Dominique Pontier

Catherine Albrieux

Jacques Joyard

Maryse Thierry Lagrange

Maryse A Block
email: mblock@cea.fr

Knock-out of the Mg protoporphyrin IX methyltransferase gene in

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Chloroplast development depends on the coordinated synthesis of chlorophylls and cognate proteins and on their specific integration into photosynthetic complexes (for a review see 1). Chlorophylls are Mg tetrapyrrole molecules resulting from condensation of δ-ALA. Insertion of Mg into protoporphyrin IX, the last common intermediate of haems and chlorophylls, is catalyzed by Mg chelatase, a membraneinteracting multisubunit enzyme containing 3 different soluble proteins: CHLH, CHLI, and CHLD [START_REF] Gibson | [END_REF](3)(4)(5). In the next step, Mg protoporphyrin IX is methylated by a membrane Ado-Met dependent methyltransferase. In Arabidopsis the gene At4g25080 or CHLM has been identified as encoding the methyltransferase enzyme and the product of this single gene has been reported to be present in the two chloroplast membrane systems: the envelope and the thylakoids (6). Whereas a sequence homologous to CHLM has been identified in tobacco (7), the existence in plants of an additional and quite dissimilar gene for Mg protoporphyrin IX methylation has not been totally excluded. Expression analysis of tobacco CHLM revealed posttranslational activation of methyltransferase activity during greening and direct interaction of CHLM with the CHLH subunit of Mg chelatase (7). Furthermore, analysis of tobacco plants expressing CHLM antisense or sense RNA led to the conclusion that methyltransferase activity is involved in the direct regulation of expression of some genes such as CHLH and LHCB (8). The meaning of the dual localization of the methyltransferase in the chloroplast envelope and the thylakoids is puzzling for different reasons. On one hand, chlorophylls are expected to be synthesized in the thylakoids where chlorophyll binding proteins are inserted into photosystems. On the other hand, the envelope is devoid of chlorophyll but contains several chlorophyll precursors from protoporphyrin IX to protochlorophyllide (9). In addition most of the enzymes of this part of the chlorophyll synthesis pathway i.e. protoporphyrinogen oxidoreductase, Mg chelatase, Mg protoporphyrin IX methylester cyclase and protochlorophyllide oxidoreductase are present in or interact with the envelope (10)(11)(12)(13). One reason for the occurrence of the chlorophyll intermediates in the envelope could be that some chlorophyll-binding proteins that are synthesized in the cytosol need to associate with chlorophyll or chlorophyll precursors during import through the envelope. Supporting this hypothesis, manipulation of Chlamydomonas in vivo systems and mutagenesis of specific residues in the LHCB protein have shown that accumulation of physiological amounts of LHCB by plastids requires interaction of the protein with chlorophyll within the inner membrane of the envelope (14,15). More recently, it was demonstrated that chlorophyllide a oxygenase (CAO) is involved in the regulated import and stabilization of the chlorophyllide b-binding light-harvesting proteins LHCB1 (LHCII) and LHCB4 (CP29) in chloroplasts (16). Beside their direct role in pigment synthesis, some chlorophyll intermediates have been proposed to play an additional role in intracellular signaling. In Chlamydomonas, Mg protoporphyrin IX and Mg protoporphyrin IX methylester were shown to substitute for light in the induction of the nuclear gene HSP70 (17). In Arabidopsis, an increased accumulation of Mg protoporphyrin IX has been reported in Norfluorazon-treated plants in which photooxidation of the plastid compartment leads to the repression of nuclear photosynthesis-related genes (18,19). Furthermore, a genetic screen based on the use of Norfluorazon has allowed the identification of a series of Arabidopsis gun (for genome-uncoupled) mutants that are deficient in chloroplast-to-nucleus signaling. Several of the corresponding mutations have been shown to affect genes coding for the protoporphyrin IX manipulating proteins CHLH , CHLD and GUN4 (19)(20)(21). In these mutants, Norfluorazon treatment induced only a moderate increase in Mg protoporphyrin IX level, correlated with partial derepression of transcription of the nuclear photosynthesis-related genes. This indicates a role of Mg protoporphyrin IX accumulation in the repression of these genes (19,22). However, this demonstration was based on manipulation of plants with Norflurazon that has obvious pleiotropic effects. Moreover, due to the possibility of substrate channeling occurring between Mg chelatase and Mg protoporphyrin IX methyltransferase, it was not possible to clearly distinguish the specific contributions of Mg Protoporphyrin IX and its methylester. Supporting the intricacy of the regulation, it has recently been shown that the barley xantha-l mutant, defective in the Mg protoporphyrin IX methylester cyclization step, has a non-gun phenotype in the presence of Norflurazon (23,24). In the absence of Norflurazon, the mutant has a high level of LHCB gene expression despite the accumulation of Mg protoporphyrin IX methylester. In the present work, we identify a chlm null mutant and analyze the role of the methyltransferase enzyme in chlorophyll synthesis and chloroplast development. We also assess the ability of the mutant to carry out plastid-to-nucleus signaling. The results reveal a high level of repression of nuclear photosynthesis-related genes and a specific accumulation of the CHLH subunit of the Mg chelatase. Functional implications of these results are discussed.

EXPERIMENTAL PROCEDURES

Plant material and growth conditions. The A. thaliana chlm mutant (ABN42 line) of the ecotype Wassilewskija (WS) was obtained from the INRA, Versailles, France. Plants were grown on Murashige and Skoog (MS, Duchefa) agar containing 0.5 % sucrose for 5 days under medium light (70 µmol photons m -2 s -1 ) before transfer to dim light (5 µmol photons m -2 s -1 ). Kanamycin selection of T-DNA containing seedlings was performed on medium supplemented with kanamycin (Sigma) at 50 mg L -1 . For Norflurazon treatment, seeds from CHLM/chlm heterozygous plants were germinated on standard MS-agar and after 5 days of culture, seedlings were transferred onto new plates including 5 µM Norflurazon and cultivated for 6 more days under strong light (100 µmol photons m -2 s -1 ). For complementation experiments, a pCambia derivative was obtained by cloning a EcoRI-HindIII fragment harboring the CaMV 35S promoter-GUS-NOST fusion from pBI221 (Clontech) in an EcoRI-HindIII digested pCambia 1200 vector. The XbaI-EcoRI CHLM cDNA from the SK+ derivative obtained by Block et al. (2002) was then inserted into an SstI-XbaI digested pCambia derivative using an EcoRI-SstI adaptor (5'-AATTAGCT-3'). The obtained binary vector was introduced into the GV3121 strain of Agrobacterium tumefaciens in order to transform CHLM/chlm heterozygous plants. The transformed plants were PCR-screened to identify CHLM/chlm heterozygous plants and self-pollinated. The LHCII-LUC construct was introduced into the chlm mutant background by crossing a heterozygous CHLM/chlm plant with a homozygous CAB-LUC transgenic plant (25). The progeny was PCR-screened for the presence of chlm T-DNA and seeds were collected from positive plants. The next generation segregates for both chlm T-DNA and the CAB-LUC transgene and was used for the experiment. The sequence encoding the putative CHLH transit peptide was amplified using TL350 (5'-GGATCCATGGCTTCGCTTGTGTATTCT CCATTC-3') and TL352 (TCTAGAGACGATTTTCACCGTTCGGA AC-3'). The corresponding PCR fragment was subcloned into pGEM-T easy vector (Promega) and sequenced. The BamHI-XbaI fragment was then ligated in frame with the XbaI-SacI EYFP gene (Clontech) into the BamHI-SacI sites of a binary vector derived from pCambia 1300 by introduction of a HindIII-EcoRI CaMV 35S promoter and Nos-T terminator cassette from EL103 (26). The obtained binary vector was introduced into the GV3121 strain of Agrobacterium tumefaciens in order to transform CHLM/chlm heterozygous plants. The transformed plants were PCR-screened to identify CHLM/chlm heterozygous plants and self-pollinated. PCR analyses. Genomic DNA was amplified using the Tag6 primer (5'-CACTCAGTCTTTCATCTACGGCA-3') and a specific primer (TL251 5'-GGAACAGCTTCGATGAGCCTAGAG-3') to screen for the presence of chlm T-DNA insertion. The endogenous copy of the gene as well as the cDNA used for complementation were followed by PCR reactions using TL251 and TL252 (5'-GTGAGACTGATGAAGTGAATCG-3'). Standard PCR conditions were used. Pigment analysis. Extraction was done according to (27). 3 mg of cotyledons of 8 day plants were ground in 200µL dimethylformamide and incubated for 16 h in the dark and on ice. The supernatant was collected by centrifugation for 15 min at 13 000xg and its absorbance in visible light recorded.

