An Overview on Stress Signals in Relation to Flowering in Litchi chinensis and Dimocarpus longan

Transcription

1 An Overview on Stress Signals in Relation to Flowering in Litchi chinensis and Dimocarpus longan Biyan Zhou and Houbin Chen College of Horticulture South China Agricultural University Guangzhou, China

2 Background Ever green woody fruit trees water stress Star fruit (Averrhoa carambola) water stress Stress produces signals Stresses promote reproductive growth What are the key stress signals? Reactive oxygen species (ROS) and Reactive nitrogen species (NO) Long life kumquat Chill, Water stress ROS and NO possibly affect flowering in ever green woody trees. Mango (Mangifera indica)

3 Litchi and longan are ever green woody fruit trees Chill, water stress What are the key stress signals? Litchi Stresses promote reproductive growth Reactive oxygen species (ROS) and Reactive nitrogen species (NO) Longan Chill Water stress Chlorate Do ROS and NO affect flowering in litchi and longan? It is very likely that ROS and NO promote flowering in litchi and longan.

4 Chlorate induces longan flowering Chlorate are highly oxidizing chemicals. When these highly oxidizing chemicals are absorbed in plants, oxidative stress may occurs. Normally, the oxidative stress induced ROS accumulation. However, there is no direct evidence showing that chlorates induce ROS accumulation by now. It seems that it is necessary as well as interesting to study the relationship between reactive species and flowering in longan.

5 Do ROS and NO promote litchi flowering? ROS and NO do affect flowering. These studies have been carried out since 2005.

6 Methyl viologen dichloride hydrate (MV) was used as ROS producer: MV accepts electrons from photosystem and transfers them to molecular oxygen, producing the destructive superoxide, O 2 -. (Halliwell, 2006), Superoxide is then transformed to H 2 O 2 by superoxide dismutase (Kraus and Fletcher, 1994). Sodium nitroprusside (SNP) was used as NO donor

7 Table Effects of MV and SNP on panicles of Feizixiao litchi Treatments No. of leaves No. of axillary panicles Ratios of axillary panicles No. of flowers per panicle per panicle to total nodes per panicle per panicle Control 5.0 ± ± ± ± mm SNP 1.1 ± 0.2 * 16.9 ± ± ± * 40 µm MV 0.9 ± 0.1 * 15.1 ± ± ± 34.2 * Feizixiao litchi trees were transferred to a growth chamber at 18 /13 (day/night temperature, 12 h day and 12 h night), a relative humidity of 75-85%, and sunlight, and were sprayed uniformly with 2 mm SNP or 40 µm MV after 1, 21 and 41 d of 18 /13 treatment. The other trees were sprayed with distilled water as control at the same time. The chamber was re-warmed to 28 /23 after panicle primordia emerged. Values are means±se from 6 replicate trees. MV and SNP treatment increased the number of flowers per panicle, decreased the number of leaves per panicle.

8 A B C Panels A: A panicle of a control tree. Panels B: A panicle of a SNP treated tree. Panels C:A panicle of a MV treated tree. SNP and MV reduced number of leaves in panicles

9 SNP and MV reduced rudimentary leaves in panicles Ad St Ad Pe St A B Ad Pe St Pe E F Ad Pe St C D Ad Pe St Pe Ad St St G H Ad Ad Pe St St Pe I J K L A LcLFY rrna C LcLFY Actin SNP [mm] B MV [µm] Control SNP SNP+PTIO MV MV+DMTU SNP+DMTU MV+PTIO SNP and MV enhanced LcLFY expression Hydrogen peroxide and nitric oxide promote reproductive growth in Litchi chinensis (Zhou et al, 2012., Bio. Plant., )

10 Conclusion based on the studies: H 2 O 2 and NO promote reproductive growth by inhibiting the growth of rudimentary leaves as well as by promoting the expression of LcLFY. What is the mechanism of the SNP- and MV-induced flowering? Why ROS or NO promote flowering in litchi? Phytohormones Carbohydrates Gene regulation

11 To investigate the mechanism of the SNPor MV-promoted flowering in litchi underlying phytohormones and carbohydrates, potted Nuomici litchi trees were transferred to a chamber at 18 /13 and spray with MV or SNP every 8 days. When panicle primordia emerged, trees were transferred to 26 /23 chamber.

