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The Plant Cel, Vol. 28: 68-80, January 2011, www. plantcellorg® 2011 American Society of Plant Biologists Cytokinin Regulates the Activity of Reproductive Meristems, Flower Organ Size, Ovule Formation, and Thus Seed Yield in Arabidopsis thaliana""= Isabel Bartrina,* Elisabeth Otto,* Miroslav Stmad,® Tor nd Thomas Schmiiling’ “Institute of Biology/Applied Genetics, Dahler Centre of Plant Sciences, Frele Universitat Berlin, D-14195 Berlin, Germany Laboratory of Growth Regulators, Palackj University and Institute of Experimental Botany Academy of Sciences of the Czech Republic, C7-78371 Olomouc, Czech Republic Werner,” The size and activity of the shoot apical meristem is regulated by transcription factors and low molecular mass signals, including the plant hormone cytokinin. The cytokinin status of the meristem depends on different factors, including lyzed by cytokinin oxidase/dehydrogenase (CKX) enzymes. H. show that CKX3 and CKXS regulate the activity of the reproductive meristems of Arabidopsis thaliana. CKXS is expressed in the central WUSCHEL (WUS) domain, while CKXS shows a broader meristematic expression. ckx3 ckxS double mutants form larger inflorescence and floral meristems. An increased size of the WUS domain and enhanced primordia formation indicate 4 dual function for eytokinin in defining the stem cell niche and delaying cellular differentiation. Consistent with this, mutation of a negative regulator gene of cytokinin signaling, ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 6, Which is expressed at the meristem flanks, caused a further delay of differentiation. Terminal cel also retarded in ckxd ckx5 flowers, which formed more cell her activity of the ckx3 ckeS placenta tissue established supernumerary Togeth reproductive development, The increased cytokinin content caused an regulating flower organ size. Furthermore, ovules leading to an increased seed set per siliqu relevance of sink strength as a yield factor. INTRODUCTION Plants grow and form new organs throughout their life cycle, ‘Shoot organs are derived from the growing tip, the shoot apical meristem (SAM) (Steeves and Sussex, 1988). The vegetative ‘SAM forms new leaves, whereas the reproductive SAM, called the inflorescence meristem. produces flowers that form seeds after fertilization. The activity of the SAM is regulated by many factors, including transcriptional regulators, receptor kinases, and plant hormones (Tucker and Laux, 2007; Veit, 2008), Among the hormones, cytokinin has a central function. Metabolism and sighal transduction of cytokinin has been slucidated during recent years (Sakakibara, 2008; Werner and Schilling, 2009) Experimental reduction of the cytokinin status, which has been achieved either by lowering the cytokinin content or by reducing cytokinin signaling, abbreviate the activity of the SAM, demon- strating that cytokinin 's a positive regulator of SAM activity (Wemer et a, 2001, 2003; Higuchi eta, 2004; Nishimura et al 1 Address comespandence to techmuedzedsti-beln. de. ‘Te author responsible fr distribution of material findings presented nth article n accordance withthe policy described Inthe Insvuctons fr Authors (www placer): Thamas Schmling (tsetmuedzedst eer. ce). ‘Dome figures in tis aricle ae spayed in color online but in black and wite inthe prt ection ontne version contains Web-only data 50pen Access articles can De viewed online without a subscription, wor plareellorg/egae/10.1105/se.110.079079, ar differentiation was the role of cytokinin in ‘and became ger, corroborating the results underpin the important role of cytokinin in ~55% increase in seed yield, highlighting the 2004; Riefler ota, 2008; Heyl ota, 2008). In ice (Oryza sativa), tlistuption of an enzymatic activation step of cytokinin by the fog. ‘mutation caused the arrest of SAM activity (Kurakawa et al 2007). By contrast, increased cytokinin production is associated with the formation of larger vegetative meristems (Chaudhury otal, 1983; Rupp et al, 1899). Similarly, mutation ofa cytokinin signaling gene in the aberrant phyllotaxy 1 fabpht) maize (Zea ‘mays) mutant increased meristem size (Giulni et al., 2004) ABPHT encodes an A-type response regulator and thus is ex- pected to play role in negative regulation of cytokinin responses, ‘The cytokinin pathway is linked to transcriptional factors regu- lating SAM activity through reciprocal interactions. involving suppression and enhancement of gene expression (Jasinski et al, 2005; Leibiried et al, 2005; Yanai et al, 2005), ‘The activty of the reproductive SAM Is one parameter deter- mining seed yield. Yield is the most important trait in plant breeding, and yield ennancement is required to meet increasing food demand. In addition, there is an increasing demand for plant-derived products for non-food purposes, such as energy production. Thus, yield enhancement is a contra issue on the dlobal agricultural agenda. Yield 's a complex walt that is gov- femed by many genes (quantitative trait loc), each contributing only a small portion tothe total yeld. Consequently, its aificu 