at SciVerse ScienceDirect

Plant Physiology and Biochemistry 60 (2012) 223e232

Contents lists available

Plant Physiology and Biochemistry

journal homepage: www.elsevier .com/locate/plaphy

Research article

Promoter activities of genes encoding b-galactosidases from Arabidopsis a1subfamily

Lucía Albornos, Ignacio Martín, Pablo Pérez, Ruth Marcos, Berta Dopico, Emilia Labrador*

Dpto. de Fisiología Vegetal, Centro Hispano-Luso de Investigaciones Agrarias (CIALE), Universidad de Salamanca, Plaza Doctores de la Reina s/n, Campus Miguel de Unamuno,37007 Salamanca, Spain

a r t i c l e i n f o

Article history:Received 13 July 2012Accepted 27 August 2012Available online 5 September 2012

Keywords:Arabidopsis thalianab-GalactosidasesCell wallDevelopmentGene promoters

Abbreviations: GH, glycosyl hydrolases; AtBGAL, ApAtBGAL, AtBGAL gene promoter.* Corresponding author. Tel.: þ34 923 294471; fax:

E-mail address: labrador@usal.es (E. Labrador).

0981-9428/$ e see front matter � 2012 Elsevier Mashttp://dx.doi.org/10.1016/j.plaphy.2012.08.012

a b s t r a c t

Promoter regions of each of the six AtBGAL gene of the subfamily a1 of Arabidopsis thaliana were used todrive the expression of the b-glucuronidase gene. The pattern of promoters (pAtBGAL) activity wasfollowed by histological staining during plant development. pAtBGAL1, pAtBGAL3 and pAtBGAL4 showeda similar activity pattern, being stronger in cells and organs in expansion, and the staining decreasingwhen cell expansion decreased with age. That indicates a consistent involvement of the encoded b-galactosidases in cells undergoing cell wall extension or remodelling in cotyledons, leaves and flowerbuds. These promoters were also active in the calyptra cells and in pollen grains. pAtBGAL2 activityshowed a clear relationship with hypocotyl elongation in both light and dark conditions and, likepAtBGAL1, pAtBGAL3 and pAtBGAL4, it was detected during the expansion of cotyledons, rosette leavesand cauline leaves. Its activity was also intense in the early stages of flower and fruit development.pAtBGAL5 was the only one among those from the subfamily a1 that was active in the trichomes thatappear throughout the plant, indicating a high specificity of the AtBGAL5 protein and its involvement inthe cell wall changes that accompany the formation of the trichome. The activity of pAtBGAL5 was alsohigh in radicles and roots, except in the meristematic area of these organs, and during seed formation.Finally, the activity of pAtBGAL12 was mainly detected in meristematic zones of the plant: the leafprimordium, emerging secondary roots and developing seeds, which indicates an involvement in thedifferentiation process.

� 2012 Elsevier Masson SAS. All rights reserved.

1. Introduction

b-Galactosidases are enzymes that hydrolyze the glycosidic bondbetween a non-reducing galactose residue and another sugar oralcohol. They can act on numerous substrates such as lactose, glyco-lipids, proteoglycans, oligosaccharides or polysaccharides. Glycosylhydrolases (GH) have been classified as families on the basis ofstructural similarity [1]. b-galactosidases fall into four glycosidehydrolase (GH) families, GH-1, GH-2, GH-35,GH-42,which are part ofsuperfamilyA (or ClanA) [2]. GH-1,GH-2andGH-42aremostly foundin microorganisms, whereas GH-35 enzymes are found in bothprokaryotes and eukaryotes. The plant b-galactosidases that havebeen described belong to GH family 35. In higher plants, the GH-35family (EC 3.2.1.23) has been implicated in carbohydrate reservemobilization and in cell wall biogenesis and modification [3,4].

rabidopsis b-galactosidases;

þ34 923 294682.

son SAS. All rights reserved.

The substrate specificities of plant b-galactosidases can vary widely,indicating functional diversity in their activity.

b-Galactosidases have been described in several plant species asmultigene families, suggesting that GH-35 gene multiplicity isubiquitous in plants. Thus, in tomato a multigene family with sevenmembers has been described, some of which are involved in fruitdevelopment and ripening [5]. In chickpea, a family of at least fourb-galactosidases is expressed differentially during seedling andplant growth [6]; one of then, the bIII-Gal protein, is associatedwith cell wall loosening during epicotyl elongation and acts on theb-(1,4) galactan side chain of rhamnogalacturonan I [7].

In the Arabidopsis genome, 17 GH-35 BGAL genes have beenidentified and designated from AtBGAL1 to AtBGAL17 [8]. The 17Arabidopsis b-galactosidases (AtBGAL) fall into two groups and sevensubfamilies [8]. The majority of these hydrolases may be glycopro-teins and, with the exception of AtBGAL14, have anN-terminal signalpeptide, and many of them appear targeted to the cell exterior, sug-gesting their potential involvement in cell wall modification.

The classification in two groups is mostly based on molecularmasses. Group 1 members are 721e731 amino acids in length.

L. Albornos et al. / Plant Physiology and Biochemistry 60 (2012) 223e232224

Group 2 BGALs are far longer (832-888 amino acids), mainly due tothe existence of a C-terminal domain that is structurally related toa sea urchin egg lectin (SUEL-lectin), which was first identified asa D-galactose-binding lectin [9], although it was later shown that itpreferentially binds to L-rhamnose [10]. With the exception ofAtBGAL14 and AtBGAL17, Arabidopsis BGALs fall into either Group 1(AtBGAL2, 4, 5, 6, 10 and 12) or Group 2 (AtBGAL1, 3, 7, 8, 9, 11, 13, 16and 17).

Most of the subfamilies contain one to three members, exceptfor the largest subfamily, a1, which consists of six proteins, AtB-GAL1, AtBGAL2, AtBGAL3, AtBGAL4, AtBGAL5 and AtBGAL12,exhibiting 60e81% sequence identity [11]. It has been predictedthat all have signal peptides and basic pIs (7,2-8,6) consistent witha cellular destination in the cell wall [8]. This localization has beenconfirmed for AtBGAL2 and AtBGAL5 [4] and for AtBGAL1 andAtBGAL12 [11].

