Zihao Wang
email: zihao.wang@inria.fr

Clair Vandersteen

Charles Raffaelli

Nicolas Guevara

François Patou

Hervé Delingette

One-shot Learning Landmarks Detection

Keywords: One-shot learning, Landmarks detection, Deep Learning

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

Introduction

Landmarks detection for target object localization plays a pivotal role in many imaging tasks. Automatic landmark detection can alleviate the challenges of image annotation by human experts and can also save time for many image processing tasks. The difficulty of landmark detection in clinical images may come from anatomical variability, or changes in body position which can lead to large differences of shape or appearance. The literature on automatic landmarks detection approaches can be roughly split into learning based versus non-learning based algorithms.

Non-Learning based landmarks detection in [START_REF] Cheung | n-sift: n-dimensional scale invariant feature transform[END_REF] is proposed the augmentation of the scale-invariant feature transform (SIFT) to arbitrary n dimensions (n-SIFT) for 3D-MRI volumes. However, the computation cost for 3D SIFT features is heavy as their complexity is a cubic function of the image size. Wörz et al. [START_REF] Wörz | Localization of anatomical point landmarks in 3D medical images by fitting 3D parametric intensity models[END_REF] leverage parametric intensity models for image landmarks detection. Ricardo et al. [START_REF] José | Detection of point landmarks in 3D medical images via phase congruency model[END_REF] use log-Gabor filters to extract frequency features for 3D Phase Congruency (PC) applied to detect head and neck landmarks. Learning based landmarks detection Probabilistic graphical models were used for bones landmark labelling in [START_REF] Schmidt | Spine detection and labeling using a parts-based graphical model[END_REF] and [START_REF] Corso | Lumbar disc localization and labeling with a probabilistic model on both pixel and object features[END_REF]. Potesil et al. [START_REF] Potesil | Personalization of pictorial structures for anatomical landmark localization[END_REF] use joint spatial priors and parts based graphical models to improve the landmarks detection accuracy of organs. Shouhei et al. [START_REF] Shouhei | Automatic detection of over 100 anatomical landmarks in medical ct images[END_REF] proposed a Bayesian inference of landmarks through a parametric stochastic landmark detector of the candidates. Donner et al. [START_REF] Donner | Global localization of 3D anatomical structures by prefiltered hough forests and discrete optimization[END_REF] applied random forest and Markov Random Field (MRF) for vertebral body landmarks detection. Mothes et al. [START_REF] Mothes | One-shot learned priors in augmented active appearance models for anatomical landmark tracking[END_REF] proposed a one-shot SVM based landmarks tracking method for X-Ray image landmark detection. Suzani et al. [START_REF] Suzani | Fast automatic vertebrae detection and localization in pathological ct scans[END_REF] proposed to train a convolutional neural network (CNN) with an annotated dataset for automatic vertebrae detection and localization. Liang et al. [START_REF] Liang | A deep learning framework for prostate localization in cone beam ct-guided radiotherapy[END_REF] proposed a two-step based residual neural network for landmarks detection. Deep reinforcement learning for landmarks detection was investigated by Ghesu et al. [START_REF] Ghesu | Multi-scale deep reinforcement learning for real-time 3D-landmark detection in ct scans[END_REF] where landmarks localization is considered as a navigation problem. The main drawback of the above deep learning based landmarks detection methods is that the creation of manually annotated dataset with 3D landmarks is time consuming and in practice very difficult to collect. To tackle this problem, Zhang et al. [START_REF] Zhang | Detecting anatomical landmarks from limited medical imaging data using t2dl[END_REF] proposed a deep learning based landmarks detection method that can be used a limited number of annotated medical images. Their framework consists of two CNNs: one for regressing the patches and the second to predict the landmark positions. Yet, this method like the rest of the learning-based methods are not suited when only one annotated image is available. Another source of difficulties is to detect landmarks that are concentrated on a small part of the image. A typical example is the detection of cochlear landmarks from CT images since the human cochlea is a tiny structure.In this paper, we tackle the problem of automatic determination of 3D landmarks in a volumetric image from a single example consisting of a reference image with its landmarks. We propose a oneshot learning approach that first localizes a Structure Of Interest (SOI) (e.g. the cochlea in a CT image of the inner ear) which lies next to the landmarks. A 2D CNN is trained offline by generating arbitrary oriented slices of a reference image with the binary mask of the SOI. Given a target image, the location of the SOI is iteratively estimated by applying the 2D CNN on 3 orthogonal sets of slices. After aligning the orientations of the two SOI on the target and reference images, a non-rigid registration algorithm is applied to propagate the landmarks to the target image. We apply this approach on 200 CT images of the temporal bone to locate 3 cochlear landmarks and show that the positioning error is within the intra-rater variability. To the best of our knowledge, this is the first one-shot learning method for landmark detection which makes it highly applicable for several clinical problems.

Method

Overview

The proposed approach is described in Fig. 1 extracted on the target image. The main challenge is to automatically extract the target image patch P target such that it is roughly aligned in position and orientation with the reference image patch in order to ease the non-rigid image registration task. To extract the centered target image patch, we first train a 2D CNN to segment the mask S ref on random slices of the reference image. This stage is performed offline and also requires an additional validation image I val where the same visible structure S val has been segmented. Given a target image, the localization stage extracts the target image patch P target by iteratively applying the segmentation network to find the center of mass of the structure and by aligning its axis of inertia. The last stage applies a registration algorithm to estimate the position of landmarks L target .

Offline one-shot CNN training

The objective is to train an algorithm that can roughly segment the structure of interest S ref ⊂ I ref . That structure must include the landmarks or must lie in the vicinity of the landmarks L ref . It should also be present in all target images and must be easy to detect int the image with some visible borders. One issue of one-shot learning is the limited amount of training data that can easily lead to overfitting [START_REF] Wu | One shot learning gesture recognition from rgbd images[END_REF][START_REF] Oriol | Matching networks for one shot learning[END_REF]. To this end, we chose to train a shallow 2D U-net f ω segmentation network in order to segment the SOI that surrounds the landmarks

Online Structure Detection

Given an input image I target , we seek to locate the structure of interest S target with the proper translation and orientation offsets in order to ease the last image registration stage.

Translation offset estimation To determine the 3D translation offset between I target and I ref , we propose to align the centers of the mass corresponding to the structures of interest S target and S ref . We rely on the trained CNN f ω () to determine S target given I target . However, with the limited training set of f ω (), we need to cope with its possible poor performance. To this end, we propose an iterative method described in algorithm 2 and Fig. 2, where the estimation of the translation offset is progressively refined. We write as f ω (I x target [k])[i, j] the output of the CNN applied on the slice k in the X direction of the volumetric image I target which is a 2D probability map. We apply the CNN on the slices of I target extracted along the X,Y,Z directions. To improve the robustness of the center of mass estimation of I target , we combine their output by multiplying the 3 probabilities outputs for each voxel. The joint output of the network at voxel [i, j, k] is then written as :

p[i, j, k] = f ω (I Z target [k])[i, j] • f ω (I Y target [j])[k, i] • f ω (I x k] (1)
The product of the 3 probability maps favors the pixels where the 3 outputs agree. This helps to filter out the false positive pixels produced by the network that are not correlated on the 3 slice orientations. The center of mass C target is then simply estimated as the barycenter of the image voxels weighted by the joint probability p[i, j, k]:

Ctarget= ( i,j,k x[i,j,k] * p[i,j,k], i,j,k y[i,j,k] * p[i,j,k], i,j,k z[i,j,k] * p[i,j,k] ) T i,j,k p[i,j,k] (2)
The target image is then cropped around the detected center C target which is written as Ptarget . When the translation offset between the target and reference images is large, the CNN segmentation performances tend to degrade since it has been trained with slices roughly centered on the center of S ref . This is why we propose to iteratively apply the same approach on I target after being centered on C target . This way, we expect the centered image to be more and more accurately segmented by the neural network since it sees slices that resemble more and more to its training set. We stop the process when the changes in the detected center C target become smaller than a threshold. Rotation offset estimation After having aligned the center of mass of the two structures of interest, the rotation offset is determined by aligning the moments of inertia of S ref and S target . More precisely, the matrix of inertia captures the ellipsoid appearance of each structure and it determines the structure orientation unambiguously if that structure does not have any axis of symmetry. Therefore the alignment of the matrices of inertia consists in applying a rotation to S target such that the eigenvectors of the 2 matrices coincide [START_REF] Jaklic | Moments of superellipsoids and their application to range image registration[END_REF][START_REF] Crisco | Efficient calculation of mass moments of inertia for segmented homogenous 3D objects[END_REF] when they are sorted according to their eigenvalues. The moments of inertia of S target are computed based on the combined probability p[i, j, k] as computed in Eq.1. Thus, after performing the eigenvalue decomposition of the 2 matrices, the rotation matrix centered on C target is applied on the image patch Ptarget to get the final target image patch P target .

Online Image Patch Registration

After the two previous stages, the estimation of the landmarks L target is achieved by performing a non-rigid registration of the reference image patch P ref onto the target image patch P target . The two image patches have the same size, are both centered on the structure of interest and their orientation roughly coincide. This is a good initialization for applying the standard diffeomorphic demons algorithm [START_REF] Vercauteren | Non-parametric diffeomorphic image registration with the demons algorithm[END_REF] as implemented in "itk::DiffeomorphicDemonsRegistrationFilter". This algorithm starts with a multi-resolution rigid registration followed by the non-rigid transformation parameterized by a stationary velocity field. It assumes that intensity distribution matches between the two images patches with a sum of square difference as similarity measure. The reference landmarks L ref are then transported to the target image patch P target through the estimated non-rigid transformation. Finally, the landmarks L target on the original target image I target are positioned by inverting the rigid transforms and cropping performed during the first two stages of the method.

Experiment

Dataset

The dataset consists of 200 volumetric CT images of the left temporal bones acquired by a GE LightSpeed CT scanner at the Nice University Center Hospital. The image dimensions are (512, 512, 160) in 3D with corresponding spacing of (0.25mm, 0.25mm, 0.24mm). In this case, the structure of interest is the cochlea, a relatively small bone having a spiral shape similar to a snail shell and without any axis of symmetry. The cochlea is easily visible on CT images. Two volumetric images were randomly selected to serve as reference and validation images and their cochlea was then segmented by an expert in a semi-automatic fashion. Three landmarks corresponding the cochlea top, center and round window were manually set on the reference image as shown in Fig. 1.

