Genetic Screen In Arabidopsis Research Articles

Control of meiotic crossover interference by a proteolytic chaperone network.

Meiosis is a specialized eukaryotic division that produces genetically diverse gametes for sexual reproduction. During meiosis, homologous chromosomes pair and undergo reciprocal exchanges, called crossovers, which recombine genetic variation. Meiotic crossovers are stringently controlled with at least one obligate exchange forming per chromosome pair, while closely spaced crossovers are inhibited by interference. In Arabidopsis, crossover positions can be explained by a diffusion-mediated coarsening model, in which large, approximately evenly spaced foci of the pro-crossover E3 ligase HEI10 grow at the expense of smaller, closely spaced clusters. However, the mechanisms that control HEI10 dynamics during meiosis remain unclear. Here, through a forward genetic screen in Arabidopsis, we identified high crossover rate3 (hcr3), a dominant-negative mutant that reduces crossover interference and increases crossovers genome-wide. HCR3 encodes J3, a co-chaperone related to HSP40, which acts to target protein aggregates and biomolecular condensates to the disassembly chaperone HSP70, thereby promoting proteasomal degradation. Consistently, we show that a network of HCR3 and HSP70 chaperones facilitates proteolysis of HEI10, thereby regulating interference and the recombination landscape. These results reveal a new role for the HSP40/J3-HSP70 chaperones in regulating chromosome-wide dynamics of recombination via control of HEI10 proteolysis.

DDT-RELATED PROTEIN4-IMITATION SWITCH alters nucleosome distribution to relieve transcriptional silencing in Arabidopsis.

DNA methylation is a conserved epigenetic modification that is typically associated with silencing of transposable elements and promoter methylated genes. However, some DNA-methylated loci are protected from silencing, allowing transcriptional flexibility in response to environmental and developmental cues. Through a genetic screen in Arabidopsis (Arabidopsis thaliana), we uncovered an antagonistic relationship between the MICRORCHIDIA (MORC) protein and the IMITATION SWITCH (ISWI) complex in regulating the DNA-methylated SUPPRESSOR OF DRM1 DRM2 CMT3 (SDC) reporter. We demonstrate that components of the plant-specific ISWI complex, including CHROMATIN REMODELING PROTEIN11 (CHR11), CHR17, DDT-RELATED PROTEIN4 (DDR4), and DDR5, function to partially de-repress silenced genes and transposable elements (TEs), through their function in regulating nucleosome distribution. This action also requires the known transcriptional activator DNAJ proteins, providing a mechanistic link between nucleosome remodeling and transcriptional activation. Genome-wide studies revealed that DDR4 causes changes in nucleosome distribution at numerous loci, a subset of which is associated with changes in DNA methylation and/or transcription. Our work reveals a mechanism for balancing transcriptional flexibility and faithful silencing of DNA-methylated loci. As both ISWI and MORC family genes are widely distributed across plant and animal species, our findings may represent a conserved eukaryotic mechanism for fine-tuning gene expression under epigenetic regulation.

A phospho-switch constrains BTL2-mediated phytocytokine signaling in plant immunity

Enabling and constraining immune activation is of fundamental importance in maintaining cellular homeostasis. Depleting BAK1 and SERK4, the co-receptors of multiple pattern recognition receptors (PRRs), abolishes pattern-triggered immunity but triggers intracellular NOD-like receptor (NLR)-mediated autoimmunity with an elusive mechanism. By deploying RNAi-based genetic screens in Arabidopsis, we identified BAK-TO-LIFE 2 (BTL2), an uncharacterized receptor kinase, sensing BAK1/SERK4 integrity. BTL2 induces autoimmunity through activating Ca2+ channel CNGC20 in a kinase-dependent manner when BAK1/SERK4 are perturbed. To compensate for BAK1 deficiency, BTL2 complexes with multiple phytocytokine receptors, leading to potent phytocytokine responses mediated by helper NLR ADR1 family immune receptors, suggesting phytocytokine signaling as a molecular link connecting PRR- and NLR-mediated immunity. Remarkably, BAK1 constrains BTL2 activation via specific phosphorylation to maintain cellular integrity. Thus, BTL2 serves as a surveillance rheostat sensing the perturbation of BAK1/SERK4 immune co-receptors in promoting NLR-mediated phytocytokine signaling to ensure plant immunity.

