Guitton B.1, Kelner J.J.2, Celton J.M.3, Renou J.P. 3, Andrés F.4, Chagné D.5, Costes E.1
RGC8 - Angers - 21 June 2016
Transcriptomic comparison between
deflowered and overloaded apple trees: toward
the understanding of biennial bearing in apple
Alternate bearing in fruit tree species
� Irregular fruit production over consecutive years
� “ON” years: high production
� “OFF” years: low production
(Jonkers, 1979; Monselise and Goldschmidt, 1982)
11935 1940 1945 1950
0
20
40
60
80
100
120
140
160 Control NPK
Yie
ld p
er tr
ee (
kg)
Year
Example of biennial bearing in apple: annual yield of ‘Dunn’s’ apple trees (Tiller et al., 1959)
Floral development in higher plants:
several time steps toward flowering
Four developmental stages in the meristem� Floral induction� Floral initiation
Meristem programmed to be floral
First morphological sign of floral commitment
Formation of floral organs
Endogenous signals
Environmental signals
- Floral differentiation
- Anthesis
6
In apple, floral initiation takes place around 39 to 53 Days After Full Bloom (DAFB)
(Hanke, 1981; Buban and Faust, 1982; Kotoda et al. 2000; Foster et al. 2003)
Floral induction Floral differentiation
Floral initiation
6853390 14 25 87 146 DAFB
3
Fruit development
What are the causes of biennial bearing in
apple ?
� A lack of flower formation rather than a poor fruit set (Singh, 1948)
� Several physiological hypotheses to explain the inhibition of floral initiation
� Flower formation inhibited by ongoing fruit development
� Hormonal inhibition from developing seeds (Chan and Cain, 1967; Neilsen and Dennis, 2000)
� Nutritional competition with developing fruits (Jonkers, 1979, Goldschmidt et al., 1985)
� Competition with vegetative growth (Dennis and Neilsen, 1999)
44
Objectives of the study� What are the genetic and physiological mechanisms underlying
biennial bearing?� Investigate biological processes involved in floral induction in apical buds
of adult apple trees� Study the effect of the presence of fruits on the differential expression of
genes involved in floral transition in apical buds of bourse shoots
� Compare gene expression between meristems of apple trees in “ON” and “OFF” conditions� RT-qPCR for targeted genes
� Flowering genes and gibberellin oxidase genes� Microarray AryANE_V1 for selected dates
� 63 011 probes (both forward and reverse sense)
5
Methods of the study
Studying the effect of the presence of fruit
on floral induction� Cultivar “Gala”: regular bearer
� Fruiting vs. deflowering treatment: “ON” vs. “OFF” trees
� contrasted situations in SAM
“OFF” tree“ON” tree 406
Expected behavior in shoot apical
meristem (SAM)
“OFF” tree“ON” tree40
Floral induction inhibited
Massive floral
induction
7
� Collected material� “ON trees”: SAM of bourse shoot of a bourse carrying fruits
� “OFF trees”: SAM of short shoot carrying no fruit
� 3 trees per treatment – 3 meristems per tree� 9 RNA samples per data point
� 3 dates from 28 to 119 DAFB� Covering floral induction, initiation
and differentiation
Bourse shoot
42
Quantification of transcript levels in SAM
8
Evaluation of trees’ return bloom the next
spring
� Situation of induction vs. non induction validated
� Meristems in contrasted situation of floral induction419
Differentially expressed genes in SAM between
“ON” and “OFF” trees in the microarray
� 3 dates after full bloom (28, 48 and 119 DAFB)
13
9
111
203
3210
28 DAFB 48 DAFB
119 DAFB
A
0 30
0
58
151
301
28 DAFB 48 DAFB
119 DAFB
B
4
Up-regulated in “ON” trees Up-regulated in “OFF” trees10
Apical buds of deflowered trees are under
active biogenesis toward floral differentiation
11
� Several Gene Ontologies were up-regulated in OFF trees
0 DAFB 119 DAFB28 48
Floral induction
Floral initiation Floral differentiation
Anatomical structure development(16 transcripts)
Cellular components biogenesis
(11 transcripts)
Oxidoreductase activityCatalytic activity
Active cell detoxification(87 transcripts)
30
0
58
151
301
28 DAFB 48 DAFB
119 DAFB
44
30
Are SPL genes involved in floral
induction of “OFF” trees?
