Regulatory mechanisms of the LBD40 transcription factor in Arabidopsis thaliana somatic embryogenesis.

Somatic embryogenesis (SE) is a process by which an embryo is derived from somatic tissue. Transcription factors (TFs) have been identified that control this process. One such TF that promotes SE is AGAMOUS-like 15 (AGL15). Prior work has shown that AGL15 can both induce and repress gene expression. One way this type of dual function TF works is via protein interactions, so a yeast 2-hybrid (Y2H) screen was undertaken. One intriguing protein with which AGL15 interacted in Y2H was LBD40. LBD40 encodes a LATERAL ORGAN BOUNDARIES (LOB)-domain TF that is unique to plants and is primarily expressed during seed development. Here, we confirm the AGL15-LBD40 interaction by quantitative assays and in planta co-immunoprecipation. We also document a role for LBD40, and the closely related protein LBD41, in supporting SE. To determine downstream genes potentially controlled by LBD40, chromatin immunoprecipitation followed by high throughput sequencing (ChIP-seq) was used. More than 400 binding regions for LBD40 were consistently found genome-wide. To determine genes responsive to LBD40/41 accumulation, RNA-seq analysis of transcriptomes of wild-type control and loss-of-function lbd40/lbd41 was performed. Combining these datasets provides insight into genes directly and indirectly controlled by these LOB domain TFs. The gene ontology (GO) enrichment analysis of these regulated genes showed an overrepresentation of biological processes that are associated with SE, further indicating the importance of LBD40 in SE. This work provides insight into SE, a poorly understood, but essential process to generate transgenic plants to meet agricultural demands or test gene function. This manuscript reports on experiments to understand the role that LDB40, a TF, plays in support of SE by investigating genes directly and indirectly controlled by LBD40 and examining physical and genetic interactions with other TFs active in SE. We uncover targets of LBD40 and an interacting TF of the MADS family and investigate targets involvement in SE.

Revisiting AGAMOUS-LIKE15, a Key Somatic Embryogenesis Regulator, Using Next Generation Sequencing Analysis in Arabidopsis.

AGAMOUS-like 15 (AGL15) is a member of the MADS-domain transcription factor (TF) family. MADS proteins are named for a conserved domain that was originally from an acronym derived from genes expressed in a variety of eukaryotes (MCM1-AGAMOUS-DEFICIENS-SERUM RESPONSE FACTOR). In plants, this family has expanded greatly, with more than one-hundred members generally found in dicots, and the proteins encoded by these genes have often been associated with developmental identity. AGL15 transcript and protein accumulate primarily in embryos and has been found to promote an important process called plant regeneration via somatic embryogenesis (SE). To understand how this TF performs this function, we have previously used microarray technologies to assess direct and indirect responsive targets of this TF. We have now revisited this question using next generation sequencing (NGS) to both characterize in vivo binding sites for AGL15 as well as response to the accumulation of AGL15. We compared these data to the prior microarray results to evaluate the different platforms. The new NGS data brought to light an interaction with brassinosteroid (BR) hormone signaling that was "missed" in prior Gene Ontology analysis from the microarray studies.

AGL15 Promotion of Somatic Embryogenesis: Role and Molecular Mechanism.

Plants have amazing regenerative properties with single somatic cells, or groups of cells able to give rise to fully formed plants. One means of regeneration is somatic embryogenesis, by which an embryonic structure is formed that "converts" into a plantlet. Somatic embryogenesis has been used as a model for zygotic processes that are buried within layers of maternal tissues. Understanding mechanisms of somatic embryo induction and development are important as a more accessible model for seed development. We rely on seed development not only for most of our caloric intake, but also as a delivery system for engineered crops to meet agricultural challenges. Regeneration of transformed cells is needed for this applied work as well as basic research to understand gene function. Here we focus on a MADS-domain transcription factor, AGAMOUS-Like15 (AGL15) that shows a positive correlation between accumulation levels and capacity for somatic embryogenesis. We relate AGL15 function to other transcription factors, hormones, and epigenetic modifiers involved in somatic embryo development.

The MADS-domain factor AGAMOUS-Like18 promotes somatic embryogenesis.

