Rebekah Kitto

Yadvender Dhillon

James Bevington

Mera Horne

Philippe Giegé

Laurence Drouard

Dimitri Heintz

Claire Villette

Nicolas Corre

Mathilde Arrivé

Mike Maneteld

Robert Bowman

Jean-Jacques Favier

Barnaby Osborne

Chris Welch
email: chris.welch@isunet.edu

Christopher P Mckay

Ming Hammond
email: ming.hammond@utah.edu

Synthetic biological circuit tested in spaceflight

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Introduction

Space exploration presents many challenges such as extreme environments, long lead times for supply chains, and risks arising from unknown unknowns. Future mission plans will likely aim for self-sufTcient crews able to implement solutions that can be adaptable to many situations. For example, 3D printers can build diverse components from the same starting materials. Such devices are currently under development for producing tools and even habitats from local materials for future exploration missions [START_REF] Cesaretti | Building components for an outpost on the Lunar soil by means of a novel 3D printing technology[END_REF][START_REF] Wong | 3D printing of surgical instruments for long-duration space missions[END_REF].

Synthetic biology is comparable to a 3D printer in its ability to produce numerous biomolecules from the same starting materials [START_REF] Menezes | Towards synthetic biological approaches to resource utilization on space missions[END_REF]. Of particular interest are medicines that have reduced shelf lives in space [START_REF] Du | Evaluation of Physical and Chemical Changes in Pharmaceuticals Flown on Space Missions[END_REF] and shelf lives shorter than proposed mission architectures for a human mission to Mars. Additionally, the ability to produce in-situ leads to reduced spacecraft masses because not every medicine or biomolecule that could possibly be required through the mission has to be taken. Plants hold particular promise for bioproduction because they already can play a critical role in life support systems through cycling CO 2 and producing food for astronauts [START_REF] Poulet | Plant's response to space environment: a comprehensive review including mechanistic modelling for future space gardeners[END_REF]. The application of synthetic biology to plants could further enhance their value to human exploration missions by making them multi-functional [START_REF] Llorente | The multiplanetary future of plant synthetic biology[END_REF]. One can envision plants designed to produce vitamins or medicines [START_REF] Zhang | Expression and functional evaluation of biopharmaceuticals made in plant chloroplasts[END_REF] critical to astronaut health on demand. These would be consumed with a normal plant-based meal. An alternate application includes plants that respond to toxins by indicating their presence and sequestering them or converting them to less hazardous compounds. Using an engineering approach, crops could be enhanced for these on-demand applications.

The effects of spaceUight on plants have been studied since the 1960s on a variety of platforms and reviewed by [START_REF] Wolverton | An update on plant space biology[END_REF], Paul et al. (2013), [START_REF] Poulet | Plant's response to space environment: a comprehensive review including mechanistic modelling for future space gardeners[END_REF], [START_REF] Vandenbrink | Space, the Tnal frontier: a critical review of recent experiments performed in microgravity[END_REF], and [START_REF] Zabel | Review and analysis of over 40 years of space plant growth systems[END_REF]. One key trend observed through several decades is the impacts of hardware design on biological results [START_REF] Poulet | Plant's response to space environment: a comprehensive review including mechanistic modelling for future space gardeners[END_REF][START_REF] Zabel | Review and analysis of over 40 years of space plant growth systems[END_REF]. Specifically, early experiments tended to lack ventilation, adequate volume, and advanced water and nutrient delivery systems seen on more modern platforms. Biological results from these early experiments often reported reduced growth and effects that could be traced to lack of convection such as hypoxic conditions in roots, reduced CO 2 exchange on leaves, and buildup of volatile organic compounds (VOCs) including ethylene [START_REF] Poulet | Plant's response to space environment: a comprehensive review including mechanistic modelling for future space gardeners[END_REF]. Modern platforms provide ventilation and environmental control which has improved plant growth in space [START_REF] Poulet | Plant's response to space environment: a comprehensive review including mechanistic modelling for future space gardeners[END_REF][START_REF] Zabel | Review and analysis of over 40 years of space plant growth systems[END_REF].

Responses to microgravity are often species-speciTc and vary depending on the stage of development of the plant (Paul et al., 2013). For example, speciTc to Arabidopsis thaliana, phototropic response is more sensitive in µG than 1 G and adequate to orient hypocotyls [START_REF] Millar | A novel phototropic response to red light is revealed in microgravity[END_REF]. [START_REF] Ferl | SpaceUight induces speciTc alterations in the proteomes of Arabidopsis[END_REF] report differential expression for 1500 proteins for space-Uown plants, [START_REF] Correll | Transcriptome analyses of Arabidopsis thaliana seedlings grown in space: implications for gravity-responsive genes[END_REF] report differential transcription (two-fold difference) of 230 genes between space-Uown and ground samples. [START_REF] Vandenbrink | RNAseq analyses of Arabidopsis thaliana seedlings after exposure to blue-light phototropic stimuli in microgravity[END_REF] report significant differential expression of 296 genes by RNA-seq. [START_REF] Paul | Arabidopsis gene expression patterns are altered during spaceUight[END_REF] report differential transcription (four-fold difference) of 182 genes including upregulation of some, but not all, heat shock genes even though growth temperatures were maintained below 25 °C. Corroborating this Tnding, microgravity experiments in parabolic Uight show increases of 0.9-1 °C in leaf surface temperature of sweet potato, barley, and wet paper replicas in microgravity [START_REF] Kitaya | The eVect of gravity on surface temperatures of plant leaves[END_REF][START_REF] Kitaya | The eVect of gravity on surface temperatures of plant leaves[END_REF]. A transgenic strain of A. thaliana where green Uuorescent protein was fused to genes of interest was Uown to space and functioned as expected [START_REF] Ferl | The eVect of spaceUight on the gravity-sensing auxin gradient of roots: GFP reporter gene microscopy on orbit[END_REF]. Though transgenic, the Uuorescent reporter monitored native activity and was not an inducible expression system in the engineering context. There are several examples of similar systems with native promotors or gene fusions in the literature [START_REF] Kittang | Exploration of plant growth and development using the European Modular Cultivation System facility on the International Space Station[END_REF][START_REF] Paul | Transgene expression patterns indicate that spaceUight aVects stress signal perception and transduction in arabidopsis[END_REF]Paul et al., , 2013b)).

Towards the demonstration of on-demand gene expression via exogenous induction, we selected transgenic Arabidopsis thaliana pX7-AtPDSi that stably expresses a synthetic inducible RNAi gene silencing system [START_REF] Guo | A chemical-regulated inducible RNAi system in plants[END_REF] (Fig. 1). The synthetic biological circuit comprises the XVE activation system [START_REF] Zuo | An estrogen receptor-based transactivator XVE mediates highly inducible gene expression in transgenic plants[END_REF] and CLX system [START_REF] Zuo | Chemical-regulated, site-speciTc DNA excision in transgenic plants[END_REF]. The XVE expression system is a transcription factor-promoter pair that is activated by estradiol [21]. A PG10-90 constitutive promotor expresses an artiTcial transcription factor called XVE that consists of a fusion of particular domains from LaxA, VP16, and the human estrogen receptor. In the presence of estradiol, the XVE transcription factors bind to eight OLexA sites on the engineered DNA promoter, thereby activating production of the downstream gene of interest, which in this case is Cre recombinase from the CLX system.

