Testing the optimal defense theory and the growth-differentiation balance hypothesis in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Two prominent theories proposed to explain patterns of chemical defense expression in plants are the optimal defense theory (ODT) and the growth-differentiation balance hypothesis (GDBH). The ODT predicts that plant parts with high fitness value will be highly defended, and the GDBH predicts that slow growing plant parts will have more resources available for defense and thus will have higher defense levels than faster growing tissues. We examined growth rate, fitness value, and defense protein levels in leaves of a wild and lab ecotype of Arabidopsis thaliana to address whether patterns of defense protein expression in this plant conform to predictions of either the ODT or the GDBH. We divided leaves of A. thaliana into six leaf classes based on three developmental stages: vegetative, bolting, and flowering; with two leaf ages at each stage: young and old. We assessed the fitness value of leaves by determining the impact of the removal of each leaf class on total seed production and germination rates. Although A. thaliana was highly tolerant to defoliation, young leaves were more valuable than old in general, and young leaves on bolting plants were the most valuable leaf class in particular. Young leaves on vegetative plants grew fastest in both ecotypes, while old leaves on bolting and flowering plants grew slowest. Finally, defense levels were assessed in each leaf class by quantifying the constitutive and inducible expression of four defense-related proteins. Expression of guaiacol peroxidase and chitinase activity conformed largely to GDBH predictions. Expression of trypsin inhibitor and polyphenoloxidase activity varied by leaf class and treatment, but conformed to neither GDBH nor ODT predictions.     

‘Real time’ genetic manipulation: a new tool for ecological field studies

Field experiments with transgenic plants often reveal the functional significance of genetic traits that are important for the performance of the plants in their natural environments. Until now, only constitutive overexpression, ectopic expression and gene silencing methods have been used to analyze gene‐related phenotypes in natural habitats. These methods do not allow sufficient control over gene expression for the study of ecological interactions in real time, of genetic traits that play essential roles in development, or of dose‐dependent effects. We applied the sensitive dexamethasone (DEX)‐inducible pOp6/LhGR expression system to the ecological model plant Nicotiana attenuata and established a lanolin‐based DEX application method to facilitate ectopic gene expression and RNA interference‐mediated gene silencing in the field and under challenging conditions (e.g. high temperature, wind and UV radiation). Fully established field‐grown plants were used to silence phytoene desaturase and thereby cause photobleaching only in specific plant sectors, and to activate expression of the cytokinin (CK) biosynthesis gene isopentenyl transferase (ipt). We used ipt expression to analyze the role of CKs in both the glasshouse and the field to understand resistance to the native herbivore Tupiocoris notatus, which attacks plants at small spatial scales. By spatially restricting ipt expression and elevating CK levels in single leaves, damage by T. notatus increased, demonstrating the role of CKs in this plant–herbivore interaction at a small scale. As the arena of most ecological interactions is highly constrained in time and space, these tools will advance the genetic analysis of dynamic traits that matter for plant performance in nature.     

Just in time: Circadian defense patterns and the optimal defense hypothesis

The optimal defense hypothesis (ODH) provides a functional explanation for the inhomogeneous distribution of defensive structures and defense metabolites throughout a plant’s body: tissues that are most valuable in terms of fitness and have the highest probability of attack are generally the best defended. In a previous review,¹ we argue that ontogenically-controlled accumulations of defense metabolites are likely regulated through an integration of developmental and defense signaling pathways. In this addendum, we extend the discussion of ODH patterns by including the recent discoveries of circadian clock-controlled defenses in plants.     

A critical role for Arabidopsis MILDEW RESISTANCE LOCUS O2 in systemic acquired resistance

Members of the MILDEW RESISTANCE LOCUS O (MLO) gene family confer susceptibility to powdery mildews in different plant species, and their existence therefore seems to be disadvantageous for the plant. We recognized that expression of the Arabidopsis MLO2 gene is induced after inoculation with the bacterial pathogen Pseudomonas syringae, promoted by salicylic acid (SA) signaling, and systemically enhanced in the foliage of plants exhibiting systemic acquired resistance (SAR). Importantly, distinct mlo2 mutant lines were unable to systemically increase resistance to bacterial infection after inoculation with P. syringae, indicating that the function of MLO2 is necessary for biologically induced SAR in Arabidopsis. Our data also suggest that the close homolog MLO6 has a supportive but less critical role in SAR. In contrast to SAR, basal resistance to bacterial infection was not affected in mlo2. Remarkably, SAR‐defective mlo2 mutants were still competent in systemically increasing the levels of the SAR‐activating metabolites pipecolic acid (Pip) and SA after inoculation, and to enhance SAR‐related gene expression in distal plant parts. Furthermore, although MLO2 was not required for SA‐ or Pip‐inducible defense gene expression, it was essential for the proper induction of disease resistance by both SAR signals. We conclude that MLO2 acts as a critical downstream component in the execution of SAR to bacterial infection, being required for the translation of elevated defense responses into disease resistance. Moreover, our data suggest a function for MLO2 in the activation of plant defense priming during challenge by P. syringae.     

