Genes uniquely expressed in vegetative and potassium chlorate induced floral buds of Dimocarpus longan

Plant Science 170 (2006) 500–510 www.elsevier.com/locate/plantsci

Genes uniquely expressed in vegetative and potassium chlorate induced floral buds of Dimocarpus longan Tracie K. Matsumoto * USDA/ARS Pacific Basin Agricultural Research Center, Pacific Basin Tropical Plant Genetic Resource Management Unit, P.O. Box 4487, Hilo, HI 96720, USA Received 24 August 2005; received in revised form 29 September 2005; accepted 30 September 2005 Available online 18 October 2005

Abstract Potassium chlorate (KClO3) induced flowering of Dimocarpus longan is a highly effective method for off-season fruit production in Southeast Asia. To begin to understand the molecular basis for longan flower induction, the suppression subtractive hybridization (SSH) technique was used to isolate genes that are differentially expressed in vegetative buds, or in KClO3 induced floral buds. Repetitive rounds of cDNA differential subtraction screening, followed by reverse northern, northern blot analysis and nucleotide sequence determination identified 65 unique genes differentially expressed in vegetative and floral buds. Many of the identified genes have been previously demonstrated to be associated with shoot and floral meristem in Arabidopsis, including Protodermal Factor 1 (LVFS-205), SHEPHERD (LVFS-100) and PISTILLATA (LVFS-199) homologs. These gene sequences validate the results of SSH procedure and represent new nucleotide sequences in longan. Hybridization of clones to different size transcripts that encode fructose-bisphosphate aldolase (LVFS-230), histone H3 (LVFS-44, LVFS-172), malonyl-CoA (LVFS-48) and SAM (sterile alpha motif) containing proteins (LVFS-290) suggest specific members of gene families or alternative splicing of RNA may be involved in longan flowering. Novel gene products not previously associated with flowering have been also identified including carbon catabolite repressor (CCR4)-associated factors (LVFS-323), SAM proteins (LVFS-290), and Notchless-like proteins (LVFS-65). These cDNA sequences represent proteins with potential regulatory roles in longan flowering. # 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Longan; Flowering; SSH; Differential expression

1. Introduction Longan, Dimocarpus longan, is a member of the Sapindaceae, a family that also includes lychee, Spanish lime and rambutan [1]. Longan trees are grown in many subtropical and tropical countries with majority of the production in Southeast Asia and Australia [1]. In the United States, production is concentrated in Florida, Hawaii and California. In 1998, the United States produced 1.4 million pounds of longan with an estimated value of $2.8 million [2]. Floral initiation in longan is often erratic and highly dependent upon the variety and weather, with cool and dry conditions favorable for flowering [1]. Even under favorable flowering conditions, alternate or biennial bearing is a serious problem in longan production [3]. The discovery of potassium

* Tel.: +1 808 959 4358; fax: +1 808 959 3537. E-mail address: [email protected]. 0168-9452/$ – see front matter # 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2005.09.016

chlorate (KClO3) induced flowering solved the problem of alternate bearing and enabled the grower to produce off-season longan [3,4]. Since this discovery, KClO3 is used to induce offseason flowers and fruits in longan trees worldwide [3–5]. Over the last few decades, understanding the mechanism of flowering has been greatly advanced by the use of molecular and genetic tools on model plants such as Arabidopsis thaliana. Flowering in Arabidopsis results from the favorable perception of various environmental influences such as: photoperiod, light quality, temperature and hormonal influences (i.e. gibberellic acid) by a developmentally receptive host [6,7]. Natural variation in flowering of Arabidopsis has been attributed to differences in the sequences of key regulator genes such as FRI (FRIGIDA) and FLC (Flowering Locus C) [8,9]. FLC is a MADS-box transcription regulator that inhibits the floral transition by repression of key ‘‘flower-time integrators’’ such as Suppressor of Overexpression of Contans 1 (SOC1) and Flowering Locus T (FT) which in turn activate floral-meristem identity genes such as LEAFY and APETALA1 [10]. FLC is a

T.K. Matsumoto / Plant Science 170 (2006) 500–510

pivotal regulator of flowering that is the convergence of the autonomous, vernalization and FRI pathways [10]. Mutations to genes that encode components of the autonomous pathway and FRI expression elevate FLC to inhibit flowering; vernalization reduces FRI expression, which in turn reduces FLC levels in part through the chromatin modification of FLC [10]. Identification of the genes involved in Arabidopsis flowering has lead to the isolation of homologous genes in other plants including MADS-box genes from woody tree species [11]. However, the function of MADS-box genes in trees remains largely unknown and there is no direct evidence that genes regulate flower timing in Arabidopsis function similarly in trees [11,12]. Interestingly, the complete sequence of the poplar genome does not contain a homolog to the Arabidopsis FLC gene suggesting trees may have an alternate methods of controlling flowering [12]. In addition to a currently unknown molecular mechanism for flowering in trees, tropical and sub-tropical fruit trees have the additional complication of not having a distinct growing season compared to their temperate counterparts. Suppressive subtractive hybridization (SSH) is a useful tool in the identification of differentially expressed genes involved in many complex processes. This method has successfully identified bract specific genes in the ornamental tree, Davidia involucrata [13] and BTH responsive genes in the tropical fruit tree papaya [14]. Genes involved in flowering have been isolated from carnation [15] and Acacia mangium [16]. The major advantage of SSH is the combination of normalization and subtraction that enriches rare transcripts while preventing the over-representation of abundant transcripts in the cDNA library [17]. Here we describe 65 unique longan genes identified by SSH and confirmed to be differentially expressed in vegetative buds or in floral buds induced by KClO3. Currently, the molecular database of longan genes is minute with less than a dozen nucleotide and protein sequences combined in NCBI (http:// www.ncbi.nlm.nih.gov/entrez). This is the first study to characterize the molecular mechanism of flowering in subtropical fruit trees; by understanding the molecular mechanism of KClO3 induced flowering in longan, we hope to build a model to identify genes involved in this novel pathway for flower induction and to understand the molecular basis of irregular flowering in sub-tropical and tropical fruit trees. This model will be used to discover alternatives to potassium chlorate for longan flower induction and develop techniques that control flowering in other sub-tropical and tropical fruit trees. Genes in the pathways will be used as functional markers in the evaluation of new germplasm accessions and crop improvement. 2. Materials and methods 2.1. Plant material Seven year old Dimocarpus longan ‘Biew Kiew’ trees grown at the USDA/ARS, Pacific Basin Agricultural Research Center (PBARC), Tropical Plant Genetic Resource Management Unit in Hilo, Hawaii located at the University of Hawaii, Waiakea

