Role of Nectar and Extrafloral Nectaries in Biotic Stress management

By KABEYA, Muamba Jerry Crop Physiology Dpt, UAS-Bangalore

Nectar Secretion complete two mutualistic interactions with animals:

Pollination Reproduction (pollinators attraction) Plant breeding program

Defense Together with Extrafloral nectaries (EFN) Anti-microbial activity (non-sterile pollinators and airborne) Anti-herbivore activity (entice predator or parasitoid insects that prey on herbivores ) Communication (plant to-plant signaling) Plant and animals co-evolution Oxidative stress mitigation

Nectar compositions Conventionally; Sugar : Suc, Glc and Fru. Sugar level on type of pollinators

I.

Generalists feeder s: attracted with nectar with high Suc

e.g. honeybees, wasps, parasitic wasp

I.

Specialists feeders : attracted with nectar with high hexose

contents e.g. ants

Amino acid:

Second most abundant known for good taste e.g. Phe, Trp, Val etc

All 20 of the normal amino acids found in protein have been identied in various nectars

Originate from phloem sap and enter secretary cells via plasmodesmata

Other nectar substances

Not carried in Phloem cells

Inorganic ions Lipid Protein

The major cations in most nectars

Phenolics
Alkaloids Vatamins Oils

Potassium 3574%
Ferric iron 23%

This rich medium with many components for cellular growth

and metabolism attract both beneficial and harmful visitors, including airborne pathogens benefit from the abundant free offerings.

However, Harmful or non-sterile feeders and airborne pathogens visiting floral nectar endanger
and are infections to gynoecium

The case study was focused in ornamental tobacco Secretion of proteins in floral nectar, termed as nectarins, in most flowering species can play very pivotal role in biological activities Nectarins are proteins secreted and accumulated in plant nectars Up to Five different proteins in tobacco nectar

Namely; Nectarin I (NEC1) Nectarin II (NEC2) Nectarin III (NEC3) Nectarin IV (NEC4) Nectarin V (NEC5) In order of increasing molecular mass In ornamental tobacco, nectarins `

` accumulate to almost 250 g/ml in nectar

These nectarins generate high levels of

hydrogen peroxide which play the
Protein indentified in plants nectars

protection role.

CA, carbonic anhydrase; SOD, superoxide dismutase

The primary function of high level of H2O 2 by nectarin is to defend the oral reproductive tract from contamination by microbes brought to the ower by non-sterile pollinators or

by airborne means but how?.

Overview of Necterin Roles

Constitutively, mature nectaries produce superoxide (deleterious)

Specifically to NEC1, possess manganese superoxise dismutase (SOD) enzymes which act on superoxide.

NEC5 (glucose oxidase) uses oxygen and DHA as electrons acceptor to generate H2O2 and ascorbate respectively

Thus, High level of H2O2 in nectar play: Defensive role against microbial attack

to gynoecium
Superoxide dismutase (SOD) enzymatic activity consumes lot of protons, there is a propensity of nectar to become basic Ovule protection Nectar palatability for visiting pollinators

carbonic anhydrase (NEC3) activity provides a pH-balanced meal to pollinators and maintains proper function of nectar enzymes

Summary of Nectar redox cycle

MDHA monodehydroascorbate DHA - Dehydroascorbate

Localization and genes expression of these nectarins Spatial and temporal expression of nectary genesa by RT-PCR

- indicates no expression; , +,++, +++ indicates ambiguous, low, medium and high levels of expression, respectively. b Plant tissues: L, leaf; R, root; S, stem; Pe, pedicel; Pi, pistil; St, stigma/style; An, anther/lament; Se, sepals; O, ovary; N, nectary. c Nectary stage: 2, Stage 2 (early oral bud); 6, Stage 6 (immature nectary, lling stage); 8, Stage 8 (immature nectary,mid-maturation stage); 10, Stage 10 (immature nectary, early maturation stage, pre-secretory); 12, Stage 12 (mature nectary, secretory stage); PF, nectaries from post-fertilized owers.

Conclusion,

The generation of high levels of hydrogen peroxides by the Necterin proteins (NEC1 &NEC5) serves primarily to protect the gynoecium from infection by microorganisms.

High levels of hydrogen peroxide in the presence of metal ions produce deleterious free radicals (hydroxyl radical, OH.).

In a novel oxidationreduction cycle, which is termed as nectar redox cycle, ascorbate, NEC3 and possibly NEC5 detoxify these free radicals

In addition, NEC3 maintains the pH of nectar by using the same buffering system found in animal sera.

Extraoral nectaries of the genus Populus were rst described by (Trelease (1881) but emphasis of its defense mechanism has been overlooked (Wooley et al., 2007). The genus Populus is a known host for many herbivorous insects and pathogens. Populus thus provides an excellent model tree to study biotic stress management Two different species of Populus: to investigate the structure, Populus tremula (Ptr) Populus tremuloides (Ptt).

nectar production and composition

in their extraoral nectaries

Procedures Populus tremula x Populus tremuloides plants (clone T89) and Populus trichocarpa (clone 93-968) were eld grown in soil under natural conditions, under long-day conditions in climate chambers (16 h of light [22C]/8 h of darkness [17C]

Plants were watered twice a week and fertilized frequently.

All experiments were performed using cultivated trees of about 4 to 5 feet height.

For effectiveness tests and visitor determination : Field-grown trees were used ( about 3 to 4 years old and 4 to 5 feet high).

Light Microscopy

Extraoral nectaries from Ptr or Ptt were harvested

Fixed in ascending grades of ethanol for 45 min each (dehydration)

Sample were embedded in Agar Scientific (2-hydroxyethyl methacrylate/glycol methacrylate )

Sections (20 mm) were made with a c-prole 16-cm knife (Leica) in a Leica RM2165 microtome and heat xed to microscope slides.

