Stéphane Verdier
email: stephaneverdier@hotmail.com

Nicole K Van Der Laak

Francis Dalard

James B Metson

Stéphane Delalande

An electrochemical and SEM study of the mechanism of formation, morphology, and composition of titanium or zirconium fluoride-based coatings

Keywords: Zirconium/titanium coatings, Magnesium alloy, SEM, Electrochemistry

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

Introduction

The need for car manufacturers to improve the overall energy efficiency of automobiles has led to the use light alloys to minimize the vehicle weight and to reduce exhaust gas emissions. Aluminium and magnesium alloys are the most widely promising materials for car body panels' weight reduction. Despite their good mechanical properties such as a high strength to weight ratio, magnesium alloys have found limited application due largely to poor corrosion resistance. The corrosion resistance of magnesium is significantly improved by alloying, in particular with aluminium and manganese or zinc [START_REF] Ghali | Uhlig's Corrosion Handbook[END_REF] and limiting the content of impurities [START_REF] Hanawalt | [END_REF]. However, such alloys must undergo surface treatment and organic coating before being incorporated in car body panels. The surface treatment aims to increase corrosion resistance and/or improve organic coating adhesion. Chromate conversion coatings typically meet these two requirements for magnesium alloy substrates. However, treatments based on hexavalent chromium are no longer acceptable due to toxicity problems.

Thus, it is necessary to develop chrome-free surface treatments before using magnesium alloys for structural applications. There are a number of alternatives to hexavalent chromium for protecting magnesium and its alloys [3]. One of these alternatives is surface treatment based on group IV-A fluorides [4]. Hexafluoro-zirconic-or hexafluoro-titanic-based treatments have been developed for aluminium alloys [5][START_REF] Hfrnstrfm | Proc. of Eurocorr[END_REF][START_REF] Schram | [END_REF][8][9][START_REF] Delalande | Proc. of The 2nd Aluminium Surface Science and Technology International Symposium[END_REF][START_REF] Lunder | Proc. of the 3rd Aluminium Surface Science and Technology International Symposium[END_REF][START_REF] Lunder | Proc. of the 15th Internat. Corrosion Congress[END_REF][START_REF] Nordlien | [END_REF] and for galvanized or galfanR coated steel [14,[START_REF] Puomi | Jyrk7s[END_REF]. On the former alloys, they lead to the formation of a thin layer that is a mixture of aluminium oxyfluoride, titanium dioxide, and zirconium oxyfluoride. However, little is know about the formation of such coatings on magnesium alloys [START_REF] Gehmecker | Proc. of The 51st World Conference of Magnesium[END_REF][START_REF] Skar | [END_REF][START_REF] Guerci | Proc. of Magnesium Alloys and Their Applications[END_REF]. Moreover, all of these studies have used commercial zirconium or titanium solutions, the exact compositions of which were not given.

In a previous work [START_REF] Verdier | [END_REF], we have studied the electrochemical behaviour and corresponding surface chemistry of AM60 magnesium alloy in fluoride solutions by XPS. The most aggressive solutions were the most concentrated ones, and the pH had no visible influence on AM60 electrochemical behaviour. The resulting film was a mixture of magnesium hydroxide and a magnesium hydroxyfluoride, approaching MgF 2 with increasing fluoration, i.e., for the most fluoride concentrated and acid solutions. We also analysed the surface chemistry of films formed on AM60 magnesium alloy after zirconium or titanium fluoride treatments by means of XPS [START_REF] Delalande | ECASIA'03 International Congress[END_REF][START_REF] Verdier | [END_REF]. The experiments showed that it is possible to form a film containing zirconium or titanium at the surface of this alloy. Zirconium is always present in its dioxide form ZrO 2 , and some hydroxy-oxide or hydroxyfluoride may also be present depending on solution composition used. Titanium was under only one chemical state, the dioxide TiO 2 . A mechanism of formation was proposed, suggesting that the zirconium or titanium complexes in solution precipitate due to a local increase of pH taking place since the reduction of water occurs at the surface of AM60.

To complete these previous works, we present here an in situ study of the electrochemical behaviour of AM60 magnesium alloy in Zr/Ti treatment solutions followed by the analysis of the resulting film morphology and composition by Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray spectroscopy (EDX).

Experimental

Samples

The magnesium alloy AM60 used contains 6% Al and 0.27% Mn by weight. Prior to the experiments, the samples were polished with SiC 1200 grit paper for electrochemical measurements and up to 4000 grit paper for surface analyses, rinsed with distilled water, cleaned in ethanol in an ultrasonic bath, and dried in a warm-pulsed air stream. When needed, the alloy microstructure was revealed by etching 20 s in a solution containing 20 mL acetic acid, 1 mL concentrated nitric acid, 60 mL ethylene glycol, and 20 mL deionised water.

Solutions

The surface treatments were performed in a 100 ml polyethylene vessel at room temperature. Solutions were prepared using hexafluoro-zirconic acid H 2 ZrF 6 and hexafluoro-titanic acid H 2 TiF 6 solutions supplied by Aldrich. In some cases, fluoride was added using potassium fluoride, KF (Acros), or hydrofluoric acid, HF (Aldrich). Potassium hydroxide, KOH (Acros), was added when an increase in pH was needed. In each case, the sample was immersed for 1 min and then rinsed with deionised water and dried in warm-pulsed air. All chemicals are reagent grade.

Electrochemical setup

The specimen was placed in a TeflonR and KELFR based device, leaving exposed an active surface 8 mm in diameter. This sample was placed in a Rotating Disk Electrode, with a rate of 1000 rpm. The auxiliary electrode was a large surface area platinum disk, and the reference electrode was a Saturated Calomel Electrode (SCE). Electrochemical experiments were carried out at constant temperature, using a 1260 Solartron potentiostat. This instrument was piloted by a computer and Scribner Corware/Corview electrochemistry software.

The rest potential E cor was monitored for 1 min, trying to begin the potential acquisition simultaneously with the electrode immersion. After this hold time, three cyclic voltammograms were plotted consecutively from E cor to +500 mV/E cor , reverse scan to À500 mV/E cor and return to E cor . The scan rate was 10 mV s À1 .

Surface analyses

Scanning Electron Microscopy was performed with a LEO Stereoscan 440 microscope equipped with an EDAX analysis apparatus. Unless otherwise specified, the primary electron beam acceleration was 20 kV. The electron microprobe analysis was performed on a Cameca Microson SU30 coupled with WDX detectors. The accelerating voltage was set at 8 keV, which gives a comparable analysis depth to that of the SEM/EDX analysis.

Results

In situ electrochemical behaviour

Table 1 shows the composition of the zirconium or titanium fluoride solutions used. The pH value was 1.9 10 À2 1.8 10 À1

1.1 10 À1 0.8 determined using pH paper because a conventional glass electrode dissolves in the acidic fluoride solutions. The pH values given below have thus to be considered with a F0.2 confidence interval.

Zirconium treatments

The rest potential showed two possible evolutions (Fig. 1). In the first, i.e., in the 10 À1 M H 2 ZrF 6 solution, the potential slightly decreased during 1 min, near À1600 mV/ SCE. In all the others solutions, the potential decreased about 50 mV during the first 2-5 s following immersion and then increased slightly from 10 to 150 mV to reach À1710 to À2000 mV/SCE after 1 min.

The comparison with the corrosion potential vs. time curves for AM60 immersed in various fluoride-based solutions [START_REF] Verdier | [END_REF] showed some similarities. For fluoride solutions, only the 10 À1 M HF solution (pH 1.9) gave a potential evolution comparable to the 10 À1 M H 2 ZrF 6 . This particular evolution is only obtained for the most acid-and fluoride-concentrated solutions and is probably related to an especially high dissolution rate of the magnesium alloy under these conditions. The two step corrosion potential evolution in most of the solutions can be related to the occurrence of a dissolution step (potential decrease during the first 2-5 s) followed by the formation of a more protective film.

The voltammograms (Fig. 2) remained the same from one scan to the next, even in the few five scan experiments performed. This indicates that there is no major evolution of the electrochemical behaviour after the 1-min hold time. Moreover, no current plateau could be observed, even for 1 V anodic or cathodic overvoltage vs. corrosion potential. The current densities increased with increasing H 2 ZrF 6 concentrations. This dependence of electrochemical behaviour of AM60 with H 2 ZrF 6 concentration is pointed out in Fig. 3. The polarization resistance R p =(dE/dj) j=0 was determined, and a linear increase in Log 10 R p of about 1 per unit pZr, with pZr=ÀLog 10 [H 2 ZrF 6 ] was observed. The most concentrated solutions are thus the most aggressive with respect to the AM60 alloy.

Titanium treatments

Fig. 4 shows the evolution of corrosion potential during 1 min in 10 À4 to 10 À1 M H 2 TiF 6 solutions. For concen- trations below 10 À1 M H 2 TiF 6 , the corrosion potential increased quickly during 5 to 10 s then at a lesser rate to reach a stable value between À1700 and À1950 mV/SCE. For the most concentrated solution, the potential decreased about 5 mV during the 10 first seconds and reached a stable value near À1420 mV/SCE. The potential drop observed in the 2-5 first seconds for zirconium-based solution was not observed in the titanium solutions. As for the zirconiumbased solutions, no time dependence of the voltammograms was observed. The current densities were found to be dependent on the fluorotitanic acid concentration, as shown in the plot of polarization resistance vs. pTi (Fig. 5). A linear increase in Log 10 R p vs. pTi is observed, as for zirconiumbased solutions, but with a lower slope (0.74). The polarization resistance of concentrated (10 À1 M) solutions of either titanic or zirconic acid are near, but for more dilute solutions, titanic ones gave smaller R p values. The most concentrated solutions are the most aggressive with respect to the substrate.

Film morphology and composition

3.2.1. Zirconium treatments 3.2.1.1. Surface morphology and composition. A film containing zirconium has been formed in each solution except 10 À3 M H 2 ZrF 6 (Fig. 6). The film consists of a thin layer (less than 1 Am thick) with spheres of 5-7 Am diameter upon it. The films did not cover the entire surface. As seen in the highest magnifications (Fig. 7), the 10 À1 M H 2 ZrF 6treated surface was very rough, as expected in light of the high aggressiveness displayed in the electrochemical analysis. The underlayer became smoother, more compact, and contained more white particles of size around 1 Am when decreasing the H 2 ZrF 6 concentration from 10 À1 to 5.10 À3 M. Only a few particles of size less than 1 Am could be seen at these magnifications for the 10 À3 M H 2 ZrF 6 treatment (Fig. 7d).

