Nondisjunction in Favor of a Chromosome: The Mechanism of Rye B Chromosome Drive during Pollen Mitosis

B chromosomes (Bs) are supernumerary components of the genome and do not confer any advantages on the organisms that harbor them. The maintenance of Bs in natural populations is possible by their transmission at higher than Mendelian frequencies. Although drive is the key for understanding B chromosomes, the mechanism is largely unknown. We provide direct insights into the cellular mechanism of B chromosome drive in the male gametophyte of rye (Secale cereale). We found that nondisjunction of Bs is accompanied by centromere activity and is likely caused by extended cohesion of the B sister chromatids. The B centromere originated from an A centromere, which accumulated B-specific repeats and rearrangements. Because of unequal spindle formation at the first pollen mitosis, nondisjoined B chromatids preferentially become located toward the generative pole. The failure to resolve pericentromeric cohesion is under the control of the B-specific nondisjunction control region. Hence, a combination of nondisjunction and unequal spindle formation at first pollen mitosis results in the accumulation of Bs in the generative nucleus and therefore ensures their transmission at a higher than expected rate to the next generation.     

oiwa,a Female Gametophytic Mutant Impaired in a Mitochondrial Manganese-Superoxide Dismutase,Reveals Crucial Roles for Reactive Oxygen Species during Embryo Sac Development and Fertilization in Arabidopsis

Reactive oxygen species (ROS) can function as signaling molecules, regulating key aspects of plant development, or as toxic compounds leading to oxidative damage. In this article, we show that the regulation of ROS production during megagametogenesis is largely dependent on MSD1, a mitochondrial Mn-superoxide dismutase. Wild-type mature embryo sacs show ROS exclusively in the central cell, which appears to be the main source of ROS before pollination. Accordingly, MSD1 shows a complementary expression pattern. MSD1 expression is elevated in the egg apparatus at maturity but is downregulated in the central cell. The oiwa mutants are characterized by high levels of ROS detectable in both the central cell and the micropylar cells. Remarkably, egg apparatus cells in oiwa show central cell features, indicating that high levels of ROS result in the expression of central cell characteristic genes. Notably, ROS are detected in synergid cells after pollination. This ROS burst depends on stigma pollination but precedes fertilization, suggesting that embryo sacs sense the imminent arrival of pollen tubes and respond by generating an oxidative environment. Altogether, we show that ROS play a crucial role during female gametogenesis and fertilization. MSD1 activity seems critical for maintaining ROS localization and important for embryo sac patterning.     

Successful Fertilization Requires the Presence of at Least One Major O-Acetylserine(thiol)lyase for Cysteine Synthesis in Pollen of Arabidopsis

