Justine Foucher

Mylène Ruh

Martial Briand

Anne Préveaux

Florian Barbazange

Tristan Boureau

Marie-Agnès Jacques

Nicolas W G Chen
email: nicolas.chen@agrocampus-ouest.fr

Improving common bacterial blight phenotyping by using

Keywords: phenotyping, plant disease, bacterial blight, Xanthomonas, TAL effectors, Phaseolus vulgaris

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

INTRODUCTION

Monitoring the impact of pathogens on plants is essential for improving knowledge on plantpathogen interactions and developing effective management practices [START_REF] Bock | Plant disease severity estimated visually, by digital photography and image analysis, and by hyperspectral imaging[END_REF].

Assessing plant symptoms is a key step in detecting plant resistance or evaluating the virulence of a pathogen. Pathogenicity tests must give homogeneous and reproducible results and use an objective method of symptom assessment to be interpretable [START_REF] Nutter | Disease assessment concepts and the advancements made in improving the accuracy and precision of plant disease data[END_REF]. Visual symptom assessment is a simple and easily accessible method used in many studies of plant-pathogens interactions. However, it lacks objectivity, accuracy and precision [START_REF] Bock | Visual rating and the use of image analysis for assessing different symptoms of citrus canker on grapefruit leaves[END_REF]Poland and 3 Nelson, 2010). The recent development of different optical techniques has allowed to automate the process of symptom assessment while ensuring a standardization of results [START_REF] Mahlein | Present and Future Trends in Plant Disease Detection[END_REF]. As such, computational image analysis provides a more objective, accurate, reproducible and quantitative measure of disease severity than visual assessment.

Common bacterial blight of bean (CBB) is a significant bacterial disease on common bean, with yield losses of more than 40% under favorable conditions [START_REF] Belete | Common Bacterial Blight (Xanthomonas axonopodis pv. phaseoli) of Beans with Special Focus on Ethiopian Condition[END_REF][START_REF] De Luque | Major constraints and trends for common bean production and commercialization; establishing priorities for future research[END_REF]. Symptoms can occur on leaves, stems, pods and seeds [START_REF] Zaumeyer | A monographic study of bean diseases and methods for their control[END_REF]. Leaf symptoms initially appear as water-soaked spots, which enlarge and can coalesce with adjacent lesions [START_REF] Goodwin | Water stress in leaves of Phaseolus vulgaris infected with Xanthomonas campestris pv. phaseoli[END_REF]. Foliar lesions are often surrounded by a chlorotic halo and evolve in necrosis, possibly resulting in the death of the entire leaf and partial defoliation of the plant. Water-soaked spots and necrosis can also be observed on pods and seeds.

These symptoms evolve in dark red-brown lesions that are generally circular and slightly sunken [START_REF] Vidaver | Xanthomonas campestris pv. phaseoli: cause of common bacterial blight of bean[END_REF].

To phenotype CBB symptoms, different organs (seeds, pods, first leaves, trifoliate leaves, stems) can be inoculated in different ways. In particular, leaves can be inoculated by dipping, spraying, rubbing, multiple needles or infiltration [START_REF] Aggour | Comparison of leaf and pod disease reactions of beans (Phaseolus vulgaris L.) inoculated by different methods with strains of Xanthomonas campestris pv. Phaseoli (Smith) dye[END_REF][START_REF] Popovic | Response of different beans against common bacterial blight disease caused by Xanthomonas axonopodis PV. phaseoli[END_REF]. Traditionally, symptoms of CBB were assessed by visual evaluation using different rating scales based on a visual estimation of the percentage of infected leaf area [START_REF] Aggour | Comparison of leaf and pod disease reactions of beans (Phaseolus vulgaris L.) inoculated by different methods with strains of Xanthomonas campestris pv. Phaseoli (Smith) dye[END_REF][START_REF] Cafati | Effect of Host on Multiplication and Distribution of Bean Common Blight Bacteria[END_REF][START_REF] Opio | Studies on Seed Transmission of Xanthomonas campestris pv phaseoli in Common Beans in Uganda[END_REF][START_REF] Pastor-Corrales | Comparing 2 Inoculation 19 Techniques for Evaluating Resistance in Beans to Xanthomonas campestrís[END_REF][START_REF] Zapata | A proposal for a uniform screening procedure for the reennhouse evalualtion of variability of Xanthomonas axonopodis pv. phaseoli and resistance on leaves of Phaseolus vulgaris[END_REF]. In 2012, it was shown that assessment of CBB symptoms by RGB image analysis was more reproducible and more objective than a rating scale, and presented a high differentiation power between plant genotypes [START_REF] Xie | Application of image analysis in studies of quantitative disease resistance, exemplified using common bacterial blight-common bean pathosystem[END_REF].

Another method was developed based on leaf inoculation by dipping combined with chlorophyll fluorescence imaging [START_REF] Rousseau | High throughput quantitative phenotyping of plant resistance using chlorophyll fluorescence image analysis[END_REF]. This assessment method was successfully used to 4 discriminate different degrees of resistance in common bean against Xpp-Xcf [START_REF] Foucher | Common bean resistance to Xanthomonas is associated with upregulation of the salicylic acid pathway and downregulation of photosynthesis[END_REF][START_REF] Rousseau | High throughput quantitative phenotyping of plant resistance using chlorophyll fluorescence image analysis[END_REF].

CBB is caused by Xanthomonas phaseoli pv. phaseoli (Xpp) and Xanthomonas citri pv. fuscans (Xcf) [START_REF] Chen | Common bacterial blight of bean: a model of seed transmission and pathological convergence[END_REF][START_REF] Constantin | Genetic characterization of strains named as Xanthomonas axonopodis pv. dieffenbachiae leads to a taxonomic revision of the X. axonopodis species complex[END_REF]. Pathological convergence of these two pathovars is probably due to extensive horizontal gene transfers, which led to genomic regions sharing 100% nucleotide identity between Xpp and Xcf [START_REF] Aritua | Genome sequencing reveals a new lineage associated with lablab bean and genetic exchange between Xanthomonas axonopodis pv. phaseoli and Xanthomonas fuscans subsp. fuscans[END_REF][START_REF] Chen | Horizontal gene transfer plays a major role in the pathological convergence of Xanthomonas lineages on common bean[END_REF]. These homologies allowed the development of specific molecular tools for detecting both Xpp and Xcf on seed lots [START_REF] Audy | Detection of the bean common blight bacteria, Xanthomonas campestris pv. phaseoli and X.c.phaseoli var. fuscans, using the polymerase chain reaction[END_REF][START_REF] De Paiva | Rapid Detection of Xanthomonas citri pv. Fuscans and Xanthomonas phaseoli pv. Phaseoli in Common Bean by Loop-Mediated Isothermal Amplification[END_REF][START_REF] Grimault | Detection of Xanthomonas axonopodis pv. phaseoli on Phaseolus vulgaris[END_REF]. Different Xpp-Xcf strains may have different levels of pathogenicity regardless of whether they belong to one or the other pathovars [START_REF] Mkandawire | Genetic Diversity and Pathogenic Variation of Common Blight Bacteria ( Xanthomonas campestris pv . phaseoli and X . campestris pv . phaseoli var . fuscans ) Suggests Pathogen Coevolution with the Common Bean[END_REF]. In addition, common bean resistance to CBB is mediated by numerous quantitative trait loci [START_REF] Monteiro | Sources, spectrum, genetics, and inheritance of phaseolus vulgaris resistance against xanthomonas citri pv. fuscans[END_REF][START_REF] Singh | Breeding Common Bean for Resistance to Common Blight : A Review[END_REF][START_REF] Yu | Development and Application of Molecular Markers to Breed Common Bean ( Phaseolus vulgaris L .) for Resistance to Common Bacterial Blight ( CBB )[END_REF]. Differences in aggressiveness combined with the presence of quantitative resistances lead to a wide range of possible disease intensities [START_REF] Duncan | Interaction of common bacterial blight bacteria with disease resistance quantitative trait Loci in common bean[END_REF]. Thus, the interaction between common bean and Xpp-Xcf must be finely phenotyped, in order to detect these variations as accurately as possible.

The pathogenicity of Xanthomonas is partly mediated by a type III secretion system (T3SS) and associated type III effectors (T3Es) [START_REF] An | Mechanistic insights into host adaptation, virulence and epidemiology of the phytopathogen Xanthomonas[END_REF][START_REF] Büttner | Regulation and secretion of Xanthomonas virulence factors[END_REF]. Among T3Es, Xanthomonas bacteria possess transcription activator-like effectors (TALEs). TALEs are injected inside the plant cell via the T3SS and migrate to the nucleus where they are able to induce targeted genes of the plant, often leading to disease enhancement [START_REF] Boch | Xanthomonas AvrBs3 family-type III effectors: Discovery and function[END_REF]. Nine different TALE-encoding genes and alleles were discovered in Xpp-Xcf [START_REF] Ruh | Xanthomonas adaptation to common bean is associated with horizontal transfers of genes encoding TAL effectors[END_REF]. Xcf strain 6165R possesses only one tal gene named tal22B while Xpp strain 6546R bears two tal genes named tal19I and tal18H.

In this study, we generated tal mutant strains 6165RΔtal22B and 6546RΔtal18H. Then, the pathogenicity of strains 6165R and 6546R were compared to each other and to their corresponding 5 tal mutants. For this, we used two different tests to phenotype CBB symptoms in controlled conditions. The first method corresponded to the method developed by [START_REF] Rousseau | High throughput quantitative phenotyping of plant resistance using chlorophyll fluorescence image analysis[END_REF], consisting of dipping the first trifoliate leaf in bacterial suspension followed by chlorophyll fluorescence imaging (CFI). The second method corresponded to a new pathogenicity test consisting of rub-inoculation of the first leaves and evaluation of symptoms by machine learningtrained imaging (MLI). MLI was performed on RGB images using the ilastik software [START_REF] Berg | Ilastik: Interactive Machine Learning for (Bio)Image Analysis[END_REF], which was recently exploited in biomedical and environmental studies as well as plant symptoms assessment [START_REF] Ilett | Application of automated electron microscopy imaging and machine learning to characterise and quantify nanoparticle dispersion in aqueous media[END_REF][START_REF] Ojeda-Martinez | Saving time maintaining reliability: a new method for quantification of Tetranychus urticae damage in Arabidopsis whole rosettes[END_REF][START_REF] Pike | An adaptable analysis workflow for characterization of platelet spreading and morphology[END_REF][START_REF] Rashid | Highly multiplexed immunofluorescence images and single-cell data of immune markers in tonsil and lung cancer[END_REF].

