Regulation of leaf organ size by the Arabidopsis RPT2a 19S proteasome subunit.

The ubiquitin/26S proteasome pathway plays a central role in the degradation of short-lived regulatory proteins, to control many cellular events. To further understand this pathway, we focused on the RPT2 subunit of the 26S proteasome regulatory particle. The Arabidopsis genome contains two genes, AtRPT2a and AtRPT2b, which encode paralog molecules of the RPT2 subunit, with a difference of only three amino acids in the protein sequences. Both genes showed similar mRNA accumulation patterns. However, the rpt2a mutant showed a specific phenotype of enlarged leaves caused by increased cell size, in correlation with increased ploidy. Detailed analyses revealed that cell expansion is increased in the rpt2a mutant by extended endoreduplication early in leaf development. The transcription of genes encoding cell cycle-related components, for DNA replication licensing and the G2/M phase, was also promoted in the rpt2a mutant, suggesting that extended endoreduplication was caused by increased DNA replication, and disrupted regulation of the G2/M checkpoint, at the proliferation stage of leaf development.

CNI1/ATL31, a RING-type ubiquitin ligase that functions in the carbon/nitrogen response for growth phase transition in Arabidopsis seedlings.

Plants are able to sense and respond to changes in the balance between carbon (C) and nitrogen (N) metabolite availability, known as the C/N response. During the transition to photoautotrophic growth following germination, growth of seedlings is arrested if a high external C/N ratio is detected. To clarify the mechanisms for C/N sensing and signaling during this transition period, we screened a large collection of FOX transgenic plants, overexpressing full-length cDNAs, for individuals able to continue post-germinative growth under severe C/N stress. One line, cni1-D (carbon/nitrogen insensitive 1-dominant), was shown to have a suppressed sensitivity to C/N conditions at both the physiological and molecular level. The CNI1 cDNA encoded a predicted RING-type ubiquitin ligase previously annotated as ATL31. Overexpression of ATL31 was confirmed to be responsible for the cni1-D phenotype, and a knock-out of this gene resulted in hypersensitivity to C/N conditions during post-germinative growth. The ATL31 protein was confirmed to contain ubiquitin ligase activity using an in vitro assay system. Moreover, removal of this ubiquitin ligase activity from the overexpressed protein resulted in the loss of the mutant phenotype. Taken together, these data demonstrated that CNI1/ATL31 activity is required for the plant C/N response during seedling growth transition.

DEAR1, a transcriptional repressor of DREB protein that mediates plant defense and freezing stress responses in Arabidopsis.

Plants have evolved intricate mechanisms to respond and adapt to a wide variety of biotic and abiotic stresses in their environment. The Arabidopsis DEAR1 (DREB and EAR motif protein 1; At3g50260) gene encodes a protein containing significant homology to the DREB1/CBF (dehydration-responsive element binding protein 1/C-repeat binding factor) domain and the EAR (ethylene response factor-associated amphiphilic repression) motif. We show here that DEAR1 mRNA accumulates in response to both pathogen infection and cold treatment. Transgenic Arabidopsis overexpressing DEAR1 (DEAR1ox) showed a dwarf phenotype and lesion-like cell death, together with constitutive expression of PR genes and accumulation of salicylic acid. DEAR1ox also showed more limited P. syringae pathogen growth compared to wild-type, consistent with an activated defense phenotype. In addition, transient expression experiments revealed that the DEAR1 protein represses DRE/CRT (dehydration-responsive element/C-repeat)-dependent transcription, which is regulated by low temperature. Furthermore, the induction of DREB1/CBF family genes by cold treatment was suppressed in DEAR1ox, leading to a reduction in freezing tolerance. These results suggest that DEAR1 has an upstream regulatory role in mediating crosstalk between signaling pathways for biotic and abiotic stress responses.

Interactome analysis reveals versatile functions of Arabidopsis COLD SHOCK DOMAIN PROTEIN 3 in RNA processing within the nucleus and cytoplasm.

Arabidopsis COLD SHOCK DOMAIN PROTEIN 3 (AtCSP3) shares an RNA chaperone function with E. coli cold shock proteins and regulates freezing tolerance during cold acclimation. Here, we screened for AtCSP3-interacting proteins using a yeast two-hybrid system and 38 candidate interactors were identified. Sixteen of these were further confirmed in planta interaction between AtCSP3 by a bi-molecular fluorescence complementation assay. We found that AtCSP3 interacts with CONSTANS-LIKE protein 15 and nuclear poly(A)-binding proteins in nuclear speckles. Three 60S ribosomal proteins (RPL26A, RPL40A/UBQ2, and RPL36aB) and the Gar1 RNA-binding protein interacted with AtCSP3 in the nucleolus and nucleoplasm, suggesting that AtCSP3 functions in ribosome biogenesis. Interactions with LOS2/enolase and glycine-rich RNA-binding protein 7 that are cold inducible, and an mRNA decapping protein 5 (DCP5) were observed in the cytoplasm. These data suggest that AtCSP3 participates in multiple complexes that reside in nuclear and cytoplasmic compartments and possibly regulates RNA processing and functioning.

SHA1, a novel RING finger protein, functions in shoot apical meristem maintenance in Arabidopsis.

