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Domain of unknown function 239 (DUF239) is a conserved sequence found in the catalytic site of Neprosins which are specific secreted prolyl endopeptidases found in the Nepenthes genus. Neprosins participate in the nitrogen cycle by digesting preys trapped in the pitcher of these carnivorous plants. Apart from that, DUF239s have been poorly documented in plants. We have identified 50 genes containing DUF239-coding sequences in the Arabidopsis genome that are distributed across six distinct phylogenetic clusters. The chromosomal distribution suggests that several genes are the result of recent duplication events, with up to eight genes found in a strict tandem distribution. In Arabidopsis, most of DUF239-containing sequences are also associated to a Neprosin-activating domain (DUF4409) and an amino-terminal α-helix which corresponds to the typical domain organization of the Neprosins described in the Nepenthes genus. Analysis of Arabidopsis transcriptomic datasets reveals that 39 genes are exclusively expressed in reproductive organs, mainly during seed development and more specifically in the endosperm (23 genes). The peculiar expression pattern of the DUF239 gene family in Arabidopsis suggests new functions of Neprosin-like proteins in plants during seed development., (© 2022 The Authors. The Plant Genome published by Wiley Periodicals LLC on behalf of Crop Science Society of America.)

Co-expression networks are essential tools to infer biological associations between gene products and predict gene annotation. Global networks can be analyzed at the transcriptome-wide scale or after querying them with a set of guide genes to capture the transcriptional landscape of a given pathway in a process named Pathway Level Coexpression (PLC). A critical step in network construction remains the definition of gene co-expression. In the present work, we compared how Pearson Correlation Coefficient (PCC), Spearman Correlation Coefficient (SCC), their respective ranked values (Highest Reciprocal Rank (HRR)), Mutual Information (MI) and Partial Correlations (PC) performed on global networks and PLCs. This evaluation was conducted on the model plant Arabidopsis thaliana using microarray and differently pre-processed RNA-seq datasets. We particularly evaluated how dataset × distance measurement combinations performed in 5 PLCs corresponding to 4 well described plant metabolic pathways (phenylpropanoid, carbohydrate, fatty acid and terpene metabolisms) and the cytokinin signaling pathway. Our present work highlights how PCC ranked with HRR is better suited for global network construction and PLC with microarray and RNA-seq data than other distance methods, especially to cluster genes in partitions similar to biological subpathways.

The molecular mechanisms underlying plant cell totipotency are largely unknown. Here, we present a protocol for the efficient regeneration of plants from Arabidopsis thaliana protoplasts. The specific liquid medium used in our study leads to a high rate of reentry into the cell cycle of most cell types, providing a powerful system to study dedifferentiation/regeneration processes in independent somatic cells. To identify the early events in the establishment of totipotency, we monitored the genome-wide transcript profiles of plantlets and protoplast-derived cells (PdCs) during the first week of culture. Plant cells rapidly dedifferentiated. Then, we observed the reinitiation and reorientation of protein synthesis, accompanied by the reinitiation of cell division and de novo cell wall synthesis. Marked changes in the expression of chromatin-associated genes, especially of those in the histone variant family, were observed during protoplast culture. Surprisingly, the epigenetic status of PdCs and well-established cell cultures differed, with PdCs exhibiting rare reactivated transposons and epigenetic changes. The differentially expressed genes identified in this study are interesting candidates for investigating the molecular mechanisms underlying plant cell plasticity and totipotency. One of these genes, the plant-specific transcription factor ABERRANT LATERAL ROOT FORMATION4, is required for the initiation of protoplast division.

Nitrate releases seed dormancy in Arabidopsis (Arabidopsis thaliana) Columbia accession seeds in part by reducing abscisic acid (ABA) levels. Nitrate led to lower levels of ABA in imbibed seeds when included in the germination medium (exogenous nitrate). Nitrate also reduced ABA levels in dry seeds when provided to the mother plant during seed development (endogenous nitrate). Transcript profiling of imbibed seeds treated with or without nitrate revealed that exogenous nitrate led to a higher expression of nitrate-responsive genes, whereas endogenous nitrate led to a profile similar to that of stratified or after-ripened seeds. Profiling experiments indicated that the expression of the ABA catabolic gene CYP707A2 was regulated by exogenous nitrate. The cyp707a2-1 mutant failed to reduce seed ABA levels in response to both endogenous and exogenous nitrate. In contrast, both endogenous and exogenous nitrate reduced ABA levels of the wild-type and cyp707a1-1 mutant seeds. The CYP707A2 mRNA levels in developing siliques were positively correlated with different nitrate doses applied to the mother plants. This was consistent with a role of the CYP707A2 gene in controlling seed ABA levels in response to endogenous nitrate. The cyp707a2-1 mutant was less sensitive to exogenous nitrate for breaking seed dormancy. Altogether, our data underline the central role of the CYP707A2 gene in the nitrate-mediated control of ABA levels during seed development and germination.

