• sparse or 'culminative' tone systems

• in which no more than one pitch pattern is pronounced per word (or longer phonological unit)

• situated within a continuum between non-tonal stress languages and tonal languages without lexical stress (cf. Hyman 2001:1377)

• hybrid tone systems

• lexical tone on monosyllabic domains; simple tone inventories (basic tonal categories H & L)

• small number of tonal patterns on polysyllabic domains

• the connection between lexical tone on monosyllabic words and tonal patterns on polysyllabic words is seen in terms of autosegmental spreading rules, that involve tone spread from its pre-specified location (mostly at the left edge of the prosodic word) rightwards, determining all or most of the tonal melody of the word (e.g. Evans 2008, Evans & Huang 2007) 韵律系统 Evans ( 2008)

单词结构

• Lizu morphemes are generally monosyllabic but words are generally disyllabic (over 80% of the collected vocabulary)

• monomorphemic words are for the most part monosyllabic, e.g. / dʒě/ 'water'

• polysyllabic words are mostly composite, e.g. /dʒê-kʰwae/ 'flood' (from /dʒě/ 'water', /kʰwae/ 'be large'), /tôNbu-mu/ 'nose hair'

(from /tôNbu/ 'nose', /mû/ 'animal hair, fur')

• there is also a handful of disyllabic and trisyllabic monomorphemic words, e.g. /mîdzə/ 'hare, rabbit'

单⾳节词

• at monosyllabic level, Lizu distinguishes two lexical tones: rising and high falling

• the two tones are fairly evenly distributed in the lexicon

• this is a robust opposition, supported by many minimal pairs

• tones are fully independent: any vowel may bear any tone and all consonants, including sonorants and voiced obstruents, can be associated with both tones HH LH

双⾳节词

• in disyllabic words, three pitch patterns are observed

• fairly evenly distributed in the lexicon 1. both syllables have a mid-to high-level pitch contour, though the first is slightly higher than the second, and the second syllable has a falling pattern, as in /mîdzə/ 'hare', /səNɡê/ 'lion' (WT seng ge), /mêlŷ/ 'thrush', and /nôNbû/ 'brain' 2. the f0 peak is typically realized before the end of the first syllable, where the pitch starts to fall and continues to fall in the second syllable, as in / mîdzə/ 'pepper', /mêlje/ 'place', and /tôNbu/ 'nose'

3. within the first syllable, there is a mid-level pitch contour with a slight rise. The f0 peak is realized within the second syllable where there is also a clear fall, as in /mutsə/ 'cat', /meljê/ 'wind', and /nɐNbô/ 'deaf person'

HH HL LH

• in contrast to the two lexical tones on monosyllabic words, the threeway contrast on disyllabic words is not supported by minimal sets • many pairwise minimal pairs 名词 动词 两个⾳节以上的词

• in tri-and tetra-syllabic words (both monomorphemic and polymorphemic): three common pitch patterns, comparable to the ones over disyllabic domains:

1. HHH: The pitch contour over all syllables remains mid or high level until the last syllable where there is a clear falling pattern, as in / ʃêNbêljê/ 'buttocks' 2. HLL: There is a rising contour on the first syllable, the peak of which varies slightly depending on the length of the word. For a three-syllable word, the peak is typically realized at the beginning of the second syllable, while for a four-syllable word, the peak is further delayed within the second syllable, after which there is a clear falling contour till the end of the word, as in /tɕjɐNtʃʰokɐ/ 'north' (WT byang phyogs)

3. LLH: The f0 peak is realized within the last syllable and the preceding syllables show a mid-level f0 contour, as in /ɲilɐɡû/ 'daytime'

HHH HLL LLH

• no contrastive minimal sets for the three patterns Phonological Word & Phonological Phrase

• a phonological word in Lizu is a unit equal to or larger than one syllable, which, when larger than one syllable, is subject to the (limited) process of vowel assimilation phonological words in Lizu coincide with grammatical words => a grammatical word is a grammatical unit between morpheme and syntactic phrase, which consists of a number of grammatical elements which always occur together, in a fixed order, and have a conventionalized coherence and meaning (Dixon & Aikhenvald 2002:35)

• a phonological phrase is a unit consisting of two or more grammatical words

• its defining characteristic in Lizu is that it can be pronounced within two pauses in an utterance and can host one of the three pitch patterns on polysyllabic domains

• in Lizu, the level of the phonological word and that of the phonological phrase are indistinguishable in terms of tone patterns

连读变调

• dynamic tone combinations

• in compounds, lexical tones undergo sandhi changes

• compounds and phrases beginning with a noun noun-noun, noun-verb, noun-classifier, noun-clitic

• the synchronically productive pattern as attested on syntactic compounds is one in which only the tonal contour of the initial word is retained and realized over the whole compound domain

连读变调

• if the domain-initial monosyllabic root or word has the rising tone, the resulting pattern is generally L(…L)H

• if the domain-initial monosyllabic root or word has the falling tone, the resulting pattern is generally H(…)H

• in domains that begin with a disyllabic root or word, the resulting contours are determined by the tonal contour of the initial disyllabic item

名+名 名+名 HL L > LLH H > HHH

名+动

• tone patterns of noun-verb compounds are derived in the same fashion as those of noun-noun compounds: the tone of the domain initial word spreads over the entire domain, whereas the tonal contrasts of all remaining words are neutralized /jê/ 'tobacco' + /tɕʰê/ 'to drink' > /jê tɕʰê/ 'to smoke' /bɹae/ 'thick rope' + /ɣǒ/ 'to twist > /bɹae ɣô/ 'to twist a rope' /waeɹae/ 'cloth' + /Ntsʰae/ 'to make' > /waeɹaê Ntsʰae/ 'to weave, to knit'

[fumae] 'tooth' + /tsě/ 'to pull up, to pull out' > /xumae tsê/ 'to pull out (teeth)'

总结

• the synchronically productive pattern: only the tonal contour of the initial word is retained and realized over the whole compound domain

• different tonal contrasts on the monosyllabic level (2 lexical tones) and on the level of words of over one syllable (3 contrastive patterns on disyllabic words) HL Tonal Pattern

• lexicalized compounds (high-frequency, disyllabic noun-verb compounds)

ɣǒ 'wine' + tɕʰê 'to drink' > ɣô tɕʰe 'to drink wine; wine-drinking'

• recent loanwords from Mandarin:

kwâtsə 'seeds', SW Mandarin /kwa⁴⁴tsə⁵³/

• tone change in compounds lêmae 'finger' > lemaê je-je 'little finger' kʰêNtsʰae 'remember' > kʰeNtsʰaê pʰaě 'be able to remember'

分析

• structure of the lexicon: Lizu morphemes (lexical and grammatical) are generally monosyllabic, but words are generally disyllabic

• preference in Lizu for syllables being grouped into binary units

• the binary preference is an instance of a strong crosslinguistic bias found in stress systems

• possibility for an analysis of Lizu as combining lexical tone and stress

• an analysis allowing for the contextualization of the phonetic properties of stress in Lizu relative to the broader literature on acoustic correlates of prominence

假设

• Lizu is a tonal language, which uses pitch contrastively to distinguish different, otherwise identical morphemes

• in contrast to omnisyllabic tone languages (such as Sinitic languages), which, according to the classical definition by Pyke (1948: 3), have "lexically significant, contrastive […] pitch on each syllable", the distribution of tones in Lizu is not entirely unrestricted

• a tone language in which every morpheme is potentially specified for tone, but in which the surface position of those tones may be determined (at least in part) by the type of positionally driven rhythmic structure typical of stress languages

重⾳

• stress (Hyman 2009: 217): a language with stress-accent is one in which there is an indication of word-level metrical structure meeting the following two central criteria A. obligatoriness: every lexical word has at least one syllable marked for the highest degree of metrical prominence (primary stress) B. culminativity: every lexical word has at most one syllable marked for the highest degree of metrical prominence C. stress-bearing unit is the syllable

• stress is a relative property arising from a relationship between two (or more) syllables, put differently, syllables are perceived as stressed only by virtue of their relationship with nonstressed syllables

表达重⾳的重要线索

• in stress languages, such as English, the principle auditory cues that correlate with word stress duration, intensity, and vowel quality (e.g. Fry, 1958;Bolinger, 1961; Gordon 2016)

• stressed syllables tend to have greater intensity, longer duration, and full (nonreduced) vowel quality, compared with nonstressed syllables

• stressed syllables also tend to have higher pitch (fundamental frequency, F0)

⾳⾼与重⾳

• van der Hulst (2014: 7): "While it was originally thought that pitch properties were an important part of the set of stress exponents [...], it has been argued that this was often an illusion, arising from the fact that stressed syllables of words 'in focus' position function as anchors for intonational pitch movements. Since descriptions of stress would often be based on the pronunciation of words in isolation, the stressed syllable would be in focus and thus be associated with an intonational pith movement. This, then, accounts for the pitch properties that are often (wrongly) argued to be an intrinsic part of the stress package. But investigation of stressed syllables in and outside of focus has shown that these pitch properties are in fact very often not part of the set of wordlevel stress properties. When stressed syllables are measured in out-offocus position they often do not include pitch as a significant factor, but rather comprise primarily the various consequences of articulatory force or hyperarticulation which typically enhance intensity ('loudness'), duration, fullness of articulation (with consequences for vowel quality and phonation)."

Teasing apart Acoustic Cues for Stress and Tone

• the difficulty of teasing the two apart is canonically due to analyzing words in isolation

• Pierrehumbert (2000): "The citation form is reconstructed as the form produced in a specific prosodic context -when the word is both phrase-final and bears the main stress of the phrase."

• when the word that contains the stressed syllable is part of a focus domain, the stressed syllable is typically linked to an intonational pitch-accent Pierrehumbert, Janet. 2000. Tonal elements and their alignment. In M. Horne (ed.), Prosody: Theory and Experiment. Studies Presented to Gosta Bruce. Kluwer, Dordrecht, 11-26.

