Opaque-2 Regulates a Complex Gene Network Associated with Cell Differentiation and Storage Functions of Maize Endosperm.

Development of the cereal endosperm involves cell differentiation processes that enable nutrient uptake from the maternal plant, accumulation of storage products, and their utilization during germination. However, little is known about the regulatory mechanisms that link cell differentiation processes with those controlling storage product synthesis and deposition, including the activation of zein genes by the maize (Zea mays) bZIP transcription factor Opaque-2 (O2). Here, we mapped in vivo binding sites of O2 in B73 endosperm and compared the results with genes differentially expressed in B73 and B73o2 We identified 186 putative direct O2 targets and 1677 indirect targets, encoding a broad set of gene functionalities. Examination of the temporal expression patterns of O2 targets revealed at least two distinct modes of O2-mediated gene activation. Two O2-activated genes, bZIP17 and NAKED ENDOSPERM2 (NKD2), encode transcription factors, which can in turn coactivate other O2 network genes with O2. NKD2 (with its paralog NKD1) was previously shown to be involved in regulation of aleurone development. Collectively, our results provide insights into the complexity of the O2-regulated network and its role in regulation of endosperm cell differentiation and function.

FERTILIZATION-INDEPENDENT SEED-Polycomb Repressive Complex 2 Plays a Dual Role in Regulating Type I MADS-Box Genes in Early Endosperm Development.

Early endosperm development presents a unique system in which to uncover epigenetic regulatory mechanisms because the contributing maternal and paternal genomes possess differential epigenetic modifications. In Arabidopsis (Arabidopsis thaliana), the initiation of endosperm coenocytic growth upon fertilization and the transition to endosperm cellularization are regulated by the FERTILIZATION-INDEPENDENT SEED (FIS)-Polycomb Repressive Complex 2 (PRC2), a putative H3K27 methyltransferase. Here, we address the possible role of the FIS-PRC2 complex in regulating the type I MADS-box gene family, which has been shown previously to regulate early endosperm development. We show that a subclass of type I MADS-box genes (C2 genes) was expressed in distinct domains of the coenocytic endosperm in wild-type seeds. Furthermore, the C2 genes were mostly up-regulated biallelically during the extended coenocytic phase of endosperm development in the FIS-PRC2 mutant background. Using allele-specific expression analysis, we also identified a small subset of C2 genes subjected to FIS-PRC2-dependent maternal or FIS-PRC2-independent paternal imprinting. Our data support a dual role for the FIS-PRC2 complex in the regulation of C2 type I MADS-box genes, as evidenced by a generalized role in the repression of gene expression at both alleles associated with endosperm cellularization and a specialized role in silencing the maternal allele of imprinted genes.

SUPPRESSOR OF FRIGIDA (SUF4) Supports Gamete Fusion via Regulating Arabidopsis EC1 Gene Expression.

The EGG CELL1 (EC1) gene family of Arabidopsis (Arabidopsis thaliana) comprises five members that are specifically expressed in the egg cell and redundantly control gamete fusion during double fertilization. We investigated the activity of all five EC1 promoters in promoter-deletion studies and identified SUF4 (SUPPRESSOR OF FRIGIDA4), a C2H2 transcription factor, as a direct regulator of the EC1 gene expression. In particular, we demonstrated that SUF4 binds to all five Arabidopsis EC1 promoters, thus regulating their expression. The down-regulation of SUF4 in homozygous suf4-1 ovules results in reduced EC1 expression and delayed sperm fusion, which can be rescued by expressing SUF4-ß-glucuronidase under the control of the SUF4 promoter. To identify more gene products able to regulate EC1 expression together with SUF4, we performed coexpression studies that led to the identification of MOM1 (MORPHEUS' MOLECULE1), a component of a silencing mechanism that is independent of DNA methylation marks. In mom1-3 ovules, both SUF4 and EC1 genes are down-regulated, and EC1 genes show higher levels of histone 3 lysine-9 acetylation, suggesting that MOM1 contributes to the regulation of SUF4 and EC1 gene expression.

RNA sequencing of laser-capture microdissected compartments of the maize kernel identifies regulatory modules associated with endosperm cell differentiation.