The differential tissue-specific expression patterns and stressresponsiveness suggest that AtBGALs are involved in diverse bio-logical processes. Several studies to determine the expression levelsof genes encoding b-galactosidases in Arabidopsis using RT-PCRand microarray techniques have been carried out in differentorgans and during several developmental stages. However, theresults obtained by both methods are sometimes contradictory.

Specifically regarding subfamily a1, Ahn et al. [8], using relativeRT-PCR analysis, reported that AtBGAL1, AtBGAL2 and AtBGAL3wereexpressed constitutively across the different organs and develop-mental stages, with the highest transcript levels in leaves, roots andflowers, while AtBGAL4 transcripts accumulate primarily in roots.AtBGAL4 and AtBGAL5 were not expressed in green seedlings. Inthese experiments, no expression of AtBGAL12 was detected.

By semi-quantitative RT-PCR using a different set of primersthan in the experiments of Ahn et al. [8], Gantulga et al. [11] re-ported that AtBGAL1 has an essentially constitutive expression,while the other five genes from subfamily a1 are expressed in most,but not all, tissues. The AtBGAL1, AtBGAL2, AtBGAL5 and AtBGAL12genes were the only ones expressed in seedlings; in the petiole,only AtBGAL1 and AtBGAL3, and in siliques, only AtBGAL1, AtBGAL3and AtBGAL4.

Perez-Almeida [12] found AtBGAL2 expression with moderatelevels of transcripts in all organs. In the case of AtBGAL5, they foundhigher levels of expression in roots than Gantulga et al. [11]. WhileGantulga [11] and Perez-Almeida [12] agree that AtBGAL5 is notdetectable in mature roots, only Perez-Almeida [12] found AtBGAL5expression in root elongation and the root hair zones of juvenileplants.

In the publicly-available microarray data, all six genes appear tobe expressed to some extent in all tissues examined although thereare notable differences in expression levels in different organs anddevelopmental stages. In addition, the use of microarrays hasrevealed that some genes are induced by biotic or abiotic stress,such as AtBGAL1, which is induced by hypoxia, salt stress or path-ogen attack.

Another method for determining the levels of expression ofa gene is to analyse the activity of its promoter. Thus, the study ofpromoters of genes encoding different b-galactosidases providesvaluable information about the places at which their genes aretranscribed, which can help to elucidate their function. The aim ofthe present work was to study the activity of the gene promotersAtBGAL1 (At3g13750), AtBGAL2 (At3g52840), AtBGAL3 (At4g36360),AtBGAL4 (At5g56870), AtBGAL5 (At1g45130) and AtBGAL12(At4g26140) from the Arabidopsis a1 subfamily that encodeb-galactosidases by construction of transgenic plants of Arabidopsisthaliana producing the enzyme b-glucuronidase (GUS) driven byAtBGAL gene promoters (pAtBGAL) and later histological localizationof GUS activity to determine the pattern of gene expression during

plant development. We decided to study the members of thesubfamily a1 of b-galactosidase since they have been described asexo-galactanases able to act on the pectic polysaccharides of the cellwall, and might therefore play important roles in cell wall remod-elling during plant growth and development [11], although theirfunctions have not yet been determined.

2. Results and discussion

2.1. Activity of the b-galactosidase promoters of the a1 subfamily

Arabidopsis transgenic plants in which the gene promoterdrives the expression of reporter proteins such as GUS wereproduced and further histological localization of GUS activityduring plant development was carried out to determine the activityof these promoters. The study was carried out in plants growingunder both dark and light conditions. Our results indicate that threeof the AtBGAL promoters studied, pAtBGAL1, pAtBGAL3 and pAtB-GAL4, showed a similar pattern of activity in the different planttissues and organs analysed; accordingly, these results will bediscussed together. In separate sections we shall analyse theactivity of the pAtBGAL2, pAtBGAL5 and pAtBGAL12 promoters.Wild type plants used as controls never showed GUS activity, andhence the images obtained are not included here.

2.1.1. Activity of pAtBGAL1, pAtBGAL3 and pAtBGAL4The activity of pBGAL1, pBGAL3 and pBGAL4 indicates a consis-

tent involvement of the encoded b-galactosidases in cells under-going wall extension or remodelling in cotyledons, leaves andflowers. These promoters exhibited the same pattern of activity,and hence for discussion as an example we shall present only theresults regarding pAtBGAL1.

pAtBGAL1 displayed strong activity in cotyledons during the firstdays after germination, progressively disappearing along thedevelopment of cotyledons (Fig. 1a,b,c). Cotyledons develop duringembryogenesis and their growth after germination is determinedby cellular expansion and not by division [13]. Thus, a degradationof cell wall pectin has been described during the expansion ofcotton cotyledons [14], probably mediated by an endopolyga-lacturonase [14] and a rhamnogalacturonan lyase [15]. Althoughthe action of b-galactosidases has not yet been described in coty-ledons, these proteins have been related to the elongation process[16e18] and could also participate in cell expansion. Similarly towhat happens in cotyledons, pAtBGAL1 shows strong activity in theleaf primordia in a development-related manner. Thus, pAtBGAL1activity was high in leaf primordia from very young seedlings(Fig. 1a), remained high in young leaves (Fig. 1b), and progressivelydecreased as the leaves matured until it finally disappearedcompletely (Fig.1c). Each newdeveloping rosette leaf, as well as thecauline leaves (Fig. 1d), followed the same pattern of GUS activity,regardless of the age of the plant.

pAtBGAL1 also displayed a strong activity in roots that persistedthroughout root development in an age-independent manner andwas located in radicles (Fig. 1e) and primary and secondary roots(Fig. 1f), except in the root elongation zone, suggesting a function inmaturation rather than in expansion in this organ. Furthermore,GUS activity was not observed in all root tissues, being limited tothe central cylinder, which stained an intense blue (Fig. 1f). It isworth noting the activity of these promoters in the root cap orcalyptra (Fig. 1f,g), although in secondary roots this activity onlystarted after they had emerged from the main root and it was notvisible when secondary roots started their development (Fig. 1h).The root cap protects the meristem by the production of mucilage,composed primarily of pectins, and by the detachment of its owncells to reduce mechanical friction against the ground, which

