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PUBLISHED: 7 DECEMBER 2015 | ARTICLE NUMBER: 15188 | DOI: 10.1038/NPLANTS.2015.188

Flexibility in the structure of spiral owers and its


underlying mechanisms
Peipei Wang1,2, Hong Liao1,2, Wengen Zhang1,2, Xianxian Yu1,2, Rui Zhang1, Hongyan Shan1,
Xiaoshan Duan1,2, Xu Yao1,2 and Hongzhi Kong1*
Spiral owers usually bear a variable number of organs, suggestive of the exibility in structure. The mechanisms
underlying the exibility, however, remain unclear. Here we show that in Nigella damascena, a species with spiral owers,
different types of oral organs show different ranges of variation in number. We also show that the total number of
organs per ower is largely dependent on the initial size of the oral meristem, whereas the respective numbers of
different types of oral organs are determined by the functional domains of corresponding genetic programmes. By
conducting extensive expression and functional studies, we further elucidate the genetic programmes that specify the
identities of different types of oral organs. Notably, the AGL6-lineage member NdAGL6, rather than the AP1-lineage
members NdFL1/2, is an A-function gene, whereas petaloidy of sepals is not controlled by AP3- or PI-lineage members.
Moreover, owing to the formation of a regulatory network, some oral organ identity genes also regulate the boundaries
between different types of oral organs. On the basis of these results, we propose that the oral organ identity
determination programme is highly dynamic and shows considerable exibility. Transitions from spiral to whorled owers,
therefore, may be explained by evolution of the mechanisms that reduce the exibility.

lowers show immense diversity in morphology, structure, composition, coloration and function. Despite the diversity, the constituent parts of the owers, the oral organs, can be classied
into very few categories (for example, sepals, petals, stamens and
carpels), suggesting that the programmes specifying the identities
of oral organs were highly conserved. The genetic programmes
specifying the identities of different types of oral organs were
rst studied in two model species, Arabidopsis thaliana and
Antirrhinum majus, based on which the famous ABC model was
proposed13. According to the model, the identities of the four
types of oral organs within a typical ower are specied by three
classes of gene functions: A alone, sepals; A and B, petals; B and
C, stamens; and C alone, carpels. Later, it was shown that another
class of gene function, namely E, is also indispensable for oral
organ identity specication, and the ABC model was extended to
form the ABCE model46. Investigations of species belonging to
various evolutionary lineages further indicated that while genes
executing the B, C and E functions are exclusively members of the
APETALA3 (AP3) and PISTILLATA (PI), AGAMOUS (AG) and
SEPALLATA (SEP) lineages of the MADS-box family7,8, respectively,
those executing the A function are quite controversial9, although
members of the APETALA1 (AP1) or AGAMOUS-LIKE 6 (AGL6)
lineages of the MADS-box family are sometimes expressed in
sepals and petals1012.
Notably, nearly all of the species investigated to date in terms of
oral organ identity specication have whorled owers, in which
ontogenetically concurrent oral organs form concentric circles
(Fig. 1ae). In owering plants, however, many species, particularly
members of basal angiosperms and lower eudicots, produce spiral
owers, in which ontogenetically subsequent oral organs are helically positioned at a regular divergent angle (that is, 137.5 or 99.5)13
(Fig. 1fj). Interestingly, probably because of the helical arrangement, the number of oral organs usually shows considerable variation in spiral owers14,15, suggestive of the exibility of the oral

structure. Yet, due to the lack of appropriate research systems, it


remains unclear how and why the number of oral organs varies
in spiral owers. Because spiral arrangement of oral organs is
very likely an ancestral condition of ower evolution13,16, concrete
answers to these questions will not only provide new insights into
the exibility of the ower in structure but also help understand
the mechanisms by which oral phyllotaxis has shifted from
spiral to whorled in such highly derived evolutionary lineages as
core eudicots and monocots.
Nigella damascena is a member of the buttercup family
(Ranunculaceae) with spiral oral phyllotaxis. Its ower contains
four types of oral organs (Fig. 1kp): sepals, which are petaloid;
petals, which form highly specialized bilabiate structures; stamens;
and carpels, which initiate separately but subsequently unite to
form a swollen capsule. Similar to the situation in many other
species with spiral oral phyllotaxis, the number of oral organs
per ower varies considerably in this species, although detailed
studies are still wanted. Yet, as an annual herb easy to cultivate
and investigate, this species is increasingly being used to address
important developmental and evolutionary questions that cannot
be answered by other existing models17,18. Here, using N. damascena
as a model, we attempt to understand (1) the range and pattern of
organ number variation in spiral owers; (2) the genetic programmes specifying the identities of different types of oral
organs, particularly petaloid sepals and bilabiate petals; (3) the
reason(s) why the number of oral organs is exible; and (4) how
the exibility in the oral organ identity specication programme
is related to the evolution of oral structure.

