F Colas

G Woodward

F J Burdon

F Guérold

E Chauvet

J Cornut

A Cébron

H Clivot

M Danger

M C Danner

C Pagnout

S D Tiegs

Towards a simple global-standard bioassay for a key ecosystem process: organic-matter decomposition using cotton strips

Keywords:

Cotton-strip bioassays are increasingly used to assess ecosystem integrity because they provide a standardized measure of organic-matter decomposition -a fundamental ecosystem process. However, several different cottonstrip assays are routinely used, complicating the interpretation of results across studies, and hindering broader synthesis. Here, we compare the decay rates and assemblages of bacteria and fungi colonizing the three most commonly used cotton materials: Artist's canvas, Calico cloth, and Empa fabric. Cotton strips from each material type were incubated in 10 streams that span a wide range of physicochemical properties across five ecoregions. Additionally, to evaluate responses to environmental stress without potentially confounding biogeographical effects, we deployed identical bioassays in five streams across an acidification gradient within a single ecoregion.

Across all streams decomposition rates (as tensile strength loss [TSL]) differed among the three cotton materials; Calico cloth decomposed fastest (time to 50% TSL [T 50 ] = 16.7 d), followed by the Empa fabric (T 50 = 18.3 d) and then Artist's canvas (T 50 = 21.4 d). Despite these differences, rates of TSL of the three cotton materials responded consistently to variation in environmental conditions; TSL of each fabric increased with stream temperature, dissolved-nutrient concentrations and acid-neutralizing capacity, although Artist's canvas and Calico cloth were more sensitive than Empa fabric. Microbial communities were similar among the materials, and values of community structure (e.g., phylotype richness and diversity) were comparable to those reported for decaying leaves in streams from the same region, the major natural basal carbon resource in forested-stream ecosystems. We present linear calibrations among pairs of assays so that past and future studies can be expressed in a "common currency" (e.g., Artist's-fabric equivalents) 'past and future studies' repeated two times in the sentence. Lastly, given its relatively low within-site variability, and the large number of streams where it has been used (> 700 across the globe), we recommend Artist's fabric for future work. These results show that cotton provides an effective and realistic standardized substrate for studying heterotrophic microbial assemblages, and acts as a reasonable proxy for more chemically complex forms of detritus. These findings add to growing evidence that cotton-strip bioassays are simple, effective and easily standardized indicators of heterotrophic microbial activity and the ecosystem processes that result.

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Introduction

Organic-matter decomposition is a fundamental ecosystem process that contributes to the global carbon cycle, governs concentrations of atmospheric CO 2 [START_REF] Battin | Biophysical controls on organic carbon fluxes in fluvial networks[END_REF][START_REF] Battin | The boundless carbon cycle[END_REF], and influences local food-web dynamics (e.g., [START_REF] Moore | Detritus, trophic dynamics and biodiversity[END_REF]. In streams and rivers this process plays a central role in ecosystem functioning (see review by [START_REF] Tank | A review of allochthonous organic matter dynamics and metabolism in streams[END_REF] and, because it responds sensitively to critical anthropogenic stressors, decomposition has been proposed as an indicator of ecosystem condition that complements commonly used structural ⁎ Corresponding author.

E-mail address: tiegs@oakland.edu (S.D. Tiegs).

https://doi.org/10.1016/j.ecolind.2019.105466 of microbial activity and community structure in streams, like those already used in terrestrial ecosystems (e.g., [START_REF] Williamson | Calico cloth decomposition as an indicator of topsoil health[END_REF][START_REF] Pankhurst | Evaluation of soil biological properties as potential bioindicators of soil health[END_REF].

The first cotton-strip assays were conducted in terrestrial ecosystems using a fabric produced by the Shirley Company (Manchester, UK), which became a benchmark industry standard in soil studies [START_REF] Latter | Use of cotton strips to indicate cellulose decomposition in the field[END_REF][START_REF] Latter | The cotton strip assay for cellulose decomposition studies in soil: history of the assay and development[END_REF][START_REF] Boulton | A simple and versatile technique for assessing cellulose decomposition potential in floodplain and riverine sediments[END_REF]. The 'Shirley Soil Burial Test Fabric' was subsequently used in aquatic ecosystems where it was found to be sensitive to variation in environmental conditions [START_REF] Hildrew | Cellulolytic decomposition in streams of contrasting pH and its relationship with invertebrate community structure[END_REF][START_REF] Boulton | A simple and versatile technique for assessing cellulose decomposition potential in floodplain and riverine sediments[END_REF][START_REF] Claret | Functional processes versus state variables: interstitial organic matter pathways in floodplain habitats[END_REF][START_REF] Tiegs | Cotton strips as a leaf surrogate to measure decomposition in river floodplain habitats[END_REF]. Unfortunately, the manufacture of Shirley material was discontinued in 2002, creating need for a new standard [START_REF] Fritz | An assessment of cellulose filters as a standardized material for measuring litter breakdown in headwater streams[END_REF]. Several materials have since been used including 'Calico' cloth [START_REF] Imberger | Searching for effective indicators of ecosystem function in urban streams: assessing cellulose decomposition potential[END_REF], 'Artist's canvas' [START_REF] Slocum | Artist canvas as a new standard for the cotton-strip assay[END_REF], also known as 'Artist's fabric' [START_REF] Tiegs | A standardized cotton-strip assay for measuring organic-matter decomposition in streams[END_REF] and 'Empa material' [START_REF] Clapcott | Exploring the response of functional indicators of stream health to land-use gradients[END_REF]. Importantly, this lack of a single standard complicates the comparison of results across studies because the physical properties of the three cotton materials differ markedly (e.g., length and fineness of cotton fiber, strength, and elastic properties). Intercalibrations are therefore needed to compare studies involving different fabric types, past and future.

Here, we compared the performance of these three materials in streams along gradients of major environmental factors that influence organic-matter decomposition rates (e.g., ecoregions, acidification, temperature, nutrient concentrations). With this approach we derived intercalibrations that allow the conversion of decomposition rates into a 'common currency' for cellulose-decomposition potential among studies using different cotton types. We also characterized bacterial and fungal communities associated with each cotton material and evaluate attributes of community structure in the context of those published for leaf litter. Our aim is to provide a standardized methodology for quantifying cellulose-decomposition potential and heterotrophic communities in streams and other habitats across the globe, so that organicmatter decomposition and the factors that influence it can be better understood.

Materials and methods

Study site description

Cotton strips were deployed in headwater streams from five ecoregions that differ in their geology and climate [START_REF] Olson | Terrestrial ecoregions of the world: a new map of life on earth[END_REF][START_REF] Omernik | Ecoregions of the conterminous united states: evolution of a hierarchical spatial framework[END_REF]: northern temperate deciduous forests of the United States (US); English lowland deciduous broadleaf woodlands (UK); Vosges Mountains in northeast of France (FRNE); western-European broadleaf forests of Switzerland (CH); and Mediterranean mountains of Pyrenees in southwestern France (FRSW). These five ecoregions encompass broad latitudinal, longitudinal and altitudinal gradients, with a wide range of environmental and climatic conditions including oceanic, humid continental and subalpine climates. A pair of streams was selected in each ecoregion. In FRNE, five additional streams were included to span an acidification gradient within a single ecoregion. Water samples were taken on the first and last day of the cotton-strip-incubation period and assessed for concentrations of ammonium, nitrate, nitrite, and phosphorus, conductivity and pH using established protocols (e.g., NF EN ISO 13395). The study streams in the five ecoregions represented a range of nitrate concentrations from 0.11 to 1.85 mg NO 3 --N/L, phosphate concentrations from 1.4 to 28.3 µg/L and ammonium concentrations from 1 to 111 µg NH 4 + -N/L. The five sites in FRNE represented a gradient of pH ranging from 4.9 to 6.9, from -910.1 to 134.4 µeq/L for acid-neutralizing capacity values (ANC) and from 52.3 to 343.3 µg/L for aluminum concentrations. Detailed information on characteristics of individual streams across the five ecoregions are available in Table 1.

indicators (e.g., invertebrate community composition) [START_REF] Boulton | A simple and versatile technique for assessing cellulose decomposition potential in floodplain and riverine sediments[END_REF][START_REF] Gessner | A case for using litter breakdown to assess functional stream integrity[END_REF]. Organic-matter decomposition is most commonly assessed using litter-bag assays, an approach that typically involves incubating a known mass of locally collected leaves in mesh bags in the field and retrieving them to determine leafmass loss over time [START_REF] Boulton | A review of methodology used to measure leaf litter decomposition in lotic environments: time to turn over an old leaf?[END_REF]. Leaf-litter breakdown has been extensively studied during the past two decades in a variety of aquatic ecosystems to evaluate the impacts of anthropogenic stressors including organic pollution, acidification, hydromorphological alterations and land-use change [START_REF] Young | Organic matter breakdown and ecosystem metabolism: functional indicators for assessing river ecosystem health[END_REF][START_REF] Tank | A review of allochthonous organic matter dynamics and metabolism in streams[END_REF][START_REF] Elosegi | Effects of hydromorphological impacts on river ecosystem functioning: a review and suggestions for assessing ecological impacts[END_REF][START_REF] Woodward | Continental-scale effects of nutrient pollution on stream ecosystem functioning[END_REF][START_REF] Chauvet | Chapter three-litter decomposition as an indicator of stream ecosystem functioning at local-to-continental scales: insights from the European RivFunction project[END_REF][START_REF] Colas | Scale dependency in the hydromorphological control of a stream ecosystem functioning[END_REF][START_REF] Ferreira | Leaf litter decomposition as a bioassessment tool of acidification effects in streams: evidence from a field study and meta-analysis[END_REF]. While understanding of the factors that influence this process has grown considerably, key knowledge gaps persist due in part to limitations of litter-bag assays.

