Functional Studies on Japanese apricot PmDAM genes

Transcription

1 Plant Dormancy Workshop, Plant Animal Genome XX , San Diego Functional Studies on Japanese apricot PmDAM genes Hisayo Yamane, Tsuyoshi Habu, Ryuta Sasaki, Ryutaro Tao Graduate School of Agriculture, Kyoto University, Kyoto, Japan

2 Annual growth and flowering cycle of Japanese apricot (Prunus mume) Growth cessation, Flowering initiation Leaf fall Vegetative growth habit Shoot tip abortion Summer Autumn Dormant season Winter Bud burst Shoot growth Growth cessation Lateral bud development Growing season Spring Fruit set Blooming Leaf bud Flower bud At leaf axil, a single leaf bud subtended by two or three flower buds was formed. Leaf buds contain shoot apical meristem, whereas flower buds contain single flower meristem.

3 Bud dormancy in temperate fruit trees Dormancy is a temporary suspension of visible growth of any plant structure containing a meristem (Lang et al., 987) Summer Autumn Winter Spring Low temperature, Short daylength, Water stress Low temperature accumulation Paradormancy Endodormancy Ecodormancy Bud burst Blooming Apical dominance, Correlative inhibition -No bud burst occurs in favorable condition -An unknown endogenous signal leads to growth inhibition -Released by low temperature (Chilling requirement) Temperature extreme, Water stress Endodormancy is regulated by an endogenous signal within (i.e., endo ) the buds Endodormant buds require a certain amounts of chilling accumulation for dormancy release

4 Agricultural problem related to bud dormancy dormant blooming bud abortion caused by incomplete dormancy release fruit set Uniform flowering is not achieved Due to global warming, farmers will not be able to grow temperate fruits in southern area of Japan!? Studies on endodormancy regulation will help to maintain uniform blooming of temperate fruit trees

5 Why Japanese apricot for dormancy study? One of the transformable plants among stone fruits Kyoto is located near one of the large Japanese apricot production areas in Japan. There are genetic resources that vary with chilling requirements Small genome size (~220Mbp) Peach (close relatives) genome has been sequenced (Apr. 200) We started Japanese apricot dormancy studies since 2005.

6 Many factors are involved in dormancy induction, maintenance and release Short daylength Low temperature Drought stress ABA????? Growth cessation, bud development Dormancy induction Prolonged low temperature Dormancy maintenance?? Long daylength (secondary effects) Dormancy release?? Positive regulator for dormancy Negative regulator for dormancy Dormancy release

7 Searching for genetic factors controlling endodormancy First, we intended to isolate the genes specifically expressed in endodormant buds among para-, endo-, and eco-dormant buds using RNA subtraction technique (Yamane et al., 2008) RNA subtraction RNA subtraction 2 Endo minus Para Endo minus Eco Still dormant but ready to bud burst Only one clone was commonly isolated from two libraries. Because this gene was similar to peach DORMANCY-ASSOCIATED MADS6, we named this gene as Prunus mume DAM6 (PmDAM6). Endodormant period Specifically expressed in endodormant leaf buds!

8 Second, we conducted EST analysis to isolate the genes specifically expressed in endodormant buds among para-, endo-, and eco-dormant buds. Total 485,376 raw reads Assembly 3,629 unique sequences 28,382 contigs and 85,247 singletons (Habu et al., submitted)

9 Leaf bud Summary of Japanese apricot dormant bud ESTs Flower bud Three genes were commonly upregulated in both leaf and flower buds at endodormant stage, including PmDAM6. Table 5. Endo-dormant-specific up-regulated unigenes in both buds EST number Similar genes in GenBank Unigene name paral endol ecol paraf endof ecof Accession Number Description PmC CBI unnamed protein product [Vitis vinifera] PmC BAH dormancy-associated MADS-box transcription factor 6 [Prunus mume] PmC ACU6624. unknown [Glycine max]

10 PmDAM6 showed endodormancy-associated expression Shoot growth Growth cessation Bud development Dormancy release Bud burst Endodormancy PmDAM6 We focused on PmDAM6 as a candidate for controlling dormancy.

