Hussein Basma
email: hussein.basma@mines-paristech.fr

Charbel Mansour

Maroun Nemer

Pascal Stabat

Marc Haddad

Sensitivity analysis of bus line electrification at different operating conditions

Keywords: battery-electric bus, capital costs, operating costs, electricity tariffs, battery technology, electric grid

The electrification of public transport buses is getting more insights, especially in urban areas. The use of Battery electric buses (BEB) results in no tailpipe emissions, which gives BEB an advantage over diesel, hybrid and natural gas buses. However, the high capital costs and fluctuating operating costs of BEB limits their market breakthrough. Minimizing the total costs of BEB is essential to ease their deployment and this can be achieved by optimally designing the battery size and managing the charging strategy of the buses. These costs incur significant fluctuations as they are highly sensitive to electricity tariffs set by local authorities. In addition, these costs are directly driven by the battery costs, battery technology, and onboard energy management. For this sake, it is essential to evaluate the effect of the mentioned parameters on BEB optimal battery sizing and charging strategy and highlight their impact on the electricity grid.

Introduction

Low-emission zones for vehicles are evolving fast in European cities amid air quality concerns. Major European cities are banning diesel vehicles as an effective measure to reduce emissions in urban areas, after the perceived failure to effectively enforce and implement the adopted vehicle emissions regulations [START_REF] Müller | City bans are spreading in Europe[END_REF]. The adoption of alternative bus technologies in public transportation helps to reduce the Green House Gas (GHG) emissions and pollutants when compared to diesel buses. Currently, there are many alternative bus technologies such as hybrid electric buses, compressed natural gas buses, fuel cell buses, and battery electric buses [START_REF] Jorgensen | Technologies for electric , hybrid and hydrogen vehicles : Electricity from renewable energy sources in transport[END_REF]. Many studies have confirmed the advantage of BEB in reducing carbon emissions when compared to diesel, hybrids and natural gas buses [START_REF] Du | Evaluating the technological evolution of battery electric buses : China as a case[END_REF][START_REF] Nylund | Fuel and Technology Alternatives for Buses Overall Energy Efficiency and Emission[END_REF].

BEBs are the most promising solution to replace the current diesel bus fleet. BEBs incur lower well-to-wheel energy consumption per driven kilometers when compared to all other alternative technologies [START_REF] Antonio | Impact of Spanish electricity mix , over the period 2008 -2030 , on the Life Cycle energy consumption and GHG emissions of Electric , Hybrid Diesel-Electric , Fuel Cell[END_REF][START_REF] Torchio | Energy , environmental and economic comparison of different powertrain / fuel options using well-towheels assessment , energy and external costs e European market analysis[END_REF]. This better energy performance is mainly due to the higher BEB powertrain efficiency. Moreover, BEBs result in the least well-to-wheel GHG emissions [START_REF] Grutter | Real World Performance of Hybrid and Electric Buses Environmental and Financial Performance of Hybrid and Battery Electric Transit Buses Based on Real World Performance of Large Operational Fleets[END_REF][START_REF] Nylund | Fuel and Technology Alternatives for Buses Overall Energy Efficiency and Emission[END_REF]. These emissions are highly dependant on the electricity generation mix.

Despite their superior environmental performance, BEBs still face many challenges that prevent their deployment at a massive scale [START_REF] Bühne | How to promote electromobility for European car drivers ? Obstacles to overcome for a broad market penetration[END_REF]. First, the costs of BEBs deployment are the highest among all other alternative bus technologies [START_REF] Lajunen | Energy consumption and cost-benefit analysis of hybrid and electric city buses[END_REF][START_REF] Mckenzie | Environmental life-cycle assessment of transit buses with alternative fuel technology[END_REF]. The costs of the battery pack result in a very high bus unit price where only fuel cell buses record a higher unit price. In addition, the costs of infrastructural modification are very high in the case of BEBs. Other challenges facing BEBs are their operation features. The two main operation features for buses are their driving range and their refueling time. BEBs suffer the lowest driving range and highest refueling time among all alternative bus technologies which may disturb their operation resulting in schedule delays [START_REF] Efthymiou | Electric vehicles charging infrastructure location : a genetic algorithm approach[END_REF][START_REF] Mahmoud | Electric buses: A review of alternative powertrains[END_REF] A proper battery sizing and charging strategy help to overcome the aforementioned challenges to ease BEBs deployment. The size of the battery directly affects the costs and the driving range. Bigger batteries result in a higher driving range, however, this yields to higher costs. On the other hand, reducing the battery size decreases the incurred costs but it reduces the driving range as well. In addition, the adopted charging strategy impacts the costs and refueling time. Utilizing fast chargers at a high power-rating shortens the refueling time, however, this comes at high chargers costs. On the other hand, using slower chargers incurs lower costs, but this yields to longer refueling time.

The choice of the optimal battery sizing and charging strategy of BEBs are highly affected by the BEBs operating conditions. Many parameters drive the total operating and capital costs of BEB and they are subject to continuous fluctuations. These parameters can be classified into three main categories: 1) Technology-related parameters: the price of Lithium-ion batteries and battery lifetime. the price of lithium-ion batteries has a direct impact on the BEB capital costs being the main battery technology used nowadays due to its energy and power density benefits [START_REF] Schmuch | Lithium-Based Rechargeable Automotive Batteries[END_REF]. 2) Energy cost-related parameters: local electricity energy and power tariffs. Energy costs parameters directly drive the BEB operating costs. Electric energy and power tariffs are determined by local authorities depending on the local electricity generation mix and ON/OFF peak hours [START_REF] Zhou | Real-world performance of battery electric buses and their lifecycle benefits with respect to energy consumption and carbon dioxide emissions[END_REF]. 3) Energy Management-related parameters: charging/discharging strategy of the battery including the minimum allowable battery state of charge (SOC) and the bus charging duration. These parameters manage the on-board energy/power source and affect both the capital and operating costs of BEB.

The mentioned parameters affect the optimal battery sizing and charging strategy of BEB leading to different total costs and charging infrastructure, in addition to different impacts on the electricity grid. That being said, it is essential to evaluate the impact of these parameters on BEB optimal battery sizing, charging strategy, and electricity grid, as they are widely varying across different countries or regions.

Methods

In order to provide an optimal battery size and charging strategy for BEB, 2 main components are necessary. First, a tool that can assess the energy consumption of BEB at all driving and weather conditions is required. Second, an optimization algorithm that will minimize the total costs of BEB by optimally sizing the battery and managing the charging strategy.

Energy Consumption Assessment

The energy consumption of BEB is assessed using a detailed bus energy model that is developed by the current author in previous work. The model considers the main energy loads encountered in BEBs covering the bus's entire energy needs. There are three main energy loads in BEBs: (1) traction energy load, (2) thermal energy load and

(3) non-mechanical auxiliaries' energy load.

Concerning the traction energy load, a powertrain model is developed to evaluate this energy load. The model includes a battery, electric machine, transmission and braking systems, and a torque controller to supply the driver's acceleration and braking demands. The considered bus is a typical single deck 12-m long bus equipped with a Lithium-ion battery that has an energy density of 85 𝑊ℎ/𝑘𝑔. A 155 kW electric machine propels the bus and recuperates the kinetic energy of the bus during braking. The transmission system is designed to meet the bus acceleration requirements.

The thermal energy load is evaluated by developing a bus cabin model and heating, ventilating and air conditioning model (HVAC). The bus cabin model estimates the thermal conditions inside the bus such as the internal temperature and humidity. The proposed model considers the different heat transfer phenomena that take place between the bus interior and external environment, internal bus components and passengers. The model is validated experimentally on an electric vehicle [START_REF] Brèque | Cabin Thermal Needs: Modeling and Assumption Analysis[END_REF]. The heat transfer due to frequent opening and closure of the bus doors is considered as well. On the other hand, an HVAC unit supplies the bus cabin with its energy needs in order to attain thermal comfort conditions inside the bus. The proposed HVAC unit is a 40 -kW reversible heat pump that operates as a heat pump and air conditioner in order to supply the bus's heating and cooling needs. The heat pump is properly sized and controlled in previous work (Al [START_REF] Haddad | Analysis of heat pump performance in battery electric buses[END_REF].

Non-mechanical auxiliaries are essential features for the bus operation. There are four main types of auxiliaries encountered in BEB: (1) Electric, (2) Pneumatic, (3) Hydraulic and (4) Thermal. Most of these auxiliaries are only found in heavy-duty vehicles and they can't b neglected. Bus doors, suspension system, and lighting are all-electric auxiliaries whereas the steering pump is a hydro-electric auxiliary. The pneumatic auxiliaries include the braking system as it operates by means of air brakes. Finally, thermal auxiliaries include Lithium-ion battery cooling and heating needs.

Fig. 1 shows the bus global energy scheme. More details on the modeling approach can be found in [START_REF] Basma | Comprehensive energy assessment of battery electric buses and diesel buses[END_REF]

Optimization Algorithm

Moreover, the authors introduce an optimization methodology that aims at minimizing the total costs of BEB by controlling the battery size, charging energy and charging power during the day while respecting the bus schedule needs to ensure undisrupted operation. The algorithm is based on the Dynamic Programming (DP) global optimization routine developed by Richard Bellman in 1954.

The cost function to be minimized is the total operating and capital costs of BEB as shown in equation ( 1)

The cost function is composed of two components: (1) energy costs representing the operating costs and (2) battery

Ω 𝑖 = ∑ 𝛼(𝑡 ). 𝐸 𝑖 . (1 + 𝜀. 𝑝 𝑖 2 𝑝 𝑚𝑎𝑥 2 ) + 𝛽. 𝑘 𝑇 𝑖=0
(1) costs representing the capital costs. The optimization algorithm minimizes the total operating and capital costs throughout the entire service life of the battery. This optimization problem is a constrained problem as there exist constraints on the state and control variables.

There are four main constraints to respect in this optimization problem: (1) Battery maximum charging power, (2) battery minimum and maximum state of charge (SOC), (3) bus schedule and (4) thermal comfort conditions inside the bus cabin.

The scope of the study covers stationary charging scenarios where the bus can only be charged when it is parked either at the depot, terminal station or stop station. That being said, there are 3 different stationary charging scenarios: (1) Depot Charging, (2) Terminal Station Charging and (3) Opportunity Charging at the bus stops. Fig. 2 graphically illustrates the mentioned scenarios.

The Optimization Algorithm is applied for each charging scenario providing optimal charging strategy and battery sizing. Moreover, the optimization algorithm provides the power rating of the required chargers referred to as the charging technology. Fig. 3 presents the optimization algorithm.

Framework

Fig. 4 shows the global overview of the proposed methodology. The proposed tool receives inputs such as bus line driving conditions, trajectory topography, weather conditions, and passengers flow to the bus stations, and evaluates the energy consumption of the bus during each trip. Afterward, the buses energy needs are fed to the In this study, the "Inputs" are manipulated to run a sensitivity analysis to study their impact on BEB optimal battery design, charging strategy, total costs, and the electricity grid. The energy cost parameter 𝛼 is a function of time, which reflects the different electric energy tariffs imposed by local authorities during the ON/OFF peak hours. In this study, the percentile difference between the peak and Off-peak hour tariffs is manipulated. This parameter is denoted by DA and is calculated according to equation (2).

