A BELL1-Like Gene of Potato Is Light Activated - Plant Physiology

A BELL1-Like Gene of Potato Is Light Activated andWound Inducible1[C][OA]

Mithu Chatterjee, Anjan K. Banerjee, and David J. Hannapel*

Department of Horticulture, Iowa State University, Ames, Iowa 50011–1100

BELL1-like transcription factors interact with their protein partners from the KNOTTED1 family to bind to target genes andregulate numerous developmental and metabolic processes. In potato (Solanum tuberosum), the BELL1 transcription factor StBEL5and its protein partner POTH1 regulate tuber formation by affecting hormone levels. Overexpression of StBEL5 in transgenic linesproduces plants that consistently exhibit enhanced tuber formation, and the mRNA of this gene moves through phloem cells in along-distance signaling pathway regulated by photoperiod. Whereas photoperiod mediates the movement of StBEL5 RNA,activation of transcription of the StBEL5 gene in leaves is regulated by white light, regardless of photoperiod or light intensity.Illumination with either red or blue light induces the StBEL5 promoter, whereas far-red light had no effect. As expected, the StBEL5promoter harbors numerous conventional light-responsive cis-acting elements like GT1, GATA, and AT1 motifs. Deletion con-structs were analyzed to determine what sequences are involved in light activation. Transcriptional activity was also mediated bywounding on stems, insect predation on leaves, and photoperiod in stolons. These results demonstrate that StBEL5 gene activity inthe leaf is correlated with wavelengths optimal for photosynthesis. The number of factors that affect the StBEL5 promoter supportsthe premise that the BELL1-like genes play a role in a wide range of functions.

Homeobox-containing genes encode transcriptionfactors that play an important role in plant morpho-genesis and development. The first homeotic gene wasdiscovered in Drosophila and subsequently isolated fromdistinct evolutionary groups like fungi, plants, and ani-mals (Chan et al., 1998). The protein encoded by homeo-box genes contains a conserved DNA-binding domain,the homeodomain consisting of three a-helices (Desplanet al., 1988; Otting et al., 1990; Laughon, 1991). The firstplant homeobox gene identified in maize (Zea mays),KNOTTED1, is involved in formation and maintenanceof the shoot apical meristem (Vollbrecht et al., 1991;Kerstetter et al., 1997; Hake et al., 2004). Another planthomeobox gene family closely related to KNOTTED1is the BELL1 family (Quaedvlieg et al., 1995; Reiser et al.,1995; Chan et al., 1998). KNOTTED1-like and BELL1-like genes belong to the TALE superclass of transcriptionfactors characterized by a three-amino-acid loop ex-tension present between the first and second a-helicesof the homeodomain (Burglin, 1997). In Arabidopsis(Arabidopsis thaliana), BELL1 (BEL1) regulates ovule de-velopment and the specification of outer and inner in-

teguments (Reiser et al., 1995). Molecular and geneticanalyses revealed that BELL1, along with the carpel-determining homeotic gene AGAMOUS, participatesin several distinct aspects of ovule morphogenesis (Rayet al., 1994; Reiser et al., 1995; Gasser et al., 1998; Westernand Haughn, 1999). Subsequent studies of BELL1-likegenes indicate that they are ubiquitous in the plantkingdom. The apple (Malus domestica) BELL1-like ho-meodomain gene, MDH1, plays an important role ingrowth, fertility, and development of carpels and fruitshape (Dong et al., 2000). Two Arabidopsis null mutantsof the BELL1-like genes PENNYWISE and POUND-FOOLISH disrupt the transition from vegetative to flo-ral development (Smith et al., 2004). Tobacco (Nicotianatabacum) plants overexpressing BELL1 of barley (Hor-deum vulgare) were dwarf with multiple shoots andexhibited malformed leaves and flowers (Muller et al.,2001). Several BELL1-like cDNAs have been identifiedin potato (Solanum tuberosum) and one, StBEL5, is in-volved in tuber development by affecting hormonelevels (Chen et al., 2003, 2004). Recently, a rice (Oryzasativa) homolog, OsBIHD1, was identified that func-tions in disease resistance and pathogen defense (Luoet al., 2005). Clearly, BELL1-like genes function in a widerange of processes in plants.