Fig. 1. GUS activity in Arabidopsis transgenic plants pAtBGAL1:GUS. Five-day-old seedlings (a); 10-day-old seedlings (b); 17-day-old plants (c); cauline leaf from 24-day-old plants(d); radicles from 3-day-old seedlings (e), roots from 12-day-old plants (f), apical part of the radicle, showing the calyptra in 5-day-old seedlings (g); emerging secondary root (h);hypocotyleradicle junction zone (i); flower buds (j); detail of flower anthers and carpels (k); developing siliques (l); detail of open silique (m). a: anthers, c: cotyledons; ca: calyptra,cl: cauline leaf, fb: flower buds, h: hypocotyl, l: leaves, o: ovaries; p: petals; pe: pedicel, r: radicle, re: receptacle, s: seed; si: silique, sp: septum, sr: secondary root, st: stigma. Theblue colour and the arrows indicate the zones with GUS activity driven by AtBGAL1 promoter. (For interpretation of the references to colour in this figure legend, the reader isreferred to the web version of this article.)

L. Albornos et al. / Plant Physiology and Biochemistry 60 (2012) 223e232 225

implies changes in the cell wall. Pectins, which are possiblesubstrates of b-galactosidases, are closely related to this process,and in this sense it has been reported that their modification in anArabidopsis mutant promotes the detachment of the lateral cells ofthe calyptra [19], which remain attached in the wild-type [8]. Theactivity of pAtBGAL in the central cylinder was also present in thehypocotyl basal zone, above the rootehypocotyl insertion zone(Fig. 1i). The GUS activity was located in the same places in plantsgrowing in both the light and the dark (data not shown).

Finally, pAtBGAL1, pAtBGAL3 and pAtBGAL4 also exhibit a similarpattern of activity in flowers. The transgenic plants showed highGUS activity in flower buds, as seen for pAtBGAL1 (Fig. 1j), that wasalmost lost when the different whorls matured, remainingrestricted to the pollen grains, the transmitting tissue of the styleand developing siliques (Fig.1k), and specific areas of the receptacle(Fig. 1l) and the septum and seed funicules (Fig. 1m). No activitywas found in seeds or mature siliques (data not shown). The b-galactosidases in pollen could act in the remodelling of the cell wallnecessary for the development of pollen grains, which may bemediated by pectinases and other enzymes of the GH families.Previously, Hrubá et al. [18] reported five AtBGAL abundantlyexpressed in pollen at different stages of Arabidopsis pollendevelopment, although only AtBGAL4 belongs to family a1. A highexpression of At5g20710 (AtBGAL7) and At1g31740 (AtBGAL15) hasbeen described in microspores and early pollen, and of At5g56870(AtBGAL4), At2g16730 (AtBGAL13) and At4g35010 (AtBGAL11) inmaturing pollen [18]. Hrubá et al. [18] suggest that early b-galac-tosidase and b-xylosidase genes may participate in cell wall loos-ening associated with young pollen expansion after microspore

mitosis and that the function of b-galactosidase in mature pollenmay play a role in cell expansion during pollen germination. Ourpresent results indicate the involvement of AtBGAL1, AtBGAL3 andAtBGAL4 in such processes.

Besides the general pattern of blue staining in flowers, there arecertain specific differences among the activities of the pAtBGAL1,pAtBGAL3 and pAtBGAL4 promoters, such as the staining observedat the base of the trichomes present in the floral stem in thepAtBGAL3:GUS transgenic plants (Fig. 2a) and the absence of GUSactivity in the bud pedicels driven by pAtBGAL4 (Fig. 2b), whichmight indicate specific functions of each of these galactosidases inthe cell wall metabolism in the differentiation of the floral whorls.In addition, pAtBGAL3 and pAtBGAL4 activities were null or verylow in the vascular tissues of petals and sepals (Fig. 2c,d), ascompared to pAtBGAL1 activity (Fig. 1k).

2.1.2. Promoter activity of AtBGAL2pAtBGAL2, as pAtBGAL1, pAtBGAL3 and pAtBGAL4 exhibited high

activity in young cotyledons and leaves, decreasing when matura-tion progressed in plants growing in both the light and the dark.Fig. 3a,b,c and d shows the decrease in staining of cotyledons andleaves in 3, 5, 10 and 17-day-old plants, respectively, grown underlight conditions. Activity was also driven by pAtBGAL2 in caulineleaves and at the base of the trichomes (Fig. 3e). The activity ofpAtBGAL2, unlike pAtBGAL1, pAtBGAL3 and pAtBGAL4, was veryweak in roots, (Fig. 3c,d,f), the stain being limited to small areasdistributed randomly in these organs (Fig. 3f).

In seedling hypocotyls, the activity of the pAtBGAL2 can berelated to the pattern of Arabidopsis hypocotyl elongation, which

Fig. 2. GUS activity in flowers of Arabidopsis pAtBGAL3:GUS (a,c) and pAtBGAL4:GUS transgenic plants (b,d). Flower buds (a,b); flowers (c,d). a: anthers, fb: flower buds, o: ovaries;p: petals; pe: pedicel, st: stigma. The blue colour and the arrows indicate the zones with GUS activity. (For interpretation of the references to colour in this figure legend, the readeris referred to the web version of this article.)

L. Albornos et al. / Plant Physiology and Biochemistry 60 (2012) 223e232226

changes depending on the growth conditions [20]. In growth underdark conditions, the hypocotyl elongation zone in young seedlingsis close to the apex of the hypocotyls while in seedlings growing inthe light, cell elongation is initially more pronounced at the base ofthe hypocotyls [20]. Thus, pAtBGAL2 activity was found just belowthe hook in young 3-day-old seedlings growing in the dark (Fig. 3g)while in seedlings growing in the light high pAtBGAL2 activity wasdetected in the basal part of the hypocotyls close to root junctionarea (Fig. 3h). In older seedlings (10 days old), pAtBGAL2 activitywas high in the hypocotyl apical zone in plants grown both in thedark (Fig. 3i) and in the light (Fig. 3j), GUS activity decreasingtowards the hypocotyl base. It is worth noting the elongated base ofcotyledons in 10-day-old plants growing in the dark (Fig. 3i) andthe high promoter activity found in this area. As developmentprogressed in leaves and the hypocotyls stem the blue colour wasclearly seen in the vascular cylinder (Fig. 3k,l).