𝐷𝐴 = 𝛼 𝐻 -𝛼 𝐿 𝛼 𝐻

(2)

Where 𝛼 𝐻 is the high cost of electricity during peak hours and 𝛼 𝐿 is the low cost of electricity during off-peak hours. 𝛼 𝐻 is fixed and 𝛼 𝐿 is varied. Opportunity charging duration (Scenario 3) 0.5-3 𝑚𝑖𝑛

Results

This section evaluates the impact of the parameters introduced in Table 2 on the optimal battery size, power demand profile during the day, charging energy and total costs.

Impact of Energy Cost-Related Parameters

• Peak/Off-Peak Hours Energy Cost Difference Fig. 5 and Fig. 6 present the variation of the optimal battery sizing and total costs function of the Peak/Off-Peak hours energy cost difference DA. As the difference between day/night electricity tariff increases, a bigger battery size is preferred. The reason is that most of the charging is done at night for bigger batteries, which reduces the operating costs despite their higher capital costs compared to smaller batteries. All 3 scenarios show a similar behavior. In addition, scenario 1 requires the biggest battery size whereas scenario 3 requires the smallest battery size, this is due to the charging frequency of the buses in each scenario. The buses can be charged once every round trip in scenario 1, twice in scenario 2 and 6 times per round trip in scenario 3. Higher charging frequency leads to smaller optimal battery size. In Fig. 9, the impact of the demand charge cost coefficient on the optimal battery size is assessed. Scenarios 1 and 2 do not show a sensitive behavior to the demand charge tariff variation. On the contrary, scenario 3 witnesses an increase in the optimal battery size as ε increases. Higher values of ε imply that the operating costs will increase with higher power demand. Smaller batteries are charged more during the day and at higher power as their initial energy level is lower compared to bigger batteries. The sensitive behavior of scenario 3 relative to ε is due to the fast chargers used in opportunity charging at the bus stops where charging takes place at high power.

Fig. 10 highlights the impact of the demand charge cost coefficient on the total costs. All 3 scenarios witness an increase in total costs as ε increases. For scenarios 1 and 2, higher values of ε result in higher operating costs but no variation in capital costs as the optimal battery size is not affected by ε, therefore, higher total costs are obtained for these 2 scenarios. Similarly, for scenario 3, higher total costs are obtained with higher values of ε, however, total costs become less sensitive to ε for values above 0.5. This is due to the bigger optimal battery size which allows charging at a lower power rate. Fig. 11 and Fig. 12 show the impact of demand charge cost coefficient on the charging energy and power respectively. At higher values of ε, less energy is charged during the day and more energy is charged during the night as night-charging takes place at lower power rates. Concerning the power demand, reducing the demand charge tariff (ε = 0.25) is resulting in higher power demand during the day which exceeds 500 kW whereas a high demand charge tariff limits the maximum power demand during the day to less than 400 kW.

Impact of Technology-Related Parameters

In this section, the presented results correspond to scenario 3, however, all other scenarios show similar behavior. Fig. 13 shows the impact of Lithium-Ion Batteries cost and service life on the total costs. At high Lithium-Ion Batteries costs, smaller batteries are preferred as they result in lower capital costs and thus lower total costs. However, when the costs of Lithium-Ion Batteries drop below a certain threshold, referred to as λ, bigger batteries incur lower total costs mainly due to their lower operating costs that compensate for the high capital costs of big Similar behavior is witnessed at all other values of the battery service life, however, there is a different value for the threshold λ at each battery service life. For example, the value of λ increases from 50 to 120 €/kWh as the service life increases from 5 to 12 years. This implies that with higher battery service life, bigger batteries are preferred as they incur lower total costs. The reason is that with higher battery service life, the total costs become more sensitive to the operating costs as their share in the total costs increases and therefore batteries that incur lower operating costs will be preferred.

Impact of Energy Management-Related Parameters

• Minimum Allowable Battery SOC Fig. 14 shows the impact of the minimum allowable battery SOC on the optimal battery sizing for the three scenarios under study. Higher values of 𝑆𝑂𝐶 𝑚𝑖𝑛 implies that the effective battery energy capacity is less, and thus bigger batteries are needed to fulfill the same energy demand resulting in higher capital costs and effectively higher total costs as shown in Fig. 15. Although higher values of 𝑆𝑂𝐶 𝑚𝑖𝑛 result in higher costs, this reduce battery aging and hence prolongs its service life.

Fig. 16 shows the impact of 𝑆𝑂𝐶 𝑚𝑖𝑛 on the power demand. The maximum power demand during the day doesn't show a sensitive behavior relative to 𝑆𝑂𝐶 𝑚𝑖𝑛 . Although the optimal battery size increases as 𝑆𝑂𝐶 𝑚𝑖𝑛 increases, the effective battery energy capacity is the same and thus the charging needs remain intact. The slight differences in the power demand during the day are due to the differences in battery size that affect the energy consumption of the bus depending on its weight. During the night, the maximum power demand is not affected by 𝑆𝑂𝐶 𝑚𝑖𝑛 because of the long charging duration that allows slow charging at a low power rate.

• Opportunity Charging Duration Fig. 17 presents the impact of opportunity charging duration on optimal battery size and this is only relevant for scenario 3. Lower durations result in higher optimal battery size as there is less time to charge the battery at the bus stop and thus the initial energy level of the battery should be higher. The need for bigger batteries at lower opportunity charging durations results in higher total costs as shown in Fig. 18. Opportunity charging duration has a significant impact on the power demand as shown in Fig. 19. With higher charging durations, the charging power rate can be reduced. The maximum power demand during the day decreases from 470 -400 kW when charging duration increases from 0.5 -1 minute. However, at a charging duration of 2 minutes, the power demand drastically increases despite higher charging duration mainly due to the smaller optimal battery size as shown in Fig. 17. This implies that the capital cost reduction resulting from decreasing the battery size outweighs the additional operating costs due to charging at higher power rates.

Fig. 2 :

 2 Fig. 2: Stationary Charging Scenarios

Fig. 4 :

 4 Fig. 4: Methodology Global Overview

Fig. 5 :

 5 Fig. 5: Impact of Peak/Off-Peak Hours Energy Cost Difference on Optimal Battery Size

Fig. 7 :

 7 Fig. 7: Impact of Peak/Off-Peak Hours Energy Cost Difference on Charging Energy Partition

Fig. 9 :

 9 Fig. 9: Impact of Demand Charge Cost Coefficient on Optimal Battery Size

Fig. 11 :

 11 Fig. 11: Impact of Demand Charge Cost Coefficient on Charging Energy Partition Fig. 12: Impact of Demand Charge Cost Coefficient on Daily Power Demand

Fig. 13 :

 13 Fig. 13: Impact of Technology-Related Parameters on Total Costs

Fig. 14 :

 14 Fig. 14: Impact of Battery Minimum Allowable State of Charge on Optimal Battery Size Fig. 15: Impact of Battery Minimum Allowable State of Charge on Total Costs

Fig. 19 :

 19 Fig. 19: Impact of Opportunity Charging Duration on Maximum Power Demand (Scenario 3)

Table 1 :

 1 Table 1 explains the cost function components. Optimization Algorithm Cost Function Components

		Variable	Definition	Unit
	Energy Costs	𝛼	Cost of unit energy	€/kWh
		𝜀	Demand Charge Cost Coefficient	-
		𝐸	Amount of Charged Energy	kWh
		𝑝	Charging Power	kW
		𝑝 𝑚𝑎𝑥	Maximum Allowable Charging Power	kW
	Battery Costs	𝛽	Cost of Lithium-ion Batteries	€/kWh
		𝑘	Battery Size (Capacity)	kWh

Fig. 1: BEB Global Energy Scheme

Table 2 :

 2 Input Parameters

		Parameter	Definition	Range	Unit
	Energy Costs	𝐷𝐴	Peak/Off-peak hours energy cost difference	0-0.9	-
		𝜀	Demand Charge Cost Coefficient	0-1	-
	Technology	𝛽	Cost of 1 kWh of Lithium-ion Batteries	50-200	€/kWh
		𝑇	Lithium-ion Batteries service life	5-12	Years
	Energy Management	𝑆𝑂𝐶 𝑚𝑖𝑛	Minimum Allowable Battery SOC	0.1-0.3	-
		𝜏


Ramanjulu Sunkar

Boaz Kaplan

Nicolas N Bouche

Tzahi Arazi

Dvora Dolev

Ina N Talke

Frans J M Maathuis

Dale Sanders

David D Bouchez

Hillel Fromm
email: h.fromm@leeds.ac.uk

Expression of a truncated tobacco NtCBP4 channel in transgenic plants and disruption of the homologous Arabidopsis CNGC1 gene confer Pb2+ tolerance

Keywords: calcium, cyclic nucleotides, heavy metals, ion channel, knockout mutant, T-DNA

établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The characterization of plant genes that code for proteins involved in the transport of heavy metals should be useful in developing appropriate biotechnologies to clean the environment (e.g. phytoremediation; [START_REF] Raskin | Plant genetic engineering may help with environmental cleanup[END_REF][START_REF] Terry | Phytoremediation of Contaminated Soil and Water[END_REF], as well as for engineering plant tolerance to toxic metals. Because certain metals such as lead (Pb 2+ ) are non-essential and toxic to plants, plants are unlikely to have transporters speci®c for these metals. Instead, the permeability of the plasma membrane to these metals is attributed to the inherently limited selectivity of certain ion transporters.

A family of channel proteins from barley (HvCBT1, [START_REF] Schuurink | Characterization of a calmodulin-binding transporter from the plasma membrane of barley aleurone[END_REF]; Arabidopsis (AtCNGC1-AtCNGC6, [START_REF] Ko È Hler | Characterization of a novel gene family of putative cyclic nucleotide-and calmodulin-regulated ion channels in Arabidopsis thaliana[END_REF][START_REF] Leng | Cloning and ®rst functional characterization of a plant cyclic nucleotide-gated cation channel[END_REF]; and tobacco (NtCBP4, [START_REF] Arazi | A tobacco plasma membrane calmodulin-binding transporter confers Ni 2+ tolerance and Pb 2+ hypersensitivity in transgenic plants[END_REF] was recently reported. In Arabidopsis, at least 19 CNGC (cyclic nucleotide-gated ion channel)-related genes are distributed among the ®ve chromosomes (N. Bouche  , unpublished results), with some of them clustered (for example, genes At2g46430, At2g46440 and At2g46450 on chromosome 2). Structurally, all these proteins contain six putative transmembrane domains, a presumed pore region located between the ®fth and sixth transmembrane domains, and a putative cyclic nucleotide monophosphatebinding domain coinciding with a calmodulin-binding site located at the C-terminus [START_REF] Arazi | A high-af®nity calmodulin-binding site in a tobacco plasma membrane channel protein coincides with a characteristic element of cyclic nucleotide-binding domains[END_REF][START_REF] Ko È Hler | Characterisation of calmodulin binding to cyclic nucleotide-gated ion channels from Arabidopsis thaliana[END_REF]. These proteins are similar in overall structure to mammalian cyclic nucleotide-gated non-selective cation channels, implicated in Ca 2+ signal transduction.

