Flowering-time_genes_modulate_meristem_determinacy_and_growth_form_in_Arabidopsis_thaliana

Flowering-time genes modulate meristem determinacy and growth form in Arabidopsis thaliana

Siegbert Melzer 1,Frederic Lens 2,3,Jero

?me Gennen 1,Steffen Vanneste 1,Antje Rohde 1&Tom Beeckman 1Plants have evolved annual and perennial life forms as

alternative strategies to adapt reproduction and survival to environmental constraints.In isolated situations,such as

islands,woody perennials have evolved repeatedly from annual ancestors 1.Although the molecular basis of the rapid evolution of insular woodiness is unknown,the molecular difference between perennials and annuals might be rather small,and a change between these life strategies might not require major genetic innovations 2,3.Developmental regulators can strongly affect evolutionary variation 4and genes involved in meristem transitions are good candidates for a switch in growth habit.We found that the MADS box proteins SUPPRESSOR OF

OVEREXPRESSION OF CONSTANS 1(SOC1)and FRUITFULL (FUL)not only control ?owering time,but also affect

determinacy of all meristems.In addition,downregulation of both proteins established phenotypes common to the lifestyle of perennial plants,suggesting their involvement in the prevention of secondary growth and longevity in annual life forms.

Plant growth originates from a small number of undifferentiated cells called meristems.Primary meristems are established during embry-ogenesis and form primary tissues from which all plant organs develop.Secondary meristems,such as axillary meristems and the cambium,originate within primary tissues.Meristems can be deter-minate—that is,consumed for the formation of an organ—or indeterminate,meaning that they are active throughout the life span of a plant.Upon ?oral induction in annual plants,vegetative shoot meristems undergo the transition to in?orescence meristems.These in?orescence meristems will remain indeterminate for some time to generate determinate ?oral meristems giving rise to ?owers.Finally,all meristems are consumed and the plants die in the same growing season.In contrast,perennial plants have evolved more elaborate life strategies to survive harsh environmental conditions for many years by forming perennial structures such as overwintering buds,bulbs or tubers,which contain at least one indeterminate meristem for the outgrowth in the next season 2.Often,perennial plants incorporate

enormous amounts of long-lived and eventually dead biomass (wood)through cambial activity (secondary growth).

Arabidopsis thaliana is a small annual herb in which ?oral induction is controlled by different ?owering-time pathways.These pathways depend on environmental cues,such as day length (photoperiod)and vernalization (cold temperature),or on plant age.Arabidopsis is a facultative long-day plant that ?owers much faster under long (16h/day)than short (8h/day)light periods.After perceiving ?owering-inducing long days,the key regulator of the photoperiodic pathway,CONSTANS (CO),activates FT (FLOWERING LOCUS T )in the leaf vasculature.The FT protein is transported to apical meristems,where it triggers the ?oral transition 5.SOC1(AGL20)and FUL (AGL8)are MADS box genes acting downstream of FT in apical meristems,but they are already expressed in leaves —indepen-dently of FT—during the vegetative phase.Early upon ?oral induction SOC1and FUL are induced in apical meristems,and,later on,both genes are also expressed in procambial strands of developing in?or-escences 6–11.FUL has been described for its role in fruit dehiscence 12,

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Table 1Flowering time,assayed by leaf numbers of the basal rosette

Long days

Short days Genotype RL CL TL s.d.RL CL TL s.d.Col 12.1 3.015.1 1.460.79.970.6 2.5ful-213.8 5.619.4 1.362.912.775.6 3.6soc1-3

23.6 4.428.0 2.282.77.990.6 2.5soc1-3ful-2

34.1 5.239.3 1.183.111.894.8 2.935S:AGL19

3.9 3.57.50.811.8 5.717.50.835S:AGL19soc1-3ful-2

4.210.91

5.1 1.411.77.319.0 1.635S:FT

3.4 1.4

4.90.8 3.5 1.8

5.20.935S:FT ful-27.9 2.510.40.98.5 2.410.9 1.335S:FT soc1-3

9.40.59.9 1.19.3 1.210.5 1.235S:FT soc1-3ful-2

21.6

4.5

26.1

1.3

32.2

8.2

40.4

1.9

RL,rosette leaves;CL,cauline leaves;TL,total leaves;s.d.,standard deviation of the mean.

