Degradome and secretome of pollination drops of Ephedra. (2024)

Introduction

Gymnosperm pollination drops are involved at some point in thecapture and delivery of pollen into ovules, followed by pollengermination and fertilization (Gelbart & von Aderkas, 2002). Therole of the pollination drop varies according to the pollinationmechanism in which it occurs (Tomlinson et al., 1997). In Ephedra,pollination drops (Fig. 1) perform both the pollen capture and deliveryfunction (Endress, 1996). Pollen can be delivered by wind or by insects,but in the latter case, pollination drops also function as anectar/reward for the pollinator (Moussel, 1980; Meeuse et al., 1990).Ephedra species are not obligately insect-pollinated, as windpollination may also occur at the same time (Karl Niklas, this volume).In this respect, Ephedra is similar to other gnetophytes (Welwitschiaand Gnetum) (Endress, 1996).

Ephedra pollination drops contain abundant sucrose, but are alsoabundant in phosphate compounds, amino acids, and polypeptides (Ziegler,1959). Until this study, no proteins have yet been documented, althoughZiegler (1959) found acid phosphatase activity in the nucellus, thesporogenous tissue that produces the pollination drop. He wrote thatsuch nucellar proteins likely are responsible for processing cellularcompounds that are secreted into the drop. We hypothesize that Ephedrapollination drops contain proteins, given that rich and diversepollination drop proteomes have been recently described from a widerange of gymnosperms (Wagner et al., 2007). To this end, we embarked onthe first proteomic study of Ephedra pollination drops. The aim was totest for the presence of proteins, and if present, to understand thevariation in protein composition in the pollination drops of Ephedra.

Ziegler (1959) also first reported the presence of mineral andorganic compounds released into pollination drops by the nucellus. Thedevelopmental stage of the nucellus at the time of pollination droprelease can vary widely among different gymnosperm taxa. In Ephedra, thenucellus is post-meiotic (Rydin et al., 2010), whereas nucellus of Taxusis premeiotic (Dupler, 1920). The nucellus of Ephedra differs from manyother gymnosperms in that a central apical portion degenerates to form apollen chamber (Rydin et al., 2010). Pollen chambers are known from theearliest fossils of Gnetales (Rothwell & Stockey, 2013). Nucellardegradation to form a pollen chamber also occurs in Ginkgo (Douglas etal., 2007) and cycads (Norstog & Nicholls, 1997). By comparison,Taxus and most other conifers have whole, undegraded nucellus throughoutpollination and into early embryo development (Singh, 1978). Thus we notonly hypothesize the presence of proteins in Ephedra pollinations drops,but we also expect that such degenerative processes in Ephedra at thetime of pollination drop formation would influence the type of proteinspresent, such as protein breakdown products that accompany tissue death.

Literature Review

Ephedra reproductive biology, of which the pollination drop is justa part, deserves detailed investigation. Gnetales (Ephedra, Gentum,Welwitschia) is a distinct lineage among the six major groups of extantgymnosperms which has occupied various and contested positions inhypothesized seed plant phytogenies (Graham & lies, 2009; Mathews,2009; Rydin and Korall, 2009). Regardless of the various possiblesister-group relationships that Gnetales may have, detailedunderstanding of this group is important for any interpretations ofevolutionary history among seed plants. Among other gymnosperms, such asthe Pinaceae and Cupressaceae, some taxa have pollination drop proteinsin common (Wagner et al., 2007). In this review, a brief history of thestudy of Ephedra pollination drops will be followed by a summary of whatis known of its pollination drop physiology and biochemistry. Tohighlight some unique and poorly understood aspects of the pollinationmechanism of Ephedra, we will compare it with other, better-studiedgymnosperm species. We will also provide a rationale for usingproteomics in the study of pollination drops. In spite of the fluidphylogeny of extant spermatophytes, it is clear that the pollinationmechanism of Ephedra is of ancient origin. Ever since Doyle'sseminal paper in 1945 in which information on extant conifer species wascombined with transformational series of key ancestral fossils,pollination drops have been considered a basic component of even theearliest pollination mechanisms of gymnosperms. Some groups have widevariation in pollination drop capture, i.e. Podocarpaceae, includingcapture of pollen by mechanisms that do not involve pollination drops,e.g. Saxegothea (Doyle, 1945). Mechanisms that lack pollination dropsare common in only two groups, Araucariaceae and some Pinaceae.Pollination drops are a prevalent feature of gymnosperm pollination andhave been documented in one fossil (Rothwell, 1977) and are suspected tobe present in most fossil groups (Doyle, 1945, 2008). Tomlinson (2012)incorporated morphological and physiological aspects of ovule behaviourin his analysis of the evolution of pollination mechanisms. In hisscheme, pollination drops are ancestral in conifers. Little andcoauthors (2014) used phytochrome gene duplication rooting of seedplants (Mathews, 2009) in combination with sister-group relations ofmajor plant extant seed plant lineages as a backbone for constraining amorphological matrix that includes extinct seed plants (Doyle, 2008).Little et al. (2014) concluded that the pollination mechanism of Ephedratraces its origins to an ancient conserved suite of traits among seedplants.

Pollination drops of Ephedra have attracted attention for over 140years. Observations of their role in pollen capture were included alongwith those of 14 other gymnosperm genera in the first detailed study ofpollination drop biology (Strasburger, 1871). Since then, Ephedra'spollination drop has been the subject of periodic investigation.Questions regarding insect-pollination (Bino et al., 1984; Porsch, 1910)and wind-pollination (Buchmann et al., 1989; Niklas & Buchmann 1987;Niklas & Kerchner, 1986; Niklas et al., 1986) have received the mostattention. More recently, a comparison of ovule morphology and anatomyamong Ephedra species (Rydin et al., 2010) has provided detailedinformation on ovule organization, including variation in pollinationdrop secretory tissue, i.e. nucellus. Ziegler (1959) compared Ephedrawith Taxits in a physiological study on some components of pollinationdrops. To put the published effort on Ephedra in perspective, Taxus, theother taxon used in Ziegler's study is, historically, thebest-studied of all gymnosperm genera. Taxus drops were not only amongthe very first to be described (Vaucher, 1841), but Strasburger (1871)provided detailed, reliable observations on their secretion andretraction. More importantly, they have an abundance of ovules witheasily accessible pollination drops that, compared with most othergymnosperm taxa, have relatively large volumes (~250 nL). Ephedraproduces an even larger drop (~1 [micro]l). Thus, given enough ovulateplants, collection is relatively easy. Early chemical analysis ofpollination drops of various conifers revealed components such ascalcium and various carbohydrates (Fujii, 1903; Schumann, 1903), whichwere later found in Ephedra also (Ziegler, 1959). Proteins of coniferpollination drops were identified by immunohistochemistry(arabinogalactans; O'Leary et al., 2004) and mass spectrometry(thaumatin-like proteins; O'Leary et al., 2007), but to datesimilar investigations have not been carried out on Ephedra.

Ephedra has a pollination mechanism that is among the most commonin gymnosperms. Pollination mechanisms can be divided into those thathave pollination drops, and a small number of species that do not(Little et al., 2014). Those with drops are classified into one of sixpollen capture mechanisms, based on how pollination drops are involvedin either pollen capture or post-capture (Little et al., 2014). Ephedrais characterized by pollination drops that both capture and delivernon-saccate pollen into the ovule (Little et al., 2014; Tomlinson,2012). A mechanism that lacks a drop is known as an "extra-ovularcapture and germination" type. In this mechanism, pollen lands nearor on the ovule where it germinates; at no point is a pollination dropinvolved. The pollen tube enters the opening of the ovule, themicropyle, and reaches the interior of the ovule to undergo spennrelease and fertilization (Endress, 1996). Gymnosperms with extra-ovularcapture and germination are known only from a small number of conifers,such as Araucariaceae, Saxegothea (Podocarpaceae), and some Pinaceae,e.g. Abies and a few species of Tsuga (Doyle, 1945). There are sixpollen capture mechanisms that have a drop, and perhaps threeextra-ovular capture and germination mechanisms that do not have drops.In the evolution of gymnosperm pollination mechanisms, extra-ovular andgermination mechanisms are derived from pollination mechanisms that havedrops (Little et al., 2014; Tomlinson, 2012). Although Ephedra'spollination mechanism is familiar, we know less about a number of itsfeatures, in particular, pollination drop composition, componentstability, and how pollen interacts with pollination drops.

