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Core Mechanisms Regulating DevelopmentallyTimed and Environmentally Triggered Abscission[OPEN]

O. Rahul Patharkar* and John C. Walker

Division of Biological Sciences and Interdisciplinary Plant Group, University of Missouri, Columbia, Missouri65211

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 poorlyunderstood at the molecular genetic level. Here, we show that Arabidopsis (Arabidopsis thaliana) can be used to elucidate thepathway controlling drought-triggered leaf shedding. We further show that much of the pathway regulating developmentallytimed floral organ abscission is conserved in regulating drought-triggered leaf abscission. Gene expression of HAESA (HAE) andINFLORESCENCE DEFICIENT IN ABSCISSION (IDA) is induced in cauline leaf abscission zones when the leaves become wilted inresponse to limited water and HAE continues to accumulate in the leaf abscission zones through the abscission process. The genesthat encode HAE/HAESA-LIKE2, IDA, NEVERSHED, and MAPK KINASE4 and 5 are all necessary for drought-induced leafabscission. Our findings offer a molecular mechanism explaining drought-triggered leaf abscission. Furthermore, the ability to studyleaf abscission in Arabidopsis opens up a new avenue to tease apart mechanisms involved in abscission that have been difficult toseparate 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 sum-mers by the process of leaf abscission to reduce thetranspiration load on the plant and to ensure youngtissues have adequate water and nutrients to survive(Street et al., 2006; Agustí et al., 2012). Abscission is theprocess enabling plants to discard unwanted organsin response to environmental stimuli or develop-mental timing (Tudela and Primo-Millo, 1992; Agustíet al., 2012; Liljegren, 2012; Niederhuth et al., 2013a).Abscission occurs at distinct boundaries, called ab-scission zones, that are laid down as the associatedorgan develops. Abscission zones have smaller cellswith smaller vacuoles than the surrounding tissue.Once abscission is triggered, the abscission zone cellsexpand and hydrolytic enzymes are released to dis-solve the abscission zone’s middle lamella, resultingin abscission (Sexton and Roberts, 1982).

Floral organ abscission in Arabidopsis (Arabidopsisthaliana) is the best studied abscission model. Thetiming of floral organ abscission is developmentallyregulated so that abscission occurs shortly after fertili-zation. Briefly, a molecular pathway has been describedin which a peptide derived from INFLORESCENCEDEFICIENT INABSCISSION (IDA) is thought to trigger

a pair of redundant receptor-like protein kinases,HAESA (HAE) and HAESA-LIKE2 (HSL2). A mitogen-activated protein kinase (MAPK) pathway, consistingof MAPK KINASE4 (MKK4)/MKK5 and MAPK3(MPK3)/MPK6, positioned genetically downstream ofHAE/HSL2, phosphorylates a MADS-domain tran-scription factor, AGAMOUS-LIKE15 (AGL15), whichresults in a transcriptional increase of HAE, therebycreating a positive feedback loop (Cho et al., 2008;Stenvik et al., 2008; Patharkar and Walker, 2015).NEVERSHED (NEV), an ADP-ribosylation factor-GTPase-activating protein, is thought to trafficHAE/HSL2 and other receptor-like protein kinases tothe correct subcellular location and is also required forabscission (Liljegren et al., 2009; Burr et al., 2011;Liljegren, 2012). Secondary mutations that suppressabscission defects of hae hsl2, ida, and nev have also beendescribed (Leslie et al., 2010; Lewis et al., 2010; Burret al., 2011; Shi et al., 2011). However, these suppressormutants cannot be clearly labeled as negative regula-tors of abscission. For example, secondary mutations inSOMATIC EMBRYOGENESIS RECEPTOR KINASE1(SERK1) can rescue the abscission defects of nev whileat the same time the triple mutant serk1 serk2 serk3 isdefective in abscission (Lewis et al., 2010; Meng et al.,2016). Additionally, when overexpressed in Arabi-dopsis protoplasts or Nicotiana benthamiana leaves,SERK3 (also known as BAK1) can form a complexwith HAE/HSL2 mediated by IDA (Meng et al.,2016). A key net transcriptional outcome of the ab-scission pathway is increased expression of cell wall-modifying enzymes that aid in cell separation at theabscission zone’s middle lamella (Niederhuth et al.,2013b).

