Greqam

Nicolas Jullien

Jean-Benoît Zimmermann

Nicolas Jullien -M@rsouin

Firms' contribution to open source software and the dominant skilled user

Free/libre or open-source software (FLOSS) is nowadays produced not only by individual benevolent developers but, in a growing proportion, by firms that hire programmers for their own objectives of development in open source or for contributing to open-source projects in the context of dedicated communities.

A recent literature has focused on the question of the business models explaining how and why firms may draw benefits from such involvement and their connected activities. They can be considered as the building blocks of a new modus operandi of an industry, built on an alternative approach to intellectual property management. Its prospects will depend on both the firms' willingness to rally and its ability to compete with the traditional "proprietary" approach. As a matter of fact, firms' involvement in FLOSS, while growing, remains very contrasting, depending on the nature of the products and the characteristics of the markets. The aim of this paper is to emphasize that, beside factors like the importance of software as a core competence of the firm, the role of users on the related markets -and more precisely their level of skills -may provide a major explanation of such diversity. We introduce the concept of the dominant skilled user and we set up a theoretical model to better understand how it may condition the nature and outcome of the competition between a FLOSS firm and a proprietary firm. We discuss these results in the light of empirical stylized facts drawn from the recent trends in the software industry.

Introduction

"Free/libre or open-source software" (FLOSS) is software whose source-code, that is the explicit expression of the programming work, remains openly accessible. Until recently, FLOSS was considered only to be of interest to programmers motivated by the building and sharing of a base of programs developed for their own needs [START_REF] Lakhani | How open source software works: Free user to user assistance[END_REF], [START_REF] Demazière | How free software developers work. The mobilization of "distant communities", working paper, M@rsouin[END_REF].

Today, open source software is increasingly integrated into many commercial offers (Novell, buying Ximian and SuSE, Sun open-sourcing its operating system, IBM open-sourcing its development tool software Eclipse, even Microsoft, which recently decided to distribute some of its software products under open license 1 ). So [START_REF] Iansiti | The Business of Free Software: Enterprise Incentives, Investment, and Motivation in the Open Source Community[END_REF] identify, amongst the various FLOSS projects, a "money-driven cluster" where "IT vendors' motives are economic. In this cluster, significant investments have been made in projects that will serve as complementary assets to drive revenues to vendors' core businesses". * The authors thank warmly the two anonymous referees for their excellent reports and suggestions and the EMR editors for the very accurate advice they gave us for the improvement of the paper. We are also indebted for helpful comments to the participants of the Conferences and seminars where we had the opportunity to present the project of this paper, more particularly the DIME London Conference "Intellectual Property Rights for Business and Society", Birkbeck University, 14 th and 15 th September 2006, the international conference "The diffusion of FLOSS and the Organization of the Software Industry: From Social Networks to Economic and Legal Models", Nice Sophia-Antipolis, May 31 st , June 1 st 2007, the Complex Markets Workshop, Warwick Business School, 31 March -2 April 2008. This research has benefited from the support of the Institut Telecom (France), the French ANR and the European STREP "Complex Markets".

1 http://solutions.journaldunet.com/0404/040407_microsoft.shtml [START_REF] Lakhani | Why Hackers Do What They Do: Understanding Motivation and Effort in Free/Open Source Software Projects[END_REF], analyzing the results of a survey of 684 software developers involved in 287 FLOSS projects, show that "a majority of (their) respondents are skilled and experienced professionals working in IT-related jobs, with approximately 40 percent being paid to participate in the FLOSS project". This situation, in which commercial business relies on the existence and durability of non-market activities, challenges traditional industrial economic theory.

As in any cooperative agreement devoted to technology or knowledge development, agents pool assets together in a "pre-competitive" phase and share the products of their efforts before returning to competition [START_REF] Crémer | Incentives and the Existence of Pareto-Optimal Revelation Mechanisms[END_REF], [START_REF] Bhattacharya | Patents vs. trade secrets: Knowledge licensing and spillover[END_REF]). But such agreements remain closed to third parties. On the contrary, a FLOSS project is an open contribution game in which the list of players is not bounded ex-ante by a cooperative agreement and the output of which is a public good that cannot be appropriated by any of the players in an exclusive way. This corresponds more to the formation of a consortium for the production of a standard2 .

In line with the seminal work of [START_REF] Teece | Profiting from technological innovation: Implications for integration, collaboration, licensing and public policy[END_REF], FLOSS can be considered as an extreme case of "open innovation" [START_REF] Chesbrough | Open innovation: The New Imperative for Creating and Profiting from Technology[END_REF], defined as "a paradigm that assumes that firms can and should use external ideas as well as internal ideas, and internal and external paths to market, as they look to advance their technology" [START_REF] Chesbrough | Open innovation: a new paradigm for understanding industrial innovation[END_REF]. [START_REF] Dahlander | A man on the inside: Unlocking communities as complementary assets[END_REF] show that firms strategically sponsor individuals who occupy a central position in a community, in order to better access distributed skills and to control the direction of development of the related projects. Those firms seem to consider communities as "a complementary asset that exists outside firm boundaries and outside their ownership or hierarchical control". This is even the sole control means when innovations, as observed by Von [START_REF] Hippel | Democratizing Innovation[END_REF], are produced by a community of innovative users that protect their innovation from private appropriation through a GPL license3 . But things often stand differently. There exists a large variety of licenses that have been designed to achieve the co-existence of open source principles with private interests [START_REF] Muselli | Licenses: strategic tools for software publishers[END_REF][START_REF] West | How open is open enough? Melding proprietary and open source platform strategies[END_REF]. But even in the case of GPL protection, intellectual property is not always shared among the members of a community. So, the database software MySQL is entirely owned by the eponymous firm 4 . Yet it has published its products under GPL license, thus abandoning any monopoly rent on them but allowing a free access for users and potential competitors.

So this is a radically brand new way of producing software that has emerged and become widespread during this last decade. It challenges the foundations and competition regime of the software industry, which was evolving more towards strengthening intellectual property protection [START_REF] Bessen | An Empirical Look at Software Patents[END_REF]. Two radically opposed ways of working are confronting each other, based on two opposing views about intellectual property management. On the one hand, the "traditional" position, referred to here as the "proprietary" approach, defends the need to strengthen the intellectual property regime to respond to a growing ease with which software codes can be appropriated and used without any fee 5 . FLOSS defenders, on the other hand, argue that that position is inefficient for innovation dynamics, reinforcing the market power of the sole dominant firms and thus the oligopolistic nature of the industry, which is economically inefficient 6 . The result of this confrontation is then of huge importance for the future of the software industry and, more widely, for the whole information and communication industry. From an even broader perspective, this case of open-source software can be viewed as a precursor of what could also take place in a wider array of industries where innovation has a strong role to play in competition and requires to access to a larger and larger range of knowledge and resources.

The outcome of this situation clearly depends both on the capability of software firms involved in FLOSS to set up business models likely to compete efficiently with those defending the "proprietary" position and on the inclination of firms incorporating software in their products either to consider FLOSS as an effective resource or even to support or contribute to their development. Currently, attitudes towards FLOSS appear to vary strongly according to the sectors of activities of what we shall refer as the information technology industry (IT industry in short).

The aim of this paper is to show that, beside factors like the importance of software as a core competence of the firm, one major explanation of such diversity can be found in the characterization of the users' skills according to the markets involved. In order to better understand this aspect, we introduce the concept of the dominant skilled user and we build a small competition model in which two firms, a proprietary one and a FLOSS one, choose their level of investment -and thus of product quality -and compete for market share. We show that the level of skill of the dominant users deeply conditions the nature and issue of the competition, as we shall illustrate through empirical stylised facts.

The paper is organised as follows. In section 2 we describe the structure of the IT industry and the variety of firms' attitude toward FLOSS. In section 3, we introduce the concept of the dominant skilled user and we present a formal competition model in which two firms, one open source and one proprietary, compete for a market characterised by a given distribution of users' skills. Section 4 provides a descriptive/empirical discussion of the application to FLOSS. Then we conclude.

The structure of the IT industry and the variety of attitudes toward FLOSS

There is a wide diversity of actors in the industry in terms of both products and size. Successive waves of innovations and company strategies have led to a progressive reshaping of the industry's borders and structure. However, the foundations of the industry have remained unchanged, since those described by [START_REF] Gérard | Concept de produit informatique et comportement des agents de l'industrie[END_REF], [START_REF] Zimmermann | Le concept de grappes technologiques[END_REF] and [START_REF] Steinmueller | The U.S. Software Industry: An Analysis and Interpretive History[END_REF]: IT products are built by assembling hardware and software units in a given architecture, and these products (isolated or integrated into networks) are used as parts of information systems and solutions. On the basis of such technical organization, it is then possible to distinguish three large types of "vertical specialization": i) component producers; ii) computers and IT devices suppliers; iii) software editors and service companies providing applications.

The basic conditions for each kind of activity (characteristics of the products, of the users, hence of the demand) that shape the main aspects of the market structure have been extensively described in the literature [START_REF] Genthon | Croissance et crise de l'industrie informatique mondiale[END_REF][START_REF] Dréan | L'industrie informatique, structure, économie, perspectives[END_REF][START_REF] Steinmueller | The U.S. Software Industry: An Analysis and Interpretive History[END_REF]. They help to explain the sources of competitive advantage and the conditions for a newcomer to enter a market, or for an incumbent firm to improve its market share.

i) Component producers supply the basic components of electronic devices like chips (Intel, Toshiba), hard disks or cards (ATI: graphic cards). The quality and performance of the main components built into an electronic device are, to an ever-increasing extent, considered as a critical feature of the performance of the product.

ii) Using these components, firms build machines more or less dedicated to specific uses. At one extreme, computers can be used for a wide scope of applications, depending on the software that is acquired and installed on them. At the other extreme, video game consoles or multimedia players are devoted to a single range of applications, while in between, mobile devices like PDAs or mobile phones are built to support a growing number of applications7 .

-Servers are designed to manage, deliver and protect information on the networks. They must be highperformance, stable, but also compatible with network standards. An archetype of server producers is SUN.