Various approaches have been used to investigate the function of this plant protein family. [START_REF] Ko È Hler | Characterization of a novel gene family of putative cyclic nucleotide-and calmodulin-regulated ion channels in Arabidopsis thaliana[END_REF] The Plant Journal (2000) 24(4), 533±542 ã 2000 Blackwell Science Ltd reported that two members of the Arabidopsis gene family (AtCNGC1 and AtCNGC2) could partially complement a K +uptake-de®cient yeast mutant. [START_REF] Leng | Cloning and ®rst functional characterization of a plant cyclic nucleotide-gated cation channel[END_REF], using heterologous expression systems, suggested that AtCNGC2 is a cyclic nucleotide-stimulated cation channel permeable to Ca 2+ and K + . Interestingly, Pb 2+ is a highly toxic metal that can bind to Ca 2+ -binding sites in regulatory proteins such as calmodulin [START_REF] Ouyang | Metal ion binding to calmodulin: NMR and ¯uorescence studies[END_REF]. The similarity in protein-binding sites for Pb 2+ and Ca 2+ is consistent with reports that Pb 2+ entry into animal cells [START_REF] Simons | Lead enters bovine adrenal medullary cells through calcium channels[END_REF][START_REF] Tomsig | Permeation of Pb 2+ through calcium channels: fura-2 measurements of voltage-and dihydropyridine-sensitive Pb 2+ entry in isolated bovine chromaf®n cells[END_REF] as well as into plant cells [START_REF] Huang | Lead phytoextraction: species variation in lead uptake and translocation[END_REF] occurs, at least in part, through Ca 2+ -permeable channels.

We have taken a reverse genetic approach to studying the function of NtCBPs and AtCNGCs. We reported on the plasma membrane localization of NtCBP4, and on the phenotype of transgenic tobacco overexpressing this protein. We found that overexpression of NtCBP4 conferred tolerance to Ni 2+ and hypersensitivity to Pb 2+ , associated with reduced Ni 2+ uptake and enhanced Pb 2+ uptake, respectively [START_REF] Arazi | A tobacco plasma membrane calmodulin-binding transporter confers Ni 2+ tolerance and Pb 2+ hypersensitivity in transgenic plants[END_REF]. Because NtCBP4 is, to date, the only identi®ed plant protein capable of modulating tolerance and accumulation of Pb 2+ , we investigated other possibilities of manipulating Pb 2+ tolerance in tobacco by engineering NtCBP4.

In addition, we tested whether NtCBP4 homologues in Arabidopsis encode proteins that are potentially involved in Pb 2+ transport. Comparison of NtCBP4 with the CNGC gene family of Arabidopsis on a phylogenetic tree reveals that NtCBP4 is most related to the Arabidopsis CNGC1 gene. At the protein level, NtCBP4 is 82% similar and 75% identical to CNGC1. Therefore AtCNGC1 is a probable orthologue of NtCBP4 in Arabidopsis. Subsequently, we isolated a CNGC1 knockout mutant and investigated its response to Pb 2+ . Here we demonstrate that expression of a truncated NtCBP4 in the transgenic tobacco, and disruption of CNGC1 in Arabidopsis resulted in improved tolerance to Pb 2+ .

Results

Transgenic tobacco plants expressing a truncated NtCBP4 exhibit improved tolerance to Pb 2+

The C-terminal half of NtCBP4 contains a structurally conserved putative cyclic nucleotide-binding domain and a high-af®nity calmodulin-binding site [START_REF] Arazi | A high-af®nity calmodulin-binding site in a tobacco plasma membrane channel protein coincides with a characteristic element of cyclic nucleotide-binding domains[END_REF]. In contrast to the mammalian cyclic nucleotide-gated channels, where the calmodulin-binding domain is near the N-terminus of the protein [START_REF] Varnum | Interdomain interactions underlying activation of cyclic-nucleotide-gated channels[END_REF], in NtCBP4 the calmodulin-binding site coincides with the predicted aC-helix structure of the cyclic nucleotidebinding domain [START_REF] Arazi | A high-af®nity calmodulin-binding site in a tobacco plasma membrane channel protein coincides with a characteristic element of cyclic nucleotide-binding domains[END_REF]. This helix is essential for cyclic nucleotide binding in related proteins from other organisms [START_REF] Shabb | Cyclic nucleotide-binding domains in proteins having diverse functions[END_REF]. We reasoned that removal of this important helix should disrupt the binding of both cyclic nucleotides and calmodulin, and could possibly render the channel inactive. Moreover, if plant NtCBP4-associated channels function as tetramers, as do mammalian cyclic nucleotide-gated channels [START_REF] Liu | Subunit stochiometry of cyclic nucleotide-gated channels and effects of subunit order on channel function[END_REF], expression of an inactive subunit might have inhibitory effects on endogenous native NtCBP4-associated channel activities. As a ®rst step to test this possibility, we prepared a truncated version of NtCBP4 from which a major part of the C-terminal half, including the aC-helix, was removed (designated NtCBP4DC; Figure 1).

Transgenic plants expressing NtCBP4DC were prepared as described in Experimental procedures, selected on kanamycin, and transferred to the greenhouse for seed production. Seeds were plated on kanamycin-containing medium to select for progeny carrying the transgene. To investigate the possible effects of NtCBP4DC expression on et al., 1999;[START_REF] Arazi | A high-af®nity calmodulin-binding site in a tobacco plasma membrane channel protein coincides with a characteristic element of cyclic nucleotide-binding domains[END_REF] and the overlapping calmodulin-binding domain (CaMBD, cross-hatched;[START_REF] Arazi | A high-af®nity calmodulin-binding site in a tobacco plasma membrane channel protein coincides with a characteristic element of cyclic nucleotide-binding domains[END_REF] are indicated. The amino-acid sequence of part of the cNBD is shown on top. The CaMBD (bold letters) and the aB and aC predicted helices (underlined) of the cNBD are according to [START_REF] Arazi | A high-af®nity calmodulin-binding site in a tobacco plasma membrane channel protein coincides with a characteristic element of cyclic nucleotide-binding domains[END_REF]. The downward arrow shows the site of the deletion used to create the C-terminal truncated protein designated NtCBP4DC (amino acids Met 1 ±Ser 593 ). Numbers denote the terminal amino-acid residues shown in the sequence, based on [START_REF] Arazi | A tobacco plasma membrane calmodulin-binding transporter confers Ni 2+ tolerance and Pb 2+ hypersensitivity in transgenic plants[END_REF] and [START_REF] Arazi | A high-af®nity calmodulin-binding site in a tobacco plasma membrane channel protein coincides with a characteristic element of cyclic nucleotide-binding domains[END_REF]. (b) The NtCBP4DC cDNA in a binary Ti plant transformation vector. The NtCBP4DC cDNA, as described in (a), was prepared by cloning a corresponding PCR-ampli®ed DNA fragment into the XhoI and EcoRI sites of the same vector used for the full-length NtCBP4.

plant response to Pb 2+ , NtCBP4DC transgenic lines were germinated in the presence of Pb(NO 3 ) 2 and their growth was compared with that of wild-type plants and of the transgenic line 49-79, which overexpresses the full-length NtCBP4 (designated NtCBP4FL) and is associated with Pb 2+ hypersensitivity [START_REF] Arazi | A tobacco plasma membrane calmodulin-binding transporter confers Ni 2+ tolerance and Pb 2+ hypersensitivity in transgenic plants[END_REF]. Eleven independent kanamycin-resistant lines (designated 42-11, 22-11, 29, 33-11, 16-6, 45-16, 52-36, 55-16, 9-2, 36-16, 55-61) that express the NtCBP4DC transgene were found to be more tolerant to Pb 2+ than wild-type plants. Figure 2 shows the phenotype of three transgenic lines expressing the NtCBP4DC gene, and the wild-type and NtCBP4FL control lines, grown with or without Pb 2+ . The NtCBP4DC plants were more tolerant to the toxic metal than the wild-type seedlings, whereas the NtCBP4FL plants were more sensitive, as previously reported [START_REF] Arazi | A tobacco plasma membrane calmodulin-binding transporter confers Ni 2+ tolerance and Pb 2+ hypersensitivity in transgenic plants[END_REF]. No differences were found between wild-type and tobacco transgenic lines in their response to other metals including Na + , Zn 2+ , Mn 2+ , Cd 2+ and La 3+ .

The expression of NtCBP4FL and NtCBP4DC in transgenic plants is driven by the same promoter. However, we wished to con®rm that their expression levels were similar, and to exclude the possibility that the expression of the NtCBP4DC mRNA abolished the expression of the endogenous native gene (for example by a gene silencing mechanism). We also wished to assess the correlation between NtCBP4DC expression and the phenotype. Northern hybridizations with total RNA samples were used for analysing steady-state mRNA levels (Figure 3a). We tested ®ve independent kanamycin-resistant transgenic lines showing a Pb 2+ -tolerant phenotype, two kanamycin-resistant transgenic lines without an apparent phenotype, the transgenic line expressing the full-length NtCBP4 [START_REF] Arazi | A tobacco plasma membrane calmodulin-binding transporter confers Ni 2+ tolerance and Pb 2+ hypersensitivity in transgenic plants[END_REF], and the wild type. In the ®ve transgenic lines that showed a Pb 2+ tolerance phenotype , the truncated-size mRNA was detected. It was easily distinguishable by its length (727 bases shorter) from the fulllength mRNA in line NtCBP4FL. Moreover, the expression levels of the full-length mRNA in NtCBP4, and the truncated mRNA in the NtCBP4DC lines were similar, with some variability in mRNA levels among the NtCBP4DC lines. Importantly, in the two kanamycin-resistant lines not showing a phenotype (lines DC-18-7, DC-12-9), expression of the truncated transgene was not detected. In addition, six independent transgenic tobacco lines expressing a non-relevant protein (glutamate decarboxylase) under the control of the 35S CaMV promoter were indistinguishable from wild-type in response to Pb 2+ (data not shown). These results indicate that the Pb 2+ tolerance phenotype is associated with expression of the truncated NtCBP4 transgene. It should be noted that the endogenous native NtCBP4 mRNA in wild-type plants was not detected in the total RNA sample, due to its very low abundance (T. Arazi and H. Fromm, unpublished results).

