Arabidopsis ASYMMETRIC LEAVES2 and Nucleolar Factors Are Coordinately Involved in the Perinucleolar Patterning of AS2 Bodies and Leaf Development.

Arabidopsis ASYMMETRIC LEAVES2 (AS2) plays a key role in the formation of flat symmetric leaves. AS2 represses the expression of the abaxial gene ETTIN/AUXIN RESPONSE FACTOR3 (ETT/ARF3). AS2 interacts in vitro with the CGCCGC sequence in ETT/ARF3 exon 1. In cells of leaf primordia, AS2 localizes at peripheral regions of the nucleolus as two AS2 bodies, which are partially overlapped with chromocenters that contain condensed 45S ribosomal DNA repeats. AS2 contains the AS2/LOB domain, which consists of three sequences conserved in the AS2/LOB family: the zinc finger (ZF) motif, the ICG sequence including the conserved glycine residue, and the LZL motif. AS2 and the genes NUCLEOLIN1 (NUC1), RNA HELICASE10 (RH10), and ROOT INITIATION DEFECTIVE2 (RID2) that encode nucleolar proteins coordinately act as repressors against the expression of ETT/ARF3. Here, we examined the formation and patterning of AS2 bodies made from as2 mutants with amino acid substitutions in the ZF motif and the ICG sequence in cells of cotyledons and leaf primordia. Our results showed that the amino acid residues next to the cysteine residues in the ZF motif were essential for both the formation of AS2 bodies and the interaction with ETT/ARF3 DNA. The conserved glycine residue in the ICG sequence was required for the formation of AS2 bodies, but not for the DNA interaction. We also examined the effects of nuc1, rh10, and rid2 mutations, which alter the metabolism of rRNA intermediates and the morphology of the nucleolus, and showed that more than two AS2 bodies were observed in the nucleolus and at its periphery. These results suggested that the patterning of AS2 bodies is tightly linked to the morphology and functions of the nucleolus and the development of flat symmetric leaves in plants.

Upstream open reading frame-mediated upregulation of ANAC082 expression in response to nucleolar stress in Arabidopsis.

Perturbations in ribosome biogenesis cause a type of cellular stress called nucleolar or ribosomal stress, which triggers adaptive responses in both animal and plant cells. The Arabidopsis ANAC082 transcription factor has been identified as a key mediator of the plant nucleolar stress response. The 5'-untranslated region (5'-UTR) of ANAC082 mRNA contains an upstream ORF (uORF) encoding an evolutionarily conserved amino acid sequence. Here, we report that this uORF mediates the upregulation of ANAC082 expression in response to nucleolar stress. When transgenic Arabidopsis plants containing a luciferase reporter gene under the control of the ANAC082 promoter and 5'-UTR were treated with reagents that induced nucleolar stress, expression of the reporter gene was enhanced in a uORF sequence-dependent manner. Additionally, we examined the effect of an endoplasmic reticulum (ER) stress-inducing reagent on reporter gene expression because the closest homolog of ANAC082 in Arabidopsis, ANAC103, is involved in the ER stress response. However, the ANAC082 uORF did not respond to ER stress. Interestingly, although ANAC103 has a uORF with an amino acid sequence similar to that of the ANAC082 uORF, the C-terminal sequence critical for regulation is not well conserved among ANAC103 homologs in Brassicaceae. Transient expression assays revealed that unlike the ANAC082 uORF, the ANAC103 uORF does not exert a sequence-dependent repressive effect. Altogether, our findings suggest that the ANAC082 uORF is important for the nucleolar stress response but not for the ER stress response, and that for this reason, the uORF sequence-dependent regulation was lost in ANAC103 during evolution.

Enhancement of shoot regeneration by treatment with inhibitors of auxin biosynthesis and transport during callus induction in tissue culture of Arabidopsis thaliana.