Despite their widespread use, litter-bag assays have shortcomings that undermine reliable comparisons among studies [START_REF] Boulton | A review of methodology used to measure leaf litter decomposition in lotic environments: time to turn over an old leaf?[END_REF] and hinder applications at large temporal and spatial scales [START_REF] Jackson | Recommendations for the next generation of global freshwater biological monitoring tools[END_REF][START_REF] Tiegs | Global patterns and drivers of ecosystem functioning in rivers and riparian zones[END_REF]. Notable among these is the difficulty in consistently obtaining leaf litter of uniform quality. Litter characteristics that influence decomposition, such as nutrient and lignin content, are highly variable both among and within tree species [START_REF] Webster | Vascular plant breakdown in freshwater ecosystems[END_REF][START_REF] Gessner | A case for using litter breakdown to assess functional stream integrity[END_REF][START_REF] Hladyz | Resource quality and stoichiometric constraints on stream ecosystem functioning[END_REF][START_REF] Lecerf | Intraspecific variability in leaf traits strongly affects alder leaf decomposition in a stream[END_REF], and even among leaves of individual trees of the same species (e.g., [START_REF] Sariyildiz | Decomposition of sun and shade leaves from three deciduous tree species, as affected by their chemical composition[END_REF]. These and other sources of variability in litter quality (e.g., interannual variation) mean that decomposition rates among studies are contingent on not only the environmental conditions in which the litter was incubated, but also the particular organic matter used. Consequently, basic understanding of how the inherent capacity of streams to process organic matter -i.e., their decomposition potential (sensu [START_REF] Imberger | Searching for effective indicators of ecosystem function in urban streams: assessing cellulose decomposition potential[END_REF] -varies through time and space, and in response to anthropogenic stressors, is impeded by a widespread lack of standardization and comparability.

Given the numerous advantages that they offer over litter-bag approaches, cotton-strip assays are a logical choice for use as a universalstandard bioassay because they enable researchers to evaluate the capacity of ecosystems to decompose organic matter. Moreover, given their ease of assembly, deployment, and portability, cotton-strip assays have potential to enable research at large spatial and temporal scales, addressing the pressing need for global monitoring and assessment [START_REF] Jackson | Recommendations for the next generation of global freshwater biological monitoring tools[END_REF][START_REF] Tiegs | Global patterns and drivers of ecosystem functioning in rivers and riparian zones[END_REF]. Consisting of ≈95% cellulose, cotton is an ecologically relevant compound given that this key carbohydrate is the main constituent of plant litter, the most abundant organic polymer on Earth, and a major basal resource in most of the Earth's food webs [START_REF] Egglishaw | An experimental study of the breakdown of cellulose in fastflowing streams[END_REF]. Cotton contains very low concentrations of nutrients such as N and P, which are highly variable among and within the litter of other plant species, and are main intrinsic determinants of decomposition rates (e.g., [START_REF] Latter | Use of cotton strips to indicate cellulose decomposition in the field[END_REF][START_REF] Claret | Functional processes versus state variables: interstitial organic matter pathways in floodplain habitats[END_REF][START_REF] Clapcott | Exploring the response of functional indicators of stream health to land-use gradients[END_REF][START_REF] Hladyz | Resource quality and stoichiometric constraints on stream ecosystem functioning[END_REF][START_REF] Tiegs | Cotton strips as a leaf surrogate to measure decomposition in river floodplain habitats[END_REF][START_REF] Tiegs | A standardized cotton-strip assay for measuring organic-matter decomposition in streams[END_REF]. Additionally, cotton fabric is less prone to fragmentation than leaves [START_REF] Egglishaw | An experimental study of the breakdown of cellulose in fastflowing streams[END_REF], thereby reducing the variability due to hydraulic conditions. Cellulose is colonized by a wide range of microorganisms, including leaf-colonizing fungi and bacteria [START_REF] Singh | Cellulose decomposition by some tropical aquatic hyphomycetes[END_REF][START_REF] Harrison | Cotton Strip Assay: An Index of Decomposition in Soils[END_REF]. They provide practical and logistical advantages over litter-bag assays, including often-shorter incubation times in the field, relative ease of use, and low cost of the measurement [START_REF] Tiegs | A standardized cotton-strip assay for measuring organic-matter decomposition in streams[END_REF]. Importantly, cotton-strip decomposition, as with leaf-litter decomposition, is sensitive to a range of anthropogenic stressors including: acidification (e.g., [START_REF] Hildrew | Cellulolytic decomposition in streams of contrasting pH and its relationship with invertebrate community structure[END_REF][START_REF] Jenkins | Long-term amelioration of acidity accelerates decomposition in headwater streams[END_REF], temperature [START_REF] Griffiths | Organic-matter decomposition along a temperature gradient in a forested headwater stream[END_REF][START_REF] Tiegs | Global patterns and drivers of ecosystem functioning in rivers and riparian zones[END_REF], metal contamination (e.g., [START_REF] Chew | Microbial cellulose decomposition in soils from a rifle range contaminated with heavy metals[END_REF][START_REF] Costello | Response of stream ecosystem function and structure to sediment metal: context-dependency and variation among endpoints[END_REF][START_REF] Gardham | Direct and indirect effects of copper-contaminated sediments on the functions of model freshwater ecosystems[END_REF], and nutrient loading (e.g., [START_REF] Boulton | A simple and versatile technique for assessing cellulose decomposition potential in floodplain and riverine sediments[END_REF][START_REF] Piggott | Multiple stressors and stream ecosystem functioning: climate warming and agricultural stressors interact to affect processing of organic matter[END_REF]. Cotton-strip assays therefore offer promising opportunities for standardized measurements

Cotton-strip bioassays

Cotton strips were prepared from bolts of 'Calico' cloth (Lincraft Pty. Ltd., Miranda, New South Wales, Australia), 'Artist's' canvas (Fredrixbrand unprimed 12-oz. heavyweight cotton fabric, Style #548, Lawrenceville, GA, USA), and 'Empa' fabric (Swissatest Product no. 222;Empa,St. Gallen,Switzerland). Detailed information on each substrate is available in Table 2. Cotton strips were prepared and deployed according to methods previously reported in the literature (e.g., [START_REF] Tiegs | A standardized cotton-strip assay for measuring organic-matter decomposition in streams[END_REF][START_REF] Tiegs | Global patterns and drivers of ecosystem functioning in rivers and riparian zones[END_REF].

In each stream, one 'block' with one strip of each material type was located in six riffle-type habitats separated by approximately 7 times the bankfull-channel width. Each block consisted of cotton strips attached to a stake hammered into the stream substrate. Strips were incubated in streams for 20-27 days beginning in November 2014, a duration predicted to yield an approximate average of 50% tensilestrength loss, an amount of decay that is believed to maximize sensitivity of the assay [START_REF] Tiegs | A standardized cotton-strip assay for measuring organic-matter decomposition in streams[END_REF]. In the five streams that spanned the acidification gradient, one additional strip of each material type located in three riffle-type habitats was incubated for 6 weeks to evaluate fungal and bacterial abundance, and to compare microbial assemblages that colonized the three types of cotton materials. Additionally, a time-series experiment was conducted in the UK and French reference streams (i.e. FRNE and FRSW) to compare the time to yield 50% tensile loss of each material. To this end, six strips for each cotton type were anchored to three additional stakes and removed weekly for 6 weeks. Temperature was recorded hourly in each stream for the duration of the experiments with a logger, except for streams in southeast UK. After incubation in the field, cotton strips were removed and placed individually into labeled plastic bags for transport to the laboratory.

Tensile loss determination

In contrast to litter-bag assays that quantify decomposition rates by measuring the loss of mass of organic matter through time, cotton-strip assays quantify the loss of tensile strength, a process that equates to the catabolism of cellulose. In the laboratory, following the protocol of [START_REF] Tiegs | A standardized cotton-strip assay for measuring organic-matter decomposition in streams[END_REF], each strip was placed in a shallow tray containing 70% ethanol and cleaned gently for 30 s with a small paint brush to remove adhering sediment and debris. The strips were then transferred to small aluminum pans, dried at 40 • C for several days, and stored in desiccators. The strips were shipped to the Aquatic Ecology Lab at Oakland University for tensile-strength determination.

The tensile strength of each strip was measured on a tensiometer (Mark-10 brand, Model #MG100, Copiague, NY, USA) mounted to a motorized test stand, and pulled at a rate of 2 cm/min. The initial tensile strength of the strips was determined using a set of control strips, i.e., for each ecoregion, seven additional strips of each cotton material that were not incubated in the streams were wetted in tap water, dried in an oven, and stored in a desiccator before being processed identically to the treatment strips (Table 2). Tensile-strength loss (TSL) was expressed as percent of the initial tensile-strength lost per day of incubation according Equation (1) (after [START_REF] Tiegs | A standardized cotton-strip assay for measuring organic-matter decomposition in streams[END_REF]:

= ◊ TSL Tensile Strength Tensile Strength Incubation Time 1 100/ treatment strips reference strips (1)
where Tensile Strength treatment strips is the maximum tensile strength recorded for each strip incubated in the field, Tensile Strength reference strips is the mean tensile strength of control strips that were not incubated in the field, and incubation time is the number of days the strips were incubated in the field. To account for differences in decomposition rate due to temperature differences across the acidification gradient, tensile strength was also expressed as a percentage of the initial TSL per degree-day by substituting degree-days for time in Eq.

(1). Degree days were estimated by summing the mean daily temperatures at each stream, based on hourly readings greater than 0 ⁰C. Additionally, the time to yield 50% TSL (T50) was estimated by fitting the tensile loss to a function of incubation time according to a linear model using the time series experiments performed in three reference streams. The linear model was used because it fits the data better than the exponential model according to AICc.

Microbial abundance and community assemblages

Before DNA extraction, strips were placed in a shallow tray filled with distilled water (not ethanol as with the other strips) and cleaned gently with a small paint brush to remove any adhering sediment. Pieces of cotton (1.5 × 1.5 cm) were crushed in Eppendorf tubes containing 750 µl of the PowerBead Tube solution from the PowerSoil DNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA). Microbial suspensions were transferred in the PowerBead Tubes and total DNA was extracted using the PowerSoil DNA Isolation Kit according to the manufacturer's instructions.

The abundance of fungi and bacteria was estimated by qPCR using the primer sets Fung5F/FF390R [START_REF] Lueders | Stable isotope probing of rRNA and DNA reveals a dynamic methylotroph community and trophic interactions with fungi and protozoa in oxic rice field soil: SIP of methylotrophs in rice field soil[END_REF], 968F/1401R [START_REF] Felske | Quantification of 16S rRNAs in complex bacterial communities by multiple competitive reverse transcription-PCR in temperature gradient gel electrophoresis fingerprints[END_REF], which target fungal 18S rRNA genes and the bacterial 16S rRNA genes, respectively. The SYBR green qPCR assays were performed as previously described [START_REF] Thion | Long-term in situ dynamics of the fungal communities in a multi-contaminated soil are mainly driven by plants[END_REF][START_REF] Cébron | Impact of clay mineral, wood sawdust or root organic matter on the bacterial and fungal community structures in two aged PAH-contaminated soils[END_REF] using a CFX96 Real-Time PCR detection system (Bio-Rad). Data were expressed as 18S to 16S rDNA copy number ratio. PCR primers ITS3GC and ITS4 were used for PCR amplification of the ITS2 region of fungal ribosomal DNA [START_REF] Nikolcheva | Seasonal and substrate preferences of fungi colonizing leaves in streams: traditional versus molecular evidence[END_REF]. PCR primers 341F-GC2/907R were used for partial amplification of the bacterial 16S rRNA genes [START_REF] Muyzer | Denaturing gradient gel electrophoresis (DGGE) in microbial ecology[END_REF][START_REF] Muyzer | Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain[END_REF].

