POPCORN functions in the auxin pathway to regulate embryonic body plan and meristem organization in Arabidopsis.

The shoot and root apical meristems (SAM and RAM) formed during embryogenesis are crucial for postembryonic plant development. We report the identification of POPCORN (PCN), a gene required for embryo development and meristem organization in Arabidopsis thaliana. Map-based cloning revealed that PCN encodes a WD-40 protein expressed both during embryo development and postembryonically in the SAM and RAM. The two pcn alleles identified in this study are temperature sensitive, showing defective embryo development when grown at 22°C that is rescued when grown at 29°C. In pcn mutants, meristem-specific expression of WUSCHEL (WUS), CLAVATA3, and WUSCHEL-RELATED HOMEOBOX5 is not maintained; SHOOTMERISTEMLESS, BODENLOS (BDL) and MONOPTEROS (MP) are misexpressed. Several findings link PCN to auxin signaling and meristem function: ectopic expression of DR5(rev):green fluorescent protein (GFP), pBDL:BDL-GFP, and pMP:MP-β-glucuronidase in the meristem; altered polarity and expression of pPIN1:PIN1-GFP in the apical domain of the developing embryo; and resistance to auxin in the pcn mutants. The bdl mutation rescued embryo lethality of pcn, suggesting that improper auxin response is involved in pcn defects. Furthermore, WUS, PINFORMED1, PINOID, and TOPLESS are dosage sensitive in pcn, suggesting functional interaction. Together, our results suggest that PCN functions in the auxin pathway, integrating auxin signaling in the organization and maintenance of the SAM and RAM.


Introduction
Embryogenesis in angiosperms begins with the division of the zygote into the precursor cells of embryo proper and suspensor. Subsequently, the establishment of apical-basal axis, radial organization of tissues, and initiation of cotyledons and apical meristems together constitute the embryonic body plan (De Smet et al., 2010). In recent years, Arabidopsis has been investigated as a model plant to address the functions of key genetic factors that contribute to embryogenesis. Auxin transport and signaling play critical roles in the establishment of embryonic body plan (Friml et al., 2003;Jenik et al., 2007).
During the transition from globular to heart embryo, the apical domain is partitioned to form the shoot apical meristem (SAM) and the cotyledons whereas the basal domain differentiates into the hypocotyl and the embryonic root apical meristem (RAM). Altered auxin signaling can result in apical patterning defects with cotyledons that are fused as seen in the long hypocotyl5 and hy5homolog (hy5 hyh) double mutant (Sibout et al., 2006) or completely absent as seen in the auxin response factor (ARF) double mutant, monopteros and nonphototropic hypocotyl4 (mp nph4) (Hardtke et al., 2004). The gnom mutant affected in auxin transport, fails to establish apical-basal patterning resulting in a ball-like embryo with no cotyledons (Mayer et al., 1993). The products of PINFORMED (PIN) gene family members, PIN1, PIN3, PIN4 and PIN7 are differentially and polarly localized to coordinate the transport of auxin during embryo patterning, and quadruple mutants of all four genes display filamentous embryos with apicalbasal polarity defects (Friml et al., 2003). Polar targeting of PIN proteins in cells is regulated via phosphorylation by the kinase PINOID (PID) (Michniewicz et al., 2007). The CUP-SHAPED COTYLEDON (CUC) genes are involved in the partitioning of the apical domain of the embryo. In the cuc1 cuc2 double mutant, the lack of or a defect in the SAM, along with fused cotyledons, result in the termination of seedling development (Aida et al., 1997). CUC gene overexpression occurs in the pin-1 pid-2 double mutant, leading to the inhibition of cotyledon primordia initiation (Furutani et al., 2004). Furthermore, CUC genes have been shown to be essential for the expression of the SHOOTMERISTEMLESS (STM) gene (Aida et al., 1999).
In the root, the plant hormone auxin has been shown to play major roles in RAM establishment and function (Overvoorde et al., 2010). The auxin response regulators BODENLOS (BDL) and MP mediate auxin signaling and provides root patterning information by activating the PLETHORA (PLT) genes (Hamann et al., 2002;Nawy et al., 2008). The two PLT genes provide positional information to establish the root stem cell niche through the auxin pathway (Aida et al., 2004).
The homeobox gene WUSCHEL-RELATED HOMEOBOX5 (WOX5) is expressed specifically in the quiescent center (QC) of the root and wox5 mutants fail to maintain stem cells in the RAM (Sarkar et al., 2007). Auxin and cytokinin together play antagonistic roles in the SAM and RAM partly by controlling the expression of ARABIDOPSIS RESPONSE REGULATOR7 (ARR7) and ARR15 (Zhao et al., 2010).
Proper organization and maintenance of both meristems depends on coordinated cell divisions that balance the number of stem cells and daughter cells that contribute to the initiation of organs. A number of transcription and signaling factors have been implicated in the functions of shoot and root meristems. These include homeobox genes STM and WUSCHEL (WUS) (Mayer et al., 1998;Lenhard et al., 2002), signaling factors CLAVATA (CLV1, CLV2 and CLV3) in the SAM (Fletcher et al., 1999;Schoof et al., 2000); and homeobox gene WOX5 and plant specific AP2 domain containing PLT transcriptional factors in the RAM (Aida et al., 2004;Sarkar et al., 2007).
Despite the number of meristem regulatory genes that have been identified thus far, mutations in the majority of these genes (i.e., WUS, CLV, STM) does not result in embryo lethality. This implies that, in addition to the possible existence of unknown genes that function both in the embryo patterning and meristem organization, there is a significant overlap and/or redundancy in the functions of these known genes. In this study we report the identification of a new and PID affecting the embryonic and postembryonic development in Arabidopsis.