The synthesis of cysteine (Cys) is a master control switch of plant primary metabolism that coordinates the flux of sulfur with carbon and nitrogen metabolism. In Arabidopsis (Arabidopsis thaliana), nine genes encode for O-acetylserine(thiol)lyase (OAS-TL)-like proteins, of which the major isoforms, OAS-TL A, OAS-TL B, and OAS-TL C, catalyze the formation of Cys by combining O-acetylserine and sulfide in the cytosol, the plastids, and the mitochondria, respectively. So far, the significance of individual OAS-TL-like enzymes is unresolved. Generation of all major OAS-TL double loss-of-function mutants in combination with radiolabeled tracer studies revealed that subcellular localization of OAS-TL proteins is more important for efficient Cys synthesis than total cellular OAS-TL activity in leaves. The absence of oastl triple embryos after targeted crosses indicated the exclusiveness of Cys synthesis by the three major OAS-TLs and ruled out alternative sulfur fixation by other OAS-TL-like proteins. Analyses of oastlABC pollen demonstrated that the presence of at least one functional OAS-TL isoform is essential for the proper function of the male gametophyte, although the synthesis of histidine, lysine, and tryptophan is dispensable in pollen. Comparisons of oastlABC pollen derived from genetically different parent plant combinations allowed us to separate distinct functions of Cys and glutathione in pollen and revealed an additional role of glutathione for pollen germination. In contrast, female gametogenesis was not affected by the absence of major OAS-TLs, indicating significant transport of Cys into the developing ovule from the mother plant.Sulfur assimilation in plants is hallmarked by two reaction sequences, namely sulfate reduction and Cys synthesis. The sulfate reduction pathway consists of three steps and produces sulfide from sulfate, which is available in the soil and transported into the roots by specific transporters (Takahashi et al., 2011). Sulfide is subsequently incorporated into the amino acid O-acetylserine (OAS) by O-acetylserine(thiol)lyase (OAS-TL; EC 2.5.1.47) to produce Cys (Hell and Wirtz, 2011). Cys then serves as the sulfur source for all organic metabolites containing reduced sulfur in plants, including proteins, cofactors, and secondary metabolites. The tripeptide glutathione (GSH) is one of the most important Cys-derived metabolites, since it has an important function in redox homeostasis and the control of development (Meyer and Rausch, 2008). Impaired GSH synthesis negatively affects growth of the shoot and root system of Arabidopsis (Arabidopsis thaliana; Vernoux et al., 2000; Xiang et al., 2001), and loss-of-function mutants for the first enzyme (GSH1, Glu-Cys ligase; EC 6.3.2.2) or the second enzyme (GSH2, glutathione synthase; EC 6.3.2.3) of the two-step pathway leading to GSH formation show an embryo- and seedling-lethal phenotype, respectively (Cairns et al., 2006; Pasternak et al., 2008).Cys synthesis by OAS-TL constitutes the direct link between carbon and nitrogen (OAS) as well as sulfur (sulfide) metabolism and, therefore, can be designated as one of the central reactions in plant primary metabolism. The genome of the model plant Arabidopsis encodes nine OAS-TL-like enzymes: OAS-TL A1 (At4g14880), OAS-TL B (At2g43750), and OAS-TL C (At3g59760) are the major isoforms and are localized in the cytosol, plastids, and mitochondria, respectively (Jost et al., 2000). OAS-TL A2 (At3g22460) encodes a truncated and nonfunctional protein (Jost et al., 2000). In the following, therefore, OAS-TL A1 is referred to as OAS-TL A. CYS D1 (At3g04940) and CYS D2 (At5g28020) show OAS-TL activity in vitro (Yamaguchi et al., 2000). Whether they contribute to net Cys synthesis in vivo is unknown (Heeg et al., 2008). CS26 (At3g03630) encodes a plastidic S-sulfocysteine synthase, which prefers thiosulfate instead of sulfide as substrate and produces S-sulfocysteine (Bermúdez et al., 2010). Whether thiosulfate is taken up from the soil or formed