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Agriculture Research Station were used for this study. The station is 9.7 km outside of Hilo, with an elevation ranging from 175 to 227 m. Mean temperature maximum and minimum is 28 and 16 8C, respectively. Annual rainfall averages 4445 mm and is most abundant during October to February. The soil consists of an extremely stony Papai muck with organic soils formed over mostly fragmental a’a lava. Trees were divided into two groups. The first group of three was treated with 300 g of granular potassium chlorate (KClO3) evenly spread below the outer edge of the canopy of the tree on April 5, 2004 and the second group of three trees was untreated controls located randomly throughout the plot. Inflorescences were visible on KClO3 treated trees 49 days after application while untreated trees remained vegetative. Samples for RNA isolation were collected from a compilation of floral buds of KClO3 treated trees and from vegetative buds of untreated trees. Flower buds were collected from the main stalk of the inflorescence prior to the elongation of the peduncle and before the emergence of the petals from the calyx. Leaf primordia were removed from vegetative buds until the bud was approximately 1 mm in length. 2.2. RNA isolation Total RNA from floral and vegetative tissues was isolated in 1 g batches using RNAqueous Small Scale Phenol-Free RNA Isolation Kit with Plant RNA Isolation Aid (Ambion Inc., Austin, TX). Each sample was dissected, weighed, ground to a fine powder in liquid nitrogen with a mortar and pestle and homogenized in 8 ml lysis/binding solution and 1 ml of Plant RNA Isolation Aid (Ambion Inc., Austin, TX). Each sample was divided into 10 aliquots and processed according to the manufacturer’s instructions. Additional samples not immediately used were stored in RNAlater-ICE at
20 8C (Ambion Inc., Austin, TX). 2.3. Suppressive subtraction hybridization (SSH) library construction One microgram of total RNA from each of the vegetative and floral buds samples was used to synthesize cDNA using the SuperSMART PCR cDNA Synthesis Kit (BD Biosciences Clontech, Palo Alto, CA). The Clontech PCR-Select (BD Biosciences Clontech, Palo Alto, CA) kit was used to construct two reciprocal subtraction cDNA libraries using the floral and vegetative cDNA as tester and driver cDNA for each respective library. Thermal cycling reactions were performed using the PTC-100 or PTC-200 (MJ Research, Waltham, MA) following the Clontech PCR-Select instructions. The resulting subtracted cDNAs obtained from the PCRSelect cDNA Subtraction Kit were ligated to pGEM-T Easy cloning vectors (Promega, Madison, WI). Plasmids containing inserts were selected on LB (Luria–Bertani) medium supplemented with 50 mg/l ampicillin, X-gal (5-bromo-4-chloro-3indolyl-b-D-galactopyranoside) and IPTG (isopropyl-beta-Dthiogalactopyranoside) for blue and white b-galactosidase colony selection. White colonies were placed in 96-well plates

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for storage at
80 8C and used as the template for colony PCR amplification for the creation of cDNA arrays. 2.4. cDNA array differential screening Two rounds of screening were used to identify differentially expressed clones. The first round was conducted using colony arrays. Putative clones identified during the first round served as templates for second round screening using PCR amplification for cDNA arrays. Colony arrays were created by growing subtracted clones in LB plus 50 mg/l ampicillin medium overnight in standard 96-well plates. The bacterial suspension was inoculated onto Hybond-N+ (Amersham Biosciences Corp., Piscataway, NJ) nylon membranes on top of LB plus 50 mg/l ampicillin using a 96-well microplate replicator (Boekel Scientific, Feasterville, PA) and grown overnight at 37 8C. Four duplicate colony arrays were prepared for each plate of subtracted clones. Membranes containing the colonies were denatured in 0.5 M NaOH (sodium hydroxide) and 1.5 M NaCl (sodium chloride) and neutralized in 0.5 M Tris–HCl (pH 7.4) and 1.5 M NaCl. DNA was cross-linked to the membranes using the Spectrolinker XL-1000 UV crosslinker (Spectronics Corporation, Westbury, NY). The cDNA arrays for second round screening were created by using 1 ml of overnight culture as a template for the PCR reaction using the Nested PCR primer 1 (5-TCGAGCG GCCGCCCGGGCAGGT-30 ) and Nested PCR primer 2R (50 AGCGTGGTCGCGGCCGAGGT-30 ) (BD Biosciences Clontech, Palo Alto, CA) and Taq polymerase (Promega Corporation, Madison, WI) using the PTC-200 thermal cycler (MJ Research, Waltham, MA). Five microliters of the PCR product was combined with 5 ml of 0.6 N NaOH and 2 ml of the resulting mixture was transferred to the Hybond-N+ (Amersham Biosciences Corp., Piscataway, NJ) nylon membranes. Blots were neutralized in 0.5 M Tris–HCl (pH 7.4) and crosslinked using the Spectrolinker XL-1000 UV crosslinker (Spectronics Corporation, Westbury, NY). Unsubtracted and subtracted cDNA probes derived from the Clontech PCR-Select (BD Biosciences Clontech, Palo Alto, CA) Kit were labeled with DIG-High Prime (Roche Applied Science, Indianapolis, IN). The blots were processed following the manufacturer’s instruction. Membranes were hybridized at 42 8C in Easy Hyb Solution and washed with DIG Wash and Block Buffer Set (Roche Applied Science, Indianapolis, IN). CDP-Star was used as the detection substrate and the resulting chemiluminescence was documented using a Lumi-Imager (Roche, Indianapolis, IN). In addition, cDNA arrays from the second screen were also hybridized to cDNA synthesized with the SMART cDNA Library Construction Kit (BD Biosciences Clontech, Palo Alto, CA) from total RNA extracted from floral and vegetative bud samples stored in RNAlater-ICE (Ambion Inc., Austin, TX). 2.5. RNA gel-blot analysis Ten micrograms of total RNA were separated on a 1.5% agarose-formaldehyde denaturing gel. RNA was transferred

