Spatially resolved gene regulatory networks in Asian rice (Oryza sativa cv. Nipponbare) leaves.

Transcriptome profiles in plants are heterogenous at every level of morphological organization. Even within organs, cells of the same type can have different patterns of gene expression depending on where they are positioned within tissues. This heterogeneity is associated with non-uniform distribution of biological processes within organs. The regulatory mechanisms that establish and sustain the spatial heterogeneity are unknown. Here, we identify regulatory modules that support functional specialization of different parts of Oryza sativa cv. Nipponbare leaves by leveraging transcriptome data, transcription factor binding motifs and global gene regulatory network prediction algorithms. We generated a global gene regulatory network in which we identified six regulatory modules that were active in different parts of the leaf. The regulatory modules were enriched for genes involved in spatially relevant biological processes, such as cell wall deposition, environmental sensing and photosynthesis. Strikingly, more than 86.9% of genes in the network were regulated by members of only five transcription factor families. We also generated targeted regulatory networks for the large MYB and bZIP/bHLH families to identify interactions that were masked in the global prediction. This analysis will provide a baseline for future single cell and array-based spatial transcriptome studies and for studying responses to environmental stress and demonstrates the extent to which seven coarse spatial transcriptome analysis can provide insight into the regulatory mechanisms supporting functional specialization within leaves.

Plant stem cells under low oxygen: metabolic rewiring by phytoglobin underlies stem cell functionality.

Root growth in maize (Zea mays L.) is regulated by the activity of the quiescent center (QC) stem cells located within the root apical meristem. Here, we show that despite being highly hypoxic under normal oxygen tension, QC stem cells are vulnerable to hypoxic stress, which causes their degradation with subsequent inhibition of root growth. Under low oxygen, QC stem cells became depleted of starch and soluble sugars and exhibited reliance on glycolytic fermentation with the impairment of the TCA cycle through the depressed activity of several enzymes, including pyruvate dehydrogenase (PDH). This finding suggests that carbohydrate delivery from the shoot might be insufficient to meet the metabolic demand of QC stem cells during stress. Some metabolic changes characteristic of the hypoxic response in mature root cells were not observed in the QC. Hypoxia-responsive genes, such as PYRUVATE DECARBOXYLASE (PDC) and ALCOHOL DEHYDROGENASE (ADH), were not activated in response to hypoxia, despite an increase in ADH activity. Increases in phosphoenolpyruvate (PEP) with little change in steady-state levels of succinate were also atypical responses to low-oxygen tensions. Overexpression of PHYTOGLOBIN 1 (ZmPgb1.1) preserved the functionality of the QC stem cells during stress. The QC stem cell preservation was underpinned by extensive metabolic rewiring centered around activation of the TCA cycle and retention of carbohydrate storage products, denoting a more efficient energy production and diminished demand for carbohydrates under conditions where nutrient transport may be limiting. Overall, this study provides an overview of metabolic responses occurring in plant stem cells during oxygen deficiency.

New interstellar laboratories in the molecular ring.

Much of what is known about chemistry in star-forming regions comes from observations of nearby (d < 500 pc) low-mass protostars. For chemistry in high-mass star-forming regions, several more distant (d ∼ 2-8 kpc), exceptionally bright molecular sources have also been the subjects of repeated observations but with concomitantly poorer linear spatial resolution. Facilities such as ALMA and JWST, however, now provide the means for observing distant sources at dramatically higher spatial resolution and sensitivity. We used the modest resolving power of the Atacama Compact Array, a dedicated subset of ALMA antennas, to carry out a pilot survey of 11 giant molecular clouds selected from the Bolocam Galactic Plane Survey [Battisti & Heyer, Astrophys. J., 2014, 780, 173] within the so-called molecular ring between about 4 and 8 kpc from the galactic center. Within our observed sample, molecular emission regions-most of which correspond to at least one (candidate) young stellar object-exhibit a range of chemical complexity and diversity. Furthermore, nine target giant molecular clouds contain well-fit methanol emission, giving us a first look at the spatial chemical variability within the objects at relatively high (compared to past observations) resolutions of ∼5''. This work lays the foundation for future high angular resolution studies of gas-phase chemistry with the full ALMA.