The clear detection of pAtBGAL2 activity in Arabidopsis seed-lings, established by histological analysis of GUS activity (Fig. 3aej),resolves the discrepancy found in the literature about if there is orthere is no accumulation of transcripts in seedling. This reporteddiscrepancy could be due to the differences in the growing condi-tions and/or in the age of the seedlings [8,11].

The high activity of pAtBGAL2 in sepals, petals, connective tissueof the anthers, filaments of the stamens, receptacle of flowers and,above all, the carpels of flower buds and mature flowers(Fig. 3m,n,o,p) agrees with the high AtBGAL2 transcript levels foundin flowers by Gantulga et al. [11] and indicates an involvement ofpAtBGAL2 in flower development. Contrariwise, in silico dataavailable via the Arabidopsis eFP browser [21] at BAR (http://www.bar.utoronto.a/) show a very low accumulation in all floral whorlsand the absence of transcripts in carpels. AtBGAL2 could contributeto the cell wall metabolism that has been reported to occur alongthe development of sepals and petals, from the flower buds tomature and senescent flowers [22]. In the stamen filaments, anactive metabolism of the cell wall hemicellulosic component has

been described that involves an a-xylosidase [23] and XTH [24],which could act in coordination with AtBGAL2. The coordinatedaction of different cell wall enzymes, such as XTH and b-galacto-sidase, in the processes of cell wall expansion and remodelling hasbeen reported previously in different species [25,26].

In the process of fruit development in Arabidopsis, Louvet et al.[27] described a decrease in the contents of galactose and arabinosein the cell walls, which could be linked to a reduction in rhamno-galacturonan I side chains, which are the substrate of many plantb-galactosidases. Regarding the involvement of AtBGAL2 in thisprocess, our results show high pAtBGAL2 activity, detected in bothimmature (Fig. 3q) and mature siliques (Fig. 3r), which disappearedin senescent fruit (Fig. 3s). During the early process of fruitformation, in immature siliques staining was high, mostly in thereceptacle and in the apical part of the siliques (Fig. 3q), while inmature siliques the staining was uniform throughout the fruit(Fig. 3r). Within the silique (Fig. 3t) it can be observed that stainingwas located mainly in valves and immature seeds, low activitybeing detected in the septum. These results indicate that AtBGAL2could be involved in the development of the fruit. Until now, thedata concerning the presence of AtBGAL2 mRNAs in Arabidopsisfruits differ, depending on the authors. Thus, Iglesias et al. [26] andAhn et al. [8] reported high levels of AtBGAL2 transcripts in siliques,while other studies have indicated that their presence is unde-tectable [11]. Also, in silico analysis detected only AtBGAL2 tran-scripts in the very early stages of fruit formation.

The pattern of GUS activity suggests that AtBGAL2 may berelated to cell expansion and elongation. Thus, we detected a veryintense staining i) at the beginning of the expansion of cotyledons,rosette leaves and cauline leaves, which decreased when theexpansion became slower, ii) in the areas of maximum elongationhypocotyls in both light and darkness and iii) in the early stages offloral organ development, before they completed their growth, aswell as in fruit. The relationship of AtBGAL2 with growth is sup-ported by its capacity to degrade cell wall galactose polymers at

Fig. 3. GUS activity in Arabidopsis transgenic plants pAtBGAL2:GUS. Cotyledons and leaves of 3-day-old (a), 5-day-old (b), 10-day-old (c) and 17-day-old (d) plants grown underconditions of light; cauline leaf and detail of trichome (e); radicle from 5-day-old seedling (f); hypocotyls from 3-day-old plants grown in the dark (g) and under light conditions (h);hypocotyls from 10-day-old plants grown in the dark (i) and under light conditions (j); rossete leaves (k) and hypocotyl (l) from 24-day-old plant; flowers buds (m); flower (n);detail of the anthers and carpels (o); detail of the receptacle (p); immature silique (q); mature silique (r) senescent silique (s); open mature silique (t). a: anthers, c: cotyledons, cl:cauline leaf, fb: flower buds, h: hypocotyl, l: leaves, o: ovaries, p: petals, r: radicle, re: receptacle, s: seed, si: silique, sp: septum, t: trichome. The blue colour and the arrows indicatethe zones with GUS activity driven by AtBGAL2 promoter. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

L. Albornos et al. / Plant Physiology and Biochemistry 60 (2012) 223e232 227

acid pH [4,11], characteristic of the cell wall in elongating cells. Asindicated above, the involvement of b-galactosidase in elongationhas been proposed by different authors for several plant materials[16e18].

2.1.3. Promoter activity of AtBGAL5The promoter of the AtBGAL5 gene is the only one among those

from the subfamily a1 that was active in the trichomes that appearthroughout the plant during its development: in the leaves of therosette (Fig. 4a,b,c,d), in cauline leaves (Fig. 4e) and in the trichomesof the floral stem and the perianth (Fig. 4f,g), indicating a high

specificity of the enzyme encoded by AtBGAL5 and its involvementin the cell wall changes that accompany the formation of thetrichome, as evidenced by the high expression of AtBGAL5 indeveloping trichomes in the proximal part of the leaves, ascompared to the null expression in the more developed trichomesin the leaf distal part (Fig. 4d,e). This pattern indicates that thegalactosidase AtBGAL5may act during trichome formation and thatit becomes inactive at the end of the process. The deposition ofcellulose that occurs at the end of the development of trichomes isaccompanied by changes in the cell wall hemicellulosic and pecticfractions; changes that are critical for the process to succeed [28].

