Stamen abscission zone transcriptome profiling reveals new candidates for abscission control: enhanced retention of floral organs in transgenic plants overexpressing Arabidopsis ZINC FINGER PROTEIN2.

Organ detachment requires cell separation within abscission zones (AZs). Physiological studies have established that ethylene and auxin contribute to cell separation control. Genetic analyses of abscission mutants have defined ethylene-independent detachment regulators. Functional genomic strategies leading to global understandings of abscission have awaited methods for isolating AZ cells of low abundance and very small size. Here, we couple laser capture microdissection of Arabidopsis thaliana stamen AZs and GeneChip profiling to reveal the AZ transcriptome responding to a developmental shedding cue. Analyses focus on 551 AZ genes (AZ(551)) regulated at the highest statistical significance (P < or = 0.0001) over five floral stages linking prepollination to stamen shed. AZ(551) includes mediators of ethylene and auxin signaling as well as receptor-like kinases and extracellular ligands thought to act independent of ethylene. We hypothesized that novel abscission regulators might reside in disproportionately represented Gene Ontology Consortium functional categories for cell wall modifying proteins, extracellular regulators, and nuclear-residing transcription factors. Promoter-beta-glucuronidase expression of one transcription factor candidate, ZINC FINGER PROTEIN2 (AtZFP2), was elevated in stamen, petal, and sepal AZs. Flower parts of transgenic lines overexpressing AtZFP2 exhibited asynchronous and delayed abscission. Abscission defects were accompanied by altered floral morphology limiting pollination and fertility. Hand-pollination restored transgenic fruit development but not the rapid abscission seen in wild-type plants, demonstrating that pollination does not assure normal rates of detachment. In wild-type stamen AZs, AtZFP2 is significantly up-regulated postanthesis. Phenotype data from transgene overexpression studies suggest that AtZFP2 participates in processes that directly or indirectly influence organ shed.

Genetic studies show abscission capacity to be influenced by disruptions in organ boundaries and other alterations in organ patterning. Partial fusion of sepals in the F-Box gene mutant Hawaiian Skirt impairs shedding of Arabidopsis floral parts in which abscission zones appear to differentiate normally (Gonzalez-Carranza et al., 2007b). Two BTB/POZ domain proteins, Blade-on-Petiole (BOP) 1 and BOP2, are expressed in regions overlapping the floral organ abscission zones (Ha et al., 2004;Hepworth et al., 2005;Norberg et al., 2005). A bop1bop2 double mutant exhibited abnormal organ patterning and loss of floral organ abscission (Hepworth et al., 2005;Norberg et al., 2005). Other transcription factors contributing to abscission competence include the structurally related MADS-box domain proteins AGL15 and AGL18; shedding of Arabidopsis floral parts is delayed in plants over-accumulating either protein (Fernandez et al., 2000;Adamczyk et al., 2007). AGL15 and AGL18 overexpressors exhibit concomitant slowing of other developmental transitions including flowering time and senescence (Fernandez et al., 2000;Adamczyk et al., 2007). Early flowering time and a slowing of floral senescence also accompany delayed abscission in plants with knocked down levels of actin-related protein 4, ARP4 (Kandasamy et al., 2005a). Similarly, knocking down ARP7 expression levels delays abscission of floral parts and alters flower development, impacting fertility (Kandasamy et al., 2005b). Pleiotropic phenotypes of ARP-deficient plants have been predicted to arise from aberrant gene transcription patterns caused by altered chromatin structure (Kandasamy et al., 2005b).
In wildtype plants, binding of ethylene to one or more ethylene receptors derepresses the ethylene signal transduction pathway leading to hormone-dependent responses including abscission. Once considered a fundamental abscission signal (Jackson and Osborne, 1970), ethylene is now viewed as an abscission rate regulator (Bleecker and Patterson, 1997;Patterson, 2001). Mutations in the etr1-1 ethylene receptor gene that render Arabidopsis plants insensitive to ethylene delay, rather than block, floral organ abscission (Patterson and Bleecker, 2004). Ethylene control of abscission is influenced in part via antagonism between ethylene and auxin. Abscission is delayed in plants with suppressed expression of the ARF2 member of the auxin response family (Ellis et al., 2005). ARF2 controls auxin signaling (Ellis et al., 2005) and ethylene synthesis is altered in arf2 mutants (Okushima et al., 2005). Non-hormone ligands controlling abscission include that encoded by inflorescence deficient in abscission (IDA). Loss-of-function ida mutants fail to abscise Arabidopsis sepals, petals and stamens (Butenko et al., 2003); transgenic lines overexpressing IDA rapidly shed all floral organs, as well as additional plant parts that do not normally abscise (Stenvik et al., 2006). The receptor for IDA is unknown but has been proposed to include receptor-like kinases (RLKs). Ant isense inhibition of gene expression corresponding to the RLK termed HAESA blocks abscission of Arabidopsis stamens, sepals and petals (Jinn et al., 2000). Expression of HAESA appears to be independent of ethylene synthesis or perception (Jinn et al., 2000). This is also the case with AGL15 (Fernandez et al., 2000) and ARP7 (Kandasamy et al., 2005b). At present, mechanisms by which putative ethylene-independent pathways contribute to abscission are poorly understood.
Many questions remain about abscission signaling. What is the primary abscission cue or cues? What regulators exert earliest control over signal perception and response?
Why do some organs abscise in response to a given stimulus while others are retained?
What genes control ethylene-dependent and independent pathways and how do pathways interact? Functional genomic approaches to addressing these questions have been hindered by an inability to obtain pure populations of AZ cells. Recently, we optimized methods for using l aser-capture microdissection (LCM) to harvest highly enriched populations of specialized cells (Cai and Lashbrook, 2006). There, replum cell harvest was linked to ATH1 GeneChip studies of fruit maturation (Cai and Lashbrook, 2006). Here, we reveal dynamic changes in global gene expression taking place in AZs of Arabidopsis stamens progressing from pre-pollination to organ shed. Functional analyses of one AZ-upregulated gene, Zinc Finger Protein 2 (AtZFP2), provide evidence that this transcription factor participates in processes that directly or indirectly influence shedding of floral organs.