A genetic screen in Arabidopsis reveals the identical roles for RBP45d and PRP39a in 5' cryptic splice site selection.

Cryptic splice sites in eukaryotic genome are generally dormant unless activated by mutation of authentic splice sites or related splicing factors. How cryptic splice sites are used remains unclear in plants. Here, we identified two cryptic splicing regulators, RBP45d and PRP39a that are homologs of yeast U1 auxiliary protein Nam8 and Prp39, respectively, via genetic screening for suppressors of the virescent sot5 mutant, which results from a point mutation at the 5' splice site (5' ss) of SOT5 intron 7. Loss-of-function mutations in RBP45d and PRP39a significantly increase the level of a cryptically spliced variant that encodes a mutated but functional sot5 protein, rescuing sot5 to the WT phenotype. We furtherly demonstrated that RBP45d and PRP39a interact with each other and also with the U1C, a core subunit of U1 snRNP. We found that RBP45d directly binds to the uridine (U)-rich RNA sequence downstream the 5' ss of SOT5 intron 7. However, other RBP45/47 members do not function redundantly with RBP45d, at least in regulation of cryptic splicing. Taken together, RBP45d promotes U1 snRNP to recognize the specific 5' ss via binding to intronic U-rich elements in plants.

HIGH CROSSOVER RATE1 encodes PROTEIN PHOSPHATASE X1 and restricts meiotic crossovers in Arabidopsis.

Meiotic crossovers are tightly restricted in most eukaryotes, despite an excess of initiating DNA double-strand breaks. The majority of plant crossovers are dependent on Class I interfering repair, with a minority formed via the Class II pathway. Class II repair is limited by anti-recombination pathways, however similar pathways repressing Class I crossovers are unknown. We performed a forward genetic screen in Arabidopsis using fluorescent crossover reporters, to identify mutants with increased or decreased recombination frequency. We identified HIGH CROSSOVER RATE1 (HCR1) as repressing crossovers and encoding PROTEIN PHOSPHATASE X1. Genome-wide analysis showed that hcr1 crossovers are increased in the distal chromosome arms. MLH1 foci significantly increase in hcr1 and crossover interference decreases, demonstrating an effect on Class I repair. Consistently, yeast two-hybrid and in planta assays show interaction between HCR1 and Class I proteins, including HEI10, PTD, MSH5, and MLH1. We propose that HCR1 plays a major role in opposition to pro-recombination kinases to restrict crossovers in Arabidopsis.

Quinone perception in plants via leucine-rich-repeat receptor-like kinases.

Quinones are produced and sensed in all kingdoms of life1-4. Plants are primary producers of quinone1,2, but the role of quinone as a signalling agent in plants remains largely unknown. One well-documented role of quinone is in the induction of haustoria (specialized feeding structures) in plants that parasitize roots, which occurs in the presence of the host-derived quinone compound 2,6-dimethoxy-1,4-benzoquinone (DMBQ)5. However, how parasitic plants sense DMBQ remains unclear, as is whether nonparasitic plants are capable of sensing quinones. Here we use Arabidopsis thaliana and DMBQ as a model plant and quinone to show that DMBQ signalling occurs in Arabidopsis via elevation of cytosolic Ca2+ concentration. We performed a forward genetic screen in Arabidopsis that isolated DMBQ-unresponsive mutants, which we named cannot respond to DMBQ1 (card1). The CANNOT RESPOND TO DMBQ1 (CARD1; At5g49760, also known as HPCA1) gene encodes a leucine-rich-repeat receptor-like kinase that is highly conserved in land plants. In Arabidopsis, DMBQ triggers defence-related gene expression, and card1 mutants show impaired immunity against bacterial pathogens. In Phtheirospermum japonicum (a plant that parasitizes roots), DMBQ initiates Ca2+ signalling in the root and is important for the development of the haustorium. Furthermore, CARD1 homologues from this parasitic plant complement DMBQ-induced elevation of cytosolic Ca2+ concentration in the card1 mutant. Our results demonstrate that plants-unlike animals and bacteria-use leucine-rich-repeat receptor-like kinases for quinone signalling. This work provides insights into the role of quinone signalling and CARD1 functions in plants that help us to better understand the signalling pathways used during the formation of the haustorium in parasitic plants and in plant immunity in nonparasitic plants.