Fig. 4. GUS activity in Arabidopsis transgenic plants pAtBGAL5:GUS. Upper part of a 5-day-old seedling (a); leaves from 10-day-old including a magnification of a trichome (b) Andfrom 12-day-old plants (c,d); cauline leaf (e), flower buds (f), flower (g); radicle from 3-day-old seedlings grown in the dark (h) And from 5-day-old seedlings grown in light (i); rootand secondary roots from 12-day-old plants (j); apical part of the radicle showing the calyptra in 3-day-old seedling (k); emerging secondary root (l); hypocotyleradicle junctionzone from plant grown in the dark (m) and in light (n); base of flower (o); open silique showing seeds of different stages of development (p). c: cotyledons; ca: calyptra, cl: caulineleaf, fb: flower buds, h: hypocotyl, l: leaves, o: ovaries, r: radicle, re: receptacle, s: seed, sp: septum, sr: secondary root, t; trichomes. The blue colour and the arrows indicate thezones with GUS activity driven by AtBGAL5 promoter. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

L. Albornos et al. / Plant Physiology and Biochemistry 60 (2012) 223e232228

AtBGAL5 could participate in these changes during the formationand elongation of the trichomes. In trichomes with a high rate ofelongation, such as from cotton fibres, a reduction in the molecularmass of polymers containing galactose has been described in thefinal stages of elongation and thickening [29].

Our results confirm the data reported by other authors, such asJakoby et al. [30], who identified AtBGAL5 as one of the genes thatare expressed at high levels in trichomes, along with genes thatencode other enzymes related to cell wall metabolism, such aspolygalacturonases, pectinesterases and glycosyltransferases. Also,Ahn et al. [8] described high levels of AtBGAL5 transcript in rosetteleaves, which could be due to the activity in trichomes. However,when the accumulation of transcripts was analysed by non-histochemical methods, low levels of AtBGAL5mRNAwere reportedfor rosette and cauline leaves [11], probably because AtBGAL5 wasonly expressed in the trichomes and these only represent a smallpart of the leaf.

pAtBGAL5was also active in radicles and roots (Fig. 4h,i,j,k,l,m,n)from plants growing under both light and in the dark conditions,except in the apical part of the organ: the meristematic area. The

subapical elongation area had the most intense blue colour(Fig. 4h,i), which decreased towards the base. Unlike other genesfrom subfamily a1, such as AtBGAL1, AtBGAL3 and AtBGAL4, AtBGAL5was not expressed in the root cap (Fig. 4k). Microarray results (theArabidopsis eFP browser [21] at BAR http://www.bar.utoronto.a/)have indicated a slight accumulation of AtBGAL5 transcripts in theroots, located in the zone of maturation and also in the root meri-stematic zone, contrary to our results. Supporting our data thatshow the absence of pAtBGAL5 activity inmeristematic zones, it canbe seen clearly that the developing secondary roots were notstained (Fig. 4l). Thus, according to our findings, AtBGAL5 could beinvolved in root elongation, as previously reported for other b-galactosidases [25]. In this sense, a differential distribution ofpectins and arabinogalactan proteins has been observed amongroot zones with different elongation rates [31]. Also, McCartneyet al. [31] described a galactan-rich transition zone between areasof division and elongation, which is lost when the cells areelongated.

The intense staining observed in the radicles could explain thehigh levels of BGAL5 transcripts detected by Gantulga et al. [11] in

L. Albornos et al. / Plant Physiology and Biochemistry 60 (2012) 223e232 229

seedlings, but differs from the results published by Ahn et al. [8],who detected a low amount of transcript in etiolated but not ingreen seedlings. Our results indicated that GUS activity exhibitedthe same pattern in seedlings grown in both the light and in thedark. Also, the activity of pAtBGAL5 in hypocotyls displayeda similar pattern in all seedlings analysed, regardless of the growthconditions. It was only possible to detect GUS activity in the basalmost area of this organ in plants grown in both the dark (Fig. 4m)and in the light (Fig. 4n).

In buds and developing flowers, GUS activity was only detectedin the trichomes (Fig. 4f,g), as mentioned above; this stainingdecreased until it eventually disappeared, as occurred in otherorgans. After fertilization, pAtBGAL5 activity was located in devel-oping seeds and in the nectaries (Fig. 4o). A decreasing activitygradient from immature seeds to the mature ones was observed, inwhich activity disappeared completely (see the three seeds in theFig. 4p). This clear staining gradient agrees with results of micro-arrays available via the Arabidopsis eFP browser [21] at BAR (http://www.bar.utoronto.a/) indicating high levels of AtBGAL5 transcriptin the seed coat (the testa) in early seed development. During theearly stages of the development of the testa, the outer layers of thisseed coat undergo strong growth regarding both division and cellexpansion [32], requiring an active cell wall metabolism in which

Fig. 5. GUS activity in Arabidopsis transgenic plants pAtBGAL12:GUS. Cotyledons and leaveslight conditions; upper zone of 3-day-old (e) and 10-day-old (f) plants growing in the dark; chypocotyleroot junction zone from 5-day-old seedling grown in light (i); root and secondabuds (l); opened flower bud (m); detail of flower anther (n); detail of flower receptacle (o)connective, h: hypocotyl, l: leave, n: nectaries, r: radicles, re: receptacle, s: seeds; si: siliqueGUS activity driven by AtBGAL12 promoter. (For interpretation of the references to colour i

AtBGAL5 could be involved. This agrees with the absence of stainingin older seeds and could indicate the involvement of AtBGAL5 inseed growth.

2.1.4. Promoter activity of AtBGAL12The activity of pAtBGAL12 in the vegetative aerial part of the

plant was detected in the leaf primordium, under both light(Fig. 5a,b,c,d) and dark (Fig. 5e,f) growth conditions, but unlikewhat happens in AtBGAL1, AtBGAL2, AtBGAL3 and AtBGAL4expression was limited to the meristematic zone and pAtBGAL12activity was not found during the later stages of leaf development(Fig. 5d,g), indicating a relationship of the b-galactosidase AtB-GAL12 with the differentiation of leaves and not with their growth.Some authors have suggested that an increase in cell wall exten-sibility caused by expansins might initiate the formation of leafprimordia in the meristem [33]. In a similar way to the abovedescribed coordinated action among other cell wall-modifyingenzymes, AtBGAL12 could participate together with the expansinsin this process. The specific location of the pAtBGAL12 activity inleaves does not agree with the high levels of transcripts detected inrosette leaves by Gantulga et al. [11]. We also detected pAtBGAL12activity at the apical end of the cotyledons, in both light (Fig. 5a,g)and in dark conditions (Fig. 5e,h), although we do not know

from 3-day-old (a); 5-day-old (b); 10-day-old (c) and 17-day-old plants growing underotyledons and hypocotyl from 10-day-old plants grown in light (g) and in the dark (h);ry roots from 10-day-old plant grown in light (j); emerging secondary root (k); flower, siliques in different maturation stages (p). a: anthers, c: cotyledons, ca: calyptra, co:, sr: secondary root, st: stigma. The blue colour and the arrows indicate the zones withn this figure legend, the reader is referred to the web version of this article.)

