Mieke Van Lijsebettens

During Arabidopsis thaliana photomorphogenesis, light promotes cotyledon expansion and inhibits hypocotyl elongation. This process involves transcriptional reprogramming controlled by various factors, including the Elongator complex, which regulates gene expression at the level of transcription and translation via epigenetic and transfer RNA modifications, respectively. The elo3–6 mutant, lacking Elongator activity, exhibited photomorphogenic defects: less open, hyponastic cotyledons and an elongated hypocotyl. RNA-Seq and miRNA-Seq revealed distinct dysregulated gene sets in the elo3–6 mutant hypocotyl and cotyledons. In the hypocotyl, the elo3–6 defect affected the expression of genes involved in chloroplast physiology, circadian regulation, and auxin responses. Impaired chloroplast biogenesis apparently triggered retrograde signaling and a hypoxia-like state, preventing full inhibition of hypocotyl elongation. The defective elo3–6 cotyledon development is likely due to compromised translation. This was supported by the presence of similar morphological defects in urm11 urm12, defective in the same type of tRNA modification as elo3–6, and the synergism observed in the elo3–6 urm11 urm12 (euu) triple mutant showing seedling lethality. Moreover, elo3–6 and urm11 urm12 showed increased tolerance to translation inhibitors, including hygromycin B, which prevented the narrower cotyledon opening in elo3–6, suggesting that strong Elongator-dependent codon–anticodon interactions are required for proper cotyledon development. Interestingly, the genes enriched in codons recognized by tRNA anticodons modified by Elongator showed decreased mRNA abundance in elo3–6, suggesting a feedback mechanism downregulating the abundance of inefficiently translated mRNAs. Our results suggest that Elongator’s transcriptional role is more important in hypocotyl growth, while its translational role is more prominent in cotyledon development.

Transcriptional enhancers participate in cell and tissue differentiation in all multicellular organisms. Here, we characterized the candidate enhancer Vegetative to generative1 (Vgt1), a major quantitative trait locus for flowering time in maize. Transgenic lines containing an inverted repeat that induces DNA methylation at Vgt1 showed early flowering and an accelerated growth rate during early development. DNA methylation of Vgt1 was associated with the downregulation of the AP2-like floral repressor ZmRap2.7 in specific leaf tissues at the early stages of maize development. In line with Vgt1 regulating ZmRap2.7, chromosome conformation capture data showed that Vgt1 physically interacts with the ZmRap2.7 transcription start site. Finally, chromatin immunoprecipitation of transiently expressed ZmRap2.7 in protoplasts indicated that this transcription factor binds to the promoters of several hundred genes. These genes include many genes that are differentially expressed in maize lines with and without extra DNA methylation at Vgt1. Altogether, we show that ZmRap2.7 is transcriptionally controlled by Vgt1 and is involved in regulating flowering time and other biological pathways, such as jasmonate signaling.

The plasma membrane aquaporin ZmPIP1;6 is expressed in maize stomatal complexes, with higher expression during the day than at night. To elucidate the role of ZmPIP1;6 in gas exchange and stomatal movement, it was expressed in maize (inbred line B104) under the control of the p35S promoter (overexpression) or its native promoter fused with mYFP (monomeric yellow fluorescent protein) cDNA (mYFP-ZmPIP1;6). In stomatal complexes of the mature zone of the leaf, mYFP–ZmPIP1;6 showed higher expression in subsidiary cells than in guard cells, and light and dark treatments influenced its subcellular localization. Notably, ZmPIP1;6 internalization increased in dark conditions compared with light. Stomatal opening was greater in ZmPIP1;6 overexpression plants than in the wild type, while closure exhibited greater sensitivity to elevated CO2 concentration or abscisic acid (ABA) treatment. Our findings revealed that reactive oxygen species (H2O2) were involved in ABA-induced stomatal closure, while ZmPIP1;6 was unable to facilitate H2O2 diffusion when expressed in yeast. Finally, ZmPIP1;6 overexpression and mYFP-ZmPIP1;6 transgenic plants exhibited higher abaxial stomatal density than the wild type. Overall, these results indicate that ZmPIP1;6 plays important roles in stomatal opening and CO2- and ABA-induced stomatal closure.

SAMBA has been identified as a plant-specific regulator of the anaphase-promoting complex/cyclosome (APC/C) that controls unidirectional cell cycle progression in Arabidopsis (Arabidopsis thaliana), but so far its role has not been studied in monocots. Here, we show the association of SAMBA with the APC/C is conserved in maize (Zea mays). Two samba genome edited mutants showed growth defects, such as reduced internode length, shortened upper leaves with erect leaf architecture, and reduced leaf size due to an altered cell division rate and cell expansion, which aggravated with plant age. The two mutants differed in the severity and developmental onset of the phenotypes, because samba-1 represented a knockout allele, while translation re-initiation in samba-3 resulted in a truncated protein that was still able to interact with the APC/C and regulate its function, albeit with altered APC/C activity and efficiency. Our data are consistent with a dosage-dependent role for SAMBA to control developmental processes for which a change in growth rate is pivotal.