10 achieve large increases in seed yield by altering single or only 1 few genes. On average, the yearly increases achieved by Classical breeding approaches in crop plants are in the range of 1 to 2% per year (Aizen etal, 2008) 70 ThePlant Cel! ‘The breakdown of cytokinin is catalyzed by cytokinin oxi- dases/dehydrogenases (CKXs). Recertly it was shown that the Gota locus of rice, which makes a major contibution to the regulation of grain yield, carries a mutation inthe promoter region ofthe Os-CKX2 gene (Ashikar| etal, 2005). This mutation caused 1 decreased expression of Os-CKX2 in the inflorescence mer~ istem, leading to increased cytokinin content and the formation ‘of more reproductive organs. The dicotyledonous model plant Arabidopsis thaliana codes for seven CKX enzymes, which differ In thelr expression domains, subcellular localization, and blo- chemical characteristics (Werner et al, 2003; Galuszka ot al 2007). Here, it i shown that in Arabidopsis, the simultaneous ‘mutation of two CKX genes delays the differentiation of cells inthe reproductive meristems, thereby causing the formation fof more and larger flowers forming more seeds. Moreover, a previously undiscovered function of cytokinin in regulating the activity of the ovule-orming placenta tissue was revealed. To- gether, these findings lend support for a central function of ‘ytoknin in regulating the growth of reproductive meristems and frgans and pinpoint the relevance of sink strength as a factor determining yield RESULTS Insertional Mutants of CKX Genes To study the functions of individual CKX genes, T-DNA insertion alleles of five of the seven CKX genes were identified by screen- ing diferent insertional populations, and homozygote ines were established (Figure 1). Arabidopsis plants harboring T-DNA in- sertions in single CKX genes showed no gross morphological changes. However, various double mutants, which all included the clo-1 allele, formed more flowers, indicating a more active inflorescence meristem. The activity was strongest when a cke5 insertion allele was combined with a mutated CKX3 gene. Therefore, further work was focused on this mutant combination All reported results were obtained with the allele combination chx3-T chxS-1 (named che ckx5) and were confirmed in the chx3-1 ckxS-2 double mutant. RT-PCR analysis showed that the T-DNA insertions in the ckx-1, chaS-1, and chx5-2 mutants completely abolished the expression of the respective gene (Figure 1). Because only a single ctx mutant allele was avalabe, We further tested the role of CKXS loss of function by transfor~ imation of the ck ckxS double mutant withthe wikl-type CKXS gene. Supplemental Figure 1 online shows that the inflores- Cconces of independent transformants resembled wild-type in- florescences. Tagether, this proves that the mutant phenotype is caused by mutation of CKX3 and CKX8. CKX Genes Regulate Inflorescence Meristem Activity, Flowers are continuously formed by the indeterminate inflores- cence meristem. The ckx3 ckxS mutant produced significant ‘more flowers than the wild type and each of the parental ines (Figures 2A to 20 and 3A), Wild-type inflorescences caried, on the average, eight flowers of stage 13-16 (Smyth et al, 1990), Whereas ckxd ckxS mutants carried 11 flowers ofthat stage, with A ea a 2. — oe Figure 1, Characterization of cx T-DNA Insertion Ales (0) Posttons of T-DNA insertions in the ckx mutans, The length af the genomic CKX gene sequences are given in base pals. The insertonal mutants were identified by PCR screening and the sit of inserton determined by DNA sequercing of the border fragments. Slack boxes represent exons, white boxes represent introns, and viangles incicate (8) CKX gene expression in the wld type (WT) and isertonal mats FRNA ‘fom 10-s-0ld seedlings was used as template forthe RT-PCR. ‘tind was chided a a corti longer pedicels (Figure 20). Scanning electron microscopy anal- ysis revealed the formation ofa larger inflorescence meristem in he ckxS ches mutant compared with the wildtype (Figures 20 to 2), The outer cell ayer of mutant meristems had smaller cells (48.3 = 3.2 cels per 500 um? compared with $0.0 * 7.1 per 500 um? in the wild type; n = 3; P < 0.08, Student's test (Figures 21 land 2J), indicating that an increased number of meristematic cells, and not cel sze, was the cause of the increase in meristem size. The mean number of floral primorcia of stage 2-8 (Smyth et al, 1990) produced by wild-ype and ckxS inflorescence ‘meristems was 9, whereas a ckxd inflorescence meristem pro- ‘duced, on average, 12, and a ckx3 ckxS inflorescence meristem produced 14 stage 2-6 flower primordia (Figures 2D to 2G). By Contrast plants overexpressing a CKX gene showed adrastically diminished Inflorescence meristem and the production of only two stage 2-6 flowers (Figure 2H). These data show that the (CKX3 and CKXS genes are negative regulators of inflorescence meristem size and activ, cckx’ ekxS Mutants Form More Siliques “The larger numberof flowers formed by the inflorescence mer- Istem would eventualy ead to a larger number of sliques. The rrumber of sliques of the