Functional annotation of proteins for signaling network inference in non-model species.

Molecular biology aims to understand cellular responses and regulatory dynamics in complex biological systems. However, these studies remain challenging in non-model species due to poor functional annotation of regulatory proteins. To overcome this limitation, we develop a multi-layer neural network that determines protein functionality directly from the protein sequence. We annotate kinases and phosphatases in Glycine max. We use the functional annotations from our neural network, Bayesian inference principles, and high resolution phosphoproteomics to infer phosphorylation signaling cascades in soybean exposed to cold, and identify Glyma.10G173000 (TOI5) and Glyma.19G007300 (TOT3) as key temperature regulators. Importantly, the signaling cascade inference does not rely upon known kinase motifs or interaction data, enabling de novo identification of kinase-substrate interactions. Conclusively, our neural network shows generalization and scalability, as such we extend our predictions to Oryza sativa, Zea mays, Sorghum bicolor, and Triticum aestivum. Taken together, we develop a signaling inference approach for non-model species leveraging our predicted kinases and phosphatases.

Changes in the herbicide sensitivity and competitive ability of Abutilon theophrasti over 28 years: Implications for hormesis and weed evolution.

BACKGROUND: The potential of weed species to respond to selection forces affecting the evolution of weedy traits such as competitive ability is poorly understood. This research characterized evolutionary growth changes in a single Abutilon theophrasti Medik. population comparing multiple generations collected from 1988 to 2016. A competition study was performed to understand changes in competitive ability, and a herbicide dose-response study was carried out to assess changes in sensitivity to acetolactate synthase-inhibiting herbicides and glyphosate over time. RESULTS: When grown in monoculture, A. theophrasti biomass production per plant increased steadily across year-lines while leaf number decreased. In replacement experiments, A. theophrasti plants from newer year-lines were more competitive and produced more biomass and leaf area than the oldest year-line. No clear differences in sensitivity to imazamox were observed among year-lines. However, starting in 1995, this A. theophrasti population exhibited a progressive increase in growth in response to a sublethal dose of glyphosate (52 g a.e. ha-1 ), with the 2009 and 2016 year-lines having more than 50% higher biomass than the nontreated control. CONCLUSION: This study demonstrates that weeds can rapidly evolve increased competitive ability. Furthermore, the results indicate the possibility of changes in glyphosate hormesis over time. These results highlight the importance of the role that rapid (i.e., subdecadal) evolution of growth traits might have on the sustainability of weed management strategies. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

The membrane-localized protein kinase MAP4K4/TOT3 regulates thermomorphogenesis.

Plants respond to mild warm temperature conditions by increased elongation growth of organs to enhance cooling capacity, in a process called thermomorphogenesis. To this date, the regulation of thermomorphogenesis has been exclusively shown to intersect with light signalling pathways. To identify regulators of thermomorphogenesis that are conserved in flowering plants, we map changes in protein phosphorylation in both dicots and monocots exposed to warm temperature. We identify MITOGEN-ACTIVATED PROTEIN KINASE KINASE KINASE KINASE4 (MAP4K4)/TARGET OF TEMPERATURE3 (TOT3) as a regulator of thermomorphogenesis that impinges on brassinosteroid signalling in Arabidopsis thaliana. In addition, we show that TOT3 plays a role in thermal response in wheat, a monocot crop. Altogether, the conserved thermal regulation by TOT3 expands our knowledge of thermomorphogenesis beyond the well-studied pathways and can contribute to ensuring food security under a changing climate.

Flood resilience loci SUBMERGENCE 1 and ANAEROBIC GERMINATION 1 interact in seedlings established underwater.

