Apart from proteins, it has become clear that certain small RNA species (sRNAs) with a length between 21 and 24 nucleotides move between cells in a regulated and biologically relevant manner (Chitwood and Timmermans2010; Van Norman et al.
2011). It is generally assumed that sRNAs move symplastically through PD. The sRNA variants differ in the way they are generated and their biological function (Chapman and Carrington2007; Voinnet2009). siRNAs are formed from perfectly matching dsRNAs, act in a noncell-autonomous fashion with at least 21-nucleotide species moving as siRNA duplexes (Dunoyer et al. 2010), and are involved in posttranscriptional gene silencing of viruses and transgenes. In addition, endoge- nous mobile siRNAs are of 24-nucleotide length, derived from transposable elements (TEs) or other methylated DNA regions, and can direct DNA methylation at target loci (Molnar et al.2010). miRNAs and transacting siRNAs (tasiRNAs) regulate gene silencing. In general, siRNAs seem to move further than miRNAs (de Felippes et al.2011).
Defects in the biogenesis of tasiRNAs result in leaf and floral phenotypes (Peragine et al.2004; Adenot et al. 2006), and it has become clear that miRNA 165/166 and miR390/tasiRNA tasiR-ARF affect leaf patterning (Husbands et al.
2009). For example, in Arabidopsis, miR390 spreads from its subSAM origin of expression to the young lateral primordia where it participates in the biogenesis of tasiRNA directed against the abaxial factors ARF3 and ARF4 (tasiR-ARF) (Chitwood et al.2009). Production of tasiR-ARF is restricted to the adaxial cell layers from which tasiR-ARF moves toward abaxial layers generating a corresponding adaxial-abaxial gradient of tasiR-ARF. This gradient likely results in the translational repression of ARF3 in adaxial cells and the presence of ARF3 protein in abaxial cells only (Husbands et al.2009). It is not known if miRNA165/66, regulating abaxial pattern- ing of lateral organs, moves in leaves or floral organs; however, this miRNA was recently shown to move from the endodermis into the vascular cylinder, thereby regulating xylem differentiation (Carlsbecker et al.2010).
In ovules, a single megaspore mother cell (MMC) originates from a group of L2- derived cells in the nucellus. The MMC undergoes meiosis resulting in a tetrad of megaspores. As a rule, three megaspores degenerate and the sole surviving func- tional megaspore further develops into the female gametophyte with the egg cell proper. Recently, it was shown that anAGO9-dependent siRNA pathway plays an essential role in singling out the MMC in a noncell-autonomous fashion (Olmedo- Monfil et al.2010). Inago9,rdr6, orsgs3mutants, several MMC-like cells develop in the nucellus, although only one continues with meiosis. Still, one or several of the other enlarged cells acquire female gametophyte identity despite the absence of meiosis, a situation resembling apospory. Interestingly, AGO9 protein could only be detected in the epidermis cells of the nucellus, was shown to preferentially associate with 24-nucleotide sRNAs, and was required for the silencing of endoge- nous TEs in the egg and synergids. Importantly, AGO9-dependent TE inactivation apparently restricts female gametophyte formation to a single precursor cell
(the MMC) through a 24-nucleotide siRNA biosynthetic pathway. The authors suggested that inactivation of TEs in all subepidermal cells of the nucellus except the MMC somehow prevents those cells to enter gametophyte development, although how this is achieved remains to be investigated. The MMC, however, appears to be somehow isolated, and thus, the silencing signal cannot enter this cell.
Indeed, the MMC is known to become symplastically isolated (Werner et al.2010), possibly due to accumulation of high levels of callose around the MMC (Schneitz et al.1995).
Movement of siRNAs also appears to be important for maintenance of genome stability in sperm cells. In the vegetative nucleus of pollen, TEs become reactivated resulting in the generation of a high level of siRNAs. By contrast, TEs in the sperm cells remain silent, possibly at least in part as a consequence of siRNAs moving from the vegetative cell into the sperm where they could act in the epigenetic silencing of the TEs (Slotkin et al.2009).
3 Receptor-Like-Kinase-Mediated Intercellular Signaling in Flowers
Cell surface receptor-like kinases (RLKs) are natural mediators of information transfer between cells and are involved in many short-range intercellular signaling processes. TheArabidopsisgenome encodes more than 600 RLK genes (Shiu and Bleecker2001); a growing number of which are known to affect several aspects of organogenesis (He´maty and H€ofte 2008; De Smet et al. 2009; Steinwand and Kieber2010; Gish and Clark2011).
