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Competing Interests: Conflict of interest statement. None declared.

Plant cells regularly encounter hypoxia (low-oxygen conditions) as part of normal growth and development, or in response to environmental stresses such as flooding. In recent years, our understanding of the multi-layered control of hypoxia-responsive gene expression has greatly increased. In this Update, we take a broad look at the epigenetic, transcriptional, translational, and post-translational mechanisms that regulate responses to low-oxygen levels. We highlight how a network of post-translational modifications (including phosphorylation), secondary messengers, transcriptional cascades, and retrograde signals from the mitochondria and endoplasmic reticulum (ER) feed into the control of transcription factor activity and hypoxia-responsive gene transcription. We discuss epigenetic mechanisms regulating the response to reduced oxygen availability, through focussing on active and repressive chromatin modifications and DNA methylation. We also describe current knowledge of the co- and post-transcriptional mechanisms that tightly regulate mRNA translation to coordinate effective gene expression under hypoxia. Finally, we present a series of outstanding questions in the field and consider how new insights into the molecular workings of the hypoxia-triggered regulatory hierarchy could pave the way for developing flood-resilient crops., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Flooded plants experience impaired gas diffusion underwater, leading to oxygen deprivation (hypoxia). The volatile plant hormone ethylene is rapidly trapped in submerged plant cells and is instrumental for enhanced hypoxia acclimation. However, the precise mechanisms underpinning ethylene-enhanced hypoxia survival remain unclear. We studied the effect of ethylene pretreatment on hypoxia survival of Arabidopsis (Arabidopsis thaliana) primary root tips. Both hypoxia itself and re-oxygenation following hypoxia are highly damaging to root tip cells, and ethylene pretreatments reduced this damage. Ethylene pretreatment alone altered the abundance of transcripts and proteins involved in hypoxia responses, root growth, translation, and reactive oxygen species (ROS) homeostasis. Through imaging and manipulating ROS abundance in planta, we demonstrated that ethylene limited excessive ROS formation during hypoxia and subsequent re-oxygenation and improved oxidative stress survival in a PHYTOGLOBIN1-dependent manner. In addition, we showed that root growth cessation via ethylene and auxin occurred rapidly and that this quiescence behavior contributed to enhanced hypoxia tolerance. Collectively, our results show that the early flooding signal ethylene modulates a variety of processes that all contribute to hypoxia survival., (© The Author(s) 2022. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Isolated nuclei provide access to early steps in gene regulation involving chromatin as well as transcript production and processing. Here, we describe transfer of the isolation of nuclei from tagged specific cell types (INTACT) to the monocot rice ( Oryza sativa L.). The purification of biotinylated nuclei was redesigned by replacing the outer nuclear-envelope-targeting domain of the nuclear tagging fusion (NTF) protein with an outer nuclear-envelope-anchored domain. This modified NTF was combined with codon-optimized Escherichia coli BirA in a single T-DNA construct. We also developed inexpensive methods for INTACT, T-DNA insertion mapping, and profiling of the complete nuclear transcriptome, including a ribosomal RNA degradation procedure that minimizes pre-ribosomal RNA (pre-rRNA) transcripts. A high-resolution comparison of nuclear and steady-state poly(A) + transcript populations of seedling root tips confirmed the capture of pre-messenger RNA (pre-mRNA) and exposed distinctions in diversity and abundance of the nuclear and total transcriptomes. This retooled INTACT can enable high-resolution monitoring of the nuclear transcriptome and chromatin in specific cell types of rice and other species., (© 2018 American Society of Plant Biologists. All Rights Reserved.)

