J Ratel

F Mercier

J Rivas

H Wang

M Angénieux

B Calmont

S Crépieux

Christelle Planche
email: christelle.planche@inrae.fr

E Engel

Planche

Fate of polychlorobiphenyls in Tenebrio molitor larvae: consequences for

Keywords: PCB, yellow mealworm, bioaccumulation, contaminants, safety 36 37

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

Introduction 44 45

The United Nations estimate that the global human population is expected to reach 9.7 Hexane, dichloromethane, acetone and toluene were organic trace analysis grade solvents 91 (Sigma-Aldrich, Saint-Quentin Fallavier, France). For PCB extraction, Florisil® and activated 92 aluminium oxide (acidic, Brockmann I) were from Sigma-Aldrich. Diatomaceous earth was 93 obtained from Thermo Fisher Scientific (Waltham, MA, USA). The certified reference material 94 AE-00059-H-2X of AccuStandard Europe (Niederbipp, Switzerland) was used for the 95 development of PCB analysis. This mix is composed of the 6 nDL-PCBs (IUPAC numbers 28, 96 52, 101, 138, 153, and 180) with individual concentrations of 20 ppm. Fluorinated internal 97 standards 3-F-PCB-52 and 5'-F- Trondheim,Norway) were used for the 98 accurate quantification of target compounds. (Supplementary Figure S1). A water spray was added to each jar containing larvae two times 130 per day. The rearing of larvae was carried out for 20 days in incubators. From the 6th day of 131 the test, the evaluation of larval weight was performed for each trial twice per week. Larvae 132 were separated by using a 1.5 mm mesh sieve. After sieving, the weight of 20 larvae was 133 determined. After each weighting, the sieved content and the larvae were put back in the jar. The PCB levels in the larvae experimentally reared were determined by µECD (Agilent) 175 coupled to a gas chromatograph (6890, Agilent Technologies). The GC parameters were the same as for the GC-MS coupling. The micro-ECD system was operated at 300 °C using data acquisition rate of 50 Hz, with nitrogen as make-up gas at a flowrate of 40 ml/min. Calibration 178 curves were preliminary built from dried larvae extracts spiked with the 6 nDL-PCBs (see Table The recovery rates of the 6 nDL-PCBs measured after spiking in dried powder of T. retainers, the PCB recoveries in larvae samples (from 98% to 129%) are of the same order of 277 magnitude as those in raw beef samples (from 87% to 133%) [START_REF] Planche | Assessment 433 of comprehensive two-dimensional gas chromatography-time-of-flight mass 434 spectrometry based methods for investigating 206 dioxin-like micropollutants in 435 animal-derived food matrices[END_REF]. This

Discussion

282

Based on this protocol, the impact of a contamination by PCBs on T. molitor was assessed 283 using a pilot experimental farming where larvae were fed for 20 days with wheat bran either accumulated) could then be used for non-food applications (biofuel production for example) 330 while the protein fraction could possibly be used for food and feed. 331 332

Conclusion 333 334

The pilot experimental farming set up enabled to assess the bioaccumulation of PCBs in T.

335

molitor larvae during rearing. A feed substrate contamination with PCBs doesn't seem to have 336 any significant impact on larval development without negative effects on growth. It could be 337 supposed that a much higher PCB level than those assessed in this study could have a 338 significant impact on the development of T. molitor, but these concentrations would be 339 unrealistic in insect feed substrate. In addition, it would be interesting to assess the impact on S1)

Table S4: Bioaccumulation factors (BAF) of PCB congeners in Tenebrio molitor larvae on a dry weight basis after 20 days of rearing on wheat bran spiked at 0.67, 4.0 or 24.4 ng PCB/g. Data represent mean BAF ± SD which is determined from 3 analytical replicates of each rearing glass jar (n=3 glass jars for each exposure condition).

Congener

Wheat S1)

Table S4: Bioaccumulation factors (BAF) of PCB congeners in Tenebrio molitor larvae on a dry weight basis after 20 days of rearing on wheat bran spiked at 0.67, 4.0 or 24.4 ng PCB/g. Data represent mean BAF ± SD which is determined from 3 analytical replicates of each rearing glass jar (n=3 glass jars for each exposure condition).

Congener

Wheat

  dry wheat bran produced by local flour mills in Puy de Dome (France). Larvae 102 were fed with non-contaminated feeds (control group) or with PCB contaminated feeds 103 (exposed groups). For exposed groups, the bran was spiked according to a protocol adapted 104 from Planche et al. (2015), based on a contaminant addition to the feed via a volatile solvent. 105 For each PCB exposure condition, the spiking was carried out on the total quantity of wheat 106 bran necessary for all the individual rearing (45g), allowing to ensure the quality of replicates. 107 Briefly, 45 g of dry wheat bran were set in a glass jar (9.5cm high×7cm wide) then immersed 108 in acetone (125 mL) containing the 6 nDL-PCBs. The mixture was evaporated down under a 109 hood and roughly homogenised. Three spiking concentrations, 0.67, 4.0 and 24.4 ppb (ng 110 PCB/g wheat bran), were tested for each PCB congener. This corresponds to levels of (6 nDL-111 PCBs) that are 0.4, 2.4 and 14.6 times the maximum level (ML) set at 10 ppb by the Regulation 112 (EU) No 277/2012 in feed materials of plant origin. The spiking concentrations are consistent 113 both with the regulation issues and with the scarce literature data dealing with PCB 114 contamination in food of plant origin, like wheat bran, with for example some concentrations 115 of PCBs in bran that can reach 1.84 ppb (Roszko et al., 2014). For the control groups, the bran 116 was either unspiked (bran without solvent or PCB) or spiked with neat solvent (bran with 117 solvent but without PCB). 118 Rearing 119 Larvae of T. molitor were produced by the startup INVERS (Saint-Ignat, France). Using a 120 balance (Precisa 410 AM-FR, Precisa Instruments Ltd., Switzerland; d: 0.0001), 10g of larvae 121 at L3 growth stage (approximatively 200 individuals) were set on a glass jar containing 45 g of 122 control or contaminated wheat bran. Three glass jars for each modality were prepared and put 123 in an 124 Scientific) customised for sufficient ventilation (300 L/h) (Figure 1). In total, 15 rearing glass 125 jars were distributed in the 5 incubators. These replicates were not randomly distributed over 126 the incubators to avoid a cross contamination between the different exposure conditions. The 127 humidity was maintained at 60% and the temperature regulated at 26°C with a monitoring 128 throughout the rearing (probe: VAISALA HMP110; data acquisition system: AOIP SA32)129

  134

Figure 1 :

 1 Figure 1: Pilot experimental farming of Tenebrio molitor larvae.

  159

Figure 2 :

 2 Figure 2: Simplified diagram of the workflow used to analyse PCBs in Tenebrio molitor

  179

1

  ) at the following concentrations: 0.2, 0.5,1.3, 3.0, 8.0, 20.0, 50.0 ng/g. Larvae extracts 180 without PCB added were run in triplicate to check the absence of targeted analytes. Each 181 concentration level was analysed 4 times by GC-µECD. Peak areas were determined, 182 normalized by internal standard, then used for calculations of the standard deviations and the 183 calibration curve equations. The linearity of the calibration curves was assessed for each PCB 184 by calculating the coefficients of determination (R 2 ). The limit of detection (LOD), using the 185 definition 3s/m (s is the standard deviation of the intercept, and m is the slope of the linear 186 calibration curve), was determined from the calibration curves for each individual PCB studied. 187 Data treatment 188 Data were processed with the TIBCO's Statistica software (version 13.0, TIBCO Software Inc.) 189 and the R software (version 3.5.1, The R Foundation for Statistical Computing). 190 (linear model) function of R was used on the calibration curve data for the determination of R 2 191 as well as s and m requested for LOD calculation. Student's t test or one-way analyses of 192 variance (ANOVA) followed by post hoc Newman Keuls test were performed to compare 193 means data. The differences were considered significant when p < 0.05.

  200 molitor (at 200 ng/g dry matter), extraction, purification and concentration are presented in 201 Table 1. All the recovery rates lay in the classically accepted range of 70 130% according to 202 the EPA Method 8000C (2003), with RSD between 10% and 18%.

  assess the fate of the 6 nDL-PCBs in T. molitor larvae during rearing with a 273 contaminated substrate, the first step of this study was to determine their recovery rates after 274 the spiking, extraction, purification, concentration and analysis protocol. Based on a protocol 275 adapted from Planche et al. (2015) with a delipidation strengthened by the addition of fat 276

  278demonstrates that the high fat level of T. molitor larvae (56-61% in whole dried T. molitor 279 (EFSA, 2021) vs 11% in raw ground beef) does not decrease the recovery rates obtained even if PCBs are hydrophobic compounds (Log Kow = 4.09 8.18 according to Hawker and Connell 281 (1988)), thereby confirming the relevance of the protocol used.