Endosperm is an absorptive structure that supports embryo development or seedling germination in angiosperms. The endosperm of cereals is a main source of food, feed, and industrial raw materials worldwide. However, the genetic networks that regulate endosperm cell differentiation remain largely unclear. As a first step toward characterizing these networks, we profiled the mRNAs in five major cell types of the differentiating endosperm and in the embryo and four maternal compartments of the maize (Zea mays) kernel. Comparisons of these mRNA populations revealed the diverged gene expression programs between filial and maternal compartments and an unexpected close correlation between embryo and the aleurone layer of endosperm. Gene coexpression network analysis identified coexpression modules associated with single or multiple kernel compartments including modules for the endosperm cell types, some of which showed enrichment of previously identified temporally activated and/or imprinted genes. Detailed analyses of a coexpression module highly correlated with the basal endosperm transfer layer (BETL) identified a regulatory module activated by MRP-1, a regulator of BETL differentiation and function. These results provide a high-resolution atlas of gene activity in the compartments of the maize kernel and help to uncover the regulatory modules associated with the differentiation of the major endosperm cell types.

Temporal patterns of gene expression in developing maize endosperm identified through transcriptome sequencing.

Endosperm is a filial structure resulting from a second fertilization event in angiosperms. As an absorptive storage organ, endosperm plays an essential role in support of embryo development and seedling germination. The accumulation of carbohydrate and protein storage products in cereal endosperm provides humanity with a major portion of its food, feed, and renewable resources. Little is known regarding the regulatory gene networks controlling endosperm proliferation and differentiation. As a first step toward understanding these networks, we profiled all mRNAs in the maize kernel and endosperm at eight successive stages during the first 12 d after pollination. Analysis of these gene sets identified temporal programs of gene expression, including hundreds of transcription-factor genes. We found a close correlation of the sequentially expressed gene sets with distinct cellular and metabolic programs in distinct compartments of the developing endosperm. The results constitute a preliminary atlas of spatiotemporal patterns of endosperm gene expression in support of future efforts for understanding the underlying mechanisms that control seed yield and quality.

MYB64 and MYB119 are required for cellularization and differentiation during female gametogenesis in Arabidopsis thaliana.

In angiosperms, the egg cell forms within the multicellular, haploid female gametophyte. Female gametophyte and egg cell development occurs through a unique process in which a haploid spore initially undergoes several rounds of synchronous nuclear divisions without cytokinesis, resulting in a single cell containing multiple nuclei. The developing gametophyte then forms cell walls (cellularization) and the resulting cells differentiate to generate the egg cell and several accessory cells. The switch between free nuclear divisions and cellularization-differentiation occurs during developmental stage FG5 in Arabidopsis, and we refer to it as the FG5 transition. The molecular regulators that initiate the FG5 transition during female gametophyte development are unknown. In this study, we show using mutant analysis that two closely related MYB transcription factors, MYB64 and MYB119, act redundantly to promote this transition. MYB64 and MYB119 are expressed during the FG5 transition, and most myb64 myb119 double mutant gametophytes fail to initiate the FG5 transition, resulting in uncellularized gametophytes with supernumerary nuclei. Analysis of cell-specific markers in myb64 myb119 gametophytes that do cellularize suggests that gametophytic polarity and differentiation are also affected. We also show using multiple-mutant analysis that MYB119 expression is regulated by the histidine kinase CKI1, the primary activator of two-component signaling (TCS) during female gametophyte development. Our data establish a molecular pathway regulating the FG5 transition and implicates CKI1-dependent TCS in the promotion of cellularization, differentiation, and gamete specification during female gametogenesis.

Comprehensive developmental profiles of gene activity in regions and subregions of the Arabidopsis seed.

Seeds are complex structures that consist of the embryo, endosperm, and seed-coat regions that are of different ontogenetic origins, and each region can be further divided into morphologically distinct subregions. Despite the importance of seeds for food, fiber, and fuel globally, little is known of the cellular processes that characterize each subregion or how these processes are integrated to permit the coordinated development of the seed. We profiled gene activity genome-wide in every organ, tissue, and cell type of Arabidopsis seeds from fertilization through maturity. The resulting mRNA datasets offer the most comprehensive description of gene activity in seeds with high spatial and temporal resolution,providing unique insights into the function of understudied seed regions. Global comparisons of mRNA populations reveal unexpected overlaps in the functional identities of seed subregions. Analyses of coexpressed gene sets suggest that processes that regulate seed size and filling are coordinated across several subregions. Predictions of gene regulatory networks based on the association of transcription factors with enriched DNA sequence motifs upstream of coexpressed genes identify regulators of seed development. These studies emphasize the utility of these data sets as an essential resource for the study of seed biology.