ATM (Ataxia Telangiectasia Mutated) is an essential checkpoint kinase that signals DNA double-strand breaks in eukaryotes. Its depletion causes meiotic and somatic defects in Arabidopsis and progressive motor impairment accompanied by several cell deficiencies in patients with ataxia telangiectasia (AT). To obtain a comprehensive view of the ATM pathway in plants, we performed a time-course analysis of seedling responses by combining confocal laser scanning microscopy studies of root development and genome-wide expression profiling of wild-type (WT) and homozygous ATM-deficient mutants challenged with a dose of gamma-rays (IR) that is sublethal for WT plants. Early morphologic defects in meristematic stem cells indicated that AtATM, an Arabidopsis homolog of the human ATM gene, is essential for maintaining the quiescent center and controlling the differentiation of initial cells after exposure to IR. Results of several microarray experiments performed with whole seedlings and roots up to 5 h post-IR were compiled in a single table, which was used to import gene information and extract gene sets. Sequence and function homology searches; import of spatio-temporal, cell cycling, and mutant-constitutive expression characteristics; and a simplified functional classification system were used to identify novel genes in all functional classes. The hundreds of radiomodulated genes identified were not a random collection, but belonged to functional pathways such as those of the cell cycle; cell death and repair; DNA replication, repair, and recombination; and transcription; translation; and signaling, indicating the strong cell reprogramming and double-strand break abrogation functions of ATM checkpoints. Accordingly, genes in all functional classes were either down or up-regulated concomitantly with downregulation of chromatin deacetylases or upregulation of acetylases and methylases, respectively. Determining the early transcriptional indicators of prolonged S-G2 phases that coincided with cell proliferation delay, or an anticipated subsequent auxin increase, accelerated cell differentiation or death, was used to link IR-regulated hallmark functions and tissue phenotypes after IR. The transcription burst was almost exclusively AtATM-dependent or weakly AtATR-dependent, and followed two major trends of expression in atm: (i)-loss or severe attenuation and delay, and (ii)-inverse and/or stochastic, as well as specific, enabling one to distinguish IR/ATM pathway constituents. Our data provide a large resource for studies on the interaction between plant checkpoints of the cell cycle, development, hormone response, and DNA repair functions, because IR-induced transcriptional changes partially overlap with the response to environmental stress. Putative connections of ATM to stem cell maintenance pathways after IR are also discussed.

NUCLEOSOME ASSEMBLY PROTEIN1 (NAP1) is conserved from yeast to human and was proposed to act as a histone chaperone. While budding yeast contains a single NAP1 gene, multicellular organisms, including plants and animals, contain several NAP1 and NAP1-RELATED PROTEIN (NRP) genes. However, the biological role of these genes has been largely unexamined. Here, we show that, in Arabidopsis thaliana, simultaneous knockout of the two NRP genes, NRP1 and NRP2, impaired postembryonic root growth. In the nrp1-1 nrp2-1 double mutant, arrest of cell cycle progression at G2/M and disordered cellular organization occurred in root tips. The mutant seedlings exhibit perturbed expression of approximately 100 genes, including some genes involved in root proliferation and patterning. The mutant plants are highly sensitive to genotoxic stress and show increased levels of DNA damage and the release of transcriptional gene silencing. NRP1 and NRP2 are localized in the nucleus and can form homomeric and heteromeric protein complexes. Both proteins specifically bind histones H2A and H2B and associate with chromatin in vivo. We propose that NRP1 and NRP2 act as H2A/H2B chaperones in the maintenance of dynamic chromatin in epigenetic inheritance.

In higher plants, PII is a nuclear-encoded plastid protein which is homologous to bacterial PII signalling proteins known to be involved in the regulation of nitrogen metabolism. A reduced ornithine, citrulline and arginine accumulation was observed in two Arabidopsis PII knock-out mutants in response to NH4+ resupply after N starvation. This difference could be explained by the regulation of a key enzyme of the arginine biosynthesis pathway, N-acetyl glutamate kinase (NAGK) by PII. In vitro assays using purified recombinant proteins showed the catalytic activation of Arabidopsis NAGK by PII giving the first evidence of a physiological role of the PII protein in higher plants. Using Arabidopsis transcriptome microarray (CATMA) and RT-PCR analyses, it was found that none of the genes involved in the arginine biosynthetic or catabolic pathways were differentially expressed in a PII knock-out mutant background. In conclusion, the observed changes in metabolite levels can be explained by the reduced activation of NAGK by PII.