to Hybond-N+ nylon membrane (Amersham Biosciences Corp., Piscataway, NJ) via upward capillary flow in 10 SSC and cross-linked to the membrane using the Spectrolinker XL-1000 UV crosslinker (Spectronics Corporation, Westbury, NY). RNA transfer efficiency and integrity were evaluated by visualization of the RNA under UV illumination. PCR products of the subtracted cDNA clones were labeled for 20 h with DIG-High Prime (Roche Applied Science, Indianapolis, IN); hybridized overnight at 50 8C in DIG Easy Hyb, washed and processed with the DIG Wash and Block Buffer Set (Roche Applied Science, Indianapolis, IN). CDP-Star was used as the detection substrate and the resulting chemiluminescence was documented using a Lumi-Imager (Roche, Indianapolis, IN). Signal intensities were quantified for pixel density using the gel analyzer plugin ImageJ 1.34j and the fold induction was determined by the ratio of floral bud intensity/vegetative bud intensity ([18]; http://www.rsb.info.nih.gov/ij/). 2.6. Sequencing and homology analysis Plasmids were purified with the Qiaprep Spin Miniprep Kit (Qiagen Inc., Valencia, CA) and sequenced with the T7 promoter or SP6 primer at the University of Hawaii at Hilo EPSCoR Genetics Core Facility. Sequence homology searches were performed at the NCBI using the BLAST program (http:// www.ncbi.nlm.nih.gov/BLAST/) [19]. Enzyme information and EC number was obtained using BRENDA the comprehensive enzyme information system (http://www.brenda.unikoeln.de/) [20]. 3. Results 3.1. Prioritization of cDNA from SSH library To identify genes involved in the promotion and repression of longan flowering, two reciprocal SSH libraries were constructed to identify genes preferentially expressed in both floral and vegetative buds. A total of 2112 clones from each SSH library, one enriched for genes expressed in floral buds and the other enriched for genes expressed in vegetative buds, were plated as colony arrays onto nylon membrane for hybridization. The membranes were hybridized with DIG labeled cDNA prior to (unsubtracted) and after (subtracted) the subtraction hybridization. To confirm these results, approximately 700 clones from each library were amplified by PCR and placed onto nylon membrane and hybridized to unsubtracted and subtracted cDNA probes. Since the unsubtracted cDNA used in the library is subjected to molecular modifications such as RsaI digestion and adaptor ligation, a reverse northern was performed to confirm differential expression. Northern blot analysis was performed on clones identified to have differential expression in floral and vegetative buds. A total of 419 northern blots were performed, 249 from the SSH library enriched for floral expressed cDNA and 170 from the SSH library enriched for vegetative expressed genes. Northern blot analysis confirmed differential expression of 360 (85%)

T.K. Matsumoto / Plant Science 170 (2006) 500–510 Table 1 Differentially expressed cDNA clones isolated multiple times Clone number Number of isolates Similarity LVFS-267 LVFS-152 LVFS-323 LVFS-10 LVFS-28 LVFS-89 LVFS-104 LVFS-411 LVFS-186 LVFS-232 LVFS-297

38 12 5 4 4 3 3 3 3 2 2

21 kD seed protein/trypsin inhibitor Pollen allergen-like protein CCR4-associated factor Elongation factor 1-a Leucoanthocyanidin reductase Metallothionein like-protein En/spm-like transposon protein Mannose/glucose-specific lectin Unknown protein At1g53560 Lipid transfer protein precursor Translation initiation factor-like protein

clones from which, 197 clones with the greatest difference in expression were selected for sequence analysis. 3.2. Sequence analysis Of the 197 sequenced clones, 63 clones did not align with sequences in the NCBI database; an E value of 10
5 or lower was considered as a significant match. Of the remaining 135 clones, 11 clones were isolated multiple times (Table 1), 65 clones aligned to unique gene sequences, and were given the designation of LVFS (Longan Vegetative Floral Subtraction) (Table 2). Clones that were isolated multiple times often appeared to represent separate cDNA ligation events since the sequence flanking that of the vector sequence was different in the majority of the clones. This suggests these clones were occurred in a greater frequency in the library after subtraction hybridization. 3.3. cDNA clones abundant in longan vegetative buds Clones that were more abundant in vegetative buds compared to floral buds include proteins involved in general metabolism, mRNA regulation, calcium and transcriptional signaling and plant defense (Table 2). Translated LVFS-254 is homologous to granule-bound starch synthase, which may contribute to the accumulation of storage reserves. Also highly expressed in vegetative buds is LVFS-22, which encodes a protein homologous to the Arabidopsis light-harvesting chlorophyll a/b binding protein (LHCB5/CP26), a monomeric antenna protein in photosystem II (PSII) [21,22]. LVFS-181 encodes an isoform of adenosine kinase (EC 2.7.1.20), which is involved in the recycling of adenosine to AMP by phosphorylation (ATP + adenosine = ADP + AMP). LVFS-195 encodes the E1 beta subunit of the pyruvate dehydrogenase complex that is involved in the oxidation and decarboxylation of pyruvate from glycolysis to form acetyl-CoA, which enters the tricarboxylic acid (TCA) cycle. LVFS-261 encodes a protein homologous to hyuC from Pseudomonas, an N-carbamyl-Lamino acid hydrolase (EC 3.5.1.87) responsible for the conversion of N-carbamyl-L-amino acids to L-amino acids [23]. Proteins associated with cell division and signaling that displayed relatively higher expression in vegetative buds than

