CCS52A2/FZR1, a cell cycle regulator, is an essential factor for shoot apical meristem maintenance in Arabidopsis thaliana
© Liu et al.; licensee BioMed Central Ltd. 2012
Received: 18 June 2012
Accepted: 3 August 2012
Published: 8 August 2012
Cell division and cell fate decisions regulate organ formation and function in plant growth and development. It is still unclear how specific meristematic regulatory networks operate with the cell cycle machinery to translate stem cell identity and maintenance into cellular behavior. In this study, we address these questions by analysis of a shoot apex defective mutant, namely xcm9.
Phenotypic analysis of the xcm9 mutant reveals concomitant premature termination of floral shoots with frequent bifurcation of the shoot apices, stems, and flowers. Microscopic observations show irregular cell organization in shoot apical meristems of xcm9. Positional cloning revealed that xcm9 is a loss of function allele of the CCS52A2/FZR1 gene, which has previously been implicated in root development. Expression analysis demonstrated that CCS52A2 maintains a higher transcriptional expression level in actively dividing tissue. Genetic studies indicated that the CCS52A2 gene functions together with WUSCHEL (WUS) and CLAVATA3 (CLV3) in regulating the development of the shoot meristem, and also contributes to this regulation together with the chromatin remodeling pathway. In addition, fewer xcm9 cells express CYCLIN B1:1, showing that cell cycle progression is disrupted in the mutant.
We propose that the CCS52A2 gene is a mediator that functions together with meristematic genes to regulate meristem organization, and cross-functions with chromatin regulators in cell cycle progression during shoot apical meristem development.
In higher plants, the vast majority of structures and organs, such as leaves, stems, roots, and flowers, are formed postembryonically from groups of undifferentiated cells, called meristems. In many species of plants, certain meristems are maintained throughout life. The activities of the root apical meristem (RAM) and the shoot apical meristem (SAM) determine root and shoot structure and function, respectively. Stem cells are confined to the centers of shoot and root apices, and their proliferation is maintained by signals that cells receive from the local environment . They undergo precisely controlled division, which must be rapid enough to replenish cells lost to differentiation, but restricted enough to prevent overproliferation of undifferentiated cells. Adjacent to the stem cells, several cells in the SAM and RAM form the organizing center (OC) and quiescent center (QC), respectively. These cells coordinate with neighboring cells to establish the balance between proliferation and differentiation in the meristem niche .
Several meristematic genes form feedback networks that control this dynamic balance. In Arabidopsis, WUS encodes a transcription factor expressed in the OC, and its expression promotes the identity of distal meristem cells as stem cells, which themselves are characterized in part by CLV3 expression [3, 4]. CLV3 encodes a peptide hormone expressed in the central apical surface of shoot and floral meristems and is necessary for controlling the size of the central zone (CZ) in SAMs [5, 6]. WUS and CLV3 form a feedback regulation loop: CLV3 acts as mobile intercellular signal to negatively regulate WUS transcription in the OC via the receptor proteins CLV1/CLV2/CORYNE (CRN), while WUS positively regulates CLV3 expression [7–9]. Similar to the shoot meristem, the root QC maintains the stem state of the surrounding cells and prevents these cells from differentiating . WOX5 is the functional homolog of WUS expressed in the QC , and CLE40 is a CLV3-related peptide expressed in differentiated stele and columella cells [12, 13]. Like the CLV3-WUS network in the shoot, CLE40 and WOX5 form a self-regulating network that controls the proliferation and differentiation of stem cells in the root .
