Making extra room for carotenoids in plant cells: New opportunities for biofortification

Plant carotenoids are essential for photosynthesis and photoprotection and provide colors in the yellow to red range to non-photosynthetic organs such as petals and ripe fruits. They are also the precursors of biologically active molecules not only in plants (including hormones and retrograde signals) but also in animals (including retinoids such as vitamin A). A carotenoid-rich diet has been associated with improved health and cognitive capacity in humans, whereas the use of carotenoids as natural pigments is widespread in the agrofood and cosmetic industries. The nutritional and economic relevance of carotenoids has spurred a large number of biotechnological strategies to enrich plant tissues with carotenoids. Most of such approaches to alter carotenoid contents in plants have been focused on manipulating their biosynthesis or degradation, whereas improving carotenoid sink capacity in plant tissues has received much less attention. Our knowledge on the molecular mechanisms influencing carotenoid storage in plants has substantially grown in the last years, opening new opportunities for carotenoid biofortification. Here we will review these advances with a particular focus on those creating extra room for carotenoids in plant cells either by promoting the differentiation of carotenoid-sequestering structures within plastids or by transferring carotenoid production to the cytosol.     

Structural modification of Quercus petraea leaf caused by Cynips quercusfolii – histological study of galls

Galls formed by the interaction of insects on plant tissues are an example of the unusual transformation and use of plants by insects. The aim of this study was to characterize the structure of galls formed by Cynips quercusfolii L. on sessile oak leaves. In the structure of galls, we distinguished the following: (1) the protective ‘first contact zone’ created by epidermal and sub-epidermal sclerenchyma rings, (2) the wide parenchymatous ring, (3) the internal protective zone created by the sclerenchyma ring, and (4) the nutritional zone consisting of cells filled with amyloplasts containing starch. A characteristic for galls in the development stage is the centripetal starch gradient in which starch accumulates in a ‘ring of amyloplasts’ in the larval chamber.     

Variation in utilization of young leaves by a swallowtail butterfly across a deer density gradient

Phytophagous insects can be affected by plant trait-mediated indirect effects of large herbivores, but little is known regarding how these effects change in response to different densities of large herbivores. To assess the response of an insect to plant qualitative change, the response of a woody vine (Aristolochia kaempferi) to browsing by sika deer (Cervus nippon) and utilization of young leaves by a swallowtail butterfly (Byasa alcinous) were investigated across a deer density gradient. Natural and simulated deer browsing stimulated the regrowth of A. kaempferi and improved nutritional and physical quality of leaves. Young leaves were frequently observed in areas with high deer densities. The proportion of young leaves among the leaves selected for oviposition was higher than their proportion of the total number of leaves. In areas with low deer densities, the utilization of young leaves by B. alcinous increased linearly with deer density, whereas in areas with high deer densities, the utilization of young leaves was around 90%.     

Autumn leaves seen through herbivore eyes

Why leaves of some trees turn red in autumn has puzzled biologists for decades, as just before leaf fall the pigments causing red coloration are newly synthesized. One idea to explain this apparently untimely investment is that red colour signals the tree's quality to herbivorous insects, particularly aphids. However, it is unclear whether red leaves are indeed less attractive to aphids than green leaves. Because aphids lack a red photoreceptor, it was conjectured that red leaves could even be indiscernable from green ones for these insects. Here we show, however, that the colour of autumnal tree leaves that appear red to humans are on average much less attractive to aphids than green leaves, whereas yellow leaves are much more attractive. We conclude that, while active avoidance of red leaves by aphids is unlikely, red coloration in autumn could still be a signal of the tree's quality, or alternatively serve to mask the over-attractive yellow that is unveiled when the green chlorophyll is recovered from senescing leaves. Our study shows that in sensory ecology, receiver physiology alone is not sufficient to reveal the whole picture. Instead, the combined analysis of behaviour and a large set of natural stimuli unexpectedly shows that animals lacking a red photoreceptor may be able to differentiate between red and green leaves.     

