Expression of a Plant Cell Wall Invertase in Roots of Arabidopsis ...

Expression of a Cell Wall Invertase in Roots Affects Flowering and Biomass Plant Biology 7 (2005) 471Fig.1 Construct for CIN1 expression in roots.Organization of the T-DNA region of the planttransformation vector pBI121 (Clontech) comprisingthe CIN1 coding sequence behind the1452-bp pyk10 promoter fragment C (AccessionAJ292756). An updated version of theCIN1 sequence was reported to GenBank (AccessionX81792).When we first grew the CIN1-expressing lines on selective mediumunder optimal conditions on plates, no significant differencesto vector only transgenic controls (with Kanamycin selection)or wild-type plants (without selection) were visible.Also, when grown on soil until 10 days after germination(DAG) the transgenic lines were indistinguishable from wildtype. In contrast, from 10 DAG on, ppyk10C::CIN1 plants displayedenhanced overall growth with respect to shoot height,shoot branching, and inflorescence number (Fig. 3A). Furthermore,while wild-type plants started flowering around 24DAG, the transgenic lines displayed an early flowering phenotype,developing the primary inflorescence 4 to 6 days earlierthan wild type (average 3 weeks after germination). After 4weeks, ppyk10C::CIN1 plants were taller and displayed moresecondary shoots (Fig. 3A). To analyze the effects of CIN1 expressionin more detail, we switched to hydroponic growthconditions (Gibeaut et al., 1997). In this hydroponic system,we could observe comparable enhanced development of theCIN1-expressing lines as seen for soil grown plants (Fig. 3B,Table 1). In comparison to wild type, transgenic lines developedshoots and flowers earlier and displayed a higher numberand increased length of secondary shoots with more flowersand siliques (Table 1).CIN1-expressing plants exhibit slightly enhancedcell wall invertase activity in rootsFig. 2 RT-PCR analyses showing CIN1 expression in roots of transgeniclines. (A) A specific 719-bp PCR product can be amplified from transgenicArabidopsis lines (ri5: lane 1, ri7: lane 2, ri10: lane 3) expressingthe heterologous invertase gene CIN1 under control of the pyk10 C promoterbut not from wild-type plants (lane 4). Actin1 control reactionsyielding a specific 391-bp PCR product are shown below each CIN1 reaction.All reactions were run with 30 (upper panel) and 35 PCR cycles(lower panel) to avoid saturating PCR conditions. (B) NoCIN1 expressioncan be detected in transgenic lines ri5 (lanes 6 and 8) and ri7(lanes 7 and 9), which showed highest CIN1 expression in roots, withRNA from total aboveground parts (17 DAG; lanes 6 and 7) or frominflorescences (29 DAG; lanes 8 and 9). Actin1 control reactions areshown below each CIN1 reaction.Expression of CIN1 in roots leads to early floweringFor the further analyses we chose lines ri5, ri7, and ri10, representingstrong, medium, and weak CIN1-expressing lines(Fig. 2A). In the hydroponic system, we determined the totalinvertase activity of cell wall extracts from roots and found,for the transgenic lines, a slight but statistically significant elevationof up to 16% (Fig. 4). However, the overall concentrationsof soluble sugars (sucrose, glucose, fructose) and starchin roots of these lines were not altered (data not shown).Expression of CIN1 in roots leads to an increasein root mass as well as whole plant biomassIn root cross-sections, the CIN1-expressing lines did not showchanges in cell size or cell number (data not shown). However,transgenic lines differed from wild type in root length andeven more pronounced in secondary root number (Fig. 3B).While in the hydroponic system wild-type plants showed anaverage number of 19.6 2.1 first order secondary roots (meanvalue of 8 single plants) with a low degree of higher order sideroots, transgenic ri5, ri7, and ri10 plants developed 27.0 4.6,34.0 3.9, and 28.8 3.7 secondary roots (mean values of 5single plants each), respectively, with a much higher degreeof higher order side roots. Since these higher order side rootswere extremely difficult to count, we wished to quantify thevisible differences in the transgenic lines by determining root

472Plant Biology 7 (2005)C. von Schweinichen and M. BüttnerFig. 3 Phenotypic changes of transgenic CIN1-expressing lines. (A)Four-week-old transgenic lines (ri3, ri5, ri7, ri10) expressing the heterologousinvertase gene CIN1 under the control of the pyk10C-promotergrown on soil, showing enhanced development of above-ground partsincluding increased shoot branching and early flowering when comparedto wild-type control plants. (B) Four-week-old transgenic lines(ri5, ri7) grown hydroponically, showing comparable increase in shootbranching and early flowering. In addition, enhanced root growth androot branching can be seen.Fig. 4 Total invertase activity of cell wall extracts from roots. Theppyk10C::CIN1 lines (ri5, ri7, ri10) were grown hydroponically and analyzedfor apoplastic invertase activity using cell wall extracts fromroots. Data represent mean values of three independent measurements(bars represent the standard error SE). Transgenic lines showslightly enhanced invertase activity over the wild-type control.mass. We measured a clear increase in fresh weight of up to60% as compared to wild-type plants (Fig. 5). Interestingly,not only the root weight was significantly higher but also theshoot fresh weight showed a similar increase (Fig. 5), resultingfrom an increase in secondary shoot number and heightFig. 5 Biomass determination of roots, shoots, and whole plants.Transgenic ppyk10C::CIN1 lines (ri5, ri7, ri10) were grown hydroponicallyand analyzed for their fresh weight of roots, shoots, and wholeplants. Data represent mean values of ten independent measurements(bars represent the standard error SE). Transgenic lines have 20±60%more biomass than the wild type.(Table 1). The relative gain in fresh weight in the analyzedtransgenic lines corresponds with the CIN1 expression levelsdetermined by semi-quantitative RT-PCR (Fig. 2A), indicatinga direct effect of invertase overexpression and root development.The same increase could be measured after freeze-dryingthe root or whole plant material and measuring the dryweight (data not shown), indicating that the increase in rootand whole plant mass is not merely due to elevated water contentbut reflects increased biomass.