Laser Capture Microdissection Facilitates AZ Transcriptome Profiling
Arabidopsis stamens, sepals and petals abscise post-pollination. Detachment is dependent upon separation of abscission zone cells residing at the bases of floral organs.
We chose stamens as a source of AZs for LCM studies because there are six stamens instead of four petals or sepals. This elevates the relative incidence of AZs in sectioned tissue and reduces the time required for cell capture. Whole flowers corresponding to five developmental stages linking pre-pollination to the onset of organ shed were fixed and paraffin-embedded and sectioned tissues were tape-transferred to In Figures 1B and 1C, scanning electron microscopy (SEM) visualizes surface features of AZ fracture planes at stages selected for abscission studies. Figure 1B depicts stamen AZ scars left on the parent plant after manual filament removal; Figure   1C shows parental sides of all floral organ fracture planes after organ detachment. The intact rounded AZ cells observed in Fig 1B (Stages 15C and 16) and Fig 1C represent proximal AZ cells that have completely separated from contiguous distal AZ cells of the leaving organ (Patterson and Bleecker, 2004). In contrast, torn AZ cells observed in Fig   1B (Stages 12 to 15C) testify that cell wall dissolution processes needed for separation from neighboring cells are not yet complete. In our studies, intact cells present on proximal fracture faces after organ removal are first evident on outermost AZ margins at Stage 15C ( Fig 1B). Thus, final stages of separation occurring within a subset of stamen AZ cells have occurred by Stage 15C. Cell separation is essentially complete between all stamen AZs of the proximal fracture plane by Stage 16, when all AZ cells are rounded and intact ( Fig 1B) and organs detach when lightly touched.
Stamen AZ cells were laser-captured from flowers at Stages 12-15C. Figures   1D and 1E show representative flower sections before and after LCM of stamen AZs, respectively. Microdissected AZs included cells from the vascular bundle that passes through all abscission layers. Approximately 10,000 floral organ AZ cells could be captured in ~1 day and the amount of RNA subsequently isolated per laser-captured cell was ~10-15 pg (Cai and Lashbrook, 2006). Total RNA served as template for preparing hybridization targets for ATH1 GeneChips (Cai and Lashbrook, 2006).