Interactions between Chromolaena odorata (Asteraceae) and Pareuchaetes pseudoinsulata (Lepidoptera: Arctiidae)

Feeding of Pareuchaetes pseudoinsulata caterpillars caused the leaves of Chromolaena odorata to turn yellow. Leaf yellowing could not be induced either by artificial removal of leaves or by drenching the plant with a solution of excreta from P. pseudoinsulata caterpillars. Yellow leaves appeared tougher but had the same energy level as that of green leaves. The amount of nitrate-nitrogen was significantly higher in yellow leaves than green leaves. P. pseudoinsulata caterpillars prefer to feed on green leaves. When forced to feed on yellow leaves, they exhibit slow growth and high mortality. Defensive factors in plants attacked by insects seemed to prevent further infestation of plants. In the field, caterpillars on the yellow plants were found during both day and night whereas on green plants they appeared to feed at night and hide in the ground at daytime.     

Leaf trait variation on Erythroxylum tortuosum (Erythroxylaceae) and its relationship with oviposition preference and stress by a host‐specific leaf miner

It has been suggested that plant physical and chemical traits vary considerably in space and time. Hence, leaf‐mining insects may adjust their oviposition in response to leaf attributes representing high quality. Moreover, herbivorous insects can modify leaf morphology by acting as stressors, increasing, for example, fluctuating asymmetry (FA) levels. Here, we investigate oviposition preference in Agnippe sp.2, a leaf‐mining moth of Erythroxylum tortuosum, in relation to differences in leaf nutritional quality (i.e. levels of water, nitrogen and tannin content), leaf area (i.e. quantity of resource hypothesis) and FA. We also verify whether temporal variation in plant nutritional quality emerges as an alternative hypothesis to explain oviposition distribution in time, and whether this leaf miner is a stress‐causing agent, increasing FA during larval development. Mined leaves and leaves with and without eggs were periodically collected from plants located in a Cerrado fragment in Brazil. In the laboratory, leaf traits were assessed (using image analysis software) and quantified (biochemical analysis) according to the aims previously determined. Oviposition probability did not change in relation to variations in nitrogen, tannins and FA of leaves. However, leaf‐miner females preferred to oviposit on leaves having large areas and low water contents. It was also verified that new leaves of E. tortuosum, which carried most leaf‐miner eggs, presented significantly lower tannins and greater levels of nitrogen and water than old leaves. The oviposition choice exhibited by leaf miners was found to be non‐random because they appear to use resource quantity and water content as cues as where to lay their eggs. The temporal variation of plant nutritional quality is likely to influence the time of leaf‐miner oviposition; and leaf FA was not increased during larval feeding, suggesting that these herbivores do not cause variations in FA levels.     

Nutritional quality of specific leaf tissues and selective feeding by a specialist leafminer

Summary Many folivorous insects are selective feeders which consume specific leaf tissues. For specialist herbivores feeding on plants of overall low nutritional quality, selective feeding may allow consumption of a high quality resource. Selective feeding may also allow insects to avoid structural or allelochemical defenses. We examined the structure and chemistry of leaves of American holly, Ilexopaca Aiton, and the feeding site of its principal insect herbivore, the native holly leafminer, Phytomyza ilicicola Loew (Diptera: Agromyzidae), to test the hypothesis that the leafminer consumes tissues which are of greater nutritional quality than the leaf as a whole. Holly leaves have a continuous layer of palisade mesophyll, uninterrupted by fibers or vascular bundles. The leafminer feeds entirely within this layer. The palisade mesophyll contained 196 mg/g dry wt extractable protein, more than twice as much as the leaf as a whole, and 375 mg/g dry wt saponins, more than 9 times that of the leaf as a whole. The water content of the palisade mesophyll was 66% higher than that of the leaf as a whole. The palisade mesophyll is 3–4 cell layers thick in leaves grown in full sun, but only 2 layers thick in shaded leaves. Crystals, probably of calcium oxalate, are abundant in the abaxial cell layer. These may impose mechanical constraints on larval feeding in shade leaves, which are thinner than sun leaves. Selective feeding on the middle palisade mesophyll of sun leaves allows the leafminer to consume a resource which is lacking in mechanical barriers and is rich in protein and water, but which contains large amounts of saponins.The investigation reported in this paper (No. 86-8-7-117) is in connection with a project of the Kentucky Agricultural Experiment Station and is published with the approval of the Director     

