Ribosomal Stress Response in plants
Nucleolar/Ribosomal Stress and Ribosome Biogenesis
All organisms in the world survive in the environments under a lot of abiotic and biotic stresses. Because of the sessile nature, plants have to sense and adapt to the surrounding environments. In addition to UV radiation, drought, and cold stress, one of the most effective abiotic stresses is known as “Nucleolar Stress” or “Ribosomal Stress”, a stress of disturbance of ribosome biogenesis that is induced by nutrient starvation, hypoxia or some mutations.
The ribosome is a highly complicated and evolutionarily conserved ribonucleoprotein machinery that executes translation reactions. The eukaryotic ribosome comprises four types of rRNA and 79–81 types of ribosomal proteins (Lecompte et al., 2002; McIntosh and Bonham-Smith, 2006; Ben-Shem et al., 2011). One rRNA (18S rRNA) and 33 ribosomal proteins constitute a small subunit (SSU) known as the 40S ribosome, and three rRNAs (the 5S, 5.8S, and 25–28S rRNAs) and 46–48 proteins constitute a large subunit (LSU) known as the 60S ribosome. The two subunits join together on mRNAs to form the 80S ribosome, which functions as a precise mechanical device that translates mRNA codons into amino acids to synthesize proteins. The biogenesis of the ribosome is finely regulated through the orchestration of the synthesis of rRNAs and their assembly with ribosomal proteins into two ribosome subunits (Henras et al., 2015; de la Cruz et al., 2015). Three of the four rRNAs, the 18S, 5.8S, and 25–28S rRNAs, are transcribed from each of the tandem repeats of rDNA as a single precursor molecule, which contains a 5’-external spacer sequence (5’-ETS), the 18S rRNA sequence, an internal spacer sequence 1 (ITS1), the 5.8S rRNA sequence, ITS2, the 25–28S rRNA sequence, and a 3’-ETS, in this order (see Fig. 3A), in the nucleolus and become mature forms after being processed in a stepwise fashion. In contrast, the 5S rRNA is transcribed separately from the other rRNAs. Pre-rRNA processing events are tightly linked with formation of the ribosomal subunits under the action of huge ribonucleoprotein particles termed processomes (Raué, 2004).
The elaborately regulated biogenesis of ribosomes is disturbed under stress conditions for various reasons, including nutrient starvation, hypoxia, chemical treatments, genetic impairment of ribosome biogenesis factors, and deficiencies in ribosomal proteins (Mayer and Grummt, 2005; Boulon et al., 2010). In animal cells, it is well known that this perturbation of ribosome biogenesis, regardless of origin, activates a specific signaling pathway leading to cell-cycle arrest or apoptosis, which is called nucleolar stress response. The main pathway of this stress response involves the tumor suppressor p53 and its destabilizer MDM2 as essential players (Deisenroth and Zhang, 2010; Golomb et al., 2014). In brief, this pathway is triggered upon the inhibition of MDM2 function by direct binding of ribosomal proteins, such as RPL5 and RPL11, and the 5S rRNA, which are unusually released from the nucleolus as a result of perturbed ribosome biogenesis; subsequently, stabilized p53 upregulates the transcription of genes that participate in cell-cycle arrest and apoptosis (Fig. 1. Left panel). The p53–MDM2 pathway is also responsible for developmental disorders and diseases caused by impaired ribosome biogenesis or ribosomal protein deficiencies (Azuma et al., 2006; Danilova et al., 2008; McGowan et al., 2008; Deisenroth and Zhang, 2010; Sondalle and Baserga, 2014).