Besides these vital meristematic regulatory genes, the organization and maintenance of cells in meristems are also modulated by several cell cycle control genes. It is still unclear whether the cell cycle machinery acts largely independently in regulating meristem organization, or acts by receiving the signals from meristematic pattern genes via unknown mechanisms. The cell division cycle protein CDC5, cyclin D3 (CYCD3), HBT/CDC27B, and the cyclin-dependent kinases A;1 (CDKA;1), CDKB2;1 and CDKB2;2 have been reported to be necessary for SAM and/or RAM development [15–19]. These proteins are among numerous cell cycle regulators, including other cyclins, CDKs, CDK inhibitors and CDCs, that precisely control the mitotic cell cycle during the four cell phases and several checkpoints to accomplish DNA replication and subsequent division. Once the need for the cell cycle regulators ends, they are degraded by ubiquitin-mediated proteolysis. Anaphase-Promoting Complex (APC), which functions as an E3 ubiquitin ligase that marks target cell cycle proteins for degradation by the 26 S proteasome, plays an important role in the phase transition of the cell cycle .
Some genes that are not considered to be part of the cell cycle regulator class of proteins also contribute to the development of meristems and are involved with the normal sequence of the cell cycle. Among these are the MGOUN 1 (MGO1) and FASCIATA 1 (FAS1) genes, which play critical roles in the fundamental organization and/or functioning of both the SAM and RAM . fas1 and mgo1 are characterized by fasciated stems and short roots [22, 23]. FAS1 is one subunit of Arabidopsis chromatin assembly factor-1 (CAF-1), which shows a conserved activity for chromatin assembly at the DNA replication fork in S phase. Loss of function of FAS1 alters the epigenetic marks at promoters of genes involved in activation of the G2 damage checkpoint, leading to inhibition of mitosis progression . MGO1 is homologous to type IB topoisomerase, which has been reported to stabilize the epigenetic state of developmentally regulated genes and to affect gene expression in conjunction with the chromatin remodeling pathway in Arabidopsis. An unknown mechanism is likely to be present that integrates the meristematic regulating network with the cell cycle machinery to translate SAM identity and maintenance into cellular behavior. The exact nature of this linkage and the mediator or mediators between these two networks is an important research question.
In this article, we report the isolation of a novel SAM defective mutant, xcm9, which displays clear signs of bifurcation and premature termination under our conditions. Positional cloning revealed that the XCM9 gene encodes an activator of APC/C, namely CCS52A2. RT-PCR, promoter analysis and in situ hybridization assays show that the CCS52A2 gene is broadly expressed in all organs tested, but has higher expression specifically in the shoot apices and root tips. Our genetic studies indicate that the CCS52A2 gene functions together with WUS and CLV3 in stem cell regulation, and also contributes to this regulation together with the chromatin remodeling pathway. Monitoring of CYCB1:1 expression revealed that cell cycle progression is disturbed in xcm9. We propose that the CCS52A2 gene is a mediator that regulates meristem organization, functions together with meristematic genes and cross-functions with chromatin regulators in cell cycle progression during SAM development.
Mutations in the XCM9gene disrupt SAM development and maintenance
Besides the defective shoots, xcm9 was reduced in height (Figure 1A), stem diameter (Figure 2E), silique length (Figure 2F) and leaf size (Figure 2G). The number of seeds per silique in xcm9 was reduced, but the seed size was about 1/5 larger in both length (448.7 ± 23.8 μm for WT, 556.6 ± 42.8 μm for xcm9, p < 0.05) and in width (252.9 ± 26 μm for WT, 321.9 ± 24.1 μm for xcm9, p < 0.05).
Cell organization of SAM is disordered in xcm9
A 2.6Kb deletion of At4g11920caused the shoot apex defect
To test if the mutant phenotype of XCM9 gene cosegregated with the kanamycin resistance harbored in the T-DNA insert, we grew the mutant seeds on MS medium with kanamycin and found that they are kanamycin-sensitive, indicating that the kanamycin resistance gene may be silenced or lost. The T-DNA website (signal.salk.edu)  indicates that the T-DNA was inserted in the 3rd exon of the At1g18960 gene, which encodes a myb-like HTH transcriptional regulator family protein. Primers were designed from the flanking sequences of the T-DNA insertion to confirm its existence. Expected amplification was obtained, indicating that the kanamycin resistance gene was silenced in the mutant. Segregation analysis indicated that the T-DNA insertion did not cosegregate with the mutant phenotype, suggesting that the mutant phenotype is not caused by the T-DNA insertion.