Selection of large host plants for oviposition by a monophagous leaf beetle: nutritional quality or enemy‐free space?

Abstract.  1. Oviposition site selection is crucial for the reproductive success of herbivorous insects. According to the preference–performance hypothesis, females should oviposit on host plants that enhance the performance of their offspring. More specifically, the plant vigour hypothesis predicts that females should prefer large and vigorously growing host plants for oviposition and that larvae should perform best on these plants.
2. The present study examined whether females of the monophagous leaf beetle Cassida canaliculata Laich. (Coleoptera: Chrysomelidae) prefer to oviposit on large host plant individuals of the meadow clary and whether large host plants are of higher nutritional quality than small host plants. Subsequently, it was tested whether the female preference correlates with offspring performance and survival.
3. In the field, females preferred large host plant individuals for oviposition and host plant quality, i.e. leaf nitrogen content, was significantly higher in leaves of large than of small host plants.
4. In the laboratory, larval development time was shorter on leaves of large host plant individuals than on small host plant individuals, but this could not be shown in the field.
5. However, a predator-exclusion experiment in the field resulted in a higher survival of larvae on large host plants than on small host plants when all predators had free access to the plants. On caged host plants there was no difference in survival of larvae between plant size categories.
6. It is concluded that females of C. canaliculata select oviposition sites that enhance both performance and survival of their offspring, which meets the predictions of the plant vigour hypothesis.     

Preference for protected feeding sites by larvae of the willow-feeding leaf beetle Galerucella lineola

1. Free-living insects are often thought of as more vulnerable to environmental hazards than concealed insects, such as galling or mining insects. The possibility that larvae of the free-living leaf beetle Galerucella lineola seek out existing plant structures and thereby become partly concealed was explored.
2. Neonate larvae of G. lineola frequently feed in rolled-in margins of young leaves of their host plant, Salix viminalis . In addition to nutritional benefits from feeding on young leaves, larvae may also gain protection against adverse weather conditions and general predators by feeding in the leaf rolls. Field and laboratory experiments were conducted to test these hypotheses.
3. Artificial shelters were constructed and cohorts of neonate larvae were placed on experimental plants. In all experiments, larvae preferred to feed in shelters, even when shelters were constructed on mature leaves.
4. In one of the experiments, fewer larvae disappeared when shelters were provided. In a predator exclusion experiment, however, no differences in predator-inflicted mortality on G. lineola were found between shelter-containing shoots and control shoots.
5. A laboratory experiment showed increased protection from desiccation when shelters were present; growth rate was higher for larvae feeding on plants with shelters.
6. Thus, free-living insects may not always be as exposed to environmental hazards as is often assumed. In particular, young larvae may take advantage of preformed structures on their host plant and feed in a concealed microhabitat. Because mortality, in general, is high during early instars, shelter-seeking behaviour may increase survival significantly. The existence of preformed shelters may therefore be a plant characteristic that should be considered when exploring the environmental risks associated with the free-living habit.     