In potato, using the KNOTTED1-like protein POTH1as bait in the yeast two-hybrid system, seven BELL1-like proteins were identified (Chen et al., 2003). Over-expression of one of the BELLs, StBEL5, and POTH1 intransgenic lines produces plants that consistently ex-hibit enhanced rates of tuber growth (Chen et al., 2003;Rosin et al., 2003). In these plants, RNA levels for GA20-oxidase1 were reduced in stolons and leaves, re-spectively. This reduction in GA 20-oxidase1 RNA accu-mulation coincides with a reduction in active GA levels

1 This work was supported by the National Science Foundation inthe Division of Integrative Organismal Biology (award no. 0344850).

* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to

the findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:David J. Hannapel ([email protected]).

[C] Some figures in this article are displayed in color online but inblack and white in the print edition.

[OA] Open Access articles can be viewed online without a sub-scription.

www.plantphysiol.org/cgi/doi/10.1104/pp.107.105924

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and an increase in tuberization. During tuberization,the levels of GA1 in swollen stolons decreases due tothe activity of key regulatory enzymes in the GA bio-synthetic pathway (Xu et al., 1998a; Kloosterman et al.,2007). Similarly, overexpression mutants of other KNOXgenes from tobacco (NTH15) and rice (OSH1) also exhib-ited reduced activity of GA 20-oxidase1 and decreasedaccumulation of GA1 (Tamaoki et al., 1997; Kusaba et al.,1998a, 1998b; Tanaka-Ueguchi et al., 1998).

During tuberization, POTH1 appears to form a tandemcomplex with its partner StBEL5 to regulate develop-ment (Chen et al., 2003, 2004). Detailed studies indi-cated that POTH1 and StBEL5 must interact to bindthe target elements and to affect transcription of theGA 20-oxidase1 gene (Chen et al., 2004). These resultsdemonstrated that both protein partners are essentialin binding the cis-element to modulate transcription.Protein-protein interaction has also been demonstratedbetween members of the BELL1 and KNOX families inArabidopsis (Bellaoui et al., 2001; Kanrar et al., 2006;Viola and Gonzalez, 2006), barley (Muller et al., 2001),and maize (Smith et al., 2002).

To gain more insight into the factors that regulate theactivity of the BELL1 genes of potato, in this study, thepromoter of StBEL5 has been characterized. Despitethe importance of BELL1-like genes in plant biology,very little is known about the regulation of their tran-scription. This is particularly significant in light of a recentreport documenting the correlation of photoperiod-mediated movement of the full-length mRNA of StBEL5with tuber formation (Banerjee et al., 2006a). BecauseStBEL5 may be involved in controlling tuberization andthis stage of development is regulated by photoperiod(Rodriguez-Falcon et al., 2006), this study focuses on thelight regulation of the StBEL5 promoter. Our resultsindicate that the StBEL5 promoter is activated by lightand is insensitive to photoperiod in the leaf and thatit harbors numerous conventional light-responsive cis-acting elements like GT1, GATA, and AT1 motifs. De-letion constructs were analyzed to determine whatsequences are involved in light activation. Promoteractivity was also mediated by wounding on stems,insect predation on leaves, and photoperiod in stolons.The number of factors that affect the StBEL5 promotersupports the premise that the BELL1-like genes play arole in a wide range of functions.

RESULTS

cis-Regulatory Elements of the StBEL5 Promoter

To elucidate how the promoter of StBEL5 regulatestranscription, the 2.29-kb upstream region (Fig. 1A) wasscreened for cis-acting regulatory elements using thesoftware PLACE (Higo et al., 1999) and the PlantCaredatabase (Lescot et al., 2002). We observed the pres-ence of several putative cis-acting light-regulatory ele-ments (LREs), including GT1 and GATA boxes (Giulianoet al., 1988; Terzaghi and Cashmore, 1995; Guilfoyle,

1997) and AT1 motifs (Fig. 1B). The G-box LRE wasidentified at positions 21,030, 2693, 327, and 94. Mostof the LREs were present in multiple copies. For ex-ample, nine GATA boxes, 11 GT1 boxes, and four AT1motifs were identified in this 2.29-kb upstream region.A careful analysis revealed the presence of a large num-ber of light elements within 500 bp of the promoterregion near the transcription start site (10 were iden-tified). Clustering of LREs within this region suggeststhat the combination of these elements may functionas a minimal light-regulatory region. The GATA boxestended to be distributed in the flanking regions of thepromoter, whereas the GT1 boxes are preponderate inthe middle region. No LREs were identified in the intron

Figure 1. StBEL5 upstream regions. A, Schematic diagram of the struc-ture consisting of promoter, 5#-UTRs, and an intron. B, DNA sequencerepresents the StBEL5 upstream region plus 150 nucleotides of the5#-UTR. Nucleotide sequences representing potential LREs are high-lighted: GATA box, gray box; GTI motifs, white box; and AT1 motifs,underlined. The divided 5#-UTR is bracketed and italicized and isflanking the intron, which is designated by light letters from nucleotides97 to 299 (B). Note: The sequence from 22,002 to 21,938 is a cloningadaptor.