Results

Ranges and patterns of oral organ number variation. To gain


some insights into the exibility of spiral owers in structure, we
inspected >500 N. damascena owers. We found that the
respective numbers of sepals, petals, stamens and carpels, as well

State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China. 2 University of
Chinese Academy of Sciences, Beijing 100049, China. These authors contributed equally to this work. * e-mail: hzkong@ibcas.ac.cn

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b

Apex

e
Base-to-apex distance

DOI: 10.1038/NPLANTS.2015.188

Base

2 3 4 5

Organ number

Apex

j
Base-to-apex distance

Base

Carpels
Stamens
Petals
Sepals
1

2 3 4 5

Organ number

Sepal
7
6
5
4

Petal

Carpel

Total

Total

10

80

100

100

60

80

80
60

40

60

20

40

40

20

20

2
1st 2nd 3rd All

Stamen

0
1st 2nd 3rd All

1st 2nd 3rd All

1st 2nd 3rd All

1st 2nd 3rd All

1st

2nd

3rd

80
60
40
20
11
2
21 0
3
31 0
4
41 0
5
51 0
6
61 0
7
71 0
8
81 0

91 90
1
00

Figure 1 | Whorled versus spiral owers. a, Diagram of a whorled ower with ve sepals (sky blue), ve petals (purple), ten stamens (orange) and ve
carpels (dark green). b, Pattern of oral organ arrangement in a; dots represent oral organs. Note that organs of adjacent whorls are positioned alternately.
c, Schematic showing the spatial distribution of oral organs on the receptacle. d, Changes in the number of oral organs along the base-to-apex axis of the
ower; because of the whorled arrangement, organs of the same whorl arise synchronously, resulting in temporal and spatial intervals between adjacent
whorls. e, Diagram of a ower in which stamens in the third whorl were homeotically transformed into petals (violet). f, Diagram of a spiral ower with the
same number of oral organs as in a; for simplicity, all ontogenetically subsequent oral organs are positioned at a divergence angle of 137.5. g, Pattern of
the arrangement of oral organs in f. h, Schematic showing the spatial distribution of oral organs on the receptacle. i, Changes in the number of oral
organs along the base-to-apex axis of the ower; because of the spiral arrangement, ontogenetically subsequent oral organs arise progressively and
alternatively, so that the intervals between different types of oral organs are not more obvious than those between organs of the same type. j, Diagram of a
ower in which two outer stamens were converted into petals (violet). kp, A ower (k and l) of Nigella damascena and its four types of oral organs: sepal
(m), petal (n), stamen (o) and carpel (p). qu, Variation in the number of sepals (q), petals (r), stamens (s) and carpels (t), as well as the total number of
oral organs (u), in wild-type plants. Data were from investigation of 129 individual plants, and 1st, 2nd and 3rd represent the rst-, second- and thirdbloomed owers, respectively. Values of the upper quartile, median and lower quartile are included in each box, and dots indicate outliers. v, Positional
effect on the total number of oral organs. w, Median numbers of sepals, petals, stamens and carpels in owers with different numbers of oral organs.
xz, Cartoons showing the structures of owers with small, medium and large numbers of oral organs.
2

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a

ARTICLES

DOI: 10.1038/NPLANTS.2015.188

NdFL1

C
St

NdAG1

St
P
S

P
S

P
S

NdAP3-1

NdAG2

NdAP3-2

NdSEP1

NdAP3-3

NdSEP2

NdPI1

NdSEP3

NdPI2

NdAGL6

Stage 2

Stage 3

Stage 4

Stage 5

Stage 6

Stage 8

NdFL1
NdAP3-1
NdAP3-2
NdAP3-3
NdPI1
NdPI2
NdAG1
NdAG2
NdSEP1
NdSEP3
NdAGL6

Figure 2 | Dynamic expression of oral organ identity genes in Nigella damascena. a, Cartoons summarizing the expression patterns of 12 genes at stages
4, 6, 8, 10 and 12 of ower development; data are based on results of qRTPCR, RNA in situ hybridization and DGE assays (for details, see Supplementary
Figs 35). NdFRUITFULL-LIKE2 (NdFL2) is not shown because its expression was negligibly low in all these stages. Colours indicate different types of oral
organ identity genes, and scales demonstrate the relative expression levels. The relative expression levels at stages 612 were taken from the DGE results
and were ranked according to the following criteria: <0.05, no expression (no colour); 0.050.25, weak expression (light colour); 0.250.5, moderate
expression (moderate colour); >0.5, high expression (dark colour). The relative expression levels before stage 6 were taken from the in situ hybridization
results and estimated according to the levels at stages 612. S, sepal; P, petal; St, stamen; C, carpel. Scale bar, 500 m. b, Dynamic changes in the expression
patterns of oral MADS-box genes at six representative developmental stages. The arched boxes and rectangles represent oral organs and the expression
domains of genes, respectively.

as the total number of all oral organs, vary considerably among


owers (Fig. 1qu). Sepals, for example, range from four to seven
in number, with ve being the predominant number (in 94.3% of
the owers examined). Notably, of the four types of oral organs,
the stamen shows the widest range of variation in number (that is,
from 3 to 84), and has been the determining factor of a ower in
structure (Fig. 1s,wz); this, in fact, is also a phenomenon found
in other species with defects in oral meristem size and/or
determinacy regulation19. More interestingly, within the same
plant, owers that bloomed earlier tended to have a larger
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number of oral organs (Fig. 1qz), and the 1st > 2nd > 3rd
pattern in terms of the total number of oral organs per ower
was observed in 90 (or 70%) of the 129 investigated plants (Fig. 1v).
To understand why some owers have more oral organs than
others and why some organ types show wider ranges of variation
in number than others, we conducted comparative morphological
analyses. We found that owers with more oral organs generally
have larger receptacles, although the shape of the receptacle
measured by the ratio of its height to its widthdoes not show
clear variation (Supplementary Fig. 1ac). In addition, the
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DOI: 10.1038/NPLANTS.2015.188

TRV2
(mock)