Fig. 6. Diagrams summarizing the main data of the activity of the gene promoters AtBGAL1, AtBGAL2, AtBGAL3, AtBGAL4, AtBGAL5 and AtBGAL12 from the Arabidopsis a1 subfamilythat encode b-galactosidases. This figure represents only general aspects in plants growing in light and in flowers and seeds (1, 2, 3 indicate different stages of maturation of seeds).More information of the promoter activities during plant development is given in Figs. 1e5. a: anthers, c: cotyledons, ca: calyptra, co: connective, h: hypocotyl, l: leave, n: nectaries,o: ovaries, p: petals, r: root, re: receptacle, s: sepals, st: stigma, t: trichomes.

L. Albornos et al. / Plant Physiology and Biochemistry 60 (2012) 223e232230

whether this zonemight represent remains of the area of formationof cotyledons.

pAtBGAL12 activity was also detected in hypocotyls (Fig. 5g,h), inthe hypocotyleroot junction zone (Fig. 5i) and in radicles and roots(Fig. 5i,j) in plants growing under both light and dark conditions.Like pAtBGAL5, pAtBGAL12 did not display activity in the apical partof radicles and roots: the meristematic zone and the root cap(Fig. 5j). However, and unlike pAtBGAL5, the emerging secondaryroot appeared intensely stained (Fig. 5k), which indicates a possibleinvolvement of AtBGAL12 at the beginning of the formation ofsecondary roots and, as happens in the formation of leaves, relatingthis protein to the early stages of organ development. A fewexamples of b-galactosidases related to rapid-division stages havebeen described: a chickpea b-galactosidase located in the cell wallsof meristematic cells in the hook and in pericycle cells [25], and a b-galactosidase from Vitis vinifera whose transcripts accumulate inthe green stage of berry fruit development [34]. Furthermore, highlevels of b-galactosidase activity have been observed in the mitoticstage during pollen development in Brassica campestris [35].Although no role for the release of galactose during these processeshas been established, a differential cell wall composition betweenmeristematic and elongating cells, including changes in pecticpolysaccharides, has been reported [36].

Regarding the reproductive organs, pAtBGAL12 activity wasdetected in the pedicels of flower buds, and the colour was more

intensewhen the flower budwas less developed (Fig. 5l), and in thestigma, which appeared as a point at the apical end of the closedfloral bud (Fig. 5l), but could be seen clearly in flower buds openedmanually (Fig. 5m). Once the flowers had developed, only a weakstaining was observed in the connective tissue of the anthers(Fig. 5n). Later, during fruit development, pAtBGAL12 activity wasdetected in the area of the receptacle, the nectaries and in the seeds(Fig. 5o). The activity in the seeds decreased along their maturationas can be observed in siliques at different stages of maturation(Fig. 5p). Thus, AtBGAL12 could be involved in the cell wallschanges occurring during the embryogenesis or maturation ofseeds in a similar way to what was previously indicated for AtB-GAL2 and AtBGAL5.

Our present results suggest that AtBGAL12 could act in the earlystages of the development of leaves and secondary roots as well asin the development of the seeds, always when a high rate of celldivision is happening. According to these results, the in silico dataavailable via the Arabidopsis eFP browser [20] at BAR (http://www.bar.utoronto.a/) show high levels of AtBGAL12 transcript at differentstages of the apical meristem.

3. Concluding remarks

Our present results from study of the promoter activities of thegenes encoding AtBGAL indicate a possible coordinated action of

L. Albornos et al. / Plant Physiology and Biochemistry 60 (2012) 223e232 231

the galactosidases of the subfamily a1 acting on the cell wall. Thepromoters of most of them (AtBGAL1, AtBGAL2, AtBGAL3 and AtB-GAL4) exhibited high activity in cells and organs in expansion,which indicated an involvement of the encoded b-galactosidases incell wall extension or remodelling in cotyledons, leaves and flowerbuds. pAtBGAL5 was the only promoter among those from thesubfamily a1 that was active in the trichomes that appearedthroughout the plants during their development. The activity ofpAtBGAL12was detectedmainly in meristematic zones of the plant,such as the leaf primordia, emerging secondary roots, and devel-oping seeds, which indicates an involvement with the differentia-tion process. Fig. 6 summarizes the main aspects of the promoteractivities of the genes AtBGAL from the subfamily a1. It is importantto note that these diagrams represent only general aspects in plantsgrowing in light and in flowers and seeds. More information of thepromoter activities during plant development is given in Figs. 1e5.

4. Methods

4.1. Plant material and growth conditions

A. thaliana ecotypeColumbiawas used as host for transformations.Seeds were washed with continuous shaking for 15e30 min in 70%ethanol with 0.1% Triton X-100 and sterilized for 8e10 min in 2.5%sodium hypochlorite with 0.05% Triton X-100 under continuousshaking. They were then exhaustively rinsed with water and incu-bated at 4 �C for 3 days before sowing.

Seeds were grown in Petri dishes on one-half-strength Mura-shige and Skoog [37] agar medium and 1% (w/v) sucrose. Theseplates were maintained in a growth chamber (Aralab, Portugal) at22 �C with both a 16-h photoperiod (provided by cool-white fluo-rescent tubes at a light intensity of approximately 80e100 mE/m2/s),or in the absence of light to obtain etiolated seedlings.

In order to obtain adult plants and seeds from the next gener-ation, 10-day-old seedlings were transferred to plastic pots con-taining a 3:1 mixture of potting soil and vermiculite and grownunder same conditions.

4.2. Construction of vectors for stable plant transformation

pAtBGAL:GUS constructs were prepared using Gateway cloningtechnology (Invitrogen, U.S.A.), according to the manufacturer’sinstructions.

