Nikola Perkovic
email: percestyler@gmail.com

Electron Mass in Gravitation and Cosmology

Fluctuations of the quantum vacuum are best understood in terms of virtual particle anti-particle pairs. In the context of the Weinberg formula that relates the mass of a pion to fundamental physical constants such as the gravitational constant and the Hubble constant, we will define the mass of an electron in the same fashion since virtual electron-positron pairs are far more common in the quantum vacuum fluctuations of Quantum Electrodynamics than virtual pion pairs. After redefining the Weinberg formula for electrons instead of pions we will replace the Hubble constant with the more suitable Hubble parameter used in modern times in an attempt to help explain why the zero point energy of the vacuum doesn't cause a much larger value of the cosmological constant.

Large number formulas pointed out by Dirac [1] have attracted the attention of physicists for almost a century, however none so much as the well-known empirical formula pointed out by Weinberg [START_REF] Weinberg | Gravitation and Cosmology[END_REF] that relates the mass of pions with certain fundamental physical constants.

m π = ( ћ 2 Gc H 0 ) 1 3
where m π is the rest mass of a pion, ћ is the reduced Planck constant, G is the gravitatopnal constant, c is the speed of light and H 0 is the Hubble constant. This would indicate that there is something special about pions in General Relativity, physical Cosmology and studies of Quantum Gravity which shouldn't be the case as pions are a rather small fraction of the matterenergy content of the universe which should mean that virtual pions in the vacuum are the dominant virtual pairs. According to Quantum Field Theory virtual particles are an inherent part of vacuum fluctuations however it is electron-positron pairs that seem to be dominant not virtual charged pions.

In modern times the Hubble constant is obsolete and the Hubble parameter H is used instead. The Hubble Parameter H is a function of the age of the universe and thus it is not a constant but it varies with cosmological time. This drew many physicists to believe that the gravitational constant and maybe the mass of the pion as well, could vary with time on a cosmological scale. Other physicists believe that the Weinberg formula from equation (1) is a mere coincidence and therefore it has no real physical meaning. We will rewrite the Weinberg formula to prove both these assumptions unfounded by relating the mass of an electron to constants in equation one to replace the pion mass.

In the Standard Model of particle physics the rest energy of an electron is defined by an empirical formula:

E e = 2Ry α 2 (0)
where 1Ry = 13.6056930099(84) eV is the Rydberg unit of energy that is a product of the Planck constant and the speed of light hc multiplied by the Ryberg constant R ∞ and α(0) is the fine structure constant that has an inverse value of α -1 (0) = 137.035999139(31). The Rydberg constant and the fine structure constant are two of the most accurately measured quantities in all of physics which is, along with the g-factor of the electron, a test of extraordinary accuracy of Quantum Electrodynamics. Pions are pseudoscalar mesons that come in three types: two charged pions that are each other's anti-particles and a neutral pion that is its own anti-particle. We will only be dealing with charged pions that are slightly more massive than the neutral one. Rest energy of a charged pion can be defined in a similar manner to equation ( 2):

(1)

(2)

E π = 4Ry α 3 (M π )
where α(M π ) is the running value of the fine structure constant. Running of the fine structure constant on the momentum/energy scale Q is denoted by α(Q) and it only becomes relevant on the electroweak scale when α -1 (M Z ) = 127.916(15) [START_REF]Review of Particle Physics[END_REF]. Having in mind that the mass of a Z boson M Z = 91.1876(21) GeV towers over that of charged pions, we can safely neglect the running of the fine structure constant in equation ( 3) without sacrificing too much accuracy as the equation then provides the charged pion rest energy of 140 MeV which is very close to the experimental value. This allows us to define the mass of a charged pion in relation to the electron mass m π = 2m e α -1 (0). Now we can rewrite the Weinberg formula as an empirical formula that relates the electron mass to the fundamental constants from equation (1):

m e = ( α 3 (0)ћ 2 8Gc H 0 ) 1 3
which is far more appropriate than equation (1) as the three quantities, namely the electron mass, the fine structure constant and the gravitational constant are the ones that are hypothesized to vary over cosmological temporal scales [START_REF] Peter | CODATA Recommended Values of the Fundamental Physical Constants[END_REF] as well as the fact that this empirical formula requires large quantities of electrons and virtual electron-positron pairs in the QED vacuum which is true unlike the case for virtual pion pairs as it is in the Weinberg formula from equation (1). As D. Slavkov Hajdukovic points out [START_REF] Hajdukovic | On the relation between mass of a pion, fundamental physical constants and cosmological parameters[END_REF] the right hand side of the Weinberg formula doesn't need to vary with time if we use the modern Hubble parameter H instead of the Hubble constant H 0 :

m e = ( α 3 (0)ћ 2 8Gc H { Ω Λ (Ω -1) 1 2 ⁄ • R 0 R }) 1 3
where R is the scale factor in the FRW metric and Ω Λ is the density parameter for the cosmological constant Λ as a candidate for dark energy. Equation ( 5) could help answer one of the largest mysteries in physics, namely why doesn't the zero point energy of the vacuum cause a larger cosmological constant [START_REF] Steinhardt | Why the Cosmological constant is so small and positive?[END_REF], [START_REF] Wang | How the huge energy of quantum vacuum to drive the slow accelerating expansion of the Universe[END_REF].

Acknowledgements: I am grateful to my professors from the Institute of Physics and Mathematics, Faculty of Science, University of Novi Sad for helpful advice and suggestions regarding literature.


Susana Saez-Aguayo

Carsten Rautengarten

Henry Temple

Dayan Sanhueza

Troy Ejsmentewicz

Omar Sandoval-Ibañez

Daniela Doñas

Juan Pablo Parra-Rojas

Berit Ebert

Arnaud Lehner

Jean-Claude Mollet

Paul Dupree

Henrik V Scheller

Joshua L Heazlewood

Francisca C Reyes
email: francisca.reyes.marquez@gmail.com

Ariel Orellana
email: aorellana@unab.cl

UUAT1 Is a Golgi-Localized UDP-Uronic Acid Transporter That Modulates the Polysaccharide Composition of Arabidopsis Seed Mucilage

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

INTRODUCTION

The plant cell wall is a complex and dynamic structure that is mainly composed of polysaccharides, with cellulose being a key component. The synthesis of noncellulosic polysaccharides (hemicellulose and pectin) occurs in the Golgi apparatus, where a number of glycosyltransferases (GTs) are located [START_REF] Liepman | Arabidopsis -a powerful model system for plant cell wall research[END_REF][START_REF] Scheible | Glycosyltransferases and cell wall biosynthesis: novel players and insights[END_REF].

GTs transfer the sugar residue from an activated nucleotide donor, in the form of a UDP-or GDP-sugar, to a growing polysaccharide chain. Most GTs are type-II membrane-bound proteins with a catalytic domain facing the Golgi lumen [START_REF] Sterling | The catalytic site of the pectin biosynthetic enzyme alpha-1,4-galacturonosyltransferase is located in the lumen of the Golgi[END_REF][START_REF] Wulff | GDP-fucose uptake into the Golgi apparatus during xyloglucan biosynthesis requires the activity of a transporter-like protein other than the UDP-glucose transporter[END_REF][START_REF] Scheible | Glycosyltransferases and cell wall biosynthesis: novel players and insights[END_REF]. However, most nucleotide sugars utilized by GTs are produced in the cytosol [START_REF] Bar-Peled | Plant nucleotide sugar formation, interconversion, and salvage by sugar recycling[END_REF][START_REF] Bonin | The MUR1 gene of Arabidopsis thaliana encodes an isoform of GDP-D-mannose-4,6-dehydratase, catalyzing the first step in the de novo synthesis of GDP-L-fucose[END_REF][START_REF] Seifert | Nucleotide sugar interconversions and cell wall biosynthesis: how to bring the inside to the outside[END_REF].

Therefore, the Golgi membrane is a physical barrier blocking access to the active GT site.

Nucleotide sugar transporters (NSTs) in the Golgi membrane overcome this topological problem and supply the substrates needed in the Golgi lumen for polysaccharide biosynthesis (Orellana et. al., 2016;[START_REF] Reyes | Golgi transporters: opening the gate to cell wall polysaccharide biosynthesis[END_REF][START_REF] Temple | The inside and outside: topological issues in plant cell wall biosynthesis and the roles of nucleotide sugar transporters[END_REF]. In Arabidopsis thaliana, the genes encoding for NSTs are similar to those encoding for plastidic triose phosphate translocators (TPTs); and together, 44 NSTs and 7 TPTs, form a gene family of 51 members [START_REF] Knappe | Analysis of the plastidic phosphate translocator gene family in Arabidopsis and identification of new phosphate translocatorhomologous transporters, classified by their putative substrate-binding site[END_REF][START_REF] Rautengarten | The Golgi localized bifunctional UDP-rhamnose/UDP-galactose transporter family of Arabidopsis[END_REF]. To date, a number of these NSTs from A. thaliana have been functionally characterized, specifically transporters for GDP-mannose (GDP-Man), GDP-fucose (GDP-Fuc), UDP-galactose (UDP-Gal), UDPglucose (UDP-Glc), UDP-rhamnose (UDP-Rha) and UDP-xylose (UDP-Xyl) [START_REF] Bakker | Molecular cloning of two Arabidopsis UDP-galactose transporters by complementation of a deficient Chinese hamster ovary cell line[END_REF][START_REF] Baldwin | Identification and characterization of GONST1, a Golgi-localized GDP-mannose transporter in Arabidopsis[END_REF][START_REF] Ebert | Identification and characterization of a Golgi-localized UDP-Xylose transporter family from Arabidopsis[END_REF][START_REF] Handford | Arabidopsis thaliana AtUTr7 encodes a golgi-localized UDP-glucose/UDP-galactose transporter that affects lateral root emergence[END_REF][START_REF] Handford | Arabidopsis thaliana expresses multiple Golgi-localised nucleotide-sugar transporters related to GONST1[END_REF][START_REF] Norambuena | Transport of UDP-galactose in plants. Identification and functional characterization of AtUTr1, an Arabidopsis thaliana UDP-galactos/UDP-glucose transporter[END_REF][START_REF] Norambuena | AtUTr2 is an Arabidopsis thaliana nucleotide sugar transporter located in the Golgi apparatus capable of transporting UDP-galactose[END_REF][START_REF] Rautengarten | The Golgi localized bifunctional UDP-rhamnose/UDP-galactose transporter family of Arabidopsis[END_REF][START_REF] Rautengarten | The Arabidopsis Golgi-localized GDP-L-fucose transporter is required for plant development[END_REF][START_REF] Rollwitz | Characterization of AtNST-KT1, a novel UDP-galactose transporter from Arabidopsis thaliana[END_REF].