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floral buds (Table 2) include LVFS-329, which encodes a protein homologous to decapping protein 2 (Dcp2) which functions with Dcp1 form a holoenzyme that is involved in cleavage of the 50 cap structure and subsequent 50 to 30 mRNA degredation [24]. LVFS-40 is homologous to proteins containing EF-hand, which are helix-loop-helix structures associated with calcium binding. Often responding to changes in intracellular calcium, these proteins undergo structural and/ or enzymatic changes to regulate different signal transduction pathways such as developmental switches or stress responses [25]. LVFS-65 is homologous to Drosophila Notchless, a WD40 repeat family protein that modulates Notch and subsequent signaling activity [26]. Also highly abundant in vegetative buds are clones involved in general plant defense (Table 2) including LVFS-267, which is homologous to a trypsin inhibitor, a protein involved in the plant defense against insects [27]. Epoxide hydrolase (LVFS310) is also involved in plant defense in the biosynthesis of the protective layer of cutin, as well as the production of anti-fungal compounds [28]. LVFS-400 encodes a protein homologous to cytochrome P450, a large family of proteins responsible for synthesis of membrane sterols and structural polymers, plant hormone homeostasis, biosynthesis of pigments, antioxidants and defense compounds and detoxification of pesticides and pollutants [29]. 3.4. cDNA clones abundant in longan floral buds Clones more abundant in floral buds include metabolic genes involved in glycolysis, oxidative phosphorylation, lignin biosynthesis, flavanoid biosynthesis, fatty acid regulation, carbon fixation and hormonal synthesis (Table 2). Clones with sequence similarity to proteins associated with glycolysis are more highly expressed in floral buds including glyceraldehyde3-phosphate dehydrogenase (EC 1.2.1.12) (LVFS-102), cytosolic triose phosphate (EC 5.3.1.1) (LVFS-163) and putative alcohol dehydrogenase (EC 1.1.1.1) (LVFS-120). LVFS-79 encodes a protein homologous to NADH-ubiquinone oxidoreductase (EC 1.6.5.3) involved in oxidative phosphorylation. Also highly expressed in floral buds is ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) (LVFS-27), which is involved in carbon fixation. Proteins involved in lignin biosynthesis, caffeoyl CoA 3-O-methyltransferase (EC 2.1.1.104) (LVFS-26) and sinapyl alcohol dehydrogenase (LVFS-241), flavanoid biosynthesis, leucoanthocyanidin reductase (LVFS-28) and isoflavone reductase (LVFS-174), fatty acid biosynthesis, 3oxoacyl-[acyl-carrier-protein] reductase (LVFS-94) fatty acid degradation, GDSL lipases (LVFS-58 and LVFS-166) and inositol biosynthesis inositol-3-phosphate synthase (EC 5.5.1.4) (LVFS-115) were expressed more in floral buds than vegetative buds. LVFS-87 is homologous to Arabidopsis At4g36470 predicted to belong to the S-adenosyl-L-methione:carboxyl methyltransferase protein family. Similar proteins in this family are the S-adenosyl-L-methione:jasmonic acid carboxyl methyltransferase (JMT) that is expressed in developing flowers, catalyzes the formation of methyl jasmonate from jasmonic acid and regulates the expression

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Table 2 List of 65 sequences of cDNA clones isolated by SSH in either floral or vegetative buds Clone number

Accession number

General metabolism Vegetative LVFS-22 DT368183 LVFS-181 DT368221

Northern veg FL

Fold induction (floral/veg)

Sequence similarity

0.13 0.31

Light-harvesting chlorophyll a/b Adenosine kinase isoform

LVFS-195 LVFS-254 LVFS-261 Floral LVFS-02 LVFS-26

DT368223 DT368230 DT368231

0.43 0.25 0.09

Putative pyruvate dehydrogenase E1 beta subunit Granule-bound starch synthase HyuC-like protein (putative N-carbamyl-L-amino acid amidohydrolase

DT368180 DT368184

3.21 3.06

Leucoanthocyanidin reductase Caffeoyl CoA 3-O-methyltransferase

LVFS-27

DT368185

3.21

Ribulose-1,5-bisphosphate carboxylase

LVFS-58 LVFS-79 LVFS-87 LVFS-94 LVFS-102 LVFS-115 LVFS-120 LVFS-163 LVFS-166 LVFS-174 LVFS-241 Both LVFS-48

DT368194 DT368198 DT368199 DT368202 DT368204 DT368244 DT368210 DT368215 DT368216 DT368219 DT368229

1.58 4.17 4.24 1.91 2.06 2.84 3.11 4.04 4.96 2.24 5.76

Putative GDSL-motif lipase/hydrolase NADH-ubiquinone oxidoreductase S-adenosyl-L-methionine:carboxyl methyltransferase family 3-Oxoacyl-[acyl-carrier-protein] reductase Glyceraldehyde-3-phosphate dehydrogenase Inositol-3-phosphate synthase Putative alcohol dehydrogenase Cytosolic triose phosphate isomerase GSDL-motif lipase Isoflavone reductase-like protein Putative sinapyl alcohol dehydrogenase

