Naoki Takata

Partners in Time: EARLY BIRD Associates with ZEITLUPE and Regulates the Speed of the Arabidopsis Clock

The circadian clock of the model plant Arabidopsis (Arabidopsis thaliana) is made up of a complex series of interacting feedback loops whereby proteins regulate their own expression across day and night. early bird (ebi) is a circadian mutation that causes the clock to speed up: ebi plants have short circadian periods, early phase of clock gene expression, and are early flowering. We show that EBI associates with ZEITLUPE (ZTL), known to act in the plant clock as a posttranslational mediator of protein degradation. However, EBI is not degraded by its interaction with ZTL. Instead, ZTL counteracts the effect of EBI during the day and increases it at night, modulating the expression of key circadian components. The partnership of EBI with ZTL reveals a novel mechanism involved in controlling the complex transcription-translation feedback loops of the clock. This work highlights the importance of cross talk between the ubiquitination pathway and transcriptional control for regulation of the plant clock.

A simple and efficient transient transformation for hybrid aspen (Populus tremula × P. tremuloides)

BackgroundThe genus Populus is accepted as a model system for molecular tree biology. To investigate gene functions in Populus spp. trees, generating stable transgenic lines is the common technique for functional genetic studies. However, a limited number of genes have been targeted due to the lengthy transgenic process. Transient transformation assays complementing stable transformation have significant advantages for rapid in vivo assessment of gene function. The aim of this study is to develop a simple and efficient transient transformation for hybrid aspen and to provide its potential applications for functional genomic approaches.ResultsWe developed an in planta transient transformation assay for young hybrid aspen cuttings using Agrobacterium-mediated vacuum infiltration. The transformation conditions such as the infiltration medium, the presence of a surfactant, the phase of bacterial growth and bacterial density were optimized to achieve a higher transformation efficiency in young aspen leaves. The Agrobacterium infiltration assay successfully transformed various cell types in leaf tissues. Intracellular localization of four aspen genes was confirmed in homologous Populus spp. using fusion constructs with the green fluorescent protein. Protein-protein interaction was detected in transiently co-transformed cells with bimolecular fluorescence complementation technique. In vivo promoter activity was monitored over a few days in aspen cuttings that were transformed with luciferase reporter gene driven by a circadian clock promoter.ConclusionsThe Agrobacterium infiltration assay developed here is a simple and enhanced throughput method that requires minimum handling and short transgenic process. This method will facilitate functional analyses of Populus genes in a homologous plant system.

Wood reinforcement of poplar by rice NAC transcription factor

Lignocellulose, composed of cellulose, hemicellulose, and lignin, in the secondary cell wall constitutes wood and is the most abundant form of biomass on Earth. Enhancement of wood accumulation may be an effective strategy to increase biomass as well as wood strength, but currently only limited research has been undertaken. Here, we demonstrated that OsSWN1, the orthologue of the rice NAC Secondary-wall Thickening factor (NST) transcription factor, effectively enhanced secondary cell wall formation in the Arabidopsis inflorescence stem and poplar (Populus tremula×Populus tremuloides) stem when expressed by the Arabidopsis NST3 promoter. Interestingly, in transgenic Arabidopsis and poplar, ectopic secondary cell wall deposition in the pith area was observed in addition to densification of the secondary cell wall in fiber cells. The cell wall content or density of the stem increased on average by up to 38% and 39% in Arabidopsis and poplar, respectively, without causing growth inhibition. As a result, physical strength of the stem increased by up to 57% in poplar. Collectively, these data suggest that the reinforcement of wood by NST3pro:OsSWN1 is a promising strategy to enhance wood-biomass production in dicotyledonous plant species.

Evolutionary Relationship and Structural Characterization of the EPF/EPFL Gene Family

EPF1-EPF2 and EPFL9/Stomagen act antagonistically in regulating leaf stomatal density. The aim of this study was to elucidate the evolutionary functional divergence of EPF/EPFL family genes. Phylogenetic analyses showed that AtEPFL9/Stomagen-like genes are conserved only in vascular plants and are closely related to AtEPF1/EPF2-like genes. Modeling showed that EPF/EPFL peptides share a common 3D structure that is constituted of a scaffold and loop. Molecular dynamics simulation suggested that AtEPF1/EPF2-like peptides form an additional disulfide bond in their loop regions and show greater flexibility in these regions than AtEPFL9/Stomagen-like peptides. This study uncovered the evolutionary relationship and the conformational divergence of proteins encoded by the EPF/EPFL family genes.