Crops with resilience to multiple climatic stresses are essential for increased yield stability. Here, we evaluate the interaction between two loci associated with flooding survival in rice (Oryza sativa L.). ANAEROBIC GERMINATION 1 (AG1), encoding trehalose 6-phosphate phosphatase 7 (TPP7), promotes mobilization of endosperm reserves to enhance the elongation of a hollow coleoptile in seeds that are seeded directly into shallow paddies. SUBMERGENCE 1 (SUB1), encoding the ethylene-responsive transcription factor SUB1A-1, confers tolerance to complete submergence by dampening carbohydrate catabolism, to enhance recovery upon desubmergence. Interactions between AG1/TPP7 and SUB1/SUB1A-1 were investigated under three flooding scenarios using four near-isogenic lines by surveying growth and survival. Pyramiding of the two loci does not negatively affect anaerobic germination or vegetative-stage submergence tolerance. However, the pyramided AG1 SUB1 genotype displays reduced survival when seeds are planted underwater and maintained under submergence for 16 d. To better understand the roles of TPP7 and SUB1A-1 and their interaction, temporal changes in carbohydrates and shoot transcriptomes were monitored in the four genotypes varying at the two loci at four developmental timeponts, from day 2 after seeding through day 14 of complete submergence. TPP7 enhances early coleoptile elongation, whereas SUB1A-1 promotes precocious photoautotrophy and then restricts underwater elongation. By contrast, pyramiding of the AG1 and SUB1 slows elongation growth, the transition to photoautotrophy, and survival. mRNA-sequencing highlights time-dependent and genotype-specific regulation of mRNAs associated with DNA repair, cell cycle, chromatin modification, plastid biogenesis, carbohydrate catabolism and transport, elongation growth, and other processes. These results suggest that interactions between AG1/TPP7 and SUB1/SUB1A-1 could impact seedling establishment if paddy depth is not effectively managed after direct seeding.

Rice SUB1A constrains remodelling of the transcriptome and metabolome during submergence to facilitate post-submergence recovery.

The rice (Oryza sativa L.) ethylene-responsive transcription factor gene SUB1A-1 confers tolerance to prolonged, complete submergence by limiting underwater elongation growth. Upon desubmergence, SUB1A-1 genotypes rapidly recover photosynthetic function and recommence development towards flowering. The underpinnings of the transition from stress amelioration to the return to homeostasis are not well known. Here, transcriptomic and metabolomic analyses were conducted to identify mechanisms by which SUB1A improves physiological function over the 24 hr following a sublethal submergence event. Evaluation of near-isogenic genotypes after submergence and over a day of reaeration demonstrated that SUB1A transiently constrains the remodelling of cellular activities associated with growth. SUB1A influenced the abundance of ca. 1,400 transcripts and had a continued impact on metabolite content, particularly free amino acids, glucose, and sucrose, throughout the recovery period. SUB1A promoted recovery of metabolic homeostasis but had limited influence on mRNAs associated with growth processes and photosynthesis. The involvement of low energy sensing during submergence and recovery was supported by dynamics in trehalose-6-phosphate and mRNAs encoding key enzymes and signalling proteins, which were modulated by SUB1A. This study provides new evidence of convergent signalling pathways critical to the rapidly reversible management of carbon and nitrogen metabolism in submergence resilient rice.

Field-grown soybean transcriptome shows diurnal patterns in photosynthesis-related processes.

Many plant physiological processes have diurnal patterns regulated by diurnal environmental changes and circadian rhythms, but the transcriptional underpinnings of many of these cycles have not been studied in major crop species under field conditions. Here, we monitored the transcriptome of field-grown soybean (Glycine max) during daylight hours in the middle of the growing season with RNA-seq. The analysis revealed 21% of soybean genes were differentially expressed over the course of the day. Expression of some circadian-related genes in field-grown soybean differed from previously reported expression patterns measured in controlled environments. Many genes in functional groups contributing to and/or depending on photosynthesis showed differential expression, with patterns particularly evident in the chlorophyll synthesis pathway. Gene regulatory network inference also revealed seven diurnally sensitive gene nodes involved with circadian rhythm, transcription regulation, cellular processes, and water transport. This study provides a diurnal overview of the transcriptome for an economically important field-grown crop and a basis for identifying pathways that could eventually be tailored to optimize diurnal regulation of carbon gain.