-Microcomputers (with a growing market share for laptop computers) are used by end users, mainly as personal computers. In order to clarify the analysis, we have split this segment into the two categories of "high quality computers" -HQC -targeted at organizations or intensive end-users8 and "low price computers" -LPC -, targeted at basic uses (Internet, for instance) and low-skilled users. Market structures and actors are dissimilar, even if some of the suppliers sell on both markets by differentiating their product ranges, in order to draw benefits from increased scale and scope economies. This appears quite similar to other industries like the automobile industry, where top-and bottom-of-the-range markets do not work similarly but can be partly held by the same manufacturers. An archetype of a "quality computers" supplier could be Dell or Toshiba, while an archetype of a "low price computer" could be Acer or Packard Bell.

-Dedicated machines such as PDAs, multimedia players, mobile phones, etc. are more and more present in information systems networking. Most of these devices are connected to the computer(s), but more and more also have access to the Internet (via Wifi, local networks or ADSL technologies), and share applications with the computer (e-mail, personal data management, etc.) Even devices such as video game consoles are increasingly connected to an Internet-based network. Archetypes of this evolution are Nokia's mobile phone, Nintendo's game console, Apple's music player (Ipod) or Palm's PDA.

iii) Since the beginning of the 1970s, some firms have specialized in software production and, since the beginning of the 1980s, in software publishing (packaged software). Three typical strategic orientations can be described that have successively emerged in two waves. These three strategic options gave rise to a market segmentation that corresponds to different degrees of user sensibility to price, variety and specificity of the offer.

The "packaged software" business solution providers: First, during the 1980s and 1990s, a growing number of users sought to reduce the costs and uncertainty related to customized development by acquiring software packages that could installed and adapted to their specific needs and constraints, with the help of service enterprises accredited by the software editor (Horn 2004, p. 98). These hybrid offers of "packages", combining standardized goods and customized services, were highly successful in the field of professional dedicated solutions, both for management needs (ERP such as SAP) and for technical software (middleware applications, compilers, development tools like those from Ilog) or branch-specific applications (like software for computer-assisted design or computer-assisted manufacturing from Dassault System or others).

With the development of networks within firms and, more recently, outside, with the diffusion of the Internet, a growing number of heterogeneous users have shared a growing number of applications (mainly those allowing information exchange and sharing). This network effect and the related need for standardization have had important consequences on further technological orientation. It was necessary to ensure the availability of a wide portfolio of software tools that could meet the needs of all these, both for expert and for unskilled users, within common or separate organizations. Two kinds of strategies can be observed as responding to this necessity:

The platform producers: On the one hand, software publishers have broadened the scope of their offer by supplying a variety of application tools that can be combined with their core product or by supplying this latter in multiple versions. This enables them to better meet users' specific needs while keeping production costs down. The archetypal example of such a "platform strategy" is Microsoft, which now offers different versions of its operating system for servers, corporate users or private individuals, as does its open-source competitor RedHat now. The same kind of strategy can be observed for Oracle, which sells professional applications developed on its database technology, and which has recently bought BEA, after other takeovers, to enlarge its applications portfolio9 . Another illustration is given by Symbian in the field of operating systems (OS) for mobile applications.

The architects: On the other hand, service companies, especially the large ones, such as IBM and Cap Gemini, have tried to master a wide range of software technologies and products they can combine and adapt to the constraints and the existing equipment of their customers. They intervene as "architects" of their customers' information systems. Smaller local services companies have a similar position, also providing infrastructure services but at a smaller scale (single server maintenance instead of a large IT infrastructure) or aimed at specific professional needs (like IT infrastructure maintenance services in the food industry).

All these segments are characterized by strong imperfect competition regimes due to diverse increasing return effects (economies of scale in production, high sunk costs -R&D, distribution channels, etc. -, technological interrelatedness and learning). One single segment (the platform market, when looking at the OS for PC computers market) can be considered as a quasi-monopoly with very high barriers to entry, controlled by a dominant leader (Microsoft, Oracle) that intends to use this dominant position in order to extend its dominance over related application tools (See, for example, Gartner for a description of the database market and its level of concentration10 ).

In such oligopolies, differentiation strategies generally play an important role. In the sole segment of low-price computers, where competition is mainly based on prices, the strategies are based on the exploitation of new market potential through vertical differentiation with the traditional PC market. In the other segments, strategies are rather of horizontal differentiation, either related to the integration of new features and high performance tools or to market segmentation through hard-soft-content bundling (mainly based on proprietary standards).

So new entries require either better performance/cost ratios (for instance lower price computers or better computational capacities for servers or high-quality computers) or innovative horizontal differentiation (e.g. new features in personal communication tools or business software).

Last but not least, horizontal differentiation can also target a sub-group of users with specific needs not fulfilled by the existing offer, as Samsung did when proposing a fold display mobile phone. This is also a possible strategy in the business software market (to propose a targeted offer at the best possible price to a "niche" market).

Since the mid-1990s, a growing number of commercial firms, both new entrants and incumbents, have decided to integrate FLOSS products into their own specific offer or toolboxes, even investing by different means in FLOSS development. Of course, these new emerging strategies must be understood in the light of the IP protection practices prevailing in each market segment and the need to strengthen competitive advantages or to rely on new ones.

Regarding the degree of involvement in FLOSS dynamics, the most active actors seems to be found in sectors where software development and use is either a core activity or a crucial condition for performances, as is the case for server manufacturers or architects (e.g. the adoption of Linux by IBM and HP since the beginning of the 2000s). At the other extreme, the weakest involvement is found amongst hardware suppliers that can only feel concerned by FLOSS for compatibility and price purposes.

When FLOSS adoption is related to marginal aspects of differentiation, it seems to have little impact on industrial structure and competition. This is generally the case for most hardware producers, when hard-softcontent is no longer bundled (servers, computers, PCTs, DVD and MP3 players, etc.)

Regarding software firms, their core competences have evolved and shifted significantly. Their main challenge is less and less to supply a "software solution" to a given problem at a given time, but increasingly to deal with short to long term uncertainty over IT system production and management. Users ask for solutions to protect them against uncertainty, granting interoperability, bug resolution, the satisfaction of new needs and the integration of technical advances. The trade-off between available solutions is not posed in terms of their cost of acquisition but of their "TCO" (total cost of ownership), in which the future costs and the costs for granting interoperability and adaptability have to be estimated. This is precisely what architects, business programs and platform producers sell to skilled users, aware of these problems and signals. On these markets the FLOSS organization seems to represent an asset for producers, who can display their involvement and succeed in building sustainable business models (see the examples of RedHat, MySQL or, in France, Linagora). But, as explained before, this is only an asset if the market regards FLOSS as providing a valueadded to the product, i.e. if it brings the users a potential for increasing their utility.

To a large extent, the differing degrees of involvement in FLOSS between different firms can be explained by the position of software in the firm's business model (as core activity or not, see [START_REF] Dahlander | Relationships Between Open Source Software Companies and Communities: Observations from Nordic Firms[END_REF] but also by the role played by skilled users in the selection of the offers on the market. If the first aspect is clearly connected to a well-developed recent literature, the second remains rather new and relatively unexplored. From that stems the need to better understand the role of the users and the way it may impact the firm's ability to achieve a viable business activity capable of competing efficiently with proprietary software firms.

To this end, we introduce in the next section the notion of the dominant user's skill and we present a theoretical competition model in which two firms with contrasting attitudes toward intellectual property protection compete on a market characterized by a given distribution of users' skills. We will see how this distribution may drive both the nature (price vs quality) and the outcome (market shares and profit levels) of the competition. For this purpose we shall give a formal conceptual base to the notion of dominant user's skill.

A competition model with customer feedback

The users, understood as the persons choosing the solution (thus not always being the "end-users"), are rather different from one market to another, causing the competitive advantage to rely on different features.

Let us distinguish between three main types of users according to their relation to the product and the technology [START_REF] Zimmermann | Le concept de grappes technologiques[END_REF][START_REF] Kogut | Open Source Software Development and Distributed Innovation[END_REF][START_REF] Hippel | The Sources of Innovation[END_REF][START_REF] Muselli | Licenses: strategic tools for software publishers[END_REF]. The first is the category of "Naïve customers or users" (that we denote N) who are not endowed with noticeable technical skills and do not individually weigh very much in economic terms. The second is the category of "Kogut-Metiu Users" (KM)11 who are not able to contribute to software development but can generate new features or innovations by revealing their own needs. Above all, they represent an irreplaceable testing and debugging base. KM users are sensitive to price and quality arguments. The third category is that of the "Von Hippel Users" (VH) who act as "sources of innovation" [START_REF] Hippel | The Sources of Innovation[END_REF] able to contribute to software development by proposing improvements or modifications, developing it by themselves or at least able to design the technical specifications.

Users play a double role, deriving from both their economic and technical standing. Depending on the market, and especially their bargaining power in it, the users are more or less able to select the (technical) offers. At one extreme, users and contracts in the global service/architects market are related to large structures, with substantial buying capacities and generally endowed with significant technical skills. So they are likely to influence economic and technical choices. At the other extreme low price computers address a mass market where individual users, in their vast majority have little budget and/or few skills. Their influence on market evolution is negligible at an individual level but of global importance in terms of elasticity to prices. So, even if they are VH or KM types, individual users in such a market can hardly make their views heard (in terms of technical choice, for instance). But this analysis should be nuanced in the case of intermediation by a "prescriber", who orders and defines the characteristics for a large number of machines destined for mass distribution by his own means (local government for secondary schools in France 12 , education in rural areas in developing countries 13 , etc.) That's the reason why, when speaking about the "user", we mean the person who negotiates or chooses the characteristics of the good, who is not always the end user.

Of course, different types of users co-exist in any given market. But the dispersion of users' skills in the related technology and more particularly in software doesn't follow the same distribution from one segment to another. Our aim here is to characterize this distribution under the assumption that it usually takes a unimodal shape, meaning that most of the users in the market segment are concentrated around a given level of skill, corresponding to what we call the dominant user's skill.