Tetrapyrole intermediate analysis.

Approximately 20 mg of frozen leaf material was homogenised in 500 µL acetone: 0.125M NH 4 OH (9:1, v/v) in the dark and centrifuged at 4°C. The supernatant was diluted with 1 mL grinding medium and extracted 3 times with 1 mL hexane. After the complete elimination of the hexane phase, the acetone phase was either directly used for fluorescence measurement or dried under argon before solubilization in methanol: 5 mM tetrabutylammonium phosphate (70:30 v/v) for HPLC analysis. Florescence emission spectra were recorded with a spectrofluorometer MOS-450 from Biologic at room temperature from 570 to 690 nm upon excitation at either 402 nm for detection of protoporphyrin IX, or 416 nm for detection of Mg protoporphyrin IX and Mg protoporphyrin IX methylester or 440 nm for detection of chlorophyllide. HPLC analysis was as described in (6) except that elution was monitored by absorbance detection at 420 nm and by fluorescence detection (λ excitation 420 nm/λ emission 595 nm or λ excitation 420 nm/λ emission 625 nm ). Standards of protorporphyrin IX (Sigma), of Mg protoporphyrin IX (Porphyrin Product Inc.), of Mg protoporphyrin IX methylester prepared and identified as described in (6) were used. When required, plants were incubated overnight with 10 mM ALA and 5 mM MgCl2 in 10 mM Hepes pH 7.0 before extraction.

Protein extraction and immunoblot. Proteins were extracted from leaf material according to the method described by (28). Protein analysis and immunoblotting were performed as described previously (6). Primary antibodies were used at the following dilution: anti CHLM 1/1000, anti KARI 1/1000, anti HSP70b 1/1000, anti CF1 1/10000, anti CRD1 1/1000, anti IE37 1/3000, anti OEP21 1/500, anti PORA-PORB 1/ 1000, anti PORC 1/1000, anti LHCB2 (Agrisera) 1/ 5000, anti LHCA2 (Agrisera) 1/2000, anti Cytf 1/5000, anti CP43 1/ 500, anti D1 (Agrisera) 1/1000, anti CHLH 1/200, anti CHLD 1/500, anti CHLI 1/200, anti-GFP (Euromedex) 1/3000. Primary antibodies were detected using peroxidase-conjugated goat antirabbit IgG (Jackson Immuno Research) or goat antimouse IgG (BioRad) and the chemiluminescent detection system (ECL, Amersham).

RT-PCR and Northern blot analysis. For RT-PCR analysis, total RNA was isolated using the Invisorb Spin Plant RNA Mini Kit following the manufacturer's instructions (Invitek) followed by a DNase treatment using the RQ1 RNase-free DNase (Promega). A PCR reaction was performed on RNA with primers for the RPL21 gene (5'-TCCACTGCGTCGACGTCTCG-3' and 5'-CTCCAACTGCAACATTGGGCG-3') to check for the absence of genomic DNA. Reverse transcription was performed on 500 ng total RNA using the ProSTAR First-Strand RT-PCR kit following the manufacturer's instructions (Stratagene). Semi-quantitative RT-PCR was calibrated by non-saturating PCR reaction (22 cycles) using primers designed to amplify the EF1-4α cDNAs (5'-CTGCTAACTTCACCTCCCAG-3' and 5'-TGGTGGGTACTCAGAGAAGG-3').

A parallel set of reactions without addition of reverse transcriptase was run as a quality control. To analyze CHLM expression, semiquantitative RT-PCR was performed with TL251 and TL252 primers (see above). The luciferase reporter gene expression was analyzed using two specific primers (5'-CCCGGCGCCATTCTATCC-3' and 5'-TGAAATCCCTGGTAATCCG-3').

For Northern blot analysis, total RNA was extracted as described by (29). 20µg total RNA were separated on agarose gel. RNA blot hybridization and membrane washing were performed on Hybond-N membrane (Amersham). The IE37 probe was obtained by PCR amplification on RT reaction by using specific primers (5'-CACCTAGACTCTCGGTGGC-3' and 5'-TTTGGGAACGATCTGATCC-3').

The 25S probe was used as a loading control (30).

Microscopy. Leaves were observed with an immersion 40x objective at room temperature by confocal laser scanning microscopy using a Leica TCS-SP2 operating system. EYFP and chlorophyll fluorescence were excited respectively at 488 nm and 633 nm. Fluorescence emission was collected from 500 to 535 nm for EYFP and from 643 to 720 nm for chlorophyll.

RESULTS

Characterization of an Mg protoporphyrin

IX methyltransferase null mutant. An Arabidopsis chlm mutant line was identified by screening the Flanking Sequence Tag database derived from the T-DNA insertion lines of the Versailles collection (31,32) using the CHLM cDNA as query sequence (6) (Fig. 1A). PCR-genotyping and kanamycin selection resulted in the selection of plants that were heterozygous for insertion of a T-DNA in the CHLM gene. Sequencing of the PCR-genotyping product and further PCR analysis showed that the insertion of the T-DNA in the single intron present in the coding sequence resulted in the deletion of the 5' part of the gene including the entire first exon (Fig. 1A). A mutant line with 3:1 kanamycin segregation as expected for a single genomic insertion was chosen for further analyses. Its progeny shows 1/3 of the kanamycin resistant plants being non green (Fig. 1B) and the yellow/white plants always exhibited a homozygous mutant genotype (Fig. 1C). Thus, the mutation in the CHLM gene is recessive and linked to the albino phenotype. In the homozygous mutant plants, RT-PCR indicated an absence of fulllength CHLM transcript (Fig. 1A,middle) and immunoblotting confirmed the absence of CHLM protein (Fig. 1A, right and 1C). Since the yellow/white plants could not produced seeds, homozygous mutants were only obtained from heterozygous plants. During silique maturation, some white seeds were visible suggesting absence of chlorophyll in the cotyledons of the homozygous seeds (Fig. 1B). However after desiccation, the seed color heterogeneity was no longer visible, making it impossible to visualize the mutant at this stage. When germinated on MS-agar containing 0.5% sucrose and under medium light (70 µmol photons m -2 s -1 ), the homozygous seedlings turned white and stopped growth after cotyledon development. However, when the plants were transferred after 5 d in medium light to dim light (5 µmol photons m -2 s -1 ), they could develop, although very slowly and with a stunted appearance; they then formed small yellow leaves (Fig. 1B). The absence of chlorophyll in the yellow homozygous plants was verified by spectrophotometry (Fig. 2A). Complementation of the mutant with the CHLM cDNA sequence resulted in expression of the CHLM protein and restoration of the green phenotype (Fig. 1C). Altogether, these results indicate that this chlm mutant is an Mg protoporphyrin IX methyltransferase null mutant and that the CHLM gene plays a unique role in the chlorophyll biosynthetic pathway in higher plants.