Map-based cloning of PCN, a WD-40 related protein
We have determined global gene expression during embryo development in Arabidopsis (Xiang et al., 2011). These datasets provide a resource for further genetic interrogation. Genetic screens and targeted reverse genetic approaches have been successful in identifying key genes in Arabidopsis embryo development. To identify hitherto unknown genes that are required for Arabidopsis embryogenesis, we analyzed T-DNA lines with insertions in selected candidate genes that are specifically or differentially expressed during zygote to globular stages of embryo development based on our global gene expression datasets (Xiang et al., 2011). One of the lines from this screen showed early embryo lethality among the segregating embryos with a phenotype resembling popped corn which we named "popcorn" (pcn). The pcn mutation segregated as a single, recessive allele. However, molecular analysis revealed that this T-DNA insertion was not linked to the mutant embryo phenotype. We therefore used a map-based cloning approach to isolate the PCN gene. A tight linkage was observed with cleaved amplified polymorphic sequences (CAPS) markers at the genomic positions 4144848 (SspI enzyme digestion) and 4369443 (RsaI enzyme digestion) with no recombination observed in the analysis of 1340 chromosomes (670 plants). The strong globular stage defects in pcn mutant suggest that the expression of gene(s) in this locus is likely critical for embryo patterning and development. We reasoned that the expressed genes in this genomic region would likely include PCN. The 224 kb region between 4144848 and 4369443 on chromosome 4 contains 53 genes of which 44 encode transposable elements. Of the remaining 9 loci, 5 are of unknown function and the other four are annotated as F-box (At4g07400), WD-40 (At4g07410), PQ loop repeat (At4g07390) and GTP-binding (At4g07524). Since TPL is also a WD-40 repeat containing protein that is critical for embryo patterning which involves specification of the apicalbasal polarity and cotyledon formation (Long et al., 2006), we predicted that At4g07410 may be associated with the defective phenotype. At4g07410 also had the highest expression level among the 53 genes in the deduced 224 kb chromosome 4 region in our microarray data for wild type (WT) globular embryos (Xiang et al., 2011).Sequencing of candidate genes in the mapped region of pcn identified a 35 bp deletion overlapping the 5 th exon and 5 th intron of At4g07410 genic region that would result in a premature stop codon of the predicted open reading frame ( Figure 1A). At4g07410 encodes 815-amino acid protein containing eight WD-40 repeats, six at the N-terminus and two at the C-terminus ( Figure 1B). The premature stop codon in pcn-1 was deduced to produce a truncated protein of 240 amino acids. In Arabidopsis, PCN, a single copy gene, shares 69% identity with At1g27470 at the amino acid level. Putative homologs of PCN are also present in other plant genera (Oryza, Sorghum, Populus, Ricinus and Vitis), yeast, zebra fish, and humans (Supplemental Figure 1). In none of these cases is any function known.
Using the At4g07410 sequence information, we screened for additional T-DNA insertional lines at this locus and identified a second allele of PCN, pcn-2 (Salk_022607) with an insertion in the 8 th exon ( Figure 1A). The pcn-2/+ plants segregated defective embryos similar in mutant phenotype to those observed in pcn-1. Heterozygous pcn-1/+ or pcn-2/+ plants did not exhibit any developmental defects other than producing 25% arrested and 75% normal embryos (n = 1028 for pcn-1, 1120 for pcn-2), indicating that pcn-1 and pcn-2 mutations are recessive. To confirm that the mutant embryo phenotypes observed in pcn-1 and pcn-2 are caused by the lesions in the At4g07410 locus, we introduced two constructs into heterozygous mutant pcn-1 and pcn-2 plants. The

Embryo patterning defects in pcn mutants
We investigated the developmental aspects of pcn using the pcn-1 allele (unless otherwise specified). Homozygous pcn seeds obtained from a pcn/+ mother plant that was grown at 22 0 C did not germinate. However, when cultured in vitro with auxin and cytokinin (Wu et al., 1992), these mutant embryos were able to form calli from which fertile plants could be regenerated. These homozygous pcn plants upon flowering produced embryos that were all defective. As this provided an advantage over the segregating heterozygote, we have used these nonsegregating homozygous lines for more detailed developmental studies (Supplemental Table 1A, Figure 2 and 3).
During WT embryogenesis, the zygote elongates and divides asymmetrically, generating a smaller apical and a larger basal cell ( Figure 3A and 3K; (Jurgens and Mayer, 1994). The first two divisions of the apical cell occur longitudinally, giving rise to the 4-cell embryo proper ( Figure 3C and 3M), followed by a round of horizontal divisions to establish the 8-cell embryo. Tangential divisions separate the protoderm from the inner cells at the dermatogen stage. Later the globular embryo initiates two cotyledonary primordia and a SAM. The descendants of the basal cell divide horizontally to generate a column of cells, the extra-embryonic suspensor. Only the uppermost cell of the basal lineage, the hypophysis contributes to the embryo. After a transverse division, the apical daughter cell of the hypophysis, the lens shape cell, gives rise to the QC of the root meristem, whereas the basal daughter cell forms the columella stem cells (Jurgens and Mayer, 1994). Development of pcn embryos was similar to WT until the 2-cell embryo stage ( Figure 3A and 3C vs. Figure 3K Table 1A). Since pcn-1 embryos displayed stronger defects than pcn-2, further analysis was done with pcn-1. The SAM region in the majority of arrested embryos was enlarged and the cotyledons lacked bilateral symmetry and appear more radialized ( Figure