实验研究

• measurement parameters: intensity, duration, and f0 (= common correlates of stress in languages of the world)

• basis: recordings of eight subjects (four female and four male), all of whom were native speakers of Lizu (the /ʃaetɕʰôpae/ dialect)

• the list of targeted words: 22 disyllabic lexical words, presented to the subjects to read in randomized order in three contexts in isolation, where the word is equivalent to a phrase in the phrasal context (phrase-initial & phrase-medial)

• in each context, the target words were read each three to four times

• measurements taken (in Praat): average intensity, duration, and average fundamental frequency over each vowel in the word

调查问卷 实验研究

• after analysis, data were subjected to statistical analysis (repeated measures ANOVAs (analyses of variance)):⽅差分析(变异数分 析):⽤于两个及两个以上样本均数差别的显著性检验

• the three tone patterns were compared as a function of (1) average intensity and duration (all speakers), and (2) average fundamental frequency (female and male speakers separately) in the three contexts (isolation, phrase-initial, phrase-final)

• post hoc comparisons using the Tukey HSD test, t-tests

• given that stress is a relative property arising from a relationship between two syllables, comparisons were based on the differences between two syllables in each word (value Syl1 -value Syl2)

实验研究

• questions what are the phonetic properties of stress (if detectable)?

the correlation between stress and tone in Lizu • post-hoc Tukey's HSD tests showed that the "HL" tonal pattern had significantly higher values; all other comparisons were insignificant

语境:句中

• MinPitch: Female F(2, 203) = 207.9, p < .001

• Male F(2, 210) = 143.7, p < .001

• post-hoc Tukey's HSD tests showed that the "HL" tonal pattern had significantly higher values (pitch), the differences in pitch (for male speakers) between the "HH" and "LH" groups were marginally significant (both at the .01 level of significance)

HL

总结

• Lizu has both lexical tone (pitch) and stress (intensity, duration)

• intonational pitch accent: "in focus" position (pitch)

• stress: on the initial syllable in the domain (trochaic):左重步

• only the initial, stress-able syllable in the domain may bear tonal contrast

• the two basic tones on monosyllabic words (H & L) only realized on the stressed syllable, spreading to the right

• intonational pitch accent is linked to the stressed syllable; BUT in domains beginning with the L tone, a combination of the tones on the domain-initial syllable is avoided, so that the intonational pitch accent shifts to the final syllable

⾥汝语中的声调与重⾳

• Lizu stress is always obligatory (all major category, words are stressed), while some words may be toneless

• HL pattern is not linked to the surface lexical tones on nouns => toneless, right-edge demarcating trochaic stress; maximum reduction in terms of duration of the second syllable

• citation form: a specific prosodic context, where the word bears the main stress of the phrase (higher pitch on the first syllable, intonational pitch accent), and it is phrase-final (final lengthening, longer duration of the second syllable)

• although future consensus might suggest that Lizu is better classified as lacking stress, the present analysis at least allows for contextualizing their phonetic properties relative to the broader literature on acoustic correlates of prominence

  • questions: are the three patterns really contrastive? how do the 2 lexical tones on monosyllabic words transform into 3 tonal patterns on words of two syllables and longer? HH > H(…)H, LH > L(L…L)H, ? > H(L…)L?

  are the three patterns on disyllabic words really contrastive? why do words of two syllables and longer have three tonal patterns, while there are only two lexical tones on monosyllabic words HH > H(…)H, LH > L(L…L)H, ? > H(L…L)L? 语境:单念 • in citation form, in which the word is both phrase-final and bears the main stress of the phrase • where stressed syllables of words 'in focus' position function as anchors for intonational pitch movements HL, HH, LH are distinct in terms of intensity and pitch a binary opposition between HL, on the one hand, vs. HH & LH, on the other hand, in terms of duration 语境:单念 • MinInt: F(2, 654) = 178.2, p < .001 • post-hoc Tukey's HSD comparisons were insignificant 语境:单念 MinPitch: F(2, 331) = 350.2, p < .001 MinPitch: F(2, 320) = 230.8, p < .001 post-hoc Tukey's HSD comparisons were insignificant 语境:单念 • MinDur: F(2, 654) = 25.73, p < .001 • post-hoc Tukey's HSD tests showed that the "HL" tonal pattern had significantly higher MinInt values than the other two groups at the .05 level of significance; all other comparisons were not significant 语境:句中 • phrase-initial & phrase-medial • stressed syllables in and outside of focus the three tonal patterns (HL, HH, LH) show a binary opposition in terms of intensity, duration, and pitch between the HL pattern, on the one hand, and the HH and the LH patterns, on the other hand HL HH LH 语境:句中 • MinInt: F(2, 416) = 31.71, p < .001 • post-hoc Tukey's HSD tests showed that the "HL" tonal pattern had significantly higher MinInt values than the other two groups at the .05 level of significance; all other comparisons were not significant HL HH LH 语境:句中 • MinDur: F(2, 416) = 19.83, p < .001

  in pitch in all three contexts • Women [F] & Men [M], mean f0 on the 1st syllable HH & LH • pairwise comparisons Women [F] & Men [M], mean f0 1st syllable • Isolation: [F] F(1, 212) = 97.48, p < .001; [M] F(1, 217) = 11.95, p < .001 • Phrase-initial: [F] F(1, 129) = 39.15, p < .001; [M] F(1, 147) = 10.97, p < .001 • Phrase-medial: [F] F(1, 62) = 46.53, p < .001; [M] F(1, 60) = 11.77, p = .001 • pairwise comparisons Women [F] & Men [M], mean f0 2nd syllable • Isolation: [F] F(1, 211) = 25.15, p < .001; [M] F(1, 219) = 3.08, p = .08 • Phrase-initial: [F] F(1, 129) =2 5,74, p < .001; [M] F(1, 149) = 3.98 , p = .05 • Phrase-medial: [F] F(1, 62)=42.24, p < .001; [M] F(1, 61) = 5.48, p = .02 • Phrase-initial HH vs HL • F(1, 143)=3.14, p=0.07 • Phrase-medial HH vs HL • F(1, 144)=3.76, p=0.05 Putting it all together • context = isolation (in citation form, in which the word is both phrase-final and bears the main stress of the phrase) significant differences between the three tonal patterns in intensity and pitch, a binary opposition between HL vs HH & LH in duration • context = phrasal (phrase-initial & phrase-medial) binary opposition between HL vs. HH & HL HL: intensity, duration (pitch = phrase-initial) HH & LH: pitch, the clearest contrast on the 1st syllable stress properties

  ://wals.info/combinations/14A#5/30.449/107.205 参考⽂献 • Evans, Jonathan P. 2008. 'African' tone in the Sinosphere. Language and Linguistics 9.3: 463-490. Evans, Jonathan P., & Huang, Chenglong. 2007. A bottom-up approach to vowel systems: the case of Yadu Qiang. Cahiers de Linguistique Asie Orientale, 36.2: 147-186. Gordon, Matthew. 2014. Disentangling stress and pitch accent: Toward a typology of prominence at different prosodic levels. In Henry van der Hulst (eds.), Word stress: Theoretical and Typological issues, 83-118. Cambridge: Cambridge University Press. Gordon, Matthew. 2016. Metrical structure and stress. Oxford Research Encyclopedia of Linguistics. Oxford: Oxford University Press. DOI: 10.1093/acrefore/ 9780199384655.013.115 • Hyman, Larry M. 2001. Tone systems. Language typology and language universals: An international Handbook, Vol. 2, pp.1367-1380. van der Hulst, Harry. 2011. Pitch accent systems. In Marc van Oostendorp, Colin Ewen, Elizabeth Hume, and Keren Rice (eds.), The Blackwell Companion to Phonology, Voll. II, 1003-1026. Oxford: Wiley-Blackwell.

• notation: prominent syllables within a word http•

•

•

• • Yip, Moira. 2002. Tone. Cambdridge: Cambridge University Press.


S Massoni

M Olteanu

P Rousset

Career-path analysis using optimal matching and self-organizing maps

This paper is devoted to the analysis of career paths and employability. The state-of-the-art on this topic is rather poor in methodologies. Some authors propose distances well adapted to the data, but are limiting their analysis to hierarchical clustering. Other authors apply sophisticated methods, but only after paying the price of transforming the categorical data into continuous, via a factorial analysis. The latter approach has an important drawback since it makes a linear assumption on the data. We propose a new methodology, inspired from biology and adapted to career paths, combining optimal matching and self-organizing maps. A complete study on real-life data will illustrate our proposal.

Introduction

The question of analyzing school-to-work transitions is a challenging topic for the economists working on the labor market. In the current economic context of the world, characterized by a significant unemployment rate of young people (in France, 19.1% of the young people under 25 were unemployed during the second semester of 2008), it is interesting to study the insertion of graduates and the evolution of their career paths. The aim of this paper is to identify and analyze career-paths typologies. Let us recall that a career path is defined as a sequence of labor-market statuses, recorded monthly. The number and the labels associated to the statuses are defined by experts. The experts take into account the fact that the more the number of labels is detailed, the more the analysis of the career paths will be accurate. We shall also remark that the labelling of the statuses is not neutral. Indeed, the choice of different criteria for identifying the statuses introduces an a priori separation between "good" and "bad" statuses. Generally, the number and the labelling of the statuses are quite similar in the literature, with different labels corresponding to employment and unemployment situations. The data "Generation 98" used for this study are labelled according to nine possible statuses, five employment statuses (permanent-labor contract, fixedterm contract, apprenticeship contract, public temporary-labor contract, on-call contract) and four unemployment statuses (unemployed, inactive, military service, education). In the state-of-the-art, two approaches seem to be currently used for clustering career paths. The first approach consists in transforming categorical variables into continuous variables by a factorial analysis and then apply usual clustering algorithms for Euclidean data ( [START_REF] Grelet | The sequence of steps in the analysis of youth trajectories[END_REF], [START_REF] Giret | Classifying qualitative time series with SOM : the typology of career paths in France, Computational and ambient intelligence[END_REF]). The second approach consists in computing adapted distances for the data ([12], [START_REF] Brzinsky-Fay | Lost in Transition? Labour Market Entry Sequences of School Leavers in Europe[END_REF]) and then cluster using an hierarchical tree and a proximity criterion based on the distance matrix only. Both approaches have some drawbacks: in the first case, the use of factorial methods implies quite strong hypothesis concerning the linearity of the data; in the second case, hierarchical clustering is not suited for large data sets and does not provide any tools for displaying and visualizing the results. In order to address these drawbacks, we propose to cluster career paths using a two-step methodology. The two steps of the algorithm are independent and quite general. First, we compute a dissimilarity matrix between the career paths. Second, a self-organizing map for dissimilarity matrices is trained. Besides identifying the main typologies of career paths, we are also looking for a graphical output representing the proximities and the evolutions in the different career paths. The rest of the document is organized as follows: Section 2 is devoted to a description of the methodology and a short state-of-the-art on the subject. Section 3 contains the results on the data "Generation 98" (CEREQ, France). The conclusion and some perspectives are presented in the last section.

