Crassulacean acid metabolism (CAM) is a water-use efficient adaptation of photosynthesis that has evolved independently many times in diverse lineages of flowering plants. We hypothesize that convergent evolution of protein sequence and temporal gene expression underpins the independent emergences of CAM from C3 photosynthesis. To test this hypothesis, we generate a de novo genome assembly and genome-wide transcript expression data for Kalanchoë fedtschenkoi, an obligate CAM species within the core eudicots with a relatively small genome (~260 Mb). Our comparative analyses identify signatures of convergence in protein sequence and re-scheduling of diel transcript expression of genes involved in nocturnal CO2 fixation, stomatal movement, heat tolerance, circadian clock, and carbohydrate metabolism in K. fedtschenkoi and other CAM species in comparison with non-CAM species. These findings provide new insights into molecular convergence and building blocks of CAM and will facilitate CAM-into-C3 photosynthesis engineering to enhance water-use efficiency in crops.

MicroRNAs (miRNAs) are regulators of gene expression in plants and animals. The biogenesis of miRNAs is precisely controlled to secure normal development of organisms. Here we report that TOUGH (TGH) is a component of the DCL1-HYL1-SERRATE complex that processes primary transcripts of miRNAs [i.e., primary miRNAs (pri-miRNAs)] into miRNAs in Arabidopsis. Lack of TGH impairs multiple DCL activities in vitro and reduces the accumulation of miRNAs and siRNAs in vivo. TGH is an RNA-binding protein, binds pri-miRNAs and precursor miRNAs in vivo, and contributes to pri-miRNA-HYL1 interaction. These results indicate that TGH might regulate abundance of miRNAs through promoting DCL1 cleavage efficiency and/or recruitment of pri-miRNAs.S mall RNAs, including microRNAs (miRNAs) and siRNAs, are sequence-specific regulators of gene expression in plants and animals (1). miRNAs are derived from imperfect stem-loop transcripts, called primary miRNAs (pri-miRNAs), which are predominately produced by DNA-dependent RNA polymerase II, whereas siRNAs are processed from perfect or near-perfect long dsRNAs (2). After generation, miRNA and siRNA are loaded into an RNA-induced silencing complex containing the Argonaute protein to guide posttranscriptional or transcriptional gene silencing (1).In animals, pri-miRNAs are first processed to precursor miRNAs (pre-miRNAs) in the nucleus by the microprocessor containing Drosha and a dsRNA-binding protein DGCR8 (1). The resulting pre-miRNAs are then processed by Dicer in the cytoplasm to produce mature miRNAs (1). It has emerged that the activities of Drosha and Dicer are controlled to regulate miRNA expression in response to developmental and environmental signals (3). In Arabidopsis, DCL1, a dsRNA-binding protein, HYL1, and a zinc finger protein, SERRATE (SE), form a complex to process pri-miRNAs in the nucleus to pre-miRNAs and then to mature miRNAs (4-6). The accumulation of miRNAs in Arabidopsis also requires DDL, which was proposed to stabilize pri-miRNAs and to facilitate their processing (7). Recently, two cap-binding proteins, CBP80/ABH1 and CBP20, were found to be required for pre-mRNA splicing and primiRNA processing (8, 9). Plants also encode several classes of endogenous siRNAs, including the natural antisense transcriptderived siRNA, siRNA derived from repetitive DNA sequences (rasiRNA), and transacting siRNA (ta-siRNA) (10). In Arabidopsis, the generation of these siRNAs from long dsRNAs involves DCL1 homologues DCL2, DCL3, and DCL4, which produce 22-nt, 24-nt, and 21-nt siRNAs, respectively (11-13).In this report, we show that TOUGH (TGH) is an important factor for miRNA and siRNA biogenesis. Loss-of-function TGH in tgh-1 reduces the activity of multiple DCLs in vitro and the accumulation of miRNA and siRNAs in vivo. In the miRNA pathway, TGH associates with the DCL1 complex and binds primiRNAs and pre-miRNAs. TGH is required for the efficient in vivo interaction between pri-miRNA and HYL1. These data suggest that TGH assists DCLs to efficiently process and/or recruit the prec...