Relationship between CH3OD Abundance and Temperature in the Orion KL Nebula.

The relative abundances of singly deuterated methanol isotopologues, [CH2DOH]/[CH3OD], in star-forming regions deviate from the statistically expected ratio of 3. In Orion KL, the nearest high-mass star-forming region to Earth, the singly deuterated methanol ratio is about 1, and the cause for this observation has been explored through theory for nearly three decades. We present high-angular resolution observations of Orion KL using the Atacama Large Millimeter/submillimeter Array to map small-scale changes in CH3OD column density across the nebula, which provide a new avenue to examine the deuterium chemistry during star and planet formation. By considering how CH3OD column densities vary with temperature, we find evidence of chemical processes that can significantly alter the observed gas-phase column densities. The astronomical data are compared with existing theoretical work and support D-H exchange between CH3OH and heavy water (i.e., HDO and D2O) at methanol's hydroxyl site in the icy mantles of dust grains. The enhanced CH3OD column densities are localized to the Hot Core-SW region, a pattern that may be linked to the coupled evolution of ice mantle chemistry and star formation in giant molecular clouds. This work provides new perspectives on deuterated methanol chemistry in Orion KL and informs considerations that may guide future theoretical, experimental, and observational work.

Single cell gene regulatory networks in plants: Opportunities for enhancing climate change stress resilience.

Global warming poses major challenges for plant survival and agricultural productivity. Thus, efforts to enhance stress resilience in plants are key strategies for protecting food security. Gene regulatory networks (GRNs) are a critical mechanism conferring stress resilience. Until recently, predicting GRNs of the individual cells that make up plants and other multicellular organisms was impeded by aggregate population scale measurements of transcriptome and other genome-scale features. With the advancement of high-throughput single cell RNA-seq and other single cell assays, learning GRNs for individual cells is now possible, in principle. In this article, we report on recent advances in experimental and analytical methodologies for single cell sequencing assays especially as they have been applied to the study of plants. We highlight recent advances and ongoing challenges for scGRN prediction, and finally, we highlight the opportunity to use scGRN discovery for studying and ultimately enhancing abiotic stress resilience in plants.

EGRINs (Environmental Gene Regulatory Influence Networks) in Rice That Function in the Response to Water Deficit, High Temperature, and Agricultural Environments.

Environmental gene regulatory influence networks (EGRINs) coordinate the timing and rate of gene expression in response to environmental signals. EGRINs encompass many layers of regulation, which culminate in changes in accumulated transcript levels. Here, we inferred EGRINs for the response of five tropical Asian rice (Oryza sativa) cultivars to high temperatures, water deficit, and agricultural field conditions by systematically integrating time-series transcriptome data, patterns of nucleosome-free chromatin, and the occurrence of known cis-regulatory elements. First, we identified 5447 putative target genes for 445 transcription factors (TFs) by connecting TFs with genes harboring known cis-regulatory motifs in nucleosome-free regions proximal to their transcriptional start sites. We then used network component analysis to estimate the regulatory activity for each TF based on the expression of its putative target genes. Finally, we inferred an EGRIN using the estimated transcription factor activity (TFA) as the regulator. The EGRINs include regulatory interactions between 4052 target genes regulated by 113 TFs. We resolved distinct regulatory roles for members of the heat shock factor family, including a putative regulatory connection between abiotic stress and the circadian clock. TFA estimation using network component analysis is an effective way of incorporating multiple genome-scale measurements into network inference.

Meta-analysis and meta-regression of transcriptomic responses to water stress in Arabidopsis.