Metabolic robustness in young roots underpins a predictive model of maize hybrid performance in the field

Heterosis has been extensively exploited for yield gain in maize (Zea mays L.). Here we conducted a comparative metabolomics‐based analysis of young roots from in vitro germinating seedlings and from leaves of field‐grown plants in a panel of inbred lines from the Dent and Flint heterotic patterns as well as selected F₁ hybrids. We found that metabolite levels in hybrids were more robust than in inbred lines. Using state‐of‐the‐art modeling techniques, the most robust metabolites from roots and leaves explained up to 37 and 44% of the variance in the biomass from plants grown in two distinct field trials. In addition, a correlation‐based analysis highlighted the trade‐off between defense‐related metabolites and hybrid performance. Therefore, our findings demonstrated the potential of metabolic profiles from young maize roots grown under tightly controlled conditions to predict hybrid performance in multiple field trials, thus bridging the greenhouse–field gap.     

What happens in the pith stays in the pith: tissue‐localized defense responses facilitate chemical niche differentiation between two spatially separated herbivores

Herbivore attack is known to elicit systemic defense responses that spread throughout the host plant and influence the performance of other herbivores. While these plant‐mediated indirect competitive interactions are well described, and the co‐existence of herbivores from different feeding guilds is common, the mechanisms of co‐existence are poorly understood. In both field and glasshouse experiments with a native tobacco, Nicotiana attenuata, we found no evidence of negative interactions when plants were simultaneously attacked by two spatially separated herbivores: a leaf chewer Manduca sexta and a stem borer Trichobaris mucorea. T. mucorea attack elicited jasmonic acid (JA) and jasmonoyl‐l ‐isoleucine bursts in the pith of attacked stems similar to those that occur in leaves when M. sexta attacks N. attenuata leaves. Pith chlorogenic acid (CGA) levels increased 1000‐fold to levels 6‐fold higher than leaf levels after T. mucorea attack; these increases in pith CGA levels, which did not occur in M. sexta‐attacked leaves, required JA signaling. With plants silenced in CGA biosynthesis (irHQT plants), CGA, as well as other caffeic acid conjugates, was demonstrated in both glasshouse and field experiments to function as a direct defense protecting piths against T. mucorea attack, but not against leaf chewers or sucking insects. T. mucorea attack does not systemically activate JA signaling in leaves, while M. sexta leaf‐attack transiently induces detectable but minor pith JA levels that are dwarfed by local responses. We conclude that tissue‐localized defense responses allow tissue‐specialized herbivores to share the same host and occupy different chemical defense niches in the same hostplant.     

Experimental demonstration of a parasite‐induced immune response in wild birds: Darwin's finches and introduced nest flies

Ecological immunology aims to explain variation among hosts in the strength and efficacy of immunological defenses. However, a shortcoming has been the failure to link host immune responses to actual parasites under natural conditions. Here, we present one of the first experimental demonstrations of a parasite‐induced immune response in a wild bird population. The recently introduced ectoparasitic nest fly Philornis downsi severely impacts the fitness of Darwin's finches and other land birds in the Galápagos Islands. An earlier study showed that female medium ground finches (Geospiza fortis) had P. downsi‐binding antibodies correlating with presumed variation in fly exposure over time. In the current study, we experimentally manipulated fly abundance to test whether the fly does, in fact, cause changes in antibody levels. We manipulated P. downsi abundance in nests and quantified P. downsi‐binding antibody levels of medium ground finch mothers, fathers, and nestlings. We also quantified host behaviors, such as preening, which can integrate with antibody‐mediated defenses against ectoparasites. Philornis downsi‐binding antibody levels were significantly higher among mothers at parasitized nests, compared to mothers at (fumigated) nonparasitized nests. Mothers with higher antibody levels tended to have fewer parasites in their nests, suggesting that antibodies play a role in defense against parasites. Mothers showed no behavioral changes that would enhance the effectiveness of the immune response. Neither adult males, nor nestlings, had P. downsi‐induced immunological or behavioral responses that would enhance defense against flies. None of the parasitized nests fledged any offspring, despite the immune response by mothers. Thus, this study shows that, while the immune response of mothers appeared to be defensive, it was not sufficient to rescue current reproductive fitness. This study further shows the importance of testing the fitness consequences of immune defenses, rather than assuming that such responses increase host fitness.     

