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Cloning and characterization of a FLORICAULA/LEAFY ortholog, PFL, in polygamous papaya

Qingyi YU1,2, Paul H MOORE3, Henrik H ALBERT3, Adrienne H K ROADER4 and Ray MING1Received 5 June 2005; Revised 27 July 2005; Accepted 3 August 2005.

Top of pageAbstractThe homologous genes FLORICAULA (FLO) in Antirrhinum and LEAFY (LFY) in Arabidopsis are known to regulate the initiation of flowering in these two distantly related plant species. These genes are necessary also for the expression of downstream genes that control floral organ identity. We used Arabidopsis LFY cDNA as a probe to clone and sequence a papaya ortholog of LFY, PFL. It encodes a protein that shares 61% identity with the Arabidopsis LFY gene and 71% identity with the LFY homologs of the two woody tree species: California sycamore (Platanus racemosa) and black cottonwood (Populus trichocarpa). Despite the high sequence similarity within two conserved regions, the N terminal proline rich motif in papaya PFL differs from other members in the family. This difference may not affect the gene function of papaya PFL, since an equally divergent but a functional LFY ortholog NEEDLY of Pinus radiata has been reported. Genomic and BAC Southern analyses indicated that there is only Elite Womens Darren Sproles jersey one copy of PFL in the papaya genome. In situ hybridization experiments demonstrated that PFL is expressed at a relatively low Mens Darren Sproles jersey level in leaf primordia, but it is expressed at a high level in the floral meristem. Quantitative PCR analyses revealed that PFL was expressed in flower buds of all three sex types male, female, and hermaphrodite with marginal difference between hermaphrodite and unisexual flowers. These data suggest that PFL may play a similar role as LFY in flower development and Elite Blue Darren Sproles jersey has limited effect on sex differentiation in papaya.

Keywords: divergence, floral meristem identity gene, flower development, gene expression, molecular phylogeny

Top of pageINTRODUCTIONFlower meristem identity genes control the developmental transition of plants from the vegetative to reproductive phase. Several flower meristem identity genes have been cloned from Arabidopsis; among them, LEAFY (LFY) is one of the earliest flower meristem identity expressed genes 1, 2. LFY encodes a plant specific transcription regulator 3, 4 and plays a major role in flower initiation 1, 2. LFY transcripts are expressed at low levels during the vegetative phase, which are increased upon the transition from the vegetative to reproductive phase 5. Youth Byron Maxwell jersey Mutations in LFY lead to the conversion of the flower meristems into inflorescence meristems and result in the replacement of flowers by lateral shoots 6, 7, 8, 9. Constitutive expression of LFY promotes reproductive transition and results in early flowering 10, 11. In the complex flower development pathway, LFY is centrally located and acts as a link between the upstream flower timing genes and downstream floral homeotic genes 5. LFY integrates environmental and endogenous signals to control flower timing 12. It has also been shown that LFY activity is required for the activation of all the three classes of floral organ identity genes 4, 13, 14, 15.

Recent studies showed that the complex regulatory network of flower development is largely conserved across distantly related plants, especially among dicotyledonous plant species 16. LFY homologs have been cloned from several species of gymnosperms and angiosperms, including both monocots and dicots 9, 17, 18, 19, 20, 21, 22, 23, 24. Their functions are highly conserved with slight variations among different species. Constitutive expression of LFY promoted flower initiation not only in Arabidopsis 11, but also in heterologous species, such as tobacco 25, aspen 11, rice 21 and citrus 26. Ectopic expression of LFY homologous genes from other plant species in the Arabidopsis lfy mutant can partially or fully complement the lfy mutation 18, 27.

Papaya (Carica papaya L.) is a fruit crop grown widely in tropical and subtropical lowland regions. It is a dicotyledonous, diploid plant species and relatively close to the fully sequenced model plant Arabidopsis from the point of view of family level 28. Papaya is one of the few plant species that produces three Authenitc Black Brandon Graham jersey sex forms, male, female and hermaphrodite, and provides a unique opportunity to examine the applicability of the floral development pathway established in hermaphrodite to dioecious plants. Our previous study demonstrated that papaya has a Authenitc Kids Chuck Bednarik jersey primitive Y chromosome controlling sex determination 29. Cloning and characterization of flower development genes from papaya will help us to understand the roles of floral meristem and organ identity genes in sex determination and differentiation in plants.

Here, we report the cloning and characterization of LFY ortholog in papaya, PFL. It shares a high similarity and identity with the LFY orthologs from other dicot species and its expression pattern is similar to that of other orthologous genes. Our results showed that PFL is a functional ortholog of the Arabidopsis LFY gene expressed in all three sex forms.

