Core Mechanisms Regulating Developmentally
Timed and Environmentally Triggered Abscission[OPEN]
O. Rahul Patharkar* and John C. Walker
Division of Biological Sciences and Interdisciplinary Plant Group, University of Missouri, Columbia, Missouri
65211
ORCID IDs: 0000-0002-1242-6549 (O.R.P.); 0000-0002-2050-1641 (J.C.W.).
Drought-triggered abscission is a strategy used by plants to avoid the full consequences of drought; however, it is poorly
understood at the molecular genetic level. Here, we show that Arabidopsis (Arabidopsis thaliana) can be used to elucidate the
pathway controlling drought-triggered leaf shedding. We further show that much of the pathway regulating developmentally
timed floral organ abscission is conserved in regulating drought-triggered leaf abscission. Gene expression of HAESA (HAE) and
INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) is induced in cauline leaf abscission zones when the leaves become wilted in
response to limited water and HAE continues to accumulate in the leaf abscission zones through the abscission process. The genes
that encode HAE/HAESA-LIKE2, IDA, NEVERSHED, and MAPK KINASE4 and 5 are all necessary for drought-induced leaf
abscission. Our findings offer a molecular mechanism explaining drought-triggered leaf abscission. Furthermore, the ability to study
leaf abscission in Arabidopsis opens up a new avenue to tease apart mechanisms involved in abscission that have been difficult to
separate from flower development as well as for understanding the mechanistic role of water and turgor pressure in abscission.
Many plants drop their leaves during hot dry summers by the process of leaf abscission to reduce the
transpiration load on the plant and to ensure young
tissues have adequate water and nutrients to survive
(Street et al., 2006; Agustí et al., 2012). Abscission is the
process enabling plants to discard unwanted organs
in response to environmental stimuli or developmental timing (Tudela and Primo-Millo, 1992; Agustí
et al., 2012; Liljegren, 2012; Niederhuth et al., 2013a).
Abscission occurs at distinct boundaries, called abscission zones, that are laid down as the associated
organ develops. Abscission zones have smaller cells
with smaller vacuoles than the surrounding tissue.
Once abscission is triggered, the abscission zone cells
expand and hydrolytic enzymes are released to dissolve the abscission zone’s middle lamella, resulting
in abscission (Sexton and Roberts, 1982).
Floral organ abscission in Arabidopsis (Arabidopsis
thaliana) is the best studied abscission model. The
timing of floral organ abscission is developmentally
regulated so that abscission occurs shortly after fertilization. Briefly, a molecular pathway has been described
in which a peptide derived from INFLORESCENCE
DEFICIENT IN ABSCISSION (IDA) is thought to trigger
* Address correspondence to [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is:
O. Rahul Patharkar ([email protected]).
O.R.P. designed and performed experiments; O.R.P. and J.C.W.
wrote the article.
[OPEN]
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510
a pair of redundant receptor-like protein kinases,
HAESA (HAE) and HAESA-LIKE2 (HSL2). A mitogenactivated protein kinase (MAPK) pathway, consisting
of MAPK KINASE4 (MKK4)/MKK5 and MAPK3
(MPK3)/MPK6, positioned genetically downstream of
HAE/HSL2, phosphorylates a MADS-domain transcription factor, AGAMOUS-LIKE15 (AGL15), which
results in a transcriptional increase of HAE, thereby
creating a positive feedback loop (Cho et al., 2008;
Stenvik et al., 2008; Patharkar and Walker, 2015).
NEVERSHED (NEV), an ADP-ribosylation factorGTPase-activating protein, is thought to traffic
HAE/HSL2 and other receptor-like protein kinases to
the correct subcellular location and is also required for
abscission (Liljegren et al., 2009; Burr et al., 2011;
Liljegren, 2012). Secondary mutations that suppress
abscission defects of hae hsl2, ida, and nev have also been
described (Leslie et al., 2010; Lewis et al., 2010; Burr
et al., 2011; Shi et al., 2011). However, these suppressor
mutants cannot be clearly labeled as negative regulators of abscission. For example, secondary mutations in
SOMATIC EMBRYOGENESIS RECEPTOR KINASE1
(SERK1) can rescue the abscission defects of nev while
at the same time the triple mutant serk1 serk2 serk3 is
defective in abscission (Lewis et al., 2010; Meng et al.,
2016). Additionally, when overexpressed in Arabidopsis protoplasts or Nicotiana benthamiana leaves,
SERK3 (also known as BAK1) can form a complex
with HAE/HSL2 mediated by IDA (Meng et al.,
2016). A key net transcriptional outcome of the abscission pathway is increased expression of cell wallmodifying enzymes that aid in cell separation at the
abscission zone’s middle lamella (Niederhuth et al.,
2013b).
