Rachid Cheddadi
email: rachid.cheddadi@um2.fr

Majda Nourelbait

Ouafaa Bouaissa

Jalal Tabel

Ali Rhoujjati

José Antonio López Sáez

Francisca Alba-Sanchez

Carla Khater

Aziz Ballouche

Laurent Dezileau

José Antonio López-Sáez

Francisca Alba-Sánchez

Henry Lamb

A History of Human Impact on Moroccan Mountain Landscapes

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

Introduction

Mediterranean ecosystems, including those in Morocco, are well known to have been strongly impacted by human activities over the last few millennia [START_REF] Ballouche | Données palynologiques et carpologiques sur la domestication des plantes et l'agriculture dans le néolithique ancien du Maroc septentrional. (site de Kaf Taht El-Ghar)[END_REF][START_REF] Lamb | Human impact on the vegetation of the Middle Atlas, Morocco, during the last 5000 years[END_REF][START_REF] Sadori | Mid-Holocene vegetation history of the central Mediterranean[END_REF][START_REF] Mercuri | Mediterranean and north-African cultural adaptations to mid-Holocene environmental and climatic changes[END_REF]. However, we still lack clear information about the extent and intensity of early human impact on the natural vegetation of Morocco. Archaeological studies have identified the sites of early human settlement, farming and industry in Morocco [START_REF] Ballouche | Données palynologiques et carpologiques sur la domestication des plantes et l'agriculture dans le néolithique ancien du Maroc septentrional. (site de Kaf Taht El-Ghar)[END_REF][START_REF] Daugas | The Earlier Neolithic in Northern Morocco: documentary, typo-chronological seriation and genetic hypothesis[END_REF][START_REF] López-Sáez | Antropización y neolitización durante el Holoceno en Marruecos: una aproximación paleopalinológica[END_REF][START_REF] López-Sáez | Paisajes neolíticos del noroeste de Marruecos: análisis arqueopalinológico de la Cueva de Boussaria[END_REF][START_REF] Morales | The origins of agriculture in North-West Africa: macro-botanical remains from Epipalaeolithic and Early Neolithic levels of Ifri Oudadane (Morocco)[END_REF][START_REF] Zapata | Holocene environmental change and human impact in NE Morocco: Palaeobotanical evidence from Ifri Oudadane[END_REF] but the impact of these human societies on the forest ecosystems and on the overall landscapes are not well known. Palaeoecological records provide insight into both potential natural and anthropogenic vegetation changes, although it is not always possible to distinguish natural disturbances, such as fires, from the small-scale disturbances caused by hunter-gatherers, or by the first pastoral and cultivating farmers. Archaeological sites provide direct evidence of the occurrence of human populations and their activities, but they do not provide information about the impact on the environment and the surrounding landscapes.

Palaeoecological records from lake and peatland sediments cannot directly show the presence of a human population, but may provide complementary information at both local and regional scales.

Moreover, a regional dataset of fossil pollen records may help reconstruct the changing spatial extent and scale of human impact through time. [START_REF] Lamb | Human impact on the vegetation of the Middle Atlas, Morocco, during the last 5000 years[END_REF] attempted a reconstruction of human impact on the vegetation of the Middle Atlas from four lacustrine pollen records spanning the last few thousand years. They identified forest exploitation in the Middle Atlas as early as 3000 BC using fossil pollen data. However, anthropogenic disturbance seems to have been slight until 2000 BC, when forest clearance and grazing became more intense. At Tigalmamine, human impact first became strongly evident at 2250 14 C yr BP (ca. 200 BC) with a strong reduction of the forest cover [START_REF] Lamb | Vegetational response to Holoene climatic change: pollen and palaeolimnological data from the Middle Atlas, Morocco[END_REF]. At Taguelmam n'Harcha [START_REF] Lamb | Human impact on the vegetation of the Middle Atlas, Morocco, during the last 5000 years[END_REF] it is even later, around 1720 14 C yr BP (ca 300 AD), when the presence of cultivated cereals is detected in the pollen record. At Sidi Ali [START_REF] Lamb | Lake evolution in a semiarid montane environment: responses to catchment change and hydroclimatic variation[END_REF], Dayat Iffir and Dayat Afourgagh [START_REF] Lamb | Human impact on the vegetation of the Middle Atlas, Morocco, during the last 5000 years[END_REF], human impact began around 3000 BP (ca. 1000 BC). According to these palaeoecological studies of the Middle Atlas, it seems that anthropogenic factors became evident only after the first millennium BC (third millennium cal BP).

Archaeobotanical data (pollen, seed and charcoal remains) provide direct evidence of human impact, tending to show that activities such as the beginning of farming started much earlier at ca.

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5600 BC in coastal or near-coastal areas [START_REF] López-Sáez | Antropización y neolitización durante el Holoceno en Marruecos: una aproximación paleopalinológica[END_REF][START_REF] Morales | The origins of agriculture in North-West Africa: macro-botanical remains from Epipalaeolithic and Early Neolithic levels of Ifri Oudadane (Morocco)[END_REF][START_REF] Zapata | Holocene environmental change and human impact in NE Morocco: Palaeobotanical evidence from Ifri Oudadane[END_REF]. This temporal difference between the palaeoecological and archaeobotanical studies in Morocco may be due either to lack of detail in the Middle Atlas pollen records (which provided "only a broad outline"; [START_REF] Lamb | Human impact on the vegetation of the Middle Atlas, Morocco, during the last 5000 years[END_REF] of human activities, or to a spatial difference between the northern Moroccan coastal sites and those at higher altitude. Indeed, it is likely that this asynchrony between human impact in mountain and coastal areas relates to the cultural capacity to conquer the high mountain areas and the historical development of the Neolithic economy in northern Africa. Other palaeobotanical studies suggest that human impact on Mediterranean ecosystems is often difficult to detect prior to the Bronze Age (around 3000 BC; [START_REF] Mercuri | Mediterranean and north-African cultural adaptations to mid-Holocene environmental and climatic changes[END_REF].