Proteomic plasticity undergirds stress responses in plants, and understanding such responses requires accurate measurement of the extent to which proteins levels are adjusted to counter external stimuli. Here, we adapt bioorthogonal noncanonical amino acid tagging (BONCAT) to interrogate protein synthesis in vegetative Arabidopsis ( Arabidopsis thaliana ) seedlings. BONCAT relies on the translational incorporation of a noncanonical amino acid probe into cellular proteins. In this study, the probe is the Met surrogate azidohomoalanine (Aha), which carries a reactive azide moiety in its amino acid side chain. The azide handle in Aha can be selectively conjugated to dyes and functionalized beads to enable visualization and enrichment of newly synthesized proteins. We show that BONCAT is sensitive enough to detect Arabidopsis proteins synthesized within a 30-min interval defined by an Aha pulse and that the method can be used to detect proteins made under conditions of light stress, osmotic shock, salt stress, heat stress, and recovery from heat stress. We further establish that BONCAT can be coupled to tandem liquid chromatography-mass spectrometry to identify and quantify proteins synthesized during heat stress and recovery from heat stress. Our results are consistent with a model in which, upon the onset of heat stress, translation is rapidly reprogrammed to enhance the synthesis of stress mitigators and is again altered during recovery. All experiments were carried out with commercially available reagents, highlighting the accessibility of the BONCAT method to researchers interested in stress responses as well as translational and posttranslational regulation in plants., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

Climate change has increased the frequency and severity of flooding events, with significant negative impact on agricultural productivity. These events often submerge plant aerial organs and roots, limiting growth and survival due to a severe reduction in light reactions and gas exchange necessary for photosynthesis and respiration, respectively. To distinguish molecular responses to the compound stress imposed by submergence, we investigated transcriptomic adjustments to darkness in air and under submerged conditions using eight Arabidopsis (Arabidopsis thaliana) accessions differing significantly in sensitivity to submergence. Evaluation of root and rosette transcriptomes revealed an early transcriptional and posttranscriptional response signature that was conserved primarily across genotypes, although flooding susceptibility-associated and genotype-specific responses also were uncovered. Posttranscriptional regulation encompassed darkness- and submergence-induced alternative splicing of transcripts from pathways involved in the alternative mobilization of energy reserves. The organ-specific transcriptome adjustments reflected the distinct physiological status of roots and shoots. Root-specific transcriptome changes included marked up-regulation of chloroplast-encoded photosynthesis and redox-related genes, whereas those of the rosette were related to the regulation of development and growth processes. We identified a novel set of tolerance genes, recognized mainly by quantitative differences. These included a transcriptome signature of more pronounced gluconeogenesis in tolerant accessions, a response that included stress-induced alternative splicing. This study provides organ-specific molecular resolution of genetic variation in submergence responses involving interactions between darkness and low-oxygen constraints of flooding stress and demonstrates that early transcriptome plasticity, including alternative splicing, is associated with the ability to cope with a compound environmental stress., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

The Arabidopsis (Arabidopsis thaliana) genome is the most well-annotated plant genome. However, transcriptome sequencing in Arabidopsis continues to suggest the presence of polyadenylated (polyA) transcripts originating from presumed intergenic regions. It is not clear whether these transcripts represent novel noncoding or protein-coding genes. To understand the nature of intergenic polyA transcription, we first assessed its abundance using multiple messenger RNA sequencing data sets. We found 6,545 intergenic transcribed fragments (ITFs) occupying 3.6% of Arabidopsis intergenic space. In contrast to transcribed fragments that map to protein-coding and RNA genes, most ITFs are significantly shorter, are expressed at significantly lower levels, and tend to be more data set specific. A surprisingly large number of ITFs (32.1%) may be protein coding based on evidence of translation. However, our results indicate that these "translated" ITFs tend to be close to and are likely associated with known genes. To investigate if ITFs are under selection and are functional, we assessed ITF conservation through cross-species as well as within-species comparisons. Our analysis reveals that 237 ITFs, including 49 with translation evidence, are under strong selective constraint and relatively distant from annotated features. These ITFs are likely parts of novel genes. However, the selective pressure imposed on most ITFs is similar to that of randomly selected, untranscribed intergenic sequences. Our findings indicate that despite the prevalence of ITFs, apart from the possibility of genomic contamination, many may be background or noisy transcripts derived from "junk" DNA, whose production may be inherent to the process of transcription and which, on rare occasions, may act as catalysts for the creation of novel genes.