5.0 0.0
6.1 2.2
25.8 10.3
4.1 0.8
41.0 11.8

TRV2
NdAP3-3

15.5 3.1
0
20.4 8.9
3.8 0.7
39.7 10.9

TRV2

6.2 0.4

NdAP3-1
NdAP3-2

6.4 1.9
0
28.0 6.5
40.7 7.5

TRV2

30.3 13.3

NdAP3-1

NdAP3-2

NdAP3-3

19.4 9.4
49.8 19.4

TRV2
NdPI1

NdPI2

15.6 4.0

27.9 12.9
43.5 13.5
TRV2

5.1 0.6

NdAG1
NdAG2

64.8 29.7

0
0

74.9 29.7

TRV2

Countless

NdAG1

NdAG2

ndap3-3

Countless

TRV2

5.0 0.0

NdFL1

7.5 0.5
27.9 12.9

NdFL2
NdANS

4.3 0.7
44.8 13.3

TRV2

5.5 0.8

NdAGL6

6.7 1.8
21.1 9.4
3.8 0.7
37.0 10.7

ndap3-3
TRV2

25.5 2.1
0

NdAGL6

19.7 5.7
4.3 0.6
50.0 9.9

TRV2

NdSEP1
NdSEP2

0
0

NdSEP3

0
42.4 15.5

Figure 3 | Phenotypes of VIGS-treated Nigella damascena owers. For each treatment, only owers with strong phenotypic changes are shown. Columns 1
and 2, top and side views of the owers, respectively; columns 3, 4, 5 and 6, wild-type or homeotically transformed sepals, petals, stamens and carpels,
respectively; and column 7, the mean numbers (boxes, with standard errors) of the different types of oral organs (for simplicity, only the 1st owers are
considered). For details, see Supplementary Figs 810 and Supplementary Table 8.
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DOI: 10.1038/NPLANTS.2015.188

differences in the size of the receptacle or, more accurately, the oral
meristem have become evident even at the early stages of ower
development (Supplementary Fig. 1d,e). Yet, at these stages, it was
difcult to determine whether larger oral meristems will eventually
develop into owers with a larger number of oral organs.
Nevertheless, we noticed that the rst-bloomed ower on each
investigated plant is usually located on the terminus of the main
stem, and that the terminal ower on the main stem generally has
the largest oral meristem. This suggests that, very likely, the total
number of oral organs per ower is determined by the initial
size of oral meristem, although growth at the late stages of development may also be critical. In addition, because different oral
organs arise sequentially from the base up to the apex of the oral
meristem, inner stamens and carpels do not appear until sepals,
petals and outer stamens become conspicuous (Supplementary
Fig. 1fi). Because of this, and because the occurrence of a small
number (usually ve) of carpels terminates the process of oral
organ initiation, the stamen became the predominant organ type
in the ower and shows the widest range of variation in number.
The observation that all four types of oral organs show considerable variation in number, however, implies that the functional
domains of the genetic programmes that specify the identities of
different types of oral organs are exible.
Floral organ identity genes and the dynamic changes of their
expression. To understand the genetic basis of oral organ
identity determination in N. damascena, we isolated orthologues of
the known and putative A-, B-, C- and E-function genes in other
species. A total of 13 genes were obtained, and phylogenetic
analyses suggested that they are members of the AP1, AP3, PI, AG,
AGL6 and SEP lineages of the MADS-box gene family
(Supplementary Tables 13 and Supplementary Fig. 2). We then
investigated the expression patterns of these genes using the
quantitative reverse transcription polymerase chain reaction (qRT
PCR), RNA in situ hybridization and digital gene expression
proling (DGE) methods (for details, see Methods, Supplementary
Figs 35 and Supplementary Tables 4 and 5). We found that the
expression patterns of all these genes change considerably during
ower development (Fig. 2), implying that no single model could
be sufcient to accurately depict the programmes specifying the
identities of different types of oral organs. The dynamic changes
of the oral organ identity genes in expression pattern, in contrast,
suggest that oral organ identity determination is a spatiotemporal
process that requires transient or persistent actions of many genes.
The variation in the functional domains of the genetic
programmes specifying the identities of different types of oral
organs, therefore, may be attributed to the exibility of the process.
Knocking down the expression of AP3- and PI-lineage members.
To understand the function of the aforementioned genes, we
attempted to knock down their expression through the virusinduced gene silencing (VIGS) technique (Supplementary Figs 6,7
and Supplementary Tables 69). We started with NdAP3-3
because it is a gene with known function and because a natural
mutant of it, Double Sepals, is available17. Tobacco rattle virus
(TRV2)NdAP3-3-treated owers with strong phenotypic changes
are morphologically indistinguishable from owers of Double
Sepals and are characterized by a lack of petals and an increase in
the number of sepals (Fig. 3 and Supplementary Fig. 8). This
suggests that the VIGS system worked well and that the TRV2
NdAP3-3 treatment has actually led to complete, or nearly so,
silencing of NdAP3-3 in some owers. Notably, in both Double
Sepals owers and TRV2NdAP3-3-treated owers with strong
phenotypic changes, the number of sepals was markedly larger
than those of the sepals plus petals in wild-type and mock-treated
owers, whereas the number of stamens decreased (Fig. 3 and
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ARTICLES
Supplementary Fig. 8). This suggests that inactivation of NdAP3-3
not only caused homeotic transformation of petals into sepals, but
also converted peripheral, or outer, stamens into sepals, thereby
shifting the petalstamen boundary. Interestingly, at the early
stages of ower development (that is, stages 3 and 4), when the
expression of NdAG1, a putative C-function gene (see below), has
not been induced, NdAP3-3 was already expressed in the region
where outer stamens would initiate (Fig. 2). This suggests that
NdAP3-3 may regulate the petalstamen boundary by inducing or
promoting the expression of NdAG1. In owers with moderate
phenotypic changes, not all petals were converted into sepals, and
the not-yet-converted petals showed a series of intermediate
features between real petals and sepals (Supplementary Fig. 8).
This suggests that the function of NdAP3-3 in petal organ identity
specication is dosage dependent.
Unlike NdAP3-3, whose expression is mainly restricted in petals,
NdAP3-1 and 2 have much broader expression domains (Fig. 2 and
Supplementary Figs 35). Knockdown of either gene, however, did
not result in obvious phenotypic change except that petals reduced
slightly in structure (Supplementary Fig. 8). In TRV2NdAP3-1
NdAP3-2-treated owers with strong phenotypic changes, the
petals reduced further, resembling sepals in shape but being slightly
smaller, and stamens were all transformed into carpels (Fig. 3 and
Supplementary Fig. 8). In owers with moderate phenotypic
changes, the stamens were all converted into carpels whereas
petals displayed a series of intermediate features as when NdAP31 or 2 was knocked down (Supplementary Fig. 8). Taken together,
these results suggest that NdAP3-1 and 2 have overlapping functions
in stamen identity specication and petal shape regulation, and that
while both functions are dosage dependent, the former requires
higher levels of NdAP3-1/2 expression than the latter.
We also treated wild-type plants with the TRV2NdAP3-1
NdAP3-2NdAP3-3 construct and Double Sepals plants with the
TRV2NdAP3-1NdAP3-2 construct. As expected, their phenotypes
were essentially the same, and owers with strong phenotypic
changes possessed only two types of organs: sepals and carpels
(Fig. 3 and Supplementary Fig. 8). A close inspection of these
owers further indicated that collective silencing of the three AP3like genes led to transformation of petals and outer stamens into
sepals, whereas inner stamens were converted into carpels. The fact
that the number of sepals in these owers was even larger than
those in the TRV2NdAP3-3-treated owers with strong phenotypic
changes or Double Sepals owers (Fig. 3 and Supplementary Fig. 8)
further implies that NdAP3-1 and/or 2 can enhance the function of
NdAP3-3 in petalstamen boundary determination.
It has been shown that proper functioning of AP3-like genes also
requires co-expression of PI-like genes1,20,21. Specically, proteins of
AP3- and PI-like genes need to form obligate heterodimers before
they bind to the control regions of downstream genes and regulate
their expression22. N. damascena has two PI-like genes, NdPI1
and 2, whose expression completely overlaps, with consistently
high levels of signals being detectable in sepals, petals and
stamens (Fig. 2 and Supplementary Figs 35). The NdPI1 and 2 proteins, however, appear to have diverged slightly in function: while
the former is able to interact with all three AP3-like proteins, the
latter is only able to interact with NdAP3-1 (Supplementary
Table 10 and Supplementary Fig. 11). When both NdPI1 and 2
were silenced by using the TRV2NdPI1NdPI2 construct, owers
with strong phenotypic changes were morphologically identical to
those in which all three AP3-like genes were silenced: petals and
stamens no longer existed, and the owers possessed only sepals
and carpels (Fig. 3 and Supplementary Fig. 8); the number of
sepals, however, was clearly smaller than that in NdAP3-1/2/3silenced owers but close to that in NdAP3-3-silenced owers
(Fig. 3 and Supplementary Fig. 8). This not only conrms that PIand AP3-like genes are functionally interdependent but also
5