12 Table 1 Effects of SNP on the rate of flowering terminal shoots in Nuomici. Treatments 26/23 +water 18/13 +water 18/13 +SNP Rate of flowering terminal shoots(%) 0.00±0.00 a 55.83±0.40 b 80.88±0.05 c Values are means ± SE (n=4). Different letters indicate statistical difference at P<0.05 level according to Duncan s multiply range test. In accordance with the previous studies, SNP also increase the rate of flowering terminal shoots. SNP promoted flowering in litchi.

13 Fig.1 Contents of auxin (IAA), gibberellin (GA1+3), cytokinin (ZRs) and abscisic acid (ABA) in leaves of watertreated or SNP-treated trees under 18/13 condition, and water-treated trees under 26/23 condition (Liu, 2012). Values represent the means of 4 replicates and bars indicate SE. SNP treatment increased the levels of IAA and ABA in leaves of Nuomici litchi during the whole stage of floral differentiation, decreased the level of GA in leaves in late stage of floral differentiation. This kind of hormonal level is essential for inhibiting vegetative growth (shoot flushing) and promoting reproductive growth. Similar results were found in the MVtreated trees.

14 Fig.2 Contents of total soluble sugar and starch in leaves of water-treated or SNPtreated trees under 18/13 condition, and water-treated trees under 26/23 condition (Liu, 2012). Values represent the means of 4 replicates and bars indicate SE. SNP could not significantly increased carbohydrate levels in leaves. Similar results were found in the MV-treated trees.

15 To investigate the mechanism of the MVpromoted flowering in litchi underlying gene expression, an MV-treated primordium cdna suppression subtractive hybridization (SSH) library was constructed.

16 Table 1 Identification of MV responsive genes isolated by SSH in litchi primordia. Clone Accession no. of matching sequence Matching sequence in public database Source of matching sequence Best e- value (BLASTx) Transport facilitation 6 XM_ hexose transporter Vitis vinifera 2.00E NM_ glycine hydroxymethyltransferase (EDA36) Arabidopsis thaliana 1.00E NM_ auxin efflux carrier component 3 (PIN3) Arabidopsis thaliana 6.00E AY putative ferredoxin protein (At1g32550) Arabidopsis thaliana 6.00E XM_ geranylgeranyl transferase type II beta subunit Ricinus communis 1.00E NM_ translocase of chloroplast 90 (AT5G20300) Arabidopsis thaliana 1.00E XM_ triose phosphate/phosphate translocator Vitis vinifera 5.00E XM_ protein transporter, putative Ricinus communis 1.00E JF Ferredoxin-2, putative Ricinus communis 2.00E U plastid phosphate/phosphoenolpyruvate translocator precursor Nicotiana tabacum 7.00E XM_ ATP binding protein, putative Ricinus communis 1.00E AK putative mitochondrial elongation factor G(EF-G) Arabidopsis thaliana 3.00E XM_ probable methyltransferase PMT14-like (LOC ) Vitis vinifera 6.00E XM_ Triose phosphate/phosphate translocator, chloroplast precursor, putative Ricinus communis 1.00E XM_ probable S-acyltransferase At1g69420-like Vitis vinifera 3.00E-15 Transcription 184 XM_ transcriptional corepressor LEUNIG-like Vitis vinifera 1.00E NM_ cyclin-dependent kinase A-1 (CDKA) Arabidopsis thaliana 1.00E NM_ intron-binding protein (EMB2765) Arabidopsis thaliana 6.00E BT putative DEAD/DEAH box RNA helicase (At3g06980) Arabidopsis thaliana 1.00E XM_ IAA-amino acid hydrolase ILR1 precursor, putative Ricinus communis 3.00E NM_ protochlorophyllide reductase B (PORB) Arabidopsis thaliana 1.00E NM_ S-adenosylmethionine decarboxylase-like protein Arabidopsis thaliana 9.00E AY casein kinase 1-like protein 1 (CKL1) Arabidopsis thaliana 2.00E NM_ somatic embryogenesis receptor-like kinase-like protein (LOC ) Glycine max 5.00E-39

