Alginate and fucoidan changes the bacterial community in different directions and the alginate or fucoidan degrading bacteria isolated from paddy soil promotes the plant growth.

Seaweed and its extracts have been developed as fertilizers because they possess plant-growth-promoting and antibacterial compounds. For use as fertilizers, the major carbohydrates in seaweed, including fucoidan and alginate, need to be efficiently digested in the soil. We isolated fucoidan/alginate degrading bacteria from paddy soil and verified its use as a biofertilizer. Results show that Stenotrophomonas pavanii has a high alginate degrading activity, and also stimulating melon, pepper, and tomato growth. The growth stimulation effect of the bacteria was enhanced by alginate treatment. Bacillus sp. was isolated as a fucoidan degrading bacterium and this bacterium was also able to stimulate melon growth. Using 16S ribosomal DNA analysis, fucoidan/alginate resistant or susceptible bacteria were successively selected. Bacteria with increased population due to fucoidan and alginate had specificity to each carbohydrate, whereas those with decreased population showed susceptibility to both carbohydrates. This report demonstrates some bacteria for their use as biofertilizers with seaweed and demonstrated that a high throughput method is efficient in identifying bacteria with specific properties.

The ten amino acids of the oxygen-evolving enhancer of tobacco is sufficient as the peptide residues for protein transport to the chloroplast thylakoid.

KEY MESSAGE: The thylakoid transit peptide of tobacco oxygen-evolving enhancer protein contains a minimal ten amino acid sequences for thylakoid lumen transports. This ten amino acids do not contain twin-arginine, which is required for typical chloroplast lumen translocation. Chloroplasts are intracellular organelles responsible for photosynthesis to produce organic carbon for all organisms. Numerous proteins must be transported from the cytosol to chloroplasts to support photosynthesis. This transport is facilitated by chloroplast transit peptides (TPs). Four chloroplast thylakoid lumen TPs were isolated from Nicotiana tabacum and were functionally analyzed as thylakoid lumen TPs. Typical chloroplast stroma-transit peptides and thylakoid lumen transit peptides (tTPs) are found in N. tabacum transit peptides (NtTPs) and the functions of these peptides are confirmed with TP-GFP fusion proteins under fluorescence microscopy and chloroplast fractionation, followed by Western blot analysis. During the functional analysis of tTPs, we uncovered the minimum 10 amino acid sequence is sufficient for thylakoid lumen transport. These ten amino acids can efficiently translocate GFP protein, even if they do not contain the twin-arginine residues required for the twin-arginine translocation (Tat) pathway, which is a typical thylakoid lumen transport. Further, thylakoid lumen transporting processes through the Tat pathway was examined by analyzing tTP sequence functions and we demonstrate that the importance of hydrophobic core for the tTP cleavage and target protein translocation.

Systemic Acquired Resistance-Mediated Control of Pine Wilt Disease by Foliar Application With Methyl Salicylate.

Pine wilt disease (PWD), caused by the pinewood nematode, is the most destructive disease in pine forest ecosystems worldwide. Extensive research has been done on PWD, but effective disease management is yet to be devised. Generally, plants can resist pathogen attack via a combination of constitutive and inducible defenses. Systemic acquired resistance (SAR) is an inducible defense that occurs by the localized infection of pathogens or treatment with elicitors. To manage PWD by SAR in pine trees, we tested previously known 12 SAR elicitors. Among them, methyl salicylate (MeSA) was found to induce resistance against PWD in Pinus densiflora seedlings. In addition, the foliar applications of the dispersible concentrate-type formulation of MeSA (MeSA 20 DC) and the emulsifiable concentrate-type formulation of MeSA (MeSA 20 EC) resulted in significantly reduced PWD in pine seedlings. In the field test using 10-year-old P. densiflora trees, MeSA 20 DC showed a 60% decrease in the development of PWD. Also, MeSA 20 EC gave the best results when applied at 0.1 mM concentration 2 and 1 weeks before pinewood nematode (PWN) inoculation in pine seedlings. qRT-PCR analysis confirmed that MeSA induced the expression of defense-related genes, indicating that MeSA can inhibit and delay the migration and reproduction of PWN in pine seedlings by modulating gene expression. These results suggest that foliar application of MeSA could reduce PWD incidence by inducing resistance and provide an economically feasible alternative to trunk-injection agents for PWD management.

Characterization of and isolation methods for plant leaf nanovesicles and small extracellular vesicles.