Plant growth-defense tradeoffs are fundamental for optimizing plant performance and fitness in a changing biotic/abiotic environment. This process is thought to involve readjusting resource allocation to different pathways. It has been frequently observed that among secondary cell wall components, alteration in lignin biosynthesis results in changes in both growth and defense. How this process is regulated, leading to growth or defense, remains largely elusive. In this article, we review the canonical lignin biosynthesis pathway, the recently discovered tyrosine shortcut pathway, and the biosynthesis of unconventional C-lignin. We summarize the current model of the hierarchical transcriptional regulation of lignin biosynthesis. Moreover, the interface between recently identified transcription factors and the hierarchical model are also discussed. We propose the existence of a transcriptional co-regulation mechanism coordinating energy allowance among growth, defense and lignin biosynthesis.

CDC5 is a MYB-related protein that exists in plants, animals, and fungi. In Arabidopsis, CDC5 regulates both growth and immunity through unknown mechanisms. Here, we show that CDC5 from Arabidopsis positively regulates the accumulation of microRNAs (miRNAs), which control many biological processes including development and adaptations to environments in plants. CDC5 interacts with both the promoters of genes encoding miRNAs (MIR) and the DNA-dependent RNA polymerase II. As a consequence, lack of CDC5 reduces the occupancy of polymerase II at MIR promoters, as well as MIR promoter activities. In addition, CDC5 is associated with the DICER-LIKE1 complex, which generates miRNAs from their primary transcripts and is required for efficient miRNA production. These results suggest that CDC5 may have dual roles in miRNA biogenesis: functioning as a positive transcription factor of MIR and/or acting as a component of the DICER-LIKE1 complex to enhance primary miRNA processing.M icroRNAs (miRNAs) and small interfering RNAs (siRNAs) are ∼22-nucleotide (nt) noncoding RNAs that regulate various biological processes including development, metabolism, and immunity in plants and animals (1-3). miRNAs are generated from primary miRNA transcripts (pri-miRNAs) containing stem-loop structure, whereas siRNAs are derived from long, perfect, doublestranded RNAs (dsRNAs) (1-3). They are associated with members of the Argonaute protein family to repress gene expression at posttranscriptional and/or transcriptional levels (1-3). In addition to miRNAs, plants encode two major classes of siRNAs: siRNAs derived from repeated DNAs (ra-siRNAs) and transacting siRNAs (ta-siRNAs) (4-6).Studies in Arabidopsis have established the framework of miRNA biogenesis in plants (1-3). In Arabidopsis, pri-miRNAs are primarily transcribed by DNA-dependent RNA polymerase II (Pol II), with assistance from the mediator complex and the transcription factor Negative on TATA less2 (NOT2) (7,8). After transcription, pri-miRNAs are processed by an RNase III enzyme called DICER-LIKE1 (DCL1) to miRNA precursors and then to mature miRNAs (9, 10). The efficient processing of pri-miRNA requires SERRATE (SE; a zinc finger protein), TOUGH (an RNA-binding protein), and a dephosphorylated HYPONASTIC LEAVES1 (HYL1; a double-stranded RNA binding protein) that form a complex with DCL1 (11-18). SE and HYL1 also promote the processing accuracy of pri-miRNAs (19). Four other proteins, DAWDLE (DDL; an RNA binding protein), Cap-binding protein 20, Cap-binding protein 80, and NOT2, which are associated with the DCL1 complex (8,(20)(21)(22), also function in miRNA biogenesis. Recent studies also reveal that the correct localization of DCL1 requires NOT2 and MODIFIER OF SNC1, 2 (an RNA binding protein) (8,23). In addition, the accumulation of a subset of miRNAs requires a proline-rich protein named SICKLE (24).The cell division cycle 5 (CDC5) protein is a conserved protein in animals, plants, and fungi (25). It was first isolated from Schizosaccharomyces pombe as a cell cycle regulat...

