Physiologia Plantarum 153: 643–653. 2015

© 2014 Scandinavian Plant Physiology Society, ISSN 0031-9317

Phytosulfokine peptide signaling controls pollen tube growth and funicular pollen tube guidance in Arabidopsis thaliana Nils Stührwohldta,† , Renate I. Dahlkea,† , Anke Kutschmara , Xiongbo Pengb , Meng-Xiang Sunb and Margret Sautera,* a b

Plant Developmental Biology and Plant Physiology, University of Kiel, 24118 Kiel, Germany Key Laboratory of MOE for Plant Developmental Biology, College of Life Science, Wuhan University, Wuhan, P. R. China

Correspondence *Corresponding author, e-mail: [email protected] Received 2 June 2014; revised 25 June 2014 doi:10.1111/ppl.12270

Phytosulfokine (PSK) is a peptide growth factor that requires tyrosine sulfation carried out by tyrosylprotein sulfotransferase (TPST) for its activity. PSK is processed from precursor proteins encoded by five genes in Arabidopsis thaliana and perceived by receptor kinases encoded by two genes in Arabidopsis. pskr1-3 pskr2-1 and tpst-1 knockout mutants displayed reduced seed production, indicative of a requirement for PSK peptide signaling in sexual plant reproduction. Expression analysis revealed PSK precursor and PSK receptor gene activity in reproductive organs with strong expression of PSK2 in pollen. In support of a role for PSK signaling in pollen, in vitro pollen tube (PT) growth was enhanced by exogenously added PSK while PTs of pskr1-3 pskr2-1 and of tpst-1 were shorter. In planta, growth of wild-type pollen in pskr1-3 pskr2-1 and tpst-1 flowers appeared slower than growth in wild-type flowers. But PTs did eventually reach the base of the style, suggesting that PT elongation rate may not be responsible for the reduced fertility. Detailed analysis of anthers, style and ovules did not reveal obvious developmental defects. By contrast, a high percentage of unfertilized ovules in pskr1-3 pskr2-1 and in tpst-1 siliques displayed loss of funicular PT guidance, suggesting that PSK signaling is required to guide the PT from the transmitting tract to the embryo sac. Cross-pollination experiments with wild-type, pskr1-3 pskr2-1 and tpst-1 male and female parents revealed that both the PT and the female sporophytic tissue and/or female gametophyte contribute to successful PT guidance via PSK signaling and to fertilization success.

Introduction Plant peptides have functions in defense signaling, growth regulation and control of root and shoot apical meristems. One structurally characteristic group of small peptides comprises small posttranslationally modified peptides such as phytosulfokine (PSK; Matsubayashi and †

Sakagami 1996), PSY1 (Amano et al. 2008), C-terminally encoded peptide 1 (Ohyama et al. 2008), CLAVATA3 (CLV3; Fletcher et al. 1999, Ohyama et al. 2009), GOLVEN (GLV/ROOT GROWTH FACTORS/CLE-like; Matsuzaki et al. 2010, Whitford et al. 2012, Fernandez et al. 2013) and tracheary element differentiation inhibitory factor (Ito et al. 2006). The peptides are

These authors contributed equally to this work.

Abbreviations – GUS, 𝛽-glucuronidase; PSK, phytosulfokine; PT, pollen tube; RT-PCR, reverse transcription-polymerase chain reaction; SKM1, STERILITY-REGULATING KINASE MEMBER1; TPST, tyrosylprotein sulfotransferase.

