Near-infrared imaging of phytochrome-derived autofluorescence in plant nuclei.

Capturing images of the nuclear dynamics within live cells is an essential technique for comprehending the intricate biological processes inherent to plant cell nuclei. While various methods exist for imaging nuclei, including combining fluorescent proteins and dyes with microscopy, there is a dearth of commercially available dyes for live-cell imaging. In Arabidopsis thaliana, we discovered that nuclei emit autofluorescence in the near-infrared (NIR) range of the spectrum and devised a non-invasive technique for the visualization of live cell nuclei using this inherent NIR autofluorescence. Our studies demonstrated the capability of the NIR imaging technique to visualize the dynamic behavior of nuclei within primary roots, root hairs, and pollen tubes, which are tissues that harbor a limited number of other organelles displaying autofluorescence. We further demonstrated the applicability of NIR autofluorescence imaging in various other tissues by incorporating fluorescence lifetime imaging techniques. Nuclear autofluorescence was also detected across a wide range of plant species, enabling analyses without the need for transformation. The nuclear autofluorescence in the NIR wavelength range was not observed in animal or yeast cells. Genetic analysis revealed that this autofluorescence was caused by the phytochrome protein. Our studies demonstrated that nuclear autofluorescence imaging can be effectively employed not only in model plants but also for studying nuclei in non-model plant species.

SWEET13 transport of sucrose, but not gibberellin, restores male fertility in Arabidopsis sweet13;14.

SWEET sucrose transporters play important roles in the allocation of sucrose in plants. Some SWEETs were shown to also mediate transport of the plant growth regulator gibberellin (GA). The close physiological relationship between sucrose and GA raised the questions of whether there is a functional connection and whether one or both of the substrates are physiologically relevant. To dissect these two activities, molecular dynamics were used to map the binding sites of sucrose and GA in the pore of SWEET13 and predicted binding interactions that might be selective for sucrose or GA. Transport assays confirmed these predictions. In transport assays, the N76Q mutant had 7x higher relative GA3 activity, and the S142N mutant only transported sucrose. The impaired pollen viability and germination in sweet13;14 double mutants were complemented by the sucrose-selective SWEET13S142N, but not by the SWEET13N76Q mutant, indicating that sucrose is the physiologically relevant substrate and that GA transport capacity is dispensable in the context of male fertility. Therefore, GA supplementation to counter male sterility may act indirectly via stimulating sucrose supply in male sterile mutants. These findings are also relevant in the context of the role of SWEETs in pathogen susceptibility.

Sphagnum capillifolium holobiont from a subarctic palsa bog aggravates the potential of nitrous oxide emissions.

Melting permafrost mounds in subarctic palsa mires are thawing under climate warming and have become a substantial source of N2O emissions. However, mechanistic insights into the permafrost thaw-induced N2O emissions in these unique habitats remain elusive. We demonstrated that N2O emission potential in palsa bogs was driven by the bacterial residents of two dominant Sphagnum mosses especially of Sphagnum capillifolium (SC) in the subarctic palsa bog, which responded to endogenous and exogenous Sphagnum factors such as secondary metabolites, nitrogen and carbon sources, temperature, and pH. SC's high N2O emission activity was linked with two classes of distinctive hyperactive N2O emitters, including Pseudomonas sp. and Enterobacteriaceae bacteria, whose hyperactive N2O emitting capability was characterized to be dominantly pH-responsive. As the nosZ gene-harboring emitter, Pseudomonas sp. SC-H2 reached a high level of N2O emissions that increased significantly with increasing pH. For emitters lacking the nosZ gene, an Enterobacteriaceae bacterium SC-L1 was more adaptive to natural acidic conditions, and N2O emissions also increased with pH. Our study revealed previously unknown hyperactive N2O emitters in Sphagnum capillifolium found in melting palsa mound environments, and provided novel insights into SC-associated N2O emissions.

OsSWEET11b, a potential sixth leaf blight susceptibility gene involved in sugar transport-dependent male fertility.