Received 10March;accepted 3September;published online 9November 2008;doi:10.1038/ng.253

1Department

of Plant Systems Biology,Flanders Institute for Biotechnology and Department of Molecular Genetics,Ghent University,Technologiepark 927,B-9052

Ghent,Belgium.2Laboratory of Plant Systematics,Institute of Botany and Microbiology,Katholieke Universiteit Leuven,Kasteelpark Arenberg 31,B-3001Leuven,Belgium.3National Herbarium of the Netherlands,Leiden University Branch,P.O.Box 9514,NL-2300RA Leiden,The Netherlands.Correspondence should be addressed to S.M.(siegbert.melzer@psb.ugent.be).

but as SOC1and FUL also interact in yeast two-hybrid experiments as homo-and heterodimers 13,FUL might have additional functions that may be in part redundant to those of SOC1in areas of overlapping expression.The soc1mutants ?ower signi?cantly later in long and short days,whereas ful mutants are only slightly delayed 7–9,12(Table 1).Flowering in double mutants with different combinations of soc1and ful mutant alleles was further delayed only in long-day conditions.In short days,the double mutants ?owered no later than soc1single mutants (Table 1and Supplementary Fig.1a ,b online),suggesting that both genes might have a redundant role for photo-periodic control of ?owering time.

During seed ripening in long days,many apically positioned in?orescence meristems of soc1-3ful-2mutants reverted to a vegeta-tive state,producing small true leaves with axillary meristems in apical rosettes (Fig.1a ,b and Supplementary Fig.2a online),which had never been observed before in Arabidopsis 14.Meristems in cauline leaf axils at the base of double-mutant in?orescences stayed in a vegetative phase and developed aerial rosettes (Supplementary Fig.2b ).Addi-tionally,the double mutants formed bracts that are normally absent in Arabidopsis 15(Supplementary Fig.3online).Wild-type and single-mutant plants senesced and died after seed maturation.In all double mutants,basal rosette leaves,cauline leaves and siliques senesced as well,whereas the apical rosettes resulting from in?orescence meristem reversions,the aerial rosettes and the stems remained alive.Of note,arrested vegetative shoots resembling dormant buds persisted in the axils of dead cauline leaves (Fig.1c ).These buds and also the aerial and apical rosettes grew out to form new in?orescences and rosettes in a next growth cycle (Supplementary Fig.2c ).Subsequently,several distinct waves of growth occurred,where again in?orescence mer-istems reverted to vegetative meristems and aerial rosettes formed at the base of the in?orescences.The soc1ful double mutants developed into highly branched shrubs in both ecotypes tested (Fig.1d and Supplementary Fig.4online).In short days,soc1ful mutants presented no distinct growth cycles and produced fewer in?orescences,but again with reversions of in?orescence meristems.Nevertheless,

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s

Figure 1Perennial features in soc1-3ful-2mutants.(a )Floral reversion at the top of a soc1-3ful-2in?orescence.(b )soc1-3ful-2apical rosettes at a later stage than shown in a .(c )Dead basal cauline leaf with a small arrested axillary shoot that will develop into an aerial rosette in the next growth wave.(d )Comparison of soc1-3ful-2mutants grown in long days for 6weeks (left),3months (middle)or 5months (right).(e )Woody stem of a 4-month-old soc1-3ful-2mutant in long days.(f )Comparison of an 8-month-old soc1-3ful-2mutant with many aerial rosettes at the base and ?oral

reversion at in?orescence meristems (left)and a 2-month-old wild-type plant (right)grown in short days.(g )A 14-month-old soc1-3ful-2mutant with rosettes on lateral branches.

