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ACKNOWLEDGMENTS
Funding:S.L., Z. Z., and A.M. acknowledge support from US
Department of Energy (DOE), BES Scientific User Facilities Division
Field Work Proposal 100317; J.D. and A. M. were supported by
the Laboratory Directed Research and Development Program in
support of the Panofsky fellowship. The contributions from T.D.,
P.H.B., A.K., A.N., J.T.O., T.J.A.W., A.L.W., and J.P.C. were
supported by the US DOE, Office of Science, Office of Basic Energy
Sciences (BES), Chemical Sciences, Geosciences, and Biosciences
Division (CSGB); E.G.C. was supported by the DOE Laboratory
Directed Research and Development program at SLAC National
Accelerator Laboratory, under contract DE-AC02-76SF00515.
P.R. and M.F.K. acknowledge support by the German Research
Foundation via KL-1439/10, and the Fellow program of the Max
Planck Society. V.A, J.C.T.B., D.G., and J.P.Mar. gratefully acknowledge
funding support from UK EPSRC grants EP/R019509/1, EP/
T006943/1, and EP/I032517/1. N.B., R.O., and A.C.L. acknowledge the
Chemical Sciences, Geosciences and Biosciences Division, US DOE,
Office of Science, BES, grant DE-SC0012376. C.B. acknowledges the
Swiss National Science Foundation and the National Center of
Competence in Research–Molecular Ultrafast Science and Technology
NCCR–MUST. L.F.D. and L.F. acknowledge support from NSF grant
1605042 and DOE DE-FG02-04ER15614. W.H. thanks the German
BMBF for funding of the project“SpeAR_XFEL”under contract
05K19PE1. Use of the Linac Coherent Light Source (LCLS), SLAC
National Accelerator Laboratory, is supported by the US DOE, Office
of Science, BES, under Contract DE-AC02-76SF00515.Author
contributions:S.L., A.M. and J.P.C. devised the experimental scheme.
S.L., A.M., and J.P.C. developed the experimental apparatus. S.L.,
J.D., J.P.Mac., Z.Z., and A.M. prepared the attosecond x-ray pulses.
M.-F.L., N.H.S., and P.W. prepared the experimental beam-line. All
Authors participated in the collection and interpretation of the
experimental data. T.D. led the data analysis. S.L., T.D., P.R., and
E.G.C. worked on the single-shot“streaking”diagnostic. S.L.,
T.D., A.M., and J.P.C. prepared an initial version of the manuscript.
All authors provided critical feedback in preparing the submitted
manuscript.Competing interests:None declared.Data and
materials availability:The partially analyzed raw data and the
raw data from the calculations is available on the Zenodo repository
( 38 ). All (other) data needed to evaluate the conclusions in the paper
are present in the paper or the supplementary materials.
SUPPLEMENTARY MATERIALS
science.org/doi/10.1126/science.abj2096
Materials and Methods
Supplementary Text
Figs. S1 to S8
References ( 39 Ð 42 )
28 April 2021; accepted 29 November 2021
Published online 6 January 2022
10.1126/science.abj2096
PLANT SCIENCERALF peptide signaling controls the polytubey block
inArabidopsis
Sheng Zhong^1 †, Ling Li^1 †, Zhijuan Wang^1 †, Zengxiang Ge^1 †‡, Qiyun Li^1 †, Andrea Bleckmann^2 ,
Jizong Wang^1 , Zihan Song^1 , Yihao Shi^1 , Tianxu Liu^1 , Luhan Li^1 , Huabin Zhou^3 , Yanyan Wang^3 , Li Zhang^1 ,
Hen-Ming Wu^4 , Luhua Lai^3 , Hongya Gu1,5, Juan Dong^6 , Alice Y. Cheung^4 ,
Thomas Dresselhaus^2 , Li-Jia Qu1,5*Fertilization of an egg by multiple sperm (polyspermy) leads to lethal genome imbalance and
chromosome segregation defects. InArabidopsis thaliana, the block to polyspermy is facilitated
by a mechanism that prevents polytubey (the arrival of multiple pollen tubes to one ovule).
