CCR4/CAF1 are widely conserved deadenylases in eukaryotes. They form a large complex that includes NOT1 as a scaffold protein and various NOT proteins that are core components of multiple levels of gene expression control. The CCR4-NOT complex also contains several RNA-binding proteins as accessory proteins, which are required for target recognition by CCR4/CAF1 deadenylases. AtCCR4a/b, orthologs of human CCR4 in Arabidopsis, have various physiological effects. AtCCR4 isoforms are likely to have specific target mRNAs related to each physiological effect; however, AtCCR4 does not have RNA-binding capability. Therefore, identifying factors that interact with AtCCR4a/b is indispensable to understand its function as a regulator of gene expression, as well as the target mRNA recognition mechanism. Here, we identified putative components of the AtCCR4-NOT complex using co-immunoprecipitation in combination with mass spectrometry using FLAG-tagged AtCCR4b and subsequent verification with a yeast two-hybrid assay. Interestingly, four of 11 AtCAF1 isoforms interacted with both AtCCR4b and AtNOT1, whereas two isoforms interacted only with AtNOT1 in yeast two-hybrid assays. These results imply that Arabidopsis has multiple CCR4-NOT complexes with various combinations of deadenylases. We also revealed that the RNA-binding protein Arabidopsis Pumilio 5 and 2 interacted with AtCCR4a/b in the cytoplasm with a few foci.

Removing the poly(A) tail is the first and rate-limiting step of mRNA degradation and apparently an effective step not only for modulating mRNA stability but also for translation of many eukaryotic transcripts. Carbon catabolite repressor 4 (CCR4) has been identified as a major cytoplasmic deadenylase in Saccharomyces cerevisiae. The Arabidopsis thaliana homologs of the yeast CCR4, AtCCR4a and AtCCR4b, were identified by sequence-based analysis; however, their role and physiological significance in plants remain to be elucidated. In this study, we revealed that AtCCR4a and AtCCR4b are localized to cytoplasmic mRNA processing bodies, which are specific granules consisting of many enzymes involved in mRNA turnover. Double mutants of AtCCR4a and AtCCR4b exhibited tolerance to sucrose application but not to glucose. The levels of sucrose in the seedlings of the atccr4a/4b double mutants were reduced, whereas no difference was observed in glucose levels. Further, amylose levels were slightly but significantly increased in the atccr4a/4b double mutants. Consistent with this observation, we found that the transcript encoding granule-bound starch synthase 1 (GBSS1), which is responsible for amylose synthesis, is accumulated to a higher level in the atccr4a/4b double mutant plants than in the control plants. Moreover, we revealed that GBSS1 has a longer poly(A) tail in the double mutant than in the control plant, suggesting that AtCCR4a and AtCCR4b can influence the poly(A) length of transcripts related to starch metabolism. Our results collectively suggested that AtCCR4a and AtCCR4b are involved in sucrose and starch metabolism in A. thaliana.

Development of pollen, the male gametophyte of flowering plants, is tightly controlled by dynamic changes in gene expression. Recent research to clarify the molecular aspects of pollen development has revealed the involvement of several transcription factors in the induction of gene expression. However, limited information is available about the factors involved in the negative regulation of gene expression to eliminate unnecessary transcripts during pollen development. In this study, we revealed that AtNOT1 is an essential protein for proper pollen development and germination capacity. AtNOT1 is a scaffold protein of the AtCCR4–NOT complex, which includes multiple components related to mRNA turnover control in Arabidopsis. Phenotypic analysis using atnot1 heterozygote mutant pollen showed that the mature mutant pollen failed to germinate and also revealed abnormal localization of nuclei and a specific protein at the tricellular pollen stage. Furthermore, transcriptome analysis of atnot1 heterozygote mutant pollen showed that the downregulation of a large number of transcripts, along with the upregulation of specific transcripts required for pollen tube germination by AtNOT1 during late microgametogenesis, is important for proper pollen development and germination. Overall, our findings provide new insights into the negative regulation of gene expression during pollen development, by showing the severely defective phonotype of atnot1 heterozygote mutant pollen.

Plants possess a cold acclimation system to acquire freezing tolerance through pre-exposure to non-freezing low temperatures. The transcriptional cascade of C-repeat-binding factors (CBFs)/dehydration response element-binding factors (DREBs) is considered a major transcriptional regulatory pathway during cold acclimation. However, little is known regarding the functional significance of mRNA stability regulation in the response of gene expression to cold stress. The actual level of individual mRNAs is determined by a balance between mRNA synthesis and degradation. Therefore, it is important to assess the regulatory steps to increase our understanding of gene regulation. Here, we analyzed temporal changes in mRNA amounts and half-lives in response to cold stress in Arabidopsis cell cultures based on genome-wide analysis. In this mRNA decay array method, mRNA half-life measurements and microarray analyses were combined. In addition, temporal changes in the integrated value of transcription rates were estimated from the above two parameters using a mathematical approach. Our results showed that several cold-responsive genes, including Cold-regulated 15a, were relatively destabilized, whereas the mRNA amounts were increased during cold treatment by accelerating the transcription rate to overcome the destabilization. Considering the kinetics of mRNA synthesis and degradation, this apparently contradictory result supports that mRNA destabilization is advantageous for the swift increase in CBF-responsive genes in response to cold stress.