Plant Hormone Modularity and the Survival-Reproduction Trade-Off.

Biological modularity refers to the organization of living systems into separate functional units that interact in different combinations to promote individual well-being and species survival. Modularity provides a framework for generating and selecting variations that can lead to adaptive evolution. While the exact mechanisms underlying the evolution of modularity are still being explored, it is believed that the pressure of conflicting demands on limited resources is a primary selection force. One prominent example of conflicting demands is the trade-off between survival and reproduction. In this review, we explore the available evidence regarding the modularity of plant hormones within the context of the survival-reproduction trade-off. Our findings reveal that the cytokinin module is dedicated to maximizing reproduction, while the remaining hormone modules function to ensure reproduction. The signaling mechanisms of these hormone modules reflect their roles in this survival-reproduction trade-off. While the cytokinin response pathway exhibits a sequence of activation events that aligns with the developmental robustness expected from a hormone focused on reproduction, the remaining hormone modules employ double-negative signaling mechanisms, which reflects the necessity to prevent the excessive allocation of resources to survival.

Friends in Arms: Flavonoids and the Auxin/Cytokinin Balance in Terrestrialization.

Land plants survive the challenges of new environments by evolving mechanisms that protect them from excess irradiation, nutrient deficiency, and temperature and water availability fluctuations. One such evolved mechanism is the regulation of the shoot/root growth ratio in response to water and nutrient availability by balancing the actions of the hormones auxin and cytokinin. Plant terrestrialization co-occurred with a dramatic expansion in secondary metabolism, particularly with the evolution and establishment of the flavonoid biosynthetic pathway. Flavonoid biosynthesis is responsive to a wide range of stresses, and the numerous synthesized flavonoid species offer two main evolutionary advantages to land plants. First, flavonoids are antioxidants and thus defend plants against those adverse conditions that lead to the overproduction of reactive oxygen species. Second, flavonoids aid in protecting plants against water and nutrient deficiency by modulating root development and establishing symbiotic relations with beneficial soil fungi and bacteria. Here, we review different aspects of the relationships between the auxin/cytokinin module and flavonoids. The current body of knowledge suggests that whereas both auxin and cytokinin regulate flavonoid biosynthesis, flavonoids act to fine-tune only auxin, which in turn regulates cytokinin action. This conclusion agrees with the established master regulatory function of auxin in controlling the shoot/root growth ratio.

Gain-of-function of the cytokinin response activator ARR1 increases heat shock tolerance in Arabidopsis thaliana.

In addition to its well-established role in plant development, the hormone cytokinin regulates plant responses to biotic and abiotic stresses. It was previously shown that cytokinin signaling acts negatively upon drought and osmotic stress tolerance and that gain-of-function of the cytokinin response regulator ARR1 causes osmotic stress hypersensitivity. Here we show that increased ARR1 action increases tolerance to heat shock and that this is correlated with increased accumulation of the heat shock proteins Hsp17.6 and Hsp70. These results show that the heat shock tolerance of plants can be elevated by increasing the expression of a cytokinin response activator.

Auxin/Cytokinin Antagonistic Control of the Shoot/Root Growth Ratio and Its Relevance for Adaptation to Drought and Nutrient Deficiency Stresses.

The hormones auxin and cytokinin regulate numerous aspects of plant development and often act as an antagonistic hormone pair. One of the more striking examples of the auxin/cytokinin antagonism involves regulation of the shoot/root growth ratio in which cytokinin promotes shoot and inhibits root growth, whereas auxin does the opposite. Control of the shoot/root growth ratio is essential for the survival of terrestrial plants because it allows growth adaptations to water and mineral nutrient availability in the soil. Because a decrease in shoot growth combined with an increase in root growth leads to survival under drought stress and nutrient limiting conditions, it was not surprising to find that auxin promotes, while cytokinin reduces, drought stress tolerance and nutrient uptake. Recent data show that drought stress and nutrient availability also alter the cytokinin and auxin signaling and biosynthesis pathways and that this stress-induced regulation affects cytokinin and auxin in the opposite manner. These antagonistic effects of cytokinin and auxin suggested that each hormone directly and negatively regulates biosynthesis or signaling of the other. However, a growing body of evidence supports unidirectional regulation, with auxin emerging as the primary regulatory component. This master regulatory role of auxin may not come as a surprise when viewed from an evolutionary perspective.