In any case, the start of human impact in northern Africa poses questions concerning population expansion either from Africa to southwestern Europe or in the opposite direction [START_REF] Zapata | Holocene environmental change and human impact in NE Morocco: Palaeobotanical evidence from Ifri Oudadane[END_REF] since there is a clear evidence of land and forest exploitation in the Iberian peninsula since ca. 5500 BC, which became even more evident in the late Holocene (ca. 2100 BC) (López-Sáez et al., 2011a, 2011b;[START_REF] Cortés | The Mesolithic-Neolithic transition in southern Iberia[END_REF][START_REF] Damblon | Contribution pollenanalytique à l'histoire des forêts de chêne liège au Maroc : la subéraie de Krimda[END_REF]. The role of human activities in the regional fire history should also be taken into account [START_REF] Damblon | Contribution pollenanalytique à l'histoire des forêts de chêne liège au Maroc : la subéraie de Krimda[END_REF][START_REF] Linstädter | Regional fire history shows abrupt responses of Mediterranean ecosystems to centennial-scale climate change (Olea-Pistacia woodlands, NE Morocco)[END_REF][START_REF] Gil-Romera | Holocene fire activity and vegetation response in south-eastern Iberia[END_REF].

In this paper we extend these earlier studies to other sites in both the Middle Atlas and the Rif mountains, from which we have obtained new, well dated records. These two mountain chains have different geological histories and therefore provide different signatures in the biogeochemical record. We use geochemical elements to identify both the contribution of the geological watersheds through time in the Rif mountains, and metallurgical activity in the Middle Atlas. The fossil pollen data provide a record of ecosystem changes in both of these montane regions, including their forest degradation and dynamics. In the Rif mountains, forest clearance led to strong soil erosion, evident as increased carbonate (Ca) in the sedimentary record. In the Middle Atlas, forest clearance is identified through geochemical elements indicative of prehistoric industrial activity.

Materials, methods and site descriptions

In the present study we use data from several late Holocene fossil pollen sequences collected in the Rif mountains and in the Middle Atlas (figure 1). The dated material (table 1) shows that these records cover a more extended time span than that of previous investigations. Here we focus on the parts of the records which show the start and the spread of human activities.

A. The Rif mountains records

During the Cenozoic, the collision between the African and European continents generated the Moroccan mountain chains [START_REF] Gómez | Role of Atlas Mountains (northwest Africa) within the African-Eurasian plate-boundary zone[END_REF][START_REF] Lamotte (de | The two main steps of the Atlas building and geodynamics of the western Mediterranean[END_REF]Teixeill et al. 2003) including the Rif range that extends along the Mediterranean Sea coast. Geologically, it belongs to the Gibraltar Arc or Alborán Sea geological region [START_REF] Michard | Pressuretemperature-time constraints on the Maghrebide mountain building: evidence from the Rif-Betic transect (Morocco, Spain), Algerian correlations, and geodynamic implications[END_REF]. Although the mountains are highly mineralized, only iron ore is mined on a large scale.

Today, the annual rainfall in some areas of the Rif reaches 2000 mm. The wettest areas in the west and central parts of the Rif support forests of Cedrus atlantica, Quercus faginea, Q. pyrenaica and Q. canariensis, as well as forests of the endangered endemic Abies pinsapo var. marocana (figure 1). The vegetation composition changes towards the drier eastern part of the Rif, where the forests are dominated by pines (Pinus halepensis, P. pinaster) with Sandarac (Tetraclinis articulata). The vegetation is heavily impacted through cutting and clearing which leads to strong soil erosion and the release of components such as hydroxides of iron, alumina, clays and the cations Ca++, Mg++ and K+. The discharge of these elements varies from one landscape to another.

As might be expected, isotopic studies using Cesium 137 ( 137 C) in northern Morocco show that agricultural lands are more extensively eroded (18-36 t/ha/yr) than the forested areas (<4.5 t/ha/yr; [START_REF] Zouagui | Application de la technique de 137 Cs à l'estimation de l'érosion hydrique dans le bassin versant de Moulay Bouchta, Rif occidental, Maroc[END_REF].

Anaasser peat bog

Anaasser peat bog is located near the rural town of Bab Berred, below Jbel Tizirène (figure 1).

This mountain shows a clear altitudinal zonation from evergreen oaks (Quercus ilex) on the lower slopes, followed by deciduous oaks (Q. canariensis and then Q. pyrenaica) and Cedrus atlantica on the very top of the mountain. The site is a floating bog surrounded by cultivated land and poplars (Populus spp). In 2012, we collected a 650 cm core (ANS) at 35°01'3.4''N, 4°59'34.5''W, 1342 masl. AMS 14 C dates on four bulk sediment samples (table 1) provide a chronology for the last 2000 years.

Dayet M'Had

Dayet M'Had is located south of the Parc Naturel de Bouhachem at the westernmost part of the Rif Mountains. The park, covering an area of about 90,000 ha, is a protected area with many wetlands, peatlands, highly diverse forest stands and matorrals that provide habitat for rare animal and plant species. The core site is currently surrounded by Pinus halepensis. Cedar forests occur at a higher altitude about 4 km from the study site. In 2012, we collected a 550 cm core at 35°7'557'N, 5°26'455'W, 754 masl (figure 1). Five 14 C AMS dates on bulk sediment (table 1) indicate that the core covers the last 5000 years.

B. The Middle Atlas records

Besides the Rif, Morocco has three other large mountain chains: the Middle, High and Anti-Atlas. They separate the Mediterranean and the Atlantic from the Saharan Desert. The Middle Atlas (800-2800 masl) is composed of Permo-Triassic bedrock covered by formations ranging from the Jurassic to the Paleogene [START_REF] Dresnay (du | Recent data on the geology of the Middle Atlas (Morocco) In[END_REF][START_REF] Herbig | Synsedimentary tectonics in the Northern Middle Atlas (Morocco) during the Late Cretaceous and Tertiary In: V. Jacobshagen, Editors[END_REF]. It includes two structural and geomorphological units, the folded and the tabular Middle Atlas, separated by the North Middle Atlassic fault [START_REF] Arboleya | A structural transect through the High and Middle Atlas of Morocco[END_REF]. Alkaline basalts of Quaternary age cover large areas especially in the centre of the two units. Subsidence due to an intense fault network, together with the karstification of dolomites [START_REF] Martin | Le Moyen Atlas central étude géomorphologique[END_REF][START_REF] Hinaje | Les Bassins Lacustres du Moyen Atlas (Maroc) : Un exemple d'Activité Tectonique Polyphasée Associée à des Structures d'Effondrement[END_REF], allowed the formation of about twenty natural permanent or semi-permanent lakes [START_REF] Chillasse | Potentialités et statuts de conservation des zones humides du Moyen-Atlas (Maroc), avec référence aux influences de la sécheresse[END_REF]. The Middle Atlas has mineral resources including iron, zinc, lead and copper [START_REF] Newman | Minerals Yearbook[END_REF].