Methodology

From a statistical point of view, several problems arise when analyzing school-to-work transitions. The data sets are usually containing categorical variables, often in high dimension and have an important sample size. In order to handle these data, we made the choice of splitting the analysis into two steps. The first step consists in defining a distance or a dissimilarity measure well-suited to the data. In the second step, a clustering method is used to build and define typologies. The clustering method has to be general enough to "forget" the initial structure of the data and determine classes on the unique basis of the dissimilarity matrix computed in the previous step. This approach has the advantage of allowing a wide choice for the dissimilarity measure in the first step.

Step 1 (Optimal matching) -Choosing a good distance

The first step of the analysis consists in choosing a dissimilarity measure between the career paths. Previous studies suggest the use of a multiple correspondence analysis ( [START_REF] Grelet | The sequence of steps in the analysis of youth trajectories[END_REF]) and a transformation of the categorical variables into continuous. This way, usual clustering algorithms based on the Euclidean distance can be trained on the factorial components. This approach has an important drawback, since it makes the assumption that data are linear. As there is no reason for this assumption to hold in our case, we prefer to use a distance which avoids this hypothesis. Optimal matching, also known as "edit distance" or "Levenshtein distance", was first introduced in biology by [START_REF] Needleman | A general method applicable to the search for similarities in the amino acid sequence of two proteins[END_REF] and used for aligning and comparing sequences. In social sciences, the first applications are due to [START_REF] Abbott | Optimal matching methods for historical sequences[END_REF]. Let us consider two sequences of different lengths a = (a1, ..., an 1 ) and b = (b1, ..., bn 2 ), ai, bj ∈ S where S is a finite state-space. How may the two sequences be aligned and what is the distance between them? The underlying idea of optimal matching is to transform the sequence a into the sequence b using three possible operations: insertion, deletion and substitution. A cost is associated to each of the three operations. The distance between a and b is computed as the cost associated to the smallest number of operations which allow to transform a into b. The method seems simple and relatively intuitive, but the choice of the costs is a delicate operation in social sciences. This topic is subject to lively debates in the literature ( [START_REF] Abbott | Sequence analysis and optimal matching methods in sociology, Review and prospect[END_REF], [START_REF] Wu | Some comments on "Sequence analysis and optimal matching methods in sociology, review and prospect[END_REF]) mostly because of the difficulties in establishing an explicit and sound theoretical frame. The interested reader may refer to [START_REF] Gauthier | How much does it cost? Optimization of costs in sequence analysis in social science data[END_REF] for a state-of-the-art. In the data set "Generation 98", all career paths have the same length, the status of the graduate students being observed during 94 months. Thus, we may suppose that there are no insertions or deletions and that the only cost to be computed is the substitution cost. The latter was computed using the transition matrix between the statuses as proposed in [12]: the less transitions between two statuses are observed, the more the statuses are different and the substitution cost is high. The cost w for transforming ai into aj is computed as a function of the observed longitudinal transitions: w (ai, aj ) = 2 -P (ai|aj) -P (aj|ai).

Step 2 -Self-organizing maps for categorical data

The methodology "optimal matching -clustering" has already been used ( [START_REF] Brzinsky-Fay | Lost in Transition? Labour Market Entry Sequences of School Leavers in Europe[END_REF], [12]) during the past few years for the analysis of career or life paths. The general approach consists in computing a dissimilarity matrix using optimal matching, build an hierarchical tree and make a description of the resulting typologies. However, hierarchical clustering is limited in terms of displaying and visualizing the results. Instead, we suggest a clustering method which provides, besides clusters, a graphical representation preserving the proximity between paths. Self-organizing maps (Kohonen algorithm, [START_REF] Kohonen | Self Organizing Maps, Springer-Berlin[END_REF]) are, at the same time, a clustering algorithm and a nonlinear projection method. The input data are projected on a grid, generally rectangular or hexagonal. The grid has the important property of topology preservation: close inputs will be projected in the same class or in neighbor classes. The algorithm was initially developed for continuous data with a Euclidean distance. Since its first application, new versions, suited for particular data structures, are regularly proposed. Let us mention [START_REF] Kohonen | Self-organizing maps for symbol strings[END_REF], who are using for the first time self-organizing maps and optimal matching for the analysis of biological sequences.

For career-paths data, we used a general self-organizing map algorithm, proposed by [START_REF] Conan-Guez | Fast algorithm and implementation of dissimilarity Self-Organizing Maps[END_REF]. Their method does not take into account the initial structure of the data and uses only a dissimilarity matrix as input. The size and the structure of the grid must also be provided as input. At the first step of the algorithm, the prototypes are chosen at random in the input data. The rest of the algorithm consists in repeating the following steps, until the partition is stable:

-Allocating step: each input is assigned to the class of the closest prototype by minimizing a criterion of intra-class variance, extended to the neighbors. During this step, the prototypes are fixed. -Representing step : once the new partition is determined, the new prototypes are computed by minimizing the same criterion. The price of the generality of the method is the poverty of the space where the inputs lie: the new prototypes have to be computed and chosen between the inputs. This algorithm is a generalization of K-means by introducing with a neighborhood relation defined between classes. Since the algorithm is BATCH, it is quite sensitive to the initializations.

Real-life data -"Generation 98" survey

For illustrating the proposed methodology, we used the data in the survey "Generation 98" from CEREQ, France (http://www.cereq.fr/). The data set contains information on 16040 young people having graduated in 1998 and monitored during 94 months after having left school. The labormarket statuses have nine categories, labelled as follows: "permanentlabor contract", "fixed-term contract", "apprenticeship contract", "public temporary-labor contract", "on-call contract", "unemployed", "inactive", "military service", "education". The following stylized facts are highlighted by a first descriptive analysis of the data (Fig. 1):

-permanent-labor contract are representing more than 20% of all statuses after one year and their ratio continues to increase until 50% after three years and almost 75% after seven years; -the ratio of fixed-terms contracts is more than 20% after one year on the labor market, but it is decreasing to 15% after three years and then seems to converge to 8%; -almost 30% of the young graduates are unemployed after one year.

This ratio is decreasing and becomes constant, 10%, after the fourth year. Considering the important ratio of permanent contracts obtained relatively fast and its absorbing character, we decided to focus our analysis on the career paths which don't enter the "permanent contract" status in less than two years. Thus, the data set is reduced to 11777 inputs, which represent almost 3/4 of the initial data. This decision is also justified by some numerical problems such as the storage of the dissimilarity matrix and the computation time. The inputs excluded from the analysis are grouped into a class corresponding to a "fast access to stable employment". The class contains 2032 inputs having obtained a permanent contract in less than a year Fig. 1. Labor market structure and 4263 inputs having obtained a permanent contract in less than two years. After having preprocessed the data, the analysis is conducted in three steps:

1. the transition matrix and the associated substitution-cost matrix are computed; 2. the dissimilarity matrix is computed by optimal matching with the substitution-cost matrix in step 1; 3. career paths are clustered with the self-organizing map algorithm, according to the dissimilarity matrix in step 2. For the first two steps, we used the R-package TraMineR, available in [START_REF] Gabadinho | Mining Sequence Data in R with TraMineR: a user's guide[END_REF]. The third step was implemented in R and is available on demand. The cost matrix computed on the 11777 input data is the following:

C =             0 1.
           
Let us remark that the values in the cost matrix are very similar. Different approaches for improving cost computation should be investigated, some perspectives are given in the conclusion.

When trying to compute the dissimilarity matrix, we were confronted to a numerical problem: the impossibility of storing a matrix of size 11777.

Actually, this numerical problem constitutes the main drawback of the proposed methodology: the sample size must be "reasonable". The size of the data has to be reduced before training the self-organizing map algorithm. In order to summarize the career-path data, we used a Kmodes algorithm, which may be trained directly on the initial data set. Thus, the 11777 career-paths were summarized by 1000 representative paths and these 1000 paths were clustered with self-organizing maps. The dissimilarity matrix between the 1000 paths was computed with the optimal matching distance and the substitution-cost matrix C. Then, we trained a self-organizing map on a rectangular grid 8 × 8. The resulting map is plotted in Fig. 2. A lecture of the map summarizes the information on the career paths. Thus, we can stress out the proximities between different paths and the evolution of the career paths.

Fig. 2. SOM 8x8

The most striking opposition appears between the career paths leading to a stable-employment situation (permanent contract and/or fixed-term contract) and the career-paths more "chaotic" (unemployment, on-call contracts, apprenticeship contracts). On the map in Fig. 2, the "stable" situations are mainly situated in the west region of the map. Thus, the first three columns contain essentially the classes where a permanent contract was rapidly obtained. However, the north and the south regions of these columns are quite different: in the north region, the access to a permanent contract is achieved after a first contact with the labor-market through a fixed-term contract, while the south classes are only subject to transitions through a military service or education periods. At the halfway between north and south regions, we may find several apprenticeship contracts or public temporary contracts. The stability of the career-paths noticed in the west region of the map is getting worse as we move to the east. In the north region, the initial fixed-term contract is getting longer until becoming a poor employment situation in the north-east corner. Thus, all the east region of the map is revealing for difficult school-to-work transitions. Let us remark the oncall contracts situations which may end by a stable contract or by unemployment. At the opposite, the career-paths starting with an apprenticeship contract are most of the time ending with a permanent contract. Finally, the south-east corner is characterized by exclusion career-paths: inactive and unemployment. The inactivity may appear immediately after the education period or after a first failure on the labor market. The clustering with SOM provided interesting results by highlighting proximities or oppositions between the career paths. In order to determine a small number of typologies, we compute an hierarchical classification tree on the 64 prototypes of the map. Ward criterion was used for aggregating classes. The clustering is represented in Fig. 3. The final configuration with nine classes allows to describe 8 career-path typologies:

1. relatively fast access to a stable situation (class 1 ) 2. transition through a fixed-term contract before obtaining a permanent contract(class 2 ) 3. fixed-term contracts (class 3 ) 4. on-call contracts with periods of unemploymentnt (class 4 ) 5. shorter or longer on-call contracts and finally a permanent contract(classes 5 and 6 ) 6. inactivity after loosing a first job or immediately after school (class 7 ) 7. apprenticeship contract ending with a fixed-term or a permanentterm contract(class 8 ) 8. long unemploymentnt period with a gradual return to employment (class 9 ) We may also add the first class which was excluded from the analysis: 9. fast access to stabl employment after leaving school. The relative importance of every class is given in Table 1.