340

  larval development of a longer rearing period on a contaminated substrate. The present study 341 revealed that there is no bioaccumulation of PCBs in T. molitor larvae during rearing.342However, PCBs can be transferred from the diet to the larvae and are then concentrated in 343 larvae during the drying process. This highlights the significant impact of the drying process 344which induces a concentration by a factor of almost 3 of PCBs in larvae. Knowing that the 345 nDL-PCB concentration maximum limit is set at 10 µg/kg either for feed materials of plant 346 origin and for feed materials of land animal origin (Commission regulation, EU No 277/2012), 347 the use as larvae feeding substrate of wheat bran containing a residual amount of nDL-PCBs 348 could result, after the rearing period, in dried larvae with an almost 3-fold higher nDL-PCB 349 concentration that could potentially exceed the maximum limit for its use as feed. This 350 highlights the importance of monitoring the quality of the feed substrates used for farming 351 insects for food and feed in order to guarantee a safe content in chemical contaminants 352 including persistent organic pollutants like PCBs. When a PCB contamination is detected in 353 larvae feed substrate, it could therefore be envisaged to use the larvae lipid fraction (where 354 PCBs are accumulated) for non-food applications. Finally, in order to ensure food safety 355 throughout the food chain, it would be interesting to assess the PCB biomagnification factors 356 if T. molitor larvae are used as feed, for example for aquaculture since it is expected that most 357 insect production will be targeted towards aquafeed by 2030 (Van Huis, 2022).

  solid waste treatment by Hermetia illucens with intestinal 378 microbiota. Environmental Pollution 242: 634-642. to Regulation (EC) No 999/2001 of the European Parliament and of the Council as 401 regards the prohibition to feed non-ruminant farmed animals, other than fur animals, 402 with protein derived from animals. 403 EU, 2017. Commission Regulation (EU) 2017/893 of 24 May 2017 amending Annexes I and 404 IV to Regulation (EC) No 999/2001 of the European Parliament and of the Council

  405 and Annexes X, XIV and XV to Commission Regulation (EU) No 142/2011 as 406 regards the provisions on processed animal protein. 407 EU, 2012. Commission Regulation (EU) No 277/2012 of 28 March 2012 amending Annexes I 408 and II to Directive 2002/32/EC of the European Parliament and of the Council as 409 regards maximum levels and action thresholds for dioxins and polychlorinated 410 biphenyls. 411 Gao, Q., Deng, W., Gao, Z., Li, M., Liu, W., Wang, X. and Zhu, F., 2019. Effect of 412 sulfonamide pollution on the growth of manure management candidate Hermetia 413 illucens. PLOS ONE 14(5): e0216086. https://doi.org/10.1371/journal.pone.0216086 414 Hawker, D.W. and Connell, D.W., 1988. Octanol-water partition coefficients of 415 polychlorinated biphenyl congeners. Environmental Science & Technology 22: 382-416 387. https://doi.org/10.1021/es00169a004 417 Meijer, N., de Rijk, T., Van Loon, J.J.A., Zoet, L. and Van der Fels-Klerx, H.J., 2021. Effects 418 of insecticides on mortality, growth and bioaccumulation in black soldier fly

Figure S1 :

 S1 Figure S1: Temperature (°C) and relative humidity (%) readings of each incubator during the larvae rearing.

Figure S2 :

 S2 Figure S2: Weight of Tenebrio molitor larvae (mg) throughout their rearing on wheat bran either unspiked (Control), spiked with neat solvent (Control + acetone) or spiked at 0.67, 4.0 or 24.4 ng PCB/g wheat bran. Data represent mean larvae weight (mg) ± SD which is determined from the 20 larvae measurement for each rearing glass jar (n=3 glass jars for each exposure condition).

Table 1 :

 1 Recovery rates (%) obtained after spiking, extraction, purification,The LOD measured with GC-µECD for the 6 nDL-PCB congeners in dried powder of T.209molitor larvae varied from 1.67 ng/g for PCB 153 to 2.98 ng/g for PCB 101, with limits of 210 quantification (LOQ) from 5.56 to 9.93 ng/g respectively (Supplementary TableS1).

	206
	207
		PCB		Recovery rates ± RSD 1
	Compound name		m/z
		congener		(%)
	2,4,4'-Trichlorobiphenyl	28	256	108 ± 17
	2,2',5,5'-Tetrachlorobiphenyl	52	292	110 ± 16
	2,2',4,5,5'-Pentachlorobiphenyl	101	326	98 ± 18
	2,2',3,4,4',5'-Hexachlorobiphenyl	138	360	107 ± 14
	2,2',4,4',5,5'-Hexachlorobiphenyl	153	360	108 ± 16
	2,2',3,4,4',5,5'-Heptachlorobiphenyl	180	394	129 ± 10

205

concentration and analysis of PCBs in dried powder of Tenebrio molitor larvae (n=3).

Table 2 and

 2 Figure S2 present the weight of T. molitor larvae at days 1, 7, 10, 14, 17 and

	218
	219	20 of rearing depending on their feeding substrate: wheat bran unspiked, spiked with neat
	220	solvent without PCB or spiked with nDL-PCBs at 0.67, 4.0 or 24.4 ng/g corresponding to 0.4,
	221	2.4 and 14.6 times the maximum level (ML) for the nDL-PCBs in feed materials of plant origin,
	222	respectively (Commission regulation (EU) No 277/2012). One-way ANOVA (p<0.05) revealed
		no significant difference between the different conditions with a mean weight of 54.1 mg per
	226
	227

223 larvae at the beginning of the study and a continuous growth throughout the rearing up to 95.6 224 mg per larvae on average after 20 days of rearing. Visual observations throughout the rearing 225 period did not reveal differences in terms of larval mortality between the different conditions.

Table 2 :

 2 [START_REF] Van Der Fels-Klerx | 451 Chemical food safety of using former foodstuffs for rearing black soldier fly larvae 452 (Hermetia illucens) for feed and food use[END_REF], with BAF = concentration of PCB in the organism (fresh or dried larvae) / 242 concentration of PCB in the feed provided (dried wheat bran).For a spiking concentration of 0.67 ng PCB/g wheat bran, concentrations in dried larvae 244 extracts of the 6 nDL-PCBs were below the detection limits detailed in Supplementary Table 245S1. For a spiking concentration of 4 ng PCB/g wheat bran, the concentrations of PCB 28, 52 246 and 101 were between LOD and LOQ (Supplementary TableS2). For all other conditions, PCB 247 congener concentrations are above the LOQ. Regarding these last values, Supplementary TableTable 3shows the BAF of the 6 nDL-PCBs congeners in fresh larvae. BAF in fresh larvae

	232
	233
			Control +
	Day	Control		0.67 ppb	4.00 ppb	24.40 ppb	Significance 1
			acetone
	1	54.5 ± 2.8	50.2 ± 1.8	54.6 ± 1.5	56.5 ± 4.5	54.6 ± 6.8	NS
	7	67.1 ± 3.5	67.2 ± 9.2	64.1 ± 3.3	65.3 ± 3.8	73.0 ± 6.4	NS
	10	85.6 ± 5.3	76.5 ± 3.3	75.4 ± 5.4	80.7 ± 5.4	82.1 ± 14.4	NS
	14	83.4 ± 4.9	81.3 ± 0.8	85.1 ± 3.3	88.9 ± 7.0	86.0 ± 2.5	NS
	17	94.1 ± 5.0	92.9 ± 5.8	96.8 ± 3.0	86.7 ± 2.1	93.7 ± 9.4	NS
	20	93.1 ± 7.2	92.2 ± 8.6	97.4 ± 5.8	97.2 ± 4.7	98.0 ± 2.4	NS
	NS: not significant (p>0.05)

Weight of Tenebrio molitor larvae (mg) throughout their rearing on wheat 228 bran either unspiked (Control), spiked with neat solvent (Control + acetone) or spiked at 229 0.67, 4.0 or 24.4 ng PCB/g wheat bran. Data represent mean larvae weight (mg) ± SD which 230 is determined from the 20 larvae measurement for each rearing glass jar (n=3 glass jars for each 231 exposure condition).

253

Table 3 :

 3 Bioaccumulation factors (BAF) of PCB congeners in Tenebrio molitor larvae on

	262
	263
	Congener	Wheat bran contamination with PCBs 0.67 ppb 4.0 ppb 24.4 ppb
	PCB 28	NA	0.80 ± 0.17 a*	0.68 ± 0.03 b
	PCB 52	NA	0.39 ± 0.07 a*	0.36 ± 0.03 a
	PCB 101	NA	0.66 ± 0.10 a*	0.67 ± 0.02 a
	PCB 138	NA	0.86 ± 0.16 a	0.96 ± 0.05 a
	PCB 153	NA	0.83 ± 0.13 a	0.99 ± 0.05 b
	PCB 180	NA	0.81 ± 0.12 a	0.97 ± 0.08 b
	267
	268
	269
	270

257 a fresh weight basis after 20 days of rearing on wheat bran spiked at 0.67, 4.0 or 24.4 ng 258 PCB/g. These values were estimated from data obtained on dried larvae, taking into account 259 the impact of drying on larval weight. Data represent mean BAF ± SD which is determined 260 from 3 analytical replicates of each rearing glass jar (n=3 glass jars for each exposure 261 condition). NA: non-available (PCBs non-detected in larvae extracts) 264 a b: different superscript letters within the same row indicate significant differences among values 265 (p<0.05). BAF are determined from quantification values in larvae extracts either between LOD and 266 LOQ (*) or greater than LOQ.

Table S1 :

 S1 Performance of GC-µECD for quantification of the 6 nDL-PCB congeners in dried powder of Tenebrio molitor larvae (linearity range: 0.2-50.0 ng/g).

	PCB	Coefficient of	Limit of detection	Limit of quantification
	congener	determination (R²)	(LOD) in ng/g	(LOQ) in ng/g
	28	0.95	2.80	9.32
	52	0.96	2.40	8.00
	101	0.94	2.98	9.93
	138	0.98	1.76	5.88
	153	0.98	1.67	5.56
	180	0.98	1.68	5.61

Table S2 :

 S2 Concentrations (ng/g) of PCB congeners in Tenebrio molitor larvae on a dried weight basis after 20 days of rearing on wheat bran spiked at 0.67, 4.0 or 24.4 ng PCB/g. For each exposure condition, three analytical replicates of each rearing glass jar were carried out (n=3 glass jars for each exposure condition).