A promoter region of each AtBGAL gene consisting of a sequenceof ca. 2000e2300 bp upstream from the translational start site wasPCR-amplified from Arabidopsis genomic DNA, adding the attB1 andattB2 sequences to the N- and C-terminals. The primers used arelisted in Supplemental table 1. The amplified products were gel-purified and used in the BP reaction with pDONR201 (Invitrogen,U.S.A.). The entry clones generated were used in LR reaction withpKGWFS7, a C-terminal GFP/GUS destination vector [38]. AllpAtBGAL:GUS constructs were verified by sequencing.

4.3. Plant transformation

All constructs generated were electroporated into Agro-bacterium tumefaciens strain C5851m. A. thaliana ecotype Col-0 was transformed with the Agrobacterium-mediated floral dipmethod [39].

Seeds harvested from infiltrated plants were screened on theappropriate antibiotic and resistant seedlings (T1) were selected. T2plants were used for the GUS assay. Images were acquired usinga Leica M205 FA stereomicroscope (Leica Microsystems, Germany).

4.4. GUS staining

GUS staining using X-glucuronide (5-bromo-4-chloro-3-indolyl-b-D-GlcUA) (Duchefa, The Netherlands) was performed asdescribed by Jefferson et al. [40].

Briefly, plant material was incubated overnight at 37 �C in GUSstaining solution (5-bromo-4-chloro-3-indolyl-b-D-GlcA, 50 mMNa phosphate buffer (pH 7.0), 2 mM potassium ferrocyanide, 2 mMpotassium ferricyanide, and 0.2% Triton X-100). GUS-stained tissueswere cleared in 70% ethanol.

GUS activity was assayed in 3, 5 and 10-day-old seedlings grownon Murashige and Skoog plates (both in light-grown and etiolatedseedlings) and in different organs from 12, 17, 24 and 38-day-oldplants growing in a potting soil and vermiculite mixture, as indi-cated above.

Experiments were performed on two plants from two inde-pendent T2 lines. Wild type control plants showed no GUS activity(data not shown). Here we only show the organs with GUS-activity.

Acknowledgement

This work was supported by grants from the Ministerio deCiencia y Tecnología, Spain (BFU2009-08769) and from the Junta deCastilla y León (SA023A10-2). Lucía Albornos was supported by FPUgrant from Spanish Ministerio de Educación, Cultura y Deporte.

Appendix A. Supplementary material

Supplementary material related to this article can be found athttp://dx.doi.org/10.1016/j.plaphy.2012.08.012.

References

[1] B. Henrissat, A classification of glycosyl hydrolases based on amino acidsequence similarities, Biochem. J. 280 (1991) 309e316.

[2] B.L. Cantarel, P.M. Coutinho, C. Rancurel, T. Bernard, V. Lombard, B. Henrissat,The Carbohydrate-Active EnZymes database (CAZy): an expert resource forglycogenomic, Nucleic Acids Res. 37 (Database issue) (2009) D233eD238.

[3] J.P. Vervelen, K. Vissenberg, in: J.P. Verbelen, K. Vissenberg (Eds.), TheExpanding Cell, Plant Cell Monographs, vol. 5, Springer, New York, 2007.

[4] D. Gantulga, Y. Turan, D.R. Bavan, A. Esen, The Arabidopsis At1g45130 andAt3g52840 genes encode b-galactosidases with activity toward cell wallpolysaccharides, Phytochemistry 69 (2008) 1661e1670.

[5] L.D. Smith, K.C. Gross, A family of at least seven b-galactosidase genes isexpressed during tomato fruit development, Plant Physiol. 123 (2000)1173e1183.

[6] R. Esteban, E. Labrador, B. Dopico, A family of b-galactosidase cDNAs related todevelopment of vegetative tissue in Cicer arietinum, Plant Sci. 168 (2005)457e466.

[7] I. Martín, B. Dopico, F.J. Muñoz, R. Esteban, R.J.F.J. Oomen, A. Driouich,J.P. Vincken, R. Visser, E. Labrador, In vivo expression of a Cicer arietinum b-galactosidase in potato tubers leads to a reduction of the galactan side-chainsin cell wall pectin, Plant Cell. Physiol. 46 (2005) 1613e1622.

[8] Y.O. Ahn, M. Zheng, D.R. Bevan, A. Esen, S.H. Shiu, J. Benson, H.P. Peng,J.T. Miller, C.L. Cheng, J.E. Poulton, M.C. Shih, Functional genomic analysis ofArabidopsis thaliana glycoside hydrolase family 35, Phytochemistry 68 (2007)1510e1520.

[9] G.A. King, K.M. Davies, Cloning of a harvest-induced b-galactosidase from tipsof harvested asparagus spears, Plant Physiol. 108 (1995) 419e420.

[10] M. Hosono, K. Ishikawa, R. Mineki, K. Murayama, C. Numata, Y. Ogawa,Y. Takayanagi, K. Nitta, Tandem repeat structure of rhamnose-bindinglectin from catfish (Silurus asotus) eggs, Biochim. Biophys. Acta 1472(1999) 668e675.

[11] D. Gantulga, Y.O. Ahn, C. Zhou, D. Battogtokh, D.R. Bevan, B.S.J. Winkel, A. Esen,Comparative characterization of the Arabidopsis subfamily a1 b-galactosi-dases, Phytochemistry 70 (2009) 1999e2009.

[12] I.B. Perez-Almeida, Arabidopsis cell wall b-galactosidase gene family:expression, catalytic activities, biological function in galactose dynamics,Botany and Plant Pathology, Ph.D. purdue, West Lafayette, 2004.

[13] H. Tsukaya, Leaf index: heteroblasty, natural variation, and the genetic controlof polar processes of leaf expansion, Plant Cell. Physiol. 43 (2002) 372e378.

[14] Z. Zhang, M.L. Pierce, J.A. Mort, Changes in homogalacturonans and enzymesdegrading them during cotton cotyledon expansion, Phytochemistry 68(2007) 1094e1103.

L. Albornos et al. / Plant Physiology and Biochemistry 60 (2012) 223e232232

[15] R. Naran, M.L. Pierce, A.J. Mort, Detection and identification of rhamnoga-lacturonan lyase activity in intercellular spaces of expanding cotton cotyle-dons, Plant J. 50 (2007) 95e107.