To verify whether the deletion in At4g11920 caused the shoot apex defects in xcm9, we next performed a complementation test by inserting a 9.7Kb genomic DNA fragment containing the 1.5Kb upstream sequence, the full-length coding region and 1.2Kb downstream sequences of At4g11920 (which includes the At4g11910 coding region) into heterozygous plants of xcm9 (Figure 4B), because homozygous xcm9 plants exhibit a strong phenotype and do not produce enough flowers for transformation. We found that all transgenic plants obtained in a homozygous xcm9 background were phenotypically indistinguishable from WT plants, confirming that the apex defect phenotypes observed in xcm9 were caused by the deletion of either At4g11910 or At4g11920. In addition, two SALK lines, SALK_001978 and SALK_073708C were identified with T-DNA insertions in exon 7 and 6 of At4g11920, respectively (Figure 4B). These two mutants showed similar phenotypes to xcm9, and were designated as xcm9-2 and xcm9-3, respectively. These results together confirmed that the mutant phenotypes were caused by the mutation of At4g11920.
The At4g11920 gene consists of nine exons and eight introns (Figure 4B) and encodes a putative CDH1/CCS52A2/FZR1 protein (CDH1, also known as HCT1 for Homolog of CDC Twenty; CCS52A2, also known as a 52 kDa protein encoded by a Cell Cycle Switch gene), which contains WD40 repeats and is a component of APC/C, acting a co-activator and substrate recognizer [27, 28]. Therefore, the xcm9 mutant was renamed ccs52a2-3. xcm9-2 corresponds to ccs52a2-1, and xcm9-3 was renamed ccs52a2-2. In Arabidopsis, CCS52A2 was reported as stabilizing root meristem maintenance by acting in the distal region of the root and regulating the mitotic state of the QC . Additionally, CCS52A2, under the regulation of the transcription factor EF1/DEL, promotes endoreduplication in cells of mature leaves . In Arabidopsis, three CDH1/FZR homologs, AtCCS52A1, AtCCS52A2 and AtCCS52B, have been previously identified . All of the CCS52 (A1, A2 and B) proteins show a similar structure, with conserved C-box, CSM (Cdh1-specific motif), the IR motifs (APC binding domain) and the CBM motif (mitotic RVL cyclin binding motif) . Importantly, ccs52a2-3 lost the conserved potential CDK phosphorylation sites, IR and CBM motifs of CCS52A2, suggesting that this allele is a possible null mutant.
CCS52A2mRNA is broadly expressed
The expression patterns of WUS and CLV3 were altered in the ccs52a2-3mutant
The disordered organization of SAM in ccs52a2-3 and high expression of CCS52A2 in the shoot apex prompted us to investigate whether the expression patterns of two essential meristematic genes, WUS and CLV3, are affected in the mutant. qRT-PCR results using dissected SAM from 10-day-old seedlings indicated that WUS was expressed at a higher level in ccs52a2-3, while CLV3 maintained slightly higher mRNA expression levels in ccs52a2-3 mutants when compared with WT (Figure 5G).
In WT, CLV3 mRNA accumulates in a small zone of cells in the first three layers at the meristem apex (Figure 6G, I) . Compared with WT, the SAMs of 8-day-old ccs52a2-3 seedlings that were examined exhibited increased CLV3 expression (Figure 6H). However, most of the 28-day-old ccs52a2-3 plants (n = 10) had a slightly wider but shallower expression of CLV3 in the reproductive SAM sections compared to limited expression in the central surface of WT SAM (Figure 6J), while some didn’t show a significant difference with WT.