The large amounts of transcriptome data available for Arabidopsis thaliana make a compelling case for the need to generalize results across studies and extract the most robust and meaningful information possible from them. The results of various studies seeking to identify water stress-responsive genes only partially overlap. The aim of this work was to combine transcriptomic studies in a systematic way that identifies commonalities in response, taking into account variation among studies due to batch effects as well as sampling variation, while also identifying the effect of study-specific variables, such as the method of applying water stress, and the part of the plant the mRNA was extracted from. We used meta-analysis, the quantitative synthesis of independent research results, to summarize expression responses to water stress across studies, and meta-regression to model the contribution of covariates that may affect gene expression. We found that some genes with small but consistent differential responses become evident only when results are synthesized across experiments, and are missed in individual studies. We also identified genes with expression responses that are attributable to use of different plant parts and alternative methods for inducing water stress. Our results indicate that meta-analysis and meta-regression provide a powerful approach for identifying a robust gene set that is less sensitive to idiosyncratic results and for quantifying study characteristics that result in contrasting gene expression responses across studies. Combining meta-analysis with individual analyses may contribute to a richer understanding of the biology of water stress responses, and may prove valuable in other gene expression studies.

Possible loss of the chloroplast genome in the parasitic flowering plant Rafflesia lagascae (Rafflesiaceae).

Rafflesia is a genus of holoparasitic plants endemic to Southeast Asia that has lost the ability to undertake photosynthesis. With short-read sequencing technology, we assembled a draft sequence of the mitochondrial genome of Rafflesia lagascae Blanco, a species endemic to the Philippine island of Luzon, with ∼350× sequencing depth coverage. Using multiple approaches, however, we were only able to identify small fragments of plastid sequences at low coverage depth (<2×) and could not recover any substantial portion of a chloroplast genome. The gene fragments we identified included photosynthesis and energy production genes (atp, ndh, pet, psa, psb, rbcL), ribosomal RNA genes (rrn16, rrn23), ribosomal protein genes (rps7, rps11, rps16), transfer RNA genes, as well as matK, accD, ycf2, and multiple nongenic regions from the inverted repeats. None of the identified plastid gene sequences had intact reading frames. Phylogenetic analysis suggests that ∼33% of these remnant plastid genes may have been horizontally transferred from the host plant genus Tetrastigma with the rest having ambiguous phylogenetic positions (<50% bootstrap support), except for psaB that was strongly allied with the plastid homolog in Nicotiana. Our inability to identify substantial plastid genome sequences from R. lagascae using multiple approaches--despite success in identifying and developing a draft assembly of the much larger mitochondrial genome--suggests that the parasitic plant genus Rafflesia may be the first plant group for which there is no recognizable plastid genome, or if present is found in cryptic form at very low levels.

Interplay between sucrose and folate modulates auxin signaling in Arabidopsis.

As sessile organisms growing in an ever-changing environment, plants must integrate multiple regulatory inputs to promote the appropriate developmental responses. One such nutritional signal is cellular sugar levels, which rise and fall throughout the day and affect a variety of developmental processes. To uncover signaling pathways that modulate sugar perception, compounds from the Library of Active Compounds in Arabidopsis were screened for the ability to perturb developmental responses to sucrose (Suc) in Arabidopsis (Arabidopsis thaliana) seedlings. This screen found that sulfonamides, which inhibit folate biosynthesis in plants, restrict hypocotyl elongation in a sugar-dependent fashion. Transcriptome analysis identified a small set of transcripts that respond to the interaction between sulfonamide and Suc, including a number of transcripts encoding Auxin/Indole-3-Acetic Acids, negative regulators of auxin signal transduction. Chemical inhibition of auxin transport or genetic disruption of auxin signaling relieved this interaction, suggesting that responses to these two nutritional stimuli are mediated by auxin. Reporter systems used to track auxin signaling and distribution showed enhanced activity in the vascular region of the hypocotyl in response to cotreatment of Suc and sulfonamide, yet no change in auxin abundance was observed. Taken together, these findings suggest that the interplay between Suc and folates acts to fine-tune auxin sensitivity and influences auxin distribution during seedling development.

PlaNet: combined sequence and expression comparisons across plant networks derived from seven species.

