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The atypical trichromatic cyanobacterial phytochrome NpTP1 from Nostoc punctiforme ATCC 29133 is a linear tetrapyrrole (bilin)-binding photoreceptor protein that possesses tandem-cysteine residues responsible for shifting its light-sensing maximum to the violet spectral region. Using bioinformatics and phylogenetic analyses, here we established that tandem-cysteine cyanobacterial phytochromes (TCCPs) compose a well-supported monophyletic phytochrome lineage distinct from prototypical red/far-red cyanobacterial phytochromes. To investigate the light-sensing diversity of this family, we compared the spectroscopic properties of NpTP1 (here renamed NpTCCP) with those of three phylogenetically diverged TCCPs identified in the draft genomes of Tolypothrix sp. PCC7910, Scytonema sp. PCC10023, and Gloeocapsa sp. PCC7513. Recombinant photosensory core modules of ToTCCP, ScTCCP, and GlTCCP exhibited violet-blue-absorbing dark-states consistent with dual thioether-linked phycocyanobilin (PCB) chromophores. Photoexcitation generated singly-linked photoproduct mixtures with variable ratios of yellow-orange and red-absorbing species. The photoproduct ratio was strongly influenced by pH and by mutagenesis of TCCP- and phytochrome-specific signature residues. Our experiments support the conclusion that both photoproduct species possess protonated 15E bilin chromophores, but differ in the ionization state of the noncanonical "second" cysteine sulfhydryl group. We found that the ionization state of this and other residues influences subsequent conformational change and downstream signal transmission. We also show that tandem-cysteine phytochromes present in eukaryotes possess similar amino acid substitutions within their chromophore-binding pocket, which tune their spectral properties in an analogous fashion. Taken together, our findings provide a roadmap for tailoring the wavelength specificity of plant phytochromes to optimize plant performance in diverse natural and artificial light environments.

Key messagePTR2 inArabidopsis thalianais negatively regulated by ABI4 and plays a key role in water uptake by seeds, ensuring that imbibed seeds proceed to germination.AbstractPeptide transporters (PTRs) transport nitrogen-containing substrates in a proton-dependent manner. Among the six PTRs inArabidopsis thaliana, the physiological role of the tonoplast-localized, seed embryo abundant PTR2 is unknown. In the present study, a molecular physiological analysis of PTR2 was conducted usingptr2mutants andPTR2COcomplementation lines. Compared with the wild type, theptr2mutant showed ca. 6 h delay in testa rupture and consequently endosperm rupture because of 17% lower water content and 10% higher free abscisic acid (ABA) content. Constitutive overexpression of thePTR2gene under the control of the Cauliflower mosaic virus (CaMV)35Spromoter inptr2mutants rescued the mutant phenotypes. After cold stratification, a transient increase inABA INSENSITIVE4(ABI4) transcript levels during induction of testa rupture was followed by a similar increase inPTR2transcript levels, which peaked prior to endosperm rupture. ThePTR2promoter region containing multiple CCAC motifs was recognized by ABI4 in electrophoretic mobility shift assays, andPTR2expression was repressed by 67% inABI4overexpression lines compared with the wild type, suggesting that PTR2 is an immediate downstream target of ABI4. Taken together, the results suggest that ABI4-dependent temporal regulation ofPTR2expression may influence water status during seed germination to promote the post-germinative growth of imbibed seeds.

An optimal size of post-embryonic root apical meristem (RAM) is achieved by a balance between cell division and differentiation. Despite extensive research, molecular mechanisms underlying the coordination of cell division and differentiation are still fragmentary. Here, we report that ORESARA 15 (ORE15), an Arabidopsis PLANT A/T-RICH SEQUENCE-AND ZINC-BINDING PROTEIN (PLATZ) transcription factor preferentially expressed in the RAM, determines RAM size. Primary root length, RAM size, cell division rate, and stem cell niche activity were reduced in an ore15 loss-of-function mutant but enhanced in an activation-tagged line overexpressing ORE15, compared with wild type. ORE15 forms mutually positive and negative feedback loops with auxin and cytokinin signalling, respectively. Collectively, our findings imply that ORE15 controls RAM size by mediating the antagonistic interaction between auxin and cytokinin signalling-related pathways.

Although the importance of chloroplast RNA splicing and ribosome maturation (CRM) domain-containing proteins has been established for chloroplast RNA metabolism and plant development, the functional role of CRM proteins in mitochondria remains largely unknown. Here, we investigated the role of a mitochondria-targeted CRM protein (At3g27550), named CFM9, in Arabidopsis thaliana. Confocal analysis revealed that CFM9 is localized in mitochondria. The cfm9 mutant exhibited delayed seed germination, retarded growth and shorter height compared with the wild type under normal conditions. The growth-defect phenotypes were more manifested upon high salinity, dehydration or ABA application. Complementation lines expressing CFM9 in the mutant background fully recovered the wild-type phenotypes. Notably, the mutant had abnormal mitochondria, increased hydrogen peroxide and reduced respiration activity, implying that CFM9 is indispensable for normal mitochondrial function. More important, the splicing of many intron-containing genes in mitochondria was defective in the mutant, suggesting that CFM9 plays a crucial role in the splicing of mitochondrial introns. Collectively, our results provide clear evidence emphasizing that CFM9 is an essential factor in the splicing of mitochondrial introns, which is crucial for mitochondrial biogenesis and function and the growth and development of Arabidopsis.