		<LOD	9.56	65.13
		<LOD	10.69	65.32
		1.77*	10.71	73.80
		<LOD	11.61	73.50
		<LOD	9.60	71.97
		1.86*	7.89	71.41
	Congener	Wheat bran contamination with PCBs <LOD 8.23 59.18 0.67 ppb 4.0 ppb 24.4 ppb <LOD 7.58 61.84
	PCB 28 PCB 180	NA <LOD NA <LOD NA <LOD NA 1.71* NA <LOD NA <LOD	5.26* 7.46 6.73* 8.91 9.98 9.92 8.98* 9.80 9.67 11.33 10.71 10.70	65.84 44.03 63.52 51.78 63.93 48.67 75.07 48.94 73.20 44.26 45.12 73.05
	NA NA: non-available (non-detected in larvae extracts) 9.94	47.06
	LOD: Limit of detection	NA	10.89	47.77
			NA	8.79*	46.21
			NA	3.28*	20.82
			NA	3.28*	25.54
			NA	5.38*	25.38
			NA	4.25*	26.93
	PCB 52		NA	4.43*	25.05
			NA	5.09*	24.18
			NA	4.28*	25.57
			NA	5.12*	25.87
			NA	4.31*	25.44
			NA	5.60*	44.90
			NA	5.77*	47.35
			NA	7.81*	46.75
			NA	6.75*	48.12
	PCB 101		NA	7.56*	44.12
			NA	8.85*	44.60
			NA	7.84*	47.83
			NA	8.88*	48.04
			NA	7.20*	46.44
		<LOD	7.17	67.28
		<LOD	7.21	62.43
		<LOD	9.32	63.24
		<LOD	8.26	69.15
	PCB 138	<LOD	10.05	64.03
		<LOD	11.55	63.56
		<LOD	10.58	70.78
		<LOD	12.40	69.11
		<LOD	10.00	68.47
		1.96*	7.65	68.68
	PCB 153	<LOD <LOD	7.70 8.84	64.16 65.71
		<LOD	7.43	70.40

* Quantification values between LOD and LOQ (see Table

  Fate of polychlorobiphenyls in Tenebrio molitor larvae: consequences for further use as food and feed J. Ratel 1 , F. Mercier 1 , J. Rivas 1 , H. Wang 1 , M. Angénieux 1 , B. Calmont 2 , S. Crépieux 3 , C. Planche 1 *, E. Engel 1

			bran contamination with PCBs
		0.67 ppb	4.0 ppb	24.4 ppb
	PCB 28	NA	2.25 ± 0.47 a**	1.93 ± 0.10 a
	PCB 52	NA	1.10 ± 0.19 a**	1.02 ± 0.07 a
	PCB 101	NA	1.84 ± 0.29 a**	1.90 ± 0.06 a
	PCB 138	NA	2.40 ± 0.45 a	2.72 ± 0. 13 a
	PCB 153	NA	2.33 ± 0.38 a	2.81 ± 0.16 b
	PCB 180	NA	2.27 ± 0.35 a	2.76 ± 0.24 b

NA: non-available (PCBs non-detected in larvae extracts) a b: different superscript letters within the same row indicate significant differences among values (p<0.05). BAF are determined from quantification values in larvae extracts between LOD and LOQ (*) or greater than LOQ.

Supplementary material

Table S1 :

 S1 Performance of GC-µECD for quantification of the 6 nDL-PCB congeners in dried powder of Tenebrio molitor larvae (linearity range: 0.2-50.0 ng/g).

	PCB	Coefficient of	Limit of detection	Limit of quantification
	congener	determination (R²)	(LOD) in ng/g	(LOQ) in ng/g
	28	0.95	2.80	9.32
	52	0.96	2.40	8.00
	101	0.94	2.98	9.93
	138	0.98	1.76	5.88
	153	0.98	1.67	5.56
	180	0.98	1.68	5.61

Table S2 :

 S2 Concentrations (ng/g) of PCB congeners in Tenebrio molitor larvae on a dried weight basis after 20 days of rearing on wheat bran spiked at 0.67, 4.0 or 24.4 ng PCB/g. For each exposure condition, three analytical replicates of each rearing glass jar were carried out (n=3 glass jars for each exposure condition).

		<LOD	9.56	65.13
		<LOD	10.69	65.32
		1.77*	10.71	73.80
		<LOD	11.61	73.50
		<LOD	9.60	71.97
		1.86*	7.89	71.41
	Congener	Wheat bran contamination with PCBs <LOD 8.23 59.18 0.67 ppb 4.0 ppb 24.4 ppb <LOD 7.58 61.84
	PCB 28 PCB 180	NA <LOD NA <LOD NA <LOD NA 1.71* NA <LOD NA <LOD	5.26* 7.46 6.73* 8.91 9.98 9.92 8.98* 9.80 9.67 11.33 10.71 10.70	65.84 44.03 63.52 51.78 63.93 48.67 75.07 48.94 73.20 44.26 45.12 73.05
		NA	9.94	47.06
		NA	10.89	47.77
		NA	8.79*	46.21
		NA	3.28*	20.82
		NA	3.28*	25.54
		NA	5.38*	25.38
		NA	4.25*	26.93
	PCB 52	NA	4.43*	25.05
		NA	5.09*	24.18
		NA	4.28*	25.57
		NA	5.12*	25.87
		NA	4.31*	25.44
		NA	5.60*	44.90
		NA	5.77*	47.35
		NA	7.81*	46.75
		NA	6.75*	48.12
	PCB 101	NA	7.56*	44.12
		NA	8.85*	44.60
		NA	7.84*	47.83
		NA	8.88*	48.04
		NA	7.20*	46.44
		<LOD	7.17	67.28
		<LOD	7.21	62.43
		<LOD	9.32	63.24
		<LOD	8.26	69.15
	PCB 138	<LOD	10.05	64.03
		<LOD	11.55	63.56
		<LOD	10.58	70.78
		<LOD	12.40	69.11
		<LOD	10.00	68.47
		1.96*	7.65	68.68
	PCB 153	<LOD <LOD	7.70 8.84	64.16 65.71
		<LOD	7.43	70.40

NA: non-available (non-detected in larvae extracts) LOD: Limit of detection * Quantification values between LOD and LOQ (see Table

Relative standard deviation (%)

3) and dried (TableS4) T. molitor larvae according to[START_REF] Truzzi | Influence of feeding substrates on the presence of toxic 441 metals (Cd, Pb, Ni, As, Hg) in larvae of Tenebrio molitor: risk assessment for human 442 consumption[END_REF] and Van der Fels-

Acknowledgements

360

This study has been implemented in the R&D Booster program funded by the Auvergne Rhône 42 Alpes Region (INSECT2FEED project).


Antoine Cannesan
email: azeddine.driouich@univ-rouen.fr

Arabinogalactan proteins in root-microbe interactions

Keywords: Arabinogalactan proteins, plant-microbe interactions, plant protection, roots, rhizosphere

Arabinogalactan proteins in root-microbe interactions

Eric Nguema-Ona, Maïté Vicré-Gibouin, Marc-Antoine Cannesan, and Azeddine Driouich Arabinogalactan proteins (AGPs) are among the most intriguing sets of macromolecules, specific to plants, structurally complex, and found abundantly in all plant organs including roots, as well as in root exudates. AGPs have been implicated in several fundamental plant processes such as development and reproduction. Recently, they have emerged as interesting actors of root-microbe interactions in the rhizosphere. Indeed, recent findings indicate that AGPs play key roles at various levels of interaction between roots and soilborne microbes, either beneficial or pathogenic. Therefore, the focus of this review is the role of AGPs in the interactions between root cells and microbes. Understanding this facet of AGP function will undoubtedly improve plant health and crop protection.

Arabinogalactan proteins

Arabinogalactan proteins (AGPs) are highly glycosylated members of the hydroxyproline-rich glycoprotein (HRGP) superfamily of plant cell wall proteins. The members of this family share common features, including their typical, but variable arabinogalactosylated glycomodules, and many other features associated with their protein and nucleic sequences such as the presence of numerous hydroxyproline (HyP)-based sites of O-glycosylation, the existence of many functional domains (often putative), or the possibility to be anchored to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor (Figure 1) [START_REF] Showalter | Arabinogalactan proteins: structure, expression and function[END_REF][START_REF] Schultz | The classical arabinogalactan protein gene family of Arabidopsis[END_REF][START_REF] Ma | Genome-wide identification, classification, and expression analysis of the arabinogalactan protein gene family in rice (Oryza sativa L.)[END_REF]. Occurrence of AGPs in almost all root cell types including root hairs, epidermal and cortical cells has been reported in most (if not all) species studied so far (Table 1). In addition, root tips release large amounts of AGP-rich rhizodeposits in the soil, including living root border cells/border-like cells (BCs/BLCs) and mucilage-rich exudates [START_REF] Hawes | Function of root border cells in plant health: pioneers in the rhizosphere[END_REF][START_REF] Hawes | The role of border cells in plant defense[END_REF][START_REF] Vicre | Root border like cells of Arabidopsis. Microscopical characterization and role in the interaction with Rhizobacteria[END_REF][START_REF] Durand | The organization pattern of root border like cells of Arabidopsis is dependent on cell wall homogalacturonan[END_REF][START_REF] Cannesan | Effect of arabinogalactan proteins from the root caps of pea and Brassica napus on Aphanomyces euteiches zoospore chemotaxis and germination[END_REF]. The use of various anti-AGP antibodies [START_REF] Knox | The use of antibodies to study the architecture and developmental regulation of plant cell walls[END_REF][START_REF] Knox | Developmentally regulated epitopes of cell surface arabinogalactan proteins and their relation to root tissue pattern formation[END_REF] and immunomicroscopy has established that AGPs are differentially distributed and developmentally regulated in root tissues (Table 1 and references therein). For example, JIM13recognized epitopes have only been found in xylem and root cap cells/BLCs in developing Arabidopsis (Arabidopsis thaliana) roots [START_REF] Vicre | Root border like cells of Arabidopsis. Microscopical characterization and role in the interaction with Rhizobacteria[END_REF][START_REF] Dolan | An AGP epitope distinguishes a central metaxylem initial from other vascular initials in the Arabidopsis roots[END_REF], whereas JIM4 epitopes have been found associated with developing pericycle cells in carrot (Daucus carota) [START_REF] Knox | Developmentally regulated epitopes of cell surface arabinogalactan proteins and their relation to root tissue pattern formation[END_REF]. Examinations of AGP-associated glycans using analytical chemistry techniques have also highlighted the strong heterogeneity in the composition and structure of AGPs in roots (Table 1). The use of Yariv reagent, known to bind and selectively precipitate AGPs [START_REF] Yariv | Precipitation of arabic acid and some seed polysaccharides by glycosylphenylazo dyes[END_REF]13], has also facilitated many of these studies (Table 1). Finally, bioinformatics and molecular tools have confirmed heterogeneity of AGP expression in roots (Figure 2). Hence, the diversity of AGP structure and localization is likely to prelude the diversity of biological functions that AGPs play in root development and survival.