The ataxia-telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) kinases coordinate the DNA damage response. The roles described for Arabidopsis thaliana ATR and ATM are assumed to be conserved over other plant species, but molecular evidence is scarce. Here, we demonstrate that the functions of ATR and ATM are only partially conserved between Arabidopsis and maize (Zea mays). In both species, ATR and ATM play a key role in DNA repair and cell cycle checkpoint activation, but whereas Arabidopsis plants do not suffer from the absence of ATR under control growth conditions, maize mutant plants accumulate replication defects, likely due to their large genome size. Moreover, contrarily to Arabidopsis, maize ATM deficiency does not trigger meiotic defects, whereas the ATR kinase appears to be crucial for the maternal fertility. Strikingly, ATR is required to repress premature endocycle onset and cell death in the maize endosperm. Its absence results in a reduction of kernel size, protein and starch content, and a stochastic death of kernels, a process being counteracted by ATM. Additionally, while Arabidopsis atr atm double mutants are viable, no such mutants could be obtained for maize. Therefore, our data highlight that the mechanisms maintaining genome integrity may be more important for vegetative and reproductive development than previously anticipated.

SAMBA has been identified as a plant-specific regulator of the anaphase-promoting complex (APC/C) which controls unidirectional cell cycle progression in Arabidopsis, but so far its role was not studied in monocots. Here, the association of SAMBA with APC/C was shown to be conserved in maize. Two samba CRISPR alleles showed growth defects that aggravated with plant age such as dwarfed plants due to shortened upper leaf length, erect leaf architecture, and reduced leaf size due to an altered cell division rate and cell expansion. Despite the fact that in both alleles the frameshift occurred at the same position, the two alleles differed in the severity and developmental onset of the phenotypes, because samba-1 represented a knock-out allele, while translation re-initiation in samba-3 resulted in a truncated protein that was still able to interact with the APC/C and regulate its function, albeit with altered APC/C activity or efficiency. Our data are consistent with a dosage-dependent role for SAMBA to control developmental processes for which a change in growth rate is pivotal.

Contrary to the conserved Elongator composition in yeast, animals, and plants, molecular functions and catalytic activities of the complex remain controversial. Elongator was identified as a component of elongating RNA polymerase II holoenzyme in yeast, animals, and plants. Furthermore, it was suggested that Elonagtor facilitates elongation of transcription via histone acetyl transferase activity. Accordingly, phenotypes of Arabidopsis elo mutants, which show development, growth, or immune response defects, correlate with transcriptional downregulation and the decreased histone acetylation in the coding regions of crucial genes. Plant Elongator was also implicated in other processes: transcription and processing of miRNA, regulation of DNA replication by histone acetylation, and acetylation of alpha-tubulin. Moreover, tRNA modification, discovered first in yeast and confirmed in plants, was claimed as the main activity of Elongator, leading to specificity in translation that might also result indirectly in a deficiency in transcription. Heterologous overexpression of individual Arabidopsis Elongator subunits and their respective phenotypes suggest that single Elongator subunits might also have another function next to being a part of the complex. In this review, we shall present the experimental evidence of all molecular mechanisms and catalytic activities performed by Elongator in nucleus and cytoplasm of plant cells, which might explain how Elongator regulates growth, development, and immune responses.

In plants, methylation at cytosines often leads to changes in gene expression and inactivation of transposable elements. Changes in cytosine methylation (epimutations) might produce epialleles with distinct phenotypes. We present a genome-wide cytosine methylation profiling method based on bisulfite conversion and next-generation sequencing, which is applicable for plant species with available reference genomes. This so-called plant-RRBS profiling method reproducibly covers specific genomic regions and enriches for coverage of cytosine positions that are suitable for comparative analyses in multi-sample studies in basic biology and breeding studies. The plant-RRBS workflow consists of genomic DNA digestion with coverage-efficient restriction endonuclease combinations followed by a performant library generation and next-generation sequencing and a straightforward, publically available methylation data processing pipeline. Plant-RRBS has a twofold higher ratio of cytosine coverage per covered genome as compared to whole-genome bisulfite sequencing, covering tens of millions of cytosine positions, and allows detection of differential cytosine methylation, which was evaluated using rice epilines.

The maize plasma membrane PIP2;5 aquaporin plays a role in controlling root radial water movement, leaf hydraulic conductivity, and plant growth.