To ascertain that the expression of the endogenous native gene in NtCBP4DC transgenic plants was not silenced, we performed RT±PCR ampli®cation with a set of primers that amplify a 404 bp region of the full-length NtCBP4 mRNA, but not of the truncated mRNA. The RT± PCR results (Figure 3b) show that a 404 bp DNA band was ampli®ed from mRNA of the NtCBP4DC plants, indicating [START_REF] Arazi | A high-af®nity calmodulin-binding site in a tobacco plasma membrane channel protein coincides with a characteristic element of cyclic nucleotide-binding domains[END_REF] were separated by SDS±PAGE and immunoblotted on a nitrocellulose membrane with polyclonal anti-NtCBP4 antibodies [START_REF] Arazi | A tobacco plasma membrane calmodulin-binding transporter confers Ni 2+ tolerance and Pb 2+ hypersensitivity in transgenic plants[END_REF][START_REF] Arazi | A high-af®nity calmodulin-binding site in a tobacco plasma membrane channel protein coincides with a characteristic element of cyclic nucleotide-binding domains[END_REF]. The positions of molecular weight markers are indicated. Immunodetection of vacuolar membrane H + -ATPase [START_REF] Ward | Dissociation and reassembly of the vacuolar H + -ATPase complex from oat roots[END_REF] served as an internal standard.

that the endogenous native NtCBP4 mRNA was indeed present in these plants. This ampli®ed band had the same mobility as the control band ampli®ed from the NtCBP4 cDNA clone. Where reverse transcriptase was omitted (±RT), no ampli®cation was detected, indicating that the observed ampli®cations described above originated from mRNA templates rather than from residual genomic DNA or from contaminating plasmid DNA in the samples. Ampli®cation with primers corresponding to the nuclear gene encoding the b-subunit of mitochondrial H + -ATPase, as a control, indicated that similar amounts of poly A + mRNA were used in all reactions. Furthermore, immunoblot analysis using anti-NtCBP4 antibodies revealed that the endogenous native protein was expressed and present in membranes of NtCBP4DC lines, and the level of expression similar to that in the wild type. An example of protein analysis of line DC-42-11, the wild type and the NtCBP4FL line is shown in Figure 3(c). It should be noted that as the anti-NtCBP4 antibodies were raised against the C-terminal half of NtCBP4 [START_REF] Arazi | A tobacco plasma membrane calmodulin-binding transporter confers Ni 2+ tolerance and Pb 2+ hypersensitivity in transgenic plants[END_REF][START_REF] Arazi | A high-af®nity calmodulin-binding site in a tobacco plasma membrane channel protein coincides with a characteristic element of cyclic nucleotide-binding domains[END_REF], the truncated NtCBP4 protein could not be detected with these antibodies. Nevertheless, this analysis revealed that expression of NtCBP4DC does not result in silencing of the endogenous NtCBP4 gene. Rather, the apparent phenotype conferred by NtCBP4DC expression occurs in the presence of the full-length native endogenous NtCBP4.

Quantitative assessment of Pb 2+ tolerance in NtCBP4DC transgenic seedlings, and Pb 2+ accumulation

The extent of growth inhibition of seedlings by Pb 2+ was evaluated by determining the fresh weight of seedlings grown in different concentrations of Pb(NO 3 ) 2 (Figure 4a). Whole-seedling fresh weight analysis revealed 50% inhibition at 0.34, 0.47, 0.58 and 0.62 mM Pb 2+ for NtCBP4FL, wild type, NtCBP4DC-22-11 and NtCBP4DC-42-11, respectively (Figure 4a). Moreover, statistical analysis revealed that the two NtCBP4DC lines were signi®cantly more tolerant (P < 0.05) than the wild-type seedlings at Pb 2+ concentrations of b0.1 mM. Pb 2+ hypersensitivity of the NtCBP4FL lines was also signi®cantly different from the response of wild-type plants (P < 0.05), as previously described [START_REF] Arazi | A tobacco plasma membrane calmodulin-binding transporter confers Ni 2+ tolerance and Pb 2+ hypersensitivity in transgenic plants[END_REF].

One of the possible explanations for the Pb 2+ -tolerant phenotype of the NtCBP4DC plants may be the attenuated uptake of the metal, as we have already demonstrated that Pb 2+ hypersensitivity in NtCBP4FL plants was associated with enhanced accumulation of Pb 2+ [START_REF] Arazi | A tobacco plasma membrane calmodulin-binding transporter confers Ni 2+ tolerance and Pb 2+ hypersensitivity in transgenic plants[END_REF]. To test this possibility, we determined Pb 2+ accumulation by ICP-AES in 12-day-old seedlings grown in the presence of 0.2 mM Pb(NO 3 ) 2 . NtCBP4DC plants (lines DC-42-11 and DC-22-11) showed a marked reduction in Pb 2+ accumulation compared with wild-type seedlings (P < 0.05), whereas NtCBP4FL plants accumulated substantially more Pb (P < 0.05) than the wild type (Figure 4b), as previously described [START_REF] Arazi | A tobacco plasma membrane calmodulin-binding transporter confers Ni 2+ tolerance and Pb 2+ hypersensitivity in transgenic plants[END_REF].

The improved Pb 2+ tolerance associated with attenuated Pb 2+ accumulation in tobacco plants expressing a truncated NtCBP4 protein raised the possibility that the truncated protein inhibits the function of endogenous NtCBP4-associated channels. When the NtCBP4 amino- (b) Lead accumulation in 12-day-old seedlings grown in the presence of 0.2 mM Pb(NO 3 ) 2 . Seedlings were dried at 80°C for 3 days and lead content was determined by ICP-AES as described [START_REF] Arazi | A tobacco plasma membrane calmodulin-binding transporter confers Ni 2+ tolerance and Pb 2+ hypersensitivity in transgenic plants[END_REF]. Data are the mean TSD of three independent experiments. acid sequence is compared to the amino-acid sequences of the proteins encoded by the Arabidopsis CNGC genes, CNGC1 emerges as most similar to NtCBP4. We reasoned that if AtCNGC1 is the orthologue of NtCBP4, then disruption of AtCNGC1 might confer Pb 2+ tolerance in Arabidopsis. To assess this possibility we chose to isolate and analyse a T-DNA insertion mutant of the Arabidopsis CNGC1.

Isolation of a CNGC1 knockout mutant and its response to Pb 2+

The full Arabidopsis CNGC1 coding sequence has recently been released and shown to be localized on chromosome 5 (BAC clone number MFH8, accession AB025622). The Versailles T-DNA library was screened by PCR for a knockout mutant using CNGC1-speci®c oligonucleotides, and T-DNA oligonucleotides localized in the left and right borders. One knockout line was isolated and shown to be disrupted in CNGC1 by sequencing the T-DNA ¯anking regions (Figure 5a). The T-DNA is inserted in front of the putative cyclic nucleotide-binding domain. The corresponding homozygous line was isolated and characterized by PCR (Figure 5b). Segregation analysis of the T-DNA insertion line revealed one insertion locus. DNA was extracted from homozygous plants, digested by several enzymes, and blotted on nylon membrane. Hybridization with a T-DNA probe revealed the presence of only one T-DNA insertion (Figure 5c). Moreover, DNA from homozygous plants was subjected to a PCR walking method [START_REF] Devic | Ef®cient PCR walking on plant genomic DNA[END_REF] Subsequently, we analysed the response of seedlings of the homozygous cncg1 mutant and wild type to different concentrations of Pb 2+ . Increasing Pb(NO 3 ) 2 concentrations adversely affected seedling development of both wild type and cngc1. However, in the presence of high concentrations of Pb 2+ (0.2 and 0.25 mM), the wild-type seedlings were chlorotic and their development was severely retarded compared with that of cngc1 seedlings (Figure 6a). Similar results were obtained with two batches of wild-type seeds and three batches of mutant seeds from plants grown at different locations.

Quantitative analysis of Pb 2+ tolerance of the cngc1 mutant is shown in Figure 6(b). This analysis revealed a statistically signi®cant higher tolerance of the mutant line compared to the wild type. Importantly, analysis of the response of the Arabidopsis wild-type and mutant lines to NiCl 2 revealed no apparent difference, suggesting that the cngc1 knockout speci®cally affects the response to Pb 2+ . However, this does not exclude possible speci®c effects on other ions, which have not been tested in this study. In addition we tested seedlings germinated and grown on agar plates. Seedlings were grown in the absence of Pb 2+ for 7 days and then transferred to agar plates containing Pb 2+ . This growth regime also revealed a phenotype of Pb 2+ tolerance of the cngc1 mutant in comparison to the wild type. Seedlings of the mutant showed less growth inhibition in the shoot, and retained more chlorophyll than the wild type at high Pb 2+ concentrations (1 and 5 mM). At these concentrations, the roots of both wild-type and cngc1 mutant ceased growing (data not shown). After 5 days at 5 mM Pb 2+ , seedlings of the cncg1 mutant retained twice as much chlorophyll as wild-type seedlings (42.8 T 5.0 and 20.6 T 1.6%, respectively), relative to the non-stressed control seedlings.

Furthermore, we tested the Pb content in Arabidopsis seedlings exposed to Pb 2+ for 2 weeks. At low concentrations of Pb 2+ (0.05 mM) there was no signi®cant difference between the cngc1 mutant and the wild type. At higher concentrations differences became apparent and signi®cant (Figure 6c). At 0.15 mM Pb 2+ , the mean content of Pb in the wild type was 30% higher than in the mutant (P < 0.05). Thus, with respect to both Pb 2+ tolerance and accumulation, the phenotype of the Arabidopsis cngc1 mutants is similar to that of transgenic tobacco plants expressing the truncated NtCBP4 protein.

Discussion

An emerging family of plant plasma membrane channel proteins resembling mammalian cyclic nucleotide-gated non-selective cation channels has been recently described in several plant species [START_REF] Arazi | A tobacco plasma membrane calmodulin-binding transporter confers Ni 2+ tolerance and Pb 2+ hypersensitivity in transgenic plants[END_REF][START_REF] Ko È Hler | Characterization of a novel gene family of putative cyclic nucleotide-and calmodulin-regulated ion channels in Arabidopsis thaliana[END_REF][START_REF] Leng | Cloning and ®rst functional characterization of a plant cyclic nucleotide-gated cation channel[END_REF][START_REF] Schuurink | Characterization of a calmodulin-binding transporter from the plasma membrane of barley aleurone[END_REF]. We chose to use transgenic plants to study the function of the tobacco NtCBP4 protein, and T-DNA insertion mutagenesis to investigate the Arabidopsis homologue CNGC1. Our previous studies revealed that overexpression of NtCBP4 in tobacco plants conferred Pb 2+ hypersensitivity and enhanced accumulation of Pb 2+ , suggesting the possible involvement of NtCBP4 in Pb 2+ uptake across the plasma membrane [START_REF] Arazi | A tobacco plasma membrane calmodulin-binding transporter confers Ni 2+ tolerance and Pb 2+ hypersensitivity in transgenic plants[END_REF]. Here we show that by expressing a modi®ed version of NtCBP4 lacking presumed regulatory domains, the phenotype with respect to Pb 2+ tolerance and accumulation is the opposite of that exhibited by plants overexpressing the full-length protein.

The mammalian cyclic nucleotide-gated channels function as tetrameric complexes [START_REF] Liu | Subunit stochiometry of cyclic nucleotide-gated channels and effects of subunit order on channel function[END_REF]. Therefore the inhibitory effect of NtCBP4DC on Pb 2+ accumulation, and the concomitant improved tolerance to Pb 2+ , may have resulted from the formation of non-functional NtCBP4/NtCBP4DC heteromeric complexes, as the endogenous native NtCBP4 gene was expressed in the background of the NtCBP4DC transgene (Figure 3c). However, as indicated, due to the fact that the anti-NtCBP4 antibodies were raised against the C-terminal half of NtCBP4 [START_REF] Arazi | A tobacco plasma membrane calmodulin-binding transporter confers Ni 2+ tolerance and Pb 2+ hypersensitivity in transgenic plants[END_REF][START_REF] Arazi | A high-af®nity calmodulin-binding site in a tobacco plasma membrane channel protein coincides with a characteristic element of cyclic nucleotide-binding domains[END_REF], the truncated NtCBP4 protein could not be detected with these antibodies. Further studies are required to assess the possible occurrence of full-length/truncated NtCBP4 heteromeric complexes in the NtCBP4DC transgenic plants. Nevertheless, we predicted that if the truncated protein causes the inactivation of endogenous NtCBP4-associated channels, then disruption of the Arabidopsis CNGC1 gene might cause a similar phenotype. Indeed, we isolated an Arabidopsis T-DNA insertion mutant of the CNGC1 gene. This mutant had characteristics similar to the tobacco plants expressing the truncated NtCBP4 with respect to the response to Pb 2+ . The phenotype of the cngc1 mutant was apparent when grown on different nutrient solutions, and irrespective of growth on liquid or solid substrate. Moreover, the phenotype was apparent when seeds were germinated in the presence of Pb 2+ , or when seedlings were exposed to Pb 2+ only 1 week after germination. These results suggest the involvement of CNGC1 in Pb 2+ transport across the plant plasma membrane, consistent with ®ndings regarding NtCBP4 in tobacco.

