Actin polymerization is essential for cell locomotion and is thought to generate the force responsible for cellular protrusions. The Arp2/3 complex is required to stimulate actin assembly at the leading edge in response to signalling. The bacteria Listeria and Shigella bypass the signalling pathway and harness the Arp2/3 complex to induce actin assembly and to propel themselves in living cells. However, the Arp2/3 complex alone is insufficient to promote movement. Here we have used pure components of the actin cytoskeleton to reconstitute sustained movement in Listeria and Shigella in vitro. Actin-based propulsion is driven by the free energy released by ATP hydrolysis linked to actin polymerization, and does not require myosin. In addition to actin and activated Arp2/3 complex, actin depolymerizing factor (ADF, or cofilin) and capping protein are also required for motility as they maintain a high steady-state level of G-actin, which controls the rate of unidirectional growth of actin filaments at the surface of the bacterium. The movement is more effective when profilin, alpha-actinin and VASP (for Listeria) are also included. These results have implications for our understanding of the mechanism of actin-based motility in cells.

Reconstitution of actin-based motility of Listeria and Shigella using pure proteins.

To grow and develop optimally, all organisms need to perceive and process information from both their biotic and abiotic surroundings. A particularly important environmental cue is light, to which organisms respond in many different ways. The responses can be simple, as in phototactic single-celled organisms, or complex, as in higher animals, which use visual inputs to modify their behavior. Many organisms can also detect cycles of light and darkness, which are exploited for seasonal and timeof-day responses. Because they are both photosynthetic and sessile, plants have to be especially plastic in response to their light environment. In addition to utilizing light as a time-keeping mechanism, plants are unique in that they use light as a source of energy and they analyze light to control such developmental decisions as when to germinate and flower. The diverse responses of plants require sophisticated sensing of intensity, direction, duration, and wavelength of light. The action spectra of light responses have provided assays to identify three photoreceptors absorbing in the ultraviolet, blue/near ultraviolet, and red/far-red (R/FR)-spectral ranges (Kendrick and Kronenberg 1994). The best characterized group of these photoreceptors is the red/far-red (R/FR) absorbing phytochromes. Phytochromes are photochromic proteins composed of a large protein (∼125 kD) covalently attached to a linear tetrapyrrole chromophore. Phytochromes are synthesized in a red-light-absorbing form, Pr (lmax = 660 nm) which, upon exposure to red light, can be phototransformed into a far-red-light-absorbing form, Pfr (lmax = 730 nm). Upon exposure to far-red light, Pfr is photoconverted to Pr (Kendrick and Kronenberg 1994). Both Pfr and Pr that has been photocycled have been shown to induce developmental responses (Shinomura et al 2000). Thus, phytochrome acts as a light-controlled developmental switch. Phytochromes have been found in all taxa of lower and higher plants examined (Mathews and Sharrock 1997; Mathews and Donoghue 1999), as well as cyanobacteria (Hughes et al. 1997; Lamparter et al. 1997; Yeh and Lagarias 1997). Phytochromes control development throughout the plant life cycle beginning with seed germination and seedling deetiolation (the transition from growth in the dark to growth in the light). They also control cotyledon/ leaf expansion and stem elongation by regulating cell division and expansion. Phytochromes enable the perception of neighboring plants or shade, and influence the transition to flowering. Because of these profound effects, phytochromes have been studied intensively for >50 years. The impact this family of photoreceptors has had on the history of Botany is extensively covered in the book, Pigment of the Imagination (Sage 1992). Many excellent reviews on phytochrome’s role in photomorphogenesis have been written recently (Mancinelli 1994; Smith 1995; Fankhauser and Chory 1997; Mustilli and Bowler 1997; Batschauer 1999; Deng and Quail 1999, and references within). In this review we will describe the complexities of phytochrome response pathways and highlight some of the recent accomplishments in elucidating the mechanisms by which phytochromes regulate so many downstream responses. Finally, we will examine the interactions between phytochrome and endogenous developmental programs.

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Light: an indicator of time and place

We have developed a highly sensitive and high-throughput method for the simultaneous analysis of 43 molecular species of cytokinins, auxins, ABA and gibberellins. This method consists of an automatic liquid handling system for solid phase extraction and ultra-performance liquid chromatography (UPLC) coupled with a tandem quadrupole mass spectrometer (qMS/MS) equipped with an electrospray interface (ESI; UPLC-ESI-qMS/MS). In order to improve the detection limit of negatively charged compounds, such as gibberellins, we chemically derivatized fractions containing auxin, ABA and gibberellins with bromocholine that has a quaternary ammonium functional group. This modification, that we call 'MS-probe', makes these hormone derivatives have a positive ion charge and permits all compounds to be measured in the positive ion mode with UPLC-ESI-qMS/MS in a single run. Consequently, quantification limits of gibberellins increased up to 50-fold. Our current method needs 180 plant samples simultaneously. Application of this method to plant hormone profiling enabled us to draw organ distribution maps of hormone species in rice and also to identify interactions among the four major hormones in the rice gibberellin signaling mutants, gid1-3, gid2-1 and slr1. Combining the results of hormone profiling data with transcriptome data in the gibberellin signaling mutants allows us to analyze relationships between changes in gene expression and hormone metabolism.