Elevated temperature rescues pcn embryo lethality
While testing the growth of pcn plants at different temperatures, we noticed that the defects in pcn embryo development were strongly alleviated after shifting the regenerated homozygous plants from 22 0 C to 29 0 C at the onset of bolting. The majority of the pcn embryos produced at 29 0 C had two WT-like cotyledons (83%, Supplemental Table 1A) and developed into normal mature embryos. The remaining embryos had no cotyledon (< 5%), one cotyledon (10%), and three cotyledons (2%) (Supplemental Table 1A). Unlike seeds from pcn homozygous plants grown at 22 0 C, 95% of pcn seeds produced at 29 0 C were able to germinate on ½ MS medium without the requirement of exogenous hormones and produced fertile plants (Supplemental Table 1B, Figure 4B). We took advantage of the temperature sensitivity characteristic of this mutant and propagated pcn and genetic combinations involving pcn by first growing the plants at 22 0 C and then shifting them to 29 0 C at bolting unless specified otherwise.
To determine at which embryo stage PCN function is essential, pcn plants were grown at 22 0 C and flower buds were first emasculated then hand pollinated with pcn pollen. A few of the developing siliques were dissected to determine the developmental stage of the embryos. The plants were then shifted to 29 0 C to complete seed development. Mature seeds were either dissected to assess the embryo phenotypes or geminated on ½ MS medium. Our results from this study showed that the shift to 29 0 C must occur at or prior to the globular stage in order to rescue the pcn embryo development (Supplemental Table 1B). A shift in temperature post-globular stage of embryo development could not suppress embryo lethality. These observations further suggest that PCN function is required already for early phases of embryo development in Arabidopsis.

pcn mutations impact post-embryonic development
To address the post-embryonic functions of PCN, 29°C temperature rescued pcn mutant seeds were germinated and plant development was analyzed at 22°C growth conditions. pcn mutant rosettes were smaller with narrower leaf shape ( Figure 4B), and abnormal vein patterns ( Figure 4D) compared to WT ( Figure 4A and 4C). Flowering pcn plants were shorter and bushier than WT. Examination of pcn roots revealed abnormal division patterns in the quiescent center (QC) and columella cells of the root cap, and root growth was retarded compared to WT ( Figure 4F-4H). Lugol staining for starch granules as a marker for columella cell differentiation revealed defects in the root cap cells in addition to the RAM defects observed in pcn ( Figure 4H). Further, the expression of the QC marker WOX5 was expanded in pcn roots ( Figure 6N and 6O). These observations suggest that PCN function is also required after germination for several processes associated with meristems both in root and shoot development.

PCN encodes a nuclear protein that is expressed broadly in embryo proper and in post-embryonic meristems
To study PCN expression and subcellular localization in detail, two different translational fusions of PCN and GFP coding regions were expressed under the control of 1.9 kb PCN putative promoter region, one with PCN cDNA (pPCN: cPCN-GFP) and the other with PCN genomic fragment (containing introns, gPCN-GFP) ( Figure 5). Independent transgenic lines of both constructs displayed identical expression patterns. For brevity we will focus on results from the gPCN-GFP reporter. Our earlier study on global gene expression patterns during Arabidopsis embryo development (Xiang et al., 2011) showed that gPCN-GFP is expressed in the ovule but not in the pollen and after fertilization is expressed in the zygote suggestive of maternal specificity. During early phases of embryogenesis, gPCN-GFP signal was detected in all the cells of the embryo proper and the suspensor until the 32-cell embryo stage ( Figure 5A and 5B).
Subsequently, gPCN-GFP expression was not detectable in the lower suspensor cells, and by the heart stage the signal was completely absent in the suspensor ( Figure 5D and 5E), but remained detectable in the embryo proper ( Figure 5E and 5F).
In seedlings, gPCN-GFP was detected in the SAM and leaf primordia ( Figure 5G), but not in the hypocotyl ( Figure 5H). In the root, gPCN-GFP is expressed in the root meristem ( Figure 5I and 5J). gPCN-GFP is also expressed in pericycle cells, both quiescent ( Figure 5K) and actively dividing ( Figure 5K and 5L) that give rise to the lateral roots and the expression continued throughout the development of lateral root primordia ( Figure 5M and 5N). These results indicate that PCN is a nuclear protein broadly expressed in embryo proper, but after germination predominantly expressed in the SAM, RAM, and in cells capable of proliferation.