Indeed, the biological roles of AGPs in a wide range of physiological plant processes have attracted the attention of plant biologists for decades (see recent reviews in [START_REF] Nguema-Ona | Arabinogalactan proteins in root and pollen tubes: distribution and functional aspects[END_REF][START_REF] Tan | Arabinogalactan-proteins and the research for these enigmatic plant cell surface proteoglycans[END_REF][START_REF] Ellis | Arabinogalactan proteins: key regulators at the cell surface[END_REF][START_REF] Seifert | The biology of arabinogalactan proteins[END_REF]) and are still the object of many exciting studies. Different possible modes of action of AGPs in general were proposed: AGPs were proposed to operate as soluble (and diffusible) signals which bind to a receptor. This mode of action is likely to occur during tracheary elements differentiation or during female gametogenesis [START_REF] Seifert | The biology of arabinogalactan proteins[END_REF][START_REF] Motose | A proteoglycan mediates inductive interaction during plant vascular development[END_REF][START_REF] Cheung | A floral transmitting tissue-specific glycoprotein attract pollen tubes and stimulate their growth[END_REF]. The precise structural motif involved in such signaling is unknown but multiple studies suggest that whole AGPs or AGP-derived glycans are good candidates.

Although not yet experimentally proven, receptor-like kinases (RLKs) and wall-associated kinases (WAKs) [START_REF] Seifert | The biology of arabinogalactan proteins[END_REF][START_REF] Gens | Arabinogalactan protein and wall-associated kinases in a plasmalemmal reticulum with specialized vertices[END_REF][START_REF] Van Hengel | AtAGP30, an arabinogalactan-protein in the cell walls of the primary root, plays a role in root regeneration and seed germination[END_REF][START_REF] Driouich | Intercourse between cell wall and cytoplasm exemplified by arabinogalactan proteins and cortical microtubules[END_REF] were proposed to act as AGP receptors. Cleavage of GPI-anchored AGPs by phospholipases (C or D) also results in the release of the GPI anchor, which was also proposed to play a role in downstream signaling [START_REF] Schultz | GPI-anchors on arabinogalactan proteins: implications for signalling in plants[END_REF].

Comparatively, little, or discrete, attention has been given to the role of AGPs in plant-microbe interaction (PMI), particularly in roots, and between root cells and microbes. Root cell AGPs have recently emerged as interesting players of PMI. Indeed, many recent studies suggest that AGPs play a crucial role at several stages of PMI, including root colonization, repelling or attraction of soil microbes, and development of infection structures. Here, we review the role and some properties AGPs display in root cells and in root exudates which, directly or indirectly, may favor or inhibit root colonization by soil microbes and enhance the ability of plants to protect themselves against Minor sugars, such as glucuronic acid or rhamnose, are also present. The backbone of the protein is enriched in hydroxyproline residues. AGPs can be anchored to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. AGP glycan structures were adapted from [START_REF] Tan | Structure of a hydroxyproline (Hyp)arabinogalactan polysaccharide from repetitive Ala-Hyp expressed in transgenic Nicotiana tabacum[END_REF][START_REF] Tryfona | Carbohydrate structural analysis of wheat flour arabinogalactan protein[END_REF][START_REF] Qi | Gum arabic glycoprotein is a twisted hairy rope[END_REF]. Note the high heterogeneity in the structure of the glycan chains. (B) AGP backbones are synthesized by members of a large multigene family and are classified into classical AGPs, hybrid AGPs, chimeric AGPs, and AG peptides (short classical AGPs) [START_REF] Schultz | The classical arabinogalactan protein gene family of Arabidopsis[END_REF][START_REF] Ma | Genome-wide identification, classification, and expression analysis of the arabinogalactan protein gene family in rice (Oryza sativa L.)[END_REF]. Classical AGPs are characterized by a signal peptide domain, a P/Hyp-rich domain, and a C-ter domain. Hybrid AGPs often consist of classical AGPs which may contain FLA, Lys-rich, nonspecific lipid transfer protein (ns-LTP), ENOD domains, or domains of unknown functions (DUFs) within their sequence. These domains can be interspersed within the sequence. Chimeric AGPs often lack the C-ter domain responsible for GPI anchorage. For recent reviews describing the chemistry of AGPs in general, see [START_REF] Nguema-Ona | Arabinogalactan proteins in root and pollen tubes: distribution and functional aspects[END_REF][START_REF] Ellis | Arabinogalactan proteins: key regulators at the cell surface[END_REF]. [START_REF] Vicre | Root border like cells of Arabidopsis. Microscopical characterization and role in the interaction with Rhizobacteria[END_REF][START_REF] Gaspar | Characterization of the Arabidopsis lysinerich arabinogalactan-protein AtAGP17 mutant (rat1) that results in a decreased efficiency of Agrobacterium transformation[END_REF][START_REF] Xie | A plant arabinogalactan-like glycoprotein promotes a novel type of polar surface attachment by Rhizobium leguminosarum[END_REF][START_REF] Berry | Arabinogalactan proteins are expressed at the symbiotic interface in root nodule of Alnus spp[END_REF][START_REF] Van Buuren | Novel genes induced during an arbuscular mycorrhizal (AM) symbiosis formed between Medicago truncatula and Glomus versiforme[END_REF][START_REF] Schultz | Novel plant and fungal AGP like proteins in the Medicago truncatula-Glomus intraradices arbuscular mycorrhizal symbiosis[END_REF][START_REF] Jackson | Arabinogalactan-proteins occur in the freeliving cyanobacterium Nostoc and in plant-Nostoc symbioses[END_REF][START_REF] Deepak | Purification and characterization of proline/ hydroxyproline-rich glycoprotein from pearl millet coleoptiles infected with downy mildew pathogen Sclerospora graminicola[END_REF][START_REF] Rathbun | Identification of a family of extensin-like glycoproteins in the lumen of Rhizobium-induced infection threads in pea root nodules[END_REF][START_REF] Olsson | Rhizobium colonization induced changes in membrane-bound and soluble hydroxyproline-rich glycoprotein composition in pea[END_REF][START_REF] Tsyganova | Distribution of legume arabinogalactan proteins-extensin (AGPE) glycoproteins in symbiotically defective pea mutants with abnormal infection threads[END_REF][START_REF] Shailasree | Accumulation of hydroxyproline rich glycoproteins in pearl millet seedlings in response to Sclerospora graminicola infection[END_REF][START_REF] Kjellbom | Oxidative crosslinking of plasma membrane arabinogalactan proteins[END_REF]. These steps include the recognition between root cells and the microbe, the colonization, and later on the formation of infectious structures [START_REF] Gage | Hanging by a thread: invasion of legume plants by rhizobia[END_REF][START_REF] Harrison | Molecular and cellular aspects of the arbuscular mycorrhizal symbiosis[END_REF].

First, at the initiation of the dialog, or recognition, between root cells and microbes, and subsequent colonization, the presence of AGPs was found to be essential. An Arabidopsis mutant with a mutation in the AGP17 gene was shown to be resistant to transformation by Agrobacterium tumefaciens (the rat1 mutant) [START_REF] Gaspar | Characterization of the Arabidopsis lysinerich arabinogalactan-protein AtAGP17 mutant (rat1) that results in a decreased efficiency of Agrobacterium transformation[END_REF]. A pretreatment of wild type roots with Yariv reagent reduced the frequency of root Indicates that the root cell type investigated in the study is not explicitly mentioned.