The amplification products were separated with Denaturing Gel Electrophoresis (DGGE) on the DCODE Mutation Detection System (Bio-Rad, Hercules, CA). Electrophoresis was performed on 8% poly-acrylamide gels with a denaturing gradient from 30 to 70% for the fungal PCR products and from 40% to 60% for the bacterial PCR products [100% denaturant corresponds to 40% (v/v) formamide and 7 M urea]. DGGE was run 16 h at 55 V and 56 °C for fungal PCR products and 16 h at 100 V and 60 °C for bacterial PCR products. The gels were stained with SYBR Green I and imaged with a STARION FLA-9000 scanner (Fujifilm Life Sciences FSVT, Courbevoie, France) before being analyzed using GelCompar II (Applied Maths, Sint-Martens-Latem, Belgium). DGGE profiles were aligned using control samples as migration markers. The molecular richness was calculated as the total number of bands/phylotypes (PR = phylotype richness) for each DGGE profile. Microbial diversity was calculated using the Shannon-Weaver index (H') and relative abundance of phylotypes (based on relative band intensity).

Statistical analyses

Nested ANOVA was used to evaluate differences in mean percent TSL per day among ecoregions and cotton materials (as a fixed effect) using streams nested within ecoregions as a random effect. A linear mixed model was used to test the variation of percent TSL per degreeday along the acidification gradient using the fabric type and the acid neutralizing capacity, or the aluminum concentrations, as fixed effects and the streams as random effect. Similarly, the relationships between percent TSL per day and nutrient concentrations were examined using linear mixed models. Nutrients concentrations and fabric type were used as fixed effects and ecoregions and streams nested within ecoregions as random effects. Variation in microbial abundance, phylotype richness and diversity among cotton fabric incubated along the acidification gradient was examined using mixed ANOVA with cotton type as fixed effect and streams as random effect. All mixed models were performed using restricted maximum likelihood (REML) and the package lmerTest [START_REF] Kuhn | Package 'lmerTest[END_REF]. Significance of fixed effects was derived using Satterthwaite approximation for degrees of freedom, as it produces acceptable Type 1 error rates for smaller samples [START_REF] Luke | Evaluating significance in linear mixed-effects models in R[END_REF]. Post-hoc comparisons of means were performed using 'glht' function of the 'multicomp' package and a Bonferroni-Holm correction [START_REF] Hothorn | Simultaneous inference in general parametric models[END_REF]. Significance of random effects was determined using 'rand' function of 'lmerTest' package. Model checking included homogeneity of variance and normal distribution of model residuals and did not reveal any obvious deviations from homoscedasticity or normality.

Non-metric multidimensional scaling (NMDS) analyses of fungal and bacterial community profiles were used to assess differences among sites and among cotton fabric using the function 'metaMDS' in vegan. The Bray-Curtis coefficient was used to quantify dissimilarity among sites and among cotton fabrics based on community profiles. Goodnessof-fit was estimated with a stress function, which ranges from 0 to 1, with values close to zero indicating a good fit. Stress < 0.15 corresponds to good ordination [START_REF] Clarke | Non-parametric multivariate analyses of changes in community structure[END_REF]. Axes values from the NMDS analyses were correlated using Spearman rank correlation with ANC and aluminum concentration values to identify variables that corresponded to among-site differences in microbial community profiles using the function 'cor.test' of the 'stats' package. Linear regression models were performed to link percent TSL per day of each cotton fabrics using the caret [START_REF] Kuhn | Package 'lmerTest[END_REF] and mass [START_REF] Venables | Modern Applied Statistics with S, fouth ed[END_REF] packages. To run the models, data were divided into two parts, a training data set (i.e. 80% of the original data set) used to build the model and a validation data set (i.e. 20% of the original data set) used to gauge the model's performance. Model performance and accuracy were estimated using root mean square error and the Pearson correlation coefficient between the observed and predicted values

Results

Tensile-strength loss across ecoregions and environmental gradients

Patterns of TSL across ecoregions were similar among the three cotton materials (Fig. 1). Across all streams, TSL ranged from 0.4 to 4.4% d -1 , with an overall mean ± SD of 2.3 ± 1.2, 2.6 ± 1.4 and 2.5 ± 1.7% for Artist's fabric, Empa and Calico, respectively (Fig. 1). Differences were observed among fabric types in terms of their overall rates of tensile-strength loss (F 2,108 = 5.6, p < 0.005) with Artist's fabric being slower to decompose than either Empa or Calico. Differences in mean rates of TSL were also observed among streams within ecoregions but not among ecoregions, although the differences were close to being statistically significant (F 4,5 = 4.8, p = 0.058).

For each of the three fabric types, TSL per degree day increased with increasing ANC (F 1,11 = 59.2, P < 0.001; Fig. 2A), and decreased with increasing aluminum concentrations (F 1,11 = 43.4, P < 0.001; Fig. 2B). No significant differences were observed among fabric types, indicating that regardless of fabric type the decomposition of cellulose is strongly affected by acidification. Similarly, fabric type did not affect TSL along gradients of NO 3 --N (Fig. 3A) and PO 4 3--P (Fig. 3B); TSL increased with concentrations of NO 3 --N (F 1,5.2 = 41.4, P < 0.005) and PO 4 3--P (F 1,6 = 9.3, P < 0.05).

Time-series experiment

Time-series experiments and linear models revealed that the time to 50% TSL (T50) differed among the fabrics type (Fig. 4). Artist's fabric decomposed more slowly than Calico and Empa with 50% tensilestrength loss achieved after 21.2 ± 2.2, 16.7 ± 3.0 and 18.3 ± 4.2, respectively. Empa fabric was the most variable at T50; Artist's fabric was the least variable (CV: 10.4% for Artist's fabric, 17.9% for Calico and 23.0% for Empa).

The TSL of the three cotton materials was strongly related (Table 3). Each linear regression model performed between TSL of each cotton type was highly significant. On average, the models accounted for 81.2 ± 4.6% of the variation with correlations between the observed and predicted values ranging from 0.88 to 0.95. The information required to convert the tensile-loss rates of each strip type to another is shown in Table 3, for example, to express measured rates of Empa TSL in terms of Artist's-fabric TSL equivalents.

Comparison of microbial communities

Heterotrophic microbial assemblages were broadly similar across cotton types, but varied significantly among streams. Across all streams and cotton materials, fungi-to-bacteria ratios ranged between 0.2 and 4.1. Mean phylotype richness (PR) ranged from 13.7 to 22 phylotypes for bacteria and from 13.7 to 23.3 for fungi. Shannon-Weaver's diversity index (H') ranged from 2.2 to 2.9 for bacteria and from 1.9 to 2.7 for fungi. NMDS ordinations performed on fungal (Fig. 5A) and bacterial communities (Fig. 5B) exhibited stress < 0.15, with Axes 1 of the NMDS being strongly correlated with ANC and total aluminum concentration for fungal (r = -0.70, p < 0.001; r = 0.66, p < 0.001, respectively) and bacterial community profiles (r = -0.61, p < 0.001; r = 0.80, p < 0.001, respectively). Detailed information on microbial abundances and assemblages across the five streams is available in Table 4.

Discussion

Ecosystem processes are rarely included as part of river-health-assessment programs, despite compelling advocacy for considering their role when gauging human impacts; part of the reason is a lack of easyto-use standardized indicators. The results presented here further support a body of evidence indicating that cotton-strip assays are sensitive to a suite of environmental factors, including those that are influenced by human activities. Adding to the utility of cotton-strip assays are close relationships among the three fabrics we examined -relationships that enable direct comparisons of past, ongoing and future studies, thereby greatly expanding the number of streams for which there are comparable data. Given that the number of fungal and bacterial phylotypes that were found colonizing the cotton strips was similar to that found on leaves, the assay holds promise as a standardized means of sampling microbes in stream and other habitats. And given that Artist's fabric had the least variability among analytical replicates, and the large number of studies and field sites where Artist's fabric has been used (e.g., including this study, over 700 different streams across the globe), we recommend Artist's fabric as the standard material for researchers who require a reliable and sensitive process-based indicator. These and other findings are key steps towards understanding large-scale variation in ecosystem functioning, and inclusion of process-based indicators in bioassessment programs across the globe.

We found consistent results across the three fabric types, and with studies using cotton-strips and litter-bags in terms of the responses to environmental gradients and stressors (e.g., [START_REF] Ferreira | Whole-stream nitrate addition affects litter decomposition and associated fungi but not invertebrates[END_REF][START_REF] Gulis | Stimulation of leaf litter decomposition and associated fungi and invertebrates by moderate eutrophication: implications for stream assessment[END_REF][START_REF] Ferreira | Future increase in temperature more than decrease in litter quality can affect microbial litter decomposition in streams[END_REF][START_REF] Fernandes | Higher temperature reduces the effects of litter quality on decomposition by aquatic fungi[END_REF][START_REF] Tiegs | A standardized cotton-strip assay for measuring organic-matter decomposition in streams[END_REF][START_REF] Tiegs | Global patterns and drivers of ecosystem functioning in rivers and riparian zones[END_REF][START_REF] Wagenhoff | Subsidy-stress and multiple-stressor effects along gradients of deposited fine sediment and dissolved nutrients in a regional set of streams and rivers[END_REF]. Organic-matter decomposition in aquatic ecosystems has typically been investigated without explicit consideration for the communities of microorganisms involved. Our study characterized bacterial and fungal communities and their role in cellulose decomposition, and paves the way for future work that will elucidate the roles of these communities in cellulose decay using next-generation molecular tools (e.g., [START_REF] Clivot | Leaf-associated fungal diversity in acidified streams: insights from combining traditional and molecular approaches[END_REF].