The model organism Arabidopsis thaliana is readily used in basic research due to resource availability and relative speed of data acquisition. A major goal is to transfer acquired knowledge from Arabidopsis to crop species. However, the identification of functional equivalents of well-characterized Arabidopsis genes in other plants is a nontrivial task. It is well documented that transcriptionally coordinated genes tend to be functionally related and that such relationships may be conserved across different species and even kingdoms. To exploit such relationships, we constructed whole-genome coexpression networks for Arabidopsis and six important plant crop species. The interactive networks, clustered using the HCCA algorithm, are provided under the banner PlaNet (http://aranet.mpimp-golm.mpg.de). We implemented a comparative network algorithm that estimates similarities between network structures. Thus, the platform can be used to swiftly infer similar coexpressed network vicinities within and across species and can predict the identity of functional homologs. We exemplify this using the PSA-D and chalcone synthase-related gene networks. Finally, we assessed how ontology terms are transcriptionally connected in the seven species and provide the corresponding MapMan term coexpression networks. The data support the contention that this platform will considerably improve transfer of knowledge generated in Arabidopsis to valuable crop species.

Clone history shapes Populus drought responses.

Just as animal monozygotic twins can experience different environmental conditions by being reared apart, individual genetically identical trees of the genus Populus can also be exposed to contrasting environmental conditions by being grown in different locations. As such, clonally propagated Populus trees provide an opportunity to interrogate the impact of individual environmental history on current response to environmental stimuli. To test the hypothesis that current responses to an environmental stimulus, drought, are contingent on environmental history, the transcriptome- level drought responses of three economically important hybrid genotypes-DN34 (Populus deltoides × Populus nigra), Walker [P. deltoides var. occidentalis × (Populus laurifolia × P. nigra)], and Okanese [Walker × (P. laurifolia × P. nigra)]-derived from two different locations were compared. Strikingly, differences in transcript abundance patterns in response to drought were based on differences in geographic origin of clones for two of the three genotypes. This observation was most pronounced for the genotypes with the longest time since establishment and last common propagation. Differences in genome-wide DNA methylation paralleled the transcriptome level trends, whereby the clones with the most divergent transcriptomes and clone history had the most marked differences in the extent of total DNA methylation, suggesting an epigenomic basis for the clone history-dependent transcriptome divergence. The data provide insights into the interplay between genotype and environment in the ecologically and economically important Populus genus, with implications for the industrial application of Populus trees and the evolution and persistence of these important tree species and their associated hybrids.

Highlights from Faraday Discussion: Astrochemistry at high resolution, Baltimore, USA, May 2023.

The Faraday Discussion 'Astrochemistry at high resolution' was held at the Space Telescope Science Institute, Baltimore, United States, and online from May 31-June 2, 2023. The meeting brought together observers, modellers, and experimentalists at different career stages and from different countries to discuss advancements in astrochemistry resulting from improved spatial resolution, spectral resolution, and sensitivity. This conference report provides highlights of the meeting and summaries of the talks presented.

AtMYB61, an R2R3-MYB transcription factor, functions as a pleiotropic regulator via a small gene network.

Throughout their lifetimes, plants must coordinate the regulation of various facets of growth and development. Previous evidence has suggested that the Arabidopsis thaliana R2R3-MYB, AtMYB61, might function as a coordinate regulator of multiple aspects of plant resource allocation. Using a combination of cell biology, transcriptome analysis and biochemistry, in conjunction with gain-of-function and loss-of-function genetics, the role of AtMYB61 in conditioning resource allocation throughout the plant life cycle was explored. In keeping with its role as a regulator of resource allocation, AtMYB61 is expressed in sink tissues, notably xylem, roots and developing seeds. Loss of AtMYB61 function decreases xylem formation, induces qualitative changes in xylem cell structure and decreases lateral root formation; in contrast, gain of AtMYB61 function has the opposite effect on these traits. AtMYB61 coordinates a small network of downstream target genes, which contain a motif in their upstream regulatory regions that is bound by AtMYB61, and AtMYB61 activates transcription from this same motif. Loss-of-function analysis supports the hypothesis that AtMYB61 targets play roles in shaping subsets of AtMYB61-related phenotypes. Taken together, these findings suggest that AtMYB61 links the transcriptional control of multiple aspects of plant resource allocation.