A Decoy Library Uncovers U-Box E3 Ubiquitin Ligases That Regulate Flowering Time in Arabidopsis.

Targeted degradation of proteins is mediated by E3 ubiquitin ligases and is important for the execution of many biological processes. Redundancy has prevented the genetic characterization of many E3 ubiquitin ligases in plants. Here, we performed a reverse genetic screen in Arabidopsis using a library of dominant-negative U-box-type E3 ubiquitin ligases to identify their roles in flowering time and reproductive development. We identified five U-box decoy transgenic populations that have defects in flowering time or the floral development program. We used additional genetic and biochemical studies to validate PLANT U-BOX 14 (PUB14), MOS4-ASSOCIATED COMPLEX 3A (MAC3A), and MAC3B as bona fide regulators of flowering time. This work demonstrates the widespread importance of E3 ubiquitin ligases in floral reproductive development. Furthermore, it reinforces the necessity of dominant-negative strategies for uncovering previously unidentified regulators of developmental transitions in an organism with widespread genetic redundancy, and provides a basis on which to model other similar studies.

NCP activates chloroplast transcription by controlling phytochrome-dependent dual nuclear and plastidial switches

Phytochromes initiate chloroplast biogenesis by activating genes encoding the photosynthetic apparatus, including photosynthesis-associated plastid-encoded genes (PhAPGs). PhAPGs are transcribed by a bacterial-type RNA polymerase (PEP), but how phytochromes in the nucleus activate chloroplast gene expression remains enigmatic. We report here a forward genetic screen in Arabidopsis that identified NUCLEAR CONTROL OF PEP ACTIVITY (NCP) as a necessary component of phytochrome signaling for PhAPG activation. NCP is dual-targeted to plastids and the nucleus. While nuclear NCP mediates the degradation of two repressors of chloroplast biogenesis, PIF1 and PIF3, NCP in plastids promotes the assembly of the PEP complex for PhAPG transcription. NCP and its paralog RCB are non-catalytic thioredoxin-like proteins that diverged in seed plants to adopt nonredundant functions in phytochrome signaling. These results support a model in which phytochromes control PhAPG expression through light-dependent double nuclear and plastidial switches that are linked by evolutionarily conserved and dual-localized regulatory proteins.

Luciferase-Based Screen for Post-translational Control Factors in the Regulation of the Pseudo-Response Regulator PRR7.

Control of protein turnover is a key post-translational control point in the oscillatory network of the circadian clock. Some elements, such as TOC1 and PRR5 are engaged by a well-described F-box protein, ZEITLUPE, dedicated to their proteolytic turnover to shape their expression profile to a specific time of night. For most other clock components the regulation of their protein abundance is unknown, though turnover is often rapid and often lags the cycling of the respective mRNA. Here we report the design and results of an unbiased genetic screen in Arabidopsis to uncover proteolytic regulatory factors of PSEUDO-RESPONSE REGULATOR 7 (PRR7), a transcriptional repressor that peaks in the late afternoon. We performed EMS mutagenesis on a transgenic line expressing a PRR7::PRR7-luciferase (PRR7-LUC) translational fusion that accurately recapitulates the diurnal and circadian oscillations of the endogenous PRR7 protein. Using continuous luciferase imaging under constant light, we recovered mutants that alter the PRR7-LUC waveform and some that change period. We have identified novel alleles of ELF3 and ELF4, core components of the ELF3-ELF4-LUX Evening Complex (EC), that dampen the oscillation of PRR7-LUC. We report the characterization of two new hypomorphic alleles of ELF3 that help to understand the relationship between molecular potency and phenotype.

