Green Phenotype Research Articles

Performance and competitiveness of red vs. green phenotypes of a cyanobacterium grown under artificial lake browning

Increasing inputs of dissolved organic matter (DOM) to northern lakes is resulting in ‘lake browning.’ Lake browning profoundly affects phytoplankton community composition by modifying two important environmental drivers—light and nutrients. The impact of increased DOM on native isolates of red and green-pigmented cyanobacteria identified as Pseudanabaena, which emerged from a Dolichospermum bloom (Dickson Lake, Algonquin Provincial Park, Ontario, Canada) in 2015, were examined under controlled laboratory conditions. The genomes were sequenced to identify phylogenetic relatedness and physiological similarities, and the physical and chemical effects of increased DOM on cellular performance and competitiveness were assessed. Our study findings were that the isolated red and green phenotypes are two distinct species belonging to the genus Pseudanabaena; that both isolates remained physiologically unaffected when grown independently under defined DOM regimes; and that neither red nor green phenotype achieved a competitive advantage when grown together under defined DOM regimes. While photosynthetic pigment diversity among phytoplankton offers niche-differentiation opportunities, the results of this study illustrate the coexistence of two distinct photosynthetic pigment phenotypes under increasing DOM conditions.

Metabolome and transcriptome profiling of Theobroma cacao provides insights into the molecular basis of pod color variation.

The Theobroma cacao presents a wide diversity in pod color among different cultivars. Although flavonoid biosynthesis has been studied in many plants, molecular mechanisms governing the diversity of coloration in cacao pods are largely unknown. The flavonoid metabolite profiles and flavonoid biosynthetic gene expression in the pod exocarps of light green pod 'TAS 410' (GW), green pod 'TAS 166' (GF), and mauve pod 'TAS 168' (PF) were determined. Changes in flavonoid metabolites, particularly the anthocyanins (cyanidin 3-O-galactoside, cyanidin 3-O-glucoside, and cyanidin O-syringic acid) were significantly up-accumulated in the mauve phenotype (PF) compared to the light green or green phenotypes, endowing the pod color change from light green or green to mauve. Consistently, the PF phenotype showed different expression patterns of flavonoid biosynthetic structural genes in comparison with GW/GF phenotypes. The expression level of LAR and ANR in GW/GF was significantly higher than PF, while the expression level of UFGT in GW/GF was lower than PF. These genes likely generated more anthocyanins in the exocarps samples of PF than that of GW/GF. Simultaneously, colorless flavan-3-ols (catechin, epicatechin and proanthocyanidin) content in the exocarp samples of PF was lower than GW/GF. Additionally, MYB (gene18079) and bHLH (gene5045 and gene21575) may participate in the regulation of the pod color. This study sheds light on the molecular basis of cacao pod color variation, which will contribute to breeding cacao varieties with enhanced flavonoid profiles for nutritional applications.

Autophagy mutants show delayed chloroplast development during de‐etiolation in carbon limiting conditions

SUMMARYAutophagy is a conserved catabolic process that plays an essential role under nutrient starvation conditions and influences different developmental processes. We observed that seedlings of autophagy mutants (atg2, atg5, atg7, and atg9) germinated in the dark showed delayed chloroplast development following illumination. The delayed chloroplast development was characterized by a decrease in photosynthetic and chlorophyll biosynthetic proteins, lower chlorophyll content, reduced chloroplast size, and increased levels of proteins involved in lipid biosynthesis. Confirming the biological impact of these differences, photosynthetic performance was impaired in autophagy mutants 12 h post‐illumination. We observed that while gene expression for photosynthetic machinery during de‐etiolation was largely unaffected in atg mutants, several genes involved in photosystem assembly were transcriptionally downregulated. We also investigated if the delayed chloroplast development could be explained by lower lipid import to the chloroplast or lower triglyceride (TAG) turnover. We observed that the limitations in the chloroplast lipid import imposed by trigalactosyldiacylglycerol1 are unlikely to explain the delay in chloroplast development. However, we found that lower TAG mobility in the triacylglycerol lipase mutant sugardependent1 significantly affected de‐etiolation. Moreover, we showed that lower levels of carbon resources exacerbated the slow greening phenotype whereas higher levels of carbon resources had an opposite effect. This work suggests a lack of autophagy machinery limits chloroplast development during de‐etiolation, and this is exacerbated by limited lipid turnover (lipophagy) that physically or energetically restrains chloroplast development.