Top of pageMATERIALS AND METHODSPlant materialsThree different papaya cultivars, SunUp, Kapoho and AU9, were germinated and planted in Kunia station, Oahu. Young leaf tissues of SunUp, Kapoho and AU9 were collected for genomic DNA Authenitc Mens Byron Maxwell jersey isolation. Hermaphrodite and female flower buds, young leaf tissues and root tissues were collected for total RNA isolation. Shoot apical meristem (SAM), floral meristem and leaf meristem tissues from SunUp and Kapoho were collected and fixed at different floral developmental stages for in situ hybridization.

cDNA library constructionTotal RNA was isolated from hermaphrodite and female flower buds using the method described by Hall et al 30. PolyA+ RNA was isolated from the total RNA using PolyATtract mRNA Isolation System (Promega, WI, USA) according to manufacturer’s instruction. About 5g polyA+ RNA was used to synthesize double stranded cDNA using the ZAP cDNA Synthesis kit (Stratagene, CA, USA). Size selected cDNA (100 ng) was ligated with 1g EcoRI/XhoI digested Uni ZAP XR lambda vector and packaged with GigapackII packaging extract (Stratagene, CA, USA). Libraries of 8106and 3.8106 recombinants, respectively, were generated. A total of 18,432 (48384) recombinant clones for each library were picked and archived in 384 well plates containing freezing medium. High density (18,432 clones per 22.5 22.5 cm2) and low density (4,608 clones per 22.522.5 cm2) membranes were made using a Genetix Q BOT (Genetix, UK).

Southern analysisIsolation of papaya genomic DNA from leaves was based on the procedure described by Tai and Tanksley 31 with minor modifications. Three restriction enzymes, HindIII, EcoRI and XbaI were used to digest papaya Authenitc Black Byron Maxwell jersey genomic DNA from the three cultivars, Kapoho, SunUp and AU9. Genomic DNA Mens Brandon Graham jersey samples (10 g) were incubated Authenitc Blue Brad Smith jersey with 5 U of restriction enzyme/g DNA in a total volume of 50 l at 37 for 4 h. Approximately 10 g digested genomic DNA was electrophoresed through 0.8% agarose gel in 1TBE buffer. After electrophoresis, the gel was blotted onto Hybond N+ membranes (Amersham, UK) using standard methods 32. The probes were labeled by random primer labeling system (RediPrimer II, Amersham, UK). The procedure for hybridization and autoradiography were as described by Chittenden et al 33.

In situ hybridizationApices, flower meristems, and leaf meristems of papaya were harvested at different stages and fixed in FAA fixation solution (3.7% Elite Darren Sproles jersey formaldehyde, 5% acetic acid, 50% ethanol). Samples were then dehydrated in increasing concentrations of ethanol and embedded in paraffin (Paraplast Plus, VWR, PA, USA). Sections (8 m thick) were cut and mounted onto ProbeOn Plus slides (Fisher Scientific, PA, USA). The amplified PFL cDNA fragment was cloned into TOPOII vector (Invitrogen, CA, USA). Antisense and sense RNA probes were generated with opposing SP6 and T7 promoters from the PFL cDNA subclone and labeled with digoxigenin (DIG) (Roche, Germany). The methods for tissue pretreatment and in situ hybridization were as described by Jackson 34.

RT PCR analysisTotal RNA was treated with RNase free DNase I (Promega, WI, USA) and quantified using a spectrophotometer (Shimadzu, Japan). About 5 g of total RNA were reverse transcribed using RETROscript Kit (Ambion, TX, USA). PCRs were performed on the cDNA using the primer Brandon Graham jersey pair PFL Sub10 3 (5′ CCA GAA CAT AGC CAA GGA GC) and PFL 2403 (5′ AAT GGC AAG ACG AGG ATG TG).

Real Time quantitative RT PCR assaysOne primer pair (A1: 5′ ATA GTG CAG GCG CCA GTC AG; A2: 5′ GGC TCA AGC TGT TCA TCA TC) had a Tm of >55 and was Elite Blue Chuck Bednarik jersey designed using Clone Manager 6 (Sci Ed Central) to produce a PCR product of 223 bp. About 2 g of total RNAs were treated with RNase free DNase I (Promega, WI, USA) and reverse transcribed using TaqMan RT kit (Applied Biosystems, CA, USA). cDNA products were diluted Elite Darren Sproles jersey 5.5 fold for use in real time RT PCR amplification. Platinum Quantitative PCR SuperMix UDG (Invitrogen, CA, USA) was used for real time RT PCR amplification. Reactions were run on a DNA Engine OPTICON 2 Real Time PCR detection system (MJ research, MA, USA). Thermocycler conditions were 2 min at 50 followed by 10 s at 95 and 40 cycles of 15 s at 95 20 s at 58 Youth Brandon Graham jersey and 30 s at 72 For each sample, an RNA sample without reverse transcriptase was included to control for genomic DNA contamination. All presented PCR data were generated from a minimum of three independent reactions for each biological replication. Actin gene was used as a standard to control for sample variation in the total amount of RNA in different reactions. Phylogenetic analyses were conducted using the Neighbor Joining (NJ) method as implemented by the PHYLIP program package. Pam matrix was used to generate distance. Bootstrap analysis (1,000 replicates) was performed to assess the support of individual branches.

