Florigen-producing cells express FPF1-LIKE PROTEIN 1 that accelerates flowering and stem growth in long days with sunlight red/far-red ratio in Arabidopsis.

Seasonal changes in spring induce flowering by expressing the florigen, FLOWERING LOCUS T (FT), in Arabidopsis. FT is expressed in unique phloem companion cells with unknown characteristics. The question of which genes are co-expressed with FT and whether they have roles in flowering remains elusive. Through tissue-specific translatome analysis, we discovered that under long-day conditions with the natural sunlight red/far-red ratio, the FT-producing cells express a gene encoding FPF1-LIKE PROTEIN 1 (FLP1). The master FT regulator, CONSTANS (CO), controls FLP1 expression, suggesting FLP1's involvement in the photoperiod pathway. FLP1 promotes early flowering independently of FT, is active in the shoot apical meristem, and induces the expression of SEPALLATA 3 (SEP3), a key E-class homeotic gene. Unlike FT, FLP1 facilitates inflorescence stem elongation. Our cumulative evidence indicates that FLP1 may act as a mobile signal. Thus, FLP1 orchestrates floral initiation together with FT and promotes inflorescence stem elongation during reproductive transitions.

Microfluidic Device for Simple Diagnosis of Plant Growth Condition by Detecting miRNAs from Filtered Plant Extracts.

Plants are exposed to a variety of environmental stress, and starvation of inorganic phosphorus can be a major constraint in crop production. In plants, in response to phosphate deficiency in soil, miR399, a type of microRNA (miRNA), is up-regulated. By detecting miR399, the early diagnosis of phosphorus deficiency stress in plants can be accomplished. However, general miRNA detection methods require complicated experimental manipulations. Therefore, simple and rapid miRNA detection methods are required for early plant nutritional diagnosis. For the simple detection of miR399, microfluidic technology is suitable for point-of-care applications because of its ability to detect target molecules in small amounts in a short time and with simple manipulation. In this study, we developed a microfluidic device to detect miRNAs from filtered plant extracts for the easy diagnosis of plant growth conditions. To fabricate the microfluidic device, verification of the amine-terminated glass as the basis of the device and the DNA probe immobilization method on the glass was conducted. In this device, the target miRNAs were detected by fluorescence of sandwich hybridization in a microfluidic channel. For plant stress diagnostics using a microfluidic device, we developed a protocol for miRNA detection by validating the sample preparation buffer, filtering, and signal amplification. Using this system, endogenous sly-miR399 in tomatoes, which is expressed in response to phosphorus deficiency, was detected before the appearance of stress symptoms. This early diagnosis system of plant growth conditions has a potential to improve food production and sustainability through cultivation management.

Oxicam-type nonsteroidal anti-inflammatory drugs enhance Agrobacterium-mediated transient transformation in plants.

Agrobacterium-mediated transformation is a key innovation for plant breeding, and routinely used in basic researches and applied biology. However, the transformation efficiency is often the limiting factor of this technique. In this study, we discovered that oxicam-type nonsteroidal anti-inflammatory drugs, including tenoxicam (TNX), increase the efficiency of Agrobacterium-mediated transient transformation. TNX treatment increased the transformation efficiency of Agrobacterium-mediated transformation of Arabidopsis thaliana mature leaves by agroinfiltration. The increase of efficiency by TNX treatment was not observed in dde2/ein2/pad4/sid2 quadruple mutant, indicating that TNX inhibits the immune system mediated by jasmonic acid, ethylene, and salicylic acid against to Agrobacterium. We also found that TNX-treatment is applicable for the transient expression and subcellular localization analysis of fluorescent-tagged proteins in Arabidopsis leaf cells. In addition, we found that TNX increases the efficiency of Agrobacterium-mediated transient transformation of Jatropha. Given that treatment with oxicam compounds is a simple and cost effective method, our findings will provide a new option to overcome limitations associated with Agrobacterium-mediated transformation of various plant species.

Analysis of plasmodesmata permeability using cultured tobacco BY-2 cells entrapped in microfluidic chips.