Network architecture and training details

We use a 2D U-net like network [START_REF] Olaf | U-net: Convolutional networks for biomedical image segmentation[END_REF] for segmenting the cochlea in 2D images. The network structure is shown in Fig: 1 and is relatively shallow in order to minimize its complexity. The network input size is [•, 100, 100, 1] followed with 4 convolutional layers (shape: [•, 100, 100, 64]). Feature maps are convoluted with a group of halved size layers but doubled in channels (shape: [•, 50, 50, 128]). Up-sampling layer applied to recover the size of the feature maps to merged with the jump concatenates feature maps (shape:[•, 100, 100, 64 + 128]). Finally, 5 convolutional layers (shape:[•, 100, 100, 64], chn = 64 for middle layers, chn = 1 for the last layer) are used for generating the final feature map. An Adam optimizer is used with a learning rate initialized to lr = 0.1 and decreasing with the number of epochs. The neural network was implemented with Tensorflow 2.0 framework and trained on one NVIDIA 1080 Ti GPU. The offline stage of the CNN takes less than 1h for training and the online stages takes around 30s.

Results

The proposed approach was evaluated qualitatively and quantitatively. In Fig: 3(a), we show the position of the center of mass of the segmented cochlea C target during three iterations of Algorithm 2. We see that the 3 points are getting closer to each other after each iteration thus demonstrating the convergence of the algorithm. In practice, we found that between 2 to 6 iterations are necessary to get a change of mass center position between two iterations less than 1mm.

For a quantitative assessment of performance, an expert positioned twice the 3 landmarks on 20 additional volumes in order to estimate the positioning error and the intra-rater variability. In addition, we also try to employ a naive registration-based landmarks detection method without the iterative localization. The setup of the naive method shares the same registration conditions as the registration steps in the proposed framework.

In Fig: 3(d) we show the 3 landmarks generated by our algorithm with the same landmarks positioned by the expert. Clearly those points are very close to each other on the 3 views. In Table 1(top), we list the average position error of the 3 landmarks on the 20 images with respect to one set of landmarks manually positioned by an expert, and in the bottom rows, we show the corresponding results obtained by the naive registration based method.

In average, the position error of L target is around 0.6mm which corresponds to a difference of position of 2 to 3 voxels. This result is satisfactory when considering the small size of the cochlea (width: 6.53 ±0.35mm, height: 3.26 ±0.24mm [START_REF] Devira | Variations in Cochlear Size of Cochlear Implant Candidates[END_REF]) within the full CT volume (128mm × 128mm × 55mm). In contrast, the naive method is almost unusable for cochlea landmarks detection as the relative error (on average 9.48mm) is too large in comparison with the size of the cochlea. For a better assessment, we also provide the intra-expert landmark position error in Table 1(middle). It shows that the algorithm error is similar to the intra-expert variability, with a lower error for two (the center and window landmarks) out of the three landmarks. When computing the landmark position error with the second set of landmarks made by the expert, or with the average of the 2 annotations, we also found that the algorithm was performing similarly to the expert. Since the intra-rater variability is in most cases lower than inter-rater variability, we believe that the proposed method is an effective way to automate landmark positioning around the cochlea on CT images. Note that the mean landmark position errors reported by Zhang et al. [START_REF] Zhang | Detecting anatomical landmarks from limited medical imaging data using t2dl[END_REF] also correspond between 2.5 to 3 times the voxel size whereas Grewal et al. [START_REF]An end-to-end deep learning approach for landmark detection and matching in medical images[END_REF] after training on 168 scans reports errors between 2 to 9 times the voxel size (2 -9mm).

Conclusion

To the best of our knowledge, the proposed method is the first one-shot learning approach for 3D landmarks detection in volumetric images. We showed that the proposed approach was effective in localizing 3D landmarks in the cochlea from CT images of the inner ear. It relies on a segmentation stage and the registration of a single user-defined image patch which makes it easy explainable and interpretable. The approach is generic and could be applied to the detection of landmarks in CT imaging and other imaging modalities. In the future, we plan to use more complex image similarity measures in the final registration algorithm and to introduce more annotated data (few-shot learning) to address challenging landmark detection problems. Other network architectures proposed in the literature for one-shot deep learning such as [START_REF] Gregory | Siamese neural networks for one-shot image recognition[END_REF][START_REF] Amirreza | One-shot learning for semantic segmentation[END_REF][START_REF] Chen | Image deformation meta-networks for one-shot learning[END_REF][START_REF] Shruti | Improving siamese networks for one shot learning using kernel based activation functions[END_REF] can be explored.

Fig. 1 .

 1 Fig. 1. Overview of the proposed framework.

  . The training set consists of slices of the reference image I ref along arbitrary rotations and translation offsets together with the associated binary masks created by slicing accordingly the reference segmentation S ref . The 2D CNN is trained by minimizing the Binary Cross-Entropy (BCE) loss function. To limit the risk of overfitting, we use a validation set consisting of another volumetric image I val and its segmentation S val . The training is stopped when the segmentation performance of f ω on the 3 orthogonal slices of I val start to decrease. The details of the training procedure are provided in algorithm 1. The CNN training can be performed offline and the 2D random image slices are centered on the center of mass C ref (for T = 0) of the segmented structure of Algorithm 1: One-shot training of CNN

Fig. 2 .

 2 Fig. 2. Iterative determination of the center of mass of the structure of interest. Steps (1) -(2) show the 2D CNN segmentation of the structure of interest from the 3 set of orthogonal slices; (3) The probability maps of the 3 views are combined; (4) Update of the center of mass from the joint probability maps; (5) The target image is cropped around the center of mass.

Fig. 3 .

 3 Fig. 3. (a) Positions of the center of mass of the cochlea during 3 iterations of the offset determination. The 3 cross marks in red, white, green correspond to the 1st, 2nd, 3rd iterations; Row (b) shows the result of the landmarks detection in the whole image Itarget; Row (c) zooms on the detected landmarks before applying the last registration stage; Row (d) zooms on the generated landmarks ('x' marks) after the registration stage and the manually positioned landmarks ('+' marks) by an expert.

  . The algorithm requires as input a reference image I ref where a set of landmarks L ref are positioned. In addition, we require that a binary mask of a visible anatomical or pathological structure S ref ⊂ I ref including the landmarks L ref ∈ S ref be provided. Given a target image I target , landmarks L target are estimated by applying an image registration algorithm between an image patch P ref ⊂ I ref centered on the reference landmarks and an image patch P target ⊂ I target

  Inputs: image: I ref , I val , segmentation: S ref , S ref S ref . Furthermore, the 2D image size of the CNN input is chosen as to cope with the translation ∆T and rotation ∆R offsets such that random slices do not include any missing pixel values.

	Output: CNN parameters ω
	Initialize: fω, ∆T, ∆R;
	while L val decreases do
	T ← (U (-1, 1)∆T ) 3 ; ;	// Uniform Random Translation
	R ← (U (-1, 1)∆R) 3 ;	// Uniform Random Rotation
	Itrans ← Resample(I ref , R, T ) ;	// Transformed Image
	Strans ← Resample(S ref , R, T ) ;	// Transformed Segmentation
	for i = 1; i < K; i + + do
	fω	ω ← -Itrans[i]|Strans[i] ;	// Train the CNN
	end
	L val ← loss(S val , fcnn(I val )) ;	// Validation loss
	end

interest

Table 1 .

 1 Position errors of the 3 cochlear landmarks ( centre, top and window) automatically generated landmarks (AUTO), a second set of manual (MANU) ones, and automatically generated landmarks of registration based naive method (REG). .42 10.95 15.78 16.49 12.83 13.04 14.28 15.09 9.66 16.21 6.82 12.91 11.06 6.96 22.69 6.16 2.22 11.68 17.79 9.74 11.84±4.97mm TOP REG 1.11 8.85 13.73 14.12 10.47 11.25 12.69 12.90 7.55 14.29 4.30 9.56 7.28 4.84 20.27 3.77 0.25 12.88 15.33 13.65 9.95±5.18mm WIN REG 2.21 2.82 8.62 9.73 4.15 5.67 5.37 7.44 2.51 7.89 1.09 6.42 3.46 1.45 15.33 1.77 4.91 16.20 9.40 16.77 6.66±4.85mm

	Image ID	0	1	2	3	4	5	6	7	8	9	10 11 12 13 14 15 16 17 18 19	µ/σ
	CEN AUTO 0.88 0.28 0.49 0.7 0.72 0.57 0.19 0.49 0.49 0.39 0.65 0.84 0.87 0.67 0.72 0.72 0.73 0.54 0.33 0.39 0.58±0.19mm
	TOP AUTO 0.7 1.33 0.56 0.73 0.72 0.37 0.31 0.78 0.35 0.2 1.63 1.15 1 0.26 1.04 0.67 0.8 1.23 0.55 0.39 0.73±0.39mm
	WIN AUTO 0.86 0.65 0.84 0.55 0.65 1.12 1.35 0.31 0.6 0.49 0.26 1.06 1.54 0.72 0.88 0.81 0.54 0.34 1.43 0.88 0.79±0.36mm
	CEN MANU 0.28 0.56 0.53 1.06 0.65 0.59 0.45 0.57 0.25 1.09 0.94 0.84 1.09 0.53 0.37 0.5 0.25 0.54 0.59 0.3 0.60±0.27mm
	TOP MANU 0.43 0.38 0.49 0.25 0.31 0.25 0.31 0.19 0.24 1.09 0 0.5 0.75 0.25 0.31 0.19 0.6 0.42 0.33 0.66 0.40±0.24mm
	WIN MANU 0.69 0.62 1.11 1.1 0.31 1.07 0.31 0.77 0.43 0.57 0.79 1.22 0.91 0.77 0.97 0.75 0.9 1.01 1.18 1.25 0.84±0.29mm
	CEN REG 4

This work was partially funded by the French government, by the National Research Agency: ANR-15-IDEX-01, and by the grant AAP Sante 06 2017-260 DGADSH.