In the Middle Atlas, Jurassic dolomitic limestone covers a large area, overlaid in turn by Quaternary basalts. Under a subhumid climate, brown fersiallitic soils have developed from this bedrock by weathering of carbonates. The alteration of basaltic rocks generates clay minerals, especially phyllosilicates, providing silica, aluminium and iron to the geochemical sedimentary record [START_REF] Dekayir | Clay minerals in hydrothermally altered basalts from Middle Atlas, Morocco[END_REF] through rainfall-induced allochthonous input of terrigenous fractions to the sedimentary basins. An earlier study [START_REF] Rhoujjati | Changements paléoenvironnementaux et paléoclimatiques depuis 21[END_REF] using XRD analyses on lacustrine sediments showed dominance of carbonates, especially detrital dolomite, followed by iron hydroxides, inosillicates (alteration of basalt), tectosilicates (ie quartz) and phyllosilicates (silicate of alumina) resulting from Triassic argillite leaching.

These geological patterns and their evolution through time have led to the deposition of minerals exploited by human populations since prehistoric times [START_REF] Akdim | Land use change and resource management in the Jnane Mas valley (Khenifra, Middle Atlas, Morocco)[END_REF]. Today, two main polymetallic districts are exploited in the Middle Atlas. The extractions concern different types of mineralization among which lead, zinc, silver, and gold in the district of Tighza and sulphides of lead, zinc, iron and copper in the district of Elhamam at about 50 km SE and 10 km North of Azrou city.

Today, the Middle Atlas has the most extensive forests of Cedrus atlantica in North Africa occupying about 140,000 ha [START_REF] Benabid | Les écosystèmes forestiers préforestiers et présteppiques du Maroc: diversité, répartition biogéographique et problèmes posés par leur aménagement[END_REF]. Other forest vegetation is composed of sclerophyllous species such as Olea europaea, Quercus ilex, Q. coccifera, and Pinus halepensis.

The climate is temperate and humid with an annual rainfall between 600 mm and 1200 mm and an average annual temperature of 10°C.

Ras El Ma marsh

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In 2011, a 3m long core was collected from Ras El Ma (REM) marsh (33°28,419'N, 05°08,325'W, 1633 masl) (figure 1) using a Russian corer. The sedimentological sequence, from the top of the core, consists of 120 cm of highly organic peat, 160 cm of compact clayey silt and 20 cm of oxidized sand with limestone concretions. Eight radiocarbon 14 C dates were obtained from organic bulk sediments (table 1). The record covers the last 18,000 years [START_REF] El Bait | An 18,000 year pollen and sedimentary record from the cedar forests of the Middle Atlas, Morocco[END_REF] but only the data of the last 3500 years are presented in the present paper.

The REM site is a wetland situated in the centre of the Middle Atlas, about 5 km south of the city of Ifrane towards Azrou. The site is formed by a slight depression oriented south west -north east at an altitude of 1633m asl. Annual rainfall varies between 860 and 1120 mm with peaks in November-December and February-March. Snowfall is variable with an annual average snow cover of 3 weeks/year. The surrounding vegetation is dominated by Cedrus atlantica mixed with evergreen stands of Olea europaea, Quercus ilex, Q. coccifera, and Pinus halepensis as well as xerophilous plants marked by the presence of herbaceous taxa (Asteraceae, Chenopodiacee, Caryophyllaceae, Liliaceae, Poaceae and Apiaceae).

Lake Tifounassine

Tifounassine is a small freshwater lake in the Middle Atlas, 40 km from Azrou on the road to Midelt, about 9 km south of the village of Timahdit (figure 1). In 2013 we collected a 550 cm long core (TIF) from Tifounassine (33°09'N, 5°06'W, 1913 masl). Ten AMS radiocarbon dates show that the record encompasses the Holocene but only the last 2500 years are discussed in the present paper

The wetland itself is composed of three parts: a deep, permanent lake occupying a small crater, then a larger but shallower spring-fed lake located to the south, often muddy and marshy;

and a large marshy zone, often dry throughout the year and used by livestock. The site is heavily overgrazed and the surrounding vegetation develops only in the wetter areas, which support many hydrophilous taxa such as Typha, Myriophyllum spicatum, Nasturtium officinale, Phragmites communis, Ranunculus aquatilis, Scirpus lacustris and Tolypella hispanica. The average annual precipitation is ca. 680 mm, mostly as snow which may persist on the upper hills until March. The average monthly temperature varies between -2°C and 30°C in winter and summer, respectively.

Palaeoecological records of human impact: the historical context

When human populations increased in Europe there was an expansion of pastoral and arable land within areas that would naturally be forested, and an increase in the use of forest products such as fuelwood and construction materials leading to an overall decline of the forest areas [START_REF] Kaplan | The prehistoric and preindustrial deforestation of Europe[END_REF]. In the Mediterranean countries of North Africa, there are very few data that provide clear evidence of such intensive and/or continuous human disturbance throughout the Holocene. In northern Morocco, there is evidence that the first agricultural societies settled around 5500 BC in the lowlands [START_REF] Zapata | Holocene environmental change and human impact in NE Morocco: Palaeobotanical evidence from Ifri Oudadane[END_REF]. In effect, most of northern Africa has undergone a succession of periods of deforestation and afforestation, or simply of abandonment, over at least the last three millennia [START_REF] Mercuri | Mediterranean and north-African cultural adaptations to mid-Holocene environmental and climatic changes[END_REF], probably restricted to areas at low altitude.

Past human disturbance in the pollen records from Morocco may be related to three types of human activity: grazing of forests by domestic goats, sheep and cattle; the expansion of cultivated lands; and by wood intake from the forests for fuel and construction, including industrial activity [START_REF] Mather | The human drivers of global land cover change: the case of forests[END_REF]. These activities may be identified in the records through:

1. reduction in arboreal taxa (oak, pines and cedars) which may be related to suppression of tree regeneration by grazing, to clearance for cultivation, or to exploitation of wood for construction, heating, and fuel for producing industrial energy; 6. strong fluctuations in arboreal pollen which may express cycles of deforestation and abandonment, followed by natural regeneration of the forest (cf. [START_REF] Iversen | The Development of Denmark's Nature since the Last Glacial[END_REF].