Intraspecific variation in plant size,secondary plant compounds,herbivory and parasitoid assemblages during secondary succession

During secondary succession on abandoned agricultural fields the diversity and abundance of insect communities often increases, whereas the performance and nutritional quality of early successional plants often declines. As the diversity and abundance of insects on a single plant are determined by characteristics of the environment as well as of the host plant, it is difficult to predict how insects associated with a single plant species will change during succession. We examined how plant characteristics of the early successional plant species ragwort (Jacobaea vulgaris), and the herbivores and parasitoids associated with these plants change during secondary succession. In ten grasslands that differed in time since abandonment (3–26 years), we measured the size and primary and secondary chemistry of individual ragwort plants. For each plant we also recorded the presence of herbivores in flowers, leaves and stems, and reared parasitoids from these plant parts. Ragwort plants were significantly larger but had lower nitrogen concentrations in recently abandoned sites than in older sites. Pyrrolizidine alkaloid (PA) composition varied among plants within sites but also differed significantly among sites. However, there was no relationship between the age of a site and PA composition. Even though plant size decreased with time since abandonment, the abundance of stem-boring insects and parasitoids emerging from stems significantly increased with site age. The proportion of plants with flower and leaf herbivory and the number of parasitoids emerging from flowers and leaves was not related to site age. Parasitoid diversity significantly increased with site age. The results of our study show that ragwort and insect characteristics both change during secondary succession, but that insect herbivore and parasitoid abundances are not directly related to plant size or nutritional quality.     

Leaf mines as visual defensive signals to herbivores

Leaf‐mining insects produce conspicuous and distinct leaf mines on various types of plant leaves. The diversity of leaf‐mine morphology has typically been explained by several factors, such as selective feeding on plant tissues, improvement of microclimate, faecal disposal, reduction in the efficiency of parasitoid search behaviour and leafminer phylogeny. Although these factors are certainly associated with mining patterns, masking the mines, rather than making them conspicuous, appears to be more advantageous for deterring parasitoids and predators of leafminers. However, here, I propose that prominent leaf mines may serve to signal or cue herbivores to avoid feeding on the mined leaves. Because most leafminers are sessile and complete their development within a single leaf, herbivory of mined leaves is detrimental to leafminer survival. Other herbivores appear to avoid consuming mined leaves for a variety of reasons: leaf mines mimic leaf variegation or mottling; mined leaves induce chemical and physical defences against herbivores; and leaf mines mimic fungal infection, animal excrement, and necrosed plant tissues. Hence, natural selection may have favoured leafminers that produce conspicuous mines because of the increased survival and fecundity of thereby reducing herbivory on mined leaves.     

VIRUS DISEASES OF CACAO IN WEST AFRICA

Increasing the number of Pseudococcus njalensis per test plant increased the infection rate with cacao virus 1A and 1M to 95 % with twenty-five insects. Young leaves with the red vein-banding symptoms were better sources of virus than mature leaves, and there was evidence of virus localization in the plant. It was estimated that Ps. njalensis nymphs took about 16 min. to penetrate plant tissues with their stylets.
Infection rates increased with time of test feeding up to 50 min. The virus was non-persistent in the feeding insect, but persisted up to 36 hr. in insects starved after the infection feed. Starvation before the infection feed increased the infection rate with infection feeds up to 10 hrs., after which the infection rate with pre-starved insects declined to the level attained by unstarved insects.     

Insect colour preference compared to flower colours in the Australian Alps

An apparent predominance of plant taxa with pale flowers in the alpine floras of Australia and New Zealand may be due to the prevalence of insects, such as flies, that prefer pale colours and the absence of other types of potential pollinators that are attracted to bright colours such as social bees and birds. In this study, the diversity of flower colours, and the preference of insects for different colours were examined for the largest contiguous alpine area in Australia, around Mt Kosciuszko. Out of an alpine flora of 204 taxa, 127 species were found to have large showy flowers. The most common flower colour among these taxa was white (53.5%), then yellow (21.3%), followed by pink (6.3%), and cream (6.3%). Only a handful of taxa had red, blue, brown, green, orange or purple flowers. When the colour preference of insects was tested using five different coloured traps (white, yellow, orange, red and purple), the most successful traps were white then yellow, with these two colours accounting for 66% of all individual insects collected. Diptera were the most common insects caught (576 insects greater than 4 mm in length, 31 morphotaxa) showing an apparent preference for white and yellow coloured traps over others. Therefore, the results add some support to the proposition that the 'white' flora of the Australian Alps may be associated with the colour preference of flies, which have previously been found to be the most common type of pollinators in the Kosciuszko alpine zone.     