Once it is produced, Cre will excise a portion of DNA between two loxP sites in the engineered CLX system, which conveniently provides a permanent change to the genomic DNA that can be analyzed post-Uight via PCR. For the purpose of in-Uight analysis, the DNA recombination event leads to production of an siRNA that silences the phytoene desaturase (PDS) gene. The result is a photobleached phenotype that is distinguishable through standard photographs. The XVE system was shown to yield 8X higher production levels than the commonly used strong constitutive promoter, CaMV 35S [START_REF] Zuo | An estrogen receptor-based transactivator XVE mediates highly inducible gene expression in transgenic plants[END_REF] and be tightly regulated, having no leaky expression of the recombinase without estradiol present [START_REF] Guo | A chemical-regulated inducible RNAi system in plants[END_REF]. Furthermore, the system could be activated at many stages of development and produce a spectrum of induction intensity.

One of the seminal discussions on the use of synthetic biology for space outlined several tasks that should be complete in a 5 yr timeframe (LanghoV et al. 2010). Among these were "testing synthetic biology techniques in microgravity", assessing the "viability and stability of [engineered] organisms in space", and the "development of RNAi technologies" (LanghoV et al. 2010). To the best of our knowledge, the experiment reported here represents the Trst space Uown experiment to address any of these recommendations. The aim of this study is to demonstrate functionality of an exogenously inducible genetic circuit in plante during spaceUight as a Trst step towards enabling synthetic biology approaches for spaceUight. While transgenes with native promotors have been demonstrated in spaceUight, exogenous induction with small molecules, and a complex synthetic circuit employing non-native promotors (G10-90), non-native regulatory sites (O LexA ), non-native proteins (Cre and XVE fusion), and RNAi have not.

While we hypothesized that all of the biochemistry would be functional, this was not necessarily a given. LanghoV et al. 2010 suggest that synthetic constructs should be tested in space stating "since organisms may behave differently in space, ground research needs to be validated in the appropriate environments." Specifically, the lack of convection manifests itself typically by reducing chemical Uux into and out of the organism and this could reduce the uptake of the estradiol induction molecule. This phenomenon has been shown to have major impacts on microorganisms (Benoit and Klaus 2007, Benoit et al. 2008, Zea et al. 2016). Additionally, the gene expression proTle of the plant is radically different in the spaceUight environment. This could lead to interactions with the genetic circuit that differ from those observed on the ground. The Uight demonstration provides Uight heritage to a suite of genetic elements that can be incorporated into future designs with reduced risk (i.e. improved Technology Readiness Level). In the process a small hermetically-sealed plant growth module was developed and the design made openly available. The design is compatible with readily available commercial platforms on the International Space Station (ISS) such as the Space Application Services ICE Cubes platform and should facilitate future plant growth experiments in space, and on the surfaces of the Moon and Mars.

Methods

Payload

A sealed chamber of 0.63 l served as the vessel which contained the experiment (Fig. 2). Seeds were Txed to Tlter paper using guar gum and hydraulically connected to the Uuid delivery port through physical contact with a piece of gauze (Absorbent Cotton Co.). Media containing the inducer was stored in a spring-pressurized bag. Once commanded, a valve released the media to the gauze, which then diffused to the Tlter papers providing water and nutrients to the seeds. Growth lighting was continuously provided by blue and red LEDs at an intensity of 140-150 µmol•m -2 •s -1 following media delivery (speciTcation sheet in supporting Tles). Images were taken every four hours in a sequence that consisted of turning oV growth lights, turning on three white imaging lights, collecting an image then returning to the original growth light stage. The entire imaging sequence takes less than Tve seconds. Details of the payload, including design, testing, and operation, are included in the supporting Tles and should be adequate to produce a replicate of the Uight hardware.

Experimental setup

Growth chamber

Whatman Tlter paper pieces (17 CHR, 70 × 90 mm) and aluminum combs were pre-measured and cut according to growth cassette slot dimensions. Seed placement was marked between comb sections with a pencil using the metal end of a mechanical pencil to create a pro nounced indent that could be viewed from the side. The growth cassette was assembled by Tlling each row with a "sandwich" of Tlter paper (seed markings), aluminum mesh, and Tlter paper (spacer, no seed markings). The seed markings were arranged to face the center of the cassette. A piece of folded gauze was placed at the bottom of an aluminum growth chamber. The gauze was centered to avoid contact with the edge of chamber and prevent blockage. Metal tweezers or forceps were used to grasp the Tlled growth cassette and lower it into the bottom of the chamber. The cassette was turned so that rows were aligned to suit the camera angle, and the cassette and gauze were pressed down to be Uush with the bottom of the chamber.

The entire chamber was covered with aluminum foil and autoclaved at 121 °C or 134 °C. Once the cycle was complete, the exterior was wiped with 70% (v/v) ethanol and then transferred to a sterile biohood. All metal tweezers and aluminum screws were autoclaved in a covered container or aluminum foil. Once the cycle was complete, materials were sprayed thoroughly with 70% ethanol and the container transferred to the biohood. All other materials were sprayed thoroughly with 70% ethanol and place in a biohood to dry under air Uow for at least 30 min. Electronic components were not autoclaved. Instead, these were sprayed with 70% ethanol and dried for at least 30 min (electronic components must be completely dry prior to assembly to avoid short-circuiting and equipment damage).

Media/guar gum/seeds

A 1% w/v guar gum solution was mixed in the biohood using the upper clear portion of the guar gum solution. A 50-100 mL container of 0.5X MS Salts, pH 5.8 was autoclaved and transferred to the biohood. Arabidopsis thaliana pX7-AtPDSi was kindly provided by Prof. Nam-Hai Chua (Rockefeller University) and Arabidopsis thaliana Col-0, the wild type strain, was originally obtained from Dr. Imara Perera (Department of Plant and Microbial Biology, North Carolina State University). Seeds were sterilized by Tve-minutes of hand swirling with 70% ethanol + 0.1% (v/v) Tween-20 followed by double rinses with 100% ethanol and ten seconds of hand swirling. The seeds were then gently suspended in 100% ethanol and ejected onto an autoclaved Whatman Tlter paper 113 (125 mm Wet Strengthened Circles) to air dry completely. Sterile seeds were collected in autoclaved tubes and stored in a dark drawer until use.

3

I have also provided the version with the leaves to see if they can make it fit. The PCB assembly is inserted into the shell after the cassette and followed by the pressure plate, water bag, and lid. (Bottom) space cube on ISS just before being placed in a bag and sealed for return to Earth.

U N C O R R E C T E D P R O O F

Assembly of growth chamber

This assembly was performed in the biohood with pre-sterilized materialsthe biohood was sprayed down with 70% ethanol and allowed to dry for at least 15 min before starting work. The autoclaved aluminum foil was also sprayed down and used as a work surface in the hood as a form of secondary containment. A sterile growth cassette was unwrapped in the hood and the blunt end of sterile tweezers was used to gently press each piece of Tlter paper down to ensure contact with the lower gauze layer. A small amount of seeds were tapped out of the tube (44 of each strain) into a sterile petri dish or onto a glass slide. A minimal amount of guar gum was added to coat the seeds. Forceps were used to gently place a seed within a pre-marked indent in Tlter paper. Seeds were placed in every indent, with pX7-AtPDSi seeds in the four rows closer to the water delivery opening (bottom of the images). A water indicator dot was added to the top edge of a piece of Tlter paper using forceps.

A total of 30 mL of 0.5X Murashige and Skoog Salts (see supplemental) was poured into a sterile 50 mL Falcon tube. 30 µL of 2 mM estradiol in ethanol (1:1000 dilution) was added for a Tnal concentration of 2 µM estradiol in 0.5X MS salts. Prior to Uight, estradiol was found to have minimal degradation after storage at room temperature for 30 days [START_REF] Kitto | Evaluating RNA-Based Gene Regulation Across DiVerent Systems[END_REF]. A sterile syringe + needle (18 G) was used to Tll the media bag with 28 mL media. Then the cube was assembled.