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Core Mechanisms Regulating Abscission
However, it is poorly understood if the pathway for
floral organ abscission has a role in abscission of other
organs and in environmentally induced abscission. For
example, are leaves shed by the same molecular mechanism as floral organ abscission? For about 20 years it
was thought that Arabidopsis only abscises it floral organs (Bleecker and Patterson, 1997; Jinn et al., 2000).
Abscission zone-like structures at the base of cauline
leaves and pedicels of Arabidopsis have been referred to
as vestigial (Stenvik et al., 2006). A few additional details
about cauline leaf abscission zones are known. It has
been reported that overexpression of IDA can trigger
unnatural abscission of cauline leaves and that that
IDA-triggered cauline leaf abscission requires BOP1
and BOP2 because they act redundantly to specify the
formation of the vestigial abscission zone (Stenvik et al.,
2006; McKim et al., 2008). Also, rounding of many cauline leaf abscission zone cells after yellow senesced
cauline leaves are forcefully removed has also been observed (McKim et al., 2008). The nev mutant’s inability to
shed its cauline leaves long after senescence has occurred
has also been described (Liljegren et al., 2009).
Shedding of leaves during periods of summer drought
is extremely common in plants. For example, woody
trees, including walnut, popular, and citrus, all shed
leaves in response to drought (Blake et al., 1984; Parker
and Pallardy, 1985; Agustí et al., 2012). Crop plants like
beans and cotton also shed leaves during drought
(Hsiao, 1973; Pandey et al., 1984). Here, we report that
Arabidopsis can abscise its cauline leaves (aerial leaves
along the stem) in response to drought (water deficit).
Unlike floral organ abscission, drought-induced cauline
leaf abscission is not triggered by developmental timing.
We further show that HAE accumulates in the abscission
zones once the leaves begin to wilt and is necessary for
leaf abscission to occur along with IDA, NEV, MKK4/5,
and BLADE ON PETIOLE1 (BOP1) and BOP2 (McKim
et al., 2008). In short, we describe a core signaling molecular mechanism that functions in abscission zones of
multiple organs with multiple triggers. These findings
contribute to a better understanding of the interplay
between drought stress and abscission.
RESULTS
Drought-Treated Arabidopsis Plants Drop Their Cauline
Leaves after They Are Rewatered
We observed Arabidopsis plants would occasionally
drop cauline leaves and were curious about this observation because previous studies suggested that Arabidopsis only abscised floral, not vegetative, organs. After
sequentially ruling out differences in genotypes, pesticide
treatment, and ethylene accumulation, we found leaf
abscission can be triggered by withholding water until
the plants began to wilt followed by rewatering (Fig. 1).
Cauline leaf abscission occurs somewhere from overnight
to 2 d after rewatering wilted plants. One to several
cauline leaves will abscise from primary and secondary
inflorescences of any plant that experiences the
drought/rewatering treatment with an average of 6.6
per plant (Fig. 1D). The first cauline leaf to develop (leaf 1;
Fig. 1) is less likely to abscise than the second or third
Figure 1. Arabidopsis abscises its cauline leaves when plants wilted by
drought (water deficit) are rewatered. A, Well-watered Arabidopsis does
not abscise its cauline leaves. The first three cauline leaves to develop
are numbered. B and C, A plant that abscised cauline leaves after being
wilted by drought and rewatered. The same plant is shown in B and C
where B is before gently touching the leaves and C is after touching. The
plants shown in A to C are laid on their side for the photo. The image
shown in C has abscised leaves placed near where they abscised from.
Red tape equals 9.5 mm in length (A–C). D, Number of leaves abscised
from well-watered or drought treatments on all inflorescences (primary
or secondary). E, Breakstrength required to remove leaves from the
plant. F, Breakstrength measured 5 d after plants reached 40% SWC (not
rewatered). Thirty percent of plants never develop a third cauline leaf on
their primary inflorescence. In these plants the third cauline leaf to
develop is on the first secondary inflorescence to develop. Therefore,
data for leaves 1 and 2 are exclusively from the primary inflorescence,
while data for leaf 3 (E and F) are a mix of leaves from both primary and
secondary inflorescences. Data are mean 6 SE; n = 10 biological replicates (one plant each; D), n = 6 (E and F); t test versus well-watered
(D and E); *P , 0.05, **P , 0.01.
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Patharkar and Walker
leaf to develop. Cauline leaf 1 only abscises by touch
30% of the time (close to 0 g force), while leaf 2 abscises
70% of the time and leaf 3 abscises 80% of the time
(Fig. 1, D and E). Cauline leaves other than the first
three to develop (labeled in Fig. 1, A–C) can also abscise. Not all ecotype Columbia (Col-0) plants develop
a third cauline leaf on the primary inflorescence.
Plants without a third cauline leaf on their primary
inflorescence (29.6%; eight of 27) develop their third
cauline leaf on the first secondary inflorescence to
emerge (Supplemental Fig. S1). Plants that are not
rewatered after wilting retain their leaves (Fig. 1F).
