Mutation in EMB1923 gene promoter is associated with chlorophyll deficiency in Chinese cabbage (Brassica campestris ssp. pekinensis)

Leaf color mutants are widespread in higher plants and can be used as markers in crop breeding or as important material in understanding the regulatory mechanisms of chlorophyll biosynthesis and chloroplast development. A stably inherited plant etiolated mutation (pem) was obtained from its wild‐type ‘FT’ (a doubled haploid line of the Chinese cabbage variety ‘Fukuda 50’) by combining ⁶⁰Co‐γ radiation and isolated microspore culture in Chinese cabbage. Compared to the wild‐type ‘FT’, the chlorophyll content in the pem mutant was decreased, the photosynthetic capacity was reduced and the chloroplast development was retarded. These physiological changes may lead to a reduction in growth and yield in the pem mutant line. Genetic analysis showed that the mutant phenotype was controlled by the single recessive nuclear pem gene. The pem gene was mapped to a 25.88 kb region on the A03 chromosome. Cloning and sequencing results showed that there was only one DNA sequence variation in this region, which was a 30 bp deletion on the promoter of Bra024218. Its homologous gene encodes EMBRYO DEFECTIVE 1923 (EMB1923) in Arabidopsis thaliana. We therefore predicted that Bra024218 was the mutated gene associated with etiolated leaves in Chinese cabbage. The pem mutant is a useful line for researching chloroplast development and the mechanism of leaf color mutation in Chinese cabbage.     

Disruption of EARLY LESION LEAF 1, encoding a cytochrome P450 monooxygenase,induces ROS accumulation and cell death in rice

Lesion-mimic mutants (LMMs) provide a valuable tool to reveal the molecular mechanisms determining programmed cell death (PCD) in plants. Despite intensive research, the mechanisms behind PCD and the formation of lesions in various LMMs still remain to be elucidated. Here, we identified a rice (Oryza sativa) LMM, early lesion leaf 1 (ell1), cloned the causal gene by map-based cloning, and verified this by complementation. ELL1 encodes a cytochrome P450 monooxygenase, and the ELL1 protein was located in the endoplasmic reticulum. The ell1 mutant exhibited decreased chlorophyll contents, serious chloroplast degradation, upregulated expression of chloroplast degradation-related genes, and attenuated photosynthetic protein activity, indicating that ELL1 is involved in chloroplast development. RNA sequencing analysis showed that genes related to oxygen binding were differentially expressed in ell1 and wild-type plants; histochemistry and paraffin sectioning results indicated that hydrogen peroxide (H₂O₂) and callose accumulated in the ell1 leaves, and the cell structure around the lesions was severely damaged, which indicated that reactive oxygen species (ROS) accumulated and cell death occurred in the mutant. TUNEL staining and comet experiments revealed that severe DNA degradation and abnormal PCD occurred in the ell1 mutants, which implied that excessive ROS accumulation may induce DNA damage and ROS-mediated cell death in the mutant. Additionally, lesion initiation in the ell1 mutant was light dependent and temperature sensitive. Our findings revealed that ELL1 affects chloroplast development or function, and that loss of ELL1 function induces ROS accumulation and lesion formation in rice.     

The chloroplastic DEVH‐box RNA helicase INCREASED SIZE EXCLUSION LIMIT 2 involved in plasmodesmata regulation is required for group II intron splicing

INCREASED SIZE EXCLUSION LIMIT 2 (ISE2) encodes a putative DEVH‐box RNA helicase originally identified through a genetic screening for Arabidopsis mutants altered in plasmodesmata (PD) aperture. Depletion of ISE2 also affects chloroplasts activity, decreases accumulation of photosynthetic pigments and alters expression of photosynthetic genes. In this work, we show the chloroplast localization of ISE2 and decipher its role in plastidic RNA processing and, consequently, PD function. Group II intron‐containing RNAs from chloroplasts exhibit defective splicing in ise2 mutants and ISE2‐silenced plants, compromising plastid viability. Furthermore, RNA immunoprecipitation suggests that ISE2 binds in vivo to several splicing‐regulated RNAs. Finally, we show that the chloroplast clpr2 mutant (defective in a subunit of a plastidic Clp protease) also exhibits abnormal PD function during embryogenesis, supporting the idea that chloroplast RNA processing is required to regulate cell–cell communication in plants.     