Plasmodesmata are unique channel structures in plants that link the fluid cytoplasm between adjacent cells. Plants have evolved these microchannels to allow trafficking of nutritious substances as well as regulatory factors for intercellular communication. However, tracking the behavior of plasmodesmata in real time is difficult because they are located inside tissues. Hence, a system was constructed to monitor the movement of substances by plasmodesmata using tobacco BY-2 cells, which are linearly organized cells, and a microfluidic device that traps them in place and facilitates observation. After targeting one cell for photobleaching, recovery of the lost H2B-GFP protein was detected within 200 min. No recovery was detected in that time frame by photobleaching the entire cell filaments. This suggested that the recovery of H2B-GFP protein was not due to de novo protein synthesis, but rather to translocation from neighboring cells. The transport of H2B-GFP protein was not observed when sodium chloride, a compound known to cause plasmodesmata closure, was present in the microfluid channel. Thus, using the microfluidic device and BY-2 cells, it was confirmed that the behavior of plasmodesmata could be observed in real time under controllable conditions.

Development of microfluidic chip for entrapping tobacco BY-2 cells.

The tobacco BY-2 cell line has been used widely as a model system in plant cell biology. BY-2 cells are nearly transparent, which facilitates cell imaging using fluorescent markers. As cultured cells are drifted in the medium, therefore, it was difficult to observe them for a long period. Hence, we developed a microfluidic device that traps BY-2 cells and fixes their positions to allow monitoring the physiological activity of cells. The device contains 112 trap zones, with parallel slots connected in series at three levels in the flow channel. BY-2 cells were cultured for 7 days and filtered using a sieve and a cell strainer before use to isolate short cell filaments consisting of only a few cells. The isolated cells were introduced into the flow channel, resulting in entrapment of cell filaments at 25 out of 112 trap zones (22.3%). The cell numbers increased through cell division from 1 to 4 days after trapping with a peak of mitotic index on day 2. Recovery experiments of fluorescent proteins after photobleaching confirmed cell survival and permeability of plasmodesmata. Thus, this microfluidic device and one-dimensional plant cell samples allowed us to observe cell activity in real time under controllable conditions.

Measurement of reactive oxygen species production by luminol-based assay in Nicotiana benthamiana, Arabidopsis thaliana and Brassica rapa ssp. rapa.

Reactive oxygen species (ROS) are critical for plant biological processes. As signaling molecules, ROS regulate plant growth and development through cell expansion, elongation, and programmed cell death. Furthermore, ROS production is induced by microbe-associated molecular patterns (MAMPs) treatment and biotic stresses, and contributes to plant resistance to pathogens. Thus, MAMP-induced ROS production has been an indicator for plant early immune responses or stress responses. One of widely used methods for the measurement is a luminol-based assay to measure extracellular ROS production with a bacterial flagellin epitope (flg22) as a MAMP elicitor. Nicotiana benthamiana is susceptible to a wide variety of plant pathogenic agents and therefore commonly used for ROS measurements. On the other hand, Arabidopsis thaliana, many of genetical lines of which are available, is also conducted to ROS measurements. Tests in an asterid N. benthamiana and a rosid A. thaliana can reveal conserved molecular mechanisms in ROS production. However, the small size of A. thaliana leaves requires many seedlings for experiments. This study examined flg22-induced ROS production in another member of the Brassicaceae family, Brassica rapa ssp. rapa (turnip), which has large and flat leaves. Our experiments indicated that 10 nM and 100 nM flg22 treatments induced high ROS levels in turnip. Turnip tended to have a lower standard deviation in multiple concentrations of flg22 treatment. Therefore, these results suggested that turnip can be a good material from the rosid clade for ROS measurement.

Micrografting in Arabidopsis Using a Silicone Chip.

The micrografting technique in the model plant Arabidopsis has been widely used in the field of plant science. Grafting experiments have demonstrated that signal transductions are systematically regulated in many plant characteristics, including defense mechanisms and responses to surrounding environments such as soil and light conditions. Hypocotyl micrografting is a powerful tool for the analysis of signal transduction between shoots and roots; however, the requirement for a high level of skill for micrografting, during which small seedlings are microdissected and micromanipulated, has limited its use. Here, we developed a silicone-made microdevice, called a micrografting chip, to perform Arabidopsis micrografting easily and uniformly. The micrografting chip has tandemly arrayed units, each of which consists of a seed pocket for seed germination and a micro-path to hold hypocotyl. All micrografting procedures are performed on the chip. This method using a micrografting chip will avoid the need for training and promote studies of systemic signaling in plants. Graphic abstract: A silicone chip for easy grafting.