Flight

Two payloads were prepared: one for Uight (Uight cube) and one to serve as the non-Uight control (ground cube). Preparation occurred in Utah and the Uight unit was mailed to the launch site without X-ray. In total, the cubes were stored for eight days prior to launch. Activation of the experiment occurred nine days after launch and the duration of the experiment was twenty days (Fig. 3). The ground cube was prepared and operated with a one-day delay compared to the Uight cube. This allowed time to respond to events as they occurred in Uight cube. Otherwise, the cubes were set up using identical batches of sterilized seeds and growth media. Fresh estradiol stocks were prepared and the hardware was assembled as described above.

Occasionally the power to a cube was cycled oV then back on for operational reasons. Power cycling causes the unit to reboot and desynchronizes the four-hour imaging sequence. Also, the Trst few images taken after rebooting are overexposed and washed out and could not be used in analysis. These images were removed from analysis. Power cycles occurred on Day 8 for the ground cube and Days 7 and 15 for the Uight cube.

At the end of the active phase, units were powered down removing light to the plants and stored at 4C until analysis. The space unit returned to the USA and was delivered as-Uown without x-ray to France for post Uight analysis. The ground unit was disassembled in Utah, biomass Uash frozen and mailed to France via normal post (with x-ray) on dry ice.

Image analysis

For quantitative analysis, every fourth image (taken approximately every eight hours) from the Uight set and the closest matching image in the ground set were analyzed through an image analysis process (supporting Tles). Each image was 2592 × 1944 pixels. First, plants were traced and cut from the original images then pasted into a blank pure white image such that only plants were present in the new image used for calculations. This was done manually in paint.net 4.1.5 by a single person for consistency. The Tle format used follows that of the original image, which was a 24-bit Portable Network Graphics (PNG), where each of three channels has eight bits (0-255). The leaf area in each image is estimated by counting the number of plant pixels in the image. Plant pixels are deTned as those pixels that are not pure white. The genetic circuit is designed to induce photobleaching in plant leaves that is observable visibly as a whitening of what would otherwise be green leaves. Thus, the effectiveness of the genetic circuit can be evaluated by quantifying the greenness of the plant leaves. The greenness of each pixel was calculated as green/(green+blue+red) where green, blue, and red are the bit values for the corresponding channels. The index reported is the median value for all pixels in a sample. Other indexes from the literature were also calculated but there were no major differences in the conclusions drawn. Two controls were conducted to verify that the greenness was not an artifact of the image itself or lighting conditions. Sections of the white Tlter paper and aluminum cassette were processed in a similar manner to the plants for comparative purposes.

Polymerase chain reaction

A polymerase chain reaction (PCR) can be used to verify the production and activity of Cre recombinase protein. Specifically, primer pairs designed to amplify around the excised portion will produce smaller fragments while primer pairs designed to amplify across a LoxP site will produce no fragments from DNA template that has successfully undergone CLX activation. Template DNA was isolated from whole plant extracts using a Qiagen DNeasy Plant Mini Kit according to manufacturer's instructions. PCR reactions were carried out following the methods and primer pairs of [START_REF] Guo | A chemical-regulated inducible RNAi system in plants[END_REF]. PCR reactions were separated by gel electrophoresis and imaged to determine the size of the ampli-Ted bands. Visible bands were also physically extracted, cleaned (using a Macherey-Nagel Nuceo-Spin PCR clean-up kit according to manufacturer's instructions), and Sanger sequenced at the IBMP "Gene Expression Analysis" platform using an ABI3130 XL sequencer, to further compare them to the expected results or identify their origin.

Results and discussion

Establishment of experimental conditions

The experimental conditions that needed to be adapted for the launch and Uight conditions are summarized here (for a detailed account, see ref. [START_REF] Kitto | Evaluating RNA-Based Gene Regulation Across DiVerent Systems[END_REF]. Important considerations included multiday storage of prepared seeds and reagents at or above room temperature, which could lead to growth of biological contaminants, loss of activity due to reagent decomposition (e.g. the inducer estradiol), and loss of seed viability and germination efTciency due to storage conditions and/or exposure to VOCs and leached chemicals from growth chamber materials. For example, instead of published conditions for seed germination on phytoagar plates (1x MS salts with 3% sucrose, 0.8% agar, 2 µM estradiol), seeds were germinated and grown afTxed to Tlter paper with 0.5X MS salts with no sucrose, 2 µM estradiol. Sucrose was removed because while it greatly enhances plant growth, it led to high incidence of biological contamination even with rigorous sterilization techniques. It was found that 0.5X MS salts was needed to promote seed germination and growth, as 2 µM of the inducer estradiol was toxic to both wildtype and transgenic seeds germinated with water alone. For this reason, the higher concentration of estradiol (10 µM) was not used, and although some decomposition of the inducer was expected under these conditions, we established in ground tests that aged seeds with aged 2 µM estradiol (30 d post-sterilization and post-stock preparation, respectively) still gave visible photobleaching.

The number of wildtype and transgenic seeds were chosen to provide adequate statistical sampling while permitting growth for enough days to visualize germination, growth, and photobleaching before depletion of CO 2 in the hermetically sealed chamber limited all plant growth. Tests were conducted to measure and adjust the light intensity of the LED light array to enable high germination rates and normal plant growth. Growth chamber materials were selected based on biocompatibility tests performed with seeds germinated with growth media containing 2 µM estradiol and oV-gassed as time permitted to reduce the exposure of the seeds or plants to VOCs.

Other conditions that could affect biological samples in general during launch include vibration and g-forces. Thus, it was beneTcial to store and launch seeds rather than pre-germinate and launch plant seedlings. In summary, these results highlight that even robust synthetic biological circuits developed for the laboratory setting may require, at minimum, adjustment or re-optimization of experimental procedures to work after prolonged storage and launch, in addition to spaceUight conditions.

Germination and growth

Images were reviewed to detect emergence of radicle, hypocotyl and cotyledon. Germination yields were higher in the ground cube than the space cube and in both cubes transgenic seeds had higher germination yields than wild type seeds (Table 1). With the exception of pX7-At-PDSi in the ground cube, germination yields were lower than the 80% threshold which is common for Col-0 [START_REF] Rivero | Handling arabidopsis plants: growth, preservation of seeds, transformation, and genetic crosses[END_REF]. In general, the resolution of the images was not adequate to distinguish between the speciTc parts but was adequate to detect activity. The green color of the cotyledon was more obvious against the white Tlter paper so the detected activity is likely weighted toward cotyledon emergence. The average time to Trst activity detected for each seed followed similar trends to the germination yield with ground seeds germinating sooner than space ones and transgenic seeds germinating sooner than wild type. [START_REF] Boyes | Growth Stage-based phenotypic analysis of arabidopsis: a model for high throughput functional genomics in plants[END_REF] report an average of 4.3 +/-0.4 days (mean +/-SD) for radicle emergence and 5.5 +/-0.6 days for hypocotyl and cotyledon emergence in Col-0 while [START_REF] Rivero | Handling arabidopsis plants: growth, preservation of seeds, transformation, and genetic crosses[END_REF] report germination of 3-5 days under continuous lighting for Arabidopsis in general. The seeds in the ground cube match these values well but the space Uown seeds are delayed. Further, there was substantial variability in activity timing. CVs for all but the Col-0 Uight seeds ranged between 22 and 28%, which is more than twice the 10.3-11.2 reported by [START_REF] Boyes | Growth Stage-based phenotypic analysis of arabidopsis: a model for high throughput functional genomics in plants[END_REF]. In the Uight cube, Col-0 seeds germinated as late as 16.75 days which is twice as late as in the ground cube. The CV of 54% reUects this.