MATERIALS AND METHODS

Bacterial strains and growth conditions

Xanthomonas phaseoli pv. phaseoli strain 6546R and Xanthomonas citri pv. fuscans strain 6165R are rifamycin-resistant derivatives of strains CFBP6546 and CFBP6165 respectively. Strains were grown at 28 °C for 48 h on trypticase soy agar (TSA) medium (17.0 g.L - 1 pancreatic digest of casein; 3.0 g.L - 1 enzymatic digest of soy bean; 5.0 g.L - 1 NaCl; 2.5 g.L - 1 K 2 HPO 4 ; 2.5 g.L - 1 glucose; 15 g.L - 1 agar), then at 28 °C for 24 h on TSA10 (a 1/10 dilution of TSA, except for agar maintained at 15 g.L - 1 ) to obtain fresh bacterial cultures. Media were supplemented by rifamycin (50 mg. L - 1 ) for selection.

Mutagenesis of tal genes and validation of mutants

Plasmid pK18mob::sacB [START_REF] Schäfer | Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutumicum[END_REF] was used to generate marker-free deletion mutants of tal22B in strain 6165R or tal18H in strain 6546R. First, specific primers (Table S1) were designed according to whole genome data [START_REF] Ruh | Xanthomonas adaptation to common bean is associated with horizontal transfers of genes encoding TAL effectors[END_REF], to amplify the flanking regions of 6 each tal gene by PCR using the PHusion® High Fidelity DNA polymerase (Finnzymes,Waltham, MA, USA) following the manufacturer's instructions. For each tal gene, PCR products were purified using the Wizard ® SV Gel and PCR Clean-Up System (Promega), then cloned in tandem into the suicide plasmid pK18mob::sacB using digestion by BsaI enzyme and ligation by T4 DNA ligase. Recombinant plasmids were electrostransferred into competent 6165R or 6546R strains.

Primary transformants were immediately grown in liquid MOKA medium (yeast extract 4 g.L - 1 ; casamino acids 8 g.L - 1 ; KH 2 PO 4 2 g.L - 1 ; MgSO 4 .7H 2 O 0.3 g.L - 1 ) for two hours at 28°C without selection, then plated on MOKA agar medium supplemented with kanamycin (50 µg.mL - 1 ). Kan r colonies were then plated on MOKA medium supplemented with kanamycin and sucrose at 10% for selecting secondary recombinants. Resulting colonies were tested for deletion of tal genes by DNA extraction followed by PCR and sequencing.

Two additional analyses were done to validate tal mutants. First, to confirm that TALE proteins corresponding to deleted genes were not produced, Western Blot assays were performed. Briefly, total proteins were extracted from 0.4 mL of overnight bacterial suspensions and migrated by Sodium Dodecyl Sulfate -Polyacrylamid Gel Electrophoresis (SDS-PAGE), then immunoblotted with a primary anti-TALE antibody. The anti-TALE antibody was raised in rabbit against an E. coli-produced designer TALE protein composed of the N-terminal domain of a TALE protein plus six repeats (I. Fuentes and L. Noël, unpublished). TALE backbone was derived from the hax3 tal gene sequence from X. campestris pv. campestris strain Xca5 as described [START_REF] Streubel | TAL effector RVD specificities and efficiencies[END_REF].

Second, to check the influence of deletion on the growth capacity of the mutants, bacterial growth control was performed. For this, liquid cultures at initial concentration of 1 × 10 6 CFU.mL -1 were prepared in TS10 broth, and the absorbance (λ = 600nm) was monitored every 8 or 30 min using a Labsystem Bioscreen C system, over an incubation period of 35 h at 28°C under shaking at 200 rpm. Each strain was controlled with technical triplicates and biological duplicates.

Genome sequencing, assembly and annotation

Genomic DNA of strains 6546RΔtal18H and 6165RΔtal22B was extracted with the Wizard ® Genomic DNA Purification Kit (Promega, Madison, USA) according to the manufacturer's recommendations. PacBio Single Molecule Real Time (SMRT) sequencing was performed at the Icahn School of Medicine at Mount Sinai (New York, USA) using one SMRT cell per strain to achieve ~100× coverage. De novo assembling was performed using the following procedure. Reads were filtered using PreAssembler Filter v1 of the SMRT Portal version 2.3 (Pacific Biosciences of California, Inc.), with Minimum Subread Length 500, Minimum Polymerase Read Quality 0.80 and Minimum Polymerase Read Length 100. Assembly was performed using Canu v1.5 [START_REF] Koren | Canu: Scalable and accurate long-read assembly via adaptive κ-mer weighting and repeat separation[END_REF]. Circularisation was done using Berokka v0.2.3 (https://github.com/tseemann/berokka).

Sequence start was fixed using the Fixstart command of Circlator v1.5.1 [START_REF] Hunt | Circlator: Automated circularization of genome assemblies using long sequencing reads[END_REF]. v1.14.6 (Seemann, 2014). Average nucleotide identity analyses between genomes of wild-type and TALE-deleted strains were performed with pyANI [START_REF] Pritchard | Genomics and taxonomy in diagnostics for food security: Soft-rotting enterobacterial plant pathogens[END_REF].

Plant materials and growing conditions

Seeds of the susceptible common bean cultivar JaloEEP558 were obtained from the Center for Tropical Agriculture (CIAT, Colombia), available under accession number G9603

(http://genebank.ciat.cgiar.org/genebank/main.do). Plants were sown in plastic pots (7 × 7 × 8 cm) containing pre-wetted soil. The sowings were covered with a P17 veil for four days for homogenized germination. Plants were grown in a growth chamber at 23°C/20°C (day/night) with a relative humidity of 80% and a photoperiod of 16 hours. Plants were watered every two days with water for the first seven days, then with a nutrient solution (7.5-5-15 N-P-K) for up to 15 days, and with a richer nutrient solution (15-10-30 N-P-K) until the end of the trial. The day before inoculation, relative humidity and temperature were increased at 95% and 28°C/25°C (day/night) to provide adequate conditions for infection. On the third day after inoculation, humidity was reduced to 80% until the end of the assay.

Pathogenicity assays

For both methods, bacterial suspensions were calibrated at 1×10 7 CFU.mL - 1 in sterile distilled water, and symptoms were monitored two weeks after inoculation. Each pathogenicity test was performed twice independently.

For phenotyping by chlorophyll fluorescence imaging (CFI), inoculations were performed at stage V1 (first trifoliolate leaf unfolded) by dipping the first trifoliate leaf for 30 s into bacterial suspensions or water as control. Symptom development was monitored by CFI at the PHENOTIC Seeds and Plants platform of the IRHS in Angers (France) as described in [START_REF] Rousseau | High throughput quantitative phenotyping of plant resistance using chlorophyll fluorescence image analysis[END_REF].

Briefly, inoculated leaflets were collected and set in the dark for 30 min. Then, for each leaflet, a first picture was taken under a modulated flash of light to measure basal fluorescence (F0) of the tissues, followed by another picture taken under a high flash of saturating light to measure the maximum fluorescence emission level (Fm). For each pixel, the maximum quantum yield of photosystem II photochemistry (Fv/Fm = (Fm-F0)/Fm) was calculated using Phenoplant (http://www.phenoplant.org) to discriminate diseased and healthy leaflet areas [START_REF] Rousseau | Phenoplant: A web resource for the exploration of large chlorophyll fluorescence image datasets[END_REF]. For each plant, results correspond to the mean disease area percentage of the three leaflets coming from the same inoculated leaf.

For phenotyping by machine learning-trained imaging (MLI), inoculations were performed by rubinoculation on the two first leaves of eight-day-old plants. Rub-inoculation consisted of one passage of a gloved finger dipped in bacterial suspension or water as control. For each leaf, two inoculations were done on the limb on each side of the central vein. To evaluate the symptoms, detached leaves were put on a LED light table of 1,600 lumen and images were taken with a fixedheight digital camera (Canon EOS 700D, Canon Inc., Taiwan) and saved as JPEG files. Each image comprised the two first leaves of a same plant, each rubbed twice, thus representing four technical replicates. Machine learning-based pixel segmentation was performed using the Pixel Classification workflow from ilastik v1.3.3 [START_REF] Berg | Ilastik: Interactive Machine Learning for (Bio)Image Analysis[END_REF]. Training was performed using 14 features including color/intensity, edge and texture, on 37 images representative of the whole dataset in terms of leaf colour and intensity of symptoms (training file available upon request).

Three labels were used for discriminating the background, leaf tissues and symptoms. Symptoms corresponded to both chlorosis and necrotic tissues, as assessed by expert eye analysis. After the training, batch processing of all images was performed. Pixel quantification was done using FiJi [START_REF] Schindelin | Fiji: An open-source platform for biological-image analysis[END_REF]. Briefly, labels were retrieved using the "Image threshold" option. Then, the "Analyse particle" command was used to quantify pixels corresponding to either the background or the symptoms. Total leaf areas were retrieved by subtracting background pixels from the total image pixels.

RESULTS

Phenotyping of strains with different degrees of pathogenicity

For both inoculation methods, more severe symptoms appeared after inoculation of strain 6546R than strain 6165R (Fig. 1). However, statistical distinction between strains was superior using rubinoculation with MLI (p < 0.01) than dip-inoculation with CFI (p > 0.05). The differences observed 10 between both methods could be explained by the inoculation method itself, which appeared as playing a major role in the homogeneity of the symptoms. Indeed, dip inoculation of trifoliates led to symptoms developing mainly from the margins, being unevenly distributed over the leaflets and evolving into more or less extended patches hardly distinguishable between leaves inoculated by one strain or the other (Fig. 1b andc). Moreover, the occurrence of symptoms was stochastic as symptoms did not appear evenly on all leaflets of the same leaf, meaning that the variability between leaflets was even higher than between individuals in some cases (not shown). In contrast, symptoms were clearly distinguishable between both strains after rub inoculation (Fig. 1e andf).

For strain 6165R, symptoms appeared as tiny spots evenly distributed across the whole inoculated area, likely caused by bacteria entering through openings corresponding to trichomes damaged by the rubbing. For strain 6546R, most of the spots coalesced, leading to large symptomatic areas. In all, rub-inoculation produced more homogeneous and reproducible symptoms than dip-inoculation.

To test if CFI could be used for assessing symptoms on rub-inoculated leaves, we compared CFI and MLI after rub-inoculation of first leaves with strain 6165R (Supp. Fig. 1). While chlorotic areas were accurately retrieved with both image acquisition methods, CFI failed to detect the tiny spots corresponding to early symptoms. Moreover, CFI tended to take into account pixels outside the inoculated area, thus not corresponding to symptoms caused by the bacteria (e.g. the petiole), which was not the case for MLI. Therefore, MLI was more suited than CFI to detect symptoms on rubinoculated plants.