Olivier Plantevin
email: plantevin@csnsm.in2p3.fr

Stéphanie Valère

Driffa Guerfa

Dr Ferdinand Lédée

Dr Gaëlle Trippé-Allard

Dr Damien Garrot

Dr Emmanuelle Deleporte

Groupe

Prof E Deleporte

Photoluminescence Tuning Through Irradiation Defects in CH_3NH_3PbI_3 Perovskites

Keywords: hybrid perovskites, photoluminescence, defects, ion irradiation

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

Introduction

In materials science, as seen in particular in semiconductor physics, it is crucial to work on the relationship between the structural, electronic and optical properties. We are considering here more specifically issues in hybrid perovskite materials. Can we understand better these semiconductor materials' properties like light emission with the help of defects engineering? [START_REF] Egger | What remains unexplained about the properties of halide perovskites?[END_REF] Recently, defect tolerance and defect passivation have become a challenge to improve efficiencies in the frame of photovoltaic devices. [START_REF] Brandt | Identifying defect-tolerant semiconductors with high minority-carrier lifetimes: beyond hybrid lead halide perovskites[END_REF][START_REF] Walsh | Instilling defect tolerance in new compounds[END_REF] In the past two decades, the family of hybrid organic-inorganic perovskites (HOP) have arisen as new functional materials firstly for their light emitting properties, with the structure A2MX4 (2D perovskites) where A is an organic cation, M is a metal cation, and X is a halide anion. More recently, since 2012, the structure AMX3 (3D perovskites) has led to the very fast emergence of a new class of solar cells, as after a few years of research effort it has led to a conversion efficiency above 23%. [4] However, a number of issues related to hysteretic behaviour, structural, thermal and UV stability, as well as moisture sensitivity have to be solved and a better knowledge of the electronic properties of such materials is obviously a prerequisite for their optimization in different opto-electronic devices. [START_REF] Green | The emergence of perovskite solar cells[END_REF][START_REF] Correa-Baena | Promises and challenges of perovskite solar cells[END_REF][START_REF] Lang | Influence of Radiation on the Properties and the Stability of Hybrid Perovskites[END_REF] On the other hand, hybrid perovskite materials have been recently shown to present very efficient radiation hardness together with self-healing properties. [START_REF] Lang | Radiation hardness and self-healing of perovskite solar cells[END_REF] The irradiation of solar cell devices with 68 MeV protons allowed to evidence that perovskite absorbers can withstand proton doses up to 10 12 cm -2 , which exceeds the damage threshold of c-Si by almost 3 orders of magnitude. In the frame of photovoltaic devices, a very good tolerance to electron irradiation (1 MeV, up to 10 16 /cm 2 ) and proton irradiation (50 keV, up to 10 15 /cm 2 ) was also shown. [START_REF] Miyazawa | Tolerance of perovskite solar cell to high-energy particle irradiations in space environment[END_REF] Theoretical calculations revealed that the electronic properties of HOP materials are tolerant to defects because the intrinsic point defects and grain boundaries do not generate gap states, but the origin of this puzzling property is still under debate. [START_REF] Egger | What remains unexplained about the properties of halide perovskites?[END_REF][START_REF] Yin | Unusual defect physics in CH3NH3PbI3 perovskite solar cell absorber[END_REF] . It might be also very interesting to use defect engineering in order to better exploit the light-emitting properties of CH3NH3PbI3. It was shown for instance that efficient amplified spontaneous emission (ASE) and continuous-wave lasing could be obtained through radiative defect inclusions in polycrystalline thin films in the orthorhombic phase below T=160 K. . [11 ,12] Interestingly, it was also shown that bulk HOP can be self-doped by defects engineering. It was actually predicted that the electronic conductivity of perovskites can be tuned between p-type and ntype by controlling growth conditions. This self-doping effect in (CH3NH3PbI3) was then reported and found to be either n or p type by changing the ratio of methylammonium iodide (MAI) and lead iodine (PbI2) which are the two precursors for perovskite formation. [START_REF] Wang | Qualifying composition dependent p and n self-doping in CH3NH3PbI3[END_REF] It was inferred that the n-doping comes from the point defects with the lower formation energy, i.e. I vacancies while the p-type behavior was attributed to Pb vacancies.

The firstly used material in the high-performing perovskites solar cells is methylammonium lead triiodide (CH3NH3PbI3) which has a tetragonal structure at room temperature (now replaced by triple cations perovskite solar cells). This material undergoes two structural phase transitions as a function of temperature : from orthorhombic to tetragonal at 160 K, and from tetragonal to cubic at about 330 K, showing important impact on charge carrier dynamics and photoluminescent properties. [START_REF] Milot | Temperature-dependent charge-carrier dynamics in CH3NH3PbI3 perovskite thin films[END_REF] We use here Helium ion irradiation at different fluences to introduce point defects with varying concentration on purpose in (CH3NH3)PbI3 processed both in single crystals and thin films. We compare the photoluminescence (PL) at varying temperature to gain understanding in the radiative recombination mechanisms and the role point defects might be playing.

Experimental Method

At first, for the materials' synthesis one needs to obtain the methylammonium iodide (CH3NH3)I (called MAI hereafter) which is further used as a precursor in the synthesis of the MAPI samples. The MAI was synthesized by adding dropwise 10,5 mL of hydriodic acid HI (57 % in water, stabilized, Sigma Aldrich) to 20 mL of methylamine CH3NH2 (2 M solution in ethanol, Sigma Aldrich) at 0°C for 2 hours. The solvent was then evaporated at 60°C under vacuum using a rotary evaporator. The powder was subsequently washed several times with diethyl ether and dried overnight at 60°C. In order to increase purity, the yellowish MAI powder was finally recrystallized in a mix of ethanol and diethyl ether.

For thin films synthesis, 159 mg of freshly made MAI and 461 mg of lead Iodide PbI2 were dissolved in 1 mL of N,N-dimethylformamide (DMF). Quartz substrates (Neyco) were cleaned with acetone and ethanol, then treated with a 10 %wt solution of KOH in ethanol in an ultrasonic bath for 15 minute each. The slides were then rinsed with distilled water and dried with pressured air. MAPbI3 thin films were obtained by spin coating the precursor solution at 2000 rpm for 15 seconds. The films were then annealed at 90°C for 20 minutes in air, allowing solvent evaporation as well as thin films crystallization. According to SEM characterizations the average grain size is expected to be about or less than 1 µm. Absorption spectroscopy measurements give an approximate thickness of 400 nm for the obtained polycrystalline thin films.

For single crystals synthesis, 636 mg of MAI and 1844 mg of lead Iodide PbI2 were dissolved in 4 mL of DMF (1:1 molar ratio) in a small Teflon capped vial. The vial was then placed in an oil bath at 105°C. Single crystals of MAPbI3 start to appear at the bottom of the vial after a couple of hours. They were recovered, dried and washed with diethyl ether several times. The typical size of the single crystals is about 3 mm. The Helium ion irradiation was performed using the IRMA implanter at CSNSM laboratory. [START_REF] Chaumont | A medium energy facility for variable temperature implantation and analysis[END_REF] We used a rather low ion energy of 30 keV and fluences between 10 14 cm -2 and 10 16 cm -2 . The ion beam size is of the order of 1 mm and is rastered over a large area in order to have a homogeneous dose deposition. These conditions could also be accessible in any laboratory using a commercial ion gun. The implantations were performed at room temperature with ion currents below 2 µA.cm -2 .

The photoluminescence was measured in reflection geometry at 45° using an argon laser (λ=488nm) at a power of 10 mW as an excitation source, and a spectrometer TRIAX320 from Horiba-JY equipped with a R928 photomultiplier (Hamamatsu) detector. For these measurements the samples were glued with silver paste in an optical closed-cycle cryostat from ARS Instruments equipped with two quartz windows. The time-resolved PL was measured using the Fluoromax equipment (Horiba Jobin-Yvon) based on TCSPC technique with an excitation from a nano-LED at 482 nm pulsed at 100 kHz (full time-range 6 µs).

Results and Discussion

According to the SRIM simulation program, the mean projected range for the Helium ions in MAPI is 275 nm at 30 keV, and the maximum implanted concentration at the highest fluence (1E16 cm -2 ) is about 0,5 at.%. [START_REF] Ziegler | The Stopping and Range of Ions in Solids[END_REF] The light penetration depth in these materials is on the same order of magnitude due to strong optical absorption. Photoluminescence spectroscopy is thus well suited to study the near-surface region that is modified with ion irradiation. The depth distribution of displaced atoms was determined using the detailed calculation with full damage cascade in the SRIM program. At the highest ion fluence (1E16 cm -2 ), the proportion of displaced atoms is about 18 at.% at about 200 nm for the iodine atoms which are the most susceptible to be displaced. The proportion of displaced atoms for the other atomic species are respectively 14%, 6% for H, Pb and 2% for both C and N atoms. So we hereby cover a range of displaced atoms between 0,18% at 1E14 cm -2 to 18% at 1E16 cm -2 for iodine atoms as estimated from the SRIM simulation.

The photoluminescence from MAPI polycrystalline thin films at room temperature is represented in Figure 1 before and after ion irradiation at 1E14, 1E15 and 1E16 He + cm -2 . The reference spectrum is centered at 1.606 eV with a Full Width at Half Maximum of 90 meV, evidencing a contribution from several in-gap states below the band gap energy at about 1.65 eV as obtained from absorption spectrum measurements and according to the known value for this material. [START_REF] Egger | What remains unexplained about the properties of halide perovskites?[END_REF] After ion irradiation we observe an intensity decrease of the overall photoluminescence spectra, however not very drastic as the intensity observed after irradiation with a high fluence of 1E16 He + cm -2 is only decreased by a factor about 20. As a matter of comparison, an irradiation of crystalline silicon with Argon ions with a number of displaced silicon atoms 10 4 times lower, ie 0.02 at.%, (equivalent to 1E12 He + cm -2 with the MAPI irradiation conditions used here) decreases the overall photoluminescence intensity together with the lifetime of minority carriers by a factor 100. Using ion irradiation in crystalline silicon with 0.2 at.% displaced atom (equivalent to 1E13 He + cm -2 with MAPI), the signal was not measurable, and lower than for a bare Si wafer. [START_REF] Defresne | Interface defects in a-Si:H/c-Si heterojunction solar cells[END_REF] This comparison emphasizes the outstanding radiation hardness of light emission properties : methylammonium lead iodide can withstand ion irradiation fluences about a factor 10 5 higher than crystalline silicon, which is consistent with what was found by other authors. [START_REF] Miyazawa | Tolerance of perovskite solar cell to high-energy particle irradiations in space environment[END_REF] It is also remarkable that the PL decay curves are drastically affected only after an irradiation at 1E16 He + cm -2 as observed in Figure 2. As a matter of comparison, we derived lifetimes from a multi-exponential model using a sum of 3 exponential functions to describe the data which were fitted up to 6 µs. Before irradiation, the reference sample shows 2 short lifetimes at 37±1 ns (17%), 200±8 ns (37%) and a long lifetime 1097±15 ns (46%). After irradiation at the lowest fluences (1E14 and 1E15 cm -2 ), only the short lifetimes are a little bit affected while the longest lifetime is not changed (34±1 ns (16%), 213±6 ns (42%) and 1097 ±15 ns (42%) at 1E14 cm -2 , and 35±1 ns (16%), 211±9 ns (35%) and 1140 ±17 ns (49%) at 1E15 cm -2 ). It is very interesting to note that only the short time decay constants are affected at moderate defect concentration, as the long lifetime being the governing one for efficient charge collection in the frame of photovoltaic devices. One can associate these short time decays to non-radiative recombinations introduced through ion irradiation as we also observe a lowering of PL intensity as shown in Figure1. One has to reach 1E16 cm -2 to observe a clear impact on the lifetimes with a decrease of all 3 time constants to 21±1 ns (18%), 92±7 ns (38%) and 777 ±23 ns (43%), which is further pronounced at 2E16 cm -2 (14±1 ns (19%), 72± ns (37%) and 458 ±25 ns (43%)).

