Arabidopsis HOT3/eIF5B1 constrains rRNA RNAi by facilitating 18S rRNA maturation.

Ribosome biogenesis is essential for protein synthesis in gene expression. Yeast eIF5B has been shown biochemically to facilitate 18S ribosomal RNA (rRNA) 3' end maturation during late-stage 40S ribosomal subunit assembly and gate the transition from translation initiation to elongation. But the genome-wide effects of eIF5B have not been studied at the single-nucleotide resolution in any organism, and 18S rRNA 3' end maturation is poorly understood in plants. Arabidopsis HOT3/eIF5B1 was found to promote development and heat stress acclimation by translational regulation, but its molecular function remained unknown. Here, we show that HOT3 is a late-stage ribosome biogenesis factor that facilitates 18S rRNA 3' end processing and is a translation initiation factor that globally impacts the transition from initiation to elongation. By developing and implementing 18S-ENDseq, we revealed previously unknown events in 18S rRNA 3' end maturation or metabolism. We quantitatively defined processing hotspots and identified adenylation as the prevalent nontemplated RNA addition at the 3' ends of pre-18S rRNAs. Aberrant 18S rRNA maturation in hot3 further activated RNA interference to generate RDR1- and DCL2/4-dependent risiRNAs mainly from a 3' portion of 18S rRNA. We further showed that risiRNAs in hot3 were predominantly localized in ribosome-free fractions and were not responsible for the 18S rRNA maturation or translation initiation defects in hot3. Our study uncovered the molecular function of HOT3/eIF5B1 in 18S rRNA maturation at the late 40S assembly stage and revealed the regulatory crosstalk among ribosome biogenesis, messenger RNA (mRNA) translation initiation, and siRNA biogenesis in plants.

Arabidopsis AAR2, a conserved splicing factor in eukaryotes, acts in microRNA biogenesis.

MicroRNAs (miRNAs) play an essential role in plant growth and development, and as such, their biogenesis is fine-tuned via regulation of the core microprocessor components. Here, we report that Arabidopsis AAR2, a homolog of a U5 snRNP assembly factor in yeast and humans, not only acts in splicing but also promotes miRNA biogenesis. AAR2 interacts with the microprocessor component hyponastic leaves 1 (HYL1) in the cytoplasm, nucleus, and dicing bodies. In aar2 mutants, abundance of nonphosphorylated HYL1, the active form of HYL1, and the number of HYL1-labeled dicing bodies are reduced. Primary miRNA (pri-miRNA) accumulation is compromised despite normal promoter activities of MIR genes in aar2 mutants. RNA decay assays show that the aar2-1 mutation leads to faster degradation of pri-miRNAs in a HYL1-dependent manner, which reveals a previously unknown and negative role of HYL1 in miRNA biogenesis. Taken together, our findings reveal a dual role of AAR2 in miRNA biogenesis and pre-messenger RNA splicing.

SPAAC-NAD-seq, a sensitive and accurate method to profile NAD+-capped transcripts.

Nicotinamide adenine diphosphate (NAD+) is a novel messenger RNA 5' cap in Escherichia coli, yeast, mammals, and Arabidopsis Transcriptome-wide identification of NAD+-capped RNAs (NAD-RNAs) was accomplished through NAD captureSeq, which combines chemoenzymatic RNA enrichment with high-throughput sequencing. NAD-RNAs are enzymatically converted to alkyne-RNAs that are then biotinylated using a copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. Originally applied to E. coli RNA, which lacks the m7G cap, NAD captureSeq was then applied to eukaryotes without extensive verification of its specificity for NAD-RNAs vs. m7G-capped RNAs (m7G-RNAs). In addition, the Cu2+ ion in the CuAAC reaction causes RNA fragmentation, leading to greatly reduced yield and loss of full-length sequence information. We developed an NAD-RNA capture scheme utilizing the copper-free, strain-promoted azide-alkyne cycloaddition reaction (SPAAC). We examined the specificity of CuAAC and SPAAC reactions toward NAD-RNAs and m7G-RNAs and found that both prefer the former, but also act on the latter. We demonstrated that SPAAC-NAD sequencing (SPAAC-NAD-seq), when combined with immunodepletion of m7G-RNAs, enables NAD-RNA identification with accuracy and sensitivity, leading to the discovery of new NAD-RNA profiles in Arabidopsis Furthermore, SPAAC-NAD-seq retained full-length sequence information. Therefore, SPAAC-NAD-seq would enable specific and efficient discovery of NAD-RNAs in prokaryotes and, when combined with m7G-RNA depletion, in eukaryotes.