Mammalian small extracellular vesicles (sEVs) can deliver diverse molecules to target cells. However, they are difficult to obtain in large quantities and can activate host immune responses. Plant-derived vesicles may help to overcome these challenges. We optimized isolation methods for two types of plant vesicles, nanovesicles from disrupted leaf and sEVs from the extracellular apoplastic space of Arabidopsis thaliana. Both preparations yielded intact vesicles of uniform size, and a mean membrane charge of approximately -25 mV. We also demonstrated applicability of these preparative methods using Brassicaceae vegetables. Proteomic analysis of a subset of vesicles with a density of 1.1-1.19 g mL-1 sheds light on the likely cellular origin and complexity of the vesicles. Both leaf nanovesicles and sEVs were taken up by cancer cells, with sEVs showing an approximately three-fold higher efficiency compared to leaf nanovesicles. These results support the potential of plant-derived vesicles as vehicles for therapeutic delivery.

In Planta Response of Arabidopsis to Photothermal Impact Mediated by Gold Nanoparticles.

Biological responses to photothermal effects of gold nanoparticles (GNPs) have been demonstrated and employed for various applications in diverse systems except for one important class - plants. Here, the uptake of GNPs through Arabidopsis thaliana roots and translocation to leaves are reported. Successful plasmonic nanobubble generation and acoustic signal detection in planta is demonstrated. Furthermore, Arabidopsis leaves harboring GNPs and exposed to continuous laser or noncoherent light show elevated temperatures across the leaf surface and induced expression of heat-shock regulated genes. Overall, these results demonstrate that Arabidopsis can readily take up GNPs through the roots and translocate the particles to leaf tissues. Once within leaves, GNPs can act as photothermal agents for on-demand remote activation of localized biological processes in plants.

Fluorescence reports intact quantum dot uptake into roots and translocation to leaves of Arabidopsis thaliana and subsequent ingestion by insect herbivores.

We explored the impact of quantum dot (QD) coat characteristics on NP stability, uptake, and translocation in Arabidopsis thaliana, and subsequent transfer to primary consumers, Trichoplusia ni (T. ni). Arabidopsis was exposed to CdSe/CdZnS QDs with three different coatings: Poly(acrylic acid-ethylene glycol) (PAA-EG), polyethylenimine (PEI) and poly(maleic anhydride-alt-1-octadecene)-poly(ethylene glycol) (PMAO-PEG), which are anionic, cationic, and relatively neutral, respectively. PAA-EG-coated QDs were relatively stable and taken up from a hydroponic medium through both Arabidopsis leaf petioles and roots, without apparent aggregation, and showed generally uniform distribution in leaves. In contrast, PEI- and PMAO-PEG-coated QDs displayed destabilization in the hydroponic medium, and generated particulate fluorescence plant tissues, suggesting aggregation. PAA-EG QDs moved faster than PEI QDs through leaf petioles; however, 8-fold more cadmium accumulated in PEI QD-treated leaves than in those exposed to PAA-EG QDs, possibly due to PEI QD dissolution and direct metal uptake. T. ni caterpillars that fed on Arabidopsis exposed to QDs had reduced performance, and QD fluorescence was detected in both T. ni bodies and frass, demonstrating trophic transfer of intact QDs from plants to insects. Overall, this paper demonstrates that QD coat properties influence plant nanoparticle uptake and translocation and can impact transfer to herbivores.

Phytostimulation of poplars and Arabidopsis exposed to silver nanoparticles and Ag⁺ at sublethal concentrations.