It is important to note that the analysis described here was con®ned to the early stages of seedling development in tobacco and Arabidopsis. The response of older plants to Pb 2+ and other metals requires further detailed analysis. In view of the fact that in Arabidopsis there are at least 19 CNGC-related genes (N. Bouche  , unpublished results), the possible formation and complexity of CNGC heteromeric channels cannot be ignored. The composition of such heteromeric complexes may vary between cell types, during development, and in response to external physical and chemical stimuli. Heteromeric complexes might account for variations in regulation as well as in ion selectivity. In addition, CNGC proteins might differentially interact with distinct calmodulin isoforms [START_REF] Ko È Hler | Characterisation of calmodulin binding to cyclic nucleotide-gated ion channels from Arabidopsis thaliana[END_REF], which could further affect their regulatory properties. Elucidating these issues requires a massive effort and a multidisciplinary research approach.

Because Pb 2+ is a non-essential toxic metal to plants, the presumed NtCBP4-and CNGC1-associated ion transport systems are likely to have other, as yet unknown, physiological roles. On the basis of their primary structure, plant CNGCs are predicted to function as components of nonselective cation channels. The involvement of CNGC1 and CNGC2 in transport of potassium has already been reported [START_REF] Ko È Hler | Characterization of a novel gene family of putative cyclic nucleotide-and calmodulin-regulated ion channels in Arabidopsis thaliana[END_REF]Leng et al., 1999, respectively). Of particular interest to us is the possible involvement of NtCBP4, CNGC1 and other related plant proteins in Ca 2+ signal transduction, either because of their expected regulation by Ca 2+ -calmodulin, and/or by being permeable to Ca 2+ , as in the case of mammalian cyclic nucleotidegated non-selective cation channels. A role in Ca 2+ transport would be consistent with earlier reports identifying Ca 2+ -permeable channels as a pathway for Pb 2+ entry into animal cells [START_REF] Simons | Lead enters bovine adrenal medullary cells through calcium channels[END_REF][START_REF] Tomsig | Permeation of Pb 2+ through calcium channels: fura-2 measurements of voltage-and dihydropyridine-sensitive Pb 2+ entry in isolated bovine chromaf®n cells[END_REF] and plants [START_REF] Huang | Lead phytoextraction: species variation in lead uptake and translocation[END_REF], and with the recent demonstration of cyclic nucleotide-dependent Ca 2+ entry into human embryonic kidney cells transfected with the Arabidopsis AtCNGC2 gene [START_REF] Leng | Cloning and ®rst functional characterization of a plant cyclic nucleotide-gated cation channel[END_REF]. We tested metal content in whole leaf extracts of wild-type and NtCBP4DC and NtCBP4FL transgenic tobacco grown in half-strength Hoagland's medium for 4 weeks (without heavy metal stress). There were no signi®cant differences between the wild type and the transgenic lines in their content of a range of metals (Ca, K, Mg, Mn, Zn and Fe). This may indicate that these CNGC channels have subtle effects on metal transport, which could be involved in signalling mechanisms.

Previously we reported that transgenic tobacco overexpressing the full-length NtCBP4 was tolerant to Ni 2+ and hypersensitive to Pb 2+ , associated with reduced accumulation of Ni 2+ and enhanced accumulation of Pb 2+ , respectively [START_REF] Arazi | A tobacco plasma membrane calmodulin-binding transporter confers Ni 2+ tolerance and Pb 2+ hypersensitivity in transgenic plants[END_REF]. We hypothesized that the hypersensitivity to Pb 2+ is the result of enhanced NtCBP4 channel activity. The opposite phenotype, resulting from the expression of a truncated NtCBP4 in tobacco and from disruption of CNGC1 in Arabidopsis, is consistent with this hypothesis. On the other hand, it would be dif®cult to associate the Ni 2+ -tolerance phenotype with channel activity. First, the Arabidopsis cngc1 mutant is not different from the wild type in response to NiCl 2 (R. Sunkar and H. Fromm, unpublished results). Secondly, we found that transgenic tobacco expressing the truncated NtCBP4 protein retains a certain degree of Ni 2+ tolerance (R. Sunkar and H. Fromm, unpublished results). Therefore it is likely that the Ni 2+ tolerance phenotype observed in transgenic tobacco [START_REF] Arazi | A tobacco plasma membrane calmodulin-binding transporter confers Ni 2+ tolerance and Pb 2+ hypersensitivity in transgenic plants[END_REF] is due to a property of the NtCBP4 protein, which is also present in the truncated protein, but is not directly associated with NtCBP4 channel activity.

In summary, we re®ned the analysis of the plasma membrane NtCBP4 tobacco protein in relation to Pb 2+ tolerance. In addition, we found similar characteristics associated with the Arabidopsis CNGC1 gene. The transgenic tobacco plants and the Arabidopsis mutant described here provide unique research tools for addressing questions related to their physiological roles, as well as for elucidating mechanisms of heavy metal uptake and tolerance in plants.

Experimental procedures

Preparation of transgenic plants

Preparation of the DNA construct and of the transgenic tobacco plants for expression of the full-length NtCBP4 protein was recently described by [START_REF] Arazi | A tobacco plasma membrane calmodulin-binding transporter confers Ni 2+ tolerance and Pb 2+ hypersensitivity in transgenic plants[END_REF]. The DNA construct for the expression of the C-terminal truncated NtCBP4 (NtCBP4DC) was prepared by cloning a PCR-ampli®ed DNA fragment using the gene-speci®c oligonucleotides 5¢-CCGCTGAGCTATGAATCACCG-CCAAGACGAG-3¢ (sense) and 5¢-GAAGGAATTCTTAAGAGGCTA-CAAGCTTTAAATCAT-3¢ (antisense), spanning the coding region Met 1 ±Ser 593 [START_REF] Arazi | A tobacco plasma membrane calmodulin-binding transporter confers Ni 2+ tolerance and Pb 2+ hypersensitivity in transgenic plants[END_REF], and containing XhoI and EcoRI restriction sites on the 5¢ and 3¢ termini, respectively. The XhoI± EcoRI-digested DNA fragment was ligated into the corresponding sites of the binary vector described by [START_REF] Arazi | A tobacco plasma membrane calmodulin-binding transporter confers Ni 2+ tolerance and Pb 2+ hypersensitivity in transgenic plants[END_REF]. Transfer of the transgene to progeny was veri®ed by germination on kanamycin, then by Northern hybridizations to con®rm the expression of the NtCNBP4DC mRNA.

Analysis of transgene expression by Northern hybridizations, RT±PCR and immunoblotting

Total RNA was isolated from 3-week-old seedlings as described [START_REF] Logemann | Improved method for the isolation of RNA from plant tissues[END_REF]. RNA fractionation, blotting and hybridization conditions were as described [START_REF] Chen | The 58-kilodalton calmodulin-binding glutamate decarboxylase is a ubiquitous protein in petunia organs and its expression is developmentally regulated[END_REF]. A HindIII fragment from the cDNA of NtCBP4 (nucleotides 1113±2024, GenBank accession number AF079872) was labelled with [ 32 P]dCTP by random priming, and used as a probe. The same membrane was hybridized with a [ 32 P]dCTP-labelled actin probe prepared by amplifying an actin gene fragment using the tomato actin gene (GenBank accession number U60480) speci®c primers 5¢-TTGTGTTGGACTCTGGTGATGG-3¢ (sense) and 5¢-AGCCAA-GATAGAGCCTCCAATC-3¢ (antisense). Total RNA was passed through an oligo-dT column to enrich for polyA mRNA, and reverse transcriptase was used to prepare the corresponding cDNA templates. PCR ampli®cation of a 404 bp NtCBP4 fulllength-speci®c sequence was performed with the primers 5¢-GACGACTTCACAGTAAGCAGC-3¢ and 5¢-CACGACTAAAAATGC-ACTCAATC-3¢ (sense and antisense, respectively), which amplify a region spanning nucleotides 2044±2447 of the NtCBP4 cDNA (GenBank accession number AF079872). PCR ampli®cation of the cDNA encoding the b-subunit of Nicotiana plumbaginifolia mitochondrial H + -ATPase (GenBank accession number X02868) served as a control, with the primers 5¢-CTTACAGGTTTG-ACCGTGGCTGAGC-3¢ (sense), and 5¢-TAGTGATCCTCTCCCA-AAATGTGAGG-3¢ (antisense). Protein extraction, membrane fractionation and immunoblotting were performed as described [START_REF] Arazi | A tobacco plasma membrane calmodulin-binding transporter confers Ni 2+ tolerance and Pb 2+ hypersensitivity in transgenic plants[END_REF][START_REF] Arazi | A high-af®nity calmodulin-binding site in a tobacco plasma membrane channel protein coincides with a characteristic element of cyclic nucleotide-binding domains[END_REF].

Plant culture, metal toxicity and metal accumulation assays

Seeds of tobacco and Arabidopsis were sterilized by washing in 70% ethanol, then with chlorine, and subsequently rinsed with distilled water and, unless otherwise indicated, placed in Petri dishes containing modi®ed Blaydes solution [START_REF] Parrot | Aluminium tolerance in alfalfa as expressed in tissue culture[END_REF] pH 4.5, with or without Pb(NO 3 ) 2 . The seedlings were grown in a controlled growth room for 10 days (Arabidopsis) or 12± 13 days (tobacco), with a day/night cycle of 16/8 h at 25°C. Seedlings were collected and their fresh weight measured, after which they were dried at 80°C for 3 days. Lead content was determined by ICP-AES as described [START_REF] Arazi | A tobacco plasma membrane calmodulin-binding transporter confers Ni 2+ tolerance and Pb 2+ hypersensitivity in transgenic plants[END_REF]. Statistical analysis of the data was performed using ANOVA and the sum of squares simultaneous test procedure. For plants grown on agar plates, seeds were sterilized as described above and germinated on plates containing 25% (v/v) Murashige and Skoog (MS) medium [START_REF] Murashige | A revised medium for rapid growth and bio assays with tobacco tissue culture[END_REF], 1% (w/v) sucrose and 0.9% (w/v) agar (Sigma A-1296). Plants were grown for 7 days on vertically positioned plates with 16/8 h light/dark cycle with temperature of 25/20°C, respectively, and light intensity of 40 mmol m ±2 sec ±1 . Subsequently seedlings were transferred to plates containing 25% (v/v) MS without CaCl 2 , with phosphate reduced to 0.01 mM and with either 0, 1 or 5 mM Pb(NO 3 ) 2 . Shoots Modulation of lead tolerance in plants 541

ã Blackwell Science Ltd, The Plant Journal, (2000), 24, 533±542 were harvested after 5 days. Chlorophyll was extracted with methanol and spectrophotometrically quanti®ed. Total chlorophyll content (mg chlorophyll ml ±1 plant extract solution) was calculated as 22.5 Q A 650 + 4.0 Q A 665 .