In this model presented below, we consider a competition game between a proprietary and an open-source software supplier on a market characterized by a given distribution of users' skills.

The users

In a pretty standard way, we consider each individual user to be characterized by a given level of preference for quality θ ∈ [0,1] 14 . In terms of the problem addressed here, this level can be considered as a good proxy of the individual user's skill: information technology products are endowed with a wide usage potential that is actually exploited more or less fully, according to the user's ability. This ability is tightly correlated with the user's level of skill in computing: the more skilled, the more he is able to benefit from the technical quality of the product. 15So for a user of level θ , the utility obtained from a product of quality s sold at price p can be written as U= θ s -p if he buys the product; = 0 otherwise.

It is considered that the weight of the user population is normalized to 1.

A given market is then characterized by the distribution of preferences given by a function

f: [0,1] → R+ : ∫ ٠ ١ f d =١ (1)
The suppliers

Let l denote the open-source firm and m the proprietary firm.

Both sell a substitutable product vertically differentiated through a quality level s > 0.

The proprietary firm chooses the quality level of its product between a low and a high level sm ∈ {sL , sH} with 0 < sL < sH The quality sl of the product delivered by the open-source firm depends on both the level of contribution cl of l to its development and the amount of the feedback effects from its user base, so that sl = q (cl , τl)

where τl is the users' feedback effect, measured as the aggregated skill level of the user base for the opensource product;

 l = ∫  l  f d  (3)
where Θl is the user base of l , assumed to be compact.

q is assumed to be twice differentiable increasing and quasi-concave in cl and τl and q(0,.) = sd where sd is the standard quality of the product as freely available on Internet.

A simple expression of q is given as linear in cl and τl by sl = λ cl (1+ τl ) (4) so that sd = 0 in so far as when it invests 0, l can only sell what it takes from the community and doesn't provide any added value in terms of interface, adaptation or services that could justify a positive price pl > 0 .

In order to avoid a trivial situation, λ is assumed to have a value such that the open-source firm needs to invest more than the proprietary firm to reach the same levels of quality sL < sH , so that The proof of proposition 1 is given in the appendix.

λ < Min (sL / cL , sH / cH ) (5

Corollary:

Let   denote the value of θ at which a user has no preference between l and m

 = p m -p l s m -s l (6)
and θ 0 the minimal level of the users entering the market.

When sl < sm ,

 ٠ = p l s l then Θl = [θ 0 ,   ] and Θm = [   , 1] When sm < sl ,  ٠ = p m s m then Θl = [   , 1] and Θlm = [θ 0 ,   ]
The game:

m chooses to invest cm = cL resp. cH for a quality sm = sL resp. sH and sets its market price pm l invests cl and gets a quality level sl at price pl , with sl either lower or greater than sm .

Respective profits can be written Πm = pm Θmcm and Πl = pl Θlcl as defined above.

So the game is the following:

m l < sm > sm sL Πm L , Πl L Πm Ḹ , Πl Ḹ sH Πm H , Πl H Πm  , Πl
The reasoning is backward:

If m chooses L, l then chooses the low strategy ( sl < sm) or high strategy ( sl > sm) that brings it the higher profit ( Πl L or Πl Ḹ ) at the equilibrium. The m profit Πm L follows. In the same way, if m chooses H, there follows a profit Πm H for m. So m will choose the strategy L or H that brings it the higher profit, and so does l.

Of course the complete resolution of the game is very heavy in the general case, due to the nature of the indifference level   that is defined through an implicit function. That's the reason we prefer to develop an approach aiming to characterize the different situations in the different quadrants of the game, and the conditions relating to the transition from one quadrant to another.

Definition: The dominant user's skill

As seen before, each market in the software industry is characterized by a dominant type of user that we have described through a typology as "naive", "K.M." or "V.H." according to their skill level in relation to the software product. Of course, this dominant type coexists with users of different skill levels, but the distribution can be considered as relatively unimodal. Hence, we characterize the level of the dominant user not through the mean value of θ among the population but through a stronger criterion of stochastic dominance, meaning that if f and g are the distribution of users' skills characterizing two markets, then

g >> f ⇔ ∀   ∈ [0,1[ , ∫   ١  g  d  ∫   ١  f  d  (7)
This criterion expresses a shifting of the mass of the users from a lower to a higher level of skills.

In order to understand how and why the dominant user's skill influences the level of investment of the opensource firm, we study two polar cases. The first (North-West quadrant) is characterized by a low skill level of the dominant user, when a large majority of users are concentrated at a low level of skills not far from 0. The opposite polar case (South-East quadrant) occurs when the population of users is dominated by highly-skilled users.

North-West Quadrant: sm = sL and sl < sm

In such a situation, m chooses a low quality while l cannot rely on a potential of users' skills large enough to reach a quality level sL without investing in a non-profitable way (assumption (5) on λ value). Then l can only aim at a low level of quality and a low price, targeting a demand that is more sensitive to price than to quality.

Proposition 2:

There exists a low threshold of the dominant user's skill under which the open-source firm invests little, and stays at a low level of quality, targeting a relatively price-sensitive market.

Proof and technical remarks about proposition 2 are given in the appendix.

The transition between the two polar cases (NW and SE quadrants) when the dominant user's skill is increasing can take two different paths: NW→ NE → SE or NW→ SW → SE, depending on the values of the parameters.

South-East Quadrant: sm = sH and sl > sm

This quadrant corresponds to a high skill level of the dominant user, when a large majority of users are concentrated at a high level of skills not far from 1. In such a situation, m will choose a high quality, while l can invest in a profitable way by relying on a potential of users' skills large enough to reach a quality level sl > sL.

The limit condition is obtained for a symmetrical duopoly situation equivalent to that of the NW case (8) with

θ 0 root of 2 cH θ 0 = sH (1 -F(θ 0 )) and ١ ٢ ∫  ٠ ١  f  d = s H  c H -١ (11)
From this stems the following proposition.

Proposition 3:

There exists a high threshold of the dominant user's skill above which the open-source firm invests a large and increasing amount, obtains a high and growing level of quality and makes a profit that increases with the level the dominant user's skill.

The proof of Proposition 3 is given in the appendix.

In conclusion, we can say that if the dominant user is unskilled, and so price-taker, FLOSS may be a good strategy if it enables the firm to supply a product at a lower price than the proprietary one, but requiring a low level of investment to adapt or improve the FLOSS solution. In the opposite situation, when the dominant user is very skilled, an offer based on FLOSS and a high level of complementary services can succeed. This result is in accordance with the work of Henkel (2006), who shows in a duopoly model that "a regime with compulsory revealing can lead not only to higher profits, but also to higher product qualities than a proprietary regime". In the intermediate situations, the FLOSS strategy may be less efficient than the proprietary one.

Discussion: The critical importance of the users' skills

How and why may those different users contribute directly or indirectly to FLOSS projects? First of all, contribution does not necessary imply code development but can take various forms in the product development and improvement. Users have to be considered as valuable "sources of innovation" (Von Hippel), not only for testing and debugging programs but also for improving product usability and performances. People decide to contribute if they get interested by the product, or if they have a problem, in which case they can either report the problem directly (VH users) or through an intermediary, the supplier for instance, that allows the user to pass from a passive to an active use of the project.

Empirical observation of firms' involvement in FLOSS development is consistent with the results of the model.

In fields where the dominant users' skill is either high or very low, firms invest in FLOSS. When the dominant user's skill is intermediate, the dominant design remains that of the classical proprietary model.

Table 1 below summarizes the main types of users likely to be found in each sub-sector of the IT industry.

Table 1. The dominant user type in each IT sub-sector.

Actors/ products

Dominant user type Comments

Components VH Component producers supply hardware manufacturers, aware of the quality and quality-price aspects of the components they will use, as well as the effects of brand reputation of these latter as a signal of quality for their own products.

Servers VH

The clients are computer-literate people, able to express needs in technical terms, to develop software for their own needs, and to innovate by themselves.

High Quality Computers KM HQC users are somewhat less computer-literate than server users; they can be characterized as "intensive frontier users". So the market is looking at a good performance-to-price ratio.

Low Price Computers

N +. KM LPC is a mass market; users have no particular skills except in the case of intermediation by a "prescriber".

PCT N + KM Players N PCT and players are relatively mass markets, but some advanced users (more in the PCT field and particularly in the PDA market) can play a constructive role in the development of new features.

Platform producers KM + N

For the OS, as for hardware components, most of the end-users buy a computer with an OS already installed. So the actual users in our sense of the term are computer manufacturers, service companies and sophisticated end-users capable of installing an alternative operating system for their proper use or the use of their customers.

On other platforms (database, middleware), the users are also computer manufacturers, service companies and highly-skilled users.

Business solution producers

VH/KM depending on the markets

In the business solutions market, users are professionals. They are able to make a technical evaluation of the product, to carry out trials and tests. This means that people may have skills in the functional domain (what they want, how the software works), and sometimes in the technical one (able to adapt or develop software to meet their own needs, especially in the tools for computer professionals market).

Architects N (+VH)

Large firms and organizations include very sophisticated users (IT division). SMEs or corporate divisions, at local or sectorial level, are clients of very heterogeneous but rather low IT skills. However, clients may be quite precise in the definition of the services they need, and so in the specification of the application characteristics.

1. In the servers market, producers have habitually provided proprietary solutions with proprietary Unix 16 .

Here, suppliers are dealing with highly-skilled VH clients that can make an essential contribution in the context of FLOSS opening. The rise of PC servers has permitted some users to avoid such a bundling problem; moreover, using Linux allows a cheaper offer (vertical advantage) reusing Unix programs (content) portfolio. Thus, some firms have been able to widen the servers market from VH users capable of managing their systems by themselves to KM clients, sensitive to prices, but also to the quality of a PC server fitted out with Linux. So new entries have been experienced like the Cobalt 17 one, but the main actors of the Unix "world" have also rapidly developed their own offers, cutting down the sources of vertical differentiation18 .