Top of pageRESULTSCloning of PFLThe Arabidopsis LFY cDNA was used as a probe to screen the papaya BAC library. Two positive BAC clones, 42B17 and 52E1, were identified and confirmed by southern hybridization. The sizes of these positive BAC Elite Black Brad Smith jersey clones were estimated as Womens Brandon Graham jersey 60 kb and 175 kb, respectively. The Mens Brad Smith jersey smaller BAC clone, 42B17, was digested with HindIII and shotgun subcloned into pPCR Script vector. The sub clones were screened with the LFY probe and the positive sub clone was sequenced from both ends. Direct sequencing of the BAC clone was carried out at the 3′ end of the sub clone where the third exon of PFL was interrupted by a HindIII site.

A full length sequence of PFL was obtained through directly sequencing of BAC clone 42B17 via a primer walking approach. Three exons were predicted based on sequence homology and conserved splicing sites. The sizes of three exons were 429 bp, 315 bp and 357 bp, respectively. Two primers were designed at the beginning of the first predicted exon and the 3′ end of the third predicted exon. The Elite Blue Brad Smith jersey cDNA fragment of PFL was obtained by amplification of the cDNA from papaya hermaphrodite flower buds using these two primers. This cDNA fragment was cloned into pCRII TOPO vector (Invitrogen, CA, USA) and sequenced. The sequence result matched exactly the predicted open reading frames. Comparison between PFL cDNA and genomic sequence confirmed that PFL has three exons and two introns and that the splicing sites are highly conserved among PFL, FLO, and LFY.

PFL cDNA was about 1.1 kb and predicted to encode a protein of 367 amino acids. The deduced amino acid sequence of PFL was aligned with other FLO/LFY like proteins (Fig. 1). This alignment revealed two highly conserved regions and two short variable regions. A proline rich region near the amino terminus and an acidic central region were found in the variable regions of most of angiosperm FLO/LFY like proteins. The proline rich region was not found in the papaya PFL. However, a highly acidic region was observed in PFL between glutamate 182 and glutamate 188 where it was preceded by a short basic region (between lysine 171 and lysine 178), which is similar to other FLO/LFY like proteins. PFL also shares with other dicot FLO/LFY like proteins a region containing leucines repeated every 7 or 8 amino acids (positions leucine 69 to leucine 99) and a region with alternating basic acidic basic stretches (positions arginine 115 to arginine 126). The leucine repeats are marked with “”. The basic and acidic regions are labeled as “+” and ” “, respectively.

Full figure and legend (372K)

The comparison of PFL proteins to other FLO/LFY like proteins showed that PFL protein is more closely related to the other dicot FLO/LFY proteins than to their monocot counterparts or to gymnosperm FLO/LFY like proteins. PFL protein shares 66% and 70% similarity with LFY and its snapdragon counterpart FLO. It shares a higher similarity with LFY homolgous proteins of two woody tree species, California sycamore (Platanus racemosa) and black cottonwood (Populus trichocarpa) at 75% and 76%, respectively (Tab. 1). Only Youth Brandon Graham jersey one hybridizing band was seen with Hind III digestion (Fig. 2). One strong band with some weak bands were seen with EcoRI digestion and XbaI digestion. Since the sequencing result showed there are EcoRI and XbaI restriction sites within the PFL gene and only one cDNA fragment was amplified from the mRNA of papaya hermaphrodite flower buds, it is likely that only one copy of PFL gene exists in the papaya genome. This same probe was also used to label the papaya BAC library (data not shown). Nine positive BAC clones were obtained. All nine positive BAC clones showed the same pattern after they were digested with HindIII and XhoI and labeled with the PFL probe. This result reinforced our conclusion that only one copy of PFL exists in the papaya genome.

Figure 2.(A) Restriction map of genomic clone of PFL of selected sites. The probe used in the southern hybridization was indicated in the line underneath. (B) Southern hybridization result of Kapoho (K) and SunUp (S) papaya genomic DNA digested with three different enzyme, EcoRI, HindIII, and XbaI, and labeled with PFL probe. S: size control. The same fragment with the probe was used as the size control.

Full figure and legend (47K)

A phylogenetic tree was constructed using the predicted protein sequences to determine the evolutionary relationships between PFLand other FLO/LFY like proteins (Tab. 2, Fig. 3). The phylogenetic tree shows three FLO/LFY like proteins from two different gymnosperm species, NLY, PrFLL and GinLFY, clustered together and quite separate from the proteins of the angiosperms. Five FLO/LFY like proteins from monocot species, RFL, LtLFY, ZFL1, ZFL2 and JunefLF cluster in a group separate from the dicot species. LFY, VcLFY and BOFH from three different species within Cruciferae formed a distinct cluster. Similar to this, NFL1, NFL2 and TOFL from two different species within the Solanaceae formed another sub cluster. The general phylogenetic relationships found in this tree are consistent with the currently accepted species phylogenies. The PFL protein is closely related to other FLO/LFY like proteins in dicots, but it is very different from its monocot or gymnosperm counterparts.