Changes in morphogen kinetics and pollen grain size are potential mechanisms of aberrant pollen aperture patterning in previously observed and novel mutants of Arabidopsis thaliana.

Pollen provides an excellent system to study pattern formation at the single-cell level. Pollen surface is covered by the pollen wall exine, whose deposition is excluded from certain surface areas, the apertures, which vary between the species in their numbers, positions, and morphology. What determines aperture patterns is not understood. Arabidopsis thaliana normally develops three apertures, equally spaced along the pollen equator. However, Arabidopsis mutants whose pollen has higher ploidy and larger volume develop four or more apertures. To explore possible mechanisms responsible for aperture patterning, we developed a mathematical model based on the Gierer-Meinhardt system of equations. This model was able to recapitulate aperture patterns observed in the wild-type and higher-ploidy pollen. We then used this model to further explore geometric and kinetic factors that may influence aperture patterns and found that pollen size, as well as certain kinetic parameters, like diffusion and decay of morphogens, could play a role in formation of aperture patterns. In conjunction with mathematical modeling, we also performed a forward genetic screen in Arabidopsis and discovered two mutants with aperture patterns that had not been previously observed in this species but were predicted by our model. The macaron mutant develops a single ring-like aperture, matching the unusual ring-like pattern produced by the model. The doughnut mutant forms two pore-like apertures at the poles of the pollen grain. Further tests on these novel mutants, motivated by the modeling results, suggested the existence of an area of inhibition around apertures that prevents formation of additional apertures in their vicinity. This work demonstrates the ability of the theoretical model to help focus experimental efforts and to provide fundamental insights into an important biological process.

Current understanding of GUN1: a key mediator involved in biogenic retrograde signaling.

Chloroplast-nucleus communication takes place via processes called anterograde and retrograde signaling pathways. Discovery of the retrograde signaling pathways from the chloroplasts to the nucleus also raised an intriguing proposition that chloroplasts may serve as environmental sensors since multitudes of environmental factors disturb chloroplastic homeostasis. Certain chloroplastic perturbations, mostly impairing transcription/translation, are coupled to the repression of photosynthesis-associated nuclear genes (PhANGs), thus finely coordinating photosynthetic and chloroplastic homeostasis. The unbiased forward genetic screen in Arabidopsis leads to the identification of six independent loci called GENOMES UNCOUPLED (GUN), whose inactivation was found to de-repress the expression of PhANGs under certain conditions promoting retrograde signaling. Of the six GUNs, five encode proteins associated with tetrapyrrole biosynthesis and one, namely GUN1, encodes a member of the pentatricopeptide repeat protein family. Despite the fact that GUN1 plays a role as a central signaling mediator for retrograde communication, the molecular details of GUN1 protein still remain to be elucidated. Here, we recapitulate our current understanding of the GUN1-mediated retrograde signaling pathway and propose a possible mode of action of GUN1 in the chloroplasts together with different aspects of GUN1 protein activity that deserve further investigation.