As was already noticed, one of the key of MAPI success are its excellent opto-electronic properties even in the presence of defects. [START_REF] Egger | What remains unexplained about the properties of halide perovskites?[END_REF][START_REF] Jia | Continuous-wave lasing in an organic-inorganic lead halide perovskite semiconductor[END_REF][START_REF] Wang | Qualifying composition dependent p and n self-doping in CH3NH3PbI3[END_REF][START_REF] Buin | Materials Processing Routes to Trap-Free Halide Perovskites[END_REF] When comparing polycrystalline thin films to single crystals samples at low temperature, we even observe an enhancement of the optical emission as can be seen from Figure 3. On the same figure we can also compare the PL spectrum at 10K from an irradiated single crystal (2E15 He + cm -2 ) with a reference, non irradiated single crystal. We observe no change in band gap energy after ion irradiation as the spectra all superimpose on the high energy side. However, we can observe some additional intensity on the low-energy side of the spectrum. A more detailed analysis allows to describe the spectra with a decomposition in 3 components that we named BE1, BE2 and BE3 for the 3 different bound exciton processes centered at three distinct energies below the band-gap, respectively 1.603 eV, 1.590 eV and 1.568 eV, as represented in Figure 4. The reference single crystal spectrum decomposition at T=10K is represented in Figure 4(a) and evidences only broad bound exciton recombination features. Indeed the free exciton signature at the energy gap position has been observed only recently with high quality single crystals, and is generally not present in PL measurements from single crystals which are rather dominated by bound exciton recombination on trap states. [START_REF] Diab | Narrow Linewidth Excitonic Emission in Organic-Inorganic Lead Iodide Perovskite Single Crystals[END_REF] One can notice that these components get broader as they are more separated from the gap, indicating a more important energy spread of the associated slightly deeper energy levels. After ion irradiation at 2E15 He + cm -2 , we can observe that the BE3 component is barely not affected, while one can observe a weight transfer from BE1 to BE2, indicating that BE2 should be related to iodine point defects. Indeed, ion irradiation promotes predominantly iodine point defects, of both vacancy or interstitial types. Following the theoretical calculations of Buin and co-workers, iodine vacancy levels should lie within the valence band. The only possible interpretation for a near-band edge level would then be iodine interstitial levels, most probably associated to the BE2 emission. [START_REF] Buin | Materials Processing Routes to Trap-Free Halide Perovskites[END_REF] This increase in BE2 intensity relatively to BE1 leads to an apparent peak position change in the overall spectrum by about 5 meV.

When we compare now the polycrystalline thin film spectrum with the irradiated single crystal in Figure 3, we also observe an important increase in BE2 contribution as can be better seen in Figure 5(a), such that the apparent peak position shift with the reference single crystal is now about 12 meV. The polycrystalline thin films are by nature more defective than the single crystals and it is interesting to notice that this is not a drawback regarding light emission as non-radiative recombination centers are not the dominant ones. In the thin films, the two increasing contributions (BE1 and BE2) are more keen to be related to grain boundaries as well as surface and interface defects and maybe lead and iodine antisites. [START_REF] Buin | Materials Processing Routes to Trap-Free Halide Perovskites[END_REF][START_REF] Agiorgousis | Strong Covalency-Induced Recombination Centers in Perovskite Solar Cell Material CH3NH3PbI3[END_REF] In Figure 6, we can observe the difference in the PL spectra between the reference polycrystalline thin film and the irradiated films at 1E15 and 1E16 He + cm -2 . Firstly, at 1E15 He + cm -2 , the total BE1 and BE2 contribution increases and broadens leading to a relative peak position lowering of about 18 meV (decomposition not shown here), while the BE3 contribution still stays almost the same. Also at the highest fluence of 1E16 He + cm -2 , as observed in Figure 5(b), this contribution is unaffected indicating a very stable component under ion irradiation.

At this fluence, a high relative increase of BE2 is seen, leading to a major spectrum contribution.

Also the BE1 contribution is shifted to higher energy at 1.631 eV. The high energy tail of the spectrum overpass the known band-gap energy at this temperature (1.64 eV), related to an important broadening of the BE2 component for this ion irradiation fluence, as evidenced from the spectral decomposition in Figure 5. This broadening is attributed to local disorder and the number of different local halide defect configurations which induces energy level broadening.

At this high fluence, we also form clusters of point defects that act as non radiative recombination centers as the integrated intensity of the spectrum is decreased by about 40% as compared to the reference MAPI polycrystalline sample.

We now consider the temperature evolution of the PL spectra between the reference thin film and the irradiated sample at 1E15 cm -2 from 10K to 150K as represented in Figure 7. In both cases, the intensity decreases with temperature due to thermal activation of non-radiative recombinations. The complex evolution of the PL spectra was already studied by several authors. [START_REF] Diab | Narrow Linewidth Excitonic Emission in Organic-Inorganic Lead Iodide Perovskite Single Crystals[END_REF][START_REF] Wehrenfennig | Charge carrier recombination channels in the low-temperature phase of organic-inorganic lead halide perovskite thin films[END_REF][START_REF] Kong | Characterization of an abnormal photoluminescence behavior upon crystal-phase transition of perovskite CH3NH3PbI3[END_REF][START_REF] Osherov | The Impact of Phase Retention on the Structural and Optoelectronic Properties of Metal Halide Perovskites[END_REF][START_REF] Wright | Electron-phonon coupling in hybrid lead halide Perovskites[END_REF][START_REF] Galkowski | Spatially resolved studies of the phases and morphology of methylammonium and formamidinium lead trihalide perovskites[END_REF] They could for instance observe a difference in the PL spectrum at low temperature when measured with low or high fluence and in general at least 2 components at low temperature that would evolve in a single broad emission feature above 120-130 K. [START_REF] Wehrenfennig | Charge carrier recombination channels in the low-temperature phase of organic-inorganic lead halide perovskite thin films[END_REF] Interestingly, the temperature dependence of the peak position changes behavior in the same range as the structural phase transition at 160 K : from room temperature to about 150 K the energy increases when the temperature is lowered, while it decreases between 150 K and 10 K. [START_REF] Wehrenfennig | Charge carrier recombination channels in the low-temperature phase of organic-inorganic lead halide perovskite thin films[END_REF][START_REF] Galkowski | Spatially resolved studies of the phases and morphology of methylammonium and formamidinium lead trihalide perovskites[END_REF] We also observe for the reference MAPI thin film such a behavior with a progressive change from a high energy peak which dominates the spectra from 10K to 100 K, progressively replaced by a lower energy feature still lowering in energy until 150 K. At temperatures higher than 150 K this component then progressively broaden and increase in energy until it reaches the room temperature spectra shown in Figure 1. After ion irradiation at 1E15 cm -2 , we observe in Figure 7(b) that the higher energy feature associated to BE1 (see Figure 5) which increases in energy with temperature for the reference MAPI sample has been completely smoothed out from the irradiated spectra. The temperature dependence in Figure 7(b) indicates rather a dominant contribution from BE2 and BE3 that progressively broaden, merge, and finally evolve towards the same kind of single-contribution in the region 100-110 K. Above 100K the spectra both from reference thin film and irradiated sample look very similar, however the intensity after ion irradiation being comparatively larger. Indeed, as we can see from the dependence of the total integrated intensity represented in Figure 8, the intensity from the reference sample has decreased by about 50% from 10K to 150K while it has decreased only by 30% in the case of the irradiated sample. This effect is thus very interesting, however limited to the orthorhombic phase at low temperature, as a steep intensity decrease is observed when approaching the phase transition temperature at 160 K. This step in the temperature dependence of the intensity was observed and discussed by other authors and attributed to electronic confinement in remaining tetragonal inclusions within the orthorhombic phase, as observed with micro-photoluminescence experiments at low temperature. [START_REF] Kong | Characterization of an abnormal photoluminescence behavior upon crystal-phase transition of perovskite CH3NH3PbI3[END_REF][START_REF] Galkowski | Spatially resolved studies of the phases and morphology of methylammonium and formamidinium lead trihalide perovskites[END_REF][START_REF] Dobrovolsky | Defect-induced local variation of crystal phase transition temperature in metal-halide perovskites[END_REF] Very interestingly, we could interpret the intensity amplification at low-temperature as due to a higher number of tetragonal inclusions in the orthorhombic phase after ion irradiation. The irradiation point defects that we created may act as anchoring centers for the tetragonal inclusions. These inclusions, having a lower band-gap than the surrounding orthorhombic phase can act as charge carrier sinks and confine efficiently the excitons. [START_REF] Wehrenfennig | Charge carrier recombination channels in the low-temperature phase of organic-inorganic lead halide perovskite thin films[END_REF][START_REF] Dobrovolsky | Defect-induced local variation of crystal phase transition temperature in metal-halide perovskites[END_REF] This effect may also be used on purpose to exploit more efficiently the radiative defect inclusions in the goal to realize amplified spontaneous emission and continuous-wave lasing that were shown to be associated to the emission from tetragonal phase inclusions . [START_REF] Neutzner | A dual-phase architecture for efficient amplified spontaneous emission in lead iodide perovskites[END_REF][START_REF] Jia | Continuous-wave lasing in an organic-inorganic lead halide perovskite semiconductor[END_REF] For defect engineering, the irradiation should not be too high, as evidenced on figure 8, where the PL intensity is shown to be lowered on the whole temperature range as observed after irradiation at 1E16 cm -2 . A small PL increase at low temperature (T<100 K) after ion irradiation is also observed with the MAPI single crystals on the same Figure 8, however we observe no intensity step at the structural phase transition. This difference indicates that the film structure influences the phase transition. The dominating emission centers at low temperature originate from surface and interface defects, that we essentially related to the BE2 contribution in the spectral decompositions discussed together with Figure 5.

Conclusion

Using Helium ion irradiation and photoluminescence spectroscopy at varying temperature, we could show that the methylammonium lead iodide samples, both in single crystals and polycrystalline thin films, present high radiation hardness as they still present light emission up to the highest irradiation fluences used in this work (2E16 He + cm -2 ). We estimated that they can withstand about 10 5 times the atomic displacements needed to quench the PL in crystalline silicon. The nature of defects giving rise to radiative recombinations and light emission was discussed. The mechanisms are very similar in single crystals and polycrystalline thin films where grain boundaries and intrinsic defects give rise to the same spectra than irradiation defects in single crystals. Ion irradiation defects are also shown to enhance light emission in the low-temperature orthorhombic phase as seen in the dependence of the total PL integrated intensity. This effect, much pronounced in thin films, was related to quantum confinement within tetragonal inclusions that may still be present in the orthorhombic phase at low-temperature (T<160 K). Another effect of the ion irradiation directly observable is the smoothing out of the emission from the less tightly bound exciton (BE1), and a strong reinforcement of the BE2 component that we tentatively attributed to Iodine interstitials when considering theoretical calculations. Finally, this work sheds some light on defects and their influence over opto-electronic properties in methylammonium lead iodide, opening new possibilities for the use of defects engineering as well as doping through ion implantation.