Leaf senescence is a natural age-dependent process that is induced prematurely by various environmental stresses. Typical alterations during leaf senescence include breakdown of chlorophyll, a shift to catabolism of energy reserves, and induction of senescence-associated genes, all of which can occur during submergence, drought, and constant darkness. Here, we evaluated the influence of the submergence tolerance regulator, SUBMERGENCE1A (SUB1A), in the acclimation responses during leaf senescence caused by prolonged darkness in rice (Oryza sativa). SUB1A messenger RNA was highly induced by prolonged darkness in a near-isogenic line containing SUB1A. Genotypes with conditional and ectopic overexpression of SUB1A significantly delayed loss of leaf color and enhanced recovery from dark stress. Physiological analysis revealed that SUB1A postpones dark-induced senescence through the maintenance of chlorophyll and carbohydrate reserves in photosynthetic tissue. This delay allowed leaves of SUB1A genotypes to recover photosynthetic activity more quickly upon reexposure to light. SUB1A also restricted the transcript accumulation of representative senescence-associated genes. Jasmonate and salicylic acid are positive regulators of leaf senescence, but ectopic overexpression of SUB1A dampened responsiveness to both hormones in the context of senescence. We found that ethylene accelerated senescence stimulated by darkness and jasmonate, although SUB1A significantly restrained dark-induced ethylene accumulation. Overall, SUB1A genotypes displayed altered responses to prolonged darkness by limiting ethylene production and responsiveness to jasmonate and salicylic acid, thereby dampening the breakdown of chlorophyll, carbohydrates, and the accumulation of senescence-associated messenger RNAs. A delay of leaf senescence conferred by SUB1A can contribute to the enhancement of tolerance to submergence, drought, and oxidative stress.

Abiotic stress, including drought, salinity, and temperature extremes, regulates gene expression at the transcriptional and posttranscriptional levels. Expression profiling of total messenger RNAs (mRNAs) from rice (Oryza sativa) leaves grown under stress conditions revealed that the transcript levels of photosynthetic genes are reduced more rapidly than others, a phenomenon referred to as stress-induced mRNA decay (SMD). By comparing RNA polymerase II engagement with the steady-state mRNA level, we show here that SMD is a posttranscriptional event. The SMD of photosynthetic genes was further verified by measuring the half-lives of the small subunit of Rubisco (RbcS1) and Chlorophyll a/b-Binding Protein1 (Cab1) mRNAs during stress conditions in the presence of the transcription inhibitor cordycepin. To discern any correlation between SMD and the process of translation, changes in total and polysome-associated mRNA levels after stress were measured. Total and polysome-associated mRNA levels of two photosynthetic (RbcS1 and Cab1) and two stress-inducible (Dehydration Stress-Inducible Protein1 and Salt-Induced Protein) genes were found to be markedly similar. This demonstrated the importance of polysome association for transcript stability under stress conditions. Microarray experiments performed on total and polysomal mRNAs indicate that approximately half of all mRNAs that undergo SMD remain polysome associated during stress treatments. To delineate the functional determinant(s) of mRNAs responsible for SMD, the RbcS1 and Cab1 transcripts were dissected into several components. The expressions of different combinations of the mRNA components were analyzed under stress conditions, revealing that both 3' and 5' untranslated regions are necessary for SMD. Our results, therefore, suggest that the posttranscriptional control of photosynthetic mRNA decay under stress conditions requires both 3' and 5' untranslated regions and correlates with differential polysome association.

