The sequencing reads from RNA-Seq were averaged for the biological replicates. For each expressed gene, a ratio of the averaged read counts in the respective experimental (trisomic or ploidy) genotype was made over the read counts in the diploid control. These ratios were plotted in bins of 0.05. The X axis notes the value for each bin and the Y axis notes the number of genes per bin. For the five trisomics, genes were partitioned into those encoded on the varied chromosome (cis) versus those encoded on the remainder of the genome that was not varied in dosage (trans). A ratio of 1.00 represents no change in the experimental genotype versus the diploid. A ratio of 1.50 represents a gene dosage effect in cis whereas 1.00 represents dosage compensation. A ratio of 0.67 represents the inverse ratio of gene expression in trans. These ratio values are demarcated with labeled vertical lines. The triploid and tetraploid ploidy series were analyzed in the same manner for all expressed genes. The vertical demarcations in this case correspond to the respective direct or inverse relationship of the ploidy comparison. The tetraploid/triploid comparison was generated by producing the respective ratios and plotting the distribution. Each comparison is labeled in the respective panel.

Changes in dosage of part of the genome (aneuploidy) have long been known to produce more severe phenotypic consequences than changes in the number of whole genomes (ploidy). To study the basis of these differences, we examined, global gene expression in mature leaf tissue for all five trisomics and in diploids, triploids and tetraploids of Arabidopsis thaliana. The trisomics produced a greater spread of expression modulation than the ploidy series. In general, expression of genes on the varied chromosome ranged from compensation to dosage effect whereas genes from the remainder of the genome ranged from no effect to reduced expression approaching the inverse level of chromosomal imbalance (2/3). Genome-wide DNA methylation was investigated in each genotype and found to shift most prominently with trisomy 4 but otherwise exhibited little change, indicating that genetic imbalance is usually mechanistically unrelated to DNA methylation. Independent inspection of gene functional classes demonstrated that ribosomal, proteasomal and gene body methylated genes were less modulated compared to all classes of genes whereas genes encoding transcription factors, signal transduction components and organelle-targeted proteins were more tightly inversely affected. Comparing transcription factors and their targets in the trisomics and in expression networks revealed considerable discordance, illustrating that altered regulatory stoichiometry is a major contributor to genetic imbalance. Re-analysis of published data on gene expression in disomic yeast and trisomic mouse cells detected similar stoichiometric effects across broad phylogenetic taxa and indicated that these responses reflect normal gene regulatory processes.

Iron is an essential mineral nutrient which severely affects the growth, yield, and nutritional quality of plants if not supplied in sufficient quantities. Wolfgang Schmidt’s group discovered a novel family of peptides in plants referred to as IRON MAN (IMA), and show that they are a sine qua non for the uptake of iron the soil. Silencing of all eight IMA genes in Arabidopsis by CRISPR-Cas9 genome editing resulted in very small, extremely chlorotic plants that died without drastic iron supplementation. IMAs are present in the genomes of all flowering plants but are missing in ferns, algae or fungi, suggesting that IMA emerged at an early stage in the evolution of land plants. Reciprocal grafting of octuple ima mutants with wild-type plants showed that IMA1 peptides in shoots positively regulate iron uptake in roots, suggesting that IMAs are the long sought-after shoot-borne signal that communicates the iron status of the leaves to tune iron uptake by roots. The discovery of IRON MAN opens a novel route of generating iron-enriched plants that may help to combat iron deficiency-induced anaemia, one of the largest nutritional disorder in humans.