HAE Expression Is Induced in Leaf Abscission Zones of
Plants with Limited Water
Since HAE expression is induced by osmotic stress
(Supplemental Fig. S2) and accumulates in floral organ
abscission zones when abscission is initiated, we asked
whether HAE was involved in cauline leaf abscission
(Jinn et al., 2000). To understand cauline leaf abscission,
plants expressing a HAE-yellow fluorescent protein
(YFP) fusion driven by the HAE promoter were observed under well-watered conditions and after a
drought/rewatering treatment. A higher HAE-YFP
signal is observed in the abscission zones of cauline
leaves from drought-treated plants than from wellwatered plants, with more HAE-YFP observable in
leaf 2 and leaf 3 than in leaf 1 (Fig. 2). Wild-type plants
not carrying the pHAE:HAE-YFP transgene do not have
fluorescence in the abscission zones; however, dried
abscised leaves do autofluoresce (Supplemental Fig.
S3). The finding of HAE accumulation in the leaf abscission zones suggests a role of HAE in leaf abscission,
possibly as the receptor for the signal to abscise.
A time course of soil drying was carried out to visualize changes in abscission zone morphology and
HAE expression as water became limiting and then was
restored. Observations focused on cauline leaf 2, which
always develops on the primary inflorescence and reliably abscises (Supplemental Fig. S1). Minimal changes
in HAE-YFP accumulation, leaf appearance, or abscission zone appearance were observed as soil dried from
well-watered conditions/88% soil water content (SWC)
to 80% SWC, despite the former having almost twice
the water (88% SWC = 9.36 g dry soil + 71.46 g water;
80% SWC = 9.36 g dry soil + 37.46 g water; Fig. 3, A and
B). At 40% SWC, the cauline leaf is visibly wilted, begins yellowing, and HAE-YFP is induced, but the abscission zone appears unchanged (Fig. 3, C and F).
Forty percent SWC has a water potential of 23.15 MPa,
which means there is essentially no available water for
the plant (Fig. 3F). One day after rewatering 40% SWC
plants, the leaves regain their full turgor but yellow
fully, a tear begins to form at the abscission zone, the
abscission zone cells become enlarged and rounded,
and HAE-YFP accumulation reaches its maximum observed level (Fig. 3D; Supplemental Fig. S4A). By 48 h,
the leaves abscise, and HAE-YFP is still present in the
Figure 2. HAE is preferentially expressed in
cauline leaf abscission zones from plants exposed to drought stress. A, Well-watered plant
and drought/rewatered plant with cauline
leaves 1 to 3 used for microscopy. B to G,
Cauline leaf abscission zones from a wellwatered plant. H to M, Cauline leaf abscission
zones from a drought/rewatered plant. Images
(B–M) were taken at the same magnification;
scale bar = 0.5 mm. Images are representative
from eight plants (n = 8).
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Core Mechanisms Regulating Abscission
Figure 3. HAE is expressed prior to cell separation when leaves become wilted. A to E, A
representative soil drying and rewatering time
course of cauline leaf 2 including wellwatered/88% SWC (A), 80% SWC (B), 40%
SWC (C), 1 d after rewatering (D), and 2 d after
rewatering (E). Top shows cauline leaf 2,
middle is a close-up image of the abscission
zone with reflected white light, and bottom is
the same as the middle where YFP fluorescence is being imaged. The time course was
repeated four times (n = 4) with similar results.
Red tape is 9.5 mm in length (A–E, top) and
scale bar is 0.5 mm (A–E, middle and bottom)
where images were taken under the same
magnification. F, Leaf relative water content at
different SWC or soil water potentials. Visible
wilting occurs at 50% SWC, and plants were
rewatered once SWC reached 40%. Relative
water content of cauline leaves is shown for
wilted cauline leaves as in C and after rewatering as in D. Data are mean 6 SE; n = 4 biological replicates (one plant each). G, Both
HAE and IDA transcripts increase in abscission
zones from wilted plants as in C compared
with well-watered controls and continue to
increase after rewatering. Data are mean 6 SE;
n = 6 biological replicates (one plant each) for
well-watered and wilted, n = 3 for rewatered;
different letters indicate statistically different
quantities within a gene target, t test P , 0.05.
abscission zone scar (Fig. 3E; Supplemental Fig. S4B).
We also found both HAE and IDA mRNAs accumulated more in the abscission zone of wilted cauline
leaves than in well-watered leaves and were further
increased once plants were rewatered (Fig. 3G). Expression dynamics of HAE are consistent with HAE
acting to prime the abscission machinery of cauline
leaves when water becomes limiting and then functioning throughout abscission after water is restored.