Transgenic Arabidopsis thaliana plants expressing cyanobacterial decarboxylation genes GLCD1 (GLYCOLATE DEHYDROGENASE I), HDH (HYDROXYACID DEHYDROGENASE), ODC (OXALATE DECARBOXYLASE) alone, and HDH::ODC simultaneously were successfully developed. Plants independently expressing GLCD1, HDH, ODC, and HDH::ODC were referred to as GD, HD, OX, and HX plants, respectively. The single-copy homozygous GD, HD, OX, and HX plants exhibited appreciable expression of chimeric genes. Phenotypic characterization demonstrated that rosette diameter of GD, HD, OX, and HX was 20, 22, 17, and 16% higher than wild-type (WT) plants. Total numbers of leaves were 32, 35, 37, and 34% more than WT plants after 32 days of sowing. Similarly, all transgenic plants produced more cauline branches than WT plants. All transgenic plants gained more height as compared to WT when recorded after 42 days of growth except HX transgenic plants. Plants vegetative dry biomass was 43% (GD), 35% (HD), 42% (OX), and 36% (HX) higher than WT plants. This is the first report on characterization of cyanobacterial decarboxylation pathway genes, which will pave the way for transformation of complete pathway in plants for better biomass accumulation.

Both obtaining high-yielding, viable protoplasts and following reliable regeneration protocols are prerequisites for the continuous expansion and development of newly emerging systems involving protoplast utilization. This study determines an efficient process from protoplast isolation to shoot regeneration in vitro. The maximum yield of protoplast extraction, which was 6.36 ± 0.51 × 106 protoplasts/g fresh weight (FW), was approximately 3.7 times higher than that previously reported for potato protoplasts. To obtain data, wounded leaves were used by partially cutting both sides of the midrib, and isolated protoplasts were purified by the sucrose cushion method, with a sucrose concentration of 20%. We confirmed a significant effect on the extraction efficiency by measuring enzymolysis during a 6 h period, with three times more washing buffer than the amount normally used. Protoplasts fixed in alginate lenses with appropriate space were successfully recovered and developed into microcalli 2 weeks after culture. In addition, to induce high efficiency regeneration from protoplasts, calli in which greening occurred for 6 weeks were induced to develop shoots in regeneration medium solidified by Gelrite, and they presented a high regeneration efficiency of 86.24 ± 11.76%.

International audience; Nannochloropsis species, unicellular industrial oleaginous microalgae, are model organisms for microalgal systems and synthetic biology. To facilitate community-based annotation and mining of the rapidly accumulating functional genomics resources, we have initiated an international consortium and present a comprehensive multi-omics resource database named Nannochloropsis Design and Synthesis (NanDeSyn; http://nandesyn.single-cell.cn). Via the Tripal toolkit, it features user-friendly interfaces hosting genomic resources with gene annotations and transcriptomic and proteomic data for six Nannochloropsis species, including two updated genomes of Nannochloropsis oceanica IMET1 and Nannochloropsis salina CCMP1776. Toolboxes for search, Blast, synteny view, enrichment analysis, metabolic pathway analysis, a genome browser, etc. are also included. In addition, functional validation of genes is indicated based on phenotypes of mutants and relevant bibliography. Furthermore, epigenomic resources are also incorporated, especially for sequencing of small RNAs including microRNAs and circular RNAs. Such comprehensive and integrated landscapes of Nannochloropsis genomics and epigenomics will promote and accelerate community efforts in systems and synthetic biology of these industrially important microalgae.

In microalgae, photosynthesis provides energy and sugar phosphates for the biosynthesis of storage and structural carbohydrates, lipids, and nitrogenous proteins. The oleaginous alga Nannochloropsis salina does not preferentially partition photoassimilates among cellulose, chrysolaminarin, and lipids in response to nitrogenous nutrient deprivation. In the present study, we investigated whether genetic impairment of the cellulose synthase gene (CesA) expression would lead to protein accumulation without the accumulation of storage C polymers in N. salina. Three cesA mutants were generated by the CRISPR/Cas9 approach. Cell wall thickness and cellulose content were reduced in the cesA1 mutant, but not in cesA2 or cesA4 cells. CesA1 mutation resulted in a reduction of chrysolaminarin and neutral lipid contents, by 66.3% and 37.1%, respectively, but increased the soluble protein content by 1.8-fold. Further, N. salina cells with a thinned cell wall were susceptible to mechanical stress, resulting in a 1.7-fold enhancement of lipid extractability. Taken together, the previous and current studies strongly suggest the presence of a controlling mechanism that regulates photoassimilate partitioning toward C and N metabolic pathways as well as the cellulose metabolism as a potential target for cost-effective microalgal cell disruption and as a useful protein production platform.