Leaf chloroplast ultrastructure and photosynthetic properties of a chlorophyll-deficient mutant of rice

Leaf chloroplast ultrastructure and photosynthetic properties of a natural, yellow-green leaf mutant (ygl1) of rice were characterized. Our results showed that chloroplast development was significantly delayed in the mutant leaves compared with the wild-type rice (WT). As leaves matured, more grana stacks formed concurrently with increasing leaf chlorophyll (Chl) content. Except for the lower intercellular CO₂ concentration, the ygl1 plants had a higher leaf net photosynthetic rate, stomatal conductance, and transpiration rate than those of the WT plants. Under equal amounts of Chl, the excitation energy of PSI and PSII was much stronger in the mutant than that in the WT. The ygl1 plants showed higher nonphotochemical quenching and lower photochemical quenching. They also exhibited higher actual photochemical efficiency of PSII with a higher electron transport rate. Under the light of 200 μmol(photon) m^?2 s^?1, the ygl1 mutant showed lesser deepoxidation of violaxanthin in the xanthophyll cycle than WT, but it increased substantially under strong light conditions. In conclusion, the photosynthetic machinery of the ygl1 remained stable during leaf development. The plants were less sensitive to photoinhibition compared with WT due to the active xanthophyll cycle. The ygl1 plants were efficient in both light harvesting and conversion of solar energy.     

Retracted: Each of the chloroplast potassium efflux antiporters affects photosynthesis and growth of fully developed Arabidopsis rosettes under short‐day photoperiod

In Arabidopsis thaliana, the chloroplast harbors three potassium efflux antiporters (KEAs), namely KEA1 and KEA2 in the inner envelope and KEA3 in the thylakoid membrane. They may play redundant physiological roles as in our previous analyses of young developing Arabidopsis rosettes under long‐day photoperiod (16 h light per day), chloroplast kea single mutants resembled the wild‐type plants, whereas kea1kea2 and kea1kea2kea3 mutants were impaired in chloroplast development and photosynthesis resulting in stunted growth. Here, we aimed to study whether chloroplast KEAs play redundant roles in chloroplast function of older Arabidopsis plants with fully developed rosettes grown under short‐day photoperiod (8 h light per day). Under these conditions, we found defects in photosynthesis and growth in the chloroplast kea single mutants, and most dramatic defects in the kea1kea2 double mutant. The mechanism behind these defects in the single mutants involves reduction in the electron transport rate (kea1 and kea3), and stomata conductance (kea1, kea2 and kea3), which in turn affect CO₂ fixation rates. The kea1kea2 mutant, in addition to these alterations, displayed reduced levels of photosynthetic machinery. Taken together, our data suggest that, in addition to the previously reported roles in chloroplast development in young rosettes, each chloroplast KEA affects photosynthesis and growth of Arabidopsis fully developed rosettes.     

Effects of light on cyanide‐resistant respiration and alternative oxidase function in Arabidopsis seedlings

Mitochondrial alternative oxidase (AOX), the unique respiratory terminal oxidase in plants, catalyzes the energy wasteful cyanide (CN)‐resistant respiration and plays a role in optimizing photosynthesis. Although it has been demonstrated that leaf AOX is upregulated after illumination, the in vivo mechanism of AOX upregulation by light and its physiological significance are still unknown. In this report, red light and blue light‐induced AOX (especially AOX1a) expressions were characterized. Phytochromes, phototropins and cryptochromes, all these photoreceptors mediate the light‐response of AOX1a gene. When aox1a mutant seedlings were grown under a high‐light (HL) condition, photobleaching was more evident in the mutant than the wild‐type plants. More reactive oxygen species (ROS) accumulation and inefficient dissipation of chloroplast reducing‐equivalents in aox1a mutant may account for its worse adaptation to HL stress. When etiolated seedlings were exposed to illumination for 4 h, chlorophyll accumulation was largely delayed in aox1a plants. We first suggest that more reduction of the photosynthetic electron transport chain and more accumulation of reducing‐equivalents in the mutant during de‐etiolation might be the main reasons.     