Pollen data allow reconstruction of the type of change, smooth or abrupt, that an ecosystem may undergo through time. The palaeoecological signal of human impact on forest ecosystems is often rapid and abrupt, especially where a settlements is close to the core site, thus allowing some discrimination between anthropogenic disturbances and longer, more gradual natural changes, except in the case of lightning-caused fires. Ancient DNA analyses, complementing the fossil pollen evidence, may help to determine more precisely both the timing and the type of human activities over the past thousands of years [START_REF] Giguet-Covex | Long livestock farming history and human landscape shaping revealed by lake sediment DNA[END_REF]. Modern DNA may also provide indirect evidence about human uses of forest resources: for instance, chloroplast microsatellites and isozymes have been used to show the potential effects of human impact vs.

natural evolutionary processes on the genetic diversity of Cedrus libani in in the eastern Mediterranean [START_REF] Fady | Genetic consequences of past climate and human impact on eastern Mediterranean Cedrus libani forests. Implications for their conservation[END_REF].

The sedimentary records may also contain valuable information concerning the watershed and its input of geochemical elements. The geological composition and the vegetation cover of the Rif mountains (dolomite) and the Middle Atlas (basalt or dolomite; [START_REF] Arboleya | A structural transect through the High and Middle Atlas of Morocco[END_REF] provide different chemical elements and amounts into the site where the records were collected. The palaeoecological records used in this study were retrieved at altitudes higher than 1500 m. Lakes and wetlands naturally draw people to the water resources, so that even in the absence of archaeological evidence for settlement, it is likely that some of the soil erosion and forest disturbance evident in the sedimentary records may be directly attributed to human agency in the local environment. In other cases, where sites were too remote or too high for lomg-lasting settlement, the sequences may record disturbance at a predominantly regional scale through fartravelled pollen, and via dust and elemental fallout from mining and metallurgical industry. Like those known from other regions (Shotyk, 1996a(Shotyk, , 1996b;;[START_REF] Mighall | Geochemical evidence for atmospheric pollution derived from prehistoric copper mining at Copa Hill, Cwmystwyth, mid-Wales[END_REF][START_REF] Bindler | Mired in the past -looking to the future: geochemistry of peat and the analysis of past environmental changes[END_REF][START_REF] Guyard | High-altitude varve records of abrupt environmental changes and mining activity over the last 4000 years in the Western French Alps (Lake Bramant, Grandes Rousses Massif)[END_REF]Pompeani et al., 2015), the sediments are not from archaeological sitesand tcannot be considered as direct records of human activity in Morocco since they do not derive directly from human environments. Nevertheless, the biological and geological indicators they contain allow the identification of provide continuous records of varying levels of industrial output, whereas archaeological data tend to provide a more episodic, discontinuous record.

Prior to the Romans, Morocco was occupied by the Phoenicians. The ancient city of Lixus, settled by the Phoenicians, dates back to the 7th century BC. In this area, it seems that anthropogenic activities (fire and grazing) over the last 3500 years did not impact strongly on a well-developed cork oak (Q. suber) forest [START_REF] Damblon | Contribution pollenanalytique à l'histoire des forêts de chêne liège au Maroc : la subéraie de Krimda[END_REF]. After the fall of Carthage, northern 1997).

Previous palynological investigations of human impact in the Middle Atlas [START_REF] Lamb | Human impact on the vegetation of the Middle Atlas, Morocco, during the last 5000 years[END_REF] and the Rif mountains [START_REF] Reille | Contribution pollenanalytique à l'histoire Holocène de la végétation des montagnes du Rif (Maroc Septentrional)[END_REF] as well as those in the present study (figures 2, 3 & 4)

show a regression of the forest taxa after 1000 BC, which suggests an increase of the human uses of the high-altitude forests. Impact of human populations on the forest ecosystems prior to 1000 BC was probably minor since anthropogenic indicators are not dominant in the sedimentary record. The early changes in the vegetation during the mid-Holocene are very probably related first to the progressive installation of Mediterranean seasonality [START_REF] Roberts | The tempo of Holocene climatic change in the eastern Mediterranean region: New high-resolution crater-lake sediment data from central Turkey[END_REF][START_REF] Jalut | Holocene circum-Mediterranean vegetation changes: Climate forcing and human impact[END_REF] and then to the gradual increase of summer aridity as observed throughout the south-western Mediterranean region [START_REF] Peyron | Contrasting patterns of climatic changes during the Holocene across the Italian Peninsula reconstructed from pollen data[END_REF]. This climate aridification allowed the expansion of true Mediterranean elements such as Olea, Pistacia and evergreen oak forest (Q. ilex, Q. coccifera).

Our data suggest that human use of forest resources was less drastic prior to the expansion of Forest degradation also led to the exposure and erosion of the soils. Calcium (Ca) content in the sedimentary record shows an increase which is very probably related to the erosion of dolomite, after the spread of the Romans in the eastern part of the Rif (figure 1, Anaasser) and later at its western edge (figure 1, M'had). Treaties signed between the Berber populations in the Rif mountains and the Romans [START_REF] Taiqui | Eléments historiques d'analyse écologique des paysages montagneux du Rif Occidental (Maroc)[END_REF] substantially reduced the Roman impact on the landscape and their intensive use of the cedar and fir trees because the Berbers remained the only users of their forests.

In the Middle Atlas, the increase of Cu, Zn and Pb at Tifounassine (figure 3) and Ras El Ma (figure 4), combined with forest regression about 2000 years ago, may be related to the presence of local metal industries for extracting lead, zinc and copper using the forest wood as a source of energy. The name of the Middle Atlas town "Rsass" (in Arabic) means literally lead, showing the importance of mineral extraction in the area, which may date back to the Roman period. The decline of zinc, lead and copper in the Middle Atlas records after the 15th century seems to suggest that Roman industrial activities ceased when they left the area. Similar changes related to the same activities are observed in the south of France [START_REF] Elbaz-Poulichet | A 3500-Year Record of Hg and Pb contamination in a Mediterranean Sedimentary Archive (The Pierre Blanche Lagoon, France[END_REF].

A major break in the landscape history is marked by the arrival of nomadic and semi-nomadic Arab tribes [START_REF] Ballouche | Contribution à l'histoire récente de la végétation du Bas-Loukkos (Province de Larache, Maroc)[END_REF]. These tribes exploited the forest but did not re-establish the industrial metallurgy as in the Roman period. In the Rif mountains the records of Pb, Zn and Cu do not show any significant changes over the last 2000 years (Bouaissa, PhD unpublished data), suggesting that the metal industry remained confined to the Middle Atlas.