SWEETs play important roles in intercellular sugar transport. Induction of SWEET sugar transporters by Transcription Activator-Like effectors (TALe) of Xanthomonas ssp. is key for virulence in rice, cassava and cotton. We identified OsSWEET11b with roles in male fertility and potential bacterial blight (BB) susceptibility in rice. While single ossweet11a or 11b mutants were fertile, double mutants were sterile. As clade III SWEETs can transport gibberellin (GA), a key hormone for spikelet fertility, sterility and BB susceptibility might be explained by GA transport deficiencies. However, in contrast with the Arabidopsis homologues, OsSWEET11b did not mediate detectable GA transport. Fertility and susceptibility therefore are likely to depend on sucrose transport activity. Ectopic induction of OsSWEET11b by designer TALe enabled TALe-free Xanthomonas oryzae pv. oryzae (Xoo) to cause disease, identifying OsSWEET11b as a potential BB susceptibility gene and demonstrating that the induction of host sucrose uniporter activity is key to virulence of Xoo. Notably, only three of six clade III SWEETs are targeted by known Xoo strains from Asia and Africa. The identification of OsSWEET11b is relevant for fertility and for protecting rice against emerging Xoo strains that target OsSWEET11b.

Advances in Synthetic Fluorescent Probe Labeling for Live-Cell Imaging in Plants.

Fluorescent probes are powerful tools for visualizing cellular and subcellular structures, their dynamics and cellular molecules in living cells and enable us to monitor cellular processes in a spatiotemporal manner within complex and crowded systems. In addition to popular fluorescent proteins, a wide variety of small-molecule dyes have been synthesized through close association with the interdisciplinary field of chemistry and biology, ranging from those suitable for labeling cellular compartments such as organelles to those for labeling intracellular biochemical and biophysical processes and signaling. In recent years, self-labeling technologies including the SNAP-tag system have allowed us to attach these dyes to cellular domains or specific proteins and are beginning to be employed in plant studies. In this mini review, we will discuss the current range of synthetic fluorescent probes that have been exploited for live-cell imaging and the recent advances in the application that enable genetical tagging of synthetic probes in plant research.

Sensors for the quantification, localization and analysis of the dynamics of plant hormones.

Plant hormones play important roles in plant growth and development and physiology, and in acclimation to environmental changes. The hormone signaling networks are highly complex and interconnected. It is thus important to not only know where the hormones are produced, how they are transported and how and where they are perceived, but also to monitor their distribution quantitatively, ideally in a non-invasive manner. Here we summarize the diverse set of tools available for quantifying and visualizing hormone distribution and dynamics. We provide an overview over the tools that are currently available, including transcriptional reporters, degradation sensors, and luciferase and fluorescent sensors, and compare the tools and their suitability for different purposes.

Transcriptional silencing of 35S driven-transgene is differentially determined depending on promoter methylation heterogeneity at specific cytosines in both plus- and minus-sense strands.

BACKGROUND: De novo DNA methylation triggered by short interfering RNAs is called RNA-directed DNA methylation (RdDM). Transcriptional gene silencing (TGS) through RdDM can be induced using a viral vector. We have previously induced RdDM on the 35S promoter in the green fluorescent protein (GFP)-expressing Nicotiana benthamiana line 16c using the cucumber mosaic virus vector. The GFP fluorescence phenotype segregated into two types, "red" and "orange" in the first self-fertilized (S1) progeny plants by the difference in degree of recovery from TGS on GFP expression. In the second self-fertilized generation (S2 plants), the phenotypes again segregated. Explaining what generates the red and orange types could answer a very important question in epigenetics: How is the robustness of TGS maintained after RdDM induction? RESULTS: In bisulfite sequencing analyses, we found a significant difference in the overall promoter hypermethylation pattern between the red and orange types in S1 plants but little difference in S2 plants. Therefore, we assumed that methylation at some specific cytosine residues might be important in determining the two phenotypes. To find the factor that discriminates stable, robust TGS from the unstable TGS with incomplete inheritance, we analyzed the direct effect of methylated cytosine residues on TGS. Because it has not yet been demonstrated that DNA methylation at a few specific cytosine residues on known sequence elements can indeed determine TGS robustness, we newly developed a method by which we can directly evaluate the effect of specific methylation on promoter activity. In this assay, we found that the effects of the specific cytosine methylation on TGS differed between the plus- and minus-strands. CONCLUSIONS: We found two distinct phenotypes, the stable and unstable TGS in the progenies of virus-induced TGS plants. Our bisulfite sequencing analyses suggested that methylation at some specific cytosine residues in the 35S promoter played a role in determining whether stable or unstable TGSs are induced. Using the developed method, we inferred that DNA methylation heterogeneity in and between the plus- and minus-strands can differentially determine TGS.

Comparison of Archaeal Communities in Mineral Soils at a Boreal Forest in Finland and a Cold-Temperate Forest in Japan.