Alleles Causing Resistance to Isoxaben and Flupoxam Highlight the Significance of Transmembrane Domains for CESA Protein Function

The cellulose synthase (CESA) proteins in Arabidopsis play an essential role in the production of cellulose in the cell walls. Herbicides such as isoxaben and flupoxam specifically target this production process and are prominent cellulose biosynthesis inhibitors (CBIs). Forward genetic screens in Arabidopsis revealed that mutations that can result in varying degrees of resistance to either isoxaben or flupoxam CBI can be attributed to single amino acid substitutions in primary wall CESAs. Missense mutations were almost exclusively present in the predicted transmembrane regions of CESA1, CESA3, and CESA6. Resistance to isoxaben was also conferred by modification to the catalytic residues of CESA3. This resulted in cellulose deficient phenotypes characterized by reduced crystallinity and dwarfism. However, mapping of mutations to the transmembrane regions also lead to growth phenotypes and altered cellulose crystallinity phenotypes. These results provide further genetic evidence supporting the involvement of CESA transmembrane regions in cellulose biosynthesis.

The Arabidopsis SAL1-PAP Pathway: A Case Study for Integrating Chloroplast Retrograde, Light and Hormonal Signaling in Modulating Plant Growth and Development?

Plant growth and development are dependent on chloroplast development and function. Constitutive high level accumulation of a chloroplast stress signal, 3′-phosphoadenosine-5′-phosphate (PAP), confers drought tolerance to plants, but slow downs and alters plant growth and development. PAP, a by-product of sulfur metabolism, is maintained at very low levels by the SAL1 phosphatase during vegetative growth of Arabidopsis and accumulates in rosettes during drought and excess light. Eight independent forward genetic screens in Arabidopsis identified SAL1 as the regulator of multiple phenotypes related to stress responses, hormonal signaling and/or perception. In this perspective article, we collate all the sal1 phenotypes published in the past two decades, and distill the different pathways affected. Our meta-analysis of publicly available sal1 microarray data coupled to preliminary hormonal treatment and profiling results on sal1 indicate that homeostasis and responses to multiple hormones in sal1 are altered during rosette growth, suggesting a potential connection between SAL1-PAP stress retrograde pathway and hormonal signaling. We propose the SAL1-PAP pathway as a case study for integrating chloroplast retrograde signaling, light signaling and hormonal signaling in plant growth and morphogenesis.

Lipopolysaccharides Trigger Two Successive Bursts of Reactive Oxygen Species at Distinct Cellular Locations.

Lipopolysaccharides (LPS) are major components of the outer membrane of gram-negative bacteria and are an important microbe-associated molecular pattern (MAMP) that triggers immune responses in plants and animals. A previous genetic screen in Arabidopsis (Arabidopsis thaliana) identified LIPOOLIGOSACCHARIDE-SPECIFIC REDUCED ELICITATION (LORE), a B-type lectin S-domain receptor kinase, as a sensor of LPS. However, the LPS-activated LORE signaling pathway and associated immune responses remain largely unknown. In this study, we found that LPS trigger biphasic production of reactive oxygen species (ROS) in Arabidopsis. The first transient ROS burst was similar to that induced by another MAMP, flagellin, whereas the second long-lasting burst was induced only by LPS. The LPS-triggered second ROS burst was found to be conserved in a variety of plant species. Microscopic observation of the generation of ROS revealed that the LPS-triggered second ROS burst was largely associated with chloroplasts, and functional chloroplasts were indispensable for this response. The lipid A moiety, the most conserved portion of LPS, appears to be responsible for the second ROS burst. Surprisingly, the LPS- and lipid A-triggered second ROS burst was only partially dependent on LORE. Together, our findings provide insight on the LPS-triggered ROS production and the associated signaling pathway.

Identification of a New Host Factor Required for Antiviral RNAi and Amplification of Viral siRNAs.