2. In the segment of low price computers (LPC) where naïve clients are the driving force behind demand, competition is overall based on prices. Installing a FLOSS OS can be considered a way to reduce prices and hence to extend the firm's market share to a wider market, following Asus' "eee PC" offer, or at least to enable a new entry by compensating for not-yet-accessible economies of scale19 . 16 See [START_REF] West | How open is open enough? Melding proprietary and open source platform strategies[END_REF] for a full discussion of FLOSS strategies in that sector. 3. Between these two cases there is the high quality computers (HQC) market. HQC producers may find it hard to switch from Windows to Linux, because this would mean either acquiring new skills (OS management and improvement), or sub-contracting this maintenance to Linux editors (e.g. RedHat or SuSE), which may lead to another dependence and to difficult relations with the dominant provider. Nevertheless, a possible future evolution in this sense is likely to arise from the pressure of customers becoming more aware of the potentialities of switching to FLOSS, as illustrated by Dell's move towards the provision of Linux-based computers. In the near future, most of the HQ will probably switch to debundling their machines from the associated OS, to give KM users the choice between a Windows and a Linux platform, or to segment more their offer between VH users with the Linux offer and KM users with Windows. It is worth noting that in this desktop market, the main push in favour of open source, for the time being, is driven by organizations or institutions (which we consider as VH users) that take decisions to equip a large number of end-users. Examples are the French "Assemblée Nationale" (French Congress) that has contracted with a service company to install Linux on all the computers provided to MPs 20 , or the initiatives of the Nigerian 21 and Macedonian 22 governments for schools.

Personal communication tools represent another intermediate case with less skilled customers (KM+N)

and a weak degree of involvement on the part of commercial actors only motivated by preoccupations of compatibility and absorptive capacity. There are lots of FLOSS products for PCT, or Mobile Computers 23 . Some are proposed by VH users, other by the constructors. At one extreme, in the games consoles segment but also to a lesser extent in the music player market, proprietary formats have introduced, as seen above, a strong bundle of hardware-software-content. Thanks to the MP3 standard or new existing or emerging open standards like Ogg, new entries are always possible in segments like the music players market, but the main actors, like Apple, remain on a strict proprietary strategy. On the contrary, barriers remain high on the video game players market due to the scarcity of independent games capable of running on Linux, unlike the PS2, Xbox and other proprietary standards games. Moreover, when they exist, such games seem harder to obtain for simple users. On the contrary, PCT suppliers have begun experiments with FLOSS products over the last couple of years:

-if the leader, Nokia only sold an Internet tablet based on Linux and a development community 24 , there are lots of open-source projects around Symbian (partly owned by Nokia, partly by Sony-Ericsson) 25 , mainly dedicated to tools for developing applications (libraries, development tools, etc.) and Samsung proposes the first smart phones based on Linux26 ; -the PDA Operating system editor Palmsource is working on the integration of its product on a Linux kernel 27 .

For the same reasons as for PC computers, we hardly see naïve or KM people switch from an installed operating system to a FLOSS one. So constructors will continue to drive the market and decide what they integrate in their offer. But operating systems are not at the heart of the product differentiation, which is more based on ergonomic aspects and hardware characteristics. In the absence of an established de facto standard, as it stands in the PC market, Linux is to be considered by PCT suppliers, as it is free of charge and benefits from a community of developer-users capable of developing new features and new products outside of any proprietary control. Implementing Linux on PCT devices may appear as a good strategy to limit differentiation to the core competences of the manufacturers. For firms like Nokia, who do not own or control any operating 20 http://www.zdnetasia.com/news/software/0,39044164,61970345,00.htm 21 http://www.zdnet.co.uk/talkback/0,1000001161,39290511-39001070c-20088736o,00.htm . system, there are strong incentives to use FLOSS. Palm is also a good example of a company that is now turning toward Linux, after having sold its OS division. 5. In the software platform market, the Linux distribution market is another very good illustration of the key role of the demand. Linux publishers, like RedHat, SuSE, Mandriva (formally Mandrakesoft), have been among the first commercial actors to enter the market using FLOSS. This could appear to be obvious on a mass market with rather naive users and a significant price-based competition. But today, the retail store sales of OS packages represent a negligible part of the revenue of such firms28 , and a major part is targeted at the business market.

One might explain this by the development of broadband connection, thanks to ADSL. But we believe a more important explanation lies in the skills of the users and the construction of the offer. Consumers buy computers with an OS already installed and few of them are skilled enough to install a different one. Additionally, there are no incentives to do so because the pre-installed OS has already been paid for with the computer. So the diffusion of FLOSS OS on desktop/laptop PCs depends more on the strategies of constructors, as discussed above, than on direct installation by users. In other words, what needs to be taken into account is the possible changing attitude of computer producers towards the dominant position of Microsoft in the OS market. For VH people wanting to install Linux on their PC, other, more technically oriented distributions exist, like Debian, and there is no need to pay for these distributions, available for download on the Web.

On the emerging OS for PC server market, things work differently. Most of the users, of VH or KM type, are aware of the technical questions involved in installing and configuring an OS. It is also easier to buy a machine without an operating system installed, and the relative price of the OS is lower. FLOSS gives these users access to a cheaper, more open and more adaptable Unix-like operating system than they could find in the traditional Unix offer. This gave FLOSS OS publishers an undeniable competitive advantage, at least until server constructors started to offer PC servers with Linux.

6. In the business software market, the more skilled the users are in terms of software development skills and (although this is a lesser driving force) in terms of expressing their functionality requirements, the more FLOSS concepts and related industrial offers are likely to spread.

It is clear that the use of open-source business software, enabling savings on the cost of licenses, offers a price advantage. Moreover, the fact that the customer can evaluate the product without buying a license is also an advantage in terms of dissemination. It may even be compulsory when dominant players already exist on the market (such as the database market where MySQL proposes software products competing against those of Oracle, IBM and Microsoft, who represent more than 80 % of the market) or when customers are highly sensitive to price (such as the ERP market which increasingly concerns SMEs and where open-source products like ERP5 or tiny ERP are now available). This strategy also enables the association of a corporate brand with a product, therefore increasing the notoriety of the firm through distribution of the latter. Moreover, on these technical markets, especially when the customers are developers, availability of the code promotes cooperation. The producer approves the contributions, ensures stability of the tool and helps developers to use it. If some individual contributor becomes important (in terms of contribution volume/quality/innovative aspect), s/he may be hired by a producer, with reduced recruitment costs and risks (ACT, MySQL and some small service companies are using this method). By contributing to innovation, the developers (and possibly companies using the tool) are therefore guaranteed that their needs will be taken into account more quickly and integrated into the product (which is a fundamental factor in reducing costs, according to [START_REF] Hippel | The Sources of Innovation[END_REF].

Obviously, capitalizing on existing products is more difficult, even if, as [START_REF] Muselli | Licenses: strategic tools for software publishers[END_REF] explained, with entire control of the software, a dual license strategy can be set up to sell the program when requested by customers (because, for example, they want to integrate it in a larger, closed package). This is what companies like Qt or MySQL offer. But, today, the main source of revenue again comes from services, more precisely what we call the "3A services" (assistance, assurance and adaptation to use). Otherwise, adaptation services must be significant enough to finance development of the product. Therefore, the objective is to transform a handicap (significant investments) into a commercial advantage, by increasing the business feedback from users and by considering openness as a way to reduce transaction costs and as a signal of quality. Currently, the main evolution for those firms is to switch from a demand pull strategy (functionalities are developed to stimulate/create the demand) to an 'on-demand' development (development when required and paid for or carried out by the users).

This explains why open-source business products are developed mainly in "business" software (ERP, computer infrastructure software such as compilers), where users ready to pay for configuration, maintenance or assistance services are numerous. But the scope could easily extend to many technical/professional software activities.

7. As far as the architects market is concerned, as [START_REF] Horn | L'économie des logiciels, collection repères[END_REF] points out, assembling components requires access to the source codes (problem of compatibility), and their adaptation to different needs (of users and other components). They must therefore be available in the form of open-source software and legally modifiable.

The competitive advantage in using free software, in addition to price, is therefore the ability to offer an assembled set of components with greater interoperability, which should increase the quality of the final product, on a market where the quality of services is one of the recurrent problems (see [START_REF] Bandt | Services aux entreprises: informations, produits, richesses[END_REF]. Revenues are generated by assembling and adaptation services, as is the case for any traditional service company.

The only uncertainty about the model concerns the availability of the components: who will develop them and who will maintain them? Moreover, the customers of these companies may already have (proprietary) programs installed that need to be taken into account. In the end, an open-source strategy could even be a guarantee of means (maximum use of free software), but not a guarantee of the results (use of only free software), unless the customer requests this, since in this situation he keeps the last word.

Two kinds of firms use FLOSS today: newcomers who specialize in FLOSS architecture, using FLOSS as a vertical (price) and horizontal differentiation asset, and incumbents, such as IBM for its service activities29 . Traditional service firms like Cap Gemini are more agnostic with regard to the technologies used and the intellectual property regime involved. They will generally follow the customers' demand, which depends on their ability to keep up with the development of the project. These customers are most often large organisations, skilled computer users that are receptive to the opportunity to integrate the most advanced software components, developed under open licenses. So they are becoming increasingly involved in FLOSS as the market grows and matures30 .

Conclusion

In application markets, all the recent new entries have been based on the competitive advantage drawn from the FLOSS label: FLOSS OS publishers (like RedHat), FLOSS database producers (MySQL), FLOSS service companies (VA Linux, or Linagora in France).

Today, incumbents are also adopting this strategy (IBM with Eclipse, SAP opening its database system, even Microsoft opening some of its technical tools.) This could shortly become the benchmark of industrial organization on these markets, inducing a growing control of FLOSS development by commercial firms and a spectacular extension of open IP regimes in the software field.

In such conditions, the open IP regime can be seen as a very efficient solution to the Schumpeterian dilemma, insofar as it permits a wide diffusion of knowledge, while encouraging innovation, as producers are incited to contribute to the development of the product they use/sell.