AGAMOUS-Like 18 (AGL18) is a MADS domain transcription factor (TF) that is structurally related to AGL15. Here we show that, like AGL15, AGL18 can promote somatic embryogenesis (SE) when ectopically expressed in Arabidopsis (Arabidopsis thaliana). Based on loss-of-function mutants, AGL15 and AGL18 have redundant functions in developmental processes such as SE. To understand the nature of this redundancy, we undertook a number of studies to look at the interaction between these factors. We studied the genome-wide direct targets of AGL18 to characterize its roles at the molecular level using chromatin immunoprecipitation (ChIP)-SEQ combined with RNA-SEQ. The results demonstrated that AGL18 binds to thousands of sites in the genome. Comparison of ChIP-SEQ data for AGL15 and AGL18 revealed substantial numbers of genes bound by both AGL15 and AGL18, but there were also differences. Gene ontology analysis revealed that target genes were enriched for seed, embryo, and reproductive development as well as hormone and stress responses. The results also demonstrated that AGL15 and AGL18 interact in a complex regulatory loop, where AGL15 inhibited transcript accumulation of AGL18, while AGL18 increased AGL15 transcript accumulation. Co-immunoprecipitation revealed an interaction between AGL18 and AGL15 in somatic embryo tissue. The binding and expression analyses revealed a complex crosstalk and interactions among embryo TFs and their target genes. In addition, our study also revealed that phosphorylation of AGL18 and AGL15 was crucial for the promotion of SE.

The Regulation of Plant Vegetative Phase Transition and Rejuvenation: miRNAs, a Key Regulator.

In contrast to animals, adult organs in plants are not formed during embryogenesis but generated from meristematic cells as plants advance through development. Plant development involves a succession of different phenotypic stages and the transition between these stages is termed phase transition. Phase transitions need to be tightly regulated and coordinated to ensure they occur under optimal seasonal, environmental conditions. Polycarpic perennials transition through vegetative stages and the mature, reproductive stage many times during their lifecycles and, in both perennial and annual species, environmental factors and culturing methods can reverse the otherwise unidirectional vector of plant development. Epigenetic factors regulating gene expression in response to internal cues and external (environmental) stimuli influencing the plant's phenotype and development have been shown to control phase transitions. How developmental and environmental cues interact to epigenetically alter gene expression and influence these transitions is not well understood, and understanding this interaction is important considering the current climate change scenarios, since epigenetic maladaptation could have catastrophic consequences for perennial plants in natural and agricultural ecosystems. Here, we review studies focusing on the epigenetic regulators of the vegetative phase change and highlight how these mechanisms might act in exogenously induced plant rejuvenation and regrowth following stress.

The EAR Motif in the Arabidopsis MADS Transcription Factor AGAMOUS-Like 15 Is Not Necessary to Promote Somatic Embryogenesis.

AGAMOUS-like 15 (AGL15) is a member of the MADS domain family of transcription factors (TFs) that can directly induce and repress target gene expression, and for which promotion of somatic embryogenesis (SE) is positively correlated with accumulation. An ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motif of form LxLxL within the carboxyl-terminal domain of AGL15 was shown to be involved in repression of gene expression. Here, we examine whether AGL15's ability to repress gene expression is needed to promote SE. While a form of AGL15 where the LxLxL is changed to AxAxA can still promote SE, another form with a strong transcriptional activator at the carboxy-terminal end, does not promote SE and, in fact, is detrimental to SE development. Select target genes were examined for response to the different forms of AGL15.

Genetic activity during early plant embryogenesis.

Seeds are essential for human civilization, so understanding the molecular events underpinning seed development and the zygotic embryo it contains is important. In addition, the approach of somatic embryogenesis is a critical propagation and regeneration strategy to increase desirable genotypes, to develop new genetically modified plants to meet agricultural challenges, and at a basic science level, to test gene function. We briefly review some of the transcription factors (TFs) involved in establishing primary and apical meristems during zygotic embryogenesis, as well as TFs necessary and/or sufficient to drive somatic embryo programs. We focus on the model plant Arabidopsis for which many tools are available, and review as well as speculate about comparisons and contrasts between zygotic and somatic embryo processes.

Direct and indirect targets of the arabidopsis seed transcription factor ABSCISIC ACID INSENSITIVE3.