In two-step culture systems for efficient shoot regeneration, explants are first cultured on auxin-rich callus-inducing medium (CIM), where cells are activated to proliferate and form calli containing root-apical meristem (RAM)-type stem cells and stem cell niche, and then cultured on cytokinin-rich shoot-inducing medium (SIM), where stem cells and stem cell niche of the shoot apical meristem (SAM) are established eventually leading to shoot regeneration. In the present study, we examined the effects of inhibitors of auxin biosynthesis and polar transport in the two-step shoot regeneration culture of Arabidopsis and found that, when they were applied during CIM culture, although callus growth was repressed, shoot regeneration in the subsequent SIM culture was significantly increased. The regeneration-stimulating effect of the auxin biosynthesis inhibitor was not linked with the reduction in the endogenous indole-3-acetic acid (IAA) level. Expression of the auxin-responsive reporter indicated that auxin response was more uniform and even stronger in the explants cultured on CIM with the inhibitors than in the control explants. These results suggested that the shoot regeneration competence of calli was enhanced somehow by the perturbation of the endogenous auxin dynamics, which we discuss in terms of the transformability between RAM and SAM stem cell niches.

Mitochondrial Pyruvate Dehydrogenase Contributes to Auxin-Regulated Organ Development.

Pyruvate dehydrogenase is the first enzyme (E1) of the PDH complex (PDC). This multienzyme complex contains E1, E2, and E3 components and controls the entry of carbon into the mitochondrial tricarboxylic acid cycle to enable cellular energy production. The E1 component of the PDC is composed of an E1α catalytic subunit and an E1ß regulatory subunit. In Arabidopsis (Arabidopsis thaliana), there are two mitochondrial E1α homologs encoded by IAA-CONJUGATE-RESISTANT 4 (IAR4) and IAR4-LIKE (IAR4L), and one mitochondrial E1ß homolog. Although IAR4 was reported to be involved in auxin conjugate sensitivity and auxin homeostasis in root development, its precise role remains unknown. Here, we provide experimental evidence that mitochondrial PDC E1 contributes to polar auxin transport during organ development. We performed genetic screens for factors involved in cotyledon development and identified an uncharacterized mutant, macchi-bou 1 (mab1). MAB1 encodes a mitochondrial PDC E1ß subunit that can form both a homodimer and a heterodimer with IAR4. The mab1 mutation impaired MAB1 homodimerization, reduced the abundance of IAR4 and IAR4L, weakened PDC enzymatic activity, and diminished mitochondrial respiration. A metabolomics analysis showed significant changes in metabolites including amino acids in mab1 and, in particular, identified an accumulation of Ala. These results suggest that MAB1 is a component of the Arabidopsis mitochondrial PDC E1. Furthermore, in mab1 mutants and seedlings where the TCA cycle was pharmacologically blocked, we found reduced abundance of the PIN-FORMED (PIN) auxin efflux carriers, possibly due to impaired PIN recycling and enhanced PIN degradation in vacuoles. Therefore, we suggest that mab1 induces defective polar auxin transport via metabolic abnormalities.

Evidence for a Role of ANAC082 as a Ribosomal Stress Response Mediator Leading to Growth Defects and Developmental Alterations in Arabidopsis.

Ribosome-related mutants in Arabidopsis thaliana share several notable characteristics regarding growth and development, which implies the existence of a common pathway that responds to disorders in ribosome biogenesis. As a first step to explore this pathway genetically, we screened a mutagenized population of root initiation defective2 (rid2), a temperature-sensitive mutant that is impaired in pre-rRNA processing, and isolated suppressor of root initiation defective two1 (sriw1), a suppressor mutant in which the defects of cell proliferation observed in rid2 at the restrictive temperature was markedly rescued. sriw1 was identified as a missense mutation of the NAC transcription factor gene ANAC082 The sriw1 mutation greatly alleviated the developmental abnormalities of rid2 and four other tested ribosome-related mutants, including rid3 However, the impaired pre-rRNA processing in rid2 and rid3 was not relieved by sriw1 Expression of ANAC082 was localized to regions where phenotypic effects of ribosome-related mutations are readily evident and was elevated in rid2 and rid3 compared with the wild type. These findings suggest that ANAC082 acts downstream of perturbation of biogenesis of the ribosome and may mediate a set of stress responses leading to developmental alterations and cell proliferation defects.

Plant Nucleolar Stress Response, a New Face in the NAC-Dependent Cellular Stress Responses.