Ecological and evolutionary factors of intraspecific variation in inducible defenses: Insights gained from Daphnia experiments

Phenotypic variation among individuals and species is a fundamental principle of natural selection. In this review, we focus on numerous experiments involving the model species Daphnia (Crustacea) and categorize the factors, especially secondary ones, affecting intraspecific variations in inducible defense. Primary factors, such as predator type and density, determine the degree to which inducible defense expresses and increases or decreases. Secondary factors, on the other hand, act together with primary factors to inducible defense or without primary factors on inducible defense. The secondary factors increase intraspecies variation in inducible defense, and thus, the level of adaptation of organisms varies within species. Future research will explore the potential for new secondary factors, as well as the relative importance between factors needs to be clarified.     

Accumulation pattern of endogenous cytokinins and phenolics in different organs of 1‐year‐old cytokinin pre‐incubated plants: implications for conservation

A better understanding of phytohormone physiology can provide an essential basis to coherently achieve a conservation drive/strategy for valuable plant species. We evaluated the distribution pattern of cytokinins (CKs) and phenolic compounds in different organs of 1‐year‐old greenhouse‐grown Tulbaghia simmleri pre‐treated (during micropropagation) with three aromatic CKs (benzyladenine = BA, meta‐topolin = mT, meta‐topolin riboside = mTR). The test species is highly valuable due to its medicinal and ornamental uses. Based on UHPLC‐MS/MS quantification, mT and mTR pre‐treated plants had the highest total CK, mostly resulting from the isoprenoid CK‐type, which occurred at highest concentrations in the roots. Although occurring in much lower concentrations when compared to isoprenoid CKs, aromatic CKs were several‐fold more abundant in the root of mT pre‐treated plants than with other treatments. Possibly related to the enhanced aromatic CKs, free bases and ribonucleotides, plants pre‐treated with mT generally displayed better morphology than the other treatments. A total of 12 bioactive phenolic compounds, including four hydroxybenzoic acids, five hydroxycinnamic acids and three flavonoids at varying concentrations, were quantified in T. simmleri. The occurrence, distribution and levels of these phenolic compounds were strongly influenced by the CK pre‐treatments, thereby confirming the importance of CKs in phenolic biosynthesis pathways.     

Microscale magnetic microparticle‐based immunopurification of cytokinins from Arabidopsis root apex

Cytokinins (CKs) are pivotal plant hormones that have crucial roles in plant growth and development. However, their isolation and quantification are usually challenging because of their extremely low levels in plant tissues (pmol g^?1 fresh weight). We have developed a simple microscale magnetic immunoaffinity‐based method for selective one‐step isolation of CKs from very small amounts of plant tissue (less than 0.1 mg fresh weight). The capacity of the immunosorbent and the effect of the complex plant matrix on the yield of the rapid one‐step purification were tested using a wide range of CK concentrations. The total recovery range of the new microscale isolation procedure was found to be 30–80% depending on individual CKs. Immunoaffinity extraction using group‐specific monoclonal CK antibodies immobilized onto magnetic microparticles was combined with a highly sensitive ultrafast mass spectrometry‐based method with a detection limit close to one attomole. This combined approach allowed metabolic profiling of a wide range of naturally occurring CKs (bases, ribosides and N⁹‐glucosides) in 1.0‐mm sections of the Arabidopsis thaliana root meristematic zone. The magnetic immunoaffinity separation method was shown to be a simple and extremely fast procedure requiring minimal amounts of plant tissue.     

