The dicing activity of DCL3 and DCL4 is negatively affected by flavonoids.

KEY MESSAGE: The dicing activities of DCL3 and DCL4 are inhibited by accumulated metabolites in soybean leaves. Epicatechin and 7,4'-dihydroxyflavone inhibited Arabidopsis DCL3 and DCL4 in vitro. Flavonoids are major secondary metabolites in plants, and soybean (Glycine max L.) is a representative plant that accumulates flavonoids, including isoflavonoids, to high levels. Naturally-occurring RNA interference (RNAi) against the chalcone synthase (CHS) gene represses flavonoid (anthocyanin) biosynthesis in an organ-specific manner, resulting in a colorless (yellow) seed coat in many soybean cultivars. To better understand seed coat-specific naturally-occurring RNAi in soybean, we characterized soybean Dicer-like (DCL) 3 and 4, which play critical roles in RNAi. Using a previously established dicing assay, two dicing activities producing 24- and 21-nt siRNAs, corresponding to DCL3 and DCL4, respectively, were detected in soybean. Dicing activity was detected in colorless seed coats where RNAi against CHS genes was found, but no dicing activity was detected in leaves where CHS expression was prevalent. Biochemical analysis revealed that soybean leaves contained two types of inhibitors effective for Arabidopsis Dicers (AtDCL3 and AtDCL4), one of which was a heat-labile high molecular weight compound of 50 to 100 kD while another was a low molecular weight substance. We found that some flavonoids, such as epicatechin and 7,4'-dihydroxyflavone, inhibited both AtDCL3 and AtDCL4, but AtDCL4 was more sensitive to these flavonoids than AtDCL3. These results suggest that flavonoids inhibit the dicing activity of DCL4 and thereby attenuate RNAi in soybean leaves.

Overexpression of maize ZmLOX6 in Arabidopsis thaliana enhances damage-induced pentyl leaf volatile emissions that affect plant growth and interaction with aphids.

Pentyl leafy volatiles (PLV) are C5 volatiles produced from polyunsaturated fatty acids by plant 13-lipoxygenases (13-LOX) in concert with other lipid metabolizing enzymes. Unlike related C6 volatiles (GLV, green leafy volatiles), little is known about the biosynthesis and physiological function of PLV in plants. Zea mays LOX6 (ZmLOX6) is an unusual plant LOX that lacks lipid oxygenation activity but acts as a hydroperoxide lyase hypothesized to be specifically involved in PLV synthesis. We overexpressed ZmLOX6 in Arabidopsis thaliana and established that it indeed produces PLVs. Overexpression of ZmLOX6 caused a mild chlorotic phenotype, and induced a similar phenotype in untransformed Col-0 plants grown in close proximity, suggesting that airborne signals, such as PLVs, are responsible for the phenotype. PLV production, dependency on the substrate from endogenous 13-LOX(s), and likely competition with endogenous 13-oxylipin pathway were consistent with the model that ZmLOX6 functions as a hydroperoxide lyase. The abundance of individual PLVs was differentially affected by ZmLOX6 overexpression, and the new profile indicated that ZmLOX6 had reaction products distinct from endogenous PLV-producing activities in the Arabidopsis host plants. ZmLOX6 overexpression also induced a new hormonal status, which is likely responsible for increased attraction and propagation of aphids, nonetheless improving host plant tolerance to aphid infestation.

Multiple Quality Control Mechanisms in the ER and TGN Determine Subcellular Dynamics and Salt-Stress Tolerance Function of KORRIGAN1.

Among many glycoproteins within the plant secretory system, KORRIGAN1 (KOR1), a membrane-anchored endo-ß-1,4-glucanase involved in cellulose biosynthesis, provides a link between N-glycosylation, cell wall biosynthesis, and abiotic stress tolerance. After insertion into the endoplasmic reticulum, KOR1 cycles between the trans-Golgi network (TGN) and the plasma membrane (PM). From the TGN, the protein is targeted to growing cell plates during cell division. These processes are governed by multiple sequence motifs and also host genotypes. Here, we investigated the interaction and hierarchy of known and newly identified sorting signals in KOR1 and how they affect KOR1 transport at various stages in the secretory pathway. Conventional steady-state localization showed that structurally compromised KOR1 variants were directed to tonoplasts. In addition, a tandem fluorescent timer technology allowed for differential visualization of young versus aged KOR1 proteins, enabling the analysis of single-pass transport through the secretory pathway. Observations suggest the presence of multiple checkpoints/branches during KOR1 trafficking, where the destination is determined based on KOR1's sequence motifs and folding status. Moreover, growth analyses of dominant PM-confined KOR1-L48L49→A48A49 variants revealed the importance of active removal of KOR1 from the PM during salt stress, which otherwise interfered with stress acclimation.