[16] H. Masuda, Y. Ozeki, S. Amino, A. Komamine, Changes in the activities ofvarious glycosidases during carrot cell elongation in a 2,4-D-free medium,Plant Cell. Physiol. 26 (1985) 995e1001.

[17] B. Dopico, G. Nicolás, E. Labrador, Changes during epicotyl growth of anautolysis-related b-galactosidases from the cell wall of Cicer arietinum, PlantSci. 72 (1990) 45e51.

[18] P. Hrubá, D. Honys, D. Twell, V. Capková, J. Tupý, Expression of b-galactosidaseand a-xylosidase genes during microspore and pollen development, Planta220 (2005) 931e940.

[19] C. Durand, M. Vicré-Gibouin, M.L. Follet-Gueye, L. Duponchel, M. Moreau,P. Lerouge, A. Driouich, The organization pattern of root border-like cells ofArabidopsis is dependent on cell wall homogalacturonan, Plant Physiol. 150(2009) 1411e1421.

[20] E. Gendreau, J. Traas, T. Desnos, O. Grandhjean, M. Caboche, H. Höfte, Cellularbasis of hypocotyls growth in Arabidopsis thaliana, Plant Physiol. 114 (1997)295e305.

[21] D. Winter, B. Vinegar, H. Nahal, R. Ammar, G.V. Wilson, N.J. Provart, An’electronic fluorescent pictograph’ browser for exploring and analyzing large-scale biological data sets, PLoS One 2 (2007) e718.

[22] E.M. O’Donoghue, S.D. Somerfield, L.M. Watson, D.A. Brummell, D.A. Hunter,Galactose metabolism in cell walls of opening and senescing petunia petals,Planta 229 (2009) 709e721.

[23] J. Sampedro, B. Pardo, C. Gianzo, E. Guitián, G. Revilla, I. Zarra, Lack ofa-xylosidase activity in Arabidopsis alters xyloglucan composition andresults in growth defects, Plant Physiol. 154 (2010) 1105e1115.

[24] K. Kurasawa, A. Matsui, R. Yokoyama, T. Kuriyama, T. Yoshizumi, M. Matsui,K. Suwabe, M. Watanabe, K. Nishitani, The AtXTH28 gene, a xyloglucanendotransglucosylase/hydrolase, is involved in automatic self-pollination inArabidopsis thaliana, Plant Cell. Physiol. 50 (2009) 413e422.

[25] I. Martín, T. Jiménez, J. Hernández-Nistal, E. Labrador, B. Dopico, The locationof the chickpea cell wall bV-galactosidase suggests involvement in the tran-sition between cell proliferation and cell elongation, J. Plant Growth Regul. 28(2009) 1e11.

[26] N. Iglesias, J.A. Abelenda, M. Rodiño, J. Sampedro, G. Revilla, I. Zarra, Apoplasticglycosidases active against xyloglucan oligosaccharides of Arabidopsis thali-ana, Plant Cell. Physiol. 47 (2006) 55e63.

[27] R. Louvet, C. Rayon, J.M. Domon, C. Rusterucci, F. Fournet, A. Leaustic,M.J. Cropeau, M.C. Ralet, C. Rihouey, M. Bardor, P. Lerouge, F. Gillet, J. Pelloux,Major changes in the cell wall during silique development in Arabidopsisthaliana, Phytochemistry 72 (2011) 59e67.

[28] T. Potikha, D.P. Delmer, A mutant of Arabidopsis thaliana displaying alteredpatterns of cellulose deposition, Plant J. 7 (1995) 453e460.

[29] H. Tokumoto, K. Wakabayashi, S. Kamisaka, T. Hoson, Changes in the sugarcomposition and molecular mass distribution of matrix polysaccharidesduring cotton fiber development, Plant Cell. Physiol. 43 (2002) 411e418.

[30] M.J. Jakoby, D. Falkenhan, M.T. Mader, G. Brininstool, E. Wischnitzki, N. Platz,A. Hudson, M. Hülskamp, J. Larkin, A. Schnittger, Transcriptional profiling ofmature Arabidopsis trichomes reveals that NOECK encodes the MIXTA-liketranscriptional regulator MYB106, Plant Physiol. 148 (2008) 1583e1602.

[31] L. McCartney, C.G. Steele-King, E. Jordan, J.P. Knox, Cell wall pectic (1-4)-b-D-galactan marks the acceleration of cell elongation in the Arabidopsis seedlingroot meristem, Plant J. 33 (2003) 447e454.

[32] G. Haughn, A. Chaudhury, All dressed up with nowhere to go: genetic analysisof seed coat development in Arabidopsis, Trends Plant Sci. 10 (2005) 472e477.

[33] S. Pien, J. Wyrzykowska, S. McQueen-Mason, C. Smart, A. Fleming, Localexpression of expansin induces the entire process of leaf development andmodifies leaf shape, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 11812e11817.

[34] L. Barnavon, T. Doco, N. Terrier, A. Ageorges, C. Romieu, P. Pellerin, Analysis ofcell wall neutral sugar composition, b-galactosidase activity and a relatedcDNA clone throughout the development of Vitis vinifera grape berries, PlantPhysiol. Biochem. 38 (2000) 289e300.

[35] M.B. Singh, R.B. Knox, A gene controlling b-galactosidase deficiency in pollenof oilseed rape Brassica campestris, J. Hered. 76 (1985) 199e201.

[36] W.G.T. Willats, C.G. Steele-King, S.E. Marcus, J.P. Knox, Side chains of pecticpolysaccharides are regulated in relation to cell proliferation and cell differ-entiation, Plant J. 20 (1999) 610e628.

[37] T. Murashige, F. Skoog, A revised medium for rapid growth and bioassays withtobacco tissue cultures, Physiol. Plant 15 (1962) 473e497.

[38] M. Karimi, D. Inzé, A. Depicker, GATEWAY vectors for Agrobacterium-mediatedplant transformation, Trends Plant Sci. 7 (2002) 193e195.

[39] S.J. Clough, A.F. Bent, Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana, Plant J. 16 (1998) 735e743.

[40] R.A. Jefferson, T.A. Kavanagh, M.W. Bevan, GUS fusions: b-glucuronidase asa sensitive and versatile gene fusion marker in higher plants, EMBO J. 6 (1987)3901e3907.