Hemicellulose and pectins have diverse structures and sugar compositions, and several nucleotide sugars are required for their synthesis. UDP-glucuronic acid (UDP-GlcA), plays a critical role in noncellulosic polysaccharide synthesis, as it is the precursor for several nucleotide sugars involved in hemicellulose and pectin synthesis. These sugars include UDP-galacturonic acid (UDP-GalA), UDP-Xyl, UDP-arabinose (UDP-Ara) and UDP-apiose (UDP-Api) [START_REF] Reboul | Down-regulation of UDP-glucuronic acid biosynthesis leads to swollen plant cell walls and severe developmental defects associated with changes in pectic polysaccharides[END_REF]. Therefore, UDP-GlcA is a major precursor required for hemicellulose and pectic polysaccharide synthesis. Substrate interconversion is required for polysaccharide biosynthesis and in Arabidopsis leaves, polysaccharides containing sugars derived from UDP-GlcA, account for nearly 50% of the cell wall biomass [START_REF] Zablackis | Characterization of the Cell-Wall Polysaccharides of Arabidopsis thaliana Leaves[END_REF]. The cytosol contains some of the enzymes for the interconversion of UDP-GlcA, such as soluble UDP-xylose synthase (UXS) [START_REF] Harper | Biosynthesis of UDP-xylose. Cloning and characterization of a novel Arabidopsis gene family, UXS, encoding soluble and putative membrane-bound UDP-glucuronic acid decarboxylase isoforms[END_REF][START_REF] Pattathil | Biosynthesis of UDP-xylose: characterization of membrane-bound AtUXS2[END_REF][START_REF] Kuang | The role of UDP-glucuronic acid decarboxylase(s) (UXS) in xylan biosynthesis in Arabidopsis[END_REF], UDP-apiose/UDP-xylose synthase [START_REF] Guyett | Real-time NMR monitoring of intermediates and labile products of the bifunctional enzyme UDP-apiose/UDP-xylose synthase[END_REF], Mølhøj et al., 2003;) and UDP-arabinose mutase [START_REF] Konishi | A plant mutase that interconverts UDP-arabinofuranose and UDParabinopyranose[END_REF][START_REF] Rautengarten | The interconversion of UDP-arabinopyranose and UDP-arabinofuranose is indispensable for plant development in Arabidopsis[END_REF]. There are also Golgi-localized interconverting enzymes. These include UDP-glucuronate epimerase (GAE), which converts UDP-GlcA into UDP-GalA [START_REF] Gu | The biosynthesis of UDP-Galacturonic Acid in plants. Functional cloning and characterization of Arabidopsis UDP-D-Glucuronic acid 4-epimerase[END_REF][START_REF] Mølhøj | The biosynthesis of D-Galacturonate in plants. functional cloning and characterization of a membrane-anchored UDP-D-Glucuronate 4-epimerase from Arabidopsis[END_REF], membrane attached UXS [START_REF] Harper | Biosynthesis of UDP-xylose. Cloning and characterization of a novel Arabidopsis gene family, UXS, encoding soluble and putative membrane-bound UDP-glucuronic acid decarboxylase isoforms[END_REF][START_REF] Kuang | The role of UDP-glucuronic acid decarboxylase(s) (UXS) in xylan biosynthesis in Arabidopsis[END_REF] and UDP-xylose-4-epimerase, which catalyzes UDP-Xyl to UDP-arabinopyranose (UDP-Arap) epimerization [START_REF] Burget | The biosynthesis of Larabinose in plants: molecular cloning and characterization of a Golgi-localized UDP-Dxylose 4-epimerase encoded by the MUR4 gene of Arabidopsis[END_REF]. All these enzymes are predicted type-II membrane proteins, and their catalytic domain faces the lumen. Therefore, the transport of UDP-GlcA into the Golgi lumen is a critical step for UDP-GalA biosynthesis, as well as for part of the luminal UDP-Xyl. Additionally, UDP-Arap produced from lumen-synthesized UDP-Xyl may rely on this transporter. UDP-GlcA transport is also important for glucuronoxylan biosynthesis because this polymer is synthesized in the Golgi lumen, where GlcA units are added to the xylan backbone. A NST that transports UDP-GlcA has been described in C. elegans and mutations in the protein responsible for this activity lead to an abnormal development in this organism [START_REF] Berninsone | Nucleotide sugar transporters of the Golgi apparatus[END_REF]. A Golgi-localized UDP-GlcA transporter is likely to play a critical role in plant cells by providing the substrate precursors needed for pectin and hemicellulose biosynthesis in the Golgi lumen. In this context, this transporter could play an important role in determining the content of plant cell wall sugars that are derived from UDP-GlcA.

To identify a UDP-GlcA transporter and analyze its role in defining cell wall composition, we took advantage of the fact that Arabidopsis seeds produce copious amounts of a pectin-rich substance that is referred to as seed coat mucilage. It is comprised of gel-like molecules that are extruded from mature seeds following water imbibition [START_REF] Saez-Aguayo | PECTIN METHYLESTERASE INHIBITOR6 promotes Arabidopsis mucilage release by limiting methylesterification of homogalacturonan in seed coat epidermal cells[END_REF][START_REF] Western | Differentiation of mucilage secretory cells of the Arabidopsis seed coat[END_REF][START_REF] Western | The sticky tale of seed coat mucilages: production, genetics, and role in seed germination and dispersal[END_REF][START_REF] Young | Analysis of the Golgi apparatus in Arabidopsis seed coat cells during polarized secretion of pectin-rich mucilage[END_REF]. Early visual examination of the mucilage provided evidence of its pectic nature and showed the presence of two distinct mucilage layers. Both layers contain large amounts of the GalA-containing polysaccharides rhamnogalacturonan I (RG-I) and homogalacturonan (HG). RG-I is mostly unbranched in the external layer, the soluble mucilage (SM), and it is branched, having arabinan and galactan side chains in the internal layer, the adherent mucilage (AM) [START_REF] Macquet | In situ, chemical and macromolecular study of the composition of Arabidopsis thaliana seed coat mucilage[END_REF][START_REF] Western | Differentiation of mucilage secretory cells of the Arabidopsis seed coat[END_REF][START_REF] Willats | In-situ analysis of pectic polysaccharides in seed mucilage and at the root surface of Arabidopsis thaliana[END_REF]. The AM also contains methylesterified HG and cellulose microfibrils [START_REF] Macquet | In situ, chemical and macromolecular study of the composition of Arabidopsis thaliana seed coat mucilage[END_REF][START_REF] Saez-Aguayo | PECTIN METHYLESTERASE INHIBITOR6 promotes Arabidopsis mucilage release by limiting methylesterification of homogalacturonan in seed coat epidermal cells[END_REF][START_REF] Western | MUCILAGE-MODIFIED4 Encodes a Putative Pectin Biosynthetic Enzyme Developmentally Regulated by APETALA2, TRANSPARENT TESTA GLABRA1, and GLABRA2 in the Arabidopsis Seed Coat[END_REF][START_REF] Willats | In-situ analysis of pectic polysaccharides in seed mucilage and at the root surface of Arabidopsis thaliana[END_REF]. Because mucilage contains high pectin levels, changes in the pathway leading to the synthesis of UDP-GalA will alter RG-I or HG synthesis, affecting the seed mucilage formation and composition. Therefore, the seed coat NST expression analysis was combined with a mucilage release-screening assay of NST mutants to select any novel NSTs potentially involved in the RG-I or HG synthesis. We identified UUAT1 (UDP-URONIC ACID TRANSPORTER 1), a gene encoding a protein that can transport UDP-GlcA and UDP-GalA in vitro. A knockout line lacking UUAT1 has less galacturonic acid (GalA) and rhamnose (Rha) in both AM and SM, and less Xyl in SM. Also, a decrease in arabinan content was observed in the seed coat. Analyses of UUAT1 expression in other organs and cells revealed differences in Ara content in uuat1 mutant vs. wild type tissue. Interestingly, besides changes in sugar content, a change in the HG methylation pattern was observed in the mucilage and more methyl groups were released from cell wall material from mucilage and stem, suggesting that HG methylation is also altered in some organs and indicating that pleiotropic changes might take place in the mutant cell wall. Our results suggest that UUAT1 transports UDP-GlcA in vivo. Furthermore, the loss of function of this transporter leads to changes in monosaccharide composition, in the cell wall, mainly in those sugars related to UDP-GlcA metabolism in the Golgi lumen. These results show the importance of the transport of UDP-GlcA in the biosynthesis of the plant cell wall.

RESULTS

Analysis of NSTs Expressed in Seed Coats and Identification of UUAT1

In silico data analyses revealed that twenty one out of the fifty one members of the NST/TPT Arabidopsis family [START_REF] Rautengarten | The Golgi localized bifunctional UDP-rhamnose/UDP-galactose transporter family of Arabidopsis[END_REF] are expressed in the seed coat during the developmental stages when mucilage is produced and accumulated in epidermal cells (Supplemental Figure 1) [START_REF] Le | Global analysis of gene activity during Arabidopsis seed development and identification of seed-specific transcription factors[END_REF]; http://seedgenenetwork.net/arabidopsis). Of these twenty one candidates, we disregarded those with reported functions (ten known NSTs) in the Arabidopsis UDP-rhamnose/UDP-galactose transporter (URGT) family [START_REF] Rautengarten | The Golgi localized bifunctional UDP-rhamnose/UDP-galactose transporter family of Arabidopsis[END_REF], UDP-galactose transporters 1, 2 and 3 UTR1, UTR2, UTR3 [START_REF] Norambuena | Transport of UDP-galactose in plants. Identification and functional characterization of AtUTr1, an Arabidopsis thaliana UDP-galactos/UDP-glucose transporter[END_REF][START_REF] Norambuena | AtUTr2 is an Arabidopsis thaliana nucleotide sugar transporter located in the Golgi apparatus capable of transporting UDP-galactose[END_REF][START_REF] Reyes | AtUTr1, a UDP-glucose/UDP-galactose transporter from Arabidopsis thaliana, is located in the endoplasmic reticulum and up-regulated by the unfolded protein response[END_REF][START_REF] Reyes | The nucleotide sugar transporters AtUTr1 and AtUTr3 are required for the incorporation of UDP-glucose into the endoplasmic reticulum, are essential for pollen development and are needed for embryo sac progress in Arabidopsis thaliana[END_REF], and GalT1 [START_REF] Bakker | Molecular cloning of two Arabidopsis UDP-galactose transporters by complementation of a deficient Chinese hamster ovary cell line[END_REF]. Among the eleven target genes that were expressed throughout seed development and lack a known function (Supplemental Figure 1), At5g17630 was discarded from the analysis because as it belongs to the triose phosphate translocator clade. Of the ten remaining genes, homozygous mutants could be obtained for seven of them, but only heterozygous mutants were obtained for the other three. Soluble mucilage content was assessed by measuring the uronic acid released following water imbibition. The results showed that a mutant allele of At5g04160, uuat1, exhibited the lowest level of mucilage uronic acid (Supplemental Figure 2). UUAT1 expression was also measured during seed development to confirm that it is expressed during the mucilage production stages (6 to 8 DAP). Supplemental Figure 3 shows a peak in UUAT1 expression at 8 DAP, a pattern similar to the expression of genes involved in mucilage synthesis [START_REF] Macquet | In situ, chemical and macromolecular study of the composition of Arabidopsis thaliana seed coat mucilage[END_REF][START_REF] Saez-Aguayo | PECTIN METHYLESTERASE INHIBITOR6 promotes Arabidopsis mucilage release by limiting methylesterification of homogalacturonan in seed coat epidermal cells[END_REF][START_REF] Rautengarten | The Golgi localized bifunctional UDP-rhamnose/UDP-galactose transporter family of Arabidopsis[END_REF]. UUAT1 encodes a polytopic transmembrane protein with ten putative membrane spanning domains (Supplemental Figure 4) and belongs to a subclade composed of five paralogues with identities ranging from 81% to 49% (Supplemental Table 1). However, their expression levels are much lower than those of UUAT1 (Supplemental Figure 3).