Conclusion and perspectives

Several typologies for career paths were highlighted by our analysis. The self-organizing map allowed a detailed characterization of the proximities, oppositions and transitions between the different career paths. The proposed methodology is thus well suited for this kind of data. However, several aspects should be improved in this approach. Concerning the labelling of the statuses, we remarked that the "military service" is not representative in a employment-unemployment discrimination. The solution is either to change the label of this status, either to delete this period. In the latter case, we would no longer have equalsized sequences and the question of computing an insertion/deletion cost would arise.

The second remark concerns the computation of the substitution-cost matrix. In this paper, it was computed using the observed transitions, but without taking into account that these transitions change in time. Indeed, in a more realistic frame we should consider the non-homogeneity of the transitions and probably use non-homogeneous Markov chains in order to estimate the costs.

Fig. 3 .

 3 Fig. 3. Final career-path typologies

Table 1 .

 1 Importance of final classes

	1. 5475	46.5%	34.1%
	2. 968	8.2%	6.0%
	3. 1082	9.2%	6.8%
	4. 514	4.4%	3.2%
	5. 1122	9.5%	7.0%
	6. 328	2.8%	2.0%
	7. 1002	8.5	6.3%
	8. 1286	10.9	8.0%
	9. 4263	NA	26.6%


Arnaud Lehner

Norbert Mamadou

Pascal Poels

Daniel Côme

Christophe Bailly

Françoise Corbineau

D C O ˆme

Changes in soluble carbohydrates, lipid peroxidation and antioxidant enzyme activities in the embryo during ageing in wheat grains

Keywords: Wheat, Ageing, Soluble carbohydrates, Antioxidant enzymes, Lipid peroxidation

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

Introduction

During storage of seeds, ageing leads to a progressive decrease in their vigour, and then in a loss of their viability [START_REF] Mcdonald | Seed deterioration: physiology, repair and assessment[END_REF][START_REF] Priestley | Seed Aging. Implications of Seed Storage and Persistence in the Soil[END_REF][START_REF] Smith | Deteriorative changes associated with the loss of viability of stored desiccation-tolerant and desiccation-sensitive seeds[END_REF]. Seed susceptibility to ageing varies among the species, but it is also dependent on the maturity stage and the conditions of development [START_REF] Hay | Seed maturity and the effects of different drying conditions on desiccation tolerance and seed longevity in Foxglove (Digitalis purpurea L.)[END_REF][START_REF] Priestley | Seed Aging. Implications of Seed Storage and Persistence in the Soil[END_REF]. As in pearl millet [START_REF] Kameswara Rao | Longevity of pearl millet (Pennisetum glaucum R. Br.) seeds harvested at different stages of maturity[END_REF], tomato [START_REF] Demir | Changes in seed quality during development and maturation in tomato[END_REF] and bean [START_REF] Bailly | Changes in oligosaccharide content and antioxydant enzyme activities in developing bean seeds as related to acquisition of drying tolerance and seed quality[END_REF], seed sensitivity to accelerated ageing decreases during the maturation drying phase of wheat grain development [START_REF] Ellis | The development of seed quality in spring and winter cultivars of barley and wheat[END_REF][START_REF] Lehner | Changes in wheat seed germination ability, and in embryo soluble carbohydrate contents and antioxidant enzyme activities during maturation drying[END_REF]. However, the rate of loss of seed viability is mainly a function of temperature and seed water content [START_REF] Mcdonald | Seed deterioration: physiology, repair and assessment[END_REF][START_REF] Roberts | Water and seed survival[END_REF]. In orthodox seeds (seeds that are tolerant to desiccation, such as wheat Abbreviations: AOS: active oxygen species; CAT: catalase; DW: dry weight; GR: glutathione reductase; MC: moisture content; MDA: malondialdehyde; P 50 : half viability period; RFO: raffinose family oligosaccharides; RH: relative humidity; Ra: raffinose; ROS: reactive oxygen species; SD: standard deviation; SOD: superoxide dismutase; Su: sucrose; T 50 : time to reach 50% of the germination percentages obtained after 14 d à Corresponding author. Tel.: +33 1 44 27 59 87; fax: +33 1 44 27 59 27. E-mail address: corbi@ccr.jussieu.fr (F. Corbineau).

grains), there is a positive relationship between viability loss and both factors (Ellis and Roberts, 1980a, b;[START_REF] Priestley | Seed Aging. Implications of Seed Storage and Persistence in the Soil[END_REF][START_REF] Roberts | Water and seed survival[END_REF]. Seed deterioration during ageing is associated with numerous cellular and biochemical alterations including loss of membrane integrity, impairment of RNA and protein syntheses, and DNA degradation [START_REF] Gidrol | Effect of accelerated aging on protein synthesis in two legume seeds[END_REF][START_REF] Gutie´rrez | Natural and artificial seed ageing in maize germination and DNA synthesis[END_REF][START_REF] Mcdonald | Seed deterioration: physiology, repair and assessment[END_REF][START_REF] Vazquez-Ramos | DNA integrity and DNA polymerase activity in deteriorated maize embryo axes[END_REF]. Although the exact mechanisms of loss of viability are by no means completely elucidated, reactive oxygen species (ROS) accumulation and lipid peroxidation are often invoked to be the major causes of seed deterioration [START_REF] Bailly | Active oxygen species and antioxidants in seed biology[END_REF][START_REF] Hendry | Oxygen, free radical processes and seed longevity[END_REF][START_REF] Priestley | Seed Aging. Implications of Seed Storage and Persistence in the Soil[END_REF]. Lipid peroxidation has been demonstrated to occur during ageing in soybean [START_REF] Sung | Lipid peroxidation and peroxide-scavenging in soybean seeds during aging[END_REF], Norway maple [START_REF] Pukacka | Changes in membrane lipid components and antioxidant level during natural aging of seeds of Acer platanoides[END_REF], peanut [START_REF] Sung | Lipid peroxidation and peroxide-scavenging enzymes associated with accelerated aging of peanut seed[END_REF], beech [START_REF] Pukacka | Production and scavenging of reactive oxygen species in Fagus sylvatica seeds during storage at varied temperature and humidity[END_REF] and sunflower seeds [START_REF] Bailly | Changes in malondialdehyde content and in superoxide dismutase, catalase and glutathione reductase activities in sunflower seeds as related to deterioration during accelerated ageing[END_REF][START_REF] Kibinza | Sunflower seed deterioration as related to moisture content during ageing, energy metabolism and active oxygen species scavenging[END_REF], but it does not seem to be involved in other species, such as pigeon pea [START_REF] Kalpana | Absence of role of lipid peroxidation during accelerated ageing of seeds of pigeon pea (Cajanus cajan L. Millsp.)[END_REF], maize [START_REF] Linn | Changes in lipids in bean seeds (Phaseolus vulgaris) and corn caryopses (Zea mays) aged in contrasting environments[END_REF] and wheat [START_REF] Girard | Absence de relation entre le taux de radicaux libres mesure´par RPE et la viabilite´des semences de ble´[END_REF]. These contradictory results might be explained partly by the differences in the conditions of ageing performed in these studies. Indeed, oxidative reactions are largely dependent on the seed moisture content (MC). At very low MC, it is likely that they are involved in the deterioration processes. Increasing MC facilitates enzymatic oxidations such as that by lipoxygenase [START_REF] Vertucci | Acquisition and loss of desiccation tolerance[END_REF], and leads to faster cellular damage. At MC higher than 0.25 g H 2 Og À1 dry matter (DM), respiration is estimated to occur [START_REF] Vertucci | Acquisition and loss of desiccation tolerance[END_REF] and might be considered to be one of the major sources of production of ROS [START_REF] Staniek | Are mitochondria a permanent source of reactive oxygen species[END_REF].

The main enzymes involved in cell detoxification are superoxide dismutase (SOD), glutathione reductase (GR), catalase (CAT) and peroxidases such as ascorbate peroxidase (APX), glutathione peroxidase (GPX) and thioredoxin peroxidase (TPX) [START_REF] Bailly | Active oxygen species and antioxidants in seed biology[END_REF][START_REF] Mittler | Oxidative stress, antioxidants and stress tolerance[END_REF][START_REF] Scandalios | Oxidative Stress and the Molecular Biology of Antioxidant Defenses[END_REF]. The ability of seeds to scavenge ROS is considered to be involved in seed storability [START_REF] Bailly | Active oxygen species and antioxidants in seed biology[END_REF][START_REF] Priestley | Seed Aging. Implications of Seed Storage and Persistence in the Soil[END_REF]. In addition, ageing is associated with a depletion of antioxidant enzyme activities in soybean [START_REF] Murthy | Nonenzymatic protein modification by the Maillard reaction reduces the activities of scavenging enzymes in Vignia radiata[END_REF], cotton [START_REF] Goel | Changes in oxidative stress enzymes during artificial aging in cotton (Gossypium hirsutum L.) seeds[END_REF], beech [START_REF] Pukacka | Production and scavenging of reactive oxygen species in Fagus sylvatica seeds during storage at varied temperature and humidity[END_REF] and sunflower seeds [START_REF] Bailly | Changes in malondialdehyde content and in superoxide dismutase, catalase and glutathione reductase activities in sunflower seeds as related to deterioration during accelerated ageing[END_REF][START_REF] Bailly | Free radical scavenging as affected by accelerated ageing and subsequent priming in sunflower seeds[END_REF][START_REF] De Paula | Function of the ascorbateglutathione cycle in aged sunflower seeds[END_REF][START_REF] Kibinza | Sunflower seed deterioration as related to moisture content during ageing, energy metabolism and active oxygen species scavenging[END_REF][START_REF] Torres | Ageing-induced changes in glutathione system of sunflower seeds[END_REF].