To verify the results from in situ hybridization, we monitored the expression of WUS and CLV3 using GUS reporters (pWUS::GUS and pCLV3::GUS) in WT and ccs52a2-3. The pWUS::GUS assay presented a similar expression pattern as RNA in situs: enlarged and sometimes dual expression domains in the ccs52a2-3 background (Figure 6K). In addition, the pCLV3::GUS location in ccs52a2-3 SAM exhibited two nearby domains of CLV3 expression in 6 of 22 8-day–old seedlings examined (Figure 6M), which indicated that two separate WUS-CLV3 supporting systems of meristem organization had been established as early as the 8-day-old stage. It was also discovered that CLV3 showed broad expression in some first layer cells that flank the major expression zone in 7 of 22 SAMs of ccs52a2-3 seedlings (Figure 6N), while the remaining 9 of 22 ccs52a2-3 SAMs exhibited a single CLV3 expression region. Additionally, the floral SAMs in main stems of ccs52a2-3 always displayed decreased CLV3 expression (Figure 6P), but in some lateral branches multiple CLV3 expression regions were observed (Figure 6Q). These were consistent with the observations of multiple SAMs in lateral branches (Figures 1G, 3I) and smaller SAMs in main stems (Figure 3F, H).
ccs52a2-3 is synergistic to wus and clvmutants
CLV1 is the direct downstream target of CLV3 in the CLV3-WUS pathway , so ccs52a2-3 clv1 was expected to have similar phenotype to ccs52a2-3 clv3. Observations of ccs52a2-3 clv1-1 and ccs52a2-3 clv1-4 double mutants showed similar fasciation in shoot apices as in the ccs52a2-3 clv3-2 double mutants. The ccs52a2-3 clv1-4 (n = 39) displayed bifurcation at a very high frequency, over 50% under our conditions, compared to 12.8% for ccs52a2-3 (n = 39). All ccs52a2-3 clv1-4 double mutants were classified into three types according to the width of fasciated stems (Figure 7G-I). Notably, type I had the widest major stem, followed by type II and then the type III. The type I and type II double mutants had no lateral branches originating from the rosette leaves, but type III did. Most of the ccs52a2-3 clv1-4 double mutants prematurely terminated in the formation of several flowers in lateral branches, which signifies that clv1-4 can’t rescue premature termination in ccs52a2-3 by enlarging SAM size in the absence of CLV function. Furthermore, flowers in both ccs52a2-3 clv3-2 and ccs52a2-3 clv1-4/clv1-1 double mutants appeared to be typical of clv flowers, with increased floral organ number, including 4–8 carpels and 5 petals. The severe SAM bifurcations and fasciation in ccs52a2-3 clv3-2 and ccs52a2-3 clv1-4 double mutants revealed a synergistic interaction between CCS52A2 and the CLV pathway, with CCS52A2 being involved in independent pathways but in the same general process of SAM development and maintenance as CLV3 and CLV1.
Shoot Meristem Defects in wus ccs52a2-3 Plants
Genotype of parent plants
CCS52A2controls cell size and ploidy in differentiated cells during cell division
CYCB1;1 was revealed as one of the possible substrates of APC/C to interact with CCS52s (A1, A2 and B) in Arabidopsis. Since CCS52A2 acts at G2/M to control mitosis exit and endocycle entry, we checked the expression of the G2/M transition reporter gene CYCB 1;1 in ccs52a2-3 mutants. We monitored GFP reporters in the root tips of marker lines harboring CYCB1;1::GFP in a ccs52a2-3 mutant background by confocal microscopy. The results showed that the number of RAM cells expressing CYCB1;1 was dramatically reduced, and the range of cells expressing CYCB1;1 in root was shorter in 7-day-old ccs52a2-3 compared with WT (Figure 5R). In addition, qRT PCR was carried out to quantify the expression levels of CYCB1;1 in the shoot. The seedling SAM of ccs52a2-3 showed lower expression level in comparison with that of WT (Figure 5S). Reduction of CYCB1;1 expression in both the SAM and RAM indicated that the cell cycle process was disturbed in ccs52a2-3 mutants.