Pollination drops are produced by the nucellus (Fujii, 1903).However, the components need not arise locally, e.g. carbohydrates foundin the drop may be the result of long distance transport as well aslocal production. In contrast to what is known about sucrose productionin flowering plant nectar (Heil, 2011), we do not know how muchpollination drop sucrose originates from extracellular or apoplastictransport versus intracellular or symplastic processes. Proteins activewithin the Ephedra nucellus have an influence on pollination dropcomposition (Ziegler, 1959). The first protein to be mentioned in thepollination drop literature was acid phosphatase, but this protein wasnot found in pollination drops; it was located by immunohistology in thenucellus of Ephedra helvetica (= E. distachya) (Ziegler, 1959). Cellularacid phosphatase was thus considered to be involved in processingcompounds destined for secretion into the pollination drop. However,acid phosphatase may not be restricted to the nucellus as it was foundby immunohistochemistry in the pollination drop of the relatedgnetophyte, Welwitschia mirabilis (Carafa et al., 1992). Later, theenzyme chitinase was identified by mass spectrometry in the drops of W.mirabilis (Wagner et al., 2007). In contrast to the only two proteinsknown from gnetophytes, there are numerous proteins known in Pinaceaeand Cupressaceae (Nepi et al., 2009).

Ephedra pollination drop secretion is not currently understood froma mechanistic standpoint. Our lack of understanding of the process ofsecretion and retraction of the pollination drop across gymnosperms ingeneral has fueled contradictory interpretations of the evolution ofpollination mechanisms (for discussion see Mugnaini et al., 2007). Somepollination drop secretion models have been proposed that are passive.Other models have been proposed that depend on the degree of activesecretion that is occurring from the nucellus (Tomlinson et al., 1997).

The passive mechanisms include both pollination drops andsubstitutes for pollination drops. At one extreme is Ziegler's(1959) suggestion that pollination drop secretion and retraction is apassive, purely physico-chemical phenomenon that lacks active cellularsecretion. He based this idea on the fact that his application ofmetabolic poisons to kill nucelli of Tax us and Ephedra did not haltpollination drop secretion. Thus, he surmised that extracellularsubstances, such as sucrose, were sufficient to draw water from nucellartissue by osmosis to form drops. Under this scheme, withdrawal wouldalso be a passive process, one driven by evaporation. However, at theother extreme, some studies suggest that pollination drops are notessential for pollen capture and delivery, but can be replaced by simplerainwater capture mechanisms that wholly or partially substitute forbiologically produced pollination drops. Various mechanisms involvingrainwater substitution of some kind have been proposed for Abies(Chandler & Owens, 2004), Cedrus (Takaso & Owens, 1995), Picea(Runions & Owens, 1996), and Pinus (Brown & Bridgewater, 1986;Greenwood, 1986), although in the latter case rainwater capture has beendismissed in a recent study by Leslie (2010). A rainwater-based capturemechanism has never been suggested for Ephedra. Drops that are exposedto the air, such as those of Ephedra, which are without surrounding orenclosing structures, are destroyed by rain. In addition, it is knownthat rain disturbs pollen uptake in species such as Taxus (Tison, 1911).Such overly exposed ovules cannot receive pollen until later, after anew drop is secreted.

In contrast to these passive models of pollen uptake, more activeroles for the ovule have been proposed. The ovule appears, at least insome cases, to be active and possibly interacting with pollen. In a widevariety of species, observations have been published in which dropsecretion and retraction occurred quickly (Jin et al., 2012; Mugnaini etal. 2007; Tomlinson et al, 1997), with retraction speed too high to beaccounted for by evaporation alone. Furthermore, in some members of thePodocarpaceae, secretion and retraction occurs repeatedly. Liquidspreads across the ovule's neighbouring surfaces, collectingbuoyant saccate pollen (Tomlinson et al., 1997). Retraction and dropemergence repeats several times to continue pollen scavenging. Mugnainiet al. (2005) proposed a two-step drop secretion mechanism for somecupressaceous species that was based on both active and passivecomponents. For example, some genera of Podocarpaceae(Podocarpus--Tomlinson et al., 1997) and some Cupressaceae,(Chamaecyparis--Owens et al. 1980) have the ability to repeatedlysecrete pollination drops, whereas ovules of other Podocarpaceae(Phyllocladus--Tomlinson et al., 1997) are able to produce a drop onlyonce, which recedes after pollen capture, never to be replaced. Thereare suggestions based on fossil evidence of pollinator-ovuleinteractions that imply gain and loss of insect pollination because ofevolutionary turnover of pollinators, possible compositional shifts ofsucrose concentrations, and changes in ovule morphological features(Labandeira et al., 2007). Among extant gymnosperms, a molecular or cellbiological mechanism needs to be developed that can account for theactive processes involved in drop secretion and retraction. The currentbottleneck to such work is the paucity of studies of molecularcomponents of pollination drops, including the lack of publishedgenomes, nucellus transcriptomes and comparative physiological studies.In Ephedra, the pollination mechanism is relatively simple: pollen iscaptured by a secreted pollination drop that subsequently recedes. If adrop is removed, the nucellus is capable of producing another one(Moussel, 1980). What is different, though not unique, about the drop inEphedra compared to that of most conifers studied is that dropproduction co-occurs with nucellus tissue breakdown. This celldegradation forms the pollen chamber where captured pollen sinks priorto germination (Moussel, 1980).

There are several reasons why the process of secretion is notclearly understood, particularly in gnetophytes. Although a functioningenzyme, i.e. acid phosphatase, was detected in the pollination drop ofWelwitschia, it is not known whether it was secreted into the drop bynucellar tissue, or it arrived in the drop after degenerative formationof the pollen chamber. Pollen chambers are found in Ephedra (Rydin etal, 2010) and some other gymnosperms, such as cycads (Norstog &Nicholls, 1997), Gingko (Douglas et al., 2007), Pinus and Picea (Singh,1978). In comparison, many gymnosperms do not have pollen chambers.Taxus has an intact nucellus, i.e. a solid dome of parenchymatous tissuethat shows no sign of degeneration before or during pollination dropformation (O'Leary et al., 2004). Since Taxus pollination dropshave proteins secreted from intact cells, it follows that ovules withcell degradation-derived pollen chambers, such as those of Ephedra,Ginkgo and Pinus, may have drops that contain proteins of two origins:1. secreted from intact cells 2. released by cell lysis.

Protein degradomics is a systems approach to mass spectrometry thatinvestigates proteases and their substrates, as well as proteolyticevents (Lopez-Ortin & Overall 2002). The portion of proteins thatoriginate from the degraded tissues are appropriately called thedegradome. However, a degradome can arise from a number of processesoccurring concurrently or independently. One source of degradome alreadyconsidered above is cellular debris due to senescence during pollenchamber formation (Roberts et al., 2012). A second source may be fromthe activity of extracellular proteases and peptidases, if present inpollination drops, that would generate breakdown products fromextracellular proteins. If this occurs then both these peptidases andproteases would be detected along with polypeptide fragments of otherproteins. Degradomes may form biochemically complex networks, but theseremain relatively unstudied in plants (Huesgen & Overall, 2012).Some of the breakdown products may function in providing signals thatregulate defense responses of living cells. Proteomics providesidentification with high confidence, but proof of functionality ofconstituents of the degradome within the pollination drop requiresfurther study of substrate processing. Furthermore, it must be shownthat these compounds are functional in situ.