* Address correspondence to [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed 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.

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http://orcid.org/0000-0002-1242-6549

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However, it is poorly understood if the pathway forfloral organ abscission has a role in abscission of otherorgans and in environmentally induced abscission. Forexample, are leaves shed by the same molecular mech-anism as floral organ abscission? For about 20 years itwas thought that Arabidopsis only abscises it floral or-gans (Bleecker and Patterson, 1997; Jinn et al., 2000).Abscission zone-like structures at the base of caulineleaves and pedicels of Arabidopsis have been referred toas vestigial (Stenvik et al., 2006). A few additional detailsabout cauline leaf abscission zones are known. It hasbeen reported that overexpression of IDA can triggerunnatural abscission of cauline leaves and that thatIDA-triggered cauline leaf abscission requires BOP1and BOP2 because they act redundantly to specify theformation of the vestigial abscission zone (Stenvik et al.,2006; McKim et al., 2008). Also, rounding of many cau-line leaf abscission zone cells after yellow senescedcauline leaves are forcefully removed has also been ob-served (McKim et al., 2008). The nevmutant’s inability toshed its cauline leaves long after senescence has occurredhas also been described (Liljegren et al., 2009).Shedding of leaves during periods of summer drought

is extremely common in plants. For example, woodytrees, including walnut, popular, and citrus, all shedleaves in response to drought (Blake et al., 1984; Parkerand Pallardy, 1985; Agustí et al., 2012). Crop plants likebeans and cotton also shed leaves during drought(Hsiao, 1973; Pandey et al., 1984). Here, we report thatArabidopsis can abscise its cauline leaves (aerial leavesalong the stem) in response to drought (water deficit).Unlike floral organ abscission, drought-induced caulineleaf abscission is not triggered by developmental timing.We further show thatHAE accumulates in the abscissionzones once the leaves begin to wilt and is necessary forleaf abscission to occur along with IDA, NEV, MKK4/5,and BLADE ON PETIOLE1 (BOP1) and BOP2 (McKimet al., 2008). In short, we describe a core signaling mo-lecular mechanism that functions in abscission zones ofmultiple organs with multiple triggers. These findingscontribute to a better understanding of the interplaybetween drought stress and abscission.

RESULTS

Drought-Treated Arabidopsis Plants Drop Their CaulineLeaves after They Are Rewatered

We observed Arabidopsis plants would occasionallydrop cauline leaves and were curious about this obser-vation because previous studies suggested that Arabi-dopsis only abscised floral, not vegetative, organs. Aftersequentially ruling out differences in genotypes, pesticidetreatment, and ethylene accumulation, we found leafabscission can be triggered by withholding water untilthe plants began to wilt followed by rewatering (Fig. 1).Cauline leaf abscission occurs somewhere from overnightto 2 d after rewatering wilted plants. One to severalcauline leaves will abscise from primary and secondaryinflorescences of any plant that experiences the

drought/rewatering treatment with an average of 6.6per 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 bydrought (water deficit) are rewatered. A,Well-watered Arabidopsis doesnot abscise its cauline leaves. The first three cauline leaves to developare numbered. B and C, A plant that abscised cauline leaves after beingwilted by drought and rewatered. The same plant is shown in B and Cwhere B is before gently touching the leaves and C is after touching. Theplants shown in A to C are laid on their side for the photo. The imageshown 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 abscisedfrom well-watered or drought treatments on all inflorescences (primaryor secondary). E, Breakstrength required to remove leaves from theplant. F, Breakstrength measured 5 d after plants reached 40% SWC (notrewatered). Thirty percent of plants never develop a third cauline leaf ontheir primary inflorescence. In these plants the third cauline leaf todevelop 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 andsecondary inflorescences. Data are mean 6 SE; n = 10 biological rep-licates (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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Core Mechanisms Regulating Abscission

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leaf to develop. Cauline leaf 1 only abscises by touch30% of the time (close to 0 g force), while leaf 2 abscises70% of the time and leaf 3 abscises 80% of the time(Fig. 1, D and E). Cauline leaves other than the firstthree to develop (labeled in Fig. 1, A–C) can also ab-scise. Not all ecotype Columbia (Col-0) plants developa third cauline leaf on the primary inflorescence.Plants without a third cauline leaf on their primaryinflorescence (29.6%; eight of 27) develop their thirdcauline leaf on the first secondary inflorescence toemerge (Supplemental Fig. S1). Plants that are notrewatered after wilting retain their leaves (Fig. 1F).