Description of tal mutants

To further evaluate the discriminating power of each symptom assessment method, we constructed two deletion mutants of tal genes named 6546RΔtal18H and 6165RΔtal22B. Both 6546RΔtal18H and 6165RΔtal22B were unable to produce TAL18H or TAL22B proteins, respectively (Fig. 2a andb). PacBio SMRT sequencing allowed us to compare the complete genome sequences of the mutants to their corresponding wild-type strains. In both cases, an average nucleotide identity of more than 99.99% was found between the wild-type strains and their mutants, indicating that no major modification occurred in the mutant strains.

As described before, both tal22B and tal18H are located on plasmids [START_REF] Ruh | Xanthomonas adaptation to common bean is associated with horizontal transfers of genes encoding TAL effectors[END_REF]. In strain 6165RΔtal22B, the deletion of tal22B was restricted to a clean gap of 3,915 bp in plasmid A corresponding to tal22B from start to stop (Fig. 2d). In strain 6546R, plasmid C comprised a gap encompassing tal18H plus 2137 bp (Fig. 2c) including three genes encoding short (60 to 107 aminoacid-long) hypothetical proteins (not shown). For this strain, directed mutagenesis failed until we tested more than 350 clones for tal18H deletion. The deletion was flanked by identical ISXac2 insertion sequences (IS) suggesting that it occurred through recombination between IS (Fig. 2c). Therefore, 6546RΔtal18H corresponded to a variant of strain 6546R presenting a spontaneous deletion of tal18H on plasmid C. For both strains, the deletion had no major effect on the bacterial growth of the mutants compared to the wild-type strains in TS10 medium (Fig. 2e andf).

Virulence evaluation of wild-type and mutant strains

With both methods, the mutant strains led to less symptoms than the corresponding wild-type strains, suggesting that tal18H and tal22B were involved in the pathogenicity of strains 6546R and 6165R, respectively (Fig. 3 and4). However, rub-inoculation with MLI significantly discriminated the two mutants from corresponding wild-type strains (p < 0.05), while dip-inoculation with CFI did not (p > 0.1). Interestingly, the accuracy of rub-inoculation followed by MLI was high enough to discriminate between strains presenting very low aggressiveness. Indeed, only 2% or 1% of the total leaf pixels corresponded to symptoms after inoculation with 6165R or 6165RΔtal22B, respectively, which further demonstrated the discriminative power of this method.

12

Phenotyping of symptoms after leaf dipping

Although training was done on images of rub-inoculated primary leaves, we tested if MLI could discriminate symptoms on trifoliate leaves after inoculation by dipping. For this, we performed both CFI and MLI on images taken from the same dip-inoculated plants (one image per leaflet).

We compared the CFI and MLI results to expert symptom assessment by eye using the reference scale of [START_REF] Opio | Studies on Seed Transmission of Xanthomonas campestris pv phaseoli in Common Beans in Uganda[END_REF]. Globally, MLI was able to detect most of the CBB symptoms retrieved by eye, while CFI detected comparatively less visible symptoms than the two other methods (Supp.

Fig. 2). In accordance with this, correlation of visual assessment with MLI was stronger than with CFI (Fig. 5). The high linear correlation found between visual assessment and MLI (R 2 > 0.96)

demonstrates that the MLI training developed in this study is a good estimator for symptom quantification on dip-inoculated leaves.

DISCUSSION

Our study highlights that rub-inoculation on first leaves combined with MLI represents a fast, simple and efficient way to quantify the fine symptom differences existing between strains differing by only one or few genes. The two tal mutants constructed here induced less symptoms than the wild-type strains, suggesting that TAL18H and TAL22B participate in the pathogenicity of strains 6546R and 6165R, respectively. However, cloning of tal genes and complementation of these strains is needed to confirm the role of these TALEs, especially because genes other than tal were missing in strain 6546RΔtal18H.

In addition to the gain of discriminating power, the rub-inoculation method presented several other advantageous characteristics compared to dip-inoculation. First, the time required to carry out a trial was reduced (Table 1). Indeed, the use of first leaves instead of first trifoliates saved one week of plant growing time, leading to a 22-day trial instead of a month. Moreover, the growth of first leaves was often more homogeneous than that of first trifoliate leaves. Consequently, for a same number of plants sown, more can be used for a pathogenicity test on first leaves than first trifoliates.

This can be an important factor for trials under controlled greenhouse conditions, where the space available is often limited. In addition, the average inoculation time per plant (including all time spent in the growth chamber) was three times longer for dip-inoculation than rub-inoculation (Table 1), as it required more handling time and a 30-second immersion of the leaf in the bacterial suspension. Furthermore, the volume of inoculum required was 20 times less for rub-inoculation than dip-inoculation. Therefore, less material was needed for rub-inoculation and the waste was easier to process. Finally, MLI requires simple materials such as a digital camera, a tripod and a LED table, which can be afforded quite easily. On the other hand, CFI requires a complete fluorescence imaging system that is much more expensive to purchase and maintain. The shooting of plants (including handling time) was also more than three time longer for chlorophyll fluorescence than RGB picture taking. This was mainly due to CFI requiring the leaves to be kept in the dark for 30 minutes prior shooting, then perform the shootings leaflet by leaflet in a dark room, while RGB pictures were done with both first leaves together.

Although the method developed here presents many advantages for symptom quantification, it is important to note that chlorophyll fluorescence imaging is able to detect non-visible symptoms resulting from a disruption of photosynthesis during the early stages of infection and has a potential to discriminate between strains differing by a single gene [START_REF] Méline | A Computation Method Based on the Combination of Chlorophyll Fluorescence Parameters to Improve the Discrimination of Visually Similar Phenotypes Induced by Bacterial Virulence Factors[END_REF]. The future development of pipelines combining different phenotyping methods will undoubtedly contribute to analyze more in-depth the complex contributions of single or multiple genes to the virulence of plant pathogens.

disease area as estimated by MLI in (a). Pixels corresponding to estimated symptoms are in red. (a)

  //github.com/PacificBiosciences/GenomicConsensus) with --algorithm best. Whole genome sequences of wild-type strains 6165R and 6546R (Briand et al., submitted) were used for comparative analyses. Annotation of whole genome assemblies was performed with Prokka

Figure 1 .

 1 Figure 1. Strain 6546R is more aggressive than strain 6165R. Images were taken at 14 days post

Figure 2 .

 2 Figure 2. Description of tal mutant strains 6546RΔtal18H (a, c, e) and 6165RΔtal22B (b, d, f).

Figure 3 .

 3 Figure 3. Pathogenicity of 6546RΔtal18H compared to wild-type strain 6546R. Images were taken

Figure 4 .

 4 Figure 4. Pathogenicity of 6165RΔtal22B compared to wild-type strain 6165R. Images were taken

Figure 5 .

 5 Figure 5. Correlation between different symptom quantification methods. Trifoliate leaves were

Figure 1 .Figure 2 .Figure 5 .Supplemental Figure 2 :

 1252 Figure 1. Strain 6546R is more aggressive than strain 6165R. Images were taken at 14 days post inoculation, and Evaluation of symptoms was done by chlorophyll fluorescence imaging on trifoliate leaves inoculated by dipping (a, b, c), or by machine learning-based imaging on first leaves inoculated by rubbing (d, e, f). Experiments were repeated twice independently, with five plants per assay, and concatenated (n=10 plants). Box-plots represent the percentage of symptomatic area per plant and p-values were calculated by Mann-Whitney test. The images correspond to leaves or leaflets presenting the closest symptom percentages to the median values. The symptoms detected appear in red on the surface of the leaf in white. White bars correspond to 1 centimeter. 266x274mm (150 x 150 DPI)

Table 1 .

 1 Characteristics of pathogenicity tests.

		Trifoliate dipping + CFI a	First leaf rubbing + MLI b
	Discriminating power	Poor	Good
	Detection of non-visible symptoms	Yes	No
	Plant growing time	15 days	8 days
	Symptom assessment date	14 days post inoculation	14 days post inoculation
	Duration of a trial	29 days	22 days
	Plants with homogeneous leaves	84%	93%
	Average time of inoculation per plant 150 seconds	43 seconds
	Volume per inoculum	500 mL	25 mL
	Equipment	PSI Open FluorCam FC 800-O	Light table and camera
		or equivalent
	Average image taking time per plant 327 seconds	45 seconds
	Image analysis software	Phenoplant	Ilastik

a CFI: chlorophyll fluorescence imaging b MLI: machine learning-based imaging

ACKNOWLEDGMENTS

We warmly thank Céline Rousseau and Guillaume Lebreton from the PHENOTIC platform (IRHS, Beaucouzé, France) for their help in the analysis of chlorophyll fluorescence images, Daniel Sochard (IRHS, Beaucouzé, France) for technical help for in planta experiments, Armelle Darrasse (IRHS, Beaucouzé, France) for valuable advices during tal mutagenesis, and Ivanna Fuentes, and Laurent Noël (LIPME, Toulouse, France) for contributing an anti-TALE antibody. The authors also thank the French Network on Xanthomonads (FNX) (https://www.reseau-xantho.org/) for recurrent scientific exchanges and methods sharing.

This study was supported by the French National Research Agency CROpTAL project (ANR-14-CE19-0002-04). JF was funded by a PhD grant (BRUTAL project) from the French National Research Institute for Agriculture (INRAE) and the region Pays de la Loire, France. MR was funded by a PhD grant (X-HOT project) from Angers-Loire Metropole, France. The funding bodies did not participate in any of the study design, data collection and analysis, or writing the manuscript.

PacBio sequencing was performed at Icahn School of Medicine at Mount Sinai (NY, USA) under the supervision of Robert Sebra, and preliminary quality control of DNA was performed at the ANAN platform from the SFR 4207 QUASAV (Angers, France). Bacterial strain preservation and supply was ensured by the CIRM-CFBP (Beaucouzé, INRAE, France).

DATA AVAILABILITY STATEMENT

The data and materials that support the findings of this study (ilastik training file and bacterial strains) are available from the corresponding author upon request. The whole genome sequences of strains 6546RΔtal18H and 6165RΔtal22B were deposited to GenBank under accession numbers CP072393-CP072395 and CP072396-CP072397, respectively.