Arabidopsis RNA polymerase II C-terminal domain phosphatase-like 1 targets mitogen-activated protein kinase cascades to suppress plant immunity.

Mitogen-activated protein kinase (MAPK) cascades play pivotal roles in plant defense against phytopathogens downstream of immune receptor complexes. The amplitude and duration of MAPK activation must be strictly controlled, but the underlying mechanism remains unclear. Here, we identified Arabidopsis CPL1 (C-terminal domain phosphatase-like 1) as a negative regulator of microbe-associated molecular pattern (MAMP)-triggered immunity via a forward-genetic screen. Disruption of CPL1 significantly enhanced plant resistance to Pseudomonas pathogens induced by the bacterial peptide flg22. Furthermore, flg22-induced MPK3/MPK4/MPK6 phosphorylation was dramatically elevated in cpl1 mutants but severely impaired in CPL1 overexpression lines, suggesting that CPL1 might interfere with flg22-induced MAPK activation. Indeed, CPL1 directly interacted with MPK3 and MPK6, as well as the upstream MKK4 and MKK5. A firefly luciferase-based complementation assay indicated that the interaction between MKK4/MKK5 and MPK3/MPK6 was significantly reduced in the presence of CPL1. These results suggest that CPL1 plays a novel regulatory role in suppressing MAMP-induced MAPK cascade activation and MAMP-triggered immunity to bacterial pathogens.

Disturbance of floral colour pattern by activation of an endogenous pararetrovirus, petunia vein clearing virus, in aged petunia plants.

White areas of star-type bicolour petals of petunia (Petunia hybrida) are caused by post-transcriptional gene silencing (PTGS) of the key enzyme of anthocyanin biosynthesis. We observed blotched flowers and a vein-clearing symptom in aged petunia plants. To determine the cause of blotched flowers, we focused on an endogenous pararetrovirus, petunia vein clearing virus (PVCV), because this virus may have a suppressor of PTGS (VSR). Transcripts and episomal DNAs derived from proviral PVCVs accumulated in aged plants, indicating that PVCV was activated as the host plant aged. Furthermore, DNA methylation of CG and CHG sites in the promoter region of proviral PVCV decreased in aged plants, suggesting that poor maintenance of DNA methylation activates PVCV. In parallel, de novo DNA methylation of CHH sites in its promoter region was also detected. Therefore, both activation and inactivation of PVCV occurred in aged plants. The accumulation of PVCV transcripts and episomal DNAs in blotched regions and the detection of VSR activity support a mechanism in which suppression of PTGS by PVCV causes blotched flowers.

Imaging-Based Resistance Assay Using Enhanced Luminescence-Tagged Pseudomonas syringae Reveals a Complex Epigenetic Network in Plant Defense Signaling Pathways.

High-throughput resistance assays in plants have a limited selection of suitable pathogens. In this study, we developed a Pseudomonas syringae strain chromosomally tagged with the Nanoluc luciferase (NL) from the deep-sea shrimp Oplophorus gracilirostris, a bioluminescent marker significantly brighter than the conventional firefly luciferase. Our reporter strain tagged with NL was more than 100 times brighter than P. syringae tagged with the luxCDABE operon from Photorhabdus luminescens, one of the existing luciferase-based strains. In planta imaging was improved by using the surfactant Silwet L-77, particularly at a lower reporter concentration. Using this imaging system, more than 30 epigenetic mutants were analyzed for their resistance traits because the defense signaling pathway is known to be epigenetically regulated. SWC1, a defense-related chromatin remodeling complex, was found to be a positive defense regulator, which supported one of two earlier conflicting reports. Compromises in DNA methylation in the CG context led to enhanced resistance against virulent Pseudomonas syringae pv. tomato. Dicer-like and Argonaute proteins, important in the biogenesis and exerting the effector function of small RNAs, respectively, showed modest but distinct requirements for effector-triggered immunity and basal resistance to P. syringae pv. tomato. In addition, the transcriptional expression of an epigenetic component was found to be a significant predictor of its immunity contribution. In summary, this study showcased how a high-throughput resistance assay enabled by a pathogen strain with an improved luminescent reporter could provide insightful knowledge about complex defense signaling pathways.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Biochemical characterization of the dicing activity of Dicer-like 2 in the model filamentous fungus Neurospora crassa.