11 Genomic DNA blot analysis Japanese apricot genome has several sequences similar to PmDAM6. Genomic library screening and shotgun sequencing 約.0kbp 約 5.7kbp 約 0.5kbp 約.5kbp 約.0kbp PmDAM PmDAM2 PmDAM3 PmDAM4 PmDAM5 PmDAM6 Japanese apricot contains six tandemly arrayed MADS-box genes (SVP/AGL24 clade) as is the case in peach (Bielenberg et al., 2008).

12 (PmDAM4/UBQ) (PmDAM5/UBQ) (PmDAM6/UBQ) (PmDAM/UBQ) (PmDAM2/UBQ) (PmDAM3/UBQ) Seasonal expression changes of PmDAM-PmDAM6 0 PmDAM 0 PmDAM2 0 PmDAM Growth cessation April May June July Aug. Sept. Oct. Nov. endodormancy 0.0 April May June July Aug. Sept. Oct. Nov. 0.0 April May June July Aug. Sept. Oct. Nov. 0 PmDAM4 0 PmDAM5 PmDAM April May June July Aug. Sept. Oct. Nov April May June July Aug. Sept. Oct. Nov. 0.0 April May June July Aug. Sept. Oct. Nov. All six PmDAM expressions were lower during active shoot growth, then up-regulated during growth cessation.

13 (PmDAM4/UBQ) (PmDAM5/UBQ) (PmDAM6/UBQ) (PmDAM/UBQ) (PmDAM2/UBQ) (PmDAM3/UBQ) Seasonal expression changes of PmDAM-PmDAM6 in high-chill, Nanko (long and deep) and low-chill, Ellching (short and shallow) 0 PmDAM 00 PmDAM2 0 PmDAM Nanko Ellching Jun Jul Aug Sept Oct Nov Dec Jan Feb Mar 0.00 Jun Jul Aug Sept Oct Nov Dec Jan Feb Mar Jun Jul Aug Sept Oct Nov Dec Jan Feb Mar Endodormancy 0 PmDAM4 PmDAM5 0 PmDAM Nanko Ellching Jun Jul Aug Sept Oct Nov Dec Jan Feb Mar Jun Jul Aug Sept Oct Nov Dec Jan Feb Mar Jun Jul Aug Sept Oct Nov Dec Jan Feb Mar All six PmDAMs were down-regulated toward spring. Negative correlations between PmDAM3-6 expressions and endodormancy release

14 Seasonal growth habit and expressions of PmDAM-6 Shoot growth Growth cessation Bud development Dormancy release Bud burst Endodormancy PmDAM4-6 PmDAM3 PmDAM,2 PmDAMs were up-regulated during growth cessation. PmDAM3-6 were downregulated during endodormancy release.

15 Cold treatment to induce endodormancy release One-year old shoots were collected in October. High-chill, Nanko Low-chill, Ellching Chilling for 32 days Chilling for 64 days Transfer to forcing condition Transfer to forcing condition Low-chill High-chill Low-chill High-chill Low-chill High-chill

16 (PmDAM4/UBQ) (PmDAM5/UBQ) (PmDAM6/UBQ) (PmDAM/UBQ) (PmDAM2/UBQ) (PmDAM3/UBQ) Decrease in PmDAM4-6 expressions coincided very well with endodormancy release 00 PmDAM PmDAM2 PmDAM Cold exposure (days) Endodormancy release Cold exposure (days) Cold exposure (days) 0 PmDAM4 PmDAM5 PmDAM Cold exposure (days) Cold exposure (days) Cold exposure (days) High-chill, Nanko (cold+) High-chill, Nanko (cold-) Low-chill, Ellching (cold+) Low-chill, Ellching (cold-)

17 Summary of expressional analyses of PmDAMs Chilling accumulation PmDAMs PmDAMs Endodormancy induction Endodormancy Endodormancy release Bud burst Downregulation of DAM-like genes during dormancy release has been reported in other temperate fruit trees including; kiwifruit (Wu et al., 20), Japanese pear (Ubi et al., 200), Peach (Jimenez et al., 200; Yamane et al., 20) Raspberry (Mazzitelli et al., 2007)

18 What are the biological functions of PmDAMs? Are PmDAMs directly involved in bud endodormancy? Endodormancy induction? Maintenance? If so, How? Which pathway?