Accumulation of Mg protoporphyrin IX.

Fluorescence analysis and HPLC separation of acetone extracts showed that Mg protoporphyrin IX was the main chlorophyll intermediate present in the mutant (Fig. 2B and2C). When the mutant was fed with ALA to increase the synthesis of intermediates, we observed an increase of Mg protoporphyrin IX whereas Mg protoporphyrin IX methylester and other downstream chlorophyll intermediates were absent (Fig. 2C). Conversely, in the wild type, fluorescent spectra indicated the presence of downstream chlorophyll intermediates and Mg protoporphyrin IX methylester was detected by HPLC when the wild type was fed with ALA. Altogether the data indicated that in the chlm mutant, the chlorophyll pathway is indeed blocked in the methyltransferase step leading to accumulation of Mg protoporphyrin IX.

Absence of chlorophyll containing protein complexes in the chlm mutant. To evaluate chloroplast development in the mutant, we analyzed the expression of a series of proteins in leaves of plants grown 20 days in medium/dim light conditions. After SDS-PAGE and Coomassie staining, we observed that the profiles of total protein extracts from wild type and mutant were similar (Fig. 3A). The large subunit of Rubisco was apparently present at a similar level in both types of plants. However a major difference was visible at the molecular size expected for LHCII proteins (Fig. 3A, arrow). Immunoblot detection confirmed considerable reduction in the accumulation of a number of major thylakoid-associated proteins including components of photosystem I (LHCI), photosystem II (LHCII, D1, CP43) and the Cytochrome b6fcomplex (Cytf) (Fig. 3B). In contrast, no significant change was visible in the levels of stromal proteins KARI and HSP70β, chloroplast envelope proteins IE37 and OEP21, or thylakoid protein CF1 from CF1-CF0 ATPase (Fig. 3B). The chlm null mutant was therefore devoid of the major chlorophyll-containing protein complexes.

The chlm mutant behaves as a "super repressor" of nuclear photosynthesis genes.

Several processes can induce depletion in chlorophyll-containing proteins, from gene expression to protein turn over. Northern blot analysis indicated that very little change was observed for the expression of chloroplast genes PSBA, PSBD and PETA while the corresponding proteins (D1, CP43 and Cytf) were absent (compare Fig. 3B and Fig. 4A,top). This result confirms that the accumulation of plastid-encoded chloroplast proteins is mostly regulated at a translational or posttranslational level. In contrast, the levels of LHCB transcripts and, to a lesser extent, RBCS transcripts, were reduced in the chlm mutant whereas the levels of mRNA for the chloroplast inner envelope protein IE37 (33) and of 25S cytosolic ribosomal RNA were not affected (Fig. 4A, bottom). In order to verify that the low amount of LHCB mRNA in the chlm mutant was related to transcriptional repression, we crossed a heterozygous CHLM/chlm plant with a line harboring an LHCB promoter-Luciferase construct and compared the LHCB promoter-dependent expression of the luciferase reporter gene in the wild-type and chlm mutant backgrounds. We observed no luciferase mRNA in the chlm mutant (Fig. 4B), indicating that the decrease of LHCB mRNA accumulation in the mutant is due to transcriptional repression. Moreover, in an assay for the gun (genome uncoupled) phenotype as defined by ( 22), we compared LHCB mRNA accumulation in wild-type and chlm mutant plants grown either in the absence or in the presence of Norfluorazon (Fig. 4C). We observed that photobleaching by Norflurazon induced even stronger repression of the LHCB gene in the chlm mutant compared to the wild-type, indicating that this mutant does not show a gun phenotype (compare Fig. 4C, lanes 2 and 4). This is not surprising since this mutant is expected to accumulate greater amounts of Mg protoporphyrin IX through a direct effect on the biosynthetic pathway. Interestingly, the extent of repression observed in the chlm mutant in the absence of Norflurazon is even stronger than that observed in wild-type plants treated with Norfluorazon. This indicates that the chlm mutant behaves more like a "super repressor" of LHCB gene expression. In conclusion, in the chlm null mutant, the chloroplast-to-nucleus retrograde signaling seems to be turned on without any additional treatment.

Up-regulation of CHLH. Besides the role of Mg protoporphyrin IX in the plastid-tonucleus signaling pathway, published data suggests an additional role for certain enzymes in the chlorophyll synthesis pathway, such as the Mg chelatase subunits (20). We analyzed how these proteins are affected in the chlm mutant. Immunoblot analysis showed that the CHLD and CHLI subunits of the Mg chelatase, the CRD1 subunit of the cyclase and the POR proteins are present in similar amounts, whereas the CHLH Mg chelatase subunit is 5 to 8 times more abundant in the mutant than in the wild-type (Fig. 5A). In the chlm complemented mutant, the level of CHLH protein is reduced, although not quite to the wild-type level (Fig. 5B), indicating that CHLM depletion leads to a specific increase in the level of the CHLH subunit of the Mg chelatase. Northern blot analyses indicated that, in the chlm mutant compared to the wild type, there were similar or barely lower levels of CHLH and CHLD transcripts (Fig. 5C). Therefore, the up-regulation of the CHLH subunit occurs at a posttranscriptional level in the chlm mutant.

CHLH is matured and accumulates inside plastids. As an initial step to elucidate the regulatory mechanism leading to this accumulation, we checked if CHLH is located inside and/or outside plastids in the chlm mutant. Since isolation of plastids from these plants was not possible, we indirectly addressed this question by testing for the presence of a unprocessed CHLH precursor in the chlm mutant. The precursor protein was expected at 154 kDa and the mature protein at 144 kDa. When running on a high molecular weight resolving gel, CHLH was visualized around 144 kDa, clearly below the 150 kDa marker and the size of the protein was similar in both the wild type and the mutant, suggesting that most of the CHLH protein, if not all, is matured and most probably addressed to plastids. The accumulation of processed CHLH protein in the chlm mutant could be related either to an increased stability of the protein in the chlm plastids or a more efficient import of the precursor form into chlm plastids. To verify the capability of the CHLH transit peptide for sorting the protein to chloroplasts, we expressed this peptide fused to the N-terminal end of EYFP in wildtype and chlm plants and analyzed the localization of the fusion protein. In the wild-type, EYFP and chlorophyll fluorescence colocalized indicating a chloroplast targeting of the EYFP protein. Aggregation of part of the EYFP in the chloroplasts is likely because additional highly fluorescent spots of EYFP were visible in the chlorophyll fluorescence area (Fig. 6B). In the chlm mutant where chlorophyll is absent, the same image of EYFP fluorescence was observed, suggesting the same plastid localization (Data not shown). To discriminate between an increased stability of CHLH or a more efficient import into chlm plastids, we compared the accumulation of the EYFP fusion protein in CHLH transit peptide-EYFP transgenic plants cosegregating for the chlm T-DNA (Fig. 6C). The size of the EYFP fusion protein in each plant was similar and corresponded to EYFP without the transit peptide and its accumulation was not different. Our conclusion is that the CHLH transit peptide is still capable of addressing the precursor protein into chloroplasts even in the chlm mutant context and that its import capacity is not modified in the chlm mutant. Therefore, the accumulation of the CHLH protein in the chlm mutant probably occurs at a posttranslocational level.

DISCUSSION

CHLM is a key gene for chlorophyll biosynthesis and chloroplast development.