Dicing of double-stranded RNA (dsRNA) into small RNA is an essential process to trigger transcriptional and post-transcriptional gene silencing. Using cell-free extracts of the model filamentous fungus Neurospora crassa, we successfully detected the dicing activity of one of two N. crassa Dicers NcDCL2. The predominant 23-nucleotide (nt) cleavage product was always detected from 30-nt to 130-nt dsRNA substrates, and additional products of approximately 18 to 28 nt were occasionally produced. The enzymatic properties of NcDCL2 are different from those of insect and plant small interfering RNA (siRNA)-producing Dicers, Drosophila melanogaster Dicer-2 and Arabidopsis thaliana DCL3 and DCL4 (AtDCL3 and AtDCL4). Whereas AtDCL3 and AtDCL4 preferentially cleave short and long dsRNAs, respectively, NcDCL2 cleaved both short and long dsRNAs. These results suggest that N. crassa has a single siRNA-producing Dicer NcDCL2, which is a prototype of plant siRNA-producing Dicers with distinct functions in diverse RNA silencing pathways. The dicing assay reported here is convenient to detect and biochemically characterize the dicing activities of both plant and fungal Dicers, and is likely applicable to other organisms.

Salt Stress and CTD PHOSPHATASE-LIKE4 Mediate the Switch between Production of Small Nuclear RNAs and mRNAs.

Phosphorylation of the RNA polymerase II (Pol II) C-terminal domain (CTD) regulates transcription of protein-coding mRNAs and noncoding RNAs. CTD function in transcription of protein-coding RNAs has been studied extensively, but its role in plant noncoding RNA transcription remains obscure. Here, using Arabidopsis thaliana CTD PHOSPHATASE-LIKE4 knockdown lines (CPL4RNAi ), we showed that CPL4 functions in genome-wide, conditional production of 3'-extensions of small nuclear RNAs (snRNAs) and biogenesis of novel transcripts from protein-coding genes downstream of the snRNAs (snRNA-downstream protein-coding genes [snR-DPGs]). Production of snR-DPGs required the Pol II snRNA promoter (PIIsnR), and CPL4RNAi plants showed increased read-through of the snRNA 3'-end processing signal, leading to continuation of transcription downstream of the snRNA gene. We also discovered an unstable, intermediate-length RNA from the SMALL SCP1-LIKE PHOSPHATASE14 locus (imRNASSP14 ), whose expression originated from the 5' region of a protein-coding gene. Expression of the imRNASSP14 was driven by a PIIsnR and was conditionally 3'-extended to produce an mRNA. In the wild type, salt stress induced the snRNA-to-snR-DPG switch, which was associated with alterations of Pol II-CTD phosphorylation at the target loci. The snR-DPG transcripts occur widely in plants, suggesting that the transcriptional snRNA-to-snR-DPG switch may be a ubiquitous mechanism to regulate plant gene expression in response to environmental stresses.

Effect of asymptomatic infection with southern tomato virus on tomato plants.

Southern tomato virus (STV) is often found infecting healthy tomato plants (Solanum lycopersicum). In this study, we compared STV-free and STV-infected plants of cultivar M82 to determine the effect of STV infection on the host plant. STV-free plants exhibited a short and bushy phenotype, whereas STV-infected plants were taller. STV-infected plants produced more fruit than STV-free plants, and the germination rate of seeds from STV-infected plants was higher than that of seeds from STV-free plants. This phenotypic difference was also observed in progeny plants (siblings) derived from a single STV-infected plant in which the transmission rate of STV to progeny plants via the seeds was approximately 86%. These results suggest that the interaction between STV and host plants is mutualistic. Transcriptome analysis revealed that STV infection affects gene expression in the host plant and results in downregulation of genes involved in ethylene biosynthesis and signaling. STV-infected tomato plants might thus be artificially selected due to their superior traits as a crop.