Previous studies on pea, maize and rape seeds assumed that soluble sugars, mainly the raffinose family oligosaccharides (RFO), might also be involved in the protection against the deleterious effects occurring during dehydration and/or ageing, suggesting that they are directly linked to storage ability (for review see: [START_REF] Obendorf | Oligosaccharides and galactosyls in seed desiccation tolerance[END_REF]. Moreover, various authors (Bernal-Lugo and Leopold, 1992;[START_REF] Horbowicz | Seed desiccation tolerance and storability: dependence on flatulence-producing oligosaccharides and cyclitols-review and survey[END_REF][START_REF] Piotrowicz-Cieslak | Changes in soluble carbohydrates in yellow lupin seed under prolonged storage[END_REF][START_REF] Sinniah | Irrigation and seed quality development in seed rapid-cycling brassica: soluble carbohydrates and heat-stable proteins[END_REF][START_REF] Steadman | Tissue-specific soluble sugars in seeds as indicators of storage category[END_REF] have demonstrated that sugar content, particularly the ratio of oligosaccharide to sucrose contents, might be used as an indicator of seed storability or ageing. In maize, for example, a value of this ratio higher than 0.2 corresponds to a good seed storability [START_REF] Bernal-Lugo | Seed stability during storage: raffinose content and seed glassy state[END_REF]. Oligosaccharides might contribute to the stabilization of intracellular glasses by increasing cytoplasm viscosity and the glass-to-liquid transition temperature, which are likely to slow down ageing alterations [START_REF] Bernal-Lugo | Seed stability during storage: raffinose content and seed glassy state[END_REF][START_REF] Leopold | The glassy state in seed: analysis and function[END_REF][START_REF] Sun | Glassy state and seed storage stability: the WLF kinetics of seed viability loss at T greater than T-g and the plasticization effect of water on storage stability[END_REF].

Most of the studies on the cellular and biochemical deterioration during seed ageing have been performed under accelerated ageing conditions, i.e., at high temperature and in saturating humidity [START_REF] Mcdonald | Seed deterioration: physiology, repair and assessment[END_REF]. Although accelerated ageing has been recognized as a good predictor of seed storability, the mechanisms involved in seed deterioration under such extreme conditions differ from those occurring under ambient conditions. In particular, the contribution of lipid peroxidation, sugar hydrolysis and Maillard reactions in seed ageing depended on water content and temperature [START_REF] Kibinza | Sunflower seed deterioration as related to moisture content during ageing, energy metabolism and active oxygen species scavenging[END_REF][START_REF] Murthy | Mechanisms of seed ageing under different storage conditions for Vigna radiata (L.) Wilczek: lipid peroxidation, sugar hydrolysis, Maillard reactions and their relationship to glass transition[END_REF][START_REF] Walters | Understanding the mechanisms and kinetics of seed aging[END_REF].

The aims of the present work were: (1) to characterize the sensitivity of wheat grains to ageing conditions, (2) to investigate whether seed deterioration is associated with H 2 O 2 accumulation and lipid peroxidation depending on temperature and seed MC during ageing, and (3) to determine whether soluble sugars and the activities of the main antioxidant enzymes might be appropriate indicators of the loss of seed viability. For this purpose, wheat seeds were artificially aged for different periods of time under two conditions: 45 1C at 100% relative humidity (RH) conditions which were generally used for accelerated ageing [START_REF] Priestley | Seed Aging. Implications of Seed Storage and Persistence in the Soil[END_REF], and 30 1C at 75% RH, conditions which were closer to those of storage during summer months in seed companies.

Experimental

Plant material

Experiments were carried out with the cultivar Charger of wheat (Triticum aestivum L.), which was one among the most cultivated cultivars in France. Plants were grown in the experimental fields of Syngenta-Agro located in Gaillon (Eure, France) in 2004. Ears were collected at the end of the maturation drying phase, i.e., when grain water content reached 10-12% fresh weight basis. Seeds were removed from the ears and placed at 20 1C and 70% RH for 5 months in order to break their dormancy. Experiments were carried out with non-dormant grains and no sprouting injury was observed.

Germination tests were performed with whole nondormant caryopses. Biochemical measurements were carried out on isolated embryos cut off the seeds with as little endosperm as possible, though still covered dorsally by the seed coat.

Water content determination

Embryos were isolated, weighed and then dried by oven drying at 105 1C for 48 h for determination of the DM. MC was calculated on DM basis. Results are expressed as gH 2 Og À1 DM and correspond to the means of the values obtained in 3 replicates of 10 embryos7SD.

Germination assays and seed viability determination

Germination assays were performed by placing whole caryopses (4 replicates of 25 grains each) in 9-cm Petri dishes on a layer of cotton wool moistened with deionized water, in darkness at 20 1C. A grain was regarded as germinated when the radicle had pierced the seed coat. Germination counts were made daily up to 14 d.

The results presented are the means of the germination percentages obtained in 4 replicates7SD. They are expressed as the germination percentages obtained after 14 d, or as the time to reach 50% of the germination percentages obtained after 14 d (T 50 ) estimated graphically from the cumulative germination curves.

Topographical tetrazolium testing was also used to verify that seeds which did not germinate at 20 1C were dead. For this purpose, seeds were cut longitudinally and were incubated in a 1% solution of 2,3,5-triphenyltetrazolium chloride [START_REF] Moore | Tetrazolium staining for assessing seed quality[END_REF] for 3-4 h at 25 1C in darkness. Seeds of which the embryo exhibited no overall carmine red staining were scored as non-viable.

Accelerated ageing treatment

Ageing treatments were carried out by placing seeds for 1-7 d at 45 1C in tightly closed boxes with free water at the bottom in order to obtain 100% RH and for 1-4.5 months at 30 1C in tightly closed boxes with saturated NaCl solution at the bottom in order to obtain 75% RH. After ageing, seeds were dried back to their original water content by placing them for 1 week at 20 1C and 70% RH, and germination was tested at 20 1C for 14 d.

Enzyme extraction and assays

Fifty isolated embryos (about 150 mg FW) were ground in liquid nitrogen and then homogenized in 1.5 ml of a potassium phosphate buffer (0.1 M, pH 7.8) containing 2mM a-dithiothreitol, 0.1 mM ethylenediaminetetraacetic acid (EDTA) and 1.25 mM polyethylene glycol 4000, and 10% (w/v) of polyvinylpolypyrrolidone. The homogenate was then centrifuged for 30 min at 14 000g and the supernatant was desalted on a PD 10 column (Amersham Biosciences, Sweden).

SOD (EC 1.15.11) and CAT (EC 1.11.16) activities were determined at 25 1C according to [START_REF] Bailly | Changes in malondialdehyde content and in superoxide dismutase, catalase and glutathione reductase activities in sunflower seeds as related to deterioration during accelerated ageing[END_REF]. The results are expressed as units mg À1 protein (SOD) and nmol H 2 O 2 decomposed min À1 mg À1 protein (CAT). GR (EC 1.6.4.2) activity was determined at 25 1Ca s described previously by [START_REF] Esterbauer | Seasonal variation of glutathione and glutathione reductase in needles of Picea abies[END_REF],b y following the rate of NADPH oxidation at 340 nm. The assay mixture contained 0.5 mM NADPH, 10 mM oxidized glutathione, 3 mM MgCl 2 in 0.1 M potassium phosphate buffer (pH 7.8), and 100 ml of enzyme extract in a total volume of 400 ml. GR activity was expressed as nmol of NADPH oxidized min À1 mg À1 protein.

The results presented correspond to the means7SD of the values obtained with three different extracts and three measurements per extract (i.e. nine measurements). Protein contents of the extracts were determined using the Bio-Rad protein assay kit with bovine serum albumin as calibration standard.

Hydrogen peroxide content

H 2 O 2 contents of isolated embryos were determined according to O' Kane et al. (1996). Fifty embryos were ground in a mortar and homogenized with 5 ml of perchloric acid (0.2 N). After 15 min of centrifugation at 13 000g at 4 1C, the resulting supernatant was neutralized to pH 7.5 with 4 N KOH and then centrifuged for 3 min at 1000g and 4 1C. The supernatant was immediately used for spectrophotometric determination of H 2 O 2 at 590 nm using a peroxidase-based assay. The reaction mixture contained 12 mM 3-dimethylaminobenzoic acid in 0.375 M phosphate buffer (pH 6.5), 1.3 mM 3-methyl-2-benzothiazolidone hydrazone, 20 ml (0.25 units) horseradish peroxidase (Sigma, USA) and 50 ml of the supernatant for a total volume of 1.5 ml. The reaction started with the addition of peroxidase. Increase in absorbance at 590 nm was monitored after 5 min at 25 1C and compared with the absorbance obtained with known amounts of H 2 O 2 . Results are expressed as nmol H 2 O 2 g À1 DW and correspond to the means of the values obtained with five different extracts7SD.

Malondialdehyde measurements

Lipid peroxidation was evaluated by measuring malondialdehyde (MDA) content from 0.5 g FW of isolated embryos, according to [START_REF] Heath | Photoperoxidation in isolated chloroplasts. I. Kinetics and stoichiometry of fatty acid peroxidation[END_REF]. Results are expressed as mmol g À1 DW and correspond to the means of measurements carried out with five different extracts7SD.

Soluble sugar measurements

Soluble sugars were extracted according to [START_REF] Black | Carbohydrate metabolism in the developing and maturing wheat embryo in relation to its desiccation tolerance[END_REF]. Ten isolated embryos were ground at room temperature in a mortar in 1 ml 80% aqueous ethanol containing melezitose (1.5 mg ml À1 ) as internal standard, and the extract was heated for 30 min at 80 1C. After centrifugation for 15 min at 14 000g and removal of the supernatant, the pellets were resuspended and reextracted twice, in 0.5 ml and then 0.35 ml 80% aqueous ethanol, at room temperature and centrifuged again. The three supernatants were combined and reduced to dryness in a centrifugal evaporator (RC 10-22, Jouan, France). The dry extracts were dissolved in 100 ml of ultra-pure water and then filtered through an acetate filter (0.45 mm pore size, Nalgene, USA) before being analysed by HPLC. Ten ml samples were injected onto a Spherisorb-NH2 column (Thermo Separation Products, France) and eluted with 70/ 30 (v/v) acetonitrile/H 2 O at a flow rate of 1 ml min À1 using a Spectra Physics 8700 pump (Spectra Physics, France). The eluents were analysed with a differential refractometer (Spectra Physics 8430) and the peak areas were integrated by a Spectra Physics 4290 integrator. Fructose, glucose, sucrose and raffinose were identified by co-elution with standards (Sigma, USA).