PCR screening strategy and knockout isolation

We used the T-DNA inserted collection developed by G. Pelletier and co-workers (INRA, Versailles, France) made by in®ltrating Arabidopsis thaliana ecotype Wassilewskija [START_REF] Bechtold | In planta Agrobacterium mediated gene transfer by in®ltration of adult Arabidopsis thaliana plants[END_REF] with Agrobacterium tumefaciens strain C58 carrying the plasmid pGKB5 [START_REF] Bouchez | A binary vector based on Basta resistance for in planta transformation of Arabidopsis thaliana[END_REF]. This library is composed of 43 584 individual lines, representing about 65 000 independent insertions (T1.5 insertion loci per line). The DNA corresponding to T 2 or T 3 lines was extracted in pools of 48 lines (908 DNA pools). To isolate the cngc1 knockout, DNA pools were screened by PCR using gene-speci®c oligonucleotides (CNGCf: 5¢-GAAGAGACG-CAGAACAATGG-3¢; CNGCr: 5¢-CTGGCTCTGCTGGTTTCTGAAG-TAGCATAG-3¢) and oligonucleotides anchored in the left and right borders of the T-DNA (RB oligonucleotide: 5¢-CTGATACCAGACGTTGCCCGCATAA-3¢; LB oligonucleotide: 5¢-CTACAAATTGCCTTTTCTTATCGAC-3¢). PCR reactions were run on an agarose gel, blotted onto nylon membranes which were hybridized with a T-DNA probe (RB plus LB) and a gene-speci®c probe (Arabidopsis EST 154M3T7 from Ohio Stock Center, corresponding to a partial CNGC1 cDNA clone). Positive PCR fragments hybridizing with both probes were isolated from agarose gel and sequenced. The 48 individual lines from the positive pool were then subjected to a new PCR and the corresponding knockout line was isolated.

Figure 1 .

 1 Figure 1. Schematic presentation of DNA constructs for expression of the full-length and C-terminal truncated NtCBP4 in transgenic tobacco. (a) Description of the full-length NtCBP4 cDNA in a binary Ti plant transformation vector, as described by Arazi et al. (1999). Regions with the 35S CaMV promoter and the transcription termination sequence are shown in hatched boxes. The NtCBP4 six-transmembrane core, putative cyclic-nucleotide monophosphate-binding domain (cNBD, in grey; Arazi et al., 1999; Arazi et al., 2000) and the overlapping calmodulin-binding domain (CaMBD, cross-hatched; Arazi et al., 2000) are indicated. The amino-acid sequence of part of the cNBD is shown on top. The CaMBD (bold letters) and the aB and aC predicted helices (underlined) of the cNBD are according to Arazi et al. (2000). The downward arrow shows the site of the deletion used to create the C-terminal truncated protein designated NtCBP4DC (amino acids Met 1 ±Ser 593 ). Numbers denote the terminal amino-acid residues shown in the sequence, based on Arazi et al. (1999) and Arazi et al. (2000). (b) The NtCBP4DC cDNA in a binary Ti plant transformation vector. The NtCBP4DC cDNA, as described in (a), was prepared by cloning a corresponding PCR-ampli®ed DNA fragment into the XhoI and EcoRI sites of the same vector used for the full-length NtCBP4.

Figure 2 .

 2 Figure 2. Transgenic tobacco seedlings expressing NtCBP4DC are tolerant to Pb 2+ . (a) Seedlings of wild-type and transgenic tobacco expressing either the full-length NtCBP4 (NtCBP4FL; Arazi et al., 1999) or the NtCBP4DC mRNA (transgenic lines DC-22-11, DC-42-11 and DC-29) were germinated and grown in the presence of the indicated concentrations of Pb(NO 3 ) 2 in modi®ed Blaydes solution (pH 4.5) for 12 days and then photographed. (b) Photo enlargements of seedlings of wild-type and representative transgenic lines in the presence of 0 and 0.75 mM Pb(NO 3 ) 2 .

Figure 3 .

 3 Figure 3. Analysis of gene and protein expression in transgenic tobacco plants. (a) Total RNA samples (20 mg) from the indicated transgenic plants and wild type were separated by gel electrophoresis, blotted and hybridized with an NtCBP4-speci®c probe (upper panel). Arrows point to the fulllength NtCBP4 (FL) and the C-terminal truncated NtCBP4 (DC) mRNAs in the upper panel. The letters on top of the upper panel refer, for the transgenic lines indicated, to the hypersensitive (S), tolerant (T) and normal (N) phenotypes regarding response to Pb 2+ relative to the wild type. The gel positions of the 25S, 18S and 16S ribosomal RNA bands are indicated. The 25S ribosomal RNA band served as an internal standard (lower panel). (b) Expression analysis of the full-length NtCBP4 mRNA in wild-type, NtCBP4FL and NtCBP4DC (line DC-42-11) plants by RT±PCR. A 404 bp DNA fragment corresponding to a region of the full-length NtCBP4 mRNA was ampli®ed with the primers designated NtCBP4. Ampli®cation of the corresponding region from the NtCBP4 cDNA clone served as a positive control (cDNA control). The amounts of polyA+ RNA were normalized using the expression of the tobacco gene encoding the bsubunit of mitochondrial H + -ATPase as a standard. Ampli®ed DNA samples and DNA size markers (indicated in bp on the left) were fractionated by agarose gel electrophoresis, stained with ethidium bromide and photographed. (c) Immunodetection of the full-length NtCBP4 in microsomes from wildtype, NtCBP4FL and NtCBP4DC (line DC-42-11) plants. Microsomes (20 mg protein) and a sample of the full-length recombinant NtCBP4 expressed in insect cells (Rec;[START_REF] Arazi | A high-af®nity calmodulin-binding site in a tobacco plasma membrane channel protein coincides with a characteristic element of cyclic nucleotide-binding domains[END_REF] were separated by SDS±PAGE and immunoblotted on a nitrocellulose membrane with polyclonal anti-NtCBP4 antibodies[START_REF] Arazi | A tobacco plasma membrane calmodulin-binding transporter confers Ni 2+ tolerance and Pb 2+ hypersensitivity in transgenic plants[END_REF][START_REF] Arazi | A high-af®nity calmodulin-binding site in a tobacco plasma membrane channel protein coincides with a characteristic element of cyclic nucleotide-binding domains[END_REF]. The positions of molecular weight markers are indicated. Immunodetection of vacuolar membrane H + -ATPase[START_REF] Ward | Dissociation and reassembly of the vacuolar H + -ATPase complex from oat roots[END_REF] served as an internal standard.

Figure 4 .

 4 Figure 4. Pb 2+ tolerance and accumulation in transgenic tobacco seedlings. (a) Relative fresh weight of wild-type and transgenic tobacco seedlings expressing the full-length NtCBP4 mRNA (NtCBP4FL; Arazi et al., 1999), and two transgenic lines expressing the truncated NtCBP4 mRNA (DC-22-11 and DC-42-11) in the presence of different concentrations of Pb(NO 3 ) 2 . Data are given as the mean FW of 150 seedlings for each concentration TSD of three independent experiments. Fresh weight of seedlings of each line grown without Pb(NO 3 ) 2 was set as 100%.(b) Lead accumulation in 12-day-old seedlings grown in the presence of 0.2 mM Pb(NO 3 ) 2 . Seedlings were dried at 80°C for 3 days and lead content was determined by ICP-AES as described[START_REF] Arazi | A tobacco plasma membrane calmodulin-binding transporter confers Ni 2+ tolerance and Pb 2+ hypersensitivity in transgenic plants[END_REF]. Data are the mean TSD of three independent experiments.

  to clone any possible T-DNA border present in this line. Importantly, all cloned borders correspond to the CNGC1 insertion. No new T-DNA ¯anking regions were cloned from the knockout line, providing evidence for a single insertion site in the CNGC1 gene.

Figure 5 .

 5 Figure 5. Genomic analysis of the T-DNA insertion in the Arabidopsis CNGC1 knockout line. (a) CNGC1 gene map and T-DNA border sequences (T-DNA sequences are underlined). Exons are represented by boxes. S1 to S6, transmembrane hydrophobic domains; P, predicted pore region; cNBD, cyclic nucleotide-binding site; CC, coiled coil helix; CaM, calmodulinbinding domain; CNGCf, CNGC1 forward oligonucleotide; CNGCr, CNGC1 reverse oligonucleotide. (b) PCR analysis of cngc1 homozygous plants. Homozygous mutant and wild type (Arabidopsis thaliana ecotype Wassilewskija) DNA were subject to PCR ampli®cation using CNGC1 gene-speci®c oligonucleotides (CNGCf and CNGCr) and T-DNA left-and right-border oligonucleotides (LB and RB). (c) Southern blot analysis of homozygous plants. DNA (2 mg per lane) was digested by different restriction enzymes (the T-DNA contains one site for PstI, EcoRI and HindIII and three sites for EcoRV), and blotted on nylon membrane. Hybridization was performed with a full-length radiolabelled T-DNA probe.

Figure 6 .

 6 Figure 6. Phenotypic analysis of the Arabidopsis cngc1 mutant in response to Pb 2+ . (a) Photographs of seedlings of wild-type and the cngc1 mutant germinated and grown in the presence of the indicated concentrations of Pb(NO 3 ) 2 for 2 weeks. (b) Relative fresh weight of wild-type and cngc1 mutant in the presence of different concentrations of Pb(NO 3 ) 2 . Data are given as the mean FW of 150 seedlings for each concentration TSD of three independent experiments. Fresh weight of seedlings of each line grown without Pb(NO 3 ) 2 was set as 100%. (c) Lead accumulation in 10-day-old seedlings grown in the presence of 0.05 and 0.15 mM Pb(NO 3 ) 2 . Lead content was determined by ICP-AES as described in Experimental procedures. Data are the mean TSD of three independent experiments.

ã Blackwell Science Ltd, The Plant Journal, (2000), 24, 533±542

Acknowledgements

This research was supported by a grant from the Israel Science Foundation to H.F., and by the French Embassy in Israel and the Acade  mie d'Agriculture de France; R.S. and T.A. were recipients of fellowships from the Sir Charles Clore Foundation; and I.N.T. was the recipient of an AstraZeneca-funded studentship. We thank H. Sze for antibodies against vacuolar ATPase.


Gérard Pogorel

Nine Regimes of Radio Spectrum Management: A 4-Step Decision Guide

Diverse radio spectrum management regimes are defined according to 4 levels of questions:

-Should frequencies be allocated according to a harmonised plan? -Should the technologies allowed be standardised? -Should spectrum usage rights be exclusive, eased, or collective? -Should usage rights be assigned through market mechanisms, administrative procedures, or hybrid procedures? This paper establishes a balanced set of decision criteria for each question. It describes nine regimes resulting from the combined answers. The taxonomy illustrates the possible rationales for a diversity of regimes broader than the usually exposed standard trilogy of Command and Control, Market and Commons: This includes Harmonised flexibility, Administered flexibility, Technology flexibility in Command and Control context, Harmonised flexibility Plus, Private Commons and California Dream. The nine regimes can also be considered as a map with which to navigate in order to accommodate institutional and technological transitions. This allows decision-makers to comeup with informed choices using all the technical information available, and based on definite criteria and a rigorous methodology.

Wireless services will undergo a major expansion in the next decade. The generally accepted view is that this will provoke an increased need for radio spectrum. Major technological changes are under way as well, which might help in improving its efficient use, but also warrant savvier management methods.