EGY3 mediates chloroplastic ROS homeostasis and promotes retrograde signaling in response to salt stress in Arabidopsis.

The chloroplast is the main organelle for stress-induced production of reactive oxygen species (ROS). However, how chloroplastic ROS homeostasis is maintained under salt stress is largely unknown. We show that EGY3, a gene encoding a chloroplast-localized protein, is induced by salt and oxidative stresses. The loss of EGY3 function causes stress hypersensitivity while EGY3 overexpression increases the tolerance to both salt and chloroplastic oxidative stresses. EGY3 interacts with chloroplastic Cu/Zn-SOD2 (CSD2) and promotes CSD2 stability under stress conditions. In egy3-1 mutant plants, the stress-induced CSD2 degradation limits H2O2 production in chloroplasts and impairs H2O2-mediated retrograde signaling, as indicated by the decreased expression of retrograde-signal-responsive genes required for stress tolerance. Both exogenous application of H2O2 (or APX inhibitor) and CSD2 overexpression can rescue the salt-stress hypersensitivity of egy3-1 mutants. Our findings reveal that EGY3 enhances the tolerance to salt stress by promoting the CSD2 stability and H2O2-mediated chloroplastic retrograde signaling.

Differential oxidative stress responses and tobacco-specific nitrosamine accumulation in two burley varieties.

Tobacco-specific nitrosamines (TSNAs) are carcinogens that accumulate in tobacco leaves during curing, storage, and processing, and their amounts in processed tobacco vary dependent on several intrinsic and extrinsic factors. Here, we assessed the hypothesis that there is a link between reactive oxygen species levels in leaves and TSNA formation during curing. First, we show that burley varieties KT 204LC and NCBH 129LC accumulate TSNAs to different levels but not as a result of a variety-specific abundance of TSNA precursors. Next, we measured the levels of reactive oxygen species, and we show that the variety that accumulates more TSNAs, NCBH 129LC, had significantly higher levels of hydrogen peroxide than KT 204LC. The NCBH 129LC also has more oxidatively damaged and glutathionylated proteins. Finally, we analyzed the antioxidant levels in KT 204LC and NCBH 129LC and their tolerance to oxidative stress. NCBH 129LC contained more of the essential antioxidant glutathione and was more tolerant to the oxidative stress-generating compound paraquat. Collectively, our data suggest that there is indeed a link between foliar oxidative stress parameters and the extent to which TSNAs accumulate in cured tobacco leaves.

Composition of the metabolomic bio-coronas isolated from Ocimum sanctum and Rubia tinctorum.

OBJECTIVE: Nanoharvesting from intact plants, organs, and cultured cells is a method in which nanoparticles are co-incubated with the target tissue, which leads to the internalization of nanoparticles. Internalized nanoparticles are coated in situ with specific metabolites that form a dynamic surface layer called a bio-corona. Our previous study showed that metabolites that form the bio-corona around anatase TiO2 nanoparticles incubated with leaves of the model plant Arabidopsis thaliana are enriched for flavonoids and lipids. The present study focused on the identification of metabolites isolated by nanoharvesting from two medicinal plants, Ocimum sanctum (Tulsi) and Rubia tinctorum (common madder). RESULTS: To identify metabolites that form the bio-corona, Tulsi leaves and madder roots were incubated with ultra-small anatase TiO2 nanoparticles, the coated nanoparticles were collected, and the adsorbed molecules were released from the nanoparticle surface and analyzed using an untargeted metabolomics approach. Similar to the results in which Arabidopsis tissue was used as a source of metabolites, TiO2 nanoparticle bio-coronas from Tulsi and madder were enriched for flavonoids and lipids, suggesting that nanoharvesting has a wide-range application potential. The third group of metabolites enriched in bio-coronas isolated from both plants were small peptides with C-terminal arginine and lysine residues.

Inhibition of Fusarium oxysporum f. sp. nicotianae Growth by Phenylpropanoid Pathway Intermediates.

