The Clock Gene TOC1 in Shoots, Not Roots, Determines Fitness of Nicotiana attenuata under Drought.

The highly conserved core circadian clock component TIMING OF CAB EXPRESSION1 (TOC1) contextualizes environmental stress responses in plants, for example by gating abscisic acid signaling and suppressing thermoresponsive growth. Selective interaction of TOC1 with PHYTOCHROME B under far-red-enriched light suggests a connection between circadian gating of light responses and sensitivity to ABA, an important regulator of growth and stress responses, including under drought. However, the fitness consequences of TOC1 function, particularly in the root, are poorly understood. Here, we used the desert annual, Nicotiana attenuata, to investigate the function of TOC1 in shoots and roots for maintaining fitness under drought, in both field and glasshouse experiments. Despite marked decreases in leaf water loss, TOC1-deficient lines failed to maintain fitness in response to drought stress as measured by total seed capsule production. Restoring TOC1 transcript levels in shoots via micrografting was sufficient to restore wild-type drought responses under field conditions. Microarrays identified a coexpression module in leaves strongly linking red and far-red light signaling to drought responses in a TOC1-dependent manner, but experiments with phytochrome-deficient lines revealed that the effects of TOC1 deficiency under drought cannot be attributed to changes in red/far-red light perception alone. Taken together, these results elucidate the sophisticated, tissue-dependent role of the circadian clock in maintaining fitness in the face of long-term abiotic stresses such as drought.

Root-expressed phytochromes B1 and B2, but not PhyA and Cry2, regulate shoot growth in nature.

Although photoreceptors are expressed throughout all plant organs, most studies have focused on their function in aerial parts with laboratory-grown plants. Photoreceptor function in naturally dark-grown roots of plants in their native habitats is lacking. We characterized patterns of photoreceptor expression in field- and glasshouse-grown Nicotiana attenuata plants, silenced the expression of PhyB1/B2/A/Cry2 whose root transcripts levels were greater/equal to those of shoots, and by micrografting combined empty vector transformed shoots onto photoreceptor-silenced roots, creating chimeric plants with "blind" roots but "sighted" shoots. Micrografting procedure was robust in both field and glasshouse, as demonstrated by transcript accumulation patterns, and a spatially-explicit lignin visual reporter chimeric line. Field- and glasshouse-grown plants with PhyB1B2, but not PhyA or Cry2, -blind roots, were delayed in stalk elongation compared with control plants, robustly for two field seasons. Wild-type plants with roots directly exposed to FR phenocopied the growth of irPhyB1B2-blind root grafts. Additionally, root-expressed PhyB1B2 was required to activate the positive photomorphogenic regulator, HY5, in response to aboveground light. We conclude that roots of plants growing deep into the soil in nature sense aboveground light, and possibly soil temperature, via PhyB1B2 to control key traits, such as stalk elongation.

Circadian clock component, LHY, tells a plant when to respond photosynthetically to light in nature.

The circadian clock is known to increase plant growth and fitness, and is thought to prepare plants for photosynthesis at dawn and dusk; whether this happens in nature was unknown. We transformed the native tobacco, Nicotiana attenuata to silence two core clock components, NaLHY (irLHY) and NaTOC1 (irTOC1). We characterized growth and light- and dark-adapted photosynthetic rates (Ac ) throughout a 24 h day in empty vector-transformed (EV), irLHY, and irTOC1 plants in the field, and in NaPhyA- and NaPhyB1-silenced plants in the glasshouse. The growth rates of irLHY plants were lower than those of EV plants in the field. While irLHY plants reduced Ac earlier at dusk, no differences between irLHY and EV plants were observed at dawn in the field. irLHY, but not EV plants, responded to light in the night by rapidly increasing Ac . Under controlled conditions, EV plants rapidly increased Ac in the day compared to dark-adapted plants at night; irLHY plants lost these time-dependent responses. The role of NaLHY in gating photosynthesis is independent of the light-dependent reactions and red light perceived by NaPhyA, but not NaPhyB1. In summary, the circadian clock allows plants not to respond photosynthetically to light at night by anticipating and gating red light-mediated in native tobacco.

Functional specialization of Nicotiana attenuata phytochromes in leaf development and flowering time.

Phytochromes mainly function in photoautotrophic organisms to adjust growth in response to fluctuating light signals. The different isoforms of plant phytochromes often display both conserved and divergent roles, presumably to fine-tune plant responses to environmental signals and optimize fitness. Here we describe the distinct, yet partially redundant, roles of phytochromes NaPHYA, NaPHYB1 and NaPHYB2 in a wild tobacco species, Nicotiana attenuata using RNAi-silenced phytochrome lines. Consistent with results reported from other species, silencing the expression of NaPHYA or NaPHYB2 in N. attenuata had mild or no influence on plant development as long as NaPHYB1 was functional; whereas silencing the expression of NaPHYB1 alone strongly altered flowering time and leaf morphology. The contribution of NaPHYB2 became significant only in the absence of NaPHYB1; plants silenced for both NaPHYB1 and NaPHYB2 largely skipped the rosette-stage of growth to rapidly produce long, slender stalks that bore flowers early: hallmarks of the shade-avoidance responses. The phenotyping of phytochrome-silenced lines, combined with sequence and transcript accumulation analysis, suggest the independent functional diversification of the phytochromes, and a dominant role of NaPHYB1 and NaPHYB2 in N. attenuata's vegetative and reproductive development.

Stem-piped light activates phytochrome B to trigger light responses in Arabidopsis thaliana roots.