Results are expressed as mg of sugars g À1 DW of embryos and correspond to the means of three measur-ements7SD.

Results

Effect of ageing treatments on embryo water content and seed germination

Fig. 1 shows the changes in embryo water content, seed viability and the time to obtain 50% of the maximal germination percentages (T 50 ) during accelerated ageing performed at 45 1C and 100% RH (Fig. 1A) and at 30 1C and 75% RH (Fig. 1B). Before ageing treatment, the embryo MC was around 10% DW, all the grains were able to germinate at 20 1C within 3 d, and the T 50 was around 1.6 d. The final germination percentage was markedly reduced after ageing for 4 d at 45 1C and 100% RH, and for 2 months at 30 1C and 75% RH. The longer the treatment and the higher the temperature and the RH, the greater was the reduction of seed viability. The half-viability period (P 50 ) of wheat grains, i.e., the time taken for germination to drop to 50% of the initial viability, was only 6 d when they were placed at 45 1C and 100% RH (Fig. 1A) as against about 3.75 months at 30 1C and 75% RH (Fig. 1B).

Ageing at 45 1C and 100% RH was associated with a progressive increase in embryo water content which reached about 40% and 60% DW after 2 and 6 d of treatment, respectively (Fig. 1A). It resulted also in a decrease in seed vigour and then in seed viability from 4 d of treatment. Forty-five % of the seed population remained able to germinate after 6 d of ageing and no germination occurred after 7 d. T 50 remained at around 1.5-1.8 d during the first 3 d of ageing, and then regularly increased, reaching 5.2 d after 6 d of treatment.

At 30 1C and 75% RH, the embryo water content reached 17% DW after 2 weeks and then remained constant throughout the 5 months of ageing (Fig. 1B). Seed viability started to decline from 2 months, and then decreased linearly as a function of the duration of storage, all the grains being dead after 4.5 months. T 50 increased regularly from 1 month of storage. It was twice as high (i.e. 3.2 d) than the T 50 of control non-aged seeds and reached 7.7 d after 3 and 4 months of storage, respectively.

The susceptibility of grains to accelerated ageing at 45 1C and 100% RH increased when they were previously treated at 30 1C and 75% RH (Table 1), and the longer the duration of the treatment the stronger was their loss of viability. All control grains placed directly at 45 1C and 100% RH remained viable after 4 d of ageing (Fig. 1A and Table 1), whereas viability of those pretreated for 1.5, 2 and 2.5 months at 30 1C and 75% RH, fell below 50% after 4, 3 and 2 d of accelerated ageing at 45 1C and 100% RH, respectively (Table 1). After 3 months of treatment at 30 1C and 75% RH, almost all the grains died when they were aged at 45 1C and 100% RH for only 2 d.

Hydrogen peroxide content and lipid peroxidation

H 2 O 2 content of the embryo of untreated control grains was 3.29 mmol g À1 DW. It remained close to this initial value, ranging from 3.10 to 3.42 mmol g À1 DW, during ageing performed at 30 1C and 75% RH (Table 2). In contrast, when grains were aged at 45 1C and 100% RH, the H 2 O 2 content of the embryo decreased during the first 3 d of the treatment, reaching a minimal value of 1.04 mmol g À1 DW, and then increased regularly up to 5.54 mmol g À1 DW at 6 d of ageing (Table 2).

MDA content of the embryo did not significantly change during either ageing treatments. Its value remained between 0.38 and 0.45 mmol g À1 DW, i.e., close to its initial value before ageing (0.42 mmol g À1 DW) (Table 2).

Antioxidant enzyme activities

Before ageing, the activities of CAT, SOD and GR in the embryo were 7.3 nmol H 2 O 2 decomposed min À1 mg À1 protein, 13.2 units mg À1 protein and 235 nmol NADPH min À1 mg À1 protein, respectively. Table 3 shows that the changes in antioxidant enzyme activities in the embryo during loss of seed viability depended on the conditions of ageing.

Accelerated ageing at 45 1C and 100% RH induced an increase from 7.3 to 9.2 nmol H 2 O 2 decomposed min À1 mg À1 protein in CAT activity during the first 2 d and then a decrease to 3.0-3.9 nmol H 2 O 2 decomposed min À1 mg À1 protein (i.e. 41-53% of its initial value) after 5-6 d (Table 3). SOD activity decreased markedly during the treatment performed at 45 1C and 100% RH; it was about 50% and 28% of its initial value after 1 and 3 d, respectively, and became nil after 5 d (Table 3). Contrary to CAT and SOD activities, GR activity increased regularly during ageing and became 4.8 times higher than its initial value (1140 nmol NADPH min À1 mg À1 protein) after 6 d (Table 3). There existed no clear relationship between the antioxidant enzyme activities and grain viability (data in Fig. 1A and Table 3). In contrast, a negative sub-linear correlation seemed to exist between CAT activity, expressed as the percentage of its initial value measured in non-aged seeds, and the time to obtain the germination of 50% of the viable seeds (T 50 )( Fig. 2), but such a correlation was not observed for SOD and GR.

As early as 0.5 month of ageing at 30 1C and 75% RH, the activity of the antioxidant enzymes studied decreased, representing only about 81%, 75% and 55% of the initial activities of CAT, SOD and GR, respectively (Table 3). These activities then remained almost unchanged until 4.5 months of ageing, i.e., when complete loss of seed viability occurred (cf. Fig. 1B). For example, CAT activity remained between 5.8 and 7.1 nmol H 2 O 2 decomposed min À1 mg À1 protein, i.e. between 79% and 97% of its initial value. SOD activity varied between 7.2 and 11.2 units SOD mg À1 protein, and GR activity fluctuated between 115 and 200 nmol NADPH mg À1 protein min À1 . There then existed no correlation between the enzyme activities and germination of seeds aged at 30 1C and 75% RH. Interestingly, Table 4 shows that the decrease in CAT activity during accelerated ageing at 45 1C and 100% RH was faster in embryos from seeds previously placed at 30 1C and 75% RH than in those from seeds aged directly at 45 1C and 100% RH. After 3 d of ageing at 45 1C and 100% RH, CAT was between 30.1% and 37% of its initial activity when seeds were previously aged at 30 1C and 75% RH. In contrast, it remained at about 92% of its initial activity when the grains were placed directly at 45 1C and 100% RH. andTable 3. However, there was no correlation in the duration of the pretreatment at 30 1C and 75% RH and the decrease in CAT activity during subsequent ageing at 45 1C and 100% RH (Table 4).

Soluble sugars

Fig. 3 shows the changes in soluble sugar contents and in the Ra/Su ratio in embryos excised from seeds aged for various times at 45 1C and 100% RH (Fig. 3A andC) and at 30 1C and 75% RH (Fig. 3B andD). In embryos of untreated control seeds, soluble sugars were mainly sucrose (Su, 59.5 mg g À1 DW) and raffinose (Ra, 49.4 mg g À1 DW), hexoses (glucose+fructose) being almost absent. During ageing at 45 1C and 100% RH, the Ra content of the embryos remained almost constant, ranging from around 40 to 50 mg g À1 DW (Fig. 3A). Su content decreased between the third and the fourth day of the treatment, and then remained almost constant at around 21-23 mg g À1 DW. Consequently, the Ra/Su ratio increased progressively during ageing, reaching the value of 2.5 after 6 d as against 0.7 for the non-aged control seeds (Fig. 3C).

When ageing was performed at 30 1C and 75% RH, both sugars increased similarly during the treatment. After 4.5-5 months, Su content was around 90 mg g À1 DW, and that of Ra reached 70 mg g À1 DW (Fig. 3B). The increase in both soluble sugars resulted in the Ra/Su ratio varying between 0.6 and 0.9, i.e. close to its initial value, during the 5 months of ageing (Fig. 3D).

Discussion

As previously shown by other authors [START_REF] Mcdonald | Seed deterioration: physiology, repair and assessment[END_REF][START_REF] Priestley | Seed Aging. Implications of Seed Storage and Persistence in the Soil[END_REF], seed ageing is characterized by a progressive decrease in seed vigour followed by a loss of seed viability, the rate of which is a positive function of temperature and water content. As expected, wheat seeds were more sensitive to accelerated ageing carried out at 45 1C and 100% RH, when water content of the embryo was 50-60% DW, than to a treatment performed at 30 1C and 75% RH, when the embryo water content was only 17% DW (Fig. 1).

Susceptibility of seeds to accelerated ageing is a good indicator of their vigour in numerous species [START_REF] Mcdonald | Seed deterioration: physiology, repair and assessment[END_REF]. In wheat, previous studies [START_REF] Ellis | The development of seed quality in spring and winter cultivars of barley and wheat[END_REF][START_REF] Lehner | Changes in wheat seed germination ability, and in embryo soluble carbohydrate contents and antioxidant enzyme activities during maturation drying[END_REF] have shown that this susceptibility decreases during the maturation-drying phase of seed development, suggesting an increase in seed vigour after the end of seed filling. The present work demonstrates that storage of grains at 30 1C and 75% RH resulted in an increase in wheat seed sensitivity to accelerated ageing at 45 1C and 100% RH, i.e., in a decrease in seed vigour, as early as after 1.5 months of storage, whereas a significant increase in T 50 was observed only after 2-3 months (Fig. 1B).