To perceive fluctuations in light quality, quantity, and timing, higher plants have evolved diverse photoreceptors including UVR8 (a UV-B photoreceptor), cryptochromes, phototropins, and phytochromes (Phys). In contrast to plants, prokaryotic oxygen-evolving photosynthetic organisms, cyanobacteria, rely mostly on bilin-based photoreceptors, namely, cyanobacterial phytochromes (Cphs) and cyanobacteriochromes (CBCRs), which exhibit structural and functional differences compared with plant Phys. CBCRs comprise varying numbers of light sensing domains with diverse color-tuning mechanisms and signal transmission pathways, allowing cyanobacteria to respond to UV-A, visible, and far-red lights. Recent genomic surveys of filamentous cyanobacteria revealed novel CBCRs with broader chromophore-binding specificity and photocycle protochromicity. Furthermore, a novel Cph lineage has been identified that absorbs blue-violet/yellow-orange light. In this minireview, we briefly discuss the diversity in color sensing and signal transmission mechanisms of Cphs and CBCRs, along with their potential utility in the field of optogenetics.

High-temperature stress related to global warming reduces the growth and productivity of seaweeds. Thus, the development of new strains is urgently required for maintaining or even enhancing the productivity of useful seaweeds such as red alga Pyropia yezoenesis in an increasingly warmer sea environment. To develop competitive high-temperature-tolerant strains of P. yezoensis (Sugwawon no. 104), we screened libraries of gamma-irradiated strains and identified high-temperature-resistant (HTR) mutants. Our results showed that HTR-1 and HTR-2 grew well at higher temperatures that inhibited the growth of the wild-type strain. The efficiency of conchosporangium maturation and conchospore release of HTR-1 was similar to or higher than the wild-type strain. Moreover, thallus growth, pigment content, photosynthetic efficiency, and monospore release from the growing thallus in HTR-1 could be maintained even at high temperature. Taken together, our data demonstrate that HTR-1 may be suitable for industrial cultivation at sea, even at elevated temperatures.

Endophytic bacteria (EB) are both a novel source of bioactive compounds that confer phytopathogen resistance and inducers of secondary metabolites in host plants. Twenty-seven EB isolated from various parts of Metasequoia glyptostroboides, Ginkgo biloba, Taxus brevifolia, Pinus densiflora, Salix babylonica, and S. chaenomeloides could produce salicylic acid (SA). The highest producers were isolates EB-44 and EB-47, identified as Pseudomonas tremae and Curtobacterium herbarum, respectively. Nicotiana benthamiana grown from EB-44-soaked seeds exhibited a 2.3-fold higher endogenous SA concentration and increased resistance against P. syringae pv. tabaci, the causative agent of tobacco wildfire disease, than plants grown from water-soaked seeds. N benthamiana and N. tabacum grown from EB-44-treated seeds developed 33% and 54% disease lesions, respectively, when infected with P. syringae pv. tabaci, and showed increased height and weight, in addition to 4.6 and 1.4-fold increases in nicotine accumulation, respectively. The results suggest that SA-producing EB-44 can successfully colonize Nicotiana spp., leading to increased endogenous SA production and resistance to tobacco wildfire disease. The newly isolated EB can offer an efficient and eco-friendly solution for controlling wildfire disease and nicotine accumulation in Nicotiana, with additional application for other important crops to increase both productivity and the generation of bioactive compounds.

Fructans are polymers of fructose that are present as storage carbohydrates in various plants. Jerusalem artichoke (Helianthus tuberosus L.) contains a high amount of inulin. Two enzymes are involved in inulin biosynthesis. The sucrose:sucrose 1-fructosyltransferase (1-SST) enzyme mainly catalyzes the synthesis of 1-kestose from sucrose. In the next step, fructan:fructan 1-fructosyltransferase (1-FFT) catalyzes the synthesis of inulin from 1-kestose. In this study, the Ht1-SST and Ht1-FFT genes were isolated from Jerusalem artichoke and expressed in potato (Solanum tuberosum L.), either separately or together, via Agrobacterium-mediated transformation. Transgenic potato tubers overexpressing Ht1-SST accumulated 1-kestose to a high level (up to 3.36 mg/g), while tubers overexpressing both Ht1-SST and Ht1-FFT accumulated up to 3.14 mg/g short-chain inulin-type fructans, with the degree of polymerization (DP) ranging from 3 to 5, excluding high DP inulins. Transgenic potato plants accumulated fructo-oligosaccharides to a high level, following the fructan biosynthetic pathway of Jerusalem artichoke, and therefore present a high potential for the mass production of inulin through established potato breeding and cultivation methods.