within the plant is unclear, but its presence in Arabidopsis was demonstrated (Tsakraklides et al., 2002). However, the synthesis of S-sulfocysteine from thiosulfate potentially constitutes an alternative sulfur fixation pathway. So far, CS26 was shown to be important for the regulation of redox homeostasis in plastids under certain stress conditions (Bermúdez et al., 2010). DES1 (At5g28030; formerly known as CS-LIKE) is a Cys desulfhydrase (EC 4.4.1.15) that releases sulfide in the cytosol (Alvarez et al., 2010). As a Cys-consuming enzyme, it contributes to Cys homeostasis, especially in late vegetative development and under certain stress conditions (Alvarez et al., 2010, 2012). CYS C1 (At3g61440), finally, encodes a mitochondrial β-cyanoalanine synthase (EC 4.4.1.9), which detoxifies cyanide by incorporation into Cys (Yamaguchi et al., 2000; Watanabe et al., 2008a; García et al., 2010). The major isoforms OAS-TL A, OAS-TL B, and OAS-TL C as well as CYS D1 and CYS D2 can interact with serine acetyltransferase (SAT; EC 2.3.1.30) in the cysteine synthase complex (CSC; Heeg et al., 2008). Although SAT acetylates Ser at the hydroxyl group to form OAS, the direct substrate of OAS-TL, formation of the CSC has no substrate-channeling function but contributes to the demand-driven regulation of Cys synthesis (Hell and Wirtz, 2011).The subcellular compartmentation of Cys precursor formation is a remarkable feature of Cys synthesis in higher plants that implies a high degree of regulation between the participating compartments: while sulfate is exclusively reduced to sulfide in plastids (Takahashi et al., 2011), the synthesis of OAS and the incorporation of sulfide take place in all three compartments where SAT and OAS-TL are present, namely in the cytosol, plastids, and mitochondria. Reverse genetics approaches proved a certain redundancy between the different SAT and OAS-TL isoforms, which demonstrates that sulfide, OAS, and Cys can be exchanged between these compartments (Haas et al., 2008; Heeg et al., 2008; Watanabe et al., 2008a, 2008b). Indeed, sulfide can easily diffuse through membranes (Mathai et al., 2009), but OAS and Cys need to be actively transported. However, the identity of these transporters is unknown. Although sulfide, OAS, and Cys can pass the mitochondrial membrane (Wirtz et al., 2012), the loss-of-function mutant for mitochondrial OAS-TL C is the only single oastl knockout mutant that displays a significant growth phenotype (Heeg et al., 2008). This result was astonishing, since OAS-TL C contributes only 5% to extractable foliar OAS-TL activity (Heeg et al., 2008). The retarded growth of the oastlC mutant, however, cannot be explained by the lack of sulfide detoxification in mitochondria by OAS-TL C, due to an alternative detoxification mechanism for sulfide in mitochondria (Birke et al., 2012). These data question the total redundancy between the different OAS-TL isoforms and suggest specific functions in the different subcellular compartments.Despite its central position in the primary metabolism of higher plants, fundamental questions about Cys synthesis are still unanswered. First, the contribution of OAS-TL-like proteins, especially CYS D1, CYS D2, and CS26, to the fixation of sulfur in planta is unknown. Second, the significance of Cys synthesis by the major OAS-TL proteins in the different subcellular compartments during sporophyte and gametophyte development is unclear. In this study, we addressed these questions using a reverse genetics approach. We were able to prove that fixation of sulfur is carried out exclusively by the major OAS-TL isoforms OAS-TL A, OAS-TL B, and OAS-TL C and elucidated specific functions for OAS-TL A in the cytosol and OAS-TL C in mitochondria of leaf cells. Furthermore, we demonstrate that Cys can be supplied by the mother plant for the development of female gametophytes lacking OAS-TL activity. In contrast, the presence of at least one functional OAS-TL isoform is essential in the male gametophyte.     