SUPPORTING INFORMATION LEGENDS

Table S1. List of primers used for tal mutagenesis Supplemental Figure 1


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"CinNapht" dyes a new Cinnoline/Naphthalimide fused hybrid fluorophore: Synthesis, Photo-physical study and use for bio-imaging

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With the increasing interest of optical molecular imaging in medicine, fluorescence microscopy has seen constant development contributing to the emergence of new technologies and probes without discontinuity for nearly 40 years. Fluorogenic probes are now considered as critical tools for the study of biological environments. 1 These sensors unmask brilliant fluorescence upon modification of their structure or of their environment, induced by multiple kind of biological stimuli. 2 Therefore, there is definite interest in creating new chemical scaffold exhibiting fluorescent behavior that could later be used for the design of chemical fluorogenic probes. One of the most important and actual challenges is the synthesis of large Stokes shift red emitting fluorophores. 3 Among the different families of fluorophores commonly used, the 1,8-naphthalimides such as 4-Amino-1,8-naphthalimide (ANI) exhibits a green emission combining good fluorescence quantum yield and large Stokes Shift (~4 000 cm -1 ) while possessing a particularly high chemical stability. 4 1,8naphthalimides based sensors in solution or as part of material have been used for the detection of multiple analytes such as anions, metals or enzymes, highlighting their interest for fluorescence imaging. 5 The easy functionalization of their imide nitrogen atom enables fine tuning of the fluorophore properties such as solubility or organelle targeting. 6 However, their uses in imaging experiments are limited to the green region of the spectrum. To overcome this drawback, some groups have focused on the synthesis of -extended naphthalimides fluorophores such as styrylnaphthalimides. 7 The latter were found very promising red to NIR emitting dyes and were used for designing fluorogenic probes. 8 Other examples including a fused pyranone, furan or carbazole rings 9 have also been reported but with less interesting photophysical properties. Nonetheless, this strategy seems promising for there are many examples of red shifted fused hybrids fluorohores. 10 We describe in this paper the synthesis of a fused ring Cinnoline/Naphthalimide hybrid here called CinNapht dye. Cinnolines are aromatic heterocycles incorporating an azobenzene moiety that usually do not emit fluorescence, mainly because of nonradiative deactivation through photoisomerization of the azo bond. 11 These molecules can nevertheless be turned into fluorescent structures by constraining the conformation of the N=N double bond like it is in cinnoline scaffold. 12 A significant number of fluorophores have recently been described incorporating this scaffold. Nevertheless many of these examples have required quaternization of the nitrogen atom to generate a Push-Pull electron effect, and induce an intramolecular charge transfer (ICT) necessary for fluorescence emission. 13 More recently, fluorogenic probes in which a cinnoline is an integral part of a push-pull backbone have been developed. In these cases the azo bond is formed in situ following nitric oxide mediated nitrosation of an aniline precursor according to the "covalent assembly" principle, firstly proposed by Anslyn in 2010 14 and then exemplified for multiple times. 15 To the best of our knowledge, only one of these probes is formed with a molecular skeleton incorporating a naphthalimide like dye. 15a, 15b However, no description of its photophysical properties is given. Moreover, it was not isolated and such strategy is limited to the NO detection. We will show in this article that this type of molecule can nevertheless possess original photophysical properties that deserve to be highlighted. We first proceeded with the synthesis of these fused naphthalimide cinnoline hybrid. 4-amino-1,8-naphthalic anhydride 1 (Scheme 1) was first synthesized from commercial 4-bromo analog by a two steps process involving an aromatic nucleophilic substitution with sodium azide followed by reduction under Staudinger conditions (Cf. Supplementary Material). This aniline was then brominated in ortho position using N-bromosuccinimide in hexafluoroisopropanol (HFIP) 16 to provide the intermediate 2 with 92% yield, and the naphthalimide 3 was formed by reaction with n-butylamine in the yield of 92%. It should be mentioned that no purification was needed for both of these two steps which greatly facilitates the synthetic process. The biaryl 4 was then obtained by Suzuki coupling of 3 with 3-(Dimethylamino)phenylboronic acid in the yield of 82%. Under the diazotization conditions, NaNO 2 /dilute HCl, 4 was converted in a mixture of two regioisomers resulting either from an azo coupling reaction in para position (5a) or in ortho position (5b) in a 84:16 ratio (determined by HPLC analysis of the crude mixture, Figure S1). The predominant CinNapht 5a was obtained with only 58% yield after purification. The minor compound 5b was obtained in the yield of 16% and exhibited a very weak fluorescence emission that we decided not investigate further. By contrast, CinNapht 5a was found to be much more emissive. In order to optimize its synthesis, azo coupling reaction of 4 was attempted with NOBF 4 as diazotization reagent in CH 3 CN. It turned out that this method enabled the almost exclusive formation of the compound 5a. HPLC analysis showed a 96:4 ratio in favor of CinNapht 5a (Figure S2) which could be isolated after purification in the yield of 73% with a high purity of 98.5% (Figure S3). The complete synthetic pathway was also reproduced with success in large scale in order to demonstrate its viability for gram scale synthesis of the CinNapht dyes. We then performed a complete analysis of photophysical properties of CinNapht 5a in different solvents. Absorption, excitation and emission spectra were recorded, molar extinction coefficient () (Figure S4), fluorescence quantum yields and fluorescence life times (Figure S5) were measured (Cf Table 1). All spectra and data are presented in Supporting Information (Figures S6 to S15). The first observation is a significant red shift in the emission wavelength relative to the initial naphthalimide fluorophores. Thus an 89nm batochromic shift of the λ max Em was observed between ANI (λ max Em= 502 nm) and CinNapht 5a (λ max Em= 591nm) in DCM (Cf Figure S16). Fluorescence quantum yields were found to be mostly modest in all tested solvents with nevertheless values up to 0.33 in DCM and a 3 571 cm -1 Stokes Shift which is quite satisfying. 19 . The QY heterogeneity might be the reflect of some kind of aggregation phenomenon for which a better solubitily observed in CHCl 3 or DCM leads also to a better quantum yield as well as a better life time decay. This could also explain the difference of Table1) or not (entry 9, Table1) with 10% of FBS. Another important characteristic of this fluorophore is the possibility of exciting it at higher energy on the S0→S2 transition band (Figure S17). This allows to significantly enlarge the difference between the absorption and emission maxima, which then reaches values exceeding 10 000 cm -1 . Finally, we also observed a solid-state fluorescence of 5a that with an orange-red emission centered at 581 nm (Figure S18). As the push-pull structure of the compound suggested an ICT type fluorescence, a solvatochromic study was carried out. By lighting at 365 nm solutions of CinNapht 5a in different solvents, a color panel ranging from green to red-pink was obtained, likely correlated with the solvent polarity (Figure 2a). This was confirmed by the linear relationship between the λ max Em of CinNapht 5a and the polarity coefficient E T (30) of the solvents with an exception for DMSO, (figure S19). 17 This partial lack of adequacy could be explained by the fact that the Dimroth and Reichardt method does not integrate any basicity parameter, which is particularly high in the case of DMSO.

In order to confirm this, the solvent effect was also analyzed with the methodology developed by Catalán 20 that relies on the description of the solute-solvent interactions with four independent parameters: polarizability (SP), dipolarity (SdP), acidity (SA) and basicity (SB). The solvent-dependent maximum emission wavenumber (𝜈 ̅) is formulated in equation ( 1) as:

𝜈 ̅ = 𝜈 ̅ 0 + 𝑎 × 𝑆𝑃 + 𝑏 × 𝑆𝑑𝑃 + 𝑐 × 𝑆𝐴 + 𝑑 × 𝑆𝐵 (1)
where 𝜈 ̅ 0 is the value of the property in the gas phase, and the coefficients a-d are the regression coefficients describing the sensitivity of the wavenumber to the different solute-solvent interactions. The fit of the experimental fluorescence wavenumbers in the various solvent gives:

𝜈 ̅ = 19950(±54) -725(±40)𝑆𝑃 -2503(±24)𝑆𝑑𝑃 -1506(±3)𝑆𝐴 -3012(±67)𝑆𝐵 (2)
showing that the fluorescence of CinNapht is mainly dependent on dipolarity, acidity and basicity parameters (See complete values Figure S20). Equation ( 2) together with the linear correlation between calculated 𝜈 ̅ and experimental 𝜈 ̅ (Graph, Figure 2) confirmed the solvatochromic behavior of the CinNapht 5a which is in accordance with its push-pull structure suggesting that ICT is responsible for the emission. To complete the experimental study and rationalize the photophysical properties, (TD)DFT calculations were carried out. The geometry of the CinNapht dye is flat and the first transition (S0→S1) happens from the HOMO to the LUMO. The HOMO is centred on the amino-cinnoline part while the LUMO is more localized on the naphthalimide part of the dye. This result together with the representation of the density difference confirm the intramolecular charge transfer (ICT) nature of the first excited state (Figure S21). The possibility of a twisted induced charge transfer (TICT) excited state was also evaluated but the energy level of that state is higher than the ICT one (E(S1-TICT)> E(S1-ICT)) even when solvent effect was included. The solvatochromic effect observed in the fluorescence spectra is thus coming from solvent effects and not from the transition from a locally excited state to a TICT one.

Finally, we have validated the potential of these new fluorophores in cell imaging experiments. CinNapht 5a was incubated at 5 µM with living A549 cells for 2h.

The images presented Figure 3 demonstrate the viability of our fluorophores for cell imaging. No pretreatment was necessary to enable cell penetration thereby strengthening the potential of CinNapht for fluorescence microscopy. Emission and excitation spectra were recorded in cell during microscopy experiments (Figure S22) and revealed that CinNapht 5a exhibits a Stokes Shift large enough to enable an excitation at 475 nm (Maximum of excitation measured in cell) while recording fluorescence across the full range of emitting fluorescence from 500 to 700 nm centered at 591 nm. We emphasis that at the concentration used for these cell imaging experiments, fluorophore 5a showed no toxicity (see Figure S23). Complete cytotoxicity study was carried out and did not reveal any significant toxicity even at high concentration (50 µM). We did not notice any significant photobleaching of the dye during these microscopy experiments. However, we cannot claim a strong photochemical stability of our fluorophore at this stage of the study. To conclude, we describe here a new fluorophore based on fused Naphthalimide Cinnoline hybrid structure so called "CinNapht". This fluorophore shows promising fluorescence properties combining a red emission and a large stokes shift. Calculations have confirmed an ICT-like behavior characteristic of a push-pull structure that is clearly identified in the skeleton of the CinNaphth's. This characteristic is reflected in the photophysical properties by a strong solvatochromism effect. This non-toxic molecule can be used for in cellulo imaging experiments. We believe that an optimization of the structure of CinNaphth's, for example by modifying the N-dimethyl moiety, should allow a significant improvement of the photophysical properties of these fluorophores, thus making them promising tools for cell imaging.