Given these results, we decided to focus on UUAT1 by analyzing its role in the biosynthesis of seed coat mucilage. Three T-DNA insertion lines were identified in the At5g04160 locus and were designated uuat1-1, uuat1-2 and uuat1-3 (Figure 1A). These mutant lines had a lower content of GalA and Rha residues in the SM fraction compared to the wild type (WT) Col-0 plants (Figure 1C and Supplemental Table 2). When compared to the other two allelic lines, uuat1-2 exhibited the most pronounced decrease in both sugars. UUAT1 transcripts were undetectable in the uuat1-2 mutant line, whereas the other two lines (uuat1-1 and uuat1-3) exhibited some UUAT1 expression, albeit at lower levels than WT Col-0 (Figure 1B). Thus, we concluded that uuat1-2 had the strongest phenotype because it was a true knock-out line, whereas the other alleles were knock-down lines and so the studies focused on the uuat1-2 allele. Molecular rescue of the uuat1-2 mutant confirmed that the absence of UUAT1 was responsible for the phenotypes observed in uuat1-2 (Supplemental Figure 5). The uuat1-2 line was transformed with a construct that contains the UUAT1 coding sequence (CDS) fused to a GFP tag and is driven by the UUAT1 endogenous promoter.

Several independent transformants were obtained and the presence of the transgene was confirmed by RT-PCR (Supplemental Figure 5A). Wild-type ruthenium red staining of the AM and sugar content levels were observed in two independent transgenic lines, indicating that UUAT1-GFP had successfully rescued the mutant (Supplemental Figure 5B and 5C).

UUAT1 is a UDP-Uronic Acid Transporter in the Golgi

To determine the substrate specificity of UUAT1 in vitro, it was expressed heterologously in Saccharomyces cerevisiae (yeast) and transport assays were conducted as reported in [START_REF] Rautengarten | The Golgi localized bifunctional UDP-rhamnose/UDP-galactose transporter family of Arabidopsis[END_REF]. Transport assays were performed using the microsomal proteins reconstituted in proteoliposomes. An immunoblotting analysis of the reconstituted protein confirmed the presence of UUAT1 in proteoliposomes (Figure 2A). Proteoliposomes were pre-loaded with uridine monophosphate (UMP), guanosine monophosphate (GMP), cytidine monophosphate (CMP) or adenosine monophosphate (AMP) and then incubated with a mixture of 15 nucleotides/nucleotide sugars to determine substrate specificity (Figure 2D, Supplemental Figure 6). Non-transported substrates were removed by gel filtration, and the proteoliposome content analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The substrate preference exhibited by UUAT1 could readily be assessed after LC-MS/MS analysis when compared to the empty vector control. UUAT1 demonstrated clear preferences for UDP-GlcA and UDP-GalA when proteo-liposomes were preloaded with UMP (Figure 2D). No significant differences in transport activity between the control and UUAT1 were observed for any other nucleotide sugar apart from UDP-Arap, although this activity was much lower than that observed for the UDP-uronic acids (Figure 2D). These substrate preferences were only specific when the proteoliposomes were preloaded with UMP and not with GMP, CMP or AMP (Supplemental Figure 6). Given the additional negative charge present in UDP-uronic acids, UDP was also tested as a potential antiporter substrate, but no transport was observed (Supplemental Figure 6). Proteoliposomes preloaded with GMP could transport GDP-sugars and some lower activity was also observed when proteoliposomes were preloaded with AMP (Supplemental Figure 6), but this is likely to be the result of the endogenous transport activities of the yeast microsomal preparation, since the proteoliposome UUAT1 expression activity did not differ from the control, as has been observed previously [START_REF] Ebert | Identification and characterization of a Golgi-localized UDP-Xylose transporter family from Arabidopsis[END_REF]. UDP-GlcA transport by UUAT1 did not achieve saturation within the concentration range utilized in the assay (Figure 2B), however, transport was affected in a time dependent manner (Figure 2C). Analysis of transport rates indicated that UUAT1 has an apparent Km of 1.5 mM for UDP-GlcA (Figure 2B).

A C-terminal translational fusion with green fluorescent protein (GFP) was used to determine the subcellular localization of UUAT1 using laser scanning confocal microscopy on transient transformed epidermal cells (Figure 3). The UUAT1-GFP distribution pattern was compared with those obtained for the cis-Golgi marker α-mannnosidase-I (Saint-Jore- [START_REF] Saint-Jore-Dupas | Plant N-glycan processing enzymes employ different targeting mechanisms for their spatial arrangement along the secretory pathway[END_REF] and an endoplasmic reticulum (ER) marker [START_REF] Nelson | A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants[END_REF]. The fluorescence signal obtained from the UUAT1-GFP protein colocalized with the punctate pattern obtained for the cis-Golgi marker α-Man-I but not with the ER marker (Figure 3A to 3F). To confirm the localization of UUAT1, trichomes of transgenic rescued plants expressing UUAT1-GFP under the endogenous promoter were analyzed, and they also showed motile structures exhibiting a punctate pattern, as has been described for Golgi resident proteins (Figure 3G) [START_REF] Boevink | Stacks on tracks: the plant Golgi apparatus traffics on an actin/ER network[END_REF]. Taking these data together, these results indicate that UUAT1 is a Golgi-localized UDP-uronic acid transporter.

Absence of UUAT1 Has Pleiotropic Effects on Seed Coat Cell Walls and Mucilage

In order to better understand the effects on the composition of cell wall polysaccharides caused by the absence of UUAT1, we analyzed the sugar content of the polysaccharides present in the seed mucilage (Table 1) in both WT Col-0 and uuat1-2 mutant plants. The SM, as expected, contained mostly GalA and Rha. However, decreases in both GalA (20%) and Rha (22%) were observed in the uuat1-2 mutant compared to the WT (Table 1).

Interestingly, despite the low Xyl levels in the sample, it displayed a similar reduction (21%).

Furthermore, the seed+AM fraction from uuat1-2 showed a 5% decrease in GalA content along with a 9% reduction in Rha (Table 1).

To further investigate the differences observed in the Seed+AM fraction and to better understand which polysaccharides might be altered in the mutant, we performed whole mount immunolabeling assays using antibodies against the epitopes present in cell wall polysaccharides, (Figure 4). Based on the measured monosaccharide composition and considering the mucilage polysaccharide composition, the labeling was performed using the following antibodies: CCRC-M36 (anti-RG-I), LM6 (anti-arabinan), JIM7 and LM20 (both antimethylated HG) [START_REF] Macquet | In situ, chemical and macromolecular study of the composition of Arabidopsis thaliana seed coat mucilage[END_REF][START_REF] Verhertbruggen | An extended set of monoclonal antibodies to pectic homogalacturonan[END_REF][START_REF] Willats | In-situ analysis of pectic polysaccharides in seed mucilage and at the root surface of Arabidopsis thaliana[END_REF]. In addition, to visualize the cell wall, we used calcofluor or propidium iodide which stain β-1,4 glucans and polysaccharides. RG-I labeling, was reduced in the AM of uuat1-2 seeds compared to WT Col-0 seeds (Figures 4A and4C, green signal). Moreover, the calcofluor labeling showed a distal cell wall defect in the mutant when compared with WT Col-0 (Figures 4B and4D, pink labeling), likely due to an abnormal cell wall rupture. The LM6 antibody showed less arabinan in the uuat1-2 AM compared to the WT Col-0, especially in the distal wall of the epidermal cells (Figures 4E and4F). The lack of staining with the LM6 antibody was restored in the transgenic plants that expressed UUAT1-GFP with the endogenous promoter (Supplemental Figure 7), providing further evidence that this phenotype is due to the UUAT1 absence.

As the rupture of the distal cell wall upon water imbibition seemed to be altered, we reasoned that the cell wall stiffness might have changed. Because the degree of HG methylation affects the stiffness of the cell wall [START_REF] Peaucelle | Arabidopsis phyllotaxis is controlled by the methyl-esterification status of cell-wall pectins[END_REF], the LM20 and JIM7 antibodies were used to look at the highly methylesterified HG distribution (Figure 5 and Supplemental Figure 8). An increase in LM20 labeling in AM was observed in the uuat1-2 mutant when compared to WT Col-0 (Figures 5 A and5B), suggesting the presence of HG with a higher degree of methyl esterification in the mutant. This result was also observed using the JIM7 antibody in the allelic lines uuat1-1 and uuat1-3 (Supplemental Figure 8).

Analysis of the uuat1-2 lines expressing UUAT1 supported this observation by showing less labelling than in the mutant, but more labelling than in the WT Col-0 when JIM7 and LM20 were used to assess the methylated HG content (Supplemental Figure 7). In addition, we performed ruthenium red staining in the presence of EDTA, a chelator that removes cations and increases the exposure of carboxylic groups, thus enhancing the ruthenium red staining (Figures 5C and5D). WT Col-0 seeds showed intense staining but mutant seeds exhibited a pale color, suggesting that fewer carboxylic groups were available for binding the dye (Figure 5D). To confirm changes in HG methylesterification in the uuat1-2 mutant, the contents of methyl groups present in the soluble mucilage and seed+AM fractions were determined by measuring the methanol released upon saponification. Both fractions displayed greater methanol release of 37% and 67%, respectively (Figure 5E). Finally, these HG methylesterification changes correlated with a 10% decrease in pectin methylesterase activity (PME), measured in dry mutant seeds (Figure 5F). All these results provide strong evidence that UUAT1 absence leads to an increase of highly methylesterified HG epitopes in mucilage.

UUAT1 Functions in Different Plant Organs

We next analyzed the UUAT1 expression pattern in organs such as roots, seedlings, rosette and cauline leaves, stems, flowers, siliques and seeds at different development stages (Supplemental Figure 9). The UUAT1 transcript was detected by qPCR in all organs analyzed, with expression peaking in stems and flowers (Supplemental Figure 9A). The GUS reporter gene [START_REF] Jefferson | The GUS reporter gene system[END_REF] was cloned under the control of the UUAT1 promoter and transformed into WT Col-0 plants to obtain spatial information regarding UUAT1 expression.

Strong GUS activity was detected in roots, seedlings, trichomes, flowers and developing seeds (Supplemental Figure 9B), confirming that UUAT1 is predominantly expressed in these organs.

All obvious phenotypes in the uuat1-2 mutant were examined first to investigate whether the UUAT1 mutation leads to changes in plant development and or cell wall composition in organs or tissues apart from the seed mucilage. Interestingly, the only change observed was in the primary stem, as uuat1-2 plants displayed an early elongation phenotype when compared to WT Col-0 plants (Figure 6A). However, this morphological difference disappeared once the plants reached a mature stage. No changes were observed in sugar composition of the stem cell wall for any of the sugars analyzed, including Xyl (Figure 6C), the most abundant sugar due to the presence of glucuronoxylan, one of the more abundant stem polymers. Because glucuronoxylan also contains GlcA in a given branching frequency, a carbohydrate gel electrophoresis (PACE) polysaccharide analysis [START_REF] Mortimer | Absence of branches from xylan in Arabidopsis gux mutants reveals potential for simplification of lignocellulosic biomass[END_REF] was used to quantify the oligosaccharides Xyl, Xyl2, and GlcAXyl4/[MeGlcA] Xyl4 released by xylanase GH11. No changes were found in the GlcA/Xyl ratio, suggesting that the glucuronoxylan structure is normal in the uuat1-2 mutant (Figure 6D). Additionally, because changes were observed in mucilage HG methylesterification, this modification was further analyzed in three development stages in stems. The methylesterification levels were observed to be significantly higher in all conditions in the uuat1-2 mutant when compared to WT Col-0 (Figure 6B).