Figure 1 :Figure 2 :

 12 Figure 1 : PL spectra from (CH3NH3)PbI3 polycrystalline thin films at room temperature after

Figure 3 :Figure 4 :

 34 Figure 3: PL spectra from (CH3NH3)PbI3 monocrystal and thin film at T=10 K, together with

Figure 5 :

 5 Figure 5: PL spectra from (CH3NH3)PbI3 thin films at T=10 K, before (a) and after irradiation

Figure 6 :Figure 7 :

 67 Figure 6: PL spectra from (CH3NH3)PbI3 thin films at T=10 K, before and after irradiation with

Figure 8 :

 8 Figure 8: Temperature dependence of the PL integrated intensity from (CH3NH3)PbI3 thin films

Acknowledgments

This work was supported by the LabEx PALM (ANR-10-LABX-0039-PALM), and IRS MOMENTOM (Université Paris-Saclay).

We acknowledge Cyril Bachelet, Stéphane Renouf and Jérôme Bourçois for the Helium ion irradiation using the IRMA implanter at CSNSM.

Received:

Revised: Published online:


Souraya Khouider

Filipe Borges

Chantal Leblanc

Alexander Ungru

Arp Schnittger

Robert Martienssen

Vincent Colot
email: vincent.colot@ens.psl.eu

Daniel Bouyer
email: daniel.bouyer@ens-lyon.fr

Male fertility in Arabidopsis requires active DNA demethylation of genes that control pollen tube function

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D

NA cytosine methylation (5-Methylcytosine) is an epigenetic mark that plays critical roles in the silencing of transposable elements (TEs) and the transcriptional regulation of genes in plants and mammals [START_REF] Law | Establishing, maintaining and modifying DNA methylation patterns in plants and animals[END_REF][START_REF] Ambrosi | Dynamics and context-dependent roles of DNA methylation[END_REF][START_REF] Luo | Dynamic DNA methylation: in the right place at the right time[END_REF] . In plants, methylation affects cytosines in the three possible contexts, CG, CHG, and CHH (where H = A, C, or T) through different molecular pathways and DNA methyltransferases (MTases). In Arabidopsis thaliana, DRM1 and DRM2, which are related to the mammalian de novo DNA MTase Dnmt3, establish DNA methylation in all sequence contexts in an RNA-dependent manner (RNA-directed DNA methylation or RdDM [START_REF] Matzke | RNA-directed DNA methylation: an epigenetic pathway of increasing complexity[END_REF] ). DRM1 and 2, together with CMT2, a plant-specific chromomethylase, are also essential for the maintenance of CHH methylation. In addition, MET1, which is the plant homolog of the maintenance DNA MTase Dnmt1 and the chromomethylase CMT3 are responsible for maintaining CG and CHG methylation, respectively [START_REF] Law | Establishing, maintaining and modifying DNA methylation patterns in plants and animals[END_REF][START_REF] Stroud | Non-CG methylation patterns shape the epigenetic landscape in Arabidopsis[END_REF] .

Although in mammals DNA methylation is reduced drastically in the early embryo and even more so in the primordial germlines, broad DNA demethylation has not been observed in the gametes or the embryo of plants, except in the CHH context [START_REF] Heard | Transgenerational epigenetic inheritance: myths and mechanisms[END_REF][START_REF] Kawashima | Epigenetic reprogramming in plant sexual reproduction[END_REF][START_REF] Ingouff | Live-cell analysis of DNA methylation during sexual reproduction in Arabidopsis reveals context and sex-specific dynamics controlled by noncanonical RdDM[END_REF] . In contrast, extensive DNA demethylation occurs in the so-called gamete companion cells that are part of the post-meiotic multicellular structures, the gametophytes, in which gametes of flowering plants are embedded. Specifically, pollen, which is the male gametophyte, contains a vegetative cell (VC), which is hypomethylated-mainly in the CG context-at specific genomic regions compared to the two sperm cells (SCs) that it encapsulates [START_REF] Ibarra | Active DNA demethylation in plant companion cells reinforces transposon methylation in gametes[END_REF][START_REF] Calarco | Reprogramming of DNA methylation in pollen guides epigenetic inheritance via small RNA[END_REF] . On the female side, the ovule includes two synergids, which play important roles for pollen tube attraction, the central cell (CC), which is globally hypomethylated-again mainly in the CG context-and the egg cell (EC) [START_REF] Park | DNA demethylation is initiated in the central cells of Arabidopsis and rice[END_REF] .

In both the VC and CC, DNA demethylation mainly depends on the activity of the DNA glycosylase DEMETER (DME) [START_REF] Ibarra | Active DNA demethylation in plant companion cells reinforces transposon methylation in gametes[END_REF] . Loss of DME leads to complete seed abortion when maternally inherited [START_REF] Choi | DEMETER, a DNA glycosylase domain protein, is required for endosperm gene imprinting and seed viability in Arabidopsis[END_REF] , possibly as a result of defects in genomic imprinting [START_REF] Satyaki | DNA methylation and imprinting in plants: machinery and mechanisms[END_REF] . On the paternal side, loss of DME leads to partial sterility, the degree of which varies between genetic backgrounds [START_REF] Schoft | Function of the DEMETER DNA glycosylase in the Arabidopsis thaliana male gametophyte[END_REF] . DNA demethylation in the VC correlates with the reactivation of TEs and the production of small RNAs (sRNAs), which may be transported to the two SCs. Based on these observations, it was proposed that demethylation in the VC serves to reinforce silencing of TEs, via sRNAs, in the SCs [START_REF] Ibarra | Active DNA demethylation in plant companion cells reinforces transposon methylation in gametes[END_REF][START_REF] Calarco | Reprogramming of DNA methylation in pollen guides epigenetic inheritance via small RNA[END_REF][START_REF] Slotkin | Epigenetic reprogramming and small RNA silencing of transposable elements in pollen[END_REF][START_REF] Martinez | Silencing in sperm cells is directed by RNA movement from the surrounding nurse cell[END_REF] . However, few TEs are reactivated in VCs [START_REF] He | Natural depletion of histone H1 in sex cells causes DNA demethylation, heterochromatin decondensation and transposon activation[END_REF] and it is unclear how defects in TE reactivation in DME-deficient pollen would cause male sterility. Thus, an alternative possibility is that active DNA demethylation is required in the VC to enable the expression of genes involved in pollen function [START_REF] Schoft | Function of the DEMETER DNA glycosylase in the Arabidopsis thaliana male gametophyte[END_REF][START_REF] He | Natural depletion of histone H1 in sex cells causes DNA demethylation, heterochromatin decondensation and transposon activation[END_REF] .

To address this possibility, we analyze pollen defective in DNA demethylase activity. We extend our analyses to situations where both DME and ROS1, which encodes the main DNA demethylase in somatic tissues [START_REF] Penterman | DNA demethylation in the Arabidopsis genome[END_REF][START_REF] Gong | ROS1, a repressor of transcriptional gene silencing in Arabidopsis, encodes a DNA glycosylase/lyase[END_REF] , are mutated. Our findings indicate that ROS1 acts together with DME in the VC to ensure proper genome-wide DNA demethylation, as well as proper activation of a small number of genes that define a signaling complex critical for pollen tube function.

Results

DME and ROS1 act together to ensure male fertility. We first measured the transmission rate of the dme mutant allele in crosses with wild-type mothers of the reference strain Columbia (Col-0). Fathers used in these crosses were heterozygous for dme (dme/+, and either wild type or homozygous mutant for ROS1 (the latter designated dme/+;ros1). Consistent with previous observations [START_REF] Schoft | Function of the DEMETER DNA glycosylase in the Arabidopsis thaliana male gametophyte[END_REF] , transmission of the dme mutant allele was reduced to 17.4% instead of the 50% expected in the case of Mendelian segregation at a single locus (Fig. 1a). In addition, we found that dme transmission was much further reduced when the dme/+;ros1 double mutant was used as pollen donor (3.2% vs. 50%; Fig. 1a), suggesting that ROS1 activity compensates partially for the lack of DME. Crosses involving double heterozygous dme/+;ros1/+ fathers confirmed these results, with a reduced transmission of dme to 11.4% when alone and to only 1.4% when combined with ros1, compared to 25% expected under Mendelian segregation in both cases. In contrast, transmission of ros1 alone (i.e., in combination with the DME wild-type allele) was not affected (Fig. 1a). Together, these results indicate that DME and ROS1 have partially redundant functions in male fertility, with DME playing a more prominent role.

DME and ROS1 are both required for proper pollen tube orientation. To identify the origin of the transmission deficiency of dme, we first examined the possibility of a meiotic defect. To this end we made use of the Fluorescent Tagged Line, FTL_1311, which harbors a transgene encoding a pollen-specific fluorescent marker as well as the quartet (qrt) mutation [START_REF] Francis | Pollen tetrad-based visual assay for meiotic recombination in Arabidopsis[END_REF] . This mutation causes failure of microspore separation during pollen development, hence providing an easy means to verify the integrity of the four haploid cells that result from meiosis. The transgene present in line FTL_1311 is closely linked (973 kb and 1.5 cM) to the DME locus (Supplementary Fig. 1a) and we first introgressed it together with the qrt mutant allele in the hemizygous state into the dme/+ and dme/+;ros1 backgrounds. We then determined the proportion of fluorescent pollen in the resulting lines, which should reflect the proportion of DME wild-type pollen given the tight linkage of the transgene to the DME wild-type allele (Supplementary Fig. 1a,b). The tetrads were intact and had two fluorescent pollen (Supplementary Fig. 1b), indicating no loss of the dme mutant allele through meiotic drive. In addition, pollen viability was not affected in the single mutant, consistent with previous observations [START_REF] Schoft | Function of the DEMETER DNA glycosylase in the Arabidopsis thaliana male gametophyte[END_REF] , nor in dme/+;ros1 (Supplementary Fig. 1c). As pollen germination deficiency can only explain part of the dme transmission defect [START_REF] Schoft | Function of the DEMETER DNA glycosylase in the Arabidopsis thaliana male gametophyte[END_REF] , we next investigated if pollen tube elongation is impaired in dme/+ and dme/+;ros1. Were this the case, we would expect less seeds carrying the dme allele in the distal part of the silique, as pollen tubes need to grow further to reach it. However, we did not detect significant differences in dme transmission rates between the upper and the lower part of siliques (Supplementary Fig. 1d), confirming previous observations made in dme single mutants [START_REF] Schoft | Function of the DEMETER DNA glycosylase in the Arabidopsis thaliana male gametophyte[END_REF] and indicating that pollen tube growth per se is not reduced in dme/+ or dme/+;ros1. We also did not observe any post-fertilization defects such as seed abortion in crosses with dme/+, consistent with a previous report [START_REF] Choi | DEMETER, a DNA glycosylase domain protein, is required for endosperm gene imprinting and seed viability in Arabidopsis[END_REF] , nor dme/+;ros1-derived pollen (Supplementary Fig. 1e). Rather, many pollen tubes followed aberrant trajectories in the transmitting tract of the pistil in crosses involving mutant fathers (Fig. 1b). Closer examination suggested that dme and dme;ros1 pollen tubes fail to orient properly in the maternal transmitting tract and thus rarely reach ovules before wild-type pollen. To determine whether dme pollen is indeed outcompeted by wildtype pollen, we applied limited amount of pollen grains to emasculated flowers, thereby providing an excess of ovules. As predicted, dme transmission was ameliorated under these conditions and increased significantly from 17% to 24% and from 2% to 12% in crossed with dme/+ and dme/+;ros1 fathers, respectively (Fig. 1c). However, mutant pollen remains less efficient compared to wild-type pollen at fertilizing ovules, irrespective of the number of ovules fertilized per silique (Supplementary Fig. 1f). Taken together, these observations suggest that DME and to a lower extent ROS1 are required for the proper journey of the pollen tube towards the ovule. DME and ROS1 together are responsible for DNA demethylation of the VC. To establish how DNA demethylation carried out by DME and ROS1 may affect pollen tube function, we analyzed the methylome of fluorescence-activated cell sorted (FACS) VCs and SCs of wild-type, single, and double mutant plants using whole-genome sequencing of bisulfite-converted genomic DNA (Supplementary Fig. 2 and Supplementary Data 1).