Our results show that chlorophyll formation is totally dependent on the CHLM gene product in Arabidopsis. The inactivation of this gene prevents setting up of chlorophyll binding proteins in the thylakoids whereas most other proteins in the chloroplast remain relatively stable. Not only photosystem I and II with their associated light harvesting complex are affected but also the cytb6f complex that contains very low amounts of chlorophyll (34)(35)(36). Chlorophyll is required for maturation of chlorophyll binding proteins, for correct folding of the complexes and for their insertion in the thylakoids (for a review 1). It also has a stabilizing effect on complexes, complexes lacking chlorophyll proteins becoming substrates for proteases. This was reported for chloroplast encoded proteins such as D1, CP43 and Cyt f (37) and it may explain the absence of these proteins in the mutant since we effectively observed that the corresponding mRNAs were expressed at relatively normal levels.

Modification of expression of nuclear encoded photosynthetic genes. On the opposite, the level of LHCB mRNAs is greatly decreased in the mutant, due to transcriptional regulation. Since the mutant specifically accumulates Mg protoporphyrin IX without any treatment, our result provides an independent indication that Mg protoporphyrin IX is a negative effector of nuclear photosynthetic gene expression, as previously suggested in Norfluorazon treated plants (19). Moreover, the chlm mutant behaves like a super-repressor of the LHCB promoter and seems more efficient in repressing LHCB expression than plants treated with Norflurazon. The extent of repression may be due to a different level of accumulation of Mg protoporphyrin IX in the plant. A second possibility may be linked to the complete absence of Mg protoporphyrin IX methylester and its derivatives in the mutant. This suggests that one of these components acts as a positive effector of nuclear photosynthetic gene expression. Mg protoporphyrin methylester itself may be a positive effector. In support of this latter hypothesis, (24) have shown that, in the absence of Norflurazon, the barley xantha l mutant that is defective in the Mg protoporphyrin methylester cyclase, accumulates Mg protoporphyrin IX methylester and has a high level of LHCB expression. In addition, (8) reported positive correlation between LHCB expression and methyltransferase activity in tobacco CHLM antisense and sense RNA mutants. The activity of Mg protoporphyrin IX methyltransferase is obviously dependent on the availability of Mg protoporphyrin IX but is also certainly adjusted to levels of Ado-Met and Ado-Hcy in the chloroplast (see for instance 38). The differential effects of Mg protoporphyrin IX and Mg protoporphyrin IX methylester on LHCB expression would consequently finely attune the synthesis of light harvesting complexes to the one-carbon metabolism.

Increase of CHLH level. In the chlm mutant we observed a strong increase in the level of mature CHLH, whereas that of CHLH mRNA was slightly reduced. Our data indicate that stabilization of the protein inside chloroplasts probably occurs in the chlm mutant. One possible mechanistic explanation for this accumulation could be traced to the increased level of Mg protoporphyrin IX in this mutant. Indeed, a tight channeling of substrate between the Mg chelatase and the methyltransferase has been suspected for a long time due to the absence of detectable Mg protoporphyrin intermediates in standard conditions (39). More recently, the physical interaction between CHLM and CHLH has been shown (40,7). During diurnal growth, the Mg chelatase activity peaks during the transfer from dark to light while the methyltransferase activity maximum follows only a few hours later. During this period, the Mg protoporphyrin IX level is transiently higher than that of Mg protoporphyrin IX methylester (7). It is possible that Mg protophorphyrin IX binds to CHLH, preventing the transitory accumulation of free Mg protoporphyrin IX when CHLM is not sufficiently present/active. This could both stabilize the rapidly turning over CHLH protein (5) and protect it against major photooxidative damage generated by free Mg protoporphyrin IX. Whatever the mechanism underlying CHLH accumulation, one can question whether its concomitant increase with Mg protoporphyrin IX plays a role in mediating the plastid-to-nucleus regulatory pathway.

  Protoporphyrin IX is the last common intermediate between the haem and chlorophyll biosynthesis pathways. The addition of Mg directs this molecule toward chlorophyll biosynthesis. The first step downstream from the branchpoint is catalyzed by the Mg chelatase and is results demonstrate that the CHLM gene is essential for the formation of chlorophyll and subsequently for the formation of photosystems I and II and cyt b6f complexes. Analysis of gene expression in the chlm mutant provides an independent indication that Mg protoporphyrin IX is a negative effector of nuclear photosynthetic gene expression, as previously reported. Moreover, it suggests the possible implication of Mg protoporphyrin IX methylester, the product of CHLM, inchloroplast-to-nucleus signaling. Finally, post-transcriptional up-regulation of the level of the CHLH subunit of the Mg chelatase has been detected in the chlm mutant and most likely corresponds to specific accumulation of this protein inside plastids. This result suggests that the CHLH subunit might play an important regulatory role when the chlorophyll biosynthetic pathway is disrupted at this particular step.

Figure 1 :

 1 Figure 1: Identification of a chlm mutant. A-Left : Structure of the CHLM gene showing the position of the T-DNA insertion in the mutant. Boxes along the gene represent exons. The deletion created by the T-DNA insertion is shown by the dotted line. Middle: CHLM gene expression in wild-type plants and homozygous mutants shown by RT-PCR on seedlings.gDNA represents the genomic DNA control. EF1-4α is used as a control to standardize the RT-PCR reactions. Right : CHLM protein accumulation in wild-type plants and homozygous mutants shown by immunoblot. B-Phenotype of the mutant. Aspect of seedlings from seeds of heterozygous plants, grown for 6 d at 10 µmol photons m -2 s -1 . On the right, the magnification shows a mutant plant grown for 5 d at 70 µmol photon m -2 s -1 , then for 15 d at 5 µmol photons m -2 s -1 . At the bottom is a picture of a green silique from a heterozygous plant. C-Phenotype, genotype determination and analysis of CHLM protein accumulation in wildtype, homozygous and complemented plants. Top : picture of 12 day-old wild-type plant (left), chlm homozygous mutant (middle) and chlm homozygous mutant harboring a 35S CaMV promoter-CHLM cDNA construct (right). Middle : PCR analysis of the genomic DNA from plants represented on the top picture using primers to detect the endogenous CHLM gene and the transgene cDNA or the T-DNA. Bottom : Immunoblot on total proteins from plants represented on the top picture using an anti-CHLM antibody.

Figure 2 :

 2 Figure 2: Analysis of pigments and chlorophyll precursors in the chlm mutant. A-Absorbance spectra of pigments extracted from the mutant (solid line) and wild type/heterozygous plants (dotted line). B-Room temperature fluorescence emission spectra of acetone extracts of mutant and wild type/heterozygous plants. The plain line indicates emission obtained with excitation light at 402 nm and the broken line at 440 nm. Plants were grown for 10 d with a 10 h light cycle at 70 µmol photons m -2 s -1 . The insert indicates

Figure 3 :

 3 Figure 3: Analysis of protein accumulation in the chlm mutant. A-Proteins extracted from leaves of wild type/heterozygous (CHLM) or chlm mutant (chlm) plants grown for 5 d at 70 µmol photon m -2 s -1 before 15 d at 5 µmol photons m -2 s -1 , were separated by SDS-PAGE and stained by Coomassie blue. The arrow indicates the position of LHCII and the asterisk the position of RubiscoL. B-Immunoblot analysis of wild type/heterozygous and homozygous mutant plant proteins. The protein against which the antibody is directed is indicated on the left. The plastidial localization of the tested proteins is indicated at the bottom of each panel.