Arabidopsis thaliana ABSCISIC ACID INSENSITIVE3 (ABI3) is a transcription factor in the B3 domain family. ABI3, along with B3 domain transcription factors LEAFY COTYLEDON2 (LEC2) and FUSCA3 (FUS3), and LEC1, a subunit of the CCAAT box-binding complex, form the so-called LAFL network to control various aspects of seed development and maturation. ABI3 also contributes to the abscisic acid (ABA) response. We report on chromatin immunoprecipitation-tiling array experiments to map binding sites for ABI3 globally. We also assessed transcriptomes in response to ABI3 by comparing developing abi3-5 and wild-type seeds and combined this information to ascertain direct and indirect responsive ABI3 target genes. ABI3 can induce and repress its transcription of target genes directly and some intriguing differences exist in cis motifs between these groups of genes. Directly regulated targets reflect the role of ABI3 in seed maturation, desiccation tolerance, entry into a quiescent state and longevity. Interestingly, ABI3 directly represses a gene encoding a microRNA (MIR160B) that targets AUXIN RESPONSE FACTOR (ARF)10 and ARF16 that are involved in establishment of dormancy. In addition, ABI3, like FUS3, regulates genes encoding MIR156 but while FUS3 only induces genes encoding this product, ABI3 induces these genes during the early stages of seed development, but represses these genes during late development. The interplay between ABI3, the other LAFL genes, and the VP1/ABI3-LIKE (VAL) genes, which are involved in the transition to seedling development are examined and reveal complex interactions controlling development.

The class III peroxidase PRX17 is a direct target of the MADS-box transcription factor AGAMOUS-LIKE15 (AGL15) and participates in lignified tissue formation.

Several physiological functions have been attributed to class III peroxidases (PRXs) in plants, but the in planta role of most members of this family still remains undetermined. Here, we report the first functional characterization of PRX17 (At2g22420), one of the 73 members of this family in Arabidopsis thaliana. Localization of PRX17 was examined by transient expression in Nicotiana benthamiana. Loss- and gain-of-function mutants in A. thaliana were studied. Regulation at the gene and protein levels was analyzed using ß-glucuronidase (GUS) activity, quantitative reverse transcriptase (qRT)-PCR, zymography, and chromatin immunoprecipitation. Phenotypes were characterized including lignin and xylan contents. PRX17 was expressed in various tissues, including vascular tissues, and PRX17 was localized to the cell wall. In prx17, the lignin content was reduced in the stem and siliques and bolting was delayed, while the opposite phenotype was observed in 35S:PRX17 plants, together with a significant increase of lignin and xylan immunofluorescence signal. Finally, we demonstrated that the transcription factor AGAMOUS-LIKE15 (AGL15) binds to the PRX17 promoter and regulates PRX17 expression level. This converging set of structural, transcriptomic and physiological data suggests that PRX17, under the control of AGL15, contributes to developmental programs by playing an essential role in regulating age-dependent lignified tissue formation, including changes in cell wall properties.

Gene Regulation by the AGL15 Transcription Factor Reveals Hormone Interactions in Somatic Embryogenesis.

The MADS box transcription factor Arabidopsis (Arabidopsis thaliana) AGAMOUS-LIKE15 (AGL15) and a putative ortholog from soybean (Glycine max), GmAGL15, are able to promote somatic embryogenesis (SE) in these plants when ectopically expressed. SE is an important means of plant regeneration, but many plants, or even particular cultivars, are recalcitrant for this process. Understanding how (Gm)AGL15 promotes SE by identifying and characterizing direct and indirect downstream regulated genes can provide means to improve regeneration by SE for crop improvement and to perform molecular tests of genes. Conserved transcription factors and the genes they regulate in common between species may provide the most promising avenue to identify targets for SE improvement. We show that (Gm)AGL15 negatively regulates auxin signaling in both Arabidopsis and soybean at many levels of the pathway, including the repression of AUXIN RESPONSE FACTOR6 (ARF6) and ARF8 and TRANSPORT INHIBITOR RESPONSE1 as well as the indirect control of components via direct expression of a microRNA-encoding gene. We demonstrate interaction between auxin and gibberellic acid in the promotion of SE and document an inverse correlation between bioactive gibberellic acid and SE in soybean, a difficult crop to transform. Finally, we relate hormone accumulation to transcript accumulation of important soybean embryo regulatory factors such as ABSCISIC ACID INSENSITIVE3 and FUSCA3 and provide a working model of hormone and transcription factor interaction in the control of SE.

Transcriptome analysis indicates that GmAGAMOUS-Like 15 may enhance somatic embryogenesis by promoting a dedifferentiated state.

Somatic embryogenesis (SE) is an important avenue for regeneration of many plants. Although documented over half a century ago, the process of SE remains poorly understood and many factors impact upon competence for SE. We recently reported that a Glycine max ortholog of a MADS-domain transcription factor that promotes SE in Arabidopsis also enhances SE in soybean. We recently assessed transcriptomes in 35Spro:GmAGL15 compared to control during an early time-course of SE and in response to 35Spro:AtAGL15. We expand here upon discussion of the types of genes regulated by overexpression of AGL15 and characterize the step of SE that may be affected by altered accumulation of AGL15.