HAE Expression Is Induced in Leaf Abscission Zones ofPlants with Limited Water

Since HAE expression is induced by osmotic stress(Supplemental Fig. S2) and accumulates in floral organabscission zones when abscission is initiated, we askedwhether 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 ob-served under well-watered conditions and after adrought/rewatering treatment. A higher HAE-YFPsignal is observed in the abscission zones of caulineleaves from drought-treated plants than from well-watered plants, with more HAE-YFP observable inleaf 2 and leaf 3 than in leaf 1 (Fig. 2). Wild-type plants

not carrying the pHAE:HAE-YFP transgene do not havefluorescence in the abscission zones; however, driedabscised leaves do autofluoresce (Supplemental Fig.S3). The finding of HAE accumulation in the leaf ab-scission 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 vi-sualize changes in abscission zone morphology andHAE expression as water became limiting and thenwasrestored. Observations focused on cauline leaf 2, whichalways develops on the primary inflorescence and re-liably abscises (Supplemental Fig. S1). Minimal changesin HAE-YFP accumulation, leaf appearance, or abscis-sion zone appearance were observed as soil dried fromwell-watered conditions/88% soil water content (SWC)to 80% SWC, despite the former having almost twicethe 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 andB). At 40% SWC, the cauline leaf is visibly wilted, be-gins yellowing, and HAE-YFP is induced, but the ab-scission zone appears unchanged (Fig. 3, C and F).Forty percent SWC has a water potential of23.15 MPa,which means there is essentially no available water forthe plant (Fig. 3F). One day after rewatering 40% SWCplants, the leaves regain their full turgor but yellowfully, a tear begins to form at the abscission zone, theabscission zone cells become enlarged and rounded,and HAE-YFP accumulation reaches its maximum ob-served 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 incauline leaf abscission zones from plants ex-posed to drought stress. A, Well-watered plantand drought/rewatered plant with caulineleaves 1 to 3 used for microscopy. B to G,Cauline leaf abscission zones from a well-watered plant. H to M, Cauline leaf abscissionzones from a drought/rewatered plant. Images(B–M) were taken at the same magnification;scale bar = 0.5 mm. Images are representativefrom eight plants (n = 8).

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abscission zone scar (Fig. 3E; Supplemental Fig. S4B).We also found both HAE and IDA mRNAs accumu-lated more in the abscission zone of wilted caulineleaves than in well-watered leaves and were furtherincreased once plants were rewatered (Fig. 3G). Ex-pression dynamics of HAE are consistent with HAEacting to prime the abscission machinery of caulineleaves when water becomes limiting and then func-tioning throughout abscission after water is restored.

Mutants Defective in Floral Organ Abscission Are AlsoDefective in Drought-Triggered Cauline Leaf Abscission

Given that bothHAE and IDA are expressed in caulineleaf abscission zones, we hypothesized that they, alongwith other components required for floral organ abscis-sion, may also be necessary for cauline leaf abscissionduring drought. We found that HAE/HSL2, IDA, NEV,and MKK4/5 are all necessary for drought-inducedcauline leaf abscission to occur (Fig. 4, A–C). The bop1bop2 double mutant, which lacks a cauline leaf abscissionzone, also fails to abscise (McKimet al., 2008). hae hsl2, ida,

nev, and mkk4/5 RNAi mutant plants all have a distinctband separating the leaf from the stem, which is the ab-scission zone (Fig. 4A).No cauline leaves abscise from haehsl2, ida, nev, mkk4/5 RNAi, and bop1 bop2 mutant plants(Fig. 4B); on average, 15 to 20 g of force are required topull off cauline leaves from themutants (Fig. 4C).We alsoobserved that cauline leaves from all mutants tested (Fig.4A) senesce following the drought treatment but do notabscise. Unlike in floral organ abscissionwhere hae singlemutants have completely wild-type abscission, hae singlemutants abscise cauline leaves at 20% of the frequency ofthe wild type (Fig. 4D). On the other hand, hsl2 singlemutant plants have leaf abscission similar to that of wild-type plants (Fig. 4E). These results suggest floral organabscission and drought-triggered leaf abscission share acommon mechanism with subtle differences.