CCS52A2 interacted with MGO1 and FAS1
CCS52A2 is necessary for the normal structure and function of the SAM in Arabidopsis
This article demonstrates that loss of CCS52A2 function in ccs52a2-3 mutants led to novel morphological defects in the SAM. The ccs52a2-3 mutant displays concomitant premature termination of floral shoots with frequent bifurcation of the shoot apices, stems, and flowers. These defects in meristems indicate that the CCS52A2 gene is essential for the proper maintenance and function of the SAM in plant development. Interestingly, the vegetative meristem and inflorescence meristem were larger and smaller than that of WT, respectively. One possible explanation for this is that that the double WUS expression zones in the 8-day-old mutant are indicative of bifurcation originating at this time. Lateral splitting results two smaller SAMs, each with a reduced WUS expression domain, and which later give rise to smaller inflorescence SAMs that terminate prematurely.
Expression changes of the major SAM regulator genes WUS and CLV3 in ccs52a2-3 were observed. Ectopic CLV3 expression was detected in the first layer of the SAM (Figure 6N), and the presence of more than one WUS and/or CLV3 expression region in a single SAM of ccs52a2-3 seedlings reveals a rare maintenance pattern in higher plant meristems. All of these abnormal expression patterns of SAM regulators indicate that malfunction of CCS52A2 affects cell identity in meristems. Previously published data show that the CCS52A2 gene plays a role in QC identity in root development, as the RAM organizing genes show irregular expression domains and locations and a failure to maintain the low mitotic activities of cells in QC zone in ccs52a2-1 root . We here provide evidence that the expression of SAM organizers are also altered in ccs52a2-3, and duplicate individual CLV3-WUS regulatory centers are sometimes generated in the mutant SAM. One possible explanation for this is that the proliferation of cells in OC zone results from a partial loss of cell division control, leading to formation of new and separate CLV3-WUS regulation systems in a single SAM of the mutant. Considering the role of CCS52A2 in the root, we give a hypothesis is that the duplicated OCs may somehow result from the failure to maintain low mitotic levels in certain SAM cells, with more cells available to fulfill certain roles.
CCS52A2synergistically interacts with SAM organizers
We suspect that CCS52A2 regulates the development of meristems by indirectly interacting with the WUS and CLV3 genes. This hypothesis is firstly supported by up-regulated expression of CLV3 and WUS in the SAM of ccs52a2-3 seedlings (Figure 5G). Additionally, the more severe phenotypes seen in ccs52a2-3 clv3-2 and ccs52a2-3 clv1-4 double mutants indicate that CCS52A2 may function in separate genetic pathways but are involved in the same process with CLV1 and CLV3, and disrupting multiple modes of cell division regulation in meristems leads to a greater likelihood of a very large loss of cell division control and massive overproliferation. The finding of more severe bifurcations from the larger SAMs (compared with any single mutants) of ccs52a2-3 clv double mutants reveals that the bigger the size of the SAM, the more bifurcations occurred. clv single mutant exhibits an expanded SAM but has not been reported to undergo bifurcation.
The enhanced wus-1 phenotype in ccs52a2-3 wus-1 double mutant indicated that CCS52A2 contributes to meristem establishment and leaf initiation together with WUS; however, CCS52A2 also acts in an independent pathway as well.
CCS52A2 functions in the SAM and stimulates the onset of endocycle in mature leaves
Previous studies suggest that CCS52A2 is an activator of APC/C, which mediates protein proteolysis during mitosis [30, 31, 34]. Therefore, it could regulate SAM maintenance and development by regulating cell division. Our hypothesis is consistent with cell cycle impairment, as indicated by the lower CYCB1;1 expression in RAM and SAM of ccs52a2-3 (Figure 5R). However, the enlargement of the SAM is mainly due to enlarged cell areas and not increased cell number, which indicates that cell proliferation is still under control in mutant SAMs even if impairment exists in the cell cycle. CCS52A2 has also been reported to promote the exit from the cell cycle into the endocycle, and leads to endoreduplication by association with APC/C in Medicago truncatula[28, 34]. In Arabidopsis, similar effects are seen in CCS52A2 knockout (KO) and overexpression (OE) lines . However, in this paper, the unchanged CV (corresponding to ploidy level) of the SAM indicates that the enlarged stem cells are not due to the entrance into endocycle in ccs52a2-3. This is consistent with previous studies that CCS52A2 is not correlated with the endocycle in the RAM in CCS52A2KO, which exhibits a normal Endoreduplication Index in the mutant . But in mature leaves, the presence of smaller cells with lower CV of cells in ccs52a2 mutants indicates a role for CCS52A2 in these tissues. Taken together, these studies further supports the idea that CCS52A2 only has obvious effects on modulating endocycle onset in differentiating cells and not in undifferentiated cells.