Given the sealed nature of the cube, a reduction in germination could lead to higher biomass yields per plant, but this is the opposite of what was observed. Estimates of the leaf area from pixel counts of photographed leaves, shows that plants in the ground cube were an order of magnitude larger than their space-Uown counterparts (Fig. 4). In the Uight cube, leaf area decreases starting at Day 9 in pX7-AtPDSi and Day 13-14 in Col-0 indicating that the growth had halted. In both conditions, the wild type strain yielded higher leaf area despite lower germi nation rates. For the transgenic strain, the reduction in leaf area is a result of leaf photobleaching, which is known to detrimentally affect plant growth. In some pX7-AtPDSi plants, later leaves do not photobleach likely because the estradiol has been depleted by the point these leaves are produced. This observation was also reported by [START_REF] Guo | A chemical-regulated inducible RNAi system in plants[END_REF] in lab experimental conditions. Taken together, the reduced growth compared to Col-0 support that the transgenic plants have reduced photosynthesis due to photobleaching. The visible difference between some early and later transgenic leaves observed in the ground cube support that estradiol is required to induce protein production (e.g. of Cre recombinase), as its depletion during the experiment leads to later leaves not being photobleached. Due to the reduced growth of plants in the Uight cube, later leaves were not visible, but we expect that estradiol would be depleted at a similar or accelerated rate (see Temperature results).

Photobleaching

Visual inspection of the images conTrms photobleached leaves and thus activation of the pX7-AtPDSi system in both ground and Uight conditions (Fig. 5, Supplementary Files 1 and 2). The ground Col-0 plants are distinctly green while the Uight Col-0 plants are more ambiguous. Quantitative image analysis however conTrms that Col-0 leaves are greener than their pX7-AtPDSi counterparts in both conditions (Fig. 6). At day 9 in both conditions, photobleaching starts and leads to a negative slope in the leaf area that arises from arrested growth. The onset of photobleaching leads to a distinct cross in the greenness proTles at day 10. Furthermore, in both conditions, the maximum difference in the greenness occurs at day 14-15. These temporal similarities between the Uight and ground cubes indicate the pX7-AtPDSi system functioned similarly despite large differences in the plants themselves.

For both the Uight and ground units, the background greenness was nearly static throughout the experiment (Supplementary Figure 1). Some peaks occur where power was cycled, causing the Trst few photos to be exposed differently. This was a known issue prior to Uight. There is a small bias between the wildtype and transgenic halves of the cube but the magnitude of 0.004 is ~6% of the observed range and within the accuracy of the methods employed.

In general, the plants in the ground cube were greener than those in the Uight cube, with the Tnal days of the experiment showing the most difference. The Uight cube exhibits a general decrease in greenness starting at Day 11 in the Col-0 plants and Day 6 in pX7-AtPDSi. In the In both Uight and ground cubes, the pX7-At-PDSi plants photobleached while the Col-0 plants did not. In the ground cube, pX7-AtPDSi plants produced green leaves later in the experiment as estradiol had been consumed. Time lapse movies are given in Supplementary Files 1 and 2 and all images are made publicly available. (For interpretation of the references to color in this Tgure legend, the reader is referred to the web version of this article.) ground cube, pX7-AtPDSi plants are generally static in greenness while the Col-0 plants are increasing throughout the experiment. Once the induction molecule becomes depleted, new leaves added by pX7-AtPDSi plants are green rather than photobleached (Supplementary File 1) [START_REF] Guo | A chemical-regulated inducible RNAi system in plants[END_REF]. Both early and later leaves were included in the quantitative analysis to avoid user bias, and the greenness index analysis reports the median of analyzed pixels. Thus the Uattening greenness in the ground condition pX7-AtPDSi plants reUects a bimodal distribution that is conTrmed by histograms (Supporting Files). Nevertheless, the observation of photobleaching visually and with quantitative analy sis in the transgenic plants provides support that Cre recombinase production and siRNA activity were induced during spaceUight, as both functions were necessary to lead to the photobleaching phenotype. One challenge of the study is the hardware constraint of a single Uuid delivery to all plants. Because of this, a key control treatment of non-induced pX7-AtPDSi plants was not conducted and thus it is possible, though, unlikely, that spaceUight alone induced the pX7-AtPDSi system.

Polymerase chain reaction

PCR reactions can be used to verify functionality of the CLX element because it relies on excision of a portion of DNA (Fig. 7) [START_REF] Guo | A chemical-regulated inducible RNAi system in plants[END_REF]. Biomass for all Col-0 and pX7-AtPDSi plants were consolidated due to the limited quantity and quality (due to time delays and room temperature storage) of the biomass that could be obtained after the experiment. None of the Col-0 wild type reactions produced PCR bands conTrming that they do not have the transgenic elements of pX7-AtPDSi. As expected for a functional pX7-AtPDSi system, there is a 992 bp band in the P1/P4 lane of both the Uight and ground samples. However, there is also a 696 bp band in the P1/P2 lane and 1331 bp band in the P3/P4 lane in both the Uight and ground samples, indicating that some DNA has not undergone Cre/LoxP recombination. For both space-Uown and ground samples, the intensity of the P1/P4 pair band is similar to that of the P1/P2 pair while the P3/P4 band is much darker, indicating similar behavior between the samples. [START_REF] Guo | A chemical-regulated inducible RNAi system in plants[END_REF] also report P3/P4 bands that have higher intensity than the P1/P2 bands. This intensity pattern is likely an artifact of the primer pair and not indicative of the biochemical functionality. The P1/P2 and P3/P4 bands in the ground sample can be explained by the later stage leaves, which were produced after estradiol had been consumed [START_REF] Guo | A chemical-regulated inducible RNAi system in plants[END_REF]. The same explanation could apply to the Uight sample, although later stage leaves were not visible, or alternatively the induction of the pX7-AtPDSi system was partial, which could be due to Uight conditions affecting the plants and/or increasing estradiol breakdown.

In both the space and ground samples, beyond the 992 bp bands in the P1/P4 lane, other bands of smaller molecular weights can also be detected. These ampliTcations cannot be explained by the pX7-AtPDSi system. The different bands were excised from gel and analyzed by Sanger sequencing. This revealed that the 992 bp fragments indeed correspond to the recombination product expected the pX7-AtPDSi system, while the lower molecular weight bands in the P1/P4 lanes are oV-target ampliTcations of a root bacteria Mesorhizobium spp genomic sequence. This contaminating prokaryote was likely present in Arabidopsis seed batches used here, as classically observed, and its genomic DNA was extracted together with plant genomic DNA [START_REF] Lundberg | DeTning the core Arabidopsis thaliana root microbiome[END_REF].

Temperature

Each cube contained a thermometer on the internal circuit board which was used to record temperature approximately every two minutes. The average temperature reported by the Uight cube was 34.29 °C while the average temperature in the ground cube was 29.13 °C. The lack of convection in the space environment can cause differences in the circuit board temperature that do not necessarily correspond to differences in temperature at the plants. Furthermore, small differences in coating thicknesses could impact the thermal distribution on the circuit boards. After the Uight, a calibration curve was produced in the Uight cube to connect outside temperatures to the thermometer output. This suggests that the Uight cube was likely operating at an external temperature 26-27 °C compared to 23-25 °C in the ground cube. The temperature range for A. thaliana growth is 16-25 °C with younger plants (pre early rosette stage) being particularly sensitive to higher temperatures [START_REF] Rivero | Handling arabidopsis plants: growth, preservation of seeds, transformation, and genetic crosses[END_REF]. [START_REF] Paul | Arabidopsis gene expression patterns are altered during spaceUight[END_REF] reported overexpression of heat shock genes in space Uown A. thaliana with temperatures in the range reported here.