HAE Is Expressed in Vestigial Pedicel Abscission ZonesWhen Fruits Become Mature

Unlike the cauline leaf abscission zone, the pedicelabscission zone in Arabidopsis is likely to be truly

Figure 3. HAE is expressed prior to cell sep-aration when leaves become wilted. A to E, Arepresentative soil drying and rewatering timecourse of cauline leaf 2 including well-watered/88% SWC (A), 80% SWC (B), 40%SWC (C), 1 d after rewatering (D), and 2 d afterrewatering (E). Top shows cauline leaf 2,middle is a close-up image of the abscissionzone with reflected white light, and bottom isthe same as the middle where YFP fluores-cence is being imaged. The time course wasrepeated four times (n = 4) with similar results.Red tape is 9.5 mm in length (A–E, top) andscale bar is 0.5 mm (A–E, middle and bottom)where images were taken under the samemagnification. F, Leaf relative water content atdifferent SWC or soil water potentials. Visiblewilting occurs at 50% SWC, and plants wererewatered once SWC reached 40%. Relativewater content of cauline leaves is shown forwilted cauline leaves as in C and after rewa-tering as in D. Data are mean 6 SE; n = 4 bio-logical replicates (one plant each). G, BothHAE and IDA transcripts increase in abscissionzones from wilted plants as in C comparedwith well-watered controls and continue toincrease after rewatering. Data are mean6 SE;n = 6 biological replicates (one plant each) forwell-watered and wilted, n = 3 for rewatered;different letters indicate statistically differentquantities within a gene target, t test P, 0.05.






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 bop2leaves yellow but do not abscise. bop1 bop2 lacks an abscission zone altogether, while hae-3 hsl2-3, ida-2, nev-3, andmkk4/5 RNAi all have a clear band (abscission zone) at the leaf-stem boundary. Images in top panels arewilted leaves (leaf 2) rightbefore rewatering. Images in middle panels are leaves 2 d after rewatering. Red tape is 9.5 mm in length. Images in bottompanels are magnified versions of middle panels showing the abscission zone. Scale bar = 0.5 mm. B, Number of leavesabscised per plant for various genotypes. C, Breakstrength required to remove leaves from the plant for various genotypes.Data are mean6 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





vestigial since the abscission zone only appears to be onthe top half of the base of the pedicel (Fig. 5D; Cho andCosgrove, 2000). HAE-YFP can be detected in the ves-tigial pedicel abscission zone once fruits have fullyelongated (floral stage 17; Fig. 5E). When siliques aremanually pulled off, the tear occurs at the vestigialabscission zone once siliques are fully elongated andHAE-YFP is present (Fig. 5, B and C). However, beforesiliques are fully elongated, the tear will occur some-where in the middle of the pedicel. This indicates thatpartial abscission occurs at the base of the pedicel oncethe silique is mature. While a drought/rewateringtreatment increased HAE-YFP expression in caulineleaf abscission zones, we did not see a similar inductionof HAE-YFP in drought/rewater-treated vestigialpedicel abscission zones (Fig. 5, E and G). Additionally,the floral position in which all following positions tearat the vestigial abscission zone was similar betweenwell-watered and drought/rewatered plants (Fig. 5, Band H), suggesting drought cannot accelerate partialabscission of the pedicel.

Mutations in HAE/HSL2 and IDA Do Not Cause SeedYield Penalty

It is theoretically possible that manipulating abscis-sion in crop plants that lose leaves or flowers duringmild drought stress might result in greater agriculturalyield. For example, if bean plants did not shed theirflowers during mild drought stress, it is possible thatthey might produce more seeds by final harvest(Pandey et al., 1984). In Arabidopsis, hae hsl2 and idamutant plants do not have reduced seed set comparedwith the wild type, while nev, mkk4/5 RNAi, and bop1bop2 mutants have a seed yield penalty (Fig. 6A). Thisindicates that homologs of HAE and IDA should bepreferentially targeted in crop plants if one is to test thehypothesis that less abscission in mild drought mayresult in greater seed set.