Consistent with previous results [START_REF] Ibarra | Active DNA demethylation in plant companion cells reinforces transposon methylation in gametes[END_REF][START_REF] Calarco | Reprogramming of DNA methylation in pollen guides epigenetic inheritance via small RNA[END_REF] , methylation in wild-type pollen is lower at many CG sites in the VCs compared to the SCs (CG-hypomethylated regions, CG-hypo DMRs (differentially methylated regions); Fig. 2a,b). A small number of predominantly short CG-hyper DMRs are also observed and these tend to exhibit also higher CHH and-to a lesser extent-CHG methylation in VCs compared to SCs (Supplementary Fig. 3a-c and Supplementary Data 2). Previous work suggests that this hypermethylation in wild-type VCs results from higher RdDM activity in the VC than in the SCs [START_REF] Slotkin | Epigenetic reprogramming and small RNA silencing of transposable elements in pollen[END_REF] . Pollen produced from single and double mutant plants showed large differences in CHG and especially CG, but not CHH methylation compared to wild-type pollen (Supplementary Fig. 3a-c). Specifically, the number and size of CG-hypo DMRs, and to a lesser extent CHG-hypo DMRs, between VCs and SCs is much reduced in mutants (Fig. 2a,b, Supplementary Fig. 3a,c, and Supplementary Data 2-5). As pollen from dme/+ and dme/+;ros1 mutant plants is an equal mix of DME wild type and dme mutants (Supplementary Fig. 1c), these results suggest that the absence of DNA demethylation at CG sites in mutant VCs was sufficient to preclude the detection in this background of CG-hypo DMRs in the VCs vs. SCs comparison.

In agreement with this explanation, CG-hypo DMRs detected in the wild type between VCs and SCs had much higher CG methylation in mutant than in wild-type VCs (Fig. 2c) and they correspond to a large extent to the CG-hypo DMRs that are detected in the comparison of VCs between wild-type and mutant plants (Supplementary Fig. 3e, f and Supplementary Data 2 and 6-8). Most CG-hypomethylation in wild-type VCs is primarily found over TEs. Moreover, protein coding genes that associate with CG-hypo DMRs between wild-type VCs and SCs overlap to about 60% with TEs within 500 bp up-and downstream of their transcription unit, in contrast to 22.5% genome-wide [START_REF] Hollister | Transposable elements and small RNAs contribute to gene expression divergence between Arabidopsis thaliana and Arabidopsis lyrata[END_REF] (Fig. 2a), and these genes showed Gene Ontology enrichments such as "pollen tube guidance," "acceptance of pollen," "recognition of pollen," and "regulation of pollen tube growth" (Tables 123and Supplementary Data 2 and 6-8). Finally, more than 1/3 of the CG-hypo DMRs detected between wild-type VCs and SCs are located within the 5′-region of the associated genes (Supplementary Fig. 3d). Together, these observations provide strong support for the proposition made previously that TE sequences contribute to reproduction-specific gene expression by providing transcription factor binding sites that become accessible in the endosperm, as well as in the VC thanks to their demethylation [START_REF] He | Natural depletion of histone H1 in sex cells causes DNA demethylation, heterochromatin decondensation and transposon activation[END_REF][START_REF] Schmitz | Patterns of population epigenomic diversity[END_REF][START_REF] Gehring | Extensive demethylation of repetitive elements during seed development underlies gene imprinting[END_REF][START_REF] Batista | The MADS-box transcription factor PHERES1 controls imprinting in the endosperm by binding to domesticated transposons[END_REF] .

DME and ROS1 are essential for expression of genes involved in pollen tube function. To investigate further the potential role of active DNA demethylation in pollen tube function, we established a list of 194 genes known or predicted to be involved in this function, based on literature research (see Supplementary Data 9). Remarkably, 108 of these genes showed VC DNA-hypomethylation, either in the comparison between wildtype VCs and SCs, and/or in the comparison between wild-type and mutant VCs (Fig. 3a and Supplementary Data 9). The gain of DNA methylation was most often over promoter regions not methylated in wild-type seedlings and pollen, except sometimes in SCs, the latter being in agreement with the observation of elevated RdDM activity in the male germline in A. thaliana [START_REF] Walker | Sexual-lineage-specific DNA methylation regulates meiosis in Arabidopsis[END_REF] (Fig. 3b, Supplementary Fig. 4 Class I, and Supplementary Data 9). In some cases, gain of DNA methylation in mutant VCs was over promoter regions, which were specifically demethylated in wild-type VCs (Fig. 3b, Supplementary Fig. 4 Class II, and Supplementary Data 9). To determine whether this gain of DNA methylation over promoter sequences in mutant VCs was linked to transcriptional downregulation of the cognate genes, we measured transcript levels by reverse transcriptase quantitative PCR (RT-qPCR) for 46 genes that show ectopic DNA methylation over the upstream region (Supplementary Data 9). We did not take into account changes in gene body methylation, as these do not appear to affect expression [START_REF] Bewick | On the origin and evolutionary consequences of gene body DNA methylation[END_REF] , nor those DMRs that are mainly dependent on ros1 due to the absence of fertility defects in this mutant (Supplementary Data 9). As dme/+ and dme/+;ros1 mutants carry 50% DME wild-type pollen, we expected to observe at most a 50% reduction in transcript levels compared to wild type if promoter hypermethylation leads to full gene repression in mutant VCs. Six genes showed reduced transcript levels in mutant pollen, with repression being more pronounced and almost complete (half of the transcript level measured in wild type Col-0) in the double mutant for three genes (Fig. 3c and Supplementary Data 10). These six genes encode signaling components or are associated with receptor-like proteins [START_REF] Chang | AtPRK2 promotes ROP1 activation via RopGEFs in the control of polarized pollen tube growth[END_REF][START_REF] Duckney | Actin-membrane interactions mediated by NETWORKED2 in Arabidopsis pollen tubes through associations with Pollen Receptor-Like Kinase 4 and 5[END_REF][START_REF] Ge | Arabidopsis pollen tube integrity and sperm release are regulated by RALF-mediated signaling[END_REF][START_REF] Lu | Pollen tubes lacking a pair of K+ transporters fail to target ovules in Arabidopsis[END_REF][START_REF] Qin | Penetration of the stigma and style elicits a novel transcriptome in pollen tubes, pointing to genes critical for growth in a pistil[END_REF][START_REF] Wang | A receptor heteromer mediates the male perception of female attractants in plants[END_REF][START_REF] Muschietti | How many receptor-like kinases are required to operate a pollen tube[END_REF] , which suggests a direct role for DME and ROS1 in enabling VCspecific expression of genes involved in pollen tube function.

A pollen tube signaling network co-regulated by active DNA demethylation. To determine experimentally that repression of these six genes in mutant VCs contributes to paternal sterility, we performed functional complementation assays using transgenes containing a pollen-specific promoter that is independent of DNA methylation changes (see Fig. 4a for a schematic description). Two promoters of different strength were chosen so as to adjust as much as possible transgene expression levels in mutant pollen with those of the native genes in wild-type pollen (Supplementary Data 10). Wild-type and ros1 plants were transformed and for each construct two independent transgenic progeny (T1) were crossed with dme/+ or dme/+;ros1 to obtain single and double mutant T2 progeny, which was then crossed with wild-type mothers to measure paternal transmission of dme in the presence of the transgene (Fig. 4b). The re-establishment of gene expression in dme/+ mutant pollen was verified by RT-qPCR (Supplementary Table 1). Control crosses without the transgene lead, as expected, to low dme transmission (12.8% in case of the dme/+ single mutant and 1.9% in case of the dme/+; ros1 double mutant; Fig. 4b). Low dme transmission was also observed when the pFIS2-mCherry transgenic line carrying the same vector backbone as for the DME/ROS1 target transgenes was used as control (12.2% for dme/+ and 2.7% for dme/+;ros1; Fig. 4b). In contrast, re-establishing expression of PRK2, PRK4, or AT2G16030 was sufficient to increase significantly dme transmission in the single and double mutant backgrounds (Fig. 4b). In addition, although forced pollen expression of RKF2 had no detectable effect in dme/+, it lead to a notable fertility increase in dme/+;ros1, similar to that observed with the other three genes (Fig. 4b). PRK2 and PRK4 encode pollen-specific receptor-like kinases (PRKs). Whereas PRK2/AT2G07040 regulates the ROP1 signaling pathway for polarized cell expansion [START_REF] Chang | AtPRK2 promotes ROP1 activation via RopGEFs in the control of polarized pollen tube growth[END_REF] , PRK4/AT3G20190 is thought to mediate the extracellular signals organizing cytoskeleton orientation during polar pollen tube growth [START_REF] Duckney | Actin-membrane interactions mediated by NETWORKED2 in Arabidopsis pollen tubes through associations with Pollen Receptor-Like Kinase 4 and 5[END_REF] . As for RKF2 (AT1G19090), it encodes a putative receptor-like serine threonine kinase that interacts with CATION/H + EXCHANGER10, which has been implied in targeting of pollen tubes to ovules [START_REF] Lu | Pollen tubes lacking a pair of K+ transporters fail to target ovules in Arabidopsis[END_REF][START_REF] Evans | The roles of the cation transporters CHX21 and CHX23 in the development of Arabidopsis thaliana[END_REF] . Finally, AT2G16030 encodes a putative pollen-specific S-adenosyl-MTase [START_REF] Muschietti | How many receptor-like kinases are required to operate a pollen tube[END_REF][START_REF] Loraine | RNA-seq of Arabidopsis pollen uncovers novel transcription and alternative splicing[END_REF] , which we have named PLOUTOS (PLOU, see "Discussion"). Remarkably, these four genes show strong pollen-specific co-expression (Supplementary Fig. 5a) and, based on publicly available membrane-bound proteininteractome surveys [START_REF] Jones | Border control-a membrane-linked interactome of Arabidopsis[END_REF][START_REF] Dong | Proteome-wide, structure-based prediction of protein-protein interactions/new molecular interactions viewer[END_REF] , they also define a protein interaction network (Supplementary Fig. 5b).

