Abstract Plants produce a large variety of lipophilic metabolites, many of which are secreted by cells and accumulated in apoplasts. These compounds often play a role to protect plants from environmental stresses. However, little is known how these lipophilic compounds are secreted into apoplastic spaces. In this study, we used shikonin-producing cultured cells of Lithospermum erythrorhizon as an experimental model system to analyze the secretion of lipophilic metabolites taking its advantages, such as the high production rate and the clear inducibility using culture conditions. Shikonin derivatives are lipophilic red naphthoquinone compounds that accumulate exclusively in apoplastic spaces of these cells and also in the root epidermis of intact plant. Microscopic analysis showed that shikonin is accumulated in a vast number of particles on the cell wall. Lipidomic analysis showed that L. erythrorhizon cultured cells secrete an appreciable portion of triacylglycerol (24 to 38% of total TAG), composed predominantly of saturated fatty acids. Moreover, in vitro reconstitution assay showed that TAG encapsulates shikonin derivatives with phospholipids to form lipid droplet-like structures. These findings suggest a novel role for TAG as a matrix lipid, a molecular component involved in the secretion of specialized lipophilic metabolites.

Abstract Plant growth and development are regulated by environmental factors, including nutrient availability and light conditions, via endogenous genetic signaling pathways. Phosphorylation-dependent protein modification plays a major role in the regulation of cell proliferation in stress conditions, and several protein kinases have been shown to function in response to nutritional status, including dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs). Although DYRKs are widely conserved in eukaryotes, the physiological functions of DYRKs in land plants are still to be elucidated. In the liverwort Marchantia polymorpha, a model bryophyte, four putative genes encoding DYRK-homologous proteins, each of which belongs to the subfamily Yak1, DYRKP, DYRK2, or PRP4K, were identified. MpYAK1-defective male and female mutant lines generated by a CRISPR/Cas9 system showed smaller sizes of thalli than did the wild-type plants, and repressed cell divisions in the apical notch regions. The Mpyak1 mutants developed rhizoids from gemmae in the gemma cup before release. The Mpyak1 lines developed sexual organs even in non-inductive short-day photoperiod conditions supplemented with far-red light. In nitrogen (N)-deficient conditions, rhizoid elongation was inhibited in the Mpyak1 mutants. In conditions with aeration with 0.08% CO2 (v/v) and N-depletion, Mpyak1 mutants accumulated higher levels of sucrose and lower levels of starch compared to wild-type. Transcriptomic analyses revealed that expression of peroxidase genes was differentially affected by MpYAK1. These results suggest that MpYAK1 is involved in the maintenance of plant growth and developmental responses to light conditions and nutrient signaling.

Photosynthetic organisms are exposed to various environmental sources of oxidative stress. Land plants have diverse mechanisms to withstand oxidative stress, but how microalgae do so remains unclear. Here, we characterized the Chlamydomonas reinhardtii basic leucine zipper (bZIP) transcription factor BLZ8, which is highly induced by oxidative stress. Oxidative stress tolerance increased with increasing BLZ8 expression levels. BLZ8 regulated the expression of genes likely involved in the carbon-concentrating mechanism (CCM): HIGH-LIGHT ACTIVATED 3 (HLA3), CARBONIC ANHYDRASE 7 (CAH7), and CARBONIC ANHYDRASE 8 (CAH8). BLZ8 expression increased the photosynthetic affinity for inorganic carbon under alkaline stress conditions, suggesting that BLZ8 induces the CCM. BLZ8 expression also increased the photosynthetic linear electron transfer rate, reducing the excitation pressure of the photosynthetic electron transport chain and in turn suppressing reactive oxygen species (ROS) production under oxidative stress conditions. A carbonic anhydrase inhibitor, ethoxzolamide, abolished the enhanced tolerance to alkaline stress conferred by BLZ8 overexpression. BLZ8 directly regulated the expression of the three target genes and required bZIP2 as a dimerization partner in activating CAH8 and HLA3. Our results suggest that a CCM-mediated increase in the CO2 supply for photosynthesis is critical to minimize oxidative damage in microalgae, since slow gas diffusion in aqueous environments limits CO2 availability for photosynthesis, which can trigger ROS formation.

Dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs) are activated via the auto-phosphorylation of conserved tyrosine residues in their activation loop during protein translation, and they then phosphorylate serine/threonine residues on substrates. The DYRK family is widely conserved in eukaryotes and is composed of six subgroups. In plant lineages, DYRK homologs are classified into four subgroups, DYRK2s, yet another kinase1s, pre-mRNA processing factor 4 kinases, and DYRKPs. Only the DYRKP subgroup is plant-specific and has been identified in a wide array of plant lineages, including land plants and green algae. It has been suggested that in Arabidopsis thaliana DYRKPs are involved in the regulation of centripetal nuclear positioning induced by dark light conditions. However, the molecular functions, such as kinase activity and the developmental and physiological roles of DYRKPs are poorly understood. Here, we focused on a sole DYRKP ortholog in the model bryophyte, Marchantia polymorpha, MpDYRKP. MpDYRKP has a highly conserved kinase domain located in the C-terminal region and shares common sequence motifs in the N-terminal region with other DYRKP members. To identify the roles of MpDYRKP in M. polymorpha, we generated loss-of-function Mpdyrkp mutants via genome editing. Mpdyrkp mutants exhibited abnormal, shrunken morphologies with less flattening in their vegetative plant bodies, thalli, and male reproductive organs, antheridial receptacles. The surfaces of the thalli in the Mpdyrkp mutants appeared uneven and disordered. Moreover, their epidermal cells were drastically altered to a narrower shape when compared to the wild type. These results suggest that MpDYRKP acts as a morphological regulator, which contributes to orderly tissue morphogenesis via the regulation of cell shape.

The advent of image-activated cell sorting and imaging-based cell picking has advanced our knowledge and exploitation of biological systems in the last decade. Unfortunately, they generally rely on fluorescent labeling for cellular phenotyping, an indirect measure of the molecular landscape in the cell, which has critical limitations. Here we demonstrate Raman image-activated cell sorting by directly probing chemically specific intracellular molecular vibrations via ultrafast multicolor stimulated Raman scattering (SRS) microscopy for cellular phenotyping. Specifically, the technology enables real-time SRS-image-based sorting of single live cells with a throughput of up to ~100 events per second without the need for fluorescent labeling. To show the broad utility of the technology, we show its applicability to diverse cell types and sizes. The technology is highly versatile and holds promise for numerous applications that are previously difficult or undesirable with fluorescence-based technologies.

Kajikawa M, Sierro N, Hashimoto T, Shoji T, Plant signaling & behavior, 2017, 2017

Kajikawa M, Abe T, Ifuku K, Furutani KI, Yan D, Okuda T, Ando A, Kishino S, Ogawa J, Fukuzawa H, Scientific reports, 2016, vol. 6, pp. 36809, 2016

Akashi K, Yoshimura K, Kajikawa M, Hanada K, Kosaka R, Kato A, Katoh A, Nanasato Y, Tsujimoto H, Yokota A, Bioscience, biotechnology, and biochemistry, 2016, vol. 80, no. 10, pp. 1907-1916, 2016

Kajikawa M, Sawaragi Y, Shinkawa H, Yamano T, Ando A, Kato M, Hirono M, Sato N, Fukuzawa H, Plant physiology, 2015, vol. 168, no. 2, pp. 752-764, 2015

Kajikawa M, Kinohira S, Ando A, Shimoyama M, Kato M, Fukuzawa H, PloS one, 2015, vol. 10, no. 3, pp. e0120446, 2015

Kihara H, Tanaka M, Yamato KT, Horibata A, Yamada A, Kita S, Ishizaki K, Kajikawa M, Fukuzawa H, Kohchi T, Akakabe Y, Matsui K, Phytochemistry, 2014, vol. 107, pp. 42-49, 2014

After the accident of the Fukushima 1 Nuclear Power Plant in March 2011, radioactive cesium was released and paddy fields in a wide area including Fukushima Prefecture were contaminated. To estimate the levels of radioactive Cs accumulation in rice produced in Fukushima, it is crucial to obtain the actual data of Cs accumulation levels in rice plants grown in the actual paddy field in Fukushima City. We herein conducted a two-year survey in 2011 and 2012 of radioactive and non-radioactive Cs accumulation in rice using a number of rice cultivars grown in the paddy field in Fukushima City. Our study demonstrated a substantial variation in Cs accumulation levels among the cultivars of rice. © 2013 The Botanical Society of Japan and Springer Japan.

