Jianfei Zhao

Transposons drive genetic diversity and evolution by altering the genomic landscape over time. Here, we describe D ELAYED AB AXIAL TRICHOMES ( DAB ), a Helitron/RC transposable element in Arabidopsis thaliana that has a role in vegetative phase change and gametogenesis. A genome-wide association study (GWAS) for the timing of abaxial trichome development (an adult leaf trait) in A. thaliana revealed a conserved haplotype of polymorphisms within DAB that delays abaxial trichome production. CRISPR-Cas9-induced deletions of DAB are gametophytic pollen-lethal, indicating that this locus is also required for pollen production. DAB produces 24-nucleotide siRNAs with sequence complementarity to genes involved in embryogenesis, gametogenesis, and seed development. DAB also impacts the expression of ARGONAUTE genes, genes involved in RNA-directed DNA methylation (RdDM), as well as genes in several key genetic pathways. This global effect on gene expression suggests that DAB may have functions beyond those identified in this study.

Key message: Loss of SPL10 and SPL11 increases penetrance of abnormal phenotypes in dcl1 embryos. The first division of the Arabidopsis zygote is asymmetric, resulting in an apical cell lineage that generates most of the embryo proper, and a basal cell lineage that produces the root meristem and the extraembryonic suspensor. Loss of function mutations in the microRNA processing enzyme genes DICER-LIKE 1 (DCL1) and SERRATE (SE) show cell division defects in the embryo proper, hypophyseal cell, and suspensor. Previous transcriptome analyses showed that the microRNA156-targeted transcription factor genes SQUAMOSA PROMOTER BINDING PROTEIN-LIKE2 (SPL2) , SPL3 and SPL11 were upregulated in both globular stage dcl1 and se embryos, while SPL10 was upregulated in dcl1 . It was previously proposed that upregulation of SPL10 and SPL11 could explain some abnormal phenotypes in dcl1 embryos. In this work, we used T-DNA and CRISPR-Cas9-induced loss of function alleles to further explore the function of SPL2 , SPL3 , SPL10 and SPL11 in early embryogenesis and their contribution to the dcl1 phenotype. On their own, spl2 , spl3 , spl10 , and spl11 single mutants and an spl10 spl11 double mutant showed no abnormal cell divisions in early embryogenesis. In the dcl1/+ background, loss of function of SPL2 or SPL3 did not change the proportion of cell division defects in hypophyseal cells or suspensors observed in dcl1/+ . Loss of spl10 or spl11 in dcl1/+ resulted in a slight decrease or increase (respectively) in the penetrance of abnormal suspensor divisions in heart stage embryos, while the spl10 spl11 double mutant caused a small increase in the penetrance of abnormal hypophyseal divisions in dcl1 embryos. The differences between our results and previous studies are likely due to genetic redundancy of miR156-targeted SPL genes, variable environmental conditions or the effect of genetic background on the penetrance of the dcl1 phenotype. In the future, analysis of higher order mutations in SPL and MIR156 genes will help to better understand the role of these important developmental regulators in early embryo development.

SUPPRESSOR OF PHYB-4#5 DOMINANT (sob5-D) was previously identified as a suppressor of the phyB-4 long-hypocotyl phenotype in Arabidopsis thaliana. Overexpression of SOB5 conferred dwarf phenotypes similar to those observed in plants containing elevated levels of cytokinin (CK) nucleotides and nucleosides. Two SOB-FIVE- LIKE (SOFL) proteins, AtSOFL1 and AtSOFL2, which are more similar at the protein level to each other than they are to SOB5, conferred similar phenotypes to the sob5-D mutant when overexpressed. We used protein sequences of founding SOFL gene family members to perform database searches and identified a total of 289 SOFL homologs in genomes of 89 angiosperm species. Phylogenetic analysis results implied that the SOFL gene family emerged during the expansion of angiosperms and later evolved into four distinct clades. Among the newly identified gene family members are four previously unreported Arabidopsis SOFLs. Multiple sequence alignment of the 289 SOFL protein sequences revealed two highly conserved domains; SOFL-A and SOFL-B. We used overexpression and site-directed mutagenesis studies to demonstrate that SOFL domains are necessary for SOB5 and AtSOFL1's overexpression phenotypes. Examination of the subcellular localization patterns of founding Arabidopsis thaliana SOFLs suggested they may be localized in the cytoplasm and/or the nucleus. Overall, we report that SOFLs are a plant-specific gene family characterized by two conserved domains that are important for function.

Correct developmental timing is essential for plant fitness and reproductive success. Two important transitions in shoot development—the juvenile-to-adult vegetative transition and the vegetative-to-reproductive transition—are mediated by a group of genes targeted by miR156, SQUAMOSA PROMOTER BINDING PROTEIN (SBP) genes. To determine the developmental functions of these genes in Arabidopsis thaliana, we characterized their expression patterns, and their gain-of-function and loss-of-function phenotypes. Our results reveal that SBP-LIKE (SPL) genes in Arabidopsis can be divided into three functionally distinct groups: 1) SPL2, SPL9, SPL10, SPL11, SPL13 and SPL15 contribute to both the juvenile-to-adult vegetative transition and the vegetative-to-reproductive transition, with SPL9, SP13 and SPL15 being more important for these processes than SPL2, SPL10 and SPL11; 2) SPL3, SPL4 and SPL5 do not play a major role in vegetative phase change or floral induction, but promote the floral meristem identity transition; 3) SPL6 does not have a major function in shoot morphogenesis, but may be important for certain physiological processes. We also found that miR156-regulated SPL genes repress adventitious root development, providing an explanation for the observation that the capacity for adventitious root production declines as the shoot ages. miR156 is expressed at very high levels in young seedlings, and declines in abundance as the shoot develops. It completely blocks the expression of its SPL targets in the first two leaves of the rosette, and represses these genes to different degrees at later stages of development, primarily by promoting their translational repression. These results provide a framework for future studies of this multifunctional family of transcription factors, and offer new insights into the role of miR156 in Arabidopsis development. In Arabidopsis, miR156 acts by repressing the expression of 10 SQUAMOSA PROMOTOR BINDING PROTEIN-LIKE (SPL) genes. The phenotype of plants over-expressing miR156 demonstrates that these genes control many aspects of plant development and physiology, but the functions of individual miR156-regulated SPL genes, and how their expression is regulated by miR156, are largely unknown. We addressed these questions by determining the phenotypes of loss-of-function mutations in these genes individually and in combination, and by comparing the expression patterns and the phenotypes of miR156-sensitive and miR156-resistant reporters for these genes. Our results reveal the unique and shared functions of the members of this gene family, and demonstrate that miR156 plays different roles in the regulation of SPL gene expression at different times in development.

SOB3 modulates seedling elongation by repressing the expression of genes associated with auxin signaling.