19 Total internode length (cm) Growth of 35S:PmDAM6 was retarded Sasaki et al., Plant Physiol. (20) emp DAM6- DAM6-2 DAM6-3 DAM6-4 DAM6-6 WT 35S: PmDAM6 Shoot growth was suppressed in all six transgenic 35S:PmDAM6 lines.

20 Growth cessation and bud set occurred in 35S:PmDAM6 poplars at non-dormancy-inducing condition (6-h daylength, 22C) WT terminal bud 35S:PmDAM6 Under LD condition, growth cessation was promoted and terminal bud set was induced in 35S:PmDAM6, whereas control plants showed continuous shoot growth. Sasaki et al. (20)

21 Endodormancy was induced in 35S:PmDAM6 poplars at non-dormancyinducing condition (6-h daylength, 22C) WT Leaf removal 35S: PmDAM6 35S:PmDAM6 poplars did not resume their growth, whereas the control poplars showed bud burst. Sasaki et al. (20)

22 PmDAM6 positively regulates endodormancy through its growth inhibitory effect Chilling accumulation Shoot growth Growth inhibitor (PmDAM6) Growth cessation Endodormancy induction Endodormancy Growth inhibitor (PmDAM6) Endodormancy release Bud burst

23 How is PmDAM6 involved in dormancy regulation? Flower buds Vegetative buds PmDAM6 is expressed not only in SAM but also in vascular tissues of shoots (leaves, stems and bud scales). Does PmDAM6 act within meristem? According to Lang et al. (987) nomenclature, factors involved in endodormancy should work within meristem (not only subtending tissues). What is the target gene of PmDAM6?

24 (PttFT2/PttACTIN) (PmFT/ACTIN) (PmDAM6/UBQ) What is the target of PmDAM6 for dormancy regulation? PtFT RNAi poplars showed bud set in LD condition (Bohlenius et al., 2006), which seems to be similar to the phenotype observed in our 35S:PmDAM6 poplars. PtFT2 expressions in 35S:PmDAM6 poplars PttACTIN PmDAM6 (32 cycles) PttFT2 (36 cycles) PttACTIN emp Seasonal expressions in Japanese apricot leaves PmDAM6 in leaves Down-regulated? weeks after acclimatization Up-regulated? WT DAM6- DAM6-5 DAM6-6 PtFT2 is down-regulated in some DAM6-,5,6 lines. 0.0 PmFT in leaves.4 April May June July Aug. Sept. Oct. Nov April May June July Aug. Sept. Oct. Nov. PmDAM6 possibly down-regulates FT in leaves?

25 (PmDAM6/UBQ) (PmFT/ACTIN) What is the target of PmDAM6 for dormancy regulation? Seasonal expressions in Japanese apricot buds PmFT in buds 0.4 PmDAM6 appeared not to repress FT expression in buds June July Aug.Sept. Oct. Nov. Dec. Jan. Feb. Mar. PmDAM6 in buds 0 0. PmDAM6 PmDAM Jun Jul Aug Sept Oct Nov Dec Jan Feb Mar FT?? We are now trying to identify the target gene of PmDAM6 in dormant buds.

26 Conclusion and prospects We identified PmDAM genes highly expressed in Japanese apricot dormant buds. PmDAM genes were down-regulated during chilling-mediated dormancy release. 35S:PmDAM6 poplars showed growth cessation and bud set under non-dormancy-inducing condition. PmDAM6 affected endodormancy release of 35S:PmDAM6 poplars. We are currently searching for the genes under the control of PmDAM6 in bud meristem.

27 Acknowledgements Lab of Pomology, Grad. Sch. of Agric., Kyoto University, Kyoto, Japan NARO Institute of fruit tree science, Japan Dr. H. Sakakibara RIKEN Plant Science Center, Yokohama, Japan Dr. M. Takagi, Dr. M. Nakata Bioproduction Research Institute, Tsukuba, Japan Dr. I. Kataoka, Dr. K. Beppu Kagawa University, Miki, Kagawa, Japan Dr. K. Yano Meiji University, Kawasaki, Japan Research funds Program for the Promotion of Basic and Applied Researches for Innovation in Biooriented Industry, Japan. ( ) Grant-in-Aid for Scientific Research (KAKENHI) from Japan Society for the Promotion of Science (JSPS), Japan. ( , 20- )