Nevertheless, the origin of the open-source rationale remains that of developer-users pooling their development efforts for their own needs, aiming at better access to efficient tools for everyone. This is the core of the opensource model and, as seen in the paper, the recent move towards commercial structuring doesn't contradict this foundation. But, as seen before, it is clear that things function differently according to the place and type of the users in the different markets of the IT industry. As we have stressed, users play a critical role in the design of FLOSS strategies in the various market segments, both in the formation and specification of the demand and in their capacity to produce efficient feedbacks that contribute to raising the quality level of the products.

For a better understanding of how this works in a competitive context, we have developed a small competition model in which one open-source software provider competes with one proprietary software firm. We have focused on two polar market situations driven by contrasting distributions of skills among users. At one extreme, users are concentrated at a very low level of skills. There are no significant feedback effects and the open-source software firm, like the proprietary one, remains at a low level of product quality, is little involved in product development and finds a competitive advantage in terms of price. At the other extreme, the more skilled the users, the higher the quality level of the open-source product, the more the open source firms invests and makes profits. The situation described in the N-W quadrant will hold as long as l does not catch up m at level sL , then as long as λ cl (1+ τl ) < sL .

At the limit, when sl = sL = sm , the two firms have to sell at the same price and profit 0, sharing the market equally.

So the demand for each of the firms can be written

d = ١ ٢ ∫  ٠ ١ f  d = ١ ٢ [١-F  ٠ ] (8)
where θ 0 = sL / p and p= cL / d (the profit of m is equal to zero) so as θ 0 is the root of 2 cL θ 0 = sL (1 -F(θ 0 )).

The profit of l is equal to zero so that Πl = d p -cl ⇔ cl = cL

The condition sl = sL can then be written as a condition on the users feedback

 l = ١ ٢ ∫  ٠ ١  f  d = s L  c L -١ (9)
Where θ 0 has been defined above.

Of course, we have only focused here on the limit conditions.

Note that ( sL , sL ) is never a Nash stable equilibrium. At the equilibrium l always chooses sl < sL so as to avoid Bertrand competition.

For a given distribution f of the users' skills, if l has only quality aims, it can reach  s l =Max  l ٠ s l so that  l   s l =٠ .

When l, as commercial competing firm, aims to maximise its profit, it chooses sl * = Arg Max Π l ( sl ) / sl ∈ ] 0 ,  s l [ With a critical distribution f c when l choosing sl = sL , neither l nor m would make any profit.

So to maximize its profit level, l will choose sl * <  s l c <  s L , so that l and m make positive profits.

Depending on the value of λ, the above threshold can be more or less high. It is decreasing with λ and cL and increasing with sL . It is then also possible that m decides to choose the high quality before l has been capable to catch up. This will happen as soon as the expected profit for m with a low strategy for l is greater for a high than for a low quality.

The limit condition is obtained with equal profits, so that We assume that the user base improves its aggregate skill level following the criterion of stochastic dominance (7) shifting from a distribution f to a distribution g such that

∀   ∈ [0,1[ , ∫   ١  g  d  ∫   ١  f  d 
The consequences of such a shift is above all and by definition a growth of the users' feedback from any population [   , 1] , ∀   ∈ [0,1[ . Ιmproving sl tends to lower, ceteris paribus, the level of the indifferent user   , enlarge the l market base, increase its profit and enable it to lower its price pl and to reinforce its level of investment cl then reinforcing again the quality level sl until reaching a new competitive equilibrium ( sl g , sH ) where sl g > sl .

.References.

) 1 :

 1 Proposition At the equilibrium, if sl < sm then for any user of level θ choosing the open-source software, ∀ θ' < θ , θ' then chooses opensource software if sl > sm then for any user of level θ choosing the open-source software, ∀ θ' > θ , θ' then chooses opensource software

  17

  www.sun.com/hardware/serverappliances/eol.html

  22 http://www.linuxtoday.com/infrastructure/2007091902626NWDPPB 23 See, for instance, http://tuxmobil.org/ a web site dedicated to Linux and mobile computers. www.nokiausa.com/770/1,7841,feat:1,00.html. Development community: http://www.maemo.org/ Palm and Linux: http://news.com.com/2102-1041_3-6175171.html?tag=st.util.print. The web site dedicated by Palm to open source: http://www.palmsource.com/opensource/.

•

  The two opposite situations show how involvement of commercial firms in FLOSS is not contradictory with the essence of open-source rationale and can combine with the way communities operate to achieve the viability of an alternative economic model based on open knowledge. The FLOSS movement has sometimes been presented as a canonical model of production for the open innovation paradigm, and even for the knowledge society. If so, open development may develop in fields where users are skilled enough to initiate the development of open knowledge and have enough market power to force the traditional producers to shift to an open model. Open initiatives have been launched in many industries, such as biotech, remote sensing and chip design. Most of the time, their chances of success are evaluated in terms of the motivation of the participants and the stability of the "community". Our contribution argues for more economic aspects of the evaluation, by taking into account the impact of the users in these productions, and their bargaining power. .Appendix Proof of the proposition 1 θ adopts the open source software if and only if Ul (θ ) > Um (θ ) ⇔ sl θ -pl > sm θ -pm when sl < sm (and pl < pm ) ⇔  p m -p l s m -s l which remains true for any θ' < θ and symmetrically for sl > sm  Proof and technical remarks about the proposition 2

[١

  ١-F    L ] p L -c L =[١-F    H ] p H -c H (10)which also depends on the differential cH -cL . Proof of the proposition 3When f dominates the condition (11) above, l and m target high quality products. So m chooses sm = sH at cost cH and l invests cl and targets a quality sl > sH with sl = λ cl (1+ τl )where l = ∫   f d  and  = p l -p m s l -s HAt prices pl and pm are such that pl > pm . The payoffs can then be written l =[١-F   ] p l -c l and  m =[ F   -F  ٠ m ] p m -c H where  ٠

What we mean is that a player offers a standard by developing software that the other players can adopt and help to develop. This "unilateral" adoption is usually called 'bandwagon' in the literature on standards (see, for instance,[START_REF] Farrell | Standardization, compatibility and innovation[END_REF].

The main principle of the GPL (General Public License) is to make its adopters disclose the source-code of the programs concerned and of any further improvement if they circulate them, as well as the free circulation of the code under the sole condition of maintaining its "open" character.

This means that any developer wanting to make a contribution to the official MySQL product has to transfer her copyright to MySQL. http://forge.mysql.com/contribute/cla.php. Once owning the whole copyright, the firm can manage a dual licensing scheme, distributing the product under the license it wants, either GPL or a more classical closed license. So a customer that wishes not to reveal further enhancements of the source code has to pay for a closed license.

See the arguments developed by the Business Software Alliance (BSA), which regroups the main actors of the software sector, such as IBM, Microsoft, Apple, Sybase, etc. on http://www.bsa.org/, especially their latest white paper (August 2008, http://www.bsa.org/country/Research%20and%20Statistics/~/media/96FC7EAFF3E84436AF62C3B393F207B1.ashx).

See the arguments of the Free Software Foundation (http://www.fsf.org), orBoldrin and Levine (2007).

This distinction between specialized and generalist devices is evolving, as Sony intends its PS3 to be the media center at home. But so far, this has not impacted on the industrial structure.

People playing games, watching video films on their computer or using it for high performance professional purposes.

"Ellison saw that if Oracle played its cards right, the confluence of the database, the Internet, and the Web browser could displace the operating system as the focal point of computing and erode Microsoft's industry dominance". Brent Schlender, CNN Money, 1999. http://money.cnn.com/magazines/fortune/fortune_archive/1999/05/24/260276/index.htm

http://www.gartner.com/it/page.jsp?id=507466

In reference to the notion of "frontier-users" put forward by[START_REF] Kogut | Open Source Software Development and Distributed Innovation[END_REF]

See for instanceBlili, Raymond and Rivard (1998)

It is worth noting that SUN, on the contrary, being the leader on the UNIX market, has been reluctant to adopt Linux and is today the server constructor which has the most difficulties in adapting its business model, with recurrent losses.

Since the middle of

2007, Dell proposes Ubuntu Linux distribution on one of its lowest-price laptops (http://www.dell.com/content/topics/segtopic.aspx/linux_3x?c=us&cs=19&l=en&s=dhs), and since 2007 Asus has been selling an ultra-portable computer running Linux, called "eee PC", at less than €300. This has been the "most wanted 2007 Christmas gift", according to the constructor, http://eeepc.asus.com/global/.

In June 2008, Nokia announced to be acquiring the whole share of Symbian and open source it under Eclipse license. See the Symbian foundation Web site: http://www.symbianfoundation.org/

http://linuxdevices.com/news/NS2854558742.html

RedHat stopped this activity (see financial report 2006, p. 31); the consumer market (including distributors, OEM sales, ecommerce and Club) represented 2.54M€ (45% of the total earnings) showing a 23.4% decrease for Mandriva in the 2005-2006 fiscal year; SuSE has been bought by Novell, so these revenues are diluted.

As explained by[START_REF] Slatter | Gambling on Growth: How to Manage the Small High-Tech Firm[END_REF], one of the main strategies for newcomers in technological markets is technological differentiation. Basing its offer on new FLOSS products can be seen as a way for new service companies to differentiate.