Chatterjee et al.

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(Fig. 1B, nucleotides 97–299). In addition to LREs, soft-ware analyses also identified GA-responsive elementslike P box, TATC box, and WRKY71OS (Gubler andJacobsen, 1992; Lanahan et al., 1992; Zhang et al., 2004),and several wound-response elements like W box,WUN motif, and G boxes (Siebertz et al., 1989; Rushtonand Somssich, 1998; Rushton et al., 2002).

Light-Activated Expression of the StBEL5 Promoter

Because StBEL5 appears to be involved in controllingtuberization and this stage of development is regu-lated by photoperiod (Rodriguez-Falcon et al., 2006),the light regulation of the StBEL5 promoter was eval-uated. Analysis of transcription was performed ontransformed potato plants containing the P-StBEL5 pro-moter construct. The design and characterization ofthis promoter:GUS construct was previously reported(Banerjee et al., 2006a). To evaluate the effect of light onthe StBEL5 promoter, GUS activity was determined us-ing in vitro-grown shoot tips of transgenic potato plants.

Promoter activity was examined in samples grownunder three different light conditions, 15 d light (16 hlight, 8 h dark), 15 d dark, or 14 d dark followed by 24 hexposure with various wavelengths of light (Fig. 2).Each set of experiments was conducted with three in-dependent lines. Here, data of one representative line

are presented. The lines containing the P-StBEL5 con-struct showed lower GUS activity when grown underdark conditions and activity increased 2- to 3-foldupon illumination with white light (Fig. 2). Light in-duction was also observed after 2 h of light treatmentfollowing the dark period (data not shown). For eval-uation of specific wavelengths, after the dark treat-ment, in vitro plants were exposed to 24 h of blue light(8 mmol m22 s21), red light (8 mmol m22 s21), or far-redlight (5 mmol m22 s21). With blue and red light, a 2- to3-fold increase in GUS activity was observed, whereasexposure to far-red light induced only a modest in-crease (Fig. 2). Because a short-day (SD) photoperiodfacilitates movement of StBEL5 mRNA and inducestuber formation, the effect of photoperiod on StBEL5promoter activity was assayed (Fig. 3). GUS expres-sion in leaves and petiole samples (Fig. 3A) harvestedfrom plants grown under either long-day (LD; 16 hlight, 8 h dark) or SD (8 h light, 16 h dark) conditionswas evaluated. Transcriptional activity was enhancedunder both photoperiod conditions. Both petioles andleaves exhibited greater levels of StBEL5 promoter ac-tivity under LD conditions (Fig. 3B). This experiment,however, does not separate the effects of photoperiodfrom light quantity because LD plants are exposed to agreater total fluence rate. To test photoperiod effects,SD (8 h light, 16 h dark) and SD plus night break (7.5 hlight with a 0.5-h night break) conditions for 12 d wereutilized. The night break simulates LD (or short night)and delivers an equivalent irradiation quantity. In thisexperiment, photoperiod had no effect on promoteractivity in leaves or petioles (Fig. 3C). To determinewhether light quantity is controlling promoter activity,P-StBEL5 plants were exposed to LD (16 h light, 8 hdark) conditions in a growth chamber under a fluencerate of 60, 200, or 400 mmol m22 s21 for 12 d. No dif-ference in promoter activity was observed in petiolesand leaf veins upon exposure to any of the three fluencerates (Fig. 3D).

To define the minimal light regulatory region, two de-letion constructs, designated P1-StBEL5 and P2-StBEL5,were designed, fused to a GUS sequence (Fig. 4A) andtransformed into potato (Banerjee et al., 2006b). P1-StBEL5 is composed of 1,052 nucleotides of promotersequence (extending from nucleotide 21,052 to the startof the untranslated region [UTR]) plus 96 nucleotidesof the 5#-UTR, whereas P2-StBEL5 is composed of 272nucleotides of the downstream promoter sequence (ex-tending from nucleotide 2272 to the start of the UTR)plus 96 nucleotides of the 5#-UTR. There were no ob-servable differences in leaf, stem, root, or stolon expres-sion between constructs with or without the intron,and this sequence was not included in constructs P1and P2. Both constructs exhibited light induction whenexposed to 24 h of white-light treatment (Fig. 4B). TheP2 construct apparently contained sufficient LREs (threeGATA, two GT1, and one AT1) to confer light induc-tion. The expression driven by this smaller fragment,however, is more than 10-fold lower than the light-driven activity of P-StBEL5. P1-StBEL5 induction was