Summary Panax ginseng C. A. Meyer, reputed as the king of medicinal herbs, has slow growth, long generation time, low seed production and complicated genome structure that hamper its study. Here, we unveil the genomic architecture of tetraploid P. ginseng by de novo genome assembly, representing 2.98 Gbp with 59 352 annotated genes. Resequencing data indicated that diploid Panax species diverged in association with global warming in Southern Asia, and two North American species evolved via two intercontinental migrations. Two whole genome duplications (WGD) occurred in the family Araliaceae (including Panax) after divergence with the Apiaceae, the more recent one contributing to the ability of P. ginseng to overwinter, enabling it to spread broadly through the Northern Hemisphere. Functional and evolutionary analyses suggest that production of pharmacologically important dammarane‐type ginsenosides originated in Panax and are produced largely in shoot tissues and transported to roots; that newly evolved P. ginseng fatty acid desaturases increase freezing tolerance; and that unprecedented retention of chlorophyll a/b binding protein genes enables efficient photosynthesis under low light. A genome‐scale metabolic network provides a holistic view of Panax ginsenoside biosynthesis. This study provides valuable resources for improving medicinal values of ginseng either through genomics‐assisted breeding or metabolic engineering.

We demonstrated successful overexpression of porcine reproductive and respiratory syndrome virus (PRRSV)-derived GP4D and GP5D antigenic proteins in Arabidopsis. Pigs immunized with transgenic plants expressing GP4D and GP5D proteins generated both humoral and cellular immune responses to PRRSV. Porcine reproductive and respiratory syndrome virus (PRRSV) causes PRRS, the most economically significant disease affecting the swine industry worldwide. However, current commercial PRRSV vaccines (killed virus or modified live vaccines) show poor efficacy and safety due to concerns such as reversion of virus to wild type and lack of cross protection. To overcome these problems, plants are considered a promising alternative to conventional platforms and as a vehicle for large-scale production of recombinant proteins. Here, we demonstrate successful production of recombinant protein vaccine by expressing codon-optimized and transmembrane-deleted recombinant glycoproteins (GP4D and GP5D) from PRRSV in planta. We generated transgenic Arabidopsis plants expressing GP4D and GP5D proteins as candidate antigens. To examine immunogenicity, pigs were fed transgenic Arabidopsis leaves expressing the GP4D and GP5D antigens (three times at 2-week intervals) and then challenged with PRRSV at 6-week post-initial treatment. Immunized pigs showed significantly lower lung lesion scores and reduced viremia and viral loads in the lung than pigs fed Arabidopsis leaves expressing mYFP (control). Immunized pigs also had higher titers of PRRSV-specific antibodies and significantly higher levels of pro-inflammatory cytokines (TNF-α and IL-12). Furthermore, the numbers of IFN-γ+-producing cells were higher, and those of regulatory T cells were lower, in GP4D and GP5D immunized pigs than in control pigs. Thus, plant-derived GP4D and GP5D proteins provide an alternative platform for producing an effective subunit vaccine against PRRSV.

Transgene expression in microalgae can be hampered by transgene silencing and unstable expression due to position effects. To overcome this, "safe harboring" transgene expression system was established for Nannochloropsis. Initially, transformants were obtained expressing a sfGFP reporter, followed by screening for high expression of sfGFP with fluorescence-activated cell sorter (FACS). 'T1' transcriptional hotspot was identified from a mutant showing best expression of sfGFP, but did not affect growth or lipid contents. By using a Cas9 editor strain, FAD12 gene, encoding Δ12-fatty acid desaturase (FAD12), was successfully knocked-in at the T1 locus, resulting in significantly higher expression of FAD12 than those of random integration. Importantly, the "safe harbored" FAD12 transformants showed four-fold higher production of linoleic acid (LA), the product of FAD12, leading to 1.5-fold increase in eicosapentaenoic acid (EPA). This safe harboring principle provide excellent proof of the concept for successful genetic/metabolic engineering of microalgae and other organisms.

Cytoplasmic male sterility (CMS) is a maternally inherited trait leading to loss of the ability to produce fertile pollen and is extensively used in hybrid crop breeding. Ogura- CMS was originally generated by insertion of orf138 upstream of atp8 in the radish mitochondrial genome and transferred to Brassica crops for hybrid breeding. Gene expression changes by dysfunctional mitochondria in Ogura-CMS result in pollen developmental defects, but little is known about gene expression patterns in vegetative tissue. To examine the interaction between nuclear and organellar regulation of gene expression, microarray and subsequent gene expression experiments were conducted with leaves of F1 hybrid Chinese cabbage derived from self-incompatible (SI) or Ogura-CMS parents (Brassica rapa ssp. pekinensis). Out of 24,000 genes deposited on a KBGP24K microarray, 66 genes were up-regulated and 26 genes were down-regulated by over 2.5 fold in the CMS leaves. Up-regulated genes included stress-response genes and mitochondrial protein genes, while genes for ascorbic acid biosynthesis and thylakoid proteins were down-regulated. Most of the major component genes for light reactions of photosynthesis were highly expressed in leaves of both SI and CMS plants, but most of the cor-responding proteins were found to be greatly reduced in leaves of CMS plants, indicating posttranscriptional regulation. Reduction in thylakoid proteins and chlorophylls led to reduction in photosynthetic efficiency and chlorosis of Ogura- CMS at low temperatures. This research provides a foundation for studying chloroplast function regulated by mitochondrial signal and for using organelle genome introgression in molecular breeding.