These late Holocene human activities had an important impact on the surrounding landscapes, but they may also have contributed to wider scale changes. At the local scale, they obviously affected ecosystem composition, structure and potentially their long-term dynamics. At the same time, basin catchments and their hydrology were also impacted by the reduction of forest cover through increasing runoff and soil erosion. At the global scale, Ruddimann ( 2003) has argued that humanrelated reduction of forest cover and the development of agriculture, especially rearing of goats, sheep and cattle, may have contributed to the early increase of atmospheric CO 2 evident from polar ice-core records. Although the North African forests are obviously much less extensive than those of Europe, Asia, the Americas and tropical Africa, their reduction may have made a contribution to the alteration of the global carbon cycle. However, some climate simulations, using transient models, tend to show that early CO 2 emissions to the atmosphere were probably not significant enough to have a clear impact on global climate [START_REF] Pongratz | Effects of anthropogenic land cover change on the carbon cycle of the last millennium[END_REF].

In fact, there may be significant contrasts in the impact of human activities at different scales.

A general decline in forest cover across North Africa masks local-scale growth or regrowth of forests. The matorralisation process during the last millennium in Morocco derives from the intensification of land clearance and grazing pressure, as well as a high frequency of fires [START_REF] Reille | Contribution pollenanalytique à l'histoire Holocène de la végétation des montagnes du Rif (Maroc Septentrional)[END_REF][START_REF] Damblon | Contribution pollenanalytique à l'histoire des forêts de chêne liège au Maroc : la subéraie de Krimda[END_REF][START_REF] Lamb | Human impact on the vegetation of the Middle Atlas, Morocco, during the last 5000 years[END_REF][START_REF] Mcgregor | Vegetation change, goats, and religion: a 2000year history of land use in southern Morocco[END_REF]). However, after the 11-13 th centuries AD, one may observe the local emergence of a new type of landscape that is poorly documented in terms of vegetation history: dehesa-like agro-sylvo-pastoral determined parklands (Vicente & Ales, 2006;[START_REF] Ballouche | Contribution à l'histoire récente de la végétation du Bas-Loukkos (Province de Larache, Maroc)[END_REF]. These original Mediterranean landscapes were then eradicated during the late 19th and early 20th century by agricultural intensification, or through reforestation.

Conclusions

Although the palaeoecological data used in this study do not originate from archaeological sites, they do provide direct and temporally continuous records of exploitation of forest, soil and mineral resources, at regional and local scales. They allow us to infer human activity during the last two millennia, from palynological and geochemical evidence. Forest disturbances had an impact on the surrounding environment through soil erosion and therefore on the release and transport of geochemical markers through the eco-hydrological systems. Analysis of these geo-indicators allows detection of the timing and varying intensity of the environmental changes. In the Middle Atlas, metals such as copper, lead and zinc were extracted by the Romans. Mining and smelting required the use of energy, obtained from the forests which served as a fuel source.

Today, there are initiatives in Morocco to preserve the forest ecosystems in the Rif and the Middle Atlas in delineated parks and reserves. Clearly, not all the forests are protected from grazing and clearing, and there is ongoing deforestation for various uses of wood in many areas in Morocco.

Besides the parks and reserves, there are also forest areas that are protected simply with fences where there is some good regeneration through natural re-sprouting and seedlings. Outside these protected areas there is very little if any natural regeneration of the forests, and in many cases the effects of the strong deforestation on soil degradation, with heavy erosion, is evident. Years cal BC/AD 1. The chronological framework of the sites appears to be robust. Details of the available radiocarbon dates should be listed in a table, with informations on the dated material, depths of samples, AMS 14C yr BP ages, etc... A new table has been added, with dated samples from each fossil record. I would also recommend not to mix uncalibrated and calibrated dates in the main text, to avoid confusion for those who do not feel comfortable with calibration curves. Following standard convention, uncalibrated dates should be reported as 14C yr BP, while calibrated dates should be reported as BC (or BCE).

All dates in the manuscript have been changed into either "14C yr BP" or "BC". "Uncal" or "cal" have been removed from the text. The time scale of all figures is now in BC/AD 2. The manuscript would benefit from some reorganization, moving most of the geological background in an expanded form of the site descriptions.

Thank you, 3. As REM, HAO1, TIF13 and Aït Ichou sites are presumably new unpublished sites, I strongly suggest that graphic logs, position of dated levels, main geochemical elements and summary percentage pollen diagrams be presented versus depth for each core. This would greatly help the reader to understand the type of changes that ecosystems underwent through time. A last figure displaying the synthesis of all data and the main cultural phases (Romans….) on a common time-scale would be appreciated.

The REM record is now published and properly cited [START_REF] El Bait | An 18,000 year pollen and sedimentary record from the cedar forests of the Middle Atlas, Morocco[END_REF]. The publication contains detailed information about stratigrahy, age/depth model, detailed pollen and XRF data .... Ait Ichou and Hachlaf data have been removed since they are not being used in the discussion (we were expecting to obtain the XRF data in the meantime but we didn't manage it). Tifounassine (TIF13) extended data are being used in another publication in preparation which includes all detailed information. The inclusion of only a part of the data (the last 3000 years) in this publication is not suitable. In effect, all necessary information is now available in an improved version of figures 2, 3 and 4. Additional details would not bring more arguments for the discussion. Instead of dropping that small paragraph, we have reworked it so that it fits the discussion.

Reviewer #2:

-Overall the background is good but this is occasionally marred by a lack of attention to detail which may be because the authors are unaware of geological studies. For example, it would be useful to include mention of the following new studies: [START_REF] Michard | Pressuretemperature-time constraints on the Maghrebide mountain building: evidence from the Rif-Betic transect (Morocco, Spain), Algerian correlations, and geodynamic implications[END_REF] on the geology of the Rif. Other recent works on the geology are uncited (pages 8 & 9) such as [START_REF] Michard | Pressuretemperature-time constraints on the Maghrebide mountain building: evidence from the Rif-Betic transect (Morocco, Spain), Algerian correlations, and geodynamic implications[END_REF]. Michard et al. (1998) deal mainly with the geological background of the Rif maounts. The publication is now properly cited -The authors make the statement that minerals were exploited since prehistoric times (page 10 lines 3 & 4) without any reference from the literature.

A reference has been added:

-I found some of the chronological information a little hard to follow. For example, apart from Ras el Ma and Aït Ichou swamp there is no indication about the dated material and this could be more clearly explained in the text. The point could also be made that the sites chronology is mainly based on AMS dating and that further work is required to explain briefly the dating method and whether it was the same as the one used for the previous work [START_REF] Lamb | Human impact on the vegetation of the Middle Atlas, Morocco, during the last 5000 years[END_REF]. It would also be useful to mention the uncalibrated dates and the identification (if any) of the samples dated. Some mention of this should be made in the text.