Figure 2. Effect of wavelength on the activity of the StBEL5 promoter.For light induction assay, in vitro transgenic potato plants containingP-StBEL5 were grown for 15 d in white light (16 h light, 8 h dark), 15 d inthe dark, or 14 d in the dark followed by blue-, red-, far-red-, or white-light irradiation for 24 h. Several shoot tips, approximately 1.0 cm inlength, were harvested for the GUS assays. GUS activity is expressed innanomoles of 4-methylumbelliferone (4-MU) produced per hour permicrogram of protein. Data represent the mean 6 SD of GUS activitymeasured with three replicates. For white light, in vitro cultures wereincubated at a light intensity of 40 mmol m22 s21 cool-white fluorescentlight at 27�C in a Percival incubator. A single incandescent bulb wasused for the far-red light treatment (5 mmol m22 s21) and a singlefluorescent tube was screened with select filters to produce blue andred wavelengths (8 mmol m22 s21).

BEL5 Promoter Activity

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about 2-fold less than P-StBEL5 activity. Histochemicalanalysis of leaves of P1 was consistent with this de-crease in GUS activity (Fig. 5A). A faint GUS reactionwas observed in the primary vein of P1 leaves (Fig. 5A,arrow). The relative promoter activities of the threeconstructs tested here are correlated to the number ofLREs identified in each construct. Accounting for GATA,GT1, and AT1 elements only, P-StBEL5, P1-StBEL5, andP2-StBEL5 contained 24, 12, and six total elements, re-spectively.

Regulation of StBEL5 Promoter Activity in

Underground Organs

Previous work has shown that, in addition to lightactivation in veins and petioles of leaves, StBEL5 pro-moter activity was observed consistently in stolons fromboth tuberizing and nontuberizing plants, in newlyformed tubers, and in roots (Banerjee et al., 2006a). Toexamine what sequences contributed to promoter ac-tivity in the dark, the three promoter constructs previ-ously described were analyzed for activity in threeunderground organs formed on potato: roots, stolons,and tubers. Histochemical analyses of roots (Fig. 5B)and stolons/tubers (Fig. 5, C–G) indicated that expres-sion of GUS was incrementally reduced in the P1 and P2constructs. Only a very low level of GUS staining wasobserved in roots of P2 transgenic lines (Fig. 5B),

whereas no GUS expression was observed in stolonsand newly formed tubers from these lines (Fig. 5, F andG). Although GUS staining was diffuse in tubers from Ptransgenic lines, promoter activity was greatest invascular connections of newly formed tubers (Fig. 5D,arrows), in internal and external phloem strands (Fig.5E, in and ex, respectively), and in the peridermal layer(Fig. 5E, pe). Activity of the StBEL5 promoter coincideswith the region of most active cell growth in a newtuber, the perimedullary region between the pith andthe phloem (Xu et al., 1998b).

To investigate further the mechanism for this acti-vation in the dark, promoter activity was assayed intuberizing stolons grown on SD plants and nontuber-izing stolons from LD plants. No signal was detectedin stolons from plants grown for 6 d in a growth cham-ber under LD conditions (Fig. 6). The overall pattern ofactivity showed no trend through 12 d of LD exposure.Activity in stolons from SD plants, however, exhibiteda steady increase from day 6 through day 12. Thisactivity corresponds to both the onset of tuber formationand the gradual accumulation of mRNA for StBEL5(Chen et al., 2003).

Wounding Effects

Previous analyses of a transverse stem section col-lected from the internodal regions of transgenic plants

Figure 3. Effect of photoperiod on StBEL5 promoteractivity. GUS expression was measured in leaf (lam-ina and veins) and petiole samples (A) grown undereither LD (16 h light, 8 h dark) and SD (8 h light, 16 hdark) conditions (B), SD (8 h light, 16 h dark) andnight break (7.5 h light, 16 h dark with a 0.5-h lightnight break) conditions for 12 d each (C), or underthree fluence rates of 60, 200, or 400 mmol m22 s21

for 12 d (D). The data represent the mean 6 SD ofGUS activity measured with three replicates. GUSactivity (B and C) was performed on soil-grown plantsincubated at a light intensity of 100 mmol m22 s21

with cool-white fluorescent bulbs plus incandescentlight at 22�C light period/18�C dark period. The sameenvironmental conditions were used for the lightintensity experiment (D) except for adjustment of thefluence rate. [See online article for color version ofthis figure.]