Post-embryonic plant growth is dependent on a functional shoot apical meristem (SAM) that provides cells for continuous development of new aerial organs. However, how the SAM is dynamically maintained during vegetative development remains largely unclear. We report here the characterization of a new SAM maintenance mutant, sha1-1 (shoot apical meristem arrest 1-1), that shows a primary SAM-deficient phenotype at the adult stage. The SHA1 gene encodes a novel RING finger protein, and is expressed most intensely in the shoot apex. We show that, in the sha1-1 mutant, the primary SAM develops normally during the juvenile vegetative stage, but cell layer structure becomes disorganized after entering the adult vegetative stage, resulting in a dysfunctional SAM that cannot initiate floral primordia. The sha1-1 SAM terminates completely at the stage when the wild-type begins to bolt, producing adult plants with a primary inflorescence-deficient phenotype. These observations indicate that SHA1, a putative E3 ligase, is required for post-embryonic SAM maintenance by controlling proper cellular organization.

Feedback regulation of the ammonium transporter gene family AMT1 by glutamine in rice.

The three members of the rice OsAMT1 gene family of ammonium transporters show distinct expression patterns; constitutive and ammonium-promoted expression in shoots and roots for OsAMT1;1; root-specific and ammonium-inducible expression for OsAMT1;2; root-specific and nitrogen-repressible expression for OsAMT1;3 [Sonoda et al. (2003), Plant Cell Physiol. 44: 726]. To clarify the feedback mechanisms, and to identify regulatory factors of the OsAMT1 genes, the accumulation of the three mRNAs and its dependence on endogenous nitrogen compounds (as quantified by capillary electrophoresis) was studied. Ammonium application to roots following a period of nitrogen starvation induced accumulation of OsAMT1;1 and OsAMT1;2 mRNA, but a decrease of OsAMT1;3 mRNA levels. The expression patterns of the three genes showed good correlation (positive in OsAMT1;1 and OsAMT1;2, negative in OsAMT1;3) with the root tissue contents of glutamine but not of ammonium. The ammonium effects on OsAMT1 expression were prevented by methionine sulfoximine, an inhibitor of glutamine synthetase. Moreover, glutamine had the same effect on transcriptional regulation of OsAMT1 genes as ammonium, indicating that glutamine rather than ammonium controls the expression of ammonium transporter genes in rice. These results imply that rice possesses unique mechanisms of adaptation to variable nitrogen sources in the soil.

Distinct expression and function of three ammonium transporter genes (OsAMT1;1-1;3) in rice.

To study the regulation of ammonium uptake into rice roots, three ammonium transporter genes (OsAMT1;1, 1;2 and 1;3; Oryza sativa ammonium transporter) were isolated and examined. OsAMT1s belong to AMT1 family, containing 11 putative transmembrane-spanning domains. Southern blot analysis and screening of the rice genome database confirmed that with OsAMT1;1-1;3 the complete AMT1 family of rice had been isolated. Heterologous expression of OsAMT1s in the yeast Saccharomyces cerevisiae mutant 31019b showed that all three OsAMT1s exhibit ammonium transport activity. Northern blot analysis showed a distinct expression pattern for the three genes; more constitutive expression in shoots and roots for OsAMT1;1, root-specific and ammonium-inducible expression for OsAMT1;2, and root-specific and nitrogen-derepressible expression for OsAMT1;3. In situ mRNA detection revealed that OsAMT1;2 is expressed in the central cylinder and cell surface of root tips. This gene expression analysis revealed a distinct nitrogen-dependent regulation for AMTs in rice, differing from that in tomato or Arabidopsis:

The slender rice mutant, with constitutively activated gibberellin signal transduction, has enhanced capacity for abscisic acid level.

The slender rice (slr1-1) mutant, carrying a lethal and recessive single mutation, has a constitutive gibberellin (GA)-response phenotype and behaves as if it were saturated with GAs [Ikeda et al. (2001) Plant Cell 13, 999]. The SLR1 gene, with sequence homology to members of the plant-specific GRAS gene family, is a mediator of the GA signal transduction process. In the slender rice, GA-inducible alpha-amylase was produced from the aleurone layer without applying GA. GA-independent alpha-amylase production in the mutant was inhibited by applying abscisic acid (ABA). Shoot elongation in the mutant was also suppressed by ABA, indicating that the slender rice responds normally to ABA. Interestingly, shoot ABA content was 10-fold higher in the mutant than in the wild type, while there was no difference in root ABA content. Expression of the Rab16A gene, which is known to be ABA inducible, was about 10-fold higher in shoots of the mutant than in those of the wild type. These results indicate that constitutive activation of the GA signal transduction pathway by the slr1-1 mutation promotes the endogenous ABA level.

Constitutive expression of a novel-type ammonium transporter OsAMT2 in rice plants.

To characterize ammonium transport pathways in rice, two cDNAs with high homology to MEP/AMT2-type ammonium transporters, OsAMT2;1 and OsAMT3;1, were isolated. Expression of OsAMT2;1 in an ammonium-uptake-defective yeast mutant showed that this gene encodes functional ammonium transporters. OsAMT2;1 was constitutively expressed in both roots and shoots irrespective of the supply of inorganic nitrogen to the medium, whereas OsAMT3;1 expression was relatively weak. A database search with the amino acid sequence of OsAMT2;1 showed that there are 10 putative OsAMT genes in rice, i.e. three each for OsAMT1, OsAMT2 and OsAMT3, respectively, and one for OsAMT4.