b

a d

c f

e SX

SP

Figure 2Secondary growth in soc1-3ful-2plants.(a )Hand section through a 5-mm-thick lateral branch of a 5-month-old double mutant with a

secondary xylem stained dark blue and a secondary phloem stained pink.The inset shows a wild-type main stem at the same magni?cation.(b )Magni?ed cross-section through a 5-mm-thick lateral branch of a

5-month-old double mutant showing radial ?les of xylem and phloem derived from secondary cambium (on the line between yellow arrowheads).(c )Cross-section through the base (1cm above the rosette)of a 6-cm-long wild-type in?orescence showing only primary tissues in the marked area.soc1and ful single mutants did not show secondary growth and were indistinguishable from wild type.(d )Cross-section through a soc1-3ful-2in?orescence at the same developmental stage as the wild-type plant in c ,with ?les of cells of secondary xylem and phloem (additional cell layers in the marked area).QJ;At this stage the interfascicular cambium was initiated and fused with QJ;the cambium from the vascular bundles to form a closed cambium.

(e )Secondary growth in a 6-cm-long 35S:AGL19soc1-3ful-2in?orescence stem 1cm above the rosette with secondary xylem and phloem.(f )Massive secondary growth in the in?orescence stem of a 5-month-old 35S:AGL19soc1-3ful-2plant 1cm above the rosette.Scale bars in a ,500m m;c ,d ,f ,100m m;e ,200m m.SP ,secondary phloem;SX,secondary xylem.

they showed a markedly increased life span and a shrub-like pheno-type through the continuous growth of vegetative aerial rosettes (Fig.1f ,g and Supplementary Fig.2d ).In contrast to wild-type plants,which showed only limited secondary growth in in?orescence stems 16,soc1ful mutants readily developed woody stems under either photoperiod (Fig.1e and Supplementary Fig.2e ).The extensive secondary growth ?nally formed a considerable wood cylinder that had never been observed before in Arabidopsis (Fig.2a ,b ).

T o ascertain whether secondary growth resulted from the loss of both SOC1and FUL or merely from the increased longevity,we analyzed the initiation of secondary growth.Wild-type and soc1-3ful-2plants were grown in short days to a stage that allowed immediate ?oral induction after transfer of the plants to long days.In soc1-3ful-2mutants,a closed cambium was established at a very early stage of in?orescence stem development.Secondary growth was already evident in the basal part of young 6-cm-long soc1ful in?orescences,and this growth gradually extended into the upper part of the in?orescence stem.No signs of such secondary growth could be recorded in wild-type plants at comparable stages (Fig.2c ,d ).At a later developmental stage,all soc1-3ful-2stems,including those of higher-order co-in?orescences,had steadily increasing amounts of secondary tissues (Supplementary Fig.5online).The early initiation of a cambium in in?orescence stems of soc1ful mutants indicates that the loss of SOC1and FUL function rather than the increased life span of the plants was responsible for the observed secondary growth.Recently it was reported that ?oral induction is a condition for xylem expansion in hypocotyls and roots 17at stages before in?orescences are formed.However,whether SOC1and FUL activity also have to be modulated in these organs is not yet known.