DT368192

1.29

Malonyl-CoA:acyl-carrier-protein transacylase

DT368227

0.47 1.276

Fructose-bisphosphate aldolase

LVFS-230

0.143 2.312 DNA replication and cell growth Vegetative LVFS-329 DT368237 Floral LVFS-10 DT368182

0.21

Decapping protein 2-like

2.84

Elongation factor 1-alpha

LVFS-37 LVFS-297 Both LVFS-44

DT368188 DT368234

2.98 2.64

Alpha-6 tubulin (TUA6) gene Translation initiation factor-like protein

DT368191

2.00

Histone H3

LVFS-172

DT368218

0.17

Histone H3

LVFS-385 LVFS-53 LVFS-76 LVFS-107

DT368239 DT368193 DT368196 DT368207

0.13 2.55 3.30 4.77

60S ribosomal protein L35 Ribosomal protein L19 Ribosomal protein S29 Ribosomal protein L11

Transport Floral LVFS-32 LVFS-90 LVFS-218 Signaling Vegetative LVFS-40 LVFS-65 Floral LVFS-6 LVFS-100 LVFS-113

DT368186

4.70

Vesicle transport protein Sec22

DT368201 DT368226

2.02 2.71

Nuclear transport factor 2 (NTF2) Protein translocation complex Sec61 gamma chain

DT368190 DT368195

0.17 0.46

EF-hand containing protein Notchless-like protein

DT368181 DT368203

2.27 2.44

Calmodulin SHEPHERD [Arabidopsis thaliana]

DT368208

5.15

WD-40 repeat family protein

T.K. Matsumoto / Plant Science 170 (2006) 500–510

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Table 2 (Continued ) Clone number

Accession number

Northern veg FL

Fold induction (floral/veg)

LVFS-176

DT368220

2.84

LVFS-199 LVFS-205 LVFS-323 Both LVFS-290

DT368224 DT368225 DT368236

16.66 4.17 2.44

DT368233

0.46

Sequence similarity Acyl-CoA binding protein MADS box PISTILLATA-like Arabidopsis thaliana protodermal factor 1 (PDF1) CCR4-associated factor Sterile alpha motif (SAM) domain-containing protein

1.16 2.18 Plant defense and stress response Vegetative LVFS-267 DT368232 LVFS-310 DT368235 LVFS-400 DT368242 Floral LVFS-35 DT368187 LVFS-89 DT368200

0.36 0.31 0.49

21 kDa seed protein/trypsin inhibitor/miraculin Epoxide hydrolase Putative cytochrome P450

1.56 2.58

Repetitive proline-rich cell wall protein 2 precursor Metallothionein-like protein Catalase Proline rich protein

LVFS-127 LVFS-128

DT368211 DT368212

2.84 1.34

LVFS-152 LVFS-370

DT368214 DT368238

7.45 53.35

LVFS-396

DT368240

2.00

Dehydration-induced protein ERD15

LVFS-399 LVFS-411 Both LVFS-232

DT368241 DT368243

2.02 1.55

Aspartic proteinase Mannose/glucose-specific lectin

DT368228

0.79

Lipid transfer protein precursor

Pollen allergen-like protein Bet v.1 Jasmonate-inducible protein ipomoelin

LVFS-168

DT368217

5.76

Lipid transfer protein III

Unknown Floral LVFS-39 LVFS-78 LVFS-104 LVFS-106 LVFS-118

DT368189 DT368197 DT368205 DT368206 DT368209

1.49 2.97 2.42 2.27 2.55

Expressed protein At3g29575 Unknown protein At2g27385 En/spm-like transposon protein Hypothetical Arabidopsis protein F22O13.12 Hypothetical Arabidopsis protein F27K19.170

LVFS-138 LVFS-186

DT368213 DT368222

4.72 2.53

Unknown protein At2g42870 Unknown protein At1g53560

Northern blot analysis confirmed differential expression of each cDNA clone and the highest scoring homologous protein is listed.

of jasmonate inducible genes [30] and S-adenosyl-L-methione:benzoic acid carboxyl methyltransferase (BAMT) which is differentially expressed during snapdragon flower development and catalyzes the formation of methyl benzoate from benzoic acid [31]. Also abundant in floral buds were clones with homology to proteins involved in cell growth (Table 2) including clones homologous to translation initiation factor (LVFS-297) and elongation factor 1a (LVFS-10), which are associated with protein synthesis and a-tubulin (LVFS-37) which is a large component of microtubules associated with cell division and growth [32]. Transport proteins more abundant in floral buds (Table 2) include LVFS-90, which is homologous to nuclear transport factor 2 (NTF2). NTF2 is associated with the Ran (small GTPase) dependent nucleocytoplasmic trafficking through the nuclear pore complex by importing RanGDP into the nucleus

[33]. LVFS-218 encodes a protein homologous to the gamma subunit of the Sec61 complex (Sss1p in yeast) which is responsible for the forward and retrograde transport of misfolded secretory and transmembrane proteins across the endoplasmic reticulum (ER); subsequent cytosolic degredation occurs at the high affinity ribosome receptor in the rough endoplasmic reticulum (RER) [34,35]. LVFS-32 encodes a protein homologous to vesicle transport protein Sec22p in yeast, which facilitates transport between the EF and Golgi complex [36]. Sec22p is an R-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) characterized by an N-terminal domain with a profilin-like fold called the longin domain; the longins are the only R-SNARES highly conserved in all eukaryotes including plants [37]. Components involved in signal pathways were identified to be differentially expressed in floral buds (Table 2) including calcium signaling by calmodulin (LVFS-6), fatty acid

