Protein modularity, cooperative binding, and hybrid regulatory states underlie transcriptional network diversification.

We examine how different transcriptional network structures can evolve from an ancestral network. By characterizing how the ancestral mode of gene regulation for genes specific to a-type cells in yeast species evolved from an activating paradigm to a repressing one, we show that regulatory protein modularity, conversion of one cis-regulatory sequence to another, distribution of binding energy among protein-protein and protein-DNA interactions, and exploitation of ancestral network features all contribute to the evolution of a novel regulatory mode. The formation of this derived mode of regulation did not disrupt the ancestral mode and thereby created a hybrid regulatory state where both means of transcription regulation (ancestral and derived) contribute to the conserved expression pattern of the network. Finally, we show how this hybrid regulatory state has resolved in different ways in different lineages to generate the diversity of regulatory network structures observed in modern species.

Time-resolved systems analysis of the induction of high photosynthetic capacity in Arabidopsis during acclimation to high light.

Induction of high photosynthetic capacity is a key acclimation response to high light (HL) for many herbaceous dicot plants; however, the signaling pathways that control this response remain largely unknown. Here, a systems biology approach was utilized to characterize the induction of high photosynthetic capacity in strongly and weakly acclimating Arabidopsis thaliana accessions. Plants were grown for 5 wk in a low light (LL) regime, and time-resolved photosynthetic physiological, metabolomic, and transcriptomic responses were measured during subsequent exposure to HL. The induction of high nitrogen (N) assimilation rates early in the HL shift was strongly predictive of the induction of photosynthetic capacity later in the HL shift. Accelerated N assimilation rates depended on the mobilization of existing organic acid (OA) reserves and increased de novo OA synthesis during the induction of high photosynthetic capacity. Enhanced sucrose biosynthesis capacity increased in tandem with the induction of high photosynthetic capacity, and increased starch biosynthetic capacity was balanced by increased starch catabolism. This systems analysis supports a model in which the efficient induction of N assimilation early in the HL shift begins the cascade of events necessary for the induction of high photosynthetic capacity acclimation in HL.

Dynamics of Rubisco regulation by sugar phosphate derivatives and their phosphatases.

Regulating the central CO2-fixing enzyme Rubisco is as complex as its ancient reaction mechanism and involves interaction with a series of cofactors and auxiliary proteins that activate catalytic sites and maintain activity. A key component among the regulatory mechanisms is the binding of sugar phosphate derivatives that inhibit activity. Removal of inhibitors via the action of Rubisco activase is required to restore catalytic competency. In addition, specific phosphatases dephosphorylate newly released inhibitors, rendering them incapable of binding to Rubisco catalytic sites. The best studied inhibitor is 2-carboxy-d-arabinitol 1-phosphate (CA1P), a naturally occurring nocturnal inhibitor that accumulates in most species during darkness and low light, progressively binding to Rubisco. As light increases, Rubisco activase removes CA1P from Rubisco, and the specific phosphatase CA1Pase dephosphorylates CA1P to CA, which cannot bind Rubisco. Misfire products of Rubisco's complex reaction chemistry can also act as inhibitors. One example is xylulose-1,5-bisphosphate (XuBP), which is dephosphorylated by XuBPase. Here we revisit key findings related to sugar phosphate derivatives and their specific phosphatases, highlighting outstanding questions and how further consideration of these inhibitors and their role is important for better understanding the regulation of carbon assimilation.

Genotype-dependent contribution of CBF transcription factors to long-term acclimation to high light and cool temperature.

When grown under cool temperature, winter annuals upregulate photosynthetic capacity as well as freezing tolerance. Here, the role of three cold-induced C-repeat-binding factor (CBF1-3) transcription factors in photosynthetic upregulation and freezing tolerance was examined in two Arabidopsis thaliana ecotypes originating from Italy (IT) or Sweden (SW), and their corresponding CBF1-3-deficient mutant lines it:cbf123 and sw:cbf123. Photosynthetic, morphological and freezing-tolerance phenotypes, as well as gene expression profiles, were characterized in plants grown from the seedling stage under different combinations of light level and temperature. Under high light and cool (HLC) growth temperature, a greater role of CBF1-3 in IT versus SW was evident from both phenotypic and transcriptomic data, especially with respect to photosynthetic upregulation and freezing tolerance of whole plants. Overall, features of SW were consistent with a different approach to HLC acclimation than seen in IT, and an ability of SW to reach the new homeostasis through the involvement of transcriptional controls other than CBF1-3. These results provide tools and direction for further mechanistic analysis of the transcriptional control of approaches to cold acclimation suitable for either persistence through brief cold spells or for maximisation of productivity in environments with continuous low temperatures.