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harboring the P-StBEL5 construct showed that GUS isnot expressed in stem cross sections (Banerjee et al.,2006a). However, we consistently observed that the cutends of stems exhibited GUS activity (Fig. 7A). Stemsamples of transgenic plants containing the P-StBEL5and P1-StBEL5 constructs exhibited a high level of GUSactivity when subjected to mechanical injuries (Fig. 7A),whereas P2-StBEL5 transgenic lines exhibited very lit-tle, if any, wound response (Fig. 7B). According to soft-ware analyses, overall, the StBEL5 promoter sequencecontains 12 G boxes, 21 WUN motifs, and nine W boxes.In the P2 construct, this number drops to two, three,and one, respectively. When leaves were subjected tothe same mechanical injury, no GUS expression wasobserved in P lines. No promoter activity was observedeven when leaves were cut into small pieces with arazor blade (Fig. 7C). The leaves, however, exhibited aresponse to insect attack. Those leaves infested withwestern flower thrips (Frankliniella occidentalis) showed

promoter activity coincident to the area of infection andat the junction of primary and secondary veins (Fig.7D, arrows). GUS expression was also observed in themesophyll region of leaves undergoing senescence(data not shown). The presence of wound-responseelements like W box, WUN motif, and G boxes in thepromoter supports the premise that StBEL5 transcrip-tion is activated by wound- and pest-induced pathways.

DISCUSSION

Because of the importance and ubiquity of the BELL1-like family (Hake et al., 2004), the lack of informationon promoter activity for any of these genes, and thereputed role of full-length StBEL5 mRNA as a mobilesignal for tuber formation (Banerjee et al., 2006a),analysis of its promoter activity would be highly in-formative. Of particular interest is the source of this

Figure 4. A, Schematic diagram of the deletion con-structs of StBEL5 promoter fused to a GUS reportergene. The upstream region is indicated by the graybox, and the 5#-UTR includes sequence from 1 to 96and 300 to 354 and is interrupted by an intron fromnucleotide 97 to 299 (diagonal-hatched box). Con-structs P1 and P2 do not include any intronic se-quence. B, Transgenic plants expressing each of thethree constructs P-StBEL5, P1-StBEL5, and P2-StBEL5exhibited light-mediated induction following a 24-hwhite-light treatment. In vitro plants were grownunder light conditions (L), 15 d in dark (D), or 14 din dark followed by white light (WL) irradiation for24 h. GUS activity was assayed on in vitro plantletsincubated at a light intensity of 40 mmol m22 s21 cool-white fluorescent light at 27�C in a Percival incubator.The lower scale of promoter activity in this experiment(compare to Fig. 3, B–D) is probably due to the qualityof light provided by the Percival unit. No incandescentbulbs were used in this analysis.

Figure 5. Expression analysis of three deletion con-structs of the StBEL5 promoter fused to the GUS re-porter gene in leaves (A), roots (B), and newly formedtubers (C–G) of transgenic potato plants containingconstruct P (C–E), P1 (C), or P2 (F–G). These constructswere described in Figure 4A. Leaves were from plantsgrown under LD conditions with white light and theroots and tubers were harvested from plants grown for10 d of SD conditions at a light intensity of 100 mmolm22 s21 with cool-white fluorescent bulbs plus incan-descent light at 22�C. D, E, and G, Internal longitudi-nal sections. The size bar in E represents 0.1 cm.Arrows in D and E indicate phloem strands. in, Internalphloem; ex, external phloem; pe, peridermal tissue.Anatomy of the newly formed tubers is based on thecareful study performed by Reeves et al. (1969).





mobile RNA and how the BEL5 promoter is regulatedin relation to light and the creation of a strong under-ground sink, the tuber. BELL1-like transcription fac-tors control numerous facets of plant development andmetabolism, but their function in light perception andsignal transduction is largely unknown. ATH1 of Arabi-dopsis is one example of a BELL1-like protein whoseexpression is tightly regulated by exposure to light(Quaedvlieg et al., 1995). Dark-grown seedlings ofphotomorphogenic mutants possessed elevated ATH1mRNA levels in comparison with etiolated wild-typeseedlings. Genetic analyses have demonstrated thatATH1 may be an important downstream componentof the DET1 and COP1 signal transduction pathwaysand involved in the expression of light-inducible genesand photomorphogenic control. Here, we have focusedon the role that light plays in regulating the transcrip-tion of a BELL1-like gene from potato, StBEL5.