Probe Set Signals Regulated at the Highest Level of Statistical Significance Define a 551-Member Slice of the AZ Transcriptome
ATH1 GeneChips contain probe sets representing ~24,000 genes of the Arabidopsis genome. Replicated hybridizations of biotin-labeled aRNAs from AZs at Stages 12-15c revealed statistically significant changes in probe set signal intensities corresponding to many AZ transcripts. We wished to restrict preliminary analyses to a manageable number of genes whose expression could provide a first glimpse of regulatory processes leading to cell separation. Restricting attention to the most significantly regulated probe sets (p-values of 0.0001 or less) generated a population of 551 transcripts (AZ 551 ) representing the Arabidopsis stamen abscission zone transcriptome. A ~ 0.2% false discovery rate was determined using the method of Storey and Tibshirani (2003). A complete list of AZ 551 genes is in Supplemental Table   1 (Table S1). Quantitative real-time PCR (Q-PCR) successfully validated microarray expression data for a subsample of 10 probe sets using stamen abscission zone RNA from laser-captured cells (data not shown). Supplemental Figure 1 (Fig. S1) depicts expression patterns of three validated transcripts related to gibberellin metabolism or action. Both Q-PCR and microarray data showed similar trends in mRNA accumulation for the GA-related mRNAs shown in Figure S1 and discussed later in the text.  (Table I).
Functional classification by GO cellular component showed that the cell wall and extracellular matrix were represented in AZs at levels exceeding those in ATH1 by ~ three to 4-fold. The nucleus was represented in AZs at levels exceeding that of ATH1 by 1.6-fold (Table I). Consistent with AZ cellular component data was the increased representation of GO categories representing molecular function (Table I).
Transcription factor activities within the AZ 551 exceeded that of ATH1 by almost 2- In Figure 2, Clusters 1-3 represent up-regulated genes within AZ 551 ; clusters 5-7 contain down-regulated genes and the remaining two clusters represent genes that are both upand down-regulated. Transcript identities are annotated in Table S1. In Table I, GO functional categories for AZ 551 transcripts were compared with Arabidopsis genome transcripts on the ATH1 GeneChip. We similarly wished to establish if certain functions were over-represented to statistically significant extents in one or more of the eight transcript clusters. In Table II (Table II).

Upregulated Cluster 1 genes encode transcriptional modulators, receptor-like kinases, cell wall modifying proteins and regulators of hormone biosynthesis, action and
transport. Cluster 1 transcripts increase in abundance between Stages 12 and 15a and remain at high levels as abscission commences near Stages 15b-c ( Fig. 2). GOannotated genes within Cluster 1 are assigned to the nuclear compartment at rates surpassing those for other transcripts represented on the ATH1 GeneChip (Table II).
~18% of Cluster 1 mRNAs are assigned to a nuclear site versus 7.6% of ATH1 Arabidopsis genes and ~12% of AZ 551 (data not shown). Myb transcription factors are well represented. Supplemental Table 2 (Table S2) (Table S2). All of these Mybs are modulated to some extent by sucrose and nitrogen status with AtMyb14 also weakly regulated by auxin (Kranz et al., 1998). AtMyb 4 and AtMyb75 gene products act as negative and positive transcriptional regulators, respectively, of phenylpropanoid biosynthesis (Borevitz et al., 2000;Jin et al., 2000;Rose et al., 2002). GO molecular function comparisons of Cluster 1 transcripts show that transcription factor activities as well as activities for DNA, RNA or nucleotide binding are over-represented (Table II). Table S2 include receptor-like kinases (RLKs) that translate extracellular signals into cellular responses (Becraft, 2002). AZ 551 RLKs present in annotations of Shiu and Bleecker (2001) (Table S2) precedes first visible signs of cell separation at Stage 15c ( We conclude tha t HAE must represent a very early contributor to abscission competence.

Upregulated genes in
HAE appears to mediate steps within an ethylene independent abscission pathway as corresponding antisense mutants have the ethylene sensitivity to evoke a hormone-dependent triple response equivalent to that of wild-type controls (Jinn et al., 2000). Ethylene likely controls the rate of a separate abscission pathway that could communicate with components of the HAE route (Butenko et al., 2006). Our data suggest that both ethylene dependent and independent pathways are operational after Stage 12 (Table S2). Components of the former include ethylene response factors (ERFs) 003 and 072 (At3g16770, At5g25190) and ethylene insensitive 3 (EIN3)binding protein 2 (EBF2; At5g25350) that contributes to SCF-dependent ubiquitination and degradation (Potuschak et al., 2003). Modulated ethylene action is accompanied by induction of multiple auxin responsive genes and accumulation of the PIN4 auxin efflux transcript (At2g01420). BR6OX2 (At3g30180) mediates the last step of brassinolide synthesis. It is upregulated as is the BR-enhanced target BEE3 (At1g73830) and GAregulated GASA5 (At3g02885; Fig. S1). Thus, expression of RLKs like HAE and HSL-2 takes place in a complex backdrop of hormone signaling.