Combination of genetic analysis and ancient literature survey reveals the divergence of traditional Brassica rapa varieties from Kyoto, Japan.

Since ancient times, humans have bred several plants that we rely on today. However, little is known about the divergence of most of these plants. In the present study, we investigated the divergence of Mibuna (Brassica rapa L. subsp. nipposinica L. H. Bailey), a traditional leafy vegetable in Kyoto (Japan), by combining genetic analysis and a survey of ancient literature. Mibuna is considered to have been bred 200 years ago from Mizuna, another traditional leafy vegetable in Kyoto. Mibuna has simple spatulate leaves, whereas Mizuna has characteristic serrated leaves. The quantitative trait loci (QTL) and gene expression analyses suggested that the downregulation of BrTCP15 expression contributed to the change in the leaf shape from serrated to simple spatulate. Interestingly, the SNP analysis indicated that the genomic region containing the BrTCP15 locus was transferred to Mibuna by introgression. Furthermore, we conducted a survey of ancient literature to reveal the divergence of Mibuna and found that hybridization between Mizuna and a simple-leaved turnip might have occurred in the past. Indeed, the genomic analysis of multiple turnip cultivars showed that one of the cultivars, Murasakihime, has almost the same sequence in the BrTCP15 region as Mibuna. These results suggest that the hybridization between Mizuna and turnip has resulted in the establishment of Mibuna.

Cell-cell adhesion in plant grafting is facilitated by ß-1,4-glucanases.

Plant grafting is conducted for fruit and vegetable propagation, whereby a piece of living tissue is attached to another through cell-cell adhesion. However, graft compatibility limits combinations to closely related species, and the mechanism is poorly understood. We found that Nicotiana is capable of graft adhesion with a diverse range of angiosperms. Comparative transcriptomic analyses on graft combinations indicated that a subclade of ß-1,4-glucanases secreted into the extracellular region facilitates cell wall reconstruction near the graft interface. Grafting was promoted by overexpression of the ß-1,4-glucanase. Using Nicotiana stem as an interscion, we produced tomato fruits on rootstocks from other plant families. These findings demonstrate that the process of cell-cell adhesion is a potential target to enhance plant grafting techniques.

Micrografting device for testing systemic signaling in Arabidopsis.

Grafting techniques have been applied in studies of systemic, long-distance signaling in several model plants. Seedling grafting in Arabidopsis, known as micrografting, enables investigation of the molecular mechanisms of systemic signaling between shoots and roots. However, conventional micrografting requires a high level of skill, limiting its use. Thus, an easier user-friendly method is needed. Here, we developed a silicone microscaled device, the micrografting chip, to obviate the need for training and to generate less stressed and more uniformly grafted seedlings. The chip has tandemly arrayed units, each of which consists of a seed pocket for seed germination and a micro-path with pairs of pillars for hypocotyl holding. Grafting, including seed germination, micrografting manipulation and establishment of tissue reunion, is performed on the chip. Using the micrografting chip, we evaluated the effect of temperature and the carbon source on grafting, and showed that a temperature of 27°C and a sucrose concentration of 0.5% were optimal. We also used the chip to investigate the mechanism of systemic signaling of iron status using a quadruple nicotianamine synthase (nas) mutant. The constitutive iron-deficiency response in the nas mutant because of iron accumulation in shoots was significantly rescued by grafting of wild-type shoots or roots, suggesting that shoot- and root-ward translocation of nicotianamine-iron complexes and/or nicotianamine is essential for iron mobilization. Thus, our micrografting chip will promote studies of long-distance signaling in plants.

An in vitro grafting method to quantify mechanical forces of adhering tissues.

Grafting is an indispensable agricultural technology for propagating useful tree varieties and obtaining beneficial traits of two varieties/species-as stock and scion-at the same time. Recent studies of molecular events during grafting have revealed dynamic physiological and transcriptomic changes. Strategies focused on specific grafting steps are needed to further associate each physiological and molecular event with those steps. In this study, we developed a method to investigate the tissue adhesion event, an early grafting step, by improving an artificial in vitro grafting system in which two pieces of 1.5-mm thick Nicotiana benthamiana cut stem sections were combined and cultured on medium. We prepared a silicone sheet containing five special cutouts for adhesion of cut stem slices. We quantitatively measured the adhesive force at these grafting interfaces using a force gauge and found that graft adhesion started 2 days after grafting, with the adhesive force gradually increasing over time. After confirming the positive effect of auxin on grafting by this method, we tested the effect of cellulase treatment and observed significant enhancement of graft tissue adhesion. Compared with the addition of auxin or cellulase individually, the adhesive force was stronger when both auxin and cellulase were added simultaneously. The in vitro grafting method developed in this study is thus useful for examining the process of graft adhesion.