High-throughput technology has facilitated genome-scale analyses of transcriptomic adjustments in response to environmental perturbations with an oxygen deprivation component, such as transient hypoxia or anoxia, root waterlogging, or complete submergence. We showed previously that Arabidopsis (Arabidopsis thaliana) seedlings elevate the levels of hundreds of transcripts, including a core group of 49 genes that are prioritized for translation across cell types of both shoots and roots. To recognize low-oxygen responses that are evolutionarily conserved versus species specific, we compared the transcriptomic reconfiguration in 21 organisms from four kingdoms (Plantae, Animalia, Fungi, and Bacteria). Sorting of organism proteomes into clusters of putative orthologs identified broadly conserved responses associated with glycolysis, fermentation, alternative respiration, metabolite transport, reactive oxygen species amelioration, chaperone activity, and ribosome biogenesis. Differentially regulated genes involved in signaling and transcriptional regulation were poorly conserved across kingdoms. Strikingly, nearly half of the induced mRNAs of Arabidopsis seedlings encode proteins of unknown function, of which over 40% had up-regulated orthologs in poplar (Populus trichocarpa), rice (Oryza sativa), or Chlamydomonas reinhardtii. Sixteen HYPOXIA-RESPONSIVE UNKNOWN PROTEIN (HUP) genes, including four that are Arabidopsis specific, were ectopically overexpressed and evaluated for their effect on seedling tolerance to oxygen deprivation. This allowed the identification of HUPs coregulated with genes associated with anaerobic metabolism and other processes that significantly enhance or reduce stress survival when ectopically overexpressed. These findings illuminate both broadly conserved and plant-specific low-oxygen stress responses and confirm that plant-specific HUPs with limited phylogenetic distribution influence low-oxygen stress endurance.

We previously characterized the rice (Oryza sativa) Submergence1 (Sub1) locus encoding three ethylene-responsive factor (ERF) transcriptional regulators. Genotypes carrying the Sub1A-1 allele are tolerant of prolonged submergence. To elucidate the mechanism of Sub1A-1-mediated tolerance, we performed transcriptome analyses comparing the temporal submergence response of Sub1A-1-containing tolerant M202(Sub1) with the intolerant isoline M202 lacking this gene. We identified 898 genes displaying Sub1A-1-dependent regulation. Integration of the expression data with publicly available metabolic pathway data identified submergence tolerance-associated pathways governing anaerobic respiration, hormone responses, and antioxidant systems. Of particular interest were a set of APETALA2 (AP2)/ERF family transcriptional regulators that are associated with the Sub1A-1-mediated response upon submergence. Visualization of expression patterns of the AP2/ERF superfamily members in a phylogenetic context resolved 12 submergence-regulated AP2/ERFs into three putative functional groups: (1) anaerobic respiration and cytokinin-mediated delay in senescence via ethylene accumulation during submergence (three ERFs); (2) negative regulation of ethylene-dependent gene expression (five ERFs); and (3) negative regulation of gibberellin-mediated shoot elongation (four ERFs). These results confirm that the presence of Sub1A-1 impacts multiple pathways of response to submergence.

About 40% of the proteins encoded in eukaryotic genomes are proteins of unknown function (PUFs). Their functional characterization remains one of the main challenges in modern biology. In this study we identified the PUF encoding genes from Arabidopsis (Arabidopsis thaliana) using a combination of sequence similarity, domain-based, and empirical approaches. Large-scale gene expression analyses of 1,310 publicly available Affymetrix chips were performed to associate the identified PUF genes with regulatory networks and biological processes of known function. To generate quality results, the study was restricted to expression sets with replicated samples. First, genome-wide clustering and gene function enrichment analysis of clusters allowed us to associate 1,541 PUF genes with tightly coexpressed genes for proteins of known function (PKFs). Over 70% of them could be assigned to more specific biological process annotations than the ones available in the current Gene Ontology release. The most highly overrepresented functional categories in the obtained clusters were ribosome assembly, photosynthesis, and cell wall pathways. Interestingly, the majority of the PUF genes appeared to be controlled by the same regulatory networks as most PKF genes, because clusters enriched in PUF genes were extremely rare. Second, large-scale analysis of differentially expressed genes was applied to identify a comprehensive set of abiotic stress-response genes. This analysis resulted in the identification of 269 PKF and 104 PUF genes that responded to a wide variety of abiotic stresses, whereas 608 PKF and 206 PUF genes responded predominantly to specific stress treatments. The provided coexpression and differentially expressed gene data represent an important resource for guiding future functional characterization experiments of PUF and PKF genes. Finally, the public Plant Gene Expression Database (http://bioweb.ucr.edu/PED) was developed as part of this project to provide efficient access and mining tools for the vast gene expression data of this study.