Fusarium wilt in tobacco caused by the fungus Fusarium oxysporum f. sp. nicotianae is a disease­management challenge worldwide, as there are few effective and environmentally benign chemical agents for its control. This challenge results in substantial losses in both the quality and yield of tobacco products. Based on an in vitro analysis of the effects of different phenylpropanoid intermediates, we found that the early intermediates trans­cinnamic acid and para­coumaric acid effectively inhibit the mycelial growth of F. oxysporum f. sp. nicotianae strain FW316F, whereas the downstream intermediates quercetin and caffeic acid exhibit no fungicidal properties. Therefore, our in vitro screen suggests that trans­cinnamic acid and para­coumaric acid are promising chemical agents and natural lead compounds for the suppression of F. oxysporum f. sp. nicotianae growth.

Cytokinin-induced protein synthesis suppresses growth and osmotic stress tolerance.

Cytokinins control critical aspects of plant development and environmental responses. Perception of cytokinin ultimately leads to the activation of proteins belonging to the type-B Response Regulator family of cytokinin response activators. In Arabidopsis thaliana, ARR1 is one of the most abundantly expressed type-B Response Regulators. We investigated the link between cytokinin signaling, protein synthesis, plant growth and osmotic stress tolerance. We show that the increased cytokinin signaling in ARR1 gain-of-function transgenic lines is associated with increased rates of protein synthesis, which lead to growth inhibition and hypersensitivity to osmotic stress. Cytokinin-induced growth inhibition and osmotic stress hypersensitivity were rescued by treatments with ABA, a hormone known to inhibit protein synthesis. We also demonstrate that cytokinin-induced protein synthesis requires isoforms of the ribosomal protein L4 encoded by the cytokinin-inducible genes RPL4A and RPL4D, and that RPL4 loss-of-function increases osmotic stress tolerance and decreases sensitivity to cytokinin-induced growth inhibition. These findings reveal that an increase in protein synthesis negatively impacts growth and osmotic stress tolerance and explain some of the adverse effects of elevated cytokinin action on plant development and stress physiology.

Metabolomic analyses of the bio-corona formed on TiO2 nanoparticles incubated with plant leaf tissues.

BACKGROUND: The surface of a nanoparticle adsorbs molecules from its surroundings with a specific affinity determined by the chemical and physical properties of the nanomaterial. When a nanoparticle is exposed to a biological system, the adsorbed molecules form a dynamic and specific surface layer called a bio-corona. The present study aimed to identify the metabolites that form the bio-corona around anatase TiO2 nanoparticles incubated with leaves of the model plant Arabidopsis thaliana. RESULTS: We used an untargeted metabolomics approach and compared the metabolites isolated from wild-type plants with plants deficient in a class of polyphenolic compounds called flavonoids. CONCLUSIONS: These analyses showed that TiO2 nanoparticle coronas are enriched for flavonoids and lipids and that these metabolite classes compete with each other for binding the nanoparticle surface.

Anatase TiO2 Nanoparticles Induce Autophagy and Chloroplast Degradation in Thale Cress (Arabidopsis thaliana).

The extensive use of TiO2 nanoparticles and their subsequent release into the environment have posed an important question about the effects of this nanomaterial on ecosystems. Here, we analyzed the link between the damaging effects of reactive oxygen species generated by TiO2 nanoparticles and autophagy, a housekeeping mechanism that removes damaged cellular constituents. We show that TiO2 nanoparticles induce autophagy in the plant model system Arabidopsis thaliana and that autophagy is an important mechanism for managing TiO2 nanoparticle-induced oxidative stress. Additionally, we find that TiO2 nanoparticles induce oxidative stress predominantly in chloroplasts and that this chloroplastic stress is mitigated by autophagy. Collectively, our results suggest that photosynthetic organisms are particularly susceptible to TiO2 nanoparticle toxicity.

Antagonistic activity of auxin and cytokinin in shoot and root organs.