1

The debate on adequate, future-oriented, spectrum management is currently reaching a critical point. A trend towards flexibility in the form of market mechanisms (auctions of spectrum property rights and trading) was initiated in New Zealand, then the U.S. in 1993, and expanded in a number of European and Asian countries as of 2000. It was consistently formalised in 2002 by the FCC Spectrum Task Force Report in the U.S.

2 and the Martin Cave Report in the UK 3 . The two reports 4 translated into a comprehensive, market-oriented spectrum management framework that has served as an underlying reference to subsequent policy initiatives in Europe since this time.

It seems, however, that this trend is encountering delays where it is already implemented or favourably considered, and facing fierce opposition in territories it has not conquered. It might consequently be useful to sort out the alternatives, list the arguments exchanged, look into the prospects offered by ongoing technological developments in wireless, and pave the way for possible transitions paths.

-Explore the whole range of choices available to regulators and industry in establishing a radio spectrum management policy by expanding beyond the standard trilogy of Command and Control, Market and Commons. -Organise and clarify the expanded set of alternatives to be considered.

-List the criteria whereby the necessary choices and decisions can be made.

Definitions: Four dimensions of spectrum management

A spectrum management regime comprises four dimensions that have to be successively analysed. 5 The concepts and alternative approaches must be explored at the following levels:

• Allocation: service harmonisation or service flexibility • Technology: standardisation or technology flexibility • Usage rights definition: alternative regimes • Assignment modes of spectrum usage rights We will successively examine the above issues and alternatives.

Allocation of frequencies: Service harmonisation or service flexibility?

Harmonisation is intended as allocating a frequency band or set of frequency bands for a service application or category of services. It consists in defining measures at international (ITU), regional (Europe, America, Asia), and national levels and poses limitations on service flexibility.

Harmonisation does not have to be implemented all over the spectrum. There can be harmonised bands where justified, and non-harmonised bands elsewhere in the spectrum. This translates into an opposition between no harmonisation all across the spectrum (or commercial spectrum) and harmonisation for distinct wireless services sets ("clusters").

Technology: Standardisation or technology flexibility

Standardisation is intended as designating a technology or set of technologies to provide a category of service. It aims to ensure that the equipment used meets the technical requirements specified in technical product standards, or specifications, in order to provide market advantage in terms of better coexistence or interoperability, cross-border roaming, economies of scale, etc. Standards can be determined by public bodies such as ETSI, or the market (industry-led), and then mandated by regulation or not. It is assumed that standardisation can only reasonably occur in a harmonised context.

For reasons of clarity, it is important to draw this distinction between harmonisation and standardisation. Harmonisation of frequency bands, is an option at frequency allocation level, standardisation takes place at technology level. There can be harmonisation with, or without, standardisation. There are obvious relationships between the two, as technical standards have to cope with the physical qualities of frequencies. Yet harmonisation and standardisation do not necessarily go hand in hand. Various technical standards, or even nonstandardised technologies, can possibly be used in harmonised bands. Although this distinction is needed, the confusion can be found in some studies on spectrum policy6 .

Usage rights definition

In the spectrum context, the following categories of usage rights can be defined:

Property rights have been widely heralded as a major factor of economic and social dynamic evolution. There can be:

-Exclusive property rights (without easements) -Property rights with easements: they make provisions for sharing, overlay, underlay, and Dynamic Frequency Selection (DFS). DFS can be intended in a restrictive sense as the possibility of shifting between a set of predetermined harmonised bands, or, more extensively, as the possibility of shifting across large areas of the spectrum. It is compatible with all nonexclusive property based regimes. The SPORT VIEWS project has examined the potential interest of introducing reasoned easements. This perspective of disaggregating property rights to benefit from technology evolution has been extensively explored by Martin Cave [START_REF] Cave | New spectrum-using technologies and the future of spectrum management: a European policy perspective[END_REF] .

Collective use, the third type of usage rights, became popular as "commons" in the late nineties with the advent of WiFi and was promoted as a far-reaching, future-oriented model. The possible extent of collective use, and conversely, of property rights, was extensively explored in a recent EU Study. 8

Assignment modes of spectrum usage rights

There are two main categories of usage right assignment modes:

• Comparative administrative procedures, which include: o Pure administrative procedures. o Hybrid modes, such as administrative procedures with a bidding price as part of a weighted multi-criterion formula. They put together the contribution to local and regional broadband development and a financial element. 9 There is a price component, but the licence remains under administrative control. o Administered incentive pricing, which also remains within the category of administrative control.

• Auctions resulting in exclusive property rights, which represent the quintessential market solution for the assignment of spectrum usage rights. Trading is a complement to this approach for secondary markets.

Assignment modes have been the subject of numerous studies. The standard trilogy is present in this table. We recognise regime 1a as the traditional Command and Control model. Regime 7b qualifies as a full propertybased market regime: one single market process combining flexible frequency allocation and technical choice. Regime 6 is the "commons" model. What this table illustrates, however, is a diversity of regimes broader that usually exposed. Before looking into the grounds on which each of these regimes could be pragmatically justified, let us provide some brief descriptions. Regime 1b, for instance, combines a hybrid of command and control at a frequency allocation and technology level, and auctions for property rights assignment. Harmonisation (at allocation level) is thus compatible with some degree of market mechanisms, at assignment level, as seen in the UMTS case in Europe. This represents some kind of limited flexibility: sticking to harmonised allocation at an upper level, with market at assignment level. It could be said to fall into a category of "harmonised flexibility."

Hybridisation can also occur between comparative administrative procedures and auctions, as in regime 4a Technology flexibility in Command and Control context exemplified by the 2006 "Wimax" authorisation procedure in France. It combined qualitative elements submitted to administrative evaluation, like the contribution to regional development, with financial bids. It can also be presented as auctions with room for negotiations and mediation by the regulator. As the regulator retains the upper hand in the mix of criteria, we consider it to fall, as a variant, into the administrative procedures category, rather than the auctions category.

Regime 5b Harmonised flexibility Plus seems like an interesting combination of harmonised frequencies, technical flexibility, and easements on property rights acquired through auctions, thus accommodating some of operators' preferences, as well as possibilities of sharing and dynamic frequency selection. Not all regimes are representative of realistic alternatives, but homage should be paid to regime 9 California Dream, which embodies the vision of a vast radio spectrum commons, supposing that the technologies exist to support it.

We have shown that flexibility and efficiency in spectrum management has to be considered at various levels and can be combined in a variety of ways. Let us now expose the iteration of pragmatic considerations at four successive levels of analysis leading to the choice of a spectrum regime.

Deciding on spectrum management regimes: The 4-step decision process

Efficiency is often quoted in relation to flexibility. We can refer to the ERO 80 Report definition of flexibility11 as:

"Increasing the ability of the spectrum regulatory framework to facilitate and adapt, in a timely manner, to user requirements and technological innovation by reducing constraints on the use of spectrum and barriers to access spectrum."

We can see that flexibility is easily accommodated within the proposed analytical taxonomy, as it takes place at all four decision levels: frequency allocation, technology, usage rights, and assignment procedure. We now have the possibility to look at spectrum efficiency against this comprehensive and articulated framework to gain a better understanding of what is at stake in the present debate.

Let us look at the criteria decision-makers can resort to when going through the four steps constitutive of spectrum policy choices.

On the two issues of harmonisation and standardisation, it is to be noted the existing literature provides neither positive nor negative compelling evidence on their overall necessity and superiority to non-harmonisation and nonstandardisation. Moreover, the existing literature does not provide either a locally applicable toolbox of criteria to make easily the proper choices. The evidence itself is not yet conclusive. For instance, in the very popular area of mobile communications services, the jury is still out on the outcome of the confrontation between the GSM-UMTS standardisation line of action in Europe and the agnostic approach adopted by the U.S. and Korea.

Industry associations, however, at least in Europe, have taken a contrarian view on service and technology flexibility in mobile services, and emphasised the benefits of harmonisation and standardisation in two recent studies (GSMA and UMTS Forum, 2006).

From this perspective, the critical questions to be answered when making choices of harmonisation versus non-harmonisation, and of standardisation versus non-standardisation, consist in the practical, future-oriented evaluation of two categories of factors. In short, the general trade-offs in matters of spectrum harmonisation and wireless technology standardisation are as follows.

Step 1: Harmonisation?

Harmonisation is meant to minimise interferences, reduce cross-border coordination requirements and ensure roaming facilities. The benefits for consumers result from the lower network planning expenses and lower prices of devices. The costs of harmonisation are the inefficiency costs incurred from local or overall suboptimal usage of the spectrum resource, administrative costs and slower innovation.

It then can be said:

Lemma 1: Harmonisation costs and benefits If the cost differential induced at network gear and device levels by lack of harmonisation is low, economies of scale apply to the whole industry, even in a non-harmonised frequency bands regime. If the differential is high, harmonisation must prevail.

In other words, the question to be answered by decision-makers is:

-How much does the lack of, or only partial, harmonisation (like in the case of GSM bands in Europe and the US) impact the cost of network equipment, terminals and services?

Step 2: Standardisation?

It is generally accepted that there is a direct negative relation between production scale and the costs of manufactured products. Standardisation leading to an increase in scale is intended to lower the costs. The argument against (costs of-) standardisation (government or even industry-led) is that it creates a lock-in, which slows or even precludes the introduction of innovative, unexpected and un-expectable technologies. The risk is then that the industry is stuck with inferior technology. At the highest conceptual level, dynamic efficiency must prevail upon static considerations: innovation being by essence largely unpredictable, government-led standardisation would have to be avoided and industry-led standardisation to be carefully monitored to avoid the establishment of barriers to entry. Eventually, the trade-off is between lower costs made possible by economies of scale on the plus side and potential barriers to entry for innovative new technologies on the minus side.

Lemma 2: Rate of technical progress and standardisation If it can be safely assumed that technical progress for network equipments and terminals in a significant period under consideration (10-15 years) can be anticipated, or accommodated within designated standards or standard categories, the benefits of standardisation apply. If too much uncertainty regarding future technologies exists, or if a careful examination of developments in the labs leads us to assume there is a risk of major disruptive changes, avoiding standardisation is the safe bet. Game theory or probabilities can be of use in the decision-making process.

-The question about standardisation is: how much is lost in terms of extra costs for consumers if terminals have to combine two or more standards? Is our grasp of the technological paradigm in wireless for the next 10-15 years strong enough to aim at the benefits of standardisation, or is there a risk we might miss valuable opportunities, like those offered for instance by sharing, collective use and DFS, and in what timeframe?

Step 3: What type of usage rights?

Exclusive property rights

Property rights, in the area of spectrum, are favourably described as fostering efficient use, allowing more players to access the resource, and, when combined with trading, introduce an element of smooth and efficient flexibility in accordance with economic optimality criteria. Many advocate that they must be exclusive (without easements), to confer the licensees the benefit of a "clean spectrum", free of interferences.

The costs and potential risks include the creation of entry barriers in access to non-replicable resources, fragmentation, hoarding, pre-emption, market dominance, foreclosure of new entrants, in a context of vertical and horizontal integration, thus creating a potentially harmful situation with no remedies.