ABNORMAL POLLEN TUBE GUIDANCE1, an Endoplasmic Reticulum-Localized Mannosyltransferase Homolog of GLYCOSYLPHOSPHATIDYLINOSITOL10 in Yeast and PHOSPHATIDYLINOSITOL GLYCAN ANCHOR BIOSYNTHESIS B in Human,Is Required for Arabidopsis Pollen Tube Micropylar Guidance and Embryo Development

The perception and response of pollen tubes to the female guidance signals are crucial for directional pollen tube growth inside female tissues, which leads to successful reproduction. In pursuing the mechanisms underlying this biological process, we identified the Arabidopsis (Arabidopsis thaliana) abnormal pollen tube guidance1 (aptg1) mutant, whose pollen tubes showed compromised micropylar guidance. In addition to its male defect, the aptg1 mutant showed embryo lethality. APTG1 encodes a putative mannosyltransferase homolog to human PHOSPHATIDYLINOSITOL GLYCAN ANCHOR BIOSYNTHESIS B and yeast (Saccharomyces cerevisiae) GLYCOSYLPHOSPHATIDYLINOSITOL10 (GPI10), both of which are involved in the biosynthesis of GPI anchors. We found that APTG1 was expressed in most plant tissues, including mature pollen, pollen tubes, mature embryo sacs, and developing embryos. By fluorescence colabeling, we showed that APTG1 was localized in the endoplasmic reticulum, where GPI anchors are synthesized. Disruption of APTG1 affected the localization of COBRA-LIKE10, a GPI-anchored protein important for pollen tube growth and guidance. The results shown here demonstrate that APTG1 is involved in both vegetative and reproductive development in Arabidopsis, likely through processing and proper targeting of GPI-anchored proteins.Double fertilization is the biological basis for seed propagation and plant reproduction in angiosperms. Pollen tubes grow through maternal tissue to deliver the immobile sperm cells into the female gametophyte (embryo sac). During this process, pollen tube guidance into the micropyle is a critical step and is precisely regulated (Dresselhaus and Franklin-Tong, 2013). Female guidance signals are generated by both sporophytic and gametophytic tissues and operate at different stages during pollen tube growth. The sporophytic signal directs the growth of pollen tubes in the stigma, style, and transmitting tract. The signal that induces pollen tubes to turn to the funiculus and grow into the micropyle is termed gametophytic guidance (Shimizu and Okada, 2000; Higashiyama et al., 2003). Extensive cellular and genetic studies have demonstrated that female gametophytes play key roles in the micropylar guidance of pollen tubes (Kasahara et al., 2005; Márton et al., 2005; Chen et al., 2007; Alandete-Saez et al., 2008; Okuda et al., 2009; Kessler and Grossniklaus, 2011; Takeuchi and Higashiyama, 2011). The molecular natures of such guidance signals have been gradually revealed in recent years (i.e. small peptides secreted by the female gametophyte, egg apparatus, or synergid cells; Márton et al., 2005; Jones-Rhoades et al., 2007; Okuda et al., 2009).Pollen tubes need to perceive the female guidance signals at the cell surface to initiate intracellular responses for directional growth. However, the mechanisms of pollen tube perception are still obscure. A few male factors involved in signal perception during pollen tube growth into ovules have been identified. For example, the Arabidopsis (Arabidopsis thaliana) sperm cell-specific protein HAPLESS2/GENERATIVE CELL-SPECIFIC1 was necessary for pollen tubes to target the micropyle (von Besser et al., 2006). Arabidopsis CATION/PROTON EXCHANGER21 (CHX21) and CHX23 encode K⁺ transporters in growing pollen tubes. Pollen grains of the chx21 chx23 double mutant germinated and extended a normal tube in the transmitting tract, but their targeting of the funiculus failed (Lu et al., 2011). Arabidopsis POLLEN DEFECTIVE IN GUIDANCE1 (POD1) was expressed in pollen grains, pollen tubes, and synergid cells. The pod1 pollen tubes showed defective micropylar guidance (Li et al., 2011). The tip of the pollen tube has been hypothesized to be the site of cue perception for micropyle-directed growth. The Arabidopsis Rab GTPase RABA4D was localized at the tips of growing pollen tubes. Pollen tubes with defective RABA4D had severely reduced growth rates and ovule targeting (Szumlanski and Nielsen, 2009). Recently, two receptor-like kinases at the apical plasma membrane (PM) of growing pollen tubes, LOST IN POLLEN TUBE GUIDANCE1 (LIP1) and LIP2, were demonstrated to guide pollen tubes to the micropyle by perceiving the AtLURE1 signal from synergid cells (Liu et al., 2013).Glycosylphosphatidylinositol (GPI) anchoring provides a strategy for targeting proteins to the outer layer of the PMs in eukaryotic cells. GPI anchors are synthesized inside the endoplasmic reticulum (ER) and are attached to proteins by posttranslational modifications in the ER. After processing, GPI-anchored proteins (GPI-APs) are transported to the cell surface following an unknown trafficking route and anchored at the cell surface (Maeda and Kinoshita, 2011). GPI-APs play very important roles in plant reproductive development (Gillmor et al., 2005; Ching et al., 2006; DeBono et al., 2009). An Arabidopsis putative GPI-AP, LORELEI, functioned in pollen tube reception of female signals, double fertilization, and early seed development (Capron et al., 2008; Tsukamoto et al., 2010). Arabidopsis COBRA-LIKE10 (COBL10), a GPI-AP, regulates the polar deposition of wall components in pollen tubes growing inside female tissues and is critical for micropylar guidance (Li et al., 2013). The conserved backbone of GPI anchors in eukaryotes is ethanolamine phosphate-6-Man-α-1,2-Man-α-1,6-Man-α-1,4-glucosamine-α-1,6-myoinositol phospholipid. During the biosynthesis of GPI anchors, monosaccharides, fatty acids, and phosphoethanolamines are sequentially added onto phosphatidylinositol. This process involves at least 16 enzymes and cofactors in mammals, including PHOSPHATIDYLINOSITOL GLYCAN ANCHOR BIOSYNTHESIS (PIG) A, B, C, F, G, H, L, M, N, O, P, Q, V, W, X, and Y (Maeda and Kinoshita, 2011). The core structure of the GPI anchor contains three Man residues donated by the substrate dolichol-phosphate-Man. GPI mannosyltransferases were required for adding the three Man residues of the GPI anchor in the ER lumen (Maeda and Kinoshita, 2011). Arabidopsis PEANUT1 (PNT1) is a homolog of the mammalian GPI mannosyltransferase PIG-M, involved in the addition of the first Man during the biosynthesis of the GPI anchor. The pnt1 mutant showed the defect of pollen viability and embryo development (Gillmor et al., 2005). PIG-B of human and GPI10 of yeast (Saccharomyces cerevisiae) encode GLYCOSYLPHOSPHATIDYLINOSITOL MANNOSYLTRANSFERASE3, involved in the addition of the third Man during the biosynthesis of the GPI anchor (Takahashi et al., 1996; Sütterlin et al., 1998). Mutation of PIG-B and GPI10 resulted in the accumulation of the GPI intermediate Man2-glucosamine-(acyl) phosphatidylinositol and led to cell death in yeast.In this study, we identified the ER-localized ABNORMAL POLLEN TUBE GUIDANCE1 (APTG1), an Arabidopsis homolog of PIG-B and GPI10. Pollen tubes of the aptg1 mutant showed compromised directional growth to the micropyle and lost the apical PM localization of COBL10. Besides the male defect, the mutant showed embryo lethality. In addition, reducing the expression of APTG1 resulted in defective seedling growth, indicating that APTG1 plays important roles in both reproductive and vegetative development.     