T o test further whether late ?owering was a prerequisite for the observed extreme longevity and secondary growth,we overexpressed the ?owering-time regulator AGL19(AGAMOUS LIKE 19)under the control of the CaMV 35S promoter in soc1-3ful-2mutants.AGL19controls ?owering downstream of a cold-perception pathway and acts independently of FT and SOC1(ref.18);therefore,it might uncouple ?owering from longevity and secondary growth.Indeed,the 35S:AGL19transgene caused the soc1-3ful-2double mutants to ?ower as early as wild-type plants containing the 35S:AGL19transgene (Table 1).Irrespective of the extremely early ?owering in long days (Supplementary Fig.6a –c online),35S:AGL19soc1-3ful-2plants showed indeterminate growth,recurrent growth cycles and the devel-opment of small aerial rosettes that formed many co-in?orescences

reminiscent of soc1-3ful-2plants (Supplementary Fig.6d –f ).More-over,secondary growth was already visible in very young stems that steadily increased in diameter as a result of the signi?cant production of secondary tissues (Fig.2e,f ).Secondary growth and longevity were established independently of pleiotropic effects of late ?owering and can be attributed to the loss of SOC1and FUL in the various soc1ful mutant combinations.Thus,SOC1and FUL not only control ?owering time,but also play a key part in determining the herbaceous growth form and the short life cycle of Arabidopsis .

Investigating whether additional ?owering time genes might be equally implicated in the adoption of perennial characteristics,we found that FT additionally modi?ed the growth form of soc1-3and ful-2mutants.The ft-1mutants ?ower late only in long days,whereas the 35S:FT transgene triggers an extremely early and photoperiod-independent ?owering in wild type 19,20.The soc1and ful single mutations slightly delayed the strong early ?owering in 35S:FT plants 21,22(Table 1).Despite the very early ?owering of 35S:FT soc1-3and 35S:FT ful-2plants,they showed a markedly increased longevity and produced through reiteration of growth many short determinate co-in?orescences,particularly in short days (Supplemen-tary Fig.6g ,h ).These results suggest that the loss of SOC1or FUL activity is suf?cient to increase the lifespan of plants independent of ?owering https://www.wendangku.net/doc/114487585.html,bination of the ft-1mutation with ful-2led to an exaggerated indeterminacy of the apical meristem,with plants reach-ing a height of up to 1m and developing only a few side branches (Fig.3a ).Finally,the in?orescence meristems of the ft-1ful-2double mutants also reverted to vegetative growth.The loss of FT function in soc1-3mutants caused the formation of multiple rosettes during the vegetative phase and of large aerial rosettes during reproduction in short days (Supplementary Fig.6i ).As these phenotypes resembled those of soc1-3ful-2double mutants,either FT (loss of FT including the downregulation of its targets SOC1or FUL)or proteins down-stream of FT act redundantly with SOC1and FUL to prevent indeterminate growth,?oral reversion and aerial rosette formation.These results suggest that FT—besides triggering ?owering—also regulates the fate of meristems and consequently affects growth,especially under short days.That FT also regulates growth processes other than ?owering has also been shown in tomato 23.

Notably,the combined soc1-3ful-2mutations suppressed early ?owering in 35S:FT plants synergistically (Table 1),indicating that SOC1and FUL have redundant and clearly more important roles in ?owering-time control by day length downstream of FT than pre-viously anticipated.35S:FT soc1-3ful-2mutants developed a short main in?orescence and few co-in?orescences that all terminated early.Again,reversions of in?orescence meristems and aerial rosettes at the base of the in?orescences allowed for the reiteration of vegetative growth.Many large rosettes formed at the bases of these co-in?or-escences,generating a cushion-plant phenotype under short-day conditions.Most of the rosette meristems remained vegetative for many months (Fig.3b ),showing that constitutive expression of the mobile ?oral stimulus FT cannot provoke the transition from vege-tative to generative meristems in the absence of SOC1and FUL.Instead,an even more exaggerated vegetative long-lived phenotype developed,indicating again that not only FUL and SOC1,but also FT,have functions beyond ?owering-time control.

In conclusion,the modulation of the activities of only three genes had a clear effect on indeterminacy of meristems and longevity of the plants,leading to the development of markedly different growth forms in Arabidopsis .Finally,aspects of the soc1-3ful-2double mutants,such as vegetative buds,recurrent growth cycles,longevity and extensive woodiness,are reminiscent of plants with a perennial life style.Similar

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Figure 3Different plant growth forms in soc1-3and ful-2mutants.