Regulations of Ribosome Biogenesis and Nucleolar Stress in Plants
In plants, particularly in the model plant Arabidopsis thaliana (Arabidopsis), many mutants that carry a mutation in a gene encoding a ribosomal protein or a ribosome biogenesis factor have been reported (Fig. 2; Byrne, 2009; Ohbayashi et al., 2011; Horiguchi et al., 2012; Tsukaya et al., 2013; Weis et al., 2015a). Most of these mutants share several notable phenotypes. In mild cases, characteristic effects are often observed in true leaves: a narrow, pointed shape, a pale green color, conspicuous indentations, obvious venations (see Fig. 4), and enhanced abaxialization of leaves under the asymmetric leaves 1 or 2 (as1 or as2, respectively) background (Van Lijsebettens et al., 1994; Ito et al., 2000; Petricka and Nelson, 2007; Yao et al., 2008; Fujikura et al., 2009; Horiguchi et al., 2011; Shinohara et al., 2014; Matsumura et al., 2016). In more severe cases, no leaf lamina is formed, root growth is retarded, and the expression of CUP-SHAPED COTYLEDON 1/2 (CUC1/2) and SHOOT MERISTEMLESS (STM) is uncontrolled; finally, in the most severe cases, all processes involving cell proliferation are strongly impeded (Szakonyl and Byrne, 2011; Shinohara et al., 2014).
The phenotypic similarity among various ribosome-related mutants suggests that a common signaling pathway connects any ribosomal trouble to certain points of plant growth and development, similar to the p53–MDM2-dependent nucleolar stress response pathway in animals. If such a pathway is present in plants, however, it should be quite different from the p53–MDM2 pathway because plants have neither p53 homologues nor MDM family proteins (Huart and Hupp, 2013).
root initiation defective 2 (rid2) is a temperature-sensitive mutant of Arabidopsis defective in the seedling growth at restrictive temperature (28℃) (Fig. 2A), which was originally isolated by screening with callus and adventitious root formation from hypocotyl explants in tissue culture as an index phenotype (Fig.2B, C; Konishi and Sugiyama, 2003; Ohbayashi et al., 2011). Under high temperature conditions, this mutant exhibits impairment of pre-rRNA processing because of a genetic lesion of the RNA methyltransferase-like protein, which has high sequence similarity to the budding yeast BUD23, known to participate in pre-rRNA modification and processing as an SSU processome-associated factor, and is cytologically characterized by cell proliferation defects accompanied by nucleolar enlargement (Fig. 2D~F; White et al., 2008; Ohbayashi et al., 2011; Sardana et al., 2013). As a first step to address genetically the hypothetical pathway of the plant-unique nucleolar stress response, we used this rid2 mutation as a nucleolar stress factor and screened for a rid2 suppressor mutant. We isolated only one suppressor mutant named suppressor of rid two 1 (sriw1) and identified a mutation in the gene encoding ANAC082, a NAC family transcription factor, as the cause of the sriw1 phenotype (Ohbayashi et al, 2017). A phenotypic comparison between the sriw1 rid2 double mutant and the rid2 single mutant indicated that all the visible defects that were characteristic of rid2 were greatly attenuated. In seedlings incubated at restriction temperature, 28°C, primary root growth and leaf development, which were very poor in rid2, were considerably improved in sriw1 rid2 (Fig. 2A). In tissue cultures carried out at 28°C, both adventitious rooting and callus formation were strongly inhibited in hypocotyl explants of rid2, but they occurred well in the sriw1 rid2 explants (Fig. 2B, C). Stele cells of hypocotyl explants of rid2 cultured on callus-inducing medium (CIM) at 28°C failed to resume cell division and underwent irregular expansion accompanied by extraordinary enlargement of the nucleolus, whereas hypocotyl stele cells of sriw1 rid2 actively divided to form callus under the same condition, and the nucleolar enlargement was much smaller in sriw1 rid2 vs. rid2 (Fig. 2D~F).
The sriw1 mutation did not affect pre-rRNA processing in the rid2 mutant
The rid2 mutation interfered with many steps of pre-rRNA processing in a temperature-dependent manner, which was reflected in the unusual accumulation of many kinds of pre-rRNA processing intermediates in rid2 (Fig. 3B). We tested if the accumulation of the intermediates is restored when sriw1, the suppressor mutation, is introduced to the rid2 mutant. RNA gel blot analysis showed that the accumulation levels and patterns of the processing intermediates were not very different between the sriw1 rid2 double mutant and the rid2 single mutant (Fig. 3B), which revealed that the suppression of the visible phenotype of rid2 by the sriw1 mutation is not tied to the recovery of rRNA biosynthesis.