Regulation of photoprotection gene expression in Chlamydomonas by a putative E3 ubiquitin ligase complex and a homolog of CONSTANS.

Photosynthetic organisms use nonphotochemical quenching (NPQ) mechanisms to dissipate excess absorbed light energy and protect themselves from photooxidation. In the model green alga Chlamydomonas reinhardtii, the capacity for rapidly reversible NPQ (qE) is induced by high light, blue light, and UV light via increased expression of LHCSR and PSBS genes that are necessary for qE. Here, we used a forward genetics approach to identify SPA1 and CUL4, components of a putative green algal E3 ubiquitin ligase complex, as critical factors in a signaling pathway that controls light-regulated expression of the LHCSR and PSBS genes in C. reinhardtii The spa1 and cul4 mutants accumulate increased levels of LHCSR1 and PSBS proteins in high light, and unlike the wild type, they express LHCSR1 and exhibit qE capacity even when grown in low light. The spa1-1 mutation resulted in constitutively high expression of LHCSR and PSBS RNAs in both low light and high light. The qE and gene expression phenotypes of spa1-1 are blocked by mutation of CrCO, a B-box Zn-finger transcription factor that is a homolog of CONSTANS, which controls flowering time in plants. CONSTANS-like cis-regulatory sequences were identified proximal to the qE genes, consistent with CrCO acting as a direct activator of qE gene expression. We conclude that SPA1 and CUL4 are components of a conserved E3 ubiquitin ligase that acts upstream of CrCO, whose regulatory function is wired differently in C. reinhardtii to control qE capacity via cis-regulatory CrCO-binding sites at key photoprotection genes.

Transcriptomic analysis of field-droughted sorghum from seedling to maturity reveals biotic and metabolic responses.

Drought is the most important environmental stress limiting crop yields. The C4 cereal sorghum [Sorghum bicolor (L.) Moench] is a critical food, forage, and emerging bioenergy crop that is notably drought-tolerant. We conducted a large-scale field experiment, imposing preflowering and postflowering drought stress on 2 genotypes of sorghum across a tightly resolved time series, from plant emergence to postanthesis, resulting in a dataset of nearly 400 transcriptomes. We observed a fast and global transcriptomic response in leaf and root tissues with clear temporal patterns, including modulation of well-known drought pathways. We also identified genotypic differences in core photosynthesis and reactive oxygen species scavenging pathways, highlighting possible mechanisms of drought tolerance and of the delayed senescence, characteristic of the stay-green phenotype. Finally, we discovered a large-scale depletion in the expression of genes critical to arbuscular mycorrhizal (AM) symbiosis, with a corresponding drop in AM fungal mass in the plants' roots.

Distinct Cold Acclimation of Productivity Traits in Arabidopsis thaliana Ecotypes.

Improvement of crop climate resilience will require an understanding of whole-plant adaptation to specific local environments. This review places features of plant form and function related to photosynthetic productivity, as well as associated gene-expression patterns, into the context of the adaptation of Arabidopsis thaliana ecotypes to local environments with different climates in Sweden and Italy. The growth of plants under common cool conditions resulted in a proportionally greater emphasis on the maintenance of photosynthetic activity in the Swedish ecotype. This is compared to a greater emphasis on downregulation of light-harvesting antenna size and upregulation of a host of antioxidant enzymes in the Italian ecotype under these conditions. This differential response is discussed in the context of the climatic patterns of the ecotypes' native habitats with substantial opportunity for photosynthetic productivity under mild temperatures in Italy but not in Sweden. The Swedish ecotype's response is likened to pushing forward at full speed with productivity under low temperature versus the Italian ecotype's response of staying safe from harm (maintaining redox homeostasis) while letting productivity decline when temperatures are transiently cold. It is concluded that either strategy can offer directions for the development of climate-resilient crops for specific locations of cultivation.