KETCH1 imports HYL1 to nucleus for miRNA biogenesis in Arabidopsis.

MicroRNA (miRNA) is processed from primary transcripts with hairpin structures (pri-miRNAs) by microprocessors in the nucleus. How cytoplasmic-borne microprocessor components are transported into the nucleus to fulfill their functions remains poorly understood. Here, we report KETCH1 (karyopherin enabling the transport of the cytoplasmic HYL1) as a partner of hyponastic leaves 1 (HYL1) protein, a core component of microprocessor in Arabidopsis and functional counterpart of DGCR8/Pasha in animals. Null mutation of ketch1 is embryonic-lethal, whereas knockdown mutation of ketch1 caused morphological defects, reminiscent of mutants in the miRNA pathway. ketch1 knockdown mutation also substantially reduced miRNA accumulation, but did not alter nuclear-cytoplasmic shuttling of miRNAs. Rather, the mutation significantly reduced nuclear portion of HYL1 protein and correspondingly compromised the pri-miRNA processing in the nucleus. We propose that KETCH1 transports HYL1 from the cytoplasm to the nucleus to constitute functional microprocessor in Arabidopsis This study provides insight into the largely unknown nuclear-cytoplasmic trafficking process of miRNA biogenesis components through eukaryotes.

Silencing Arabidopsis CARBOXYL-TERMINAL DOMAIN PHOSPHATASE-LIKE 4 induces cytokinin-oversensitive de novo shoot organogenesis.

De novo shoot organogenesis (DNSO) is a post-embryonic development programme that has been widely exploited by plant biotechnology. DNSO is a hormonally regulated process in which auxin and cytokinin (CK) coordinate suites of genes encoding transcription factors, general transcription factors, and RNA metabolism machinery. Here we report that silencing Arabidopsis thaliana carboxyl-terminal domain (CTD) phosphatase-like 4 (CPL4RNAi ) resulted in increased phosphorylation levels of RNA polymerase II (pol II) CTD and altered lateral root development and DNSO efficiency of the host plants. Under standard growth conditions, CPL4RNAi lines produced no or few lateral roots. When induced by high concentrations of auxin, CPL4RNAi lines failed to produce focused auxin maxima at the meristem of lateral root primordia, and produced fasciated lateral roots. In contrast, root explants of CPL4RNAi lines were highly competent for DNSO. Efficient DNSO of CPL4RNAi lines was observed even under 10 times less the CK required for the wild-type explants. Transcriptome analysis showed that CPL4RNAi , but not wild-type explants, expressed high levels of shoot meristem-related genes even during priming on medium with a high auxin/CK ratio, and during subsequent shoot induction with a lower auxin/CK ratio. Conversely, CPL4RNAi enhanced the inhibitory phenotype of the shoot redifferentiation defective2-1 mutation, which affected snRNA biogenesis and formation of the auxin gradient. These results indicated that CPL4 functions in multiple regulatory pathways that positively and negatively affect DNSO.

Purification and characterization of Arabidopsis thaliana oligosaccharyltransferase complexes from the native host: a protein super-expression system for structural studies.

The oligosaccharyltransferase (OT) complex catalyzes N-glycosylation of nascent secretory polypeptides in the lumen of the endoplasmic reticulum. Despite their importance, little is known about the structure and function of plant OT complexes, mainly due to lack of efficient recombinant protein production systems suitable for studies on large plant protein complexes. Here, we purified Arabidopsis OT complexes using the tandem affinity-tagged OT subunit STAUROSPORINE AND TEMPERATURE SENSITIVE3a (STT3a) expressed by an Arabidopsis protein super-expression platform. Mass-spectrometry analysis of the purified complexes identified three essential OT subunits, OLIGOSACCHARYLTRANSFERASE1 (OST1), HAPLESS6 (HAP6), DEFECTIVE GLYCOSYLATION1 (DGL1), and a number of ribosomal subunits. Transmission-electron microscopy showed that STT3a becomes incorporated into OT-ribosome super-complexes formed in vivo, demonstrating that this expression/purification platform is suitable for analysis of large protein complexes. Pairwise in planta interaction analyses of individual OT subunits demonstrated that all subunits identified in animal OT complexes are conserved in Arabidopsis and physically interact with STT3a. Genetic analysis of newly established OT subunit mutants for OST1 and DEFENDER AGAINST APOTOTIC DEATH (DAD) family genes revealed that OST1 and DAD1/2 subunits are essential for the plant life cycle. However, mutations in these individual isoforms produced much milder growth/underglycosylation phenotypes than previously reported for mutations in DGL1, OST3/6 and STT3a.