Summary Despite the importance of pentatricopeptide repeat (PPR) proteins in organellar RNA metabolism and plant development, the functions of many PPR proteins remain unknown. Here, we determined the role of a mitochondrial PPR protein (At1g52620) comprising 19 PPR motifs, thus named PPR19, in Arabidopsis thaliana. The ppr19 mutant displayed abnormal seed development, reduced seed yield, delayed seed germination, and retarded growth, indicating that PPR19 is indispensable for normal growth and development of Arabidopsis thaliana. Splicing pattern analysis of mitochondrial genes revealed that PPR19 specifically binds to the specific sequence in the 3′-terminus of the NADH dehydrogenase 1 (nad1) transcript and stabilizes transcripts containing the second and third exons of nad1. Loss of these transcripts in ppr19 leads to multiple secondary effects on accumulation and splicing of other nad1 transcripts, from which we can infer the order in which cis- and trans-spliced nad1 transcripts are normally processed. Improper splicing of nad1 transcripts leads to the absence of mitochondrial complex I and alteration of the nuclear transcriptome, notably influencing the alternative splicing of a variety of nuclear genes. Our results indicate that the mitochondrial PPR19 is an essential component in the splicing of nad1 transcripts, which is crucial for mitochondrial function and plant development.

Like other halophilic cyanobacterial genomes, the de novo-assembled genome of Euhalothece sp. Z-M001 lacks genes encoding keto-carotenoid biosynthesis enzymes, despite the presence of genes encoding carotenoid-binding proteins (CBPs). Consistent with this, HPLC analysis of carotenoids identified β-carotene and zeaxanthin as the dominant carotenoids. CBPs coexpressed with the zeaxanthin biosynthesis gene increased the survival rates of Escherichia coli strains by preventing antibiotic-induced accumulation of reactive oxygen species (ROS). RNA-seq analysis of Euhalothece revealed that among various salt resistance-related genes, those encoding the Na+ transporting multiple resistance and pH adaptation (Mrp) systems, glycine betaine biosynthesis enzymes, exopolysaccharide metabolic enzymes, and CBPs were highly upregulated, suggesting their importance in hypersaline habitats. During the early phase of salt deprivation, the amounts of β-carotene and zeaxanthin showed a negative correlation with ROS content. Overall, we propose that in some halophilic cyanobacteria, β-carotene and zeaxanthin, rather than keto-carotenoids, serve as the major chromophores for CBPs, which in turn act as effective antioxidants.

Transgenic potato (Solanum tuberosum L. cv. Desiree) plants expressing components of a novel cyanobacterial photorespiratory glycolate catabolism pathway were developed. Transgenic plant expressing glcD1 (glycolate dehydrogenase I) gene was referred to as synGDH and transgenic plants expressing gcl (glyoxylate carboligase) and tsr (tartronic semialdehyde reductase) genes simultaneously were designated as synGT. Both synGDH and synGT plants showed stable gene transformation, integration and expression. Enhanced glyoxylate contents in synGDH plants were detected as compared to synGT and non-transgenic (NT) plants. Phenotypic evaluation revealed that synGDH plants accumulated 11 % higher dry weight, while, tuber weight was 38 and 16 % higher than NT and synGT, respectively. Upon challenging the plants in high temperature and high light conditions synGDH plants maintained higher Fv/Fm and showed less bleaching of chlorophyll as compared to synGT and NT plants. These results indicate that genetic transformation of complete pathway in one plant holds promising outcomes in terms of biomass accumulation to meet future needs for food and energy.

At present geological epoch, the carbon concentrating mechanism (CCM) of cyanobacteria represents the obligatory tool for adaptation to low content of CO2 in the atmosphere and for the maintenance of sufficient photosynthetic activity. Functional CCM was found in modern cyanobacteria from different ecological niches. However, the presence of such mechanism in species that inhabit soda lakes is not obvious due to high content of inorganic carbon (C i) in the environment. Here we analyze CCM components that have been identified by sequencing of the whole genome of the alkaliphilic cyanobacterium Microcoleus sp. IPPAS B-353. The composition of the CCM components of Microcoleus is similar to that of ‘model’ β-cyanobacteria, freshwater and marine Synechococcus or Synechocystis spp. However, CahB1 protein of Microcoleus, which is the homolog of CcaA, the carboxysomal β-type carbonic anhydrase (CA) of β-cyanobacteria, appeared to be the only active CA located in cell envelopes. The conservative regions of CcmM, CahG (a homolog of archeal γ-CAs, Cam/CamH), and ChpX of Microcoleus possess single amino acid substitutions that may cause a lack of CA activities. Unlike model cyanobacteria, Microcoleus induces only one BicA-type bicarbonate transporter in response to C i limitation. The differences in the appearance of CCM components and in their characteristics between alkaliphilic Microcoleus and freshwater or marine cyanobacteria are described. The possible reasons for the maintenance of CCM components in cyanobacteria, which permanently live at high concentrations of C i in soda lakes, are discussed.