Regulation of stem cell maintenance in SAMs and FMs is mediated through an autoregulatory feedback loop involving the signal peptide CLAVATA3 (CLV3), the leucine-rich repeat (LRR) RLK CLV1, and the homeobox transcription factor WUSCHEL (WUS) (Clark et al.1997; Mayer et al. 1998; Fletcher et al.1999;
Brand et al.2000; Schoof et al.2000). WUS is an indirect positive regulator of stem cells which in turn express CLV3 that negatively regulates WUS through CLV1 and the plasma membrane–localized phosphatases POLTERGEIST (POL) and PLL1 (Yu et al. 2003; Gagne and Clark 2010). More recently, it was found that this feedback loop also involves the direct negative control ofCLV1by WUS (Busch et al.2010). Apart from regulatingCLV1expression, WUS seems to foster stem cell development by influencing the hormonal control of the stem cell niche in the SAM (Leibfried et al.2005; Gordon et al.2009; Zhao et al.2010). Interestingly, WUS also regulates chalaza formation in a nonautonomous fashion (Gross-Hardt et al.
2002; Sieber et al.2004). The mechanism is not understood but does not involve the CLVgenes.
Perception of the CLV3 peptide has proven to be more complex than initially appreciated. First, it was realized that a processed form of the CLV3 peptide directly binds to CLV1 and CLV2 (Kondo et al.2006; Ogawa et al.2008; Guo et al.2010).
Second, it is now apparent that several receptor complexes act in parallel in the perception of CLV3 at the cell surface. One receptor complex consists of constitu- tive CLV1 homodimers. In addition, CLV1 can form heterodimers with the closely related and redundantly acting BAM receptors (DeYoung et al.2006; DeYoung and Clark2008; Bleckmann et al.2010; Guo et al.2010; Zhu et al.2010). Furthermore, the receptor-like protein CLV2 (Kayes and Clark1998; Jeong et al.1999), which carries but a small cytoplasmic domain, forms a receptor complex with the trans- membrane putative kinase CORYNE (CRN) which itself carries a transmembrane domain but only a small extracellular domain (Miwa et al.2008; M€uller et al.2008).
In addition, homo-oligomers formed by the RLK RECEPTOR-LIKE PROTEIN KINASE 2 (RPK2)/TOADSTOOL 2 (TOAD2) represent a third CLV3-transmitting receptor complex (Kinoshita et al.2010).
RLKs are also involved in interhistogenic-layer communication in the SAM, FM, and the organs derived from those meristems. The underlying communication can go in two directions. For example, the epidermis has an important influence on subepidermal cell behavior (Reinhardt et al.2003). The brassinosteroid receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1) (Li and Chory 1997; Kinoshita et al.2005) was demonstrated to participate in the communication between epider- mis and subepidermis in the control of cell morphogenesis by providing an epider- mis-derived nonautonomous signal (Savaldi-Goldstein et al.2007). An “inside to outside” mechanism of intercell-layer communication in floral organs is suggested by the subcellular localization of the epidermally expressed RLK ARABIDOPSIS CRINKLY 4 (ACR4) (Gifford et al.2003). ACR4 is theArabidopsishomolog of maize CRINKLY 4 (CR4) (Becraft et al.1996; Becraft et al.2001) and involved in the regulation of epidermal cell organization in ovule integuments, sepals, and leaves (Gifford et al. 2003; Watanabe et al. 2004; Gifford et al.2005). ACR4- dependent control of epidermis development also involves the RLK ABNORMAL LEAF SHAPE 2 (ALE2) (Tanaka et al.2007).
The STRUBBELIG (SUB) locus encodes a LRR-RLK that was implied in intercell-layer communication in flowers as well (Chevalier et al. 2005; Yadav et al.2008).SUB, also known asSCRAMBLED(SCM) (Kwak et al.2005), regulates cell morphogenesis in FMs and ovules in a noncell-autonomous fashion. In the FM, expression of functional SUB:GFP fusion protein from the L1 was sufficient to rescue cellular defects in the L2 while nucellar expression of SUB:GFP was able to rescue integument defects to a large extent in ovules.SUBinteracts with the RLK geneERECTA(ER) (Torii et al.1996) in a synergistic fashion in stem development but interestingly not in ovules (Vaddepalli et al. 2011). How SUB affects the behavior of neighboring cells is currently being investigated. With QUIRKY (QKY),ZERZAUST(ZET), andDETORQEO(DOQ), three additional components of the SUB signaling pathway have recently been identified genetically (Fulton et al.2009).QKYwas found to encode a putative membrane-anchored C2-domain protein. On the basis of related domain architecture in animal proteins such as synaptotagmins or ferlins, QKY was hypothesized to function in membrane traf- ficking. Additional postulated scenarios include a role ofSUBandQKYin cell wall biology or the regulation of PD function.