Figure 1 :

 1 Figure 1: Structure of Naphthamidide dyes and extended analogues.

Figure 2 :

 2 Figure 2: Solvatochromism Study: Visual representation of CinNapht solution under 365 nm light. (1. Hexane, 2. Toluene, 3. CHCl 3 , 4. Dioxane, 5. DCM, 6. EtOH, 7. MeOH, 8. DMSO) and Plot of experimental and predicted emission wavenumber (Catalan method)

Figure 3 :

 3 Figure 3: Isocontour plot of the HO and LU molecular orbitals (isovalue 0.004)

Figure 4 :

 4 Figure 4: Confocal microscopic images of A549 lung cancer cells treated with CinNapht 5a at 5µM for 2h at 37 °C using a 40× oil immersion objective. (Exc: 475nm Em: 500 -700 nm).

Table 1 :

 1 Photophysical properties of CinNapht 5a

  Polarity coefficient of solvents based on literature17 , b Values corresponding to S 0 -S 1 transition but strong S 0 -S 2 transition is also observed (see Supporting Information Fig S6 to S13),c Relative QY determined at 25 °C using [Ru(bpy) 3 ]Cl 2 (QY = 0.04 in air Saturated H2O)18 , d Fitted by a biexponential function, the value indicated is an average lifetime. (For more details see supporting information Fig S5 and experimental section). In Dioxane the two lifetime were 1.66 ns and 3.31 ns. In DMSO the lifetime were 1.13ns and 2.21 ns. In MeOH the lifetime were 2.68 ns and 0.42 ns. e Solubility in aqueous medium such as RPMI culture medium (Roswell Park Memorial Institute medium), without phenol red, was found to be quite poor and required 5% of DMSO, f A small part of precipitation was observed that could not enable an accurate determination of .

	Entry	Solvent	ET(30) a	λmax Abs b (nm)	max (M -1 .cm -1 )	λmax Em (nm)	Stokes Shift (cm -1 )	QY c	Life Time (ns)
	1	Hexane	31	469	12 200	520	2091	0.01	0.37
	2	Toluene	33,9	480	12 000	550	2652	0.09	0.88
	3	Dioxane	36	477	21 000	572	3482	0.16	1.89 d
	4	CHCl3	39,1	489	15 700	566	2782	0.25	2.23
	5	DCM	40,7	488	16 400	591	3571	0.33	3.67
	6	DMSO	45,1	496	15 500	682	5499	0.06	1.26 d
	7	EtOH	51,9	493	15 200	667	5291	0.05	0.84
	8	MeOH	55,4	492	15 100	681	5640	0.02	0.47 d
	9	RPMI e	n.a.	496	n.d. f	675	5346	< 0.01	n.d.
	10	RPMI e + 10% FBS	n.a.	493	n.d. f	651	4923	0.02 g	n.d.
	11	In Cell	n.a.	475	n.a.	590	4104	n.d.	n.d.
	12	Solid	n.a.	491	n.a.	581	3446	n.d.	n.d.

a g Values observed by adding 10% of Foetal Bovine Serum (FBS) in RPMI Medium Scheme 1: Synthesis of CinNapht 5a end 5b. a Isolated yield using NOBF 4 . b Isolated yield using NaNO 2 /HCl fluorescence quantum yield observed in RPMI medium supplemented (entry 10,

Acknowledgments

This project has received funding by the French National Research Agency under the program CHARMMMAT ANR-11-LABX-0039-grant. The present work has benefited from Imagerie-Gif light microscopy core facility supported by French National Research Agency (ANR-11-EQPX-0029/Morphoscope, ANR-10-INBS-04/FranceBioImaging ; ANR-11-IDEX-0003-02/ Saclay Plant Sciences ). This work was granted access to HPC resources from the "Mésocentre" computing center of CentraleSupélec and École Normale Supérieure Paris-Saclay supported by CNRS and Région Île-de-France (http://mesocentre.centralesupelec.fr/). We thanks Univ. Paris-Saclay NanoPE platform at DER Chimie, Ecole Normale Supérieure Paris-Saclay for using Xenius SAFAS fluorimeter. We thanks the Région Ile-de-France and DIM NanoK for financial support. The University Paris-Saclay and the CNRS are acknowledged for funding.

Notes and references ‡ These authors contributed equally to this work.

Conflicts of interest

There are no conflicts to declare.


Li Zhang

Vincent Montigaud

Boris Le Guennic

Peng Zhang

Jinkui Tang

Dysprosium(III) compounds assembled via a versatile ligand incorporating salicylic hydrazide and 8-hydroxyquinolin units: Syntheses, structures and magnetic properties

Dysprosium(III) compounds assembled via a versatile

ligand incorporating salicylic hydrazide and 8-hydroxyquinolin units: syntheses, structures and magnetic properties

Introduction

Single-molecule magnets (SMMs) showing slow relaxation of magnetization deriving from molecular-based blocking anisotropy have been a burgeoning field of intense interest of physical, chemical and material scientists over the past two decades on account of their exotic technological applications in high-density information storage, quantum computing, and spintronics. [1][2][3][4][5][6][7][8] Since the seminal discovery that the doubledecker complex [Tb(Pc)2] -•(Bu4N) + (Pc 2-= phthalocyaninic) shows slow relaxation of the magnetization at low temperatures, 9 considerable attention is being paid to elaborate high-barrier SMMs via using 4f ions as a result of their unparalleled magnetic anisotropy arising from the unquenched orbital angular momentum in ligand field. 10,11 Certainly, dysprosium(III) ion plays a crucial role in the exploitation of lanthanide-based SMMs because of the joint cooperation of large magnetic ground states, single-ion anisotropy and spinparity effect in Kramers ions (odd number of 4f electrons), which thus are responsible for numerous ground-breaking results. [12][13][14][15][16][17][18][19][20][21] In particular, judicious optimization of the crystal field resulted in magnetic bistability persisting up to 80 K recently in a dysprosium metallocene, which allows access to the operating temperature above liquid nitrogen, a critical point to the practical application. 22 Notably, Dy-based SMMs of varying nuclearities and topologies range over as single, dimeric, [23][24][25][26] triangular, 5,27 cube, 28,29 rhombus, 30 defect-dicubane, [31][32][33] tetrahedron, 34,35 square-based pyramid, 20,36 wheel, [37][38][39] nanotubes, 40 and so forth. To the best of our knowledge, one of the most important factors that realize the tailor-made of functional SMMs is the design of organic ligands. Progress in the field makes clear that hydrazone Schiff-base ligands are quite suitable to construct aesthetically structural dysprosium-based SMMs showing typical relaxation dynamics. 41,42 Such rigid multidentate ligands usually possess several coordination pockets to encapsulate lanthanide ions formed by a sequence of oxygen and nitrogen donors, while various conformations can be derived from the facile keto-enol tautomerism and isomerism from the rotatable C-C bond which favours versatile bridging modes through μ-O connections. Here we designed a aroylhydrazone ligand (2hydroxy)-N'-((8-hydroxyquinolin-2-yl)methylene)benzohydrazide (H3L, Scheme 1) possessing a flexible phenoxo group of salicylic hydrazide with various coordination behaviours relying on the reaction conditions. Therefore, a series of mononuclear, dinuclear and tetranuclear Dy III assemblies as well as 1D linear Dy III polymer, formulated as [Dy(H2L)(HL)]•CH3OH

(1), 5), were isolated. The magnetic measurements were carried out on all complexes. In particular, dc magnetic susceptibility data evidences distinct magnetic coupling interactions in the dinuclear complexes 2 and 3, and only complex 3 shows slow relaxation behaviour of magnetization despite the similar structures. To get insight into the key factors of hydrazone Schiff-base ligands in determining magnetic properties of Dybased SMMs, ab initio calculations and electrostatic potential analysis are performed on complexes 2, 3, and three other complexes (6, 7, 8), where four kinds of ligands with similar structural features but different charge distribution and bridging donors are incorporated specially into deprotonated enolic, quinolinic/phenoxo, and alkoxido oxygen donors showing the gradually increasing electron-donating ability. The results reveal the important interrelationship of magnetic anisotropy, magnetic coupling interactions and SMM properties. Here the magnetic anisotropy is determined by charge distribution on ligands, thus the angles of easy axes to Dy•••Dy connecting line allow the calculations of magnetic dipolar interactions (Jdip) and further affect the magnitude of magnetic exchange coupling Jexch, which results in a combined effect on magnetic coupling interactions between Dy centres (Jtotal).

Scheme 1 Structure of ligand H3L and the related ligands as well as their coordination modes.

Experimental section

Materials and Physical Measurements. All reagents and solvents were of analytical reagent grade and were used as commercially obtained without further purification. Dy(C6H5COO)3 were prepared according to a literature procedure described elsewhere. 43 Elemental analysis (C, H, and N) were performed with a Perkin-Elmer 2400 analyzer. FTIR spectra were recorded with a Perkin-Elmer Fourier transform infrared spectrophotometer using the reflectance technique (4000-300 cm -1 ). Samples were prepared as KBr disks. Magnetic susceptibility measurements were carried out on a Quantum Design MPMS-XL7 SQUID magnetometer equipped with a 7 T magnet. The direct current (dc) measurements were collected with an external magnetic field of 1000 Oe in the temperature range 1.9-300 K, and the alternating-current (ac) measurements were carried out in a 3.0 Oe ac field oscillating at different frequencies from 1 to 1500 Hz. The experimental magnetic susceptibility data are corrected for the diamagnetism estimated from Pascal's tables 44 and sample holder calibration.

Crystallographic data collection and refinement. Crystallographic data of complexes 1-5 were collected on a Bruker Apex II CCD diffractometer with graphite monochromated Mo-Kα radiation (λ = 0.71073 Å) at 296 (2) K. The structure was solved by direct methods by means of SHELXS-97 [START_REF] Sheldrick | SHELXS-97, Program for Crystal Structure Solution[END_REF] and refined on F 2 by full-matrix least squares in the Olex2 package. [START_REF] Dolomanov | [END_REF] The location of Dy atoms was easily determined, and O, N, and C atoms were subsequently determined from the difference Fourier maps. All non-hydrogen atoms were refined with anisotropic thermal parameters. The H atoms were introduced in calculated positions and refined with fixed geometry with respect to their carrier atoms. CCDC 1021519 (1), 1021520 (2), 1021521 (3), 1021522 (4), and 1021523 (5) contain the supplementary crystallographic data for this paper. These data can be obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif. Synthesis of H3L. The Schiff-base ligand H3L ((2-hydroxy)-N'-((8-hydroxyquinolin-2-yl)methylene)-benzohydrazide) was synthesized by condensation of 2-hydroxybenzohydrazide and 8-hydroxyquinoline-2-carbaldehyde in a 1:1 ratio in methanol. 47 The resultant orange precipitate was filtered off, washed with diethyl ether and dried to obtain orange powder in 70%.