In 2005, Gartner forecasted that « 2008, 95 percent of Global 2000 organizations will have formal open-source acquisition and management strategies » (http://www.gartner.com/DisplayDocument?doc_cd=125868)


Rossitza Atanassova

Noëlle Favet

Françoise Martz

Brigitte Chabbert

Marie-Thérèse Tollier

Bernard B Monties

Bernard Fritig

Michel Legrand

Noelle Fa Vet

Fran�ise Martz

Brigitte Chabbertz

Marie-Therese Tollier2

Bernard Monties2

Michel Lagrand1

Altered lignin composition in transgenic tobacco expressing O-methyltransferase sequences in sense and antisense orientation

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

Introduction

Lignin is the second most abundant organic compound on earth, after cellulose. It is a complex aromatic polymer which provides mechanical strength to plant cell walls and hydrophobicity to conducting vessels (Lewis and Ya mamoto, 1990;[START_REF] Monties | Lignins[END_REF] In addition, lignin deposi tion is thought to represent a defense reaction against pathogen invasion (Legrand, 1983;Matern and Kneusel, 1988;[START_REF] Nicholson | Phenolic compounds and their role in disease resistance[END_REF][START_REF] Vance | Lignification as a mechanism of disease resistance[END_REF]. However, lignin may have a negative impact on the utilization of the biomass [START_REF] Buxton | Lignin constituents and cell wall digestibility of grass and legume stems[END_REF][START_REF] Dean | Biotechnological modification of lignin structure and composition in forest trees[END_REF]Whetten and Sederoff, 1991). During paper pulping, for example, lignin must be eliminated from wood by chemical treatments that are expensive and polluting. The quantity of lignin in forage crop is also negatively correlated to digestibility and appetence [START_REF] Buxton | Lignin constituents and cell wall digestibility of grass and legume stems[END_REF][START_REF] Jung | Influence of lignin on digestibility of forage cell wall material[END_REF]. Therefore the modification of lignin content and/or composition by genetic manipulation would be of great economic interest.

The phenylpropanoid pathway provides the building units of lignin (Figure 1 ). Lignin is a complex polymer arising from the dehydrogenative polymerization of the three monolignols, namely p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol (Grisebach, 1981). The two later monomers bear, respectively, one and two methyl groups introduced by o-diphenol-0-methyltransferases (OMT; EC 2.1.1.6). OMTs of angiosperm species, were considered as bifunctional since the same enzymes methylate both caffeic acid and 5-hydroxyferulic acid in vitro to yield ferulic acid and sinapic acid, respectively (Figure 1). Recently, it has been suggested that another enzyme, caffeoyl-coA 0-methyltransferase, is implicated in lignin biosynthesis (Ye et al., 19941 and the analysis of lignin from transgenic tobacco plants that we describe here, supports this hypothesis. After synthesis of the respective coA esters by coA ligases, reduction steps catalyzed by cinnamoyl-coA reductase (CCR) and cinnamyl alcohol dehydrogenase (CADI lead to the corresponding alcohols. Consequently, angiosperm lignin contains both guaiacyl (coniferyl-derived) and syringyl (sinapyl-derived) units. This is in contrast to the situation in gymnosperm species, whose lignin is mainly constituted of guaiacyl units because their OMTs do not accept 5-hydroxyferulic acid as substrate [START_REF] Kuroda | Purification and properties of 0-methyltransferase involved in the biosynthesis of Gymnosperm lignin[END_REF]. Hence, OMTs are regulatory enzymes controlling lignin content and com position.

To bacco has been shown to possess two classes of OMTs differing by substrate specificities and expression patterns (Hermann et al., 1987;[START_REF] Jaeck | Regulation of enzymes involved in lignin biosynthesis: induction of 0-methyltrans ferase mRNAs during the hypersensitive reaction of tobacco to tobacco mosaic virus[END_REF][START_REF] Legrand | 0-diphenol 0methyltransferases of healthy and tobacco-mosaic-virus infected hypersensitive tobacco[END_REF][START_REF] Pellegrini | Molecular cloning and expression of a new class of ortho diphenol-0-methyltransferases induced in tobacco leaves by infection or elicitor treatment[END_REF]. Class I OMT is highly expressed 6, p-coumaroyl-coA 3-hydroxylasei 7, caffeoyl-coA 0-methyltransferase; 8, cinnamoyl-coA reductase; 9. cinnamyl alcohol dehydrogenase.

in lignified tissues whereas class II OMTs are barely detect able in healthy plants but are strongly induced upon infection, Class I enzymes, specifically involved in lignin biosynthesis, have been cloned recently from several plant species [START_REF] Bugos | cDNA cloning, sequence analysis and seasonal expression of lignin bispecific caffeic acid/5-hydroxyferulic acid 0-methyltrans ferase[END_REF]Collazo et al,, 1992;[START_REF] Dumas | Nucleotide sequence of a complementary DNA encoding 0-methyltransferase from poplar[END_REF][START_REF] Gowri | Stress responses in alfalfa (Medicago sativa L.1.X. Molecular cloning and expression of 5-adenosyl-L methionine:caffeic acid 3-0.methyltra nsferase, a key enzyme of lignin biosynthesis[END_REF][START_REF] Poeydomenge | A cDNA encoding S-adenosyl-L-methionine : caffeic acid 3-0. methyltransferase from Eucalyptus[END_REF] and a class II OMT clone has been isolated from tobacco [START_REF] Pellegrini | Molecular cloning and expression of a new class of ortho diphenol-0-methyltransferases induced in tobacco leaves by infection or elicitor treatment[END_REF].

Mutants lines of maize, sorghum and pearl millet have been shown to have a reduced lignin content and better digestibility (Cherney et al., 1991). These mutants are called brown midrib (bm) mutants because they show a characteristic brown pigmentation of lignified tissues. It has been shown that the bm 3 maize mutant has a reduced OMT activity [START_REF] Grand | Comparison of lignins and of enzymes involved in lignification in normal and brown-midrib (bm3) mutant corn seedlings[END_REF] due to alterations of OMT gene [START_REF] Vignols | The brown midrib3 (bm3} mutation in maize occurs in the gene encoding caffeic acid 0methyltransferase[END_REF]. The bm 6 mutant of sorghum bicolor has a depression in both OMT and CAD activities [START_REF] Pillonel | Involvement of cinnamoyl-alcohol dehydrogenase in the control of lignin formation in Sorghum bicolor L. Moench[END_REF]. Recently, an Arabidopsis mutant was

shown to be affected in the ferulate 5-hydroxylation step and to have an altered lignin composition [START_REF] Chapple | An Arabidopsis mutant defective in the general phenylpropanoid pathway[END_REF]. These findings suggest that a modulation of the activity of lignin biosynthetic enzymes by genetic engineer was achieved, the lignin composition was strongly modi fied. To tal lignin content was not significantly affected but the novel lignin had a remarkably decreased content in syringyl units and contained a new building unit derived from 5-hydroxyferulic acid (one of the OMT substrates, see Figure 1). These features are also characteristic of lignin of bm3 mutants of maize [START_REF] Lapierre | Mise en evidence d'un nouveau type d'unite constitutive dans les lignines d'un mutant de mals bm3[END_REF] and the findings presented here demonstrate the suitability of OMT genes as targets for genetic manipulation of lignin quality.

Results

Comparative efficiencies of the different OMT constructs

The expression of several plant genes has been efficiently and specifically decreased by expressing their antisense counterpart (for review see have been used successfully. In other experiments, the expression of transgenes in sense orientation has also been shown to lead to the silencing of the endogenous gene (Flavell, 1994;[START_REF] Matzke | Genomic imprinting in plants : parental effects and trans-inactivation phenomena[END_REF]Napoli eta/,, 1990).

Here, we used five different fragments of class I OMT cDNA (depicted in Figure 2) in sense or antisense orienta tion to modulate OMT activity in transgenic plants and to study, in turn, the consequences upon lignin biosynthesis,

The T-DNA of the pFB8 binary vector (Figure 2) also carried the �-glucuronidase (uidAI gene that served as an internal control gene with no homologous sequence in the plant genome. Tw enty independent transformants selected on kanamycin medium were regenerated in each case.

Twe nty-one untransformed regenerants were also grown under the same conditions (but without kanamycin selec tion) and constituted the control population.

In fact, to evaluate the potential effects of gene trans formation on endogenous gene expression, it is necessary to know the natural variability of gene expression in the control population. It has been reported previously that the basal level of OMT activity was highly variable in healthy tobacco leaves [START_REF] Legrand | 0-diphenol 0methyltransferases of healthy and tobacco-mosaic-virus infected hypersensitive tobacco[END_REF]. Class I OMT which is involved in lignin biosynthesis, has been shown to be specifically expressed in lignified tissues of stems [START_REF] Jaeck | Regulation of enzymes involved in lignin biosynthesis: induction of 0-methyltrans ferase mRNAs during the hypersensitive reaction of tobacco to tobacco mosaic virus[END_REF] 10 -5 nkat µg-1 was found which was not significantly different as compared with untransformed plants. There fore, the activity measured for each individual transformant was compared with the activity mean value calculated from the untransformed population.

OMT activities were measured in populations of 20 plants transformed with each of the 10 constructs depicted in Figure 2. The distribution of the values was found to be Normal as in the case of control population (Figure 3) and ";!<. :.;. ingly, in the progeny of parents with strongly modulated

�200 � "> -� 142 I- � 100 -• --1--•••-.... ---� -------------------- CD >
OMT activity, a number of plants displayed similar changes in enzyme activity (data not shown). Taken together, these results demonstrate that the transgenes were stably inte grated in the plant genome and are transmitted to the progeny.

The steady-state level of endogenous and transgene mRNAs was analyzed by Northern blot. For this purpose, we used three types of probes with different specificities (Figure 6).

(i) A probe derived from OMT cDNA hybridizing to sense or antisense transcripts from the OMT transgene and also to those from the resident OMT gene.

(ii) A probe derived from the 35S polyadenylation signal specific to the OMT transgene transcripts.

(iii) A probe synthesized from the uidA gene.

The level of uidA transcripts was used as an internal control of T-DNA expression with no interference with the expression of plant homologous sequences. Finally, hybridization was carried out with a 255 rRNA probe to ensure that the same quantity of total RNA had been loaded on the gel for each sample (data not shown). reduced CAD activity [START_REF] Halpin | Manipulation of lignin quality by down-regulation of cinnamyl-alcohol dehydrogenase[END_REF] and in natural mutants of maize and sorghum [START_REF] Kuc | The abnormal lignins produced by the brown-midrib mutants of maize[END_REF][START_REF] Porter | Phenotype, fiber composition, and in vitro dry matter disappearance of chemically induced brown-midrib (bmr) mutants of Sorghum[END_REF]. Transverse sections from the stem of plants exhibiting a wide range of OMT levels were stained with Wiesner and Maule solutions. Wiesner stain is known to react with cinnamaldehyde residues in lignin and the color intensity roughly reflects the total lignin content (Monties, 1989). No difference was detected with this stain between control and inhibited plants (Figure 9a andb)

indicating no important variation in lignin quantity. In contrast, the MaOle reaction, which is specific for free syringyl units (Monties, 1989), revealed striking variations in staining intensity (Figure 9c-h). Xylem and phloem tissues of control plants (Figure 9c and el and of an overexpressing plant (Figure 9g) stained bright red.