Secretome proteins characteristically have signal cleavage peptidesthat permit their active export across the plasmalemma. In gymnosperms,such cleavage signal peptides have been described for thaumatin-likeproteins found in pollination drops (O'Leary et al., 2007). Sincecleavage peptides are removed during export of the proteins from cells,confirmation requires querying peptide sequences against gene databases,and then isolating the gene from the plant material to verify thepresence of a cleavage peptide coding sequence. The identification ofenzymes has altered our view of how pollination drops function andprovided new insights into the biochemical role played by pollinationdrops during reproduction (Prior et al., 2013). A wide variety ofproteins have been identified in P. menziesii (Poulis et al., 2005),Larix x marschlinsii (O'Leary et al., 2007), Juniperus communis, J.oxycedrus, Welwitschia mirabilis, and Chamaecyparis lawsoniana (Wagneret al., 2007). Protein identifications from these taxa suggest roles inantimicrobial defense, carbohydrate modification, alteration ormaintenance of osmotic levels, and pollen selection (Nepi et al. 2009).Some of these roles have been confirmed with enzyme assays. Douglas-firpollination drop proteins identified as invertases have, after closerbiochemical study, been proven to cleave sucrose in situ. Invertases inthis system act as regulators of the pollination drop'scarbohydrate composition. In turn, this change in solute concentrationof the major pool of molecules in the drop has a direct influence on theselection of conspecific over heterospecific pollen in Douglas-fir andlarch (von Aderkas et al., 2012). In comparison with conspecific pollenthat prefer these osmotic conditions and readily germinate,heterospecific pollen germinate much less frequently, and show poorgermination rates. Another example of enzyme assay confirmation ofidentified proteins is that of chitinases. These were proven to processchitin substrates in situ (Coulter et al., 2012), suggesting that theseproteins have a defensive role during reproduction, defending the ovuleand pollen against airborne pathogenic fungi.

The chemical composition of the pollination drop of Ephedra speciesmust be considered in a biological and ecological context. Certaincomponents may qualitatively enhance the ecological services alreadyprovided by the plant, e.g. the quality of the nectar reward for insects(Fig. 2). In Ephedra, the high amounts of sucrose attract insects, as wehave ourselves seen on many occasions, confirming published studies(Bino et al. 1984; Meeuse et al., 1990; Moussel, 1980). Ephedra also isrelatively rich in amino acids, especially glutamine and glutamic acid(Ziegler, 1959). In angiosperm nectar, free amino acids are the nextmost abundant group of compounds after carbohydrates. Free amino acidsinfluence sensory preferences in insects (Linander et al., 2012). Avariety of insects have been recorded from Ephedra spp., includingdipterans, as well as hymenopterans such as vespids, braconids andchalcids, but not bees (reviewed in Bino et al., 1984). It is likelythat pollination drop composition, like plant nectar composition, may behighly influenced by plant phylogeny versus pollinator preferences(Nicolson, 2011). Drops in Ephedra having evolved in an aridenvironment, it is also possible that high solute concentrations, i.e.sucrose, are necessary to prevent drops from evaporating too quickly,which may have been a possible pre-adaptation to insect pollination. Ifadvances are to be expected in the study of chemecological aspects ofinsect pollination in Ephedra, more thorough chemical analysis as wellas insect behavioural studies will be required.

Many components of pollination drops influence pollen growth anddevelopment. Sucrose has a universal role in Ephedra of also providing anutrient source for pollen germination and pollen tube growth,regardless of whether the species is insect- or wind-pollinated. Ephedrapollen germinates rapidly and the pollen tube grows quickly, reachingthe egg in 14 h, which is much faster than other gymnosperms (El-Ghazalyet al., 1997; Williams, 2012). The pollen can even germinate while inthe pollination drop outside the micropyle (Bino et al., 1984). It wouldappear that the tubes do not have to be in close proximity of thenucellus to be able to grow long distances. The pollination drop withits carbohydrate and other substances is able to support long distancegrowth of these tubes (Bino et al., 1984). Sucrose is also the majorcontributor to the osmotic potential of the drop. In vitro assays ofother gymnosperms have also shown that carbohydrate concentrations canplay a critical role in germination success (Dumont-BeBoux et al.,1999). Ziegler (1959) showed that calcium is present in Ephedra. Becausecalcium is critically important in pollen gennination for most seedplants, it is a major component of pollen gennination media (Brewbaker& Kwack, 1963). Ephedra also contains a variety of amino acids(Ziegler, 1959), which may contribute to pollen germination as suggestedin studies of Junipenis pollen growth in vitro on media supplementedwith the major amino acids found in pollination drops (Duhoux & PhamThi, 1980; Seridi-Benkaddour & Chesnoy, 1988). The other compoundsthat Ziegler (1959) found in Ephedra include polypeptides andphosphate-rich compounds. These compounds were only identified as togeneral class, and remain uncharacterized. As should be apparent withEphedra, there are many unrealized opportunities for researchers whowould like to enter this field. We would like to reiterate that Ephedrahas a pollination drop of enormous volume compared to some gymnosperms(Ephedra ~1000 nL versus Chamaecyparis lawsoniana ~10 nL). A consequenceis that many thousand fewer drops need to be collected for a chemicalanalysis. This advantage is multiplied by the fact that several speciesof Ephedra are small easy-to-grow plants, some becoming sexuallyproductive in a less than a year if vegetatively propagated.

In spite of a history of study of various aspects Ephedrapollination drop biology, including secretion and retraction(Strasburger, 1871), ecological features (Bino et al., 1984, Buchmann etal., 1989), nucellus morphology (Rydin et al., 2010) and physiology, andcomposition (Ziegler, 1959), we still need to address fundamentalquestions concerning drop composition and the influence, if any, ofpollen chamber formation in this composition. A more detailed andthorough analysis of components, especially protein composition, needsto be undertaken before the ecological services that Ephedra pollinationdrops provide can be considered.

Materials and Methods

A--Sample Collection.. Ephedra pollination drop samples werecollected by touching the drops with a micropipette tip. Drops wereexpelled into an Eppendoif tube and stored at -20[degrees]C untilanalysed. Ephedra likiangensis and E. minuta drops were collected fromplants in the botanical greenhouse at Stockholm University from January17 through February 16, 2012 and December 21 through January 10, 2012respectively. E. foeminea drops were collected in Asprovalta, Greece inJuly 2011. E. distachya drops were collected in Nea Vrasna, Greece May30 and June 2, 2011. E. trijurca drops were collected at the Aqua FriaRiver Bottom, Maricopa County, Arizona, U.S.A. on March 17, 2012. E.monosperma drops were collected from March to April, 2011 fromgreenhouse-grown plants at the Orchard Park Facility, University ofCalifornia at Davis. E. compacta drops were collected in Laguna deAlchichica, Puebla, Mexico from April 10 to 23, 2012. In addition,samples of Ginkgo biloba and Larix x marschlinsii were collected fromtrees growing outdoors on the campuses of University of California atDavis and University of Victoria, respectively. A separate comparativestudy was carried out on pollination drops of E. monosperma collected onthree sample dates, March 9, 24 and April 10, 2011.

B--ID SDS PAGE.. 20 [micro]L of pollination drop sample was mixedwith 5 [micro]L NuPage MES SDS Buffer and 1 [micro]L of 1 M DDT. Sampleswere boiled at 99[degrees]C for 10 min, and then loaded on to a NuPageNovex 4-12 % Bis-Tris precast gel. 5 [micro]L of BLUeye PrestainedProtein Ladder was run alongside the samples. The gel was fixed with a40 % ethanol /10 % acetic acid solution for 10 min, and then stainedwith 0.1 % G250 Coomassie Brilliant Blue overnight. The gel was thendestained with 10 % acetic acid solution.