Mutation in PCN perturbs the expression domains of SAM organizing genes
To better understand the SAM defects in pcn, we investigated the expression patterns of well-characterized SAM organizing genes using whole mount embryo in situ hybridization and reporter genes. In the WT shoot meristem, WUS and CLV3 expression domains mark the three apical layers of stem cells and the underlying organizing center, respectively ( Figure 6A and 6G) (Fletcher et al., 1999;Schoof et al., 2000). In pcn embryos, CLV3 was expressed in an enlarged domain encompassing almost the whole apex and was also ectopically expressed in the cotyledons ( Figure 6H and 6I). Compared to WT, WUS mRNA expression had expanded into L1 and L2 layers of the SAM and towards the inner layers of the cotyledons ( Figure 6B).

Expression of STM marks SAM cells that do not enter into lateral organ formation.
During WT embryogenesis, STM is first expressed in a band of cells around the central apical region of late globular-stage embryos. During the transition to heart stage, the expression becomes restricted to the central core of the SAM ( Figure   6C) (Long and Barton, 1998). In pcn, STM was variably expressed below the L3 layer of the SAM. In the majority of the embryos examined (30 out of 44), STM expression was detected in a large apical region of the embryo ( Figure 6D). CLV1 is expressed in the L2 and L3 layers of the WT SAM (Long and Barton, 1998). In pcn, CLV1 expression was still limited to the L2 and L3 layers but the expression domain had expanded to a slightly broader region compared to WT ( Figure 6E and 6F). Furthermore, qRT-PCR results revealed that WUS, CLV3, CUC and STM in SAM are significantly upregulated in pcn embryos (Supplemental Table 3). These results suggest that PCN function is required to restrict the expression of STM, WUS and CLV genes within their respective specified boundaries in the meristem.

PCN and WUS interact genetically
To determine whether PCN genetically interacts with WUS, we analyzed pcn wus-1 double mutants. wus-1 embryos develop normally except for the absence of a shoot meristem in the seedlings after germination (Laux et al., 1996). Double heterozygous pcn/+ wus/+ plants produced homozygous recessive pcn wus double mutant embryos at a ratio of 1:15 that did not initiate cotyledon primordia and the seeds did not germinate, even when the mother plant was grown at 29 0 C. However, pcn wus-1/+ seeds germinated and produced plants with rosette leaves, albeit at a slower rate than WT. After bolting, however, the SAM terminated as pin-like structures with arrested lateral organ primordia and differentiation of the epidermal cells into trichomes near the SAM region, indicating haploinsufficiency for WUS in pcn background ( Figure 7J-7N). These observations indicate that wus embryos cannot complete their development unless PCN function is present.
Whereas the lack of PCN can be remedied by high temperature insofar as embryo development is concerned. Such a rescue requires WUS. These synergistic defects suggest partial compensatory functions for WUS and PCN during embryo and postembryonic development.

Auxin distribution and response are altered in pcn
Defective cotyledon patterning in pcn embryos raised the question whether auxin response and/or auxin transport were affected in pcn embryos. To address this question, we introduced the auxin response marker DR5 rev :GFP, and as a readout of polarized auxin transport machinery, pPIN1:PIN1-GFP and pPIN7:PIN7-GUS into pcn mutant. At 22°C, DR5 rev :GFP expression was observed throughout the embryo proper in pcn similar to WT up to the octant stage ( Figure 8A, 8F and 8G) (Friml et al., 2003). However, from 32-cell globular stage onwards, the basal auxin maximum, which is restricted to the hypophyseal region in WT, expanded into the lower suspensor cells in arrested globular pcn embryos. The two auxin maxima corresponding to the two emerging cotyledon primordia in WT ( Figure 8B-8E) were absent in the pcn embryos that did not produce cotyledons ( Figure 8I, 8J and 8L). In pcn embryos that did form cotyledons, the auxin maxima correlated with the number of cotyledons ( Figure   8M-8P). In a few mutant embryos several auxin maxima were also observed in the apical region of late torpedo stage embryos ( Figure 8K). In contrast to DR5 rev :GFP expression in the vascular procambial cells of later stage WT embryos ( Figure 8E), the GFP signal was discontinuous or absent in the differentiating procambial cells of pcn embryos ( Figure 8K-8P). Unlike WT, DR5 rev :GFP was ectopically expressed in the SAM of pcn embryos ( Figure 8P and 8U). Consistent with the phenotypic recovery, this ectopic DR5 expression was suppressed when pcn embryos were subjected to 29 0 C treatment ( Figure   8U and 8V). In the RAM, the expression became restricted to the columella region similar to WT ( Figure 8Q  showed that PIN7 was expressed 0.6 fold lower than WT (Supplemental Table 3).
Taken together, pcn mutants displayed altered PIN1 localization and PIN7 expression.
To test whether auxin response is altered in pcn mutants, we performed a lateral root induction assay by application of the auxin analog NAA (Evans et al., 1994) to seedlings. In WT, lateral root primordia induced by 2 mg/l NAA displayed normal DR5 rev :GFP expression (Figure 9-3 A and 3B). However, in pcn, NAA induced abnormal bulges of pericycle cells with diffused expression of DR5:GFP that did not differentiate into lateral root primordia (Figure 9-3C-3E). Thus, while auxin response as measured by DR5 appeared in place, subsequent root development required PCN activity. In an alternate assay to measure the strength of the auxin response in pcn mutants, we used the pHS: AXR3NT-GUS reporter (Gray et al., 2001). In the pHS:AXR3NT-GUS construct, the GUS gene is fused to the amino-terminal domains I and II of the AXR3/IAA17 and its expression is controlled by a heat shock promoter. Due to the inclusion of the degron containing domain II of AXR3, the associated GUS is subject to degradation in an auxin-responsive manner (Gray et al., 2001). As shown in Figure 9-3I-3K and 3M-3N, the fusion protein was more stable in pcn seedlings than in WT as visualized by GUS, suggesting that IAA protein turnover is affected in pcn mutant. These results collectively show that PCN plays a role in auxin response pathway.