Abscisic Acid Is Unlikely to Directly Regulate Abscissionin Arabidopsis

As its name would suggest, abscisic acid (ABA) wasnamed for its ability to promote abscission. However,currently ABA’s ability to promote abscission is viewedcontroversially. Current thinking is thatABAmay not beimportant for abscission in many plants. Initial studieson ABA and abscission may have been flawed in thatsuch high concentrations of ABA were used that thetreatment resulted in production of ethylene that in turncaused abscission (Sexton and Roberts, 1982). Wedesigned experiments using Arabidopsis mutants in

ABA synthesis and signaling to understand if ABAplaysa direct role in abscission inArabidopsis. First, ABAdoesnot appear to be necessary for floral organ abscissionsince aba insensitive1 (abi1) and aba deficient1 (aba1) do notretain their floral organs (Supplemental Fig. S5A). Sim-ilar results were obtained with aba2 mutants (Ogawaet 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 mu-tants because 0% of them can survive the treatment(Supplemental Fig. S5B). Therefore, we modified ourassay so that the level of visible wiltingwas controlled tobe similar between wild-type Landsberg erecta (Ler-0)and ABA mutants rather than exposing both the wildtype and ABA mutants to the same SWC. When abi1-1 and aba1-1 mutants wilted and rehydrated similar tothe wild type, they also abscised statistically similarnumber of leaves as the wild type (Supplemental Fig. S5,C andD). abi1-1 and aba1-1 abscised slightly fewer leavesthan did the wild type, although not statistically differ-ent. However, abi1-1 and aba1-1 are smaller than thewildtype and have fewer cauline leaves. The percentage oftotal cauline leaves abscised by abi1-1 was almost iden-tical to the wild type, while aba1-1 actually abscised ahigher percentage, although not statistically different(Supplemental Fig. S5D). The abscission zone scar leftafter abscission was fairly similar between abi1-1 and thewild type (Supplemental Fig. S5E). We conclude thatABA is not essential for abscission and therefore unlikelyto directly regulate abscission but could be placed farupstream of the abscission process in Arabidopsis.

DISCUSSION

The drought-induced cauline leaf abscission path-way is summarized in the model shown in Figure 6B.BOP1 and BOP2 redundantly specify the formation ofan abscission zone between the stem and cauline leaves(McKim et al., 2008). IDA, HAE/HSL2, MKK4/5, andNEV are all required for cauline leaf abscission that istriggered by drought. We speculate that IDA, HAE/HSL2, and MKK4/5 would be ordered the same as theyare in the genetic pathway that governs floral organabscission; however, we have yet to perform any ex-periments in cauline leaves to confirm this order.

The significance of our findings at the physiologicallevel is that Arabidopsis can shed its cauline leaves afterone bout with drought, potentially placing it in a betterposition to deal with subsequent occurrences ofdrought due to reduced leaf area for transpiration(Kozlowski, 1973; Tschaplinski et al., 1998; Escuderoet al., 2008). The nutrients drawn out of leaves beforeabscission (the leaves become yellow and presumablynitrogen 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 versusthe wild type; ***P , 0.005.











Figure 5. HAE-YFP expression correlates with partial fruit abscission at the vestigial pedicel abscission zone. A, pHAE:HAE-YFPinflorescence with flowers/siliques numbered by floral position. B, Same inflorescence as in A after flowers or siliques weremanually 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-lengthsilique. The top half tears off relatively smoothly, while the bottom half has a rough surface due to the abscission zone only beingpresent at the upper part of the base of the pedicel (Cho and Cosgrove, 2000). E, Close-up view of vestigial abscission zones frominflorescence in Awith floral position and stage indicated. Top panels are extended depth of field bright-field images, and bottompanels showHAE-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 treatedwith drought followed by rewatering. All siliques break at the baseof 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). Experimentswere repeated three times (n = 3) with similar results.