� SPL (SQUAMOSA PROMOTER BINDING PROTEIN-LIKE)� Involved in vegetative to reproductive phase transition
� SPLs are involved in the age, photoperiodic and GA flowering pathways of Arabidopsis thaliana (Jung et al. 2011; Torti et al. 2012, Hyun et al.
2016)
SPL5 SPL9 SPL9
12
Meristems of trees bearing a heavy crop are
under stress conditions
13
� Several transcripts involved in response to starvation were up-regulated in ON trees
Response to stress (5 transcripts)
ASN1 (GLUTAMINE-DEPENDENT ASPARAGINE SYNTHASE 1) response to sucrose starvation (Rose et al. 2006)
SEN1 (SENESCENCE 1) induced by phosphate starvation (Xiao et al. 2010)
Gluconeogenesis and autophagyPCK1 (PHOSPHOENOLPYRUVATE CARBOXYKINASE): regulation of gluconeogenesisCA1 and CA2 (CARBONIC ANHYDRASE): carbon utilizationGlutaredoxin family proteins: cellular homosteasisGroup of putative lipases: lipid degradation during starvation
0 DAFB 119 DAFB28 48
Floral induction
Floral initiation Floral differentiation
13
9
111
203
3210
28 DAFB 48 DAFB
119 DAFB
0
932
Meristems of trees bearing a heavy crop are
under unfavorable redox status
14
� Oxidoreductase molecular function significantly over-represented� 8, 21 and 25 up-regulated in ‘ON’ trees at 28, 48 and
119 DAFB
Response to oxidative stress (4 transcripts)OXS3 (OXYDATIVE STRESS 3): tolerance to oxidative stressQuinone oxidoreductase-like protein: detoxification of reactive carbonylsAPR1 and APR2 (5′ADENYLYLPHOSPHOSULFATE REDUCTASE): necessary to combat oxidative stress
Hormonal regulation in response to stress (17 transcripts)
ABR1, ATHB-7 and RD26: response to abscisic acid stimulus9 Leucine-Rich-Repeat protein kinases: cellular communication and signal transductionGA20ox, GA2ox: Gibberellin metabolism / catabolism4 SNAK1: response to gibberellin stimulus
0 DAFB 119 DAFB28 48
Floral induction
Floral initiation Floral differentiation
13
9
111
203
3210
28 DAFB 48 DAFB
119 DAFB
0
93210
Tempranillo 1 (RAV family), a floral
repressor is up-regulated in ‘ON’ trees
� Known to link photoperiodic and GA pathways� Directly repress FT expression
(Castillejo et al. 2008)
� Regulate GA biosynthesis via GA3ox (Osnato et al. 2012)
� Involved in photoperiod and sucrose control of flowering in soybean (Lu et al.
2014)
TEM1
15
Summary of microarray analysis
� Meristems of trees bearing heavy crop are under stress conditions� Starvation, unfavorable redox status
� Sugar, hormonal and redox status involved in floral induction� Control through sugar signaling (Wahl et al. 2013) or via a direct control
through redox status, as recently demonstrated? (Blanvillain et al. 2011; Kocsyet al. 2013)
� SAM of “OFF” trees could be induced to flower through GA pathway involving SPL genes� As recently described in Arabidopsis thaliana under long days (Jung et
al. 2011; Tori et al. 2012)
16
�European collaborative project « AlternApp »: ANR-DFJ projectINRA, AFEF Montpellier, France INRA, UMR 547 PIAF, Clermont-Ferrand, FranceJulius Kuehn-Institute (JKI), Dresden, Germany University of Hohenheim, Stuttgart, Germany Foundation E. Mach (IASMA), San Michele all’Adige, Italy
17
Microarray results recently publishedGuitton et al. 2016 in BMC
http://bmcplantbiol.biomedcentral.com/articles/10.1186/s12870-016-0739-y