Conclusions

Despite the interest in synthetic biology for space applications, very little, if any at all, has been reported about synthetic biology deployment in the actual space environment. As synthetic biology functions within the context of an organism, the organism's microgravity phenotype is paramount to a successful synthetic biology output. In this experiment, an in plante synthetic biological circuit was evaluated in space-Uight. The circuit was designed to produce a protein (Cre recombinase) and an siRNA whose activities in sequence lead to leaf photobleaching for easy visualization and measurement on orbit. Visual inspection of the images illustrates that pX7-AtPDSi leaves are lighter than their wild type Col-0 counterparts. This is despite challenges with plant growth on orbit. Calculating the greenness index enables quantitative analysis and helps to remove bias. The greenness index conTrms that pX7-AtPDSi plants were less green than their Col-0 counterparts. Furthermore, the temporal patterns in greenness index are similar between the Uight and ground cubes. Finally, since Cre causes a DNA recombination event, PCR after sample retrieval could be used to conTrm at a molecular level that the pX7-AtPDSi system functioned in space as it did on the ground.

The differences in germination and growth patterns between the Uight and space cubes can be explained by higher than optimal temperatures in the space cube. This experiment was among a trial batch intended to test the newly-installed ICE Cubes platform on the Columbus module of the ISS. As such, detailed thermal proTles of the ICE Cubes platform on orbit were not available prior to Uight. A thermometer installed on the internal circuit board indicates that the external temperature -thus the temperature most closely experienced by the plants -was 26-27 °C. This is 1-2 °C above A. thaliana's optimum growth range. [START_REF] Rivero | Handling arabidopsis plants: growth, preservation of seeds, transformation, and genetic crosses[END_REF] report negative responses particularly through germination and early rosette stages when exposing A. thaliana to temperatures above 25 °C. Microgravity further compounds the overheating issue by reducing the plants' ability to regulate their temperature lower [START_REF] Kitaya | The eVect of gravity on surface temperatures of plant leaves[END_REF][START_REF] Kitaya | The eVect of gravity on surface temperatures of plant leaves[END_REF] due to a lack of convection and others have reported over expression of heat shock proteins in space Uown A. thaliana [START_REF] Paul | Arabidopsis gene expression patterns are altered during spaceUight[END_REF].

Another environmental stress may have been high humidity. While the humidity was not measured, condensation visible on the walls and cassette are consistent with a high relative humidity as would be expected in a sealed chamber. The humidity would likely be similar in both cubes so does not explain the difference in germination yields or growth but may serve to push the plants into a stressed state where other stresses arising from spaceUight could act more prominently. [START_REF] Rivero | Handling arabidopsis plants: growth, preservation of seeds, transformation, and genetic crosses[END_REF] report seed sterility can result from humidity over 90%. Despite the combinatorial stresses from spaceUight and higher than optimal temperatures, the synthetic biological circuit was still functional, demonstrating the circuit's robustness.

Demonstrating a functional synthetic biological circuit in the space-Uight environment is the Trst step toward development of synthetic biology applications for spaceUight. The pX7-AtPDSi system has several unique aspects that each had to function in order to obtain the observed result like exogenous induction with a small molecule and non-native regulatory elements. Specifically, the XVE estradiol-inducible transcription factor, Cre/LoxP recombination system, and RNAi post-transcriptional gene silencing must have functioned as subcomponents to the combined pX7-AtPDSi system. XVE can be used to control level and timing of expression of any gene of interest in response to the inducer estradiol, while RNAi can be used to repress expression of any target gene(s). Cre/LoxP can be used to up regulate, down regulate, delete, exchange, and reverse genes and DNA segments which provides many design opportunities when combined with a tightly regulated activator. Despite loss of convection which could impact small molecule uptake and mod-iTed gene expression proTles resulting from spaceUight, all of these biological control circuits appear to have functioned as intended though suboptimal growth hinders a more conclusive determination. Synthetic biology projects intended for space can incorporate these elements in their designs to mitigate the risk of unknown behaviors during space-Uight. Future studies should verify that spaceUight alone, without chemical induction, does not induce the circuit.

In order to facilitate future plant studies in space, the design developed as part of the experiment has been made open and available (Supplemental online material. The cost to construct the payload is less than 10,000 USD dominated by the cost of the machining and hermetic seal. In-house machining could bring the unit cost down to ~2000 USD. Space Applications Services and other providers are advertising Uights for less than 70,000 USD, which is an attainable price point for many academic and research groups. Further, the design now has Uight heritage reducing the risk for future Uights.

The experiment reported here is an early example of synthetic biological elements being tested in space. As the biological elements must work within organisms, context is an important consideration during design. SpaceUight is known to produce different phenotypes so working with space Uown elements is ideal. Alternatively, new elements can be tested in the spaceUight environment using an open design.

Uncited references

, .

Declaration of Competing Interest

The authors declare that they have no known competing Tnancial interests or personal relationships that could have appeared to inUuence the work reported in this paper.

Funding

Funding at NASA Ames was provided by the Ames Center Innovation Fund. Work at UC Berkeley and University of Utah was funded by BWF CASI award 1007224 (to MCH), NIH P50 GM102706 (to Center for RNA Systems Biology), and University of Utah (to MCH). ISU received funding from Eurometropole (the City of Strasbourg) and Groupe des Assurances Mutuelles Agricoles of Strasbourg (Groupama). The spaceUight was provided by Space Applications Services.

Fig. 1 .

 1 Fig.1. Schematic of pX7-AtPDSi genetic circuit which is induced by exogenous addition of estradiol. When induced with estradiol, the circuit permanently reconTgures itself to constitutively expresses RNAi (intron-containing self-complementary hairpin RNA) which bind to the mRNA of the gene of interest (PDS) marking it for degradation by native systems and thus preventing translation. PDS (Phytoene desaturase) is carotenoid biosynthetic enzyme whose activity as implications for plant color. (For interpretation of the references to color in this Tgure legend, the reader is referred to the web version of this article.)

Fig. 2 .

 2 Fig. 2. (Top) diagram of the hardware unassembled. Water bag and seed cassette are not pictured. The PCB assembly is inserted into the shell after the cassette and followed by the pressure plate, water bag, and lid. (Bottom) space cube on ISS just before being placed in a bag and sealed for return to Earth.

Fig. 3 .

 3 Fig. 3. Experimental timeline of the space cube. The cube was active on orbit for twenty days. The ground cube was operated on a one-day delay.

Fig. 4 .

 4 Fig. 4. Total leaf area as estimated by pixel counts. Patterns observed reUect germinations rates and physiological impacts of photobleaching. Growth was better by an order of magnitude in the ground cube than the space cube indicating stresses in the Uight cube.

Fig. 5 .

 5 Fig. 5. Images of plants from the experiment: top two rows are Uight plants (Day 15), bottom two rows are ground plants (Day 14). The Uight plants were smaller than the ground plants indicating stresses from spaceUight. In both Uight and ground cubes, the pX7-At-PDSi plants photobleached while the Col-0 plants did not. In the ground cube, pX7-AtPDSi plants produced green leaves later in the experiment as estradiol had been consumed. Time lapse movies are given in Supplementary Files 1 and 2 and all images are made publicly available. (For interpretation of the references to color in this Tgure legend, the reader is referred to the web version of this article.)

Fig. 6 .

 6 Fig. 6. Normalized greenness index (green/(green+red+blue)) is calculated for each plant pixel and the median for each sample reported. In both Uight (left) and ground (right) units transgenic plants start greener but Uip at about Day 9. (For interpretation of the references to color in this Tgure legend, the reader is referred to the web version of this article.)