Changes in the Chlorophyll Content and Cytokinin Levels in the Top Three Leaves of New Plant Type Rice During Grain Filling

This paper reports the ways that the differences in leaf senescence are related to grain filling, grain yield, and the dynamics of cytokinins (CKs) in the top three leaves of four field-grown new plant type (NPT) rice, a tropical japonica developed at the International Rice Research Institute, Philippines, to increase the yield potential of rice. The chlorophyll content in leaves decreased from flowering to maturity in all the NPT lines, whereas the grain filling percentage was higher in the fast-senescing NPT line than in slow-senescing NPT line. Grain yield was positively correlated with senescence in the flag leaf. Rapid changes in the CK levels were recorded in the leaves of the fast-senescing line, whereas the CK levels were relatively stable in leaves of the slow-senescing line, suggesting that the dynamics of CKs in the fast-senescing line are vital for fast senescence. There were no significant changes in bioactive CKs, CK O-glucosides (storage CKs), and cis-zeatin derivatives in different leaves of the slow-senescing NPT line between 0 and 3 weeks after flowering, suggesting that the content of these CKs is relatively stable during grain filling. A progressive increase in levels of bioactive CKs was positively correlated with gradual accumulation of CK N-glucosides (inactive CKs) in the top three leaves of the slow-senescing NPT line, whereas the decrease of bioactive CKs in the flag leaf of the fast-senescing line was accompanied by accumulation of CK O-glucosides. These results suggest that there is a higher rate of biosynthesis and/or import of bioactive CKs as well as their turnover which may favor delay of leaf senescence in the slow-senescing NPT line.     

Basipetal auxin versus acropetal cytokinin transport,and their interaction with NO3 fertilisation in cotyledon senescence and sink:source relationships in cucumber (Cucumis sativus L.)

The paramount role of cytokinins (CKs) in initiation, as well as prevention, of senescence is well established. In recent years, experimental methods have become available to raise and lower the CK concentration and experimentally manipulate senescence. Decapitating the apical shoot and adding the synthetic auxin naphthylacetic acid to the cut stem reduced endogenous CKs to low levels. Conversely, if no auxin was applied, xylem and leaf CK levels increased dramatically, indicating that basipolar auxin transport is a key determinant in the synthesis of CKs and is potentially more important than NO₃. Manipulating the concentration of applied NO₃ caused considerable variation in leaf CK levels and concomitant changes in senescence. These and other results suggest that the frequently discussed decrease in nitrogen use efficiency (NUE) may be more highly regulated by CKs than by NO₃. Analysis of the re‐metabolisation and re‐allocation of chlorophyll, proteins, amino acids and starch in three different cucumber cultivars indirectly showed that these metabolites were significantly affected by the concentration of CKs in the leaves. Further research in this area may allow leaf senescence and plant yield to be more efficiently regulated by manipulating CKs and/or basipolar auxin transport instead of nitrate.     

Developmental Changes in Direct and Indirect Defenses in the Young Leaves of the Neotropical Tree Genus Inga (Fabaceae)

Plant fitness is affected by herbivory, and in moist tropical forests, 70 percent of herbivore damage occurs on young leaves. Thus, to understand the effects of herbivory on tropical plant fitness, it is necessary to understand how tropical young leaves survive the brief, but critical, period of susceptibility. In this study, we surveyed three species of Inga during young leaf expansion. Three classes of toxic secondary metabolites (phenolics, saponins, and tyrosine), extrafloral nectar production, leaf area, and extrafloral nectary area were measured at randomly assigned young leaf sizes. In addition, all defenses were compared for potential trade‐offs during leaf expansion. No trade‐offs among defenses were found, and the concentration of all defenses, except tyrosine, decreased during leaf expansion. We suggest that plants continued to increase phenolic and saponin content, but at a rate that resulted in decreasing concentrations. In contrast, tyrosine content per leaf steadily increased such that a constant concentration was maintained regardless of young leaf size. Nectar production remained constant during leaf expansion, but, because young leaf area increased by tenfold, the investment in extrafloral nectar per leaf area significantly decreased. In addition, nectary area did not change during leaf expansion and therefore the relative size of the nectary significantly decreased during young leaf expansion. These results support the predictions of the optimal defense hypothesis and demonstrate that the youngest leaves have the highest investment in multiple defenses, most likely because they have the highest nitrogen content and are most susceptible to a diversity of herbivores.     