Mutants Defective in Floral Organ Abscission Are Also
Defective in Drought-Triggered Cauline Leaf Abscission
Given that both HAE and IDA are expressed in cauline
leaf abscission zones, we hypothesized that they, along
with other components required for floral organ abscission, may also be necessary for cauline leaf abscission
during drought. We found that HAE/HSL2, IDA, NEV,
and MKK4/5 are all necessary for drought-induced
cauline leaf abscission to occur (Fig. 4, A–C). The bop1
bop2 double mutant, which lacks a cauline leaf abscission
zone, also fails to abscise (McKim et al., 2008). hae hsl2, ida,
nev, and mkk4/5 RNAi mutant plants all have a distinct
band separating the leaf from the stem, which is the abscission zone (Fig. 4A). No cauline leaves abscise from hae
hsl2, ida, nev, mkk4/5 RNAi, and bop1 bop2 mutant plants
(Fig. 4B); on average, 15 to 20 g of force are required to
pull off cauline leaves from the mutants (Fig. 4C). We also
observed that cauline leaves from all mutants tested (Fig.
4A) senesce following the drought treatment but do not
abscise. Unlike in floral organ abscission where hae single
mutants have completely wild-type abscission, hae single
mutants abscise cauline leaves at 20% of the frequency of
the wild type (Fig. 4D). On the other hand, hsl2 single
mutant plants have leaf abscission similar to that of wildtype plants (Fig. 4E). These results suggest floral organ
abscission and drought-triggered leaf abscission share a
common mechanism with subtle differences.
HAE Is Expressed in Vestigial Pedicel Abscission Zones
When Fruits Become Mature
Unlike the cauline leaf abscission zone, the pedicel
abscission zone in Arabidopsis is likely to be truly
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Patharkar and Walker
Figure 4. Mutants defective in floral organ abscission are also defective in drought-induced leaf abscission. A, Wild type
(WT; Col-0) plants abscise after drought/rewatering, while hae-3 hsl2-3, ida-2, nev-3, mkk4/5 RNAi, and bop1 bop2
leaves yellow but do not abscise. bop1 bop2 lacks an abscission zone altogether, while hae-3 hsl2-3, ida-2, nev-3, and
mkk4/5 RNAi all have a clear band (abscission zone) at the leaf-stem boundary. Images in top panels are wilted leaves (leaf 2) right
before rewatering. Images in middle panels are leaves 2 d after rewatering. Red tape is 9.5 mm in length. Images in bottom
panels are magnified versions of middle panels showing the abscission zone. Scale bar = 0.5 mm. B, Number of leaves
abscised per plant for various genotypes. C, Breakstrength required to remove leaves from the plant for various genotypes.
Data are mean 6 SE ; n = 5 biological replicates (one plant each; B and C); t test versus the wild type; *P , 0.1, **P , 0.05,
***P , 0.01. D and E, Number of abscised cauline leaves in hae mutant plants (D) and hsl2 mutant plants (E) after a
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Core Mechanisms Regulating Abscission
vestigial since the abscission zone only appears to be on
the top half of the base of the pedicel (Fig. 5D; Cho and
Cosgrove, 2000). HAE-YFP can be detected in the vestigial pedicel abscission zone once fruits have fully
elongated (floral stage 17; Fig. 5E). When siliques are
manually pulled off, the tear occurs at the vestigial
abscission zone once siliques are fully elongated and
HAE-YFP is present (Fig. 5, B and C). However, before
siliques are fully elongated, the tear will occur somewhere in the middle of the pedicel. This indicates that
partial abscission occurs at the base of the pedicel once
the silique is mature. While a drought/rewatering
treatment increased HAE-YFP expression in cauline
leaf abscission zones, we did not see a similar induction
of HAE-YFP in drought/rewater-treated vestigial
pedicel abscission zones (Fig. 5, E and G). Additionally,
the floral position in which all following positions tear
at the vestigial abscission zone was similar between
well-watered and drought/rewatered plants (Fig. 5, B
and H), suggesting drought cannot accelerate partial
abscission of the pedicel.
Mutations in HAE/HSL2 and IDA Do Not Cause Seed
Yield Penalty
It is theoretically possible that manipulating abscission in crop plants that lose leaves or flowers during
mild drought stress might result in greater agricultural
yield. For example, if bean plants did not shed their
flowers during mild drought stress, it is possible that
they might produce more seeds by final harvest
(Pandey et al., 1984). In Arabidopsis, hae hsl2 and ida
mutant plants do not have reduced seed set compared
with the wild type, while nev, mkk4/5 RNAi, and bop1
bop2 mutants have a seed yield penalty (Fig. 6A). This
indicates that homologs of HAE and IDA should be
preferentially targeted in crop plants if one is to test the
hypothesis that less abscission in mild drought may
result in greater seed set.