CCS52A1, a homolog of CCS52A2, has also been reported to control the endocycle of cells in mature leaves . The similar ploidy changes in null mutants of CCS52A1 and CCS52A2 signify these two genes overlap in function in endoredupliction onset, but that they are not redundant.
CCS52A2was involved in chromatin remodeling
Plants carrying a mutation in the MGO1 gene exhibit some phenotypes similar to ccs52a2-3 mutants , such as bifurcation of stems. MGO1 encodes an Arabidopsis Type IB DNA Topoisomerase, which functions in the relaxation of supercoiled DNA and acts in a number of different DNA metabolic processes, including replication, transcription, repair, and chromatin compaction [36, 37]. It has been reported that MGO1 cooperates with chromatin regulators and is necessary for the maintenance of several epigenetically regulated genes . The indistinguishable phenotypes of ccs52a2-3 mgo1-1 double mutants compared to the mgo1-1 single mutant revealed that these two genes might be involved in the same pathway. Recently, it was found that mutation of MGO1 suppresses ectopic WUS activity and enhances stem cell defects in wus mutants . Likewise, lack of CCS52A2 enhances the SAM defect in wus-1, which further supports a functional relationship between MGO1 and CCS52A2, and imply that their functions may converge to a common downstream process with WUS in stem cell regulation . MGO1 and CCS52A2 proteins are both factors regulating cell cycle processes. It would be interesting to further investigate the molecular nature of the link between CCS52A2 and MGO1.
fas1 is another fasciated mutant that displays stem bifurcation, as do ccs52a2-3 and mgo1. FAS1 encodes one subunit of the chromatin assembly factor (CAF-1), which coordinates nucleosome assembly during DNA replication and repair by depositing newly synthesized histone H3 and H4 . Defects in FAS1 lead to a halt in the progression through G2 by up-regulating several genes involved in the G2 DNA checkpoint, which it accomplishes by changing epigenetic marks of their promoters . The novel phenotypes of ccs52a2-3 fas1-1 double mutants demonstrate the two genes have a synergistic interaction in SAM development. The shared phenotypes of plants that have defects in either FAS1, MGO1 or CCS52A2 reveal a possibility that these three genes might be involved in the same downstream processes. Thus, FAS1 modulates the epigenetic states of genes involved in the G2 damage checkpoint, while MGO1 acts in DNA repair and maintains the expression of several epigenetically regulated genes [24, 25]. Additionally, studies on nodule organogenesis indicate that CCS52A proteins mediate the degradation of mitotic cyclins in the proper phases of the cell cycle to inactivate CDKs, eventually blocking the G2 to M transition and thereby triggering endoreduplication . Previous expression profile analysis of different cell samples in the SAM reveals an enrichment of DNA repair and chromatin modification pathways in stem cells, which suggests that the maintenance of flexible chromatin may facilitate the dynamic balance of gene expression during SAM development . These results lend further evidence to the hypothesis that CCS52A2 might have a function related to triggering the G2/M transition, and that the signal pathways of CCS52A2, FAS1, and MGO1 are all involved in epigenetic maintenance and the activation of the G2 checkpoint.