Hopefully, this issue has now been (1) properly addressed in the manuscript, (2) a table with all dated samples has been added and (3) the dated samples are now plotted in red in each figure (2, 3 and 4).

-The description of the vegetation is straight-forward and clear and has been previously published but again some previous research on this topic are note cited (Benabid, 2000).

Thank you, indeed Benabid's work was unfortunately not cited. It is now However, in addressing the human impact on the vegetation through time the authors propose new thoughts about the decrease of the forest cover and the increase of the geochemical markers based on relatively sparse evidence. While the authors show clearly the Response to Reviewer Comments forest reduction in both Rif and Middle Atlas, their interpretation of the lack of the Cu, Pb and Zn in the Rif can be challenged by the rare "important" Palaeozoic formations unlike the Middle Atlas.

We agree that the geological background of the Rif may be a good explanation to the low presence (and consequently low exploitation) of Cu, Pb and Zn -The human impact on the vegetation should have been one of the central highlights of this paper but unfortunately the data are not presented in a way that would allow to understand it. Until now there is no clear archaeological evidence of the Roman presence in the Moroccan mountains including those of the eastern Rif and the Middle Atlas as it was published more than 25 years ago (Euzennat, 1989). The authors claim also that Morocco was occupied by the Phoenicians (page 13, line 1) and the understanding of this reviewer is that such presence was very limited to the coast. It might also have been valuable for the authors to have undertaken a research on the archaeological literature which is in this paper sadly very brief and uninformative.

We fully agree with these statements. However, as stated in the manuscript, the fossil pollen data do not allow to tackle archaeological issues. Thus, a thorough archaeological discussion is difficult to carry in such context.

  2. the occurrence of plant taxa that are either used and/or cultivated by man such as cereals, vines and olive trees, or ruderal plant taxa that often accompany human disturbances such as Cistus,Urtica, Plantago, Mercurialis, Rumex, and Asphodelus (López-Sáez et al., 2003; Sadori 2011); 3. increased abundance of grass (Poaceae) pollen co-occurring with ruderal taxa and declining arboreal abundance; 4. the co-occurrence of peak abundances of charcoal with pollen indications of disturbance; 5. the co-occurrence of geochemical and geophysical indicators of allochthonous input of sediment from the site catchments with pollen indications of disturbance;

  Morocco was annexed to the Roman Empire in 40 AD. Roman influence expanded into the Middle Atlas, as Roman populations settled the town of Volubilis, near Meknes (figure 1), then the capital of Morocco (called Mauretania at that time). Morocco remained a part of the Roman Empire until 429 AD when the Vandals displaced the Romans, who recovered it and then left several times. Thus, after ca. 500 AD the Rif and the Middle Atlas mountains remained occupied by the indigenous Berber inhabitants and the Byzantines until the arrival of the Arabs in 681 AD (Sadiki,

  the Roman Empire throughout the whole Mediterranean region. In Morocco, pollen records from both the Middle Atlas and the Rif mountains tend to confirm such strengthening of human activities during the spread of the Roman Empire. As stated by[START_REF] Mather | The human drivers of global land cover change: the case of forests[END_REF], tThe deforestation trend expressed, particularly in the pollen records of Ras El Ma and Tifounassine in the Middle Atlas, is coherent with the pattern observed by[START_REF] Mcevedy | Atlas of World Population History[END_REF] and more recently simulated by[START_REF] Kaplan | The prehistoric and preindustrial deforestation of Europe[END_REF] in a vegetation model that shows a clear decline since 1000 BC.

Figure 1 .

 1 Figure 1. Map of forest vegetation in the Rif and the Middle Atlas mountains, showing locations of palaeoecological sites.

Figure 2 .

 2 Figure 2. Arboreal pollen and calcium content of sediment cores from Anaasser (left) and M'Had (right) in the Rif mountains, plotted against a calibrated radiocarbon timescale. Dated bulk sediment samples are indicated by red stars.

Figure 3 .

 3 Figure 3. Arboreal pollen, selected anthropogenic indicator pollen taxa, and metal element content of a sediment core from Tifounassine, Middle Atlas, plotted against a calibrated radiocarbon timescale. Dated bulk sediment samples are indicated by red stars.

Figure 4 .

 4 Figure 4. Arboreal pollen, selected anthropogenic indicator pollen taxa, and metal element content of a sediment core from Ras El Ma, Middle Atlas. Dated bulk sediment samples are indicated by red stars.

Figure_1

  Figure_1Click here to download high resolution image

  4. I was left wondering to what extent the paragraph about « Ruddiman's hypothesis » is useful and relevant in this paper. It should be cut in my opinion.

Table 1 .

 1 Radiocarbon dates from bulk sediment samples from four sites in the Middle Atlas (Ras el Ma and Tifounassine) and Rif (M'Had and Anasser) mountains. 14C dates were calibrated with Calib 7.1[START_REF] Reimer | IntCal13 and MARINE13 radiocarbon age calibration curves 0-50000 years calBP[END_REF]) using the calibration data set intcal13.

Acknowledgements

RC thanks Jean Maley for the friendly incitement to write the present paper. This work was supported by the French national program EC2CO-Biohefect, "Variabilité paléoclimatique et impact sur les forêts de conifères au Maroc depuis la dernière période glaciaire". We acknowledge support from the Excellence Research Projects funding programme of Andalusian government: "Relic-Flora (RNM-7033)". We thank the LMC14 staff (Laboratoire de Mesure du Carbone-14), ARTEMIS national facility, UMS 2572 CNRS-CEA-IRD-IRSN-MCC, for the results obtained with the Accelerator Mass Spectroscopy method. This is an ISEM contribution n° xxx-xxxx


Franck Charmantray

Virgil Hélaine

Bertrand Legeret

Laurence Hecquet

Preparative scale enzymatic synthesis of d-sedoheptulose-7-phosphate from βhydroxypyruvate and d-ribose-5-phosphate

Keywords: d-Sedoheptulose-7-phosphate, Biocatalysis, Transketolase

An enzymatic method for ready access to D-sedoheptulose-7-phosphate on a preparative scale was developed, based on the irreversible transketolase-catalyzed reaction: β-hydroxypyruvate + D-ribose-5phosphate → D-sedoheptulose-7-phosphate. D-Sedoheptulose-7-phosphate disodium salt was obtained in 81% overall yield determined using a standard curve obtained by LC/MS/MS.