Global analysis of gene activity during Arabidopsis seed development and identification of seed-specific transcription factors.

Most of the transcription factors (TFs) responsible for controlling seed development are not yet known. To identify TF genes expressed at specific stages of seed development, including those unique to seeds, we used Affymetrix GeneChips to profile Arabidopsis genes active in seeds from fertilization through maturation and at other times of the plant life cycle. Seed gene sets were compared with those expressed in prefertilization ovules, germinating seedlings, and leaves, roots, stems, and floral buds of the mature plant. Most genes active in seeds are shared by all stages of seed development, although significant quantitative changes in gene activity occur. Each stage of seed development has a small gene set that is either specific at the level of the GeneChip or up-regulated with respect to genes active at other stages, including those that encode TFs. We identified 289 seed-specific genes, including 48 that encode TFs. Seven of the seed-specific TF genes are known regulators of seed development and include the LEAFY COTYLEDON (LEC) genes LEC1, LEC1-LIKE, LEC2, and FUS3. The rest represent different classes of TFs with unknown roles in seed development. Promoter-beta-glucuronidase (GUS) fusion experiments and seed mRNA localization GeneChip datasets showed that the seed-specific TF genes are active in different compartments and tissues of the seed at unique times of development. Collectively, these seed-specific TF genes should facilitate the identification of regulatory networks that are important for programming seed development.

Identification of genes expressed in the angiosperm female gametophyte.

Until recently, identification of gene regulatory networks controlling the development of the angiosperm female gametophyte has presented a significant challenge to the plant biology community. The angiosperm female gametophyte is fairly inaccessible because it is a highly reduced structure relative to the sporophyte and is embedded within multiple layers of the sporophytic tissue of the ovule. Moreover, although mutations affecting the female gametophyte can be readily isolated, their analysis can be difficult because most affect genes involved in basic cellular processes that are also required in the diploid sporophyte. In recent years, expression-based approaches in multiple species have begun to uncover gene sets expressed in specific female gametophyte cells as a means of identifying regulatory networks controlling cell differentiation in the female gametophyte. Here, recent efforts to identify and analyse gene expression programmes in the Arabidopsis female gametophyte are reviewed.

Identification of transcription-factor genes expressed in the Arabidopsis female gametophyte.

BACKGROUND: In flowering plants, the female gametophyte is typically a seven-celled structure with four cell types: the egg cell, the central cell, the synergid cells, and the antipodal cells. These cells perform essential functions required for double fertilization and early seed development. Differentiation of these distinct cell types likely involves coordinated changes in gene expression regulated by transcription factors. Therefore, understanding female gametophyte cell differentiation and function will require dissection of the gene regulatory networks operating in each of the cell types. These efforts have been hampered because few transcription factor genes expressed in the female gametophyte have been identified. To identify such genes, we undertook a large-scale differential expression screen followed by promoter-fusion analysis to detect transcription-factor genes transcribed in the Arabidopsis female gametophyte. RESULTS: Using quantitative reverse-transcriptase PCR, we analyzed 1,482 Arabidopsis transcription-factor genes and identified 26 genes exhibiting reduced mRNA levels in determinate infertile 1 mutant ovaries, which lack female gametophytes, relative to ovaries containing female gametophytes. Spatial patterns of gene transcription within the mature female gametophyte were identified for 17 transcription-factor genes using promoter-fusion analysis. Of these, ten genes were predominantly expressed in a single cell type of the female gametophyte including the egg cell, central cell and the antipodal cells whereas the remaining seven genes were expressed in two or more cell types. After fertilization, 12 genes were transcriptionally active in the developing embryo and/or endosperm. CONCLUSIONS: We have shown that our quantitative reverse-transcriptase PCR differential-expression screen is sufficiently sensitive to detect transcription-factor genes transcribed in the female gametophyte. Most of the genes identified in this study have not been reported previously as being expressed in the female gametophyte. Therefore, they might represent novel regulators and provide entry points for reverse genetic and molecular approaches to uncover the gene regulatory networks underlying female gametophyte development.

The MYB98 subcircuit of the synergid gene regulatory network includes genes directly and indirectly regulated by MYB98.