Small interfering RNAs (siRNAs) are processed from virus-specific dsRNA to direct antiviral RNA interference (RNAi) in diverse eukaryotic hosts. We have recently performed a sensitized genetic screen in Arabidopsis (Arabidopsis thaliana) and identified two related phospholipid flippases required for antiviral RNAi and the amplification of virus-derived siRNAs by plant RNA-dependent RNA polymerase1 (RDR1) and RDR6. Here we report the identification and cloning of ANTIVIRAL RNAI-DEFECTIVE2 (AVI2) from the same genetic screen. AVI2 encodes a multispan transmembrane protein broadly conserved in plants and animals with two homologous human proteins known as magnesium transporters. We show that avi2 mutant plants display no developmental defects and develop severe disease symptoms after infection with a mutant Cucumber mosaic virus (CMV) defective in RNAi suppression. AVI2 is induced by CMV infection, particularly in veins, and is required for antiviral RNAi and RDR6-dependent biogenesis of viral siRNAs. AVI2 is also necessary for Dicer-like2-mediated amplification of 22-nucleotide viral siRNAs induced in dcl4 mutant plants by infection, but dispensable for RDR6-dependent biogenesis of endogenous transacting siRNAs. Further genetic studies illustrate that AVI2 plays a partially redundant role with AVI2H, the most closely related member in the AVI2 gene family, in RDR1-dependent biogenesis of viral siRNAs and the endogenous virus-activated siRNAs (vasi-RNAs). Interestingly, we discovered a specific genetic interaction of AVI2 with AVI1 flippase that is critical for plant development. We propose that AVI1 and AVI2 participate in the virus-induced formation of the RDR1/RDR6-specific, membrane-bound RNA synthesis compartment, essential for the biogenesis of highly abundant viral siRNAs and vasi-RNAs.

An Arabidopsis mutant with high operating efficiency of Photosystem II and low chlorophyll fluorescence

The overall light energy to biomass conversion efficiency of plant photosynthesis is generally regarded as low. Forward genetic screens in Arabidopsis have yielded very few mutants with substantially enhanced photochemistry. Here, we report the isolation of a novel Arabidopsis mutant with a high operating efficiency of Photosystem II (φPSII) and low chlorophyll fluorescence from a library of lines harboring T-DNA constructs encoding artificial transcription factors. This mutant was named Low Chlorophyll Fluorescence 1 (LCF1). Only a single T-DNA insertion was detected in LCF1, which interrupted the expression of the full length mRNA of the gene At4g36280 (MORC2). We demonstrate that the high φPSII and low levels of chlorophyll fluorescence were due to a decrease in PSII:PSI ratio. Although LCF1 plants had decreased rosette surface area and biomass under normal growth conditions, they contained more starch per gram fresh weight. The growth defect of LCF1 was alleviated by low light and short day conditions, and growth could even be enhanced after a period of dark-induced senescence, showing that the plant can utilize its excess photosynthetic conversion capacity as a resource when needed.

TNL-mediated immunity in Arabidopsis requires complex regulation of the redundant ADR1 gene family.

Nucleotide-binding leucine-rich repeat proteins (NLRs) serve as intracellular immune receptors in animals and plants. Sensor NLRs perceive pathogen-derived effector molecules and trigger robust host defense. Recent studies revealed the role of three coiled-coil-type NLRs (CNLs) of the ADR1 family - ADR1, ADR1-L1 and ADR1-L2 - as redundant helper NLRs, whose function is required for defense mediated by multiple sensor NLRs. From a mutant snc1-enhancing (MUSE) forward genetic screen in Arabidopsis targeted to identify negative regulators of snc1 that encodes a TIR-type NLR (TNL), we isolated two alleles of muse15, both carrying mutations in ADR1-L1. Interestingly, loss of ADR1-L1 also enhances immunity-related phenotypes in other autoimmune mutants including cpr1, bal and lsd1. This immunity-enhancing effect is not mediated by increased SNC1 protein stability, nor is it fully dependent on the accumulation of the defense hormone salicylic acid (SA). Transcriptional analysis revealed an upregulation of ADR1 and ADR1-L2 in the adr1-L1 background, which may overcompensate the loss of ADR1-L1, resulting in enhanced immunity. Interestingly, autoimmunity of snc1 and chs2, which encode typical TNLs, is fully suppressed by the adr1 triple mutant, suggesting that the ADRs are required for TNL downstream signaling. This study extends our knowledge on the interplay among ADRs and reveals their complexity in defense regulation.