The MADS-Domain Factors AGAMOUS-LIKE15 and AGAMOUS-LIKE18, along with SHORT VEGETATIVE PHASE and AGAMOUS-LIKE24, Are Necessary to Block Floral Gene Expression during the Vegetative Phase.

Multiple factors, including the MADS-domain proteins AGAMOUS-LIKE15 (AGL15) and AGL18, contribute to the regulation of the transition from vegetative to reproductive growth. AGL15 and AGL18 were previously shown to act redundantly as floral repressors and upstream of FLOWERING LOCUS T (FT) in Arabidopsis (Arabidopsis thaliana). A series of genetic and molecular experiments, primarily focused on AGL15, was performed to more clearly define their role. agl15 agl18 mutations fail to suppress ft mutations but show additive interactions with short vegetative phase (svp) mutations in ft and suppressor of constans1 (soc1) backgrounds. Chromatin immunoprecipitation analyses with AGL15-specific antibodies indicate that AGL15 binds directly to the FT locus at sites that partially overlap those bound by SVP and FLOWERING LOCUS C. In addition, expression of AGL15 in the phloem effectively restores wild-type flowering times in agl15 agl18 mutants. When agl15 agl18 mutations are combined with agl24 svp mutations, the plants show upward curling of rosette and cauline leaves, in addition to early flowering. The change in leaf morphology is associated with elevated levels of FT and ectopic expression of SEPALLATA3 (SEP3), leading to ectopic expression of floral genes. Leaf curling is suppressed by sep3 and ft mutations and enhanced by soc1 mutations. Thus, AGL15 and AGL18, along with SVP and AGL24, are necessary to block initiation of floral programs in vegetative organs.

Alterations in the transcriptome of soybean in response to enhanced somatic embryogenesis promoted by orthologs of Agamous-like15 and Agamous-like18.

Somatic embryogenesis (SE) is a poorly understood process during which competent cells respond to inducing conditions, allowing the development of somatic embryos. It is important for the regeneration of transgenic plants, including for soybean (Glycine max). We report here that constitutive expression of soybean orthologs of the Arabidopsis (Arabidopsis thaliana) MADS box genes Agamous-like15 (GmAGL15) and GmAGL18 increased embryogenic competence of explants from these transgenic soybean plants. To understand how GmAGL15 promotes SE, expression studies were performed. Particular genes of interest involved in embryogenesis (abscisic acid-insensitive3 and FUSCA3) were found to be directly up-regulated by GmAGL15 by using a combination of quantitative reverse transcription-polymerase chain reaction and chromatin immunoprecipitation. To look more broadly at changes in gene expression in response to GmAGL15, we assessed the transcriptome using the Affymetrix Soybean Genome Array. Interestingly, the gene expression profile of 35Spro:GmAGL15 explants (0 d in culture) was found to resemble nontransgenic tissue that had been induced for SE by being placed on induction medium for 3 d, possibly explaining the more rapid SE development observed on 35Spro:GmAGL15 tissue. In particular, transcripts from genes related to the stress response showed increased transcript accumulation in explants from 35Spro:GmAGL15 tissue. These same genes also showed increased transcript accumulation in response to culturing nontransgenic soybean explants on the medium used to induce SE. Overexpression of GmAGL15 may enhance SE by making the tissue more competent to respond to 2,4-dichlorophenoxyacetic acid induction by differential regulation of genes such as those involved in the stress response, resulting in more rapid and prolific SE.

Ectopic expression of the phosphomimic mutant version of Arabidopsis response regulator 1 promotes a constitutive cytokinin response phenotype.

BACKGROUND: Cytokinins control numerous plant developmental processes, including meristem formation and activity, nutrient distribution, senescence timing and responses to both the abiotic and biotic environments. Cytokinin signaling leads to the activation of type-B response regulators (RRBs), Myb-like transcription factors that are activated by the phosphorylation of a conserved aspartate residue in their response receiver domain. Consistent with this, overexpression of RRBs does not substantially alter plant development, but instead leads to cytokinin hypersensitivity. RESULTS: Here we present comparative analysis of plants overexpressing Arabidopsis RRB 1 (ARR1) or a phosphomimic ARR1D94E mutant in which the conserved aspartate-94 (D94) is replaced by the phosphomimic residue glutamate (E). The D94E substitution causes a 100-fold increase in response activation and instigates developmental and physiological changes that characterize wild-type plants treated with cytokinins or transgenic plants with increased cytokinin content. CONCLUSION: The current model of cytokinin signaling emphasizes the essential role of conserved aspartate residue phosphorylation of RRBs in promoting cytokinin responses. Our comparative analyses of developmental and physiological traits of ARR1 and ARR1D94E overexpressing plants revealed that the ARR1D94E protein is indeed a constitutive and wide-spectrum cytokinin response activator.