PCN genetically interacts with the auxin response module comprising BDL and MP
The IAA/AUX-ARF auxin module BDL/MP mediates auxin transcriptional response and together with PLT1 and PLT2 positively regulate PIN expression in root and vascular development (Szemenyei et al., 2008). In pcn, the expression domains of MP and PLT1 in the embryonic RAM were more restricted compared to WT ( Figure 6J-6M); also in the vascular initials of the pcn embryo, MP expression was discontinuous and ectopic in the SAM region of the torpedo embryo ( Figure 6K). Thus aberrations in the expression of MP and PLT1 were more pronounced in the pcn embryos that showed stronger developmental defects. Consistent with this observation, MP-GUS in the pcn mutant showed ectopic expression in the apical region of the embryo that included the SAM ( Figure 10A4-6) and the cotyledon primordia ( Figure 10A5). Furthermore, qRT-PCR showed that MP, WOX5, PLT1, PLT2 and PLT3 were significantly upregulated in pcn embryo (Supplemental Table 3). These findings prompted us to investigate potential interactions between pcn and auxin mutants. mp single mutants develop a basal stump instead of a root and one or two cotyledons (Berleth and Jurgens, 1993). In the progeny of pcn/+ mp/+ plants, we identified putative double mutants at a ratio 1:15 with a novel embryo phenotype.
These double mutant embryos showed cup-like cotyledon with a narrow embryo axis compared to WT ( Figure 10A1 and A2). These embryos were arrested not only at 22 0 C but also when grown at 29 0 C, similar to the observations with double mutants of pcn and wus-1 suggesting that MP functions are required for the rescue of pcn mutant at 29 0 C. pcn mp/+ plants were small, had delayed rosette leaf and root growth and the SAM terminated into pin-like structures  Table S3). The loss-of-function mutant, drn-1, shows defective embryo patterning and functional post-embryonic SAM (Chandler et al., 2007), whereas the gain-of-function drn-D mutant, show defective SAM and upregulated expression of both WUS and CLV3 as seen in pcn (Kirch et al., 2003). pcn drn-1 double mutant showed the pcn embryo phenotype. However, after rescue at 29 o C and during post-embryonic development, the young rosettes terminated growth with arrested SAM and radialized leaf primordia which were not observed in the respective single mutants ( Figure 10E1-3 (Friml et al., 2004;Kaplinsky and Barton, 2004). Unlike in pcn or pin1-1/+ and pid-2/+ heterozygous plants, in pcn pin1-1/+ ( Figure 7A) and pcn pid-2/+ plants ( Figure 7F) inflorescence shoot meristem terminated in a pin-like structure and lateral organ primordia were arrested ( Figure 7E, 7H, and 7I). In all cases, trichomes and guard cells, which are normally restricted to leaves or leaf primordia, were found at the tip of the pin-like structure in the meristem region ( Figure 7H and 7I), which is indicative of precocious differentiation of meristem cells. As one of its functions, TPL acts as a corepressor with BDL in a complex with MP (Szemenyei et al., 2008). tpl-1 and tpl-1/+ plants developed similar to WT after bolting whereas the pcn tpl-1/+ inflorescence meristems also terminated in a pin-like inflorescence ( Figure 7Q and 7R). Taken together, in the absence of PCN function, inflorescence meristem development becomes more sensitive to the gene dosage of PIN1, PID, and TPL.

Discussion
We have identified PCN as an essential gene for Arabidopsis embryo development. Mutations in the PCN locus resulted in developmental defects that link maintenance of the embryonic meristem and cotyledon formation to auxinmediated processes that regulate early to late patterning events during embryogenesis. Embryo defects in pcn were detectable as early as the four-cell stage and progressively displayed more defects both in the apical and basal programs resulting in striking cotyledon phenotypes, enlarged SAM and abnormal RAM suggesting the importance of PCN for embryo development including stem cell organization and maintenance. Our studies indicate a significant contribution of PCN in the early steps of auxin signaling by its ability to genetically interact with BDL and therefore likely functions as a repressor to regulate several key pathways that operate during embryo patterning (Supplemental Table 3). TPL, another well studied corepressor functions with BDL (Szemenyei et al., 2008), also genetically interacts with PCN. Auxin maxima and distribution were also altered in the pcn mutant suggesting a link between auxin mediated embryo patterning and PCN function. Another interesting feature of the pcn mutant embryo is its ability to recover at 29 0 C which enabled investigation into the post-embryonic roles of PCN. The defects in NAA-induced lateral root formation in pcn seedlings along with the prolonged IAA protein stability in this mutant suggest that PCN is required for auxin induced response and downstream signaling.