Introduction

D-Sedoheptulose-7-phosphate (Scheme 1) is an extremely important compound that is of great interest in fields such as metabolism studies and the synthesis of compounds with biological properties. It is a wellknown key metabolite in the non-oxidative pentose phosphate pathway and in the Calvin cycle.

Scheme 1

Recently, Mahmud and co-workers reported that d-sedoheptulose-7-phosphate was the only precursor of the mC 7 N units of both the α-glucosidase inhibitor acarbose [1] used for the treatment of type II insulinindependent diabetes, and validamycin A [2] an antibiotic used to control sheath blight, a phytopathogenic disease of rice plants. Also, jointly with the Naganawa group, the same authors reported on the biosynthetic pathway to the generic family of pyralomycin antibiotics [3] from D-sedoheptulose-7phosphate.

The role of D-sedoheptulose-7-phosphate as the precursor of the l-glycero-D-manno-heptose (L,D hep) segment in the lipopolysaccharides (LPS) of Gram-negative bacteria such as E. coli, Salmonella, and Shigella is also of particular interest [4].

Studies in progress in all these fields prompted researchers to seek a practical route for the synthesis of this phosphorylated sugar. Thiem and co-workers explored the hexokinase-mediated phosphorylation of D-sedoheptulose [5], but surprisingly, they recovered only the secondary phosphate, D-sedoheptulose-6phosphate. Floss and co-workers, who were interested in elucidating the biosynthetic pathways that lead to the aminocyclitol moieties of acarbose, validamycin, and pyralomycin, proposed an enzymatic synthesis of isotopically labelled D-sedoheptulose-7-phosphate. This was obtained using a multienzymatic system with alanine racemase, D-amino acid oxidase, catalase and transketolase [6], from unlabeled D-ribose-5-phosphate and l-[3-13 C]serine via β-hydroxypyruvate as the C2 fragment donor. This multi-step synthesis afforded labelled d-sedoheptulose-7-phosphate with 69% yield based on its radioactivity.

Our goal was to develop an efficient, scalable synthesis of D-sedoheptulose-7-phosphate. We were also interested in finding a reliable method to determine its purity. One approach was to take advantage of the irreversible reaction catalyzed by transketolase (TK: EC 2.2.1.1). When β-hydroxypyruvate (HPA) was used as the donor substrate, the reaction went to completion owing to the release of carbon dioxide (Scheme 2). Scheme 2.

TK is thus a useful catalyst to obtain D-threo ketoses, and its synthetic potential has been largely studied with various aldehydes [7]. In our group, we used a Saccharomyces cerevisiae recombinant strain to overproduce TK enzyme [8].

Here we describe a method to obtain, in only one step and on a preparative scale (multigram synthesis), highly pure D-sedoheptulose-7-phosphate, quantified by the calibration curve obtained by LC/MS/MS.

Experimental

Materials

Chemistry

Chemicals and solvents were purchased from Aldrich and Acros and were reagent grade. All solvents for analysis were LC/MS grade (Riedel de Haen, Sophyc-Socolab, France). 1 H, 31 P and 13 C NMR spectra were recorded on a Bruker Avance 400 spectrometer in D 2 O; δ values are given in ppm and J values in Hertz. For 31 P NMR we used H 3 PO 4 (5%) as the external standard in a sealed capillary. For the quantification of D-sedoheptulose-7-phosphate by 1 H NMR, we used TSP-d 4 as an internal standard.

LC-MS and LC-MS/MS (ESI) analyses

The LC/MS system consisted of a Waters ® Alliance ® 2695 high-performance liquid chromatography (HPLC) set-up (Waters SA, St-Quentin-en-Yvelines, France) coupled to a quadrupole time-of-flight (micro-QTOF) mass spectrometer (Micromass, Manchester, UK). This was fitted with a Z-spray source operating in negative ionization mode ESI(-)). Source and desolvation temperatures were set to 110 and 350 °C, respectively. Gaseous N 2 was used as both nebulizer gas (35 L h -1 ) and desolvation gas (350 L h -1 ). The voltages were 2.1 kV for the stainless-steel capillary and 35 V for the sample cone. For MS-MS experiments, the collision energy used depended on the analyte assayed. It was set at 12 eV for D-ribose-5-phosphate and 17 eV for D-sedoheptulose-7-phosphate. Analyses were performed as follows: 20 µL aliquots from reaction mixtures were quenched with 1% formic acid in water (180 µL) and acetonitrile (800 µL). LC analyses of D-ribose-5-phosphate and D-sedoheptulose-7-phosphate were carried out using an analytical zwitterionic and hydrophilic phase (Zic ® -Hilic column, 2.1 mm × 100 mm; with 5.0 µm particle size from SeQuant AB, Sweden) at a flow rate of 0.2 mL min -1 . The mobile phase was composed of (A) acetonitrile and (B) 2 mM ammonium formate pH 3.5. Isocratic elution at A/B, 80/20, v/v was performed for 10 min. The injection volume was 5 µL.

For MS/MS experiments transition settings for the different sugar phosphates were: l-glycerol-3-phosphate (r.t: 3.9 min), m/z -169/-97, D-ribose-5-phosphate (r.t: 4.7 min), m/z -229/-97, and D-sedoheptulose-7-phosphate (r.t: 6.0 min), m/z -289/-97. Data were acquired and processed using MassLynx™ software. For the sugar phosphates, the loss of the sugar moiety in the collision cell was unambiguously detected by the m/z -97 fragment (phosphate group).

Expression and purification of transketolase

Transketolase from S. cerevisiae was produced from S. cerevisiae strain H402xpTKL1 [8]. TK crude extract was obtained following our own procedure. The cells were collected by centrifugation at 3000 × g for 4 min. 10 g of cells were resuspended in 100 mL of Tris buffer (0.1 M) at pH 7.4 and placed in a oneshot cell disrupter at 2 kBar. The solution was centrifuged at 15,000 × g for 20 min. Once the precipitate was discarded, the pH of the supernatant was adjusted to 7.4 and the TK crude extract was kept at -20 °C until use. TK activity was determined thanks to a spectrophotometric assay as described earlier [9].

Determination of β-hydroxypyruvate content

The initial concentration of β-hydroxypyruvate was enzymatically assayed by spectrophotometry using NADH-dependent lactate dehydrogenase [10].