The female gametophyte contains two synergid cells that play a role in many steps of the angiosperm reproductive process, including pollen tube guidance. At their micropylar poles, the synergid cells have a thickened and elaborated cell wall: the filiform apparatus that is thought to play a role in the secretion of the pollen tube attractant(s). MYB98 regulates an important subcircuit of the synergid gene regulatory network (GRN) that functions to activate the expression of genes required for pollen tube guidance and filiform apparatus formation. The MYB98 subcircuit comprises at least 83 downstream genes, including 48 genes within four gene families (CRP810, CRP3700, CRP3730 and CRP3740) that encode Cys-rich proteins. We show that the 11 CRP3700 genes, which include DD11 and DD18, are regulated by a common cis-element, GTAACNT, and that a multimer of this sequence confers MYB98-dependent synergid expression. The GTAACNT element contains the MYB98-binding site identified in vitro, suggesting that the 11 CRP3700 genes are direct targets of MYB98. We also show that five of the CRP810 genes, which include DD2, lack a functional GTAACNT element, suggesting that they are not directly regulated by MYB98. In addition, we show that the five CRP810 genes are regulated by the cis-element AACGT, and that a multimer of this sequence confers synergid expression. Together, these results suggest that the MYB98 branch of the synergid GRN is multi-tiered and, therefore, contains at least one additional downstream transcription factor.

The female gametophyte.

The angiosperm female gametophyte is critical for plant reproduction. It contains the egg cell and central cell that become fertilized and give rise to the embryo and endosperm of the seed, respectively. Female gametophyte development begins early in ovule development with the formation of a diploid megaspore mother cell that undergoes meiosis. One resulting haploid megaspore then develops into the female gametophyte. Genetic and epigenetic processes mediate specification of megaspore mother cell identity and limit megaspore mother cell formation to a single cell per ovule. Auxin gradients influence female gametophyte polarity and a battery of transcription factors mediate female gametophyte cell specification and differentiation. The mature female gametophyte secretes peptides that guide the pollen tube to the embryo sac and contains protein complexes that prevent seed development before fertilization. Post-fertilization, the female gametophyte influences seed development through maternal-effect genes and by regulating parental contributions. Female gametophytes can form by an asexual process called gametophytic apomixis, which involves formation of a diploid female gametophyte and fertilization-independent development of the egg into the embryo. These functions collectively underscore the important role of the female gametophyte in seed and food production.

The AGL62 MADS domain protein regulates cellularization during endosperm development in Arabidopsis.

Endosperm, a storage tissue in the angiosperm seed, provides nutrients to the embryo during seed development and/or to the developing seedling during germination. A major event in endosperm development is the transition between the syncytial phase, during which the endosperm nuclei undergo many rounds of mitosis without cytokinesis, and the cellularized phase, during which cell walls form around the endosperm nuclei. The molecular processes controlling this phase transition are not understood. In agl62 seeds, the endosperm cellularizes prematurely, indicating that AGL62 is required for suppression of cellularization during the syncytial phase. AGL62 encodes a Type I MADS domain protein that likely functions as a transcription factor. During seed development, AGL62 is expressed exclusively in the endosperm. During wild-type endosperm development, AGL62 expression is strong during the syncytial phase and then declines abruptly just before cellularization. By contrast, in mutant seeds containing defects in some FERTILIZATION-INDEPENDENT SEED (FIS) class Polycomb group genes, the endosperm fails to cellularize and AGL62 expression fails to decline. Together, these data suggest that AGL62 suppresses cellularization during the syncytial phase of endosperm development and that endosperm cellularization is triggered via direct or indirect AGL62 inactivation by the FIS polycomb complex.

AGL61 interacts with AGL80 and is required for central cell development in Arabidopsis.

The central cell of the female gametophyte plays a role in pollen tube guidance and in regulating the initiation of endosperm development. Following fertilization, the central cell gives rise to the seed's endosperm, which nourishes the developing embryo within the seed. The molecular mechanisms controlling specification and differentiation of the central cell are poorly understood. We identified AGL61 in a screen for transcription factor genes expressed in the female gametophyte. AGL61 encodes a Type I MADS domain protein, which likely functions as a transcription factor. Consistent with this, an AGL61-green fluorescent protein fusion protein is localized to the nucleus. In the context of the ovule and seed, AGL61 is expressed exclusively in the central cell and early endosperm. agl61 female gametophytes are affected in the central cell specifically. The morphological defects include an overall reduction in size of the central cell and a reduced or absent central cell vacuole. When fertilized with wild-type pollen, agl61 central cells fail to give rise to endosperm. In addition, synergid- and antipodal-expressed genes are ectopically expressed in agl61 central cells. The expression pattern and mutant phenotype of AGL61 are similar to those of AGL80, suggesting that AGL61 may function as a heterodimer with AGL80 within the central cell; consistent with this, AGL61 and AGL80 interact in yeast two-hybrid assays. Together, these data suggest that AGL61 functions as a transcription factor and controls the expression of downstream genes during central cell development.