17 Stress response 440 XM_ vitamin-b12 independent methionine synthase Populus trichocarpa 2.00E AF cell-type guard cell proline-rich protein (GPP1) Nicotiana glauca 5.90E XM_ structural constituent of cell wall, putative Ricinus communis 5.00E XM_ metal ion binding protein Ricinus communis 6.00E-111 Carbohydrate metabolism 228 XM_ GATA zinc finger protein regulating nitrogen assimilation (GATA-1) Populus trichocarpa 2.00E HM neoxanthin synthase (NSY) Citrus sinensis 4.00E AK beta Galactosidase - like protein Arabidopsis thaliana 1.00E XM_ probable glycosyltransferase At5g03795-like (LOC ) Vitis vinifera 2.00E NM_ pfkb-type carbohydrate kinase-like protein (AT1G19600) Arabidopsis thaliana 4.00E NM_ mitochondrial malate dehydrogenase (mmdh) Solanum lycopersicum 6.00E NM_ Glucose-6-phosphate/phosphate translocator-like protein (CUE1) Arabidopsis thaliana 2.00E NM_ Glucan endo-1,3-beta-glucosidase precursor, putative Arabidopsis thaliana 8.00E XM_ malic enzyme, putative Ricinus communis 0.00E+00 Secondary metabolism 454 EF anthocyanidin synthase (ANS) Vitis vinifera 2.20E AB flavonol synthase Citrus unshiu 1.00E-119 Intracellular signaling 249 XM_ auxin-induced protein AUX28-like Vitis vinifera 1.00E XM_ B-box type zinc finger protein CONSTANS-LIKE 4-like (Bbox1) Vitis vinifera 1.00E AK cell division cycle protein-related, Arabidopsis thaliana 9.00E EU SKIP interacting protein 21 (SKIP21) Oryza sativa 2.00E EU receptor-like kinase FER Poncirus trifoliata FER 3.00E XM_ serine/threonine protein phosphatase 2A Vitis vinifera 1.00E EU putative ADP-ribosylation factor Brassica napus 8.00E NM_ calreticulin-2 (CRT1b) Arabidopsis thaliana 2.00E AY putative calcium-binding protein(at1g18210) Arabidopsis thaliana 1.40E NM_ aluminum induced protein with YGL and LRDR motifs (AT4G27450) Arabidopsis thaliana 2.00E NM_ remorin-like protein Arabidopsis thaliana 4.00E NM_ putative LRR receptor-like serine/threonine-protein kinase (AT1G53440) Arabidopsis thaliana 1.00E NM_ peptide chain release factor erf subunit 1 (ERF1-1) Arabidopsis thaliana 7.00E-22

18 Others 156 NM_ polyubiquitin 14 (UBQ14) Arabidopsis thaliana 1.00E DQ acidic proline-rich protein PRP25 precursor-like Arachis hypogaea 7.00E XM_ Aspartic proteinase nepenthesin-2 precursor Ricinus communis 3.00E XM_ S ribosomal protein L27 Vitis vinifera 1.00E XM_ S ribosomal protein L19-like Vitis vinifera 9.00E XM_ palmitoyl-acyl carrier protein thioesterase Ricinus communis 2.00E AF ubiquitin extension protein(uep1 或 ubiquitin/ribosomal protein 27a (Prunus avium 6.00E NM_ chaperone binding / ATPase activator Arabidopsis thaliana 7.20E XM_ Adenosine kinase, putative Ricinus communis 2.00E BT E2 ubiquitin-conjugating-like enzyme Ahus5 (At3g57870) Arabidopsis thaliana 2.00E XM_ Polyadenylation factor subunit, putative Ricinus communis 8.00E NM_ fatty acyl-acp thioesterases B (FATB) Arabidopsis thaliana 6.00E AY aspartate aminotransferase (AAT1) (At2g30970; F7F1.18) Arabidopsis thaliana 2.00E XM_ Aspartic proteinase 2 precursor, putative Ricinus communis 6.00E NM_ microtubule-associated protein-related protein (AT5G57210) Arabidopsis thaliana 9.70E XR_ zinc finger CCCH domain-containing protein Vitis vinifera 4.00E FJ alpha-tubulin Dimocarpus longan 5.00E NM_ ketol-acid reductoisomerase (AT3G58610) Arabidopsis thaliana 5.00E XM_ ferredoxin-1-like (LOC ) Vitis vinifera 9.00E NM_ polyubiquitin 10 (UBQ10) Arabidopsis thaliana 4.00E EU S ribosomal protein L12 Zea mays 6.00E NM_ senescence-associated protein 13 (SAG13) Arabidopsis thaliana 5.00E AF N-myristoyltransferase-like protein Arabidopsis thaliana 2.00E-45 Unknown protein 188 AY unknown protein (At1g21380) Arabidopsis thaliana 5.80E BT unknown protein (At1g76120) Arabidopsis thaliana 2.00E AK predicted protein Hordeum vulgare 4.00E XM_ predicted protein (MYB091) Populus trichocarpa 2.00E XM_ hypothetical protein LOC Vitis vinifera 1.00E XM_ hypothetical protein LOC Vitis vinifera 2.00E XM_ predicted protein, Populus trichocarpa 6.00E XM_ conserved hypothetical protein Ricinus communis 1.00E-67 No matching sequences were found in the NCBI database: 152,153,170,177,198,200,215,636,670,694 The nested primers are not included