High N availability decreases N uptake and yield under limited water availability in maize.

Water and nitrogen (N) are the most limiting factors to plant productivity globally, but we lack a critical understanding of how water availability impacts N dynamics in agricultural systems. Plant N requirements are particularly uncertain when water is limited because of the interactive effect of water and N on plant growth, N demand, and plant uptake. We investigated impacts of N application and water availability on plant growth and N movement, including above and belowground growth, water productivity, N productivity, N uptake, N recovery, and greenhouse gas emissions within a semi-arid system in northeastern Colorado, USA. Moderately high soil N availability depressed grain yield and shoot growth under both limited and full water availability, despite no indication of physical toxicity, and came with additional risk of deleterious N losses. Under low N availability, plant N concentrations in aboveground tissues showed greater recovery of N than what was applied in the low N treatments under both full and limited water availability. This enhanced recovery underscores the need to better understand both plant soil foraging and processes governing resource availability under these conditions. Finally, limited water availability reduced N uptake across all N treatments and left 30% more soil nitrate (NO3-) deep in the soil profile at the end of the season than under full water availability. Our results show that plant N needs are not linearly related to water use and emphasize the need for an integrated understanding of water and N interactions, plant foraging for these resources, and the dynamics of processes that make N available to plants.

Deficit irrigation impacts on greenhouse gas emissions under drip-fertigated maize in the Great Plains of Colorado.

Precise water and fertilizer application can increase crop water productivity and reduce agricultural contributions to greenhouse gas (GHG) emissions. Regulated deficit irrigation (DI) and drip fertigation control the amount, location, and timing of water and nutrient application. Yet, few studies have measured GHG emissions under these practices, especially for maize (Zea mays L.). The objective was to quantify N2 O and CO2 emission from DI and full irrigation (FI) within a drip-fertigated maize system in northeastern Colorado. During two growing seasons of measurement, treatments consisted of mild, moderate, and extreme DI and FI. Deficit irrigation was managed based on growth stage so that full evapotranspiration (ET) was met during the yield-sensitive reproductive stage, but less than full crop ET was applied during the late vegetative and maturation growth stages. In the first year, mild DI (90% ET) reduced N2 O emissions by 50% compared with FI. In the second year, compared with FI, moderate DI (69-80% ET) reduced N2 O emissions by 15%, and extreme DI (54-68% ET) reduced N2 O emissions by 40%. Only extreme DI in the second year significantly reduced CO2 emissions (by 30%) compared with FI. Mild DI reduced yield-scaled emissions in the first year, but moderate and extreme DI had similar yield-scaled emissions as FI in the second year. The surface drip fertigation resulted in total GHG emissions that were one-tenth of literature-based measurements from sprinkler-irrigated maize systems. This study illustrates the potential of DI and drip fertigation to reduce N2 O and CO2 emissions in irrigated cropping systems.

Drought-Induced Root Pressure in Sorghum bicolor.

Root pressure, also manifested as profusive sap flowing from cut stems, is a phenomenon in some species that has perplexed biologists for much of the last century. It is associated with increased crop production under drought, but its function and regulation remain largely unknown. In this study, we investigated the initiation, mechanisms, and possible adaptive function of root pressure in six genotypes of Sorghum bicolor during a drought experiment in the greenhouse. We observed that root pressure was induced in plants exposed to drought followed by re-watering but possibly inhibited by 100% re-watering in some genotypes. We found that root pressure in drought stressed and re-watered plants was associated with greater ratio of fine: coarse root length and shoot biomass production, indicating a possible role of root allocation in creating root pressure and adaptive benefit of root pressure for shoot biomass production. Using RNA-Seq, we identified gene transcripts that were up- and down-regulated in plants with root pressure expression, focusing on genes for aquaporins, membrane transporters, and ATPases that could regulate inter- and intra-cellular transport of water and ions to generate positive xylem pressure in root tissue.