In this study, we showed that mutations in the CCS52A2 gene disrupted the normal structure and function of the SAM in Arabidopsis. CCS52A2 modulates the expression domains of the meristem regulatory gene WUS and CLV3. Moreover, double mutant analyses illustrates that CCS52A2 synergistically interacts with SAM organizers. The cell cycle was disturbed in the absence of CCS52A2, as indicated by reduced expression of CYCB1; 1. CCS52A2 is involved in control of cell size and ploidy in differentiated cells during cell division. Furthermore, CCS52A2 is involved in chromatin remodeling with FAS1 and MGO1, which were previously reported to be involved in the activation of the G2 checkpoint and epigenetic maintenance. We propose that CCS52A2 regulates meristem organization, functions together with meristematic genes and cross-functions with chromatin regulators in cell cycle progression during SAM development.
Plant materials and growth conditions
ccs52a2-3, ccs52a2-1 and ccs52a2-2 mutants (Col ecotype) in the CCS52A2 gene were obtained from the SALK_074403, SALK_073708 and SALK_001978, respectively, from the Arabidopsis Biological Resource Center (ABRC). wus-1, clv1-1, clv1-4, clv3-2,CLV::GUS marker line, WUS::GUS marker line (all in Ler ecotypes), mgo1-1 (Wassilewskija, Ws ecotpye) and fas1-1(Ler ecotype) were used. In the case of double mutants with wus-1, clv1-1, clv1-4, clv3-2, CLV::GUS marker line, and WUS::GUS marker line, we analyzed only plants that also harbored the erecta mutation. To ensure there were no background effects with the ccs52a2-3 phenotype, we crossed ccs52a2-3 with wildtype Ler accession, and selected ccs52a2-3 erecta plants for examination. ccs52a2-3 mutation showed no significant difference in phenotype between these two ecotypes. All of the plants for morphological analysis were grown in soil in growth chambers under 14-h-light/10-h-dark photoperiods (120μmolm-2 s-1) at 24/22 degrees C. Seedlings used for microscopic studies were grown on Murashige and Skoog (MS) medium on vertically oriented plates in growth chambers under the same light conditions. The F1 of all double mutants were morphologically similar to WT. Seeds from plants with the ccs52a2-3 phenotype in the F2 population were chosen to harvest, and clv, wus, fas1 or mgo1 phenotypic plants were selected as homozygous double mutants in the F3 population. In F1 and F2 population of the double mutant, the clv, wus and ccs52a2-3 mutation was also genotyped by sequencing, which were carried out with the following primers: DWUSL: 5’-CTACCACCGTTGATGTGATC-3’; DWUSR: 5’-TCATGCAAGCTCAGGTACTG-3’; DCLV1L: 5’-GACTTCTTGGTTACGTAGCG-3’; DCLV1R: 5’-ACATCACTCTTCTCGTCCAC-3’; ccs52a2-3 mutation was genotype by checking the PCR product in agarose gelwith primers: 35R-2: 5’-GGTGATTAGTTACCCATCCACGTGTTATAC-3’; 38 F-2: 5’-GCACTTGCTAGCTTACTTCACCGAATCATA-3’.
RT-PCR and Real-time PCR
RNA was extracted from various tissues using the Trizol method, and 2 ug of RNA was used with M-MLV reverse transcriptase (Promega) in an RT reaction to cDNA. The expression level of each gene was normalized against the expression level of ACTIN2. Reverse-transcript PCR was carried out with the following primers: CCS52A2R: 5’-TCGTAACACATCCCATATCT-3’; CCS52A2F:5’-TAAGACGGAAACGCAGAGGT-3’; and ACTINF: 5’-TGCGACAATGGAACTGGAATG’-3; ACTINR: 5’- CAAbGACGTAGGATAGCATGTG-3’. Primers for qRT PCR reactions: qWUSR/F, qCLV3R/F were synethesised by previously report ; qCCS52A2F: 5’-ACACGCCGCAGCAGTGA-3’; qCCS52A2R: 5’-AGCAGTGCCACCACCAGAAG-3’; qACTIN2F: 5’-GGCTCCTCTTAACCCAAAGGC-3’; qACTIN2R :5’-CACACCATCACCAGAATCCAGC-3’.