植物的诱导抗虫性

植物对植食性昆虫的抗性可包括两个方面,即植物的组成抗性（constitutiveresistance）和诱导抗性（inducedresistance）。组成抗性是指植物在遭受植食性昆虫进攻前就已存在的抗虫特性;而诱导抗性是指植物在遭受植食性昆虫进攻后所表现出来的一种抗虫特性[1,2]。根据作用世代的不同,诱导抗性又分为迅速的诱导抗性（rapidlyinducedre－sistance,RIR）和滞后的诱导抗性（delayedinducedresistance,DIR）。前者是指对当前世代的植食性昆虫的影响,而后者是指对后续的1～几个世代的植食性昆虫的影响[2]。研究植物的诱导抗虫性,不仅能在…     

Physiological factors affecting the rapid decrease in protein assimilation efficiency by a caterpillar on newly‐mature tree leaves

Lymantria dispar L. caterpillars have a decreased ability to assimilate protein from mature leaves of red oak (Quercus rubra) compared with young, expanding leaves. The present study determines whether the drop in protein assimilation efficiency (PAE) occurs during the rapid phase of leaf maturation. Several mechanisms that might account for decreased PAE are also examined: mature leaf tissues could resist being chewed efficiently, protein in mature leaf tissues could become difficult to extract, and other nutrients in mature leaves might become growth limiting. The entire seasonal decrease in PAE occurs rapidly (in less than 2 weeks), when the leaves finished expanding. The maturation process is characterized by increased levels of fibre and decreased levels of water but no significant changes in the levels of protein or carbohydrates. Despite increased fibre in mature leaves, they are not chewed into larger food particles than are immature leaves. Carbohydrate assimilation efficiencies remain high on mature leaves, and signs of limiting water levels in larvae of L. dispar on mature leaves are not observed. The most important finding in the present study is the decreased extractability of protein in food particles from mature leaves, which plays a major role in explaining the rapid decrease in PAE. It is hypothesized that structural changes in cell walls during the rapid process of leaf maturation decrease protein extractability, which, in turn, greatly decreases the nutritional quality of mature oak leaves for caterpillars. The results of the present study therefore suggest a general mechanism to help explain the widely documented decrease in the nutritional quality of the mature leaves of many tree species for herbivorous insects.     

Factors affecting calculation of nutritional induces for foliage-fed insects: an experimental approach

To determine how nutritional indices for insects fed leaves are affected by the experimental conditions and the physiology of the plant material, we used larvae of the buckmoth, Hemileuca lucina Hy. Ed. (Saturniidae) and their hostplant Spiraea latifolia Ait. Bork (Rosaceae). Under experimental conditions identical to those used to determine larval nutritional indices, we found that the age of leaves (new versus mature) significantly affected their metabolism and water loss, but simulated herbivory did not directly affect leaf metabolism. Over a 6-day test, nitrogen concentration showed an initial increase followed by a gradual decline, and was higher in new leaves compared to mature leaves. New leaves increased in protein concentration and then gradually returned to the initial level, whereas mature leaves changed little over the 6-day test. These changes in percent nitrogen and protein may largely reflect the disproportional changes in non-nitrogenous materials. Solitary and grouped larvae had similar growth rates on new leaves, but they differed on mature leaves. Deliberate manipulation of larvae during the course of an experiment significantly reduced relative growth rates by increasing duration of the stadium rather than by decreasing biomass gained. Two methods of estimating larval gut contents at mid-stadium were compared: weight of frass produced and weight of digestive tract and contents. After the end of the 4-day test period used to determine nutritional indices, the digestive tracts with food accounted for 10.8% of the larval dry weight. Larval frass produced in 24 h after the end of the test period comprised 9.3% of the larval dry weight. Correction factors for plant metabolism changed nutritional indices by 1 to 8%, while those for larval gut contents altered indices by 2 to 15%.     