Intensifying drought eliminates the expected benefits of elevated carbon dioxide for soybean.

Stimulation of C3 crop yield by rising concentrations of atmospheric carbon dioxide ([CO2]) is widely expected to counteract crop losses that are due to greater drought this century. But these expectations come from sparse field trials that have been biased towards mesic growth conditions. This eight-year study used precipitation manipulation and year-to-year variation in weather conditions at a unique open-air field facility to show that the stimulation of soybean yield by elevated [CO2] diminished to zero as drought intensified. Contrary to the prevalent expectation in the literature, rising [CO2] did not counteract the effect of strong drought on photosynthesis and yield because elevated [CO2] interacted with drought to modify stomatal function and canopy energy balance. This new insight from field experimentation under hot and dry conditions, which will become increasingly prevalent in the coming decades, highlights the likelihood of negative impacts from interacting global change factors on a key global commodity crop in its primary region of production.

Heat waves imposed during early pod development in soybean (Glycine max) cause significant yield loss despite a rapid recovery from oxidative stress.

Heat waves already have a large impact on crops and are predicted to become more intense and more frequent in the future. In this study, heat waves were imposed on soybean using infrared heating technology in a fully open-air field experiment. Five separate heat waves were applied to field-grown soybean (Glycine max) in central Illinois, three in 2010 and two in 2011. Thirty years of historical weather data from Illinois were analyzed to determine the length and intensity of a regionally realistic heat wave resulting in experimental heat wave treatments during which day and night canopy temperatures were elevated 6 °C above ambient for 3 days. Heat waves were applied during early or late reproductive stages to determine whether and when heat waves had an impact on carbon metabolism and seed yield. By the third day of each heat wave, net photosynthesis (A), specific leaf weight (SLW), and leaf total nonstructural carbohydrate concentration (TNC) were decreased, while leaf oxidative stress was increased. However, A, SLW, TNC, and measures of oxidative stress were no different than the control ca. 12 h after the heat waves ended, indicating rapid physiological recovery from the high-temperature stress. That end of season seed yield was reduced (~10%) only when heat waves were applied during early pod developmental stages indicates the yield loss had more to do with direct impacts of the heat waves on reproductive process than on photosynthesis. Soybean was unable to mitigate yield loss after heat waves given during late reproductive stages. This study shows that short high-temperature stress events that reduce photosynthesis and increase oxidative stress resulted in significant losses to soybean production in the Midwest, U.S. The study also suggests that to mitigate heat wave-induced yield loss, soybean needs improved reproductive and photosynthetic tolerance to high but increasingly common temperatures.

Leaf hydraulic conductance declines in coordination with photosynthesis, transpiration and leaf water status as soybean leaves age regardless of soil moisture.

Photosynthesis requires sufficient water transport through leaves for stomata to remain open as water transpires from the leaf, allowing CO2 to diffuse into the leaf. The leaf water needs of soybean change over time because of large microenvironment changes over their lifespan, as leaves mature in full sun at the top of the canopy and then become progressively shaded by younger leaves developing above. Leaf hydraulic conductance (K(leaf)), a measure of the leaf's water transport capacity, can often be linked to changes in microenvironment and transpiration demand. In this study, we tested the hypothesis that K(leaf) would decline in coordination with transpiration demand as soybean leaves matured and aged. Photosynthesis (A), stomatal conductance (g(s)) and leaf water potential (Ψ(leaf)) were also measured at various leaf ages with both field- and chamber-grown soybeans to assess transpiration demand. K(leaf) was found to decrease as soybean leaves aged from maturity to shading to senescence, and this decrease was strongly correlated with midday A. Decreases in K(leaf) were further correlated with decreases in g(s), although the relationship was not as strong as that with A. Separate experiments investigating the response of K(leaf) to drought demonstrated no acclimation of K(leaf) to drought conditions to protect against cavitation or loss of g(s) during drought and confirmed the effect of leaf age in K(leaf) observed in the field. These results suggest that the decline of leaf hydraulic conductance as leaves age keeps hydraulic supply in balance with demand without K(leaf)becoming limiting to transpiration water flux.

