Search for: All records

SUMMARY WUSCHEL (WUS) is transcription factor vital for stem cell proliferation in plant meristems. In maize,ZmWUS1is expressed in the inflorescence meristem, including the central zone, the reservoir of stem cells.ZmWUS1overexpression in theBarren inflorescence3mutant leads to defects in inflorescence development. Here, single-cell ATAC-seq analysis shows thatZmWUS1overexpression alters chromatin accessibility throughout the central zone. The CAATAATGC motif, a known homeodomain recognition site, is predominantly observed in the regions with increased chromatin accessibility suggesting ZmWUS1 is an activator in the central zone. Regions with decreased chromatin accessibility feature various motifs and are adjacent toAUXIN RESPONSE FACTORgenes, revealing negative regulation of auxin signaling in the central zone. DAP-seq of ZmWUS1 identified the TGAATGAA motif, abundant in epidermal accessible chromatin compared to the central zone. These findings highlight ZmWUS1’s context-dependent mechanisms for stem cell maintenance in the inflorescence meristem. 

ABSTRACT The formation of the plant body proceeds in a sequential post-embryonic manner through the action of meristems. Tightly coordinated meristem regulation is required for development and reproductive success, eventually determining yield in crop species. In maize, the REL2 family of transcriptional corepressors includes four members, REL2, RELK1 (REL2-LIKE1), RELK2, and RELK3. In a screen forrel2enhancers, we identified shorter double mutants with enlarged female inflorescence meristems (IMs) carrying mutations inRELK1. Expression and genetic analysis indicate thatREL2andRELK1cooperatively regulate female IM development by controlling genes involved in redox balance, hormone homeostasis, and differentiation, ultimately tipping the meristem toward an environment favorable to expanded expression of theZmWUSCHEL1gene, a key stem-cell promoting transcription factor. We further demonstrate thatRELKgenes have partially redundant yet diverse functions in the maintenance of various meristem types during development. By exploiting subtle increases in ear IM size inrel2heterozygous plants, we also show that extra rows of kernels are formed across a diverse set of F1 hybrids. Our findings reveal that the REL2 family maintains development from embryonic initiation to reproductive growth and can potentially be harnessed for increasing seed yield in a major crop species. One sentence summaryREL2-RELKs fine tune hormone and chemical cues to prevent expanded expression of ZmWUSCHEL1 in maize inflorescence meristems, and can potentially be harnessed for increasing seed yield in hybrids. 

Abstract The formation of the plant body proceeds in a sequential postembryonic manner through the action of meristems. Tightly coordinated meristem regulation is required for development and reproductive success, eventually determining yield in crop species. In maize (Zea mays), the RAMOSA1 ENHANCER LOCUS2 (REL2) family of transcriptional corepressors includes four members, REL2, RELK1 (REL2-LIKE1), RELK2, and RELK3. In a screen for rel2 enhancers, we identified shorter double mutants with enlarged ear inflorescence meristems (IMs) carrying mutations in RELK1. Expression and genetic analysis indicated that REL2 and RELK1 cooperatively regulate ear IM development by controlling genes involved in redox balance, hormone homeostasis, and differentiation, ultimately tipping the meristem toward an environment favorable to expanded expression of the ZmWUSCHEL1 gene, which encodes a key stem-cell promoting transcription factor. We further demonstrated that RELK genes have partially redundant yet diverse functions in the maintenance of various meristem types during development. By exploiting subtle increases in ear IM size in rel2 heterozygous plants, we also showed that extra rows of kernels are formed across a diverse set of F1 hybrids. Our findings reveal that the REL2 family maintains development from embryonic initiation to reproductive growth and can potentially be harnessed for increasing seed yield in a major crop species. 

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. 

SUMMARY Stem cells in plant shoots are a rare population of cells that produce leaves, fruits and seeds, vital sources for food and bioethanol. Uncovering regulators expressed in these stem cells will inform crop engineering to boost productivity. Single-cell analysis is a powerful tool for identifying regulators expressed in specific groups of cells. However, accessing plant shoot stem cells is challenging. Recent single-cell analyses of plant shoots have not captured these cells, and failed to detect stem cell regulators likeCLAVATA3andWUSCHEL. In this study, we finely dissected stem cell-enriched shoot tissues from both maize and arabidopsis for single-cell RNA-seq profiling. We optimized protocols to efficiently recover thousands ofCLAVATA3andWUSCHELexpressed cells. A cross-species comparison identified conserved stem cell regulators between maize and arabidopsis. We also performed single-cell RNA-seq on maize stem cell overproliferation mutants to find additional candidate regulators. Expression of candidate stem cell genes was validated using spatial transcriptomics, and we functionally confirmed roles in shoot development. These candidates include a family of ribosome-associated RNA-binding proteins, and two families of sugar kinase genes related to hypoxia signaling and cytokinin hormone homeostasis. These large-scale single-cell profiling of stem cells provide a resource for mining stem cell regulators, which show significant association with yield traits. Overall, our discoveries advance the understanding of shoot development and open avenues for manipulating diverse crops to enhance food and energy security. 

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.