Cotton differs from leaf litter (the most commonly used substrate in organic-matter decomposition studies and a key source of carbon in streams) in that it does not contain appreciable quantities of nutrients such as nitrogen and phosphorus that can promote microbial activity, nor does it contain lignin or secondary compounds that can inhibit it [START_REF] French | Some effects of changing soil chemistry on decomposition of plant litters and cellulose on a Scottish moor[END_REF]. Because of these and other differences in quality, different heterotrophic microbial communities between leaves and cotton strips might be expected. However, given the extremely wide ranging quality of litter and other organic matter that has been documented, including that from riparian trees (e.g., [START_REF] Boyero | Riparian plant litter quality increases with latitude[END_REF], microbes can also be expected to harbor adaptations that allow them to exploit diverse types of resources. Despite the differences between cellulose fabric and more-complex organic matter, the microbial communities from cotton strips were similar to those reported for leaf-litter in terms of phylotype richness and diversity [START_REF] Manerkar | Q-RT-PCR for assessing archaea, bacteria, and fungi during leaf decomposition in a stream[END_REF][START_REF] Clivot | Leaf-associated fungal diversity in acidified streams: insights from combining traditional and molecular approaches[END_REF]. Here we found between 14 and 23 OTUs of fungi along the gradient of acidification; in the same geographic same area, [START_REF] Clivot | Leaf-associated fungal diversity in acidified streams: insights from combining traditional and molecular approaches[END_REF] reported between 17 and 22 OTUs of fungi on decaying alder (Alnus glutinosa) leaves in five streams across an acidification gradient. Regarding bacterial community profiles, [START_REF] Clivot | Leaf-associated fungal diversity in acidified streams: insights from combining traditional and molecular approaches[END_REF] found a range of 15 to 28 OTUs on maple leaves in streams spanning an acidification gradient, compared to the range of 14 and 22 OTUs in our study. In a first-order stream in Nova Scotia (Canada), [START_REF] Manerkar | Q-RT-PCR for assessing archaea, bacteria, and fungi during leaf decomposition in a stream[END_REF] found a total of 23 OTUs of fungi on leaf disks of Tilia cordata. Our results, in the context of previously reported findings, suggest that in addition to providing a useful method for quantifying decomposition rates, cotton-strip assays offer a standardized means of sampling heterotrophic microbial communitie.

The community structure of microorganisms found on cotton strips, and any organic matter in streams, stems from several processes, including inoculum transport from the upstream environment, colonization on the substrate and subsequent microbial activity and growth. Of these, inoculum pressure and colonization would seem to be the least sensitive to substrate quality; this might explain the similar levels of phylotype diversity found between cotton strips and leaves. Regardless of the mechanisms behind the similarity in phylotype diversity between cotton strips and leaves, cotton strips seem to function as a standardized substrate for obtaining a representative sample of microbial heterotroph communities.

While the three cotton materials displayed similar responses to variable environmental conditions and stressors, such as acidification and nutrient loading, TSL differed among them; this demonstrates the need for intercalibrations across studies that use different assays. Since the microbial communities that colonize cotton strips were similar, simple physical differences in the properties of the cotton types may be driving the differences in TSL (e.g., thread density, thread strength). Artist's canvas is far denser than the Empa and Calico cotton (i.e., 407 g/m 2 for Artist's fabric and 205 and 115 g/m 2 for Empa and Calico, respectively), an observation that may explain why the Artist's fabric had the least within-site variability of the three fabric types examined. A notable finding was that the Empa material was less sensitive to environmental conditions than the other fabric types given its greater coefficient of variation. Despite the differences among them we nonetheless found very strong positive linear relationships between the TSL values. This enables direct comparisons of studies by providing researchers with information to convert data to a common currency. For example, with the calibration parameters provided in Table 3, TSL of Calico and Empa materials can easily and accurately be converted to Artist's-fabric equivalents.

Tensile-strength loss is the most commonly applied method for quantifying cotton-strip decomposition, an approach that requires a tensiometer. Tensiometers are common in materials-testing facilities across the globe, and in engineering departments at universities. Additionally, some laboratories perform tensile-strength determination on a contract basis for researchers who do not have ready access to a tensiometer. These instruments range widely in their degree of sophistication and price. For researchers who opt not to determine tensile strength, the cotton-strip assay can still be applied, and in lieu of tensile strength, closed chamber measurements of respiration can be performed (see Tiegs et al., 2013 for details). And as mentioned above, the assay can be used as a standard means of sampling microbial community structure. Lastly, mass loss can be measured on the strips, which relates to tensile-strength loss [START_REF] Tiegs | Cotton strips as a leaf surrogate to measure decomposition in river floodplain habitats[END_REF]. However, we urge

Table 4

Phylotype richness (PR) and diversity index (H') of bacteria and fungi for three cotton fabrics based on qPCR and DGGE performed on 16S and 18S rRNA. PR-Bac: Phylotype richness of bacteria, H-Bac: Shannon-Weaver diversity index of bacteria, PR-fungi: Phylotype richness of fungi, H-fungi: Shannon and Weaver diversity index of fungi, F/B ratio: fungi-to-bacteria ratio from qPCR. Streams are ordered according to the acidification gradient, from 1 (circumneutral stream) to 5 (acidified stream). caution with this approach since significantly longer incubations may be required, and the strips become prone to mass loss through unraveling, rather than carbon mineralization. Whether through determination of tensile strength, respiration, or heterotrophic-community structure, the cotton-strip assay offers utility and value to ecologists interested in organic-matter decomposition.

Here we helped develop the cotton-strip assay by deploying it in streams, however, it is increasingly used to evaluate microbial activity in other ecosystems, terrestrial and aquatic. For example, cotton strips have been deployed in shallow hyporheic sediments [START_REF] Boulton | A simple and versatile technique for assessing cellulose decomposition potential in floodplain and riverine sediments[END_REF][START_REF] Burrows | High rates of organic carbon processing in the hyporheic zone of intermittent streams[END_REF] and deep groundwater [START_REF] Lategan | Is cotton-strip tensile strength a surrogate for microbial activity in groundwater?[END_REF]. Artist's fabric has been deployed in remote high-latitude wetlands [START_REF] Vizza | Landscape patterns shape wetland pond ecosystem function from glacial headwaters to ocean[END_REF], in the flocculant sediments of lakes, wetlands and streams (Kincaid et al., in revision), and in spruce peatlands as part of a large scale warming and carbon-dioxide-enrichment experiment (SPRUCE). Additionally, the assay has tremendous potential to improve understanding of how decomposition rates vary in marine habitats, and to date, strips have been deployed in estuaries [START_REF] Bierschenk | Intensity of land use in the catchment influences ecosystem functioning along a freshwater-marine continuum[END_REF], kelp forests (Filbee-Dexter unpublished data), and intertidal mangrove creek systems (Tiegs unpublished data). Lastly, cotton strips made of Artist's fabric have been deployed in hundreds of riparian zones across the globe to better understand the drivers of carbon processing in these ecosystems [START_REF] Tiegs | Global patterns and drivers of ecosystem functioning in rivers and riparian zones[END_REF]. Through time, and with the continued used of standardized decomposition assays, a finer resolution picture will emerge of how carbon is processed in Earth's ecosystems, such as identifying its hot spots and hot moments (sensu [START_REF] Mcclain | Biogeochemical hot spots and hot moments at the interface of terrestrial and aquatic ecosystems[END_REF].

The cotton-strip assay is believed to be most sensitive to variation in environmental conditions when values of tensile-strength loss are near 50% [START_REF] Harrison | Cotton Strip Assay: An Index of Decomposition in Soils[END_REF] and the research presented here is useful for optimizing incubation durations in order to hit this target. These durations are 21d, 16d, 18d for Artist's fabric, calico and Empa material, respecively. Temperature and nutrient status are additional factors to consider when estimating an appropriate incubation duration, with moderate levels of enrichment stimulating decay rates. However, very high nutrient concentrations, such as those associated with agriculture and urbanization, can be associated with slowed decay [START_REF] Woodward | Continental-scale effects of nutrient pollution on stream ecosystem functioning[END_REF], and incubation duration may need to be adjusted accordingly. Notably, most of the streams studied here have somewhat high nutrient levels, and longer incubations with more degree day accumulations will be required to hit the target decay level of 50% in other systems. Moisture is believed to be limiting to microbial activity in terrestrial habitats [START_REF] Tiegs | Global patterns and drivers of ecosystem functioning in rivers and riparian zones[END_REF], and incubation durations may need to be lengthened considerably (e.g., up to many months) when aridity is a factor. While 50% TSL should be the aim in most instances, appreciable deviations from this target will not invalidate studies, but may limit the sensitivity of the assay.

Our results validate the utility of cotton strips as effective indicators of both ecosystem functioning and the community structure of the microbes that drive it. These are needed steps towards a more universal, global bioassay. Hundreds of streams have now had their decomposition potential quantified with the cotton-strip assay and this data constitutes a substantial foundation for establishing baselines to track environmental change. And if developed within the framework of new global biomonitoring initiatives (e.g., Future Earth, Intergovernmental Science-Policy Platform on Biodiversity and Ecosystem Services), the cotton-strip assay holds promise as a means to track the impacts of global change on vital aspects of carbon cycling, such as decomposition rates and the structure of heterotrophic microbial communities.

Fig. 1 .

 1 Fig.1. Mean TSL per day ( ± SD) across fabric types and ecoregions. CH: western European broadleaf forests in Switzerland; FRSW: the Mediterranean mountains of Pyrenees in southwestern France; FRNE: the Vosges mountain in northeastern France; UK: English lowland beech forests; US: temperate deciduous forests of the northern United States. Continuous black line indicates the mean percent tensile strength loss per day for all cotton fabrics; the dashed line indicates the mean value for each of the three fabric types across all streams.

Fig 2 .

 2 Fig 2. Mean TSL per degree day ( ± SD) along acid-neutralizing capacity (A) and aluminum concentrations (B) gradients. N = 5 streams in each panel.

Fig. 4 .

 4 Fig.4. Barplot of T50 ( ± calculated on three reference sites using linear models for Artist's fabric, Calico cloth and Empa's fabric. N = 3 streams.

Table 1

 1 Ranges of values of main physicochemical characteristics of water among streams across the five ecoregions examined: western European broadleaf forests in Switzerland (CH); the Mediterranean mountains of Pyrenees in southwestern of France (FRSW); the Vosges mountains in northeastern of France (FRNE); the English lowlands deciduous forests (UK); the temperate deciduous forests of the northern United States (US). Concentrations of Al and acid neutralizing capacity (ANC) were determined following the methods detailed in[START_REF] Ferreira | Leaf litter decomposition as a bioassessment tool of acidification effects in streams: evidence from a field study and meta-analysis[END_REF]. Nutrient gradient (n = 10). ANC, pH and Al were determined in 5 streams in FRNE that span an acidification gradient.