C--LC-MS/MS Analysis.. Samples were reduced with dithiothreitol (30min at 37[degrees]C), and cysteine sulfhydryls were alkylated withiodoacetamide (30 min at 37[degrees]C in darkness). Trypsin (2 [micro]g;Promega) was added to each sample, which was digested at 37[degrees]Cfor 16 h. The samples were de-salted on a Waters HLB Oasis column, speedvac-concentrated and then stored at -80[degrees]C prior to LC-MSanalysis. Peptide mixtures were rehydrated to 100 [micro]L with 2 %acetonitrile/water/2 % formic acid and separated by on-line reversedphase chromatography using a Thermo Scientific EASY-nLC II system with areversed-phase pre-column Magic C-18AQ (100 [micro]m internal diameter,2 cm length, 5 [micro]m, 100 [Angstrom], Michrom BioResources Inc,Auburn, CA) pre-column and a reversed phase nano-analytical column MagicC-18AQ (75 tun internal diameter, 15 cm length, 5 [micro]m, 100[Angstrom], Michrom BioResources Inc, Auburn, CA) both in-houseprepared, at a flow rate of300 nl/min. The chromatography system wascoupled to an LTQ Orbitrap Velos mass spectrometer equipped with aNanospray II source (Thermo Fisher Scientific). Solvents were A: 2 %acetonitrile, 0.1 % formic acid; B: 90 % acetonitrile, 0.1 % formicacid. After a 249 bar (~5 [micro]L) precolumn equilibration and 249 bar(~8 [micro]L) nanocolumn equilibration, samples were separated by a 90min gradient (0 min: 5 % B; 80 min: 45 % B; 2 min: 90 % B; 8 min: 90%B).

D--Data Analysis Parameters.. Raw LCMS files were converted toMascot Generic Format and processed with PEAKS Client 6 (BioinformaticsSofware Inc, Waterloo, ON, Canada) with Peaks DB and Spider searchesenabled against the Uniprot/Trembl and Uniprot/Swiss-Prot Allspeciestaxonomy databases. Only plant species were selected. Settings were asfollows: instrument type set as FT-ICR/Orbitrap; high energy CID asfragmentation mode; parent ion error tolerance 8 ppm; fragment ion errortolerance 0.03 Da; trypsin as enzyme; up to one missed cleavage allowed;carbamidomethylation as a fixed modification; deamidation and oxidationas variable modifications. Peptide spectrum match false discovery rate(FDR), peptide FDR and protein FDR all set to [less than or equal to] 1%. The quality of the spectra were verified for proteins that wereidentified by only a single peptide sequence.

E--Scanning Electron Microscopy.. Ephedra monosperma ovules werecollected from the Bev Glover Greenhouse, University of Victoria. Ovuleswere removed from branches and mounted on a Deben MIG cold stage in aHitachi S-3500 N variable pressure scanning electron microscope (VPSEM). The microscope was operated at 20 kV, 50 Pa variable pressure inbackscattered electron mode using a Robinson BSE detector.

Results

A--Comparative Study of Seven Ephedra Species.. All Ephedrapollination drops contained proteins (Fig. 3). The relatively lightbands of Ephedra proteins run at native concentrations indicate loweramounts of protein, compared to that of larch and Ginkgo (Fig. 4).Proteins identified from liquid extractions of pollination drops can beseparated into degradome and secretome proteins (Tables 1 and 2). We didnot include proteins that had good spectra that matched uncharacterizedproteins, e.g. inferred protein from Picea sitchensis cDNA, althoughthese could be as many as a third of the high quality identities for anyone species, e.g. E. foeminea pollination drops contained 29 proteins,of which only 20 were characterized.

The number of characterized proteins in pollination drops ofEphedra species ranged from 6 to 20, averaging 13.4[+ or -]5.3identified proteins/species (Table 3). Ephedra foeminea and E. trifurcacontained more proteins (20), compared to E. distachya (15), E. compacta(13), E. minuta (11), E. likiangensis (9), and E. monosperma (6). Theseproteins could be divided into intracellular (64 %) and extracellularproteins (36 %). The percentage of intracellular proteins ranged from 44to 100 %: E. likiangensis (44 %), E. minuta (45 %), E. trifurca (50 %),E. compacta (54 %), E. monosperma (67 %), Ephedra foeminea (80 %) and E.distachya (100 %).

In all pollination drops a variety of intracellular proteins weredetected (Tables 1 and 3). The most frequently detected intracellularproteins--ubiquitin and polyubiquitin--were in five species (Table 1).Dessication-related proteins were detected in four species.Cyclophilin-[alpha], histones, and elongation factor 1-ct were detectedin three different species. Four of the most common proteins, i.e.detected in more than three or more species, were detected in drops ofE. foeminea. However, this might be expected given that the E. foemineahad the most proteins of any species in this comparative analysis. E.compacta had three of the commonly shared proteins. The remainingproteins on Table 1 were detected one or two times only.

Extracellular proteins were less abundant than intracellularproteins (Tables 2 and 3). The most commonly shared extracellularproteins were xylosidases (Table 2), which were detected in drops offour Ephedra species. Aspartic protease, [beta]-galactosidase,peroxidase and serine carboxypeptidase were detected in three Ephedraspecies. The remaining seven proteins on Table 2 were detected only onceor twice.

On a species level, proteins detected in drops represented a widevariety of enzymes. The proteins are either water-soluble proteinssecreted into the pollination drop, or are from the water-solubleportion of plant cells: no membrane-anchored proteins were detected inany samples. Ephedra foeminea drops had a probable defense protein(chitinase), two carbohydrate-modifying enzymes ([beta]-xylosidase,glycosyl-hydrolase-like protein), and proteases (aspartic protease,serine carboxypeptidase). The largest number of proteins were associatedwith the cytoplasm, including histone proteins, citrate synthase,elongation-factor-1-[alpha], cyclophilin, calreticulin, luminal-bindingprotein 4, a probable glycerophosphoryl diester phosphodiesterase,polyubiquitin, peptidyl-prolyl cis-trans isomerase, BIP isoform A, andgranule bound starch synthase. Ephedra trifurca had a similar number ofcharacterized proteins as E. foeminea, divided evenly between secretomeand degradome. Ephedra trifurca had some of the same proteins as E.foeminea (histone, elongation-factor-1-[alpha], ubiquitin, chitinase,[beta]-xylosidase, aspartic proteinase, serine carboxypeptidase). Theproteins found in drops of E. trifurca were divided evenly betweendegradome and secretome. Ephedra trifurca had defense proteins,including a chitinase and an alpha amylase inhibitor, peroxidase andendoglucanases, as well as a carbohydrate-modifying enzymes, e.g.[beta]-D-xylosidase and [beta]-galactosidase, and a serinecarboxypeptidase. Some other apoplastic enzymes, such as malatedehydrogenase, were detected.

In drops of E. likiangensis, intracellular and extracellularproteins were equally present; among the symplastic proteins, ubiquitinand proteases were predominant. Ephedra minuta drops had abundantsymplastic ubiquitins (Table 3), as well as apoplasticcarbohydrate-modifying enzymes ([beta]-xylosidase, [beta]-glucosidase)and defense proteins (thaumatin-like proteins). Cellular proteins notnormally found in the apoplast included Elongation factor 1-a,ubiquitin, acyl-CoA-binding domain-containing protein, actin. E.compacta had a number of ubiquitin and polyubiquitin proteins, as wellas acyl-CoA-binding domain-containing protein, calmodulin, a peptidase,and [alpha]-amylase; all of these were degradome proteins. Among thesecretome proteins were [beta]-xylosidase, [beta]-galactosidase, SOD,aspartic protease and peroxidase. Ephedra monosperma had mostlydegradome proteins (profilins, desiccation-related protein, theGTP-binding protein RAN-1, and ceramidase) and had only two secretomeproteins that we could detect in this initial comparative study--serinecarboxypeptidase and glucan endo-1,3-[beta]-glucosidase. Ephedradistachya was unique among the species sampled, because all of its 15proteins were degradome proteins (Table 3).

B--Comparative Study of Ephedra monosperma Drops from Three Dates..We were able to get samples of Ephedra monosperma pollination drops fromthree different dates (Table 4). Thirty-two proteins were identifiedfrom these samples, more than four times the number found in E.monosperma sample used in the comparative study of different Ephedraspecies (Table 3). The number of proteins declined with time, with thelargest number of proteins (22) found in the first sample (Mar. 9),which was not long after pollination drops began to be produced in thegreenhouse. On the next two dates, progressively fewer proteins werefound until only 14 proteins could be detected on the final date (Apr.10). Four proteins, hom*ologs of serine carboxypeptidase-like 32 proteinin hom*ologous to one found in Arabidopsis thaliana, histone 4 in Pisuinsativum, [alpha]-galactosidase and a predicted protein hom*ologous to onein Populus trichocarpa, were found at all three time points. Fourteenproteins were detected at two time points and 14 were only found at onetime. Most proteins (20/32) were degradome proteins. The exceptions wereextracellular proteins, such as serine carboxypeptidase, thaumatin-likeprotein, acid [alpha]- and [beta]-galactosidase, peroxidase, as well as[alpha]-xylosidase.