Mapping of a Pale Green Mutant Gene and Its Functional Verification by Allelic Mutations in Chinese Cabbage (Brassica rapa L. ssp. pekinensis).

Leaves are the main organ for photosynthesis, and variations in leaf color affect photosynthesis and plant biomass formation. We created two similar whole-plant pale green mutants (pem1 and pem2) from the double haploid (DH) Chinese cabbage line “FT” through ethyl methanesulfonate (EMS) mutagenesis of seeds. Photosynthetic pigment contents and net photosynthetic rates were significantly lower in the mutants than in the wild-type “FT,” and the chloroplast thylakoid endomembrane system was poor. Genetic analysis showed that the mutated phenotypes of pem1 and pem2 were caused by a single nuclear gene. Allelism tests showed that pem1 and pem2 were alleles. We mapped Brpem1 to a 64.25 kb region on chromosome A10, using BSR-Seq and map-based cloning of 979 F2 recessive individuals. Whole-genome re-sequencing revealed a single nucleotide polymorphism (SNP) transition from guanine to adenosine on BraA10g021490.3C in pem1, causing an amino acid shift from glycine to glutamic acid (G to E); in addition, BraA10g021490.3C in pem2 was found to have a single nucleotide substitution from guanine to adenosine, causing an amino acid change from E to lysine (K). BraA10g021490.3C is a homolog of the Arabidopsisdivinyl chlorophyllide a 8-vinyl-reductase (DVR) gene that encodes 3,8-divinyl protochlorophyllide a 8-vinyl reductase, which is a key enzyme in chlorophyll biosynthesis. Enzyme activity assay and chlorophyll composition analysis demonstrated that impaired DVR had partial loss of function. These results provide a basis to understand chlorophyll metabolism and explore the mechanism of a pale green phenotype in Chinese cabbage.

BnaA02.YTG1, encoding a tetratricopeptide repeat protein, is required for early chloroplast biogenesis in Brassica napus

Chloroplasts are essential for plant growth and development, as they play a key role in photosynthesis. The chloroplast biogenesis process is complex and its regulatory mechanism remains elusive. We characterized a spontaneous Brassica napus (rapeseed) mutant, ytg, that showed a delayed greening phenotype in all green organs and retarded growth. We identified BnaA02.YTG1 encoding a chloroplast-localized tetratricopeptide repeat protein widely expressed in rapeseed tissues. We speculated that the ytg phenotype was caused by the deletion of BnaA02.YTG1 based on sequence comparison of 4608 (with normal green leaves, isolated from the elite Chinese rapeseed cultivar ZS11) and ytg combined with transcriptome data and CRISPR/Cas9 gene editing results. The homologous gene (BnaC02.YTG1) restored the phenotype of the mutant. BnaA02.YTG1 interacted with MORF2, MORF8, and OZ1. RNA editing of the ndhD-2, ndhF-290, petL-5, and ndhG-50 plastid transcripts was affected in ytg. These findings suggested that BnaA02.YTG1 participates in RNA editing events. We predicted 29 RNA editing sites in the chloroplast of Brassica napus by comparison with the Arabidopsis chloroplast genome. We conclude that BnaA02.YTG1 affects the posttranscriptional regulation of plastid gene expression and suggest that a tetratricopeptide repeat protein is involved in the chloroplast RNA editing in rapeseed.

CRISPR/Cas9-mediated targeted mutagenesis in Japanese cedar (Cryptomeria japonica D. Don)

Cryptomeria japonica (Japanese cedar or sugi) is one of the most important coniferous tree species in Japan and breeding programs for this species have been launched since 1950s. Genome editing technology can be used to shorten the breeding period. In this study, we performed targeted mutagenesis using the CRISPR/Cas9 system in C. japonica. First, the CRISPR/Cas9 system was tested using green fluorescent protein (GFP)-expressing transgenic embryogenic tissue lines. Knock-out efficiency of GFP ranged from 3.1 to 41.4% depending on U6 promoters and target sequences. The GFP knock-out region was mottled in many lines, indicating genome editing in individual cells. However, in 101 of 102 mutated individuals (> 99%) from 6 GFP knock-out lines, embryos had a single mutation pattern. Next, we knocked out the endogenous C. japonica magnesium chelatase subunit I (CjChlI) gene using two guide RNA targets. Green, pale green, and albino phenotypes were obtained in the gene-edited cell lines. Sequence analysis revealed random deletions, insertions, and replacements in the target region. Thus, targeted mutagenesis using the CRISPR/Cas9 system can be used to modify the C. japonica genome.