		FRNE		CH		UK		FRSW		US
		Min	Max	Min	Max	Min	Max	Min	Max	Min	Max
	Degree days NO 3 --N (mg/L) NO 2 --N (µg/L) NH 4 + -N (µg/L) PO 4 3--P (µg/L)	135.2 0.11 < 10 < 10 2.0	172.0 0.73 < 10 < 10 5.0	135.2 0.93 2.0 6.0 14.0	186.8 1.66 45.0 10.0 17.0	n.a. 0.26 0.60 1.00 1.40	n.a 1.34 2.00 7.00 1.60	205.1 0.74 < 10 45.0 14.0	231.1 1.85 37.0 111.0 28.3	52.9 n.a n.a n.a n.a	60.4 n.a n.a n.a n.a
	Acidification gradient (n = 5)
		TH		MR		BR			RR		CE
	ANC (µEq/L) pH Al (µg/L)	134.4 6.9 52.3	38.9 6.3 174.3	10.1 5.6 207.1		9.4 5.7 176.0		-10.0 4.9 343.3

Table 2

 2 General characteristics of the cotton materials before they were incubated in the field.

	Calico	Empa	Artist's

(Secretaría de Educación Superior Ciencia, Tecnología e Innovación, SENESCYT) through the PROMETEO scholar-exchange program, the Oakland University Research Development grant program, and a research grant from the Huron Mountain Wildlife Foundation.

Acknowledgements

We thank Mariana Burchi, Diana Ethaiya, Chivonne Gammon, Mahnoor Moeen and Ghania Moeen for laboratory assistance and for performing tensile-strength measurements. This research was supported by an award to S.D.T. from the Ecuadorian Ministry of Science


Rituparna Goswami

Atef Asnacios

Olivier Hamant
email: olivier.hamant@ens-lyon.fr

Marie-Edith Chabouté
email: marie-edith.chaboute@ibmp-cnrs.unistra.fr

Cell signaling and gene regulation Current Opinion in Plant

Beyond its biochemical nature, the nucleus is also a physical object. There is accumulating evidence that its mechanics plays a key role in gene expression, cytoskeleton organization, and more generally in cell and developmental biology. Building on data mainly obtained from the animal literature, we show how nuclear mechanics may orchestrate development and gene expression. In other words, the nucleus may play the additional role of a mechanical rheostat.

Although data from plant systems are still scarce, we pinpoint recent advances and highlight some differences with animal systems. Building on this survey, we propose a list of prospects for future research in plant nuclear mechanotransduction and development.

Introduction

Living organisms adapt their physiology and development to environmental cues, notably through transcriptional control. They also respond to internal cues. In particular, growth and shape generate patterns of mechanical stress. In turn, cells resist or yield to mechanical forces by modulating their mechanical properties. This applies to the matrix [START_REF] Engler | Matrix elasticity directs stem cell lineage specification[END_REF], the cytoskeleton [START_REF] Durand-Smet | A comparative mechanical analysis of plant and animal cells reveals convergence across kingdoms[END_REF], as well as the nucleus [START_REF] Lovett | Modulation of Nuclear Shape by Substrate Rigidity[END_REF], with important consequences on gene expression and development [START_REF] Alam | The mammalian LINC complex regulates genome transcriptional responses to substrate rigidity[END_REF]. We recently found that nucleus shape and mechanics depend on osmotic conditions, and that a nuclear regulator controls this response [START_REF] Goswami | Mechanical Shielding in Plant Nuclei[END_REF]. Here we review key features of nuclear mechanics and mechanotransduction based on data in mammals, yeast, and more recently plants, to highlight shared principles and plant specificities.

The three structural compartments of the nucleus

The nucleus is surrounded by the nuclear envelope (NE). The NE consists of two lipid bilayer membranes, namely outer (ONM) and inner (INM) nuclear membranes, which are separated by a ∼35-50 nm perinuclear space. These membranes fuse together at the sites of Nuclear Pore Complex (NPC) giving rise to an ultra-donut topology [START_REF] Torbati | Ultradonut topology of the nuclear envelope[END_REF]. Both NE components, notably LINC complexes (Linker of Nucleoskeleton and Cytoskeleton) and NPC may contribute to changes of mechanical properties of the nucleus in animals [START_REF] Kirby | Emerging views of the nucleus as a cellular mechanosensor[END_REF]. In plants LINC complexes and NPC have been identified [START_REF] Fal | Nuclear envelope: a new frontier in plant mechanosensing?[END_REF].

The nucleoskeleton is connected to the NE by the inner nuclear membrane. It forms a filament meshwork of 10-30 nm composed of coiled-coiled proteins, called lamina in animals and plamina in plants [START_REF] Turgay | The molecular architecture of lamins in somatic cells[END_REF][START_REF] Ciska | The intriguing plant nuclear lamina[END_REF]. While lamins form the lamina in animals, no orthologs of lamins but rather functional homologs have been identified in plants [START_REF] Dittmer | LITTLE NUCLEI Genes Affecting Nuclear Morphology in Arabidopsis thaliana[END_REF][START_REF] Ciska | NMCP/LINC proteins: Putative lamin analogs in plants?[END_REF][START_REF] Ciska | Evolutionary history and structure of nuclear matrix constituent proteins, the plant analogues of lamins[END_REF].

Last, the nucleus contains DNA wrapped around histones to form the nucleosomes. The nucleosomes are organized into 3D higher-order structures, where each chromosome occupies a territory called CT (for chromosome territory) in all eukaryotes, the organization of which can be modulated by mechanical forces [START_REF] Tajik | Transcription upregulation via force-induced direct stretching of chromatin[END_REF]. Particularly in Arabidopsis, each CT forms a rosette like structure where transcriptionally active euchromatin loops emanate from the central cluster of condensed inactive heterochromatin which form chromocenters, located at the nuclear periphery [START_REF] Fransz | Interphase chromosomes in Arabidopsis are organized as well defined chromocenters from which euchromatin loops emanate[END_REF][START_REF] Bi | Nonrandom domain organization of the Arabidopsis genome at the nuclear periphery[END_REF].

Given their direct and indirect roles in transcription, these three compartments have attracted considerable attention in biology. Because they are also physical components, they are the targets of mechanical deformation and regulation, and have become a hot topic of study in biophysics too.

The nucleus as a physical object

The nucleus has specific mechanical features. When probed through the diffusion of internalized nanoparticles, the nuclear structure reveals transient ~300 nm sized microdomains with elastic trapping and low elastic moduli (~20 Pa) and viscosities (~50 Pa.s) [START_REF] Tseng | Micro-organization and visco-elasticity of the interphase nucleus revealed by particle nanotracking[END_REF].

When the whole nucleus is deformed under constant loading (creep experiment), it displays a time-scale free weak power law behavior, with a viscoelastic modulus ranging from 1 to 10 -0.3 [START_REF] Dahl | Power-law rheology of isolated nuclei with deformation mapping of nuclear substructures[END_REF]. This corresponds to a mechanical behavior dominated by elasticity, with, at a typical time scale of a second, an apparent elastic modulus of about 5 kPa, and an apparent viscosity of ~300 Pa.s. More generally, this kind of behavior is observed in many out-of-equilibrium complex systems with multiscale structure and dynamics [START_REF] Sollich | Rheology of soft glassy materials[END_REF](), like dense suspensions of colloidal particles (muds, shaving foams, emulsions) [START_REF] Höhler | Rheological memory effect in aqueous foam[END_REF][START_REF] Purnomo | Glass transition and aging in dense suspensions of thermosensitive microgel particles[END_REF].

Importantly, the nucleus viscoelastic modulus correlates with cell identity. For example, during human stem cell differentiation, nuclei stiffen ~6-fold relative to cell cytoplasm.

Consistently, nuclei of primary human fibroblasts are ~2 times stiffer than nuclei from Marrow-derived human stem cells and their stiffness correlates with the level of expression of lamins [START_REF] Pajerowski | Physical plasticity of the nucleus in stem cell differentiation[END_REF].

The ability of the nucleus to react to forces also entails energy requirements and dissipation.

In that sense, and like most biological objects, the nucleus belongs to the family of active materials [START_REF] Fletcher | Active Biological Materials[END_REF]. The idea that the nucleus may be an active mechanosensitive element has gained more and more momentum in the past decades [START_REF] Wang | Mechanotransduction at a distance: mechanically coupling the extracellular matrix with the nucleus[END_REF], also building on the characterization of its structural elements and their physical link to the cytoskeleton [START_REF] Tajik | Transcription upregulation via force-induced direct stretching of chromatin[END_REF][START_REF] Poh | Dynamic force-induced direct dissociation of protein complexes in a nuclear body in living cells[END_REF].

In sum, there is now ample evidence that the mechanical properties of the main mechanicallyrelevant elements of the nucleus (nuclear envelope, lamins and chromatin) modulate nuclear mechanosensing, leading to the concept of a nuclear rheostat ( [START_REF] Thorpe | Dynamic regulation of nuclear architecture and mechanics-a rheostatic role for the nucleus in tailoring cellular mechanosensitivity[END_REF], Figure 1).

The dynamic mechanical properties of chromatin

The nucleus displays a plastic behavior and strain hardening (stiffening with increased deformation) reminiscent of the non-linear stress-strain relationship of chromatin fibers [START_REF] Dahl | Power-law rheology of isolated nuclei with deformation mapping of nuclear substructures[END_REF]. This suggests that chromatin may play a dominating role in nuclear mechanics. In fact, in contrast to the classical view of nuclear mechanics depending mainly on a stiff nucleoskeleton [START_REF] Stephens | Chromatin histone modifications and rigidity affect nuclear morphology independent of lamins[END_REF]. Nuclear mechanical properties have also been measured in the plant model Arabidopsis and may be linked to nuclear shape and chromatin status [START_REF] Goswami | Mechanical Shielding in Plant Nuclei[END_REF].