Upregulated Cluster 2 transcripts encode proteins with roles in pectin modification, hormone synthesis and degradation, receptor binding and signal transduction.
Cluster 2 transcript levels rise throughout all stages assayed whereas Cluster 1 mRNA abundance stabilizes after Stage 15a (Fig. 2). It is possible that gene expression at later stages in Cluster 2 contributes to processes necessary for final stages of organ detachment. Cluster 2 is enriched in transcripts with likely roles in cell wall structural modification (Table II). Regulated cell wall genes encode two glycosyl hydrolases with structural similarities to endo-ß-1,3-glucanases (At4g18340) and endo-ß-1,4-glucanases (At2g32990). However, most upregulated cell wall genes encode pectolytic enzymes,    , 1997;Schaller et al., 2000).

Likely functions of Cluster 3 and 4 genes overlap with those of Clusters 1 and 2.
Multiple  suggests that RLK signaling is central to cell separation control. Expression data for two additional RLKs in Cluster 4 are listed in Table S2.
GO cellular component analysis suggests that the cell wall is an overrepresented site of Cluster 3 gene expression (Table II). Cell wall modifying protein candidates (Table S2) are At-XTH12 (At5g57530) and At-Extensin 4 (At1g76930).
GO categorization assigns Cluster 6 transcripts to the nucleus more often than it assigns all transcripts represented on the ATH1 GeneChip (Table II). Just as multiple Myb genes were upregulated elsewhere, many Mybs are downregulated in Cluster 6 (Table S2) (Table I).