Gall aphids do not select optimal galling sites (Smynthurodes betae; Pemphigidae)

Abstract. 1. In studies of insect-host plant interaction it is often suggested that insects preferentially colonize host plants (or sites within plants) on which their fitness is maximized (a positive covariance of preference and performance). This suggestion stems from the assumption that natural selection has driven the system toward optimal use of resources.
2. Our study of the galling aphid Smynthurodes betae Westw. demonstrates that the distribution of galls on leaves is not due to preference, and can be altered by manipulating the aphid arrival time or the shoot growth rate.
3. We found no correlation between gall density and performance (aphid clone size) at different positions along the shoot.
4. Because leaves on the growing shoot are not equally responsive to aphid stimulation, the colonizers have no choice but to settle on leaves that are at the right stage when they arrive.
5. S.betae colonizers did not discriminate between shoots of their host and a congeneric non-host, on which their fitness is invariably zero.
6. Synchronization between galler and host plant phenologies seems to be the key to the observed distribution of galls on the tree. The data give no support to the preference-performance hypothesis.     

Colorado potato beetle manipulates plant defenses in local and systemic leaves

Herbivore microbial associates can affect diverse interactions between plants and insect herbivores. Some insect symbionts enable herbivores to expand host plant range or to facilitate host plant use by modifying plant physiology. However, little attention has been paid to the role of herbivore-associated microbes in manipulating plant defenses. We have recently shown that Colorado potato beetle secrete the symbiotic bacteria to suppress plant defenses. The bacteria in oral secretions from the beetle hijack defense signaling pathways of host plants and the suppression of induced plant defenses benefits the beetle’s performance. While the defense suppression by the beetle-associated bacteria has been investigated in local damaged leaves, little is known about the effects of the symbiotic bacteria on the manipulation of plant defenses in systemic undamaged leaves. Here, we demonstrate that the symbiotic bacteria suppress plant defenses in both local and systemic tissues when plants are attacked by antibiotic-untreated larvae.     

Nutritional ecology of insect–plant interactions: persistent handicaps and the need for innovative approaches

Quantifying the flow of matter and energy in food webs is indispensable when assessing the effects of increases in atmospheric carbon dioxide, ozone level and temperature as a result of global climate change. In insect nutritional ecology, quantification of digestive and metabolic efficiency is performed using gravimetric methods in all published cases. A few cases combined these methods with calorimetric and respirometric techniques. Since 1986, methodological pitfalls and sources of error inherent to applying gravimetry as the only method to construct nutrient budgets have been addressed in a number of papers without noticeable impact on subsequent research. Especially for insects feeding on living plant tissues, the gravimetric method has inherent handicaps as it can only be used with excised plant tissues and does not allow for the dynamics of plant metabolism. We discuss the major constraints of the gravimetric method as it pertains to the physiological processes of both the insect and plant. We apply a relationship between relative metabolic rate and relative growth rate of the insect for an analysis of the gravimetric literature. The analysis reveals that gravimetry has given rise to physiologically unlikely results for poikilothermic insects. This points to serious constraints on progress in this field. We identify plant respiration as the major source of error in gravimetric studies. We establish that no single study has, thus far, determined the metabolic efficiency of a herbivore feeding on a photosynthetically active plant with its phyllosphere microclimate. We argue that a quantitative understanding of the ecophysiology and nutritional ecology of insect–plant interactions must rely on the adoption of a combination of existing and complementary methods such as the double labelled water method and infrared gas analysis.