keep younger tissues healthy (Dela Fuente and Leo-pold, 1968; Addicott, 1982; Harvey and van denDriessche, 1999). This study shows at the molecularlevel how leaves are abscised in response to drought.Floral organ abscission has served as an excellentmodelfor understanding abscission at the molecular level.However, as with all model systems, the question re-mains of how far the results can be extrapolated to othersystems. We show that genes first described to be nec-essary for floral organ abscission (HAE, IDA, NEV, andMKK4/5) also have a role in drought-induced organabscission (Jinn et al., 2000; Butenko et al., 2003; Choet al., 2008; Liljegren et al., 2009). The requirement ofrewatering for leaf abscission to occur could be a reasonwhy Arabidopsis leaf abscission has not been charac-terized despite more than 20 years of Arabidopsis ab-scission research. HAE also likely functions in partialabscission of mature fruits at the vestigial pedicel ab-scission zone since its expression is correlated withpartial abscission; however, we have not tested if HAEis genetically necessary for partial abscission of fruits.The Arabidopsis pedicel abscission zone is referred toas vestigial because it only spans half of the pediceldiameter, which does not allow spontaneous abscission(Cho and Cosgrove, 2000). HAE-YFP accumulates inthe upper portion of base of the pedicel once the siliqueis at floral stage 17 (Fig. 5E; Alvarez-Buylla et al., 2010).However, partial abscission of the pedicel appears to becorrelated with fruit development rather than withwater 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 moremolecular components that regulate abscission. Whileregulators of leaf abscission appear to be similar tothose of floral organ abscission, drought-triggered leafabscission seems to be more quantitative than floralorgan abscission. Single mutants ofHAE are identical towild-type plants in terms of floral organ abscission butare clearly defective in drought-triggered leaf abscis-sion. To date, no mutations have been discovered thatmake floral organ abscission occur before pollination,although the double mutant agl15 agl18 and IDAoverexpressors abscise at an earlier floral position(Stenvik et al., 2006; Patharkar and Walker, 2015). Per-haps the more quantitative nature of leaf abscissionwillallow the discovery ofmutations that causemore leavesto fall off when a plant experiences drought. Currently,most drought studies in Arabidopsis focus on plantsurvival or plant wilting as a physiological response todrought (Fujita et al., 2011). Cauline leaf abscissioncould be used as a quantitative and easily measurabledrought response assay.

How HAE and IDA expression is activated whencauline leaves wilt is still unknown. HAE can be tran-scriptionally increased by ABA in seedlings (Winteret al., 2007; Goda et al., 2008). However, since we foundABA is not essential for abscission in Arabidopsis, it ismore likely ABA acts as an additive factor rather thanan initiator. HAE coexpressed genes have overrepre-sented AtMYC2 binding sites in their promoters(Patharkar and Walker, 2015). AtMYC2 is a basic helix-loop-helix transcription factor that can activate tran-scription of ABA-responsive genes (Abe et al., 2003).Another possible way HAE transcription could beactivated in abscission zones of wilted cauline leaves isby activation of the MAPK cascade that leads to HAEexpression through inactivation of the transcriptionalrepressor AGL15 (Patharkar and Walker, 2015;Patharkar et al., 2016).

We do not know why Arabidopsis abscises caulineleaves but not rosette leaves. It is likely due to lack of afunctional abscission zone. The broad spectrum inducerof abscission, overexpression of IDA, does not triggerrosette leaf abscission (Stenvik et al., 2006). About halfof the large rosette leaves do senesce in our assay so thattranspiration from the rosette would be reduced. It maybe more beneficial for a plant to shed cauline leavesthan rosette leaves because attached dead caulineleaves can block some light from lower leaves whiledead rosette leaves would not. We also do not knowwhy the first cauline leaf abscises at lower frequencythan leaf 2 or 3. We speculate that it could simply bebecause the first leaf is close to the soil; therefore, the potshields it from some of the airflow in the growthchamber, thus reducing the water stress on it.

From an agricultural perspective, the conservedmolecular mechanisms uncovered here offer greatleads for agricultural improvement. Abscission can bemanipulated in many ways to increase agriculturalyield. For example, the canned tomato industry rou-tinely grows tomatoes carrying the jointless mutation,

Figure 6. Mutations in HAE/HSL2 or IDA have minimal seed yieldpenalty compared with other abscission-defective mutants. A, Seedyield 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 inArabidopsis. BOP1 and BOP2 are required for cauline leaf abscissionzone development. IDA,HAE/HSL2,MKK4/5, andNEV are all requiredfor drought-induced leaf abscission as well as floral organ abscission.