PCN genetically interacts with WUS and the transcriptional corepressor TPL.
WUS, which functions in a negative feedback loop to specify stem cells of the SAM, has been shown to physically interact with a TPL ortholog in Antirrhinum to repress differentiation pathways in the SAM (Schoof et al., 2000;Kieffer et al., 2006). In addition, pcn also genetically interacts with PIN and PID as both pcn pin1-1/+ and pcn pid-2/+ developed pin-like inflorescence with terminal differentiation. This suggests that PCN is required for sustained meristem activity during postembryonic development. Interestingly, the respective homozygous double mutants displayed strong developmental arrest at the globular stage.
These observations suggest PCN functions are coordinated with PIN and PID both during embryo development and postembryonically in developmental programs associated with meristems. The post-embryonic phenotypes also suggest that PCN has a significant role in the maintenance of a functional SAM through the auxin signaling pathway; previously the auxin signaling pathway has been shown to be essential for RAM functions (Benjamins and Scheres, 2008).
Based on these observations, it is tempting to speculate that PCN likely functions in the BDL/TPL repressive pathway to regulate auxin-mediated embryo patterning and meristem maintenance.

PCN plays an important role in restricting the meristem size
An enlarged SAM ( Figure 2J and 2M) and ectopic WUS expression in the L1 and L2 layers ( Figure 6B) were both observed in pcn mutant. WUS is normally restricted to a few cells in the L3 layer of WT ( Figure 6A); these WUS-expressing cells act as an organizing center (OC) and signal the overlying cells to function as stem cells (Mayer et al., 1998). Ectopic expression of WUS in the L1 layer using the CLV3 promoter results in an abnormally swollen SAM that is arrested in the seedling (Brand et al., 2002) and a recent study showed that WUS directly activates CLV3 expression by binding to its promoter region (Yadav et al., 2011). Therefore, WUS mis-expression in the L1 and L2 layers followed by its selfamplification is partly responsible for the meristem defects in pcn. pcn wus-1 double mutants could not develop past the globular stage either at 22 0 C or 29 0 C, indicating that WUS and PCN together are essential for early embryogenesis.
The double mutant embryos of pcn tpl-1 and pcn wus-1 are arrested at the globular stage even when grown at 29 0 C. TPL and WUS mutations are haploinsufficient in the pcn background, as the pcn tpl-1/+ and pcn wus-1/+ seeds were viable if grown at 29 0 C, but developed similar pin-like inflorescence phenotypes post-germination. These observations are consistent with the proposed function of TPL as a corepressor and its physical interactions with WUS (Kieffer et al., 2006). Further, these results also suggest that PCN and TPL likely mediate alternative roles in regulating WUS repression and activation to maintain the SAM functions. The replacement of inflorescences with pin-like structures is usually considered as an indicator of the disruption in auxin movement and gradient (Okada et al., 1991;Friml et al., 2004;Kaplinsky and Barton, 2004). Disordered divisions in the SAM and RAM occur at the same time as the loss of OC and QC identity in the pcn mutant. Abnormal SAM, RAM and auxin maxima observed in pcn indicate that PCN plays an important role in maintaining meristem functions through auxin signaling. WUS expression is negatively regulated by the CLV signal transduction pathway (Brand et al., 2000;Schoof et al., 2000). Surprisingly, our study shows a broader expression of CLV3 in pcn ( Figure 6G-6I) although WUS expression was also expanded. One possible explanation is that in pcn mutants, CLV signaling is compromised resulting in enlarged meristem. Therefore, PCN likely provides a link between genetic regulation of meristem maintenance and auxin signaling pathway.

PCN functions in the auxin pathway
Regulated degradation of Aux/IAA proteins that repress ARF transcription factors bound to auxin-response elements play a crucial role in auxin-mediated signaling (Mockaitis and Estelle, 2008). BDL, an IAA protein, along with the corepressor TPL (Szemenyei et al., 2008), is thought to form a complex with the ARF transcription factor MP (Weijers et al., 2006) to regulate auxin responsive target genes. BDL and MP are expressed from zygote to early globular stages and are subsequently restricted to the hypophyseal cell derivatives and the developing vascular initials (Hamann et al., 2002). Our study indicates that the pcn mutation interferes with auxin response similar to mutations in the well-characterized bdl (Hamann et al., 1999). Like bdl, mutations in PCN cause abnormal cell divisions in the basal domain during early embryo development. Although pcn enhanced the early embryo phenotypes of bdl mutant, surprisingly bdl rescued the pcn mutant embryo even when grown at 22 0 C, suggesting that BDL activity is involved in pcn embryo lethality. Consistent with this, the ectopic expression of BDL-GFP and DR5 rev :GFP in the SAM of pcn embryos was suppressed in pcn plants grown at 29 0 C for seed/embryo rescue ( Figure 10D6 and D7, Figure 8U and 8V). Further, the mis-expression of STM outside the SAM (Figure 6D), and the auxin maxima observed in the SAM of the pcn embryo ( Figure 8P) suggests that PCN is required for tight regulation of auxin response and maxima in the SAM. Therefore, it is likely that ectopic BDL activity in the SAM of pcn embryos contributes to the striking pcn embryo phenotypes suggesting that BDL function is required for the arrested pcn embryo phenotype.
Since the bdl pcn double mutant is not completely reverted to WT, it is likely that other genes may be involved in pcn rescue at 22 0 C. Our results suggest that TPL activity may provide some regulatory functions in the pcn mutant background. Consistent with this, the pcn tpl-1 double mutant is embryo lethal with embryos arrested at the globular stage when grown either at either 22 0 C or 29 0 C. Furthermore, the heterozygous tpl-1 in pcn background (pcn tpl-1/+) had a partially terminated SAM whereas heterozygous pcn in tpl-1 background (pcn/+ tpl-1) showed tpl-1 post-embryonic phenotypes ( Figure 7O-7R). These observations suggest that PCN and TPL have both distinct and overlapping functions. Since PCN appears to function together with BDL and TPL to regulate downstream targets, disruption of the BDL containing complex may cause an unknown alternative pathway to rescue pcn in the bdl mutant background.
Although the pcn mp/+ mutant was not arrested at 29 0 C, it showed severe cotyledon phenotypes compared to the mp single mutant and post-embryonically the SAM terminated in a pin-like structure ( Figure 10A1-3). As our results indicate that PCN genetically interacts with BDL, it is likely that PCN works with BDL and TPL to negatively regulate MP and its targets. It is interesting to note that both of these genes that genetically interact with BDL encode WD40 repeat containing proteins (Szemenyei et al., 2008). In addition to the involvement of PCN in the BDL/TPL repression of MP, the pHS:AXR3NT-GUS based assay (Gray et al., 2001) in the pcn showed that IAA proteins are more stable in the pcn mutant suggesting that PCN has a broader role in auxin signaling.