main stem after formation of the last flower was compared. The single mutants ckxd and cha pro- duced about as many sliques as wild-type plants. Mutants Cytokinin Action in Reproductive Tissues 71 Cy a MARANA A Figure 2. The Formation of Flower Primaria Increased inthe ck cha Mutant (A) ana (8 Irorescences ofthe wl ype (WT) (A) and la ota (8 (©) Algnment of flowers from wik-type an cad ckxs nforescences, approximately stage 19-16 (Smyth st al, 199). (0) e Scanning etetron mlcrogranhs of the main fewerng apex ofthe wid ype (0), che () kas (kx chs (G), and 355:.CKX(H). The arrow in Q indicates the inforescence meristem, Stage 2-6 floral bude a numbered from the youngest to edest. (@ ard (9) Close-up pictures of wi-ype Band ckxd cha i) inflorescence meristems. Bars = 100 um in () 0 (H) and 10 pm in) ane harboring insertions in the GKX2, CKX4, CKXS, or CKXS genes, as well as various combinations thereot, did not show enhanced numbers of siiques (Figure 3A). However, combination ofa che ‘mutant allele with ckx2, chad, ckxS, oF che significantly in- creased the formation of sliques. The clad cles double mutant showed the largest increase, generating ~40% more sliques than did the wild type. More flowers were formed per time unit (Figure 36), indicating a shorter floral plastochron. Its notewor- thy thatthe length ofthe reproductive phase in lo ckxS plants was not altered. In the later stages of development, mutant plants often showed an irregular pattern of siiques along the stem, indicating that the larger meristem size interfered wi positional signals regulating the spacing andor timing of pri- ‘mordium initiation (Figure 3C), ckxd ckxS mutants formed a stronger stem, whion hadi about a 15% larger diameter com- pared with the wild type (Figure 4). Ths difference is most likely due to increased cambial actity, which is known to be affected by cytokinin (Matsumoto-Kitano et al, 2008; Nieminen ot al 2008), ytokinin Content in ckx8 ckxS Mutants The oytokinin profile of cfxS ckxS mutant inflorescences was compared with that of wild-type inflorescences. Table 1 shows that the levels of the biologically active trans-zeatin and trans-zeatinriboside were approximately fourfold higher ininflo- rescences of ckx3 ckxS mutarts compared with the wild type. The levels of biologically Inactive trans-zeatin-type cytokinins (.e., glucosides and nucleotides) also increased up to eightfold “The levels of the iP-type cytokinins were low, and among thern, only the level of isopentenyladenosine 5'-monophosphate in- creased about threefold, These results indicate that CKXS and (CKXS regulate inflorescence mer'stem activity by modulating the cytokinin concentration and that frans-zeatin-lype cytokinins are the predominant form of the hormone in the inflorescence. Flowor Development of ckx ckxS Mutants, Development of kx3 cha mutant flowers also differed from the wild type. Figure 5A shows that flowers of ckx3 ckxS mutants Were significantly larger compared with wilk-type flowers and flowers of single mutants. The petal surface increased by ~40% (Figure 58), which was due to a larger cell number (Figure SC) Growth of ckxd ckx5 gynoecia, in particular, was enhanced and ner final size was strongly increased (Figure 5D), The increase in stamen sizeof ckx3 ckxS mutants was less pronounced, causing reduced sel-polination of mutant flowers, With low frequency (~10 to 15%6), lowers showed ather morphological abnormalities toa varying degree (e.g., an altered number of floral organs) 72 ThePlant Cel 0 c 35 30 20 5 10 5 ° No.of Flowers and Siiques SP Pe e Figure 3 Lateral Organ Iriiaton and Phllotaxis ef che Mutar (A) The number of silques on the main stem during one Ife cycle is showin (t= 15) Wila-ype (W7) plants formed 587 sigue (100%) (@) Numver o Howers and silques (sage 12-18) of Saveek-ola plans ‘The plants nad slated follower atthe same time. Staging of foral merstems was according to Smyth eta. 1890} =). (6) regular pattem of sigues onthe man stem ofthe chad ckxS mutant {Hight ineated by arows compared wih the wid ype tet). Data represents mean value = £0. “P< 0.01, calulated by Student's test. [See online ate for color version ofthis gure (Figure SE; see Supplemental Figure 2 online). The increased size and cell number of floral organs suggest that cytokinin prolongs the duration ofthe cel division phase during organ growth, ‘ckx’ ckx5 Mutants Form More Seeds Inspection of ckx3 clx5 mutant gynoecia revealed that they Contained almost twice as many ovules as wild-type gynoesia did Figure 5G), indicating an enhanced activity of the mutant placenta tissue (Figure SF). The mutant gynoecia were densely filed, and some ovules were differently shaped, probably due to spatial constraints (see Supplemental Figure 2 online). The ch And ckxS parents also showed an increased number of ovules per gynoecium, with a stronger effect in cx (Figure 5G), To test Whether the increased ovule number together with the larger flower number would lead to the formation of more seeds, chr cha mutants were grown in agrowth chamber, which faciltated sel-polination. Siiques of ckx3 cheS mutants were longer