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Summary Chloroplast Unusual Positioning 1 (CHUP1) plays an important role in the chloroplast avoidance and accumulation responses in mesophyll cells. In epidermal cells, prior research showed silencingCHUP1‐induced chloroplast stromules and amplified effector‐triggered immunity (ETI); however, the underlying mechanisms remain largely unknown.CHUP1 has a dual function in anchoring chloroplasts and recruiting chloroplast‐associated actin (cp‐actin) filaments for blue light‐induced movement. To determine which function is critical for ETI, we developed an approach to quantify chloroplast anchoring and movement in epidermal cells. Our data show that silencingNbCHUP1inNicotiana benthamianaplants increased epidermal chloroplast de‐anchoring and basal movement but did not fully disrupt blue light‐induced chloroplast movement.SilencingNbCHUP1auto‐activated epidermal chloroplast defense (ECD) responses including stromule formation, perinuclear chloroplast clustering, the epidermal chloroplast response (ECR), and the chloroplast reactive oxygen species (ROS), hydrogen peroxide (H₂O₂). These findings show chloroplast anchoring restricts a multifaceted ECD response.Our results also show that the accumulated chloroplastic H₂O₂inNbCHUP1‐silenced plants was not required for the increased basal epidermal chloroplast movement but was essential for increased stromules and enhanced ETI. This finding indicates that chloroplast de‐anchoring and H₂O₂play separate but essential roles during ETI. 

Abstract In plants, cytoplasmic mRNA decay is critical for posttranscriptionally controlling gene expression and for maintaining cellular RNA homeostasis. Arabidopsis DCP1-ASSOCIATED NYN ENDORIBONUCLEASE 1 (DNE1) is a cytoplasmic mRNA decay factor that interacts with proteins involved in mRNA decapping and nonsense-mediated mRNA decay (NMD). There is limited information on the functional role of DNE1 in RNA turnover, and the identities of its endogenous targets are unknown. In this study, we utilized RNA degradome approaches to globally investigate DNE1 substrates. Monophosphorylated 5′ ends, produced by DNE1, should accumulate in mutants lacking the cytoplasmic exoribonuclease XRN4, but be absent from DNE1 and XRN4 double mutants. In seedlings, we identified over 200 such transcripts, most of which reflect cleavage within coding regions. While most DNE1 targets were NMD-insensitive, some were upstream ORF (uORF)-containing and NMD-sensitive transcripts, indicating that this endoribonuclease is required for turnover of a diverse set of mRNAs. Transgenic plants expressing DNE1 cDNA with an active-site mutation in the endoribonuclease domain abolished the in planta cleavage of transcripts, demonstrating that DNE1 endoribonuclease activity is required for cleavage. Our work provides key insights into the identity of DNE1 substrates and enhances our understanding of DNE1-mediated mRNA decay. 

Abstract Plant small RNAs are important regulatory elements that fine-tune gene expression and maintain genome integrity by silencing transposons. Reproductive organs of monocots produce abundant phased, small interfering RNAs (phasiRNAs). The 21-nt reproductive phasiRNAs triggered by miR2118 are highly enriched in pre-meiotic anthers, and have been found in multiple eudicot species, in contrast with prior reports of monocot specificity. The 24-nt reproductive phasiRNAs are triggered by miR2275, and are highly enriched during meiosis in many angiosperms. Here, we report the widespread presence of the 21-nt reproductive phasiRNA pathway in eudicots including canonical and non-canonical microRNA (miRNA) triggers of this pathway. In eudicots, these 21-nt phasiRNAs are enriched in pre-meiotic stages, a spatiotemporal distribution consistent with that of monocots and suggesting a role in anther development. Although this pathway is apparently absent in well-studied eudicot families including the Brassicaceae, Solanaceae and Fabaceae, our work in eudicots supports an earlier singular finding in spruce, a gymnosperm, indicating that the pathway of 21-nt reproductive phasiRNAs emerged in seed plants and was lost in some lineages. 

Abstract Small RNAs are non-coding RNAs that play important roles in the lives of both animals and plants. They are 21- to 24-nt in length and ∼10 nm in size. Their small size and high diversity have made it challenging to develop detection methods that have sufficient resolution and specificity to multiplex and quantify. We created a method, sRNA-PAINT, for the detection of small RNAs with 20 nm resolution by combining the super-resolution method, DNA-based points accumulation in nanoscale topography (DNA-PAINT), and the specificity of locked nucleic acid (LNA) probes for the in situ detection of multiple small RNAs. The method relies on designing probes to target small RNAs that combine DNA oligonucleotides (oligos) for PAINT with LNA-containing oligos for hybridization; therefore, we developed an online tool called ‘Vetting & Analysis of RNA for in situ Hybridization probes’ (VARNISH) for probe design. Our method utilizes advances in DNA-PAINT methodologies, including qPAINT for quantification, and Exchange-PAINT for multiplexing. We demonstrated these capabilities of sRNA-PAINT by detecting and quantifying small RNAs in different cell layers of early developmental stage maize anthers that are important for male sexual reproduction.