The hormones auxin and cytokinin are essential for plant growth and development. Because of the central importance of root and shoot apical meristems in plant growth, auxin/cytokinin interactions have been predominantly analyzed in relation to apical meristem formation and function. In contrast, the auxin/cytokinin interactions during organ growth have remained largely unexplored. Here, we show that a specific interaction between auxin and cytokinin operates in both the root and the shoot where it serves as an additional determinant of plant development. We found that auxin at low concentrations limits the action of cytokinin. An increase in cytokinin level counteracts this inhibitory effect and leads to an inhibition of auxin signaling. At higher concentrations of both hormones, these antagonistic interactions between cytokinin and auxin are absent. Thus, our results reveal a bidirectional and asymmetrical interaction of auxin and cytokinin beyond the bounds of apical meristems. The relation is bidirectional in that both hormones exert inhibitory effects on each other's signaling mechanisms. However, this relation is also asymmetrical because under controlled growth conditions, auxin present in nontreated plants suppresses cytokinin signaling, whereas the reverse is not the case.

trans-Cinnamic acid-induced leaf expansion involves an auxin-independent component.

The phenylpropanoid pathway, the source of a large array of compounds with diverse functions, starts with the synthesis of trans-cinnamic acid (t-CA) that is converted by cinnamate-4-hydroxylase (C4H) into p-coumaric acid. We have recently shown that in Arabidopsis, exogenous t-CA promotes leaf growth by increasing cell expansion and that this response requires auxin signaling. We have also shown that cell expansion is increased in C4H loss-of-function mutants. Here we provide further evidence that leaf growth is enhanced by either t-CA or a t-CA derivative that accumulates upstream of C4H. We also show that this growth response pathway has two components: one that requires auxin signaling and another which employs a currently unknown mechanism.

Oxidative stress-induced formation of covalently linked ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit dimer in tobacco plants.

OBJECTIVE: Many abiotic stresses cause the excessive accumulation of reactive oxygen species known as oxidative stress. While analyzing the effects of oxidative stress on tobacco, we noticed the increased accumulation of a specific protein in extracts from plants treated with the oxidative-stress inducing herbicide paraquat which promotes the generation of reactive oxygen species primarily in chloroplasts. The primary objectives of this study were to identify this protein and to determine if its accumulation is indeed a result of oxidative stress. RESULTS: Here we show that the paraquat-induced protein is a covalently linked dimer of the large subunit of ribulose-1,5-bisphosphate carboxylase (LSU). Increased accumulation of this LSU dimer was also observed in tobacco plants exposed to ultra-small anatase titanium dioxide nanoparticles (nTiO2), which because of their surface reactivity cause oxidative stress by promoting the generation of superoxide anion. nTiO2 nanoparticle treatments also caused a decline in the chloroplast thylakoid proteins cytochrome f and chlorophyll a/b binding protein, thus confirming that covalent LSU dimer formation coincides with loss of chloroplast function.

Modulation of auxin and cytokinin responses by early steps of the phenylpropanoid pathway.

BACKGROUND: The phenylpropanoid pathway is responsible for the synthesis of numerous compounds important for plant growth and responses to the environment. In the first committed step of phenylpropanoid biosynthesis, the enzyme phenylalanine ammonia-lyase (PAL) deaminates L-phenylalanine into trans-cinnamic acid that is then converted into p-coumaric acid by cinnamate-4-hydroxylase (C4H). Recent studies showed that the Kelch repeat F-box (KFB) protein family of ubiquitin ligases control phenylpropanoid biosynthesis by promoting the proteolysis of PAL. However, this ubiquitin ligase family, alternatively named Kiss Me Deadly (KMD), was also implicated in cytokinin signaling as it was shown to promote the degradation of type-B ARRs, including the key response activator ARR1. Considering that ubiquitin ligases typically have narrow target specificity, this dual targeting of structurally and functionally unrelated proteins appeared unusual. RESULTS: Here we show that the KFBs indeed target PAL but not ARR1. Moreover, we show that changes in early phenylpropanoid biosynthesis alter cytokinin sensitivity - as reported earlier - but that the previously documented cytokinin growth response changes are primarily the result of altered auxin signaling. We found that reduced PAL accumulation decreased, whereas the loss of C4H function increased the strength of the auxin response. The combined loss of function of both enzymes led to a decrease in auxin sensitivity, indicating that metabolic events upstream of C4H control auxin sensitivity. This auxin/phenylpropanoid interaction impacts both shoot and root development and revealed an auxin-dependent stimulatory effect of trans-cinnamic acid feeding on leaf expansion and thus biomass accumulation. CONCLUSIONS: Collectively, our results show that auxin-regulated plant growth is fine-tuned by early steps in phenylpropanoid biosynthesis and suggest that metabolites accumulating upstream of the C4H step impact the auxin response mechanism.