Synthesis of [Dy(H2L)(HL)]•CH3OH (1)

. Dy(ClO4)3•6H2O (0.1 mmol, 0.057 g) was added to a suspension of H3L (0.1 mmol, 0.031 g) in CH3OH/CH2Cl2 (10 mL/5 mL), followed by the adding of KOH (0.1 mmol, 0.006 g). The resultant dark red solution was stirred for 5 h and subsequently filtered. The filtrate was exposed to air to allow the slow evaporation of the solvent. Dark red blocks of 1 suitable for X-ray diffraction analysis were collected after 10 days. Yield: Please do not adjust margins Please do not adjust margins H3L (0.1 mmol, 0.031 g) in CH3OH/CH2Cl2 (10 mL/5 mL), followed by the adding of Et3N (0.2 mmol, 0.028 mL). The resultant orange solution was stirred for 5 h and subsequently filtered. The filtrate was exposed to air to allow the slow evaporation of the solvent. Dark orange blocks of 2 suitable for X-ray diffraction analysis were collected after 5 days. Yield: 20 mg (30%, based on Dy). Elemental analysis (%) calcd for C53H52Dy2N6O15: C, 47.58, H, 3.92, N, 6.28; found: C, 47.35, H, 3.97, N, 6.34. IR (KBr, cm -1 ): 3615 (w), 3353 (br), 1598 (m), 1580 (m), 1544 (w), 1489 (s), 1474 (s), 1446 (m), 1404 (s), 1362 (s), 1332 (s), 1309 (m), 1256 (m), 1190 (w), 1175 (w), 1154 (m), 1099 (m), 1076 (w), 1030 (w), 970 (w), 938 (w), 910 (w), 858 (w), 835 (m), 761 (m), 736 (w), 714 (m), 671 (w), 566 (w).

Synthesis of [Dy2(HL)2(NO3)2(DMF)4]•4DMF

(3). Dy(NO3)3•6H2O (0.1 mmol, 0.046 g) was added to a suspension of H3L (0.1 mmol, 0.031 g) in CH3OH/CH2Cl2 (10 mL/5 mL), followed by the adding of Et3N (0.3 mmol, 0.042 mL). The resultant orange solution was stirred for 5 h and subsequently filtered. The resulting precipitation was dried and redissolved in DMF (10 mL). Diethyl ether was allowed to diffuse slowly into the solution at room temperature and orange rhombic single crystals of 3 were formed in 5 days. Yield: 28 mg (34%, based on Dy). Elemental analysis (%) calcd for C58H78Dy2N16O20: C, 42.36, H, 4.78, N, 13.63; found: C, 42.21, H, 4.82, N, 13.75. IR (KBr, cm - 1 ): 2931 (w), 2809 (w), 2602 (w), 1650 (s), 1597 (m), 1547 (w), 1489 (s), 1449 (s), 1384 (m), 1360 (s), 1332 (s), 1304 (s), 1273 (m), 1254 (m), 1216 (m), 1180 (w), 1160 (m), 1103 (m), 1082 (m), 1038 (w), 966 (w), 927 (w), 900 (w), 849 (w), 787 (w), 768 (m), 737 (m), 702 (w), 674 (m), 569 (w).

Synthesis of [Dy4L4(CH3OH)4]•2CH3OH•4H2O (4).

The orange filtrate obtained in 3 was exposed to air to allow the slow evaporation of the solvent. Red blocks of 4 suitable for X-ray diffraction analysis were collected after one week. Yield: 12 mg (23%, based on Dy). Elemental analysis (%) calcd for C74H72Dy4N12O22: C, 41.70, H, 3.40, N, 7.89; found: C, 41.50, H, 3.37, N, 7.93. IR (KBr, cm -1 ): 3368 (br), 1629 (w), 1585 (m), 1563 (m), 1481 (s), 1453 (m), 1426 (m), 1381 (m), 1334 (m), 1309 (m), 1274 (w), 1254 (m), 1142 (w), 1104 (m), 1062 (m), 973 (w), 923 (w), 870 (w), 838 (m), 756 (m), 671 (m), 582 (w).

Synthesis of ([Dy4(HL)4(C6H5COO)4(CH3OH)(H2O)]•2CH3OH•CH3CN•H2O)n (5).

Dy(C6H5COO)3 (0.1 mmol, 0.053 g) was added to a suspension of H3L (0.1 mmol, 0.031 g) in CH3OH/CH3CN (5 mL/10 mL), followed by the adding of NaHCO3 (0.1 mmol, 0.008 g). The resultant orange solution was stirred for 5 h and subsequently filtered. The filtrate was exposed to air to allow the slow evaporation of the solvent. Red needles of 5 suitable for X-ray diffraction analysis were collected after two weeks. Yield: 8 mg (13%, based on Dy). Elemental analysis (%) calcd for C101H83Dy4N13O25: C, 47.97, H, 3.31, N, 7.

Results and Discussion

Syntheses.

The o-vanilloylhydrazone and picolinoylhydrazone ligands along with their derivatives have been widely used and proven to be an effective strategy for developing Dy-SMMs. [49][50][51] On the other hand, the 8hydroxyquinoline unit possessing one pyridine donor and one phenolate unit can function as chelating and bridging agent, and is thus an ideal building block in molecular multimetallic ensembles. 52 Therefore, the ligands combining the above two components are expected to blossom into achieving pure dysprosium-based systems with pleasing structural motif and magnetic properties. In fact, several typical examples have been reported with the ligands in Scheme 1 by our group and others, respectively, showing a wide range of nuclearities from two to eight metal centres. 25,30,[53][54][55] However, systematic investigations on this kind of ligands have not been performed. Here complexes 1-5 in Fig. 1 were successfully synthesized by reacting H3L with the corresponding dysprosium salts in different solvents under various bases. Among these, the interesting structural feature lies in the flexible coordination patterns of phenoxo groups from salicylic hydrazide (red rectangle in Fig. 1), depending on the reaction conditions. The reaction of H3L and Dy(NO3)3•6H2O, in the presence of three equivalents of triethylamine in a mixture of methanol and dichloromethane (v/v = 2:1) produces [2×2] grid-like Dy4 molecule (4), in which fully deprotonated ligand L 3-bridges three dysprosium centres through their enolic oxygen and phenoxo oxygen atoms (pink). In contrast, C-C rotation leads to the trans-conformation isomerism of phenoxo oxygen atoms to enolic oxygen atom in the formation of other compounds. In particular, a linear 1D Dy III chain (5) was obtained via connecting two distant Dy2 fragments by the phenoxo oxygen atoms when base NaHCO3 was present. The phenoxo groups remain uncoordinated and offer little assistance in the course of assembly of mononuclear and dinuclear complexes 1-3. The structural data and refinement details are summarized in Table S1, and selected bond lengths and angles are listed in Table S2.

Crystal structure. Crystal structure of 1. The reaction of Dy(ClO4)3•6H2O with H3L in methanol/dichloromethane (2:1 ratio), in the presence of KOH, affords dark red crystals of 1, which crystallizes in the monoclinic space group P21/c with Z = The metal centre is chelated concurrently by two N2O2 coordination pockets (Scheme 2a) of H2L -and HL 2-ligands in a crisscross fashion, leading to a pseudo-2-fold axis (Fig. 2b). Two tetradentated ligands saturate the eight-coordinated sphere with an effective geometry of distorted shape among triangular dodecahedron (D2d), snub disphenoid (D2d), and biaugmented trigonal prism (C2v) but closer to snub disphenoid (Fig. 2c), as determined by continuous shape measure theory and SHAPE software (Table S3). 56 The crystal packing arrangement of complex 1 along the crystallographic c-axis exhibits a zigzag pattern with the nearest intermolecular Dy•••Dy distance of 7.544 Å, which is shown in Fig. S1. 57 Please do not adjust margins Please do not adjust margins N2O7 coordination sphere, resulting in a geometry of distorted muffin (Cs) (Fig. 4b) with SHAPE software 58 (Table S4). Structurally, the major difference of complex 3 compare to 2 is the coordinating solvent molecules, leading to different local environments on Dy III ion, which turns out to remarkably affect the magnetic behaviour (see below). 32,59 The Dy-O and Dy-N distances fall in the range of 2.354(4)-2.611(5) Å and 2.480(4)-2.515(5) Å, respectively. Crystal structure of 5. A synthesis similar to that of 2 except that Et3N and CH3OH/CH2Cl2 were replaced by NaHCO3 and CH3OH/CH3CN leads to red needle crystals of complex 5 after two weeks which crystallizes in the triclinic space group P with Z = 2. As shown in Fig. 6a, the structure is arranged in a nearly linear one-dimensional (1D) chain composed of Dy4 asymmetric units (Fig. 6b) which can be regarded as two similar Dy2 subunits. Within the Dy2 subunits, the dysprosium centres are held together by two quinolinic oxygen groups (O1 and O4) of two HL 2-ligands in an antiparallel fashion and a η 1 :η 1 :μ2benzoate bridge with the Dy•••Dy distance of 3.7303(6) and 3.7494(6) Å, respectively. The required environment of Dy1 and Dy3 are completed by a bidentated benzoate ion, while the remaining sites of Dy2 and Dy4 are methanol and water molecules, respectively, affording the prime difference between the two Dy2 subunits. Each Dy III ion has a N2O6 coordination sphere, with Dy-N and Dy-O distances in the range 2.455(6)-2.519(7) Å and 2.262(5)-2.481(6) Å, respectively. It's worth noting that conformational flexibility of ligands HL 2-(as a result of C-C rotation between C11-C12 and C45-C46) functions as a long-distance linkage in 3.21212131212 mode (Scheme 2d) and facilitates extension of the molecular size from Dy2 units to an infinite 1D polymer. Furthermore, a detailed analysis of the crystal packing of 5 reveals that these 1D chains are connected to engender a 2D supramolecular architecture by means of weak π-π stacking interactions between two phenyl rings of coordinated benzoate group with the distance between two centroids defined by phenyl ring of 3.9 Å and the dihedral angle equal to 16.7° (Fig. S3). The determination of the exact geometry of the Dy III centres was ascertained by continuous shape measures analysis in SHAPE software. It reveals that Dy1 and Dy3 exhibit an intermediate geometry between biaugmented trigonal prism (C2v) and triangular dodecahedron (D2d), while the actual geometry around Dy2 and Dy4 is among triangular dodecahedron (D2d), snub disphenoid (D2d) and biaugmented trigonal prism (C2v) (Table S3).