than inthe wildtype (20 mm compared with 17 mm) and contained up to110 seeds, compared with an average of 5 seeds in wild-type silaues, due to more densely packed seeds. The total seed yield ‘of ckx8 ckxS mutants increased by 55% compared with the wile ‘ype (Figure SH). (CKXS and CKXS Expression Pattern during Inflorescence Development (CKxs transcripts were detected n the center ofthe inflorescence ‘meristem and in the floral meristems of stage 2to stage S flowers (Figures 6A to 6D). This coincides with the expression of WUSCHEL (WUS) Figures 6E and GF). However, CKXS expres sion appeared later in flower development than that of WUS, Which is seen already in stage 1 flowers (Mayer etal, 1988), and its expression domain became less detined, appeating in stage '5 mainly in the region between long stamen primorcia and gy- roecia primordia Figure 60) and disappearing thereafter. CKXS ‘expression was strong in the procambium of the inflorescence stems and flowers (Figures 6G to 6), Weaker CKXS expression was also detected in the inlorescence and floral meristems. In developing flowers, CKXS transcripts were detected in stamen ‘and gynoecia primordia during different developmental stages (Figures 6J and 6K). During gynoecia development, CKXS RNA ‘accumulated in the medial cells of the cylinder where the two ‘main vascular bundles bagin to ifferertiate at stage 7 (Figure 6) (Forréndiz ata, 1988). At stage 8, the transcripts accumulated specifically atthe flanks of the medial ridges marking the devel- ‘oping placentas, and they were detected in the inflated ovule primordia during stage @ (Figures 6M and 6N). During ovule sitferertation at stage 10-12, CKXS was expressedin the ces of the chalazal region crectly adjacent tothe nucells but tin the sitferertating integuments (Figure 60) Gr Figure 4, Phenotype ofthe Inforescence Stem of ckx? ckaS Mutants Compared wth the Ws Type. (0) Te intorescence stem of eka cha mutant (ght thier than in the wild type (WT, The insets show a close-up of the inflorescence (@) ana (0) Hana-cut transverse section of the inflorescence stem ofthe wi ype (B) and the ck cke5 mutant (C) stained with tohine bse Sections wore made the bate ofthe stm of Seweek-old plats Cytokinin Action in Reproductive Tissues 73, Table 1. Ojoknin Contert of Iforesvences Genotype (yiokinn Metaborte z ry RMP. a ‘zoa wits type 4a tor=s9 1746 = 794 23203 34>08 9 clos 165297 4605.4 11280 + 2958 180220 wast 12208 P ier ieRMP ipos Wile type age 028 = 009 016 = 013, 300 = 102 0.20024 fad os 2628 028 = 010, 035 = 010 1005 + 250 0.20 = 008 ‘Approximately 05 g of Arabidopsis inferescences per sample was harvest were harvested for each genotype. Data shown are pmolgresh weight = 30,7 and pooled 20 after germination Five independent ological samples Fibatige 5'-monephorphate; 1296, trans-reatn &-ghcoside; tZOG, trane-reatn O-glucoide, ZROG, tanz-reatn riboside O-ghcosie” iP, N (a%isopentenyfacerre; (PR, N{A%sopenteryadenesine; RMP, N*Aisopentenyaserosne 5 -monopnosphale;IPBG, N-(Wisopentenyaderine CKX Genes Regulate the Expression Domain of WUS The WUS/CLAVATA (CLV) pathway regulates SAM size, and the domain of WUS expression becomes larger in mutants with a larger meristem (Carles et al, 2004). To study whether this Central regulatory module was affected by the ckx3 ckxS muta- tions or whether meristem enlargement occurred independently, the WUS expression domain was analyzed by in situ hybriciza- tion, Figure 7 shows that the WUS domain marking the organl2- Ing center of the SAM was enlarged in che3 clxS mutants, Suggesting that the cytokinin status regulates the size of the WUS expression domain and, eventually, the WUS expression level ‘Mutation of AHP6 Causes a Higher Activity of the Inflorescence Meristem "Next, we investigated whether ather components of the cytokinin system exert an addltive negative regulatory activity in the inflorescence meristem, To this end, the afpS mutation was infrogressed into the clesd ckxS mutant background. ARABI- DOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 6 (AHP6) joncodes a pseudo-phosphotransfer protein that has been shown to act as an inhibitor of cytokinin signaling during forma- tion of root vascular tissue (Mahdnen et al, 2006). However. its regulatory actvity in other developmental programs is unknown, In situ hybridization revealed expression of AHPS inthe inflores- ‘cence meristem atthe position where the nex lower primordium will arise (stage Po) and in early stage 1 primordium, in floral organ primordia of stage 3 flowers, and in the distal part of developing gynoecia in stage 6 flowers (Figures 8A to 8C) Analysis of mutants harboring the afp6-1 or ahpé-3 null alleles (Mahénen et l., 2008) showed that the size ofthe inflorescence meristem was only marginally increased (similar results were ‘obtained for both ahs mutant alleles). However, the combin: tion of ahps with ckxd and, in particular, clo3 ckx6 increased further the size ofthe inflorescence meristems and formation of flower primordia (Figures 8D to 8) At the end of ther ife cycle, cha ches ahps triple mutants hac 23% more siiques on the main