Determination of the standard curves for the phosphorylated sugars by LC/MS/MS

The D-ribose-5-phosphate standard curve was obtained from diluted solutions of commercial D-ribose-5phosphate (■, Fig. 1) in the 0-200 µM concentration range (l-glycerol-3-phosphate at 20 µM was used as the internal standard (IS). In the conditions tested, the detection limit was found to be 5 µM (i.e., 5 pmol, injection volume 5 µL). As D-sedoheptulose-7-phosphate was not commercially available, we decided to generate it in situ from d-ribose-5-phosphate by TK-catalyzed reaction. It was possible to plot the D-sedoheptulose-7-phosphate standard curve only under two conditions. First, the reaction had to be quantitative. Second, the reaction had to be one-to-one (no side reactions). The reaction was carried out as follows: D-ribose-5-phosphate in the 0-10 mM range reacted with a large excess of HPA (165 mM; K M = 33 mM) in the presence of TK for 2 h.

We observed a total conversion of D-ribose-5-phosphate as evidenced by LC/MS/MS (data not shown). Similar slopes were observed for D-ribose-5-phosphate disappearance (♦, Fig. 2) and D-sedoheptulose-7phosphate appearance (■, Fig. 2). Our approach thus provided a very accurate D-sedoheptulose-7phosphate standard curve. After sampling as previously described (dilution factor: 50), the D-sedoheptulose-7-phosphate standard curve was plotted from the corresponding means and standard deviations (S.D.) from five measurements for each concentration of D-sedoheptulose-7-phosphate studied (♦, Fig. 1). Under the conditions tested, the detection limit was found to be 3 µM (i.e., 15 pmol, injection volume 5 µL).

Results

Principle

In the reaction with transketolase, the C1-C2 ketol unit was transferred from β-hydroxypyruvate to Dribose-5-phosphate in the presence of thiamine pyrophosphate (ThDP) and divalent cation (Mg 2+ ) to give D-sedoheptulose-7-phosphate via an irreversible reaction.

Synthesis of sedoheptulose-7-phosphate barium salt

D-ribose-5-phosphate as its disodium salt (15.1 g, 55 mmol) was dissolved in distilled water (230 mL, 240 mM). ThDP (200 mg, 0.44 mmol, 2 mM) and MgCl 2 (63 mg, 0.66 mmol, 3 mM) were then added.

After adjustment of the pH to 7.5 with 1 N NaOH, β-hydroxypyruvate [11] (60% pure) as its lithium salt (12.1 g, 66 mmol, 285 mM) was added. The reaction was initiated by addition of TK (300 units) and the pH was maintained at 7.5 by addition of a 0.5 N HCl solution (pH stat apparatus). The reaction progress was monitored by determining β-hydroxypyruvate content (see Section 2.3). As shown in Fig. 3, the control without TK exhibited an intrinsic slight degradation of β-hydroxypyruvate in the conditions of the reaction. This degradation was estimated to be 20% of the starting material in 8 h. This problem was circumvented by adding a slight excess (1.2 equivalent) of β-hydroxypyruvate versus D-ribose-5phosphate. After 8 h under the conditions tested, β-hydroxypyruvate consumption was 4 times higher in the reaction (with TK) than in the blank (without TK). After complete reaction of β-hydroxypyruvate and D-ribose-5-phosphate the mixture was cooled in ice.

Three volumes of MeOH were then added and the mixture was stirred. The suspension was then centrifuged at 4500 rpm for 15 min. The precipitate was discarded and the supernatant collected. Aqueous BaCl 2 solution (75 mmol, 50 mL) at pH 6.0 was poured in with stirring and the solution cooled in an ice bath. The precipitate formed was discarded after centrifugation. Five volumes of ethanol were gently added to the supernatant with continued stirring. The precipitate formed was washed twice with ethanol and once with diethyl ether, and air-dried. D-Sedoheptulose-7-phosphate (29.9 g) was isolated as its Ba salt. Its organic purity was high, neither β-hydroxypyruvate nor D-ribose-5-phosphate being detected by 1 H, 13 C or 31 P NMR experiments. 31 P and 13 C NMR data fitted with those published earlier [12] and [13].

Due to slight excess in BaCl 2 used (1.35 equivalent) to achieve the precipitation of D-sedoheptulose-7phosphate as its Ba salt, the sample purity was 65% [START_REF]α-pyranose) was found to be 65% pure using TSP-d4 as an internal standard in 1 H NMR[END_REF] from the D-sedoheptulose-7-phosphate standard curve obtained by LC/MS/MS (see Section 2.4). Presumably, the sample was contaminated by barium hydroxide. The reaction yield was 90% based on d-ribose-5-phosphate.

Conversion to sodium d-sedoheptulose-7-phosphate

A sample salt of D-sedoheptulose-7-phosphate isolated as its barium salt (500 mg, 0.8 mmol) was converted into its disodium salt with gentle stirring with a Dowex 50 × 8 H + resin. After filtration, the supernatant was adjusted to pH 7.0 using a 1 N NaOH solution, followed by lyophilization. D-Sedoheptulose-7-phosphate as its disodium salt was recovered in 90% yield (251 mg, 0.72 mmol).

Conclusion

We describe a one-pot, straightforward, multigram-scale synthesis giving D-sedoheptulose-7-phosphate, isolated as its disodium salt, in high overall yield (81%). Considering more general aspects about carbohydrate synthesis, our approach highlights enzymatic carbon-carbon bond formation is an attractive alternative to conventional chemical methods. It offers stereochemical control, mild conditions and needs no protecting group.

Fig. 1 .

 1 Fig. 1. D-Ribose-5-phosphate (■) and d-sedoheptulose-7-phosphate (♦) standard curves.

Fig. 2 .

 2 Fig. 2. TK-catalyzed reaction causing a fall in Dribose-5-phosphate (♦) and the formation of D-sedoheptulose-7-phosphate (■) in time.

Fig. 3 .

 3 Fig. 3. Consumption and stability of β-hydroxypyruvate in time at pH 7.5, 25 °C: HPA, D-ribose-5phosphate, ThDP, MgCl 2 (with TK) (♦); versus blank reaction: HPA, D-ribose-5-phosphate, ThDP, MgCl 2 (without TK) (■).

Acknowledgments

We thank Professor G. Schneider (Karolinska Institut, Division of Molecular Biology, S-17177 Stockholm, Sweden) for the gift of the yeast strain H402 transformed with pTKL1 plasmid.

Supplementary data

1 H, 31 P and 13 C NMR data for D-sedoheptulose-7-phosphate synthesized are available as supplementary data.