Efficient knockout of the phytoene desaturase gene in a hybrid poplar (Populus alba × Populus glandulosa) using the CRISPR/Cas9 system with a single gRNA.

The CRISPR/Cas9 system has been used for genome editing in several plant species; however, there are few reports on its use in trees. Here, CRISPR/Cas9 was used to mutate a target gene in Populus alba × Populus glandulosa hybrid poplars. The hybrid poplar is routinely used in molecular biological studies due to the well-established Agrobacterium-mediated transformation method. A single guide RNA (sgRNA) with reported high mutation efficiency in other popular species was designed with a protospacer adjacent motif sequence for the phytoene desaturase 1 (PagPDS1) gene. The pHSE/Cas9-PagPDS1 sgRNA vector was delivered into hybrid poplar cells using Agrobacterium-mediated transformation. The transgenic plants were propagated and classified them into three groups according to their phenotypes. Among a total of 110 lines of transgenic hybrid poplars, 82 lines showed either an albino or a pale green phenotype, indicating around 74.5% phenotypic mutation efficiency of the PagPDS1 gene. The albino phenotypes were observed when the CRISPR/Cas9-mediated mutations in both PagPDS1 alleles in the transgenic plants. There was no off-target modification of the PagPDS2 gene, which has a potential sgRNA target sequence with two mismatches. The results confirmed that the sgRNA can specifically edit PagPDS1 rather than PagPDS2, indicating that CRISPR/Cas9-mediated genome editing can effectively induce target mutations in the hybrid poplar. This technique will be useful to improve tree quality in hybrid poplars (P. alba × P. glandulosa); for example, by enhancing biomass or stress tolerance.

Status of the largest extant population of the critically endangered Aeolian lizard Podarcis raffonei (Capo Grosso, Vulcano island).

The Aeolian wall lizard Podarcis raffonei is an island endemic that survives only on three tiny islets, and on the Capo Grosso peninsula of the Vulcano island, thus is among the European vertebrates with the smallest range and one of the most threatened by extinction. This species is declining due to competition and hybridization with the non-native lizard Podarcis siculus, but a regular monitoring program is lacking. Here we assessed the size and status of the Capo Grosso population of P. raffonei on Vulcano. In September 2015 we captured 30 individuals showing the typical brown phenotype of P. raffonei, while one single male showed a green phenotype, apparently intermediate between P. raffonei and the non-native Podarcis siculus. In May 2017, only 47% of 131 individuals showed the typical brown phenotype (P. raffonei-like) and 53% showed the green phenotype (P. siculus-like). Based on N-mixture models and removal sampling the estimated size of the Capo Grosso population was of 800–1300 individuals in 2017, being similar to 2015; available data suggest that the total range of the species could be as small as 2 ha. The frequency of individuals with the typical P. raffonei phenotype dramatically dropped between two samplings with a parallel increase of individuals displaying the green phenotype. Observation on outdoor captive-bred individuals demonstrates plasticity for colouration in P. raffonei individuals from Capo Grosso, with several individuals showing the typical brown pattern in September 2017 and a green pattern in March 2021. Non-exclusive hypotheses, including hybridization with P. siculus and plasticity in colour pattern of P. raffonei, are discussed to explain the phenotypic shifts of the P. raffonei population of Capo Grosso. While genomic evidence is required to reach conclusions and investigate eventual hybridization, it is urgent to undertake a programme for the monitoring and management of this lizard.