The EDX analyses integrated over large areas for each H 2 ZrF 6 treatment investigated are reported in Table 2. The EDX Zr L peaks for 10 À1 M H 2 ZrF 6 -treated samples were weak for high-concentration solutions and became more intense as the solution concentration decreased to 5.10 À3 M, as seen by the Zr atomic concentrations which increased from 0.4% to 1.5%. At this point, we have to be careful with respect to the EDX results since the depth resolution of this technique is in a first approximation a few microns using a 20 kV incident electron beam voltage. The concentrations should thus not be considered as reflecting the exact composition of the film, but one can assume that if the morphology and thickness of the various films are similar from one sample to another, then one can compare the respective EDX analyses. The results presented in Table 2 suggest that an optimum H 2 ZrF 6 concentration exists that lead to the formation of a zirconium film. The fluoride concentration in the film remained low even for the films of highest zirconium content, which suggests that zirconium is mostly in an oxide or oxyhydroxide form rather than fluoride.

Local composition.

To investigate the local composition of the film, some EDX point analyses were carried out. Results from these experiments are given in Table 3. Zirconium is present after 10 À3 M H 2 ZrF 6 treatment under the form of dispersed submicron scale particles or over the manganese-rich particles, previously observed [22] at the surface of this alloy. For the other solutions, the EDX results confirm the absence of zirconium on the uncovered surface. Moreover, aluminium depletion is associated with these areas, indicating that they are a primary a solid solution [22]. SEM observations of AM60 after revealing the microstructure by etching (Fig. 8) and the corresponding line analysis show that this segregation effect is due to the eutectic solidification.

Microprobe analysis after treatment in 10 À3 M HF+ 5Â10 À3 M H 2 ZrF 6 (which corresponds approximately to the 5Â10 À3 M H 2 ZrF 6 treatment) was performed. It was observed (Fig. 9) that the conversion species, i.e., fluorine and zirconium, laterally segregated in the film, and that the segregation appeared to be controlled by the underlying microstructure. Fluorine deposited predominantly over the magnesium-rich primary a phase, while the zirconium appears to deposit over the aluminium-rich phases.

Zirconium is also present in the 6-7 Am spheres. In all the cases, a gaseous evolution took place at the sample surface during immersion in the solutions. It is interesting to note that a gel was seen at the surface of the sample when immersed in the 10 À1 , 10 À2 , and 5.10 À3 M H 2 ZrF 6 solutions, which moves along the sample in the 10 À1 M solution for which the gaseous evolution is the most vigorous. Moreover, we observed brighter vertical paths (Fig. 6a) slightly enriched in zirconium at the surface of this latter sample by SEM. These observations will be discussed below.

Titanium treatments

3.2.2.1. Surface morphology and composition. The surface resulting from the 10 À1 M H 2 TiF 6 treatment (Fig. 10) is apparently not covered by a film, contrary to the 10 À2 and 10 À3 M solutions. Numerous white spots cover the entire surface after these three treatments. As seen in Fig. 10a, some of them are undermining and are thus cathodic with respect to the alloy matrix. Titanium is absent in the global analyses (Table 4) for 10 À1 and 10 À2 M treatments, although a film is observed for the latter. For the 10 À3 M treatment, titanium is present in the global analysis. The fluorine concentration decreased with Particles (2) 87.4F1.3 3.7F2.3 5.3F1.8 3.0F1.7 0.6F0.1 -Mn (2) 41.0F0.4 12.1F6.8 29.9F8.7 10.8F1.4 0.7F0.4 5.5F4.1

The concentrations are in at.%. The various investigated areas correspond to US-uncovered surface, UL-under-layer particles, S-white spheres, and Mnmanganese-rich particles. The numbers between brackets give the number of points to obtain the mean value and standard deviation. decreasing H 2 TiF 6 concentration, in agreement with our previous XPS results [START_REF] Delalande | ECASIA'03 International Congress[END_REF][START_REF] Verdier | [END_REF].

Local composition.

The white cathodic particles contain manganese (Table 3). These particles are also titanium-and fluorine-enriched. As pointed out in Fig. 10d, the film observed after 10 À3 M H 2 TiF 6 treatment is heterogeneous both in a lateral and vertical direction. EDX maps not presented here [22] and local analyses showed that titanium is absent in the dendrites of the primary a solid solution (Fig. 10d-arrow 1), and the film is thicker in some areas (Fig. 10d-arrow 2) than in others (Fig. 10d-arrow 3). Some spherical particles of size of a few microns are also observed (Fig. 10d-arrow 4) and were found to contain titanium, oxygen, and fluorine.

Influence of pH

An increase in zirconium or titanium concentration in the film has previously been observed by XPS [START_REF] Delalande | ECASIA'03 International Congress[END_REF][START_REF] Verdier | [END_REF] when increasing the pH of 5.10 À3 M H 2 ZrF 6 and 10 À3 M H 2 TiF 6 solutions. The corrosion potential increase observed in this case is heightened (Fig. 11) by increasing the pH (addition of KOH). This evolution could be related to the formation of a more passive or thicker film at the surface of AM60. We simultaneously observed smaller current densities on the voltammograms (Fig. 12). Contrary to the general trend observed in the other solutions, the successive voltammograms (from first to third scan) showed a decrease in anodic current density. This suggests that, for this solution, a more protective film formed that could provide even better insulating properties for longer immersion time.

Influence of fluoride ions

Fig. 13 illustrates the negative effect of fluoride ions' addition on the film formation. For small KF or HF additions (10 À3 M), the film has the overall same morphology than without any fluoride addition, but with increasing fluoride concentration, the film progressively disappeared.

These observations are supported by the electrochemical behaviour of AM60. The voltammograms in the 5.10 À3 M H 2 ZrF 6 solution with KF or HF additions are indeed similar to those obtained without addition of fluoride for low (10 À3 M) HF or KF concentrations, and far from the zirconium-free corresponding fluoride solutions, whereas for high (10 À1 M) HF or KF concentrations, the voltammograms are similar to those in the zirconium-free HF or KF solutions, and far from the corresponding one without fluoride addition. For medium fluoride additions (10 À2 M), the electrochemical behaviour is intermediate between fluoride-free and zirconiumfree solution. The electrochemical behaviour of AM60 is thus driven by its reactivity with respect to fluoride ions when these latter are present in high concentration. The SEM observations provided no evidence of film formation for more concentrated H 2 ZrF 6 solutions, i.e., 10 À1 or 10 À2 with addition of 10 À2 -10 À1 M KF or HF. The addition of fluoride ions to the 10 À3 M H 2 TiF 6 solution led to the same observations. With increasing the concentration of fluoride ions, the solutions became thus more aggressive with respect to the AM60 alloy, and less zirconium or titanium was deposited at the surface.

Discussion

The zirconium and titanium solutions have a wide range of aggressiveness towards the AM60 alloy. For both zirconium and titanium, the more concentrated solutions are more aggressive. One can think that these observations are related to the pH of the solutions. However, we have shown in a previous study [START_REF] Verdier | [END_REF] that the polarization resistance of AM60 in fluoride solutions was not influenced by the acidity of the solution in a range of pH 2-7. On the contrary, the logarithm of polarization resistance increased to about 0.65 per pF unit, meaning that the most concentrated fluoride solutions were also the most aggressive with respect to AM60. Some of the solutions used here are slightly more acidic than in this previous study, and moreover, titanium solutions are slightly more acidic than the equivalent zirconium solutions since titanium IV ion is smaller than Zr IV ion. However, we think that the dependence of R p with zirconium or titanium concentrations observed here is linked to the free fluoride ions concentration in the solutions. The zirconium and titanium ions have different coordination numbers [START_REF] Jolivet | De la solution a `l'oxyde[END_REF] in aqueous solutions (8 for Zr IV and 6 for Ti IV) and certainly different complex dissociation constants. This explains the different Log R p vs. pZr or pTi slopes observed more likely than their slight differences in pH.

Low H 2 ZrF 6 or H 2 TiF 6 concentrations favour the formation of a zirconium or titanium film, in agreement with XPS measurements [START_REF] Delalande | ECASIA'03 International Congress[END_REF][START_REF] Verdier | [END_REF], except for the 10 À3 M H 2 ZrF 6 solution. XPS analyses showed that this solution led to the films of highest zirconium content, whereas almost no film has been observed by SEM. For that solution, the film might be very thin and homogeneous, and thus, the SEM technique is not appropriate. The most effective solutions are also the least aggressive with respect to the substrate, which indicates that significant substrate dissolution is required, to obtain a film and might even prevent its formation.

Even if the SEM/EDX is not adequate to analyse the exact composition of the films due to its poor depth resolution, its lateral resolution was found to be helpful. Here, we saw that the film forms preferentially in the cathodic phases, i.e., primary solid solution and manganeserich particles. The preferential nucleation of a zirconium film on cathodic particles on aluminium alloys has recently been pointed out in Refs. [START_REF] Lunder | Proc. of the 3rd Aluminium Surface Science and Technology International Symposium[END_REF][START_REF] Lunder | Proc. of the 15th Internat. Corrosion Congress[END_REF][START_REF] Nordlien | [END_REF]. The film formation proceeds through the reduction of water:

H 2 O þ 2e À Y 1=2 H 2 þ OH À :
This reaction occurs, by a galvanic coupling effect, mostly on the cathodic phases and causes the interfacial pH to increase locally. This increase in pH will lead to the acidbase precipitation of zirconium or titanium complexes, which are stable only in limited pH ranges [START_REF] Jolivet | De la solution a `l'oxyde[END_REF][START_REF] Pourbaix | [END_REF][START_REF] Pourbaix | [END_REF]. Moreover, these observations are in good agreement with the surface chemistry found by our previous XPS work [START_REF] Delalande | ECASIA'03 International Congress[END_REF][START_REF] Verdier | [END_REF].

The ineffectiveness of most concentrated H 2 ZrF 6 or H 2 TiF 6 solutions is probably related to their high aggressiveness due to fluoride ions and the nature of the zirconium and titanium complexes under these conditions.

Further study will be needed in order to determine the level of corrosion protection provided by these films. However, we suggest that the primary a phase which is not covered by the film and has a tendency to corrode first in aggressive media [22] will act as defect points.