Discussion

Pollination drops of Ephedra contain proteins. Although this hasnot been reported previously in Ephedra, it was expected, as all otherpollination drops analyzed to date contain proteins. However, theprotein profiles in this study exhibit some notable differences fromthose of other gymnosperms we have measured, most of which were conifers(Wagner et al., 2007). Ephedra spp. not only have lower concentrationsof protein, judging from the lightness of the bands in the gels, butalso contain fewer total proteins. In addition, the protein profiles ofEphedra show substantial amounts of intracellular proteins not found inconifer pollination drops. In short, Ephedra has a degradome, consistingof proteins, and presumably shorter peptide fragments. The most likelysource of the protein degradome is from nucellar degeneration whichforms the flask-shaped pollen chamber during pollination dropproduction, causing intracellular proteins to be added to the otherpollination drop compounds. This assumption is logical, since pollenchamber formation occurs prior to and during pollination drop secretion(Rydin et ah, 2010). A protein that is characteristic of this degradomeis ubiquitin, which plays a major role in recycling proteins inside acell. It is not known to function outside of the cytoplasm, i.e. in theapoplastic fluids of plants. Protein profiles of both degradome andsecretome are composed of a few dozen proteins at most. Compared toother gymnosperms, the average number of proteins, which is about adozen per Ephedra species, is slightly greater than in pollination dropsof the Cupressaceae sampled to date, which range from half-a-dozen to adozen (Wagner et al., 2007), but much less than those of pinaceousspecies, such as Pseudotsuga menziesii (Poulis et al., 2005) and Larix xmarschlinsii (O'Leary et al., 2007), which have many dozens each.

In Ephedra pollination drops there are also proteins that are notpart of the degradome. These proteins are likely formed inside cells anddischarged into the apoplastic fluid by active cellular processes, andtogether these constitute a secretome of substances exported intopollination drops, similar to what has been found in most gymnospermsinvestigated using proteomics. Chitinase is an example of a protein thatbelongs to the secretome. In the results reported here, chitinases werepresent in both E. foeminea and E. trifurca. Chitinase is also found inpollination drops of another gnetophyte, Welwitschia mirabilis, as wellas a number of conifers (Wagner et al., 2007). In Douglas-fir drops,chitinases are able to process chitin substrates in situ, which suggeststhat they are active in anti-fungal defense during sexual reproduction(Coulter et al., 2012). Should the chitinases in the pollination drop ofEphedra prove functional, they may also protect ovules, which like thoseof other gymnosperms are exposed to the elements and are, therefore,more vulnerable to wind-bome pathogens than those of angiosperms whichare enclosed within a protective ovary.

The percentage of characterized cellular versus secretory proteinsin the drops ranged from 44 to 100 %, depending on species. Othergymnosperms, such as Juniperus, typically have no intracellular proteinsin their pollination drops (Wagner et al., 2007). The most commonintracellular protein found in Ephedra pollination drops is ubiquitin,which is found in five of the seven species. Of the 24 intracellularproteins detected, only 10 are found in more than one species. Thisimplies that although a degradome is universal in Ephedra pollinationdrops, its composition may widely differ among the species. To provide abetter idea of variation of protein profiles, studies need to beundertaken that focus on variation among individual plants as well asover the period of pollination drop secretion.

A measure of the variation in degradome is given by our samples ofE. monosperma plants from the same greenhouse population over three timepoints from the early to late in the pollination drop period. There weremore proteins at the beginning of the period than at the end, whichimplies that proteins initially present in drops are broken down overtime. Most of the proteins were clearly intracellular proteins, e.g.GTP-binding nuclear protein RAN 1, confirming that a degradome isconstantly present in the drops. Only a few proteins are found acrossall time points, e.g. histone 4, the majority varying widely. This wasequally true for secretome and degradome profiles. Ephedra monospermahas as much variation over time as there is among all species of Ephedra(Table 3). Investigations into variation within a species are important,as they will better allow us to isolate proteins that may havebiological function.

The question of function must be considered carefully. Caution mustbe exercised for many reasons. These drops not only capture pollen, butfungi, bacteria, viruses and dust. We have been able to show in previousstudies that enzymes in the drop, in particular, chitinases andinvertases are able to function in situ, but this work is difficultbecause of the small amount of liquid with which one has to work. As aconsequence, it is one thing to find proteins with identities andtherefore, functions, but it is quite another to prove that the proteinsfunction as expected from their sequence-based identities.

We assume that the degradome proteins, for example, ubiquitin, andhistones are not functional in the drop, because they are outside thecell where they are normally located. Cytoplasmic proteins such asubiquitin are involved in recycling proteins and peptides targeted forbreakdown inside the cell. Ubiquitin has not been previously found inpollination drops of Pinaceae in which pollen chambers are not formedand the nucelli do not undergo a degradation at the time of droprelease, e.g. Pseudotsuga and Larix. Other proteins that are strictlycytoplasmic include cyclophilin A (a plant immunophilin), which isrestricted to cell organelles: its presence in the drop is likely due tocell death and subsequent leakage of cellular contents.

Focusing on two species in the comparative study, E. foeminea andE. distachya, E.foeminea had the most detected proteins, half of whichare degradome proteins, where E. distachya had only degradome proteins.Having about 50 % degradome proteins is close to the average for theseven species that we measured. In addition to ubiquitin, justdiscussed, notable degradome proteins in E. foeminea are histones whichare normally restricted to the nucleus and involved in chromosomeorganization, Granule-bound starch synthase which synthesizes amylose inthe chloroplast, BIP isoform A which is a molecular chaperone located onthe endoplasmic reticulum, and immunophilins such as peptidyl-prolylcis-trans isomerase which are found in a number of locations within thecell. The predominance of these cytoplasmic proteins among thedegradomic fraction is probably due to either their abundance indegrading cells, and/or in their slower rate of degradation compared tothat of other proteins (i.e. already reduced to small peptides or aminoacids). The profile of proteins detected in pollination drops of E.distachya consists entirely of intracellular proteins, none of which arenormally found in apoplastic secretions including: proteins involved insignal transduction, e.g. small Ran-related GTP-binding protein;calmodulin 4 which is a regulatory protein controlled by calcium;nucleoside diphosphate kinase that regulates metabolic pools ofnucleoside diphosphates; histones that control chromosome organization;heat-shock proteins that regulate a plant cell's response tostress. Recently, there have been papers that suggest a few of theseproteins may function in the apoplast. For example, root border cells ofangiosperms and gymnosperms (Wen et al, 2008b) upregulate geneexpression that results in secretion of intracellular proteins such asDNA-bound histones that act a trap for pathogens (Hawes et al., 2012).

In other gymnosperm pollination drops, most proteins do not appearto be related to a degradome, but are secreted by cells directly intothe apoplast. In these cases the collective secreted protein componentis known as a secretome. The secretome proteins that we have been ableto identify from our analyses of various species of gymnosperms werefrom many classes of enzymes. We detected a variety of defense proteins,including among others, thaumatin-like protein, peroxidase,glucan-endo-[beta]-1,3-glucanase, and superoxide dismutase. However, theproteins of the secretome are probably not all involved in defense. Inaddition there are carbohydrate-modifying enzymes such as [alpha]- and[beta]-galactosidase proteins. In roots of peas, galactosidases operateon cell wall fragments to produce galactose, which is inhibitory to rootgrowth (Wen et al., 2008a). All of the proteins that we have designatedas part of the secretome, e.g. peroxidase, malate dehydrogenase,superoxide dismutase and thaumatin-like proteins, have been foundapoplastically in other plants. Some protein classes have many membersthat have diverse functions, e.g. serine carboxypeptidases. Theseinclude serine carboxypeptidases that have regulatory functions both inthe cytoplasm, as well as in the extracellular spaces. Until theseproteins are shown to function in situ in the pollination drop, theyare, like all other enzymes included in our lists, assigned to thesecretome because they or members of their class of protein have beendetected in the apoplast of other plants. In our survey of Ephedrapresented here, no proteins are common to the secretomes of all species.The number of proteins ranges among the Ephedra species between 2 and 10per pollination drop/species.