Protein, in Processes Affecting Abscission Capacity
A subset o f AZ 551 transcripts was considered for potential functional analyses; one such target was Zinc Finger Protein 2 (AtZFP2; At5g57520).
Zinc finger proteins regulate many developmental and stress responses (Takatsuji, 1998(Takatsuji, , 1999. At least 33 Arabidopsis ZFPs contain only one zinc-finger domain (Englbrecht et al., 2004). Twenty-eight members of this so-called C1-l1 class of zinc finger proteins share a conserved QALGGH sequence within a putative DNAcontacting surface and a C-terminal leucine-rich sequence (Tague and Goodman, 1995;Takatsuji, 1998Takatsuji, , 1999Englbrecht et al., 2004). However, sequence alignments show low similarity elsewhere. Thus, ZFPs may carry out both distinct and overlapping functions. AtZFP2 is a single copy, single zinc finger domain gene (Tague and Goodman, 1995). Our transcriptional profiling revealed that AtZFP2 was up-regulated in Cluster 3 of AZ 551 (Table S1). Promoter-GUS assays revealed enhanced potential contribution of AtZFP2 to abscission. However, the AtZFP2 gene product appears to participate in non-AZ localized processes as well (Fig. 4D-I). Promoter: GUS assays show that sites of AtZFP2::GUS transgene expression include stamens and carpels (Fig. 4D, E), cotyledons and major veins of rosette leaves (Fig. 4F), trichomes of inflorescence leaves (Fig. 4G, H) and stems (Fig. 4G, I).
As the first step towards testing AtZFP2's potential role in floral organ abscission, we constitutively expressed a 35S::AtZFP2 transgene in Arabidopsis. Ninety-five hygromycin resistant T1 plant lines were transplanted; almost half of these lines subsequently showed distinct phenotypes relative to wildtype controls. Quantitative real-time PCR analysis of transgenic flowers was used to assess the combined level of 35S::AtZFP2 and endogenous AtZFP2 (Fig. S2-A) and the 35S::AtZFP2 transgene level alone ( Fig. S2-B). Data in Figure S2 show that transgenic flowers exhibit significant increases in total AtZFP2 transcript level relative to wildtype controls.
To analyze potential AtZFP2 functions, transgenic phenotypes were visually classified into three types: mild, strong and severe. In all types, stamen filaments appeared shorter than in wildtype plants (data not shown) and plants were sterile.
Transgenic lines were characterized in the T1 generation due to sterility. Other  (Fig. 5B). In strong phenotype transgenic plants, organ retention is very prolonged (Fig. 5C, E). Over 40 closed buds are present on the 'strong' inflorescence depicted in Figure 5E. The extremely slow inflorescence elongation rates of severe phenotype plants like that in Figure 4D   In wildtype Arabidopsis, developmental abscission follows pollination and fertilization. We wished to establish whether 35S::AtZFP2 abscission delays could be due to impairment of these functions, particularly because the short stamen filaments of transgenic plants (data not shown) would be expected to limit effective pollination.
Pollen grains from wildtype flowers were used to pollinate 35S::AtZFP2 stigma; results are shown in Figure 5J-L. Four days after pollination, the mild phenotype plant in Figure 5J has elongated siliques but still retains floral parts. Floral parts are retained even longer in strong phenotype plants ( Fig. 5K; 12 days post-pollination) and severe phenotype plants ( Fig. 5L; 32  The increased floral organ retention seen in Figure 5 has several possible causes, including a failure to differentiate AZs or a failure to separate AZ cells once differentiated. In theory, such failures could occur in all AZs of the floral whorl or just a subset. For example, failure to differentiate or separate only the AZs of sepals would entrap petals and stamens and lead to unobservable abscission. To distinguish between these possibilities, SEM was used to visualize the parental faces of AZ fracture planes after manual removal of wildtype and transgenic floral parts (Fig. 6). When organs were removed from flowers at Position 3 of wildtype plants or transgenic plants with mild, strong or severe phenotypes, cells at bases of petals, sepals and stamens were torn.
Tearing is thought to reflect damage to cells that were tightly linked to their neighbors via cell wall connections at the time of organ removal, i.e. were non-abscised. At position 5, cells at the bases of all wildtype floral organs are intact, suggesting that middle lamellar connectio ns to adjacent cells were at least partially dissolved due to initiation of stamen, sepal and petal abscission. In Figure 6 al., 2002;Becker and Theißen, 2003). The mechanism whereby modulation of repressor activity could alter abscission capacity is uncertain but it is clear that pedicel abscission is blocked in jointless mutants (Butler, 1936) Figure S4 is in Table S2. Some expressed genes have been shown by others to be associated with the abscission and/or dehiscence processes, suggesting that the AZ 551 population may also represent a valuable source of new cell separation determinants. This hypothesis was confirmed via functional analyses showing an influence of one upregulated AZ 551 gene, AtZFP2, on abscission. Abscission of 35S::AtZFP2 floral organs was asynchronous and delayed; in plants with severe pleiotropic phenotypes abscission did not occur over time periods of two to three weeks.
Interestingly, it appears that 35S::AtZFP2 expression does not similarly inhibit cell separation during dehiscence as transgenic silique splitting was observed in Figure 5G and I.
Comparison of AZ 551 to the sequenced Arabidopsis genome reveals that cell wall proteins are disproportionately represented in AZs. Transcripts for PGs, pectate lyases, PMEs and PME inhibitors are especially well-represented, not surprising given the known roles of pectin modifying enzymes in reducing adhesion between contiguous AZ cells. Our data set would actually be expected to underestimate the potential contributions of pectin modification to abscission control as the size of AZ 551 was limited by F-testing to transcripts with p-values less than 0.0001. Indeed, additional pectin-related proteins exhibiting modulated accumulation prior to abscission are represented by signals lying just outside that window of statistical significance. These transcripts include PMEs and PGs not listed in Tables S 1-S2. Interestingly, the significant up-regulation reported in Table S2 for one PG ( At3g07970) before abscission was at odds with prior reports that this transcript was not expressed in Arabidopsis AZs (Gonzalez-Carranza et al., 2007a). We therefore investigated whether the At3g07970 probe set might be cross-hybridizing to another structurally similar PG present in the stamen AZ sample. BLAST analysis defined one structural relative of this PG as abscission-related PGAZAT/At2g41850 (Gonzalez-Carranza et al., 2002).
However, our expression profiles for At3g07970 are markedly different from those of PGAZAT (data not shown) and do not support the presence of substantial crosshybridization.
A second close structural relative of the At3g07970 PG is Early induction of abscission pathways presents both opportunities and challenges to researchers interested in understanding abscission control. Opportunities include the potential to identify novel, early regulators suitable for use in plant improvement. Crops including soybean and cotton exhibit high rates of precocious shed prior to fruit set, limiting yield potential (Wiebold et al., 1981;Guinn, 1982).
Others like citrus resist detachment at harvest and require chemical harvesting aids that may bring undesirable side effects (Kender et al., 2001). Our studies should help identify gene targets whose modification may reduce or promote shedding of commercially important organs. However, a key challenge will be devising methods to select targets that preferentially control abscission rather than multiple plant processes such as were observed with AtZFP2. For example, distinguishing genes controlling abscission from those co-regulating unrelated processes like anther dehiscence, pollination and/or floral senescence will be important. Of course, it is possible that the primary abscission cue that initiates ethylene-independent and/or -dependent abscission pathways may be derived from these or other events.
terminator of the transgene. The QuantiTect® primer assay, At_ZFP2-1-SG (Qiagen Inc, Cat#: QT00844144), was then used to collectively PCR amplify 35S::AtZP2 and endogenous AtZFP2. Relative expression was calculated according to Livak and Schmittgen (2001) with 18S rRNA as internal control and wild type expression normalized to 1. Three biological replications were conducted for wild type flowers; eight biological replications were conducted for 35S::AtZFP2 flowers.