A GLABRA1 ortholog on LG A9 controls trichome number in the Japanese leafy vegetables Mizuna and Mibuna (Brassica rapa L. subsp. nipposinica L. H. Bailey): evidence from QTL analysis.

Brassica rapa show a wide range of morphological variations. In particular, the leaf morphologies of the Japanese traditional leafy vegetables Mizuna and Mibuna (Brassica rapa L. subsp. nipposinica L. H. Bailey) are distinctly different, even though they are closely related cultivars that are easy to cross. In addition to the differences in the gross morphology of leaves, some cultivars of Mibuna (Kyo-nishiki) have many trichomes on its leaves, whereas Mizuna (Kyo-mizore) does not. To identify the genes responsible for the different number of trichomes, we performed a quantitative trait loci (QTL) analysis of Mizuna and Mibuna. To construct linkage maps for these cultivars, we used RNA-seq data to develop cleaved amplified polymorphic sequence (CAPS) markers. We also performed a restriction site-associated DNA sequencing (RAD-seq) analysis to detect single-nucleotide polymorphisms (SNPs). Two QTL analyses were performed in different years, and both analyses indicated that the largest effect was found on LG A9. Expression analyses showed that a gene homologous to GLABRA1 (GL1), a transcription factor implicated in trichome development in Arabidopsis thaliana, and the sequences 3'-flanking (downstream) of BrGL1, differed considerably between Mizuna (Kyo-mizore) and Mibuna (Kyo-nishiki). These results indicate that BrGL1 on LG A9 is one of the candidate genes responsible for the difference in trichome number between Mizuna and Mibuna. Detecting genes that are responsible for morphological variations allows us to better understand the breeding history of Mizuna and Mibuna.

A Decrease in Ambient Temperature Induces Post-Mitotic Enlargement of Palisade Cells in North American Lake Cress.

In order to maintain organs and structures at their appropriate sizes, multicellular organisms orchestrate cell proliferation and post-mitotic cell expansion during morphogenesis. Recent studies using Arabidopsis leaves have shown that compensation, which is defined as post-mitotic cell expansion induced by a decrease in the number of cells during lateral organ development, is one example of such orchestration. Some of the basic molecular mechanisms underlying compensation have been revealed by genetic and chimeric analyses. However, to date, compensation had been observed only in mutants, transgenics, and γ-ray-treated plants, and it was unclear whether it occurs in plants under natural conditions. Here, we illustrate that a shift in ambient temperature could induce compensation in Rorippa aquatica (Brassicaceae), a semi-aquatic plant found in North America. The results suggest that compensation is a universal phenomenon among angiosperms and that the mechanism underlying compensation is shared, in part, between Arabidopsis and R. aquatica.

Multimodal biopanning of T7 phage-displayed peptides reveals angiomotin as a potential receptor of the anti-angiogenic macrolide Roxithromycin.

Roxithromycin (RXM) is a semi-synthetic fourteen-membered macrolide antibiotic that shows anti-angiogenic activity in solid tumors. In the present study, we conducted biopanning of T7 phage-displayed peptides either on a 96-well formatted microplate, a flow injection-type quartz-crystal microbalance (QCM) biosensor, or a cuvette-type QCM. RXM-selected peptides of different sequence, length and number were obtained from each mode of screening. Subsequent bioinformatics analysis of the RXM-selected peptides consistently gave positive scores for the extracellular domain (E458-T596) of angiomotin (Amot), indicating that this may comprise a binding region for RXM. Bead pull down assay and QCM analysis confirmed that RXM directly interacts with Amot via the screen-guided region, which also corresponds to the binding site for the endogenous anti-angiogenic inhibitor angiostatin (Anst). Thus, multimodal biopanning of T7PD revealed that RXM binds to the extracellular domain on Amot as a common binding site with Anst, leading to inhibition of angiogenesis-dependent tumor growth and metastasis. These data might explain the molecular basis underlying the mechanism of action for the anti-angiogenic activity of RXM.