stem compared with ckx3 ckeS and 47% more compared with the wild type (Figure 8). These results clearly show that AHP negatively regulates the activ of the inflorescence meristem depending on the cytokinin status, By contrast, ower size and the number of ovules per gynosclum was similarin the chad che and ckr3 ckxS ahp6 mutants (Figure 8K, indicating that AHPS ray not be involved in regulating lower size and the activity of the placenta. DISCUSSION ‘Several actiities of cytokinin and in particular of CKX and CKXS in regulating organ size were revealed in this study. Fist, the size ofthe reproductive meristems of Arabidopsis was promoted by cytokinin. An Increase of endogenous cytokinins in inflores- ccences caused the formation of larger inflorescence meristems ‘capable ofintiating more flower primordia. The larger number of meristematic cells indicates that cytokinin delays the develop ‘mental switch that causes cells to exit from thelr meristematic cell cycle activity. Second, the enhanced cytokinin content in- creased the cell number and, thus, the size of floral organs and particularly ofthe ovule-bearing gynoecia. Third, the capacity of the placenta tissue to initiate ovule primordia was enhanced, resulting in a higher density of ovules and seeds within the ‘carpels. Together, these changes led to an increase in seed yield ‘of ~55%. An even higher increase could potentially be achieved but was probably restricted by a heterostylous phenotype that reduced sell-feriizaton Cytokinin Regulates the Activity ofthe Reproductive SAM by Distinct Mechanisms Iwas found that CKX8 and other CKX genes regulate the size of the reproductive SAM ina partially redundant fashion. The CKX3 ‘expression domain was similar to that of WUS in inflorescence and floral meristems, although in the latter its expression appeared later and ceased earler than that of WUS. Therefore, (CKX3 may be part of the circuy regulating the cytokinin status and, thus, the siz of the WUS domain. Indeed, itwas shown re- cently that cytokinin acts as a positional cue for patterning WUS expression in both a CLV1-dependent and CLV1-independent fashion (Gordon et al, 2008). WUS, in tum, ctectly represses ne expression of several A-type response regulator (ARR) genes that code for feedback repressors of the cytokinin signaling pathway and, tnus, increases the cytokinin status in the organizing conter (Lelbftied t al., 2005; Zhao etal, 2010) 74 ThePlant Cel Petal Surface (mm!) No. of Flower Organs WZ “WT ed oles “ae —sageav Figure 5, Flower Phonetype and Seed Vel of che Mutants. uBee 25 ‘No. of cells/Surface Area Sepals Petals Stamen Carpels iG Hg 150 gus Eos $00 F100 Bos Bos & 50 zs dos 5s 8 6 Boo Se ee we ° ° (0) Stage 13 flowers. From let orght the wld type, che, has, and clad cs, (6) Petal surtace of cha mutants, stage 14 flowers, 38 DAG (n (6) Cell rurmber per surace area in wie-ype and clas cheS mutant petals 30) WT, wits ype 1) (0) The coresponing gynoeca of lowers shown in (A). From kt right the wil ype, ck, cS, and eh eo, (G Floral organ number per fewer the wi type, ced, eka, and efx che (n= 50) {F Young ovale ofthe wild type and clx3 cls. Staging of ovules is accoring o Schnetz otal. (1895). Bars (G) Number of eves per gyraeceum 12. 0 wm. (0 Seed yield ofthe wld type an cd ca undergrowth chamber condtions fo = 20. Data represents mean vale = 82, < 0.01, ealeulted by Student's test, Furthermore, cytokinin downregulates the expression of the CCLVT gene, which functions to restric the WUS domain (Brand ft al, 2000; Schoot et al, 2000; Lindsay et a, 2008; Gordon et al, 2008). Together, this indicates that the cytokinin status of the organizing canter is relevant for determining the size of the stem cell niche and of the meristem, However, mutation of CKX3 alone was not sufficient to increase strongly meristem size, and (CKXS was expressed more broadly in the meristem. Thus, the cytokinin status outside ofthe central domain might be relevant as well, and another aspect of cytokinin activity in the SAM may ‘operate independently of the WUS pathway. An essential tran- scription factor for SAM function, STM, which is required for cell division in the peripheral zone and to maintaln meristem cells in he uncitferentiated state (Endiizl etal, 1986; Long etal, 1996) induces cytoxinin-synthesizing IPT genes (Jasinski et al. 2005; Yanai etal, 2005) and is itself subject to regulation by cytokinin (Rupp etal, 1999). An enhanced cytokinin satus ofthe SAM as a Consequence of CKX gene mutation may enhance STM expres- sion and thus increase meristem actvity. The links between cytokinin and the network of transcription factors and receptor kinases regulating SAM ientty and size are summarized in a ‘model shown in Figure 8 Enlargement of the SAM may be the result of ciferent causes. It may be due to a disturbed transition of cells ‘rom the central zone to the periphery or due to an accumulation of cells atthe periphery (Laufs et al, 1988). Thus, It could be that cytokinin Cytokinin Action in Reproductive Tissues 75, Figure 6, C18 ancl CKXS mRNA Expression Patem in Inforescence and Flower Tissues