We show here that FERONIA, ANJEA, and HERCULES RECEPTOR KINASE 1 receptor-like kinases
located at the septum interact with pollen tubeÐspecific RALF6, 7, 16, 36, and 37 peptide ligands
to establish this polytubey block. The same combination of RALF (rapid alkalinization factor)
peptides and receptor complexes controls pollen tube reception and rupture inside the targeted ovule.
Pollen tube rupture releases the polytubey block at the septum, which allows the emergence
of secondary pollen tubes upon fertilization failure. Thus, orchestrated steps in the fertilization
process inArabidopsisare coordinated by the same signaling components to guarantee and
optimize reproductive success.S
eed plants rely on tightly regulated fer-
tilization mechanisms to secure fertility
and reproductive success. Like in animals,
the entrance of supernumerary sperm
into a single egg—i.e., polyspermy—is
restricted to ensure chromosomal balance and
progeny health ( 1 , 2 ). Fertilization in angio-
sperms is more complex because two sperm
cells are carried by one pollen tube that grows
in the maternal pistil tissues and ultimately
releases its sperm cell cargo inside the ovule
( 3 ). Although hundreds of pollen tubes may
grow into the transmitting tract of a pistil,
usually only a single tube, in response to at-
tractants, emerges from the septum in the
vicinity of each ovule to target the ovule (Fig. 1A)
( 4 ). The block to polytubey (i.e., the emer-
gence of multiple pollen tubes targeting an
ovule) prevents the occurrence of polyspermy.
This polytubey block is likely further reinforced
by successful gamete fusion that triggers prog-
rammed cell death (PCD) of the persistent syn-
ergid cell, which leads to the elimination ofpollen tube attractants. Thus, the first pollen
tube that emerges from the septum will have
the privilege of fertilizing the female gametes,
providing a precondition for conspecific pol-
len precedence (i.e., the preferential use of
pollen from the same species for fertilization)
( 5 ). If the first pollen tube fails, however, fer-
tilization success will be ensured by fertili-
zation recovery ( 6 , 7 ), in which the polytubey
block is suspended to allow the emergence of
secondary pollen tubes for another chance of
fertilization. Therefore, plants can (i) restrict
polyspermy by enforcing the polytubey block
at the septum under normal circumstances
and (ii) salvage fertility by removing the poly-
tubey block when fertilization fails. Here, we
report the molecular mechanisms by which
the polytubey block is implemented or sus-
pended, when needed.FERONIA, ANJEA, and HERCULES RECEPTOR
KINASE 1 receptor kinases are required to
establish the polytubey block
To identify factors that may establish the poly-
tubey block, we conducted RNA sequencing
(RNA-seq) analysis using transmitting tract
and septum tissues and searched for candi-
date receptors that may perceive signals from
the pollen tube. We found that seven malectin-
like domain-containing receptor-like kinase
(MLD-RLK) (also known asCatharanthus
roseusRLK1-LIKE or CrRLK1L) genes were
highly expressed [verified by real-time quanti-
tative polymerase chain reaction (qPCR); fig.
S1, A and B]. Transcriptional and translational
markers showed that three of these genes—
FERONIA (FER), ANJEA (ANJ), and HERCULES
RECEPTOR KINASE 1 (HERK1)—were expressed
in the ovule, transmitting tract, and septum
epidermis (Fig. 1B and fig. S2). We further290 21 JANUARY 2022•VOL 375 ISSUE 6578 science.orgSCIENCE
(^1) State Key Laboratory for Protein and Plant Gene
Research, Peking-Tsinghua Center for Life Sciences at
College of Life Sciences, Peking University, Beijing 100871,
People’s Republic of China.^2 Cell Biology and Plant
Biochemistry, University of Regensburg, 93053
Regensburg, Germany.^3 College of Chemistry and
Molecular Engineering, Peking University, Beijing 100871,
People’s Republic of China.^4 Department of Biochemistry
and Molecular Biology, Molecular and Cell Biology
Program, Plant Biology Program, University of
Massachusetts, Amherst, MA 01003, USA.^5 The National
Plant Gene Research Center (Beijing), Beijing 100101,
People’s Republic of China.^6 The Waksman Institute of
Microbiology, Rutgers the State University of New Jersey,
Piscataway, NJ 08854, USA.
*Corresponding author. Email: [email protected]
†These authors contributed equally to this work.
‡Present address: Institute of Science and Technology Austria,
Klosterneuburg 3400, Austria.
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