PCN integrates auxin signaling and meristem functions
The pcn mutant phenotype, its genetic interactions with factors associated with auxin signaling and meristem fate, and the qRT-PCR results of genes that are differentially regulated in the pcn embryo, indicate that PCN functions along with BDL and TPL to mediate MP repression (Figure 11). MP protein regulates both its own expression and that of BDL thus functioning as a genetic switch triggered in response to auxin in a threshold-specific manner to degrade BDL and relieve the repression of its target genes (Lau et al., 2011). MP, which is upregulated in the pcn embryo, through negative regulation of ARR7 and ARR15 (downregulated in pcn embryo; Supplemental Table 3), likely controls the WUS / CLV3 feedback loop in the SAM as previously suggested (Zhao et al., 2010), thus revealing a link between auxin signaling and meristem function. Several studies have shown that PIN polarity and the resulting auxin localization is essential for embryo patterning (Friml et al., 2003;Geldner et al., 2003;Michniewicz et al., 2007;Kitakura et al., 2011  Furthermore, in pcn embryos, several genes encoding AP2-domain transcription factors and members of the WOX family that have been shown to maintain the activity of stem cells and control embryo patterning (Haecker et al., 2004;Wurschum et al., 2006;Chandler et al., 2007;Galinha et al., 2007) are significantly upregulated as shown by our qRT-PCR analysis (Supplemental Table 3). In the embryonic and post-embryonic RAM, auxin induces the expression of AP2 transcription factors, PLT1 and PLT2, to specify the quiescent center (Aida et al., 2004), and both of these genes as well as WOX5 are upregulated in the pcn embryo (Supplemental Table 3). Collectively, these lines of evidence indicate that PCN functions as a negative regulator or repressor with key roles in embryo patterning and meristem organization / maintenance through auxin mediated signals (Figure 11).
In conclusion, based on our developmental and genetic studies, the earliest signs of embryonic pcn defects are associated with auxin transport and response pathway. These mutant phenotypes are further enhanced by mutations in several key components of auxin transport and signaling involving pin-1, pid, tpl and mp ( Figure 11). These genes also have a dosage dependent auxin transport and signaling defect-associated pin phenotypes after bolting ( Figure 11). Interestingly, genetic studies with meristem factor WUS also revealed similar embryonic and postembryonic phenotypes linking the auxin signaling defects with embryonic and postembryonic meristems ( Figure 11). The genetic and functional framework developed for PCN in the present study ( Figure 11) will help future experiments to dissect other components of PCN mediated pathways that operate during embryonic and post-embryonic development in Arabidopsis.

Plant growth, mutant lines, and map based cloning
Arabidopsis thaliana plants were grown on soil or in sterile culture on 1/2 MS medium containing Murashige and Skoog salts. After incubation for at least 4 days at 4 0 C in darkness, plants were grown in growth chambers under long-day conditions (16 hr light and 8 hr dark) at 22 0 C or 29 0 C. Homozygous pcn plants and double mutants of pcn (except pcn bdl) were grown at 22°C until they began to flower and then shifted to 29°C. Details of mutants and marker lines used in this study are listed in Supplemental Table 2.
Prior to mapping, the pcn-1 mutant was backcrossed four times to wild type Columbia (Col). pcn-1/+ (BC4, Col) x wild type Landsberg erecta (Ler), and , 670 F2 plants were used for mapping. Crosses with known genetic markers indicated that PCN mapped close to the centromere of chromosome 4. Because no known genetic markers were available in this region, we designed 20 Cleaved Amplified Polymorphic Sequence (CAPS) markers and 87 F2 plants were assayed for these markers on the long arm close to the centromere of chromosome 4. The initial mapping was done using SSR markers from The Arabidopsis Information Resource (http://www.arabidopsis.org). The dCAPS markers used for fine mapping of the pcn-1 mutation were generated based on the sequence information of Col and Ler.