Expression of a Cell Wall Invertase in Roots Affects Flowering and Biomass Plant Biology 7 (2005) 473Table 1 Enhanced development of transgenic Arabidopsis plants expressing the cell wall invertase CIN1 gene under control of the pyk10C promoterin comparison to wild-type plants. Mean values standard errors from at least nine independent plants per line are shown. Probability valuesas determined by Students t-tests are indicated by asterisks: * p < 0.05, ** p < 0.01Line 21 DAG 24 DAG 26 DAG 27 DAG 28 DAG 29 DAGNumber ofshootsShoot length(mm)Total numberof flowersTotal numberof siliquesWT 0.25 0.13 0.83 0.11 2.42 0.63 4.00 0.77 6.08 0.92 7.08 1.04ri2 0.75 0.16* 2.63 0.75* 6.38 1.53* 8.00 1.70* 9.88 1.44* 10.88 1.19*ri3 0.56 0.18 1.78 0.64 5.44 1.28* 7.56 1.31* 9.56 1.02* 11.33 0.76**ri5 0.56 0.18 1.89 0.54 5.11 0.82** 7.44 1.02* 9.89 0.82** 11.33 1.01*ri7 0.70 0.21 2.70 0.78* 6.00 1.03** 7.30 1.19* 8.70 1.18 10.30 1.18ri10 0.78 0.32 2.56 1.08 3.67 1.15 5.00 1.46 6.67 1.60 7.67 1.76WT 0.42 0.26 4.08 1.59 24.75 7.88 47.08 12.84 79.00 17.30 112.42 18.80ri2 8.63 4.31 45.63 17.26* 104.00 26.34* 134.63 28.43* 169.38 28.90* 197.13 26.77*ri3 1.44 0.71 16.89 6.69 66.44 17.60* 103.00 20.27* 147.33 20.70* 189.11 19.58**ri5 4.33 2.71 29.56 11.81 76.44 17.98* 116.11 18.66** 157.44 17.85** 187.56 15.77**ri7 8.70 4.35 50.10 18.33 104.30 27.34* 138.00 31.10* 167.80 31.88* 196.80 30.36*ri10 6.78 5.69 23.89 15.55 53.11 23.06 74.67 26.49 100.11 30.53 123.44 32.65WT 0.00 0.00 0.00 0.00 0.33 0.26 0.92 0.48 2.42 0.91 4.58 1.42ri2 0.00 0.00 1.13 0.64 4.00 1.61 8.13 3.29 13.25 5.02 22.63 7.36*ri3 0.00 0.00 0.22 0.22 1.44 0.99 3.33 1.64 7.89 3.17 17.11 6.43ri5 0.00 0.00 0.78 0.46 2.67 1.19 4.89 1.80 11.33 3.31* 18.67 5.26*ri7 0.00 0.00 1.00 0.63 4.90 1.82 * 8.60 3.43* 16.80 5.82* 27.40 8.62*ri10 0.22 0.22 1.00 0.88 4.11 3.20 6.89 4.64 11.00 6.59 15.78 8.93WT 0.00 0.00 0.00 0.00 0.00 0.00 0.08 0.08 0.67 0.36 1.33 0.54ri2 0.00 0.00 0.50 0.38 1.75 0.90 3.00 1.32 5.75 2.14* 10.38 4.06ri3 0.00 0.00 0.00 0.00 0.33 0.33 0.67 0.47 2.11 1.40 4.56 1.96ri5 0.00 0.00 0.22 0.22 0.89 0.51 1.89 0.93 3.78 1.53 6.33 2.14*ri7 0.00 0.00 0.20 0.20 2.00 0.94 3.80 1.43* 6.60 2.41* 12.10 4.95ri10 0.00 0.00 0.57 0.57 2.43 1.97 4.43 3.66 6.29 4.62 10.29 6.78DiscussionCell wall invertases seem to play an important role in determiningsink strength by increasing the sucrose gradient betweenthe phloem and the sink apoplast. In order to modulateassimilate partitioning and to enhance sink strength weexpressed a plant cell wall invertase under the control of thepyk10C promoter fragment. This truncated promoter drivesa characteristic pattern of GUS expression in transgenic Arabidopsisplants during plant development, resulting in rootspecificity in advanced developmental stages (Nitz et al.,2001). When we used this truncated promoter to drive CIN1expression, transgenic ppyk10C::CIN1 plants showed apparentchanges with respect to their flowering time, morphology, andbiomass. Most studies addressing related questions so far haveused a yeast cell wall invertase with a signal peptide for apoplastictargeting (von Schaewen et al., 1990; Sonnewald et al.,1991; Bussis et al., 1997; Sonnewald et al., 1997; Weber et al.,1998; Neubohn et al., 2000; Zuther et al., 2004; Heyer et al.,2004). However, when we expressed the same yeast cell wallinvertase (kindly provided by Prof. U. Sonnewald) under controlof the pyk10C promoter, we could not observe any effectscomparable with those found for the ppyk10C::CIN1 plants, asdescribed below. Furthermore, we used