Role of Aminoalcoholphosphotransferases 1 and 2 in Phospholipid Homeostasis in Arabidopsis

Aminoalcoholphosphotransferase (AAPT) catalyzes the synthesis of phosphatidylcholine (PC) and phosphotidylethanolamine (PE), which are the most prevalent membrane phospholipids in all eukaryotic cells. Here, we show that suppression of AAPTs results in extensive membrane phospholipid remodeling in Arabidopsis thaliana. Double knockout (KO) mutants that are hemizygous for either aapt1 or aapt2 display impaired pollen and seed development, leading to embryotic lethality of the double KO plants, whereas aapt1 or aapt2 single KO plants show no overt phenotypic alterations. The growth rate and seed yield of AAPT RNA interference (RNAi) plants are greatly reduced. Lipid profiling shows decreased total galactolipid and phospholipid content in aapt1-containing mutants, including aapt1, aapt1/aapt1 aapt2/AAPT2, aapt1/AAPT1 aapt2/aapt2, and AAPT RNAi plants. The level of PC in leaves was unchanged, whereas that of PE was reduced in all AAPT-deficient plants, except aapt2 KO. However, the acyl species of PC was altered, with increased levels of C34 species and decreased C36 species. Conversely, the levels of PE and phosphatidylinositol were decreased in C34 species. In seeds, all AAPT-deficient plants, including aapt2 KO, displayed a decrease in PE. The data show that AAPT1 and AAPT2 are essential to plant vegetative growth and reproduction and have overlapping functions but that AAPT1 contributes more than AAPT2 to PC production in vegetative tissues. The opposite changes in molecular species between PC and PE and unchanged PC level indicate the existence of additional pathways that maintain homeostatic levels of PC, which are crucial for the survival and proper development of plants.     