Genetic diversity, linkage disequilibrium, and population structure analysis of the tea plant (Camellia sinensis) from an origin center, Guizhou plateau, using genome-wide SNPs developed by genotyping-by-sequencing.

BACKGROUND: To efficiently protect and exploit germplasm resources for marker development and breeding purposes, we must accurately depict the features of the tea populations. This study focuses on the Camellia sinensis (C. sinensis) population and aims to (i) identify single nucleotide polymorphisms (SNPs) on the genome level, (ii) investigate the genetic diversity and population structure, and (iii) characterize the linkage disequilibrium (LD) pattern to facilitate next genome-wide association mapping and marker-assisted selection. RESULTS: We collected 415 tea accessions from the Origin Center and analyzed the genetic diversity, population structure and LD pattern using the genotyping-by-sequencing (GBS) approach. A total of 79,016 high-quality SNPs were identified; the polymorphism information content (PIC) and genetic diversity (GD) based on these SNPs showed a higher level of genetic diversity in cultivated type than in wild type. The 415 accessions were clustered into three groups by STRUCTURE software and confirmed using principal component analyses (PCA)-wild type, cultivated type, and admixed wild type. However, unweighted pair group method with arithmetic mean (UPGMA) trees indicated the accessions should be grouped into more clusters. Further analyses identified four groups, the Pure Wild Type, Admixed Wild Type, ancient landraces and modern landraces using STRUCTURE, and the results were confirmed by PCA and UPGMA tree method. A higher level of genetic diversity was detected in ancient landraces and Admixed Wild Type than that in the Pure Wild Type and modern landraces. The highest differentiation was between the Pure Wild Type and modern landraces. A relatively fast LD decay with a short range (kb) was observed, and the LD decays of four inferred populations were different. CONCLUSIONS: This study is, to our knowledge, the first population genetic analysis of tea germplasm from the Origin Center, Guizhou Plateau, using GBS. The LD pattern, population structure and genetic differentiation of the tea population revealed by our study will benefit further genetic studies, germplasm protection, and breeding.

Post-Translational Regulation of the Dicing Activities of Arabidopsis DICER-LIKE 3 and 4 by Inorganic Phosphate and the Redox State.

In Arabidopsis thaliana, small interfering RNAs (siRNAs) generated by two Dicer isoforms, DCL3 and DCL4, function in distinct epigenetic processes, i.e. RNA-directed DNA methylation and post-transcriptional gene silencing, respectively. Plants often respond to their environment by producing a distinct set of small RNAs; however, the mechanism for controlling the production of different siRNAs from the same dsRNA substrate remains unclear. We established a simple biochemical method to visualize the dsRNA-cleaving activities of DCL3 and DCL4 in cell-free extracts prepared from Arabidopsis seedlings. Here, we demonstrate that different nutrient statuses of a host plant affect the post-translational regulation of the dicing activity of DCL3 and DCL4. Phosphate deficiency inhibited DCL3, and the activity of DCL3 was directly activated by inorganic phosphate. Sulfur deficiency inhibited DCL4 but not DCL3, and the activity of DCL4 was recovered by supplementation of the cell-free extracts with reductants containing a thiol group. Immunopurified DCL4 was activated by recombinant Arabidopsis thioredoxin-h1 with dithiothreitol. Therefore, DCL4 is subject to redox regulation. These results demonstrate that post-translational regulation of DCL activities fine-tunes the balance between branches of the gene silencing pathway according to the growth environment.

Multiple N-glycans cooperate in the subcellular targeting and functioning of Arabidopsis KORRIGAN1.