19 To further confirm if the differentially expressed genes are induced by MV, six genes were selected from the library.

20 Table 1 Identification of MV responsive genes isolated by SSH in litchi primordia. Clone Accession no. of matching sequence Matching sequence in public database Source of matching sequence Best e- value (BLASTx) Transport facilitation 6 XM_ hexose transporter Vitis vinifera 2.00E NM_ glycine hydroxymethyltransferase (EDA36) Arabidopsis thaliana 1.00E NM_ auxin efflux carrier component 3 (PIN3) Arabidopsis thaliana 6.00E AY putative ferredoxin protein (At1g32550) Arabidopsis thaliana 6.00E XM_ geranylgeranyl transferase type II beta subunit Ricinus communis 1.00E NM_ translocase of chloroplast 90 (AT5G20300) Arabidopsis thaliana 1.00E XM_ triose phosphate/phosphate translocator Vitis vinifera 5.00E XM_ protein transporter, putative Ricinus communis 1.00E JF Ferredoxin-2, putative Ricinus communis 2.00E U plastid phosphate/phosphoenolpyruvate translocator precursor Nicotiana tabacum 7.00E XM_ ATP binding protein, putative Ricinus communis 1.00E AK putative mitochondrial elongation factor G(EF-G) Arabidopsis thaliana 3.00E XM_ probable methyltransferase PMT14-like (LOC ) Vitis vinifera 6.00E XM_ Triose phosphate/phosphate translocator, chloroplast precursor, putative Ricinus communis 1.00E XM_ probable S-acyltransferase At1g69420-like Vitis vinifera 3.00E-15 Transcription 184 XM_ transcriptional corepressor LEUNIG-like Vitis vinifera 1.00E NM_ cyclin-dependent kinase A-1 (CDKA) Arabidopsis thaliana 1.00E NM_ intron-binding protein (EMB2765) Arabidopsis thaliana 6.00E BT putative DEAD/DEAH box RNA helicase (At3g06980) Arabidopsis thaliana 1.00E XM_ IAA-amino acid hydrolase ILR1 precursor, putative Ricinus communis 3.00E NM_ protochlorophyllide reductase B (PORB) Arabidopsis thaliana 1.00E NM_ S-adenosylmethionine decarboxylase-like protein Arabidopsis thaliana 9.00E AY casein kinase 1-like protein 1 (CKL1) Arabidopsis thaliana 2.00E NM_ somatic embryogenesis receptor-like kinase-like protein (LOC ) Glycine max 5.00E-39

21 Stress response 440 XM_ vitamin-b12 independent methionine synthase Populus trichocarpa 2.00E AF cell-type guard cell proline-rich protein (GPP1) Nicotiana glauca 5.90E XM_ structural constituent of cell wall, putative Ricinus communis 5.00E XM_ metal ion binding protein Ricinus communis 6.00E-111 Carbohydrate metabolism 228 XM_ GATA zinc finger protein regulating nitrogen assimilation (GATA-1) Populus trichocarpa 2.00E HM neoxanthin synthase (NSY) Citrus sinensis 4.00E AK beta Galactosidase - like protein Arabidopsis thaliana 1.00E XM_ probable glycosyltransferase At5g03795-like (LOC ) Vitis vinifera 2.00E NM_ pfkb-type carbohydrate kinase-like protein (AT1G19600) Arabidopsis thaliana 4.00E NM_ mitochondrial malate dehydrogenase (mmdh) Solanum lycopersicum 6.00E NM_ Glucose-6-phosphate/phosphate translocator-like protein (CUE1) Arabidopsis thaliana 2.00E NM_ Glucan endo-1,3-beta-glucosidase precursor, putative Arabidopsis thaliana 8.00E XM_ malic enzyme, putative Ricinus communis 0.00E+00 Secondary metabolism 454 EF anthocyanidin synthase (ANS) Vitis vinifera 2.20E AB flavonol synthase Citrus unshiu 1.00E-119 Intracellular signaling 249 XM_ auxin-induced protein AUX28-like Vitis vinifera 1.00E XM_ B-box type zinc finger protein CONSTANS-LIKE 4-like (Bbox1) Vitis vinifera 1.00E AK cell division cycle protein-related, Arabidopsis thaliana 9.00E EU SKIP interacting protein 21 (SKIP21) Oryza sativa 2.00E EU receptor-like kinase FER Poncirus trifoliata FER 3.00E XM_ serine/threonine protein phosphatase 2A Vitis vinifera 1.00E EU putative ADP-ribosylation factor Brassica napus 8.00E NM_ calreticulin-2 (CRT1b) Arabidopsis thaliana 2.00E AY putative calcium-binding protein(at1g18210) Arabidopsis thaliana 1.40E NM_ aluminum induced protein with YGL and LRDR motifs (AT4G27450) Arabidopsis thaliana 2.00E NM_ remorin-like protein Arabidopsis thaliana 4.00E NM_ putative LRR receptor-like serine/threonine-protein kinase (AT1G53440) Arabidopsis thaliana 1.00E NM_ peptide chain release factor erf subunit 1 (ERF1-1) Arabidopsis thaliana 7.00E-22