A dominant spotted leaf gene TaSpl1 activates endocytosis and defense-related genes causing cell death in the absence of dominant inhibitors

The spotted leaf lesion mimic trait simulates cell death in a plant responding to pathogen infection. Some spotted leaf genes are recessive, while others are dominant. A small number of plants with a lesion mimic phenotype appeared in a segregating population obtained by crossing two normal green wheat strains, XN509 and N07216. Here, we clarified the genetic model and its breeding value. Phenotyping of the consecutive progeny populations over six cropping seasons showed that the spotted leaf lesion mimic phenotype was controlled by a dominant gene designated TaSpl1, which was inhibited by two other dominant genes, designated TaSpl1-I1 and TaSpl1-I2. Using bulked segregant analysis RNA-seq (BSR-Seq) and newly developed KASP-PCR markers, the TaSpl1 and TaSpl1-I1 loci in N07216 were mapped to the end of chromosomes 3DS and 3BS, respectively. Plants with the spotted phenotype showed lower levels of stripe rust and powdery mildew than those with the normal green phenotype. Compared with normal leaves, the differentially expressed genes in spotted leaves were significantly enriched in plant-pathogen interaction and endocytosis pathways. There were no differences in the yield parameters of the F5 and F6 sister lines, N13039S with TaSpl1 and N13039 N without TaSpl1. These results provide a greater understanding of spotted leaf phenotyping and the breeding value of the lesion mimic allele in developing disease-resistance varieties.

Partial and full mycoheterotrophy in green and albino phenotypes of the slipper orchid Cypripedium debile.

Most green orchids form mycorrhizal associations with rhizoctonia fungi, a polyphyletic group including Serendipitaceae, Ceratobasidiaceae, and Tulasnellaceae. Although accumulating evidence indicated that partial mycoheterotrophy occurs in such so-called rhizoctonia-associated orchids, it remains unclear how much nutrition rhizoctonia-associated orchids obtain via mycoheterotrophic relationships. We investigated the physiological ecology of green and albino individuals of a rhizoctonia-associated orchid Cypripedium debile, by using molecular barcoding of the mycobionts and stable isotope (13C and 15N) analysis. Molecular barcoding of the mycobionts indicated that the green and albino individuals harbored Tulasnella spp., which formed a clade with the previously reported C. debile mycobionts. In addition, stable isotope analysis showed that both phenotypes were significantly enriched in 13C but not in 15N. Therefore, green and albino individuals were recognized as partial and full mycoheterotrophs, respectively. The green variants were estimated to obtain 42.5 ± 8.2% of their C from fungal sources, using the 13C enrichment factor of albino individuals as a mycoheterotrophic endpoint. The proportion of fungal-derived C in green C. debile was higher than that reported in other rhizoctonia-associated orchids. The high fungal dependence may facilitate the emergence of albino mutants. Our study provides the first evidence of partial mycoheterotrophy in the subfamily Cypripedioideae. Partial mycoheterotrophy may be more general than previously recognized in the family Orchidaceae.

Cold acclimation can specifically inhibit chlorophyll biosynthesis in young leaves of Pakchoi

BackgroundLeaf color is an important trait in breeding of leafy vegetables. Y-05, a pakchoi (Brassica rapa ssp. chinensis) cultivar, displays yellow inner (YIN) and green outer leaves (GOU) after cold acclimation. However, the mechanism of this special phenotype remains elusive.ResultsWe assumed that the yellow leaf phenotype of Y-05 maybe caused by low chlorophyll content. Pigments measurements and transmission electron microscopy (TEM) analysis showed that the yellow phenotype is closely related with decreased chlorophyll content and undeveloped thylakoids in chloroplast. Transcriptomes and metabolomes sequencing were next performed on YIN and GOU. The transcriptomes data showed that 4887 differentially expressed genes (DEGs) between the YIN and GOU leaves were mostly enriched in the chloroplast- and chlorophyll-related categories, indicating that the chlorophyll biosynthesis is mainly affected during cold acclimation. Together with metabolomes data, the inhibition of chlorophyll biosynthesis is contributed by blocked 5-aminolevulinic acid (ALA) synthesis in yellow inner leaves, which is further verified by complementary and inhibitory experiments of ALA. Furthermore, we found that the blocked ALA is closely associated with increased BrFLU expression, which is indirectly altered by cold acclimation. In BrFLU-silenced pakchoi Y-05, cold-acclimated leaves still showed green phenotype and higher chlorophyll content compared with control, meaning silencing of BrFLU can rescue the leaf yellowing induced by cold acclimation.ConclusionsOur findings suggested that cold acclimation can indirectly promote the expression of BrFLU in inner leaves of Y-05 to block ALA synthesis, resulting in decreased chlorophyll content and leaf yellowing. This study revealed the underlying mechanisms of leaves color change in cold-acclimated Y-05.

