated in pylm. Therefore, these results provide a basis for understanding the physiological and biochemical causes of the mutant phenotype of pylm.

Modulation of biosynthesis of photosynthetic pigments and light-harvesting complex in wild-type and gun5 mutant of Arabidopsis thaliana during impaired chloroplast development.

Plants in response to different environmental cues need to modulate the expression of nuclear and chloroplast genomes that are in constant communication. To understand the signals that are responsible for inter-organellar communication, levulinic acid (LA), an inhibitor of 5-aminolevulinic acid dehydratase, was used to suppress the synthesis of pyrrole-derived tetrapyrroles chlorophylls. Although, it does not specifically inhibit carotenoid biosynthesis enzymes, LA reduced the carotenoid contents during photomorphogenesis of etiolated Arabidopsis seedlings. The expression of nuclear genes involved in carotenoid biosynthesis, i.e., geranylgeranyl diphosphate synthase, phytoene synthase, and phytoene desaturase, was downregulated in LA-treated seedlings. Similarly, the transcript abundance of nuclear genes, i.e., Lhcb1, PsbO, and RcbS, coding for chloroplastic proteins was severely attenuated in LA-treated samples. In contrast, LA treatment did not affect the transcript abundance of chalcone synthase, a marker gene for cytoplasm, and β-ATP synthase, a marker gene for mitochondria. This demonstrates the retrograde signaling from chloroplast to nucleus to suppress chloroplastic proteins during impaired chloroplast development. However, under identical conditions in LA-treated tetrapyrrole-deficient gun5 mutant, retrograde signal continued. The tetrapyrrole biosynthesis inhibitor LA suppressed formation of all tetrapyrroles both in WT and gun5. This rules out the role of tetrapyrroles as signaling molecules in WT and gun5. The removal of LA from the Arabidopsis seedlings restored the chlorophyll and carotenoid contents and expression of nuclear genes coding for chloroplastic proteins involved in chloroplast biogenesis. Therefore, LA could be used to modulate chloroplast biogenesis at a desired phase of chloroplast development.