(a )A ft-1ful-2double mutant with an exaggerated indeterminate growth.(b )A 35S:FT soc1-3ful-2plant showing a cushion-plant growth habit after 8months of growth in short days.The inset shows a 35S:FT plant grown in short days.Scale bar,10cm.

phenotypic traits have also been acquired in Sy-0(refs.24,25),a naturally occurring Arabidopsis accession from the Isle of Skye.In Sy-0,the expression of SOC1and FUL is strongly reduced (Supplementary Fig.7online).

In angiosperms,the perennial woody habit is believed to be the ancestral condition,from which annual herbaceous lineages have evolved several times independently.Conversely,evolution from annual herbaceous ancestors to perennial woody taxa has also repeatedly occurred 1.For example,in various annual herbaceous lineages,such as Sonchus and Echium ,woody perennial species evolved on isolated islands from their continental annual ancestors 26–28.Here we have shown the strong impact of only three genes (FT ,SOC1and FUL )on plant growth form and longevity in Arabidopsis .The rapid parallel evolution of ‘insular woodiness’28might also have been established through mutations in a small number of developmental genes.METHODS

Seeds of Arabidopsis thaliana (L.)Heyhn.We obtained Columbia (Col-0)and Landsberg erecta (L er )ecotypes as well as ful-1and ful-2mutants from the Nottingham Arabidopsis Stock Centre.Seeds of 35S:FT plants were provided by P .Wigge (John Innes Centre),seeds of the soc1-1allele were provided by G.Coupland (Max Planck Institute for Plant Breeding Research)and seeds of soc1-2in Col and L er backgrounds by I.Lee (University of Seoul).The soc1-3mutant has been described previously as agl20-3(ref.7).Plants were grown in soil,either under short-day (8h light/16h dark)or long-day conditions (16h light/8h dark)at 221C under ?uorescent tubes emitting a photon ?ux density of 150m mol m –2sec –1.We assayed ?owering time by counting leaf number.Reciprocal crosses of soc1-3and ful-2,soc1-2and ful-2in Col backgrounds and soc1-1and soc1-2with ful-1in L er backgrounds generated identical phenotypes in the homozygous progenies.We monitored the ful-1and ful-2mutations by the silique phenotype and we genotyped the soc1mutations by PCR.

For RT-PCR,we isolated total RNA from lower stem parts of Col-0and Sy-0plants and prepared cDNA as previously described 7.Quantitative real-time PCR was run on an iCycler (BioRad).As a control,a fragment from the gene encoding the eukaryotic protein synthesis initiation factor 4A (eIF4A )was ampli?ed and used to normalize the data (Supplementary Table 1online).

For microscopy,stem pieces of 3–5mm were ?xed overnight in 4%formaldehyde in 50mM phosphate (pH 7).The stems were dehydrated in a graded ethanol series and transferred to T echnovit 7100(Kulzer)or LR white (medium grade)(London Resin)embedding medium according to the supplier instructions.Sections of 6–8m m were cut with a rotary microtome and stained with toluidine blue before viewing under a Leica microscope.Note:Supplementary information is available on the Nature Genetics website.ACKNOWLEDGMENTS

We appreciate the continuous support of K.Apel,D.Inze

′and E.Smets.We thank J.Chandler and M.De Cock for critical reading of the manuscript.S.V.is indebted to the Institute for the Promotion of Innovation through Science and T echnology in Flanders for a predoctoral fellowship.F.L.and A.R.are

postdoctoral fellows of the Research Foundation-Flanders (FWO).Seeds of 35S:FT plants were provided by P .Wigge (John Innes Centre),seeds of the soc1-1allele were provided by G.Coupland (Max Planck Institute for Plant Breeding Research)and seeds of soc1-2in Col and L er backgrounds by I.Lee (University of Seoul).

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