Adaptation of the Photosynthetic Apparatus of Scenedesmus obliquus to Strong and Weak Light Conditions

Homocontinuous cultures of the unicellular green alga Scenedesmus obliquus were grown under strong (28 W/m²～28,000 lux) and weak (5 W/m²～5000 lux) light conditions to simulate the conditions of ‘sun’ and ‘shade’ plants. As in higher plants the cells adapted to strong light had less chlorophyll but demonstrated a higher photosynthetic capacity and a higher respiration rate, so that their compensation point was reached at three times higher energy than in the cells grown under low light intensities. The CO₂ fixation rate and the RuDP carboxylase activity under saturating light intensities were both higher in the cells grown in strong light. In spite of the differences in the pigment content and in the light saturated photosynthetic capacities for both cultures, the quantum yields of photosynthetic oxygen evolution were equal. As documented for some species of higher plants Scenedesmus is not genetically determined to be either a ‘sun’ or ‘shade’ organism but can adapt its photosynthetic apparatus to the different light intensities.     

ERIL1, the plant homologue of ERI‐1, is involved in the processing of chloroplastic rRNAs

Proteins belonging to the enhancer of RNA interference‐1 subfamily of 3′–5′ exoribonucleases participate in divergent RNA pathways. They degrade small interfering RNAs (siRNAs), thus suppressing RNA interference, and are involved in the maturation of ribosomal RNAs and the degradation of histone messenger RNAs (mRNAs). Here, we report evidence for the role of the plant homologue of these proteins, which we termed ENHANCED RNA INTERFERENCE‐1‐LIKE‐1 (ERIL1), in chloroplast function. In vitro assays with AtERIL1 proved that the conserved 3′–5′ exonuclease activity is shared among all homologues studied. Confocal microscopy revealed that ERL1, a nucleus‐encoded protein, is targeted to the chloroplast. To gain insight into its role in plants, we used Nicotiana benthamiana and Arabidopsis thaliana plants that constitutively overexpress or suppress ERIL1. In the mutant lines of both species we observed malfunctions in photosynthetic ability. Molecular analysis showed that ERIL1 participates in the processing of chloroplastic ribosomal RNAs (rRNAs). Lastly, our results suggest that the missexpression of ERIL1 may have an indirect effect on the microRNA (miRNA) pathway. Altogether our data point to an additional piece of the puzzle in the complex RNA metabolism of chloroplasts.     

Analysis of the expression of CLA1, a gene that encodes the 1-deoxyxylulose 5-phosphate synthase of the 2-C-methyl-D-erythritol-4-phosphate pathway in Arabidopsis

The discovery of the 2-C-methyl-D-erythritol-4-phosphate pathway for the biosynthesis of isoprenoids raises the important question of the nature and regulation of the enzymes involved in this pathway. CLA1, a gene previously isolated from Arabidopsis, encodes the first enzyme of the 2-C-methyl-D-erythritol-4-phosphate pathway, 1-deoxy-D-xylulose-5-phosphate synthase. We demonstrate this enzyme activity by complementation of the cla1-1 mutant phenotype and by direct enzymatic assays. Based on mRNA and protein expression patterns this enzyme is expressed mainly in developing photosynthetic and non-photosynthetic tissues. The beta-glucuronidase expression pattern driven from the CLA1 gene regulatory region supports the northern and protein data while also showing that this gene has some level of expression in most tissues of the plant. A mutation in the CLA1 gene interferes with the normal development of chloroplasts and etioplasts, but does not seem to affect amyloplast structure. Microscopic analysis also shows a pleiotropic effect of the CLA1 gene mutation in mesophyll tissue formation.     

The Arabidopsis nuclear DAL gene encodes a chloroplast protein which is required for the maturation of the plastid ribosomal RNAs and is essential for chloroplast differentiation

Altered pigmentation is an easily scored and sensitive monitor of plastid function. We analyzed in detail a yellow colored transposon-tagged mutant (dal1-2) that is allelic to the dal mutant previously identified (Babiychuk et al., 1997). Mesophyll cells of mutant plants possess abnormal nucleoids and more but smaller plastids than wild type cells. Plastid development in dal1-2 is not altered in the dark but is arrested at the early steps of thylakoid assembly. The amino acid sequence of the protein deduced from our cDNA clone is 21 amino acids longer than the previously published DAL sequence (Babiychuk et al., 1997) and allowed us to show that DAL codes for a chloroplast protein. The dal1-2 mutation has a global negative effect on plastid RNA accumulation and on expression of nuclear encoded photosynthetic genes. We show that the plastid RNA polymerases, the nuclear-encoded NEP and the plastid-encoded PEP, are functional in the mutant. Precursor 16S and 23S rRNA species specifically accumulate at a high level in the mutant but the 5-end and the long 3-end trailer are not modified. We suggest that the dal mutation is involved in plastid rRNA processing and consequently in translation and early chloroplast differentiation.     