Looking at exclusive property rights assigned through market mechanisms, whatever the harmonisation and standardisation context, many question their ability to foster competition and efficiency in spectrum usage on the grounds that strategic use and significant market power lurk around the corner. They dispute the view held by the FCC and OFCOM that, should Significant Market Power situations arise, they could be dealt with through standard generic competition monitoring rules and procedures. They argue exclusive property rights on limited resources intrinsically build up to barriers to entry and have a negative impact on flexibility.

An interpretation of the obligations formally or informally imposed on operators by regulators to subcontract part of their capacity to MVNOs is precisely that they have had to mitigate the exclusive character of the licenses by imposing or inducing some kind of sharing.

Property rights with easements

The introduction of easements would be justified by recent advances in low power and dynamic frequency selection (DFS), also called dynamic spectrum access networks (DySpaN), They have led some to think that spectrum efficiency would be increased by easements permitting sharing, overlay, underlay, without harmful interference.

Collective use

Collective use refers to access to license-exempt bands, an "etiquette" limiting, but not preventing interferences.

-The benefits of collective use include:

-Low entry barriers -Quickly addressed niche applications -Certainty of obtaining access -Lower demand for licensed spectrum -Innovation (anti-monopoly) -Public infrastructure -Freedom of speech/cultural diversity -Light licensing -Possibility of Private commons or Experimental commons -The collective use costs are technical restrictions and higher risks of interference.

The EU study on collective use presents an up-to-date assessment of the potential extent of this category of usage rights.

12

Deciding on spectrum usage rights

In deciding on the nature of spectrum usage rights, two levels of analysis have to be considered. The assumptions made here are that:

-Sticking to exclusive property rights if flexible technologies are feasible leads to an inferior outcome in terms of consumer welfare.

-Flexible technologies are better accommodated by a regime-mix strategy that would include easements and collective use.

Decision theory illustrates how choices to be made under those conditions depend on the applied methodologies and the values of variables.

-If scenarios can be assigned probabilities, the outcome will depend on them.

No objective probability being available, the exercise consisting in subjectively assigning probabilities to the scenarios, based on our "best knowledge" can significantly enlighten the decision process. Basically, if the "Advent of flexible technologies scenario" is considered the most probable, the "Policy-Mix" strategy wins. On the contrary, if this advent is considered improbable, "Exclusive Property Rights Strategy" wins, unless the relative value of the "High consumer welfare" is very high.

-Alternatively, a very basic game-theoretic approach provides the following outcomes:

Posture

2.4.

Step 4: What kind of spectrum assignment mode should be adopted?

Administrative assignment procedures/hybrid procedures

Regulators have made significant efforts over the last decade to keep up with market changes and innovations. Some contend, however, that administrative methods might have reached a limit and that the situation and inconsistencies in spectrum management regimes actually hinder the deployment of new technologies. Advocates of administered flexibility, intended as flexibility achieved within an administered setting, argue that the complexity of spectrum usage, namely the prevention of interferences, demands strong control mechanisms. They add that even if it represents some costs, those are smaller than those induced by the multiplication of conflicts and litigations that would occur in their absence. There should be a trade-off between the costs induced by sub-optimal administrative procedures and the technical monitoring, adjustment costs and litigation costs they help avoid.

Hybrid procedures (administrative with bidding as a criterion among others) have the positive effect of combining:

-Welfare considerations -Domestic and international security concerns -Preserving national or regional champions to a certain degree -Capturing rents (possibly by maintaining them?).

Some governments, not all, are willing to implement this set of criteria and design their procedures accordingly. Others favour a more radical market approach across the board.

Auctions/trading

Whether market mechanisms in the form of auctions and trading can bring competition and efficiency in spectrum usage is hotly debated. Some agencies are optimistic that competition will take place. The pro-market line of reasoning taken by the FCC Task Force 2002 report and the Radio Agency Martin Cave 2002 reports refer to the respectable consideration that the introduction of market mechanisms optimises the usage of spectrum, as of any other resource. It would basically seem that the same holds for the policy orientations upheld by the European Commission: what they propose, not without merit, is an extension to spectrum of the general internal market principles, which are the backbone of the EU economic propositions. As for trading, it is usually thought of as a way either to correct initial flaws in allocation or assignment, or to allow for changes over time.

On the minus side, the existence of a budgetary bias is a case in point: in situations of doubt as to the most efficient spectrum management regime, budgetary considerations will have assignment methods providing the maximum income for the government budget prevail. Governments have often clashed with regulators as the consideration of cash-strapped budgets has overwhelmed any other, including consumer welfare.

Navigating the nine spectrum management regimes: migrations and transitions

It is too early to provide a fact-based assessment of each of the nine spectrum regimes presented. It is also true that most agencies in charge of spectrum, even those advocating a strong market-orientation like OFCOM or the FCC, tend to adopt a careful and progressive approach to changes in management methods; the road to a competitive spectrum market is more evolutionary than revolutionary. The specifications and restrictions attached to the frequency usage plan are progressively softened or lifted, and possibly changed into an orientation framework. In this sense, the variety of spectrum regimes presented in this paper can provide a map on which an evolutionary path from one spectrum regime to another may be traced, alongside evolutions in wireless technologies and spectrum usage. The actual implementation of cognitive radio, dynamic frequency selection, for instance, could warrant an extension of easements over time.

Given the strong interaction of decisions on frequency bands, service applications, and technology assessments, which constitutes the basic foundation of the choice of a spectrum management regime, it is advisable to operate those choices for relevant sets of bands, services, and technology areas, which we call wireless clusters. Such clusters would include, for instance, mobile voice and television, fixed wireless access, professional mobile radio, collective use, public safety, etc. 13 From this perspective, there might be migrations over time from one spectrum regime to another, to implement changes required by demonstrated positive technological changes. Instead of a war of doctrines between command and control, market and commons, we could have, when required for a cluster of wireless services, an evolutionary process over time between progressive spectrum regimes.

It should be remembered throughout this evolutionary process, however, that like at a higher level of policy consideration, efficiency lies in tough choices. If we look at the strongest performing economies over the last 50 years, good performances have been achieved by market economies like the U.S. and UK on the one hand, as well as co-ordinated social market economies like Germany, Japan, Ireland and the Netherlands on the other. Countries in the middle have performed less strongly. This may indicate that the problem lies not with market mechanisms, or more social models per se, but with distortions resulting from their application being only partial: a significant part of our present spectrum situation is due to the fact that part of the commercial spectrum is managed in an un-commercial way. If the broadcasting industry had been offered the possibility to trade (sell) its spectrum, maybe it would have found more profitable to do so and use the proceeds to expand its cable, satellite, and ADSL TV operation, instead of switching from analogue to digital on terrestrial.

Legacy and institutional factors might play a bigger role than markets and technologies, especially as the time horizon and gradient are superior to that usually encountered in business and industry. How can you have trading, or auctions for that matter, if this is an anathema to powerful and vocal players? What could then be a second-best approach if market-induced flexibility cannot be extended right across the EU? One first element of an answer is easements, sharing, collective use, and dynamic access. Some sharing will have to take place due to the expected limitations of spectrum available for IMT and beyond. Furthermore, new technologies will help bridge the gap between static (shortterm) and dynamic (long-term) efficiency as embedded in the standardisation versus non-standardisation dilemma. They substitute families of contiguous technologies to single technologies at any single time. They also facilitate smooth evolutionary transitions over time, enabling dynamic efficiency, and lifting the curse of necessary disruptions between discrete standards. The same evolutionary vision could apply to spectrum management regimes.

. Nine spectrum management regimes: a 4-step decision guide Frequency Allocation: Harmonisation Or Not STEP 1 Technologies Standardisation or not STEP 2 Usage rights STEP 3 Spectrum assignment mode STEP 4 Spectrum Management Regime #

	1.5Harmonised	Standardisation	Property	a/Administrative
	spectrum (no	Service	(no flexibility)	techno	rights/-Exclusive	Assignment Procedure/Hybrid
	flexibility)					b/ Auctions/Trading
						10
	The table below summarises the overall alternatives arranged in a 4-step
	decision tree designing nine spectrum management regimes:

1a Standard Command and Control (CC) 1b Technology Control/Property rights (PR) Market

	Property	a/Administrative
	rights/with	Assignment
	Easements	Procedure/Hybrid
		b/ Auctions/Trading

2a Mitigated CC with easements 2b Technical CC+ Mitigated Market Collective use 3 CC Collective

	Harmonised	Techno	Property	a/Administrative
	spectrum		flexibility	rights	Assignment
	(no	Service	NO	Exclusive	Procedure/Hybrid
	flexibility)		Standardisation		b/ Auctions/Trading

4a Technology flexibility in CC context 4b Harmonised flexibility

	Property	a/ Administrative
	rights with.	Assignment
	Easements	Procedure/Hybrid
		b/ Auctions/Trading

5a Controlled flexibility 5b Harmonised flexibility Plus Collective Use 6 Standard 'Commons" Regime

	Service	Techno	Property	a/Administrative
	flexibility)	flexibility	rights	Assignment
	NO	NO	Exclusive	Procedure/Hybrid
	Harmonisation	Standardisation		b/ Auctions/Trading

7a Administered Flexibility 7b Pure market regime: libertarian

	Property	a/Administrative
	rights with.	Assignment
	Easements	Procedure/Hybrid
		b/ Auctions/Trading

8a Technology Flexibility/Administered semi-PR Market 8b Mitigated Market regime: semi-libertarian Collective Use 9 California Dream

  The first is the relevance and institutional of the property-rights framework in the radio frequency spectrum area. It actually confronts decisions-makers with difficult social and political choices, very much related to the general institutional setting and mood in each country: the extension of property-rights meets less resistance in the UK and U.S. than in other countries. A second level of analysis rests at the technology level: it concerns the reality and feasibility of the technologies justifying easements in the property rights category, and of collective use. If we generically call those

	Strategies & scenarios	Scenario 1	Scenario 2
	for the decade ahead
		Advent of flexible	No significant difference In
		use technologies	trend with present technology
			mix
	Exclusive Property	Consumer Welfare	Consumer Welfare
	Rights Strategy	LOW	MEDIUM
	Policy-Mix (Easements	Consumer Welfare	Consumer Welfare
	& Collective Use)	HIGH	LOW
	Strategy
	We then have two strategies:
	-Allow for the possible advent and significant extension of "flexible
	technologies", and then either derive the greatest possible benefits if they
	work -or be left with an awkward and inefficient regulation framework if
	they don't.
	-Stick to what we know best: property rights, and then be right if "flexible
	technologies" don't work, or miss a significant opportunity if they do.
	Those scenarios, strategies and outcomes are summarised in the simple table
	below:

"flexible technologies", we can envisage two simple scenarios: -Scenario 1: "flexible technologies" work -Scenario 2: "flexible technologies" don't work.

Johann es M. Bauer, A Co mpa rativ e Analysis of S pectru m Manag e me nt Regimes, Quello Working Papers, 200 2

Booz-Allen, Study for the UMTS Forum, 2006

CEPT-ERO Report 80: Enhancing harmonisation and increasing Flexibility in Spectrum Management, www.ero.dk/, March 2006

Study on Legal, economic and technical aspects of collective use of spectrum in the European Community by Mott MacDonald Ltd, Aegis Systems Limited, IDATE, Indepen Ltd and Wik Consult

SPORT VIEWS Study Report March 2007, www.sportviews.org

1 This paper has strongly benefited from the research being carried out within the European Commission Specific Support Action SPORT VIEWS (Spectrum