In mammals, when the balance shifts towards an increase of the transcriptionally less active state, condensed heterochromatin, and decrease of the transcriptional state of the gene-rich active less condensed euchromatin, nuclei become stiffer [START_REF] Stephens | Chromatin histone modifications and rigidity affect nuclear morphology independent of lamins[END_REF][START_REF] Furusawa | Chromatin decompaction by the nucleosomal binding protein HMGN5 impairs nuclear sturdiness[END_REF][START_REF] Stephens | Chromatin and lamin A determine two different mechanical response regimes of the cell nucleus[END_REF]. This response was revealed by playing on chromatin compaction and cytological studies through quantification of relative euchromatin/heterochromatin levels, using either inhibitors of histone post-translational modifications (histone deacetylation, histone methylation, histone demethylation) or genetics (overexpression of nucleosomal binding proteins). In plants, only one study was conducted in differentiated tissues using mutants exhibiting decondensed heterochromatin (ddm1, atxr5atxr6) and no obvious nuclear deformation was observed (nuclear sphericity and volumes). However, the analyses were conducted on fixed nuclei, which can modify the mechanical properties of the nucleus [START_REF] Poulet | The LINC complex contributes to heterochromatin organisation and transcriptional gene silencing in plants[END_REF]. The nucleus also follows a power-law rheology when subjected to osmosis regulation [START_REF] Dahl | Power-law rheology of isolated nuclei with deformation mapping of nuclear substructures[END_REF]: isolated nuclei from epidermal cells increase their volume up to 200% and dilute the DNA content when removing salts in the media, whereas the nucleus becomes wrinkled, due to chromatin condensation, in high salt concentrations.

The dynamic mechanical properties of lamins

Lamins are nuclear intermediate filaments which can form the fibrous layer in the lamina but they are also found in soluble form in the nucleoplasm [START_REF] Gesson | A-type Lamins bind both hetero-and euchromatin, the latter being regulated by laminaassociated polypeptide 2 alpha[END_REF]. Depending on their locations, lamins have different functions. While lamin A/C and B interact with heterochromatin at the nuclear periphery [START_REF] Gruenbaum | Lamins: Nuclear Intermediate Filament Proteins with Fundamental Functions in Nuclear Mechanics and Genome Regulation[END_REF], only lamin A/C interacts with euchromatin via lamin-associated polypeptide 2α allowing a more direct regulation of gene expression [START_REF] Gesson | A-type Lamins bind both hetero-and euchromatin, the latter being regulated by laminaassociated polypeptide 2 alpha[END_REF]. Because lamin A interacts with chromatin, they can regulate nuclear viscosity, whereas lamin B rather modulate nuclear elasticity [START_REF] Swift | Nuclear lamin-A scales with tissue stiffness and enhances matrix-directed differentiation[END_REF][START_REF] Swift | The nuclear lamina is mechano-responsive to ECM elasticity in mature tissue[END_REF]. Interestingly using super-resolution microscopy, lamin B1 was shown to form an outer concentric ring, in a curvature-dependent localization to restrain outward protrusions of the stiff lamin A/C network facing the chromatin [START_REF] Nmezi | Concentric organization of A-and B-type lamins predicts their distinct roles in the spatial organization and stability of the nuclear lamina[END_REF]. Such organization may help to explain the differential mechanical properties of lamins [START_REF] Swift | Nuclear lamin-A scales with tissue stiffness and enhances matrix-directed differentiation[END_REF][START_REF] Swift | The nuclear lamina is mechano-responsive to ECM elasticity in mature tissue[END_REF]. Both protein levels and folding degree of lamin A seem to scale with tissue stiffness: protein levels are increasing during differentiation in human cells [START_REF] Swift | Nuclear lamin-A scales with tissue stiffness and enhances matrix-directed differentiation[END_REF] whereas lamins unfolding, through their phosphorylation, was induced in cells growing on soft matrix [START_REF] Buxboim | Matrix elasticity regulates lamin-A,C phosphorylation and turnover with feedback to actomyosin[END_REF].

In plants, while nucleoskeleton structure was identified by SEM and TEM [START_REF] Fiserova | Nuclear envelope and nuclear pore complex structure and organization in tobacco BY-2 cells[END_REF][START_REF] Moreno Díaz De La Espina | Isolation and ultrastructural characterization of the residual nuclear matrix in a plant cell system[END_REF] no true lamin exists. Yet specific functional homologs are present [START_REF] Ciska | The intriguing plant nuclear lamina[END_REF]. This includes the CRWN (CROWDED NUCLEI) coiled-coil domain proteins found initially in carrot as NMPC (Nuclear Matrix Constituent proteins) [START_REF] Masuda | Peripheral framework of carrot cell nucleus contains a novel protein predicted to exhibit a long alpha-helical domain[END_REF] and further identified in Arabidopsis as CRWN1-4 [START_REF] Dittmer | LITTLE NUCLEI Genes Affecting Nuclear Morphology in Arabidopsis thaliana[END_REF][START_REF] Wang | Arabidopsis CROWDED NUCLEI (CRWN) proteins are required for nuclear size control and heterochromatin organization[END_REF]. While CRWN1 and CRWN4 are exclusively located at the nuclear periphery to mediate chromatin tethering, CRWN2 and CRWN3 are found in the nucleoplasm [START_REF] Wang | Arabidopsis CROWDED NUCLEI (CRWN) proteins are required for nuclear size control and heterochromatin organization[END_REF]. Using FRET experiments, CRWN1 was shown to interact with the nucleoplasm N-terminus domain of SUN1 (Sad1-UNC-84, [START_REF] Graumann | Evidence for LINC1-SUN associations at the plant nuclear periphery[END_REF]). Other proteins were shown to interact physically with SUN1 using yeast two hybrid, such as the plant specific lamin-like KAKU4 [START_REF] Goto | The Novel Nuclear Envelope Protein KAKU4 Modulates Nuclear Morphology in Arabidopsis[END_REF] or the chromatin associated protein PWO1 (PROLINE-TRYPTOPHANE-TRYPTOPHANEPROLINE INTERACTOR OF POLYCOMBS1, [START_REF] Mikulski | The chromatin-associated protein pwo1 interacts with plant nuclear lamin-like components to regulate nuclear size[END_REF]). In Arabidopsis while CRWN1 and KAKU4 may maintain nuclear morphology through their interaction with the NE [START_REF] Goto | The Novel Nuclear Envelope Protein KAKU4 Modulates Nuclear Morphology in Arabidopsis[END_REF][START_REF] Sakamoto | LITTLE NUCLEI 1 and 4 regulate nuclear morphology in Arabidopsis thaliana[END_REF], they can deform the NE independently [START_REF] Goto | The Novel Nuclear Envelope Protein KAKU4 Modulates Nuclear Morphology in Arabidopsis[END_REF]. Interestingly, genetic interactions between single and double mutants, reveal that PWO1 and CRWN1 control nuclear size and shape in the same genetic pathway and control expression of a similar set of target genes repressed by the repressive histone marks H3K27me3. [START_REF] Mikulski | The chromatin-associated protein pwo1 interacts with plant nuclear lamin-like components to regulate nuclear size[END_REF]. In the same trend, LINC complexes were shown to contribute to heterochromatin organisation and transcriptional gene silencing in plants [START_REF] Poulet | The LINC complex contributes to heterochromatin organisation and transcriptional gene silencing in plants[END_REF]. Yet, very little is known about the contribution of these factors to nuclear mechanics. With the current data, only nuclear shape can be used as a proxy of nuclear mechanical properties. As a future direction of research, the scaling of nuclear deformation and chromatin organization with mechanical stress levels should be investigated in plants. Further studies could also correlate changes in plamina and nuclear mechanics to gene expression, including HI-C data and epigenetic landscape, as well as nuclear translocation of specific transcription factors.

The nuclear envelope as a transducer of cytoplasmic cues

The NE has its own mechanical properties. The elasticity, viscosity and plasticity of the NE is supported and modulated by several key proteins [START_REF] Rowat | Characterization of the elastic properties of the nuclear envelope[END_REF]. The geometric gap between the nuclear membranes is mostly maintained via the SUN1 and SUN2 proteins [START_REF] Crisp | Coupling of the nucleus and cytoplasm: role of the LINC complex[END_REF]. SUN1 and SUN2 [START_REF] Crisp | Coupling of the nucleus and cytoplasm: role of the LINC complex[END_REF][START_REF] Graumann | Characterization of SUN-domain proteins at the higher plant nuclear envelope[END_REF][START_REF] Zhou | Novel plant SUN-KASH bridges are involved in RanGAP anchoring and nuclear shape determination[END_REF][START_REF] Zhou | Identification of unique SUN-interacting nuclear envelope proteins with diverse functions in plants[END_REF][START_REF] Murphy | Structure and expression of the maize (Zea mays L.) SUN-domain protein gene family: evidence for the existence of two divergent classes of SUN proteins in plants[END_REF]. In the case of vertebrates, SUN1 is also connected to lamins A/C [START_REF] Haque | Cell signaling and gene regulation Current Opinion in Plant Biology 17 a physical connection between the nuclear lamina and the cytoskeleton[END_REF].

In a cellular context, the dynamics of actomyosin and microtubules can generate compressive and pulling stresses on the nucleus [START_REF] Gundersen | Nuclear positioning[END_REF]. The NE plays a key intermediary role. For instance, disrupting the perinuclear actin cap through actin depolymerization leads to an increase of nuclear volume in mouse fibroblasts [START_REF] Kim | Volume regulation and shape bifurcation in the cell nucleus[END_REF]. Similarly, untethering chromatin from the inner nuclear membrane induces nuclear deformation and reduces nuclear rigidity in S. pombe [START_REF] Schreiner | The tethering of chromatin to the nuclear envelope supports nuclear mechanics[END_REF].

In plants, the presence of a large vacuole may further constrain the nucleus in a thin cytoplasmic compartment. Consistently, actin depolymerization in leaf epidermal cells does not change the elongated shape of nuclei [START_REF] Sakamoto | LITTLE NUCLEI 1 and 4 regulate nuclear morphology in Arabidopsis thaliana[END_REF]. Yet, LINC complexes are associated to actin via Myosin XI-i in plant cells [START_REF] Tamura | Myosin XI-i links the nuclear membrane to the cytoskeleton to control nuclear movement and shape in Arabidopsis[END_REF] and impaired LINC functions prevent nuclear elongation in differentiated tissues [START_REF] Tamura | Recent advances in understanding plant nuclear envelope proteins involved in nuclear morphology[END_REF][START_REF] Meier | Cell Biology of the Plant Nucleus. TL -68[END_REF]. Therefore, the shape of plant nuclei depends on NE factors and the cytoskeleton.