The nucleolus is the most prominent nuclear domain, where the core processes of ribosome biogenesis occur vigorously. All these processes are finely orchestrated by many nucleolar factors to build precisely ribosome particles. In animal cells, perturbations of ribosome biogenesis, mostly accompanied by structural disorders of the nucleolus, cause a kind of cellular stress to induce cell cycle arrest, senescence, or apoptosis, which is called nucleolar stress response. The best-characterized pathway of this stress response involves p53 and MDM2 as key players. p53 is a crucial transcription factor that functions in response to not only nucleolar stress but also other cellular stresses such as DNA damage stress. These cellular stresses release p53 from the inhibition by MDM2, an E3 ubiquitin ligase targeting p53, in various ways, which leads to p53-dependent activation of a set of genes. In plants, genetic impairments of ribosome biogenesis factors or ribosome components have been shown to cause characteristic phenotypes, including a narrow and pointed leaf shape, implying a common signaling pathway connecting ribosomal perturbations and certain aspects of growth and development. Unlike animals, however, plants have neither p53 nor MDM2 family proteins. Then the question arises whether plant cells have a nucleolar stress response pathway. In recent years, it has been reported that several members of the plant-specific transcription factor family NAC play critical roles in the pathways responsive to various cellular stresses. In this mini review, we outline the plant cellular stress response pathways involving NAC transcription factors with reference to the p53-MDM2-dependent pathways of animal cells, and discuss the possible involvement of a plant-unique, NAC-mediated pathway in the nucleolar stress response in plants.

A genetic link between epigenetic repressor AS1-AS2 and a putative small subunit processome in leaf polarity establishment of Arabidopsis.

Although the DEAD-box RNA helicase family is ubiquitous in eukaryotes, its developmental role remains unelucidated. Here, we report that cooperative action between the Arabidopsis nucleolar protein RH10, an ortholog of human DEAD-box RNA helicase DDX47, and the epigenetic repressor complex of ASYMMETRIC-LEAVES1 (AS1) and AS2 (AS1-AS2) is critical to repress abaxial (ventral) genes ETT/ARF3 and ARF4, which leads to adaxial (dorsal) development in leaf primordia at shoot apices. Double mutations of rh10-1 and as2 (or as1) synergistically up-regulated the abaxial genes, which generated abaxialized filamentous leaves with loss of the adaxial domain. DDX47 is part of the small subunit processome (SSUP) that mediates rRNA biogenesis. In rh10-1 we found various defects in SSUP-related events, such as: accumulation of 35S/33S rRNA precursors; reduction in the 18S/25S ratio; and nucleolar hypertrophy. Double mutants of as2 with mutations of genes that encode other candidate SSUP-related components such as nucleolin and putative rRNA methyltransferase exhibited similar synergistic defects caused by up-regulation of ETT/ARF3 and ARF4 These results suggest a tight link between putative SSUP and AS1-AS2 in repression of the abaxial-determining genes for cell fate decisions for adaxial development.

The conflict between cell proliferation and expansion primarily affects stem organogenesis in Arabidopsis.

Plant shoot organs such as stems, leaves and flowers are derived from specialized groups of stem cells organized at the shoot apical meristem (SAM). Organogenesis involves two major processes, namely cell proliferation and differentiation, whereby the former contributes to increasing the cell number and the latter involves substantial increases in cell volume through cell expansion. Co-ordination between the above processes in time and space is essential for proper organogenesis. To identify regulatory factors involved in proper organogenesis, heavy-ion beam-irradiated de-etiolated (det) 3-1 seeds have been used to identify striking phenotypes in the A#26-2; det3-1 mutant. In addition to the stunted plant stature mimicking det3-1, the A#26-2; det3-1 mutant exhibited stem thickening, increased floral organ number and a fruit shape reminiscent of clavata (clv) mutants. DNA sequencing analysis demonstrated that A#26-2; det3-1 harbors a mutation in the CLV3 gene. Importantly, A#26-2; det3-1 displayed cracks that randomly occurred on the main stem with a frequency of approximately 50%. Furthermore, the double mutants clv3-8 det3-1, clv1-4 det3-1 and clv2-1 det3-1 consistently showed stem cracks with frequencies of approximately 97, 38 and 35%, respectively. Cross-sections of stems further revealed an increase in vascular bundle number, cell number and size in the pith of clv3-8 det3-1 compared with det3-1. These findings suggest that the stem inner volume increase due to clv mutations exerts an outward mechanical stress; that in a det3-1 background (defective in cell expansion) resulted in cracking of the outermost layer of epidermal cells.