The alcohol insoluble residue (AIR) sugar composition in uuat1-2 mutant roots, trichome, and pollen tube preparations was analyzed to uncover any possible changes in cell wall composition in other UUAT1 expressing organs (Figure 7). Ara levels were reduced in roots and pollen tubes. A slight decrease was observed in trichomes but it was not significant. No decrease in GalA or Xyl was observed in any other of the tissues analyzed; thus, Ara was the only sugar whose content consistently decreased in the mutant and this decrease was tissue-specific. In conclusion, our results indicate that plants lacking UUAT1 show changes in the composition of cell wall monosaccharides derived from the metabolism of UDP-GlcA in the Golgi, with arabinose being the most affected. Furthermore, mutants in UUAT1 show enhanced levels of methylesterification in cell wall polysaccharides, a likely response to cope with changes in cell wall composition.

DISCUSSION

UDP-GlcA is synthesized and utilized in the plant cell cytosol, but it is also required in the Golgi lumen for synthesis of UDP-GalA, UDP-Xyl and UDP-Arap. Therefore, UDP-GlcA needs to be transported from the cytosol across the Golgi membrane into the Golgi lumen to be converted into these nucleotide sugars. Our work led to the identification of UUAT1, a protein that can transport UDP-GlcA, UDP-GalA and low levels of UDP-Arap in vitro.

However, UUAT1 is unlikely to transport significant amounts of UDP-GalA or UDP-Arap in vivo, because both UDP-GlcA 4-epimerase and UDP-Xyl 4-epimerase, the enzymes involved in the synthesis of UDP-GalA and UDP-Arap respectively, are located in the Golgi lumen [START_REF] Burget | The biosynthesis of Larabinose in plants: molecular cloning and characterization of a Golgi-localized UDP-Dxylose 4-epimerase encoded by the MUR4 gene of Arabidopsis[END_REF][START_REF] Gu | The biosynthesis of UDP-Galacturonic Acid in plants. Functional cloning and characterization of Arabidopsis UDP-D-Glucuronic acid 4-epimerase[END_REF][START_REF] Mølhøj | The biosynthesis of D-Galacturonate in plants. functional cloning and characterization of a membrane-anchored UDP-D-Glucuronate 4-epimerase from Arabidopsis[END_REF]. A cytosolic salvage pathway for UDP-GalA has been reported [START_REF] Yang | Identification of Galacturonic Acid-1-phosphate Kinase, a New Member of the GHMP Kinase Superfamily in Plants, and Comparison with Galactose-1-phosphate Kinase[END_REF], but requires release from the cell wall of GalA, which can then be converted into UDP-GalA by the enzymes GalA kinase and Sloppy, a promiscuous UDP-sugar pyrophosphorylase [START_REF] Kotake | Properties and physiological functions of UDP-sugar pyrophosphorylase in Arabidopsis[END_REF].

Therefore, this salvage pathway may be active only under certain circumstances and perhaps cytosolic UDP-GalA formed via this pathway could be transported by UUAT1. UDP-Arap can also be biosynthesized by UGE1 and UGE3 in the cytoplasm, using UDP-Xyl to form UDP-Arap. This would presumably be in addition to their roles in the UDP-Glc to UDP-Gal epimerization. However, mutations in these genes suggest that this cytosolic pathway is less important than that located in the Golgi [START_REF] Rösti | UDP-glucose 4-epimerase isoforms UGE2 and UGE4 cooperate in providing UDP-galactose for cell wall biosynthesis and growth of Arabidopsis thaliana[END_REF][START_REF] Kotake | Bifunctional cytosolic UDP-glucose 4-epimerases catalyse the interconversion between UDP-D-xylose and UDP-L-arabinose in plants[END_REF][START_REF] Rautengarten | The interconversion of UDP-arabinopyranose and UDP-arabinofuranose is indispensable for plant development in Arabidopsis[END_REF][START_REF] Kotake | Metabolism of larabinose in plants[END_REF]. Furthermore, because UUAT1 did not exhibit in vitro transport activity for UDP-Araf (arabinose in its furanose form), we postulate that the main in vivo role for UUAT1 is to transport UDP-GlcA to the Golgi lumen. Finally, we believe the low UDP-Arap activity observed is not a function specific to UUAT1, because it is also observed in other nucleotide sugar transporters such as the UDP-Rha/UDP-Gal transporters and the UDP-Xyl transporter [START_REF] Ebert | Identification and characterization of a Golgi-localized UDP-Xylose transporter family from Arabidopsis[END_REF][START_REF] Rautengarten | The Golgi localized bifunctional UDP-rhamnose/UDP-galactose transporter family of Arabidopsis[END_REF] UUAT1 is expressed in seed coat epidermal cells and knockout plants showed a number of mucilage-related phenotypes. The uuat1-2 mutant displayed reduced GalA and Rha content in both the SM and the seed+AM fraction. Thus, the GalA decrease could be explained by a lower UDP-GlcA transport rate to the Golgi lumen, the substrate required for the UDP-GalA synthesis. Thus, its reduced transport rate could lead to lower levels of this nucleotide sugar in the mutant. On the other hand, UUAT1 does not transport UDP-Rha, so the Rha decrease is likely due to an impairment in synthesis of the RG-I backbone, which is composed of repeating (GalA-Rha)n disaccharide units. Because one of the substrates (UDP-GalA) is reduced, it is likely that Rha incorporation has been also affected, leading to lower levels of this sugar in SM and the seed+AM fraction. Something similar occurs in mutants in URGT2, a UDP-Rha transporter that is also expressed in seed coat epidermal cells. SM in this mutant also exhibits lowered Rha and GalA, even though URGT2 does not transport UDPuronic acids [START_REF] Rautengarten | The Golgi localized bifunctional UDP-rhamnose/UDP-galactose transporter family of Arabidopsis[END_REF]. The GalA and Rha decrease observed in mucilage (a RG-I enriched matrix) from UDP-Rha and UDP-GlcA transporter mutants suggests a coordination in the supply of both nucleotide sugars during RG-I biosynthesis.

The uuat1 mutants exhibit a decrease in arabinan in AM, as detected by LM6 antibody immunolabeling. Ara is an abundant sugar in the seed+AM fraction and can be present in other polysaccharides [START_REF] Western | Isolation and Characterization of Mutants Defective in Seed Coat Mucilage Secretory Cell Development in Arabidopsis[END_REF], so changes in the total Ara content may not reveal the differences in a low-abundant Ara-containing polymer. The use of antibodies can detect precise changes in arabinan, which is present in the wild type and almost undetectable in the mutant, suggesting that UUAT1 plays an important role in providing the precursor for arabinan synthesis. The absence of UUAT1 also results in a reduction in Xyl in SM. However, this phenotype is different from the one observed for muci21 and irx14, xylosyltransferases mutants that show alterations in epidermal cell mucilage adhesion [START_REF] Hu | Xylan synthesized by Irregular Xylem 14 (IRX14) maintains the structure of seed coat mucilage in Arabidopsis[END_REF][START_REF] Voiniciuc | Highly Branched Xylan Made by IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis Seeds[END_REF][START_REF] Ralet | Xylans provide the structural driving force for mucilage adhesion to the Arabidopsis seed coat[END_REF]. On the other hand, no Xyl changes were observed in the seed+AM fraction, suggesting a precise and discrete role for UUAT1 in Xylcontaining polymer synthesis.

The results show that both the SM and seed+AM matrices in the uuat1 mutants have lower levels of sugars (GalA, Xyl and Ara) provided by UDP-sugars derived from the metabolism of UDP-GlcA in the Golgi. This supports the role of UUAT1 as an in vivo UDP-GlcA transporter.

Because these sugars are not completely diminished, it suggests that other UDP-GlcA transporters are present in seed coat epidermal cells. Alternatively, some compensatory mechanisms, such as UDP-Xyl transport by specific transporters could be activated [START_REF] Ebert | Identification and characterization of a Golgi-localized UDP-Xylose transporter family from Arabidopsis[END_REF].

In addition to the changes in cell wall composition, our calcofluor staining studies revealed that distal cell walls of seed coat epidermal cells exhibited an abnormal rupture upon imbibition. Whether the changes observed in GalA, Rha, Xyl and arabinan are responsible for this feature remains to be determined. However, an interesting observation was that the uuat1-2 mutant exhibits an increase in the level of HG methylation, a feature that was partially rescued in plants expressing UUAT1. The increase in labeling by the LM20 antibody in mucilage correlated with reduced ruthenium red staining, a dye that binds to the HG carboxyl groups, suggesting their blockage by methyl groups. In addition, more methyl groups were released from mucilage derived from uuat1-2 mutant seeds and lower PME activity was detected. This methylation increase may be the result of the adaptation that takes place in the uuat1-2 mutant to compensate for changes in its cell wall. Because the degree of HG methylation correlates to cell wall stiffness, methylation changes may contribute to the altered rupture of distal cell walls during seed imbibition. Regarding HG methylation and arabinan content, it is interesting to note that mutants containing lower HG methylation levels due to defective pectin methyltransferase activity exhibit an increase in Ara [START_REF] Kim | CGR2 and CGR3 have critical overlapping roles in pectin methylesterification and plant growth in Arabidopsis thaliana[END_REF], which could account for higher arabinan levels. By contrast, uuat1-2 plants show a greater HG methylation and lower arabinan levels, suggesting that an inverse correlation may exist between arabinan content and the degree of HG methylation.

The expression pattern of UUAT1 indicates that it might have additional functional roles in other organs or cell types. Indeed, an evaluation of the cell wall sugar composition of other organs and cells from the uuat1-2 mutant showed lower levels of Ara in roots and pollen tubes, but not a decrease in Xyl or GalA. These results suggest that changes in the mutant plant cell wall composition are organ-dependent. In this sense, Ara is the sugar most affected in the organs evaluated, suggesting that the supply of UDP-Ara for arabinosyltransferases is more affected in uuat1-2 plants. On the other hand, the levels of GalA and Xyl did not exhibit significant differences in the mutant, except in seeds. These results could be explained due to redundancy of paralogue genes present in subclade V of the NST gene family [START_REF] Rautengarten | The Golgi localized bifunctional UDP-rhamnose/UDP-galactose transporter family of Arabidopsis[END_REF], which could supply UDP-GlcA for the UDP-GalA synthesis. This explanation is also valid for the absence of Xyl changes, but it is important to mention that UDP-Xyl levels in the Golgi lumen are also directly controlled by a UDP-Xyl transporter (UXT1), described recently by [START_REF] Ebert | Identification and characterization of a Golgi-localized UDP-Xylose transporter family from Arabidopsis[END_REF]. Mutants in this transporter had decreased Xyl content in stems, and glucuronoxylan was strongly affected [START_REF] Ebert | Identification and characterization of a Golgi-localized UDP-Xylose transporter family from Arabidopsis[END_REF], suggesting that the UDP-Xyl biosynthesized by cytosolic UXS and transported by UXT1 may be required for xylan synthesis. Furthermore, an Arabidopsis cytosolic UXS triple mutant was shown to have an irregular xylem phenotype, while the lumenal UXS triple mutant had no Xyl-associated phenotype [START_REF] Kuang | The role of UDP-glucuronic acid decarboxylase(s) (UXS) in xylan biosynthesis in Arabidopsis[END_REF]. These findings suggest that UUAT1 is less important in the synthesis of Xyl-containing polysaccharides. Furthermore, it is likely that the UDP-Xyl made in the cytosol is used to synthesize Xyl-containing polysaccharides, whereas the UDP-Xyl made in the Golgi lumen could be used for the synthesis of UDP-Arap in a tissue-specific manner. However, more data will be required to confirm this hypothesis. No changes were observed in the GlcA/Xyl ratio of glucuronoxylan, one of the main polymers containing GlcA, supporting the idea of redundancy in the UDP-GlcA transport.