Brassinosteroid Regulates Cell Elongation by Modulating Gibberellin Metabolism in Rice

Brassinosteroid (BR) and gibberellin (GA) are two predominant hormones regulating plant cell elongation. A defect in either of these leads to reduced plant growth and dwarfism. However, their relationship remains unknown in rice (Oryza sativa). Here, we demonstrated that BR regulates cell elongation by modulating GA metabolism in rice. Under physiological conditions, BR promotes GA accumulation by regulating the expression of GA metabolic genes to stimulate cell elongation. BR greatly induces the expression of D18/GA3ox-2, one of the GA biosynthetic genes, leading to increased GA₁ levels, the bioactive GA in rice seedlings. Consequently, both d18 and loss-of-function GA-signaling mutants have decreased BR sensitivity. When excessive active BR is applied, the hormone mostly induces GA inactivation through upregulation of the GA inactivation gene GA2ox-3 and also represses BR biosynthesis, resulting in decreased hormone levels and growth inhibition. As a feedback mechanism, GA extensively inhibits BR biosynthesis and the BR response. GA treatment decreases the enlarged leaf angles in plants with enhanced BR biosynthesis or signaling. Our results revealed a previously unknown mechanism underlying BR and GA crosstalk depending on tissues and hormone levels, which greatly advances our understanding of hormone actions in crop plants and appears much different from that in Arabidopsis thaliana.     

The Arabidopsis Ethylene/Jasmonic Acid-NRT Signaling Module Coordinates Nitrate Reallocation and the Trade-Off between Growth and Environmental Adaptation

Stresses decouple nitrate assimilation and photosynthesis through stress-initiated nitrate allocation to roots (SINAR), which is mediated by the nitrate transporters NRT1.8 and NRT1.5 and functions to promote stress tolerance. However, how SINAR communicates with the environment remains unknown. Here, we present biochemical and genetic evidence demonstrating that in Arabidopsis thaliana, ethylene (ET) and jasmonic acid (JA) affect the crosstalk between SINAR and the environment. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays showed that ethylene response factors (ERFs), including OCTADECANOID-RESPONSIVE ARABIDOPSIS AP2/ERF59, bind to the GCC boxes in the NRT1.8 promoter region, while ETHYLENE INSENSITIVE3 (EIN3) binds to the EIN3 binding site motifs in the NRT1.5 promoter. Genetic assays showed that cadmium and sodium stresses initiated ET/JA signaling, which converged at EIN3/EIN3-Like1 (EIL1) to modulate ERF expression and hence to upregulate NRT1.8. By contrast, ET and JA signaling mediated the downregulation of NRT1.5 via EIN3/EIL1 and other, unknown component(s). SINAR enhanced stress tolerance and decreased plant growth under nonstressed conditions through the ET/JA-NRT1.5/NRT1.8 signaling module. Interestingly, when nitrate reductase was impaired, SINAR failed to affect either stress tolerance or plant growth. These data suggest that SINAR responds to environmental conditions through the ET/JA-NRT signaling module, which further modulates stress tolerance and plant growth in a nitrate reductase-dependent manner.     

A Chimeric Arabinogalactan Protein Promotes Somatic Embryogenesis in Cotton Cell Culture