22 Others 156 NM_ polyubiquitin 14 (UBQ14) Arabidopsis thaliana 1.00E DQ acidic proline-rich protein PRP25 precursor-like Arachis hypogaea 7.00E XM_ Aspartic proteinase nepenthesin-2 precursor Ricinus communis 3.00E XM_ S ribosomal protein L27 Vitis vinifera 1.00E XM_ S ribosomal protein L19-like Vitis vinifera 9.00E XM_ palmitoyl-acyl carrier protein thioesterase Ricinus communis 2.00E AF ubiquitin extension protein(uep1 或 ubiquitin/ribosomal protein 27a (Prunus avium 6.00E NM_ chaperone binding / ATPase activator Arabidopsis thaliana 7.20E XM_ Adenosine kinase, putative Ricinus communis 2.00E BT E2 ubiquitin-conjugating-like enzyme Ahus5 (At3g57870) Arabidopsis thaliana 2.00E XM_ Polyadenylation factor subunit, putative Ricinus communis 8.00E NM_ fatty acyl-acp thioesterases B (FATB) Arabidopsis thaliana 6.00E AY aspartate aminotransferase (AAT1) (At2g30970; F7F1.18) Arabidopsis thaliana 2.00E XM_ Aspartic proteinase 2 precursor, putative Ricinus communis 6.00E NM_ microtubule-associated protein-related protein (AT5G57210) Arabidopsis thaliana 9.70E XR_ zinc finger CCCH domain-containing protein Vitis vinifera 4.00E FJ alpha-tubulin Dimocarpus longan 5.00E NM_ ketol-acid reductoisomerase (AT3G58610) Arabidopsis thaliana 5.00E XM_ ferredoxin-1-like (LOC ) Vitis vinifera 9.00E NM_ polyubiquitin 10 (UBQ10) Arabidopsis thaliana 4.00E EU S ribosomal protein L12 Zea mays 6.00E NM_ senescence-associated protein 13 (SAG13) Arabidopsis thaliana 5.00E AF N-myristoyltransferase-like protein Arabidopsis thaliana 2.00E-45 Unknown protein 188 AY unknown protein (At1g21380) Arabidopsis thaliana 5.80E BT unknown protein (At1g76120) Arabidopsis thaliana 2.00E AK predicted protein Hordeum vulgare 4.00E XM_ predicted protein (MYB091) Populus trichocarpa 2.00E XM_ hypothetical protein LOC Vitis vinifera 1.00E XM_ hypothetical protein LOC Vitis vinifera 2.00E XM_ predicted protein, Populus trichocarpa 6.00E XM_ conserved hypothetical protein Ricinus communis 1.00E-67 No matching sequences were found in the NCBI database: 152,153,170,177,198,200,215,636,670,694 The nested primers are not included

23 Relative expression of the six representative genes after 10 h of MV treatment. Homology gene CO4 AUX28 UEP1 PIN3 CDKA Fd1 Control 0.91± ± ± ± ± ±0.16 Treatment 1.88±0.27* 1.98±0.18* 2.78±0.18* 3.87±0.29* 2.56±0.11* 3.30±0.23 Terminal shoots with floral buds at the millet stage were cut off from litchi trees and immediately placed in 40 µm MV solution or distilled water as a control. All the treated shoot cuttings were placed in a growth chamber at 20. Ten hours later, panicle primordia were sampled for qpcr analysis. Values are means ± SE from 3 replicates. Statistically significant differences (P<0.05) between the treatment and the control are indicated by asterisks according to Student s t-test. Five out of the six representative genes were significantly induced by MV after 10 h of treatment.