Involvement of rRNA biosynthesis in the regulation of CUC1 gene expression and pre-meristematic cell mound formation during shoot regeneration.

At an early stage of shoot regeneration from calli of Arabidopsis, pre-meristematic cell mounds develop in association with localized strong expression of CUP-SHAPED COTYLEDON (CUC) genes. Previous characterization of root initiation-defective 3 (rid3), an Arabidopsis mutant originally isolated as being temperature-sensitive for adventitious root formation, with respect to shoot regeneration implicated RID3 in the negative regulation of CUC1 expression and the restriction of cell division in pre-meristematic cell mounds. Positional cloning has identified RID3 as a WD40 repeat protein gene whose molecular function was not investigated before. Here we performed in silico analysis of RID3 and found that RID3 is orthologous to IPI3, which mediates pre-rRNA processing in Saccharomyces cerevisiae. In the rid3 mutant, rRNA precursors accumulated to a very high level in a temperature-dependent manner. This result indicates that RID3 is required for pre-rRNA processing as is IPI3. We compared rid3 with rid2, a temperature-sensitive mutant that is mutated in a putative RNA methyltransferase gene and is impaired in pre-rRNA processing, for seedling morphology, shoot regeneration, and CUC1 expression. The rid2 and rid3 seedlings shared various developmental alterations, such as a pointed-leaf phenotype, which is often observed in ribosome-related mutants. In tissue culture for the induction of shoot regeneration, both rid2 and rid3 mutations perturbed cell-mound formation and elevated CUC1 expression. Together, our findings suggest that rRNA biosynthesis may be involved in the regulation of CUC1 gene expression and pre-meristematic cell-mound formation during shoot regeneration.

Nuclear PP2A-Cdc55 prevents APC-Cdc20 activation during the spindle assembly checkpoint.

Cdc55, a regulatory B-subunit of protein phosphatase 2A (PP2A) complex, is essential for the spindle assembly checkpoint (SAC) in budding yeast, but the regulation and molecular targets of PP2A-Cdc55 have not been clearly defined or are controversial. Here, we show that an important target of Cdc55 in the SAC is the anaphase-promoting complex (APC) coupled with Cdc20 and that APC-Cdc20 is kept inactive by dephosphorylation by nuclear PP2A-Cdc55 when spindle is damaged. By isolating a new class of Cdc55 mutants specifically defective in the SAC and by artificially manipulating nucleocytoplasmic distribution of Cdc55, we further show that nuclear Cdc55 is essential for the SAC. Because the Cdc55-binding proteins Zds1 and Zds2 inhibit both nuclear accumulation of Cdc55 and SAC activity, we propose that spatial control of PP2A by Zds1 family proteins is important for tight control of SAC and mitotic progression.

Genetic identification of Arabidopsis RID2 as an essential factor involved in pre-rRNA processing.

A temperature-sensitive mutant of Arabidopsis, root initiation defective 2-1 (rid2-1), is characterized by peculiar defects in callus formation. To gain insights into the requirements for the reactivation of cell division, we analyzed this mutant and isolated the gene responsible, RID2. The phenotypes of rid2-1 in tissue culture and in seedlings indicated that the rid2 mutation has various (acute and non-acute) inhibitory effects on different aspects of cell proliferation. This suggests that the RID2 function is not directly involved in every cycle of cell division, but is related to 'vitality', supporting cell proliferation. The rid2-1 mutation was shown to cause nucleolar vacuolation and excessive accumulation of various intermediates of pre-rRNA processing. Positional cloning of the RID2 gene revealed that it encodes an evolutionarily conserved methyltransferase-like protein, which was found to localize in the nucleus, with accumulation being most evident in the nucleolus. It can be inferred from these findings that RID2 contributes to the nucleolar activity for pre-rRNA processing, probably through some methylation reaction.