In plants, methylation catalyzed by HEN1 (small RNA methyl transferase) prevents microRNAs (miRNAs) from degradation triggered by uridylation. How methylation antagonizes uridylation of miRNAs in vivo is not well understood. In addition, 5′ RNA fragments (5′ fragments) produced by miRNA-mediated RNA cleavage can be uridylated in plants and animals. However, the biological significance of this modification is unknown, and enzymes uridylating 5′ fragments remain to be identified. Here, we report that in Arabidopsis, HEN1 suppressor 1 (HESO1, a miRNA nucleotidyl transferase) uridylates 5′ fragments to trigger their degradation. We also show that Argonaute 1 (AGO1), the effector protein of miRNAs, interacts with HESO1 through its Piwi/Argonaute/Zwille and PIWI domains, which bind the 3′ end of miRNA and cleave the target mRNAs, respectively. Furthermore, HESO1 is able to uridylate AGO1-bound miRNAs in vitro. miRNA uridylation in vivo requires a functional AGO1 in hen1, in which miRNA methylation is impaired, demonstrating that HESO1 can recognize its substrates in the AGO1 complex. On the basis of these results, we propose that methylation is required to protect miRNAs from AGO1-associated HESO1 activity that normally uridylates 5′ fragments. S mall interfering RNAs (siRNAs) and microRNAs (miRNAs), ∼20-25 nucleotides (nt) in size, are important regulators of gene expression. miRNAs and siRNAs are derived from imperfect hairpin transcripts and perfect long double-stranded RNAs, respectively (1, 2). miRNAs and siRNAs are then associated with Argonaute (AGO) proteins to repress gene expression through target cleavage and/or translational inhibition (3). The cleavage of target mRNAs usually occurs at a position opposite the tenth and eleventh nucleotides of miRNAs, resulting in a 5′ RNA fragment (5′ fragment) and a 3′ fragment (4). In Arabidopsis, the major effector protein for miRNA-mediated gene silencing is AGO1, which possesses the endonuclease activity required for target cleavage (5-7). In Drosophila, the exosome removes the 5′ fragments through its 3′-to-5′ exoribonuclease activity (8). How 5′ fragments are degraded in higher plants remains unknown. It has been shown that the 5′ fragments are subject to untemplated uridine addition at their 3′ termini (uridylation) in both animals and plants (9). However, the biological significance of this modification remains unknown because of a lack of knowledge of the enzymes targeting 5′ fragments for uridylation.Uridylation plays important roles in regulating miRNA biogenesis. In animals, TUT4, a terminal uridyl transferase, is recruited by Lin-28 (an RNA binding protein) to the let-7 precursor (prelet-7), resulting in uridylation of prelet-7 (10, 11). This modification impairs the stability of prelet-7, resulting in reduced levels of let-7. In addition, monouridylation has been shown to be required for the processing of some miRNA precursors (12). Deep sequencing analysis reveals that precursor uridylation is a widespread phenomenon occurring in many miRNA families in ani...

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3-O-caffeoylquinic acid, also known as chlorogenic acid (CGA), functions as an intermediate in lignin biosynthesis in the phenylpropanoid pathway. It is widely distributed among numerous plant species and acts as an antioxidant in both plants and animals. Using GC-MS, we discovered consistent and extreme variation in CGA content across a population of 739 4-yr-old Populus trichocarpa accessions. We performed genome-wide association studies (GWAS) from 917 P. trichocarpa accessions and expression-based quantitative trait loci (eQTL) analyses to identify key regulators. The GWAS and eQTL analyses resolved an overlapped interval encompassing a hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyl transferase 2 (PtHCT2) that was significantly associated with CGA and partially characterized metabolite abundances. PtHCT2 leaf expression was significantly correlated with CGA abundance and it was regulated by cis-eQTLs containing W-box for WRKY binding. Among all nine PtHCT homologs, PtHCT2 is the only one that responds to infection by the fungal pathogen Sphaerulina musiva (a Populus pathogen). Validation using protoplast-based transient expression system suggests that PtHCT2 is regulated by the defense-responsive WRKY. These results are consistent with reports of CGA functioning as an antioxidant in response to biotic stress. This study provides insights into data-driven and omics-based inference of gene function in woody species.

Knowledge about the impact of different geographical environments on rhesus macaque gut microbiota is limited. In this study, we compared the characteristics of gut microbiota in six different Chinese rhesus macaque populations, including Hainan, Nanning, Guizhou, Xichang, Jianchuan and Tibet. Through the composition analysis of operational taxonomic units (OTUs), we found that there were significant differences in the abundance of core overlapping OTUs in the six Chinese groups. Specifically, the Tibet population exhibited the highest gut microbial diversity and the most unique OTUs. Statistically significant differences in the composition of gut microbiota among the six groups at phylum and family level were evident. Specifically, Tibet had higher abundances of Firmicutes and lower abundances of Bacteroidetes than the other geographical groups, and the higher abundance of Firmicutes in the Tibetan group was mainly caused by a significant increase in the family Ruminococcaceae and Christensenellaceae. Phylogenetic investigation of communities by reconstruction of unobserved state analysis showed that the enrichment ratio for environmental information processing and organismal systems was the highest in the Tibet population. Additionally, our results suggested that in the adaptation process of rhesus macaques to different geographical environments, the abundance of the core common flora of the intestinal microbes had undergone varying degree of change and produced new and unique flora, both of which helped to reshape the gut microbiota of rhesus macaques. In particular, this change was more obvious for animals in the high-altitude environments.