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synthesis and degradation by acetyl-CoA binding proteins (ACBP) (LVFS-176) and global gene regulation by CCR4-associated factor (LVFS-323). WD-40 repeat family protein (LVFS-113) associated with protein–protein interactions and meristem identity genes PISTILLATA-like protein (LVFS-199), SHEPHERD-like (LVFS-100) and Protodermal Factor 1 (PDF1) (LVFS-205) were also differentially expressed in floral buds. PDF1 is a putative extracellular proline-rich protein associated with the L1 layer of meristems [38]. Other proline-rich cell wall proteins (LVFS-35 and LVFS-128) are also highly expressed in floral buds and may be associated with meristem layers. SHD is an ortholog of GRP94 located in the endoplasmic reticulum (ER) and is a HSP90-like protein that is responsible for correct folding and formation of complex of CLAVATA (CLV) proteins [39]. CLV genes promote the formation of organs from peripheral stem daughter cells in shoot and floral meristems; mutations in CLV results in the accumulation of stem cells in the meristem, which increases the size of the meristem dome (Ishiguro et al., 2002). A similar protein was also identified to be highly expressed in floral buds; LVFS-218 is homologous to the gamma subunit of the Sec61p, which is responsible for the transport of misfolded secretory and transmembrane proteins across the ER. LVFS-199 encodes a MADS-box transcription factor related to PISTILLATA (PI) (LVFS-199) which is associated with parthenocarpic fruits in apples [40]. Clones similar to proteins involved in plant defense or stress responses are also differentially expressed in floral buds (Table 2) including two proline-rich cell wall proteins (LVFS35 and LVFS-128) associated with developmental properties such as germination, pod formation, nodulation and xylem differentiation or lignification, as well as stress responses to wounding, fungal elicitors, ethylene, drought and light [41]. LVFS-89 contains sequences homologous to metallothionein (MT)-like proteins, which functions in cadmium and zinc toxicity in animals and is implicated in copper tolerance in plants [42]. Catalase (EC 1.11.1.6) (LVFS-127) responds to reactive oxygen species (ROS) stress and is responsible for the breakdown of hydrogen peroxide to oxygen and water [43]. LVFS-152 encodes a pollen allergen-like protein homologous to the Bet v.1 protein that belongs to the pathogenesis-related plant protein family PR-10, produced in response to various pathogens but with unknown function [44]. ERD15 (LVFS396) is a protein highly induced by dehydration and cesium toxicity with a putative PAM2 (PABP-interacting motif) domain which is a binding site for PABC (poly (A)-binding protein C-terminal) containing proteins whose function is implicated in RNA metabolism and DNA damage repair [45,46]. Aspartic proteinase (LVFS-399) is implicated in protein processing and or degradation, plant senescence, abiotic and biotic stress response, programmed cell death (PCD) and pistil–pollen interactions in plant reproduction [47]. Mannoseglucose specific lectin (LVFS-411) is implicated in the recognition of high-mannose glucan from foreign microorganisms and plant predators [48]. A clone with sequence homology to ipomoelin, a wound and methyl jasmonate induced lectin regulated by dephosphorylation and calcium

which reduces the growth and development of silkworms, is exclusively induced in longan floral buds [49]. 3.5. cDNA clones abundant in both longan floral and vegetative buds Clones homologous to ribosomal protein L11 (LVFS-107), L19 (LVFS-53) and S29 (LVFS-76) are more abundant in floral buds while ribosomal protein L35 (LVFS-385) is more abundant in vegetative buds (Table 2). Specific regulation of ribosomal proteins are common even in gene family members such as the Arabidopsis L11 genes where RPL11C is observed in shoot and primary root meristems and leaf primordial, while the RPL11A is observed in more specific cell types [50]. The clone for [acyl-carrier-protein] malonyltransferase (EC 2.3.1.39) (LVFS-48) hybridizes to two different size transcripts, the larger more abundant in the floral buds and the smaller more abundant in the vegetative buds. It is not clear if the difference in size is due to hybridization of the probe to distinct genes or alternatively spliced transcripts of the same gene and will need further investigation. The presence of multiple transcripts is also evident in northern blot analysis of LVFS-290 which contains sequences homologous to the sterile alpha motif (SAM) domain which is responsible for protein–protein interaction between SAM domains and non-SAM domains, formation of multiple self-associated architecture and binding of RNA [51]. LVFS-44 and LVFS-172 encode two histone H3like proteins involved in chromatin remodeling, the larger transcript more abundant in vegetative buds and the smaller transcript in floral buds. This expression pattern is also evident in northern blot analysis of LVFS-230, which is homologous to fructose-bisphosphate aldolase (EC 4.1.2.13), a glycolytic enzyme that catalyzes the reversible aldol cleavage of fructose 1,6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. Lipid transfer proteins (LTP) are small, basic protein that bind fatty acids and transfer phospholipids between membranes and have been implicated in cutin and wax assembly, as well as pathogen defense [52]. LVFS-168 is homologous to LTPIII and is abundant in floral buds. Two transcripts of different sizes hybridize to LVFS-232, which is homologous to a lipid transfer protein precursor. LTP proteins in Arabidopsis have been isolated to specific cell types, suggesting different transcripts of LTP observed in longan may also play a role in plant development [53]. 4. Discussion An SSH library was created from longan and 65 clones were identified that are abundant in either vegetative buds of untreated controls, or floral buds induced by KClO3 treatment. While many genes isolated in this study have been previously characterized to be associated with flowering in other plants, many new genes with novel functions have been isolated and will be further characterized. Majority of the clones were recovered only once with the exception of 12 clones, suggesting that the library may still be a source of additional flowering genes and will be further evaluated.