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translated as pre-proproteins into the endoplasmic reticulum and are subsequently processed and modified in the secretory pathway. PSK is a disulfated pentapeptide that is processed from a 90–100 amino acid precursor (Yang et al. 1999). Sulfotransfer is catalyzed by a tyrosylprotein sulfotransferase (TPST) in the trans-Golgi (Komori et al. 2009). tpst-1 knockout plants are severely dwarfed with stunted roots as a result of a disorganized root apical meristem and reduced cell growth (Komori et al. 2009, Stührwohldt et al. 2011). In Arabidopsis, five genes encode for PSK precursors. PSK precursor genes are present in monocot and dicot genomes and appear to be ubiquitous in higher plants (Yang et al. 2000, Lorbiecke and Sauter 2002). The PSK4 precursor protein was shown to be proteolytically cleaved by a subtilisin-like serine protease in the apoplast (Srivastava et al. 2009). PSK is perceived by plasma membrane-bound leucine-rich repeat receptor proteins (Matsubayashi and Sakagami 1999, 2000). In Arabidopsis, PSK receptors are encoded by two genes, PSKR1 and PSKR2. They possess a region of extracellular leucine-rich repeats, separated by an island domain, a single transmembrane region and an intracellular Ser/Thr kinase domain (Matsubayashi et al. 2002). PSK was first identified as a bioactive compound responsible for the feeder effect (Matsubayashi and Sakagami 1996). Plant cells kept in liquid culture require a minimum density to undergo cell division. This can be substituted for by medium from cultured cells. PSK was identified as the chemical compound that exerts this feeder effect and was hence classified as a growth factor. In Arabidopsis, root growth is promoted by PSK signaling through its receptors, and growth is supported by nanomolar concentrations of PSK (Matsubayashi et al. 2006, Amano et al. 2008, Kutschmar et al. 2009). PSK promotes root growth by stimulating cell elongation rather than cell division (Kutschmar et al. 2009). PSK signaling in the epidermis is sufficient to drive growth in a non-cell autonomous manner (Hartmann et al. 2013). PSKR1 appeared to be expressed ubiquitously at low levels and PSK genes were shown to be expressed in vegetative tissues and in flowers (Matsubayashi et al. 2006), possibly indicating a role for PSK signaling in reproduction. Similar to the feeder effect observed in cultured plant cells, the pollen population effect indicates that pollen kept in vitro at low density will not germinate but can be induced to do so by addition of medium from a high-density pollen culture. In tobacco (Nicotiana tabacum L.), addition of PSK to pollen cultured at low density promoted germination in a dose-dependent manner (Chen et al. 2000). In maize (Zea mays L.), two PSK precursor genes were shown to be specifically expressed in male and female 644

gametophytes including male and female gametes (Lorbiecke et al. 2005). These observations indicated that PSK may have a function in plant reproduction. For successful fertilization, the pollen tube (PT) has to negotiate its way across a long distance through the pistil, and along the funiculus before it enters the micropyle of the embryo sac. PT growth from the stigma through the transmitting tract to the embryo sac can be separated into five phases (Kandasamy et al. 1994, Johnson and Preuss 2002, Dresselhaus and Franklin-Tong 2013). In phase I, the pollen attaches to the stigma and germinates. In phase II, the PTs grow along the papillar cells in direction of the transmitting tract. In phase III, PTs enter the transmitting tract and grow intercellularly toward the ovules. PTs can cover distances of up to 2000 μm in about 10 h (Kandasamy et al. 1994). At phase IV, PTs leave the transmitting tract and grow along the funiculus toward the micropyle. Finally, at phase V, the PT bursts and the sperm cells enter the embryo sac. Fertilization occurs and embryo and seed development start. To elucidate the role of PSK in reproduction, we performed a detailed analysis of PSK precursor genes and PSK receptor gene expression in Arabidopsis flowers. We further analyzed PT growth, PT orientation and fertilization success making use of the receptor null mutant pskr1-3 pskr2-1 and of the tpst-1 mutant. Analysis of self- and cross-pollinated wild-type, pskr1-3 pskr2-1 and tpst-1 siliques indicated a function of PSK in short-distance signaling of funicular PT guidance.

Materials and methods Plant material and growth conditions Experiments were performed with Arabidopsis thaliana ecotype Columbia (Col-0). Plants were grown in a 2:3 sand-humus mixture that was frozen at −80∘ C for 2 days to avoid insect contamination and watered regularly with tap water. Prior to germination, seeds were stratified at 4∘ C in the dark for 2 days and then transferred to a growth chamber under long day (16 h light/8 h dark; 70 μmol photons m−2 s−1 ) conditions. The PSK receptor double knockout mutant pskr1-3 pskr2-1 and the tpst-1 mutant which has an insertion within the coding sequence of the TPST responsible for tyrosine sulfation were previously described (Kutschmar et al. 2009, Stührwohldt et al. 2011). All experiments were carried out with homozygous lines. Reverse transcription-polymerase chain reaction For reverse transcription-polymerase chain reaction (RT-PCR) analysis, total RNA was isolated from shoots

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and roots of 8-day-old seedlings and 14-day-old plants, from juvenile leaves, the first and second inflorescence (

Phytosulfokine peptide signaling controls pollen tube growth and funicular pollen tube guidance in Arabidopsis thaliana.

Phytosulfokine (PSK) is a peptide growth factor that requires tyrosine sulfation carried out by tyrosylprotein sulfotransferase (TPST) for its activit...
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