A c c e p t e d M a n u s c r i p t

Please do not adjust margins Please do not adjust margins Static Magnetic Properties. The direct current (dc) magnetic susceptibilities of all five complexes were investigated under a 1 kOe field over the temperature range 2-300 K. As shown in Fig. 7a, the observed χMT product for 1 at 300 K is 13.68 cm 3 K mol - 1 , in agreement with the theoretical value of 6 H15/2 ground state of Dy III ion (S = 5/2, L = 5, 6 H15/2, g = 4/3). The χMT value demonstrates a slight drop on lowering the temperature, which can be attributed to the thermally depopulation of excited Stark sublevels of anisotropic Dy III ion. Then the value declines sharply down to 2.0 K, reaching a minimum value of 10.42 cm 3 K mol -1 , suggesting possible intermolecular magnetic interactions.

Variable temperature dc magnetic data for 2 and 3 (Fig. 7b) reveal reasonable χMT values of 31.85 and 29.24 cm 3 K mol -1 at 300 K for two uncoupled Dy III ions (S = 5/2, L = 5, 6 H15/2, g = 4/3). For 2, The χMT product remains relatively constant above 50 K and shows a rapid drop at lower temperatures to 21.85 cm 3 K mol -1 at 2 K, which is mostly ascribed to the thermal depopulation of Stark sublevels and possible intramolecular antiferromagnetic interactions. In contrast, the roughly invariable χMT product above 26 K was followed by a sharp increase to a maximum value of 33.24 cm 3 K mol -1 at 2 K for 3, indicating the presence of strongly ferromagnetic interaction between the Dy III spin carriers. 57 The distinct static magnetic behaviors are derived from the changes of local coordination environments around Dy ions. The detailed investigations are discussed in the quantum chemical calculations Section (vide infra). For 4 and 5, the observed χMT products at room temperature are 56.45 and 56.91 cm 3 K mol -1 , exactly consistent with the expected value of 56.68 cm 3 K mol -1 for four non-interacting Dy III ions. Upon lowering the temperature, both values of χMT gently decrease until approximately 70 K, while χMT of 5 decreases faster than that of 4 at lower temperatures, to reach a value of 44.33 and 29.34 cm 3 K mol -1 at 2 K for 4 and 5, respectively.

M versus H/T data (inset of Fig. 7top and Fig. S4 in the Supporting Information) for complexes 1-5 are far from reaching saturation in the 0-70 kOe field range and the corresponding magnetization data do not lie on a single mastercurve below 5 K, confirming the presence of significant magneto-anisotropy and/or low lying excited states in all systems. 60

Dynamic Magnetic Properties.

Alternating current (ac) susceptibility measurements were carried out for 1-5 under zero dc field to gain insight into the dynamics of magnetization using a 3 Oe oscillating ac field. As indicated by the temperature-dependent ac susceptibility data (Fig. S5), complexes 1, 2, 4 and 5 do not display any out-of-phase ac signal, implying a very fast relaxation of magnetization as a result of fast zero-field quantum tunneling of the magnetization (QTM). However, a significant sharp peak around an external dc field of 600 Oe where the QTM is minimized is clearly observed at 997 Hz and 2 K for complex 1, as seen from preliminary fielddependent ac measurements (Fig. S6). The application of an optimal field H = 600 Oe thus induces the appearance of temperature-dependent signals of out-of-phase ac susceptibility (χ'') below 8 K for 1 (Fig. S7), suggesting fieldinduced slow relaxation of magnetization. 34,[61][62][63][64] Because of the lack of out-of-phase maxima above the temperature of 1.9 K for 1, which is the lowest limit of our magnetometer, we plotted ln(χ"/χ') vs. 1/T over the frequency range 3-1200 Hz and extracted the estimated activation energy Ea = 4.9 K and

A c c e p t e d M a n u s c r i p t

Please do not adjust margins Please do not adjust margins characteristic times τ0 = 2.2 × 10 -5 s by applying linear fits (Fig. S8). 65 This method is reasonable with the assumption that there is only one characteristic relaxation process of the Debye type with one barrier and one time constant, which had been applied earlier to the Mn12 acetate system. 66 As shown in Fig. 8, both in-phase (χ') and out-of-phase (χ") ac susceptibilities are strongly frequency and temperature dependent where the χ" component clearly exhibits a frequency dependence maximum for 3 in the absence of dc field, indicating the blocking behavior of magnetization characteristic for SMMs. 57 The relaxation times were extracted from the fitting of frequency-dependent χ" susceptibility in the range of 1.9-6.5 K using the generalized Debye model (Table S5), and were plotted as a function of 1/T (Fig. 8). The fitting of the hightemperature data (5.0 to 6.5 K) with the Arrhenius law (τ = τ0exp(Ueff/kBT)) gives an energy gap of 43.2 K and a preexponential factor of 6.43×10 -8 s, which is typical of Orbach relaxation process. We note, however, relaxation times deviate from the linearity and trace out an exponential growth at T < 5.0 K, suggesting the occurrence of other phonon-assisted relaxation processes. 57 The Cole-Cole plots (Fig. 8) shows a nicely symmetrical semicircular shape in the range of 1.9 K-6. S9). These last compounds that contain different types of ligands (Scheme 3) were previously described. 25,57,67 The crystal field energy splitting and their corresponding g factors are listed in Tables S6-10. A highly uniaxial anisotropy indicated by gz > 19.20 (gz = 20 for a pure 6 H15/2 ground multiplet) is found for all complexes, but the slightly lower transverse components of anisotropy (gx, gy) on the ground doublet states of complexes 3, 6, and 7 could explain the occurrence of SMM behaviours as a result of less mixing microstates which slow down the quantum tunneling relaxation of magnetization. Despite the terminal solvent molecules and anions, the anisotropy axes of Dy centers in all complexes almost completely lie within the Dy2O2 plane formed via the coordination of two antiparallel ligands with two Dy ions while the directions are determined by charge distribution on the ligands, which is consistent with the electrostatic model developed by Long. 11 Here mapping the molecular electrostatic potential provides a powerful visualization of the effect of charge distribution on ligands on determining anisotropy properties of Dy centres (Fig. 9). As shown in Scheme 3, the four ligands demonstrate different charge distribution and bridging donors which specially contain deprotonated enolic, quinolinic/phenoxo, and alkoxido oxygen donors showing the gradually increasing electron-donating ability, but it is also of importance to identify the terminal and bridging coordinate atoms which exhibit different electrostatic effects, i.e. the terminal charge has stronger electrostatic effects than bridging one in the same situation. Apparently in complexes 6 and 7 the bond to terminal phenoxo oxygen atom dominates the crystal field, thus leading to the lowest electrostatic potential and the resultant preferential direction of anisotropic axis, as shown in Fig. 9. Interestingly, in comparison with enolic bridging oxygen atoms in complex 6 the stronger electron-donating ability of alkoxido oxygen bridges in complex 7 generates other low potential sites on the axial direction which efficiently improves the uniaxiality and crystal field splitting on Dy ions indicated by the lower gx and gy factors as well as the larger total energy separation (Tables S8,S9). As a result, complex 7 shows the best SMM behaviour with the highest effective barrier, Ueff = 94 K (Ueff = 69 K for complex 6). In contrast, the quinolinic oxygen atoms on ligand L 2-play the bridging role while the terminal enolic oxygen delivers relatively weak bonding interaction to Dy center in complexes 2 and 3, thus weakening the uniaxiality of crystal field compared with that in complexes 6 and 7 (Tables S6,S7), which is evidenced by the average distribution of electrostatic potential on the coordination spheres of Dy ions. Such effects appear more pronounced when the phenol group in L2 2-is substituted by pyridyl group to L3 -in Fig. 9 owing to the disappearance of terminal charge. It is shown that the lowest electrostatic potential site disappears on the site of pyridyl nitrogen site. Since the above calculations are not able to describe the lowtemperature static magnetic behaviour of these complexes which demonstrate different magnetic coupling interactions between Dy centres based on χMT vs. T plots, the intramolecular magnetic interactions are considered in the calculations within the Lines model with an effective spin s =1/2 for each magnetic centre, as implemented in the POLY_ANISO code, Ĥ = Jdipŝ1ŝ2 + Jexchŝ1ŝ2. The exchange Hamiltonian is built on the basis of the ground state Kramers Doublet (KD) of the Dy III centre leading to 2 2 = 4 exchange states (2 KDs). The intermolecular magnetic interactions are neglected due to the large distances between the [Dy2] dimers. In a first attempt to describe these interactions, only the dipolar term, Jdip, is computed (Fig. S10-14, green curves) which is associated with the Dy…Dy distances and the calculated orientations of main anisotropy axes and g tensors. As one can see, all dimers exhibit ferromagnetic dipolar coupling terms (Jdip> 0) with a narrow range of magnitude from 0.94 to 1.37 cm -1 (Table 1) owing to their similar directions of main anisotropic axes and the small deviation angles from Dy…Dy connecting line. In contrast, a qualitative picture of the exchange term, Jexch, is realized through a fitting to obtain the best agreement with experimental data (Fig. S10-14, red curves). A description of the exchange states is given in Table S11, and the values of Jdip, Jexch, and Jtotal are listed in Table 1. In complexes 3, 6, and 7 the ferromagnetic Jdip terms are balanced using small antiferromagnetic Jexch terms, thus leading to the observed ferromagnetic interactions (Jtotal> 0), while a stronger antiferromagnetic Jexch term is required to be able to stabilize the antiferromagnetic configuration in complexes 2 and 8, respectively. A closer look at structural details reveals that the stronger antiferromagnetic exchange interaction in complexes 2 and 8 is mainly related with the larger angles of easy axes to Dy•••Dy connecting line which can modulate the superexchange path between two Dy centres in terms of similar Dy-O-Dy angles in all complexes. Furthermore, the Dy•••Dy distances is also affecting the magnitude of Jexch term, i.e. the antiferromagnetic interaction is gradually improved from 3, 6, to 7 with the decreasing Dy•••Dy distances.

Conclusions

The versatile ligand H3L combining the 8-hydroxyquinoline unit with the multidentate hydrazone ligand was rationally designed by us to construct the novel multinuclear lanthanide SMMs, and the resulting complexes vary from unique mononuclear, dinuclear, grid-like tetranuclear to linear 1D chain. Their formations are largely ascribed to the versatile and flexible coordination modes of phenoxo groups from salicylic hydrazide depending on reaction conditions. The presented results confirm that the elaborate design of ligand H3L provide an extremely effective strategy for preparation of new molecular architectures with novel type of metal topologies. Furthermore, the magnetic measurements were performed on all complexes.