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4.1. Carbon utilization associated clones In subtropical and tropical fruit trees, cultural practices play an important role in controlling vegetative growth, which in turn influences floral induction. Unlike temperate fruit trees, where growth, dormancy and break of dormancy is determined by the seasons, sub-tropical and tropical fruit trees rely on more subtle changes in rainfall, temperature or nutrient availability [54]. Optimizing flowering in sub-tropical and tropical fruit trees are often more reliant on cultural management techniques, often involving the promotion or restriction of vegetative growth [1,55]. Restriction of vegetative growth is often associated with higher sugar accumulation, which in turn has been implicated in flowering [56]. The carbon to nitrogen (C:N) ratio in flowering has been a dogma of physiological studies on flowering since proposed by Klebs in 1913. High carbohydrates and low nitrogen results in a high C:N ratio which favors flowering and high nitrogen results in a lower C:N ratio, which favors vegetative growth. Molecular and biochemical evidence has revealed an intricate and integrated nitrogen and carbon sensing and signaling network to control various developmental processes [56,57]. Recent studies suggest photoperiods conducive to flowering lead to a high C:N ratio in the phloem, which may be important to the floral transition in the meristem of Sinapsis alba and Arabidopsis thailiana [58]. In this study, we have isolated clones that encode proteins similar to three sequential enzymes from glycolysis: (1) fructose-bisphosphate aldolase (LVFS-230) catalyzes the reaction of fructose 1,6-bisphosphate to dihydroxyacetonephosphate and glyceraldehyde 3-phosphate, (2) triose phosphate isomerase (LVFS-163) catalyzes the isomerization of dihydroxyacetone-phosphate and glyceraldehyde 3-phosphate and (3) glyceraldehyde 3-phosphate dehydrogenase (LVFS102) catalyzes glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate that are more abundant in floral buds. Fructose 1,6-bisphosphate aldolase was also isolated from a fluorescent differential display method to identify genes involved in the short day photoperiodic response to flowering in rice [59]. The expression of this enzyme is also circadian-regulated in Arabidopsis [60]. Anti-sense plants that expressed lower levels of plastid fructose 1,6-bisphosphate aldolase have decreased photosynthesis, starch accumulation and sucrose content [61]. Interestingly, we have identified three variants of fructose 1,6bisphosphate aldolase (LVFS-230) in longan. It is not presently known if these variants represent different members of a gene family or alternative splicing events. Alternative splicing of FCA (gene involved in the autonomous flowering pathway) at intron3 produces a negative feedback loop to limit the amount of FCA protein, which controls the level of FLC mRNA and flowering time [7]. In Drosophila, alternative splicing of fructose 1,6-bisphosphate aldolase produces three different transcripts, which are differentially expressed during different stages of development from embryo, larvae, pupae and adults [62]. In strawberries, cytosolic fructose 1,6-bisphosphate aldolase activity and expression increased during fruit ripening from the green to red stages of fruit development [63]. Together

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these data suggest that fructose 1,6-bisphosphate aldolase may be a link between sugar metabolism and flowering. A clone homologous to pyruvate dehydrogenase complex (PDC) E1 subunit (LVFS-195) was isolated in the SSH screen and found to be more abundant in vegetative buds compared to floral buds. The PDC is a multi-subunit enzyme that catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA into the tricarboxylic acid (TCA) cycle. LVFS-195 is homologous to Arabidopsis gene At1g30120, which is a plastid PDC that is distinct from the mitochondrial PDC (mtPDC). Although both plastid and mitochondrial PDC are subject to feedback inhibition by acetyl-CoA and NADH, only the mitochondrial PDC is subjected to regulation by phosphorylation from pyruvate dehydrogenase kinase (PDHK) and dephosphorylation by pyruvate dehydrogenase phosphatase (PDHP). Antisense PDHK plants, a negative regulator of mtPDC, resulted in increased activity of PDC, reduced accumulation of vegetative growth, early flower development and shorter generation time [64,65]. Cytoplasmic male sterility in sugar beets has also been observed in anti-sense PDH expressed in tapetum cells [66]. These results suggest the PDC complex is important in flower induction and development; however, the role of the plastid PDC in flower induction is currently unknown. 4.2. Fatty acid and lipid associated clones Seeds from anti-sense PDHK Arabidopsis plants have increased oil content and seed weight implicating a role for mtPDC as a link between glycolysis and the TCA cycle and fatty acid biosynthesis [65]. Floral longan cDNA associated with fatty acid biosynthesis include a variant of LVFS-48, a malonyl-CoA-acyl-carrier-protein transacylase and LVFS-94, a 3-oxoacyl-[acyl-carrier-protein] reductase. LVFS-176 is an acyl-CoA binding protein, which serves as a pool former and transporter for acyl-CoA esters in fatty lipid synthesis and fatty acid degradation [67]. LVFS-58 and LVFS-166 are GDSLmotif lipases involved in fatty acid degradation. GDSL-motif lipases were identified as downstream genes regulated by APETALA3 and PISTILLATA floral homeotic genes [68]. Fatty acids are biological signals for cell death, induction of pathogenesis proteins and oxidative stress genes and serve as a precursor for jasmonate, which is required for anther dehiscence and pollen development [69]. A S-adenosyl-Lmethionine:carboxyl methyltransferase was isolated from longan floral buds and is homologous to the Arabidopsis gene At4g36470 which belongs to a family of proteins that catalyzes the transfer to methyl groups to form volatile compounds such as methyl benzoate and methyl jasmonate [30,31]. 4.3. Plant defense associated clones A highly expressed protein LVFS-370, which is homologous to the jasmonate inducible protein ipomoelin, was identified in the SSH screen. Ipomoelin expression is induced by wounding, methyl jasmonate, ethylene and hydrogen peroxide and reduced by salicylic acid and nitric oxide [70,71]. The ipomoelin protein is thought to be a lectin involved in reducing