transformation events, thus suggesting that AGPs are involved in the recognition and initial attachment of rhizobia to the root [START_REF] Gaspar | Characterization of the Arabidopsis lysinerich arabinogalactan-protein AtAGP17 mutant (rat1) that results in a decreased efficiency of Agrobacterium transformation[END_REF]. The complementation of rat1 mutants with the wild type AtAGP17 gene restored the wild type phenotype [START_REF] Gaspar | Characterization of the Arabidopsis lysinerich arabinogalactan-protein AtAGP17 mutant (rat1) that results in a decreased efficiency of Agrobacterium transformation[END_REF]. The role of AGPs in recognition and attachment of rhizobia to root surface was also proven with Rhizobium species: it has been shown that AGPs secreted by Arabidopsis root cap cells and BLCs influence attraction and/or attachment of Rhizobium sp. [START_REF] Vicre | Root border like cells of Arabidopsis. Microscopical characterization and role in the interaction with Rhizobacteria[END_REF]. Pretreatment of Arabidopsis roots with Yariv reagent significantly reduced the ability of Rhizobium sp. to colonize root tip cells [START_REF] Vicre | Root border like cells of Arabidopsis. Microscopical characterization and role in the interaction with Rhizobacteria[END_REF]. Taking this finding further, the authors showed that inhibiting AGP Oglycosylation with a chemical analog of proline (3,4-dehydroprolin), also altered the ability of Rhizobium sp. to attach to the root cap cells and BLCs [START_REF] Vicre | Root border like cells of Arabidopsis. Microscopical characterization and role in the interaction with Rhizobacteria[END_REF]. More recently, it has been elegantly demonstrated that a novel molecular mechanism involving AGPs of pea (Pisum sativum) root mucilage and an unknown Rhizobium leguminosarum factor were responsible for the polar attachment of R. leguminosarum to the root surface [START_REF] Xie | A plant arabinogalactan-like glycoprotein promotes a novel type of polar surface attachment by Rhizobium leguminosarum[END_REF]. Interestingly, R. leguminosarum mutants lacking plasmidic nodulation genes or extracellular glucomannan were still able to show this polar attachment [START_REF] Xie | A plant arabinogalactan-like glycoprotein promotes a novel type of polar surface attachment by Rhizobium leguminosarum[END_REF][START_REF] Downie | The roles of extracellular proteins, polysaccharides and signals in the interactions of rhizobia with legumes roots[END_REF][START_REF] Williams | Glucomannan-mediated attachment of Rhizobium leguminosarum to pea root hairs is required for competitive nodule infection[END_REF], supporting the idea of a novel molecular mechanism. Together, these studies show that an AGP-based recognition system is required and complementary to other modes of recognition of host roots by beneficial microbes and contribute to the success of root colonization by symbiotic bacteria. By contrast, it is not known if secreted AGPs play a role during early phases (recognition, attraction) of root colonization by symbiotic fungi forming mycorrhizae. It has been shown that an agglutinin isolated from stinging nettle (Urtica dioica agglutinin, UDA [START_REF] Vierheilig | Resistance of Urtica dioica to mycorrhizal colonization: a possible involvement of Urtica dioica agglutinin[END_REF][START_REF] Lerner | The gene for stinging nettle lectin (Urtica dioica agglutinin) encodes both a lectin and a chitinase[END_REF]), and shown to display antifungal properties, inhibits the development of a symbiotic fungi forming vesicular arbuscular mycorrhizae, Glomus mosseae. However, whether UDA interacts with plant AGPs is not known. Finally, AGPs have also been frequently found at the interface of microbe infectious structures and root cells.

Microbial infection is often mediated by the formation of infectious structures where microbes and root cells meet. Examples of such structures are infection threads [START_REF] Gage | Hanging by a thread: invasion of legume plants by rhizobia[END_REF], actinorhizal nodules [START_REF] Berry | Arabinogalactan proteins are expressed at the symbiotic interface in root nodule of Alnus spp[END_REF], arbuscular mycorrhizae [START_REF] Van Buuren | Novel genes induced during an arbuscular mycorrhizal (AM) symbiosis formed between Medicago truncatula and Glomus versiforme[END_REF][START_REF] Schultz | Novel plant and fungal AGP like proteins in the Medicago truncatula-Glomus intraradices arbuscular mycorrhizal symbiosis[END_REF], and cyanobacterial stem gland symbioses [START_REF] Jackson | Arabinogalactan-proteins occur in the freeliving cyanobacterium Nostoc and in plant-Nostoc symbioses[END_REF]. A chimeric population of AGPs (called arabinogalactan protein extensins, AGPEs), shown to be enriched in arabinose and galactose [START_REF] Deepak | Purification and characterization of proline/ hydroxyproline-rich glycoprotein from pearl millet coleoptiles infected with downy mildew pathogen Sclerospora graminicola[END_REF], and recognized by the monoclonal antibody (mAb) MAC265 [START_REF] Bradley | Isolation of monoclonal antibodies reacting with peribacteroid membranes and other components of pea root nodules containing Rhizobium leguminosarum[END_REF][START_REF] Van Den Bosch | Common components of the infection thread matrix and the intercellular space identified by immunocytochemical analysis of pea nodules and uninfected roots[END_REF], has been identified as the major component of infection thread lumen of the rhizobium-pea symbiosis [START_REF] Rathbun | Identification of a family of extensin-like glycoproteins in the lumen of Rhizobium-induced infection threads in pea root nodules[END_REF][START_REF] Olsson | Rhizobium colonization induced changes in membrane-bound and soluble hydroxyproline-rich glycoprotein composition in pea[END_REF][START_REF] Tsyganova | Distribution of legume arabinogalactan proteins-extensin (AGPE) glycoproteins in symbiotically defective pea mutants with abnormal infection threads[END_REF]. It has been hypothesized that physical and biochemical properties of AGPEs may have an important influence on the progress of tissue and cell colonization by Rhizobium, probably by surrounding the bacteria in the infection thread or by regulating the growth of the infection thread itself [START_REF] Rathbun | Identification of a family of extensin-like glycoproteins in the lumen of Rhizobium-induced infection threads in pea root nodules[END_REF]. Interestingly, AGPE epitopes recognized by the mAb MAC265 have also been detected in a pathogenesis context. Indeed, these epitopes were shown to be more abundantly present in the cell wall of a pearl millet (Pennisetum glaucum) cultivar resistant to infection by the pathogenic oomycete Sclerospora graminicola, when compared to a susceptible cultivar [START_REF] Shailasree | Accumulation of hydroxyproline rich glycoproteins in pearl millet seedlings in response to Sclerospora graminicola infection[END_REF]. The authors proposed that these AGPEs may crosslink to each other and form a network which might provide anchorage for lignification and create a barrier impermeable to fungal hyphae. Interestingly, they also observed an increase in peroxidase and H 2 O 2 contents required for crosslinking (see also [START_REF] Kjellbom | Oxidative crosslinking of plasma membrane arabinogalactan proteins[END_REF] for oxidative crosslinking of AGPs).

AGPs were also found to play an active role during the formation of root actinorhizal nodules in alder (Alnus glutinosa)-Frankia symbiosis [START_REF] Berry | Arabinogalactan proteins are expressed at the symbiotic interface in root nodule of Alnus spp[END_REF]. Using immunocytochemistry coupled to electron microscopy, Berry et al. [START_REF] Berry | Arabinogalactan proteins are expressed at the symbiotic interface in root nodule of Alnus spp[END_REF] showed that AGP-associated epitopes were abundantly present in nodule-infected tissues. AGP epitopes recognized by the mAb JIM4 were found associated with pectic polysaccharides in the cell walls, whereas those recognized by JIM13 were abundantly found at the membrane-cell wall border along the symbiotic interface at the early

AGP1 AGP2 AGP3 AGP4 AGP5 AGP6 AGP7 AGP9 AGP10 AGP11 AGP12 AGP13 AGP14 AGP15 AGP16 AGP17 AGP18 AGP20 AGP21 AGP22 AGP23 AGP24 AGP25 AGP26 AGP27 AGP31

TRENDS in Plant Science

Figure 2. Relative level of expression of arabinogalactan protein (AGP) genes in different root tissues of Arabidopsis thaliana (modified from [START_REF] Nguema-Ona | Arabinogalactan proteins in root and pollen tubes: distribution and functional aspects[END_REF]). The figure was generated using Genevestigator [110]. Reproduced, with permission, from Annals of Botany ß Oxford Press.

infection stage [START_REF] Berry | Arabinogalactan proteins are expressed at the symbiotic interface in root nodule of Alnus spp[END_REF]. The authors proposed that the JIM13 antigen may function in directing Frankia growth during early infection, or that the antigen could participate in the formation of new plant cell walls at the interface that accompany the colonization of the host cells by Frankia. Interestingly, in mature-infected cells, JIM13-associated epitopes were found in the host cytoplasm and vacuole [START_REF] Berry | Arabinogalactan proteins are expressed at the symbiotic interface in root nodule of Alnus spp[END_REF].

Here, it was suggested that this location may indicate a turnover of this epitope after cell wall synthesis has ceased.

In symbiotic fungi-root associations between Medicago truncatula roots and symbiotic fungi of the genera Glomus, two independent studies have shown that the transcript of an AGP was particularly abundant in cells containing arbuscular mycorrhizae [START_REF] Van Buuren | Novel genes induced during an arbuscular mycorrhizal (AM) symbiosis formed between Medicago truncatula and Glomus versiforme[END_REF][START_REF] Schultz | Novel plant and fungal AGP like proteins in the Medicago truncatula-Glomus intraradices arbuscular mycorrhizal symbiosis[END_REF]. The authors speculated that this AGP may be a structural component of the interface compartment or, alternatively, it might be involved in mediating the interaction between the plant cortical cells and fungal hypha during arbuscule development [START_REF] Van Buuren | Novel genes induced during an arbuscular mycorrhizal (AM) symbiosis formed between Medicago truncatula and Glomus versiforme[END_REF]. It is also noteworthy that AGP-like proteins from Glomus intraradices were shown to be expressed during root colonization where they are believed to facilitate the formation of arbuscular mycorrhizae [START_REF] Schultz | Novel plant and fungal AGP like proteins in the Medicago truncatula-Glomus intraradices arbuscular mycorrhizal symbiosis[END_REF]. Interestingly, Nostoc spp., cyanobacteria species which also develop symbioses with plants, have been shown to contain several consensus domains defining AGP genes and to exhibit glycan epitopes associated with higher plant AGPs [START_REF] Jackson | Arabinogalactan-proteins occur in the freeliving cyanobacterium Nostoc and in plant-Nostoc symbioses[END_REF]. The role of these AGP-like proteins in symbiosis remains to be clearly established.

Apart from microbe-plant infection, it was also shown that AGPs were required for a successful infection of plant tissues, by plant parasites. For example, the holoparasite plant Cuscuta reflexa was shown to induce a localized (restricted to the infection sites) synthesis of an AGP, termed 'attachment AGP: attAGP' by the host, required for parasite attachment [START_REF] Albert | An attack of the plant parasite Cuscuta reflexa induces the expression of attAGP, an attachment protein of the host tomato[END_REF]. Using the RNA interference approach, a correlation between the level of expression of the attAGP and the force of attachment of the parasite to its host was observed: the lower the level of attAGP, the lower was the force of attachment of the parasite to its host [START_REF] Albert | An attack of the plant parasite Cuscuta reflexa induces the expression of attAGP, an attachment protein of the host tomato[END_REF].

Together, these studies link AGPs to the formation of infectious structures of either beneficial or pathogenic microbes, and of plant parasites. They also suggest that AGPs could be possible targets for strategies aiming at controlling root infections.