A single SNP in Brcer1 results in wax deficiency in Chinese cabbage (Brassica campestris L. ssp. pekinensis)

Cuticle wax covering the surface of plants is mainly composed of derivatives of long-chain saturated fatty acids. Chinese cabbage leaves with wax deficiency has a bright green phenotype, which makes it attractive and improves its commercial value. In this study, a wax deficiency mutant, cer1, was obtained from an ethyl methanesulfonate (EMS) mutagenesis population in Chinese cabbage. Scanning electron microscopy (SEM) revealed that cuticular wax in the leaves and stems of mutants was significantly less than that in the wild type (WT). The mutants decreased the total wax, alkanes, ketones and alcohols contents in leaves, but aldehydes content increased compared to WT. Genetic analysis showed that the mutant cer1 phenotype was controlled by a single recessive nuclear gene. MutMap and Kompetitive Allele-Specific PCR (KASP) analyses indicated that BraA09g066480.3C, which encoded aldehyde decarboxylase, might be the causal gene. A single nucleotide substitution (SNP) from C to T in the fourth exon of BraA09g066480.3C resulted in an amino acid substitution from serine to phenylalanine, which led to changes in the three-dimensional structure of proteins. SNP 45,148,866 occurred in the highly conserved fatty acid hydroxylase domain. This study revealed that SNP is effective for marker-assisted selection of wax deficient Chinese cabbage and provides convenience for breeding new varieties exhibiting a wax deficient phenotype.

The effect of water extract from bentonite on embryonic and larval development of the green toad (bufoviridis) with different phenotypes

Ontogenesis is a complex process of morphophysiological transformations. Embryonic and postembryonic development is continuously affected by both endogenous and environmental factors, which lead to variability in morphometric parameters. The research was conducted in the laboratory of ecological embryology of the Federal State Budgetary Educational Institution of Higher Education of the North Osetian Federal University (Vladikavkaz). The results showed that under the influence of bentonite extract, the morphological parameters of green toad larvae with a brown phenotype are more variable than in larvae with an olive green phenotype in both the control and experimental groups. Thus, the embryos and larvae of the green toad with the olive green phenotype are more stable than the larvae that had the brown phenotype, both in natural circumstances and under the influence of bentonite clay extracts.

Parental bleaching susceptibility leads to differences in larval fluorescence and dispersal potential in Pocillopora acuta corals

Coral reef ecosystems are declining at an alarming rate. Increasing seawater temperatures and occurrence of extreme warming events can impair sexual reproduction in reef-building corals and inhibit the ability for coral communities to replenish and persist. Here, we investigated the role of photophysiology on the reproductive ecology of Pocillopora acuta coral colonies by focusing on the impacts of bleaching susceptibility of parents on reproduction and larval performance, during an El Niño Southern Oscillation event in Mo'orea, French Polynesia. Elevated temperature conditions at that time induced bleaching phenotypic differences among P. acuta individuals: certain colonies became pale (from the loss of pigments and/or decline in symbiont cell density), while others remained pigmented (normal/high symbiont cell density). More specifically, we studied the impact of parental phenotypes on offspring's fluorescence by counting released larvae and sorting them by fluorescence types, we assessed survival to thermal stress, recruitment success and post-recruitment survival of released larvae from each fluorescent phenotype, during summer months (February to April 2016). Our results showed that red and green fluorescent larvae released by P. acuta had distinct physiological performances: red fluorescent larvae exhibited a higher survival into the pelagic phase regardless temperature conditions, with lower capacity to settle and survive post-recruitment, compared to green larvae that settle within a short period. Interestingly, pale colonies released two-to seven-fold more red fluorescent larvae than pigmented colonies did. In the light of our results, photophysiological profiles of the brooding P. acuta parental colonies may modulate the fluorescence features of released larvae, and thus influence the dispersal strategy of their offspring, the green fluorescent larval phenotypes being more performant in the benthic than pelagic phase.