Construction of pPCN:PCN-GFP and complementation vectors, and transformation into Arabidopsis
pPCN:PCN-GFP constructs were derived as follows: first we used forward primer1: 5'-AACACTGCAGCGCAATCGCACCTATTTTTAATC-3 and reverse primer1: 5'-AAAAGCGGCCGCGACGGAAGCAGAAGGAGAAGTGT-3' to amplify the PCN promoter sequence from genomic DNA, and the PCN cDNA was amplified by PCR from a WT Columbia cDNA library using forward primer 2: 5'-AAAAGCGGCCGCATGCTCGAGTACCGTTGCAGCTC-3' and reverse primer2: 5'-AAAAGGATCCAGTTCCAAAAATATGTCTGTC-3'. Both promoter and cDNA PCR products were digested with NotI and then the fragments were ligated.

PCR-based genotyping, qRT-PCR experiment
Genotypes of the pcn-1 and pcn-2 loci in the transgenic plants were determined by PCR. To genotype the pcn-1, PCR primers (forward primer:5'-TCTGAGTAACTGTTTCTTCTGTTGC-3', reverse primer:5'-CCACTCACAAGAACTGAACACCT-3') were used, the PCR fragments size is 218 bp (mutant) and 254 bp (Col wt). To genotype pcn-2 (Salk_022607), forward 5'-TTCCGGATGATATACTGCCAG,-3', reverse primer 5'-CCCCAGGAAACTCTTGATACC-3' and middle primer 5'-GCGTGGACCGCTTGCTGCAACT-3' primers were used. Other primers and methods used for PCR genotyping of double mutants in this study are listed in Supplemental Table 2. qRT-PCR experiment was performed as described by Xiang et al. (Xiang et al., 2011). Both globular to heart stage WT and pcn embryos were isolated and total RNA samples were isolated as described in RNAqueous-micro kit (Ambion, catalog no. 1927) and the respective double-strand cDNAs were produced using amplified aRNA following the protocol of MessageAmpTM II aRNA kit (Ambion, catalog no. 1751). Gene-specific primers were designed using Primer 3 software.
qRT-PCR reactions were performed using the protocol and equipment of Applied Biosystem Step One. qRT-PCR primers and results are listed in Supplemental Table 3.

Assay for BFA, FM4-64 staining, auxin response and IAA degradation experiment
For BFA treatment and FM4-64 staining, the WT and pcn embryos were isolated and treated as described earlier (Ganguly et al., 2010). To determine the auxin response of pcn, both mutants and WT (Col-0) seeds were surface-sterilized.
These seeds were incubated at 4 0 C for 3 days and then were germinated on ½ MS for 4 days. These seedlings were transferred to ½ MS supplemented with 0.2 mg/l NAA. Seedling phenotypes were examined at 2 and 4 days with NAA in the medium. The response was evaluated by analyzing lateral root development as well as DR5 rev :GFP expression. Both pHS:AXR3NT-GUS and pcn / pHS:AXR3NT-GUS seeds were germinated in ½ MS for 5 days, then heatshocked for 2h at 37 0 C and incubated at 20 0 C for 1-3h to check the degradation of GUS (Gray et al., 2001).

Y2H assay
Y2H was performed as described in ProQuest™ Two-Hybrid System with Gateway® Technology Kit protocol (Invitrogen, Cat# PQ10001-01). Genes and primers are listed in Supplemental Table 4.

Microscopy
Whole-mount embryos were prepared by clearing the ovules in chloral hydrate solution [8:1:2 chloral hydrate:glycerol:water (w:v:v)] (Christensen et al., 1998) for 4 h. Slides were viewed using a Leica microscope at differential interference contrast optics. Confocal laser scanning microscopy was performed using Leica SP2 and Zeiss LSM-510 microscopes. Histochemical staining for GUS activity in this study was performed following the protocol of Gray et al. (Gray et al., 2001).
Lugol staining of the roots was performed as described in (van den Berg et al., 1997). Whole mount in situ hybridization was performed following the protocol of Hejatko et al (Hejatko et al., 2006). Scanning electron microscopy was performed as described in (Venglat et al., 2002).              indicate auxin maxima as shown by DR5-GFP expression; and blue highlighted regions show WOX5 expression. pcn mutant embryos are able to recover at 29 0 C, produce rosettes with narrow leaves and, after flowering, set seeds. However, loss of function of the auxin and meristem associated TPL, PIN1, PID, MP and lethality that cannot be rescued even with the 29 0 C treatment. Heterozygous mutations in these same genes and homozygous recessive drn in the pcn background are able to progress through embryogenesis and produce viable seeds. However, after germination and production of rosette leaves, the inflorescence meristems terminate into pin-like structures. 44

drn-1 John Chandler
Homozygous mutants were confirmed by the absence of the wild-type gene using the following primers DRNF: 5'-ATGGAAAAAGCCTTGAGAAAC-3'; DRNR: 5'-CTATCCCCACGATCTTCGGCA-3' Note: Three biological replicates were used for qRT-PCR analysis; tubulin (At5g12250) was used as internal control and its cycle threshold (CT) mean is 31. 4 -31.6. Red indicates genes are significantly up-regulated in pcn, blue indicated genes are significantly down-regulated in pcn.* indicate statistically significant (1.5 fold) and pvalue less than 0.05.