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Abstract Mutations in cis-regulatory regions play an important role in the domestication and improvement of crops by altering gene expression. However, assessing the in vivo impact of cis-regulatory elements (CREs) on transcriptional regulation and phenotypic outcomes remains challenging. Previously, we showed that the dominant Barren inflorescence3 (Bif3) mutant of maize (Zea mays) contains a duplicated copy of the homeobox transcription factor gene ZmWUSCHEL1 (ZmWUS1), named ZmWUS1-B. ZmWUS1-B is controlled by a spontaneously generated novel promoter region that dramatically increases its expression and alters patterning and development of young ears. Overexpression of ZmWUS1-B is caused by a unique enhancer region containing multimerized binding sites for type B RESPONSE REGULATORs (RRs), key transcription factors in cytokinin signaling. To better understand how the enhancer increases the expression of ZmWUS1 in vivo, we specifically targeted the ZmWUS1-B enhancer region by CRISPR-Cas9-mediated editing. A series of deletion events with different numbers of type B RR DNA binding motifs (AGATAT) enabled us to determine how the number of AGATAT motifs impacts in vivo expression of ZmWUS1-B and consequently ear development. In combination with dual-luciferase assays in maize protoplasts, our analysis reveals that AGATAT motifs have an additive effect on ZmWUS1-B expression, while the distance separating AGATAT motifs does not appear to have a meaningful impact, indicating that the enhancer activity derives from the sum of individual CREs. These results also suggest that in maize inflorescence development, there is a threshold of buffering capacity for ZmWUS1 overexpression. 

Abstract NAKED ENDOSPERM1 (NKD1), NKD2, and OPAQUE2 (O2) are transcription factors important for cell patterning and nutrient storage in maize (Zea mays) endosperm. To study the complex regulatory interrelationships among these 3 factors in coregulating gene networks, we developed a set of nkd1, nkd2, and o2 homozygous lines, including all combinations of mutant and wild-type genes. Among the 8 genotypes tested, we observed diverse phenotypes and gene interactions affecting cell patterning, starch content, and storage proteins. From ∼8 to ∼16 d after pollination, maize endosperm undergoes a transition from cellular development to nutrient accumulation for grain filling. Gene network analysis showed that NKD1, NKD2, and O2 dynamically regulate a hierarchical gene network during this period, directing cellular development early and then transitioning to constrain cellular development while promoting the biosynthesis and storage of starch, proteins, and lipids. Genetic interactions regulating this network are also dynamic. The assay for transposase-accessible chromatin using sequencing (ATAC-seq) showed that O2 influences the global regulatory landscape, decreasing NKD1 and NKD2 target site accessibility, while NKD1 and NKD2 increase O2 target site accessibility. In summary, interactions of NKD1, NKD2, and O2 dynamically affect the hierarchical gene network and regulatory landscape during the transition from cellular development to grain filling in maize endosperm. 

Abstract Many eukaryotic transcription factors (TF) form homodimer or heterodimer complexes to regulate gene expression. Dimerization of BASIC LEUCINE ZIPPER (bZIP) TFs are critical for their functions, but the molecular mechanism underlying the DNA binding and functional specificity of homo-versusheterodimers remains elusive. To address this gap, we present the double DNA Affinity Purification-sequencing (dDAP-seq) technique that maps heterodimer binding sites on endogenous genomic DNA. Using dDAP-seq we profile twenty pairs of C/S1 bZIP heterodimers and S1 homodimers inArabidopsisand show that heterodimerization significantly expands the DNA binding preferences of these TFs. Analysis of dDAP-seq binding sites reveals the function of bZIP9 in abscisic acid response and the role of bZIP53 heterodimer-specific binding in seed maturation. The C/S1 heterodimers show distinct preferences for the ACGT elements recognized by plant bZIPs and motifs resembling the yeast GCN4cis-elements. This study demonstrates the potential of dDAP-seq in deciphering the DNA binding specificities of interacting TFs that are key for combinatorial gene regulation. 

Abstract Historically, xenia effects were hypothesized to be unique genetic contributions of pollen to seed phenotype, but most examples represent standard complementation of Mendelian traits. We identified the imprinteddosage-effect defective1(ded1) locus in maize (Zea mays) as a paternal regulator of seed size and development. Hypomorphic alleles show a 5–10% seed weight reduction whended1is transmitted through the male, while homozygous mutants are defective with a 70–90% seed weight reduction.Ded1encodes an R2R3-MYB transcription factor expressed specifically during early endosperm development with paternal allele bias. DED1 directly activates early endosperm genes and endosperm adjacent to scutellum cell layer genes, while directly repressing late grain-fill genes. These results demonstrate xenia as originally defined: Imprinting ofDed1causes the paternal allele to set the pace of endosperm development thereby influencing grain set and size. 