Conclusions

The zirconium or titanium-containing film formed at the surface of AM60 magnesium alloy is in most cases heterogeneous. The film formation proceeds by precipitation of the Zr and Ti complexes initiated by the increase of interfacial pH related to the reduction of water. The more dilute solutions are most effective in the formation of a film. This is related to the fluoride ions' influence on the electrochemical behaviour of AM60 and also to the nature of Ti and Zr complexes. An alkalinization of the solution seems to favour the formation of this film, whereas fluoride additions were found to have a detrimental effect.

Fig. 1 .

 1 Fig. 1. Corrosion potential E cor vs. time in (4) 10 À1 M HF, (+) 10 À2 M KF, (5) 10 À1 M, and (o) 10 À3 M H 2 ZrF 6 .

Fig. 3 .

 3 Fig. 3. Polarization resistance for various pZr solutions, first scan, after a preliminary hold time of 1 min at E cor .

Fig. 5 .Fig. 6 .

 56 Fig. 5. Polarization resistance for various pTi solutions, first scan, after a preliminary hold time of 1 min at E cor .

Fig. 7 .

 7 Fig. 7. Film underlayer aspect after (a) 10 À1 M, (b) 10 À2 M, (c) 5.10 À3 M, and (d) 10 À3 M H 2 ZrF 6 treatments.

Fig. 8 .

 8 Fig. 8. (a) Microprobe image of AM60 surface after revealing the microstructure by etching and (b) corresponding line analysis of Mg, Al, and O.

Fig. 9 .Fig. 10 .

 910 Fig. 9. Microprobe maps of AM60 surface after 10 À3 M HF+5.10 À3 M H 2 ZrF 6 treatment, (a) Mg, (b) F, (c) Al, and (d) Zr.

Fig. 12 .

 12 Fig. 12. Current-voltage curves plotted after a preliminary hold time of 1 min at E cor . (a) (-) 5.10 À3 M H 2 ZrF 6 , pHE2.2 (5) 5.10 À3 M H 2 ZrF 6 , pHE5.3; (b) (o) 10 À3 M H 2 TiF 6 , pHE2.2, (4) 10 À3 M H 2 TiF 6 , pHE4.4.

Fig. 13 .

 13 Fig. 13. AM60 surface after 5.10 À3 M H 2 ZrF 6 treatments with KF additions. (a) 10 À3 M, (b) 10 À2 M, and (c) 10 À1 M.

Table 1

 1

	Composition of Zr/Ti fluoride solutions
	[H 2 ZrF 6 ] (M)	pH	[H 2 TiF 6 ] (M)	pH
	10 À3	2.9	10 À4	3.5
	5.10 À3	2.2	10 À3	2.2
	10 À2

Table 2

 2

	EDX analyses after zirconium treatments
	H 2 ZrF 6 (M)	Mg	Al	O	F	Zr	Mn
	10 À1	85.1	6.9	5.9	1.7	0.42	-
	10 À2	84.1	8.2	5.5	1.4	0.8	-
	5.10 À3	80.9	6.7	9.1	1.8	1.5	-
	10 À3	86.5	5.7	5.7	2.1	-	-

The concentrations are in at.%.

Table 3

 3

	EDX local analyses after zirconium treatments
	H 2 ZrF 6 (M)	Area	Mg	Al	O	F	Zr	Mn
	10 À1	US	92.9	2.1	3.2	1.8	-	-
		UL	59.7	6.5	24.1	7.8	1.9	-
		Mn (2)	27.2F12.5	23.7F8.2	23.2F3.7	12.7F2.7	1.8F0.2	11.4F5.5
	10 À2	S (2)	21.9F8.8	3.4F0.3	61.1F8.0	4.9F0.1	8.7F0.8	-
	5.10 À3	US (3)	94.3F0.6	2.0F0.3	2.2F0.4	1.5F0.1	-	-
		S (2)	22.8F8.9	4.1F0.3	54.5F5.2	4.0F0.6	13.7F2.4	0.9F0.3
		UL (2)	55.7F2.5	4.5F2.5	30.1F1.9	2.7F0.8	7.0F0.6	-
	10 À3

Table 4

 4

	EDX analysis after titanium treatments
	H 2 TiF 6 (M)	Area	Mg	Al	O	F	Ti	Mn
	10 À1	Large	77.7	5.1	9.8	7.4	-	-
		Mn	38.8	12.0	29.0	14.9	0.3	5.0
	10 À2	Large	81.9	6.8	7.2	4.1	-	-
		Mn	45.4	22.1	13.3	8.4	0.6	10.2
	5.10 À3	Large	83.9	7.2	6.8	1.6	0.5	-
		Sphere	36.2	0.9	48.5	10.7	3.7	-
	The concentrations are in at.%.


Alexandre Grondin
email: alexandre.grondin@ird.fr

Pablo Affortit

Christine Tranchant-Dubreuil

Carla De La Fuente-Canto

Ce ´dric Mariac

Pascal Gantet

Vincent Vadez

Yves Vigouroux

Laurent Laplaze

Aquaporins are main contributors to root hydraulic conductivity in pearl millet [Pennisetum glaucum (L) R. Br.]

Pearl millet is a key cereal for food security in arid and semi-arid regions but its yield is increasingly threatened by water stress. Physiological mechanisms relating to conservation of soil water or increased water use efficiency can alleviate that stress. Aquaporins (AQP) are water channels that mediate root water transport, thereby influencing plant hydraulics, transpiration and soil water conservation. However, AQP remain largely uncharacterized in pearl millet. Here, we studied AQP function in root water transport in two pearl millet lines contrasting for water use efficiency (WUE). We observed that these lines also contrasted for root hydraulic conductivity (Lpr) and AQP contribution to Lpr. The line with lower WUE showed significantly higher AQP contribution to Lpr. To investigate AQP isoforms contributing to Lpr, we developed genomic approaches to first identify the entire AQP family in pearl millet and secondly, characterize the plasma membrane intrinsic proteins (PIP) gene expression profile. We identified and annotated 33 AQP genes in pearl millet, among which ten encoded PIP isoforms. PgPIP1-3 and PgPIP1-4 were significantly more expressed in the line showing lower WUE, higher Lpr and higher AQP contribution to Lpr. Overall, our study suggests that the PIP1 AQP family are the main regulators of Lpr in pearl millet and may possibly be associated with mechanisms associated to whole plant water use. This study paves the way for further investigations on AQP functions in pearl millet hydraulics and adaptation to environmental stresses.

Introduction

Plant hydraulics depends on soil water capture by roots, transport to the leaves and diffusion as vapor from the stomatal cavity to the atmosphere. In this plant hydraulic continuum, radial water transport from the soil solution to the xylem vessels uses two paths: the apoplastic path where water flows along the cell wall structures, and the cell to cell path where water can flow across cell membranes (transcellular) or along cytoplasmic continuities formed by plasmodesmata (symplastic) [START_REF] Steudle | The cohesion-tension mechanism and the acquisition of water by plant roots[END_REF]. Extracellular hydrophobic barriers of lignin and suberin located in the endodermis are thought to restrict diffusion of water along the apoplastic path [START_REF] Geldner | The Endodermis[END_REF]. In the presence of such barriers, water channels present in cell membranes called aquaporins enable cell to cell transport [START_REF] Wang | Surveillance of cell wall diffusion barrier integrity modulates water and solute transport in plants[END_REF].

Aquaporins (AQP) are present across life forms, with the exception of thermophilic Archaea and intracellular bacteria [START_REF] Abascal | Diversity and evolution of membrane intrinsic proteins[END_REF]. They belong to the Major Intrinsic Proteins (MIP) superfamily and are characterized by six transmembrane domains and two highly conserved Asn-Pro-Ala (NPA) motifs [START_REF] Verma | Major intrinsic protein superfamily: Channels with unique structural features and diverse selectivity filters[END_REF]. Another typical AQP signature is selectivity filters structuring the pore, which are composed of aromatic/arginine (ar/R) motifs and Froger's residues [START_REF] Froger | Prediction of functional residues in water channels and related proteins[END_REF][START_REF] Hub | Mechanism of selectivity in aquaporins and aquaglyceroporins[END_REF]. In higher plants, AQP isoforms fall into five families; Plasma membrane Intrinsic Proteins (PIP), Tonoplast Intrinsic Proteins (TIP), Nodulin26-like Intrinsic Proteins (NIP), Small Intrinsic Proteins (SIP) and uncharacterized (X) Intrinsic Proteins (XIP) [START_REF] Maurel | Aquaporins in plants[END_REF]. Although plant AQP are localized throughout the cell secretory system, PIP, NIP and XIP are preferentially located in plasma membranes, while TIP accumulate in the tonoplast and SIP in the endoplasmic reticulum [START_REF] Ishikawa | Novel type aquaporin SIPs are mainly localized to the ER membrane and show cell-specific expression in Arabidopsis thaliana[END_REF][START_REF] Wudick | A look inside: Localization patterns and functions of intracellular plant aquaporins[END_REF][START_REF] Bienert | Solanaceae XIPs are plasma membrane aquaporins that facilitate the transport of many uncharged substrates[END_REF]. Functional studies, combined with modelling approaches demonstrated that in addition to being permeable to water, AQP also transport other substrates [START_REF] Bansal | Homology modeling of major intrinsic proteins in rice, maize and Arabidopsis: Comparative analysis of transmembrane helix association and aromatic/arginine selectivity filters[END_REF][START_REF] Wallace | Homology modeling of representative subfamilies of Arabidopsis major intrinsic proteins. Classification based on the aromatic/arginine selectivity filter[END_REF]. Some PIP isoforms are permeable to hydrogen peroxide (H 2 O 2 ), nitrate (NO 3 -) and carbon dioxide (CO 2 ), some TIP isoforms to ammonia (NH 3 ) and urea, and some NIP to small organic solutes or mineral nutrients [START_REF] Rodrigues | Aquaporins facilitate hydrogen peroxide entry into guard cells to mediate ABA-and pathogen-triggered stomatal closure[END_REF][START_REF] Uehleln | The tobacco aquaporin NtAQP1 is a membrane CO 2 pore with physiological functions[END_REF][START_REF] Loque | Tonoplast intrinsic proteins AtTIP2;1 and AtTIP2;3 facilitate NH 3 transport into the vacuole[END_REF][START_REF] Biela | The Nicotiana tabacum plasma membrane aquaporin NtAQP1 is mercury-insensitive and permeable for glycerol[END_REF][START_REF] Ma | A silicon transporter in rice[END_REF][START_REF] Liu | Ectopic expression of a rice plasma membrane intrinsic protein (OsPIP1;3) promotes plant growth and water uptake[END_REF]. AQP possess wide range of physiological functions and have now been identified in a number of crop species such as rice, maize, tomato, cotton, sorghum, foxtail millet, watermelon and cannabis [START_REF] Chaumont | Aquaporins constitute a large and highly divergent protein family in maize[END_REF][START_REF] Azad | Genome-wide characterization of major intrinsic proteins in four grass plants and their non-aqua transport selectivity profiles with comparative perspective[END_REF][START_REF] Reddy | Genome-wide identification and characterization of the aquaporin gene family in Sorghum bicolor (L.)[END_REF][START_REF] Sakurai | Identification of 33 rice aquaporin genes and analysis of their expression and function[END_REF][START_REF] Guerriero | Identification of the aquaporin gene family in Cannabis sativa and evidence for the accumulation of silicon in its tissues[END_REF][START_REF] Reuscher | Genome-wide identification and expression analysis of aquaporins in tomato[END_REF].