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Wild type (WT)

ndap3-3

SEP1/2/3
AG1

AGL6
AP3-3

AG2

AP3-1/2
PI1/2

ndap3-3 TRV2

SEP1/2/3
AGL6
AG1
AP3-1/2
PI1/2

SEP1/2/3
AGL6

SEP1/2/3
AGL6

SEP1/2/3
AG1
AGL6
AG2

SEP1/2/3
AG1
AP3-3
AG2

AGL6
AG2

AP3-3

AP3-1/2
PI1/2

AG1
AG2

TRV2NdAP3-1NdAP3-2NdAP3-3

TRV2NdAP3-1NdAP3-2

SEP1/2/3
AG1

AG2

ndap3-3 TRV2NdAP3-1NdAP3-2

SEP1/2/3
AGL6
AG1
AP3-1/2 AG2
PI1/2

AGL6

AG1
AP3-1/2
PI1/2

AG2

TRV2NdAP3-3

TRV2 (mock)

DOI: 10.1038/NPLANTS.2015.188

PI1/2

TRV2NdPI1NdPI2

ndap3-3 TRV2NdPI1NdPI2

SEP1/2/3

SEP1/2/3
AG1
AGL6
AG2

AG1
AG2

AGL6

TRV2NdSEP1NdSEP2NdSEP3

TRV2NdAG1NdAG2

SEP1/2/3
AGL6
AP3-3
AP3-1/2
PI1/2

Organ legend

TRV2NdFL1NdFL2

ndap3-3 TRV2NdAG1NdAG2

SEP1/2/3
AGL6

TRV2NdAGL6NdFL1NdFL2

Sepal
Petal
Stamen

AGL6

SEP1/2/3
AG1

AP3-3

SEP1/2/3
AG1
AG2

AP3-1/2
PI1/2

Carpel

AG2

AP3-3

AP3-1/2
PI1/2

Bract
Sepal, deeply lobed

TRV2NdAGL6

ndap3-3 TRV2NdAGL6

Petal, highly reduced


Bract, linear

SEP1/2/3
AG1
AP3-3

AP3-1/2
PI1/2

SEP1/2/3
AG1
AG2

AP3-1/2 AG2

PI1/2

Figure 4 | Floral organ identity determination in wild-type, Double Sepals and VIGS-treated Nigella damascena owers. Arched boxes represent oral
organs, with their widths being proportional to the median numbers of corresponding organs in owers with strong phenotypic changes. Rectangles indicate
the deduced functional domains of the oral organ identity genes. Inferred positive and negative regulatory relationships between genes are indicated by
arrows and bars, respectively, in the wild-type condition.