Cell walls of the uuat1-2 mutant also exhibited other pleiotropic changes in sugar composition and HG methylation depending on the tissue analyzed. For instance, a Gal decrease was observed in roots, and a GalA increase occurred in pollen tubes. HGs can modulate cell wall stiffness in pollen tubes (Parre and Geitmann, 2005a), and callose content changes may also have an impact on the cell wall mechanics of pollen tubes (Parre and Geitmann 2005b). Consequently, these changes may be a response to the Ara change observed in the uuat1-2 mutant. Stems also showed methylation increases at different development stages, which correlate with an early plant bolting phenotype. These pleiotropic events may correspond to adaptations of the mutant due to the absence of UUAT1 and are an indication of the plasticity displayed by the plant cell wall.

METHODS

Plant Material and Growth Conditions

Unless specified otherwise, Arabidopsis (Arabidopsis thaliana (L.) Heynh) and tobacco (Nicotiana benthamiana Domin) plants were germinated and grown in a growth chamber using a long-day regime (16 h photoperiod), the light intensity was 100 μmol m -2 s -1 and the temperature 21°C. For seeds and aerial tissue collection, plants were grown in soil (Terracult blue substrate, Terracult GmbH) supplemented with fertilizer (Basacote plus 6M, Compo Expert) in a relative humidity (RH) of 60%. The plants were grown in MS media (Duchefa) (2.155 g/L), 1% sucrose and 0.8% agar to obtain root material. T-DNA insertion lines for UUAT1 (SALK_124146C/ uuat1-1, SALK_105023C/ uuat1-2 and SALK_048507/ uuat1-3) were obtained from the Arabidopsis Biological Resource Center (ABRC, http://abrc.osu.edu/) using the SIGnAL Salk collection [START_REF] Alonso | Genome-wide insertional mutagenesis of Arabidopsis thaliana[END_REF]. SALK_105023C was annotated as uuat1-2. Wild type Columbia-0 (WT Col-0) and mutants were transformed using Agrobacterium tumefaciens (GV3101::pMP90) carrying the specified vectors, using the standard floral dip method [START_REF] Clough | Floral dip: a simplified method for Agrobacteriummediated transformation of Arabidopsis thaliana[END_REF].

Infiltration of Tobacco Leaves and Subcellular Localization of UUAT1-GFP

Six week-old tobacco leaves were infiltrated with Agrobacterium tumefaciens, strain GV3101::pMP90 as described in [START_REF] Batoko | A Rab1 GTPase is required for transport between the endoplasmic reticulum and Golgi apparatus and for normal Golgi movement in plants[END_REF]. Two independent transformations were undertaken for analyzing UUAT1 subcellular localization. ER localization was analyzed using UUAT1-GFP and the ER marker AtWAK-mCherry-HDEL (Wall Associated Kinase-2 carrying an ER retention signal). Golgi localization was analyzed by cotransformation of UUAT1-GFP and the Golgi localized protein -mannosidase I (-mannosidase I-mCherry; [START_REF] Nelson | A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants[END_REF]. Fluorescent signals were analyzed 60 h after infiltration by confocal laser scanning microscopy, using an Olympus FluoView FV1000 spectral microscope.

GUS Staining

The histochemical localization of β-glucuronidase (GUS) activity was performed as described in [START_REF] Jefferson | The GUS reporter gene system[END_REF]. Tissues were imaged using an Olympus SZ61 stereoscopic microscope and seeds were analyzed with an Olympus Fluoview FV1000 confocal microscope. 488 nm excitation and emission of 485 nm and 491 nm were used for the analysis of seed GUS staining by confocal microscopy, [START_REF] Truernit | High-resolution whole-mount imaging of three-dimensional tissue organization and gene expression enables the study of Phloem development and structure in Arabidopsis[END_REF].

Ruthenium Red Staining

Mucilage released from mature dry seeds was stained either directly with 0.03% (w/v) ruthenium red or after imbibition in 0.5 M EDTA, pH 8.0, for 90 min. After EDTA treatment, seeds were stained for 2 min and observed with a light microscope (Olympus SZ61).

Genotyping

Genomic DNA was extracted from Arabidopsis 7 d-old cotyledons as described in [START_REF] Edwards | A simple and rapid method for the preparation of plant genomic DNA for PCR analysis[END_REF]. PCR was conducted to amplify the wild type and mutant alleles using the primers described in Supplementary Table 3.

Cloning Procedures

The UUAT1 coding sequence (CDS) without the stop codon was amplified from cDNA prepared from Arabidopsis leaf RNA, using the primers described in Supplemental Table 3.

Resulting PCR products were introduced into the pENTR/D TOPO vector according to standard protocols (Life Technologies) to generate the entry clone pENTR-UUAT1. The Cterminal GFP fusion under the control of the cauliflower mosaic virus 35S promoter was generated by introducing the UUAT1 CDS from the entry clone into the gateway destination vector pK7FWG2.0 [START_REF] Karimi | GATEWAY vectors for Agrobacteriummediated plant transformation[END_REF] using LR clonase (Thermo Fisher Scientific). For the rescue construct, the intergenic region (653 bp) between At5g04170 and At5g04160 was defined as the UUAT1 promoter (pUUAT1) and was amplified from Arabidopsis genomic DNA using the primers described in Supplemental Table 3. Resultant PCR products were introduced into the pENTR 5-TOPO vector (Thermo Fisher Scientific) to generate the pENTR5-pUUAT1 entry clone. Both, C-terminal GFP and HA fusions were obtained by recombining the entry clones pENTR-UUAT1 and pENTR5-pUUAT1 with destination vectors R4pGWB504 and R4pGWB513, respectively. For the transcriptional fusion of pUUAT1 to the GUS reporter gene, the entry clone pENTR5-pUUAT1 and the destination vector pKGWFS7 [START_REF] Karimi | GATEWAY vectors for Agrobacteriummediated plant transformation[END_REF] were recombined using LR clonase (Thermo Fisher Scientific).

Expression Analysis

Total RNA from stems, rosette and cauline leaves, and roots was extracted using Trizol (Thermo Fisher Scientific). For developing seeds, the pollination time in days after pollination (DAP) was defined phenotypically as the time at which the flowers are just starting to open and the long stamens grow over the gynoecium, as previously described in [START_REF] Western | Differentiation of mucilage secretory cells of the Arabidopsis seed coat[END_REF]. Seeds were dissected from approximately nine siliques in each DAP for further RNA extraction. RNA extractions were performed using RNeasy Plus Micro Kit, according to manufacturer instructions (Quiagen). 1 μg of total RNA was used as a template for firststrand cDNA synthesis with an oligo (dT) primer and SuperScript II (Thermo Fisher Scientific), according to the manufacturer instructions. The primers described in Supplemental Table 3 were used to amplify PCR products from single-stranded cDNA in the wild type and uuat1-2 samples for the CDS of UUAT1 CDS; EF1αA4, primers were described in [START_REF] North | The Arabidopsis ABA-deficient mutant aba4 demonstrates that the major route for stress-induced ABA accumulation is via neoxanthin isomers[END_REF] Quantitative PCR (qPCR) was performed using the Fast EvaGreen qPCR Master Mix kit (Mx3000P, Stratagene). Reactions contained 1 μL of 1:2 diluted cDNA in a total volume of 10 μL. Reactions were carried out using primers that has been previously tested for their efficiency rates and sensitivity in a cDNA dilution series. The quantification and normalization procedures were done using the following equation, as described by Stratagene:

Normalized Unknown Control = (1+𝐸 target) -∆𝐶𝑡 target (1+𝐸 norm) -∆𝐶𝑡 norm
where E corresponds to the efficiency of amplification of the target gene, Ct = threshold cycle (Ct), "Control" represents the calibrator sample and norm refers to the reference or normalizer gene. Primers for UUAT1, UUAT2, UUAT3, UUAT4, UUAT5, EF1α [START_REF] Hong | Identification and testing of superior reference genes for a starting pool of transcript normalization in Arabidopsis[END_REF], UBC9 and seed reference gene At4g12590 [START_REF] Hong | Identification and testing of superior reference genes for a starting pool of transcript normalization in Arabidopsis[END_REF] were those described in Supplemental Table 3.

Analysis of In Vitro UUAT1 Transport

For heterologous expression, we used the uracil-auxotrophic Saccharomyces cerevisiae strain INVSc-1 (MATa his3D1 leu2 trp1-289 ura3-52 MAT his3D1 leu2 trp1-289 ura3-52, Thermo Fisher Scientific). The UUAT1 coding sequence was cloned onto the Gateway expression vector pYES-DEST52 (Thermo Fisher Scientific) and then introduced into the yeast strain with the S.c. EasyComp Transformation Kit (Thermo Fisher Scientific). The control was the yeast strain transformed with the empty vector pYES-DEST52. Microsomal fractions were obtained from 200 mL cultures grown at 30°C. Yeast cells were pelleted and spheroplasts produced in 10 mL resuspension buffer (50 mM potassium phosphate, pH 7.1, 1.4 M sorbitol, 10 mM NaN3, and 40 mM 2-mercaptoethanol, 6,000 U Lyticase (Sigma-Aldrich) for 1 h at 37°C. Spheroplasts were harvested by centrifugation and washed with 0.8 M sorbitol, 10 mM triethanolamine / acetic acid pH 7.2, 1 mM EDTA. The spheroplasts were lysed with glass beads in 5 mL 0.8 M sorbitol, 10 mM triethanolamine/acetic acid pH 7.2, 1 mM EDTA, protease inhibitor cocktail from SIGMA ALDRICH and 1 mM PMSF. Microsomes were isolated by sequential centrifugation (8,000 g for 10 min (F1), and 100,000 g for 75 min (F2)). The F2 fraction was reconstituted in 10 mM Tricine-KOH pH 7.5, 50 mM potassium gluconate, 20% glycerol. Proteoliposomes were generated with acetone-washed soybean Lα-phosphatidylcholine (Avanti Polar Lipids) in reconstitution buffer (10 mM Tricine-KOH pH 7.5, 50 mM potassium gluconate and 20% glycerol). Reconstitution of microsomal membranes obtained from the UUAT1-expressing yeast or the control cells was undertaken using approximately 400 µg microsomal protein in reconstitution buffer, lipid at a ratio of 13:1 (lipid:protein), 10 mM exchange substrate and 50 mM octyl-β-glucoside. Unincorporated components were removed from reconstituted liposomes using Sephadex G50 columns (GE Healthcare). 200 µL Aliquots were incubated with nucleotide sugar substrates at 25°C for the indicated times to assess transporter activities. Kinetic parameters were calculated with nonlinear regression using the Prism 7 application (GraphPad Software, La Jolla, CA).