Ohmori Y, Kajikawa M, Nishida S, Tanaka N, Kobayashi NI, Tanoi K, Furukawa J, Fujiwara T, Journal of plant research, 2014, vol. 127, no. 1, pp. 67-71, 2014

Tanaka N, Uraguchi S, Saito A, Kajikawa M, Kasai K, Sato Y, Nagamura Y, Fujiwara T, Plant & cell physiology, 2013, vol. 54, no. 12, pp. 2011-2019, 2013

Minamisawa N, Sato M, Cho KH, Ueno H, Takechi K, Kajikawa M, Yamato KT, Ohyama K, Toyooka K, Kim GT, Horiguchi G, Takano H, Ueda T, Tsukaya H, The Plant journal : for cell and molecular biology, 2011, vol. 68, no. 5, pp. 788-799, 2011

Akashi K, Yoshida K, Kuwano M, Kajikawa M, Yoshimura K, Hoshiyasu S, Inagaki N, Yokota A, Planta, 2011, vol. 233, no. 5, pp. 947-960, 2011

Kajikawa M, Shoji T, Kato A, Hashimoto T, Plant physiology, 2011, vol. 155, no. 4, pp. 2010-2022, 2011

Kajikawa M, Fujibe T, Uraguchi S, Miwa K, Fujiwara T, Bioscience, biotechnology, and biochemistry, 2011, vol. 75, no. 12, pp. 2421-2423, 2011

Shoji T, Kajikawa M, Hashimoto T, The Plant cell, 2010, vol. 22, no. 10, pp. 3390-3409, 2010

Kajikawa M, Morikawa K, Abe Y, Yokota A, Akashi K, Plant cell reports, 2010, vol. 29, no. 7, pp. 771-778, 2010

Uchida H, Yamashita H, Kajikawa M, Ohyama K, Nakayachi O, Sugiyama R, Yamato KT, Muranaka T, Fukuzawa H, Takemura M, Ohyama K, Planta, 2009, vol. 229, no. 6, pp. 1243-1252, 2009

Kajikawa M, Hirai N, Hashimoto T, Plant molecular biology, 2009, vol. 69, no. 3, pp. 287-298, 2009

Kajikawa M, Matsui K, Ochiai M, Tanaka Y, Kita Y, Ishimoto M, Kohzu Y, Shoji S, Yamato KT, Ohyama K, Fukuzawa H, Kohchi T, Bioscience, biotechnology, and biochemistry, 2008, vol. 72, no. 2, pp. 435-444, 2008

Kajikawa M, Yamato KT, Sakai Y, Fukuzawa H, Ohyama K, Kohchi T, FEBS letters, 2006, vol. 580, no. 1, pp. 149-154, 2006

Kajikawa M, Yamato KT, Kohzu Y, Shoji S, Matsui K, Tanaka Y, Sakai Y, Fukuzawa H, Plant & cell physiology, 2006, vol. 47, no. 1, pp. 64-73, 2006

Kajikawa M, Yamato KT, Fukuzawa H, Sakai Y, Uchida H, Ohyama K, Phytochemistry, 2005, vol. 66, no. 15, pp. 1759-1766, 2005

Kajikawa M, Yamato KT, Kanamaru H, Sakuradani E, Shimizu S, Fukuzawa H, Sakai Y, Ohyama K, Bioscience, biotechnology, and biochemistry, 2003, vol. 67, no. 8, pp. 1667-1674, 2003

Kajikawa M, Yamaoka S, Yamato KT, Kanamaru H, Sakuradani E, Shimizu S, Fukuzawa H, Ohyama K, Bioscience, biotechnology, and biochemistry, 2003, vol. 67, no. 3, pp. 605-612, 2003

In the haploid dioecious liverwort, Marchantia polymorpha, the X chromosome, but not the Y, carries a cluster of ribosomal RNA genes (rDNAs). Here we show that sequences of 5S, 17S, 5.8S and 26S rDNAs are highly conserved(> 99% identity) between the X chromosomal and autosomal rDNA repeat units, but the intergenic spacer sequences differ considerably. The most prominent difference is the presence of a 615-bp DNA fragment in the intergenic spacer, X615, which has accumulated predominantly in the rDNA cluster of the X chromosome. These observations suggest that the rDNA repeat unit on the X chromosome evolved independently of that on autosomes, incorporating sex chromosome-specific sequences.