Interestingly, cortical microtubules align with maximal tensile stress directions in plant cell walls [START_REF] Hamant | Developmental patterning by mechanical signals in Arabidopsis[END_REF][START_REF] Hamant | Are microtubules tension sensors?[END_REF] and cytoplasmic microtubules are connected to the nuclear envelope through the microtubule nucleation complexes and regulatory factors [START_REF] Janski | Identification of a novel small Arabidopsis protein interacting with gamma-tubulin complex protein 3[END_REF][START_REF] Janski | The GCP3-interacting proteins GIP1 and GIP2 are required for gamma-tubulin complex protein localization, spindle integrity, and chromosomal stability[END_REF]. Yet, how cortical stress may affect microtubule nucleation or NE properties is unknown.

The mechanical regulation of gene expression through the nucleus properties

Because the cytoskeleton and NE components are relatively stiff, cortical mechanical cues can change nucleus shape rapidly, leading to subsequent chromatin changes (Figure 2). For instance, the perinuclear actin cap can induce nuclear deformation mediated by LINC complexes and lamins A/C in 30 seconds [START_REF] Poh | Dynamic force-induced direct dissociation of protein complexes in a nuclear body in living cells[END_REF][START_REF] Chambliss | The LINC-anchored actin cap connects the extracellular milieu to the nucleus for ultrafast mechanotransduction[END_REF][START_REF] Lombardi | The interaction between nesprins and sun proteins at the nuclear envelope is critical for force transmission between the nucleus and cytoskeleton[END_REF]. Disrupting LINC complexes through SUN1 perturbation in fibroblasts cells leads to different transcriptional responses on soft versus stiff substrates [START_REF] Alam | The mammalian LINC complex regulates genome transcriptional responses to substrate rigidity[END_REF]. Unfolding of lamins through their dephosphorylation affect gene expression within 10 minutes [START_REF] Buxboim | Matrix elasticity regulates lamin-A,C phosphorylation and turnover with feedback to actomyosin[END_REF]. Last, destabilization of lamins under stress induces global chromatin reorganization, release of sequestrated transcription factors as well as In parallel to the propagation of stress through the internal structural elements of the cell, cortical cues may indirectly reach the nucleus through protein translocation to the nucleus. In particular, the well-known Hippo pathway-YAP/TAZ involves the traffic of the transcriptional activator ß-catenin from the cytoplasm to the nucleoplasm through the NPC [START_REF] Dupont | Role of YAP/TAZ in mechanotransduction[END_REF]. Note that because NPCs are directly connected to nucleoskeleton, chromatin, and LINC complexes [START_REF] Jahed | The LINC and NPC relationship -it's complicated! TL -129[END_REF], the passive nuclear entry of the co-transcriptional factor YAP also depends on the physical deformation of the nucleus [START_REF] Elosegui-Artola | Force Triggers YAP Nuclear Entry by Regulating Transport across Nuclear Pores[END_REF].

Chromatin is actually a site where mechanical and biochemical cues largely overlap [START_REF] Pajerowski | Physical plasticity of the nucleus in stem cell differentiation[END_REF][START_REF] Stephens | Chromatin histone modifications and rigidity affect nuclear morphology independent of lamins[END_REF][START_REF] Stephens | Chromatin and lamin A determine two different mechanical response regimes of the cell nucleus[END_REF][START_REF] Schreiner | The tethering of chromatin to the nuclear envelope supports nuclear mechanics[END_REF]. In response to mechanical stress, changes in gene transcription may be modified either by changes of gene position inside the nucleus or by changes of the chromatin state itself. For instance, direct stretching of chromatin through mechanical forces (using RGD-coated magnetic beads) can induce expression of a GFP-tagged gene in hamster cellular line, and this involves actin and LINC complexes. This is triggered through a change in the spatial chromatin organization, i.e. a change in the location of the transgene [START_REF] Tajik | Transcription upregulation via force-induced direct stretching of chromatin[END_REF]. Upon cell mechanical constraint, actomyosin induces the nuclear translocation of HDAC3 leading to decreased level of histone acetylation (permissive mark for gene expression) in mouse embryo fibroblast cells leading to a geometry-dependent transcriptional response. Such response is reversible [START_REF] Jain | Cell geometric constraints induce modular gene-expression patterns via redistribution of HDAC3 regulated by actomyosin contractility[END_REF][START_REF] Damodaran | Compressive force induces reversible chromatin condensation and cell geometry-dependent transcriptional response[END_REF]. On the contrary, inhibition of actin leads to the recruitment of the histone methyltransferase G9a to the NE and thus increases the level of repressive marks H3K9me2/3 in T-lymphocyte cells [START_REF] Zhang | Integrin α4β1 controls G9a activity that regulates epigenetic changes and nuclear properties required for lymphocyte migration[END_REF]. Interestingly, using an active 3D mechano-chemical model, also integrating cell geometric constraints, one can predict nuclear mechanics and architecture patterns [START_REF] Alisafaei | Regulation of nuclear architecture, mechanics, and nucleocytoplasmic shuttling of epigenetic factors by cell geometric constraints[END_REF].

In plants, the situation might be different at the molecular level, knowing for instance that there is no plant homolog of the YAP/TAZ pathway. However, the relationship between nuclear mechanics and gene expression might apply to all kingdoms. In this respect, we recently unraveled a link between nucleus, shape, mechanics and touch-response gene expression in response to hyperosmotic stress [START_REF] Goswami | Mechanical Shielding in Plant Nuclei[END_REF]. Interestingly, touch-response genes are also induced in response to conditions of changes in light conditions [START_REF] Lee | Genome-wide identification of touch-and darknessregulated Arabidopsis genes: a focus on calmodulin-like and XTH genes[END_REF]. This is also true when plants are subjected to sound vibrations [START_REF] Ghosh | Exposure to Sound Vibrations Lead to Transcriptomic, Proteomic and Hormonal Changes in Arabidopsis[END_REF]. Even though nuclear shape changes have not been explored in these latter cases, one could speculate that the plant nucleus integrate multiple environmental cues in part through its mechanics to regulate gene expression.

Functional implications of nuclear shaping in development

During development, tissue change their shape or mechanical status, and the resulting mechanical conflicts can transduce mechanical cues to cells and, arguably, nuclei. Although most of the knowledge on nuclear mechanics and mechanotransduction has been generated from studies in single cells in culture, this should also apply to cells in a tissue context. This starts to be addressed [START_REF] Fernandez-Sanchez | Mechanotransduction's Impact on Animal Development, Evolution, and Tumorigenesis[END_REF].

For instance, during epidermal morphogenesis in humans, actin-driven force decreased the level of the lamin-binding protein emerin at the INM inducing a loss of anchoring of heterochromatin at the NE and changes in transcriptional programs mediated by the polycomb PRC2, with important consequences for cell identity [START_REF] Le | Mechanical regulation of transcription controls Polycomb-mediated gene silencing during lineage commitment[END_REF]. Furthermore, impaired NE functions, notably lamins A, lead to a wide range of diseases called laminopathies, such as muscular dystrophies and progeria [START_REF] Horn | The LINC complex is essential for hearing[END_REF]. Thus, nuclear mechanics shows functional regulation or correlation through a range of physical and pathological condition.

Plant tissues are usually much stiffer than animal ones, because plant cells are highly pressurized with a turgor pressure in the MPa range, and surrounded by a stiff cell wall. Yet, plant cells are still experiencing mechanical conflicts, the pattern of which is determined by tissue shape and growth. Cortical microtubule arrays play a major role in guiding cellulose synthase complex (CSC) to regulate the cell wall and its physical properties. As mentioned above, cortical microtubules align with the direction of maximal tensile stress in the cell wall, thereby reinforcing the cell wall to resist tensile stress [START_REF] Hamant | Developmental patterning by mechanical signals in Arabidopsis[END_REF]. γ tubulins are present at the NE, where they nucleate cytoplasmic microtubules, together with GIP proteins (gamma-tubulin complex protein 3 (GCP3) interacting Proteins) [START_REF] Batzenschlager | The GIP gamma-tubulin complex-associated proteins are involved in nuclear architecture in Arabidopsis thaliana[END_REF]. Interestingly, γ tubulins are also found at the INM in a complex with SUN1 and GIPs which form a complex with the centromeric chromatin via CENH3 [START_REF] Batzenschlager | Arabidopsis MZT1 homologs GIP1 and GIP2 are essential for centromere architecture[END_REF][START_REF] Chumová | Microtubular and Nuclear Functions of γ-Tubulin: Are They LINCed?[END_REF]. Although GIPs are conserved across species, they are found on both sides of the NE only in plants. Indeed, GIPs are not only associated with microtubule nucleation complexes outside the nucleus (like their human homolog MOZART1 [START_REF] Hutchins | Systematic analysis of human protein complexes identifies chromosome segregation proteins[END_REF]), but also with the centromeres close to the inner side of the NE [START_REF] Batzenschlager | The GIP gamma-tubulin complex-associated proteins are involved in nuclear architecture in Arabidopsis thaliana[END_REF][START_REF] Batzenschlager | Arabidopsis MZT1 homologs GIP1 and GIP2 are essential for centromere architecture[END_REF]. The GIP-γ tubulin hub might thus have a specific role in nuclear mechanotransduction in plants. Consistently, the gip1gip2 mutant exhibits constitutive induction of touch-response genes [START_REF] Goswami | Mechanical Shielding in Plant Nuclei[END_REF].

Impaired GIP activity lead to irregular shape and lobulated nuclei in both meristems and differentiated root tissues [START_REF] Batzenschlager | The GIP gamma-tubulin complex-associated proteins are involved in nuclear architecture in Arabidopsis thaliana[END_REF] as well as severe developmental defects, with the presence of callus on differentiated tissues [START_REF] Janski | The GCP3-interacting proteins GIP1 and GIP2 are required for gamma-tubulin complex protein localization, spindle integrity, and chromosomal stability[END_REF]. Such effects are not shared with other NE proteins. In particular, most of the nuclear envelope proteins characterized in LINC complexes identified so far regulate nuclear shape, yet, they do not necessarily induce major developmental defects when impaired [START_REF] Tamura | Recent advances in understanding plant nuclear envelope proteins involved in nuclear morphology[END_REF][START_REF] Meier | Cell Biology of the Plant Nucleus. TL -68[END_REF]. This may in part be due to genetic redundancy. For instance, single crwn mutants have no growth defects, whereas crwn1crwn2 and crwn1crwn2crwn4 exhibit defective development, like stunted plants [START_REF] Wang | Arabidopsis CROWDED NUCLEI (CRWN) proteins are required for nuclear size control and heterochromatin organization[END_REF]. When compared to wild type, these mutants exhibit smaller and denser nuclei with fewer, more aggregated, chromocenters. Interestingly, only CRWN1 and CRWN4 allow tethering of heterochromatin at the nuclear periphery [START_REF] Hu | Plant lamin-like proteins mediate chromatin tethering at the nuclear periphery[END_REF].