Growth-limiting drought stress induces time-of-day-dependent transcriptome and physiological responses in hybrid poplar.

Drought stress negatively impacts the health of long-lived trees. Understanding the genetic mechanisms that underpin response to drought stress is requisite for selecting or enhancing climate change resilience. We aimed to determine how hybrid poplars respond to prolonged and uniform exposure to drought; how responses to moderate and more severe growth-limiting drought stresses differed; and how drought responses change throughout the day. We established hybrid poplar trees (Populus × 'Okanese') from unrooted stem cutting with abundant soil moisture for 6 weeks. We then withheld water to establish well-watered, moderate and severe growth-limiting drought conditions. These conditions were maintained for 3 weeks during which growth was monitored. We then measured photosynthetic rates and transcriptomes of leaves that had developed during the drought treatments at two times of day. The moderate and severe drought treatments elicited distinct changes in growth and development, photosynthetic rates and global transcriptome profiles. Notably, the time of day of sampling produced the strongest effect in the transcriptome data. The moderate drought treatment elicited global transcriptome changes that were intermediate to the severe and well-watered treatments in the early evening but did not elicit a strong drought response in the morning. Stable drought conditions that are sufficient to limit plant growth elicit distinct transcriptional profiles depending on the degree of water limitation and on the time of day at which they are measured. There appears to be a limited number of genes and functional gene categories that are responsive to all of the tested drought conditions in this study emphasizing the complex nature of drought regulation in long-lived trees.

Host induced gene silencing of the Sclerotinia sclerotiorum ABHYDROLASE-3 gene reduces disease severity in Brassica napus.

Sclerotinia sclerotiorum is a pathogenic fungus that infects hundreds of crop species, causing extensive yield loss every year. Chemical fungicides are used to control this phytopathogen, but with concerns about increasing resistance and impacts on non-target species, there is a need to develop alternative control measures. In the present study, we engineered Brassica napus to constitutively express a hairpin (hp)RNA molecule to silence ABHYRDOLASE-3 in S. sclerotiorum. We demonstrate the potential for Host Induced Gene Silencing (HIGS) to protect B. napus from S. sclerotiorum using leaf, stem and whole plant infection assays. The interaction between the transgenic host plant and invading pathogen was further characterized at the molecular level using dual-RNA sequencing and at the anatomical level through microscopy to understand the processes and possible mechanisms leading to increased tolerance to this damaging necrotroph. We observed significant shifts in the expression of genes relating to plant defense as well as cellular differences in the form of structural barriers around the site of infection in the HIGS-protected plants. Our results provide proof-of-concept that HIGS is an effective means of limiting damage caused by S. sclerotiorum to the plant and demonstrates the utility of this biotechnology in the development of resistance against fungal pathogens.

Time of day shapes Arabidopsis drought transcriptomes.

Under natural conditions, it is common for plants to experience water deprivation (drought) for periods of days or longer. Plants respond to drought stress by reconfiguring their transcriptome activity. Transcriptome changes in response to drought are dynamic, and are shaped by mitigating factors like time during the diurnal cycle. To date, analyses of drought-induced transcriptome remodelling have concentrated on dynamic changes induced by rapid desiccation, or changes at a single time point following gradual water stress. To gain insights into the dynamics of transcriptome reconfiguration in response to gradual drying of the soil, the drought-induced transcriptomes of Arabidopsis thaliana were examined at four time points over a single diel period - midday, late day, midnight, and pre-dawn. Transcriptome reconfigurations were induced by drought in advance of changes to relative water content, leaf water loss, and chlorophyll content. Comparative analyses support the hypothesis that the drought-responsive transcriptomes were shaped by invocation of distinct hormonal and stress response pathways at different times of the day. While a core set of genes were drought responsive at multiple time points throughout the day, the magnitude of the response varied in a manner dependent on the time of day. Moreover, analysis of a single time point would fail to identify suites of drought-responsive genes that can only be detected through assessment of the dynamics of diurnal changes, emphasising the value of characterising multiple time-of-day-specific drought transcriptomes.