In plants, AQP are involved in water transport in both roots and shoots. In Arabidopsis, isoforms AtPIP2-2 and AtPIP1-2 contribute around 14% and 20% of the root osmotic conductivity and shoot hydraulic conductivity, respectively [START_REF] Javot | Role of a single aquaporin isoform in root water uptake[END_REF][START_REF] Postaire | A PIP1 aquaporin contributes to hydrostatic pressure-induced water transport in both the root and rosette of Arabidopsis[END_REF]. AQP also have important roles in plant growth, CO 2 fixation, nutrient allocation, reproduction or biotic interactions [START_REF] Maurel | Aquaporins in plants[END_REF]. In recent years, AQP functions in plant water relations have received more attention as a potential target for crop improvement [START_REF] Vadez | Root hydraulics: The forgotten side of roots in drought adaptation[END_REF][START_REF] Moshelion | Role of aquaporins in determining transpiration and photosynthesis in water-stressed plants: Crop water-use efficiency, growth and yield[END_REF][START_REF] Shekoofa | Aquaporin activity to improve crop drought tolerance[END_REF][START_REF] Reddy | Molecular cloning and expression analysis of aquaporin genes in pearl millet [Pennisetum glaucum (L) R. Br.] genotypes contrasting in their transpiration response to high vapour pressure deficits[END_REF][START_REF] Zhang | Root foraging elicits niche complementarity-dependent yield advantage in the ancient 'three sisters' (maize/bean/squash) polyculture[END_REF][START_REF] Tharanya | Quantitative trait loci (QTLs) for water use and crop production traits co-locate with major QTL for tolerance to water deficit in a finemapping population of pearl millet (Pennisetum glaucum L. R.Br.)[END_REF]. For instance, AQP could contribute to balancing root water transport with transpiration in soybean under high evaporative demand [START_REF] Sadok | Transpiration response of "slow-wilting" and commercial soybean (Glycine max (L.) Merr.) genotypes to three aquaporin inhibitors[END_REF]. This hypothesis is supported by the increased AQP expression and root hydraulic conductivity upon high transpiration demand in rice and grapevine [START_REF] Vandeleur | The role of plasma membrane intrinsic protein aquaporins in water transport through roots: Diurnal and drought stress responses reveal different strategies between isohydric and anisohydric cultivars of grapevine[END_REF][START_REF] Sakurai-Ishikawa | Transpiration from shoots triggers diurnal changes in root aquaporin expression[END_REF]. Furthermore, overexpression of SiTIP2-2 in tomato increased transpiration rate and was associated with improved fruit yield upon moderate soil water stress [START_REF] Sade | Improving plant stress tolerance and yield production: Is the tonoplast aquaporin SiTIP2;2 a key to isohydric to anisohydric conversion?[END_REF]. Conversely, in other crops more adapted to hot and dry climates such as pearl millet, lower transpiration under high vapor pressure deficit (VPD) has been associated with AQP function and proposed to be beneficial for crop yield under such conditions [START_REF] Reddy | Molecular cloning and expression analysis of aquaporin genes in pearl millet [Pennisetum glaucum (L) R. Br.] genotypes contrasting in their transpiration response to high vapour pressure deficits[END_REF][START_REF] Kholova | Constitutive water-conserving mechanisms are correlated with the terminal drought tolerance of pearl millet [Pennisetum glaucum (L.) R. Br.][END_REF][START_REF] Kholova | Water saving traits comap with a major terminal drought tolerance quantitative trait locus in pearl millet [Pennisetum glaucum (L.) R. Br.][END_REF]. Therefore, AQP may be involved in different physiological mechanisms that determine the rate and pattern of plant water usage [START_REF] Vadez | Water: The most important "molecular" component of water stress tolerance research[END_REF]. In fact, specific isoforms might be involved in different scenarios, calling for a better understanding of AQP family members and their functions in crops [START_REF] Moshelion | Role of aquaporins in determining transpiration and photosynthesis in water-stressed plants: Crop water-use efficiency, growth and yield[END_REF].

Pearl millet [Pennisetum glaucum (L) R Br.] is a key cereal for food security in arid and semi-arid regions [START_REF] Debieu | Pearl millet genome: Lessons from a tough crop[END_REF]. However, its yields are low and are often affected by climate unpredictability (heat waves and dry spells), which is forecast to worsen [START_REF] Sultan | Evidence of crop production losses in West Africa due to historical global warming in two crop models[END_REF][START_REF] Debieu | Response to early drought stress and identification of QTLs controlling biomass production under drought in pearl millet[END_REF]. The recent release of the pearl millet genome has opened new opportunities for functional genomic-based efforts to improve pearl millet yield and abiotic stress tolerance [START_REF] Varshney | Pearl millet genome sequence provides a resource to improve agronomic traits in arid environments[END_REF]. In this study, links between AQP function in roots and water use were investigated by measuring AQP contribution to Lpr in two pearl millet inbred lines contrasting for water use efficiency. The entire AQP family in pearl millet was characterized using a genomic approach and analyses of root AQP gene expression provided insights into how AQP isoform contribute to root hydraulics.

Materials and methods

Plant material and growth conditions

IP4952 and IP17150, two pearl millet inbred lines that are part of the Pearl Millet inbred Germplasm Association Panel (PMiGAP) were used in this study [START_REF] Varshney | Pearl millet genome sequence provides a resource to improve agronomic traits in arid environments[END_REF]. The water use efficiency (expressed as a plant biomass produced per amount of water transpired in g.kg -1 ) of these two lines was characterized in two lysimeters experiments performed under well-irrigated conditions at the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT, India) using the protocol outlined in [START_REF] Vadez | Stay-green quantitative trait loci's effects on water extraction, transpiration efficiency and seed yield depend on recipient parent background[END_REF]. These experiments indicated that IP4952 and IP17150 displayed relatively low and high water use efficiency, respectively (2.35 versus 3.72 g. kg -1 ) [START_REF] Vadez | Transpiration efficiency: New insights into an old story[END_REF]. These two lines were used for root hydraulic conductivity measurements and AQP expression analyses in which plants were grown in hydroponic conditions in a nethouse (greenhouse made of shade nets) at the IRD/ISRA Bel Air research station (Dakar, Senegal; 14.701615 N-17.425359 W). Plants were germinated in Petri dishes in a dark chamber at 37˚C for two days. Plants were exposed to light (37˚C, 12h day/night cycle) for one day before being transplanted to a black mat covering a 30L container (45x40x24 cm containing 40 plants in total) filled with half strength Hoagland solution [START_REF] Hoagland | The water-culture method for growing plants without soil[END_REF]. The system allowed roots to grow in the nutrient solution without been exposed to light. Oxygen was constantly supplied to the root zone using an air pump.

Root hydraulic conductivity and aquaporin contribution

Root hydraulic conductivity (Lpr) was measured in April-May 2019 from 9AM to 12PM on 15 day old plants. Minimum and maximum temperature and humidity over the period of plant growth was 17/34˚C and 34/100%. At the time of measurements, average temperature, relative humidity and VDP were 24.4 ± 0.3˚C, 75.1 ± 1.2% and 0.8 ± 0.1, respectively. Plants replicates were grown sequentially to allow analysis at the same age, in a randomized design taking into account the time of measurement. Lpr measurements were performed using a pressure bomb (model 1000, PMS instrument company, USA) according to [START_REF] Boursiac | Early effects of salinity on water transport in Arabidopsis roots. Molecular and cellular features of aquaporin expression[END_REF] and [START_REF] Henry | Root attributes affecting water uptake of rice (Oryza sativa) under drought[END_REF]. Briefly, plants were inserted into the pressure chamber filled with nutrient solution with our without 2mM azide (NaN 3 ). The hypocotyl was carefully threated through the silicone grommet of the pressure chamber lid so the intact root system was sealed into the chamber. Roots were pressurized with compressed air at 0.4 MPa for 5 min to equilibrate, followed by xylem sap collection at 0.1, 0.2 and 0.3 MPa for 5 min using pre-weighed 2 ml Eppendorf tubes filled with cotton placed on top of the stem. The mass of xylem sap exuded at each pressure was determined by weighing and was used to calculate root conductance (L 0 ; slope of xylem sap weight at each pressure). After the measurements, roots systems were scanned to determine average root diameter, length and root surface area using WinRhizo Pro version 2012b (Regent Instruments, Canada). Root conductance was divided by root surface area to calculate Lpr. AQP contribution to Lpr was estimated using relative Lpr inhibition by azide calculated as: 100 À ððLpr azide individual replicate x 100Þ=Lpr no azide variety mean Þ ð1Þ

Genome-wide aquaporin identification

A total of 772 AQP protein sequences from 19 plant species (Arabidopsis thaliana, Beta vulgaris, Brachypodium distachyon, Cicer arietinum, Gossypium hirsutum, Glycine max, Hordeum vulgare, Linum usitatissimum, Musa acuminate, Panicum virgatum, Pennisetum glaucum, Populus tremula, Oryza sativa, Setaria italica, Solanum lycopersicum, Solanum tuberosum, Sorghum bicolor, Vitis vinifera and Zea mays) were aligned against the pearl millet genome (ASM217483v2) and the non-assembled pearl millet scaffolds [START_REF] Varshney | Pearl millet genome sequence provides a resource to improve agronomic traits in arid environments[END_REF] using tblastn with an e-value of 10 -5 as initial cut-off to identify high scoring pairs. High scoring pairs were further filtered to keep those with a bit score �100. Hot-spots of high scoring pairs were identified and redundant high scoring pairs were filtered to keep those with highest bit-score for further analysis (S1 Table ). The locations of filtered high scoring pairs in the pearl millet genome were used to identify regions with homologies to AQP genes.