Mitochondrial intron splicing is a plant-specific feature that was acquired during the coevolution of eukaryotic host cells and a-proteobacteria. The elimination of these introns is facilitated by mitochondrial-targeted proteins encoded by its host, nucleus. What’s this factor 9 (WTF9), a nucleus-encoded plant organelle RNA recognition (PORR) protein, is involved in the splicing of the mitochondrial group II introns rpl2 and ccmFC. Disruption of WTF9 causes developmental defects associated with the loss of cytochromes c and c1 in Arabidopsis. Using a co-immunoprecipitation assay, Hsu et al. from Dr. Guang-Yuh Jauh’s laboratory found that HSP60s interacted with WTF9, which was further confirmed by a pull-down assay. HSP60s are molecular chaperones that assist with protein folding in both eukaryotic and prokaryotic cells. However, accumulating evidence suggests that HSP60s also participate in other biological functions such as RNA metabolism and RNA protection. In this study, we found that consistently with their interaction with WTF9, HSP60s interacted with 48 nucleotides of the ccmFC intron. In mutant studies, the double mutant hsp60-3a1hsp60-3b1 exhibited a small stature phenotype and reduced splicing efficiency for rpl2 and ccmFC. These observations were similar to those in wtf9 mutants and suggest that HSP60s are involved in the RNA splicing of rpl2 and ccmFC introns in mitochondria. Our findings suggest that HSP60s participate in mitochondrial RNA splicing through their RNA-binding ability. (Hsu et al. Plant and Cell Physiology 2018 )

https://doi.org/10.1093/pcp/pcy199

Splicing of pre-mRNAs is an essential step in the expression of most eukaryotic genes. Both constitutive splicing and alternative splicing, which produces multiple mRNA isoforms from a single primary transcript, are modulated by reversible protein phosphorylation. Although the plant splicing machinery is known to be a target for phosphorylation, the protein kinases involved remain to be fully defined. We report here the identification of PRP4 KINASE A (PRP4KA) in a forward genetic screen based on an alternatively-spliced GFP reporter gene in Arabidopsis thaliana (Arabidopsis). Prp4 kinase is the first spliceosome-associated kinase shown to regulate splicing in fungi and mammals but it has not yet been studied in plants. We identified in the same screen mutants defective in SAC3A, a putative mRNA export factor that is highly co-expressed with PRP4KA in Arabidopsis. Whereas the sac3a mutants appear normal, the prp4ka mutants display a pleiotropic phenotype featuring atypical rosettes, late flowering, tall final stature, reduced branching and lowered seed set. Analysis of RNA-sequencing data from prp4ka and sac3a mutants identified widespread and partially overlapping perturbations in alternative splicing in the two mutants. Quantitative phosphoproteomic profiling of a prp4ka mutant detected phosphorylation changes in several serine/arginine-rich proteins, which regulate constitutive and alternative splicing, and other splicing-related factors. Tests of PRP4KB, the paralog of PRP4KA, indicated that the two genes are not functionally redundant. The results demonstrate the importance of PRP4KA for alternative splicing and plant phenotype, and suggest that PRP4KA may influence alternative splicing patterns by phosphorylating a subset of splicing regulators. Contributors to this work include Tatsuo Kanno, Peter Venhuizen, Tuan-Nan Wen, Wen-Dar Lin, Phebe Chiou, Maria Kalyna, Antonius J.M. Matzke and Marjori Matzke. The paper is published in Genetics Society of America – Genetics (https://doi.org/10.1534/genetics.118.301515).

9 - 11 December 2018 | Taipei, Taiwan

Phosphatidylcholine (PtdCho) is the conserved membrane lipid component among eukaryotic cells yet playing multiple biological roles beyond a constituent of cellular membranes. In plants, the phospho-base N-methyltransferases (PMTs) catalyze the biosynthesis of phosphocholine (PCho), a prerequisite for PtdCho biosynthesis. However, it remains elusive whether specific PMT isoform is involved in PtdCho biosynthesis in Arabidopsis. A research project led by Dr. Yuki Nakamura revealed that two isoforms of PMT, PMT1 and PMT3, redundantly play an essential role in PCho biosynthesis in Arabidopsis. Double mutation of PMT1 and PMT3 contained trace amount of PCho and affected PtdCho biosynthesis in vivo, showing severe growth defect due to defective leaf vein patterning. This study highlights the importance of PMT1 and PMT3 in PCho biosynthesis, which are required for PtdCho biosynthesis, and possible roles of these metabolites in vascular development in Arabidopsis.