MYB98 positively regulates a battery of synergid-expressed genes encoding filiform apparatus localized proteins.

The synergid cells within the female gametophyte are essential for reproduction in angiosperms. MYB98 encodes an R2R3-MYB protein required for pollen tube guidance and filiform apparatus formation by the synergid cells. To test the predicted function of MYB98 as a transcriptional regulator, we determined its subcellular localization and examined its DNA binding properties. We show that MYB98 binds to a specific DNA sequence (TAAC) and that a MYB98-green fluorescent protein fusion protein localizes to the nucleus, consistent with a role in transcriptional regulation. To identify genes regulated by MYB98, we tested previously identified synergid-expressed genes for reduced expression in myb98 female gametophytes and identified 16 such genes. We dissected the promoter of one of the downstream genes, DD11, and show that it contains a MYB98 binding site required for synergid expression, suggesting that DD11 is regulated directly by MYB98. To gain insight into the functions of the downstream genes, we chose five genes and determined the subcellular localization of the encoded proteins. We show that these five proteins are secreted into the filiform apparatus, suggesting that they play a role in either the formation or the function of this unique structure. Together, these data suggest that MYB98 functions as a transcriptional regulator in the synergid cells and activates the expression of genes required for pollen tube guidance and filiform apparatus formation.

Synergid cell death in Arabidopsis is triggered following direct interaction with the pollen tube.

During angiosperm reproduction, one of the two synergid cells within the female gametophyte undergoes cell death prior to fertilization. The pollen tube enters the female gametophyte by growing into the synergid cell that undergoes cell death and releases its two sperm cells within the degenerating synergid cytoplasm to effect double fertilization. In Arabidopsis (Arabidopsis thaliana) and many other species, synergid cell death is dependent upon pollination. However, the mechanism by which the pollen tube causes synergid cell death is not understood. As a first step toward understanding this mechanism, we defined the temporal relationship between pollen tube arrival at the female gametophyte and synergid cell death in Arabidopsis. Using confocal laser scanning microscopy, light microscopy, transmission electron microscopy, and real-time observation of these two events in vitro, we demonstrate that synergid cell death initiates after the pollen tube arrives at the female gametophyte but before pollen tube discharge. Our results support a model in which a signaling cascade triggered by pollen tube-synergid cell contact induces synergid cell death in Arabidopsis.

Identification of genes expressed in the Arabidopsis female gametophyte.

The angiosperm female gametophyte typically consists of one egg cell, two synergid cells, one central cell, and three antipodal cells. Each of these four cell types has unique structural features and performs unique functions that are essential for the reproductive process. The gene regulatory networks conferring these four phenotypic states are largely uncharacterized. As a first step towards dissecting the gene regulatory networks of the female gametophyte, we have identified a large collection of genes expressed in specific cells of the Arabidopsis thaliana female gametophyte. We identified these genes using a differential expression screen based on reduced expression in determinant infertile1 (dif1) ovules, which lack female gametophytes. We hybridized ovule RNA probes with Affymetrix ATH1 genome arrays and validated the identified genes using real-time RT-PCR. These assays identified 71 genes exhibiting reduced expression in dif1 ovules. We further validated 45 of these genes using promoter::GFP fusions and 43 were expressed in the female gametophyte. In the context of the ovule, 11 genes were expressed exclusively in the antipodal cells, 11 genes were expressed exclusively or predominantly in the central cell, 17 genes were expressed exclusively or predominantly in the synergid cells, one gene was expressed exclusively in the egg cell, and three genes were expressed strongly in multiple cells of the female gametophyte. These genes provide insights into the molecular processes functioning in the female gametophyte and can be used as starting points to dissect the gene regulatory networks functioning during differentiation of the four female gametophyte cell types.