Arabidopsis thaliana KORRIGAN1 (KOR1) is an integral membrane endo-ß1,4-glucanase in the trans-Golgi network and plasma membrane that is essential for cellulose biosynthesis. The extracellular domain of KOR1 contains eight N-glycosylation sites, N1 to N8, of which only N3 to N7 are highly conserved. Genetic evidence indicated that cellular defects in attachment and maturation of these N-glycans affect KOR1 function in vivo, whereas the manner by which N-glycans modulate KOR1 function remained obscure. Site-directed mutagenesis analysis of green fluorescent protein (GFP)-KOR1 expressed from its native regulatory sequences established that all eight N-glycosylation sites (N1 to N8) are used in the wild type, whereas stt3a-2 cells could only inefficiently add N-glycans to less conserved sites. GFP-KOR1 variants with a single N-glycan at nonconserved sites were less effective than those with one at a highly conserved site in rescuing the root growth phenotype of rsw2-1 (kor1 allele). When functionally compromised, GFP-KOR1 tended to accumulate at the tonoplast. GFP-KOR1Δall (without any N-glycan) exhibited partial complementation of rsw2-1; however, root growth of this line was still negatively affected by the absence of complex-type N-glycan modifications in the host plants. These results suggest that one or several additional factor(s) carrying complex N-glycans cooperate(s) with KOR1 in trans to grant proper targeting/functioning in plant cells.

High throughput selection of antibiotic-resistant transgenic Arabidopsis plants.

Kanamycin resistance is the most frequently used antibiotic-resistance marker for Arabidopsis transformations, however, this method frequently causes escape of untransformed plants, particularly at the high seedling density during the selection. Here we developed a robust high-density selection method using top agar for Arabidopsis thaliana. Top agar effectively suppressed growth of untransformed wild-type plants on selection media at high density. Survival of the transformed plants during the selection were confirmed by production of green true leaves and expression of a firefly luciferase reporter gene. Top agar method allowed selection using a large amount of seeds in Arabidopsis transformation.

Arabidopsis CPL4 is an essential C-terminal domain phosphatase that suppresses xenobiotic stress responses.

Eukaryotic gene expression is both promoted and inhibited by the reversible phosphorylation of the C-terminal domain of RNA polymerase II (pol II CTD). More than 20 Arabidopsis genes encode CTD phosphatase homologs, including four CTD phosphatase-like (CPL) family members. Although in vitro CTD phosphatase activity has been established for some CPLs, none have been shown to be involved in the phosphoregulation of pol II in vivo. Here we report that CPL4 is a CTD phosphatase essential for the viability of Arabidopsis thaliana. Mass spectrometry analysis identified the pol II subunits RPB1, RPB2 and RPB3 in the affinity-purified CPL4 complex. CPL4 dephosphorylates both Ser2- and Ser5-PO(4) of the CTD in vitro, with a preference for Ser2-PO(4). Arabidopsis plants overexpressing CPL4 accumulated hypophosphorylated pol II, whereas RNA interference-mediated silencing of CPL4 promoted hyperphosphorylation of pol II. A D128A mutation in the conserved DXDXT motif of the CPL4 catalytic domain resulted in a dominant negative form of CPL4, the overexpression of which inhibited transgene expression in transient assays. Inhibition was abolished by truncation of the phosphoprotein-binding Breast Cancer 1 C-terminal domain of CPL4, suggesting that both catalytic function and protein-protein interaction are essential for CPL4-mediated regulation of gene expression. We were unable to recover a homozygous cpl4 mutant, probably due to the zygotic lethality of this mutation. The reduction in CPL4 levels in CPL4(RNAi) plants increased transcript levels of a suite of herbicide/xenobiotic-responsive genes and improved herbicide tolerance, thus suggesting an additional role for CPL4 as a negative regulator of the xenobiotic detoxification pathway.

Loss of function of Arabidopsis C-terminal domain phosphatase-like1 activates iron deficiency responses at the transcriptional level.