suggests that, unlike NdAP3-1 or 2, NdPI1 and 2 cannot enhance the


function of NdAP3-3 in petalstamen boundary determination. The
observation that silencing of either AP3- or PI-like genes does not
affect the appearance of sepals, however, suggests that petaloidy
of sepals in N. damascena may have nothing to do with AP3- or
PI-lineage members, as is generally assumed23,24.
Knocking down the expression of AG-lineage members. To
analyse the functions of the two AG-lineage members, NdAG1
6

and 2, we rst attempted to knock down each of them with the


TRV2NdAG1NdANS and TRV2NdAG2NdANS constructs,
respectively. No obvious phenotypic change was observed even in
anthocyanidin-free owers (Supplementary Fig. 9), suggesting that
(1) the downregulation did not lead to complete (or nearly so)
silencing of either gene; or (2) the two genes have overlapping
and redundant functions if NdAG2 function in a non-cell
autonomous fashion in stamens. In TRV2NdAG1NdAG2treated owers with strong phenotypic changes, stamens and
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DOI: 10.1038/NPLANTS.2015.188

carpels no longer existed, whereas sepals and petals formed owerwithin-ower structures: ve normal-looking sepals and an
unstable number of petals were positioned at the peripheral
regions of the ower, followed by recurrent occurrences of ve
deeply lobed sepals and an unstable number of petals (Fig. 3 and
Supplementary Fig. 9). This suggests that silencing of NdAG1 and
2 has led to the transformation of stamens into petals and carpels
into sepals, as well as the loss of oral determinacy. Notably, the
number of petals in the outer owers was obviously larger than
the total number of petals plus stamens in mock-treated owers
(Fig. 3 and Supplementary Fig. 9), suggesting that the stamen
carpel boundary shifted inward. Given that NdAG1 is expressed in
both stamens and carpels whereas NdAG2 is only expressed in
carpels, these results suggest that (1) NdAG1 is a C-function gene
that species the identities of stamens and carpels; (2) NdAG2 is
involved in both carpel identity specication and oral determinacy
control; (3) NdAG1 can repress the expression of NdAP3-3 such
that silencing of NdAG1 led to inward expansion of NdAP3-3
into stamens; and (4) NdAG2 can specify the stamencarpel
boundary, probably through regulating the expression of
B-function genes.
TRV2NdAG1NdAG2-treated owers with moderate phenotypic changes can be categorized into two classes (Supplementary
Fig. 9). In the rst class, owers also formed ower-withinower structures, and the stamens of the outer owers displayed
a transitional state between stamens and petals, suggestive of incomplete stamen-to-petal transformation. In the second class, stamens
were completely transformed into petals, whereas carpels still
existed, suggestive of the failure of the carpel-to-sepal transformation; yet, additional carpels arose from the middle of the ower,
suggesting that the oral determinacy was broken. Taken together,
these results further suggest that the functions of NdAG1/2 in
stamen/carpel identity specication and oral determinacy control
are separable, with this latter function requiring higher levels of
expression than the former.
We also treated the Double Sepals plants with the TRV2
NdAG1NdAG2 construct. The resultant owers with strong phenotypic changes only bore one type of oral organs, sepals, and
the owers became ower-within-ower structures (Fig. 3 and
Supplementary Fig. 9). In addition, similar to the situation in
TRV2NdAG1NdAG2-treated wild-type owers with strong phenotypic changes, newly converted sepals sometimes have lobed
margins, suggesting that the shape of sepals was also affected.
Knocking down the expression of AP1- and AGL6-lineage
members. To determine whether the two AP1-lineage members
are A-function genes, we treated wild-type plants with the TRV2
NdFL1NdFL2NdANS construct. The resultant anthocyanidinfree owers, however, remained unchanged in either gross
morphology or organ number (Fig. 3 and Supplementary Fig. 10).
This, together with the observation that the expression levels of
both NdFL1 and 2 are generally low in all oral organs (Fig. 2
and Supplementary Figs 35), suggests that the contributions of
the two genes to oral organ identity specication are negligible.
Because AGL6-lineage members in some basal angiosperms have
been proposed to be A-function genes11,25, we then knocked down
the expression of NdAGL6 using the TRV2NdAGL6 construct. In
owers with strong phenotypic changes, sepals became bract-like,
with the margins being seriously lobed and the lobes being
narrow and linear. Petals, which did exist, were no longer
bilabiate but became linear-to-obovate-shaped structures, in which
upper lips, pseudonectaries, long hairs and short trichomes no
longer existed (Fig. 3 and Supplementary Fig. 10). Interestingly,
stamens and carpels remained unchanged in both appearance and
number, suggesting that, as a key regulator of sepal and petal
development, NdAGL6 plays a minor, if any, role in stamen or
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ARTICLES
carpel development, although its expression can be detected in
those organs (Fig. 2 and Supplementary Figs 35). The
observation that simultaneous silencing of NdFL1, NdFL2 and
NdAGL6 using the TRV2NdFL1NdFL2NdAGL6 construct
resulted in the same phenotype as when NdAGL6 was silenced
(Supplementary Fig. 10) further suggests that NdAGL6, rather
than NdFL1 or 2, is the A-function gene involved in sepal and
petal identity determination.
In TRV2NdAGL6 and TRV2NdFL1NdFL2NdAGL6-treated
owers with less strong phenotypic changes, sepals and petals
showed a series of transformation (Supplementary Fig. 10). Sepals
with moderate phenotypic changes were bract-like, but had irregularly lobed margins, whereas those with weak phenotypic changes
were lanceolate and entirely margined. Petals with nearly strong
phenotypic changes were at and lanceolate, and the upper lip, the
hairs and the nectar on the lower lip were absent. Petals with
moderate phenotypic changes also lacked the upper lip but possessed
a nearly intact lower lip, although pseudonectaries were still absent.
Upper lips were only observed in petals with weak phenotypic
changes. Taken together, these results suggest that the functions of
NdAGL6 in sepal and petal development are also dosage dependent.
TRV2NdAGL6-treated Double Sepals owers with strong phenotypic changes are composed of pinnately lobed, bract-like sepals
and normal-looking stamens and carpels (Fig. 3 and Supplementary
Fig. 10). As expected, the number of bract-like sepals increased
markedly compared with that of sepals plus petals in wild-type
and NdAGL6-silenced owers, suggesting that in addition to
sepals and petals, some outer stamens were converted into bractlike organs. Clearly, as a sepal and petal identity gene, NdAGL6
plays key roles in generating the entirely margined petaloid sepals
in wild-type plants and, working together with NdAP3-3,
determines the basic structure of the petals.
Knocking down the expression of SEP-lineage members. To
determine the function of the three SEP-like genes, we treated
wild-type plants with the TRV2NdSEP1NdSEP2NdSEP3
construct. As expected, owers with strong phenotypic changes
only had one type of organ, bracts (Fig. 3 and Supplementary
Fig. 10). In addition, similar to the situation when two AGlineage members were knocked down, the determinacy of the
ower was broken, and a ower-within-ower phenotype was
produced. When only the outer owers were taken into
consideration, the number of oral organs was clearly larger than
those of mock-treated plants, suggesting that knockdown of the
three SEP-lineage members affected the structure of the ower in
the same way as knockdown of the AG-lineage members
(Supplementary Fig. 10). Clearly, SEP-lineage members are
E-function genes and play indispensable roles in both oral organ
identity specication and oral determinacy control. Consistent
with this, proteins of all three SEP-lineage members can interact
with those of NdAGL6, NdAG1 and NdAG2 assays, suggestive of
their abilities to form heterodimers (Supplementary Fig. 11).
TRV2NdSEP1NdSEP2NdSEP3-treated owers with moderate
phenotypic changes can be classied into three grades
(Supplementary Fig. 10). In the rst grade, the owers were indeterminate, and all oral organs were transformed into bract-like structures except that some stamens remained unchanged. In the second
grade, the owers were also indeterminate and sepals and petals
became bract-like, whereas stamens and carpels still existed. In the
third grade, sepals and petals were bract-like, whereas stamens,
carpels and the determinacy of the ower remained unchanged.
Taken together, these results suggest that sepal and petal development requires the highest levels of SEP gene expression, followed
by oral determinacy control and carpel and stamen development,
and that the functions of SEP genes in oral organ identity specication and oral determinacy control are separable.
7