本研究では、ハイスループットな変異株スクリーニングによる順遺伝学解析系が確立されたモデル緑藻クラミドモナスを活用し、植物界で先駆けて従来型のオートファジー因子とは異なる新奇な細胞生存を制御する分子機構の包括的な解明を目指している。本年は、表現型の緩やかなオートファジー変異体（atg9）への更なる変異導入により選抜されたN・S・Pのいずれの栄養欠乏条件においても生存性が親株よりも早期に低下する変異体1C7および21B1変異株の解析を進めた。昨年度、これらの変異体ではそれぞれ異なるタンパク質リン酸化酵素遺伝子（1C7遺伝子、21B1遺伝子と名付けた）に挿入変異を持つことを明らかにしている。それぞれの変異体と野生型との交配により、ATG9への変異とそれぞれのタンパク質リン酸化酵素遺伝子の変異を分離した。1C7遺伝子あるいは21B1遺伝子にのみ変異をもつ子孫系統の生存率に関する表現型は、元の1C7および21B1変異株と同等であった。このことから、1C7および21B1変異株における生存率低下の表現型にはATG9の変異は寄与せず、もっぱら1C7遺伝子あるいは21B1遺伝子への変異によるものであることが示唆された。それぞれの単独変異体に野生型の1C7遺伝子および21B1遺伝子を導入すると変異表現型は野生型と同程度まで回復した。21B1遺伝子に蛍光タンパク質Venus遺伝子およびFLAGタグを連結したプラスミドを構築し、21B1遺伝子の単独変異体に導入したところ、表現型の回復とともにFLAG抗体を用いたウェスタン解析において予想サイズにシグナルが見られた。このシグナルの強度は栄養欠乏条件の切り替えの前後で変化しなかったことから、21B1タンパク質の発現レベルは栄養欠乏による制御を受けていないことが示唆された。今後、細胞内のVenus由来蛍光パターンの観察により細胞内局在の推定を進める。

エストライド脂質は水酸基をもつ脂肪酸に他の脂肪酸が１分子エステル結合して連結した二量体構造を含む脂質である。エストライド脂質の生産と活用のため、ツノケイソウのエストライド化を担う生合成遺伝子を同定することを目的とした。そのため、ツノケイソウのcDNA発現ライブラリーを発現させた分裂酵母を用いた機能スクリーニングを行った。培養温度20℃での振盪培養において、4 mg/mlのリシノール酸を培養液に添加した場合には60日以上完全に生育を抑制することがわかった。cDNAライブラリーを導入した分裂酵母を4 mg/mlのリシノール酸添加条件でバルク培養したところ、38日後に増殖が始まり45日後には定常期まで達した。また細胞からはエストライドTAGが検出された。さらに増殖した細胞はツノケイソウの機能未知の遺伝子が導入されていることがわかった。 ツノケイソウの中性脂質蓄積量の底上げを目的とし、ノケイソウの変異株集団の中から脂質蓄積異常株を探索して新規制御因子の単離を試みた。ツノケイソウで順遺伝学的解析を行うための工夫として高発現プロモーターのゲノムへのランダム挿入によるアクティベーションタギングを用いた。高発現プロモーターとしてツノケイソウの内在性の硝酸還元酵素遺伝子CgNRのプロモーターを用いた。約5,000株の独立した形質転換株を単離し、ナイルレッド蛍光を指標に中性脂質蓄積異常を示す変異株、すなわち野生株と比べて脂質蓄積量の高い株および低い株の両方を選抜した。その結果、ナイルレッド蛍光値、中性脂質蓄積量がともに野生株と比べて約50％まで減少した46C1株が単離された。46C1株ではプロテアーゼドメインをもつ遺伝子の転写開始点上流に導入したプロモーター配列が順向きに挿入されていた。

When nutrient deprived, microalgae accumulate triacylglycerol (TAG) in lipid droplets. Dual-specificity kinase, TAG-accumulation-regulator-1 (TAR1) has been reported to regulate carbon metabolism in a green alga, Chlamydomonas reinhardtii. We identified total 426 TAR1-dependent phosphoproteins by phosphoproteomic analysis (Shinkawa et al. 2019). We focused a novel protein kinase from these TAR's target proteins. It prevents the excess accumulation of triacylglycerol (TAG) in C/N-stress conditions. Phosphorylation of the continuous serine residues is TAR1-dependently induced in the C/N-stress conditions. Expression of mutated protein with Alanine substitution of these Ser residues could not complement the mutant phenotype of the kinase, suggesting the importance of phosphorylation of these serine residues for function of the kinase. We also showed this kinase protein located at plasma membrane.