Stimulation of isoprene emissions and electron transport rates as key mechanisms of thermal tolerance in the tropical species Vismia guianensis.

Tropical forests absorb large amounts of atmospheric CO2 through photosynthesis, but high surface temperatures suppress this absorption while promoting isoprene emissions. While mechanistic isoprene emission models predict a tight coupling to photosynthetic electron transport (ETR) as a function of temperature, direct field observations of this phenomenon are lacking in the tropics and are necessary to assess the impact of a warming climate on global isoprene emissions. Here we demonstrate that in the early successional species Vismia guianensis in the central Amazon, ETR rates increased with temperature in concert with isoprene emissions, even as stomatal conductance (gs ) and net photosynthetic carbon fixation (Pn ) declined. We observed the highest temperatures of continually increasing isoprene emissions yet reported (50°C). While Pn showed an optimum value of 32.6 ± 0.4°C, isoprene emissions, ETR, and the oxidation state of PSII reaction centers (qL ) increased with leaf temperature with strong linear correlations for ETR (Æ¿ = 0.98) and qL (Æ¿ = 0.99) with leaf isoprene emissions. In contrast, other photoprotective mechanisms, such as non-photochemical quenching, were not activated at elevated temperatures. Inhibition of isoprenoid biosynthesis repressed Pn at high temperatures through a mechanism that was independent of stomatal closure. While extreme warming will decrease gs and Pn in tropical species, our observations support a thermal tolerance mechanism where the maintenance of high photosynthetic capacity under extreme warming is assisted by the simultaneous stimulation of ETR and metabolic pathways that consume the direct products of ETR including photorespiration and the biosynthesis of thermoprotective isoprenoids. Our results confirm that models which link isoprene emissions to the rate of ETR hold true in tropical species and provide necessary "ground-truthing" for simulations of the large predicted increases in tropical isoprene emissions with climate warming.

Optimization of Photosynthetic Productivity in Contrasting Environments by Regulons Controlling Plant Form and Function.

We review the role of a family of transcription factors and their regulons in maintaining high photosynthetic performance across a range of challenging environments with a focus on extreme temperatures and water availability. Specifically, these transcription factors include CBFs (C-repeat binding factors) and DREBs (dehydration-responsive element-binding), with CBF/DREB1 primarily orchestrating cold adaptation and other DREBs serving in heat, drought, and salinity adaptation. The central role of these modulators in plant performance under challenging environments is based on (i) interweaving of these regulators with other key signaling networks (plant hormones and redox signals) as well as (ii) their function in integrating responses across the whole plant, from light-harvesting and sugar-production in the leaf to foliar sugar export and water import and on to the plant's sugar-consuming sinks (growth, storage, and reproduction). The example of Arabidopsisthaliana ecotypes from geographic origins with contrasting climates is used to describe the links between natural genetic variation in CBF transcription factors and the differential acclimation of plant anatomical and functional features needed to support superior photosynthetic performance in contrasting environments. Emphasis is placed on considering different temperature environments (hot versus cold) and light environments (limiting versus high light), on trade-offs between adaptations to contrasting environments, and on plant lines minimizing such trade-offs.

Effects of Foliar Redox Status on Leaf Vascular Organization Suggest Avenues for Cooptimization of Photosynthesis and Heat Tolerance.

