A complex role of Arabidopsis CDKD;3 in meiotic progression and cytokinesis.

Meiosis is a specialized cell division that halves the number of chromosomes in two consecutive rounds of chromosome segregation. In angiosperm plants is meiosis followed by mitotic divisions to form rudimentary haploid gametophytes. In Arabidopsis, termination of meiosis and transition to gametophytic development are governed by TDM1 and SMG7 that mediate inhibition of translation. Mutants deficient in this mechanism do not form tetrads but instead undergo multiple cycles of aberrant nuclear divisions that are likely caused by the failure to downregulate cyclin dependent kinases during meiotic exit. A suppressor screen to identify genes that contribute to meiotic exit uncovered a mutation in cyclin-dependent kinase D;3 (CDKD;3) that alleviates meiotic defects in smg7 deficient plants. The CDKD;3 deficiency prevents aberrant meiotic divisions observed in smg7 mutants or delays their onset after initiation of cytokinesis, which permits formation of functional microspores. Although CDKD;3 acts as an activator of cyclin-dependent kinase A;1 (CDKA;1), the main cyclin dependent kinase that regulates meiosis, cdkd;3 mutation appears to promote meiotic exit independently of CDKA;1. Furthermore, analysis of CDKD;3 interactome revealed enrichment for proteins implicated in cytokinesis, suggesting a more complex function of CDKD;3 in cell cycle regulation.

The biological functions of nonsense-mediated mRNA decay in plants: RNA quality control and beyond.

Nonsense-mediated mRNA decay (NMD) is an evolutionarily conserved quality control pathway that inhibits the expression of transcripts containing premature termination codon. Transcriptome and phenotypic studies across a range of organisms indicate roles of NMD beyond RNA quality control and imply its involvement in regulating gene expression in a wide range of physiological processes. Studies in moss Physcomitrella patens and Arabidopsis thaliana have shown that NMD is also important in plants where it contributes to the regulation of pathogen defence, hormonal signalling, circadian clock, reproduction and gene evolution. Here, we provide up to date overview of the biological functions of NMD in plants. In addition, we discuss several biological processes where NMD factors implement their function through NMD-independent mechanisms.

A CENH3 mutation promotes meiotic exit and restores fertility in SMG7-deficient Arabidopsis.

Meiosis in angiosperm plants is followed by mitotic divisions to form multicellular haploid gametophytes. Termination of meiosis and transition to gametophytic development is, in Arabidopsis, governed by a dedicated mechanism that involves SMG7 and TDM1 proteins. Mutants carrying the smg7-6 allele are semi-fertile due to reduced pollen production. We found that instead of forming tetrads, smg7-6 pollen mother cells undergo multiple rounds of chromosome condensation and spindle assembly at the end of meiosis, resembling aberrant attempts to undergo additional meiotic divisions. A suppressor screen uncovered a mutation in centromeric histone H3 (CENH3) that increased fertility and promoted meiotic exit in smg7-6 plants. The mutation led to inefficient splicing of the CENH3 mRNA and a substantial decrease of CENH3, resulting in smaller centromeres. The reduced level of CENH3 delayed formation of the mitotic spindle but did not have an apparent effect on plant growth and development. We suggest that impaired spindle re-assembly at the end of meiosis limits aberrant divisions in smg7-6 plants and promotes formation of tetrads and viable pollen. Furthermore, the mutant with reduced level of CENH3 was very inefficient haploid inducer indicating that differences in centromere size is not the key determinant of centromere-mediated genome elimination.

Nonsense-Mediated RNA Decay Factor UPF1 Is Critical for Posttranscriptional and Translational Gene Regulation in Arabidopsis.