Interestingly, kinase activity of SUB is not required for its function in vivo (Chevalier et al.2005; Vaddepalli et al.2011). In vitro kinase assays were negative, but critically, transgenes carrying several well-characterized mutations in the SUB kinase domain were able to rescue thesubmutant phenotype. Thus, SUB is a plant representative of the unusual class of atypical or “dead” kinases that is best studied in animals (Kroiher et al.2001; Boudeau et al. 2006; Castells and Casacuberta 2007). However, it should be noted that in some instances even the signaling mechanism of biochemically active RLKs, such as ACR4 or FEI1, may not absolutely require a functional kinase domain in vivo (Gifford et al. 2005; Xu et al.2008). Thus, it is conceivable that redundant activities exist in multiprotein receptor complexes that could substitute for the absence of kinase activity of a single receptor. In any case, it is an exciting challenge to unravel a signaling pathway mediated by an atypical RLK.
Anthers are the male reproductive tissues of plants. They constitute micro- sporangia within which the male germline develops. The pollen mother cells (PMCs), or microsporocytes, which will undergo meiosis, are contained in the four corners of the anther and within concentric cell somatic layers, the tapetum, the middle layer, and the endothecium subjacent to the epidermis. The PMCs and the cell layers are derived from an archesporial cell through a set of regulated stereotypic cell divisions. As a model system to study organogenesis, early anther development has met with considerable interest and it has become apparent that a number of RLKs are involved in the establishment of the different cell layers during early anther ontogenesis (Feng and Dickinson2007; Feng and Dickinson2010a).
Somatic cell fate in general appears to be under the control of the redundantly acting CLV1 homologs BAM1 and BAM2 (Hord et al. 2006). It was suggested thatBAM1/2restrict proliferation of sporogenous cells and/or promote differentiation of the peripheral somatic cells. Formation of the tapetum is under the control of another set of LRR-RLKs. Defects in the RLK genesEXTRA MICROSPOROCYTES1 (EMS1)/EXTRA SPOROGENOUS CELLS (EXS) (Canales et al. 2002; Zhao et al.
2002) and SOMATIC EMBRYOGENESIS RECEPTOR KINASE1 (SERK1) and SERK2(Albrecht et al.2005; Colcombet et al.2005) result in an overproliferation of PMCs and the absence of the tapetum. A similar phenotype is observed in mutants with a defect inTAPETUM DETERMINANT1(TPD1) predicted to encode a small and secreted protein (Yang et al.2003). Interestingly, the function of theEMS1/EXS andTPD1genes is conserved in evolution (Nonomura et al.2003; Zhao et al.2008).
The similar phenotypes suggest that all genes act in the same process, and genetic and biochemical data indicate that EMS1/EXS and TPD1 constitute a receptor-ligand pair (Jia et al.2008). There is evidence that SERK1/2 can form homodimers in plant cells (Albrecht et al.2005) but it remains to be shown if SERK1/2 are part of the EMS1/
EXS receptor complex or if they act in parallel to EMS1/EXS.
The mutant phenotype ofems1/exs,serk1/2, andtpd1mutants suggests that this signaling pathway either regulates PMC proliferation or the specification of tapetal cells. Recent evidence, however, indicates thatEMS1/EXSregulates cell proliferation in the tapetal cell monolayer (Feng and Dickinson2010b). Tapetum development, and middle layer formation, is also regulated by the RLK RPK2 (Mizuno et al.2007).
4 Conclusions
With this chapter, we have provided a brief account of some of the better under- stood intercellular signaling aspects of floral development. The last 20 years have witnessed a series of landmark papers and an overall impressive body of exciting work. The observation that TFs and sRNAs can move between cells has revolutionized our thinking about how plant cells communicate. After decades of hard but fruitless work, the molecular nature of the florigen is finally being unraveled. While 25 years ago some people would argue that the cell wall would make it unlikely that plants possess RLKs, we now know that RLKs do exist and in truly staggering numbers which by far exceed the number of different RLKs in humans. As one may expect, however, a number of questions remain. For example, why do some TFs and sRNAs move through PD and others don’t? There is a perhaps surprising specificity in the mechanisms that regulate transport of molecules through PD. How is this achieved? With respect to RLK signaling mechanisms, a major area requiring even more research relates to the identification and analysis of their ligands, as we know only a handful of specific ligands. In addition, the downstream signaling components have been identified for only a few RLKs, and the function of only a comparably small number of RLKs is known at all. Finally, how is the information flow mediated by different RLKs integrated to direct proper cellular behavior? As already indicated, we have come a long way.
These are exciting times in plant signal transduction, and no doubt research will be very rewarding for many years to come.
Acknowledgments We thank members of our lab for fruitful discussions. We apologize to colleagues whose work we could not cite due to space restrictions. Work on signaling in floral organs in the Schneitz lab is funded by grants SCHN 723/1-3, SCHN 723/3-2, and SCHN 723/6-1 from the German Research Council (DFG) and by the Free State of Bavaria.
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