Discussion

Our results reveal that the activity of the DNA glycosylase ROS1 is not confined to somatic tissues, and that it contributes to the demethylation of the genome in the VC. Moreover, they demonstrate that DME/ROS1-mediated DNA demethylation plays an essential role in the regulation of genes required for pollen tube function, thus providing a molecular explanation for the male sterility phenotypes observed in dme/+ and dme/+;ros1 mutants.

It has been reported that the pollen sterility in dme/+ mutants is more pronounced in the accession Col-0 than in Ler [START_REF] Choi | DEMETER, a DNA glycosylase domain protein, is required for endosperm gene imprinting and seed viability in Arabidopsis[END_REF][START_REF] Schoft | Function of the DEMETER DNA glycosylase in the Arabidopsis thaliana male gametophyte[END_REF] . Furthermore, ROS1-dependent DNA demethylation varies between accessions in vegetative tissues [START_REF] Tang | The DNA demethylase ROS1 targets genomic regions with distinct chromatin modifications[END_REF] and DNA methylation levels in VCs are higher in Col-0 compared to Ler [START_REF] Borges | Transposon-derived small RNAs triggered by miR845 mediate genome dosage response in Arabidopsis[END_REF] . This higher DNA methylation level affects mainly the CHH context, consistent with RdDM being especially aggressive in male sex cells, where it targets numerous genes [START_REF] Walker | Sexual-lineage-specific DNA methylation regulates meiosis in Arabidopsis[END_REF] . Thus, we can expect demethylase activity to be more critical in Col-0 than in Ler to counterbalance the potential silencing of genes required for pollen function. Moreover, genes with strain-independent dense, TE-like DNA methylation tend to be silenced during vegetative growth and expressed specifically in pollen and seeds, presumably as a result of active DNA demethylation in the respective gametophytes [START_REF] Schmitz | Patterns of population epigenomic diversity[END_REF][START_REF] You | Atypical DNA methylation of genes encoding cysteine-rich peptides in Arabidopsis thaliana[END_REF] . Remarkably, three of the genes that are required for male fertility (AT2G16030/PLOU, PRK2, and RKF2) show dense TE-like DNA methylation outside the VC (Fig. 3b and Supplementary Fig. 4) and are specifically activated in pollen (Supplementary Fig. 5a). Such heterochomatization of reproductive genes might contribute to their rapid evolution [START_REF] Dapper | Relaxed selection and the rapid evolution of reproductive genes[END_REF] , as cytosines show higher mutation rates when methylated [START_REF] Ossowski | The rate and molecular spectrum of spontaneous mutations in Arabidopsis thaliana[END_REF] . Be that as it may, whether the demethylation of TEs during reproduction is a side effect of a gene regulatory role or vice versa remains to be determined.

Among the four pollen-specific genes regulated by DME and ROS1, AT2G16030 was previously uncharacterized. Nonetheless, its protein product is thought to interact with MALE DIS-COVERER 2, a receptor-like kinase involved in pollen tube guidance [START_REF] Wang | A receptor heteromer mediates the male perception of female attractants in plants[END_REF][START_REF] Muschietti | How many receptor-like kinases are required to operate a pollen tube[END_REF] . Given that dme mutant pollen tubes appear to be blind to maternal signals that direct pollen tube progression, we propose to name gene AT2G16030 as PLOUTOS (PLOU), after the son of Demeter and god of wealth, who was blinded by Zeus in Greek mythology [START_REF] Hesiod | Theogony[END_REF] . Pollen tube progression is governed by multiple molecular interactions with female tissues, through preovular (sporophytic) and ovular (gametophytic) signaling [START_REF] Higashiyama | Gametophytic pollen tube guidance: attractant peptides, gametic controls, and receptors[END_REF][START_REF] Johnson | A fruitful journey: pollen ttube navigation from germination to fertilization[END_REF][START_REF] Zhong | Peptide/receptor-like kinase-mediated signaling involved in male-female interactions[END_REF] and it has been hypothesized that DNA demethylation is required for the ovule-specific expression of genes encoding cysteine-rich peptides, which could act as pollen tube attractants [START_REF] You | Atypical DNA methylation of genes encoding cysteine-rich peptides in Arabidopsis thaliana[END_REF] . Thus, it is tempting to speculate that active DNA demethylation has been co-opted in the male and female reproductive organs to enable the expression of genes underpinning parental communication and reproductive success.

Methods

Plant material and growth conditions. Mutant alleles dme-6 (GK-252E03-014577) and ros1-3 (WiscDsLox469C11) used in this study are in the Col-0 background and have been characterized previously [START_REF] Penterman | DNA demethylation in the Arabidopsis genome[END_REF][START_REF] Shirzadi | Genome-wide transcript profiling of endosperm without paternal contribution identifies parent-of-origin-dependent regulation of AGAMOUS-LIKE36[END_REF] . Seeds where surfacesterilized and sowed on 1/2 Murashige and Skoog (MS) medium plates containing sulfadiazine in case of dme-6/+ mutant lines or plates without antibiotic selection under sterile conditions. After few days of 4 °C stratification the plates where transferred to 22.5 °C/16 h light-17 °C/8 h dark growth conditions for 10-15 days before transfer to soil under the same growth conditions with 75% humidity control.

Quantification of dme transmission rate. Pre-emasculated Col-0 wild-type flowers were pollinated with pollen from either dme/+, ros1/+, or dme/+;ros1 mutants. As the T-DNA insertion responsible for the dme mutation confers resistance to sulfadiazine, mature seeds derived from these crosses were sterilized and sowed on 1/2 MS medium containing sulfadiazine, for selection of the dme allele-containing plants. To verify proper pollination, siliques were harvested separately and only crosses with at least 20 seeds/silique were taken into account. In general, the total number of seeds per silique did not differ in crosses with Col-0, ros1, dme/+, and dme/+;ros1 fathers. We corroborated sulfadiazine resistance with the presence of the T-DNA insertion in the DME locus by comparing the numbers of antibiotic resistant plants with those identified as dme/+ lines by PCR genotyping of two separate populations of the progeny of the same cross (Col_x_dme/+): Sulfadizine-resistant: 17.5% (n = 480); PCR genotyping: 17.8% (n = 185); p = 0.9115, Fisher's exact test. The ros1 mutant allele segregation was measured by PCR genotyping (see Supplementary Data 11 for primer information). In the mutant vs. wildtype pollen competition assays, limited amount of pollen derived from dme/+ and dme/+;ros1 mutants was applied to the stigmata of the pistils. In both cases, the anthers contain approximately half DME wild-type and half dme mutant pollen (see Supplementary Fig. 1a,b). To verify that fertilization was indeed restricted to few pollen grains, only siliques with less than 25 seeds were taken into account, which corresponds to about half of the normal seed set in Col-0. Mutant allele transmission frequencies were established using Microsoft Excel software version 2013.

In vivo pollen tube visualization. Pistils from 24 h fertilized flowers were fixed for 2 h in acetic acid/EtOH (1:3) solution, under vacuum at room temperature after which pistils were re-hydrated with successive incubation with 70% EtOH, 50% EtOH, 30% EtOH, and finally distilled water. Pistils were then transferred to 8 M NaOH solution overnight and washed three times with distilled water. Staining was performed by incubating pollinated pistils in Methyl-Blue solution (0.2 mg/ml methyl blue in 100 mM KH 2 PO 4 pH 11), 2 h, under dark condition at room temperature. After transfer on microscopic slide in 50% glycerol, fluorescence was observed under UV light. Microscopy acquisition were performed on a Zeiss Axioskop 2 plus microscope and images were treated using ImageJ v1.52.

Pollen viability assay. Alexander's staining were performed to assess pollen viability. Stamens containing mature pollen were treated with few microliter of Alexander solution and observed under light microscope.

Pollen isolation and RT-qPCR We used a published protocol (BioProtocols site: Bio-101: e67. DOI: 10.21769/BioProtoc.67) with minor changes [START_REF] Lu | RNA isolation from Arabidopsis pollen grains[END_REF] . Mature flower of about 20 plants were collected and put into 150 mL of cold mannitol (0.3 M) for quadruplicates of Col-0, ros1, dme/+, and dme/+;ros1 each. In the case of the transgene complementation lines, duplicates were used. After 2 min of agitation by thorough shaking, the solution was filtered using a 100 µm nylon mesh. Isolated pollen was collected after 5 min centrifugation (3500 × g at 4 °C), the pellet rinsed with water and centrifuged again at 10,000 × g for 5 min. Purified pollen was stored at -80 °C and the material homogenized with metallic beads using a Qiagen tissue-lyser (2 × 1′ 30 Hz) to perform total RNA extraction using a Qiagen RNeasy Mini Kit, following the manufacturer's instruction. RNA integrity was verified on gel after speZZctrometric quantification and cDNA synthesis was carried out with 10 μg of RNA for Col-0 wild type and mutants and 1 μg of RNA in the case of the transgene complementation lines using the Invitrogen Superscript IV Reverse Transcriptase, following the manufacturer's instructions. qPCR was done employing Roche SYBR Green reagent and a Roche LightCycler 480 with technical duplicates for each sample and primers (Supplementary Data 11) were tested using the Roche LightCycler Software 4.1 for specificity and >90% efficiency beforehand.

Cell sorting of VCs and SCs. Pollen nuclei of wild-type (Col-0) and mutant dme/+, ros1, and dme/+;ros1 plants were purified by FACS using SYBR Green staining as previously described [START_REF] Schoft | SYBR green-activated sorting of Arabidopsis pollen nuclei based on different DNA/RNA content[END_REF][START_REF] Borges | FACS-based purification of Arabidopsis microspores, sperm cells and vegetative nuclei[END_REF] . Briefly, open flowers were collected into a 50 mL falcon tubes and vortexed in 10 mL of Galbraith buffer (45 mM MgCl 2 , 30 mM Sodium Citrate, 20 mM MOPS, 1% Triton-100 pH 7.0) for 3 min, at room temperature. This crude fraction was then filtered through Miracloth (Calbiochem) and centrifuged for 1 min at 2600 × g to concentrate the pollen fraction. The pollen was then transferred to a 1.5 mL Eppendorf tube containing ~100 μL of acid-washed glass beads (425-600 μm, Sigma) and vortexed continuously at maximum speed for 3 min, to break the pollen cell wall. The fraction containing the released nuclei was then filtered through a 10 μm mesh (Celltrics, Sysmex-Partec) to exclude pollen debris and stained with SYBR Green dye (Lonza). FACS was performed using a FACSAria IIU cell sorter (BD Biosciences), using the integrated FACSDiva v6.1 software with 70 µ nozzle at 70 psi. A 488 nm laser was used for SYBR Green excitation, which was detected by a 530/30 nm band-pass filter (Supplementary Fig. 2). Approximately 500,000 nuclei from each genotype and cell type were purified, and genomic DNA was extracted using the Masterpure kit (Epicenter).