Abscisic Acid Is Unlikely to Directly Regulate Abscission
in Arabidopsis
As its name would suggest, abscisic acid (ABA) was
named for its ability to promote abscission. However,
currently ABA’s ability to promote abscission is viewed
controversially. Current thinking is that ABA may not be
important for abscission in many plants. Initial studies
on ABA and abscission may have been flawed in that
such high concentrations of ABA were used that the
treatment resulted in production of ethylene that in turn
caused abscission (Sexton and Roberts, 1982). We
designed experiments using Arabidopsis mutants in
ABA synthesis and signaling to understand if ABA plays
a direct role in abscission in Arabidopsis. First, ABA does
not appear to be necessary for floral organ abscission
since aba insensitive1 (abi1) and aba deficient1 (aba1) do not
retain their floral organs (Supplemental Fig. S5A). Similar results were obtained with aba2 mutants (Ogawa
et al., 2009). We found that our assay for leaf abscission,
in which we withheld water until SWC reaches 40%
and then rewatered, could not be used with ABA mutants because 0% of them can survive the treatment
(Supplemental Fig. S5B). Therefore, we modified our
assay so that the level of visible wilting was controlled to
be similar between wild-type Landsberg erecta (Ler-0)
and ABA mutants rather than exposing both the wild
type and ABA mutants to the same SWC. When abi11 and aba1-1 mutants wilted and rehydrated similar to
the wild type, they also abscised statistically similar
number of leaves as the wild type (Supplemental Fig. S5,
C and D). abi1-1 and aba1-1 abscised slightly fewer leaves
than did the wild type, although not statistically different. However, abi1-1 and aba1-1 are smaller than the wild
type and have fewer cauline leaves. The percentage of
total cauline leaves abscised by abi1-1 was almost identical to the wild type, while aba1-1 actually abscised a
higher percentage, although not statistically different
(Supplemental Fig. S5D). The abscission zone scar left
after abscission was fairly similar between abi1-1 and the
wild type (Supplemental Fig. S5E). We conclude that
ABA is not essential for abscission and therefore unlikely
to directly regulate abscission but could be placed far
upstream of the abscission process in Arabidopsis.
DISCUSSION
The drought-induced cauline leaf abscission pathway is summarized in the model shown in Figure 6B.
BOP1 and BOP2 redundantly specify the formation of
an abscission zone between the stem and cauline leaves
(McKim et al., 2008). IDA, HAE/HSL2, MKK4/5, and
NEV are all required for cauline leaf abscission that is
triggered by drought. We speculate that IDA, HAE/
HSL2, and MKK4/5 would be ordered the same as they
are in the genetic pathway that governs floral organ
abscission; however, we have yet to perform any experiments in cauline leaves to confirm this order.
The significance of our findings at the physiological
level is that Arabidopsis can shed its cauline leaves after
one bout with drought, potentially placing it in a better
position to deal with subsequent occurrences of
drought due to reduced leaf area for transpiration
(Kozlowski, 1973; Tschaplinski et al., 1998; Escudero
et al., 2008). The nutrients drawn out of leaves before
abscission (the leaves become yellow and presumably
nitrogen is being mobilized out of them) may also act to
Figure 4. (Continued.)
drought and rewatering treatment. Data are mean 6 SE ; n = 6 biological replicates (one plant each; D and E); t test versus
the wild type; ***P , 0.005.
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Figure 5. HAE-YFP expression correlates with partial fruit abscission at the vestigial pedicel abscission zone. A, pHAE:HAE-YFP
inflorescence with flowers/siliques numbered by floral position. B, Same inflorescence as in A after flowers or siliques were
manually pulled off. After position 7 all siliques tear off at the base of the pedicel, indicating partial abscission has occurred.
Before position 8 tearing occurs in the middle of the pedicel. C, Close-up view of B. D, Scar left after pulling off a full-length
silique. The top half tears off relatively smoothly, while the bottom half has a rough surface due to the abscission zone only being
present at the upper part of the base of the pedicel (Cho and Cosgrove, 2000). E, Close-up view of vestigial abscission zones from
inflorescence in A with floral position and stage indicated. Top panels are extended depth of field bright-field images, and bottom
panels show HAE-YFP fluorescence. F and G, Pedicel abscission is not drought-inducible. Bright-field (F) or HAE-YFP florescence
(G) of a position 11 pedicel abscission zone from plants treated with drought followed by rewatering. All siliques break at the base
of the pedicel when pulled after position 8 (H). Red tape is 9.5 mm in length (A–C, H). Scale bar = 100 mm (D–G). Experiments
were repeated three times (n = 3) with similar results.
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Core Mechanisms Regulating Abscission
Figure 6. Mutations in HAE/HSL2 or IDA have minimal seed yield
penalty compared with other abscission-defective mutants. A, Seed
yield grown under nonstressed conditions. Data are mean 6 SE;
n = 3 biological replicates (one plant each); t test versus the wild type;
*P , 0.05. B, A model of drought-induced cauline leaf abscission in
Arabidopsis. BOP1 and BOP2 are required for cauline leaf abscission
zone development. IDA, HAE/HSL2, MKK4/5, and NEV are all required
for drought-induced leaf abscission as well as floral organ abscission.
keep younger tissues healthy (Dela Fuente and Leopold, 1968; Addicott, 1982; Harvey and van den
Driessche, 1999). This study shows at the molecular
level how leaves are abscised in response to drought.