24 (a) LcCO4 Control Treatment LcAux28 LcUEP1 LcPIN3 Time of treatment Time of treatment Fig. 4 Relative quantities of the LcCO4 (a), LcAUX28 (b), LcUEP1(c), LcPIN3(d) in the MV-treated or water-treated floral buds of Nuomichi from 0 to 15 h of treatment. The relative expression of these genes increased to the highest level at 10 h of treatment then decreased at 15 h of treatment.

25 To investigate whether the differentially expressed genes express during floral differentiation, relative expression of the genes in buds of the terminal shoots was determined at a stage from floral induction to floral organ development.

26 Fig. 5 a-g, Images of the floral buds on 25 December 2011 (a) showing buds at floral induction stage, on 13 January 2012 (b) showing buds at floral initiation stage, on 1 February (c), 5 February (d) and 12 February (e) showing rhachis developmental stage, on 20 February (f) showing the floral organ developmental stage. g-k, relative quantities of the LcCO4 (g), LcAUX28 (h), LcUEP1(i), LcPIN3(j) and LcCDKA(k) in the MV-treated or water-treated floral buds of Nuomichi. Expression level of LcCO4 (g) at floral induction stage was higher than that at the latter developmental stages. The relative expression of LcAux28 (h) increased to the highest level at floral initiation stage and decreased at the latter developmental stage. LcUEP1(i) and LcCDKA(k) transcrition was low at induction stage and relatively higher at initiation, rhachis developmental stages and floral organ developmental stage. LcPIN3(i) expression was low at floral induction, initiation and the beginning of rhachis developmental stage, and high at the latter rhachis and floral organ developmental.

27 Which genes were regulated by NO? An SNP-treated primordium cdna SSH library was constructed to identify NO responsive genes.

28 Table 3 Identification of SNP responsive genes isolated by SSH in litchi primordia. Clone Accession no. of matching sequence Matching sequence in public database Source of matching sequence Transport facilitation Best e- value (BLASTx) 12 HQ Lipid transfer protein (LTP) Dimocarpus longan 6.00E JN Aquaporin PIP1 (PIP1) Dimocarpus longan 2.00E NM_ Flavonol sulfotransferase-like protein (SOT12) Arabidopsis thaliana 2.00E JF Tubulin beta-6 Brassica oleracea var. 1.40E XM_ Putative,tonoplast intrinsic protein Arabidopsis lyrata 1.00E NM_ Oligopeptide transporter 7 (OPT7) Arabidopsis thaliana 3.00E NM_ Ribosome associated membrane protein RAMP4 (AT1G27330) Arabidopsis thaliana 2.00E XM_ Electron carrier Ricinus communis 8.00E AJ Mitochondrial half-abc transporter (STA1 gene) Arabidopsis thaliana 1.00E-16 Q176 NM_ Phosphatidylinositol transfer-like protein Arabidopsis thaliana 5.00E-110 Transcription 566 NM_ S-adenosylmethionine decarboxylase-like protein Arabidopsis thaliana 2.00E EU Metallothionein-like protein Zea mays 2.00E NM_ Gibberellin-regulated family protein (AT2G18420) Arabidopsis thaliana 2.00E NM_ S-adenosylmethionine decarboxylase-like protein Arabidopsis thaliana 4.00E EU Metallothionein-like protein Zea mays 3.00E-20 Stress response 2 DQ Vegetative storage protein (VSP1) Litchi chinensis 3.00E JF Mannose/glucose-specific lectin Litchi chinensis 2.00E AJ Putative proline-rich cell wall protein Vitis vinifera 3.20E HQ Oxygen evolving enhancer protein 1 (OEE1) Litchi chinensis 2.00E AF Dehydration-responsive protein Prunus persica 1.00E EU Antimicrobial peptide snakin Capsicum annuum 2.00E-09 Unknown protein 6 AY Unknown protein Arabidopsis thaliana 1.00E XM_ Conserved hypothetical protein Ricinus communis 5.00E XM_ Conserved hypothetical protein Ricinus communis 2.00E AY Unknown protein (At3g08030) Arabidopsis thaliana 1.00E XM_ Hypothetical protein Arabidopsis lyrata 4.00E AY Unknown protein (At1g32460) Arabidopsis thaliana 8.00E AY Unknown protein (At5g64400) Arabidopsis thaliana 1.00E-32 Q158 EU Hypothetical protein Zea mays 1.40E-01