Immunoaffinity purification of polyribosomes (polysomes) from crude leaf extracts of Arabidopsis (Arabidopsis thaliana) was achieved with transgenic genotypes that overexpress a translational fusion of a ribosomal protein (RP) with a His(6)-FLAG dual epitope tag. In plants with a cauliflower mosaic virus 35S:HF-RPL18 transgene immunopurification with anti-FLAG agarose beads yielded 60-Svedberg ribosomal subunits, intact 80-Svedberg monosomes and polysomes. Sucrose density gradient fractionation of the purified complexes demonstrated that the distribution of polysome size was similar in crude cell extracts and the purified complexes. The immunopurified complexes included putative cytosolic RPs of Arabidopsis and ribosome-associated proteins, as well as full-length transcripts of high and low abundance. Whole-genome profiling using long DNA oligonucleotide-based microarrays provided a high level of reproducibility between polysomal mRNA samples immunopurified from two independent biological replicates (r approximately 0.90). Comparison of immunopurified and total cellular RNA samples revealed that for most of the genes, the mRNAs were associated with the epitope-tagged polysomal complexes, with an average relative level of association of 62.06% +/- 4.39%. The results demonstrate that the immunopurification of polysomes can be a valuable tool for the quantification of mRNAs present in translation complexes in plant cells. This technology can be extended to evaluation of mRNA populations at the cell- or tissue-specific level by regulation of the tagged RP with distinct promoters.

The genome-wide protein sequences from Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) spp. japonica were clustered into families using sequence similarity and domain-based clustering. The two fundamentally different methods resulted in separate cluster sets with complementary properties to compensate the limitations for accurate family analysis. Functional names for the identified families were assigned with an efficient computational approach that uses the description of the most common molecular function gene ontology node within each cluster. Subsequently, multiple alignments and phylogenetic trees were calculated for the assembled families. All clustering results and their underlying sequences were organized in the Web-accessible Genome Cluster Database (http://bioinfo.ucr.edu/projects/GCD) with rich interactive and user-friendly sequence family mining tools to facilitate the analysis of any given family of interest for the plant science community. An automated clustering pipeline ensures current information for future updates in the annotations of the two genomes and clustering improvements. The analysis allowed the first systematic identification of family and singlet proteins present in both organisms as well as those restricted to one of them. In addition, the established Web resources for mining these data provide a road map for future studies of the composition and structure of protein families between the two species.

Analysis of 80S ribosomes of Arabidopsis (Arabidopsis thaliana) by use of high-speed centrifugation, sucrose gradient fractionation, one- and two-dimensional gel electrophoresis, liquid chromatography purification, and mass spectrometry (matrix-assisted laser desorption/ionization time-of-flight and electrospray ionization) identified 74 ribosomal proteins (r-proteins), of which 73 are orthologs of rat r-proteins and one is the plant-specific r-protein P3. Thirty small (40S) subunit and 44 large (60S) subunit r-proteins were confirmed. In addition, an ortholog of the mammalian receptor for activated protein kinase C, a tryptophan-aspartic acid-domain repeat protein, was found to be associated with the 40S subunit and polysomes. Based on the prediction that each r-protein is present in a single copy, the mass of the Arabidopsis 80S ribosome was estimated as 3.2 MD (1,159 kD 40S; 2,010 kD 60S), with the 4 single-copy rRNAs (18S, 26S, 5.8S, and 5S) contributing 53% of the mass. Despite strong evolutionary conservation in r-protein composition among eukaryotes, Arabidopsis 80S ribosomes are variable in composition due to distinctions in mass or charge of approximately 25% of the r-proteins. This is a consequence of amino acid sequence divergence within r-protein gene families and posttranslational modification of individual r-proteins (e.g. amino-terminal acetylation, phosphorylation). For example, distinct types of r-proteins S15a and P2 accumulate in ribosomes due to evolutionarily divergence of r-protein genes. Ribosome variation is also due to amino acid sequence divergence and differential phosphorylation of the carboxy terminus of r-protein S6. The role of ribosome heterogeneity in differential mRNA translation is discussed.