We expected to find acid phosphatase in the drop, since twodifferent laboratories have reported its presence via activity assays inGnetales. Ziegler (1959) detected it in the nucellus of E. helvetica(=E. distachya subsp. helvetica) as well as in the nongnetalean Taxusbaccata (Taxaceae) and Carafa et al. (1992) reported its presence inpollination drops of W. mirabilis. However, we did not detect thisenzyme in any pollination drops of the seven Ephedra species that weanalyzed using proteomics methods. We have never found it in anyconifers, but a proteomic analysis of the nucellus has yet to becompleted.

There are more proteins in these species than we have been able todescribe. In all Ephedra species, there was a relatively high percentageof uncharacterized proteins. Although the mass spectra of proteins towhich no identity can be assigned are of high quality, the databasesagainst which we search this information often have insufficient depth,particularly with regards to gymnosperms. This situation should improveif, in future, databases improve. For example, genomes of Picea abies(Nystedt et al., 2013) and P. glauca (Birol et al., 2013) will be usefulonce they are annotated. As more gymnosperms are covered, the improveddepth of the databases will assist in protein identification. Molecularbiologists will be able to use these databases to make better proteinidentifications and to improve the prediction of functions for theseproteins. However studies of the distantly related Gnetales may notbenefit to as a large degree compared to those of conifers.

Ephedra pollination drops may be acting as nectar. However, inspite of high sucrose concentrations among all Ephedra pollination dropsmeasured to date, not all species are insect-pollinated, e.g. E.campylopoda (Porsch, 1910): some are insect- and wind-pollinated, e.g.E. aphylla (Meeuse et al., 1990), and others are only wind-pollinated,e.g. E. trifurca (Buchmann et al., 1989). Insects that are notpollinators, such as ants, are also attracted to Ephedra drops (Porsch,1910). Ziegler (1959) mentioned the high concentrations of amino acidsin drops, which would influence the palatability of these drops to sometypes of insects. Insect pollination is certainly widespread amonggnetophytes (Endress, 1996), although it may not be obligate in anyEphedra species.

Until this study, any proteins in pollination drops were consideredto probably be a functional portion of the drop (Nepi et al., 2009). Thepossibility that proteins may also be byproducts of pollen chamberformation that have been washed into the drop has never been explored.This is due to the fact that the species investigated to date did nothave pollen chambers formed front nucellar breakdown. Thus thepollination drops of Ephedra are probably a mixture of functional andformerly functional, as well as biologically inactive proteins and/orpeptides. As such, Ephedra differs from conifers analyzed to date, suchas Pinaceae and Cupressaceae. It will be interesting to expandpollination drop analysis into Pirns, Ginkgo and cycads, all of whichhave pollen chambers. The low amount of protein in Ephedra dropssuggests a less important role, if any, for these proteins duringreproduction. The higher sucrose concentrations in these drops result inhigher osmotic pressure in these drops, which may prevent foreign pollenfrom germinating (von Aderkas et al., 2012) and pathogens fromestablishing and growing.

Ephedra pollination drops have proteins that can be divided intothose that belong to the degradome, itself a result of pollen chamberformation, and those that are exported by the cytoplasm into the dropand form an active part of the secretome that is, based on similarity toother gymnosperms, involved in carbohydrate modification, defense andother apoplastic activities.

DOI 10.1007/s12229-014-9147-x

Acknowledgments The authors would like to thank University ofVictoria (UVic), UVic-Gcnomc BC Protcomics Centre (UVic-GBCP), GenomeCanada, and Genome BC for their support, as well as the Natural Sciencesand Engineering Research Council of Canada (NSERC) for a DiscoveryResearch Grant Program (PvA), and a Post-graduate Scholarship ProgramGrant (NAP). Wc acknowledge the expert assistance of B. Gowcn (UVic), D.Smith (UVic) and Carol Parker (UVic-GBCP), as well as the invaluableassistance in sample collections from S. Ickcrt-Bond, C Rydin, K.Bolindcr, A. Rydberg, J. Jcmstcdt and I. Locra-Carrizalcs.

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Patrick von Aderkas (1,6) * Natalie Prior (1) * Susannah Gagnon (1)* Stefan Little (1,2) * Tyra Cross (3) * Darryl Hardie (3) * ChristophBorchers (3) * Robert Thornburg (4) * Chen Hou (5) * Alexandra Lunny (1)

(1) Centre for Forest Biology, Department of Biology, University ofVictoria, Victoria, BC V8W 3N2, Canada

(2) Department of Plant Sciences, Mail Stop 1, University ofCalifornia, Davis, One Shields Avenue, Davis, CA 95616, USA

(3) UVic--Genome BC Protcomics Centre, University of Victoria,Victoria, BC V8W 3N2, Canada

(4) Department of Biochemistry, Biophysics & Molecular Biology,Iowa State University, Ames, 1A 50011, USA

(5) Department of Ecology, Environment and Plant Science, StockholmUniversity, SE-106 91 Stockholm, Sweden

(6) Author for Correspondence; e-mail: [emailprotected]