Fig. 7 .

 7 Fig. 7. PCR conTrmation of DNA excision from CLX element of pX7-AtPDSi. Expected band sizes for the primer pairs is listed left and ladder bands on the right. Col-0 plants have no bands because the transgenic elements are not present. The P1/P4 lane conTrms successful excision. Smaller bands in the P3/P4 lane were due to contamination.

  et al. Life Sciences in Space Research xxx (xxxx) xxx-xxx co-authors reviewed and approved the Tnal draft.

Table 1

 1 Germination yield and general statistics of Trst activity timing.

		Flight		Ground
		Col-0	pX7-AtPDSi	Col-0	pX7-AtPDSi
	Geminated/total	22/43	26/40	25/43	41/43
	Gemination Yield (%) Mean (d) ⁎ ⁎ Std Err of Mean (d) ⁎ ⁎ Std Dev (d) ⁎ ⁎ CV (%) ⁎ ⁎ Min (d) ⁎ ⁎ Max (d) ⁎ ⁎ Range (d) ⁎ ⁎	0.51 6.66 0.55 3.60 0.54 2.67 16.75 14.08	0.65 5.69 0.21 1.30 0.23 3.17 7.50 4.33	0.58 4.39 0.19 1.25 0.28 3.00 7.67 4.67	0.93 3.43 0.12 0.80 0.23 2.33 6.00 3.67

⁎⁎ time to Trst detectable activity in images.

Author contributions

Conceptualization: RK, YD, JB, MFH, RB, JJF, BO, CPM, CW, MCH. Methodology: RK, YD, JB, MFH. Formal analysis: JB, YD, MM, PG, LD, DH, CV, NC, MA. Writing-original draft: JB, RK, YD, MCH, CPM. All

Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.lssr.2020.09.002.


Qi Wang

Jean-François Halet

Samia Kahlal

Alvaro Muñoz-Castro

Jean-Yves Saillard

Electron count and electronic structure of bare icosahedral Au 32 and Au 33 ionic nanoclusters and ligated derivatives. Stable models with intermediate superatomic shell fillings †

a DFT calculations were carried out on bare Au32 and Au33 nanoclusters with various charges, in order to analyze their stability with respect to different cluster electron numbers. Results indicate that in addition to the neutral Au32 hollow species, significant HOMO-LUMO gaps are computed for [Au32] 8+ (hollow) and [Au32] 4+ (two-shell structure). Species with smaller HOMO-LUMO gaps can reach stability upon "passivation" by a ligand shell, as experimentally exemplified. Icosahedral frameworks of Ih or lower symmetry are favored for the cationic nanoclusters whereas different structures are computed for the anionic ones.

Introduction

Gold clusters have attracted increased interest due to their sizedependent characteristics making them useful templates for nanosized species, for further applications in several fields ranging from catalysis, nanoelectronics, among others. 1 In 2004, Johansson et al. 2 and Gu et al. 3 predicted simultaneously the thermal stability of the bare neutral golden hollow cluster Au 32 , the structure of which adopts a fullerene-type geometry of I h symmetry (the socalled golden-fullerene cage). Although there is no direct experimental determination of its structure so far -only photoelectron spectroscopy (PES) experiments were carried out 4 it is likely to be one of the very lowest energy minima of bare Au 32 , although the structure of its true ground state remains controversial. 5 This is a spherical deltahedral arrangement describing a regular 32-vertex-60-face pentakis dodecahedron, a Catalan solid which is the dual polyhedron of the truncated icosahedron (the framework of C 60 ). Such a polyhedron displays two types of symmetry-equivalent vertices, divided in a group of 12 equivalent vertices Au ico describing an icosahedron and a group of 20 equivalent Au dod vertices describing a regular dodecahedron. The latter polyhedron, which exhibits 12 pentagonal faces, is the dual polyhedron of the icosahedron (top of Figure 1). Thus, the Au 32 cluster can be described as made of two nested polyhedra (Au 12 and Au 20 ) of approximately equal center-to-vertex radius. In this pseudo-spherical arrangement, there are Au dod -Au dod and Au dod -Au ico contacts, but no Au ico -Au ico contacts.

Figure 1. Top: The one-shell neutral Au 32 hollow cluster, as theoretically predicted. 2,3 The nested Au 12 icosahedron and Au 20 dodecahedron are shown in blue and orange, respectively. Bottom: The 2-shell Au 12 @Au 20 core, as found in Au 32 Cl 8 (PR 3 ) 12 (R = Et, n Pr, n Bu) 14 and [Au 32 (PPh 3 ) 8 (dpa) 6 ] 2+ (Hdpa = 2,2'-bipyridylamine).

15

The stability of Au 32 supported by a few theoretical studies 6,7 is related to its valence cluster electron number (cen), i.e., 32 -5d electrons are not considered -which provides the cluster with shell closure and spherical aromaticity. This electron count is in line with Mingos' electron counting rules for hollow spherical clusters in which the bonding is predominantly ensured by σ-type (radial-type) orbitals, gold 6s AOs in the present case. 8,[START_REF] Mingos | Introduction to cluster chemistry[END_REF] The model on which are based these rules is that of nearly free electrons constrained to move on the surface of a sphere of a given radius. Within this It is noteworthy that this cen is not a superatom "magic" number. [START_REF] Khanna | [END_REF][START_REF] Reveles | Proc. Natl. Acad. Sci[END_REF][START_REF] Castleman | [END_REF][13] . Thus, from the 32-cen hollow system to a 34-cen non-hollow one, the difference lies in the occupied 2S level in the latter that does not exist in the former (which is a single pseudo-spherical shell).

One should also take into account that the F-type orbitals, which are degenerate in the ideal R 3 symmetry of the sphere, split into two levels, namely t 2u and g u , in I h symmetry. Thus, the possibility for closed-shell electron counts with partial filling of the F shell, namely 24 In this paper, we investigate the possibility of stabilizing an icosahedral Au 32 architecture with seven potential closed-shell electron counts, corresponding to partial filling of the 1F shell, but also for possible filling of the 2S orbital, owing to the fact that a two-shell onion-like structure could be sufficiently compact for behaving as a superatom with a filled 2S orbital. The ten following tentative closed-shell configurations are summarized in Table 1

Remark

Au@Au12@Au20 0 20 +13 Au@Au12@Au20 0.26 Ih Au@Au12@Au20 6 26 +7 Au@Au12@Au20 0.27 C2-distorted Au@Au12@Au20 8 28 +5 Au@Au12@Au20 0.86

Ci-distorted

Au@Au12@Au20 14 34 -1 Non-icosahedral structure 1.08 Encapsulated triangle Au3@Au30 (C1) below). The possibility to add a supplementary Au atom at the cluster center is also investigated. Such a situation is expected to favor the occupation of the 2S orbital, thus suggesting the possible closed-shell configurations given in Table 2. Indeed the icosahedral X@Y 12 @Z 20 matryoshka arrangement is well documented in cluster chemistry.

17-19

Computational details

Geometry optimizations and subsequent calculations were performed on bare and ligated Au 32 and Au 33 clusters at the Density Functional Theory (DFT) level, [START_REF] Dyall | Introduction to Relativistic Quantum Chemistry[END_REF] with the Amsterdam Density Functional (ADF) code, [START_REF] Velde | [END_REF]22 employing the Becke-Perdew (BP86) exchange-correlation functional within the generalized gradient approximation (GGA), 23,24 and the valence triple zeta basis set, plus 2 polarization function (TZ2P). 25 The relativistic effects were treated at the scalar level by using the Zeroth Order Regular Approximation (ZORA) Hamiltonian. 26 Empirical Grimme DFT-D3 corrections were included to account for dispersion forces. 27 Vibrational frequency analysis was performed to check that optimized geometries were minima on their potential energy. Clusters models with large positive charges (see Table 1) were computed both as isolated species in vacuum and in solution using the dielectric Conductor like Screening Model (COSMO) of solvation.