Ribosomal protein S6 (RPS6) is located in the mRNA binding site of the 40S subunit of cytosolic ribosomes. Two maize (Zea mays) rps6 genes were identified that encode polypeptides (30 kD, 11.4 pI) with strong primary amino acid sequence and predicted secondary structure similarity to RPS6 of other eukaryotes. Maize RPS6 was analyzed by the use of two-dimensional gel electrophoresis systems, in vivo labeling with [(32)P]P(i) and immunological detection. Nine RPS6 isoforms were resolved in a two-dimensional basic-urea/sodium dodecyl sulfate-polyacrylamide gel electrophoresis system. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry performed on trypsin-digested isoforms identified four serine (Ser) and one threonine (Thr) residue in the carboxy-terminal region as phosphorylation sites (RRS(238)KLS(241)AAAKAS(247)AAT(250)S(251)A-COOH). Heterogeneity in RPS6 phosphorylation was a consequence of the presence of zero to five phosphorylated residues. Phosphorylated isoforms fell into two groups characterized by (a) sequential phosphorylation of Ser-238 and Ser-241 and (b) the absence of phospho-Ser-238 and presence of phospho-Ser-241. The accumulation of hyper-phosphorylated isoforms with phospho-Ser-238 was reduced in response to oxygen deprivation and heat shock, whereas accumulation of these isoforms was elevated by cold stress. Salt and osmotic stress had no reproducible effect on RPS6 phosphorylation. The reduction in hyper-phosphorylated isoforms under oxygen deprivation was blocked by okadaic acid, a Ser/Thr phosphatase inhibitor. By contrast, the recovery of hyper-phosphorylated isoforms upon re-oxygenation was blocked by LY-294002, an inhibitor of phosphatidylinositol 3-kinases. Thus, differential activity of phosphatase(s) and kinase(s) determine complex heterogeneity in RPS6 phosphorylation.

Cytosolic isozymes of 6-phosphogluconate dehydrogenase were purified from roots of maize (Zea mays L.). The final preparation contained two 55-kD proteins. Affinity-purified dehydrogenases from a maize line that is null for both cytosolic 6-phosphogluconate dehydrogenase isozymes (Pgd1-null, Pgd2-null) lacked the 55-kD proteins. The substrate kinetics of the purified enzyme were determined.

The hypoxic stress response of Zea mays L. seedling roots involves regulation of gene expression at transcriptional and posttranscriptional levels. We investigated the effect of hypoxia on the translational machinery of seedling roots. The levels of monoribosomes and ribosomal subunits increased dramatically within 1 hour of stress. Prolonged hypoxia resulted in continued accumulation of nontranslating ribosomes, as well as increased levels of small polyribosomes. The return of seedlings to normal aerobic conditions resulted in recovery of normal polyribosome levels. Comparison of ribosomal proteins from control and hypoxic roots revealed differences in quantity and electrophoretic mobility. In vivo labeling of roots with [(35)S]methionine revealed variations in newly synthesized ribosomal proteins. In vivo labeling of roots with [(32)P]orthophosphate revealed a major reduction in the phosphorylation of a 31 kilodalton ribosomal protein in hypoxic stressed roots. In vitro phosphorylation of ribosomal proteins by endogenous kinases was used to probe for differences in ribosome structure and composition. The patterns of in vitro kinased phosphoproteins of ribosomes from control and hypoxic roots were not identical. Variation in phosphoproteins of polyribosomes from control and hypoxic roots, as well as among polyribosomes from hypoxic roots were observed. These results indicate that modification of the translational machinery occurs in response to hypoxic stress.