Published online: 17 December 2014

Table 1 Degradome proteinsfound in pollination drops ofEphedra sppProtein SpeciesUbiquitins E. compacta E. foeminea E. likiangensis E. minuta E. trifurcaDessication-related protein E. compacta E. likiangensis E. minuta E. monospermaCyclophilin A E. distachya E. foeminea E. minutaElongation factor 1-[alpha] E. distachya E. foeminea E. trifurcaHistones E. distachya E. foeminea E. trifurcaAcyl-CoA-binding domain- E. compactacontaining protein 6 E. trifurca[alpha]-Amylase E. compacta E. likiangensisCalmodulin E. compacta E. distachyaGlycosyl hydrolase E. foeminea E. trifurcaGTP-binding nuclear protein E. distachya E. monospenna[alpha]-Amylase inhibitor E. trifurcaAuxin response factor E. distachyaCalreticulin E. foemineaCeramidase E. monospennaCitrate synthase E. foemineaCysteine proteinase E. likiangensis[alpha]-Gliadin E. trifurcaGlyccrophosphoryl diester phosphodiesterase E. foemineaGranule-bound starch synthase E. foemineaHeat shock proteins E. distachyaLactoylglutathione lyase E. trifurcaLuminal-binding protein E. foemineaProfilin E. monospennaThiol protease alcurain E. likiangensisTable 2 Secretome proteins foundin pollination drops of Ephedra spp.Proteins that could also be consid-ered degradome are marked with anasteriskProtein SpeciesXylosidases E. compacta E. foeminea E. minuta E. trifurcaAspartic proteinase * E. compacta E. likiangensis E. trifurcaGalactosidases E. compacta E. minuta E. trifurcaPeroxidase E. compacta E. likiangensis E. trifurcaSerine carboxypeptidases * E. foeminea E. monospe- rma E. trifurcaChitinase E. foeminea E. trifurcaGlucan endo-1,3- [beta]-glucosidase E. monospe- rma E. trifurcaMalate dehydrogenase E. trifurcaPeptidase * E. likiangensisSuperoxide dismutase * E. compactaThaumatin-like protein E. minutaTable 3 Peptide sequences and identities of pollination dropproteins found in Ephedra spp. Proteins in red are degradomeproteins, those in black are secretomeSpecies Peptide amino acid Protein identification sentience obtainedE. compacta K.SSEEAME(sub N)DYITK.V Acyl-CoA-binding M.GLKEEFEEY(sub H)AEK.V domain-containing protein R.AKWDAWK.A 6 OS=Arcibidopsis thaliana K.EGIPPVQQR.L Ubiquitin-NEDDS-like R.TLADYNIQK.E protein RUB2 OS=Oryza E.VESSDTIDNVK.A sativa subsp. japonica R.TLADYNIQK.E Polyubiquitin 2 OS=Zea K.EGIPPVQQR.L mays R.TLADYNIQK.E Putative polyubiquitin E.VESSN(+.98)T1DNVK.A (Fragment) OS=Arabidopsis thaliana R.NIQVVDGSNNLKAPK.G Putative carboxyl-terminal peptidase OS=Arabidopsis thaliana R.VFDKDQNGFISAAELR.H Calmodulin (Fragment) OS=Pyrus communis K.AVADIVINHR.C Alpha amylase (Fragment) OS=Cuscuta reflexa L.GVESGQDAVIR.G Dessication-related R.TPEEILR.I protein putative; 70055- 71849 OS=Arabidopsis thaliana K.VTEQDLE(sub Putative beta-xylosidase AJDTYNPPFK.S (Fragment) OS=Triticum aeslivum R.STPEMWPDIIQK.A Beta-galactosidase OS=Picea sitchensis R.AVVVHADPDDLGK.G Superoxide dismutase [Cu- Zn] OS=Pinus sylvestris K.GEHTYVPVTK.K Aspartic proteinase (Fragment) OS=Cucumis sativus R.FDNNYYK.D Peroxidase (Fragment) OS=Lupinus polyphyllusE. distachya K.ATAGDTHLGGEDFDNR.M Heat shock 70 kDa protein R.IINEPTAAAIAYGLDKK.A OS=Glycine max R.VEIIPNDQGNR.T K.NKITITNDKGR.L K.ATAGDTHLGGEDFDNR.M Heat shock cognate 70 kDa R.IINEPTAAAIAYGLDKK.A protein 1 OS=Solanun R.VEIIANDQGNR.T lycopersicum K.NKITITNDKGR.L R.ELISNSSDALDKIR.F Heat shock protein 81-2 K.ADLVNNLGTIAR.S OS=Arabidopsis thaliana D.AIDEYAIGQLK.E R.FESLTDK.S K.IGGIGTVPVGR.V Elongation factor 1-alpha N.IVVIGHVDSGK.S OS=Zea mays R.VETGVIKPG.M F.DKDQNGFISA.A Calmodulin 4 (Fragment) MADQLTDDQISEFK.E OS=Dancus carota FDKDGDGC(+57.02)ITTK.E Calmodulin protein (Fragment) OS=Pinus taeda R.DNIQGITKPAIR.R Histone H4 OS=Solanum melongena L.FEDTNLC(+57.02)AIHAK.R Histone H3-like 1 OS=-Arabidopsis thaliana R.NVIHGSDAVESAQ(sub Nucleoside diphosphate R)K.E kinase OS=Arabidopsis lyraia subsp. lyrata K.AGfa*gDDAPR.A Actin-3 0S=Arabidopsis R.GNGTGGESIYGEK.F Peptidyl-prolyl cis-trans isomerase OS=Zea mays R.VLQISGER.N 18.1 kDa class I heat shock protein (Fragment) OS=Medicago sativa R.VLQISGER.S Small heat shock protein hspl 0.4 (Fragment) OS=Quercus suber K.LVIVGDGGTGK.T GTP-binding nuclear protein Ran2 OS=Solanum lycopersicum K.LVIVGDGGTGK.T Small Ran-rclated GTP- binding protein OS=Triticum aestivum R.TFVKVYK.S Auxin response factor 12 OS=Oryza sativa subsp. indicaE. foeminea K.EALQAEVGLPVDR.N Granule-bound starch K.VVGTPAYEEM(+15.99)VR.N synthase 1 R.FAFSDYPELNLPER.F chloroplastic/amyloplastic K.SSFDFIDGYEKPVEGR.K OS=Zea mays K.MGDGYETVR.F R.VLTVSPYYAEELISGIAR.G R.FAFSDYPELNLPER.F Granule-bound starch K.VVGTPAYEEM(+15.99)VR.N synthase OS=Zea mays K.EALQAEVGLPVDR.N subsp. mays K.MGDGYETVR.F K.SSFDFIDGYEKPVEGR.K R.VLTVSPYYAEELISGIAR.G R.EAEEFAEEDKK.V BiP isoform A OS=Glycine K.FELSGIPPAPR.G max R.VEIESLFDGVDFSEPLTR.A K.DYFDGKEPNK.G R.LSQEEIER.M K.EAEEFAEEDKK.V Luminal-binding protein 4 R.VEIESLFDGVDFSEPLTR.A OS=Nicoriana tabacum K.DYFDGKEPNK.G R.LSQEEIER.M K.TFASGILVPK.S Probable glycerophosphoryl diester phosphodiesterase 3 OS=Arabidopsis thaliana DNIQGITKPAIR.R Histone H4 (Fragment) R.ISGLIYEETR.G OS=Danicus carota R.DNIQGITKPAIR.R Histone H4 OS=Silene R.ISGLIYEETR.G lanfolia K.KPEGYDDIPK.E Calreticulin OS=Zea mays K.LDC(+57.02)GGGYVK.L K.KPEGYDDIPK.E Calreticulin OS=Prunus R.FEDGWDKR.W armeniaca R.EIAQDFK.T Histone H3-like 1 QS=Arabidopsis thaliana R.TLADYNIQK.E Polybiquitin 9 OS=Arabidopsis thalinia R.ALGLPLERPK.S Citrate synthase OS=Picea sitchensis R.ALGLPLERPK.S Citrate synthase 5 mitochondrial OS=Arabidopsis haliana R.GNGTGGESIYGEK.F Peptidyl-prolylecis-trans isomerase OS=Zea mays R.IGGIGTVPVGR Elongation factor 1-alpha (Fragments) OS=Pxeudotsuga menziesii K.AGLQFPVGR.I Histone H2A OS=Euphorbia esula V.VTQ(+.98)QDLDDTYQPPFK.S Beta-xylosidase/alpha-L- arabinofuranosidase 2 OS=Medicago varia R.VWVYSGDTDGR.V Serine carboxypeptidase- like 32 OS=Arabidopsis thaliana R.AINSM(+l5.99) Class IV chitinase ECNGGNPSAVQ(subD)DR.V OS=Nepenlhes alata R.C(+57.02)YESYSEDPS Glycosyl hydrolase-like (sub K)IVK.A protein (Fragment) OS=Picea sitchensisE. K.IQDKEGIPPDQQR.L Ubiquitin