which results in tomatoes with no pedicel abscissionzone (Mao et al., 2000). When picked, jointless tomatoesleave their calyx and stem behind on the plant, result-ing in less puncture damage to other picked tomatoes(Zahara and Scheuerman, 1988). Additionally, syn-thetic auxins and ethylene blockers are used in appleand Citrus orchards to prevent preharvest drop(Anthony and Coggins, 1999; Yuan and Carbaugh,2007). We found that HAE/HSL2 and IDA could betop candidates for agricultural manipulation sinceknocking them out results in minimal yield penaltycompared with nev, mkk4/5 RNAi, and bop1 bop2 (Fig.5A). Manipulation of HAE and IDA for agriculturecould be achieved by breeding for weak alleles, geneticediting by CRISPR/Cas9, or devising chemicals thatblock IDA peptide function. HAE and IDA homologslikely play the same pivotal role, regulating abscissionin a wide range of species. For example,HAE homologsare also up-regulated in poplar abscission zones whenabscission is activated by shading leaves (Jin et al.,2015), and IDA homologs are induced in soybean andtomato abscission zones prior to abscission (Tucker andYang, 2012). In fact, the active peptide from IDA isconserved in all flowering plants (Stø et al., 2015). Also,Citrus IDA3 can complement abscission deficiency inArabidopsis ida mutant plants (Estornell et al., 2015).Interestingly, Citrus-like Arabidopsis abscises its leavesafter it is rewatered following drought that causeswilting (Agustí et al., 2012).

A lot of recent attention has been given to activationof cell wall-modifying enzymes that hydrolyze themiddle lamella of the abscission zone as the impliedmechanism of cell separation (Niederhuth et al., 2013a,2013b; Liu et al., 2013). Our findings strongly suggestthat while drought triggers abscission, water isrequired for abscission to occur. While hydrolytic en-zymes likely dissolve the middle lamella of the abscis-sion zone, water is probably necessary for abscissionzone cell expansion. In the early 1900s, mechanicalshearing from the enlargement of abscission zone cells,driven by turgor, was thought to be the major mecha-nism leading to abscission (Fitting, 1911; Sexton andRoberts, 1982), and perhaps this should be given morethought again.

CONCLUSION

Plants lose their leaves by an active process, calledabscission, in which leaves are cut free when it is ben-eficial to the plant. Leaves are cut free at a specializedlayer of cells, called the abscission zone, that serves asscissors for the plant. In our work, we have identifiedthe genes that tell the abscission zone to cut leaves freeduring drought. Interestingly, in Arabidopsis many ofthe genes required for drought-triggered abscission arealso necessary for abscission of floral organs after pol-lination, which suggests a common mechanism. Thiswork connects the floral organ abscission field to thedrought field and lays the groundwork for a new fieldin 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 wildtype (Arabidopsis Biological Resource Center stock no. CS70000), except ofexperiment 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; Choet al., 2008). Plants were grown in Promix BX (Premier Tech Horticulture) at23°C, 16 h light/8 h dark, 100 to 150mE$m22$s21, and 50% to 70% humidity. Thestarting SWC of fresh soil from bags after autoclaving was calculated byweighing, drying at 80°C for 2 d, and then reweighing. Plants were grown in-dividually in 5-ounce plastic cups with the equivalent of 9.36 g of completelydry soil and 0.0787 g of Peters Peat Lite Special 20-10-20 fertilizer (Scotts). Wedefined well-watered growing conditions as watering plants to saturation andallowing them to grow until SWC reached 80%, at which point plants wererewatered to saturation. Plants watered to saturation on a daily basis are un-healthy. SWC was determined daily by weighing the pots. Drought (waterdeficit) was imposed once plants had inflorescences 15 to 20 cm tall (5–6 weeksold) 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 hlater SWC was 40%. Plants were planted in a randomized complete block ex-perimental design.

To make ABA mutants wilt similar to Ler-0 controls, ABA mutants wereshielded from air flow in the growth chamber by blocking the vents in thegrowth chamber behind the ABA mutants and shielding on right and left ofplants (shielded from three sides while top and side away from vents was open).Other growth conditions were identical to those described above, except thatvisiblewilting first occurred at 75%SWCand severewilting (similar towild-typeplants at 40% SWC) occurred at 65% SWC; therefore, ABA mutant plants wererewatered once SWC reached 65%. Even with the gentler drought treatment,ABA mutants could not regain full turgor by rewatering alone; therefore, onceABA plants were rewatered, they were covered with a clear dome for 1 d fol-lowed by 3 d in restricted air flow (as above). Because ABA mutants recovermore slowly than the wild type after drought treatment, leaf abscission wasscored 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 havingwater 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 chamberwas not sufficient to blow off either abscised floral organs or abscised caulineleaves. A petal breakstrength meter was used to determine the force required topull cauline leaves free (Lease et al., 2006).