RNAocalzaton by insta hybridztion with GKXS (At [}), US (E] ans (FD, and CKXS (1] to [O} antisonse probes hyisized to wikttype iss. ) to () and () 0 (0 show longitusinal sectons though inforescence meristems, feral merstems, accorsing to Smyth et al (1980, Bars =25 wm. (A) anal (8) CHS is expressed inthe center of the in lowers. Staging of Heal meristems is scence meristem (A) and nthe center af the floral meristem at stage 2 (8 (6) in stage 4 lowers, CKXS is expressed in a broader central area ofthe fal meristem. (0) n stage 5 flowers, the CKX signal appears batwo ong stamen primerdia and gyroecia primera. (ard WLS is expressed nthe ergaizng center ofthe ierescence (E) and forl meristems (F) durng ferent developmental stages (a stage 4 flower is shown hes). (6) to () CKXS is expressed inthe procamblum of inflorescence stems ((G] and (HD and flowers () H) shows a transverse seeton trough an inflorescence stem below the meristem. (1) 01 is expressed in stage 6 lowers between long stamen primordia ard gynoscla primordia (arowheadk) (19 C1065 is expressed in stage 7 loners. (to 0) Transverse section rough ovaries. CKXS ransripts are detected in he developing placentas of stage 7 (L). stage & M), stage 8 (Mand sage 11 (0) owers ‘operates through aifferent pathways to regulate SAM size. Itis ‘tempting to speculate that cio loss-of-function causes an enhanced oytokinin concentration in the central zone and alters ‘a morphogenic gradient that decreases toward the flanks of the ‘meristem and regulates organ initiation andor the establishment ‘oforgan boundary demain. Interestingly, the highest expression of CKXS was foundin the procambial cells, suggesting thatit also has, beside its possible role in regulating vascular differentiation (Mahénen etal, 2000), the function of restricting cytokinin flow: derived trom the vasculature, Together, cytokinin gradientsin the ‘SAM could provide positional information regulating cell difer- entiation ‘Consistent with a role for eytokinin atthe meristem borders is the expression of AHPS in differentiating cells at the meristem flanks and the increased inflorescence mer'stem size caused by _ahp6 mutation inthe efx mutant and even more so in the ck ‘hx double mutant, The sight increase of meristem size caused by the simultaneous mutation of the CKX3 and AHPS genes, hich are expressed instinct meristematic domains, indicates the existence of a non-cel-autonomaus regulation of meristem Ze by these genes. The consequences of a/p6 mutation were rot strong and consistent in the wild type under our growth Conditions, indicating that the cytokinin system is normally well buttered. Interestingly, another negative regulator of cytokinin signaling, the maize A-type response regulator-encoging ABPHT ‘gene, is expressed also atthe site othe incipient leat pimordium atthe meristem border, and its mutation causes the formation of Figure 7, Detection of WUS mANA Expression in the Inflorescence Merster, (9 Tre wild ype, (8) The chs cia5 mutant “Transerpts were ented by in stu hybridization, Bas = 25 wm, 7% ThePlant Cell J 00 K 90 ie 5 200 0 He go 20 2 30 2 gz ° S&F 3 Pr SC CK Fe e Figure 8. Enhanced Cytokinin Signaling Leads to Even More Active nferescence Meristems (090 (C) mANA localzation by n sit hybridization with AHPE antisense probes hybridizes to wik-ype tissues. AHPS is expressedin the nforescence erste a the postion where the nex lower primordia wilaise athe youngest primordia (A). APS transcripts ean also be detected in oral organ primordia of stage 3 towers (B) and in stage 6 flowers a he dstal end of tre developing gyneecium (C) (0) fF Scanning electron micrographs afinforescerce meristems of 4-weok-ol8 wike-ype (), ckx3 ch (E),ahp6 (Fc ap (), kx ap (H, and old cog ah () plants Floral buds are numbered rm the tt prmordum of stage 2 tothe fst primordizm stage 2 (9) ck cha anos mutants form more slques on th main stem than do ck clas mutans a = 20). (09 The gynoecia of ck eka aps ipl mutants do nol form more ovules than cad eke mutants do (p= 18) ‘ars ~20 um (A] to [Ch an 80 yan QD] te [F Data represents mean value= £9, Values otmutant ines were compared wit he wid ype. “P< 00, caleulated by Students Ctest wus ———> meristem activity he eee ARR <— oytokinin < pT< STM Via (CKX3) CKXS) other CKXs) Figure 8. Mode! of CKX and AHPE Gene Function in Regulating Ino rescence Mefstom Size and Acti “The model shows the action of CKX and AHPS genes and cytokinin in concert with other known factors reguing shoot meristem size in ‘Arabidopsis, The model integrates results ofthis arte ang ther wore Sescribd in tho text The gones stud inthis work are shown in gray oes. larger meristems (Giulini et a, 2004). In Arabidopsis, mutation of several A-type response regulator genes was necessary to alter SAM activity (To etal, 2004; Leibtied et al, 2005). Thus, negative regulation of cytokini-induced expansion of the shoot ‘meristem at the meristem borders is relevant in both monocots and dicots. The regulatory modules might have been at least partly conserved since the menocot-cicot divergence. Cytokinin Regulates Gynoecium Size and Placenta Activity