Lipid peroxidation and oxidative stress have been widely indicated as the major causes of seed deterioration during ageing [START_REF] Mcdonald | Seed deterioration: physiology, repair and assessment[END_REF][START_REF] Priestley | Seed Aging. Implications of Seed Storage and Persistence in the Soil[END_REF]. Previous data obtained with various species, such as sunflower [START_REF] Bailly | Changes in malondialdehyde content and in superoxide dismutase, catalase and glutathione reductase activities in sunflower seeds as related to deterioration during accelerated ageing[END_REF][START_REF] Kibinza | Sunflower seed deterioration as related to moisture content during ageing, energy metabolism and active oxygen species scavenging[END_REF], soybean [START_REF] Sung | Lipid peroxidation and peroxide-scavenging in soybean seeds during aging[END_REF], Norway maple [START_REF] Pukacka | Changes in membrane lipid components and antioxidant level during natural aging of seeds of Acer platanoides[END_REF], watermelon [START_REF] Chiu | Lipid peroxidation and peroxide-scavenging enzymes associated with accelerated aging and hydration of watermelon seeds differing in ploidy[END_REF] and peanut [START_REF] Sung | Lipid peroxidation and peroxide-scavenging enzymes associated with accelerated aging of peanut seed[END_REF], have demonstrated that loss of seed viability during accelerated ageing is associated with an accumulation of MDA. On the contrary, the results presented here with wheat (Table 2) and those obtained by [START_REF] Pearce | Changes in fatty acid content of polar lipids during aging of seeds of peanuts (Arachis hypogea L.)[END_REF] with peanut, by [START_REF] Priestley | Absence of lipid oxidation during accelerated aging of soybean seeds[END_REF] with soybean and by [START_REF] Kalpana | Absence of role of lipid peroxidation during accelerated ageing of seeds of pigeon pea (Cajanus cajan L. Millsp.)[END_REF] with pigeon pea show that loss of seed viability is not related to an increase in lipid peroxidation evaluated by MDA content. Furthermore, [START_REF] Girard | Absence de relation entre le taux de radicaux libres mesure´par RPE et la viabilite´des semences de ble´[END_REF] did not observe any correlation between free radical content and the viability of wheat grains. Although loss of seed viability results from cellular damage, different mechanisms might be involved in mortality depending on ageing conditions, mainly on water content [START_REF] Kibinza | Sunflower seed deterioration as related to moisture content during ageing, energy metabolism and active oxygen species scavenging[END_REF][START_REF] Murthy | Mechanisms of seed ageing under different storage conditions for Vigna radiata (L.) Wilczek: lipid peroxidation, sugar hydrolysis, Maillard reactions and their relationship to glass transition[END_REF][START_REF] Walters | Understanding the mechanisms and kinetics of seed aging[END_REF], which might explain why the lipid peroxidation model has often been the subject of debate. In sunflower seeds, for example, [START_REF] Kibinza | Sunflower seed deterioration as related to moisture content during ageing, energy metabolism and active oxygen species scavenging[END_REF] showed that loss of seed viability at 35 1C is associated with lipid peroxidation when MC of the embryo axis is higher than 29.9% DW, whereas below this critical MC it results from accumulation of H 2 O 2 without lipid peroxidation. In wheat grains placed at 45 1C and 100% RH, i.e., at embryo MC close to 50-60% DW, loss of viability might also be attributed to H 2 O 2 production without MDA accumulation (Table 2). H 2 O 2 , or its derivatives such as the hydroxyl radical, are known to react with other molecules than lipids, such as proteins and nucleic acids [START_REF] Beckman | Oxidants, antioxidants, and aging[END_REF]. These oxidative attacks can lead to inactivation of key enzymes and/or damage to transport protein receptors or ion channels, thus resulting in extensive cellular dysfunction [START_REF] Halliwell | Free Radicals in Biology and Medecine[END_REF]. At 30 1C and 75% RH, i.e. at low seed MC, loss of viability occurred without accumulation of H 2 O 2 or MDA, but we cannot exclude a possible role of secondary products, such as free radicals, aldehydes and hydroperoxides, produced through autooxidation reactions, in cell deterioration [START_REF] Mcdonald | Seed deterioration: physiology, repair and assessment[END_REF].

In addition, our results show that loss of wheat seed viability at 45 1C and 100% RH is concomitant with a progressive impairment of the efficiency of the antioxidant defence system (Table 3). It is in particular associated with a decrease in CAT and SOD activities and in an increase in GR activity (Table 3), the latter being not marked enough to limit H 2 O 2 accumulation from the fifth day of the treatment (Table 2). Depletion of antioxidant enzyme activities was also observed during seed ageing in soybean [START_REF] Murthy | Nonenzymatic protein modification by the Maillard reaction reduces the activities of scavenging enzymes in Vignia radiata[END_REF], cotton [START_REF] Goel | Changes in oxidative stress enzymes during artificial aging in cotton (Gossypium hirsutum L.) seeds[END_REF], beech [START_REF] Pukacka | Production and scavenging of reactive oxygen species in Fagus sylvatica seeds during storage at varied temperature and humidity[END_REF] and sunflower [START_REF] Bailly | Changes in malondialdehyde content and in superoxide dismutase, catalase and glutathione reductase activities in sunflower seeds as related to deterioration during accelerated ageing[END_REF][START_REF] Bailly | Free radical scavenging as affected by accelerated ageing and subsequent priming in sunflower seeds[END_REF][START_REF] De Paula | Function of the ascorbateglutathione cycle in aged sunflower seeds[END_REF][START_REF] Kibinza | Sunflower seed deterioration as related to moisture content during ageing, energy metabolism and active oxygen species scavenging[END_REF][START_REF] Torres | Ageing-induced changes in glutathione system of sunflower seeds[END_REF]. However, the enzymes involved in the antioxidant defence system exhibit different sensitivities to ageing depending on the species and the conditions of storage. In wheat seeds, SOD was more sensitive than CAT to a treatment at 45 1C and 100% RH (Table 3), whereas in sunflower seeds placed in the same conditions CAT activity was more sensitive than that of GR, SOD being almost unchanged [START_REF] Bailly | Changes in malondialdehyde content and in superoxide dismutase, catalase and glutathione reductase activities in sunflower seeds as related to deterioration during accelerated ageing[END_REF]. Alteration of SOD activity was also observed during ageing of seeds of Acer platanoides [START_REF] Pukacka | Changes in membrane lipid components and antioxidant level during natural aging of seeds of Acer platanoides[END_REF] and soybean [START_REF] Sung | Lipid peroxidation and peroxide-scavenging in soybean seeds during aging[END_REF], but an increase in GR activity during ageing treatment is in disagreement with data obtained by some authors [START_REF] Bailly | Changes in malondialdehyde content and in superoxide dismutase, catalase and glutathione reductase activities in sunflower seeds as related to deterioration during accelerated ageing[END_REF][START_REF] De Paula | Function of the ascorbateglutathione cycle in aged sunflower seeds[END_REF][START_REF] De Vos | Ageing of tomato seeds involves glutathione oxidation[END_REF][START_REF] Kibinza | Sunflower seed deterioration as related to moisture content during ageing, energy metabolism and active oxygen species scavenging[END_REF]. Such an increase in GR activity measured in vitro probably did not reflect the in vivo activity since H 2 O 2 strongly accumulated after 4 d of ageing. Although H 2 O 2 did not accumulate during the first day of ageing at 45 1C and 100% RH (Table 2), there existed a good correlation between seed vigour, evaluated by T 50 at 20 1C, and CAT activity, but such a correlation between T 50 , or seed viability, and both SOD and GR activities was less obvious (Fig. 2). CAT activity was also suggested to be a good indicator of seed vigour in sunflower [START_REF] Bailly | Free radical scavenging as affected by accelerated ageing and subsequent priming in sunflower seeds[END_REF] and rice [START_REF] Tanida | Catalase activity of rice seed embryo and its relation to germination rate at a low temperature[END_REF].

After 0.5 month of accelerated ageing performed at 30 1C and 75% RH, the activities of CAT, GR and SOD were lower than those measured in control non-aged seeds. Thereafter, they remained almost unchanged (Table 3). There was no correlation between the enzyme activities measured in vitro and seed viability. The ability of wheat embryos to maintain the enzyme activities was not then a good indicator of ageing, which was in disagreement with the suggestion of [START_REF] Bernal-Lugo | Effect of seed ageing on the enzymatic antioxidant system of maize cultivars[END_REF] with maize caryopses. However, we cannot exclude the possibility that the antioxidant machinery became unable to remove the ROS produced during ageing and by resumed metabolism during the imbibition phase of germination [START_REF] Bailly | Active oxygen species and antioxidants in seed biology[END_REF]. In addition, our results show that the sensitivity of CAT to accelerated ageing at 45 1C and 100% RH increased with the duration of previous treatment at 30 1C and 75% RH.