Structural annotation

Correspondence between selected high scoring pairs and annotated genes in the pearl millet genome was investigated. Potential AQP genes were identified and their genomic sequence ± 1000pb upstream and downstream of the start/end position was retrieved as well as the predicted gene structure [START_REF] Varshney | Pearl millet genome sequence provides a resource to improve agronomic traits in arid environments[END_REF]. When predicted high scoring pairs did not correspond to previously annotated genes, the GENSCAN Web Server (http://hollywood.mit.edu/ GENSCAN.html) was used to predict the exon-intron structure of the genomic region. Putative AQP genomic sequences were aligned against the Plant EST (downloaded in August 2018) and the UniProt/Swiss-Prot plant protein (February 2016) databases and were manually annotated using the Artemis software (version 17.0.1, Sanger Institute, UK; S2 Table ). Annotation was confirmed by aligning reads from pearl millet transcriptomes [START_REF] Sarah | A large set of 26 new reference transcriptomes dedicated to comparative population genomics in crops and wild relatives[END_REF] against the pearl millet genome using the Tablet software (version 1.19.09.03) [START_REF] Milne | Using tablet for visual exploration of second-generation sequencing data[END_REF]. Coding and protein sequences were then generated. AQP gene structure were visualized using GSDS2.0 software [START_REF] Hu | GSDS 2.0: An upgraded gene feature visualization server[END_REF]. The genomic, coding and protein sequences of the pearl millet AQP genes are available at the following address: https://dataverse.ird.fr/dataset.xhtml?persistentId=doi:10.23708/WVCG5O.

Sequencing

AQP genes with incomplete sequences in coding regions were resequenced (S2 Table ) using genomic DNA or cDNA from Tift 23D2B1 (genotype used to draft the pearl millet whole genome sequence). DNA was prepared using DNeasy Plant mini extraction kit (Qiagen, Germany). cDNA was prepared by first extracting RNA using the RNeasy Plant mini extraction kit (Qiagen, Germany), followed by DNAse treatment (RNase-free DNase set; Qiagen, Germany) and then reverse transcription reaction (Omniscript RT kit; Qiagen, Germany), according to the manufacturer's instructions. Corresponding DNA/cDNA fragments were amplified using the Phusion high-fidelity DNA polymerase (Thermo Scientific, USA), purified (Geneclean turbo kit, MP Biomedicals, USA) and sent for sequencing (Eurofins Genomics, Germany). These new sequences are referenced in Genbank under the accession number MT474859 for Pennisetum glaucum (Pg) PIP2-8, MT474860 for PgTIP3-1, MT474861 for PgTIP4-1, MT474862 for PgTIP4-2, MT474863 for PgTIP4-3, MT474866 for PgNIP1-2, MT474867 for PgNIP3-5, MT474868 for PgSIP1-2 and MT474869 for PgSIP2-1. Primers used for amplification are presented in S3 Table . Difficulties in amplifying the missing sequence of PgTIP5-1 were encountered. In that specific case, unpublished MINION reads (Mariac, Vigouroux, Berthouly-Salazar; unpublished) were used to complete its sequence (Genbank accessions MT474864 and MT474865). Missing nucleotides were assembled onto the pearl millet genomic sequence and the coding frame of the new protein was determined.

Identification of functional motifs and transmembrane domains

The NCBI conserved domain database (CDD) was used to identify Asn-Pro-Ala (NPA) motifs and aromatic/arginine (ar/R) selectivity filters in the putative AQP protein sequences. Froger's residues were identified according to [START_REF] Froger | Prediction of functional residues in water channels and related proteins[END_REF]. The number and location of the transmembrane domains were studied using TMHMM (http://www.cbs.dtu.dk/services/TMHMM/), TMpred (https://embnet.vital-it.ch/software/TMPRED_form.html) and Phyre2 [START_REF] Kelley | The Phyre2 web portal for protein modeling, prediction and analysis[END_REF]. Protein sequences were aligned using the CLUSTALW alignment function in the Maga7 software (version 7.0.26) [START_REF] Kumar | MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets[END_REF]. Alignments were colored using the Color Align Properties program (https://www. bioinformatics.org/sms2/color_align_prop.html). Conserved domains, as well as transmembrane domains, were further manually analyzed to detect sequence alterations. Phyre2 was also used to predict Pennisetum glaucum PIP (PgPIPs) structure based on homologies with aquaporins of known structure. This generated Protein Data Bank (PDB) files that were used for studying three-dimensional geometric structure and pore morphology of PIP using Pore-Walker software (https://www.ebi.ac.uk/thornton-srv/software/PoreWalker/). The PDB files are available at the following address: https://dataverse.ird.fr/dataset.xhtml?persistentId= doi:10.23708/WVCG5O.

Phylogenetic analysis

Phylogenetic analyses of P. glaucum AQP (PgAQP) was conducted including AQP identified in A. thaliana (AtAQP), O. sativa (OsAQP) and P. tremula (PtAQP) using the Mega7 software (version 7.0.26) [START_REF] Kumar | MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets[END_REF]. PgAQP, AtAQP, OsAQP and PtAQP protein sequences were aligned using the CLUSTAW function and a phylogenetic tree was built using the maximum likelihood method with 1000 reiterations. This allowed determination of the statistical stability of each node. Based on their position in the phylogenetic tree, PgAQP isoforms were classified into PIP, SIP, TIP and NIP families and named according to their close homologs.

Expression profiling

Quantitative polymerase chain reaction (RT-PCR) was used to measure PgPIP gene expression in 15 day old plants grown in hydroponic conditions. One seminal and one crown root were collected between 10AM and 12PM. Sampled roots were immediately frozen using liquid nitrogen and later ground using a TissueLyser II (Qiagen). RNA and cDNA (from 1μg of RNA) were prepared using extraction kits as described above. RT-PCR was performed with 1μL of diluted cDNA (1:9) in a Brillant III ultra fast SYBRgreen QPCR master mix (Agilent Technologies, USA) using a StepOnePlus Real-Time PCR System (Applied biosystems, USA). Primers used to amplify the different PgPIP genes were checked for specificity and efficiency prior to the experiment (S4 Table ). The pearl millet Ubiquitin gene (Pgl_GLEAN_10001684) was used as a reference and PgPIP relative expression was calculated according to the deltadelta ct method. Data to characterize PgAQP shoot expression profiles were collected from [START_REF] Sarah | A large set of 26 new reference transcriptomes dedicated to comparative population genomics in crops and wild relatives[END_REF]. Leaves and inflorescence of ten open pollinated cultivated pearl millet varieties were used [START_REF] Sarah | A large set of 26 new reference transcriptomes dedicated to comparative population genomics in crops and wild relatives[END_REF].

Statistics

Statistical analyses were performed using R version 3.5.2 (R Development Core Team, 2018) using ANOVA (aov script) to detect significant differences. The Least Significant Difference (LSD) test, within the Agricolae package, was used to group differences in letter classes.

Results

Pearl millet aquaporin contribution to root hydraulic conductivity

In order to determine if AQP function and root radial water flow could be associated with water use in pearl millet, we measured root hydraulic conductivity (Lpr) in IP4952 and IP17150, previously described as low and high water use efficiency lines, respectively. These lines did not show significant differences in root diameter and root surface area at the time of Lpr measurement, although root length was significantly higher in IP17150 as compared to IP4952 (p < 0.05; S5 Table ). No significant differences in L 0 and Lpr (p = 0.148) were observed between IP4952 and IP17150 (Fig 1 ). In both IP4952 and IP17150, treatment with azide, an inhibitor of AQP activity, led to significant L 0 and Lpr reduction (p < 0.001; S6 Table). Significant differences in L 0 were observed between lines in the presence of azide (S6 Table ), indicating genotypic differences in anatomical traits. This effect of azide application on Lpr was mostly reversible after treating the same roots with azide-free solution (S1 Fig) . AQP contribution to Lpr was significantly higher in IP4952 (low water use efficiency) as compared to IP17150 (high water use efficiency; 84.64 ± 1.98% versus 76.40 ± 2.61%, p < 0.05; S6 Table ). Therefore, Lpr inhibition by azide indicates that AQP could contribute more than 75% to Lpr in pearl millet.

and S6 Table

Aquaporin identification and annotation

To gain insight on the AQP isoforms contributing to Lpr in pearl millet, we characterized AQP gene families in the pearl millet genome. We blasted 772 AQP protein sequences identified in 19 different species on the pearl millet reference genome (chromosome assembly and scaffolds) [START_REF] Varshney | Pearl millet genome sequence provides a resource to improve agronomic traits in arid environments[END_REF]. A total of 7005 sequences with bit score >100, representing 50 specific hits were identified (S1 Table ). Forty-seven of the hits fell within previously annotated genes, one fell in a non-annotated part of the genome on chromosome 5, and two in non-assembled parts of the genome (scaffold763 and scaffold8428).

Manual de novo annotation of the 50 putative AQP genes allowed the identification of eight genes with no start or with early stop codons in the first exon that were classified as pseudogenes (S2 Table ). Nine genes did not encode AQP isoforms but rather zinc-finger protein/LRR receptor-like serine-threonine protein kinase families or DEAD-like helicase-N protein superfamilies. The absence of AQP signature domains (NPA and Ar/R motifs) in their protein sequence confirmed their non-affiliation to the AQP family. A number of genes showed an excessive number of exons or longer first exon and were re-annotated on the basis of alignment with transcriptome sequences or with close protein homologs (Uniprot/Swiss-Prot blast results) and presence of AQP isoform conserved domains. In addition, ten genes showing missing sequences in coding regions were newly assembled and annotated after sequencing. Overall, 33 Pennisetum glaucum AQP (PgAQP) genes were identified in the pearl millet genome, sixteen of which were annotated de novo (Table 1).