Floral organ abscission has served as an excellent model
for understanding abscission at the molecular level.
However, as with all model systems, the question remains of how far the results can be extrapolated to other
systems. We show that genes first described to be necessary for floral organ abscission (HAE, IDA, NEV, and
MKK4/5) also have a role in drought-induced organ
abscission (Jinn et al., 2000; Butenko et al., 2003; Cho
et al., 2008; Liljegren et al., 2009). The requirement of
rewatering for leaf abscission to occur could be a reason
why Arabidopsis leaf abscission has not been characterized despite more than 20 years of Arabidopsis abscission research. HAE also likely functions in partial
abscission of mature fruits at the vestigial pedicel abscission zone since its expression is correlated with
partial abscission; however, we have not tested if HAE
is genetically necessary for partial abscission of fruits.
The Arabidopsis pedicel abscission zone is referred to
as vestigial because it only spans half of the pedicel
diameter, which does not allow spontaneous abscission
(Cho and Cosgrove, 2000). HAE-YFP accumulates in
the upper portion of base of the pedicel once the silique
is at floral stage 17 (Fig. 5E; Alvarez-Buylla et al., 2010).
However, partial abscission of the pedicel appears to be
correlated with fruit development rather than with
water availability (Fig. 5, A–D and F–H).
The ability to examine abscission in floral organs and
leaves triggered by development or environmental cues
will undoubtedly aid researchers in discovering more
molecular components that regulate abscission. While
regulators of leaf abscission appear to be similar to
those of floral organ abscission, drought-triggered leaf
abscission seems to be more quantitative than floral
organ abscission. Single mutants of HAE are identical to
wild-type plants in terms of floral organ abscission but
are clearly defective in drought-triggered leaf abscission. To date, no mutations have been discovered that
make floral organ abscission occur before pollination,
although the double mutant agl15 agl18 and IDA
overexpressors abscise at an earlier floral position
(Stenvik et al., 2006; Patharkar and Walker, 2015). Perhaps the more quantitative nature of leaf abscission will
allow the discovery of mutations that cause more leaves
to fall off when a plant experiences drought. Currently,
most drought studies in Arabidopsis focus on plant
survival or plant wilting as a physiological response to
drought (Fujita et al., 2011). Cauline leaf abscission
could be used as a quantitative and easily measurable
drought response assay.
How HAE and IDA expression is activated when
cauline leaves wilt is still unknown. HAE can be transcriptionally increased by ABA in seedlings (Winter
et al., 2007; Goda et al., 2008). However, since we found
ABA is not essential for abscission in Arabidopsis, it is
more likely ABA acts as an additive factor rather than
an initiator. HAE coexpressed genes have overrepresented AtMYC2 binding sites in their promoters
(Patharkar and Walker, 2015). AtMYC2 is a basic helixloop-helix transcription factor that can activate transcription of ABA-responsive genes (Abe et al., 2003).
Another possible way HAE transcription could be
activated in abscission zones of wilted cauline leaves is
by activation of the MAPK cascade that leads to HAE
expression through inactivation of the transcriptional
repressor AGL15 (Patharkar and Walker, 2015;
Patharkar et al., 2016).
We do not know why Arabidopsis abscises cauline
leaves but not rosette leaves. It is likely due to lack of a
functional abscission zone. The broad spectrum inducer
of abscission, overexpression of IDA, does not trigger
rosette leaf abscission (Stenvik et al., 2006). About half
of the large rosette leaves do senesce in our assay so that
transpiration from the rosette would be reduced. It may
be more beneficial for a plant to shed cauline leaves
than rosette leaves because attached dead cauline
leaves can block some light from lower leaves while
dead rosette leaves would not. We also do not know
why the first cauline leaf abscises at lower frequency
than leaf 2 or 3. We speculate that it could simply be
because the first leaf is close to the soil; therefore, the pot
shields it from some of the airflow in the growth
chamber, thus reducing the water stress on it.
From an agricultural perspective, the conserved
molecular mechanisms uncovered here offer great
leads for agricultural improvement. Abscission can be
manipulated in many ways to increase agricultural
yield. For example, the canned tomato industry routinely grows tomatoes carrying the jointless mutation,
Plant Physiol. Vol. 172, 2016
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Patharkar and Walker
which results in tomatoes with no pedicel abscission
zone (Mao et al., 2000). When picked, jointless tomatoes
leave their calyx and stem behind on the plant, resulting in less puncture damage to other picked tomatoes
(Zahara and Scheuerman, 1988). Additionally, synthetic auxins and ethylene blockers are used in apple
and Citrus orchards to prevent preharvest drop
(Anthony and Coggins, 1999; Yuan and Carbaugh,
2007). We found that HAE/HSL2 and IDA could be
top candidates for agricultural manipulation since
knocking them out results in minimal yield penalty
compared with nev, mkk4/5 RNAi, and bop1 bop2 (Fig.