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Discussion

Determining factors of oral structure. In this study, using


N. damascena as a model, we investigated the ranges, patterns and
mechanisms of oral organ number variation in spiral owers.
We found that the respective numbers of sepals, petals, stamens
and carpels, as well as the total number of all oral organs, vary
considerably among owers, conrming the notion that the
structures of spiral owers are usually exible. We also found that
the ranges of variation in the number of different types of oral
organs are by no means the same: some organ types show much
wider ranges of variation than others. Interestingly, this seems to
be a common feature of spiral owers, although detailed studies
of other species are still lacking. By conducting extensive
morphological, expression and functional analyses, we further
demonstrated that the total number of organs per ower is largely
dependent on the initial size of oral meristem, whereas the
respective numbers of different types of oral organs are
determined by the functional domains of corresponding genetic
programmes. Based on this and other studies26, it becomes clear
that the basic structure of a ower is determined by three main
factors: (1) the initial size of the oral meristem, which determines
the total number of oral organs19; (2) the spatiotemporal process
of oral organ initiation, which determines the pattern (spiral,
decussate, whorled or irregular), rate/pace (slow or fast and
constant or irregular) and number (small or large and lowly or
highly variable) of the organs formed2729; and (3) the activities of
oral organ identity genes, which determine the type and
number of the organs formed and affect the composition,
sexuality and symmetry of the ower1,30,31. Clearly, investigations
of N. damascena not only uncovered the commonalities and
peculiarities of whorled and spiral owers but also shed new
light on the underlying mechanisms of ower development and
evolution. Dissection of evolutionary developmental biological
(Evo-Devo) questions like this, therefore, should start with
careful selection of suitable non-model systems, followed by best
use of the existing knowledge, available resources and cuttingedge techniques (for example, qRTPCR, DGE, VIGS or even
virus-induced gene complementation).
Floral organ identity determination in N. damascena. Our
expression and knockdown results allow us to propose a model for
oral organ identity determination in N. damascena. In this model,
sepals are specied by NdAGL6 and NdSEP1/2/3, petals by NdSEP1/
2/3, NdAGL6, NdAP3-1/2/3 and NdPI1/2, stamens by NdAP3-1/2,
NdPI1/2, NdAG1 and NdSEP1/2/3, and carpels by NdAG1/2 and
NdSEP1/2/3 (Fig. 4). Interestingly, inconsistent with the expression
data, NdFL1/2 are unlikely to function in oral organ identity
determination, NdAGL6 is not directly involved in stamen or carpel
development, and petaloidy of sepals does not require function of
AP3- or PI-lineage members. This, together with the observation
that the functions of most, if not all, oral organ identity genes are
dosage dependent1,3234, strongly suggests that oral organ identity
determination is a complex process that cannot be elucidated unless
detailed functional studies are conducted. The observation that
incomplete downregulation of the NdAP3-3 gene generated a wide
array of reduced petals that resemble those of wild-type Nigella
species further implies that diversication of oral organs may have
been caused by modication of the expression levels of some oral
organ identity genes.
Notably, the programmes specifying the identities of different
types of oral organs in N. damascena are similar to, but slightly
different from, those in other investigated species. First, the
AGL6-lineage member NdAGL6, rather than the AP1-lineage
members NdFL1/2, is the A-function gene that species the identities of sepals and petals. Second, B function is collectively fullled by
ve genes, two of which are PI-like and three of which are AP3-like.
8