Arabinogalactan proteins (AGPs) are a family of extracellular plant proteoglycans implicated in many aspects of plant growth and development, including in vitro somatic embryogenesis (SE). We found that specific AGPs were produced by cotton (Gossypium hirsutum) calli undergoing SE and that when these AGPs were isolated and incorporated into tissue culture medium, cotton SE was promoted. When the AGPs were partly or fully deglycosylated, SE-promoting activity was not diminished. Testing of AGPs separated by reverse-phase high-performance liquid chromatography revealed that the SE-promoting activity resided in a hydrophobic fraction. We cloned a full-length complementary DNA (cotton PHYTOCYANIN-LIKE ARABINOGALACTAN-PROTEIN1 [GhPLA1]) that encoded the protein backbone of an AGP in the active fraction. It has a chimeric structure comprising an amino-terminal signal sequence, a phytocyanin-like domain, an AGP-like domain, and a hydrophobic carboxyl-terminal domain. Recombinant production of GhPLA1 in tobacco (Nicotiana tabacum) cells enabled us to purify and analyze a single glycosylated AGP and to demonstrate that this chimeric AGP promotes cotton SE. Furthermore, the nonglycosylated phytocyanin-like domain from GhPLA1, which was bacterially produced, also promoted SE, indicating that the glycosylated AGP domain was unnecessary for in vitro activity.Arabinogalactan proteins (AGPs) comprise a diverse group of plant proteoglycans (for review, see Fincher et al., 1993; Nothnagel, 1997; Seifert and Roberts, 2007; Ellis et al., 2010). They are structurally complex, generally consisting of a Pro-, Ala-, Ser-, and Thr-rich protein backbone that is extensively modified, principally by hydroxylation of Pro residues (to Hyp) and subsequent glycosylation through O-linkages with type II arabinogalactans (Tan et al., 2003; Shimizu et al., 2005). Many AGPs also have a C-terminal hydrophobic domain that is processed and replaced with a glycosylphosphatidylinositol (GPI) anchor, which acts to tether the molecule to the extracellular face of the plasma membrane (Schultz et al., 1998). AGPs are also defined by their ability to be bound and precipitated by the synthetic dye β-glucosyl Yariv reagent (β-GlcY) and related molecules (Yariv et al., 1967). These dyes have been useful in isolating, localizing, and quantifying AGPs.AGPs are grouped into three subclasses (Schultz et al., 2002): AGPs have an N-terminal signal sequence, an arabinogalactosylated domain, and a hydrophobic C-terminal domain; “chimeric AGPs” contain at least one arabinogalactosylated domain and a domain with an unrelated motif; while “hybrid AGPs” contain arabinogalactosylated as well as different Pro/Hyp-rich glycoprotein motifs.AGPs are implicated in many aspects of plant cell growth and development. Historically, it was not possible to assign roles to individual AGPs, as tests were conducted with unfractionated mixtures of AGPs. More recently, individual AGPs, mainly from Arabidopsis (Arabidopsis thaliana), have been studied using techniques such as mutant analysis and gene knockout/silencing, providing evidence for roles of individual AGPs in cell expansion, root and seed regeneration, the coordination of vascular development, both male and female gametogenesis, the development of cotton fibers, and as contributors to plant stem strength (Shi et al., 2003; van Hengel and Roberts, 2003; Acosta-García and Vielle-Calzada, 2004; Motose et al., 2004; Yang et al., 2007; Levitin et al., 2008; Coimbra et al., 2009; Li et al., 2010; MacMillan et al., 2010).Conditioned media from in vitro embryogenic cultures contain factors that can promote somatic embryogenesis (SE), implying the presence of secreted signaling molecules (de Vries et al., 1988). There is evidence that secreted AGPs, which are components of conditioned media, are involved in SE. For example, SE in carrot (Daucus carota) and spruce (Picea abies) cell cultures was promoted when AGPs from conditioned media were added exogenously (Kreuger and van Holst, 1993; Egertsdotter and von Arnold, 1995). Subsequent studies showed the association of particular AGP epitopes with SE-promoting activity and the involvement of AGPs in SE for several other species (Kreuger et al., 1995; McCabe et al., 1997; Toonen et al., 1997; Chapman et al., 2000; Saare-Surminski et al., 2000; Ben Amar et al., 2007). There is also evidence that SE-promoting AGPs may be cleaved by an endochitinase (Egertsdotter and von Arnold, 1988; Domon et al., 2000; van Hengel et al., 2001, 2002), but neither the identity of the individual AGP(s) involved in promoting SE nor the mechanism of action has been established.In this study, we focused on SE in cotton (Gossypium hirsutum ‘Coker 315’), which is a limiting step in cotton transformation, and the potential role of AGPs in this process. We show that cotton calli undergoing somatic embryogenesis secrete an AGP fraction that promotes SE when incorporated back into the growth medium. We report the cloning and sequencing of a complementary DNA (cDNA) encoding a chimeric AGP present in this fraction and show that this molecule promotes SE.