In addition to the above-mentioned roles at the interface of plants and microbes, it was proposed that AGPs may contribute to a signaling cascade responsible for the modulation of plant immune response [START_REF] Gaspar | Characterization of the Arabidopsis lysinerich arabinogalactan-protein AtAGP17 mutant (rat1) that results in a decreased efficiency of Agrobacterium transformation[END_REF]. Such a modulation of the plant immune system upon infection by soil microbes is well documented and contributes to the success or the failure of root infection and disease establishment [START_REF] Zamioudis | Modulation of host immunity by beneficial microbes[END_REF]. In a study on the rat1 Arabidopsis mutant, it was shown that, prior and after root infection by A. tumefaciens, the content of salicylic acid (SA) and pathogenesis-related proteins1 (PR-1) remains unchanged in the mutant roots [START_REF] Gaspar | Characterization of the Arabidopsis lysinerich arabinogalactan-protein AtAGP17 mutant (rat1) that results in a decreased efficiency of Agrobacterium transformation[END_REF]. By contrast, SA and PR-1 contents are reduced in the wild type roots upon infection and this reduction is likely to favor successful infections by A. tumefaciens. The authors proposed that AGPs were required for the modulation of the content of SA and PR1, thus allowing colonization of roots by A. tumefaciens. They have also suggested that certain structural features (likely to be glycans) of AtAGP17 may be responsible for such modulation [START_REF] Gaspar | Characterization of the Arabidopsis lysinerich arabinogalactan-protein AtAGP17 mutant (rat1) that results in a decreased efficiency of Agrobacterium transformation[END_REF]. The presence of a GPI anchor would allow AtAGP17 to interact with wall kinases such as WAKs located in the plasma membrane [START_REF] Gens | Arabinogalactan protein and wall-associated kinases in a plasmalemmal reticulum with specialized vertices[END_REF], or to be cleaved by specific phospholipases and released as a soluble-signaling molecule [START_REF] Schultz | GPI-anchors on arabinogalactan proteins: implications for signalling in plants[END_REF]. The role of AGPs as soluble-signaling molecules has previously been demonstrated in several studies: xylogen, for example, is a diffusible, high molecular weight AGP, able to induce differentiation of Zinnia (Zinnia elegans L.) mesophyll cells into tracheary elements [START_REF] Motose | A proteoglycan mediates inductive interaction during plant vascular development[END_REF].

AGPs secreted by root cap cells and BCs/BLCs: a role in plant protection

AGPs are also synthesized by root cap cells and root capderived BCs and BLCs. BCs and BLCs are released within the rhizosphere and are required for the survival and protection of the root in the soil [START_REF] Hawes | Function of root border cells in plant health: pioneers in the rhizosphere[END_REF][START_REF] Hawes | The role of border cells in plant defense[END_REF][START_REF] Vicre | Root border like cells of Arabidopsis. Microscopical characterization and role in the interaction with Rhizobacteria[END_REF][START_REF] Driouich | Unity is strength: the power of border cells and border like cells in relation with plant defense[END_REF][47][48]. AGPs are highly expressed at the cell surface of BCs and BLCs [START_REF] Vicre | Root border like cells of Arabidopsis. Microscopical characterization and role in the interaction with Rhizobacteria[END_REF][START_REF] Durand | The organization pattern of root border like cells of Arabidopsis is dependent on cell wall homogalacturonan[END_REF][START_REF] Cannesan | Effect of arabinogalactan proteins from the root caps of pea and Brassica napus on Aphanomyces euteiches zoospore chemotaxis and germination[END_REF], but are also abundantly secreted into the rhizosphere by the same cells, as components of the polysaccharide-rich mucilage [START_REF] Vicre | Root border like cells of Arabidopsis. Microscopical characterization and role in the interaction with Rhizobacteria[END_REF][START_REF] Durand | The organization pattern of root border like cells of Arabidopsis is dependent on cell wall homogalacturonan[END_REF][START_REF] Xie | A plant arabinogalactan-like glycoprotein promotes a novel type of polar surface attachment by Rhizobium leguminosarum[END_REF][START_REF] Ma | The mucilage proteome of maize (Zea mays L.) primary roots[END_REF]]. An interesting study has shown that an Arabidopsis mutant unable to form root BLCs released BCs [START_REF] Durand | The organization pattern of root border like cells of Arabidopsis is dependent on cell wall homogalacturonan[END_REF]. Surprisingly, this unexpected release of BCs, instead of BLCs, was accompanied by a secretion of a thick layer of mucilage termed 'BC biofilm' [START_REF] Driouich | Border cells versus border-like cells: are they alike?[END_REF], mostly consisting of AGPs and pectic xylogalacturonan (XGA). Secreted XGA and AGPs were both proposed to contribute to root cap protection: XGA is described as highly resistant to degradation by microbial pectin-hydrolyzing enzymes [START_REF] Jensen | Identification of a xylogalacturonan xylosyltransferase involved in pectin biosynthesis in Arabidopsis[END_REF], whereas AGPs would help hold the cells together like a 'glue', thus allowing them to remain close to the root tip to ensure its protection [START_REF] Durand | The organization pattern of root border like cells of Arabidopsis is dependent on cell wall homogalacturonan[END_REF][START_REF] Driouich | Unity is strength: the power of border cells and border like cells in relation with plant defense[END_REF][START_REF] Driouich | Border cells versus border-like cells: are they alike?[END_REF].

The importance of root cap cells and BCs/BLCs in recognition and attraction of beneficial soil microbes has been described previously [START_REF] Vicre | Root border like cells of Arabidopsis. Microscopical characterization and role in the interaction with Rhizobacteria[END_REF][START_REF] Gaspar | Characterization of the Arabidopsis lysinerich arabinogalactan-protein AtAGP17 mutant (rat1) that results in a decreased efficiency of Agrobacterium transformation[END_REF][START_REF] Xie | A plant arabinogalactan-like glycoprotein promotes a novel type of polar surface attachment by Rhizobium leguminosarum[END_REF]. These studies suggest that AGPs secreted by root cap cells and BCs/BLCs are required for successful infection of roots by beneficial microbes. By contrast, attraction of pathogenic microbes by AGPs seems to be a strategy of entrapment of the pathogen, followed by their subsequent neutralization [START_REF] Cannesan | Effect of arabinogalactan proteins from the root caps of pea and Brassica napus on Aphanomyces euteiches zoospore chemotaxis and germination[END_REF]. Using an in vitro assay, it has been shown that AGPs synthesized by pea root cap cells and BCs were able to inhibit the development of the pathogenic oomycete Aphanomyces euteiches, thus providing, to the best of our knowledge, the first report of antimicrobial properties of AGPs [START_REF] Cannesan | Effect of arabinogalactan proteins from the root caps of pea and Brassica napus on Aphanomyces euteiches zoospore chemotaxis and germination[END_REF]. Infection by oomycetes involves zoospore attraction by chemotaxis, followed by encystment, and subsequent cyst germination [START_REF] Tyler | Molecular basis of recognition between Phytophtora pathogens and their hosts[END_REF]. A purified AGP fraction extracted from pea root cap cells and BCs has been found effective in attracting, by chemotaxis, A. euteiches zoospores [START_REF] Cannesan | Effect of arabinogalactan proteins from the root caps of pea and Brassica napus on Aphanomyces euteiches zoospore chemotaxis and germination[END_REF]. Interestingly, the purified AGP fraction also provoked zoospore encystment (immobilization of deflagellated zoospores or cysts). Therefore, it is probable that AGPs may contribute to protection against root infection by immobilizing zoospores at the periphery of the tip or in the surrounding environment. It has been reported that many antimicrobial compounds and extracellular DNA are secreted by root cap cells and BCs into the so-called 'extracellular traps' that neutralize the immobilized pathogen much like the ones formed by human neutrophil cells [START_REF] Hawes | The role of border cells in plant defense[END_REF]47]. AGPs have been proposed to be part of the trap complex [START_REF] Driouich | Unity is strength: the power of border cells and border like cells in relation with plant defense[END_REF]. Finally, the purified AGP fraction was shown to significantly reduce cyst germination and hyphal proliferation [START_REF] Cannesan | Effect of arabinogalactan proteins from the root caps of pea and Brassica napus on Aphanomyces euteiches zoospore chemotaxis and germination[END_REF]. The precise mode of action of AGPs on A. euteiches development is unknown. However, it is noteworthy that certain AGPs harbor a nonspecific lipid transfer protein domain (ns-LTPlike AGPs) [START_REF] Kobayashi | Expression and genome-wide analysis of the xylogen-type gene family[END_REF], which may physically target microbe membranes and inhibit microbe development. It has been shown that ns-LTP proteins, which are classified as pathogenesis-related proteins 13 [START_REF] Van Loon | The families of pathogenesis-related proteins, their activities, and comparative analysis of PR-1 type proteins[END_REF], were able to exhibit cytotoxic and membrane permeabilization properties towards bacterial and fungal plant pathogens [55][START_REF] Garcia-Olmedo | The defensive role of nonspecific lipidtransfer proteins in plants[END_REF][START_REF] Molina | Lipid transfer proteins (nsLTPs) from barley and maize leaves are potent inhibitors of bacterial and fungal plant pathogens[END_REF]. That ns-LTP-like AGPs have the same effects on A. euteiches as pea root AGPs remains to be proven. Similarly, it is not known whether the Yariv-precipitated AGPs in the pea root cap study [START_REF] Cannesan | Effect of arabinogalactan proteins from the root caps of pea and Brassica napus on Aphanomyces euteiches zoospore chemotaxis and germination[END_REF] contain LTP-like proteins.