Polyacrylamide gel electrophoresis was carried out with 2.5 µg protein of proteoliposomes on a 7-15% SDS-PAGE gel. Immunoblotting was conducted with the anti-V5 antibody, using a 1:10,000 dilution (Thermo Fisher Scientific).

Nucleotide Sugar Quantification Using Tandem Mass Spectrometry

The transport assay reactions were purified using ENVI-Carb SPE columns (Sigma-Aldrich) and then lyophilized overnight, as outlined in [START_REF] Ito | Organic cation transporter/solute carrier family 22a is involved in drug transfer into milk in mice[END_REF], and then analyzed by tandem mass spectrometry (LC-MS/MS). Nucleotide sugars were separated using a Hypercarb column (150 mm × 1 mm, 5 μm) at a flow rate of 50 μL min -1 with an 1100 series HPLC system (Agilent Technologies, CA) and a 4000 QTRAP LC-MS/MS system (Sciex, CA) equipped with a TurboIonSpray ion source. Initial conditions were 95% buffer A (LC-MS grade water with 0.3% formic acid, pH 9.0 with ammonia) and 5% buffer B (100 % acetonitrile) for 1 min followed by a gradient to 75% (A) in 20 min, then 50% (A) in 5 min before returning to 95% (A) in 5 min. The instrument was operated in negative ion mode, using the multiple reaction monitoring (MRM) scan type. A declustering potential (DP) of -40, entrance potential (EP) of -10, collision cell exit potential (CXP) was -15. The ion spray voltage was set at -4200 V, source temperature (TEM) at 425 °C, collision gas (CAD) was set to High and source gas 1 (GS1) and 2 (GS2) were both set to 20. A time of 100 ms was applied for each transition, resulting in a duty cycle of 1.0501 s with both Q1 and Q3 resolution set to Unit. All data were acquired using Analyst 1.6 Build 3773 (Sciex, CA).

Nucleotide sugars were quantified using MultiQuant 2.1 (build 2.1.1296.02.1) software (Sciex, CA) by integrating the signal peak areas of samples against a range of nucleotide sugar standards (2.5 to 20 pmol).

Seed Immunolabeling

Immunolabeling was performed with four monoclonal antibodies, CCRC-M36 (labels rhamnogalacturonan-I), LM6 (arabinan), JIM7 (partially methyl-esterified homogalacturonan) and LM20 (highly methyl-esterified homogalacturonan) [START_REF] Saez-Aguayo | PECTIN METHYLESTERASE INHIBITOR6 promotes Arabidopsis mucilage release by limiting methylesterification of homogalacturonan in seed coat epidermal cells[END_REF]. A double labeling with an antibody plus calcofluor white (0.01%) or propidium iodide (20 µg mL -1 ) was performed as indicated for each antibody to observe the seed surface and AM layer. Optical sections were obtained using an Olympus LX81 spectral confocal laserscanning microscope. A 488 nm argon laser line was used to excite Alexa Fluor 488, a 405 nm diode laser line was used to excite calcofluor white and a 543 nm neon laser line was used to excite propidium iodide. Fluorescence emission was detected between 504 and 579 nm for Alexa Fluor 488, 412 and 490 nm for calcofluor white, and 550 nm and 725 nm for propidium iodide. For comparisons of the signal intensity within one experiment, the laser gain values were fixed.

Trichome Isolation

Trichome isolation was performed as described in [START_REF] Marks | A new method for isolating large quantities of Arabidopsis trichomes for transcriptome, cell wall and other types of analyses[END_REF], with slight modifications. The aerial parts of 18 d-old seedlings were placed in a 50 mL tube with 15 mL of preheated (37ºC) phosphate-buffered saline (137 mM KCl, 10 mM K2HPO4, 2 mM KH2PO4) containing 100 mM EGTA-KOH (pH 7.5) and 50 mg of glass beads 425-600 µm (Sigma-Aldrich). The plant material was then subjected to four cycles at maximum vortex speed for 30 s and on ice for 30 s. The trichomes were recovered using a nylon cell strainer (pore size: 70 µm, BD Falcon) and re-suspended in PBS buffer without EGTA.

Alcohol-Insoluble Residue (AIR) Preparation

Plant tissues were ground in liquid nitrogen and extracted twice in 80% ethanol with agitation for 1 h at room temperature followed by removal of lipids by washing twice with methanol:chloroform (1:1) and twice with acetone. The final alcohol insoluble residue (AIR) was dried overnight at room temperature. A sequential extraction procedure was used for determining the sugar composition of SM and seed+AM,. 20 mg of seeds were imbibed 3 times with 1 mL of water for 20 min; the SM was separated by 10 min of centrifugation at 12,000 g, lyophilized and resuspended in 300 µL of water before hydrolysis. The AM + seed fraction was lyophilized and AIR preparation was prepared as described above.

Acid Hydrolysis

Two mg AIR were hydrolyzed for 20 min for soluble mucilage and 1h for other tissues with 450 µL 2 M trifluoroacetic acid (TFA) at 121 °C. TFA was evaporated at 60°C with nitrogen and the samples were washed twice in 250 µL of 100% isopropanol and dried in a speedvac. The suspension was clarified by passing through a syringe filter (pore size: 0.45 µm), transferred to a new tube and used for HPAEC-PAD analysis as described below. Inositol was used as the internal control for TFA hydrolysis.

In Vitro Pollen Tube Growth and Cell Wall Extraction

Pollen was grown in vitro in a liquid medium according to the method described in [START_REF] Boavida | Temperature as a determinant factor for increased and reproducible in vitro pollen germination in Arabidopsis thaliana[END_REF], [START_REF] Dardelle | Biochemical and immunocytological characterizations of Arabidopsis pollen tube cell wall[END_REF]. Forty freshly opened flowers were submerged in 1 mL of germination medium containing 5 mM CaCl2•, 0.01% (w/v) H3BO3, 5 mM KCl, 1 mM MgSO4, and 10% (w/v) sucrose (pH 7.5), and tubes were shaken with a vortex to release the pollen grains from the anthers. Flowers were removed with a pair of tweezers, and the pollen suspension was then pelleted at 3,200 g for 6 min. New GM (250 µL) was added to the pellet, and pollen grains were grown in a growth chamber in the dark at 22°C for 6 h. Before any further manipulation, pollen germination and pollen tube growth were assessed with an inverted microscope. After 6 h, three volumes of 95% ethanol were added to the GM and stored at 4°C until use. Six h-old pollen tubes from 480 flowers were pooled, centrifuged at 5,000 g and rinsed three times with 1 mL of 70% ethanol to remove salts and sucrose. The insoluble material was ground and treated three times with 70% ethanol at 70°C for 15 min followed by an incubation with 1 mL of a mixture of chloroform:methanol (1:1, v/v) for 15 min. After centrifugation (12,000 g for 10 min), the remaining insoluble material was dried to yield the AIR fraction (about 1 mg). This experiment was performed three times.

Methylesterification Analysis of AIR Samples

The degree of methylesterification of WT Col-0 and uuat1-2 was analyzed in 2 mg of AIR preparations from roots or seeds with AM. For SM, 5 mg of seeds were imbibed in 200 µL of ultrapure water for 6 h, as described in [START_REF] Anthon | Comparison of three colorimetric reagents in the determination of methanol with Alcohol Oxidase. Application to the assay of Pectin Methylesterase[END_REF]. All experiments were done using 3 technical replicates and at least 2 biological replicates.

High Performance Anion Exchange Chromatography with Pulsed Amperometric

Detection (HPAEC-PAD)

A Dionex ICS3000 ion chromatography system, equipped with a pulsed amperometric detector, a CarboPac PA1 (4 mm x 250 mm) analytical column and a CarboPac PA1 (4 mm x 50 mm) guard column was used to quantify sugars. The separation of neutral sugars was performed at 40ºC with a flow rate of 1 mL/min using an isocratic gradient of 20 mM NaOH for 20 min followed by a wash with 200 mM NaOH for 10 min. After every run, the column was equilibrated in 20 mM NaOH for 10 min. Separation of acidic sugars was performed using 150 mM NaOAc and 100 mM NaOH for 10 min at a flow rate of 1 mL/min at 40°C. Standard curves of neutral sugars (D-Fuc, L-Rha, L-Ara, D-Gal, D-Glc, D-Xyl, and D-Man) or acidic sugars (D-GalA and D-GlcA) were used for quantification.

Determination of Monosaccharide Composition of Pollen Tubes Using Gas

Chromatography-Flame Ionization Detection (GC-FID)

Samples were prepared as described in [START_REF] Dardelle | Pollen tube cell walls of wild and domesticated tomatoes contain arabinosylated and fucosylated xyloglucan[END_REF]. Approximately 0.5 mg of sample was hydrolyzed with 2 M TFA for 2 h at 110°C. Monosaccharides were then derivatized with 1 M methanol-HCl at 80°C overnight followed by a mixture of hexamethyldisiloxan:trimethyldisiloxan:pyridine (3:1:9) at 110°C for 20 min. After drying, derivatives were dissolved in 1 mL of cyclohexane and injected into the 3800 GC system equipped with a CP-Sil5-CB column. A temperature gradient from 120 to 160°C at 10°C min - 1 , 160 to 220°C at 1.5°C min -1 and 220 to 280°C at 20°C min -1 was used. Quantification was based on the internal standard and response factors previously determined for each monosaccharide.

Determination of PME Activity

Total protein extraction and PME activity assays were performed as described in [START_REF] Saez-Aguayo | PECTIN METHYLESTERASE INHIBITOR6 promotes Arabidopsis mucilage release by limiting methylesterification of homogalacturonan in seed coat epidermal cells[END_REF]. Measurements of stained areas to determine PME activity were obtained using the ImageJ software [START_REF] Abramoff | Image processing with ImageJ[END_REF] Analysis of Stem Xylan Using PACE AIR preparations and PACE were performed as described by [START_REF] Mortimer | Absence of branches from xylan in Arabidopsis gux mutants reveals potential for simplification of lignocellulosic biomass[END_REF]. One mg of AIR from basal stems were incubated overnight in 0.1 M ammonium acetate buffer (pH5.5) with an excess of Neocallimastix patriciarum Xyn11A xylanase at 21ºC. Samples were derivatized with 8-aminonapthalene-1,3,6-trisulphonic acid (ANTS; Invitrogen). After drying in vacuo, the samples were resuspended in 3 M urea (100 μL), of which 5 μL was loaded onto the PACE gels. Samples were electrophoresed for 30 min at 200 V and then for 100 min at 1,000 V. Gels were visualized using a Genebox (Syngene) equipped with a transilluminator with long-wave tubes emitting at 365 nm and a short-pass (500-600 nm) filter. The quantity of each of the oligosaccharides released by Xyn11A [Xyl, (Xyl)2, GlcA-(Xyl)4/ Me-GlcA(Xyl)4] as well as the GlcA/Xyl ratio could be calculated by using the analytical software Genetools To analyze monosaccharide composition, a water-soluble extraction was used to isolate the SM fraction. The adherent mucilage cannot be detached from the seed and form the seed+ soluble mucilage fraction (seed + MA). Sugar content was obtained using HPAEC-PAD from seed + AM and from SM. Values are in mg/g of dry seeds and are the means of 3 biological replicates. Standard errors are shown in parentheses for 2 technical replicates each. (*) Significant statistical differences using the Wilcoxon test (p <0.05).