Figure 4 :

 4 Figure 4: Analysis of plastidial protein encoding gene expression in the chlm mutant. A-Northern blot analysis of RNA extracted from wild type/heterozygous (CHLM) and homozygous mutant (chlm) plants. Genes encoding chloroplast-encoded chloroplast proteins (CECP) have been tested in the top panel and are indicated on the left. Genes encoding nucleus-encoded chloroplast proteins (NECP) have been tested in the bottom panel and are indicated on the left. B-RT-PCR analysis of RNA extracted from segregating wild type/heterozygous (CHLM) and homozygous mutant (chlm) plants harboring the LHCII promoter-luciferase fusion. Specific primers have been used to analyse luciferase (LUC) expression. The absence of genomic DNA contamination is indicated by the -RT panel, using L21 primers. The standardization has been done using both L21 and EF1-4α specific primers. C-Northern blot analysis of RNA extracted from wild type/heterozygous (CHLM) and homozygous mutant (chlm) plants grown in the absence or in the presence of Norfluorazon (NF). The probes used are indicated on the left and figures below each panel indicate relative quantification of the signals.

Figure 5 :

 5 Figure5: Analysis of the expression of proteins from the chlorophyll biosynthesis pathway in the chlm mutant. A-Immunoblot analysis of wild type/heterozygous (CHLM) and homozygous mutant (chlm) plant proteins. The protein against which the antibody is directed is indicated on the left. B-Immunoblot analysis of wild type/heterozygous (CHLM), homozygous mutant (chlm) and complemented homozygous mutant (cp. chlm) plant proteins. The protein against which the antibody is directed is indicated on the left. C-Northern blot analysis of RNA extracted from wild type/heterozygous (CHLM) and homozygous mutant (chlm) plants. Probes against the CHLH and CHLD genes were used and are indicated on the left.

Figure 6 :

 6 Figure 6: Analysis of the CHLH protein in the chlm mutant. A-Wild type/heterozygous (CHLM) and homozygous mutant (chlm) plant proteins were run on a high molecular weight resolving gel and analyzed with an anti-CHLH antibody. B-Confocal microscopy analysis of wild type seedlings harboring the CHLH transit peptide-EYFP fusion (TpCHLH/EYFP). The EYFP fluorescence picture is shown at the top and merging between this picture and a chlorophyll fluorescence picture at the bottom. The bar corresponds to 5 µm. C-Immunoblot

This manuscript has been published in Journal of Biological Chemistry 282(4): 2297-2304 -Doi:10.1074/jbc.M610286200

Acknowledgements:

Particular thanks are due to researchers who gave us aliquots of antibodies we used in this work: R. Dumas, M. Schroda, S. Merchant, T. Masuda, F.-A. Wollman and M. Hansson. We acknowledge D. Vega for excellent technical assistance and F.-Y. Bouget for the LHCII-LUC line. We thank Dr. R.Cooke for copy editing of the manuscript.


Alexandre Govin
email: govin@emse.fr

Marie-Claude Bartholin

Barbara Biasotti

Max Giudici

Valentina Langella

Philippe Grosseau

Impact of Guar Gum Derivatives on Properties of Freshly-Mixed Cement-Based Mortars

Keywords: Cement, HydroxyPropyl Guar, Mortar, Rheology, Water retention

Mortars are traditionally made from a mixture of sand, a binder and water. However, modern factory-made mortars are currently, very complex materials. Indeed, to exhibit various properties from the fresh paste to the hardened material, mortar formulations are composed of many mineral and organic admixtures. Among organic admixtures, polysaccharides, such as cellulose ethers (CE), are widely used into mortar formulation to improve water retention capacity of the freshly-mixed materials. The water retention capacity is an essential property of mortars leading to enhance the cement hydration and its adhesion to a substrate. Moreover, many polysaccharide admixtures, acting as viscosity-enhancing agents (VMA), prevent segregation and improve the homogeneity and workability of cement-based system. Indeed, the viscosity of the system strongly increases using polysaccharides and the rheological behavior of the cementitious paste may be completely modified and adapted to the application. Nevertheless, polysaccharides, as sugars, act on cement hydration. The main drawback is the retarding effect in hydration mechanism and setting-time of the cement. The present study examines the influence of chemical composition and structure of guar gum derivatives on fresh state properties of Portland cement-based mortars, such as water retention capacity (WR) and rheological behavior of fresh state Portland-based mortars. This work focuses on the guar gum derivatives since their manufacturing process is low pollutant and they provide very good properties to cement-based mortars. The investigation was also completed by adsorption isotherms. For this purpose, a native guar gum, some HydroxyProplyl Guars (HPG) and some hydrophobically modified HPGs were selected. The effect of the molar substitution (MS HP ) and the degree of substitution (DS AC ) was investigated. The results highlight that chemical composition of HPGs has a remarkable effect on fresh state properties of mortars. The native guar gum does not impact both WR and rheological behavior. Increasing MS HP leads to an improvement of the WR and the stability of mortars while the hydrophobic units further enhance WR and conduct to a decrease in the yield stress and an increase in the resistance to the flow of admixed mortars.

Originality

Hydroxypropyl guars (HPGs) are very interesting molecules because they are able to induce water retention properties comparable to those obtained with cellulose ethers, which are the widely used water-retaining agent. Moreover, by adapting the chemical composition and structure of the Hydroxypropyl guars it is possible to improve the water retention capacity of mortars, to modify the rheological behavior of mortars and to decrease the impact of the admixtures on the hydration delay of cement. It is important to underline that manufacturing of HPG causes less pollution than cellulose derivatives. Therefore the use of HPGs is in fact already well-established in construction chemistry. Nevertheless, studies about HPG as admixtures for cementitious materials are very scarce in the published literature.

Introduction

Modern factory-made mortars are complex materials, in which several kinds of admixtures are added in order to obtain specific properties, from the fresh state to the hardened material. Indeed, since many years, concretes, mortars or cement grouts with high fluidity have been developed, since their use implies many economical and technical advantages. However, the use of highly flowable mixtures may lead to segregation or excessive bleeding and subsequently, durability issues. In order to overcome this problem by enhancing the sedimentation resistance while maintaining high fluidity, viscosity-enhancing admixtures (VEA) are frequently introduced within the formulation (Rols S. et al., 1999;Khayat K. H. et al., 1997;Lachemi M. et al., 2004;Sonebi M., 2006). Among these admixtures, natural polysaccharides or their derivatives (such as welan gum, starch derivatives or cellulose ethers) are the most widely used. Moreover, the incorporation of these VEAs in shotcrete or render mortar is useful to ensure sagging resistance for thick application on vertical support, and to allow sufficient fluidity for normal pumpability by supplying shear thinning rheological behavior (Paiva H. et al., 2006). Indeed, these admixtures provide, generally, high yield stress and apparent viscosity at low shear rate but low resistance to flow at high shear rate (Khayat K. H., 1998). However, their mode of action is not fully understood, since results are sometimes contradictory. Water retention (WR) is another essential property of monolayer render at fresh state. Indeed, high water retention improves the cement hydration and limits the absorption of the mixing water by a substrate and thus provides good mechanical and adhesive properties to the mortar (Bertrand L. et al., 2004;Jenni A. et al., 2005). Among admixtures enhancing water retention capacity of the freshlymixed mortars, cellulose ethers (CE) are the most widely used. Nevertheless, hydroxypropyl guar (HPG) are now also well-established in the construction industry as water retention agent for mortars (Biasotti B. et al., 2011;Poinot T. et al., 2013;Poinot T. et al., 2014;Cappellari M. et al., 2013). Moreover, HPGs are already widely used in various industrial fields, such as textile printing, hydraulic fracturing process, oil production or paper manufacturing, due to their thickening effect (Risica D. et al., 2010;Lapasin R. et al., 1995). Consequently, since HPGs improve the two main properties of mortar, they appear as suitable admixtures to be used in render formulation. The aim of this study is to provide an understanding of the effect of chemical composition and structure of HPGs and its dosage on macroscopic properties of mortars. For this purpose, an original guar gum and five HPGs with specific chemical modifications, such as increase in MSHP or substitutions by hydrophobic units, were selected. The impact of admixtures on the water retention capacity and on the rheological behavior of mortars was investigated.