Forward chemical genetic screens in Arabidopsis identify genes that influence sensitivity to the phytotoxic compound sulfamethoxazole

BackgroundThe sulfanilamide family comprises a clinically important group of antimicrobial compounds which also display bioactivity in plants. While there is evidence that sulfanilamides inhibit folate biosynthesis in both bacteria and plants, the complete network of plant responses to these compounds remains to be characterized. As such, we initiated two forward genetic screens in Arabidopsis in order to identify mutants that exhibit altered sensitivity to sulfanilamide compounds. These screens were based on the growth phenotype of seedlings germinated in the presence of the compound sulfamethoxazole (Smex).ResultsWe identified a mutant with reduced sensitivity to Smex, and subsequent mapping indicated that a gene encoding 5-oxoprolinase was responsible for this phenotype. A mutation causing enhanced sensitivity to Smex was mapped to a gene lacking any functional annotation.ConclusionsThe genes identified through our forward genetic screens represent novel mediators of Arabidopsis responses to sulfanilamides and suggest that these responses extend beyond the perturbation of folate biosynthesis.

Targeted parallel sequencing of large genetically-defined genomic regions for identifying mutations in Arabidopsis

Large-scale genetic screens in Arabidopsis are a powerful approach for molecular dissection of complex signaling networks. However, map-based cloning can be time-consuming or even hampered due to low chromosomal recombination. Current strategies using next generation sequencing for molecular identification of mutations require whole genome sequencing and advanced computational devises and skills, which are not readily accessible or affordable to every laboratory. We have developed a streamlined method using parallel massive sequencing for mutant identification in which only targeted regions are sequenced. This targeted parallel sequencing (TPSeq) method is more cost-effective, straightforward enough to be easily done without specialized bioinformatics expertise, and reliable for identifying multiple mutations simultaneously. Here, we demonstrate its use by identifying three novel nitrate-signaling mutants in Arabidopsis.

A large-scale genetic screen in Arabidopsis to identify genes involved in pollen exine production.

Exine, the outer plant pollen wall, has elaborate species-specific patterns, provides a protective barrier for male gametophytes, and serves as a mediator of strong and species-specific pollen-stigma adhesion. Exine is made of sporopollenin, a material remarkable for its strength, elasticity, and chemical durability. The chemical nature of sporopollenin, as well as the developmental mechanisms that govern its assembly into diverse patterns in different species, are poorly understood. Here, we describe a simple yet effective genetic screen in Arabidopsis (Arabidopsis thaliana) that was undertaken to advance our understanding of sporopollenin synthesis and exine assembly. This screen led to the recovery of mutants with a variety of defects in exine structure, including multiple mutants with novel phenotypes. Fifty-six mutants were selected for further characterization and are reported here. In 14 cases, we have mapped defects to specific genes, including four with previously demonstrated or suggested roles in exine development (MALE STERILITY2, CYP703A2, ANTHER-SPECIFIC PROTEIN6, TETRAKETIDE α-PYRONE REDUCTASE/DIHYDROFLAVONOL-4-REDUCTASE-LIKE1), and a number of genes that have not been implicated in exine production prior to this screen (among them, fatty acid ω-hydroxylase CYP704B1, putative glycosyl transferases At1g27600 and At1g33430, 4-coumarate-coenzyme A ligase 4CL3, polygalacturonase QUARTET3, novel gene At5g58100, and nucleotide-sugar transporter At5g65000). Our study illustrates that morphological screens of pollen can be extremely fruitful in identifying previously unknown exine genes and lays the foundation for biochemical, developmental, and evolutionary studies of exine production.