In particular, it provides the opportunity to get insight into the interrelationship of magnetic anisotropy, magnetic coupling interactions and SMM properties, when the dinuclear complexes A c c e p t e d M a n u s c r i p t

  (HL)4(C6H5COO)4(CH3OH)(H2O)]•2CH3OH•CH3CN•H2O)n (

  20; found: C, 47.86, H, 3.35, N, 7.32. IR (KBr, cm -1 ): 3616 (w), 3370 (br), 3064 (w), 1601 (m), 1579 (m), 1544 (m), 1488 (s), 1446 (m), 1403 (s), 1362 (s), 1331 (s), 1309 (m), 1255 (s), 1189 (w), 1154 (m), 1098 (m), 1076 (w), 1025 (w), 938 (w), 910 (w), 857 (w), 834 (m), 760 (m), 714 (m), 670 (w), 565 (w).

Scheme 2

 2 Scheme 2 Coordination modes of H3L indicated by the Harris Notation 48 in complexes 1-5.

  A c c e p t e d M a n u s c r i p t Please do not adjust margins Please do not adjust margins

4 .

 4 The single-crystal X-ray structure of complex 1 is represented in Fig.2aand the asymmetric molecule consists of a Dy III ion, a mono-deprotonated H2L -ligand, a di-deprotonated HL 2-ligand together with an uncoordinated methanol molecule.

Fig. 1

 1 Fig. 1 Various products from ligand H3L in different conditions, highlighting three diverse coordination patterns of phenoxo groups with vivid colours.

Fig. 2 a

 2 Fig. 2 a) Molecular structure of complex 1 with solvents and hydrogen atoms omitted for clarity; b) Arrangement of H2L -and HL 2-ligands in a crisscross fashion; c) Eight-coordinate distorted geometry around Dy ion.Crystal structure of 2. A procedure similar to that of 1 except that Dy(ClO4)3•6H2O and KOH were replaced by Dy(C6H5COO)3 and Et3N yields dark orange blocks of 2 after 5 days. X-ray analysis performed on single crystals of complex 2 reveal that it crystallizes in the triclinic space group P-1 with Z = 1. As shown in Fig.3a, two dysprosium centres of the centrosymmetric 2 are bridged by two quinolinic oxygen groups (O1 and O1A) of two antiparallel HL 2-ligands with Dy-O-Dy angles being 107.79(14)°

Fig. 3 a

 3 Fig. 3 a) Dinuclear structure of complex 2 with solvents and hydrogen atoms omitted for clarity; b) Distorted triangular dodecahedral geometry around the Dy1 ion in 2. Symmetry codes: A, -x+1, -y, -z+1. Crystal structure of 3. A method of allowing diethyl ether to slowly diffuse into DMF solution of precipitation from the reaction of Dy(NO3)3•6H2O with H3L and Et3N produces orange rhombic crystals of 3. Complex 3 crystallizes in the monoclinic space group P21/c with Z = 2, whose structure is depicted in Fig. 4a. A pair of Dy III ions individually residing in N2O2 coordination pocket are linked by two quinolinic oxygen atoms (O1 and O1A) of two antiparallel HL 2-ligands with Dy-O-Dy angles equal to 111.89(14)° and Dy•••Dy distance being 3.9523(7) Å. One bidentated nitrate and two terminal DMF molecules fulfil the

Fig. 4

 4 Fig. 4 a) Molecular structure of 3 with solvents and hydrogen atoms omitted for clarity. (b) Coordination polyhedra observed in 3, a distorted muffin for Dy1 and Dy2. Symmetry codes: A, -x, -y+1, -z+1. Crystal structure of 4. Complex 4 is isolated from the slow evaporation of the filtrate from the same reaction of complex 3 in one week. It crystallizes in monoclinic space group C2/c with Z = 4 and its partially labelled structure is illustrated in Fig. 5a. A [2×2] grid arrangement forms via four dysprosium centres assembled by four fully deprotonated ligands L 3-arranged in antiparallel pairs above and below the metallic pseudo-plane (Fig. 5c). Adjacent Dy III ions are bridged by enolic oxygen (O2 and O5) and phenoxo oxygen atoms (O6 and O3) from four ligands with the 3.112122231111 bridging mode (Scheme 2c), which form a [Dy4(µ2-O)8] central core (Fig. 5b) with Dy-O-Dy angles falling in the range 109.6(3)-114.5(3)° and average Dy•••Dy distance of 3.912 Å. All eight-coordinate Dy ions have a N2O6 coordination sphere, adopting a triangular dodecahedral geometry (D2d) (Fig. 5d) and the remaining sphere is completed by a methanol molecule. The average Dy-N and Dy-O distances are 2.496 and 2.354 Å, respectively. The close inspection of the crystal packing arrangements exhibits a channel containing the coordinated methanol molecules and aromatic rings of L 3-or was considered as a bigger [2×2] grid with the whole Dy4 molecule as the vertex (Fig. S2).

Fig. 5 a

 5 Fig. 5 a) Partially labeled structure of 4 with solvents and hydrogen atoms omitted for clarity; b) Top view of coordination environment around the Dy III ions in 4; c) Side view of the nonplanar Dy4 core; d) Coordination polyhedron of Dy1 with a

Fig. 6

 6 Fig. 6 a) 1D linear chain structure of 5 running along a axis with solvents and hydrogen atoms omitted for clarity; b) Structure of the Dy4 asymmetric unit of 5.

1

 1

Fig. 7 a

 7 Fig. 7 a) Temperature dependence of the χMT product at 1000 Oe for 1. Inset: M vs. H/T plots for 1 at low temperatures. b) Temperature dependence of the χMT products at 1000 Oe for 2-5.

Fig. 8 a

 8 Fig. 8 a) Frequency and temperature dependence of the inphase (top) and out-of-phase (bottom) ac susceptibility of 3 under zero dc field. b) Magnetization relaxation time, lnτ versus T -1 (Arrhenius plot using ac data) for 3 under zero dc field. The solid line corresponds to the fit. c) Cole-Cole plots measured at 1.9-6.5 K under zero dc field, with the best fit to the generalized Debye model.

  5 K and can be fitted to the generalized Debye model, yielding α parameters of 0.019-0.084, suggesting a fairly narrow distribution of relaxation time at different temperatures. Quantum Chemical Calculations. In order to get insight into the interrelationship of magnetic anisotropy, magnetic coupling interactions and SMM properties, ab initio SA-CASSCF/RASSI-SO (see Computational details in SI) on the basis of X-ray determined structures of complexes 2, 3 and three other Dy2 complexes, namely [Dy2(H2O)2(NO3)2(L1)2] (6), [Dy2(CH3OH)2(PhCOO)2(L2)2] (7) and [Dy2(NO3)2(L3)2] (8) (Fig.

Fig. 9

 9 Fig. 9 The gz axial directions (top) and molecular electrostatic potential (bottom) computed for the ground states KD of complexes 2 (a), 3 (b), 6 (c), 7 (d), 8 (e). See Computational details in SI.

A c c e 3

 3 Scheme 3 The charge distribution on four different kinds of Schiff-base ligands. The arrows indicate the bridging donors.

2 and 3

 3 are compared with three other Dy2 complexes (6, 7, 8) with another two kinds of similar Schiff-base ligands showing the different bridging donors and charge distribution. Therefore, detailed ab initio calculations and electrostatic potential analysis reveal that the anisotropic electronic structures of Dy ions indicated by g factors and axial directions are associated with charge distribution on those ligands, which further determines the magnetic coupling interactions including dipolar and exchange terms, Jdip and Jexch, between Dy centres and the resultant SMM behaviours of different complexes. This work presents a promising example for understanding the relationship between the anisotropic electronic structures and charge distribution on the ligands, thus offers new strategies for modulating the behavior of SMMs.

A c c e p t e d M a n u s c r i p t

  Please do not adjust marginsPlease do not adjust margins support through the 66 th China Postdoctoral Science Foundation Funded Project (2019M663946XB). V. M. and B. L. G. thank the French GENCI/IDRIS-CINES centres for high-performance computing resources.Notes and referencesA c c e p t e d M a n u s c r i p tPlease do not adjust margins Please do not adjust marginsSYNOPSIS TOCAssembly of dysprosium(III) salts with hydrazone H3L affords a variety of molecular magnetic compounds from mononuclear, dinuclear, grid-like tetranuclear to linear 1D chain, due to the versatile and flexible coordination modes of phenoxo groups from salicylic hydrazide.

Synthesis of [Dy2(HL)2(C6H5COO)2(CH3OH)2]•3CH3OH (2).

	15 mg (19%, based on Dy).
	Elemental analysis (%) calcd for C35H27DyN6O7: C, 52.15, H, 3.38,
	N, 10.43; found: C, 52.29, H, 3.29, N, 10.63. IR (KBr, cm -1 ): 3645
	(w), 3182 (br), 1606 (m), 1588 (m), 1542 (w), 1484 (s), 1442 (m),
	1358 (s), 1336 (s), 1306 (m), 1252 (m), 1216 (m), 1171 (w), 1153
	(m), 1131 (m), 1104 (m), 1036 (w), 938 (w), 901 (w), 830 (w),
	737 (m), 664 (w), 592 (w), 563 (w), 534 (w).
	Dy(C6H5COO)3 (0.1 mmol, 0.053 g) was added to a suspension of

Table 1

 1 Intramolecular distances, angles and magnetic interaction constants. (*The angles between easy axes and Dy•••Dy connecting line)

	complexes	2	3	6	7	8
	Dy•••Dy/ Å	3.8	3.95	3.83	3.77	3.73
	Dy-O-Dy/ °	108	112	110	112	112
	gz angle*/ °	30	21	22	18	29
	Jdip/ cm -1	0.94	1.08	1.19	1.37	1.01
	Jexch/ cm -1	-1.20	-0.29	-0.59 -0.66	-1.08
	Jtotal/ cm -1	-0.26	0.79	0.6	0.71	-0.07

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Acknowledgements

We thank the National Natural Science Foundation of China (Grant 21871247) and Key Research Program of Frontier Sciences, CAS (Grant ZDBS-LY-SLH023) for financial support. J. T. gratefully acknowledges support of the Royal Society-Newton Advanced Fellowship (NA160075). L. Z. is grateful for the

Conflicts of interest

There are no conflicts to declare.