Rapid nitrogen fixation contributes to a similar growth and photosynthetic rate of Robinia pseudoacacia supplied with different levels of nitrogen

Robinia pseudoacacia L. is a legume species that is widely used in afforestation, which has high N2 fixation capacity and rapid growth rate. Both nitrogen (N) supply and phenology affect plant growth, photosynthesis and leaf senescence. The aim of this study was to determine how N supply affects N2 fixation, leaf photosynthesis and senescence of R. pseudoacacia at different phenological stages. Seedlings of R. pseudoacacia were supplied with different levels of 15N-labelled NH4NO3 solution, with seedlings of Sophora japonica Linn. as reference plants to calculate the percentage of N derived from the atmospheric N2 (%Ndfa). Compared with plants supplied with a high N level, those with a low N supply had a higher %Ndfa at an early developmental stage. Nitrogen fixation compensated the effect of a low N supply on plant growth in R. pseudoacacia. A high N supply decreased biomass allocation to lateral roots and nodules, and increased the relative growth rate of plant height as well as specific leaf area. The eighth mature compound leaf of R. pseudoacacia tended to have a higher net photosynthetic rate than the fourth leaf, and the leaves still maintained a moderate photosynthetic rate in early autumn. Plants tended to allocate more biomass to leaves at an early developmental stage and to stems and roots at a later developmental stage (3 months old). The N level did not affect leaf photosynthesis at different phenological stages, primarily due to (i) a high %Ndfa under low N supply at early growing stage, and a similar high %Ndfa under all N supplies at a late growing stage, and (ii) the delayed greening phenotype of expanding leaves to save nutrients for mature leaves.

Physicochemical Variability and Biodiesel Potential of Seed Oils of Two Hibiscus sabdariffa L. Phenotypes.

Considerable interest is being focused on vegetable oils as fuel. Due to their characteristics being close to diesel and their renewable potential, studies recommend their use for agricultural applications. Hibiscus sabdariffa var. sabdariffa is widely studied for the nutritional properties of its calyces. Although the seeds of this species are known to be rich in fatty acids, their use is little known in Benin Republic. Similarly, a few studies have attempted to characterize the seeds of the green phenotype of this plant species. By following standard methods, the fatty acid profiles of oils extracted from the seeds of the two varieties (red phenotype, sabdariffa (HSS), and green phenotype, altissima (HSA)) of H. sabdariffaL. were established. A comparative study of their physicochemical properties was also performed to highlight their potential use as fuel. It follows that HSS seed oil is yellow while HSA seed oil is dark green. For the two varieties, values obtained for the kinematic viscosity (∼4 mm2/s), cetane number (∼55), and density (0.87 g/cm3) are in accordance with the U.S. and European standards. However, it is observed that HSA oil is significantly more acidic (23.10 ± 0.22 for HSS vs 18.20 ± 0.40 mg KOH/g oil for HSS) with a higher peroxide value (HSA: 0.280 ± 0.002 vs HSS: 0.140 ± 0.001). The major fatty acids are the following: palmitic (HSA: 27.09 vs HSS: 25.48%), oleic (HSA: 31.81 vs HSS: 35.21%), and linoleic (HSA: 31.43 vs HSS: 29.70%) acids. These fatty acid profiles give to the two oils calorific values (∼39.45 MJ/kg) lower than that of diesel but good oxidative stability and cold filter plugging. The two oils could be used as fuel oil, after their transesterification to improve their properties.

Identification of a delayed leaf greening gene from a mutation of pummelo.

