Differential degradation of RNA species by autophagy-related pathways in Arabidopsis.

The plant vacuole recycles proteins and RNA delivered to it by autophagy. In this study, by isolating intact vacuoles from Arabidopsis plants, followed by subsequent RNA purification, and deep sequencing, we provide a comprehensive characterization of Arabidopsis vacuolar RNAome. In the vacuolar RNAome, we detected ribosomal RNAs, transfer RNAs, including those of chloroplast origin, and in addition small RNA types. As autophagy is a main mechanism for the transport of RNA to the vacuole, atg5-1 mutants deficient in autophagy were included in our analysis. We observed severely reduced amounts of most chloroplast-derived RNA species in these mutants. Comparisons with cellular RNA composition provided an indication of possible up-regulation of alternative RNA breakdown pathways. By contrast, vacuolar RNA processing and composition in plants lacking vacuolar ribonuclease 2, involved in cellular RNA homeostasis, only showed minor alterations, possibly because of the presence of further so far unknown vacuolar RNase species. Among the small RNA types, we detected mature miRNAs in all vacuolar preparations but at much lower frequency in atg5-1, raising the possibility of a biological role for vacuolar miRNAs.

Characterization of filament-forming CTP synthases from Arabidopsis thaliana.

Cytidine triphosphate (CTP) is essential for DNA, RNA and phospholipid biosynthesis. De novo synthesis is catalyzed by CTP synthases (CTPS). Arabidopsis encodes five CTPS isoforms that unanimously share conserved motifs found across kingdoms, suggesting all five are functional enzymes. Whereas CTPS1-4 are expressed throughout Arabidopsis tissues, CTPS5 reveals exclusive expression in developing embryos. CTPS activity and substrates affinities were determined for a representative plant enzyme on purified recombinant CTPS3 protein. As demonstrated in model organisms such as yeast, fruit fly and mammals, CTPS show the capacity to assemble into large filaments called cytoophidia. Transient expression of N- and C-terminal YFP-CTPS fusion proteins in Nicotiana benthamiana allowed to monitor such filament formation. Interestingly, CTPS1 and 2 always appeared as soluble proteins, whereas filaments were observed for CTPS3, 4 and 5 independent of the YFP-tag location. However, when similar constructs were expressed in Saccharomyces cerevisiae, no filaments were observed, pointing to a requirement for organism-specific factors in vivo. Indications for filament assembly were also obtained in vitro when recombinant CTPS3 protein was incubated in the presence of CTP. T-DNA-insertion mutants in four CTPS loci revealed no apparent phenotypical alteration. In contrast, CTPS2 T-DNA-insertion mutants did not produce homozygous progenies. An initial characterization of the CTPS protein family members from Arabidopsis is presented. We provide evidence for their involvement in nucleotide de novo synthesis and show that only three of the five CTPS isoforms were able to form filamentous structures in the transient tobacco expression system. This represents a striking difference from previous observations in prokaryotes, yeast, Drosophila and mammalian cells. This finding will be highly valuable to further understand the role of filament formation to regulate CTPS activity.

Cytidine Triphosphate Synthase Four From Arabidopsis thaliana Attenuates Drought Stress Effects.

Cytidine triphosphate synthase (CTPS) catalyzes the final step in pyrimidine de novo synthesis. In Arabidopsis, this protein family consists of five members (CTPS1-5), and all of them localize to the cytosol. Specifically, CTPS4 showed a massive upregulation of transcript levels during abiotic stress, in line with increased staining of CTPS4 promoter:GUS lines in hypocotyl, root and to lesser extend leaf tissues. In a setup to study progressive drought stress, CTPS4 knockout mutants accumulated less fresh and dry weight at days 5-7 and showed impaired ability to recover from this stress after 3 days of rewatering. Surprisingly, a thorough physiological characterization of corresponding plants only revealed alterations in assimilation and accumulation of soluble sugars including those related to drought stress in the mutant. Bimolecular fluorescence complementation (BiFC) studies indicated the interaction of CTPS4 with other isoforms, possibly affecting cytoophidia (filaments formed by CTPS formation. Although the function of these structures has not been thoroughly investigated in plants, altered enzyme activity and effects on cell structure are reported in other organisms. CTPS activity is required for cell cycle progression and growth. Furthermore, drought can lead to the accumulation of reactive oxygen species (ROS) and by this, to DNA damage. We hypothesize that effects on the cell cycle or DNA repair might be relevant for the observed impaired reduced drought stress tolerance of CTPS4 mutants.

CTP Synthase 2 From Arabidopsis thaliana Is Required for Complete Embryo Development.

Pyrimidine de novo synthesis is an essential pathway in all organisms. The final and rate-limiting step in the synthesis of the nucleotide cytidine triphosphate (CTP) is catalyzed by CTP synthase (CTPS), and Arabidopsis harbors five isoforms. Single mutant lines defective in each one of the four isoforms do not show apparent phenotypical alterations in comparison to wild-type plants. However, Arabidopsis lines that contain T-DNA insertions in the CTPS2 gene were unable to produce homozygous offspring. Here, we show that CTPS2 exhibits a distinct expression pattern throughout embryo development, and loss-of-function mutants are embryo lethal, as siliques from +/ctps2 plants contained nearly 25% aborted seeds. This phenotype was rescued by complementation with CTPS2 under control of its endogenous promoter. CTPS2::GFP lines revealed expression only in the tip of columella cells in embryo root tips of the heart and later stages. Furthermore, CTPS2 expression in mature roots, most pronounced in the columella cells, shoots, and vasculature tissue of young seedlings, was observed. Filial generations of +/ctps2 plants did not germinate properly, even under external cytidine supply. During embryo development, the CTPS2 expression pattern resembled the established auxin reporter DR5::GFP. Indeed, the cloned promoter region we used in this study possesses a repeat of an auxin response element, and auxin supply increased CTPS2 expression in a cell-type-specific manner. Thus, we conclude that CTPS2 is essential for CTP supply in developing embryos, and loss-of-function mutants in CTPS2 are embryo lethal.

Cytosolic CTP Production Limits the Establishment of Photosynthesis in Arabidopsis.

CTP synthases (CTPS) comprise a protein family of the five members CTPS1-CTPS5 in Arabidopsis, all located in the cytosol. Specifically, downregulation of CTPS2 by amiRNA technology results in plants with defects in chlorophyll accumulation and photosynthetic performance early in development. CTP and its deoxy form dCTP are present at low levels in developing seedlings. Thus, under conditions of fast proliferation, the synthesis of CTP (dCTP) can become a limiting factor for RNA and DNA synthesis. The higher sensitivity of ami-CTPS2 lines toward the DNA-Gyrase inhibitor ciprofloxacin, together with reduced plastid DNA copy number and 16S and 23S chloroplast ribosomal RNA support this view. High expression and proposed beneficial biochemical features render CTPS2 the most important isoform for early seedling development. In addition, CTPS2 was identified as an essential enzyme in embryo development before, as knock-out mutants were embryo lethal. In line with this, ami-CTPS2 lines also exhibited reduced seed numbers per plant.