Table 1 .

 1 Sugar Composition of Seeds Plus Adherent Mucilage (seed + AM) and Extracted Soluble Mucilage (SM) from WT Col-0 and uuat1-2 Plants.

	Structure	Sugar	WT Col-0	uuat1-2
	Seed +AM	GalA 20.67 (0.21)* 19.68 (0.42)*
		Rha	21.77 (0.77)* 20.02 (0.45)*
		Fuc	1.74 (0.05)	1.72 (0.03)
		Ara	41.21 (1.59)	39.62 (1.06)
		Xyl	11.37 (0.56)	11.45 (0.47)
		Man	3.44 (0.12)	3.69 (0.16)
		Gal	30.23 (0.40)	29.06 (1.13)
		Glc	7.43 (0.26)	8.18 (0.29)
		GlcA	2.44 (0.08)	2.55 (0.07)
	Total Seed + AM		140.30 (2.80) 135.96 (1.92)
	SM	GalA	5.66 (0.21)*	4,56 (0.22)*
		Rha	8,65 (0.49)*	6.76 (0.43) *
		Ara	0.08 (0.02)	0.09 (0.01)
		Xyl	0.43 (0.02)*	0.34 (0.02)*
		Gal	0.31 (0.04)	0.26 (0.02)
	Total SM		15.15 (0.64)	11.99 (0.95)

ACKNOWLEDGEMENTS

This work was supported by FONDECYT 11130498 (to F.C.R), FONDECYT 3140415 (to S.S.A), FONDECYT 1151335, Fondo de Areas Prioritarias-Centro de Regulacion del Genoma-15090007, ECOS-CONICYT C14B02 and PFB-16 (to A.O) and a CONICYT fellowship to H.T. Work conducted by the Joint BioEnergy Institute was supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research, through contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the U. S. Department of Energy. J.L.H. is supported by an Australian Research Council Future Fellowship [FT130101165]. Work conducted by Glyco-MEV (J-C.M. and A.L.) was in part supported by the VASI research network from the Upper Normandy region and the French ministry of research. We would like to thank Miriam Barros for her advice and expertise in confocal microscopy. We are also grateful to Hernan Salinas and Alvaro Miquel for technical assistance with HPAEC analysis, to Flavien Dardelle and François Le Mauff for technical assistance with GC analyses of pollen tube cell walls.

 (Syngene). Results presented correspond to 4 biological replicates. The enzyme was a kind gift of Harry Gilbert (University of Newcastle, UK).

Accession Numbers

Nucleotide sequences for Arabidopsis UUAT1 have been deposited in GenBank [START_REF] Benson | GenBank[END_REF] under accession numbers KT923621 (At5g04160, coding sequence) and KT923622 (At5g04160, promoter). T-DNA insertion lines in the At5g04160 locus were obtained from the Arabidopsis Biological Resource Center: uuat1-1 (SALK_124146C), uuat1-2, (SALK_105023C) and uuat1-3 (SALK_048507).

Supplemental

Supplemental


Yuhao Wu
email: wuyuhao@ist.osaka-u.ac.jp

Yuki Manabe
email: y-manabe@cs.kumamoto-u.ac.jp

Daniel M German

Katsuro Inoue
email: inoue@ist.osaka-u.ac.jp

How Are Developers Treating License Inconsistency Issues? A Case Study on License Inconsistency Evolution in FOSS Projects

Keywords: software license, code clone, license inconsistency

A license inconsistency is the presence of two or more source files that evolved from the same original file containing different licenses.

In our previous study, we have shown that license inconsistencies do exist in open source projects and may lead to potential license violation problems. In this study, we try to find out whether the issues of license inconsistencies are properly solved by analyzing two versions of a FOSS distribution-Debian-and investigate the evolution patterns of license inconsistencies. Findings are: license inconsistencies occur mostly because the original copyright owner updated the license while the reusers were still using the old version of the source files with the old license; most license inconsistencies would disappear when the reusers synchronize their project from the upstream, while some would exist permanently if reusers decide not to synchronize anymore. Legally suspicious cases have not been found yet in those Debian distributions.

Introduction

Free and open source software (FOSS) is software that can be freely used, changed, and shared (in modified or unmodified form) by anyone. FOSS should always be distributed under certain open source software licenses, otherwise they cannot be reused by others. The license of a source file usually resides in its header comment.

Many studies in software engineering have been done on software license. Some approaches for software license identification have been proposed [START_REF] German | A sentence-matching method for automatic license identification of source code files[END_REF], [START_REF] Gobeille | The FOSSology project[END_REF], [START_REF] Tuunanen | Automated software license analysis[END_REF]. Vendome et al. investigated the reasons on when and why developers adopt and change licenses by conducting a survey with the relevant developers [START_REF] Vendome | When and why developers adopt and change software licenses[END_REF].

We have developed a method to detect license inconsistencies in large-scale FOSS projects [START_REF] Wu | Analysis of license inconsistency in large collections of open source projects[END_REF]. A license inconsistency is defined as two or more source files that evolved from the same original file containing different licenses. For example, file A and file B have the same program code, but the header of A includes GPL-2.0 and B includes Apache-2.04 . We have applied our detection method to Debian 7.5 5 , one of the Linux distributions, and discovered different reasons that caused license inconsistencies and determined whether they are potentially illegal or not.

Although this work has provided us clues that some license inconsistencies indicate potential legal issues, this one-time analysis is not enough to understand how developers are dealing with the issues: whether developers are putting their efforts on eliminating license inconsistencies or they simply ignore the issues. The investigation of the evolution of license inconsistencies can help us understand whether the legal issues of license inconsistency discovered in the previous work are handled by developers. Therefore, we conducted this research and try to answer the following questions:

-RQ1. What are the evolution patterns of license inconsistencies and the underlying reasons? Analyzing the evolution patterns of license inconsistencies might gives us insight on the reasons that caused them to appear and disappear. The findings are: license inconsistencies appear, persist and disappear due to different reasons. They appear mostly because the original author updates the license while the reusers still use the old version of the files; they persist mostly because the downstream project is not synchronized with the upstream project yet; and they disappear when the downstream project is synchronized with the upstream project. -RQ2. Is the issue of license inconsistencies properly handled by developers?

The findings are: license inconsistencies are mainly caused by distribution latency, and they will disappear when the developers synchronize their projects from the upstream projects. They persist because the reusers are still using an old version of the files and do not perform the synchronization. We do not consider this as a legal issue.

In order to address these questions, we apply our license inconsistency detection method to Debian 8.2 (which was the newest version when we started this study) in addition to Debian 7.5, and investigate the evolution of license inconsistencies between theses two versions.

The rest of this paper is organized as follows: Section 2 introduces the methodology we employ to address our research questions. Section 3 describes the result we got from our analysis. A discussion of this result is given in Section 4. Section 5 introduces the related work. Finally, we conclude our paper in Section 6.

Methodology

In this section we describe how we design our study. A license inconsistency group is a group of multiple files that have same code contents but with different licenses. As introduced in our previous paper [START_REF] Wu | Analysis of license inconsistency in large collections of open source projects[END_REF], we use the following steps to reveal license inconsistency groups:

1. Create groups of file clones: For all the source files in the target projects, we apply CCFinder [START_REF] Kamiya | CCFinder: A multilinguistic token-based code clone detection system for large scale source code[END_REF] to extract the normalized token sequences of each file. The normalized token sequences is a token sequence of the source code, removing comments and whitespaces and changing all user-defined identifiers to a special token. Note that, although CCFinder itself is a clone detection tool, we do not utilize the full functionality of CCFinder and we only use it to generate the normalized token sequences of source files. By computing and categorizing the hash value of these token sequences, we then create a group for files that have the same normalized token sequences. We call them license inconsistency groups, or group for short in the rest of this paper. Each group contains at least two different files; i.e., a unique file is not contained in any group. 2. Identify licenses for files in each group: For each group of file clones, Ninka [START_REF] German | A sentence-matching method for automatic license identification of source code files[END_REF] is used to identify the license(s) of each file. Ninka identifies the license sentences in the comment parts of each file, and compares those with its license database. It can identify more than 110 different OSS licenses and their different versions with 93% accuracy. Meanwhile, it will report "NONE" if the file has no license and "UNKNOWN" if the license sentence of the file does not match the database. The result is a list of licenses for each file group. 3. Report groups that contain a license inconsistency and calculate the inconsistency metrics: We compare the license list of each file group. File groups are reported to have license inconsistencies unless all the licenses on the list are exactly the same. The result is a list of file groups that contain one or more types of license inconsistencies.

We apply these steps to Debian 7.5 and 8.2 respectively and obtain the file groups that contain license inconsistencies.

Compare the difference of groups

All the files in one group have the same token sequences, from which we calculate the hash value as the id for that group. We then compare the id of the groups in each version and get the set of groups that exist: a) only in Debian 7.5; b) only in Debian 8.2; c) in both versions.

The result of this step would be three sets of file groups. ii Groups that exist in either version only.

Investigate the groups manually

The groups that only exist in Debian 7.5 imply that they are eliminated from the new version; those that only exist in Debian 8.2 imply that new license inconsistency cases appears in this new version; those that exist in both versions indicate that they remain during these two versions. We manually examine the different groups and present the results in the next section.

Results

The number of packages and files in Debian 7.5 and 8.2 are shown in Table 1. We only consider .c, .cpp and .java files which are the supported types of our detection method. The detection result of license inconsistency groups in Debian 7.5 and 8.2 is shown in Table 2. In this table, intersection means that both two versions of Debian contain that group of license inconsistency; relative complement means only that version of Debian contain that group of license inconsistency. Thus there are 4062 groups of license inconsistencies detected in both versions; 2701 groups only in Debian 7.5; and 2947 groups only in Debian 8.2. By examining the groups that are only in Debian 7.5 we can find out how and why license inconsistencies disappeared in Debian 8.2; while examining the groups that are only in Debian 8.2 we can understand how and why license inconsistencies appeared. The intersection part indicates that these groups of license inconsistencies persisted in Debian 8.2.

Why do license inconsistencies appear?

There are several reasons that license inconsistencies appear. The key point is that there are multiple copies of the same source file with different licenses in make Debian License upgraded here Reuse Fig. 1: A case of license inconsistency evolution which involves project make, remake and kbuild. the same distribution. We describe the general reasons that license inconsistency appears, thus the examples given in this section are not limited to those that appear in Debian 8.2. We categorize them into three types according to the reason that caused the copy of files.

-Internal copy-and-paste of source files but their licenses are different. If copies of the same file exist in one project (we call these internal copies), they should also exist in the final distribution (e.g. Debian 7.5). Thus a case of license inconsistency will be reported by our method if they contain different licenses.