In inhibited plants a good correlation was found between the intensity of the red stain and the level of OMT residual activity: the red color turned orange and yellow when OMT decreased (compare Figure 9d, f and h from plants Bia, Bi o and A17 which had 12%, 8% and 2% residual activity, respectively, Ta ble 1). These data strongly suggest that OMT inhibition markedly decreased the syringyl content of lignin without significantly affecting the amount of total lignin.

Chemical analysis of transgenic lignin

To confirm the above observations lignin from control and transgenic plants was analyzed by the thioacidolysis method. This procedure has the major advantage of pre serving the C6-C3 skeleton characteristic of lignin mon omers (Monties, 1989) and discriminates between products recovered from guaiacyl (G) and syringyl (5) units involved in �-0-4 bonds [START_REF] Lapierre | Thioacidolysis of poplar lignins: identification of monomeric syringyl products and characterization of guaiacyl-syri ngyl rich fractions[END_REF]. Both unit types being major constituents of angiosperm lignin, their molar ratio

(5/G) is a characteristic feature of lignin. Figure 10 shows typical gas chromatography profiles obtained from a con trol 1Ti2l and a transgenic plant (Bil which had only 6% residual OMT activity. In the inhibited plant a sharp decrease in the syringyl peak and the appearance of a new peak corresponding to the 5-0H guaiacyl unit [START_REF] Lapierre | Mise en evidence d'un nouveau type d'unite constitutive dans les lignines d'un mutant de mals bm3[END_REF] were observed as compared with the control profile. Most interestingly, the same situation has been reported in a bm3 natural mutant of maize [START_REF] Lapierre | Mise en evidence d'un nouveau type d'unite constitutive dans les lignines d'un mutant de mals bm3[END_REF] which had a low level of OMT activity [START_REF] Grand | Comparison of lignins and of enzymes involved in lignification in normal and brown-midrib (bm3) mutant corn seedlings[END_REF]. Several transformed and untransformed plants with varying levels of OMT activity were analyzed and the data are presented in Table 1. The total amount of lignin was estimated by Klason's method and found to be similar in control and transgenic plants. Thus, as already suggested by our observations with Wiesner's stain (Figure 9), OMT inhibition did not significantly affect lignin quantity.

Chemical analysis also confirmed that the syringyl unit biosynthesis is profoundly affected by OMT inhibition.

Indeed, the S/G ratio measured in transgenic lignin of the most inhibited plants is less than one-tenth of the value determined in control plants. The four control plants were chosen because they were representative of natural fluctuations in OMT activity. Their S/G ratio was found to be rather constant and close to 1 (Table 1 ). It is also noteworthy that an increase in OMT activity due to over expression of the OMT gene in plant A2, had no significant effect on lignin composition, indicating that 0-methylation is not a rate-limiting step in lignin monomer biosynthesis.

When 5/G values measured from plants showing a range of OMT reduction were compared, a direct correlation between the level of OMT inhibition and the amplitude of the effects on lignin synthesis was found: when OMT activity reached 56% of the control mean value (plant B17,

Table 1) no effect on S/G and no 5-0H G units were observed; when OMT activity fell to 12% of the control (plant Bia) the S/G value was decreased by threefold and a significant amount of 5-0H G was detected; finally, for residual activity of 8% or less (plants Bi0, Bi and A17) S quantities were strongly reduced, S/G ratios were very low and the highest amounts of 5-0H G were measured.

Moreover, the quantities measured for 5-0H G and S units were negatively correlated and this is in accordance with expectation for derivatives of, respectively, the substrate and the product of an GMT-catalyzed reaction (see Figure 1).

In contrast to the steep decrease in S unit content, G content was not reduced in transgenic lignin, indicating that the first methylation step leading to ferulic acid was not inhibited. On the contrary, slightly higher amounts of G unit were measured in several plants. A similar observa tion has been made in the bm3 mutant of maize [START_REF] Lapierre | Mise en evidence d'un nouveau type d'unite constitutive dans les lignines d'un mutant de mals bm3[END_REF] and represents another feature common to lignin of engineered tobacco and of natural maize mutants.

Ta ken together, these data demonstrate th at the modulation of OMT activity in transgenic plants influences lignin com position directly as already observed for natural mutations. For each degradation product, erythro and three isomers are observed. antisense transformant (out of seven) with 20% residual OMT activity compared with controls and with a reduced lignin content but no change in lignin composition. We never found a significant decrease in lignin content of transformants, even in plants with only 2-3% residual enzyme activity (Table 1 ). On the other hand, Dwivedi et al.

Discussion

( Indeed, our data show that transgenic lignin resembles the lignin of mutants lines when compared with lignin of wild plants. Hence, one may assume that similar perturbations affect lignin synthesis in both cases. Therefore, one can expect to increase by genetic engineering the economic value of crops whose digestibility could be improved, as is the case for natural mutants of maize. Similarly, the alteration of lignin composition of woody species and particularly, the decreased content in methoxyl groups which give rise to polluting mercaptans during the pulping process, would be of great economic and ecological interest. On the other hand, the reduced proportion of syringyl unit in transgenic lignin is likely to lead to a more condensed polymer very rich in carbon-carbon linkages (Monties, 1989). Transgenic tobacco will enable us to test whether the synthesis of such a modified lignin offers a better protection to the plant against pathogen infection.

Plant transformation and regeneration

To bacco (Nicotiana tabacum cv. Samsun NN) was transformed by a modification of the leaf disk method [START_REF] Horsch | A simple and general method for transfering genes into plants[END_REF].

Murashige and Skoog IMSI medium was used with an NH4N03 concentration lowered twofold. Kanamycin (Sigma) at 150 mg m1-1 and cefotaxime (Duchefa) at 350 mg m1-1 were used as selective agents during the in vitro regeneration and propagation procedures. Calli formation and shoot differentiation were carried out on MS medium supplemented with 6-benzylaminopurine (Serva} (2 mg m1-11 and naphthalene acetic acid (Serva} (0.05 mg ml -\ Rooting was obtained on MS medium with half sucrose concentration (15 g 1-11. Transformed plants were grown in vitro for 5 weeks under a light-dark regime of 12 h (20 -30 µE m-2 sec'. 24"C}-12 h (20°C}, then the basal part of the stem was assayed for OMT activity, and the upper part was micropropagated under the same conditions. For each construct, 20 independent transformants were regenerated, as well as 21 untransformed plants as controls (without kanamycin selection}.

Once tested for OMT activity, subcultured plantlets of the clones of interest were transferred to the greenhouse and cultivated in soil under a light-dark period of 16 h/8 h, 22:t4°C, till flowering.

Plants were allowed to self-pollinate and F, seeds were harvested and further selected on germination medium containing kanamycin.

Enzym e assays

OMT and GUS assays were conducted on 5-week-old plantlets regenerated in vitro. The bottom of the stem ( 1 cm high} was ground in liquid nitrogen, extracted with 100 mM sodium phos phate buffer pH 7.5, 10 mM OTT (2/1 v/w}, with the addition of polyclar AT (Serva) and quartz. The crude extract was clarified by centrifugation and filtered on glass wool. For OMT assay, 50 µI of the protein extract was added to 1 ml phosphate buffer containing 3 mM caffeic acid, 50 µM tritiated S-adenosyl-L-methionine (0.1 µCi ml-1) and incubated for 1 ha t 37°C in scintillation vials.

The reaction was stopped with 100 µI 9 M H 2 S04, 5 ml NA scintillation solution (Beckman) was mixed in by vortexing for 45 sec, and the radioactivity of ferulic acid extracted by the scintillation solution, was counted in a Beckman LS 9000 appar atus. When OMT activity was measured on adult plants, an additional step of exclusion chromatography on PD-10 columns (Pharmacia) was performed to eliminate the endogenous o diphenolic substrates. GUS fluorimetric tests were carried out on the same samples according to the procedure of [START_REF] Jefferson | GUS fusions: �-glucuronidase as a sensitive and versatile gene fusion marker in higher plants[END_REF]. The protein content was determined by the method of [START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein-dye binding[END_REF] using the Bio-Rad reagent.

RNA analysis

RNA was isolated from 1-2 g frozen plant material by phenol extraction [START_REF] Howell | Replication of cauliflower mosaic virus and transcription of its genome in turnip leaf protoplasts[END_REF] followed by selective precipita tion with 4 M LiCI (Verwoerd et al., 1989). For Northern blots, RNA samples (10 µg) separated on formaldehyde gels were transferred on to Hybond N filters (Amersham} and hybridized to randomly primed radiolabeled probes synthesized from: (i} 1217 bp OMT cDNA; (ii} 230 bp 35S polyadenylation signal; (iii) 1800 bp uidA coding region; (iv) 300 bp ribosomal 25S gene (as a control of the amount of RNA loaded). mRNA was quantified on Northern blots using a Fujix Bio-Imaging Analyzer (BAS 1000 MacBas).

Histology

Stem sections were hand cut with a razor blade from the 19th or 20th internode of plants grown for 3 months in the greenhouse, and treated with hot ethanol to eliminate tissue pigmentation.

Wiesner and Maule reactions were performed according to standard protocols (Strivastava, 1966). For MaOle staining, stem sections were immersed for 5 min in 1% KMn04, rinsed, destained in 30% HCI, washed and mounted in concentrated NH40H. For the Wiesner reaction, stem sections were incubated for 2 min in phloroglucine solution (2% in ethanol/water 95/5 v/v}, then mounted in 50% HCI. Bright field photographs were taken at sevenfold magnification with a Schimadzu SMZ-U 300m (Nikon) binocular microscope.