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DOI: 10.1038/NPLANTS.2015.188

Interestingly, although the two PI-like genes have largely overlapping functions, the three AP3-like genes have diverged considerably
in expression and function. Third, C function is provided by the two
functionally redundant but distinguishable AG-lineage members.
No STK-lineage member has ever been identied in N. damascena
or any other species of the Ranunculales35,36; thus, it has been
hypothesized that one of the two AG-like genes, AG2, species
ovule identity37,38. In N. damascena, the exact function of NdAG2
remains unclear, yet the available data suggest that it plays key
roles in carpel identity specication, stamencarpel boundary determination and oral determinacy regulation.
Genes affecting the boundaries between different types of oral
organs. Our studies have also uncovered the regulatory
relationships between some oral organ identity genes (Fig. 4 and
Supplementary Fig. 12). First, NdAP3-3 induces the expression of
NdAG1 in outer stamens: silencing of NdAP3-3 led to delayed
expression of NdAG1 in oral meristem and transformation of outer
stamens into sepals. Second, NdAP3-3 promotes the expression of
NdAP3-1 and NdAP3-2 in outer stamens: knockdown of NdAP3-3
led to decreased expression of the two genes in these organs. Third,
NdAP3-1/2 represses the expression of NdAG2 in stamens:
knockdown of NdAP3-1/2 led to outward expansion of NdAG2
expression from carpels to stamens and transformation of stamens
into carpels. Fourth, AG-like genes, possibly NdAG1, repress the
expression of NdAP3-3 in stamens: knockdown of NdAG1/2 led
to inward expansion of NdAP3-3 into stamens and transformation
of stamens into petals. Fifth, AG-like genes prevent the expression
of NdAGL6 in stamens and carpels: knockdown of NdAG1/2 led
to inward expansion of NdAGL6 from sepals and petals to
stamens and carpels. Taken together, these observations suggest
that the seemingly simple and modularized programme of oral
organ identity determination is controlled by a relatively complex
regulatory network in which a change in one gene may cause
subsequent changes in others.
Probably because of the formation of the regulatory network,
some genes become the regulators of the boundaries between different types of oral organs. NdAP3-3, for example, not only species
the identity of petals but also determines the boundary between
petals and stamens. When NdAP3-3 was mutated or silenced, all
petals and outer stamens were transformed into sepals, and thus
the boundary between petals and stamens shifted inwards. When
all three AP3-like genes were silenced, the boundary between
petals and stamens shifted further inwards, suggesting that the function of NdAP3-3 in regulating the boundary between petals and
stamens can be strengthened by NdAP3-1 and 2. A similar phenomenon was observed in TRV2NdAG1NdAG2-treated owers with
strong phenotypic changes: the number of petals in outer owers
was signicantly higher than those of petals plus stamens in
mock-treated owers, suggesting that in addition to stamen-topetal and carpel-to-sepal transformations, the boundary between
stamens and carpels shifted inwards, so that additional petals
were produced.
Spiral versus whorled owers. Two models (that is, spatial and
sequential) have been proposed to explain how the identities of
oral organs are specied, with neither of them being completely
satisfactory3942. From this study, it is clear that oral organ
identity determination is a highly dynamic process, in which
different oral organ identity genes act sequentially and the
functions of late-acting genes depend on those of early-acting
ones. In addition, the oral organ developmental process is tightly
associated with other processes, such as oral meristem formation
and oral organ initiation, so that the exibility in oral structure
is further strengthened. The exibility, however, is not so obvious
in whorled owers, probably due to the formation of oral organs
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DOI: 10.1038/NPLANTS.2015.188

into whorls. In spiral owers, because the distances between organs


of different types are not as distinct from those between organs of
the same type as in whorled owers, expansion or retraction of
the expression domain of one gene (such as NdAP3-3 or NdAG2)
may easily shift the boundaries between different types of organs.
In whorled owers, however, this does not occur unless the
change affects the entire whorl. Clearly, whorled owers are less
susceptible of the exibility of the programmes specifying oral
organ identities than spiral ones.
It remains unclear whether the most recent common ancestor of
angiosperms produced spiral or whorled owers. However, it is
known that both spiral and whorled owers are widespread in
basal angiosperms and that members of several highly derived owering plant lineages, such as core eudicots and monocots, generally
produce whorled owers12,13. This suggests that canalization in oral
structure must have occurred during owering plant evolution and
that whorled owers may have been evolutionarily more advantageous than spiral ones14,43. One explanation for this is that when
the number and arrangement of oral organs are xed, the structure
of the ower becomes stabilized and can efciently avoid the impact
of environmental changes. However, to achieve this, the ower
needs to evolve some mechanisms to stabilize the number of, as
well as the boundaries between, different types of oral organs.
The way the owers are borne or assembled on the plant may also
matter because positional effects can affect the initial size of oral
meristem. Therefore, if the transitions from spiral to whorled
oral phyllotaxis did occur during evolution, mechanisms that
reduced the exibility of the oral organ identity determination programme must have been critical.