The expression of genes that control iron (Fe) uptake and distribution (i.e. Fe utilization-related genes) is tightly regulated. Fe deficiency strongly induces Fe utilization-related gene expression; however, little is known about the mechanisms that regulate this response in plants. Transcriptome analysis of an Arabidopsis (Arabidopsis thaliana) mutant defective in RNA polymerase II C-terminal domain-phosphatase-like1 (CPL1) revealed significant up-regulation of Fe utilization-related genes (e.g. IRON-REGULATED TRANSPORTER1), suggesting the importance of RNA metabolism in Fe signaling. An analysis using multiple cpl1 alleles established that cpl1 mutations enhanced specific transcriptional responses to low Fe availability. Changes in protein level were less prominent than those in transcript level, indicating that cpl1-2 mainly affects the Fe deficiency response at the transcriptional level. However, Fe content was significantly increased in the roots and decreased in the shoots of cpl1-2 plants, indicating that the cpl1 mutations do indeed affect Fe homeostasis. Furthermore, root growth of cpl1-2 showed improved tolerance to Fe deficiency and cadmium (Cd) toxicity. cpl1-2 plants accumulated more Cd in the shoots, suggesting that Cd toxicity in the roots of this mutant is averted by the transport of excess Cd to the shoots. Genetic data indicate that cpl1-2 likely activates Fe deficiency responses upstream of both FE-DEFICIENCY-INDUCED TRANSCRIPTION FACTOR-dependent and -independent signaling pathways. Interestingly, various osmotic stress/abscisic acid (ABA)-inducible genes were up-regulated in cpl1-2, and the expression of some ABA-inducible genes was controlled by Fe availability. We propose that the cpl1 mutations enhance Fe deficiency signaling and promote cross talk with a branch of the osmotic stress/ABA signaling pathway.

Structured 3' UTRs destabilize mRNAs in plants.

BACKGROUND: RNA secondary structure (RSS) can influence the regulation of transcription, RNA processing, and protein synthesis, among other processes. 3' untranslated regions (3' UTRs) of mRNA also hold the key for many aspects of gene regulation. However, there are often contradictory results regarding the roles of RSS in 3' UTRs in gene expression in different organisms and/or contexts. RESULTS: Here, we incidentally observe that the primary substrate of miR159a (pri-miR159a), when embedded in a 3' UTR, could promote mRNA accumulation. The enhanced expression is attributed to the earlier polyadenylation of the transcript within the hybrid pri-miR159a-3' UTR and, resultantly, a poorly structured 3' UTR. RNA decay assays indicate that poorly structured 3' UTRs could promote mRNA stability, whereas highly structured 3' UTRs destabilize mRNA in vivo. Genome-wide DMS-MaPseq also reveals the prevailing inverse relationship between 3' UTRs' RSS and transcript accumulation in the transcriptomes of Arabidopsis, rice, and even human. Mechanistically, transcripts with highly structured 3' UTRs are preferentially degraded by 3'-5' exoribonuclease SOV and 5'-3' exoribonuclease XRN4, leading to decreased expression in Arabidopsis. Finally, we engineer different structured 3' UTRs to an endogenous FT gene and alter the FT-regulated flowering time in Arabidopsis. CONCLUSIONS: We conclude that highly structured 3' UTRs typically cause reduced accumulation of the harbored transcripts in Arabidopsis. This pattern extends to rice and even mammals. Furthermore, our study provides a new strategy of engineering the 3' UTRs' RSS to modify plant traits in agricultural production and mRNA stability in biotechnology.

Bacterial diversity and composition on the rinds of specific melon cultivars and hybrids from across different growing regions in the United States.

The goal of this study was to characterize the bacterial diversity on different melon varieties grown in different regions of the US, and determine the influence that region, rind netting, and variety of melon has on the composition of the melon microbiome. Assessing the bacterial diversity of the microbiome on the melon rind can identify antagonistic and protagonistic bacteria for foodborne pathogens and spoilage organisms to improve melon safety, prolong shelf-life, and/or improve overall plant health. Bacterial community composition of melons (n = 603) grown in seven locations over a four-year period were used for 16S rRNA gene amplicon sequencing and analysis to identify bacterial diversity and constituents. Statistically significant differences in alpha diversity based on the rind netting and growing region (p < 0.01) were found among the melon samples. Principal Coordinate Analysis based on the Bray-Curtis dissimilarity distance matrix found that the melon bacterial communities clustered more by region rather than melon variety (R2 value: 0.09 & R2 value: 0.02 respectively). Taxonomic profiling among the growing regions found Enterobacteriaceae, Bacillaceae, Microbacteriaceae, and Pseudomonadaceae present on the different melon rinds at an abundance of ≥ 0.1%, but no specific core microbiome was found for netted melons. However, a core of Pseudomonadaceae, Bacillaceae, and Exiguobacteraceae were found for non-netted melons. The results of this study indicate that bacterial diversity is driven more by the region that the melons were grown in compared to rind netting or melon type. Establishing the foundation for regional differences could improve melon safety, shelf-life, and quality as well as the consumers' health.