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Highly Sensitive and High-Throughput Analysis of Plant Hormones Using MS-Probe Modification and Liquid Chromatography–Tandem Mass Spectrometry: An Application for Hormone Profiling in Oryza sativa

Because plants are sessile, they have developed intricate strategies to adapt to changing environmental variables, including light. Their growth and development, from germination to flowering, is critically influenced by light, particularly at red (660 nm) and far-red (730 nm) wavelengths. Higher plants perceive red and far-red light by means of specific light sensors called phytochromes(A-E). However, very little is known about how light signals are transduced to elicit responses in plants. Here we report that nucleoside diphosphate kinase 2 (NDPK2) is an upstream component in the phytochrome signalling pathway in the plant Arabidopsis thaliana. In animal and human cells, NDPK acts as a tumour suppressor. We show that recombinant NDPK2 in Arabidopsis preferentially binds to the red-light-activated form of phytochrome in vitro and that this interaction increases the activity of recombinant NDPK2. Furthermore, a mutant lacking NDPK2 showed a partial defect in responses to both red and farred light, including cotyledon opening and greening. These results indicate that NDPK2 is a positive signalling component of the phytochrome-mediated light-signal-transduction pathway in Arabidopsis.

Phytochrome signalling is mediated through nucleoside diphosphate kinase 2

Low temperature is one of the important environmental changes that affect plant growth and agricultural production. To investigate the responses of rice to cold stress, changes in protein expression were analyzed using a proteomic approach. Two-week-old rice seedlings were exposed to 5 degrees C for 48 h, then total crude proteins were extracted from leaf blades, leaf sheaths and roots, separated by 2-DE and stained with CBB. Of the 250-400 protein spots from each organ, 39 proteins changed in abundance after cold stress, with 19 proteins increasing, and 20 proteins decreasing. In leaf blades, it was difficult to detect the changes in stress-responsive proteins due to the presence of an abundant protein, ribulose bisphosphate carboxylase/oxygenase large subunit (RuBisCO LSU), which accounted for about 50% of the total proteins. To overcome this problem, an antibody-affinity column was prepared to trap RuBisCO LSU, and the remaining proteins in the flow through from the column were subsequently separated using 2-DE. As a result, slight changes in stress responsive proteins were clearly displayed, and four proteins were newly detected after cold stress. From identified proteins, it was concluded that proteins related to energy metabolism were up-regulated, and defense-related proteins were down-regulated in leaf blades, by cold stress. These results suggest that energy production is activated in the chilling environment; furthermore, stress-related proteins are rapidly up-regulated, while defense-related proteins disappear, under long-term cold stress.

Proteomic analysis of rice seedlings during cold stress

Protein databases constructed using high-resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) were used to explore the proteome expressed in various rice tissues. Proteins from leaf sheath, root, and cultured suspension cells were systematically analyzed using 2D-PAGE, mass spectrometry and Edman sequencing, followed by database searching. In all, 79 of the 431 spots detected by 2D-PAGE in the leaf sheath, 73 of the 508 spots in the root and 140 of the 962 spots in the cultured suspension cells could be identified. Protein lists were constructed for each tissue and used to investigate the effects of gibberellin (GA) treatment. In the leaf sheath, root and cultured suspension cells, 8, 21, and 14 of the identified proteins, respectively, were regulated by GA. These proteins included polypeptides involved in general metabolism, energy production, transcriptional regulation and signal transduction in the leaf sheath; in metabolism and defense in the root; and in metabolism, energy production, cell growth, defense and signal transduction in the cultured suspension cells. These results indicate that the proteome databases assembled in these studies will be useful for the rapid assessment of changes in protein content in specific tissues, and that proteins regulated by GA may play a significant role in tissue growth.

Proteome analysis of rice tissues by two-dimensional electrophoresis: an approach to the investigation of gibberellin regulated proteins.