AGPs secreted into the rhizosphere: role in communication with soil microbes? Along with organic acids, secondary metabolites, and proteins, AGP-containing mucilages are abundantly secreted into the rhizosphere by root tips. Organic acids [START_REF] Rudrappa | Root-secreted malic acid recruits beneficial soil bacteria[END_REF], secondary metabolites [START_REF] Neal | Benzoxazinoids in roots exudates of maize attract Pseudomonas putida to the rhizosphere[END_REF], and proteins [60,61] are known to play a role in microbial cooperation in the rhizosphere [START_REF] Berendsen | The rhizosphere microbiome and plant health[END_REF][START_REF] Badri | Rhizosphere chemical dialogues: plantmicrobe interactions[END_REF][START_REF] Bais | The role of root exudates in rhizosphere interactions with plants and other organisms[END_REF][START_REF] Barea | Microbial co-operation in the rhizosphere[END_REF], but how AGPs affect the rhizosphere microbiome has been neglected. Evidence for AGP occurrence in root exudates was initially provided by two studies [START_REF] Bacic | Structural analysis of secreted root slime from maize (Zea mays L.)[END_REF][START_REF] Moody | Structural analysis of secreted slime from wheat and cowpea roots[END_REF], when chemical structures typical of AGPs were found in maize (Zea mays), wheat (Triticum aestivum), and cowpea (Vigna unguiculata) mucilage. AGP occurrence in root exudates was later reported in several other species including pea [START_REF] Xie | A plant arabinogalactan-like glycoprotein promotes a novel type of polar surface attachment by Rhizobium leguminosarum[END_REF][START_REF] Knee | Root mucilage from pea and its utilization by rhizosphere bacteria as a sole carbon source[END_REF], soybean (Glycine max L.) [START_REF] Timotiwu | Identification of mono-, oligo-, and polysaccharides secreted from soybean roots[END_REF], Arabidopsis [START_REF] Vicre | Root border like cells of Arabidopsis. Microscopical characterization and role in the interaction with Rhizobacteria[END_REF][START_REF] Durand | The organization pattern of root border like cells of Arabidopsis is dependent on cell wall homogalacturonan[END_REF], and maize [START_REF] Ma | The mucilage proteome of maize (Zea mays L.) primary roots[END_REF]. AGP-containing root exudates were frequently proposed to serve as lubricants protecting the root tip as it pushes through the soil [70-72], to stabilize soil aggregates [START_REF] Gregory | Plant Roots: Their Growth, Activity, and Interactions with Soils[END_REF]73], and protect root tips against toxicity of aluminum and other heavy metals [START_REF] Gregory | Plant Roots: Their Growth, Activity, and Interactions with Soils[END_REF][START_REF] Mensch | Metal binding properties of high molecular weight soluble exudates from maize (Zea mays L.) roots[END_REF]. AGP-rich mucilage also facilitates water retention in the rhizosphere [START_REF] Carminati | A model of root water uptake coupled with rhizosphere dynamics[END_REF] and may indirectly contribute to the attraction of living soil microbes towards a water-rich and carbon-rich microenvironment. Chemotaxis, for example, is a phenomenon that is impacted by AGPs [START_REF] Cannesan | Effect of arabinogalactan proteins from the root caps of pea and Brassica napus on Aphanomyces euteiches zoospore chemotaxis and germination[END_REF].

In the rhizosphere, the microbiome consists of commensal, pathogenic, and beneficial microbes [START_REF] Berendsen | The rhizosphere microbiome and plant health[END_REF][START_REF] Barea | Microbial co-operation in the rhizosphere[END_REF]. To colonize a given rhizosphere, microbes have to, among other things, be able to use available nutrients. Interestingly, it was shown that many soil microbes including the biocontrol agents Trichoderma viride [START_REF] Kotake | Molecular cloning and expression in Escherichia coli of a Trichoderma viride endo-b-1,6-galactanase gene[END_REF] and Streptomyces avermitilis [START_REF] Yuan | Characterization of Streptomyces lydicus WYEC108 as a potential biocontrol agent against fungal root and seed rots[END_REF][START_REF] Ichinose | Characterization of an endo-b-1,6galactanase from Streptomyces avermitilis NBRC14893[END_REF][START_REF] Ling | An exo-b-1,3-galactanase from Streptomyces sp. provides insights into type II arabinogalactan structure[END_REF], the soil-borne pathogenic fungus Fusarium oxysporum [START_REF] Sakamato | Characterization of Fusarium oxysporum b-1,6-galactanase, an enzyme that hydrolyses larch wood arabinogalactan[END_REF], as well as many other microbes (Aspergillus niger [START_REF] Haque | Mode of action of b-glucuronidase from Aspergillus niger on the sugar chains of arabinogalactan proteins[END_REF] and Neurospora crassa [START_REF] Takata | Degradation of carbohydrate moieties of arabinogalactan proteins by glycoside hydrolases from Neurospora crassa[END_REF]) all produce AGP glycan-degrading enzymes (e.g., b-1,3-galactanases, b-1,6-galactanases, arabinofuranosidases, b-glucuronidases). Furthermore, a set of experiments showed that rhizobacteria were able to grow on an AGP-rich mucilage, suggesting their ability to hydrolyze and metabolize AGP-derived sugars for their growth in the rhizosphere [START_REF] Knee | Root mucilage from pea and its utilization by rhizosphere bacteria as a sole carbon source[END_REF]. In support of this is a study [START_REF] Benizri | Additions of maize root mucilage to soil changed the structure of the bacterial community[END_REF] that showed that maize root mucilage (particularly enriched in AGPs) [START_REF] Ma | The mucilage proteome of maize (Zea mays L.) primary roots[END_REF][START_REF] Bacic | Structural analysis of secreted root slime from maize (Zea mays L.)[END_REF] was able to influence the composition of bacterial communities in soil. Consequently, root AGPs seem to significantly contribute to the shaping of the rhizosphere microbiome. It is possible that AGPs would select for specific antagonist microbes to help the root prosper within the rhizosphere. Microbial interactions occurring in such a microenvironment have been extensively reviewed [START_REF] Berendsen | The rhizosphere microbiome and plant health[END_REF][START_REF] Bais | The role of root exudates in rhizosphere interactions with plants and other organisms[END_REF][START_REF] Barea | Microbial co-operation in the rhizosphere[END_REF][START_REF] Raaijmakers | The rhizosphere: a playground and battlefield for soilborne pathogens and beneficial microorganisms[END_REF]. Different types of antagonism behavior including mycoparasitism, antibiosis, and competition can develop between inhabitants and lead to soil suppressiveness towards a disease caused by a specific soil-borne plant pathogen [START_REF] Hiddink | Effect of mixed and single crops on disease suppressiveness of soils[END_REF]. How root-secreted AGPs of a given plant species would affect the establishment of beneficial microbes (e.g., plant growth promoting bacteria/fungi) within a rhizosphere is an interesting issue to unravel.

Also the susceptibility of AGPs to degradation by microbial depolymerases may result in the production of AGPderived oligosaccharides. Given the diversity of AGP glycans in plants, a high diversity of AGP-derived oligosaccharide structures can be generated from secreted AGPs, and may have interesting biological functions. The ability of cell wall-derived oligosaccharides to modulate or activate plant defense mechanisms has been studied for decades and was demonstrated for plant-derived as well as for fungal-derived cell wall degradation products (the oligosaccharin theory) [START_REF] Albersheim | Oligosaccharins: oligosaccharide regulatory molecules[END_REF]87]. It was shown, for example, that upon plant infection by pathogenic microbes, endopolygalacturonase-mediated degradation of pectin homogalacturonans yielded oligogalacturonans, which were found to activate plant defense mechanisms [START_REF] Cervone | Host-pathogen interactions. XXXIII. A plant protein converts a fungal pathogenesis factor into an elicitor of plant defense responses[END_REF][START_REF] Vorwerk | The role of plant cell wall polysaccharide composition in disease resistance[END_REF]. Recently, a hybrid kinase consisting of the extracellular domain of WALL-ASSOCIATED KINASE1 and the intracellular domain of the Elongation Factor Tu-receptor kinase was shown to bind oligogalacturonans and activate defense responses [START_REF] Brutus | A domain swap approach reveals a role of the plant wall-associated kinase 1 (WAK1) as a receptor of oligogalacturonides[END_REF]. It is therefore tempting to speculate on the ability of AGP-derived oligosaccharides to act as elicitors [START_REF] Seifert | The biology of arabinogalactan proteins[END_REF]. Plant cell wall-degrading enzymes of unknown functions, including b-galactosidases, were found to be released into the rhizosphere by BCs/BLCs [START_REF] Wen | Extracellular proteins in pea root tip and border cell exudates[END_REF][START_REF] Kotake | Molecular cloning of a b-galactosidase from radish that specifically hydrolyze b-1,3 and b-1,6 galactosyl residues of arabinogalactan protein[END_REF]. That such AGP-derived fragments would act as damage-associated molecular patterns (DAMPs) [START_REF] Boller | A renaissance of elicitors: perception of microbe-associated molecular patterns and danger signals by pattern recognition receptors[END_REF] with the ability to modulate the plant immune system is plausible but remains to be demonstrated.