The interaction of heat stress with internal signaling networks was investigated through Arabidopsisthaliana mutants that were deficient in either tocopherols (vte1 mutant) or non-photochemical fluorescence quenching (NPQ; npq1, npq4, and npq1 npq4 mutants). Leaves of both vte1 and npq1 npq4 mutants that developed at a high temperature exhibited a significantly different leaf vascular organization compared to wild-type Col-0. Both mutants had significantly smaller water conduits (tracheary elements) of the xylem, but the total apparent foliar water-transport capacity and intrinsic photosynthetic capacity were similarly high in mutants and wild-type Col-0. This was accomplished through a combination of more numerous (albeit narrower) water conduits per vein, and a significantly greater vein density in both mutants relative to wild-type Col-0. The similarity of the phenotypes of tocopherol-deficient and NPQ-deficient mutants suggests that leaf vasculature organization is modulated by the foliar redox state. These results are evaluated in the context of interactions between redox-signaling pathways and other key regulators of plant acclimation to growth temperature, such as the C-repeat binding factor (CBF) transcription factors, several of which were upregulated in the antioxidant-deficient mutants. Possibilities for the future manipulation of the interaction between CBF and redox-signaling networks for the purpose of cooptimizing plant productivity and plant tolerance to extreme temperatures are discussed.

Extensive DNA-binding specificity divergence of a conserved transcription regulator.

The DNA sequence recognized by a transcription regulator can be conserved across large evolutionary distances. For example, it is known that many homologous regulators in yeasts and mammals can recognize the same (or closely related) DNA sequences. In contrast to this paradigm, we describe a case in which the DNA-binding specificity of a transcription regulator has changed so extensively (and over a much smaller evolutionary distance) that its cis-regulatory sequence appears unrelated in different species. Bioinformatic, genetic, and biochemical approaches were used to document and analyze a major change in the DNA-binding specificity of Matα1, a regulator of cell-type specification in ascomycete fungi. Despite this change, Matα1 controls the same core set of genes in the hemiascomycetes because its DNA recognition site has evolved with it, preserving the protein-DNA interaction but significantly changing its molecular details. Matα1 and its recognition sequence diverged most dramatically in the common ancestor of the CTG-clade (Candida albicans, Candida lusitaniae, and related species), apparently without the aid of a gene duplication event. Our findings suggest that DNA-binding specificity divergence between orthologous transcription regulators may be more prevalent than previously thought and that seemingly unrelated cis-regulatory sequences can nonetheless be homologous. These findings have important implications for understanding transcriptional network evolution and for the bioinformatic analysis of regulatory circuits.

Metabolomic, photoprotective, and photosynthetic acclimatory responses to post-flowering drought in sorghum.

Climate change is globally affecting rainfall patterns, necessitating the improvement of drought tolerance in crops. Sorghum bicolor is a relatively drought-tolerant cereal. Functional stay-green sorghum genotypes can maintain green leaf area and efficient grain filling during terminal post-flowering water deprivation, a period of ~10 weeks. To obtain molecular insights into these characteristics, two drought-tolerant genotypes, BTx642 and RTx430, were grown in replicated control and terminal post-flowering drought field plots in California's Central Valley. Photosynthetic, photoprotective, and water dynamics traits were quantified and correlated with metabolomic data collected from leaves, stems, and roots at multiple timepoints during control and drought conditions. Physiological and metabolomic data were then compared to longitudinal RNA sequencing data collected from these two genotypes. The unique metabolic and transcriptomic response to post-flowering drought in sorghum supports a role for the metabolite galactinol in controlling photosynthetic activity through regulating stomatal closure in post-flowering drought. Additionally, in the functional stay-green genotype BTx642, photoprotective responses were specifically induced in post-flowering drought, supporting a role for photoprotection in the molecular response associated with the functional stay-green trait. From these insights, new pathways are identified that can be targeted to maximize yields under growth conditions with limited water.

Following gene duplication, paralog interference constrains transcriptional circuit evolution.

Most models of gene duplication assume that the ancestral functions of the preduplication gene are independent and can therefore be neatly partitioned between descendant paralogs. However, many gene products, such as transcriptional regulators, are components within cooperative assemblies; here, we show that a natural consequence of duplication and divergence of such proteins can be competitive interference between the paralogs. Our example is based on the duplication of the essential MADS-box transcriptional regulator Mcm1, which is found in all fungi and regulates a large set of genes. We show that a set of historical amino acid sequence substitutions minimized paralog interference in contemporary species and, in doing so, increased the molecular complexity of this gene regulatory network. We propose that paralog interference is a common constraint on gene duplicate evolution, and its resolution, which can generate additional regulatory complexity, is needed to stabilize duplicated genes in the genome.