The solvent (dichloromethane and dimethylformamide) effects were supposed to take into account, at least partly, the effects of the counteranion. 29 It turns out that, for all the computed cations, calculations both in vacuum and solvent provided similar results. Thus, calculations taking into account solvent effects are not further discussed below. Fragment decomposition analyses were carried out by single-point calculations using the optimized geometries. Time-dependent density functional theory (TD-DFT) calculations 30 were performed with the Gaussian 16 package 31 (chosen for CPU time efficiency) using the hybrid B3LYP functional 32 and the split valence Def2-TZVP basis set. 33 As suggested by a reviewer, the longrange corrected CAM-B3LYP functional 34 was also tested (Figure S3). It provided results in a less satisfying agreement with the experimental spectrum of Au 32 Cl 8 (PR 3 ) 12 14 than with B3LYP.

Results and discussion

Relevant computed results corresponding to the various computed configurations of bare [Au 32 ] q models are gathered in Table 1. Their optimized structures are shown in Figure 2 and metrical data are given in Table 3. Ground-state closed-shell configurations were found for all the computed systems, except for the [Au 32 ] 14+ model (cen = 18), which yielded a dissociated structure, presumably because of its large cationic charge. Including solvent effects in the calculations provided similar results. As expected, the neutral [Au 32 ] (cen = 32) one-shell hollow cluster of I h symmetry exhibits the largest HOMO-LUMO gap among all the computed systems, in agreement with the prediction of Johansson and of Gu. 2). Its significant HOMO-LUMO gap lets expecting potential stability for this structure. The non-stability of the icosahedral (pseudo-spherical) structure for this 34-cen count can be tentatively explained from the fact that more than half of the cluster 6s combinations, mainly responsible for cluster bonding, are filled, thus implying occupation of antibonding orbital(s) and favoring a structure with less connectivity and lower symmetry.

Among the considered electron counts for which a lower HOMO-LUMO gap was obtained, two of them, namely that of [Au 32 ]

6+ and [Au 32 ] 8+ deserve some comments. Independently from its starting geometry, the former converged to a unique structure corresponding to the 2S 0 1F 8 configuration that is best described as Au 12 @Au 20 rather than the expected one-shell arrangement, although some of the Au-Au distances within the Au 12 icosahedron are fairly large (3.07 Å). It is also distorted away from I h to C i symmetry (but close to T h ), presumably due to second-order Jahn-Teller instability in relation with its small HOMO-LUMO gap (0.35 eV).

The [Au 32 ]

8+ case is more striking. As said above, an I h one-shell structure with the 2S 0 1F 6 configuration and a large HOMO-LUMO gap was found for this species (Table 1). At first sight, an Au 12 @Au 20 two-shell structure appears unfavorable for this 24-cen count since it would likely lead to the 2S 2 1F 4 configuration, i.e., to partial filling of one of the t 2u or g u levels of 1F parentage. 16 However, as mentioned earlier, diamagnetic ligated clusters with an [Au 32 ] 8+ kernel were experimentally characterized, exhibiting the Au 12 @Au 20 two-shell arrangement. 14,15 The solid state structure of these clusters departs from ideal I h to approximate S 6 symmetry, a feature which was interpreted by Schnepf and collaborators 14 as a Jahn-Teller effect allowing the stabilization of two of the 1F orbitals and the possibility of the 2S 2 1F 4 closed-shell configuration. We thus looked for this putative configuration for a two-shell [Au 32 ] 8+ structure and found a minimum of D 5d symmetry in which the four 1F electrons occupy an e 1u level, all the other 1F levels are vacant (Figure 4). The rather small HOMO-LUMO gap (0.27 eV) obtained suggested possible HOMO-LUMO level crossings (thus electron configuration changes) upon moderate structural variations. As a matter of fact, when the starting structure used for the geometry optimization is an S 6 arrangement derived from the Au 32 kernel present in the X-ray structure of the 24-cen Au 32 Cl 8 (PEt 3 ) 12 nanocluster, 14 then the closed-shell 2S 0 1F 6 configuration was found instead (Figure 4). It is noteworthy that the corresponding optimized structure of exact C i symmetry departs significantly from S 6 . It is only 0.08 eV more stable than the D 5d structure and surprisingly 0.55 eV more stable than the one-shell icosahedral structure, despite its small HOMO-LUMO gap (0.33 eV). This result led us to wonder about the electronic structure of the Schnepf's 14 The simulated spectrum (Figure 6) shows a good agreement with the experiment. 14 The intense high-energy band computed at 449 nm (exp. 480 nm) 14 mostly involves 5d → 1G electronic transition.

The wide low-energy band is made of two major components at 620 and 685 nm (exp. 662 and 727nm) 14 mainly due to 1F → 1G and 1F → 1F electronic transitions, respectively. Similar calculations on the bare C i [Au 32 ] 8+ species led to a similar shape of the simulated UV-Vis spectrum somewhat red-shifted (Figure 6). The computed spectra of the two other [Au 32 ] 8+ cores (I h and D 5d ) are also reported in Figure 6. Although the I h structure exhibits a 5d → 1G band at a similar energy than its C i relative, the shape of its absorption curve is different higher energies. This result suggests that the simulated UV-Vis spectrum of the bare metal core ion should contain the optical signature of its real ligand-protected counterpart. For this reason, we also performed TD-DFT calculations on all the other A c c e p t e d M a n u s c r i p t models listed in Table 1 and gathered their simulated spectra in Figure S1. Pertinent results corresponding to some tested configurations of the atom-centered Matryoshka [Au 33 ] q model are gathered in Table 2. Their optimized structures are shown in Figure 7 and their metrical data are given in Table 4. Their TD-DFT-simulated UV-Vis spectra are shown in Figure S2, together with their non-centered isoelectronic relatives, for comparison. The frontier orbital diagrams of the three cationic species are plotted in Figure 8.

Conclusions

DFT calculations carried out on various bare Au 32 nanoclusters with various charges indicated that the hollow icosahedral arrangement is possible not only for the cluster electron number of 32 as initially predicted, but also for lower cluster electron numbers. In particular, when compared to their neutral [Au 32 ] reference, 5+ species has the largest HOMO-LUMO gap and is likely to be observed in the future. This 28-cen count appears to be favored for both hollow and atom-centered arrangements.

A c c e

  p t e d M a n u s c r i p t model, the solutions of the Schrödinger equation are simply the spherical harmonics expressed with respect to an origin situated at the center of the cluster sphere. For 32 electrons, the corresponding closed-shell configuration is S 2

A c c e

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

2 - 4

 24 Incidentally, this structure was found slightly distorted to D 2h in a recent DFT investigation. 5c Substantial HOMO-LUMO gaps are also found for the one-shell [Au 32 ] 8+ (cen = 24), [Au 32 ] 4+ (cen = 28) and [Au 32 ] 2-(cen = 34) clusters, indicating particular chemical stability for these electron counts. Both one-shell [Au 32 ] 8+ and [Au 32 ] 4+ systems are of icosahedral parentage, 16 although the symmetry obtained for our optimized geometry of [Au 32 ] 4+ is lowered to C i . Its major feature is a lengthening of six among the 30 edges of the Au 20 dodecahedron (3.84 Å vs. 3.15 Å avg.). The frontier orbital diagrams of [Au 32 ] and [Au 32 ] 4+ are shown in Figure 3, and that of the oneshell [Au 32 ] 8+ on the left hand-side of Figure 4.