Soybean leaf hydraulic conductance does not acclimate to growth at elevated [CO2] or temperature in growth chambers or in the field.

BACKGROUND AND AIMS: Leaf hydraulic properties are strongly linked with transpiration and photosynthesis in many species. However, it is not known if gas exchange and hydraulics will have co-ordinated responses to climate change. The objective of this study was to investigate the responses of leaf hydraulic conductance (Kleaf) in Glycine max (soybean) to growth at elevated [CO2] and increased temperature compared with the responses of leaf gas exchange and leaf water status. METHODS: Two controlled-environment growth chamber experiments were conducted with soybean to measure Kleaf, stomatal conductance (gs) and photosynthesis (A) during growth at elevated [CO2] and temperature relative to ambient levels. These results were validated with field experiments on soybean grown under free-air elevated [CO2] (FACE) and canopy warming. KEY RESULTS: In chamber studies, Kleaf did not acclimate to growth at elevated [CO2], even though stomatal conductance decreased and photosynthesis increased. Growth at elevated temperature also did not affect Kleaf, although gs and A showed significant but inconsistent decreases. The lack of response of Kleaf to growth at increased [CO2] and temperature in chamber-grown plants was confirmed with field-grown soybean at a FACE facility. CONCLUSIONS: Leaf hydraulic and leaf gas exchange responses to these two climate change factors were not strongly linked in soybean, although gs responded to [CO2] and increased temperature as previously reported. This differential behaviour could lead to an imbalance between hydraulic supply and transpiration demand under extreme environmental conditions likely to become more common as global climate continues to change.

Over-expressing the C(3) photosynthesis cycle enzyme Sedoheptulose-1-7 Bisphosphatase improves photosynthetic carbon gain and yield under fully open air CO(2) fumigation (FACE).

BACKGROUND: Biochemical models predict that photosynthesis in C(3) plants is most frequently limited by the slower of two processes, the maximum capacity of the enzyme Rubisco to carboxylate RuBP (V(c,max)), or the regeneration of RuBP via electron transport (J). At current atmospheric [CO(2)] levels Rubisco is not saturated; consequently, elevating [CO(2)] increases the velocity of carboxylation and inhibits the competing oxygenation reaction which is also catalyzed by Rubisco. In the future, leaf photosynthesis (A) should be increasingly limited by RuBP regeneration, as [CO(2)] is predicted to exceed 550 ppm by 2050. The C(3) cycle enzyme sedoheptulose-1,7 bisphosphatase (SBPase, EC 3.1.3.17) has been shown to exert strong metabolic control over RuBP regeneration at light saturation. RESULTS: We tested the hypothesis that tobacco transformed to overexpressing SBPase will exhibit greater stimulation of A than wild type (WT) tobacco when grown under field conditions at elevated [CO(2)] (585 ppm) under fully open air fumigation. Growth under elevated [CO(2)] stimulated instantaneous A and the diurnal photosynthetic integral (A') more in transformants than WT. There was evidence of photosynthetic acclimation to elevated [CO(2)] via downregulation of V(c,max) in both WT and transformants. Nevertheless, greater carbon assimilation and electron transport rates (J and J(max)) for transformants led to greater yield increases than WT at elevated [CO(2)] compared to ambient grown plants. CONCLUSION: These results provide proof of concept that increasing content and activity of a single photosynthesis enzyme can enhance carbon assimilation and yield of C(3) crops grown at [CO(2)] expected by the middle of the 21st century.