Cytokinin signaling promotes differential stability of type-B ARRs.

Cytokinins control key aspects of plant growth, including shoot and root meristem development and the timing of senescence of leaves and stems. Cytokinin perception triggers a 2-component signaling mechanism that ultimately leads to phosphorylation-dependent activation of a class of transcriptional regulators called type-B ARRs (RRBs). We have recently shown that the stability of the RRB family member ARR1 is increased in response to elevated cytokinin concentrations. In contrast, cytokinin decreases the stability of the closely related RRB member ARR2. The molecular mechanism governing the differential stability regulation of these 2 closely related RRBs remains unknown.

Cytokinin signaling stabilizes the response activator ARR1.

The cytokinins play essential roles in the development and environmental responses of higher plants. Cytokinin signaling leads to the phosphorylation-dependent activation of two classes of Arabidopsis response regulators (RRs): the type-B RR (RRB) transcriptional activators that promote the expression of cytokinin response genes and the type-A RRs (RRAs) that are encoded by primary cytokinin response genes and function as response inhibitors. We show that cytokinin signaling increases the abundance of ARR1, a ubiquitously expressed RRB, by preventing its degradation by the 26S proteasome. We also show that the RRAs act to suppress ARR1 accumulation, thus providing an explanation for their inhibitory action in cytokinin signaling. Collectively, our results reveal an additional regulatory mechanism in the cytokinin response pathway that involves the cytokinin-dependent stability control of a major RRB response activator.

Ectopic expression of the phosphomimic mutant version of Arabidopsis response regulator 1 promotes a constitutive cytokinin response phenotype.

BACKGROUND: Cytokinins control numerous plant developmental processes, including meristem formation and activity, nutrient distribution, senescence timing and responses to both the abiotic and biotic environments. Cytokinin signaling leads to the activation of type-B response regulators (RRBs), Myb-like transcription factors that are activated by the phosphorylation of a conserved aspartate residue in their response receiver domain. Consistent with this, overexpression of RRBs does not substantially alter plant development, but instead leads to cytokinin hypersensitivity. RESULTS: Here we present comparative analysis of plants overexpressing Arabidopsis RRB 1 (ARR1) or a phosphomimic ARR1D94E mutant in which the conserved aspartate-94 (D94) is replaced by the phosphomimic residue glutamate (E). The D94E substitution causes a 100-fold increase in response activation and instigates developmental and physiological changes that characterize wild-type plants treated with cytokinins or transgenic plants with increased cytokinin content. CONCLUSION: The current model of cytokinin signaling emphasizes the essential role of conserved aspartate residue phosphorylation of RRBs in promoting cytokinin responses. Our comparative analyses of developmental and physiological traits of ARR1 and ARR1D94E overexpressing plants revealed that the ARR1D94E protein is indeed a constitutive and wide-spectrum cytokinin response activator.

Direct isolation of flavonoids from plants using ultra-small anatase TiO2 nanoparticles.

Surface functionalization of nanoparticles has become an important tool for in vivo delivery of bioactive agents to their target sites. Here we describe the reverse strategy, nanoharvesting, in which nanoparticles are used as a tool to isolate bioactive compounds from living cells. Anatase TiO2 nanoparticles smaller than 20 nm form strong bonds with molecules bearing enediol and especially catechol groups. We show that these nanoparticles enter plant cells, conjugate enediol and catechol group-rich flavonoids in situ, and exit plant cells as flavonoid-nanoparticle conjugates. The source plant tissues remain viable after treatment. As predicted by the surface chemistry of anatase TiO2 nanoparticles, quercetin-based flavonoids were enriched amongst the nanoharvested flavonoid species. Nanoharvesting eliminates the use of organic solvents, allows spectral identification of the isolated compounds, and opens new avenues for use of nanomaterials for coupled isolation and testing of bioactive properties of plant-synthesized compounds.