Plant genome analyses, including Arabidopsis thaliana showed a large gene family of plant receptor kinases with various extracellular ligand-binding domain. Now intensively studies to understand physiological and cellular functions for higher plant receptor kinases in diverse and complex biological processes including plant growth, development, ligands perception including steroid hormone and plant-microbe interactions. Brassinosteroids (BRs) as a one of well know steroid hormone are plant growth hormones that control biomass accumulation and also tolerance to many biotic and abiotic stress conditions and hence are of relevance to agriculture. BRI1 receptor kinase, which is localized in plasma membrane in the cell sense BRs and it bind to a receptor protein known as BRASSINOSTEROID INSENSITIVE 1 (BRI1). Recently, we reported that BRI1 and its co-receptor, BRI1-ASSOCIATED KINASE (BAK1) autophosphorylated on tyrosine residue (s) in vitro and in vivo and thus are dual-specificity kinases. Other plant receptor kinases are also phosphorylated on tyrosine residue (s). Post-translational modifications (PTMs) can be studied by altering the residue modified by directed mutagenesis to mimic the modified state or to prevent the modification. These approaches are useful to not only characterize the regulatory role of a given modification, but may also provide opportunities for plant improvement.

Most aquatic photosynthetic organisms have developed inorganic carbon-concentrating mechanisms (CCMs) to compensate for the kinetic constraints of CO2 concentration in the vicinity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), which functions in the first steps of carbon fixation. CCMs, which are widespread in various types of algae, evolved independently among algal lineages and accordingly play a fundamental role in algal photosynthesis, metabolism, growth, and biomass production. Nannochloropsis, a marine eustigmatophycean microalga, is a candidate organism for biofuel production due to its high lipid content; however, inorganic carbon (Ci) availability in Nannochloropsis cells is not sufficient for complete carbon fixation due to inherently weak CCM machinery, including a CO2-leaking HCO3− pump. CrLCIA, a member of the formate-nitrite transporter family, functions as a HCO3− transporter in Chlamydomonas reinhardti. Thus, in this study, we overexpressed the CrLCIA gene heterologously in N. salina CCMP1776 in an attempt to reinforce its bicarbonate transport activity. CrLCIA expression in N. salina increased intracellular Ci and carbonic anhydrase (CA) activity, resulting in increased growth (30%) and biomass (2-fold). These results indicate that constitutively expressed CrLCIA leads to increased Ci uptake and CA activity, contributing to high availability of CO2 for photosynthesis under low CO2 conditions. The total fatty acid (FA) per cell mass in the transgenic lines was similar to that of wild-type Nannochloropsis cells, indicating that total FA productivity in the transgenic lines is increased approximately 2-fold. These findings will be useful to improve the CCM function for high biomass and lipid production using genetic modification tools in Nannochloropsis species, contributing to improved biodiesel production.

Plants of wheat cultivar Iksan370, which was recently produced by crossing paternal line Keumkang with maternal line Xian83, accumulate anthocyanins in their coleoptiles and seeds. However, the spatio-temporal regulation of anthocyanin biosynthesis genes in wheat remains poorly understood. Therefore, in the present study, we characterized wheat dihydroflavonol 4-reductase (DFR), which catalyzes the conversion of dihydroflavonol to leucoanthocyanidins during anthocyanin biosynthesis, using a heterologous expression system. The deduced amino acid sequence from full-length cDNA of wheat DFR cloned from young seedlings of Iksan370 (TaDFR-I) includes a well-conserved substrate-binding domain compared with previously identified DFRs. Furthermore, as expected, phylogenetic tree analysis placed TaDFR-I in the monocotyledonous clade. Introduction of TaDFR-I into the wild-type Arabidopsis Col-0 and dfr mutant backgrounds resulted in significant accumulation of anthocyanins in the respective transgenic plants. Similarly, TaDFR transcripts highly accumulated in Iksan370 wheat seedlings under various anthocyanin-inducing conditions, including low temperature, high salt and sugar levels, and UV-B illumination. Thus, we functionally characterized TaDFR-I from Iksan370 lines using a heterologous expression system and revealed the presence of transcriptional regulatory factors similar to those of Arabidopsis that regulate TaDFR expression in response to various abiotic stresses.

The Korean wheat line Iksan370 was recently bred by crossing the paternal line Keumkang with the maternal line Xian83. Iksan370 has a giant phenotype in terms of its whole body architecture, and its height and leaf and spikelet length are higher than those of the paternal line Keumkang. From young seedlings to adult plants, the photosynthetic pigmentation and photosystem II efficiency of Iksan370 and Keumkang lines did not differ significantly; however, Iksan370 lines had significantly larger leaf epidermal cells than Keumkang, resulting in enlarged leaf architecture. Consistently, RNA sequencing analysis and subsequent quantitative real-time PCR analysis indicated that expression of target of rapamycin (TOR) was several fold higher in Iksan370 than in Keumkang, while expression of cell division- and photosynthesis-related genes did not differ among the lines. These data suggest that the giant characteristics of Iksan370 leaves are owing to enhanced expression of TOR, which is involved in cell expansion.