An Arabidopsis ATP-dependent, DEAD-box RNA helicase loses activity upon IsoAsp formation but is restored by PROTEIN ISOASPARTYL METHYLTRANSFERASE.

Orthodox seeds are capable of withstanding severe dehydration. However, in the dehydrated state, Asn and Asp residues in proteins can convert to succinimide residues that can further react to predominantly form isomerized isoAsp residues upon rehydration (imbibition). IsoAsp residues can impair protein function and can render seeds nonviable, but PROTEIN ISOASPARTYL METHYLTRANSFERASE (PIMT) can initiate isoAsp conversion to Asp residues. The proteins necessary for translation upon imbibition in orthodox seeds may be particularly important to maintain in an active state. One such protein is the large, multidomain protein, Arabidopsis thaliana PLANT RNA HELICASE75 (PRH75), a DEAD-box helicase known to be susceptible to isoAsp residue accumulation. However, the consequences of such isomerization on PRH75 catalysis and for the plant are unknown. Here, it is demonstrated that PRH75 is necessary for successful seed development. It acquires isoAsp rapidly during heat stress, which eliminates RNA unwinding (but not rewinding) competence. The repair by PIMT is able to restore PRH75's complex biochemical activity provided isoAsp formation has not led to subsequent, destabilizing conformational alterations. For PRH75, an important enzymatic activity associated with translation would be eliminated unless rapidly repaired by PIMT prior to additional, deleterious conformational changes that would compromise seed vitality and germination.

Decreased GmAGL15 expression and reduced ethylene synthesis may contribute to reduced somatic embryogenesis in a poorly embryogenic cultivar of Glycine max.

Somatic embryogenesis (SE) is the process by which cells become dedifferentiated and reprogram to follow an embryogenic pathway. It is important for regeneration of transgenic plants as well as for propagation of certain genotypes. However, competence for SE varies, even among genotypes of a species, and the basis for this variation is not understood. We have found that the MADS-box transcription factor (Glycine max) AGAMOUS-Like 15 [(Gm)AGL15] promotes SE in Arabidopsis and in soybean when overexpressed. In soybean, part of the promotion of SE is via GmAGL15-mediated control of ethylene biosynthesis and response. Addition of ACC, the precursor to ethylene, to culture media enhanced SE in Arabidopsis and soybean. Transcription factors important for embryogenesis responded directly to GmAGL15 and to ethylene accumulation. Here we correlate ethylene production and patterns of gene expression with SE potential of soybean genotypes. However, other results indicate that there is not a complete positive correlation between ethylene production and SE, indicating that the interactions between hormones, gene expression and developmental outcomes are complex.

AGAMOUS-Like15 promotes somatic embryogenesis in Arabidopsis and soybean in part by the control of ethylene biosynthesis and response.

Many of the regulatory processes occurring during plant embryogenesis are still unknown. Relatively few cells are involved, and they are embedded within maternal tissues, making this developmental phase difficult to study. Somatic embryogenesis is a more accessible system, and many important regulatory genes appear to function similar to zygotic development, making somatic embryogenesis a valuable model for the study of zygotic processes. To better understand the role of the Arabidopsis (Arabidopsis thaliana) MADS factor AGAMOUS-Like15 (AGL15) in the promotion of somatic embryogenesis, direct target genes were identified by chromatin immunoprecipitation-tiling arrays and expression arrays. One potential directly up-regulated target was At5g61590, which encodes a member of the ethylene response factor subfamily B-3 of APETALA2/ethylene response factor transcription factors and is related to Medicago truncatula somatic embryo-related factor1 (MtSERF1), which has been shown to be required for somatic embryogenesis in M. truncatula. Here, we report confirmation that At5g61590 is a directly expressed target of AGL15 and that At5g61590 is essential for AGL15's promotion of somatic embryogenesis. Because At5g61590 is a member of the ethylene response factor family, effects of ethylene on somatic embryogenesis were investigated. Precursors to ethylene stimulate somatic embryogenesis, whereas inhibitors of ethylene synthesis or perception reduce somatic embryogenesis. To extend findings to a crop plant, we investigated the effects of ethylene on somatic embryogenesis in soybean (Glycine max). Furthermore, we found that a potential ortholog of AGL15 in soybean (GmAGL15) up-regulates ethylene biosynthesis and response, including direct regulation of soybean orthologs of At5g61590/MtSERF1 named here GmSERF1 and GmSERF2, in concordance with the M. truncatula nomenclature.