Methods

Plant materials and growth conditions. Seeds of N. damascena and its Double
Sepal mutant were purchased from B & T World Seeds (Paguignan, France), sown
in nutrient soil and grown under conditions of 12-h day/12-h night at 24 C and 60%
relative humidity.
Gene isolation. Total RNA was extracted from oral buds at various developmental
stages using the SV Total RNA Isolation System (Promega) and reverse-transcribed
into cDNA with the SuperScript III First-Strand Synthesis System (Life
Technologies). The 3-end sequences of the oral organ identity genes were
amplied through a two-step nested PCR technique using lineage-specic
degenerate forward primers and a universal reverse primer (Supplementary Table 2).
Amplied fragments with expected sizes were puried, cloned into the pEASY-T3
vector (TransGen Biotech) and sequenced. Full-length coding sequences of the
genes were retrieved from transcriptomic data.
Phylogenetic analyses. Coding sequences of the genes from other species were
retrieved through BLAST searches against publicly available databases
(Supplementary Table 3). Phylogenetic analyses were performed for DNA sequences
in PhyML 3.0 using the maximum-likelihood method44. The general time
reversible (GTR) + I + model was applied, and 1,000 bootstrap replicates were
performed. Genes isolated from this study were named according to their
phylogenetic belongingness and sequence features.
Quantitative reverse-transcription PCR. Floral buds, bracts and individual oral
organs at stage 12 were collected from wild-type, Double Sepals and VIGS-treated
plants. qRTPCR was conducted using the PrimeScript RT Reagent Kit (Perfect Real
Time) (Takara) in an Applied Biosystems ViiA 7 Real-Time PCR System (Life
Technologies). Three biological replicates for each sample were used, and all
reactions were run with three technical replicates. The housekeeping gene NdACTIN
was used as an internal quantitative control. The relative expression values were
calculated using the comparative CT (2CT) method45. Primers used are listed in
Supplementary Table 4.
RNA in situ hybridization. Floral buds at various developmental stages were xed
overnight in fresh FAA (3.7% formaldehyde, 5% acetic acid, and 50% ethanol) at
4 C and then embedded in Paraplast (Sigma). Gene-specic fragments covering the
C-terminal end of the coding sequence and the 3 untranslated region were used as
templates for synthesis of antisense and sense digoxigenin-labelled RNA probes
using the DIG RNA labelling kit (Roche) (Supplementary Fig. 6 and Supplementary
Table 5). Sectioning and subsequent treatments were performed as described46.
Images were captured with a Leica DM5000 B microscope.
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Transcriptomic and digital gene expression analyses. For transcriptomic analysis,


total RNA was extracted from inorescence, and owers at different developmental
stages were mixed to generate a pool for library construction. Paired-end 100-bplong reads were then sequenced by Illumina HiSeq2000 and assembled using
Trinity47. For DGE proling, organs at stages 6/7, 9, 12 and 16 of ower development
were subjected to RNA extraction and library preparation, and Illumina single-end
sequencing with the 100-bp-long reads was performed. The obtained reads from
each library were mapped to the reference transcriptome, and the mappable reads
were used to estimate the transcription level by the reads per kilobase per million
mapped reads method48.
Virus-induced gene silencing. Fragments specic for each gene were used for VIGS
experiments (Supplementary Fig. 6 and Supplementary Table 6), with the specicity
of the fragments being determined in R (Supplementary Table 7). The VIGS
fragments of the genes were then introduced into the TRV2-based pYL156 vector
(Supplementary Fig. 6). Wild-type or Double Sepals plants were treated as
described49, and the efciency of the silencing was checked by using qRTPCR
methods (Supplementary Fig. 7). As many as possible plants were treated for each
construct to make sure that owers with the strongest phenotypic changes were seen
in at least ten individuals. Two or three rounds of treatment were conducted for most
constructs (Supplementary Table 8) to ensure that the results were consistent and
reliable. When silencing of a gene or gene combination failed to generate any
observable phenotypic change, a fragment of the NdANS gene was added as a
positive control. Here, ANS is the reporter gene ANTHOCYANIDIN SYNTHASE,
silencing of which can cause loss of anthocyanidin. Flowers showing signs of
phenotypic changes or NdANS silencing were photo-documented, and the oral
organs were counted and compared (Supplementary Table 9). Because the number
of oral organs varies considerably among owers, only the rst owers of the
individual plants were considered in phenotyping.
For other methods, see Supplementary Information.

Received 14 September 2015; accepted 29 October 2015;


published 7 December 2015

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Acknowledgements
We thank members of the Kong laboratory for helpful discussion, and E. M. Kramer, S.-H.
Shiu and three anonymous reviewers for valuable comments. This work was supported by
National Natural Science Foundation of China Grants 31125005 and 31330007 and CAS
Interdisciplinary Innovation Team.

Author contributions
P.W. and H.K. designed the research. P.W., H.L. and W.Z. performed the qRTPCR, in situ
hybridization and VIGS experiments, with the help of R.Z. and H.S. for analyzing the data.
R.Z. and X. Yu provided the RNA-seq and yeast two-hybrid results, respectively, and P.W.,
X.D. and X. Yao conducted the morphological analyses. P.W., H.S. and H.K. wrote
the manuscript.

Additional information
Supplementary information is available online. Reprints and permissions information is
available online at www.nature.com/reprints. Correspondence and requests for materials should
be addressed to H.K.

Competing interests
The authors declare no competing nancial interests.

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