The role of cytokinins in responses to water deficit in tobacco plants over-expressing trans-zeatin O-glucosyltransferase gene under 35S or SAG12 promoters

The impact of water deficit progression on cytokinin (CK), auxin and abscisic acid (ABA) levels was followed in upper, middle and lower leaves and roots of Nicotiana tabacum L. cv. Wisconsin 38 plants [wild type (WT)]. ABA content was strongly increased during drought stress, especially in upper leaves. In plants with a uniformly elevated total CK content, expressing constitutively the trans -zeatin O-glucosyltransferase gene ( 35S::ZOG1 ), a delay in the increase of ABA was observed; later on, ABA levels were comparable with those of WT.
As drought progressed, the bioactive CK content in leaves gradually decreased, being maintained longer in the upper leaves of all tested genotypes. Under severe stress (11 d dehydration), a large stimulation of cytokinin oxidase/dehydrogenase (CKX) activity was monitored in lower leaves, which correlated well with the decrease in bioactive CK levels. This suggests that a gradient of bioactive CKs in favour of upper leaves is established during drought stress, which might be beneficial for the preferential protection of these leaves.
During drought, significant accumulation of CKs occurred in roots, partially because of decreased CKX activity. Simultaneously, auxin increased in roots and lower leaves. This indicates that both CKs and auxin play a role in root response to severe drought, which involves the stimulation of primary root growth and branching inhibition.     

Cytokinin dehydrogenase differentially regulates cytokinin and indirectly affects hydrogen peroxide accumulation in tomato leaf

Cytokinin dehydrogenase (CKX) catalyzes the irreversible degradation of cytokinins (CKs). CKs play a role in the regulation of hydrogen peroxide (H(2)O(2)) accumulation while H(2)O(2) is involved in chlorophyll degradation. Here, we elucidated how CKX differentially regulates representative CK levels and indirectly influence H(2)O(2) accumulation in tomato leaves. We induced drought, salt and iron-deficiency stresses in tomato plants and found that chlorosis-inducing stresses (salt and iron-deficiency) induced lower chlorophyll levels as compared to drought stress and control. Protein gel blot analysis detected two CKXs in tomato leaves: a 35 kDa protein (CKX35) found in chlorotic leaves associated with a reduction in representative CK levels and high H(2)O(2) concentrations, and a 37 kDa protein (CKX37) found in green leaves associated with representative CKs and H(2)O(2) at normal levels. In summary, CKX isoforms in the tomato leaf appear to have distinct roles in differentially regulating CK levels and indirectly influencing H(2)O(2) accumulation.     

Asynchronous hatching in the burying beetle,Nicrophorus quadripunctatus,maxmizes parental fitness

Life history theory predicts that natural selection favours parents who balance investment across offspring to maximize fitness. Theoretical studies have shown that the optimal level of parental investment from the offspring's perspective exceeds that of its parents, and the disparity between the two generates evolutionary conflict for the allocation of parental investment. In various species, the offspring hatch asynchronously. The age hierarchy of the offspring usually establishes competitive asymmetries within the brood and determines the allocation of parental investment among offspring. However, it is not clear whether the allocation of parental investment determined by hatching pattern is optimal for parent or offspring. Here, we manipulated the hatching pattern of the burying beetle Nicrophorus quadripunctatus to demonstrate the influence of hatching pattern on the allocation of parental investment. We found that the total weight of a brood was largest in the group that mimicked the natural hatching pattern, with the offspring skewed towards early hatchers. This increases parental fitness. However, hatching patterns with more later hatchers had heavier individual offspring weights, which increases offspring fitness, but this hatching pattern is not observed in the wild. Thus, our study suggests that the natural hatching pattern optimizes parental fitness, rather than offspring fitness.     

Effects of elevated [CO(2)] and nitrogen nutrition on cytokinins in the xylem sap and leaves of cotton

We measured the level of xylem-derived cytokinins (CKs) entering a cotton leaf, and the CK levels in the same leaf, thus enabling xylem sap and foliar CKs to be compared concurrently. Although zeatin was the dominant CK in xylem sap, zeatin, dihydrozeatin, and N(6)-(2-isopentenyl) adenine were present in approximately equimolar levels in leaves. Elevated [CO(2)] (EC) has an effect on the levels of cytokinins in sap and leaf tissues. This effect was modulated by the two levels of root nitrogen nutrition (2 and 12 mM nitrate). Growth enhancement (70%) in EC over plants in ambient [CO(2)] (AC) was observed for both nitrogen nutrition treatments. Low-nitrogen leaves growing in EC exhibited photosynthetic acclimation, whereas there was no sign of photosynthetic acclimation in high-nitrogen grown leaves. Under these prevailing conditions, xylem sap and leaf tissues were obtained for CK analysis. Higher nitrogen nutrition increased the delivery per unit leaf area of CKs to the leaf at AC. EC caused a greater increase in CK delivery to the leaf at low nitrogen conditions (106%) than at high nitrogen conditions (17%). EC induced a significant increase in CK content in low-nitrogen leaves, whereas CK content in leaf tissues was similar for high-nitrogen leaves growing in AC and EC.