Materials and methods

Mineral products

Mineral products used in this study consist in blend Portland cement (Holcim), lime (Holcim), calcium carbonate (Calcitec V60, Mineraria Sacilese S.p.A.) and dolomite (Bombardieri and Leidi 0.1-0.4mm). The mineral compositions of the commercial Portland cement, CEM II/B-LL 32.5 R according to the European standard EN 197-1 (Standard EN 197-1, 2012), used are given in Tab. 1. The phase composition was determined by Rietveld refinement method (Siroquant V2.5 software) after XRD analysis (D5000, Siemens) and the oxide composition was quantified by means of X-ray fluorescence spectroscopy.

Organic admixtures

Guar gum is a natural polysaccharide extracted from the seed endosperm of Cyamopsis tetragonolobus. This polymer consists in a β(1-4)-linked D-mannopyranose backbone with random branchpoints of galactose via an α(1-6) linkage (Figure 1a). Hydroxypropyl guars (HPGs) are obtained from the original guar gum via an irreversible nucleophilic substitution, using propylene oxide in the presence of an alkaline catalyst (Figure 1b). The manufacture of HPGs has the advantage of having a more reduced impact on the environment than cellulose derivatives. Indeed, guar gum is extracted by simple thermo-mechanical process, exhibits a higher chemical reactivity and is soluble in cold water thanks to its branched-chain structure with a lot of hydroxyl groups. Thus, the chemical modification of the original guar gum requires normal reaction conditions of temperature and pressure, does not generate large quantity of by-products, and requires minimal purification procedure (Biasotti B. et al., 2011). In this paper, three HPGs and an original guar gum provided by Lamberti S.p.A were studied. They exhibit roughly the same molecular weight, around 2.10 6 g.mol -1 since they are all from the same original guar gum (noted HPG 6 in the paper) (Poinot T. et al., 2013). Tab. 3 provides a qualitative description of the polymers used. The qualitative substitution degrees are provided by the manufacturers. The molar substitution ratio (MS HP ) represents the number of hydroxypropyl units per anhydroglucose unit and is less than 3 for the investigated HPGs. The degree of substitution (DS AC ) represents the amount of alkyl chain per anhydroglucose unit. The only difference between HPGs 1 and 3 is the molar substitution ratio, which increases, while HPG 4 exhibits an additional substitution (short alkyl chains).

Methodology

Mortars were prepared according to the following mixture proportions: 12% of cement, 3% of lime, 18% of calcium carbonate, 43% of dolomite Bombardieri and 24% of dolomite Leidi (by weight). The admixtures (0.05, 0.075, 0.1, 0.125 and 0.15%) were in addition to the total dry mixture (i.e. cement, lime, calcium carbonate and dolomite) and is expressed in weight percent by weight of binder (% bwob). Dry mixture was blended in a shaker (Wab, Turbula, Germany) for 10 min. Deionised water was added in order to obtained a liquid-to-solid ratio L/S = 0.22. The mixing procedure was in accordance with EN 196-1 (Standard EN 196-1, 2006).

The experimental methodology consisted in dividing the freshly mixed mortar into three parts in order to characterize several properties from the same mixing. A first part was used to characterize the rheological behavior of the mortar, the water retention study was performed on the second part and the third part of the freshly mixed mortar was centrifuged in order to determine the adsorption isotherms and the polymer concentration within the pore solution following a procedure described later.

In order to improve the understanding of the mechanism involved in Water Retention, additional dosages were tested for WR and adsorption measurements: 0.03 and 0.06% bwob for HPG 4, 0.06% bwob for HPG 3, 0.175 and 0.2% bwob in the case of HPG 1 and 0.2% bwob for the native guar gum (HPG 6).

All tests were carried out, at least, in triplicate and at a controlled temperature because water retention, rheological behavior of the mortar and adsorption isotherms are temperature-dependent. A control test was also performed with a mortar without admixture.

Adsorption curves of HPGs into mortars

The adsorption isotherms were determined using the depletion method. The non-adsorbed polymer remaining within the pore solution was quantified by means of Total Organic Carbon (TOC) measurements. Prior to analysis, the pore solution was extracted from admixed or non-admixed mortar.

The extraction was performed by means of two centrifugation steps. The first step consisted in the centrifugation of around 150 g of mortar at 5000 rpm for 5 min. The supernatant was, afterward, centrifuged again at 14500 rpm for 10 min in order to avoid the presence of mineral particles within the solution. The supernatant was diluted with hydrochloric acid solution at 0.1 mol.L -1 . The total organic carbon was determined by combustion at 850 °C with a Vario-TOC Cube (Elementar). The adsorbed amount of polysaccharides was calculated from the difference of TOC content of the HPG reference solution and the TOC content of the supernatant.

Water Retention of mortars

The water retention capacity of freshly-mixed mortar can be assessed using different tests where the removed water after suction or depression is measured (Patural L. et al., 2001). In this study, the standard method used to estimate the water retention capacity of a mortar, was the test described in ASTM C1506-09 and shown in figure 2 (Standard ASTM C1506-09, 2009). It had to be performed 15 min after mixing to measure the water loss of a mortar under depression.

The standardized apparatus was submitted to a vacuum pressure of 50 mm of mercury (6.6 10 3 Pa) for 15 min. Then, the water retention capacity, WR, was calculated using the following equation:

100 W W W WR(%) 0 1 0   
where W 0 represents the initial mass of mixing water; W 1 is the loss of water mass after aspiration. All the experiments were carried out at 20 °C. Three classes of water retention (measured by ASTM method) of a fresh mortar can be specified according to the DTU 26.1 (Standard NF DTU 26.1, 2008.). The first class (low water retention category) contains mortars that exhibit water retention lower than 86%. The second class (intermediate) corresponds to values ranging from 86% to 94%. The last one (strong) is defined by water retention higher than 94%, corresponding to the required values in the field of rendering application.

Rheological behavior of mortars

The rheological measurements were performed with Rheometer MCR 302 (Anton-Paar), thermostated at 20 °C. The rheological properties of fresh mortars were investigated with vane-cylinder geometry (figure 3a) since this system is suitable for granular pastes like mortars (Bouras R. et al., 2012;Barnes H. A. et al., 2001). The gap thickness, distance between the periphery of the vane tool and the outer cylinder, was set at 8.5 mm, in order to be less sensitive to the heterogeneity of the mortar. Using a Couette analogy, the shear stress and shear rate were calculated from the torque and the applied rotational velocity respectively, after calibration with glycerol (Ait- Kadi A. et al., 2002). The mortar was introduced into the measurement system at the end of the mixing cycle. At 10 min, the mortar was pre-sheared for 30 s at 100 s -1 in order to re-homogenize the sample and to eliminate its shear history because of thixotropic character of cementitious materials (Phan T. H. et al., 2006;Roussel N. et al., 2012). After a period of rest of 5 min, the rheological measurements were started (total time = 15 min). At this time, the hydration rate is low enough which allows overcoming the irreversible effect of cement hydration on rheological behavior, especially at low shear rate (Phan T. H. et al., 2006). The imposed shear rate was decreased by step from 300 to 0.06 s -1 (16 steps). At each shear rate, the measuring time was adjusted in order to obtain a steady state whatever the formulation. The samples were systematically submitted to high shear rate (100 s -1 ) for 30 s before each imposed shear rate in order to resuspend particles of mortar within the mortar mixtures (figure 3b). The results were expressed as shear stress  according to shear rate  and the Herschel-Bulkley (HB) model was applied to fit the experimental data and used to describe mortars rheological behavior (Herschel W. M. et al., 1926):

n K      0
where  0 correspond to the yield stress, K the consistency coefficient and n the fluidity index.