The molecular mechanism of light signal perception via phytochrome was analysed using the third internodes of etiolated pea seedlings irradiated with red or red followed by far-red light. A crude membrane fraction prepared from the tissue was labelled by [gamma-32P]ATP at 4 x 10(-8) M for 15 s at 0 degree C, and the proteins were separated by two-dimensional gel electrophoresis. The phosphorylation of a protein with a molecular mass of about 15 kDa in the crude membrane fraction increased with an increase in the intensity of red light irradiation (10, 50 and 100 mumol m-2 s-1) for 20 s. Successive irradiation with red light (100 mumol m-2 s-1 for 20 s) and subsequent irradiation with far-red light reduced the phosphorylation of the protein, depending on the intensity of the far-red light (from 0.1 to 8 mumol m-2 s-1 for 180 s). A plasma membrane purified from the crude membrane fraction from red light irradiated tissue showed a rapid phosphorylation of the 15 kDa protein by 4 x 10(-8) M [gamma-32P]ATP at 0 degree C for 7 s, and subsequent addition of ATP, GTP, ADP or GDP at 10(-5) or 10(-6) M efficiently removed the phosphoryl group of the 15 kDa protein. The 15 kDa protein was autophosphorylated in the gel following separation by sodium dodecylsulphate (SDS) polyacrylamide gel electrophoresis. The partially purified 15 kDa protein included nucleoside diphosphate kinase (NDP kinase) activity, as well as cross-reactivity with an antibody specific to rat NDP kinase as assayed by immunostaining and crossreactivity with an antibody specific to ricet NDP kinase as assayed by immunoprecipitation.

Phytochrome regulates phosphorylation of a protein with characteristics of a nucleoside diphosphate kinase in the crude membrane fraction from stem sections of etiolated pea seedlings.

— At 5 days after sowing of pea seeds in darkness, intact seedlings were either irradiated with red light for 40 s at 50 μmol/m2/s at the third internode or with red light as above and then with far-red light for 180 s at 0.4 μmol/m2/s, and the stems were sectioned from below the hook (mainly the third internodes) and placed in liquid N2 in a mortar. The samples were well ground, and after the addition of extraction buffer, homogenates were centrifuged to prepare the crude membrane and soluble fractions. Red-light irradiation increased the phosphorylation of an 18 kDa protein, while far-red-light irradiation decreased it. The 18 kDa protein (formerly 15 kDa protein) was identified as nucleoside diphosphate kinase (EC 2.4.6) (NDP kinase) by western blotting using an NDP kinase-specific antibody. The membrane and the soluble fractions of the red-light-irradiated samples were separated by native polyacrylamide gel electrophoresis. The protein complexes prepared from the membrane and soluble fractions differed in their mobilities, as determined by two-dimensional electrophoresis and nonequilibrium pH gradient electrophoresis. The major protein spots from both samples were cut out from the gel and tested for NDP kinase and protein kinase activity. Both protein preparations showed NDP kinase activity and changes from nucleoside diphosphates and deoxynucleoside diphosphates to nucleoside triphosphates and deoxynucleoside triphosphates in the presence of [γ-32P]ATP. Both preparations showed protein kinase phosphorylation of myelin basic protein (MBP) rather than histone H1 as protein substrates, suggesting that NDP kinase possesses a function similar to that of MAP kinase.

Characterization of Protein Complexes Containing Nucleoside Diphosphate Kinase with Characteristics of Light Signal Transduction through Phytochrome in Etiolated Pea Seedlings

根深ネギの晩抽性遺伝資源，晩ネギの分げつ性に関する遺伝解析のため，はじめに14品種・系統を用いて分げつ数と諸形質の相関分析を行った．相関分析の結果より，‘分げつ数’は‘開花日’と有意な相関が見られず，分げつ性と晩抽性は独立して選抜できる可能性が示唆された．次に相関分析での‘分げつ数’の多少および‘開花日’の早晩について変異が大きくなるように6品種・系統を選定し，片側ダイアレル分析を実施した．解析集団は生育段階に応じて3回分げつ数を調査した．片側ダイアレル分散分析の結果，全調査回で相加効果を示すa項のF値がb項を大きく上回ったこと，平均優性度が1以下であったことから，分げつ数は相加効果が大きい不完全優性の形質であると推定された．また優性遺伝子の平均的作用方向は分げつ数の少ない方向と考えられた．3回の分げつ数調査およびダイアレル分析結果を比較すると，親系統の優性順序，親系統の優性および劣性遺伝子の有する割合が生育段階によって変動することが示され，親系統間で分げつを支配する遺伝子の種類や作用機構が異なると考えられた．さらに狭義の遺伝率がいずれの調査回でも0.78以上と高かったことから，晩ネギから一本性の晩抽系統を育成するには，初期世代での選抜が重要であることが示唆された． 

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Norifumi Tanaka

Papers

Proteome analysis of rice tissues by two-dimensional electrophoresis: an approach to the investigation of gibberellin regulated proteins.

Phytochrome regulates phosphorylation of a protein with characteristics of a nucleoside diphosphate kinase in the crude membrane fraction from stem sections of etiolated pea seedlings.

Characterization of Protein Complexes Containing Nucleoside Diphosphate Kinase with Characteristics of Light Signal Transduction through Phytochrome in Etiolated Pea Seedlings

Correlation analysis and half-diallel analysis of tiller number in the bunching onion