29 Carbohydrate metabolism 80 NM_ Fructose-bisphosphate aldolase, class I (AT2G36460) Arabidopsis thaliana 3.00E AY Carbonic anhydrase Arabidopsis thaliana 1.00E HQ Chloroplast ribulose-1,5-bisphosphate carboxylase/oxygelitchi chinensis 3.00E EF Chloroplast protein Arnebia euchroma 2.00E NM_ Long-chain-alcohol oxidase Arabidopsis thaliana 2.00E-52 Others 14 FJ Chanthaburi ribosomal protein L9 gene Sonneratia ovata 8.00E NM_ Chlorophyll a-b binding protein CP29.1 (LHCB4.1) Arabidopsis thaliana 5.00E NM_ Polyubiquitin 10(UBQ10) Arabidopsis thaliana 3.00E AB Self-incompatibility (S-) locus region Ipomoea trifida 1.60E XM_ Ripening-induced protein 1-like (MRIP1) Vitis vinifera 2.90E NM_ S ribosomal protein S8-1 (AT5G20290) Arabidopsis thaliana 4.00E XM_ ATP binding protein Ricinus communis 2.00E BT Nodulin-like protein Arabidopsis thaliana 3.00E EU Polyubiquitin (WubiG) Triticum aestivum 2.00E NM_ Ribosome associated membrane protein RAMP4 Arabidopsis thaliana 3.00E AF S ribosomal protein L10 Vitis riparia 9.00E NM_ Nucleic acid-binding, OB-fold-like protein Arabidopsis thaliana 9.00E XM_ Ubiquitin-conjugating enzyme E2 Vitis vinifera 8.00E XM_ Small nuclear ribonucleoprotein Ricinus communis 2.00E XM_ S ribosomal protein S8-like (LOC ) Vitis vinifera 4.00E EF Ubiquitin ligase-like protein Oryza sativa Japonica 7.00E GQ Transgenic GMO cassette Glycine max 8.00E-34 Q1309 HM Miraculin-like protein 1 Citrus limonia 2.00E-18 Secondary metabolism 21 XM_ Similar to anthocyanidin reductase Vitis vinifera 2.00E AB Chalcone synthase CHS3 Citrus sinensis 7.00E FJ Peroxidase 4 Litchi chinensis 2.00E AK Monodehydroascorbate reductase (NADH) - like protein Arabidopsis thaliana 2.00E EU Laccase Litchi chinensis 1.00E BT Putative copper amine oxidase (At2g42490) Arabidopsis thaliana 7.00E-20 Intracellular signaling 67 AY Ethylene receptor (ERS) Litchi chinensis 1.00E NM_ Putative gibberellin-regulated protein Arabidopsis thaliana 1.00E XM_ SKP1-like protein 1A (LOC ), Vitis vinifera 8.00E AB G protein beta-subunit-like protein Nicotiana tabacum 4.00E AY Aux/IAA protein Vitis vinifera 4.00E D Diacylglycerol kinase Arabidopsis thaliana 1.80E-01 Q163 AJ ABA and calcium induced protein phosphatase 2C (PP2C) Fagus sylvatica 8.00E-17 No matching sequences were found in the NCBI database: 817,891,923,1036,1410 The nested primers are not included