Acclimation of photosynthesis to irradiance and spectral quality in British plant species: chlorophyll content, photosynthetic capacity and habitat preference

Twenty-two common British angiosperms were examined for their ability to acclimate photosynthetically to sun and shade conditions. Plants were grown under low irradiance, far-red enriched light (50 μmol m^?2 s^?1), selected to mimic as closely as possible natural canopy shade, and moderately high light of insufficient irradiance to induce photoinhibitory or photoprotective responses (300 μmol m^?2 s^?1). Light-and CO²-saturated photosynthetic rates of oxygen evolution (P_max) and chlorophyll content were measured. Large variation was found in both parameters, and two ‘strategies’ for long-term acclimation were identified: firstly a change in chlorophyll per unit leaf area which was found to correlate positively with photosynthetic capacity, and secondly changes in chlorophyll alb ratio and P_max, indicative of alterations at the chloroplast level, which were not associated with a change in chlorophyll content per unit leaf area. Combinations of these two strategies may occur, giving rise to the observed diversity in photosynthetic acclimation. The extent and nature of photosynthetic acclimation were compared with an index of shade association, calculated from the association each species has with woodland. It was found that the greatest flexibility for change at the chloroplast level was found in those species possessing an intermediate shade association, whilst acclimation in ‘sun’ species proceeded by a change in chlorophyll content; obligate shade species showed little capacity for acclimation at either the chloroplast or leaf level. A framework for explaining the variation between plant species in leaf-level photosynthetic capacity, in relation to the natural light environment, is presented. This is the first time the potential for light acclimation of photosynthesis in different plant species has been satisfactorily linked to habitat distribution.     

Blue light effects on rose photosynthesis and photomorphogenesis

Through its impact on photosynthesis and morphogenesis, light is the environmental factor that most affects plant architecture. Using light rather than chemicals to manage plant architecture could reduce the impact on the environment. However, the understanding of how light modulates plant architecture is still poor and further research is needed. To address this question, we examined the development of two rose cultivars, Rosa hybrida‘Radrazz’ and Rosa chinensis‘Old Blush’, cultivated under two light qualities. Plants were grown from one‐node cuttings for 6 weeks under white or blue light at equal photosynthetic efficiencies. While plant development was totally inhibited in darkness, blue light could sustain full development from bud burst until flowering. Blue light reduced the net CO₂ assimilation rate of fully expanded leaves in both cultivars, despite increasing stomatal conductance and intercellular CO₂ concentrations. In ‘Radrazz’, the reduction in CO₂ assimilation under blue light was related to a decrease in photosynthetic pigment content, while in both cultivars, the chl a/b ratio increased. Surprisingly, blue light could induce the same organogenetic activity of the shoot apical meristem, growth of the metamers and flower development as white light. The normal development of rose plants under blue light reveals the strong adaptive properties of rose plants to their light environment. It also indicates that photomorphogenetic processes can all be triggered by blue wavelengths and that despite a lower assimilation rate, blue light can provide sufficient energy via photosynthesis to sustain normal growth and development in roses.     