This analysis revealed an as yet unanticipated role for cytokinin in regulating gynoeclum size and the activty of the placenta. The molecular events that govern development of the placenta Which isthe intemal surface of the carpels giving rise to ovule primordia, and ovule formation are not well understood. Re- rmarkably, however, the same genes, including WUS, involved in regulating SAM activity seem to play a role, suggesting the ‘operation of related mechanisms (Skinner etal, 2004).CKXS and CKXS may regulate the activity of meristematic cells in the placenta and thus influence organogenic capacity and ovule primordia formation. In particular, the distance between two ovule primordia is regulated by cytokinin. A second distinct ‘mechanism may be relevant as well Given hat a defined number Cf cells in the floral meristem give rise to each carpel (Bossinger and Smyth, 1896), CKX activity in the floral meristem may affect the population of cells that are recrutted into the gynoeclum primordium and thus determine the final gynoecium se. ‘The results discussed above highlight the role of cytokinin in regulating shoot organ initiation and size. A recurrent theme is the regulation of the ext of cells from their meristematic phase. Cytokinin retards this diferentiation step, resulting in an in- creased organ cell number. interestingly, this activity inthe shoot forgans is opposite to that in the root, where cytokinin causes an cater cellular dierentition of meristematic cells (Werner et al 2008; Dello loio et al, 2007). Obviously, the final size of plant organs is influenced by numerous factors (Bégre et al, 2008; Krizek, 2008), including AINTEGUMENTA, KLUH, and ARGOS, mediating auxin activity (Mizukami and Fischer, 2000; Hu etal 2008; Anastasiou et al, 2007), and BIG BROTHER and DA1 Cytokinin Action in Reproductive Tissues 77 functioning in the ubiquitin proteasome pathway (Disch et al. 2006; Leta, 2008). Iwill be interesting to study how cytokinin is linked to these other factors regulating organ size, Cytokinin Is an Evolutionary Conserved Yield-Regulating Factor ‘The resuits reported here extend the concept of cytokinin as a factor regulating yield, which was first noted during the analysis of the Gnta (Os-CKX2} gene in rice (Ashikari et al, 2008), to a dicotyledonous plant. In contrast with rice, where mutation of a single gene was sutficient for yield enhancement, mutation of 1wo CKX genes was needed in Arabidopsis. One of these, CKXS, Is the closest Arabidopsis homolog of Os-CKX2, but GKXS is ‘more distantly related. There are some fundamental ctferences between the developmental events leading to flower or spikelat formation in Arabidopsis and monocotyledonous plants such as rice. Arabidopsis produces floral meristems directly from the inflorescence meristem with indeterminate growth habit. By Contrast. inrice the inflorescence (rachis) meristem forms bracts and inflorescence branches, and eventually aborts, The flowers are then formed from spikelet meristems developed from primary and secondary branch meristems, which are axillary meristems {toh et a, 2005). An additional cifference is that the highest expression of Os-CKX2 was found in the vascular tissues of developing culms, suggesting that Os-CKX2 plays a role in regulating cytokinin levels in the vascular system, hence cytoki- rin transport to the inflorescence meristems (Ashikari et al 2005). By contrast, the expression of AI-CKX3 in the organizing Center suggests its role in meristem-autonomous modulation of the cytokinin concentration, Iwi be interesting to study how the lexpression and regulatory functions of CKX genes have been adapted during evolution to cope withthe specific developmen- tal differences between the reproductive tissues of Arabidopsis and rice, Despite these differences, the role of CKX genes in regulating seed yield nas been evolutionary conserved, suggest- ing functional relevance for all or most flowering plants Importantly, yield enhancement in ckx mutants was obtained without increasing the strength ofthe CO,-fixing source but was {dependent on the strength of the sink issue its capacity to make use of the fixed carbon). This resuit supports models ‘considering the sink strength as an important factor in regulating allocation of fxed carbon (Marcel's and Heuvelink, 2007) and, more specifcaly, underpins the relevance of regulating the differentiation and activity of reproductive meristems as a factor termining sink strength ‘The increase in seed yield obtained by chor gene mutation Is high compared with an average annual increase of 1 to 29% achieved by classical breeding strategies (Aizen et al, 2008) land may be instructive for application. Increasing plant yield is an important agranomical goa, in paticular in view of the chal- lenging future increase of demand for food and plant-derved products for industial applications (Borlaug. 