Genome editing techniques are critical for manipulating genes not only to investigate their functions in biology but also to improve traits for genetic engineering in biotechnology. Genome editing has been greatly facilitated by engineered nucleases, dubbed molecular scissors, including zinc-finger nuclease (ZFN), TAL effector endonuclease (TALEN) and clustered regularly interspaced palindromic sequences (CRISPR)/Cas9. In particular, CRISPR/Cas9 has revolutionized genome editing fields with its simplicity, efficiency and accuracy compared to previous nucleases. CRISPR/Cas9-induced genome editing is being used in numerous organisms including microalgae. Microalgae have been subjected to extensive genetic and biological engineering due to their great potential as sustainable biofuel and chemical feedstocks. However, progress in microalgal engineering is slow mainly due to a lack of a proper transformation toolbox, and the same problem also applies to genome editing techniques. Given these problems, there are a few reports on successful genome editing in microalgae. It is, thus, time to consider the problems and solutions of genome editing in microalgae as well as further applications of this exciting technology for other scientific and engineering purposes.

It is necessary for molecular breeders to overcome the difficulties in applying abundant genomic information to crop breeding. Candidate orthologs would be discovered more efficiently in less-studied crops if the information gained from studies of related crops were used. We developed a comparative analysis tool and web-based genome viewer to identify orthologous genes based synteny as well as sequence similarity between tomato, pepper and potato. The tool has a step-by-step interface with multiple viewing levels to support the easy and accurate exploration of functional orthologs. Furthermore, it provides access to single nucleotide-polymorphism markers from the massive genetic resource pool in order to accelerate the development of molecular markers for candidate orthologs in the Solanaceae. This tool provides a bridge between genome data and breeding by supporting effective marker development, data utilization and communication. Database URL: http://tgsol.seeders.co.kr/scomp/

Bioechnology of microalgae takes much attention because of their ability to utilize light energy and fix CO2. Research in biotechnology of microalgae including eukaryotic algae and cyanobacteria is an important and attractive topic which attracts the interests of the public widely. This Research Topic aims to create a collection approaching biotechnology and biology of eukaryotic algae and cyanobacteria. Basic science of molecular biology and biochemistry is indispensable for proceeding future application of microalgae, and hence, the title includes "molecular biology" and "biochemistry". Broad range of basic and applied science of microalgae is appreciated in this special topic.

Microalgal photosynthesis provides energy and carbon-containing precursors for the biosynthesis of storage carbohydrates such as starch, chrysolaminarin, lipids, and cell wall components. Under mild nitrogen deficiency (N−), some Nannochloropsis species accumulate lipid by augmenting cytosolic fatty acid biosynthesis with a temporary increase in laminarin. Accordingly, biosynthesis of the cellulose-rich cell wall should change in response to N− stress because this biosynthetic pathway begins with utilisation of the hexose phosphate pool supplied from photosynthesis. However, few studies have characterised microalgal cell wall metabolism, including oleaginous Nannochloropsis sp. microalgae subjected to nitrogen deficiency. Here, we investigated N-induced changes in cellulose biosynthesis in N. salina. We observed that N− induced cell wall thickening, concurrently increased the transcript levels of genes coding for UDPG pyrophosphorylase and cellulose synthases, and increased cellulose content. Nannochloropsis salina cells with thickened cell wall were more susceptible to mechanical stress such as bead-beating and sonication, implicating cellulose metabolism as a potential target for cost-effective microalgal cell disruption.

Cyanobacteriochromes (CBCRs) are a subfamily of phytochrome photoreceptors found exclusively in photosynthetic cyanobacteria. Four CBCRs containing a second Cys in the insert region (insert-Cys) have been identified from the nonheterocystous cyanobacterium Microcoleus B353 (Mbr3854g4 and Mbl3738g2) and the nitrogen fixing, heterocystous cyanobacterium Nostoc punctiforme (NpF2164g3 and NpR1597g2). These insert-Cys CBCRs can sense light in the near-UV to orange range, but key residues responsible for tuning their colour sensitivity have not been reported. In the present study, near-UV/Green (UG) photosensors Mbr3854g4 (UG1) and Mbl3738g2 (UG2) were chosen for further spectroscopic analysis of their spectral sensitivity and tuning. Consistent with most dual-Cys CBCRs, both UGs formed a second thioether linkage to the phycocyanobilin (PCB) chromophore via the insert-Cys. This bond is subject to breakage and relinkage during forward and reverse photoconversions. Variations in residues equivalent to Phe that are in close contact with the PCB chromophore D-ring in canonical red/green CBCRs are responsible for tuning the light absorption peaks of both dark and photoproducts. This is the first time these key residues that govern light absorption in insert-Cys family CBCRs have been identified and characterised.

Arctic microalgae are considered alternative sources for biodiesel production in cold climates. In this study, we describe the isolation of a novel Chlamydomonas sp. from Arctic sea ice, which we named ArM0029A, and the development of a new culture medium, HAP3, that allows greater cell growth and density than seawater-based media. The fatty acid composition of ArM0029A was characterized at various sodium chloride (NaCl) concentrations. Culture at low NaCl concentration (7 ‰), relative to seawater, enhanced growth and fatty acid accumulation of ArM0029A. Total fatty acid content reached a maximum of approximately 27 % of dry weight at 7 ‰ NaCl without nitrogen, and unsaturated fatty acids, such as C16:4, C18:2, and C18:3, were predominant. Interestingly, the proportion of C16:3 was significantly induced in ArM0029A by salt stress. These findings indicate that use of ArM0029A may be feasible for production of lipids, especially polyunsaturated fatty acids, under cold climatic conditions via adjustment of NaCl concentrations.