Results and Discussion

Adsorption curves of HPGs into mortars

Figure 4 shows the adsorption isotherms of the hydroxypropyl guar and the original guar gum on Portland based-mortars. The results confirm that adsorption of original guar gum and HPGs on cementitious materials occurs. According to Wang et al. the adsorption mechanism of galactomannose polysaccharides at solid-liquid interfaces involves strong hydrogen bonding (Wang J. et al., 2005). In the range of polymer dosage used in the present study, no plateau was reached, and this, whatever the admixture. For the original guar gum (HPG 6), the adsorption appears totally linear and is the highest of all the tested polymers, corresponding to a total adsorption higher than 98.5% of the introduced polymer. The presence of hydroxypropyl substitutions on the guar leads to a decrease in the affinity of the polymer with the binder since the amount of HPG 1 adsorbed is lower than that of the original guar gum. The adsorption is further reduced by the increasing values of MS HP (HPG 3). This tendency is consistent with previous studies on HPGs and cellulose ethers (CE) (Poinot T. et al., 2013;Brumaud C. et al., 2014). Figure 4 highlights also the effect of the additional alkyl chain on the adsorption. It appears that the hydrophobic side chains slightly intensify the adsorption of the hydrophobically modified HPG on surface of grains with respect to HPG 3. Consequently, the polymer concentration within the extracted pore solution rises following this order: HPG 6 < HPG 1 < HPG 4 < HPG 3. Excepted HPG 6, the amount of non-adsorbed polymer increases with increasing polymer dosage (results not shown here).

Impact of HPGs on the water retention property of fresh mortars

The evolution of the water retention capacity of fresh admixed mortars, according to the polymer dosage, is shown in figure 5. The non-admixed mortar exhibits a low water retention capacity of about 72% ± 0.3%. The effect of the original guar gum (HPG 6) on the WR property seems negligible, regardless of dosage. This result is coherent with the adsorption of the polymer. Indeed, since the adsorption is higher than 98.5% of the initial amount of polymer, very few molecules are still in the pore solution (figure 4). The composition of pore solution is thus very close to that of the non-admixed mortar, leading to similar WR. The water retention increases with the use of HPGs. Moreover, the WR rises according to polymer dosage, until reaching a plateau with very high WR values (>97%) for HPG 4. In the range of polymer dosage studied, the WR values reached for HPGs 1, 3 and 4 are greater than 94% and therefore belong to the strong WR class. These results suggest that the substitution of hydroxyl units from original guar gum by hydroxypropyl units leads to an increase in WR of mortars. Furthermore, the increase in the MS HP (from HPG 1 to 3) improves the WR capacity of mortar, since HPG 3 provides a higher WR despite a lower dosage. Moreover, an abrupt change in slope can be noticed for mortars admixed with HPGs 1 and 3. This occurs for a decreasing polymer dosage from HPG 1 to HPG 3. The results are consistent with those of previous studies performed with HPGs or CEs and with the proposed mechanism (Poinot T. et al., 2014;Bülichen D. et al., 2012;Marliere C. et al., 2012). Indeed, the WR of admixed mortars is mainly governed by the ability of polysaccharidic admixtures to form a hydrocolloidal associated polymer molecules network and to induce overlapping of polymer coils within the pore solution (Poinot T. et al., 2014;Bülichen D. et al., 2012;Marliere C. et al., 2012).

When the concentration of polymer increases into the solution, the isolated polymer coils, existing at low polymer concentration, begin to come into contact each other. This concentration is defined as the coil-overlap concentration (noted C*). Above this critical concentration, the polysaccharide aggregates stop the water flow by plugging the porous network of a thin polysaccharide-enriched filter cake at the interface mortar-substrate resulting in a sudden and sharp rise in WR curves (Poinot T. et al., 2014). The abrupt change in slope is reached for a decreasing polymer dosage from HPG 1 (0.15% bwob) to HPG 3 (0.05% bwob). The only difference between these HPGs is the increasing substitution degree.

According to literature, the increase in MS HP does not lead to a change in the C* (Volpert E. et al., 1996). However, the increasing substitution degree leads to a decrease in polymer adsorption on cementitious materials (figure 4) and hence an increase in polymer amount in pore solution.

Consequently, the coil overlapping occurs at lower dosage.

The results furthermore highlight the positive impact of additional alkyl chain on WR. Indeed, the highest WR was obtained with HPG 4 for the lowest polymer dosages. Moreover, the abrupt change in slope occurs for a decreasing polymer dosage from HPG 3 (0.05% bwob) to HPG 4 (0.03% bwob).

The presence of additional alkyl chains (HPG 4), despite slightly higher adsorption than HPG 3, leads to the formation of polymer associates at lower polymer dosage. Indeed, the interconnection between alkyl chains creates intramolecular and intermolecular interactions through specific hydrophobic interactions which cause a decrease in the coil-overlapping concentration (Cheng Y. et al., 2002;Semenov A.N. et al., 1995;Simon S. et al., 2003). Consequently, the abrupt change in slope is reached for a lower polymer dosage of HPG 4 than HPG 3.

Rheological behavior of fresh mortars

The evolution of the yield stress, extracted from Herschel-Bulkley model, is shown in figure 6. The mortar without admixture exhibits a yield stress value of around 45 Pa. From the results, three classes of HPG, inducing different evolution of the yield stress with the polymer dosage, can be highlighted for admixed mortars. The first category only contains the original guar gum (HPG 6), which induces a low and quasi linear decrease in the yield stress of mortar when HPG dosage increases. On the contrary, HPGs 1 and 3 lead to a continuous rise of the yield stress of mortars from 50-60 Pa to around 120 Pa with the increase in the HPG dosage from 0.05% to 0.15%. Finally, HPG 4 constitutes the third class of admixture. The use of this admixture leads an improvement of the yield stress compared to the non-admixed mortar, whatever the dosages tested in the study. However, the improvement is not proportional to the admixture dosage. Indeed, our first dosage (0.05% bwob) leads to a higher yield stress than non-admixed mortar. Beyond this dosage, increasing the dosage from 0.05% to 0.1% bowb provides a slow and low decrease in yield stress, before reaching a plateau for polymer dosages higher than 0.1% bwob. It is worth noting that the value of the yield stress reached on the plateau is still higher than that of the mortar without admixture.