The promoter sequence of StBEL5 contains severalLREs and the presence of these elements was corre-lated with light elicitation (Fig. 4). Dark-grown plantsexhibited enhanced promoter activity within 2 h of ex-posure to white light (data not shown). This enhancedactivity was wavelength dependent, with blue and redlight eliciting significant activation (Fig. 2). These re-sults imply that promoter activity may be selectivelymediated by phytochrome and/or blue-light recep-tors. Perception and transduction of light are achievedby at least two principal groups of photoreceptors, phy-tochromes and cryptochromes (for review, see Jiao et al.,2007). Phytochromes are red/far-red light-absorbingreceptors encoded by a gene family of five members(phyA–phyE) in Arabidopsis. Cryptochrome 1, crypto-chrome 2, and phototropin are the blue-light receptorsthat have currently been identified. The phototropins

primarily regulate processes optimizing photosynthesis,whereas transcriptional and developmental changesare attributed to the phytochromes and the crypto-chromes. Blue light (390–500 nm) elicits a variety ofphysiological responses in plants, including four thatmaximize photosynthetic potential. These include pho-totropism, light-induced opening of stomata, chloroplastmigration in response to changes in light intensity, andsolar tracking (Kinoshita et al., 2001; Briggs and Christie,2002).

Cryptochromes interact genetically with multiplephytochromes (Neff et al., 2000). For example, phyto-chrome B and cryptochrome 2 have been shown totightly colocalize in vivo, bind in vitro, and interact inthe control of flowering time, hypocotyl elongation,and circadian clocks (Mas et al., 2000). Blue- and red-light fluence rates as low as 8 mmol m22 s21 were aseffective as white-light treatments of 40 mmol m22 s21

in inducing the StBEL5 promoter (Fig. 2). In addition,the red and blue filters delivered light at 650 and 450nm, both of which are well within the spectral range

Figure 6. Promoter activity of StBEL5:GUS in stolons from plants grownunder SD (8 h light, 16 h dark) or LD (16 h light, 8 h dark) days. GUSquantification was performed on stolon tips harvested from transgeniclines containing the P-StBEL5construct. Stolon tips (approximately1.0 cmin length) were harvested after 6, 8, and 12 d from whole plants grown at alight intensity of 100 mmol m22 s21 with cool-white fluorescent bulbs plusincandescent light at 22�C. ND, Not detected. Three replicates wereaveraged for each harvest and error bars are indicated.

Figure 7. Wound response of the StBEL5 promoter. A, Stem segments oftransgenic plants containing P1-StBEL5 subjected to mechanical injurywith a razor blade (left-side segment) or a forceps (right-side segment)and immediately incubated in GUS buffer as described in ‘‘Materialsand Methods.’’ The middle segment is an excised stem portion that wasnot wounded. Similar results were obtained from transgenic linesharboring the P-StBEL5 construct. B, Wounded stem segments fromtransgenic lines harboring the P2-StBEL5 construct. C, Leaves fromtransgenic lines of P-StBEL5 subjected to mechanical wounding. D,Leaf from transgenic plants of P-StBEL5 infested with the western flowerthrip. Note sites of insect feeding (arrows).

Chatterjee et al.




for both photosynthetic activity and light absorption(Taiz and Zeiger, 2006).

Although there are examples where the minimallight-driven regulatory region on a promoter can be asshort as 52 bp (Schafer et al., 1997; Martinez-Hernandezet al., 2002), several studies have confirmed that the min-imal light-driven region is generally found within 250 bpupstream from the transcriptional start site (Terzaghiand Cashmore, 1995). Deletion analysis of the StBEL5upstream region revealed that the construct containing272 nucleotides of promoter plus 96 nucleotides of the5#-UTR P2-StBEL5 was sufficient for light-mediatedactivation (Fig. 4). It is possible that the combinatorialinteraction of the three GATA, two GT1, and one AT1elements in this sequence is sufficient to participate inlight induction, allowing the P2-StBEL5 construct to actas a minimal light regulatory sequence.