Plant receptor-like kinases (RLKs) are proteins that are involved in the regulation of development, hormone signaling, abiotic, and biotic stress responses. It has been suggested that cysteine-rich receptor-like kinases (CRKs), which are one of the largest RLK groups, is significant in pathogen defense and programmed cell death. The CRK1 gene is isolated and characterized from tomato (Solanum lycopersicum L.). The SlCRK1 has two C-X8-C-X2-C motifs: a trans-membrane region and a kinase domain similar to other CRKs. The semi-quantitative RT-PCR exhibits the specific expression of SlCRK1 in the flower, but not in the root, leaf, seed, and fruit of the tomato. In addition, SlCRK1 exhibits pollen-specific expression in the floral organ. SlCRK1 has pollen-specific cis-acting elements in the promoter region, and its promoter has pollen-specific activity in the homozygous transgenic plants of tomato and Arabidopsis as confirmed through histochemical GUS assays. Moreover, the expression of SlCRK1 is not detected via stress treatment or hormone treatment. In this study, SlCRK1 from tomato is characterized and its promoter can be useful in developing transgenic plants with foreign genes that should be expressed in pollens.

Kim, W. B., Lim, C. J., Jang, H. A., Yi, S. Y., Oh, S.-K., Lee, H. Y., Kim, H. A., Park, Y.-I. and Kwon, S.-Y. 2014. SlPMEI, a pollen-specific gene in tomato. Can. J. Plant Sci. 94: 73–83. Pectin is one of the main components of plant cell walls, and its biosynthesis is controlled by pectin methylesterase (PME). Pectin methylesterase inhibitors (PMEIs) are key regulators of PME. We report here the cloning and characterization of a novel Solanum lycopersicum L. PMEI gene, SlPMEI. RT-PCR studies of leaf, seed, fruit, flower, and flower organs confirmed that SlPMEI is expressed specifically in pollen. Promoter analysis of SlPMEI revealed pollen-specific cis-acting elements (pollen lat52 and g10). In addition, SlPMEI is expressed independently of abiotic stress, pathogen exposure, and growth stage in tomato, and a histochemical analysis of promoter activity revealed pollen-specific expression in both Arabidopsis and tomato. Under the microscope, we observed pollen-specific GUS expression in the stamen of transgenic tomato plant. These results indicate that the promoter of SlPMEI has strong pollen-specific activity, and could therefore be useful for development of industrially and agronomically important transgenic plants.

Despite the importance of pentatricopeptide repeat (PPR) proteins in organellar RNA metabolism and plant development, the functions of many PPR proteins remain unknown. Here, we determined the role of a mitochondrial PPR protein (At1g52620) comprising 19 PPR motifs, thus named PPR19, in Arabidopsis thaliana. The ppr19 mutant displayed abnormal seed development, reduced seed yield, delayed seed germination, and retarded growth, indicating that PPR19 is indispensable for normal growth and development of Arabidopsis thaliana. Splicing pattern analysis of mitochondrial genes revealed that PPR19 specifically binds to the specific sequence in the 3'-terminus of the NADH dehydrogenase 1 (nad1) transcript and stabilizes transcripts containing the second and third exons of nad1. Loss of these transcripts in ppr19 leads to multiple secondary effects on accumulation and splicing of other nad1 transcripts, from which we can infer the order in which cis- and trans-spliced nad1 transcripts are normally processed. Improper splicing of nad1 transcripts leads to the absence of mitochondrial complex I and alteration of the nuclear transcriptome, notably influencing the alternative splicing of a variety of nuclear genes. Our results indicate that the mitochondrial PPR19 is an essential component in the splicing of nad1 transcripts, which is crucial for mitochondrial function and plant development.

Summary Pumilio RNA-binding proteins are largely involved in mRNA degradation and translation repression. However, a few evolutionarily divergent Pumilios are also responsible for proper pre-rRNA processing in human and yeast. Here, we describe an essential Arabidopsis nucleolar Pumilio, APUM24, that is expressed in tissues undergoing rapid proliferation and cell division. A T-DNA insertion for APUM24 did not affect the male and female gametogenesis, but instead resulted in a negative female gametophytic effect on zygotic cell division immediately after fertilization. Additionally, the mutant embryos displayed defects in cell patterning from pro-embryo through globular stages. The mutant embryos were marked by altered auxin maxima, which were substantiated by the mislocalization of PIN1 and PIN7 transporters in the defective embryos. Homozygous apum24 callus accumulates rRNA processing intermediates, including uridylated and adenylated 5.8S and 25S rRNA precursors. An RNA–protein interaction assay showed that the histidine-tagged recombinant APUM24 binds RNAin vitro with no apparent specificity. Overall, our results demonstrated that APUM24 is required for rRNA processing and early embryogenesis in Arabidopsis.