Figure 2 .

 2 Figure 2. Optimized geometries of the computed bare [Au 32 ] q

Figure 3 .

 3 Figure 3. Frontier orbital diagrams of [Au 32 ] (left) and [Au 32 ] 4+

A c c e

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

Figure 4 .

 4 Figure 4. Frontier orbital diagrams of the structures corresponding to the three minima computed for bare [Au 32 ] 8+ : one-shell I h (left), two-shell D 5d (middle), and two-shell C i (right). Only 1F and 2S Kohn-Sham orbitals are plotted.

14

 14

Figure 5 .

 5 Figure 5. Frontier orbital diagram of the Au 32 Cl 8 (PH 3 ) 12 model. Only 1F and 2S Kohn-Sham orbitals are plotted.

Figure 6 .

 6 Figure 6. Simulated UV-Vis spectra of Au 32 Cl 8 (PH 3 ) 12 and of the various computed [Au 32 ]8+ structures.

Figure 7 .

 7 Figure 7. Optimized geometries of the computed [Au 33 ]q models.

Figure 8 .

 8 Figure 8. Frontier orbital diagrams of the [Au 33 ] q cations of icosahedral parentage: [Au 33 ] 13+ (left), [Au 33 ] 7+ (middle), and [Au 33 ] 5+

  Indeed, superatoms are not only spherical, but also

	close-packed (non-hollow) clusters, or at least multi-shell (onion-
	like) systems. Their electron counting rules are based on the
	spherical jellium model, which is related to that of nearly free
	electrons constrained to move inside a sphere. The nearest
	superatom closed-shell ("magic") number is 34, i.e., 1S 2 1P	6 1D 10 2S 2
	1F 14

Table 1

 1 . It turns out that while we were writing this paper, an elegant theoretical work by Lin and collaborators on bare and ligated compounds containing the [Au 32 ] 8+ and [Au 32 ] 4+ kernel came out.

		. The various 1S 2 1P	6 1D 10 2S x 1F y closed-shell electron
	configurations investigated for different bare icosahedral [Au 32 ]
			Expected
	Expected structure	electron configuration 2S x 1F y x y	cen = 18 + x + y	Cluster charge (q)	Optimized structure	Computed results HOMO-gap (eV) LUMO	Remark
	One shell	0	0	18		+14	-	-	Unbound structure
	One shell	0	6	24		+8	One shell	1.13	Ih
	One shell	0	8	26		+6	Au12@Au20 (see below)	0.35	Ci-distorted (see below)
	One shell	0	14	32		0	One shell	1.55	Ih
	Au12@Au20	2	0	20		+12	Au12@Au20	0.20	Ih
	Au12@Au20	2	4	24		+8	Au12@Au20	0.27	D5d
									Ci-distorted
	Au12@Au20	2	4	24		+8	Au12@Au20	0.33	with 2S 0 1F	6
								configuration
									Ci-distorted
	Au12@Au20	2	6	26		+6	Au12@Au20	0.35	with 2S 0 1F	8
								configuration
	Au12@Au20	2	8	28		+4	Au12@Au20	0.99	Ci-distorted
							Non-	Encapsulated
	Au12@Au20	2	14	34		-2	icosahedral	0.93	triangle
							structure	Au3@Au29 (C1)

16

Our results on these two particular electron counts are in large part similar to theirs (see q architectures.

Table 2 .

 2 The

		various 1S 2 1P	6 1D 10 2S x 1F
	Expected structure	1F y	y	cen = 20 + y	Cluster charge (q)	Optimized structure	Computed results HOMO-LUMO gap (eV)

y closed-shell electron configurations investigated for bare icosahedral atom-centered [Au 33 ]

q architectures.

  and Wang's 15 ligated clusters, as compared to bare [Au 32 ] 8+ . The ligated Au 32 Cl 8 (PH 3 ) 12 cluster model was then computed to mimic Schnepf's systems Au 32 Cl 8 (PR 3 ) 12 (R = Et, As a supplementary proof for our computed electronic structure, we performed TD-DFT calculations on Au 32 Cl 8 (PH 3 ) 12 to simulate the UV-Vis spectrum of Au 32 Cl 8 (PR 3 ) 12 (R = Et,

				n Pr,
	n Bu).	14 As for bare [Au 32 ]	8+ , its C i optimized structure cannot be
	approximated to S 6 . Its Kohn-Sham orbital diagram is shown in
	Figure 5. It is consistent with that of the C i two-shell [Au 32 ]	8+ model
	and thus to the 2S	0 1F 6 configuration in contrary with the 2S 2 1F 4
	closed-shell configuration proposed by Schnepf.	14 Interestingly, its
	HOMO-LUMO gap (0.87 eV) is larger than that of its isoelectronic
	bare metallic relative (0.33 eV). This finding suggests that bare
	[Au 32 ]	q models with rather small HOMO-LUMO gaps can get
	supplementary chemical stability by interaction with a specific
	ligand sphere. Our results on Au 32 Cl 8 (PH 3 ) 12 are in turn consistent
	with previous calculations by Lin and colls. on the 24-cen
	[Au 32 (PPh 3 ) 8 (dpa) 6 ] 2+ cluster.	15,16

n Pr, n Bu).

Table 4 .

 4 Au-Au distances (Å) of the atom-centered [Au 33 ]

	Small HOMO-

q clusters of icosahedral parentage. Average and range values are given for non-I h symmetry.

  As several of our computed icosahedral models, this cation exhibits a distorted icosahedral arrangement. We suggest that this (possibly distorted) icosahedral structure is disfavored for electron counts larger than 32, as exemplified by the [Au 32 ]2-anion which exhibits an Au 3 @Au 29 flattened . Interestingly, almost the same structure is obtained for the isoelectronic atom-centered [Au 33 ] -anion. Stability is expected for these two non-icosahedral structures, which suggests the possibility to stabilize them with neutral ligands.It should be noted that previous calculations on [Au 33 ] -found a similar structure as one of its low-energy minima.35,36 As we are interested in icosahedral structures we did not explore the whole energy surface of [Au 33 ]

	2,3 the significant HOMO-LUMO gaps 8+ (one-shell) and [Au 32 ] 4+ (two-shell) led us to think that these architectures should also exhibit chemical obtained for [Au 32 ] stability, once protected by specific anionic ligand shells. Moreover, even with rather small HOMO-LUMO gaps, Au 32 cations can reach stability when "passivated" by a ligand shell, as exemplified by the [Au 32 ] 8+ (two-shell) cation, stabilized as ligated species, 14,15 with the same metal electron configuration (2S 0 1F 6 ). -. In any case, a fragment decomposition analysis as provided by the ADF program 22 allowed us to described [Au 32 ] 2-and [Au 33 ] -as consisting of a 2-electron [Au 3 ] + triangle encapsulated within 32-electron [Au 29 ] 3-and [Au 30 ] 2-envelopes, respectively. Both [Au 3 ] + and its host cage were found to exhibit significant HOMO-LUMO gaps as isolated fragments. Incidentally, the triangular [Au 3 ] + cation is isolobal to H 3 + . Among the tested atom-centered icosahedral systems, the [Au 33 ]

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

Acknowledgements

The authors are grateful to the Chilean-French ECOS-CONYCYT program (project C18E04) and to the French-Chilean International Associated Laboratory for "Multifunctional Molecules and Materials" (LIA-CNRS N°1027). The GENCI French national computer resource is acknowledged for its support (project a0010807367). Q. W. thanks the China Scholarship Council for a Ph.D. grant. A.M.-C. thanks funding from FONDECYT 1180683.

Conflicts of interest

There are no conflicts to declare.