But how can the activity of the StBEL5 promoter thatoccurs in stolon tips and tubers in the dark (Fig. 5, C–E)be reconciled with light induction? Similar to the lightresponse, this activity in the dark is progressively re-duced in the P1 and P2 constructs (Fig. 5, B and C, andF and G). Photoperiod only affects promoter activity instolon tips (Fig. 6). In this system, white-light expo-sure, regardless of intensity or photoperiod, activatedthe BEL5 promoter to its greatest levels in leaf veinsand petioles. In response to SDs, phloem transport offull-length StBEL5 mRNA is enhanced, delivering thismRNA to stolon tips (Banerjee et al., 2006a). StBEL5promoter activity in stolon tips increases in response toSDs as the tuber develops (Fig. 6). BEL5 and its Knoxpartner bind to a specific tandem TTGAC motif on theirtarget promoters to affect transcription (Chen et al.,2004). Such a double motif is present on the promoterof StBEL5 on opposite strands beginning at nucleotide2820 (GTCAATGCTTGAC; Fig. 1B, dotted line). Basedon these observations, it is feasible that StBEL5 protein(with its Knox partner) autoregulates its own pro-moter to transduce a light-mediated signal from theleaf to the underground storage organ, the tuber. Thiswould be consistent with the observations that SDsactivate the StBEL5 promoter in stolon tips (Fig. 6) andthat the P2 construct (missing the putative BEL5/Knoxmotif) is not active in stolons and newly formed tubers(Fig. 5, F and G).

The StBEL5 promoter appears to be regulated by adiverse and complex array of factors. A wound re-sponse was observed on stems, but not leaves, whereasinsect predation activated the promoter in leaves. Pro-moter activity in roots, stolons, and tubers is subject toa process active in the dark, whereas leaves respond tolight. These results are consistent with the fact that theBELL1 family, along with its respective Knox partners,is involved in a number of developmental and meta-bolic processes. Clearly, BELL1-like genes function inmore than just ovule and inflorescence development.For example, Brevipedicellus, a KNOTTED1-like tran-scription factor of Arabidopsis, regulates several genesinvolved in lignin biosynthesis, an important biochem-ical pathway of secondary cell wall growth (Mele et al.,

2003). A BELL1-like gene from rice, OsBIHD1, was iden-tified that functions in disease resistance and pathogendefense (Luo et al., 2005).

Of the numerous signaling pathways mediated bylight or photoperiod, the one most similar to the in-duction of tuber formation is flowering (for review, seeRodriguez-Falcon et al., 2006). Both processes are con-trolled by the action of phytochrome A, phytochromeB, and blue-light receptors (Endo et al., 2007). It hasbeen clearly established that phytochrome B plays apivotal role in regulating tuber formation (Jackson et al.,1996; Jackson and Prat, 1996). Both pathways involvethe mobilization of a photoperiod-mediated signal thatmoves long distance to activate a newly forming sink(Banerjee et al., 2006a; Lin et al., 2007). There are im-portant differences, however. During flowering, the sig-nal moves from a light organ, the leaf, to another organin the light, the shoot tip. Potato tuberization is un-usual in that it involves a signal that arises in the lightand is transported to an underground organ in the dark,the stolon tip. In this light-to-dark model, white lightactivates transcription in the leaf in coordination withphotosynthesis. Both blue and red light, in the optimalranges for the action spectrum of photosynthesis, ac-tivate the StBEL5 promoter (Fig. 2). Under the LDs ofthe growing season, both StBEL5 transcripts and photo-assimilate accumulate in the leaves, ready for delivery.Late in the season, as days shorten, StBEL5 mRNA istransported to the stolon tip, translated, and importedinto the nucleus. In tandem with its Knox partner,StBEL5 could then target genes that activate cell divi-sion and expansion in the subapical region of the stolontip. Adequate supplies of sugar in the leaf are thentranslocated to the newly formed sink for the synthesisof starch in the tuber. In this way, the trigger for thedevelopment of tuber formation, a bioenergic-expensiveprocess, is coordinated with the production of sugarsin photosynthesis.

MATERIALS AND METHODS

Plant Material and Growth Condition

To assess photoperiod effects, transformation was implemented on the

photoperiod-responsive potato (Solanum tuberosum) subsp. andigena (Banerjee

et al., 2006b). In SD-adapted genotypes like subsp. andigena, SD photoperiods

(,12 h light) are required for tuber formation, whereas under LD conditions

no tubers are produced. In vitro transgenic potato plants were grown at 27�C

with a photoperiod of a 16-h-light (fluence rate of 40 mmol m22 s21) and 8-h-

dark cycle in a growth chamber (Percival Scientific). Soil-grown plants were

grown at 22�C with a photoperiod of 16-h-light (fluence rate of 100 mmol m22 s21)

and 8-h-dark cycle for LD conditions and, for a SD photoperiod, plants were

transferred for 14 to 16 d under a 16-h-dark and 8-h-light cycle in a growth

chamber. For light induction experiment, one set of in vitro-grown plants were

kept in a 16-h-light and 8-h-dark cycle for 15 d, one set in dark for 15 d, and

one set in dark for 14 d followed by light treatment for 24 h. For dark treat-

ment, plants were transferred to a dark incubator maintained at 27�C.