5A). Manipulation of HAE and IDA for agriculture
could be achieved by breeding for weak alleles, genetic
editing by CRISPR/Cas9, or devising chemicals that
block IDA peptide function. HAE and IDA homologs
likely play the same pivotal role, regulating abscission
in a wide range of species. For example, HAE homologs
are also up-regulated in poplar abscission zones when
abscission is activated by shading leaves (Jin et al.,
2015), and IDA homologs are induced in soybean and
tomato abscission zones prior to abscission (Tucker and
Yang, 2012). In fact, the active peptide from IDA is
conserved in all flowering plants (Stø et al., 2015). Also,
Citrus IDA3 can complement abscission deficiency in
Arabidopsis ida mutant plants (Estornell et al., 2015).
Interestingly, Citrus-like Arabidopsis abscises its leaves
after it is rewatered following drought that causes
wilting (Agustí et al., 2012).
A lot of recent attention has been given to activation
of cell wall-modifying enzymes that hydrolyze the
middle lamella of the abscission zone as the implied
mechanism of cell separation (Niederhuth et al., 2013a,
2013b; Liu et al., 2013). Our findings strongly suggest
that while drought triggers abscission, water is
required for abscission to occur. While hydrolytic enzymes likely dissolve the middle lamella of the abscission zone, water is probably necessary for abscission
zone cell expansion. In the early 1900s, mechanical
shearing from the enlargement of abscission zone cells,
driven by turgor, was thought to be the major mechanism leading to abscission (Fitting, 1911; Sexton and
Roberts, 1982), and perhaps this should be given more
thought again.
CONCLUSION
Plants lose their leaves by an active process, called
abscission, in which leaves are cut free when it is beneficial to the plant. Leaves are cut free at a specialized
layer of cells, called the abscission zone, that serves as
scissors for the plant. In our work, we have identified
the genes that tell the abscission zone to cut leaves free
during drought. Interestingly, in Arabidopsis many of
the genes required for drought-triggered abscission are
also necessary for abscission of floral organs after pollination, which suggests a common mechanism. This
work connects the floral organ abscission field to the
drought field and lays the groundwork for a new field
in Arabidopsis, drought-triggered leaf abscission.
MATERIALS AND METHODS
Plant Material and Growth Conditions
The Col-0 ecotype of Arabidopsis (Arabidopsis thaliana) was used as the wild
type (Arabidopsis Biological Resource Center stock no. CS70000), except of
experiment with ABA mutants, in which Ler-0 was used as the wild type.
Mutants used were the indicated allele: hae-3 hsl2-3, ida-2, nev-3, bop1-3 bop2-1,
abi1-1, aba1-1, aba1-3, and aba1-4 (Koornneef et al., 1982; Leung et al., 1997;
Hepworth et al., 2005; Cho et al., 2008; Liljegren et al., 2009; Niederhuth et al.,
2013b). Tandem mkk4/5 RNAi was described previously (Ren et al., 2002; Cho
et al., 2008). Plants were grown in Promix BX (Premier Tech Horticulture) at
23°C, 16 h light/8 h dark, 100 to 150 mE$m22$s21, and 50% to 70% humidity. The
starting SWC of fresh soil from bags after autoclaving was calculated by
weighing, drying at 80°C for 2 d, and then reweighing. Plants were grown individually in 5-ounce plastic cups with the equivalent of 9.36 g of completely
dry soil and 0.0787 g of Peters Peat Lite Special 20-10-20 fertilizer (Scotts). We
defined well-watered growing conditions as watering plants to saturation and
allowing them to grow until SWC reached 80%, at which point plants were
rewatered to saturation. Plants watered to saturation on a daily basis are unhealthy. SWC was determined daily by weighing the pots. Drought (water
deficit) was imposed once plants had inflorescences 15 to 20 cm tall (5–6 weeks
old) by not rewatering plants once SWC reached 80%. For drought treatments,
SWC was allowed to fall to 40% and then plants were rewatered to saturation.
Visible wilting was first observed at 50% SWC and approximately 24 to 36 h
later SWC was 40%. Plants were planted in a randomized complete block experimental design.
To make ABA mutants wilt similar to Ler-0 controls, ABA mutants were
shielded from air flow in the growth chamber by blocking the vents in the
growth chamber behind the ABA mutants and shielding on right and left of
plants (shielded from three sides while top and side away from vents was open).
Other growth conditions were identical to those described above, except that
visible wilting first occurred at 75% SWC and severe wilting (similar to wild-type
plants at 40% SWC) occurred at 65% SWC; therefore, ABA mutant plants were
rewatered once SWC reached 65%. Even with the gentler drought treatment,
ABA mutants could not regain full turgor by rewatering alone; therefore, once
ABA plants were rewatered, they were covered with a clear dome for 1 d followed by 3 d in restricted air flow (as above). Because ABA mutants recover
more slowly than the wild type after drought treatment, leaf abscission was
scored 4 d after rewatering for experiments with Ler-0 and ABA mutants.