Determining Position of Partial Pedicel Abscission

Flowers or siliqueswere gently pulled by hand as described previously (Choand 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 againstSuc standards (Boyer, 1995). Soil at 40% relative water content was drier thanthe driest Suc standard of22.702MPa andwas extrapolated past that standard.

Leaf Relative Water Content

Leaf relative water content was measured on whole leaves as describedpreviously (Barrs and Weatherley, 1962).

Microscopy

Images were collected with a Zeiss SteREO Discovery v12 epifluorescencemicroscope equipped with a Canon EOS 6D camera. Bright-field images are





extended depth of field images processed with Zerene Stacker using the Pmaxalgorithm. Bright-field images were taken with autoexposure and identicalsettings for white balance. YFP images were acquiredwith a KSC 295-823D YFPCube (excitation 490–510 nm; dichroic beamsplitter 515 long pass; emission520–550 nm). All YFP images within an experiment were taken with identicalsettings for exposure, white balance, and ISO digital film speed. YFP images areof a single depth of field unless noted otherwise.

Constructs and Transgenic Plants

The HAE promoter HAE-YFP construct was made by PCR amplifying the1885 bp upstream of the start codon to the last codon before the stop codon andinserting the resulting PCR product into the NotI site of pE6c (Dubin et al.,2008). Gateway LR reaction was performed to move the HAE promoterHAE-YFP insert to the pBiB-Basta binary vector. The binary vector was usedwith Agrobacterium strain GV3101 to introduce the HAE promoter HAE-YFPinto hae-3 hsl2-3 via the floral dip method. The construct complemented theabscission defect of hae hsl2. Plants used in this study had a single HAEpromoter HAE-YFP insertion in the intergenic space between At4g11280 andAt4g11290 and had close to native expression levels (Supplemental Fig. S6).

Quantitative Reverse Transcription PCR

Cauline leaf abscission zoneswere handdissectedbymaking three straight cutswith a razor blade from cauline leaf 2. A single abscission zone was used perreplicate. RNA isolation, reverse transcription, and quantitative PCR were per-formedasdescribedpreviously (Patharkar andWalker, 2015), exceptHot Start Taq(New England Biolabs) was used in place of Platinum Taq. Primers for HAE andthe reference gene At5g46630 were described previously (Patharkar and Walker,2015). Forward 59-GAGTAGTCCTTGTGTAGCGG-39 and reverse 59-TCTTA-GAAGGAGCAGAAGGAG-39 were used as primers for IDA quantification.

Gene Expression during Osmotic Stress

Publicly availablemicroarray data formannitol treatmentwere downloadedfrom Gene Expression Omnibus (accession GSE5622; Kilian et al., 2007) andreanalyzed with RobiNA using the PLIER algorithm (Lohse et al., 2010).

Seed Yield

Plants were planted in a randomized complete block experimental designwith one plant per pot and given optimal watering. Plants were tied to a stake toprevent tangling of inflorescences. Once the majority of plants in an experimenthad 10%yellow siliques, wateringwas discontinued and plantswere allowed todry. Seeds were purified by passing them through a sieve three times and rolledonce 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 arealways on the primary inflorescence, while the third cauline leaf todevelop can occur on either the primary or the secondary inflorescence.

Supplemental Figure S2. HAE expression is induced by osmotic stress inshoots (Kilian et al., 2007).

Supplemental Figure S3. Dry cauline leaves autofluoresce.

Supplemental Figure S4. Cauline leaf abscission zone cells become en-larged and rounded at the time of abscission.

Supplemental Figure S5. ABA does not regulate abscission directly inArabidopsis.

Supplemental Figure S6. pHAE:HAE-YFP position and expression levels.

ACKNOWLEDGMENTS

We thank Stefan Bennewitz for generating the pHAE:HAE-YFP transgenicline, Melody Kroll and Catherine Espinoza for reading and editing the

manuscript, and Robert Sharp for use of his psychrometer for water potentialmeasurements.

Received June 23, 2016; accepted July 27, 2016; published July 28, 2016.

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