Light Source and Energy Measurements

For various light wavelength treatments of transgenic in vitro plants, the

light filters from Carolina Biological Supply (CBS) red 650, CBS far-red 750,

and CBS blue 450 were used. The source of light from the plant material was





adjusted to yield 8 mmol m22 s21 (red and blue) and 5 mmol m22 s21 (far red) of

uniform irradiation as measured by the LI-189 radiometer (LI-COR). White

light was provided from a bank of fluorescent tubes with a fluence rate of

40 mmol m22 s21.

Promoter Deletion Constructs and Transformationof Potato

For the two deletion constructs P1-StBEL5 and P2-StBEL5 (Fig. 4A), genomic

fragments were amplified using PCR with primers 5#-GCTCTAGAAACCT-

GTGGTCGGAGTGGAC-3#, 5#-GCTCTAGACACTCCACAATCTACCGAA-

ACA-3#, and 5#-TACCCGGGATGTAACTAACTGTCTATCTTCGGG-3#. For

amplification of the P1-StBEL5 product, the PCR cycle was as follows: 3 min

at 94�C; 35 cycles (30 s at 94�C; 30 s at 58�C; 1.20 min at 68�C); followed by 68�C

for 10 min. For the P2-StBEL5 product, PCR reaction was performed as fol-

lows: 3 min at 94�C; 35 cycles (30 s at 94�C; 30 s at 58�C; 30 s at 68�C); with a 10-min

extension step at 68�C. The amplified promoter fragments were cloned in

pBI101 vector (Jefferson et al., 1987) and mobilized to Agrobacterium tumefaciens

strain GV2260 by chemical transformation (An et al., 1988). These deletion

constructs were then transferred to potato subsp. andigena employing an

Agrobacterium-mediated transformation protocol (Banerjee et al., 2006b).

Kanamycin-resistant and GUS-positive transgenic plants were selected for

further analysis. The isolation and characterization of the P-StBEL5 construct

was previously reported (Banerjee et al., 2006a). The 2.29-kb upstream frag-

ment was sequenced and analyzed with software PLACE, plant cis-acting

regulatory elements (Higo et al., 1999), and PlantCARE (Lescot et al., 2002).

Histochemical and Fluorometric Analyses

Expression of the GUS reporter gene was analyzed by incubating the

samples overnight at 37�C in GUS buffer containing 1.0 M NaHPO4, pH 7.0,

0.25 M EDTA, pH 8.0, 0.5 mM potassium ferrocyanide, 0.5 mM potassium

ferricyanide, 10% Triton X-100, 1 mg/mL X-gluc (5-bromo-4-chloro-3-indolyl-

b-D-GlcUA). Samples were cleared with 100% ethanol and photographed

employing a Nikon COOLPXX995 digital camera.

Frozen tissue samples were homogenized using 100 mL of chilled extrac-

tion buffer containing 50 mM sodium phosphate buffer, pH 7.0, 10 mM EDTA,

0.1% sarcosine, 10 mM 2-malic enzyme. Samples were then centrifuged at

13,000 rpm for 15 min, at 4�C. The supernatant obtained was used for protein

quantification (Bradford, 1976). Extracts containing approximately 10 mg of

protein were aliquoted and their volume made up to 50 mL with extraction

buffer containing 2.0 mM 4-methylumbelliferyl-b-D-glucuronide and incu-

bated at 37�C for 16 h. The reaction was stopped by adding 200 mL of stop

buffer (0.2 M Na2CO3). The relative fluorescence was measured using a fluo-

rometer (FluroMax-2) with an excitation wavelength of 355 nm and an emis-

sion wavelength at 460 nm. The specific activity of GUS was calculated using a

calibration curve for 4-methylumbelliferone.

Sequence data from this article can be found in the GenBank/EMBL data

libraries under accession number EU200938.

ACKNOWLEDGMENT

We thank Dr. Suqin Cai for her assistance with photodocumentation.

Received July 19, 2007; accepted September 26, 2007; published October 5,

2007.

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A BELL1-Like Gene of Potato Is Light Activated - Plant Physiology

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