Figure 7 presents the evolution of the consistency coefficient (K from Herschel-Bulkley equation). As in the case of the yield stress, the results can be divided into three classes of polymer. The first class is only composed of the original guar gum which provides a very low or negligible modification of the consistency coefficient with increasing polymer dosage compared to non-admixed mortar. HPGs 1 and 3, constituting the second group, induce first a low increase from in the consistency coefficient, followed by a plateau. Finally, HPG 4 leads to a continuous increase in the consistency coefficient of admixed mortars. Figure 8 shows the evolution of the fluidity index (n) versus the polymer dosage for all the studied mortars. It is worth to note that whatever the mortars (non-admixed and admixed), the values of the fluidity index are lower than 1, meaning that they are all shear thinning. Due to the high standard deviation, the value of the fluidity index of mortars admixed with HPGs 1 and 3 and HPG 6 seem to be unchanged as the dosage of HPGs increase. However, HPG 4 leads to a low increase followed by a continuous decrease in the fluidity index until reaching values around 0.5. This means that the shear thinning behavior of mortars becomes more and more pronounced. The rheological results (figures 6, 7 and 8) highlight that HPGs 1 and 3, HPG 4 and HPG 6 behave quite differently. Indeed, HPGs 1 to 3 lead to a continuous increase in the yield stress, while HPG 4 modifies mainly the consistency coefficient and the fluidity index. This means that HPGs 1-3 increase the stability of mortars while HPG 4 increases the resistance to the flow of admixed mortars. HPGs 1 and 3 affect the rheological behavior of the admixed mortars in the same way, i.e. an increase in the yield stress, a low increase followed by a plateau in the consistency coefficient and a negligible modification of the fluidity index when the polymer dosage rises. Figure 4 shows that HPGs adsorb onto particles constituting the mortar. Prima facie, this adsorption could be responsible for the increase in the yield stress because of bridging flocculation (Brumaud C. et al., 2014). However, despite a strong drop of the adsorption (50%) with the increase in the MS HP (from HPG 1 to 3) the yield stress also increases. This suggests that the non-adsorbed polymer may be responsible for the yield stress increase. The potential loss of bridging can be compensated by an increase in the pore solution viscosity induced by the rise in the polymer concentration and/or by the depletion flocculation induced by the non-adsorbed coils (Palacios M. et al., 2012). Moreover, the presence of HPG coils within the pore solution leads to an increase in the consistency coefficient (K) compared to non-admixed mortars. However, the expected increase in K due to the rise of pore solution viscosity with the polymer dosage can be compensated by steric hindrance, leading to a plateau for K.

Since the adsorption of original guar gum onto the surface of the binder is higher than 98.5%, one expects to detect a very strong increase in the yield stress compared to non-admixed mortar. However, the rheological behavior of the admixed mortar with the original guar gum is very close to that of the non-admixed mortar. This result suggests that the entire molecule of the original guar gum could be mainly adsorbed onto the surface of only one particle, limiting therefore the bridging flocculation. The very high concentration of free hydroxide groups on the backbone of the guar could be responsible of this mechanism. Moreover, the adsorption of the guar molecule onto a single particle leads to an increase in the steric hindrance and in the dispersion and lubrication effects, leading to a low but continuous decrease in the yield stress. For dosages higher than 0.1% bwob, the concentration in polymer coils (HPG 6) into the pore solution begins to slightly increase, leading to the beginning of the increase in the consistency coefficient and of the decrease in the fluidity index. The additional alkyl chain also modifies the rheological properties of mortars. Contrary to HPG 3, HPG 4 leads to a strong and continuous increase in the consistency coefficient and a decrease in the fluidity index. These results highlight that mortars become more and more shear-thinning since the fluidity index decreases from 0.8 to 0.5. This rheological behavior gets more pronounced as the HPG dosage increases. These results are consistent with the fact that the hydrophobically modified HPG leads to the formation of coil overlapping at lower HPG dosage (between 0.03 and 0.05% bwob) since the presence of additional alkyl chains enhances the entanglement. Above this dosage, entanglement occurs between polymer coils, inducing a shear thinning behavior to the solution. At low shear rate, the entanglement of polymer coils leads to a higher pore solution viscosity and thus higher mortar viscosity. When the shear rate increases, the polysaccharide chains align in the direction of the flow resulting in less and less effect on mortar fluidity. The shear thinning behavior of the solution, and thus of the mortars, amplify with the increasing polymer dosage. The yield stress is also impacted by the additional alkyl chain. Indeed,  0 , of mortar admixed with HPG 4, increases for a dosage equal to 0.05% bwob then slowly decreases for dosages ranging from 0.05% to 0.075% bwob, before reaching a plateau for higher dosages ( 0 reached is still higher than that of the non-admixed mortar). This result could be explained by a change in the HPG conformation due to the presence of additional alkyl chains. Indeed, as previously mentioned, alkyl chain creates intramolecular and intermolecular interactions through specific hydrophobic interactions. Intramolecular association of hydrophobic units tends to force the polymer chain into a more compact conformation (Simon S. et al., 2003;Aubry T. et al., 1994). Moreover, the conformation of hydrophobically modified polymers in aqueous solution involves the presence of alkyl chains mainly inside the coils in order to limit contacts between hydrophobic chains and water (Simon S. et al., 2003). Consequently, the hydrophilic groups, such as hydroxyl and hydroxypropyl, are preferentially on the outskirts of the coils, promoting the adsorption onto the surface of particles (figure 4) and therefore steric hindrance which implies a prevention of direct contacts between particles. Moreover, due to a more compact conformation and avoidance between water and hydrophobic units, the bridging ability of hydrophobically modified HPG should decrease. All these points should lead to a decrease in the yield stress.

Conclusions

In this paper, we studied the effect of several guar gum derivatives on water retention property and rheological behavior of mortars. Based upon the results, it was found that the original guar gum was totally adsorbed onto particle surface, leading to a negligible modification of WR and rheological behavior with respect to the non-admixed mortar. Depending of the chemical structure of HPGs, it is possible to promote the water retention according to two different ways. First, by increasing the MS HP of HPGs, the amount of adsorbed polymer drops, which leads to an increase in the HPG concentration within the pore solution and therefore to lower HPG dosage necessary to reach coil overlapping. Second, by enhancing overlapping, the hydrophobically modified HPGs improve the effectiveness of WR agent at low dosage. HPGs also modify the rheological behavior of the mortars. As in the case of WR, the hydrophobic characteristic of HPGs is the preponderant parameter. Indeed, it was shown that additional alkyl chain mainly leads to a more shear thinning behavior of the mortar and to a rise in the consistency coefficient, while classical HPGs strongly increases the yield stress.

-Rols S., Ambroise J., Péra J., 1999

Figure 1

 1 Figure 1 Molecular structure of original guar gum (a) and HydroxyPropyl Guar (b)

Figure 2

 2 Figure 2 Device for Water Retention measurements

Figure 3

 3 Figure 3 Experimental device (a) and experimental protocol for rheological measurements

Figure 4

 4 Figure 4 Adsorption isotherms of HPGs 1, 3, 4 and original guar gum (HPG 6)

Figure 5

 5 Figure 5 Impact of polymer dosage on water retention capacity of fresh admixed mortars

Figure 6

 6 Figure 6 Impact of polymer dosage on yield stress of fresh admixed mortars (HPGs 1, 3, 4 and original guar gum (HPG 6))

Figure 7

 7 Figure 7 Impact of polymer dosage on consistency coefficient of fresh admixed mortars (HPGs 1, 3, 4 and original guar gum (HPG 6))

Figure 8

 8 Figure 8 Impact of polymer dosage on fluidity index of fresh admixed mortars (HPGs 1, 3, 4 and original guar gum (HPG 6))

  Tab. 2 Median particle diameters by volume and specific surface area of the mineral phases.

		CEM II/B-	Lime	Dolomite	Dolomite	Calcite
		LL		Leidi	Bombardieri
	d 50 (µm)	15	5	300	600	20
	BET specific surface area (m 2 /g)	2.40	5.67	0.43	0.34	1.44

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Francoise Berthoud

Laurent Lefèvre

Cédric Gossart

ICT is part of

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

ICT is part of climate change !

Françoise Berthoud 1 , Laurent Lefevre 2 , Cédric Gossart 3 -EcoInfo 1 CNRS-LPMMC, Grenoble, France -francoise.berthoud@grenoble.cnrs.fr 2 Inria -Avalon Team -LIP Laboratory -Ecole Normale Supérieure de Lyon, France -laurent.lefevre@inria.fr 3 Institut Mines-Télécom/Télécom École de Management, Évry, France -cedric.gossart@telecom-em.eu http://ecoinfo.cnrs.fr Can we reduce its impact and apply good practices and tools to other society domains ?