CRWN1 interacts with PWO1, and both control nuclear size [START_REF] Mikulski | The chromatin-associated protein pwo1 interacts with plant nuclear lamin-like components to regulate nuclear size[END_REF]. PWO1 physically interacts with the polycomb complex PRC2 mediating H3K27me3 repressive marks [START_REF] Hohenstatt | PWWP-DOMAIN INTERACTOR OF POLYCOMBS1 interacts with polycomb-group proteins and histones and regulates arabidopsis flowering and development[END_REF]. Whereas mechanical regulation of nuclear architecture was linked to cell fate decisions in mammals [START_REF] Shivashankar | Mechanical regulation of genome architecture and cell-fate decisions[END_REF][START_REF] Miroshnikova | Epigenetic gene regulation, chromatin structure, and force-induced chromatin remodelling in epidermal development and homeostasis[END_REF], this remains to be explored in plants.

Perspectives for plant science: the role of the nucleus in multi-stress responses

In contrast to animals, plants exhibit a plastic postembryonic development that largely depends on environmental cues. In line with the established role of nuclear mechanics in animal development, plant development also relies on nuclear mechanotransduction. Yet the exact role of nuclear shape, mechanics and structural elements in signaling and development remains to be investigated. Conducting this research in plants will not only unravel conserved and divergent mechanisms in a walled context, it will also help to understand how the nucleus integrates cues from the environment.

In a recent study in Arabidopsis, we show the importance of nuclear mechanics in roots exposed to osmotic stresses. Throughout mechanical assessment of nucleus stiffness by atomic force microscopy and microrheometry, we revealed that hyperosmotic stress leads to nucleus shrinking, stiffening and chromatin remodeling. We also observed a strong induction of touch-response genes. Importantly, these responses were reversible upon return to isoosmotic conditions. We propose that chromatin behaves like a mechanosensitive gel in response to osmotic stresses (Figure 3) [START_REF] Goswami | Mechanical Shielding in Plant Nuclei[END_REF].

Interestingly, the gip1gip2 mutant nuclei mimicked a hyper-osmotic phenotype. This further suggests that not only NE envelope proteins play an important role in the plant response to mechanical cues, whether such cues are emanating internally from growth or externally from the environment. Consistently, we found that gip1gip2 plants are more resistant to harsh osmotic conditions. This suggests that the absence of GIP may actually prime the plant to hyperosmotic conditions (Figure 3).

Based on these recent results, and building on the comparative analysis of nucleus mechanics and mechanotransduction in animals, we thus propose that the nucleus also acts as a mechanical rheostat in plants. Why would plants be competitive model systems to address this question? Plant cells do not move. Thus the relation between tissue stress and nucleus behavior is more amenable to be investigated in plants. While turgor pressure and the presence of a stiff plant cell wall represent obvious mechanical differences between plant and animal cells, two important remarks regarding mechanotransduction can be made. First, plant protoplasts, i.e. plant cells without their walls, display mechanical properties that are remarkably similar to that of animal cells. However, these features are dependent on the microtubule cytoskeleton, while they are mainly supported by the actin network in animal cells [START_REF] Durand-Smet | A comparative mechanical analysis of plant and animal cells reveals convergence across kingdoms[END_REF]. Second, in plants, the microtubule network organization and orientation is dependent on both cell shape [START_REF] Ambrose | A CLASP-modulated cell edge barrier mechanism drives cell-wide cortical microtubule organization in Arabidopsis[END_REF] [START_REF] Durand-Smet | Cytoskeletal organization in isolated plant cells under geometry control[END_REF] and the direction of maximal stress in the cell wall [START_REF] Hamant | Developmental patterning by mechanical signals in Arabidopsis[END_REF]. Thus, any external or internal cues leading to cell shape changes and/or mechanical stresses in the cell wall, may lead to signal propagation through the microtubule network and the NE to the internal nuclear structure. In this context, the nuclear sensitivity to cell shape [START_REF] Versaevel | Spatial coordination between cell and nuclear shape within micropatterned endothelial cells[END_REF] and subsequent chromatin modifications [START_REF] Jain | Cell geometric constraints induce modular gene-expression patterns via redistribution of HDAC3 regulated by actomyosin contractility[END_REF][START_REF] Alisafaei | Regulation of nuclear architecture, mechanics, and nucleocytoplasmic shuttling of epigenetic factors by cell geometric constraints[END_REF] observed in animal cells could also be at play in plant cells. However, in plant cells, the link between cell shape and nuclear shape and activity might be dependent on microtubules rather than actin, as in animal cells, and the forces needed to induce cell shape modifications and mechanotransduction should be orders of magnitude higher than those involved in animal cell mechanosensing.

Manuscript

  Ref.: Ms. No. COPLBI-D-19-00047 57: Cell signaling and gene regulation Current Opinion in Plant Biology 2

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  Ref.: Ms. No. COPLBI-D-19-00047 57: Cell signaling and gene regulation Current Opinion in Plant Biology 6 proteins are located at the INM and interact, via their C-terminus SUN domain, with the KASH (Klarsicht/ANC-1/SYNE homology) domain of KASH family proteins located at the ONM. These SUN-KASH complexes form the LINC complexes and are found across all eukaryotes

  Ref.: Ms. No. COPLBI-D-19-00047 57: Cell signaling and gene regulation Current Opinion in Plant Biology 7 modifications in setting repressive histone marks H3K27me3 mediated by the polycomb complex PRC2 [64] (Figure 2).

  Ref.: Ms. No. COPLBI-D-19-00047 57: Cell signaling and gene regulation Current Opinion in Plant Biology

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  Ref.: Ms. No. COPLBI-D-19-00047 57: Cell signaling and gene regulation Current Opinion in Plant Biology 11 Finally, the analysis of multi-stress responses is also facilitated in plants due to their plastic development and sessile nature, meaning that eco-devo study of nuclear mechanotransduction can be envisioned in plants. This may also help to understand plant responses to abiotic and biotic stresses too [88,89]. Altogether, nuclear mechanotrasnduction appears as a new field of research in the plant community, with implications going far beyond a comparison with animal nuclei. REFERENCES AND RECOMMENDED READING Papers of particular interest, published within the period of review, have been highlighted as: *of special interest Bi et al., 2017 Genome Res.: Using the nucleoporin NUP136/NUP1 fused to GFP in RE-ChIP experiments in different Arabidopsis tissues, the authors show that 10 to 20% of chromosome regions are anchored at the nuclear periphery and are enriched in TE and H3K27me3-marked genes. Nmezi et al., 2019 PNAS: Using high resolution microscopy STORM, the authors show that Lamin B1 forms an outer concentring ring adjacent to the INM, the localization of which is strain-dependent. Lamins B stabilize nuclear shape and prevents protusions from the underlying Lamins A/C network. Chumova et al., 2019 Cells: In Arabidopsis, the γ tubulin initially associated with the microtubule nucleation complexes at the ONM, are found to co-localize with SUN at the INM. They are also present in complexes associated with GIP, suggesting new roles of these proteins at the NE. Damodaran et al., 2018, Mol Biol Cell: The authors show the importance of fibroblast geometry in changing the chromatin condensation state and the transcriptional response to compressive forces. Shivashankar, 2019, Curr. Opin. Cell Biol.: In this review, the author discusses the contribution of mechanical cues in changing nuclear architecture and cell fate transitions in mammals.

  Ref.: Ms. No. COPLBI-D-19-00047 57: Cell signaling and gene regulation Current Opinion in Plant Biology 12 Miroshnikova et al., 2019, Curr. Opin Gen. Dev: The authors present the latest advances linking mechanics and epigenetic gene regulation during differentiation. ** of outstanding interest Stephens et al., 2018 Mol Biol. Cell: The authors show that modifications of histone state are sufficient to dictate nuclear rigidity independently of lamin perturbation. For instance, chromatin decompaction via increased acetylation is sufficient to decrease nuclear stiffness without perturbing lamins. Mikulski et al., 2019 Plant Cell: The authors show that PWO1, which interacts with the polycomb complex PRC2, also interact physically with lamin-like CRWN1. This reveals mechanisms for repressing chromatin at the nuclear periphery in plants. Hu et al., 2019 Genome biol.: The authors show that lamin-like CRWN1 is needed to anchor repressed chromatin domains to the nuclear periphery in Arabidopsis. CRWN1 has a direct interaction with repressive heterochromatin regions, highlighting chromatin interaction with the plamina in Arabidopsis. Alisafaei et al., 2019, Proc Natl Acad Sci U S A.: The authors develop a 3D chemomechanical model considering the reciprocity in dynamics between extracellular matrix, cytoskeleton and nucleus using micropatterning experiments. Goswami et al., 2020 Current Biol. : In this paper, the authors propose that plant nuclei may act as a mechanical rheostat in response to osmotic stress with concomitant changes in nuclear rigidity, shape and mRNA levels of touch-response gene. The absence of GIP may prime the plant to respond to hyperosmotic conditions.

20 Figure legends Figure 1 :

 20legends1 Figure legends

Figure 2 :

 2 Figure 2: The features of chromatin under mechanical stress Changes of nuclear architecture under mechanical stress lead to chromatin condensation, clustering of chromocenters, repressive histone mark modifications inducing altered accessibility for the transcription of differentiated gene, and release of transcription factors from the nuclear periphery

Figure 3 :

 3 Figure 3: Nuclear mechanical response in plants under hyperosmotic stress Upon hyperosmotic stress, the nucleus is more compact with clustered chromocenters and induction of touch-response gene expression. The nuclear envelope proteins GIPs may act as negative regulators of this response.

ACKNOWLEDGMENTS

Ref.: Ms. No. COPLBI-D-19-00047 57: Cell signaling and gene regulation Current Opinion in Plant Biology 13 The authors were supported by the Centre National de la Recherche Scientifique (CNRS, defi Mecanobio, NEstress 2016-2018), by Fondation Schlumberger pour l'Education et la Recherche (FSER 2016-2018), by the European Research Council Grants ERC-2013-CoG-615739 "MechanoDevo", by the IdEX international PhD program (unista, Strasbourg) and by HFSP Grant 2018, RGP, 009. This study was partially supported by the labex «Who AM I ?», labex ANR-11-LABX-0071 and the Université de Paris, Idex ANR-18-IDEX-0001 funded

by the French Government through its «investments for the future» program. We apologize to our colleagues whose work we were not able to discuss because of space constraints.