微細藻の一種、珪藻は地球上の光合成の25%を担っています。本研究では、独自開発の高効率形質転換系を用い、弱光適応型の珪藻の機能を強化して、明環境下でも迅速に増殖し、効率的な有用代謝産物・油脂生産をする細胞へと分子育種します。そして、社会実装のための関連技術を確立して、自然光による光合成を通じてCO2を有用物質に転換することにより、エコフレンドリーな低炭素化社会の実現を目指します。

When nutrient deprived, microalgae accumulate triacylglycerol (TAG) in lipid droplets. Dual-specificity kinase, TAG-accumulation-regulator-1 (TAR1) has been reported to regulate carbon metabolism in a green alga, Chlamydomonas reinhardtii. TAR1 is required for acetate-dependent TAG accumulation and degradation of chlorophyll and photosynthesis-related proteins in photomixotrophic nitrogen (N)-deficient conditions. We report that the tar1-1 mutant maintained high levels of cell viability and lower generation of H2O2 compared with wild-type (WT) cells in photoautotrophic N-deficient conditions. Mating efficiency and mRNA abundance of key regulators in gametogenesis were lower in tar1-1 than those in WT. In addition, tar1-1 accumulated higher levels of TAG and starch per cell volumes compared with WT. We identified total 426 TAR1-dependent phosphoproteins by phosphoproteomic analysis. Several protein kinases and enzymes related to N assimilation and carbon metabolism were included.

Although microalgae accumulate triacylglycerol (TAG) in response to N-deficient (-N) conditions, the regulatory mechanisms are poorly understood. I identified a kinase, TAG accumulation regulator 1 (TAR1), which is a member of dual-specificity tyrosine-phosphorylation-regulated kinase family in a green alga, Chlamydomonas reinhardtii. Kinase domain of TAR1 demonstrated auto- and trans-phosphorylation activities. A tar1-1 mutant accumulated TAG to levels 0.1-fold of those of a wild-type strain in -N conditions. In -N conditions, tar1-1 maintained chlorophyll and photosynthetic activity. In -N conditions, global changes in expression levels of -N responsive genes in N assimilation and tetrapyrrole metabolism were noted between tar1-1 and wild-type cells. These results indicated that TAR1 is a regulator of TAG accumulation in -N conditions, and it functions in cell growth and repression of photosynthesis in -N conditions.

クラミドモナスに対する高感染性アグロバクテリウム株の選定と形質転換条件の最適化クラミドモナス細胞核中の染色体DNAにT-DNAが安定に組み込まれる条件をつきとめるため、以下の諸条件を組み合わせてアグロバクテリウム感染の最適化を試みた。a, 高感染性アグロバクテリウム菌株の選定 単子葉植物や酵母などアグロバクテリウムの宿主範囲外生物の形質転換で使用されている高感染性（supervirulent）菌株を用いて、安定形質転換体が取得できるかを調べ、最も頻度が高く薬剤耐性能を示す菌株を絞り込んだ。b, 感染に最適なクラミドモナス株と細胞ステージの決定 クラミドモナスには細胞壁が厚い系統と薄い系統が存在する。細胞壁の厚さが感染効率に影響するかを細胞壁の厚さの異なる3種類の系統（C9、5D、cw-15）を用いて検証した。しかし、細胞壁の有無は薬剤耐性個体の出現頻度に影響を及ぼさなかった。c, 適切な選抜マーカー遺伝子とアグロバクテリウムの除菌薬剤の選定 クラミドモナスの従来法による形質転換体選抜にはaph7’遺伝子（ハイグロマイシン選抜）、aphVIII 遺伝子（パラモマイシン選抜）、ble遺伝子（ゼオシン選抜）などの選抜システムが使用されている。これらの薬剤選抜系の中でアグロバクテリウム法に適したものを調べるため、各遺伝子発現カセットを既にバイナリーベクターpCAMBIA-1300に組み込み、感染実験に用いた。また、感染後のアグロバクテリウム菌体の除菌に、meropenemやmoxalactamといった新規eta-lactam系抗生物質がクラミドモナスでも効果的であることが示された。

Algal photosynthesis, which is base for production of bio-materials, is supported by carbon-concentrating mechanism (CCM). To identify CCM-related genes, RNA-seq analyses using different RNA samples prepared from cells cultured in various stress conditions including low-CO2 or nitrogen starved conditions. Using RNA-seq data and genomic sequence data, we have developed a new expression database for green alga, Chlamydomonas reinhardtii. Genes which are induced in low-CO2 stress conditions, nitrogen-starved conditions, and anaerobic conditions were identified by comparing the RNA expression profiles. Several CCM-related genes were artificially overexpressed in transgenic Chlamydomonas cells and the photosynthetic characteristics were show to be significantly improved than wild type cells.