Concluding remarks and future outlook

It is clear that AGPs are abundantly synthesized by root cells and secreted into the rhizosphere. However, current understanding of AGP function in PMI is limited. Studies discussed in this review have clearly shown that AGPs play important roles in mediating many root cell-microbe interactions. First, AGPs are involved in attracting and initiating root tip colonization by beneficial microbes. They were also found expressed at the interface of infectious structures that are formed between various beneficial microbes and root cells, and which allow the exchange of nutrients between the root and its symbiont. At these physical interfaces, they are likely to be important as structural components and/or signaling molecules. Interestingly, in a pathogenesis context, they are also likely to set the scene for mounting an efficient and localized defense response. Based on recent finding of their antimicrobial properties, AGPs are directly involved in controlling some pathogenic microbes. Purified AGPs from pea root cap cells and BCs were shown to effectively inhibit the development of a devastative pea pathogenic microbe [START_REF] Cannesan | Effect of arabinogalactan proteins from the root caps of pea and Brassica napus on Aphanomyces euteiches zoospore chemotaxis and germination[END_REF]. However, many questions remain to be answered: what are the mechanisms involved? Can root AGPs perform in a similar manner with other soil-borne pathogens? Are AGPs from a given species (e.g., pea root AGPs) only effective on their, or some of their, natural microbial enemies (A. euteiches for pea)? A more general point to unravel is the promising role AGPs may play in the dialog between roots and soil microbes. Evidence points towards their involvement in this dialog as supporting players, or facilitators, of colonization of the rhizosphere by specific groups of microbes [START_REF] Vicre | Root border like cells of Arabidopsis. Microscopical characterization and role in the interaction with Rhizobacteria[END_REF][START_REF] Cannesan | Effect of arabinogalactan proteins from the root caps of pea and Brassica napus on Aphanomyces euteiches zoospore chemotaxis and germination[END_REF][START_REF] Knee | Root mucilage from pea and its utilization by rhizosphere bacteria as a sole carbon source[END_REF][START_REF] Benizri | Additions of maize root mucilage to soil changed the structure of the bacterial community[END_REF]. Additional research is needed to further understand how AGPs precisely inhibit or stimulate members of the microbial community. A recent review has proposed a model summarizing the many interactions occurring between roots and microbes in the rhizosphere [START_REF] Berendsen | The rhizosphere microbiome and plant health[END_REF], and it is tempting to include AGPs, or AGPderived oligosaccharides and/or glycopeptides, in such a model. Here, we highlight in Figure 3 the possible roles played by AGPs in such processes. However, the precise modes of action of AGPs in some of these possible scenarios remain to be investigated, as these glycoproteins harbor complex chemical structures, and for most of the possible scenarios mentioned above it is unknown whether all AGP populations, specific families, or specific sequences (oligosaccharides or glycopeptides) are responsible for biological activity. An interesting study [START_REF] Van Hengel | N-Acetylglucosamine and glucosaminecontaining arabinogalactan proteins control somatic embryogenesis[END_REF] showed that a given AGP can contain in its structure (the chitinase-sensitive motif) the ability to inhibit carrot somatic embryogenesis, whereas other motifs (i.e., glycopeptides remaining after endochitinase treatment and repurified using Yariv reagent) yielded the opposite result (reviewed in [START_REF] Seifert | The biology of arabinogalactan proteins[END_REF]). This supports that AGPs are able to play dual roles in different processes including interactions of roots with microbes.

Clearly, interactions between roots and microbes are crucial for plant health and AGPs play a significant role in such interactions. Basic and strategic studies, as well as the development of novel tools, should help not only to further understand the function of AGPs within the rhizosphere but also to facilitate their use, or the use of molecules derived from them (or modeled on them), as natural compounds for crop protection in a sustainable manner.

Figure 1 .

 1 Figure1. Structure of arabinogalactan proteins (AGPs). (A) AGPs are heavily glycosylated cell wall proteins and their glycans predominantly consist of arabinose and galactose. Minor sugars, such as glucuronic acid or rhamnose, are also present. The backbone of the protein is enriched in hydroxyproline residues. AGPs can be anchored to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. AGP glycan structures were adapted from[START_REF] Tan | Structure of a hydroxyproline (Hyp)arabinogalactan polysaccharide from repetitive Ala-Hyp expressed in transgenic Nicotiana tabacum[END_REF][START_REF] Tryfona | Carbohydrate structural analysis of wheat flour arabinogalactan protein[END_REF][START_REF] Qi | Gum arabic glycoprotein is a twisted hairy rope[END_REF]. Note the high heterogeneity in the structure of the glycan chains. (B) AGP backbones are synthesized by members of a large multigene family and are classified into classical AGPs, hybrid AGPs, chimeric AGPs, and AG peptides (short classical AGPs)[START_REF] Schultz | The classical arabinogalactan protein gene family of Arabidopsis[END_REF][START_REF] Ma | Genome-wide identification, classification, and expression analysis of the arabinogalactan protein gene family in rice (Oryza sativa L.)[END_REF]. Classical AGPs are characterized by a signal peptide domain, a P/Hyp-rich domain, and a C-ter domain. Hybrid AGPs often consist of classical AGPs which may contain FLA, Lys-rich, nonspecific lipid transfer protein (ns-LTP), ENOD domains, or domains of unknown functions (DUFs) within their sequence. These domains can be interspersed within the sequence. Chimeric AGPs often lack the C-ter domain responsible for GPI anchorage. For recent reviews describing the chemistry of AGPs in general, see[START_REF] Nguema-Ona | Arabinogalactan proteins in root and pollen tubes: distribution and functional aspects[END_REF][START_REF] Ellis | Arabinogalactan proteins: key regulators at the cell surface[END_REF].

+

Figure 3 .

 3 Figure 3. Model summarizing possible roles for arabinogalactan proteins (AGPs) during root cell interaction with microbes. (A) AGPs are able to attract symbiotic microbes (bacteria and fungi) that will later infect roots and develop infection structures. They are abundantly found at the physical interface of root cells and microbe infectious structures where they might control the formation of these structures and promote microbe adherence and progression into the root. AGPs are also able to repel root pathogens or to inhibit their development. (B) Soil microbes are able to degrade root AGPs, potentially releasing oligosaccharides and/or glycopeptides as damageassociated molecular patterns (DAMPs) that would activate plant defense mechanisms (red arrows). (C) Secreted AGPs may also favor the colonization of the rhizosphere by beneficial microbes (i.e., plant growth promoting rhizobia/fungi, PGPR/PGPF; biocontrol agent, BCA) which are able either to activate plant defense responses such as induced systemic resistance (ISR, [45]; blue arrows) or to antagonize pathogenic microbes and suppress a disease. Different microbes inhabiting the rhizosphere (i.e., symbiotic, commensal, and pathogenic microbes) may interact with each other. AGPs can also modulate the plant immune system to favor root colonization by soil microbes. Purple arrows indicate possible induction and/or modulation of defense mechanisms in the aerial parts of the plant, as a consequence of either the activation of defense mechanisms by AGP degradation products (B) or by beneficial microbes (PGPF, PGPR, or BCA) (C). (À) repression of plant immune system (A); (+) activation of plant immune system (B, C).

Table 1 .

 1 Distribution of arabinogalactan proteins in roots of a range of plant species a

	Species	Root cell type	Use of antibodies/lectins Use of	Analytical chemistry Molecular	Refs
						genetic
			Immunohistochemistry/	Yariv reagent		(mutant)
			blotting
	Arabidopsis thaliana Root cap and BLCs	JIM13, JIM14, MAC207	Root treatment			[6]
	A. thaliana	Root cap and BLCs	JIM13				[7]
	A. thaliana	Epidermal cells	JIM13, JIM14	Root treatment			[98]
	A. thaliana	Epidermal cells	LM2, JIM14				[99]
	A. thaliana	Epidermal cells		Root treatment		AtAGP17	[24]
	A. thaliana	Epidermal cells		Root treatment and			[100]
				electrophoresis
				techniques
	A. thaliana	Differentiating cells	Eel anti-H agglutinin	Electrophoresis	Sugar composition		[101]
				techniques
	A. thaliana	Elongating cells	JIM8, MAC207, JIM16		Sugar composition	AtAGP30	[21]
	A. thaliana	Epidermal, cortical				AtFLA4/SOS5 [102]
		and endodermal cells
	A. thaliana	Young xylem cells	JIM13, JIM14				[11]
	Pisum sativum	Root cap and BCs	JIM13, JIM14, JIM8	Electrophoresis	Sugar composition		[8]
				techniques	and glycosidic
					linkage analyses
	P. sativum	Root mucilage	JIM13		Sugar composition		[25]
	P. sativum	Root cells b and root	JIM8, MAC207, MAC265				[31]
		infection structure
	P. sativum	Root mucilage			Sugar composition		[68]
					and glycosidic l
					inkage analyses
	Zea mays	Root mucilage			Sugar composition		[66]
					and glycosidic
					linkage analyses
	Z. mays	Root mucilage			Proteomic analysis		[49]
	Z. mays	Root epidermal cells	LM2				[103]
		and mucilage
	Daucus carota	Root pericycle cells	MAC207, JIM4				[10]
	D. carota	Root pericycle cells	JIM4				[104]
	D. carota	Root apical meristem	MAC207, JIM4, JIM15,				[105]
			JIM8, JIM14, JIM16
	Raphanus sativus	Root tip/cap cells and BLCs Gal4-BSA				[106]
	R. sativus	Primary/mature roots b			Sugar composition		[107]
					and glycosidic
					linkage analyses
	Brassica napus	Root cap and BLCs	JIM13, JIM14, JIM8	Electrophoresis	Sugar composition		[8]
				techniques	and glycosidic
					linkage analyses
	Benincasa hispida	Epidermal cells	LM2, JIM14, JIM16,				[108]
			JIM15, JIM17, JIM101,
			MAC265, MAC266
	Triticum spp.	Root mucilage			Sugar composition		[67]
					and glycosidic l
					inkage analyses
	Vigna unguiculata	Root mucilage			Sugar composition		[67]
					and glycosidic
					linkage analyses
	Alnus spp.	Cortical cells and root	JIM13, JIM4				[26]
		infection structure
	Oryza sativa	Root apex b	JIM8				[109]

a Different approaches have been used to study AGP distribution, including analytical chemistry, immuno-based methods, electrophoretic techniques, bioinformatics, and molecular tools.

b

Acknowledgments

We are grateful to La Re ´gion de Haute Normandie and le Grand Re ´seau de Recherche-Ve ´ge ´tal, Agronomie, Sol et Innovation, l'Universite ´de Rouen, Le Fonds Europe ´en FEDER, and l'ANR for financial support. A.D. is grateful to all former students who have participated in different 'AGP biology' projects over the years, in his laboratory.