Pearl millet aquaporins phylogenic analysis

To classify the PgAQP into families and name them, a phylogenetic tree was built using the PgAQP protein sequences along with protein sequences from Arabidopsis thaliana, Oryza sativa and Populus tremula (Fig 2). The PgAQP were named according to their grouping into families (PIP, TIP, SIP or NIP) and close homologs (Table 1). Ten isoforms showed homologies to the PIP family with three isoforms falling in the PIP1 sub-family (PgPIP1-1, PgPIP1-3 and PgPIP1-4) and seven isoforms falling in the PIP2 sub-family (PgPIP2-1, PgPIP2-2, PgPIP2-3, PgPIP2-5, PgPIP2-6, PgPIP2-7 and PIP2-8). Nine isoforms from the P. glaucum TIP family (PgTIP), eleven isoforms from the P. glaucum NIP family (PgNIP) and three isoforms from the P. glaucum SIP family (PgSIP) were further identified. No isoforms from the XIP family were identified in pearl millet. This analysis further confirmed the classification of PgPIP1-1, PgPIP2-1, PgPIP2-3, PgPIP2-6, PgTIP1-1 and PgTIP2-2 cloned by [START_REF] Reddy | Molecular cloning and expression analysis of aquaporin genes in pearl millet [Pennisetum glaucum (L) R. Br.] genotypes contrasting in their transpiration response to high vapour pressure deficits[END_REF]. However, PgPIP1-2 from [START_REF] Reddy | Molecular cloning and expression analysis of aquaporin genes in pearl millet [Pennisetum glaucum (L) R. Br.] genotypes contrasting in their transpiration response to high vapour pressure deficits[END_REF] was renamed as PgPIP1-3 in this study.

Most PgAQP genes were localized on chromosome 3 and none were localized on chromosome 7 (Fig 3). Two PgAQP hot-spots were observed, one in a region of 11899bp on chromosome 3 containing PgPIP2-1, PgPIP2-5 and PgPIP2-6 and the other in a region of 141812bp on chromosome 4 containing PgNIP3-2, PgNIP3-3, PgNIP3-4 and PgNIP3-5.

Aquaporin gene structure in pearl millet

PgAQP genes showed large variation in gene length (ranging from 837bp for PgPIP2-8 to 3855bp for PgNIP3;1; Table 1). Transcript lengths were less variable and relatively conserved within families, with lengths of around 800-900bp for the PgPIP and PgNIP genes, 750-800bp for the PgTIP genes and 700-750bp for the PgSIP genes.

To further confirm the phylogenetic classification, gene structure of the PgAQP genes was analyzed (S2 Fig) . PgAQP genes displayed between one (PgPIP2-8) and five exons (PgNIP2-1, PgNIP2-2 and PgNIP4-1). Except for the NIP family, intron-exon organization was generally conserved within families with 5 of 9 PgPIP genes displaying 3 exons, 6 of 9 PgTIP genes displaying 2 exons and all PgSIP genes displaying 3 exons, supporting their phylogenetic distribution. Furthermore, PgPIP2-5/PgPIP2-6 and PgNIP3-2/PgNIP3-3 were found to be close homologs in the phylogenetic analysis (Fig 2 ), and were located near each other on the pearl millet genome (Fig 3). They showed similar gene and transcript length (Table 1) as well as gene structure (S2 Fig) .  PgAQP coding regions encoded proteins with length varying between 250 to 300 amino acids, with molecular weight of around 30kD for the PgPIP and PgNIP and 25kD for the PgTIP and PgSIP isoforms (Table 1).

Pearl millet AQP conserved domains

Analysis of selectivity filters and transmembrane domains was performed to investigate polymorphisms that could impact substrate selectivity of the PgAQP. Analysis of conserved domains showed that all PgAQP isoforms belonged to the MIP super-family and displayed typical double NPA motifs (S7 Table andTable 2). Although some polymorphisms in NPA motifs were observed in some isoforms (particularly from the NIP family), subsequent aminoacids had the same chemical properties (generally neutral and non-polar; Table 2, Fig 4 and

S3-S5 Figs)

. Ar/R selectivity filters and Froger's residues were well conserved in the PgPIP isoforms, except for the Froger's residue on position 1 (P1; Table 2,Fig 4). More polymorphisms were observed in these residues for the PgTIP, PgNIP and PgSIP isoforms although the ar/R residue on Loop E (R on LE2) and the Froger's residues at positions 3 (A) and 4 (F/Y) were well conserved across all isoforms (Table 2). Plasma membrane intrinsic proteins (PIP)

PgPIP1-1 NPA NPA F H T R Q S A F W PgPIP1-3 NPA NPA F H T R Q S A F W PgPIP1-4 NPA NPA F H T R V S A F W PgPIP2-1 NPA NPA F H T R Q S A F W PgPIP2-2 NPA NPA F H T R Q S A F W PgPIP2-3 NPA NPA F H T R Q S A F W PgPIP2-5 NPA NPA F H T R - S A F W PgPIP2-6 NPA NPA F H T R Q S A F W PgPIP2-7 NPA NPA F H T R T S A F W PgPIP2-8 NPA NPA F H T R H S A F W
Tonoplast intrinsic proteins (TIP)

PgTIP1-1 NPA NPA H I A V T S A Y W PgTIP2-1 NPA TPA H I G R T S A Y W PgTIP2-2 NPA NPA H I G R T S A Y W PgTIP2-3 NPA NPA H I G R T S A Y W PgTIP3-1 NPA NPA H V A R T V A Y W PgTIP4-1 NPS NPA N S A R T S A Y W PgTIP4-2 NPA NPA Q S A R T S A Y W PgTIP4-3 NPA NPA H I A H T S A Y W PgTIP5-1 NPA NPA Q V A R R S A Y W

Noduline-26 like intrinsic proteins (NIP)

PgNIP1-1 NPA NPA W V A R F T A Y V PgNIP1-2 NPA NPA W V A R F T A Y F PgNIP1-4 NPA NPV W A A R F S A Y I PgNIP2-1 NPA NPA G S G R L T A Y F PgNIP2-2 NPA NPA G S G R L T A Y F PgNIP3-1 NPS NPV A I G R F T A Y L PgNIP3-2 NPA NPA A A A R Y T A Y M PgNIP3-3 NPA NPA A A A R Y T A Y M PgNIP3-4 NPA NPA A A A R Y T A Y M PgNIP3-5 NPA NPA A A A R Y T A Y M PgNIP4-1 NPA NPI M G G R M T A Y L

Small intrinsic proteins (SIP)

PgSIP1-1 NPT NPA V V P N M A A Y W PgSIP1-2 NPT NPA L I P N M A A Y W PgSIP2-1 NPL NPA S H G S F A A Y W
The two NPA (Asparagine-Proline-Alanine) motifs are located on loop B (LB) and loop E (LE). Aromatic/Arginine motifs (Ar/R) are located on helix 2 (H2), helix 5 (H5), and loop E (LE1 and LE2). P1-5 designed the five Froger's position.

https://doi.org/10.1371/journal.pone.0233481.t002

Transmembrane domain analyses using three different types of prediction software suggested a high probability that all identified PgAQP possess six transmembrane domains with cytoplasmic N-terminal and C-terminal ends, as is typically observed for AQP (S8 Table ). Predictions of 3-dimensional geometric structure and pore morphology suggested a continuous pore that runs longitudinally across both sides of the membranes for all PgPIP (S6 Fig) . Two constrictions in the pore center were typically observed at both extremities, illustrating the two constraints caused by the NPA motifs.

Aquaporin expression profiling in pearl millet

PIP isoforms are thought to play major roles in root hydraulic conductivity because of their localization at the plasma membrane [START_REF] Maurel | Aquaporins in plants[END_REF]. To infer PgPIP isoforms putative importance to Lpr in pearl millet, we analyzed PgPIP gene expression pattern in roots of IP4952 and IP17150 using quantitative PCR. PgPIP genes were generally more expressed in IP4952 as compared to IP17150 (Fig 5). In both lines, PgPIP1-1, PgPIP1-3, PgPIP1-4, and PgPIP2-3 were most expressed while PgPIP2-5, PgPIP2-6, PgPIP2-7 and PgPIP2-8 were less expressed. PgPIP1-3 and PgPIP1-4 were the only genes significantly differently expressed between both lines.

Expression of PgPIP in shoots (leaves and inflorescence) were retrieved from [START_REF] Sarah | A large set of 26 new reference transcriptomes dedicated to comparative population genomics in crops and wild relatives[END_REF]. Transcriptomic analyses from ten pearl millet varieties suggest that PgPIP1-4 and PgPIP2-3, two of

Discussion

Investigation of the role of AQP in root water transport in pearl millet, a heat and droughtadapted crop, suggest that PgAQP are major contributors to root water flow. Root hydraulic conductivity (Lpr) varied around 1E-07 m 3 m -2 s -1 MPa -1 , within the previously report range of other plants [START_REF] Javot | The role of aquaporins in root water uptake[END_REF]. Treatment of roots with a common AQP inhibitor (azide) suggested that AQP contribute up to 84% of Lpr in pearl millet (S6 Table ). This figure is higher than what has been observed in Arabidopsis (57 to 64%) [START_REF] Sutka | Natural variation of root hydraulics in Arabidopsis grown in normal and salt-stressed conditions[END_REF] and rice (42 to 79%) [START_REF] Grondin | Root aquaporins contribute to whole plant water fluxes under drought stress in rice (Oryza sativa L.)[END_REF]. However, complete Lpr recovery following inhibition by azide (S1 Fig) suggests AQP contribution was not overestimated due to off-target effects of azide.

The AQP gene family of the pearl millet genome was characterized and thirty-three putatively functional AQP isoforms (based on conserved domains and protein topology) were identified, belonging to the PIP [START_REF] Wudick | A look inside: Localization patterns and functions of intracellular plant aquaporins[END_REF], TIP [START_REF] Ishikawa | Novel type aquaporin SIPs are mainly localized to the ER membrane and show cell-specific expression in Arabidopsis thaliana[END_REF], SIP (3) and NIP [START_REF] Bienert | Solanaceae XIPs are plasma membrane aquaporins that facilitate the transport of many uncharged substrates[END_REF] families. No XIP were identified, which confirm the absence of isoforms from this family in the monocotyledon clade [START_REF] Maurel | Aquaporins in plants[END_REF]. The number of AQP identified in pearl millet is similar to what has been observed in Arabidopsis [START_REF] Vandeleur | The role of plasma membrane intrinsic protein aquaporins in water transport through roots: Diurnal and drought stress responses reveal different strategies between isohydric and anisohydric cultivars of grapevine[END_REF] [START_REF] Johanson | The complete set of genes encoding major intrinsic proteins in Arabidopsis provides a framework for a new nomenclature for major intrinsic proteins in plants[END_REF], rice [START_REF] Tharanya | Quantitative trait loci (QTLs) for water use and crop production traits co-locate with major QTL for tolerance to water deficit in a finemapping population of pearl millet (Pennisetum glaucum L. R.Br.)[END_REF] [START_REF] Sakurai | Identification of 33 rice aquaporin genes and analysis of their expression and function[END_REF] and maize [START_REF] Reddy | Molecular cloning and expression analysis of aquaporin genes in pearl millet [Pennisetum glaucum (L) R. Br.] genotypes contrasting in their transpiration response to high vapour pressure deficits[END_REF] [START_REF] Chaumont | Aquaporins constitute a large and highly divergent protein family in maize[END_REF]. Interestingly, AQP genes were over-represented on Chromosome 3 with fourteen genes (Fig 3) [START_REF] Tattini | Detection of genomic structural variants from next-generation sequencing data[END_REF]. Similarly, PgNIP3-2 and PgNIP3-3, located on Chromosome 4 may be the result of duplication events. Furthermore, PgPIP2-7 was identified in scaffold763 suggesting that genes may be missing on parts of the pearl millet genome assembly.