30 To further confirm if the differential expressed genes are induced by SNP, five genes were selected from the library.

31 Carbohydrate metabolism 80 NM_ Fructose-bisphosphate aldolase, class I (AT2G36460) Arabidopsis thaliana 3.00E AY Carbonic anhydrase Arabidopsis thaliana 1.00E HQ Chloroplast ribulose-1,5-bisphosphate carboxylase/oxygelitchi chinensis 3.00E EF Chloroplast protein Arnebia euchroma 2.00E NM_ Long-chain-alcohol oxidase Arabidopsis thaliana 2.00E-52 Others 14 FJ Chanthaburi ribosomal protein L9 gene Sonneratia ovata 8.00E NM_ Chlorophyll a-b binding protein CP29.1 (LHCB4.1) Arabidopsis thaliana 5.00E NM_ Polyubiquitin 10(UBQ10) Arabidopsis thaliana 3.00E AB Self-incompatibility (S-) locus region Ipomoea trifida 1.60E XM_ Ripening-induced protein 1-like (MRIP1) Vitis vinifera 2.90E NM_ S ribosomal protein S8-1 (AT5G20290) Arabidopsis thaliana 4.00E XM_ ATP binding protein Ricinus communis 2.00E BT Nodulin-like protein Arabidopsis thaliana 3.00E EU Polyubiquitin (WubiG) Triticum aestivum 2.00E NM_ Ribosome associated membrane protein RAMP4 Arabidopsis thaliana 3.00E AF S ribosomal protein L10 Vitis riparia 9.00E NM_ Nucleic acid-binding, OB-fold-like protein Arabidopsis thaliana 9.00E XM_ Ubiquitin-conjugating enzyme E2 Vitis vinifera 8.00E XM_ Small nuclear ribonucleoprotein Ricinus communis 2.00E XM_ S ribosomal protein S8-like (LOC ) Vitis vinifera 4.00E EF Ubiquitin ligase-like protein Oryza sativa Japonica 7.00E GQ Transgenic GMO cassette Glycine max 8.00E-34 Q1309 HM Miraculin-like protein 1 Citrus limonia 2.00E-18 Secondary metabolism 21 XM_ Similar to anthocyanidin reductase Vitis vinifera 2.00E AB Chalcone synthase CHS3 Citrus sinensis 7.00E FJ Peroxidase 4 Litchi chinensis 2.00E AK Monodehydroascorbate reductase (NADH) - like protein Arabidopsis thaliana 2.00E EU Laccase Litchi chinensis 1.00E BT Putative copper amine oxidase (At2g42490) Arabidopsis thaliana 7.00E-20 Intracellular signaling 67 AY Ethylene receptor (ERS) Litchi chinensis 1.00E NM_ Putative gibberellin-regulated protein Arabidopsis thaliana 1.00E XM_ SKP1-like protein 1A (LOC ), Vitis vinifera 8.00E AB G protein beta-subunit-like protein Nicotiana tabacum 4.00E AY Aux/IAA protein Vitis vinifera 4.00E D Diacylglycerol kinase Arabidopsis thaliana 1.80E-01 Q163 AJ ABA and calcium induced protein phosphatase 2C (PP2C) Fagus sylvatica 8.00E-17 No matching sequences were found in the NCBI database: 817,891,923,1036,1410 The nested primers are not included

32 Table 3 Relative expression of the five representative genes after 10 h of SNP treatment. Homology gene ANR CHS GR AUX/IAA E3 Control Treatment 5.1* 4.51* 4.23* 3.47* 1.47 Terminal shoots with floral buds at the millet stage were cut off from litchi trees and immediately placed in SNP solution or distilled water as a control. All the treated shoot cuttings were placed in a growth chamber at 20. Ten hours later, panicle primordia were sampled for qpcr analysis. Values are means ± SE from 3 replicates. Statistically significant differences (P<0.05) between the treatment and the control are indicated by asterisks according to Student s t-test. Four out of the five representative genes were significantly induced by SNP after 10 h of treatment.

33 To investigate whether the differentially expressed genes express during floral differentiation, relative expression of the genes in buds of the terminal shoots was determined at a stage from floral induction to floral organ development.

34 a b c d e f (a) showing buds at floral induction stage, on 13 January (b) showing buds at floral initiation stage, on 1 February. (c), (d) and (e) showing rhachis developmental stage. (f) showing the floral organ developmental stage.

35 Time of treatment (Y-M-D ) Time of treatment (Y-M-D ) Fig. 7 Relative quantities of the LcANR (a), LcCHS (b), LcGR(c), LcAUX/IAA (d) in the SNP-treated or water-treated floral buds of Nuomichi. The differentially expressed genes were expressed to some extend during floral differentiation.

36 What will be done in the future study? According to the MV and SNP SSH cdna libraries, we isolated many phytohormone related genes, such as transportation components LcPIN1, AUX28, signaling transduction pathway component such as the AUX/IAA. Future works will focus on functions of these phytohormone related genes in relation to litchi flowering. Establish signal transduction networks between stress signals and pytohormone underlying litchi flowering.

37 Acknowledgments The project was funded by National Natural Science Foundation ( ) The Agricultural Industry Project (CARS-33-08) by the Ministry of Agriculture. Master student: Wei-Wei Liu and Xin-Yu Lu.

38 Thank you!