Counting Abscised Leaves and Leaf Breakstrength
Two days after plants were rewatered after having water withheld until 40%
SWC, each cauline leaf was touched gently so that the plant was barely moved.
Leaves that fell off were counted as abscised. The airflow in the growth chamber
was not sufficient to blow off either abscised floral organs or abscised cauline
leaves. A petal breakstrength meter was used to determine the force required to
pull cauline leaves free (Lease et al., 2006).
Determining Position of Partial Pedicel Abscission
Flowers or siliques were gently pulled by hand as described previously (Cho
and Cosgrove, 2000), except that we report floral position (Alvarez-Buylla et al.,
2010) rather than silique position.
Soil Water Potential
SWC was measured with an isopiestic thermocouple psychrometer against
Suc standards (Boyer, 1995). Soil at 40% relative water content was drier than
the driest Suc standard of 22.702 MPa and was extrapolated past that standard.
Leaf Relative Water Content
Leaf relative water content was measured on whole leaves as described
previously (Barrs and Weatherley, 1962).
Microscopy
Images were collected with a Zeiss SteREO Discovery v12 epifluorescence
microscope equipped with a Canon EOS 6D camera. Bright-field images are
518
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Core Mechanisms Regulating Abscission
extended depth of field images processed with Zerene Stacker using the Pmax
algorithm. Bright-field images were taken with autoexposure and identical
settings for white balance. YFP images were acquired with a KSC 295-823D YFP
Cube (excitation 490–510 nm; dichroic beamsplitter 515 long pass; emission
520–550 nm). All YFP images within an experiment were taken with identical
settings for exposure, white balance, and ISO digital film speed. YFP images are
of a single depth of field unless noted otherwise.
Constructs and Transgenic Plants
The HAE promoter HAE-YFP construct was made by PCR amplifying the
1885 bp upstream of the start codon to the last codon before the stop codon and
inserting the resulting PCR product into the NotI site of pE6c (Dubin et al.,
2008). Gateway LR reaction was performed to move the HAE promoter
HAE-YFP insert to the pBiB-Basta binary vector. The binary vector was used
with Agrobacterium strain GV3101 to introduce the HAE promoter HAE-YFP
into hae-3 hsl2-3 via the floral dip method. The construct complemented the
abscission defect of hae hsl2. Plants used in this study had a single HAE
promoter HAE-YFP insertion in the intergenic space between At4g11280 and
At4g11290 and had close to native expression levels (Supplemental Fig. S6).
Quantitative Reverse Transcription PCR
Cauline leaf abscission zones were hand dissected by making three straight cuts
with a razor blade from cauline leaf 2. A single abscission zone was used per
replicate. RNA isolation, reverse transcription, and quantitative PCR were performed as described previously (Patharkar and Walker, 2015), except Hot Start Taq
(New England Biolabs) was used in place of Platinum Taq. Primers for HAE and
the reference gene At5g46630 were described previously (Patharkar and Walker,
2015). Forward 59-GAGTAGTCCTTGTGTAGCGG-39 and reverse 59-TCTTAGAAGGAGCAGAAGGAG-39 were used as primers for IDA quantification.
Gene Expression during Osmotic Stress
Publicly available microarray data for mannitol treatment were downloaded
from Gene Expression Omnibus (accession GSE5622; Kilian et al., 2007) and
reanalyzed with RobiNA using the PLIER algorithm (Lohse et al., 2010).
Seed Yield
Plants were planted in a randomized complete block experimental design
with one plant per pot and given optimal watering. Plants were tied to a stake to
prevent tangling of inflorescences. Once the majority of plants in an experiment
had 10% yellow siliques, watering was discontinued and plants were allowed to
dry. Seeds were purified by passing them through a sieve three times and rolled
once over paper so that debris that could not roll was excluded.
Supplemental Data
The following supplemental materials are available.
Supplemental Figure S1. The first two cauline leaves to develop are
always on the primary inflorescence, while the third cauline leaf to
develop can occur on either the primary or the secondary inflorescence.
Supplemental Figure S2. HAE expression is induced by osmotic stress in
shoots (Kilian et al., 2007).
Supplemental Figure S3. Dry cauline leaves autofluoresce.
Supplemental Figure S4. Cauline leaf abscission zone cells become enlarged and rounded at the time of abscission.
Supplemental Figure S5. ABA does not regulate abscission directly in
Arabidopsis.
Supplemental Figure S6. pHAE:HAE-YFP position and expression levels.
ACKNOWLEDGMENTS
We thank Stefan Bennewitz for generating the pHAE:HAE-YFP transgenic
line, Melody Kroll and Catherine Espinoza for reading and editing the
manuscript, and Robert Sharp for use of his psychrometer for water potential
measurements.
Received June 23, 2016; accepted July 27, 2016; published July 28, 2016.
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