2007; Edgerton, 2008), The findings reported here might be especially relevant to achieve yield enhancement in closely related crop species such 1s ciseed rape (Brassica napus), one of the most important sources of vegetable cil, However, it should be noted that crop plants have undergone selection for yeld enhancement, which is 78 ThePlant Cell not the case in Arabidopsis. Therefore it could be that beneficial alleles of CKX genes have already been accumulated in crop plants during the breeding process, METHODS: Plant Material and Growth Conditions The Columbia-0 ecolype of Avabidopstthalana was Used as the wit ‘ype. Line 35S:CKXt was described previously (emer et al. 2003). The T-DNA insertion mutants cln2-1 (SALK 068485), ckx3-1(SALK_050838, fad-1 (SALK 055204), chaS-1 (SALK_084909), and ckx6-2 (SALK_ 1070071) were fom the Sak lnsttute Genomic Analysis Laboratory (Alonso etal, 2003), and cixs-2 ine 1D 832810) was from the GAB KAT ellction (Rosso et al, 2003). Te aNpé-1 andahp6-s mutants were Seseribed by Mihénen etal, (2008). Genotyping was performed after [DNA tsolaton by PCR with gene-specife and T-DNA border arimers listed in Sunplemental Table 1 online or derived cleaved-amplfed palymorshic sequence markers in the case of ahp6-1 (Mahan et 2006), Downle ant trple mutant Were obtained by crossing. Plants were ‘Foun in ne greenhouse on soi at 22°C under long-day conditions (6h lghvh dar. Plan's were grown ingrowth chambers (Percval AR-66l) fonsollat24'C alightintonsty of ~100 ymolm=ts-*, and65% humity Linder long-ty conditions for seed yield measurement [Analysis of CKX Gene Expression Total RNA was extracted trom seedings according to Verwoerd t aL. ($99, The RNA was treated with RNase-free ONase | Fermentas) at 37°C for30 min, One merlterof25 mM EDTA was ade at 65°C for 10 min, RNA (0.5) wae used for RT-PCR, Primers uted forthe respective (kx gene are Istedin Supplemental Table 1 online. Alloximer pas span the respective T-DNA insertion ste. all RT-PCR reactions, the Actin? {gone was used asa contl, RT-PCR was performed wth the One-Step BRT-PR wi (Qiagen) accoraing tothe manufacturer's nstuetons, The PCR comprised 35 cycles of 30s at94'C, 40.at57°C, and 2minat 72°C. Scanning Electron Microscopy ‘Scanning electron microscopy was performed as described (Krupkové ‘tal, 2007 using a LEO 480 meraxcope (Zeiss, Determination ofthe Cytokinin Status Plants were grown on solunt the maln infrescence was ~10 em high (~S0dater germination), Foreacn sample, -0.5gotinforescences wth stage 1 to stage 15 flowers (Smyth ota, 1980) were pooled, and fve Independent samples were collected and analyzed for each genotype The oytokhin content was determined by uulrapertormance. igh ehvomatography-electaspray tandem mass. spectrometry Novak etal, 2008, Petal Surface and Cell Size Measurement ‘The aoa of petals was measured from digtal images of cssocted organs with the Scion mage program. Petals wee cleared (Malamy and Beniey, 1907), and average cell szes were cakeulated fom trerumber ofcells per Lust area of gal micrographs. In situ Hybridization “Te CK probe fori stu hybratization was amplified by RT-PCR fom {otalmANA of 10-d-0k wi-ype seedings. RT-PCR was performed with the One-Step RT-PCR kt (Giager) according to tho manutactuy's Instuctions. The CKXS probe was amped from the clone RAFL24-08 Kos (GenBank number AK176978.), and the WUS probe from the cDNA. clone PENTR221-AT2G17850 (GenBank number D465). Primer sequences se detaledin Supplemental Table Tenne. Ariizense probes wore produced byin vir anseription with igoxigerin-11-UTP using T7 FRNA polymerase Roche). In situ hybrdkzation was performed! according to stanaar protocols (Jackson eta. 1894 Light Microscopy ‘Gynoecia were cleared anc mounted as described (Malay and Bente, 1997) for ove counting art observation, Hand-cut cross sections of the base of stems ftom S-weekld plants were stained for 5 min 0.023% aqueous ohicine blue 0, rinsed, and mountedinwater,Allsamples were ‘wowed with an Axioskop 7 plus merotcepe (Zee), (CKX3 Complementation Construct “Tre 35 promoter of plasmid pBINSMGFP (Werner et al, 2008) was replaced by # synthete elgonuetetise containing EeoRt, Av, Smal and Kenl resvction ses (Werrer etal, 2070).ACCKXS genomic fragment was sunclored via Kpnlthel retretion tes tram pBS-CKXS (Werner al, 2001) to generate an N-terminal fusion with green fluorescent pratein (SFP. Then, a2088-bp promoter agrent ofthe CAS gene was mpl by POR ram DNA at Arabidopsis Calumbla-Dusing appropriate timers (see Supplemental Tabo1 online) and inserted into the Avl’Kpr Ses upstream ofthe CKXG-GFP fasion gone, resuling inte vector DCH (CKXE-GFP, whch was use fr transformation of the ca cls mutant Sequence data from thi artele can be foundin the Arabidopsis Genome Intiatve oF GenBank/EMBL databases urder te fllowing accession numbers: CKX1 (At2g41510), CKX2 (AL2gT9500), CKXS (At5956970 (CK (A0g28740), CXS (A'TQ7S450), CRXE (A19962440}, and AHPE (atigsor00, ‘Supplemental Data ‘Tre following material ave aval inthe online version of this atte, Supplemental Figure 1. Complementation of the ckxS ckxS Mutant Phenotype by the GKXS Gere. Supplemental Figure 2. Developmental Abenations of Floral Organs and Ovules in the cb ofa Mutant. ‘Supplemental Table 1, Primers Used in This Stay ACKNOWLEDGMENTS. 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