Effects of the sriw1 mutation in seedlings of other ribosome-related mutants
In Arabidopsis, in addition to rid2, rid3 and rh10 are also available as temperature-sensitive, rRNA biosynthesis-deficient mutations. rid3 and rh10 are missense mutations of genes encoding a WD40 repeat protein and an RNA helicase-like protein, respectively, which primarily affect different aspects of pre-rRNA processing. rid3 and rh10 were employed as tester mutants. The strong defects of primary root growth and leaf development observed in the rid3 and rh10 seedlings were markedly reduced by the introduction of the sriw1 mutation. The levels of the pre-rRNA intermediates in the sriw1 single mutant were stably low and equivalent to those of the wild type. Aberrantly increased accumulation of the intermediates was detected similarly in rid3 and sriw1 rid3 at 28°C (Ohbayashi et al., 2017). It was thus shown that the recovery of the seedling morphology of rid3 was achieved by the sriw1 mutation, without recovery of pre-rRNA processing.
Leaf development is highly susceptible to various ribosome-related mutations, including ribosomal protein gene mutations, and characteristic effects of these mutations can be easily detected in leaf morphology. In an attempt to further investigate the effectiveness of sriw1, oligocellula 5 (oli5) and rpl4d which carries a mutation in a gene that encodes the ribosomal protein RPL5A and RPL4D, respectively, were added to tester mutants, and leaf morphology was examined for single tester mutants and double mutants carrying tester and sriw1 mutations (Fig. 4). When grown at 22°C, seedlings of the rid2, rid3, oli5, and rh10 single mutants formed pointed and slightly to moderately narrow true leaves with a little simpler and more obvious venation compared with the wild type. At 28°C, leaves of the temperature-sensitive mutants rid2, rid3, and rh10 were much narrower in shape and venations were much simpler. These phenotypes were clearly suppressed in every mutant by the introduction of the sriw1 mutation, indicating that sriw1 is effective as a general suppressor of developmental disorders in a broad range of ribosome-related mutants.
Hyposensitivity of Callus Formation of the sriw1 Mutant to Agents That Interfere with Ribosome Biogenesis or Ribosomal Function
A variety of agents, such as antibiotics and synthetic substrate analogs, interfere with ribosome biogenesis or ribosomal function. The sriw1 single mutant was compared with the wild type regarding sensitivity of callus formation to these agents. The first group of agents used in this experiment included actinomycin D, 5-fluorouracil, and camptothecin, which act on different targets to prevent rRNA biosynthesis and are known to trigger a nucleolar stress response in animal cells. As the second group of agents, cycloheximide and puromycin, which inhibit translation via different mechanisms, were employed. The results showed that sriw1 was somewhat less sensitive to all of these agents, regardless of group, with respect to callus formation than the wild type, i.e., the sriw1 mutation relieved the repression of cell proliferation caused by the pharmaceutical perturbation of ribosomes.
Expression Patterns of ANAC082 as Influenced by the rid2 and rid3 Mutations
The spatial patterns of ANAC082 expression were analyzed using ANAC082p::GUS as a reporter gene. In young wild-type seedlings, a relatively strong GUS activity was detected in the shoot apical region, particularly around the vascular or provascular strands of developing leaves and leaf primordia, and modest activity was observed in the root apical meristem (Fig. 5A). In the rid2 mutant, GUS activity in these regions was markedly elevated (Fig. 5A), which indicated that the expression of ANAC082 is increased by the rid2 mutation. The expression level of ANAC082 was also examined using quantitative reverse transcription polymerase chain reaction (qRT–PCR) analysis of rid2 and rid3 (Fig. 5B), which showed an enhancement of ANAC082 expression in both rid2 and rid3. Consequently, ANAC082 expression proved to be stimulated in response to the impairment of rRNA biosynthesis.
All these data stated above imply that ANAC082 is a key mediator which responds to nucleolar stress signal and regulate the downstream developmental processes (i.e. repression of cell proliferation) thorough the upregulation of the expression level.