ABC1K1/PGR6 kinase: a regulatory link between photosynthetic activity and chloroplast metabolism

Arabidopsis proton gradient regulation (pgr) mutants have high chlorophyll fluorescence and reduced non‐photochemical quenching (NPQ) caused by defects in photosynthetic electron transport. Here, we identify PGR6 as the chloroplast lipid droplet (plastoglobule, PG) kinase ABC1K1 (activity of bc1 complex kinase 1). The members of the ABC1/ADCK/UbiB family of atypical kinases regulate ubiquinone synthesis in bacteria and mitochondria, and impact various metabolic pathways in plant chloroplasts. Here, we demonstrate that abc1k1 has a unique photosynthetic and metabolic phenotype that is distinct from that of the abc1k3 homolog. The abc1k1/pgr6 single mutant is specifically deficient in the electron carrier plastoquinone, as well as in β–carotene and the xanthophyll lutein, and is defective in membrane antioxidant tocopherol metabolism. After 2 days of continuous high light stress, abc1k1/pgr6 plants suffer extensive photosynthetic and metabolic perturbations, strongly affecting carbohydrate metabolism. Remarkably, however, the mutant acclimates to high light after 7 days together with a recovery of carotenoid levels and a drastic alteration in the starch‐to‐sucrose ratio. Moreover, ABC1K1 behaves as an active kinase and phosphorylates VTE1, a key enzyme of tocopherol (vitamin E) metabolism in vitro. Our results indicate that the ABC1K1 kinase constitutes a new type of regulatory link between photosynthetic activity and chloroplast metabolism.     

SOT1, a pentatricopeptide repeat protein with a small MutS‐related domain,is required for correct processing of plastid 23S–4.5S rRNA precursors in Arabidopsis thaliana

Ribosomal RNA processing is essential for plastid ribosome biogenesis, but is still poorly understood in higher plants. Here, we show that SUPPRESSOR OF THYLAKOID FORMATION1 (SOT1), a plastid‐localized pentatricopeptide repeat (PPR) protein with a small MutS‐related domain, is required for maturation of the 23S–4.5S rRNA dicistron. Loss of SOT1 function leads to slower chloroplast development, suppression of leaf variegation, and abnormal 23S and 4.5S processing. Predictions based on the PPR motif sequences identified the 5′ end of the 23S–4.5S rRNA dicistronic precursor as a putative SOT1 binding site. This was confirmed by electrophoretic mobility shift assay, and by loss of the abundant small RNA ‘footprint’ associated with this site in sot1 mutants. We found that more than half of the 23S–4.5S rRNA dicistrons in sot1 mutants contain eroded and/or unprocessed 5′ and 3′ ends, and that the endonucleolytic cleavage product normally released from the 5′ end of the precursor is absent in a sot1 null mutant. We postulate that SOT1 binding protects the 5′ extremity of the 23S–4.5S rRNA dicistron from exonucleolytic attack, and favours formation of the RNA structure that allows endonucleolytic processing of its 5′ and 3′ ends.     

Non‐invasive,whole‐plant imaging of chloroplast movement and chlorophyll fluorescence reveals photosynthetic phenotypes independent of chloroplast photorelocation defects in chloroplast division mutants

Leaf chloroplast movement is thought to optimize light capture and to minimize photodamage. To better understand the impact of chloroplast movement on photosynthesis, we developed a technique based on the imaging of reflectance from leaf surfaces that enables continuous, high‐sensitivity, non‐invasive measurements of chloroplast movement in multiple intact plants under white actinic light. We validated the method by measuring photorelocation responses in Arabidopsis chloroplast division mutants with drastically enlarged chloroplasts, and in phototropin mutants with impaired photorelocation but normal chloroplast morphology, under different light regimes. Additionally, we expanded our platform to permit simultaneous image‐based measurements of chlorophyll fluorescence and chloroplast movement. We show that chloroplast division mutants with enlarged, less‐mobile chloroplasts exhibit greater photosystem II photodamage than is observed in the wild type, particularly under fluctuating high levels of light. Comparison between division mutants and the severe photorelocation mutant phot1‐5 phot2‐1 showed that these effects are not entirely attributable to diminished photorelocation responses, as previously hypothesized, implying that altered chloroplast morphology affects other photosynthetic processes. Our dual‐imaging platform also allowed us to develop a straightforward approach to correct non‐photochemical quenching (NPQ) calculations for interference from chloroplast movement. This correction method should be generally useful when fluorescence and reflectance are measured in the same experiments. The corrected data indicate that the energy‐dependent (qE) and photoinhibitory (qI) components of NPQ contribute differentially to the NPQ phenotypes of the chloroplast division and photorelocation mutants. This imaging technology thus provides a platform for analyzing the contributions of chloroplast movement, chloroplast morphology and other phenotypic attributes to the overall photosynthetic performance of higher plants.