SUMMARY The stilbenoid pathway is responsible for the production of resveratrol in grapevine (Vitis viniferaL.). A few transcription factors (TFs) have been identified as regulators of this pathway but the extent of this control has not been deeply studied. Here we show how DNA affinity purification sequencing (DAP‐Seq) allows for the genome‐wide TF‐binding site interrogation in grape. We obtained 5190 and 4443 binding events assigned to 4041 and 3626 genes for MYB14 and MYB15, respectively (approximately 40% of peaks located within −10 kb of transcription start sites). DAP‐Seq of MYB14/MYB15 was combined with aggregate gene co‐expression networks (GCNs) built from more than 1400 transcriptomic datasets from leaves, fruits, and flowers to narrow down bound genes to a set of high confidence targets. The analysis of MYB14, MYB15, and MYB13, a third uncharacterized member of Subgroup 2 (S2), showed that in addition to the few previously known stilbene synthase (STS) targets, these regulators bind to 30 of 47STSfamily genes. Moreover, all three MYBs bind to severalPAL,C4H, and4CLgenes, in addition to shikimate pathway genes, theWRKY03stilbenoid co‐regulator and resveratrol‐modifying gene candidates among which ROMT2‐3 were validated enzymatically. A high proportion of DAP‐Seq bound genes were induced in the activated transcriptomes of transientMYB15‐overexpressing grapevine leaves, validating our methodological approach for delimiting TF targets. Overall, Subgroup 2 R2R3‐MYBs appear to play a key role in binding and directly regulating several primary and secondary metabolic steps leading to an increased flux towards stilbenoid production. The integration of DAP‐Seq and reciprocal GCNs offers a rapid framework for gene function characterization using genome‐wide approaches in the context of non‐model plant species and stands up as a valid first approach for identifying gene regulatory networks of specialized metabolism. 

Summary Plants undergo several developmental transitions during their life cycle. In grapevine, a perennial woody fruit crop, the transition from vegetative/green‐to‐mature/woody growth involves transcriptomic reprogramming orchestrated by a small group of genes encoding regulators, but the underlying molecular mechanisms are not fully understood.We investigated the function of the transcriptional regulatorVviNAC33by generating and characterizing transgenic overexpressing grapevine lines and a chimeric repressor, and by exploring its putative targets through a DNA affinity purification sequencing (DAP‐seq) approach combined with transcriptomic data.We demonstrated that VviNAC33 induces leaf de‐greening, inhibits organ growth and directly activates the expression ofSTAY‐GREEN PROTEIN 1(SGR1), which is involved in Chl and photosystem degradation, andAUTOPHAGY 8f(ATG8f), which is involved in the maturation of autophagosomes. Furthermore, we show that VviNAC33 directly inhibitsAUXIN EFFLUX FACILITATOR PIN1,RopGEF1andATP SYNTHASE GAMMA CHAIN 1T(ATPC1), which are involved in photosystem II integrity and activity.Our results show that VviNAC33 plays a major role in terminating photosynthetic activity and organ growth as part of a regulatory network governing the vegetative‐to‐mature phase transition. 

Variegation is a rare type of mosaicism not fully studied in plants, especially fruits. We examined red and white sections of grape (Vitis vinifera cv. ‘Béquignol’) variegated berries and found that accumulation of products from branches of the phenylpropanoid and isoprenoid pathways showed an opposite tendency. Light-responsive flavonol and monoterpene levels increased in anthocyanin-depleted areas in correlation with increasing MYB24 expression. Cistrome analysis suggested that MYB24 binds to the promoters of 22 terpene synthase (TPS) genes, as well as 32 photosynthesis/light-related genes, including carotenoid pathway members, the flavonol regulator HY5 HOMOLOGUE (HYH), and other radiation response genes. Indeed, TPS35, TPS09, the carotenoid isomerase gene CRTISO2, and HYH were activated in the presence of MYB24 and MYC2. We suggest that MYB24 modulates ultraviolet and high-intensity visible light stress responses that include terpene and flavonol synthesis and potentially affects carotenoids. The MYB24 regulatory network is developmentally triggered after the onset of berry ripening, while the absence of anthocyanin sunscreens accelerates its activation, likely in a dose-dependent manner due to increased radiation exposure. Anthocyanins and flavonols in variegated berry skins act as effective sunscreens but for different wavelength ranges. The expression patterns of stress marker genes in red and white sections of ‘Béquignol’ berries strongly suggest that MYB24 promotes light stress amelioration but only partly succeeds during late ripening.