Delayed greening of young leaves is an unusual phenomenon of plants in nature. Citrus are mostly evergreen tree species. Here, a natural mutant of "Guanxi" pummelo (Citrus maxima), which shows yellow leaves at the young stage, was characterized to identify the genes underlying the trait of delayed leaf greening in plants. A segregating population with this mutant as the seed parent and a normal genotype as the pollen parent was generated. Two DNA pools respectively from the leaves of segregating seedlings with extreme phenotypes of normal leaf greening and delayed leaf greening were collected for sequencing. Bulked segregant analysis (BSA) and InDel marker analysis demonstrated that the delayed leaf greening trait is governed by a 0.3 Mb candidate region on chromosome 6. Gene expression analysis further identified a key candidate gene (Citrus Delayed Greening gene 1, CDG1) in the 0.3 Mb region, which showed significantly differential expression between the genotypes with delayed and normal leaf greening phenotypes. There was a 67 bp InDel region difference in the CDG1 promoter and the InDel region contains a TATA-box element. Confocal laser-scanning microscopy revealed that the CDG1-GFP fusion protein signals were co-localized with the chloroplast signals in the protoplasts. Overexpression of CDG1 in tobacco and Arabidopsis led to the phenotype of delayed leaf greening. These results suggest that the CDG1 gene is involved in controlling the delayed leaf greening phenotype with important functions in chloroplast development.

Investigating the effects of nest shading on the green turtle (Chelonia mydas) hatchling phenotype in the Ogasawara islands using a field-based split clutch experiment.

The Ogasawara Islands are an important rookery for the green turtle (Chelonia mydas) in the North Pacific. Green turtles possess temperature-dependent sex determination, and warmer incubation temperatures produce more females than males. Therefore, conservation practices such as nest shading may be required for this population to mitigate the effect of global warming on their sex ratio. To consider the application of such conservation practices in the Ogasawara population, it is fundamental to understand how artificially modified nest environments will affect green turtle hatchling phenotypes that influence their fitness. Here, we investigated the effects of nest shading on green turtle hatchling phenotypes in the Ogasawara population by using a split clutch experimentequally separating the clutch, relocating each half-clutch into an outdoor hatchery area either with or without shading, and observing the subsequent hatchling phenotype. Our results showed that the shading treatment produced hatchlings with a better self-righting response and a larger carapace size. Additionally, the shading treatment mostly reduced the production of hatchlings with a nonmodal scute pattern and produced hatchlings with a smaller unabsorbed yolk sac, which may be associated with their residual yolk mass. These results suggest that conservation practices such as shading could alter not only the sex ratio but also the hatchling phenotype that influences their fitness. Hence, our results suggest that applications of such conservation strategies must be carefully considered.

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.

Sequence analysis and protein interactions of Arabidopsis CIA2 and CIL proteins

BackgroundA previous screening of Arabidopsis thaliana for mutants exhibiting dysfunctional chloroplast protein transport identified the chloroplast import apparatus (cia) gene. The cia2 mutant has a pale green phenotype and reduced rate of protein import into chloroplasts, but leaf shape and size are similar to wild-type plants of the same developmental stage. Microarray analysis showed that nuclear CIA2 protein enhances expression of the Toc75, Toc33, CPN10 and cpRPs genes, thereby up-regulating protein import and synthesis efficiency in chloroplasts. CIA2-like (CIL) shares 65% sequence identity to CIA2, suggesting that CIL and CIA2 are homologous proteins in Arabidopsis. Here, we further assess the protein interactions and sequence features of CIA2 and CIL.ResultsSubcellular localizations of truncated CIA2 protein fragments in our onion transient assay demonstrate that CIA2 contains two nuclear localization signals (NLS) located at amino acids (aa) 62-65 and 291-308, whereas CIL has only one NLS at aa 47-50. We screened a yeast two-hybrid (Y2H) Arabidopsis cDNA library to search for putative CIA2-interacting proteins and identified 12 nuclear proteins, including itself, CIL, and flowering-control proteins (such as CO, NF-YB1, NF-YC1, NF-YC9 and ABI3). Additional Y2H experiments demonstrate that CIA2 and CIL mainly interact with flowering-control proteins via their N-termini, but preferentially form homo- or hetero-dimers through their C-termini. Moreover, sequence alignment showed that the N-terminal sequences of CIA2, CIL and NF-YA are highly conserved. Therefore, NF-YA in the NF-Y complex could be substituted by CIA2 or CIL.ConclusionsWe show that Arabidopsis CIA2 and CIL can interact with CO and NF-Y complex, so not only may they contribute to regulate chloroplast function but also to modulate flower development.