Tissue culture of two medicinal trees native to Japan

Wadatsuminoki (Nothapodytes amamianus) is an endangered tree species observed in only Amami Oshima Island located in southern part of Japan. According to the Red list published by Ministry of Environment, it is classified as 1A (Critically endangered) and naturally remaining number is only 20. It contains camptotesin which is used for anti-cancer drugs. Kagikazura (Uncaria rhynchophylla) is an medicinal tree species observed widely in Japan and China. It contains alkaloids (rhynchophylline, iso-rhynchophylline, hirstine and so on) which are good for remedy of high blood pressure and dementia. For the purpose of micropropagation and development of basis of useful substance production by cell culture as well as conservation of endangered species, tissue culture procedure was developed for those two species. Excised shoots of 2 years old seedling of Wadatsuminoki rooted in the 1/2DCR medium containing 3 g/l activated charcoal powder. Newly shoots were induced from in vitro root segments subcultured to 1/2MS medium containing 2uM BAP. This cycle can be used for micropropagation of Wadatsuminoki. We have succeeded in micropropagation by tissue culture of Wadatsuminoki (Figure 1). Callus proliferation from stem or root segments was observed on the 1/2LP medium containing 0.5uM BAP and 1 uM 2,4-D, this subculture cell line may be used for the possible production of secondary metabolites in vitro. Shoots were induced from stem spine (thorn) of Kagikazura in the 1/2MS medium containing BAP or Zeatin. Regenerated plants were obtained by rooting of these shoots on 1/2MS medium containing 1 uM IBA. Callus induced around the stem segments were continuously subcultured in the fresh 1/2LP medium containing 0.5 uM BAP and 1 uM 2, 4-D. These cell lines can be used for the possible secondary metabolite production and for breeding by somaclonal variation or genetic engineering.

The Arabidopsis NST3/SND1 promoter is active in secondary woody tissue in poplar

Wood biomass is one of the promising future materials for biofuels with no competing food uses. However, the higher cost to produce bioethanol from wood feedstocks is regarded as a priority issue. Genetic engineering techniques have been proposed to enhance the quality and quantity of wood materials to overcome the cost problem. Although many genetically engineered trees with applicable traits such as low lignin, a high syringyl to guaiacyl ratio and high cellulose content are generated, ectopic expression of an effector gene under a constitutive promoter can sometimes induce untoward side effects on plant growth and development. Our recent study demonstrated that AtNST3/SND1 promoter of Arabidopsis thaliana is a candidate tool for driving a potent activator to enhance wood biomass production in poplar without any growth retardation. However, the tissue- and cell-dependent activity of the promoter remains to be elucidated. In the present study, we generated transgenic poplar expressing AtNST3/SND1promoter::GUS to examine in detail the activity of the AtNST3/SND1 promoter. Histochemical analysis revealed that the promoter was predominantly active in secondary woody tissue. Our result indicates that the AtNST3/SND1 promoter is an option for expressing an effector gene to modify secondary cell wall components and wood biomass.

Iron-deficiency response and expression of genes related to iron homeostasis in poplars