Chemical analysis of lignin

Thioacidolysis method and product separation by gas chromato graphy have been described previously [START_REF] Lapierre | Thioacidolysis of poplar lignins: identification of monomeric syringyl products and characterization of guaiacyl-syri ngyl rich fractions[END_REF][START_REF] Lapierre | Mise en evidence d'un nouveau type d'unite constitutive dans les lignines d'un mutant de mals bm3[END_REF].

Figure 1 .

 1 Figure 1. OMT-catalyzed reactions in the general phenylpropanoid pathway. Nomenclature of units obtained after chemical degradation by thio acidalysis is indicated in brackets. Italics show the pathway from 5-0H ferulate to 5-0H guaiacyl unit characteristic of transgenics with reduced OMT activity. 1, phenylalanine ammonia-lyase; 2, cinnamate 4-hydroxylase; 3, p-coumarate 3-hydroxylase; 4, ferulate 5-hydroxylase; 5, coA-ligase;

  ing has the potential to change lignin biosynthesis in transgenic plants. A few attempts to repress OMT or CAD genes of tobacco by expressing antisense RNAs have been reported recently. Halpin et al, ( 1994) have demonstrated that transgenic tobacco with low CAD activity have lignin modified by the incorporation of cinnamyl aldehydes, The effects of antisense expression of a lucerne OMT gene (Ni et al., 1994) and of an aspen OMT gene (Dwivedi et al,, 1994) on lignin biosynthesis of transgenic tobacco plants have also been investigated. The results of the two studies differed considerably from ours (see below), perhaps due to the heterologous nature of the cDNAs introduced in tobacco, Expression of the lucerne sequence led to a reduced lignin content without affecting monomer com position (Ni et a/,, 1994) whereas expression of the aspen sequence was claimed to slightly change lignin composi tion of transgenic tobacco (Dwivedi et al., 1994). In this paper we have investigated the possibility of downregulating class I OMT expression in tobacco by expressing different parts of the homologous sequence in sense and antiserise orientation, A total of 200 plants transformed with 10 different constructs were analyzed for OMT activity and compared with the control population. OMT modulation was obtained with every construct tested but with highly variable effici ency, as evidenced by the varying proportions of plants with elevated or reduced activity in the different transgenic populations. Inhibition was sustained through the adult stage only in plants transformed with the complete cDNA and, when a high level of OMT inhibition (less than 20% residual activity)

.Figure 2 .

 2 Figure 2. The pFBB binary vector and the sense and antisense constructs containing different fragments of OMT cDNA.

  and therefore we have measured OMT activity in the basal part of the stems of control plants. The control population consisted of 21 5-week-old untrans formed, in vitro regenerated plants. The data are presented in Figure 3 and show important fluctuations in enzyme activity levels. The distribution of the values was proven to be Normal by Probit analysis, with a mean value of 2.7X10-5 nkat µg-1 protein and a standard deviation of 1.2x 10 -5 nkat µg-1 protein. Plants transformed with an empty plasmid (without OMT insert) were also generated and analyzed. An enzyme activity value of 3.7 ::+:: 1.7x

�

  Figure 3. OMT activity in untransformed control plants. The activity value of each individual is expressed as the percentage of the mean value of the population !dashed line). The two solid lines indicate the s1andard deviation.

00 Figure 5 .

 005 Figure 5. Comparison of OMT activity between 5-week-old plantlets (solid barsl cultured in vitro and the corresponding adult plants (shaded bars) grown for 3 additional months in the greenhouse.

Figure 6

 6 Figure 6 presents the data obtained for A and B plants which displayed OMT alterations at the adult stage. The 35S terminator probe allowed specific detection of the transcripts of the OMT transgene only in some trans formants, those with a high level of OMT activity arising from OMT gene overexpression (plants A2, A 5 , As, A7l. As expected, no signal was detected in untransformed controls IT plants), but more surprisingly, a very weak signal or no T4 Al A5 A6 A7 A17 Bl BlO B18 BU B17 T8

Figurm 4 .

 4 Figurm 4. OMT activity in sense and antisense transgenic plants transformed with the different constructs shown in Figure 2. En2yme activity was measured in 5-wee k-old, in virro cultured plantlets, and expressed as a percentage of the mean value of the control population. Hori20ntal lines correspond to the standard deviation of the OMT values of control population. The shaded letters indicate the populations which significantly differ from the control.

Figure 7 .

 7 Figure 7. Relationship between OMT inhibition level, OMT mRNA and uidA mRNA amounts in stem tissue of antisense-inhibited trsnsformants. mRNA was quantified by profile analysis of the Northern blots (Figure 61 using a Bio-Imaging Allalyzer. Mean value from four control plants (T) was taken as 100% OMT mRNA. In B plants (Figure 6, upper panell, endogenous and transgene OMT tnmscripts could be discriminated because the latter is longer, due to the presence of an additive polyadenylation signal {see Figure 21. uidA mRNA level was the highest in B1 plant and was taken as 100% value.

Figure 10 .

 10 Figure 10. Gas chromatograms showing the main degradation products recovered from thioacidolysis of lignin of a control plant �T12) and an antisense transformant (81) with very low residual activity.

  ) obtained one transformant (out of four) with 54% residual OMT activity, which displayed no change in lignin content and had a SN ratio of 0.77. This is 10-fold higher than the 0.07 value determined in the present study for the most inhibited transgenics (Table11. The spectacular decrease we found in S/G ratio demonstrates unequivocally the effects of OMT manipulation on lignin composition. Moreover, the thioacidolysis method enabled us to demon strate the accumulation of 5-0H G. This component cannot be detected with the alkaline nitrobenzene oxidation pro cedure used in the other studies (Owivedi et al., 1994; Ni et al., 1994). The study of plants with varying degrees of inhibition enabled us to establish a direct correlation between the quantity of OMT mRNA detected on blots and the level of residual enzyme activity. Moreover, in stems of inhibited lignin in adult transgenic and control plants with different levels of OMT activity (% OMT). Total lignin content was estimated by Klason's method and is expressed as percentage of cell wall residues (nd, not determined). Monomeric composition of lignin was determined after thioacidolysis: G, guaiacyl unit; S, syringyl unit and 5-0H G, 5•0H guaiacyl unit. Unit amounts are expressed as µmoles per gram of Klason lignin.

Figure 9 .

 9 Figure 9. Histochemical reactions on transverse stem sections of control and transgenic plants. Wiesner's staining was performed on: (al a control plant (T12); (b) an antisense transformant (87) with reduced activity. Maule's staining was performed on: {c} and le) control plants IT19 and T11, respectively); (di and (fl antisense transformants with different levels of OMT inhibition {818 and 810, respectively}; {g) a sense overexpressing transformant IA21; lhl a sense inhibited transformant (A17).

Finally

  techniques were performed essentially as described bySambrook et al. (t989). The binary vector pFB8 was derived from pBI 101 ![START_REF] Jefferson | GUS fusions: �-glucuronidase as a sensitive and versatile gene fusion marker in higher plants[END_REF]. The 420 bp fragment of CaMV 35S RNA promoter corresponding to bp 7016--7436 of CM 1841(Gardner et al., 1981) was inserted at the Smal site in front of the uidA gene to produce a transcriptional fusion. A 1.2 kb Hindlll fragment of pRT99 (Tepfer et al., 1988) carrying the CAT gene flanked by the 420 bp CaMV 35S promoter and terminator sequences was inserted at the Hind lll site of the pBI 101 polylinker. The CAT coding region has been deleted as a BamHI fragment and the resulting vector pFB8 (Figure 21 carried three cassettes from the right to the left border of T-DNA: Ii) the npt-11 gene for kanamycin resistance; Iii) the cassette for sense or antisense expression controlled by 355 5' and 3' sequences; (iii) the uidA gene, also driven by the 35S promoter, as internal control. The class I OMT cDNA fragment was isolated from a pKS II plasmid (Stratagene) containing the tobacco class I OMT cDNA, inserted at the EcoRI site. Different fragments of OMT I cDNA, depicted in Figure 2, were used: (i) a full-length cDNA 1457 bp (EcoRV-SmaU; (ii) a 1217 bp IHindlll-Hind lll) segment lacking the 5' untranslated region, the first 18 codons of the ORF, and 94 bp from the extreme 3' untranslated region; !iii) a 710 bp (EcoRl-Hincll) fragment corresponding to the 5' half of the cDNA; (iv) a 430 bp (H in cll-EcoRI) fragment corresponding to the 3' part of the ORF; (v) a 300 bp IEcoRl-EcoRI) fragment carrying the lastthree codons of the ORF and the 3' untranslated region. All five DNA fragments were purified, blunt ended by filling in with the Kienow fragment of DNA polymerase I and ligated to the Smal site of pFB8. The orientation of the inserts was determined by restriction digestion of plasmids recovered from transformed Escherichia coli DH5a.The binary vectors were transferred to Agrobacterium tume fa ciens LBA4404 by the triparental mating procedure with pRK2013 {in E. coli HB101) as helper plasmid {Bevan, 1984). The integrity of the vectors was verified by analyzing total DNA from Agro bacterium according to the method of Dhaese et al. {1979).

Table 1 .

 1 Chemical analysis of lignin

	Plant	o/oOMT	Klason
	A2	322	nd
	T12	191	nd
	T11	172	17.7:!:0.09
	T19	80	19.1 :!:0.09
	T7	69	nd
	817	56	nd
	818	12	18.7:!:0.05
	810	8	18.8:!:0.03
	81	6	17.6±0.03
	A17	2	18.5±0.04

transgenic plants should prove to be valuable tools to investigate regulation mechanisms controlling lignin bio synthesis. It should be stressed that the data of the present work contradict most of those obtained by introducing an heterologous OMT sequence into tobacco plants (Owivedi et al., 1994; Ni et al., 19941. Ni et al. (1994) obtained one

Acknowledgments

We thank Dr K. Richards for careful reading of the manuscript.

We are grateful to P. Keltz and R. Wagner for taking good care of tobacco plants. This work was supported by a grant from the Commission of European Communities (ECLAIR-OPLIGE AGRE 0021).