OS=Triticumlikiangensis E.VESSDTIDNVK.A aestivum R.TLADYNIQK.E K.YNGGlDTEEA(sub S)YPYK.G Thiol protease alcurain R.EDGIVSPVK.N OS=Hordeum vulgare L.GVESGQDAVIR.G Dessication-related R.TPEEILR.I protein_putative; 70055- 71K49 OS=Arabidopsis thaliana K.AVADIVINHR.C Alpha amylase (Fragment) OS=Cuscuta reflexa R.FDNNYYK.D Peroxidase (Fragment) OS=Lupinus polyphyllus R.NIQVVDGSNNLKAPK.G Putative carboxyl-terminal peptidase OS=Arabidopsis thaliana A.Q(+.98)GSGEYFTR.I Aspartic proteinase nepenthesin-l_putative OS=Ricinus communis K.GEHTYVPVTK.K Aspartic proteinase (Fragment) OS=Cucumis salivus R.EDGIVSPVK.D Cysteine proteinase OS=Elaeis guineensis var. teneraE. minuta R.LIfa*gKQLEDGR.T Ubiquitin-NEDD8-like K.EGIPPVQQR.L protein RUB2 OS=Oryza R.TLADYNIQK.E sativa subsp. japonica E.VESSDTIDNVKAK.I K.VESSDTIDNVKAK.I Ubiquitin OS=Musa R.LIfa*gKQLEDGR.T acuminata R.TLADYNIQK.E R.LIfa*gKQLEDGR.T Polyubiquitin 2 OS=Zea R.TLADYNIQK.E mays K.EGIPPVQQR.L L.GVESGQDAVIR.G Dessication-related R.TPEEILR.I protein_putative; 70055- 71849 QS=Arubiddpsis thaliana R.GNGTGGESIYGEK.F Cyclophilin A (Fragment) OS=Tritieum aestivum K.FFKGQC(+57.02) Thaumatin-like protein PQAYSYAK.D K.DDATSV(sub T)FTC OS=Cryptomeria japonica (+57.02)PSP(sub G)TNYK.V K.GQC(+57.02)PQAYSYAK.D Thaumatin-like protein OS=Pinus taeda R.STPEMWPDIIQK.A Beta-galactosidase K.NVVFNTAK.I OS=Picea sitchensis K.WGHLKEL.H R.YAVNYVR.G Beta-glucosidase_putative OS=Ricinus communis A.VNQDSLGVQGK.K Alpha-galactosidase K.ALADYVHAK.G OS=Oryza saliva subsp. japonica R.WEVPYNLLPR.E Alpha-xylosidase OS=Arabidopsis thalianaE. K.YM(+15.99)VIQGEPGVVIR.G Profilin-1 (Fragment)monosperma OS=Triticum aestivum K.YM(+15,99)VIQGEPG Profilin OS=Zea mays VVIR.G L.LGVESGQDAVIR.G Dessication-related protein_putative; 70055- 71849 OS=Arabidopsis thaliana K.LVIVGDGGTGKT.T GTP-binding nuclear protein Rail-A1 OS=Nicotiana tabacum R.SPSAYLNNPP(sub A)EER.N Ceramidase putative OS=Ricinus communis R.VWVYSGDTDGRVP.V Serine carboxypeptidase 1 OS=Zea mays L.FNENLKPGPTG(sub SJER.N Glucan endo-1 3-beta- glucosidase 11 OS =Arabidopsis thalianaE. trifurca K.SSEEAME(sub N)DYITK.V Acyl-CoA-binding M.GLKEEFEEY(sub H)AEK.V domain-containing protein R.AKWDAWK.A 6 OS=Arabidopsis thaliama K.EGIPPVQQR.L Ubiquitin-NEDD8-like R.TLADYNIQK.E protein RUB2 OS=Oryza K.IQDKEGIPPDQQR.L sativa subftp. japonica E.VESSDTIDNVK.A K.EGIPPVQQR.L Ubiquitin putative R.TLADYNIQK.E OS=Ricinus communis K.IQDKEGIPPDQQR.L L.EVESSDTIDNVK.A K.ITSFLDPDGWK.T Lactoylglutathione lyase K.V(sub T)VLVDNEDFLK.E OS=Gossypium hirsutum Q.QLPQFEEIR.N Alpba-gliadin OS=Triticum aestivum K.VTE(sub L) Os 11 g0291000 protein QDLEDTYNPPFK.S OS=Oryza sativa subsp. japonica R.IGGIGTVPVGR Elongation factor 1-alpha (Fragments) OS=Pseudotsuga menziesii K.EHGAQEGQAGTGAFPR.C Alpha-amylase inhibitor 0.19 OS--Triticum aestivum K.EHGAQEGQAGTGAFPR.C Dimeric alpha-amylase inhibitor OS=Aegiiops umbellulata K.AGLQFPVGR.I Probable histone H2A.1 OS=Oryza sativa subsp. japonica K.VTQ(+.98)QDLEDTYNP Beta-D-xylosidase 1 (sub V)PFK.S E.TMIGNYAGK.A OS=Arabidopsis thaliana E.WWSEALHGISDVGPGT (sub A)K.F H.T(sub S)AITSGQGFGGTIK.A Class IV chitinase Chia4- R.ELAAFFANVMHETS Pa2 variant (Fragment) (sub G)GL.C S.WNYNYGAAGK.S OS--Picea abies R.STPEMWPDLIQK.A Beta-galactosidase A.FRTDNEPFKA.A OS=Pyrus communis R.STPEMWPDLIR.K Beta-galactosidase (Fragment) OS=Mangifera indica K.MELIDAAFPLLK.G Malate dehydrogenase OS=Picea sitchensis R.VWVYSGDTDGRVPVT.S Serine carboxypeptidase II- 3 OS=Hordeum vulgare I.GGYYDAGDNVK.F Endoglucanase 20 OS=Arabidopsis thaliana GGYYDAGDNVK.F Putative endo-1_4_-beta- glucanase (Fragment) OS=Solamtm lycopersicum R.FDNNYYK.D Peroxidase (Fragment) OS=Lupinus polyphyllus K.GEHTYVPVTK.K Aspartic proteinase (Fragment) OS=Cucumis sativusTable 4 Ephedra monosperma pollination drop proteins fromthree collection dates. Degradome proteins arc at the top ofthe list, marked by a red "x"Protein Mar Mar Apr 9 24 10Histone H4 OS=Pisum sativum PE=1 SV=2 x x xPredicted protein OS=Populus trichocarpa x x xGN=POPTRDRAFT_642406 PE=4 SV=1Putative uncharacterized protein OS=Selaginella x xmoellendorffii GN=SELMODRAFT 143620 PE=4 SV=1Putative uncharacterized protein OS=Glvcine max x xPE=2 SV=1Acyl-CoA-binding protein (Fragment) OS =Jatropha x xcurcas PE=2 SV=1Glycosyl hydrolase family-like protein OS=Salvia x xmiltiorrhiza PE=2 SV=1GTP-binding nuclear protein Ran-AI OS=Nicotiana x xtabacum GN=RAN-A 1 PE=2 SV=1Eukaryotic initiation factor 4A OS=Triticum x xaestivum PE=2 SV=1RAS-like protein (Fragment) OS =Arabidopsis x xthaliana PE=2 SV=1Translation initiation factor OS=Zea mays x xGN=eIF-4A PE=2 SV=1Acid beta-fructofuranosidase OS=Solanum xlycopersicum GN=T1V1 PE=2 SV=1Alpha-glucosidase 0S= Hordeum vulgare PE=2 SV=1 xMulticystatin OS=Helianthus annum GN=smc PE=2 SV=1 xPolyubiquitin 11 OS=Arabidopsis thaliana GN=UBQ11 xPE=1 SV=1Predicted protein OS=Populus trichocarpa xGN=POPTRDRAFT_1090916 PE=4 SV=1Endoglucanase 23 0S=Oryza sativa subsp. japonica xGN=GLU12 PE=2 SV=1NtPRp27-like protein OS=Solanum tuberosum PE=2 xSV=1Ubiquitin-like protein (Fragment) OS=Solatium xlycopersicum GN=ubiquitin-like PE=2 SV=1Cyclophilin A (Fragment) OS=Triticum aestivum xGN=CYPI8-3 PE=3 SV=1Photosystem II Q(B) protein (Fragment) OS=Kochia xscoparia GN=psbA PE=4 SV=1Alpha-galactosidase OS=Coffea arabica PE=I SV=1 x x xSerine carboxypeptidase-like 32 OS=Arabidopsis x x xthaliana GN=SCPL32 PE=2 SV=1Alpha-galactosidase 0S=Oryza sativa subsp. x xjaponica GN=Osl0g0493600 PE=1 SV=1Acid alpha galactosidase 1 OS=Cucumis sativus PE=2 x xSV=1Alpha-xylosidase OS=Arabidopsis lyrata x xGN=ARALYDRAFT_894626 PE=4 SV=1Peroxidase (Fragment) OS=Lupinus polvphyllus PE=2 x xSV=2Alpha-xylosidase OS=Arabidopsis thaliana GN=XYLI x xPE=1 SV=1Beta-galactosidase 1 OS=Oryza sativa subsp. x xjaponica GN=Os01g0533400 PE=2 SV=1Beta-galactosidase 8 OS =Arabidopsis thaliana xGN=BGAL8 PE=2 SV=2Thaumatin-like protein OS=Cryptomeriajaponica xGN=Cry j 3.1 PE=2 SV=1Alpha-galactosidase OS=Coffea canephora GN=gal 1 xPE=2 SV=1Beta-galactosidase 9 OS=Oryza sativa subsp x.japonica GN=Os06g0573600 PE=2 SV=1