Whole-genome bisulfite sequencing and DMR calling. Genomic DNA was fragmented by Covaris to ~300 bp and fragments were end-repaired, A-tailed, and ligated to methylated Illumina adaptors (Bioo Scientific). Ligated fragments were bisulfite treated with the EZ DNA Methylation-Gold Kit (Zymo) and PCR-enriched with KAPA high-fidelity Uracil+ polymerase (Roche). Reads were preprocessed with Trimmomatic v0.32 [START_REF] Bolger | Trimmomatic: a flexible trimmer for Illumina sequence data[END_REF] to remove adaptors and Bismark v0.16.3 [START_REF] Krueger | a flexible aligner and methylation caller for Bisulfite-Seq applications[END_REF] was used to map filtered reads to the Arabidopsis TAIR10 genome reference, to calculate methylation at each individual cytosine. DMRs were defined as 50 bp bins containing at least 2, 3, or 4 differentially methylated mCGs, mCHGs or mCHHs with an absolute methylation difference of 0.40, 0.25, and 0.10, respectively. Bins localizing within 400 bp of each other were merged. A summary of all genome-wide bisulfite sequencing data generated in this study is presented in Supplementary Data 1 and is accessible through the NCBI's Gene Expression Omnibus (GSE141154).

GO-term analysis. The DAVID gene functional classification tool [START_REF] Huang | Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources[END_REF] was used to identify categories of biological function of genes associated to hypo-CG-DMRs detected in the comparison between wild-type VCs vs. SCs, as well as wild-type vs. dme/+ and dme/+;ros1 VCs. TAIR10 genome was used as background. We used the standard parameters to detect over-represented GO terms for biological function, giving Fisher's exact p-values, fold-enrichment scores, and number of genes from our gene-list found in a given term.

Plasmid construction and plant transformation. Functional complementation assay was performed by using the Multisite Gateway Technology (Invitrogen). Promoters were PCR-amplified from genomic DNA and inserted into the pENTR5'-TOPO vector using the pENTR5′-TOPO TA Cloning Kit (Invitrogen). CDSs of candidate genes were amplified by PCR on cDNA extracted from mature pollen, using forward and reverse primers containing attB1 and attB2 sites, respectively (Supplementary Data 11). Gateway BP reaction was used to insert amplified CDS into the pDONR207 vector. We used Gateway LR reaction to clone the ANX2 (AT5G28680) promoter region in combination with the CDS of PRK2, PRK4, and RALFL15, as well as the regulatory sequence of Raba4D (AT3G12160) in front of RKF2, TRX5, and PLOU (AT2G16030) into the pDestination vector pB7m24GW3. Transformation of Agrobacterium tumefaciens (strain GV3101) were then performed by incubation with 1 µg of the produced vector (5 min on ice), followed by 5 min freezing in liquid nitrogen. After 1 min recovery at 37 °C, 1 mL of Luria-Bertani (LB) medium were added for a 2 h incubation at 28 °C (with shaking), plated on LB containing gentamycin, rifampicin + spectinomycin, and incubated 48 h at 28 °C. Plants were transformed by dipping in A. tumefaciens solution. Primers used to generate constructs are listed in Supplementary Data 11.

Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Fig. 1

 1 Fig. 1 DME and ROS1 act semi-redundantly to promote pollen tube progression. a Transmission frequency of the dme and ros1 alleles in crosses between Col-0 wild-type mothers and ros1/+, dme/+, dme/+;ros1/+, and dme/+;ros1 pollen donors as bar plot giving the mean ± SD as error bars and individual data points shown as gray dots. Number of individual plants and biological replicates is given below the graph and pairwise comparisons of dme transmission rate between the different crosses revealed significant differences (p-values of two-sided T-tests in pairwise comparison is shown in the graph). b Fluorescent microscopy of aniline blue stained pistils 24 h after crosses of wild-type Col-0 mothers with Col-0, dme/+ and dme/+;ros1 fathers, respectively, revealing pollen tubes in the transmitting tract. Red channel shows autofluorescence of maternal tissues, including the ovules. White arrow indicates aberrant pollen tube trajectory in the mutants; white scale bar equals 20 µm in all photographs. c Violin plots showing the distribution of the transmission frequency of the dme allele in individual siliques from crosses between wild-type Col-0 mother plants and dme/+, as well as dme/+;ros1 fathers with either excess of pollen or excess of ovules (few pollen grains used in the respective cross). Inserted boxplots represent 50% of all values with the second and third quartile separated by the median value as black line, mean value is shown as a red rhomb together with the number (in red). Number of individual plants and siliques (which equals replicates) as well as p-value of two-sided T-test is given. Source data underlying Fig. 1a, c are provided as a Source Data file.

Fig. 2

 2 Fig. 2 DME and ROS1 are responsible for DNA demethylation of the vegetative cell in pollen. a Number and association with genome annotations of CG-DMRs detected in the comparison between VCs and SCs in wild-type (Col), ros1, dme/+ and dme/+;ros1 mutant plants (hypomethylated = lower DNA methylation level in VCs). b Venn diagram showing the overlap of CG-hypo DMRs detected between the VCs and the SCs of Col-0 wild-type, dme/+, and dme/+;ros1. c Heatmap of absolute CG methylation level in SCs and VCs of CG-hypo DMRs detected between VCs and SCs in Col-0 wild type (11,107 regions). The lines represent the indicated genotype for SCs and VCs, respectively. DMRs in the columns were sorted by complete linkage hierarchical clustering using Euclidean distance.

Fig. 3

 3 Fig. 3 DME and ROS1 are required for the expression of genes involved in pollen tube function. a Venn diagram giving the number and overlap of genes implied in pollen tube function (see Supplementary Data 9) associated with hyper DMRs in the respective mutant VCs compared to wild-type VCs. b Genome-browser view of two genes involved in pollen tube function (PRK4/AT3G20190 and PLOU/AT2G16030), showing the absolute DNA methylation level for each cytosine context as indicated in the graph for five samples (wild-type [Col-WT] seedlings and VCs of Col-0 wild type, ros1, dme/+ and dme/+;ros1). c RT-qPCR from pollen of the given genotypes of transcripts showing lower abundance in dme/+ and dme/+;ros1 compared to wild-type (Col-0) pollen. For each sample four biological replicates have been used and relative expression level calculated with respect to the reference genes shown on the left. Bars represent mean values with SD shown as error bars. p-values of two-sided T-test in the respective comparison with wild-type level are indicated by stars above the bars; absence of stars indicates no significant difference (p > 0.05; see Supplementary Data 10 for exact p-values). Source data underlying Fig. 3c are provided as a Source Data file.

Fig. 4

 4 Fig. 4 DME/ROS1 target genes are required for male fertility. a Strategy to functionally validate the role of DME/ROS1 target genes for pollen tube function. In the wild-type DME/ROS1 target genes become demethylated in VCs, a prerequisite for proper gene activation. In dme mutant VCs the demethylation is absent and hence prevents gene expression of the underlying gene. By introducing DME/ROS1 target genes under the control of a DNA methylation-independent, pollen-specific promoter, the gene activity is restored in dme VCs and should, at least partially, rescue the male dme transmission defect. b Box plot showing male dme mutant allele transmission frequency of crosses of wild-type (Col-0) mothers with dme/+ (left graph) and dme/+;ros1 (right graph) fathers, carrying transgenes to express DME/ROS1 targets independently of DNA methylation in pollen. Transgenes are indicated below the X-axis. pFIS2:RFP is a negative control, containing a FIS2 promoter expressing a reporter gene in the same vector backbone as the other constructs. Individual data points are shown as gray dots and boxes contain 50% of all values, with the second and third quartile separated by the median level; red lines indicate the mean level with corresponding number given in red. The total number of progeny, as well as the replicates (in bold) is given below the graph, representing single siliques in the case of dme/+ or 1-4 siliques in the case of dme/+;ros1 for each cross. Pairwise comparison with the dme-only crosses revealed significantly elevated dme transmission in the case of AT2G16030/PLOU, PRK4, and PRK2 in the dme/+ and dme/+;ros1 mutant background and RKF2 in the dme/+;ros1 background (red stars indicate statistical significance in pairwise comparison with control crosses without transgene: ***p < 0.001; *p < 0.05; the exact p-value derived from two-sided T-test is shown above the respective cross). Source data underlying Fig. 4b are provided as a Source Data file.

Table 1

 1 GO-term enrichment for genes associated with hypo-CG-DMRs detected between Col VC and SC.

	Term

a Number of genes from list. b Percentage of genes in this category/Total number of genes in the list. c Fisher's exact p-value; <0.05 is considered strongly enriched (only values <0.02 are shown). ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-20606-1 4 NATURE COMMUNICATIONS | (2021) 12:410 | https://doi.org/10.1038/s41467-020-20606-1 | www.nature.com/naturecommunications

Table 3

 3

	Term

GO-term enrichment for genes associated with hypo-CG-DMRs detected between Col and dme;ros1 VCs. a Number of genes from list. b Percentage of genes in this category/Total number of genes in the list. c Fisher's exact p-value; <0.05 is considered strongly enriched (only values <0.02 are shown).

Table 2

 2 GO-term enrichment for genes associated with hypo-CG-DMRs detected between Col and dme VCs.

	Term

a Number of genes from list. b Percentage of genes in this category/Total number of genes in the list. c Fisher's exact p-value; <0.05 is considered strongly enriched (only values <0.02 are shown).

NATURE COMMUNICATIONS | (2021) 12:410 | https://doi.org/10.1038/s41467-020-20606-1 | www.nature.com/naturecommunications

Acknowledgements

We thank members of the Colot lab for helpful discussions and critical reading of the manuscript, as well as Erwann Caillieux and Sevrine Larney for technical assistance in plant care and collecting pollen material. Gregory Copenhaver is kindly acknowledged for providing the FTL1311 line. This work was supported in part by the European Union Seventh Framework Program Network of Excellence EpiGeneSys (HEALTH-F4-2010-257082, to V.C.) and the Investissements d'Avenir ANR-10-LABX-54 MEMOLIFE, ANR-11-IDEX-0001-02 PSL*Research University (both to V.C.). S.K. was supported by a PhD studentship from PSL University, with additional funding provided by MEMOLIFE. Research in the Martienssen laboratory is supported by the US National Institutes of Health (NIH) grant R01 GM067014 and by the Howard Hughes Medical Institute. We acknowledge assistance from the Cold Spring Harbor Laboratory Shared Resources, which are funded in part by the Cancer Center Support Grant (5PP30CA045508). Further financial support includes a European Research Council Starting Independent Researcher Grant (210867-2) to A.S.

Data availability

Data supporting the findings of this work are available within the paper and its

Supplementary Information files. A reporting summary for this article is available as a Supplementary Information file. The datasets and plant materials generated and analyzed during the current study are available from the corresponding author (D.B.) upon request. WGBS data that support the findings of this study have been deposited at NCBI's Gene Expression Omnibus under accession number GSE141154. Source Data are provided with this paper.

Author contributions

Competing interests

The authors declare no competing interests.