Nonsense-mediated RNA decay (NMD) is an RNA control mechanism that has also been implicated in the broader regulation of gene expression. Nevertheless, a role for NMD in genome regulation has not yet been fully assessed, partially because NMD inactivation is lethal in many organisms. Here, we performed an in-depth comparative analysis of Arabidopsis (Arabidopsis thaliana) mutants lacking the NMD-related proteins UPF3, UPF1, and SMG7. We found different impacts of these proteins on NMD and the Arabidopsis transcriptome, with UPF1 having the biggest effect. Transcriptome assembly in UPF1-null plants revealed genome-wide changes in alternative splicing, suggesting that UPF1 functions in splicing. The inactivation of UPF1 led to translational repression, as manifested by a global shift in mRNAs from polysomes to monosomes and the downregulation of genes involved in translation and ribosome biogenesis. Despite these global changes, NMD targets and mRNAs expressed at low levels with short half-lives were enriched in the polysomes of upf1 mutants, indicating that UPF1/NMD suppresses the translation of aberrant RNAs. Particularly striking was an increase in the translation of TIR domain-containing, nucleotide binding, leucine-rich repeat (TNL) immune receptors. The regulation of TNLs via UPF1/NMD-mediated mRNA stability and translational derepression offers a dynamic mechanism for the rapid activation of TNLs in response to pathogen attack.

artMAP: A user-friendly tool for mapping ethyl methanesulfonate-induced mutations in Arabidopsis.

Mapping-by-sequencing is a rapid method for identifying both natural as well as induced variations in the genome. However, it requires extensive bioinformatics expertise along with the computational infrastructure to analyze the sequencing data and these requirements have limited its widespread adoption. In the current study, we develop an easy to use tool, artMAP, to discover ethyl methanesulfonate (EMS) induced mutations in the Arabidopsis genome. The artMAP pipeline consists of well-established tools including TrimGalore, BWA, BEDTools, SAMtools, and SnpEff which were integrated in a Docker container. artMAP provides a graphical user interface and can be run on a regular laptop and desktop, thereby limiting the bioinformatics expertise required. artMAP can process input sequencing files generated from single or paired-end sequencing. The results of the analysis are presented in interactive graphs which display the annotation details of each mutation. Due to its ease of use, artMAP makes the identification of EMS-induced mutations in Arabidopsis possible with only a few mouse click. The source code of artMAP is available on Github (https://github.com/RihaLab/artMAP).

Sequencing and De Novo Assembly of the Complete Chloroplast Genome of the Peruvian Carrot (Arracacia xanthorrhiza Bancroft).

Arracacia xanthorrhiza is an important secondary food crop in South America and Puerto Rico. The lack of crop protection and improvement strategies leads to infections damaging the storage roots. Here, we report the annotated complete chloroplast genome sequence of A. xanthorrhiza as a step toward developing genomic resources for this crop.

De novo transcriptome profiling of cold-stressed siliques during pod filling stages in Indian mustard (Brassica juncea L.).

Low temperature is a major abiotic stress that impedes plant growth and development. Brassica juncea is an economically important oil seed crop and is sensitive to freezing stress during pod filling subsequently leading to abortion of seeds. To understand the cold stress mediated global perturbations in gene expression, whole transcriptome of B. juncea siliques that were exposed to sub-optimal temperature was sequenced. Manually self-pollinated siliques at different stages of development were subjected to either short (6 h) or long (12 h) durations of chilling stress followed by construction of RNA-seq libraries and deep sequencing using Illumina's NGS platform. De-novo assembly of B. juncea transcriptome resulted in 133,641 transcripts, whose combined length was 117 Mb and N50 value was 1428 bp. We identified 13,342 differentially regulated transcripts by pair-wise comparison of 18 transcriptome libraries. Hierarchical clustering along with Spearman correlation analysis identified that the differentially expressed genes segregated in two major clusters representing early (5-15 DAP) and late stages (20-30 DAP) of silique development. Further analysis led to the discovery of sub-clusters having similar patterns of gene expression. Two of the sub-clusters (one each from the early and late stages) comprised of genes that were inducible by both the durations of cold stress. Comparison of transcripts from these clusters led to identification of 283 transcripts that were commonly induced by cold stress, and were referred to as "core cold-inducible" transcripts. Additionally, we found that 689 and 100 transcripts were specifically up-regulated by cold stress in early and late stages, respectively. We further explored the expression patterns of gene families encoding for transcription factors (TFs), transcription regulators (TRs) and kinases, and found that cold stress induced protein kinases only during early silique development. We validated the digital gene expression profiles of selected transcripts by qPCR and found a high degree of concordance between the two analyses. To our knowledge this is the first report of transcriptome sequencing of cold-stressed B. juncea siliques. The data generated in this study would be a valuable resource for not only understanding the cold stress signaling pathway but also for introducing cold hardiness in B. juncea.