Different authors have demonstrated that soluble sugars, mainly oligosaccharides, and the oligosaccharides/sucrose ratio are positively correlated with potential seed longevity (Bernal-Lugo and [START_REF] Bernal-Lugo | Seed stability during storage: raffinose content and seed glassy state[END_REF][START_REF] Horbowicz | Seed desiccation tolerance and storability: dependence on flatulence-producing oligosaccharides and cyclitols-review and survey[END_REF][START_REF] Lin | The relationship between carbohydrate composition of some tree seeds and their longevity[END_REF][START_REF] Sinniah | Irrigation and seed quality development in seed rapid-cycling brassica: soluble carbohydrates and heat-stable proteins[END_REF][START_REF] Steadman | Tissue-specific soluble sugars in seeds as indicators of storage category[END_REF] and/or are good indicators of seed ageing [START_REF] Bernal-Lugo | Changes in soluble carbohydrates during seed storage[END_REF][START_REF] Petruzelli | Wheat aging: the contribution of embryonic and non embryonic lesions to loss of seed viability[END_REF][START_REF] Piotrowicz-Cieslak | Changes in soluble carbohydrates in yellow lupin seed under prolonged storage[END_REF][START_REF] Yaklick | Effect of aging on soluble oligosaccharide content in soybean seeds[END_REF]. It has been suggested that oligosaccharides inhibit crystallization of sucrose during desiccation, and thus permit formation of a glassy state which is likely to slow down ageing alterations [START_REF] Bernal-Lugo | Seed stability during storage: raffinose content and seed glassy state[END_REF]Buitink et al., 1998b;[START_REF] Leopold | The glassy state in seed: analysis and function[END_REF][START_REF] Leprince | A calorimetric study of glass transition behaviors in axes of bean with relevance to storage stability[END_REF][START_REF] Walters | Understanding the mechanisms and kinetics of seed aging[END_REF]. There is, for example, a good correlation between the longevity of pollen of Typha latifolia or pea seeds and the molecular mobility in the cytoplasm (Buitink et al., 1998a). This seems to be confirmed for wheat seeds when pre-harvest sprouting occurs, since a decrease in raffinose is associated with a loss of storage ability [START_REF] Lehner | Recherche d'indicateurs physiologiques et biochimiques de la qualite´germinative et de l'aptitude a`la conservation des semences de ble´[END_REF]. According to previous data [START_REF] Black | Carbohydrate metabolism in the developing and maturing wheat embryo in relation to its desiccation tolerance[END_REF][START_REF] Lehner | Changes in wheat seed germination ability, and in embryo soluble carbohydrate contents and antioxidant enzyme activities during maturation drying[END_REF], raffinose and sucrose are the main soluble sugars in the embryo of wheat and the value of the raffinose/sucrose ratio is about 0.7 in embryo of mature grains (Fig. 3). Our results demonstrate clearly that sugar metabolism in wheat embryo largely depends on ageing conditions (Fig. 3). Loss of seed viability at 45 1C and 100% RH was associated with a marked increase in the Ra/Su ratio from the third day of accelerated ageing (Fig. 3C), due to a decrease in Su content (Fig. 3A). Decrease in soluble sugars which has already been observed in wheat [START_REF] Petruzelli | Wheat aging: the contribution of embryonic and non embryonic lesions to loss of seed viability[END_REF], maize [START_REF] Bernal-Lugo | Changes in soluble carbohydrates during seed storage[END_REF]a n d soybean seeds [START_REF] Yaklick | Effect of aging on soluble oligosaccharide content in soybean seeds[END_REF] during accelerated ageing, indicates that activity of hydrolytic enzymes might occur during the treatment. In fact, a-galactosidase and invertase are active as soon as the water activity (Wa) is higher than 0.50 and 0.58, respectively [START_REF] Drapon | Enzyme activity as a function of water activity[END_REF][START_REF] Silver | The behavior of invertase in model systems at low moisture content[END_REF]. In addition, soluble sugars can be metabolized through respiration which occurs when the water content is higher than 25% DW [START_REF] Bruni | Hydration, protons and onset of physiological activities in maize seeds[END_REF][START_REF] Vertucci | Acquisition and loss of desiccation tolerance[END_REF] and the rate of Maillard reactions increases as water content rises above 0.14 g H 2 Og À1 DW [START_REF] Murthy | Mechanisms of seed ageing under different storage conditions for Vigna radiata (L.) Wilczek: lipid peroxidation, sugar hydrolysis, Maillard reactions and their relationship to glass transition[END_REF]. In wheat embryo, a value of the Ra/Su ratio exceeding 1, due to a decrease in Su, was associated with the outset of loss of viability at 45 1C and 100% RH. The marked decrease in sucrose content observed after 3 d of ageing might reflect an increase in respiratory metabolism leading to an increase in H 2 O 2 production (Table 2). It is well known that mitochondria participate for a large part in the production of AOS [START_REF] Kowaltowski | Mitochondrial implication in apoptosis: towards an endosymbiont hypothesis of apoptosis evolution[END_REF], and [START_REF] Kibinza | Sunflower seed deterioration as related to moisture content during ageing, energy metabolism and active oxygen species scavenging[END_REF] showed in sunflower embryo that the higher the energy metabolism during ageing the lower was the seed viability.

In contrast, loss of viability of wheat grains placed at 30 1C and 75% RH was associated with a progressive increase in both sucrose and raffinose contents in the embryo (Fig. 3B). Increase in sucrose might result from the hydrolysis of starch which is present in the embryo of mature grains at harvest [START_REF] Black | Carbohydrate metabolism in the developing and maturing wheat embryo in relation to its desiccation tolerance[END_REF]. Increase in sucrose was also observed in lupin seeds stored for several years under laboratory conditions (about 20 1C), but it was concomitant with a decrease in oligosaccharide content [START_REF] Piotrowicz-Cieslak | Changes in soluble carbohydrates in yellow lupin seed under prolonged storage[END_REF]. Such data suggest that biochemical (enzymatic) changes can occur at low MC. Reducing sugars are also known to play an important role in the Amadori and Maillard reaction [START_REF] Wettlaufer | Relevance of Amadori and Maillard products to seed deterioration[END_REF], the products of which are involved in various alterations during ageing [START_REF] Kristal | An emerging hypothesis: synergistic induction of ageing by free radical and Maillard reactions[END_REF][START_REF] Monnier | Non enzymatic glycosylation, the Maillard reaction and the aging process[END_REF][START_REF] Murthy | Mechanisms of seed ageing under different storage conditions for Vigna radiata (L.) Wilczek: lipid peroxidation, sugar hydrolysis, Maillard reactions and their relationship to glass transition[END_REF]. However, no increase in fructose, glucose or galactose has been noticed in wheat embryo during either type of ageing (data not shown).

In conclusion, our results demonstrate that ageing of wheat grains (cv Charger) is associated with changes in the antioxidant defence system and in soluble sugar content in the embryo. Similar results were obtained with other cultivars such as Tre´mie and Apache (data not shown). They give evidence that these biochemical events largely depend on the conditions of ageing and underline the likely key role of the embryo water content in the mechanisms involved in seed deterioration or protection against ageing damage. Consequently, accelerated ageing cannot be used as the only model to understand the mechanisms involved in seed ageing, and we cannot establish a clear relationship between either active oxygen species scavenging, or soluble sugars and seed ageing independently on the conditions of storage. This absence of correlation might result from the fact that all measurements were carried out in embryos isolated from aged seeds before their imbibition. Further investigations are required for elucidating the effects of storage conditions on the subsequent efficiency of the antioxidant system during the critical step of imbibition.

  Fig.1. Changes, during accelerated ageing at 45 1C and 100% RH (A) and at 30 1C and 75% RH (B), in grain viability ('), embryo moisture content expressed on dry weight basis (n) and the time to reach 50% of the germination percentages obtained after 14 d (T 50 )( J). Means of 3 replicates7SD. Where no bars are shown, the spread of SD is less than the size of the symbols.

  Fig.2. Relationships between the time to obtain 50% germination (T 50 )at201C with grains aged for various times at 45 1C and 100% RH, and catalase (CAT), superoxide dismutase (SOD) and glutathione reductase (GR) activities measured in the embryos. Values from Fig.1Aand Table3.

Fig. 3 .

 3 Fig. 3. Changes in sucrose (') and raffinose (m) contents (A, B) and in the Ra/Su ratio (C, D) in embryos from grains aged at 45 1C and 100% RH (A, C) and at 30 1C and 75% RH (B, D). Means of three measurements7SD. Where no bars are shown, the spread of SD is less than the size of the symbols.

Table 1

 1 Effect of the duration of pretreatment at 30 1C and 75% RH on the viability of seeds subsequently aged for 2-4 d at 45 1C and 100% RH

	Duration of treatment at 30 1C and 75% RH (months)	Seed viability (%) after ageing at 45 1C and 100% RH for (d)
		0234
	0 (control)	100	100	100	9970.7
	1.5	100	8072.0	7671.4	2672.8
	2	100	6476.3	4071.4	1870.7
	2.5	100	4072.1	872.1	1271.4
	39 2 71.1	471.9	471.8	672.3
	3.5	8672.1	270.7	872.2	271.0

Control seeds were placed directly at 45 1C and 100% RH. Means of 4 replicates7SD.

Table 2

 2 Effect of the duration of ageing on hydrogen peroxide (H 2 O 2 ) and malondialdehyde (MDA) contents in wheat embryos

	Ageing treatment	H 2 O 2 (mmol g À1 DW) MDA (mmol g À1 DW)
	Conditions Duration
	45 1C	0 d	3.2970.02	0.4270.03
	100% RH 1 d	2.5671.20	0.4170.03
		2 d	1.6470.51	0.3970.05
		3 d	1.0470.00	0.4370.01
		4 d	1.4270.20	0.4370.02
		5 d	2.4270.80	0.4270.03
		6 d	5.5470.97	0.3970.04
	30 1C	0 month	3.2970.02	0.4270.03
	75% RH	1 month	-	0.4370.05
		2 months	3.1270.10	0.4570.05
		2.5 months -	0.4370.02
		3.5 months -	0.4170.10
		4 months	3.4270.12	0.4370.03
		4.5 months 3.1070.25	0.3970.01
	Embryos were isolated from grains aged for various times at 45 1C and
	100% RH and at 30 1C and 75% RH. Means of 5 replicates7SD.

  À1 mg À1 protein. SOD activity: units mg À1 protein. GR activity: nmol NADPH min À1 mg À1 protein. Means7SD of the values obtained with three different extracts and three measurements per extract.

	Table 3
	Effect of the duration of ageing on catalase (CAT), superoxide dismutase
	(SOD) and glutathione reductase (GR) activities in wheat embryos
	Ageing treatment	Activity of
	Conditions	Duration	CAT	SOD	GR
	45 1C	0 d	7.371.1	13.272.6	235749
	100% RH	1 d	9.673.2	6.672.1	29677
		2 d	9.271.0	5.470.7	6497100
		3 d	6.773.1	3.870.8	401740
		4 d	6.170.5	1.671.2	505750
		5 d	3.071.5	0	11507360
		6 d	3.971.2	0	11407200
	30 1C	0 month	7.371.1	13.272.6	235749
	75% RH	0.5 month	5.970.6	9.871.1	130730
		1 month	6.670.7	9.772.6	115760
		2 months	6.570.7	8.171.8	-
		2.5 months	5.970.1	7.271.9	200760
		3.5 months	6.370.3	11.273.1	125740
		4 months	5.870.5	9.972.2	160732
		4.5 months	6.471.0	10.172.0	180721
	Embryos were isolated from grains aged for various durations at 45 1C
	and 100% RH and at 30 1C and 75% RH. CAT activity: nmol H 2 O 2
	decomposed min

Table 4

 4 Effect of the duration of pretreatment at 30 1C and 75% RH on CAT activity in the embryo of seeds subsequently aged at 45 1C and 100% RH for 2-4 d Control seeds were placed directly at 45 1C and 100% RH. Means of the values obtained with three different extracts and three measurements per extract. CAT activity in the embryo of untreated control seeds was 7.371.1 nmol H 2 O 2 decomposed min À1 mg À1 protein.

	Duration of ageing at 30 1C and 75% RH (months)	CAT activity (% initial value) after subsequent ageing at 45 1C and 100% RH for (d)
		023	4
	0 (control)	100	126.1	91.8	83.6
	1.5	87.7	103.6	37	26.5
	2.5	80.8	91.8	30.2	20
	3	80.8	80.8	30.1	17.8
	3.5	86.3	94.5	31.5	17.8