Identification of direct targets of FUSCA3, a key regulator of Arabidopsis seed development.

FUSCA3 (FUS3) is a B3 domain transcription factor that is a member of the LEAFY COTYLEDON (LEC) group of genes. The LEC genes encode proteins that also include LEC2, a B3 domain factor related to FUS3, and LEC1, a CCAAT box-binding factor. LEC1, LEC2, and FUS3 are essential for plant embryo development. All three loss-of-function mutants in Arabidopsis (Arabidopsis thaliana) prematurely exit embryogenesis and enter seedling developmental programs. When ectopically expressed, these genes promote embryo programs in seedlings. We report on chromatin immunoprecipitation-tiling array experiments to globally map binding sites for FUS3 that, along with other published work to assess transcriptomes in response to FUS3, allow us to determine direct from indirect targets. Many transcription factors associated with embryogenesis are direct targets of FUS3, as are genes involved in the seed maturation program. FUS3 regulates genes encoding microRNAs that, in turn, control transcripts encoding transcription factors involved in developmental phase changes. Examination of direct targets of FUS3 reveals that FUS3 acts primarily or exclusively as a transcriptional activator. Regulation of microRNA-encoding genes is one mechanism by which FUS3 may repress indirect target genes. FUS3 also directly up-regulates VP1/ABI3-LIKE1 (VAL1), encoding a B3 domain protein that functions as a repressor of transcription. VAL1, along with VAL2 and VAL3, is involved in the transition from embryo to seedling development. Many genes are responsive to FUS3 and to VAL1/VAL2 but with opposite regulatory consequences. The emerging picture is one of complex cross talk and interactions among embryo transcription factors and their target genes.

Chromatin immunoprecipitation to verify or to identify in vivo protein-DNA interactions.

Chromatin immunoprecipitation (ChIP) is a valuable tool to detect the interaction in vivo between a DNA-associated protein and DNA fragments. Combined with approaches to assess gene expression in response to accumulation of a transcription factor, it is possible to identify direct responsive targets from targets that are indirectly responsive to accumulation of the transcription factor. ChIP may be used to confirm in vivo association of a transcriptional regulator with suspected target DNA fragments. ChIP may also be used to discover new targets, and when combined with high-throughput approaches to identify DNA fragments associated with a transcription factor, it may provide a tool to study the gene regulatory networks active during plant development and/or response to the environment. Furthermore, ChIP is also a powerful means to map epigenetic modifications within a genome.

Substrates of the Arabidopsis thaliana protein isoaspartyl methyltransferase 1 identified using phage display and biopanning.

The role of protein isoaspartyl methyltransferase (PIMT) in repairing a wide assortment of damaged proteins in a host of organisms has been inferred from the affinity of the enzyme for isoaspartyl residues in a plethora of amino acid contexts. The identification of PIMT target proteins in plant seeds, where the enzyme is highly active and proteome long-lived, has been hindered by large amounts of isoaspartate-containing storage proteins. Mature seed phage display libraries circumvented this problem. Inclusion of the PIMT co-substrate, S-adenosylmethionine (AdoMet), during panning permitted PIMT to retain aged phage in greater numbers than controls lacking co-substrate or when PIMT protein binding was poisoned with S-adenosyl homocysteine. After four rounds, phage titer plateaued in AdoMet-containing pans, whereas titer declined in both controls. This strategy identified 17 in-frame PIMT target proteins, including a cupin-family protein similar to those identified previously using on-blot methylation. All recovered phage had at least one susceptible Asp or Asn residue. Five targets were recovered independently. Two in-frame targets were produced in Escherichia coli as recombinant proteins and shown by on-blot methylation to acquire isoAsp, becoming a PIMT target. Both gained isoAsp rapidly in solution upon thermal insult. Mutant analysis of plants deficient in any of three in-frame PIMT targets resulted in demonstrable phenotypes. An over-representation of clones encoding proteins involved in protein production suggests that the translational apparatus comprises a subgroup for which PIMT-mediated repair is vital for orthodox seed longevity. Impaired PIMT activity would hinder protein function in these targets, possibly resulting in poor seed performance.