AGL80 is required for central cell and endosperm development in Arabidopsis.

During plant reproduction, the central cell of the female gametophyte becomes fertilized to produce the endosperm, a storage tissue that nourishes the developing embryo within the seed. The molecular mechanisms controlling the specification and differentiation of the central cell are poorly understood. We identified a female gametophyte mutant in Arabidopsis thaliana, fem111, that is affected in central cell development. In fem111 female gametophytes, the central cell's nucleolus and vacuole fail to mature properly. In addition, endosperm development is not initiated after fertilization of fem111 female gametophytes. fem111 contains a T-DNA insertion in AGAMOUS-LIKE80 (AGL80). FEM111/AGL80 is a member of the MADS box family of genes that likely encode transcription factors. An AGL80-green fluorescent protein fusion protein is localized to the nucleus. Within the ovule and seed, FEM111/AGL80 is expressed exclusively in the central cell and uncellularized endosperm. FEM111/AGL80 expression is also detected in roots, leaves, floral stems, anthers, and young flowers by real-time RT-PCR. FEM111/AGL80 is required for the expression of two central cell-expressed genes, DEMETER and DD46, but not for a third central cell-expressed gene, FERTILIZATION-INDEPENDENT SEED2. Together, these data suggest that FEM111/AGL80 functions as a transcription factor within the central cell gene regulatory network and controls the expression of downstream genes required for central cell development and function.

NUCLEAR FUSION DEFECTIVE1 encodes the Arabidopsis RPL21M protein and is required for karyogamy during female gametophyte development and fertilization.

Karyogamy, or nuclear fusion, is essential for sexual reproduction. In angiosperms, karyogamy occurs three times: twice during double fertilization of the egg cell and the central cell and once during female gametophyte development when the two polar nuclei fuse to form the diploid central cell nucleus. The molecular mechanisms controlling karyogamy are poorly understood. We have identified nine female gametophyte mutants in Arabidopsis (Arabidopsis thaliana), nuclear fusion defective1 (nfd1) to nfd9, that are defective in fusion of the polar nuclei. In the nfd1 to nfd6 mutants, failure of fusion of the polar nuclei is the only defect detected during megagametogenesis. nfd1 is also affected in karyogamy during double fertilization. Using transmission electron microscopy, we showed that nfd1 nuclei fail to undergo fusion of the outer nuclear membranes. nfd1 contains a T-DNA insertion in RPL21M that is predicted to encode the mitochondrial 50S ribosomal subunit L21, and a wild-type copy of this gene rescues the mutant phenotype. Consistent with the predicted function of this gene, an NFD1-green fluorescent protein fusion protein localizes to mitochondria and the NFD1/RPL21M gene is expressed throughout the plant. The nfd3, nfd4, nfd5, and nfd6 mutants also contain T-DNA insertions in genes predicted to encode proteins that localize to mitochondria, suggesting a role for this organelle in nuclear fusion.

Targeted mutagenesis using zinc-finger nucleases in Arabidopsis.

Targeted mutagenesis is an essential tool of reverse genetics that could be used experimentally to investigate basic plant biology or modify crop plants for improvement of important agricultural traits. Although targeted mutagenesis is routine in several model organisms including yeast and mouse, efficient and widely usable methods to generate targeted modifications in plant genes are not currently available. In this study we investigated the efficacy of a targeted-mutagenesis approach based on zinc-finger nucleases (ZFNs). In this procedure, ZFNs are used to generate double-strand breaks at specific genomic sites, and subsequent repair produces mutations at the break site. To determine whether ZFNs can cleave and induce mutations at specific sites within higher plant genomes, we introduced a construct carrying both a ZFN gene, driven by a heat-shock promoter, and its target into the Arabidopsis genome. Induction of ZFN expression by heat shock during seedling development resulted in mutations at the ZFN recognition sequence at frequencies as high as 0.2 mutations per target. Of 106 ZFN-induced mutations characterized, 83 (78%) were simple deletions of 1-52 bp (median of 4 bp), 14 (13%) were simple insertions of 1-4 bp, and 9 (8%) were deletions accompanied by insertions. In 10% of induced individuals, mutants were present in the subsequent generation, thus demonstrating efficient transmission of the ZFN-induced mutations. These data indicate that ZFNs can form the basis of a highly efficient method for targeted mutagenesis of plant genes.