The increasing likelihood of silver nanoparticle (AgNP) releases to the environment highlights the importance of understanding AgNP interactions with plants, which are cornerstones of most ecosystems. In this study, poplars (Populus deltoides × nigra) and Arabidopsis thaliana were exposed hydroponically to nanoparticles of different sizes (PEG-coated 5 and 10 nm AgNPs, and carbon-coated 25 nm AgNPs) or silver ions (Ag(+), added as AgNO3) at a wide range of concentrations (0.01 to 100 mg/L). Whereas all forms of silver were phytotoxic above a specific concentration, a stimulatory effect was observed on root elongation, fresh weight, and evapotranspiration of both plants at a narrow range of sublethal concentrations (e.g., 1 mg/L of 25 nm AgNPs for poplar). Plants were most susceptible to the toxic effects of Ag(+) (1 mg/L for poplar, 0.05 mg/L for Arabidopsis), but AgNPs also showed some toxicity at higher concentrations (e.g., 100 mg/L of 25 nm AgNPs for poplar, 1 mg/L of 5 nm AgNPs for Arabidopsis) and this susceptibility increased with decreasing AgNP size. Both poplars and Arabidopsis accumulated silver, but silver distribution in shoot organs varied between plant species. Arabidopsis accumulated silver primarily in leaves (at 10-fold higher concentrations than in the stem or flower tissues), whereas poplars accumulated silver at similar concentrations in leaves and stems. Within the particle subinhibitory concentration range, silver accumulation in poplar tissues increased with exposure concentration and with smaller AgNP size. However, compared to larger AgNPs, the faster silver uptake associated with smaller AgNPs was offset by their toxic effect on evapotranspiration, which was exerted at lower concentrations (e.g., 1 mg/L of 5 nm AgNPs for poplar). Overall, the observed phytostimulatory effects preclude generalizations about the phytotoxicity of AgNPs and encourage further mechanistic research.

Sugar promotes vegetative phase change in Arabidopsis thaliana by repressing the expression of MIR156A and MIR156C.

Nutrients shape the growth, maturation, and aging of plants and animals. In plants, the juvenile to adult transition (vegetative phase change) is initiated by a decrease in miR156. In Arabidopsis, we found that exogenous sugar decreased the abundance of miR156, whereas reduced photosynthesis increased the level of this miRNA. This effect was correlated with a change in the timing of vegetative phase change, and was primarily attributable to a change in the expression of two genes, MIR156A and MIR156C, which were found to play dominant roles in this transition. The glucose-induced repression of miR156 was dependent on the signaling activity of HEXOKINASE1. We also show that the defoliation-induced increase in miR156 levels can be suppressed by exogenous glucose. These results provide a molecular link between nutrient availability and developmental timing in plants, and suggest that sugar is a component of the leaf signal that mediates vegetative phase change. DOI:http://dx.doi.org/10.7554/eLife.00260.001.

Calmodulin-related CML24 interacts with ATG4b and affects autophagy progression in Arabidopsis.

Plants encounter environmental stress challenges that are distinct from those of other eukaryotes because of their relative immobility. Therefore, plants may have evolved distinct regulatory mechanisms for conserved cellular functions. Plants, like other eukaryotes, share aspects of both calcium- and calmodulin-based cellular signaling and the autophagic process of cellular renewal. Here, we report a novel function for an Arabidopsis calmodulin-related protein, CML24, and insight into ATG4-regulated autophagy. CML24 interacts with ATG4b in yeast two-hybrid, in vitro pull-down and transient tobacco cell transformation assays. Mutants with missense mutations in CML24 have aberrant ATG4 activity patterns in in vitro extract assays, altered ATG8 accumulation levels, an altered pattern of GFP-ATG8-decorated cellular structures, and altered recovery from darkness-induced starvation. Together, these results support the conclusion that CML24 affects autophagy progression through interactions with ATG4.

miRNA control of vegetative phase change in trees.

After germination, plants enter juvenile vegetative phase and then transition to an adult vegetative phase before producing reproductive structures. The character and timing of the juvenile-to-adult transition vary widely between species. In annual plants, this transition occurs soon after germination and usually involves relatively minor morphological changes, whereas in trees and other perennial woody plants it occurs after months or years and can involve major changes in shoot architecture. Whether this transition is controlled by the same mechanism in annual and perennial plants is unknown. In the annual forb Arabidopsis thaliana and in maize (Zea mays), vegetative phase change is controlled by the sequential activity of microRNAs miR156 and miR172. miR156 is highly abundant in seedlings and decreases during the juvenile-to-adult transition, while miR172 has an opposite expression pattern. We observed similar changes in the expression of these genes in woody species with highly differentiated, well-characterized juvenile and adult phases (Acacia confusa, Acacia colei, Eucalyptus globulus, Hedera helix, Quercus acutissima), as well as in the tree Populus x canadensis, where vegetative phase change is marked by relatively minor changes in leaf morphology and internode length. Overexpression of miR156 in transgenic P. x canadensis reduced the expression of miR156-targeted SPL genes and miR172, and it drastically prolonged the juvenile phase. Our results indicate that miR156 is an evolutionarily conserved regulator of vegetative phase change in both annual herbaceous plants and perennial trees.