The selectivity of an AQP is defined by the three dimensional structure of the amino-acids constituting its pore. The NPA motifs on loops B and E contribute to the dipole moment of the α-helices and prevent proton permeation [START_REF] Chakrabarti | Molecular basis of proton blockage in aquaporins[END_REF][START_REF] To ¨rnroth-Horsefield | Structural mechanism of plant aquaporin gating[END_REF]. These motifs were strictly conserved in PgPIP but showed some polymorphisms for other isoforms (Table 2). However, these substitutions did not drastically change the positive electrostatic potential at the NPA motifs, suggesting that proton exclusion from AQP pores is conserved in pearl millet. Furthermore, the Isoleucine (I) preceding the Froger's residue P4 and P5 at the end of Loop E, shown to be essential for CO 2 transport in PIP [START_REF] Mori | CO 2 transport by PIP2 aquaporins of barley[END_REF], is conserved in PgPIP (Fig 4).

The ar/R motifs, composed of four amino-acids, restrict extracytosolic access to the pore and constitute the main selectivity filter. Modelling approaches based on ar/R signatures were used to predict permeability of plant AQP [START_REF] Bansal | Homology modeling of major intrinsic proteins in rice, maize and Arabidopsis: Comparative analysis of transmembrane helix association and aromatic/arginine selectivity filters[END_REF][START_REF] Wallace | Homology modeling of representative subfamilies of Arabidopsis major intrinsic proteins. Classification based on the aromatic/arginine selectivity filter[END_REF]. For instance, the F-H-T-R signature observed in the PgPIP (Table 2), which seems strictly conserved across PIP from different species, has been associated with water and H 2 O 2 permeability [START_REF] Rodrigues | Aquaporins facilitate hydrogen peroxide entry into guard cells to mediate ABA-and pathogen-triggered stomatal closure[END_REF][START_REF] Javot | Role of a single aquaporin isoform in root water uptake[END_REF][START_REF] Postaire | A PIP1 aquaporin contributes to hydrostatic pressure-induced water transport in both the root and rosette of Arabidopsis[END_REF][START_REF] Tournaire-Roux | Cytosolic pH regulates root water transport during anoxic stress through gating of aquaporins[END_REF]. Furthermore, PgPIP1-3 carries the same ar/R motif and similar Froger's residues as OsPIP1-3, which has been recently shown to transport nitrate (NO 3 -) [START_REF] Liu | Ectopic expression of a rice plasma membrane intrinsic protein (OsPIP1;3) promotes plant growth and water uptake[END_REF]. The H-I-G-R signature observed in PgTIP2-1, PgTIP2-2 and PgTIP2-3, that is conserved in TIP2 from Arabidopsis, maize and rice, supposedly allows permeability to water, NH 3 , urea and H 2 O 2 . The H-I-A-V signature, observed in PgTIP1 (PgTIP1-1), may allow permeability to NH 3, urea and H 2 O 2 but restrict water permeability [START_REF] Maurel | The vacuolar membrane protein gamma-TIP creates water specific channels in Xenopus oocytes[END_REF][START_REF] Dynowski | Plant plasma membrane water channels conduct the signalling molecule H 2 O 2[END_REF][START_REF] Jahn | Aquaporin homologues in plants and mammals transport ammonia[END_REF]. In PgNIP, the W-V-A-R signature have been associated with water, NH 3 and H 2 O 2 permeability, while the A-I/A-G/A-R signatures were associated with restricted water and NH 3 permeability. Interestingly, PgNIP2-1 and PgNIP2-2 showed ar/R signatures (G-S-G-R) similar to OsNIP2-1, which is permeable to silicon [START_REF] Ma | A silicon transporter in rice[END_REF], and also possesses precisely 108 amino-acids between the two NPA motifs, which is assumed to be essential for silicon permeability [START_REF] Deshmukh | A precise spacing between the NPA domains of aquaporins is essential for silicon permeability in plants[END_REF].

Apart from the selectivity filters, a numbers of residues involved in PIP molecular gating mechanisms or subcellular localization were also conserved in PgPIP [START_REF] Maurel | Aquaporins in plants[END_REF]. For instance, the Histidine (H) on Loop D (H199 in AtPIP2-1), which can be protonated to stabilize the pore in a closed conformation, was conserved in most PgPIP [START_REF] To ¨rnroth-Horsefield | Structural mechanism of plant aquaporin gating[END_REF][START_REF] Tournaire-Roux | Cytosolic pH regulates root water transport during anoxic stress through gating of aquaporins[END_REF][START_REF] Verdoucq | Structure-function analysis of plant aquaporin AtPIP2;1 gating by divalent cations and protons[END_REF]. Furthermore, two Serine residues (S), located on Loop B and C-ter (S121 and S280 in AtPIP2-1) that would favor an openpore conformation when phosphorylated, were conserved in PgPIP2 [START_REF] To ¨rnroth-Horsefield | Structural mechanism of plant aquaporin gating[END_REF][START_REF] Johansson | Water transport activity of plasma membrane aquaporin PM28A is regulated by phosphorylation[END_REF]. Another Serine residue located on C-ter (S283 in AtPIP2-1), involved in PIP trafficking, was conserved in the PgPIP, with the exception of PgPIP2-7 [START_REF] Prak | Multiple phosphorylations in the C-terminal tail of plant plasma membrane aquaporins: Role in subcellular trafficking of AtPIP2;1 in response to salt stress[END_REF]. Other residues located on the N-ter (Lysine 3 and Glutamate 6 in AtPIP2-1), subjected to methylation and involved in PIP trafficking were also conserved in most PgPIP [START_REF] Santoni | Methylation of aquaporins in plant plasma membrane[END_REF]. Some of these post-translational modification mechanisms can affect aquaporin function in response to particular abiotic or nutritional stimuli and may well be conserved in pearl millet [START_REF] Tournaire-Roux | Cytosolic pH regulates root water transport during anoxic stress through gating of aquaporins[END_REF][START_REF] Prak | Multiple phosphorylations in the C-terminal tail of plant plasma membrane aquaporins: Role in subcellular trafficking of AtPIP2;1 in response to salt stress[END_REF][START_REF] Grondin | Aquaporins contribute to ABAtriggered stomatal closure through OST1-mediated phosphorylation[END_REF][START_REF] Di Pietro | Coordinated post-translational responses of aquaporins to abiotic and nutritional stimuli in Arabidopsis roots[END_REF].

AQP in plants are expressed in roots and shoots, including inflorescence and pollen. Some PgPIP genes show tissue-specific expression with PgPIP1-4 and PgPIP2-3 having higher expression levels in roots, while PgPIP2-1 is expressed more in shoots and PgPIP1-1 and PgPIP1-3 are expressed in both roots and shoots (Fig 5 and S7 Fig). It has been shown that PIP isoforms agglomerate as tetramers in the plasma membrane, each monomer forming functional units. Functional studies as well as protein-protein interactions studies suggest that PIP tetramers can be formed of heteromers of PIP1 and PIP2 with distinct functional properties depending on the isoform combination [START_REF] Fetter | Interactions between plasma membrane aquaporins modulate their water channel activity[END_REF][START_REF] Zelazny | FRET imaging in living maize cells reveals that plasma membrane aquaporins interact to regulate their subcellular localization[END_REF][START_REF] Yaneff | Heteromerization of PIP aquaporins affects their intrinsic permeability[END_REF][START_REF] Bellati | Intracellular pH sensing is altered by plasma membrane PIP aquaporin co-expression[END_REF]. Based on PgPIP gene expression in pearl millet, PgPIP1-1, PgPIP1-3 and PgPIP1-4 might interact with PgPIP2-3 to form heteromers in roots while PgPIP1-1, PgPIP1-3 and PgPIP2-1 might form different combinations of heteromers in shoots.

Intraspecific diversity in AQP isoform expression has been observed in rice and Arabidopsis [START_REF] Sutka | Natural variation of root hydraulics in Arabidopsis grown in normal and salt-stressed conditions[END_REF][START_REF] Grondin | Root aquaporins contribute to whole plant water fluxes under drought stress in rice (Oryza sativa L.)[END_REF][START_REF] Lian | Upland rice and lowland rice exhibited different PIP expression under water deficit and ABA treatment[END_REF][START_REF] Alexandersson | Transcriptional regulation of aquaporins in accessions of Arabidopsis in response to drought stress[END_REF]. In our study, diversity in expression patterns of PgAQP genes were observed between two pearl millet inbred lines contrasting for water use strategy (Fig 5). IP4952 showed significantly greater AQP contribution to Lpr as compared to IP17150 and also showed significantly higher PgPIP1-3 and PgPIP1-4 gene expression. These results suggest that differences in expression of these AQP genes may reflect differences in AQP contribution to Lpr in pearl millet. These observations are in line with results from [START_REF] Vandeleur | The role of plasma membrane intrinsic protein aquaporins in water transport through roots: Diurnal and drought stress responses reveal different strategies between isohydric and anisohydric cultivars of grapevine[END_REF] showing that the expression of VvPIP1-1 is associated with root hydraulics and response to water stress in two isohydric and anisohydric grapevine (Vitis vinifera) cultivars. Transpiration response to high VPD in four pearl millet recombinant inbred lines has been linked to PgPIP gene expression in roots [START_REF] Reddy | Molecular cloning and expression analysis of aquaporin genes in pearl millet [Pennisetum glaucum (L) R. Br.] genotypes contrasting in their transpiration response to high vapour pressure deficits[END_REF]. The same work suggested that a down-regulation of PgPIP genes under high VPD induced reduction in transpiration and water savings. Our results show lower expression of PgPIP1-3 and PgPIP1-4 in IP17150 as well as a reduced role of AQP in Lpr in IP17150, the line with higher water use efficiency. These findings support the observations of [START_REF] Reddy | Molecular cloning and expression analysis of aquaporin genes in pearl millet [Pennisetum glaucum (L) R. Br.] genotypes contrasting in their transpiration response to high vapour pressure deficits[END_REF]. More recently, overexpression of rice OsPIP1-3 enhanced water use efficiency in transgenic tobacco plants [START_REF] Liu | Ectopic expression of a rice plasma membrane intrinsic protein (OsPIP1;3) promotes plant growth and water uptake[END_REF]. However, this response may well be linked to higher photosynthesis rates due to improved nitrate uptake and transport rather than hydraulics. Overall, expression profiling suggests that APQ may have different physiological functions across the pearl millet plant and contribute to its response to the environment. However, expression alone is certainly not fully representative of AQP function due to various types of post-translational regulations affecting AQP function [START_REF] Maurel | Aquaporins in plants[END_REF]. Further investigations are needed to better understand the links between reduced transpiration under high VPD, improved water use efficiency and AQP function in roots.