ABSTRACT Iron (Fe) deficiency is a serious agricultural problem, especially in calcareous soils, which are distributed worldwide. Poplar trees are an important biomass plant, and overcoming Fe deficiency in poplars will increase biomass productivity worldwide. The poplar Fe-deficiency response and the genes involved in poplar Fe homeostasis remain largely unknown. To identify these genes and processes, we cultivated poplar plants under Fe-deficient conditions, both in calcareous soil and hydroponically, and analyzed their growth rates, leaf Soil and Plant Analyzer Development (SPAD) values, and metal concentrations. The data clearly showed that poplars have notable growth defects in both calcareous soil and a Fe-deficient hydroponic culture. They exhibited serious chlorosis of young leaves after 3 weeks of Fe-deficient hydroponic culture. The Fe concentrations in old leaves with high SPAD values were markedly lower in Fe-deficient poplars, suggesting that poplars may have good translocation capability from old to new leaves. The Zn concentration in new leaves increased in Fe-deficient poplars. The pH of the hydroponic solution decreased in the Fe-deficient culture compared to the Fe-sufficient culture. This finding shows that poplars may be able to adjust the pH of a culture solution to better take up Fe. We also analyzed the expression of Fe homeostasis-related genes in the roots and leaves of Fe-sufficient and Fe-deficient poplars. Our results demonstrate that PtIRT1, PtNAS2, PtFRO2, PtFRO5, and PtFIT were induced in Fe-deficient roots. PtYSL2 and PtNAS4 were induced in Fe-deficient leaves. PtYSL3 was induced in both Fe-deficient leaves and roots. These genes may be involved in the Fe uptake and/or translocation mechanisms in poplars under Fe-deficient conditions. Our results will increase a better understanding of the Fe-deficiency response of poplars and hence improve the breeding of Fe-deficiency-tolerant poplars for improved biomass production, the greening of high pH soils, and combatting global warming.

Expression analysis of cellulose synthases that comprise the Type II complex in hybrid aspen

Gene duplication in plants occurs via several different mechanisms, including whole genome duplication, and the copied genes acquire various forms and types. The cellulose synthase (CesA) family functions in cellulose synthesis complex (CSC) formation, which is involved in the synthesis of primary and secondary cell walls in plants. In the genome of Populus, 17 CesA have been annotated, and some of them appeared through whole genome duplication. The nucleotide sequence of the duplicated genes changed during subsequent evolution, and functional differentiation of genes might have occurred. To gain insight into the evolutionary fate of the duplicated CesA, expression analysis with quantitative reverse transcription polymerase chain reactions and promoter-reporter assays was performed on three duplicated gene pairs whose products have been reported to form a single CSC. Changes in expression of each gene at different developmental stages were detected and divergent expression patterns in different organs and tissues observed between the gene pairs. Among the tested genes, expression of PttCesA3-C was apparently lower than that of its counterpart, PttCesA3-D. The results suggest that the six CesA are approaching sub-functionalisation or non-functionalisation. Furthermore, the level of functionalisation may vary among the three pairs of genes, and functional specialisation of each CesA should have been achieved, at least partially, through differences in expression of genes.

Role of the Circadian Clock in Cold Acclimation and Winter Dormancy in Perennial Plants

Seasonal variation is a strong cue directing the growth and development of plants. It is particularly important for perennials growing in temperate and boreal regions where woody plants must become dormant to survive freezing winter temperatures. Shortening of the photoperiod induces growth cessation, bud set and a first degree of cold acclimation in most woody plants. The subsequent drop in temperature then produces a greater tolerance to cold and, in deciduous trees, leaf senescence and fall. Trees must time their periods of dormancy accurately with their environment. Circadian clocks underlie this ability, allowing organisms to predict regular, daily changes in their environment as well as longer term seasonal changes. This chapter provides an update on the plant clock in a model annual, thale cress (Arabidopsis thaliana), and further summarizes recent advances about the clock in perennial plants and its involvement in their annual growth cycles, which allows trees to withstand cold and freezing temperatures. Moreover, we outline our views on areas where future work on the circadian clock is necessary to gain insight into the life of a tree.

Monitoring Seasonal Bud Set, Bud Burst, and Cold Hardiness in Populus

Using a perennial model plant allows the study of reoccurring seasonal events in a way that is not possible using a fast-growing annual such as Arabidopsis thaliana (Arabidopsis). In this study, we present a hybrid aspen (Populus tremula × P. tremuloides) as our perennial model plant. These plants can be grown in growth chambers to shorten growth periods and manipulate day length and temperature in ways that would be impossible under natural conditions. In addition, the use of growth chambers allows easy monitoring of height and diameter expansion, accelerating the collection of data from new strategies that allow evaluation of promoters or inhibitors of growth. Here, we describe how to study and quantify responses to seasonal changes (mainly using P. tremula × P. tremuloides) by measuring growth rate and key events under different photoperiodic cycles.