For example, in a project named FreeMedForms, some source files in a plugins directory are under BSD3 license. These files are copied to other directories with their licenses changed to GPL-3.0+.

This type of license inconsistency could exist for just a short time, if the difference of license was due to mistakes and was later on fixed by developers. On the other hand, if developers decided to distribute these source files under different licenses, it would exist permanently.

-Different versions of the same project are included in the same distribution. Developers decide to never depend on the upstream project. project which causes license inconsistencies are included in the final distribution, those license inconsistencies will be reported by our method.

For example, there is a project named groovy which contains files that had no license in version 1.7.2, and were then added a Apache-2.0 license in version 1.8.6. Both of these two versions of this project are included in Debian 7.5, thus this license inconsistency is reported.

-Upstream and downstream projects both exist in the same distribution. In this research, if project B reuses source files from project A by copy-and-paste, we call project B the downstream project and project A the upstream project. While the previous two reasons are about the same project, this reason involves multiple projects. Files from the upstream project are reused in the downstream project, and the license of these files were changed either by the original author or the reuser. If both of these projects are included in the same distribution, the license inconsistency will be reported by our method.

An example is shown in Figure 1. As we can see from this graph, several source files in remake and kbuild project were originally from the make project, where license upgrade occurred in year 2010. Debian 7.5 includes older versions of make and remake, where the license was still GPL-2.0, while the newer version of kbuild contains the GPL-3.0 license. Thus license inconsistency is reported for this case.

Why do license inconsistencies persist?

License inconsistencies usually exist in several continuous versions of a distribution. Some of them disappear soon, some exist for a long time, while some even persist forever. From the result in this research, we can summarize them into two reasons.

-Source files are not yet synchronized, but license inconsistencies will eventually disappear when they are. This type of license inconsistencies occurred because the downstream project has not yet been synchronized with the upstream project where the license of source files were changed. However, since the developers of the downstream projects are still synchronizing the project from the upstream regularly, this type of license inconsistencies will be eliminated eventually.

For example, in the case of the project JSON-lib and jenkins-json described earlier, although license inconsistencies appeared in Debian 7.5 (where jenkins-json still uses source files under Apache-2.0/MIT dual license), they disappeared in Debian 8.2, where developers of jenkins-json project synchronized from the upstream project and the license all become Apache-2.0 only. Though this case of license inconsistency disappeared in Debian 8.2, we consider it as a typical example to explain why license inconsistencies would exist for only a period of time and disappear when the source files are synchronized.

-Downstream project no longer synchronizes from upstream, and license inconsistencies will likely exist permanently. In this case, developers of downstream project chose to no longer synchronize from the upstream project, thus the license inconsistencies are likely to exist forever, unless the synchronization resumes.

As shown in Figure 2, among the results we found a project named Mockito which copy-and-owned several source files from a project named EasyMock. The license of these files in the upstream project were changed from MIT to Apache-2.0 in year 2009, however the Mockito project still uses the original MIT license. Besides, Mockito project made some changes to the source code of these files by their own, and never again synchronized from EasyMock. After checking the history of these files in Mockito project, we found that one of the commit in year 2007 contains the following commit message: "umbilical cord between mockito package and easymock package is cut! ", which implies that they will never synchronize from the upstream project. Thus this case of license inconsistency is likely to exist permanently, unless Mockito project decides to synchronize from EasyMock again.

Why do license inconsistencies disappear?

We observed several cases where license inconsistencies disappeared from Debian 8.2. The reasons are summarize as follows.

-Downstream project synchronized from upstream project. When the downstream project synchronized from the upstream project, the license of the source files becomes the same, thus the license inconsistencies disappear.

Again, from Figure 1 we can see that Debian 8.2 updated all these three projects to a newer version where all of their licenses are upgraded to GPL-3.0, thus this license inconsistency disappears.

The case of project JSON-lib and jenkins-json discussed earlier also applies here.

-The source code that contained the license inconsistency was removed or changed-thus no longer identical. In this research since we only inspect identical files, only files that contain the same token sequences are considered that they are from the same origin. If the source code of a file changed dramatically which made their token sequences different from the corresponding files, or if the relevant source file was removed in the new version, then the license inconsistencies will no longer be reported by our method.

For example, there is a file in project icu which is under IBM copyrights in both versions of Debian. This file was reused in project openjdk-7 but with a GPL-2.0 license in Debian 7.5. Thus this case of license inconsistency was reported in Debian 7.5. However, the source code of the file in openjdk-7 was changed in Debian 8.2 while the license remained the same. Our method no longer not consider them as file clones since they have different token sequences, thus the license inconsistency disappears in the newer version of Debian.

Discussion

Revisiting the research questions

The answer to our research questions are as follows:

RQ1: What are the evolution patterns of license inconsistencies and the underlying reasons? -Appear. i) Internal copy-and-paste of source files in a project but their licenses are different; ii) Different versions of the same project are included in the same distribution; iii) Upstream and downstream projects both exist in the same distribution. -Persist. i) Source files are not yet synchronized, but license inconsistencies will eventually disappear when they are; ii) Downstream project no longer synchronize from upstream, and license inconsistencies are likely to persist permanently. -Disappear. i) Downstream project synchronized from upstream project;

ii) Source code which contained license inconsistency was removed or changed and we consider them as different files.

RQ2: Is the issue of license inconsistency properly handled by developers? The evolution of license inconsistencies in Debian shows that, they are mainly caused by distribution latency and will be eliminated when the downstream projects get synchronized. We found license inconsistencies appear mostly because the original author modified the license while the reusers were still using the old version of the file; they disappear because the files are synchronized with the upstream projects; and they persist if the downstream projects were not synchronized.

Effectiveness of this approach

This approach is effective to analyze how developers are addressing the issues of license inconsistency in a certain software ecosystem. As shown in the results, we could use this method to reveal how license inconsistency groups evolved over time. By analyzing different groups we could have a basic idea on how developers are treating license inconsistency issues. For example, by analyzing the license inconsistency groups that only exists in the old version, we could learn why license inconsistencies disappeared in the newer version and what efforts did the developers put to eliminate them.

We could apply this same approach to other software systems to study how different communities are dealing with license inconsistency issues.

Threats to validity

Internal validity. We use the same methodology as our previous study to detect license inconsistency cases, which relies on the token sequence generation from CCFinder and the license identification from Ninka. For file clone detection, we use normalized token sequences as the metric to decide clones. If source files are modified a lot (e.g. add/remove several statements), they might not be recognized as clones. We could use approaches that detect similar source files to mitigate this problem. Regarding license identification, Ninka is state-of-the-art license identification tool which has an accuracy of 93% [START_REF] German | A sentence-matching method for automatic license identification of source code files[END_REF]. As shown in Section 3, our manual analysis also proves its high accuracy and precision.

External validity. This study focuses on the license inconsistency issues in Debian. The results and analysis may not be generated to other software systems. And we plan to study other software systems in the future.

Related Work

Many studies in software engineering have been done on software license. Some approaches for software license identification have been proposed [START_REF] German | A sentence-matching method for automatic license identification of source code files[END_REF][START_REF] Gobeille | The FOSSology project[END_REF][START_REF] Tuunanen | Automated software license analysis[END_REF]. Using these approaches, some researches analyzed software licenses in open source projects and revealed some license issues. Di Penta et al. provided an automatic method to track changes occurring in the licensing terms of a system and did an exploratory study on license evolution in six open source systems and explained the impact of such evolution on the projects [START_REF] Di Penta | An exploratory study of the evolution of software licensing[END_REF]. German et al. proposed a method to understand licensing compatibility issues in software packages [START_REF] German | Understanding and auditing the licensing of open source software distributions[END_REF]. They mainly focused on the compatibility between license declared in packages and those in source files. In another research, they analyzed license inconsistencies of code siblings (a code clone that evolves in a different system than the code from which it originates) between Linux, FreeBSD and OpenBSD, but they did not explain the reasons underlying these inconsistencies [START_REF] German | Code siblings: Technical and legal implications of copying code between applications[END_REF]. Alspaugh et al. proposed an approach for calculating conflicts between licenses in terms of their conditions [START_REF] Alspaugh | Intellectual property rights requirements for heterogeneously-licensed systems[END_REF]. Vendome et al. performed a large empirical study of Java applications and found that changing license is a common event and a lack of traceability between when and why the license of a system changes [START_REF] Vendome | License usage and changes: A large-scale study of java projects on github[END_REF]. In their following research [START_REF] Vendome | When and why developers adopt and change software licenses[END_REF], they investigated the reasons on when and why developers adopt and change licenses during evolution of FOSS Java projects on GitHub by conducting a survey with the relevant developers. They concluded that developers consider licensing as an important task in software development.

In our previous research [START_REF] Wu | Analysis of license inconsistency in large collections of open source projects[END_REF], we proposed a method to detect license inconsistencies in large-scale FOSS projects. We then applied this method to Debian 7.5 and examined the results manually and discovered various reasons that caused these license inconsistencies, among which some were legally suspicious and deserved further investigation. As far as we know, no research has been done to investigate the evolution of license inconsistencies.

Conclusions

In this research we have applied our license inconsistency detection method to both Debian 7.5 and Debian 8.2. By comparing the results of these two versions of Debian, we identified three evolution patterns of license inconsistencies. With a manual analysis of the license inconsistency cases we discovered various reasons that caused the evolution. Although license inconsistencies are detected in both versions of Debian, from our manual analysis we concluded that these reported license inconsistencies are caused because the upstream project updated the license while the downstream project has not been synchronized yet. These findings suggest that in our target ecosystem, Debian, license inconsistencies are caused by distribution latency and they will disappear when the downstream projects get synchronized.

For future work, we would apply this approach to other software ecosystems and see whether there are different patterns of license inconsistency evolution and whether the license inconsistency issues are properly handled.

2. 1

 1 Obtain license inconsistency groups for Debian 7.5 and Debian 8.2

Fig. 2 :

 2 Fig. 2: A case of license inconsistency evolution which involves project EasyMock and Mockito.

Table 1 :

 1 Number of packages and files in Debian 7.5 and 8.2.

	Number of	Debian 7.5 Debian 8.2
	Source Packages	17,160	20,577
	Total files	6,136,637 13,124,700
	.c files	472,861	767,006
	.cpp files	224,267	335,269
	.java files	365,213	477,154

Table 2 :

 2 License inconsistency groups between two versions on Debian.

	Number of groups Debian 7.5 Debian 8.2
	Intersection i	4062	4062
	Relative complement ii	2701	2947
	Total	6763	7009

i Groups that exist in both versions.

  Similar to the previous reason, if different versions of the same

		MIT			Apache-2.0
			License changed here
				2009.12.17	2013.7.11
	EasyMock	…	2.4	2.5.1	3.2	…
		Reuse
			2007.11.17	2011.12.17	2012.6.4
	Mockito	…	…	1.9.0	1.9.5	…
				2014.04.26	2015.09.05
				Debian 7.5	Debian 8.2

Note that pairs of no license and any license, and different license versions are reported as license inconsistency.

[START_REF] German | Code siblings: Technical and legal implications of copying code between applications[END_REF] https://www.debian.org/

Acknowledgments

This work is supported by Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (S) "Collecting, Analyzing, and Evaluating Software Assets for Effective Reuse"(No.25220003) and Osaka University Program for Promoting International Joint Research, "Software License Evolution Analysis".