Tissue sectioning and GUS staining
Tissue was fixed in FAA (3.7% formaldehyde: 50% ethanol: 5% acetic acid) overnight and embedded with Technovit 7100 (Heraeus Kulzer) following the protocol from manufacturer. The sections were cut to 2um and stained with 0.1% toluidine blue. The slides were washed with 70% and 100% ethanol and embedded with a coverslip by neutral resins after drying in air. Histochemical GUS was performed as described previously (Schoof et al., 2000) with minor modifications (elongated the staining time to 8–10 hours).
The shoot apices of 8-day-old seedlings and 28-day-old plants were fixed for 3 h in FAA (3.7% formaldehyde: 50% ethanol: 5% acetic acid) solution after vacuum filtration and embedded in paraplast embedding medium (Oxford Labware, St. Louis, MO). Samples were sectioned at 8um thickness and placed onto Probe-On Plus microscope slides (Fisher Scientific). Tissue preparation, hybridization and detection procedures were performed as described [13, 42]. All the probes were generated with the DIG RNA Labeling Kit T7/SP6 (Roche) and T3 RNA polymerase (Promega), following the manufacturer’s instructions. The template of the CLV3 probe was the first 291 bp of the CLV3 coding sequence and was cloned into the pCRBlunt vector (Invitrogen). The template of the WUS probe was the first 664 bp of the WUS coding sequence and was cloned into the pBluescript KS vector (Fermentas). The CCS52A2 probe was generated by cloning the 340 bp of 5’ UTR sequence into pGEMTeasy (Promega), with the primers CCS525UL: 5’-CCCGGGAAAGAAAAAAAAAAC-3’ and CCS525UR: 5’-ACTCGAGTCACGAATCAA-3’. All templates for the probes were sequenced before the probes were synthesized.
Construction of Transgenes
To generate the transgene for complementation, the full-length genomic sequence (9.7 Kb) of CCS52A2, including the CCS52A2 coding region (including introns), plus a 1.5-Kb promoter and 1.2-Kb terminator, was excised from BAC T26M18 (provided by ABRC) with restriction enzymes XbaI and EcoRV and inserted into the XbaI-SmaI site of the pCambia 3301 vector. The plasmids were introduced into Agrobacterium tumefaciens strain GV3101 by electroporation and transformed into ccs52a2-3 heterozygote plants by the floral dip method [43, 44]. The positive transgenic ccs52a2-3−/− plants were screened from 12-day-old T1 generation by genotyping and spraying 54 mg/ml Basta.
To generate the CCS52A2P::CCS52A2CDS::GUS constructs, the 1.6 Kb promoter region of CCS52A2 was amplified using primers PG1R: 5’-GTCAAGCTTTGTTAACCGTGAAGGCTCTG-3’ and PG1L: 5’-CCGTCTAGATTACTGTTTCGTTC CTCCAG-3’, digested with Hind III and XbaI and inserted into pBI121. Then a 1.5Kb CDS region was amplified from cDNA using primers p1ATGL2: 5’-GCGTCTAGATGGAAGAAGATGAATCAAC-3’ and p1ATGR2: 5’-TATGGATCCACACCG GATTGTTGTT CT-3’, digested with BamHI and Xbal and inserted into pBI121 after the promoter. All the plasmids were introduced into Agrobacterium tumefaciens strain GV3101 by electroporation and transformed into wild type (Col) or ccs52a2-3 heterozygote by the floral dip method [43, 44].
We thank Zhongchi Liu, Thomas Laux, Jian Xu and the Arabidopsis Biological Resource Center for providing mutants and reporter lines. We thank Howard Berg for guidance with microscope photographs and Mayank Gautam for editing the manuscript. We also thank the members in Kede Liu’s Lab and Mark P. Running’s Lab for support in daily work. This work was supported by grants from Chinese 111 Project (B07041) award to K.D.L. and NSF IOS-0744895 awarded to M.P.R.
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