a heterologous enzymefrom a different plant species (CIN1 from C. rubrum) tocircumvent possible effects of endogenous Arabidopsis invertaseinhibitors (Greiner et al., 1998), since recent studies revealedonly limited sequence conservation between invertaseinhibitors from different plant species (Rausch and Greiner,2004).When we examined the ppyk10C::CIN1 plants in detail, wefirst found changes in flowering time. Flowering started 4 to6 days earlier than in wild-type control plants and a higherinflorescence number developed due to more secondaryshoots. A similar phenotype was observed when a yeast cellwall invertase gene (ScSUC2) was expressed under control ofthe meristem-specific KnAT1 gene promoter (Heyer et al.,2004). The pKnAT1::ScSUC2 plants develop more siliques and,in turn, a higher seed yield resulting from enhanced branchingof the secondary shoots (axillary inflorescences). While theppyk10C::CIN1 plants in our study did not show enhancedbranching of secondary shoots, they developed a higher numberof secondary shoots and thus more siliques (Table 1).As an additional phenotypic change in the ppyk10C::CIN1plants, we observed an increase in root fresh and dry weightresulting from longer roots and a higher number of secondaryroots. Furthermore, transgenic plants developed more andfaster-growing secondary shoots leading to a higher freshweight as compared to wild-type control plants. These effectson aerial parts, where the pyk10C promoter is not active, maybe more indirect. Possibly, the increase in root mass, and consequentlyin root surface, allows more effective absorption of

474Plant Biology 7 (2005)C. von Schweinichen and M. Büttnernutrients from the medium, resulting in enhanced overall development.Although we found a slight increase in total cell wall invertaseactivity, we could not measure significant differences in theconcentrations of soluble sugars in roots of the transgenicplants in comparison to wild type, suggesting that the observedphenotypes do not result from changes in the steadystatelevels of these sugars. However, minor changes in theratio of soluble sugars might lead to a short temporal or localincrease in the apoplastic hexose concentration, resulting inhigher cell division activity and, in turn, to enhanced rootlength and root branching. Such prolonged cell division wasfound in large-seeded V. faba genotypes, where prolongedcell wall invertase activity in the seed coat and the resultinghigh hexose conditions, finally lead to an increased cell numberin the embryo (Weber et al., 1996). This idea is further supportedby the finding that CIN1 expression also shows similareffects when driven by a different sink-specific promoter (vonSchweinichen, unpublished).The parallel overexpression of a monosaccharide transporter(AtSTP6; Scholz-Starke et al., 2003) under control of the samepromoter did not further affect the observed phenotypes ofthe ppyk10C::CIN1 plants (data not shown), suggesting that inroots such a transporter is constitutively expressed and thatroot cells always have a high capacity to take up hexoses. SuchAtSTPs have already been identified in Arabidopsis roots(AtSTP1: Sauer et al., 1990; Sherson et al., 2000; AtSTP4: Truernitet al., 1996; AtSTP13: Büttner, unpublished).Taken together, the presented data suggest that the sink capacityto take up monosaccharides is not limited and that thedecision whether sinks are provided with sucrose or hexosesis regulated by the activity of cell wall invertases. 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