The roles of photoreceptors and their associated signaling mechanisms have been extensively studied in plant photomorphogenesis with a major focus on the photoresponses of the shoot system. Accumulating evidence indicates that light also influences root growth and development through the light-induced release of signaling molecules that travel from the shoot to the root. We explored whether aboveground light directly influences the root system of Arabidopsis thaliana Light was efficiently conducted through the stems to the roots, where photoactivated phytochrome B (phyB) triggered expression of ELONGATED HYPOCOTYL 5 (HY5) and accumulation of HY5 protein, a transcription factor that promotes root growth in response to light. Stimulation of HY5 in response to illumination of only the shoot was reduced when root tissues carried a loss-of-function mutation in PHYB, and HY5 mutant roots exhibited alterations in root growth and gravitropism in response to shoot illumination. These findings demonstrate that the underground roots directly sense stem-piped light to monitor the aboveground light environment during plant environmental adaptation.

Root jasmonic acid synthesis and perception regulate folivore-induced shoot metabolites and increase Nicotiana attenuata resistance.

While jasmonic acid (JA) signaling is widely accepted as mediating plant resistance to herbivores, and the importance of the roots in plant defenses is recently being recognized, the role of root JA in the defense of above-ground parts remains unstudied. To restrict JA impairment to the roots, we micrografted wildtype Nicotiana attenuata shoots to the roots of transgenic plants impaired in JA signaling and evaluated ecologically relevant traits in the glasshouse and in nature. Root JA synthesis and perception are involved in regulating nicotine production in roots. Strikingly, systemic root JA regulated local leaf JA and abscisic acid (ABA) concentrations, which were associated with differences in nicotine transport from roots to leaves via the transpiration stream. Root JA signaling also regulated the accumulation of other shoot metabolites; together these account for differences in resistance against a generalist, Spodoptera littoralis, and a specialist herbivore, Manduca sexta. In N. attenuata's native habitat, silencing root JA synthesis increased the shoot damage inflicted by Empoasca leafhoppers, which are able to select natural jasmonate mutants. Silencing JA perception in roots also increased damage by Tupiocoris notatus. We conclude that attack from above-ground herbivores recruits root JA signaling to launch the full complement of plant defense responses.

A simple and efficient micrografting method for stably transformed Nicotiana attenuata plants to examine shoot-root signaling.

To adjust their development to the environment, plants rely on specific signals that travel from shoot to root and vice versa. Here we describe an efficient micrografting protocol for Nicotiana attenuata, a useful tool for identifying these signals and understanding their functions. Additionally we analyzed transcript accumulation profiles of scions and rootstocks of grafts performed with wild-type and stably transformed N. attenuata. Our results are consistent with the source-to-sink movement of an sRNA silencing signal.

Antioxidant and antimutagenic properties of the monoterpene indole alkaloid psychollatine and the crude foliar extract of Psychotria umbellata Vell.

Psychollatine is a monoterpene indole alkaloid produced and accumulated by Psychotria umbellata Vell. (Rubiaceae) leaves in relatively high amounts (approximately 3% of the dry weight). The alkaloid has been shown to display opioid-like analgesic, anxiolytic, antidepressive and antipsychotic activities in rodents. In vitro assays suggested a protective role for this molecule in plant oxidative stress responses. This work reports antioxidant properties of psychollatine and the crude foliar extract from P. umbellata in strains of Saccharomyces cerevisiae proficient and deficient in antioxidant defenses exposed to H2O2 and paraquat. The antimutagenic activity of P. umbellata and its main alkaloid were assayed in S. cerevisiae N123 strain in presence of H2O2. Moreover, the antioxidant capacity of these substances on the hydroxyl radical (OH.) was investigated, using the hypoxanthine/xanthine oxidase assay. Psychollatine and the crude foliar extract of P. umbellata showed protective effect against oxidative stress in yeast, acting both as antioxidant and antimutagenic agents.

Antioxidant and antimutagenic effects of the crude foliar extract and the alkaloid brachycerine of Psychotria brachyceras.

The monoterpene indole alkaloid brachycerine from Psychotria brachyceras has been shown to be induced by UV and to have in vitro antioxidant activity, indicating a possible protective role against the secondary effects of this radiation. In this work, we have studied the antioxidant properties of brachycerine and a crude foliar extract from P. brachyceras by using Saccharomyces cerevisiae strains proficient and deficient in antioxidant defenses. The mutagenic and antimutagenic potential of these substances were assayed in S.cerevisiae N123 strain in the presence and absence of H2O2. In addition, we tested the antioxidant capacity of brachycerine and a crude foliar extract from P. brachyceras on hydroxyl radicals (OH) using the hypoxanthine/xanthine oxidase assay. The results show that brachycerine and the crude foliar extract of P. brachyceras have antioxidant and antimutagenic effects in yeast and probably this action is mainly due to the scavenging of OH radicals.

Regeneration of Psychotria umbellata and production of the analgesic indole alkaloid umbellatine.

Psychotria umbellata Vell. (Rubiaceae), a Brazilian coastal woody species, produces umbellatine (also known as psychollatine), an analgesic indole alkaloid. An in vitro embryogenic regeneration protocol capable of yielding alkaloid-accumulating plants was developed. Rhizogenic calli, which were obtained from stem segments derived from rooted apical cuttings, were cultured on Murashige and Skoog's (MS) medium containing either 1 mg l(-1) NAA (naphthalene acetic acid) and no kinetin, or 5 mg l(-1) NAA + 1 mg l(-1) kinetin. Calli did not accumulate umbellatine. Segments of rhizogenic callus were cultured on complete MS medium with various concentrations of kinetin and sucrose. Plant regeneration was best in the light with 0.25 mg l(-1) of kinetin and 1.5% sucrose. After 3 months of acclimatization in soil mixture, plant survival was 81%. Leaves of 10-month-old regenerated plants yielded umbellatine concentrations equivalent to those of adult forest-grown plants.