Small nucleolar RNAs (snoRNAs) are noncoding RNAs (ncRNAs) that guide chemical modifications of structural RNAs, which are essential for ribosome assembly and function in eukaryotes. Although numerous snoRNAs have been identified in plants by high-throughput sequencing, the biological functions of most of these snoRNAs remain unclear. Here, we identified box C/D SnoR28.1s as important regulators of plant growth and development by screening a CRISPR/Cas9-generated ncRNA deletion mutant library in Arabidopsis thaliana. Deletion of the SnoR28.1 locus, which contains a cluster of three genes producing SnoR28.1s, resulted in defects in root and shoot growth. SnoR28.1s guide 2′-O-ribose methylation of 25S rRNA at G2396. SnoR28.1s facilitate proper and efficient pre-rRNA processing, as the SnoR28.1 deletion mutants also showed impaired ribosome assembly and function, which may account for the growth defects. SnoR28 contains a 7-bp antisense box, which is required for 2′-O-ribose methylation of 25S rRNA at G2396, and an 8-bp extra box that is complementary to a nearby rRNA methylation site and is partially responsible for methylation of G2396. Both of these motifs are required for proper and efficient pre-rRNA processing. Finally, we show that SnoR28.1s genetically interact with HIDDEN TREASURE2 and NUCLEOLIN1. Our results advance our understanding of the roles of snoRNAs in Arabidopsis.

Regulation of translation is a crucial step in gene expression. Developmental signals and environmental stimuli dynamically regulate translation via upstream small open reading frames (uORFs) and ribosome pausing. Recent studies have revealed many plant genes that are specifically regulated by uORF translation following changes in growth conditions, but ribosome-pausing events are less well understood. In this study, we performed ribosome profiling (Ribo-seq) of etiolated maize (Zea mays) seedlings exposed to light for different durations, revealing hundreds of genes specifically regulated at the translation level during the early period of light exposure. We identified over 400 ribosome-pausing events in the dark that were rapidly released after illumination. These results suggested that ribosome pausing negatively regulates translation from specific genes, a conclusion that was supported by a non-targeted proteomics analysis. Importantly, we identified a conserved nucleotide motif downstream of the pausing sites. Our results elucidate the role of ribosome pausing in the control of gene expression in plants; the identification of the cis-element at the pausing sites provides insight into the mechanisms behind translation regulation and potential targets for artificial control of plant translation.

Starch is an indispensable energy reserve for pollen and failure of starch biosynthesis in pollen leads to male sterility in flowering crops. Nonetheless, the regulatory mechanisms underlying starch biosynthesis in rice (Oryza sativa) pollen remain unclear. Here, we identified a target of the microRNA OsmiR159, SPOROCYTELESS ETHYLENE-RESPONSIVE ELEMENT BINDING FACTOR-ASSOCIATED AMPHIPHILIC-REPRESSION 2 (OsSPEAR2). OsSPEAR2 is predominantly expressed in mature pollen and OsSPEAR2 possesses transcriptional repressor activity and localizes in the nucleus. Disruption of OsSPEAR2 results in severely shrunken pollen grains and male sterility. OsSPEAR2 interacts with multiple OsTCPs, including OsTCP14. OsTCP14 is a target of OsmiR319 and a knockout mutation in OsTCP14 partially rescues the defective pollen phenotype of Osspear2. In addition, transcriptome analyses revealed significant downregulation of numerous genes associated with carbohydrate metabolism, specifically in Osspear2 anthers, including several genes critical for starch biosynthesis. Moreover, OsTCP14 directly represses the expression of the essential starch biosynthesis gene OsUGP2; however, this repression could be alleviated by OsSPEAR2. Noteworthily, embryophyte-specific SPEAR2 and SPOROCYTELESS were also identified as miR159 targets involved in regulating plant growth and development in Arabidopsis (Arabidopsis thaliana), indicating that the miR159-SPEAR regulatory module may be conserved among embryophytes. Collectively, our findings reveal OsmiR159-OsSPEAR2-OsTCP14-OsUGP2 as a regulatory cascade that modulates starch biosynthesis during pollen development in rice.

Reproductive phasiRNAs (phased, small interfering RNAs), produced from numerous PHAS loci, are essential for plant anther development. PHAS transcripts are enriched on endoplasmic reticulum-bound ribosomes in maize (Zea mays), but the impact of ribosome binding on phasiRNA biogenesis remains elusive. Through ribosome profiling of maize anthers at 10 developmental stages, we demonstrated that 24-PHAS transcripts are bound by ribosomes, with patterns corresponding to the timing and abundance of 24-PHAS transcripts. Ribosome binding to 24-PHAS transcripts is conserved among different maize inbred lines, with ribosomes enriched upstream of miR2275 target sites. We detected short open reading frames (sORFs) in the ribosome-binding regions of some 24-PHAS transcripts and observed a 3-nt periodicity in most sORFs, but mass spectrometry failed to detect peptides corresponding to the sORFs. Deletion of the entire ribosome-binding region of 24PHAS_NO296 locus eliminated ribosome binding and decreased 24-nt phasiRNA production, without affecting 24PHAS_NO296 transcript levels. In contrast, disrupting only the sORFs in 24PHAS_NO296 did not substantially affect the generation of 24-nt phasiRNAs. A newly formed sORF in these mutants may have re-directed ribosome binding to its transcripts. Overall, these findings demonstrate that sORFs facilitate ribosome binding to 24-PHAS transcripts, thereby promoting phasiRNA biogenesis in meiotic anthers.

MicroRNA160 plays a crucial role in plant development by negatively regulating the auxin response factors (ARFs). In this manuscript, we design an automatic molecule machine (AMM) based on the dual catalytic hairpin assembly (D-CHA) strategy for the signal amplification detection of miRNA160. The detection system contains four hairpin-shaped DNA probes (HP1, HP2, HP3, and HP4). For HP1, the loop is designed to be complementary to miRNA160. A fragment of DNA with the same sequences as miRNA160 is separated into two pieces that are connected at the 3′ end of HP2 and 5′ end of HP3, respectively. In the presence of the target, four HPs are successively dissolved by the first catalytic hairpin assembly (CHA1), forming a four-way DNA junction (F-DJ) that enables the rearrangement of separated DNA fragments at the end of HP2 and HP3 and serving as an integrated target analogue for initiating the second CHA reaction, generating an enhanced fluorescence signal. Assay experiments demonstrate that D-CHA has a better performance compared with traditional CHA, achieving the detection limit as low as 10 pM for miRNA160 as deduced from its corresponding DNA surrogates. Moreover, non-target miRNAs, as well as single-base mutation targets, can be detected. Overall, the D-CHA strategy provides a competitive method for plant miRNAs detection.