Pearl millet is a drought-adapted crop that will play a major role in the adaptation of agriculture to future climates in arid and semi-arid regions of Africa and India. Here, we provide a comprehensive view of the AQP genes and isoforms present in pearl millet as well as their contribution in root radial water transport. Our results suggest that differential expression of PgPIP1-3 and PgPIP1-4 may be associated with different water use strategies. Therefore, our study supports a potential role of these two AQP isoforms in regulating pearl millet hydraulics and potentially adaptation to challenging environmental conditions.

Fig 1 .

 1 Fig 1. Root hydraulic conductivity and aquaporin contribution in roots of IP4952 and IP17150. Root hydraulic conductivity (Lpr) was measured in plants grown in hydroponic conditions between 9AM to 12PM with or without 2mM azide. (A) Lpr values were measured without azide. (B) Aquaporin contribution to Lpr shown as relative difference between Lpr of azide treated and non-azide treated plants. Bars represent mean values ± se of n = 10-15 plants. � : p < 0.05. https://doi.org/10.1371/journal.pone.0233481.g001

Fig 2 .

 2 Fig 2. Phylogenetic relationship among aquaporins isoforms from pearl millet, Arabidopsis, rice and poplar. A phylogenetic tree was generated using the Maximum Likelihood method with 1000 reiterations in MEGA7. Bootstrap values above 50% are represented. The PIP, TIP, SIP, NIP and XIP family clades are represented by blue, grey, purple, green and red, respectively. Pearl millet sequences are indicated by colored dots. https://doi.org/10.1371/journal.pone.0233481.g002

Fig 3 .

 3 Fig 3. Distribution of aquaporin genes in the pearl millet genome. The seven chromosomes (Chr) of the pearl millet genome are represented according to their size in megabase (Mb). Positions of PIP, TIP, SIP and NIP genes are represented in blue, purple, orange and green, respectively. PgPIP2-7, which is localized on scaffold763, is not represented. https://doi.org/10.1371/journal.pone.0233481.g003

Fig 4 .

 4 Fig 4. Conserved domains and membrane topology of the PIP isoforms from pearl millet. Alignment of the PIP isoforms were obtained using ClustalW in Mega7. Sequence identities and similarities (80%) are highlighted in colors. The transmembrane domains are represented by orange bars with the N-terminal and C-terminal ends of the protein located in the cytosol. NPA: Asparagine-Proline-Alanine motifs; � : Aromatic/Arginine selectivity filters. #: Froger's residues. https://doi.org/10.1371/journal.pone.0233481.g004

  . The proximal localization of PgPIP2-5 and PgPIP2-6 in Chromosome 3, their phylogenetical relatedness and similar gene structure (Fig 3 and S2 Fig), suggest possible tandem duplication events in this region

Fig 5 .

 5 Fig 5. Relative expression of PIP genes in roots of IP4952 and IP17150. Transcript abundance of each PIP genes were measured between 9AM and 12PM on plants grown in hydroponic conditions and normalized to the expression of PgPIP2-5 in IP4952. Bars show mean values ± se of n = 6-8 biological replicates, each with three technical replicates. Letters indicate significance difference among groups. https://doi.org/10.1371/journal.pone.0233481.g005

Table 1 . Description and distribution of pearl millet aquaporin genes. ID Gene Pgl_Glean ID Gene length (bp) Transcript length (bp) Protein length (aa) Protein MW (kD) Chr Start End Plasma membrane intrinsic proteins (PIP)

 1

	1	PgPIP1-1 Pgl_GLEAN_10001520 3097	867	288	30.70	3	5721622	5724718
	2	PgPIP1-3 Pgl_GLEAN_10010809 1601	867	288	30.76	3	274573998 274575598
	3	PgPIP1-4 Pgl_GLEAN_10005724 992	900	298	31.38	2	104247932 104248932
	4	PgPIP2-1 Pgl_GLEAN_10028064 2920	873	290	30.35	3	12453415 12456334
	5	PgPIP2-2 Pgl_GLEAN_10028876 2830	867	288	30.12	3	45603209 45606038
	6	PgPIP2-3 Pgl_GLEAN_10035675 1903	873	290	30.39	3	257669631 257671533
	7	PgPIP2-5 Pgl_GLEAN_10028056 1062	834	277	28.93	3	12167791 12168852
	8	PgPIP2-6 Pgl_GLEAN_10028055 1053	861	286	29.94	3	12156953 12158005
	9	PgPIP2-7 Pgl_GLEAN_10010255 1170	861	286	29.79	Scaffold763 240584	241753
	10 PgPIP2-8 Pgl_GLEAN_10009812 837	837	278	29.17	2	64966663 64967453
	Tonoplast intrinsic proteins (TIP)
	11 PgTIP1-1 Pgl_GLEAN_10002147 1499	750	249	25.72	5	153303973 153305471
	13 PgTIP2-1 Pgl_GLEAN_10000631 844	744	247	24.88	2	30657555 30658398
	16 PgTIP2-2 Pgl_GLEAN_10030617 933	747	248	25.03	3	100540239 100541171
	12 PgTIP2-3 Pgl_GLEAN_10009584 851	750	249	25.06	3	33622445 33623295
	15 PgTIP3-1 Pgl_GLEAN_10028702 911	801	266	27.41	2	44624536 44625481
	14 PgTIP4-1 Pgl_GLEAN_10002901 2557	738	245	25.58	1	263875434 263878070
	18 PgTIP4-2 Pgl_GLEAN_10003219 1455	744	247	25.15	3	148813464 148814777
	17 PgTIP4-3 Pgl_GLEAN_10003218 844	747	248	25.10	3	148807632 148808412
	19 PgTIP5-1 Pgl_GLEAN_10033583 1087	813	270	26.69	3	272599938 272601028
	Noduline-

26 like intrinsic proteins (NIP)

	20 PgNIP1-1 Pgl_GLEAN_10012175 2316	846	281	29.52	2	195368171 195370486
	21 PgNIP1-2 Pgl_GLEAN_10028618 2560	846	281	29.53	1	261387113 261389753
	22 PgNIP1-4 Pgl_GLEAN_10028339 1137	837	278	29.36	3	145385690 145386826
	23 PgNIP2-1 Pgl_GLEAN_10018521 3364	891	296	31.82	3	14105646 14109009
	24 PgNIP2-2 Pgl_GLEAN_10019286 3821	894	297	31.57	2	103033018 103036838
	25 PgNIP3-1 Pgl_GLEAN_10034621 3855	909	302	31.43	2	40497062 40500916
	26 PgNIP3-2 Pgl_GLEAN_10030882 1151	846	281	29.60	4	55609410 55610656
	27 PgNIP3-3 Pgl_GLEAN_10030883 1087	780	259	27.05	4	55648949 55650374
	28 PgNIP3-4 Pgl_GLEAN_10030881 1258	750	249	25.15	4	55573901 55575272
	29 PgNIP3-5 Pgl_GLEAN_10030872 934	837	278	27.79	4	55508562 55509419
	30 PgNIP4-1 Pgl_GLEAN_10012100 1319	921	306	31.52	6	110090448 110091766
	Small intrinsic proteins (SIP)
	31 PgSIP1-1 Pgl_GLEAN_10003744 2818	726	241	25.32	1	175152169 175154986
	32 PgSIP1-2 Pgl_GLEAN_10014008 3375	759	252	25.91	4	93394125 93397493
	33 PgSIP2-1 Pgl_GLEAN_10026167 1899	759	252	27.10	5	126489209 126491163

PgPIP2-7 is located on scaffold763, which was not assembled to the pearl millet genome. bp: base pairs; aa: amino-acids; MW: molecular weight; kD: kilo Dalton; Chr: chromosome; Start: position of the ATG; End: position of the stop codon. https://doi.org/10.1371/journal.pone.0233481.t001

Table 2 . Amino-acids residues in conserved domains of pearl millet aquaporins isoforms.

 2

	Isoform	NPA (LB)	NPA (LE)	Ar/R selectivity filters			Froger's residue
				H2	H5	LE1	LE2	P1	P2	P3	P4	P5

PLOS ONE | https://doi.org/10.1371/journal.pone.0233481 October 1, 2020

Acknowledgments

The authors are grateful to Dr Prakash Gangashetty (ICRISAT, Niger) for providing seeds of the inbred lines used in this study, Dr James Burridge (IRD, France) for critical reading and editing of our manuscript and Dr Kwanho Jeong (IRD, France) for his kind support in the preparation of this manuscript. The authors acknowledge the IRD iTrop HPC (South Green Platform) at IRD Montpellier for providing HPC resources that have contributed to the research results reported in this paper (https://bioinfo.ird.fr/-http://www.southgreen.fr).

This work was supported by the CGIAR Research Programme on Grain Legumes and Dryland Cereals (GLDC). PA was supported by the Cultivar program from the Agropolis Foundation as part of the "Investissement d'Avenir" (ANR-l0-LABX-0001-0l) under the frame of I-SITE MUSE (ANR-16-IDEX-0006). The French Agence Nationale de la Recherche supports the postdoctoral fellowship of CFC (ANR Grant RootAdapt n ˚ANR17-CE20-0022-01 to LL).

All relevant data are within the manuscript and its Supporting Information files. All sequences and Protein Data Bank files are available at: https://dataverse.ird.fr/ dataset.xhtml?persistentId=doi:10.23708/ WVCG5O. All new sequences are also available from the GenBank database (accession number MT474859 for PgPIP2-8, MT474860 for PgTIP3

Supporting information

S1 Table. High scoring pairs with highest bit score at the fifty hot-spots. (PDF)

S2 Table . Functional annotation of the pearl millet genomic regions corresponding to hotspots of high scoring pairs. (PDF)

S3