Archaeal communities in mineral soils were compared between a boreal forest in Finland and cold-temperate forest in Japan using 16S rRNA gene-targeted high-throughput sequencing. In boreal soils, Thaumarchaeota Group 1.1c archaea predominated and Thaumarchaeota Group 1.1a-associated and Group 1.1b archaea were also detected. In temperate soils, Thaumarchaeota Group 1.1a-associated and Group 1.1b archaea were dominant members at the subsurface, whereas their dominancy was replaced by Thermoplasmata archaea at the subsoil. An analysis of the ammonia monooxygenase subunit A gene of Archaea also indicated the distribution of Thaumarchaeota Group 1.1a-associated and Group 1.1b archaea in these soils.

Physiological and Genotypic Characteristics of Nitrous Oxide (N2O)-Emitting Pseudomonas Species Isolated from Dent Corn Andisol Farmland in Hokkaido, Japan.

Dent corn Andisol at the Hokkaido University Shizunai Livestock Experimental Farm actively emits nitrous oxide (N2O). In order to screen for culturable and active N2O emitters with high N2O emission potential, soft gel medium containing excess KNO3 was inoculated with soil suspensions from farm soil samples collected at different land managements. Dominant bacterial colonies were searched for among 20 of the actively N2O-emitting cultures from post-harvest soil and 19 from pre-tilled soil, and all isolates were subjected to the culture-based N2O emission assay. Ten active N2O-emitting bacteria, four from post-harvest soil and six from pre-tilled soil, out of 156 isolates were identified as genus Pseudomonas by 16S rRNA gene sequencing. These N2O emitters showed clear responses to NO3(-) within a neutral pH range (5.5-6.7), and accelerated N2O production with 1.5-15 mM sucrose supplementation, suggesting the production of N2O during the denitrification process. However, the negative responses of 6 active N2O emitters, 3 from post-harvest soil and 3 from pre-tilled soil, out of the 10 isolates in the acetylene-blocking assay suggest that these 6 N2O emitters are incomplete denitrifiers that have lost their N2O reductase (N2OR) activity. Although the PCR assay for the denitrification-associated genes, narG and nirK/S, was positive in all 10 Pseudomonas isolates, those negative in the acetylene-blocking assay were nosZ-negative. Therefore, these results imply that the high N2O emission potential of dent corn Andisol is partly attributed to saprophytic, nosZ gene-missing pseudomonad denitrifiers.

Distinct metabolites for photoreactive L-phenylalanine derivatives in Klebsiella sp. CK6 isolated from rhizosphere of a wild dipterocarp sapling.

Photoaffinity labeling is a reliable analytical method for biological functional analysis. Three major photophores--aryl azide, benzophenone and trifluoromethyldiazirine--are utilized in analysis. Photophore-bearing L-phenylalanine derivatives, which are used for biological functional analysis, were inoculated into a Klebsiella sp. isolated from the rhizosphere of a wild dipterocarp sapling in Central Kalimantan, Indonesia, under nitrogen-limiting conditions. The proportions of metabolites were quite distinct for each photophore. These results indicated that photophores affected substrate recognition in rhizobacterial metabolic pathways, and differential photoaffinity labeling could be achieved using different photophore-containing L-phenylalanine derivatives.

Trace amounts of furan-2-carboxylic acids determine the quality of solid agar plates for bacterial culture.

BACKGROUND: Many investigators have recognised that a significant proportion of environmental bacteria exist in a viable but non-culturable state on agar plates, and some researchers have also noticed that some of such bacteria clearly recover their growth on matrices other than agar. However, the reason why agar is unsuitable for the growth of some bacteria has not been addressed. METHODOLOGY/PRINCIPAL FINDINGS: According to the guide of a bioassay for swarming inhibition, we identified 5-hydroxymethylfuran-2-carboxylic acid (5-HMFA) and furan-2-carboxylic acid (FA) as factors that inhibit bacterial swarming and likely inhibit extracellular polysaccharide production on agar. The furan-2-carboxylic acids 5-HMFA and FA effectively inhibited the swarming and swimming of several environmental bacteria at concentrations of 1.8 and 2.3 µg L(-1) (13 and 21 nmol L(-1)), respectively, which are equivalent to the concentrations of these compounds in 0.3% agar. On Luria-Bertani (LB) plates containing 1.0% agar that had been previously washed with MeOH, a mixture of 5-HMFA and FA in amounts equivalent to their original concentrations in the unwashed agar repressed the swarming of Escherichia coli K12 strain W3110, a representative swarming bacterium. CONCLUSIONS/SIGNIFICANCE: Agar that contains trace amounts of 5-HMFA and FA inhibits the proliferation of some slow-growing or difficult-to-culture bacteria on the plates, but it is useful for single colony isolation due to the ease of identification of swarmable bacteria as the non-swarmed colonies.