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the growth and survival of silkworms [49]. Genes identified during flower development often include proteins involved in plant defense against pathogens and insects or stress responsive genes; these genes are hypothesized to protect the developing flower and seeds against pathogens and predators [15,16,59,68]. In this study we have identified proteins implicated in plant defense in both vegetative and floral buds indicating plant defense proteins may be produced to protect actively growing organs. Insect defense related proteins include a trypsin inhibitor (LVFS-267), ipomoelin (LVFS-370) and a mannose-glucose lectin (LVFS-411). Pathogen related proteins include an epoxide hydrolase (LVFS-310), proline-rich cell wall proteins (LVFS-35 and LVFS-128), pollen allergen-like Bet v.1 (LVFS-152) and lipid transfer proteins (LVFS-232 and LVFS-168). Stress regulated proteins include a cytochrome P450 (LVFS-400), metallothionein-like protein (LVFS-89), catalase (LVFS-127), dehydration induced protein ERD15 (LVFS-396) and aspartic proteinase (LVFS-399). 4.4. Vegetative/floral associated clones Another explanation for the high expression of plant defense related proteins may be that these proteins are downstream components that share a common signal transduction pathway essential for flower development. A flowering model for subtropical and tropical fruit trees proposed by Davenport [72] suggests that phytohormones and a ‘‘florigenic’’ promoter and inhibitor control the balance between vegetative and flowering flushes. This can be related on the molecular level to the regulation of FLC of LEAFY on flower promotion or repression in Arabidopsis [73,74]. Although a homolog of FLC is not present in the genome of poplar [12], an alternate or functional equivalent of FLC may exist in subtropical and tropical fruit trees. We have identified histone H3 proteins that are differentially expressed in vegetative and floral buds. In Arabidopsis, histone modification of chromatin structure is responsible for expression of FLC [10]. In Arabidopsis, Vernalization-Insensitive (VIN) 3 is induced by cold and necessary for deacetylation of histone H3 which allows dimethylation at Lys-9 and Lys-27 by Vernalization (VRN) 1 and 2 to lower FLC expression and allow flowering [75,76]. Conversely, Early Flowering (ELF) 7 and 8 are homologs of the PAF complex in yeast and function in the trimethylation at Lys-4 of histone H3 that result in an increased expression of the FLC clade of flowering repressors (FLC, MAF2 and FLM) to delay flowering [10]. In longan, two variants of histone H3 are present in vegetative and floral buds; the larger band more abundant in vegetative buds and the smaller in floral buds. Alternative splicing of histone macro2A1 occurs in mice and is expressed at similar levels in adult male and female tissues and differentially expressed in testes and differentiating male and female embryonic stem cells suggesting that the alternatively spliced mH2A1 protein is involved in multiple functions including X chromosome inactivaton [77]. It is currently not known if the longan histone H3 proteins are encoded by different genes or are products of alternative splicing and will be further investigated.

4.5. Global transcription/regulation associated clones Here we describe the identification of two genes complexes that may serve a role in global transcription regulation. The CCR4-NOT complex is a regulator of gene expression whose function is evolutionarily conserved in yeast, mice and humans. Composed of at least nine subunits, the CCR4-NOT complex regulates transcription, protein modification, ubiquitination and mRNA degradation in yeast [78]. The human counterpart of yeast CAF1, a component of the CCR4-NOT complex, was isolated in genetic screens for suppressors of anti-proliferative activity in various cell types and is localized in chromosomes frequently deleted in human tumors, suggesting this complex is an important factor in human disease [78]. Another putative regulatory protein (LVFS-290) contains a sterile alpha motif (SAM) domain, which is involved in protein–protein interactions. Diverse biological functions have been associated with proteins containing SAM domains, including translational repression, apoptosis, chromatin remodeling, receptor tyrosine kinase signaling, small ubiquitin-like modifier (SUMO) localization and developmental regulation [51,79]. The ubiquitous nature of the SAM domain may account for the hybridization of LVFS-290 to multiple transcripts in vegetative and floral buds. Currently, plant proteins containing SAM domains have not been characterized in flower induction. Notch signaling is an important signaling pathway that regulates cell fate choices by promoting the development and/ or proliferation of some cell types and influencing multiple developmental stages in a cell lineage [80]. Notch signaling is cell dependent where Notch signals are oncogenic in pre-T cells but suppress tumors in keratinocytes [81]. Notchless was isolated from Drosophila in a genetic screen for modifiers of Notch signaling [26]. Notchless contains WD-40 repeats that directly bind to the cytoplasmic domain in Notch and modifies Notch signaling [26]. Although Notch is a common signal pathway in vertebrates, flies and worms, the complete genome sequence of Arabidopsis does not include a homolog for Notch [82]. However, Arabidopsis does contain a homolog to Notchless, but the function is currently unknown. Identification of a Notchless homolog in longan (LVFS-65) that is abundant in vegetative buds suggests that Notchless in plants may have a role in suppression of flowering in longan through a functional equivalent of Notch. 4.6. Meristem associated clones Meristem associated genes were also identified in the screen including Protodermal Factor 1 (PDF1) (LVFS-205) [38] and SHEPHERD (SHD) (LVFS-100) [39] proteins associated with meristem integrity. Isolation of MdPI (Malus domestica PISTILLA) from apple [40] suggests the longan homolog LVFS-199 may have potential agricultural implication in seedless longan production through genetic engineering. The natural occurrence of retrotransposon insertions within the PI locus in apple also suggests that natural variation may also occur in longan and other species, which may be useful in

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characterizing new longan cultivars for parthenocarpic fruit production. In conclusion, we have identified 65 cDNA clones from an SSH library of vegetative and potassium chlorate induced floral buds from longan. Many of the genes identified have been previously reported in floral induction in other plant species while others represent novel genes not previously characterized in plants or associated with flowering. Potassium chlorate induced flowering in longan provides us with a unique opportunity to study flowering in a sub-tropical fruit tree. Molecular data obtained from this study will be used to build a model to identify the key regulators of flowering in a nontemperate climate and understand the genetic variation in subtropical fruit and nut species.

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