Differential staining with toluidine blue, mounted in immersion oil under coverslips, and examined with a VHX-100k digital microscope

Nectary Appearance and its Effectiveness Nectary appearance and leaf damage were quantied using leaves (1,257 leaves) of different trees (Ptt) Damage was classied as : Severe (more than 75% damage of the leaf surface Mild (less than 75%)

Healthy (no damage)

Transmission Electron Microscopy Small sections of leaf tissue (12 mm) were cut with a razor blade Fixation for 4 hrs (1% (w/v) formaldehyde, 1 mM EGTA, 50 mM cacodylate buffer, and 5% glutaraldehyde) Post-xation overnight at room temperature ( 2% (w/v) osmium tetroxide)

Pre staining ( 3% (w/v) uranyl acetate in 20% ethanol for 1 h) Dehydration in series of graded ethanol Embedment in Spurrs epoxy resin Sectioning with ultramicrotome and stained with lead citrate Sections were examined using a Zeiss EM 10c transmission electron microscope at 80 kV. Induction of EFN Young and fully expanded leaves of each plant were induced: Manual Induction puncturing 100 times with a needle (1 mm diameter) cutting the leaf tip (about 10% of the total leaf area) Herbivory induction Caterpillars (Spodoptera littoralis, Spodoptera exigua, and Lymantria dispar) were placed

on Ptr leaves.

Mechanical Induction with MecWorm

Individual leaves of the intact test plant were continuously damaged by the robotic MecWorm system over a period of 24 h
Resulted in 333mm2 of damaged area using four punches per minute. Volatiles were then collected
Findings

Positioning of poplar extrafloral nectaries In both species, nectary pairs localized at the base of the leaf blade. Ptr A and D, arrows denote nectary positions. In the leaf base enlargements, nectaries in detail (arrows in B and E),

Ptt

Ptr release of large nectar amounts (C), and large Ptt nectaries releasing small nectar amounts(F)

In spring, many emerging Ptt and Ptr leaves harbor nectaries

To test whether this phenomenon results from heritable genetics, the extraoral nectary densities of eld-grown Ptt trees were determined

Effectiveness of nectaries against herbivores in Ptt

The populations of leaves with

(38%) and without (62%) nectaries

were highly conserved among

individual trees

Effectiveness of nectaries against herbivores in Ptr

Nectary-free leaves, however, exhibited a higher percentage of damage with Ptt and Ptr

 Naturally nectaries appeared with the onset of leaf emergence on both poplar species. Nectar on Ptt was continuously released over a week, unlike Ptr for few

days and later died (nectaries)

Under stress after first nectar secretion Secondary nectaries is produced : new nectaries occurred side by side or on top of dead ones () with the same secretion characteristics as the initial population

1st holocrine nectar secretion

Dead of nectaries after holocrine secretion

20 nectar-secreting nectaries on top of dead ones

So, how does the secondary nectar production of Ptr induced?

To test whether EFN production in Ptr results from manual stress, leaves of Ptr were wounded by puncturing the leaf

blade with a needle.

But After this procedure, however, nectar production was not observed.

To test whether persistent wounding initiates nectar production under stress

An automated damage procedure was applied using MecWorm mechanical caterpillar (computer-controlled), which mimics the damage caused by herbivores in terms of the spatiotemporal pattern of leaf destruction

Even these near natural wounding settings did not trigger nectar production. This indicates that Ptr nectar production seems not to be initiated simply by wounding of leaves but requires another stimulus instead

Which stimulant then ?

Caterpillars (S. littoralis, Spodoptera exigua, and Lymantria dispar) were placed on Ptr leaves to feed on.

Again,

neither

nectary

nor

nectar
observed

production
within 48 h

was
of

caterpillar feeding.

This clearly showed that herbivore-evoked leaf damage is not causing Ptr nectar secretion.

But,

When intact plants of Ptr were subjected to mealy bugs which, in contrast to caterpillars, feed on phloem sap, nectar secretion set in Nectaries statistics

B4 experiment < 10%

Four days after infestation, 42% of all leaves were

equipped with nectaries

about 50% of them (42%) produced nectar

In most cases, the secondary nectar production occurred upon attack by sucking insects (confined to specific types of herbivores)

Visitors attracted by extrafloral Ptt and Ptr nectaries. (A) honey bee at Ptt Generalist

(B) honey bee at Ptr. - Generalist

Sugar content in nectars of (A) Ptt (B) Ptr Ratios were constant within the single species but differed between both poplar ecotypes. (Mean SD, n=5) (C) wasps at Ptt. - Generalist (D) ant at Ptr. - Specialist

Conclusions Ptt continuously secretes nectar from long-living nectaries, and excess nectar is reabsorbed via endocytosis. Ptr, only in the case of special insect attack, produces secondary nectar by extraoral nectaries. Both Ptt and Ptr trees seem to protect their delicate rst leaves in spring against herbivores by nectar production.

Ptr nectar seems to attract rather specialist visitors, than Ptt which provides nectar for generalists.

TRITROPHIC INTERACTIONS
Three eating (trophic) levels thatoften link several species in a community through herbivory, predation and/or parasitism.

A sudden decrease in Daphnia would result in a decrease in damsel fly larvae and an increase in unicellular algae.

Hover flies sip nectar from flowers and in turn are eaten by a spiders. This is a version of: Predators eat herbivores, and herbivores eat plants.

Daphnia are herbivores on a unicellular algae and the prey of damsel flies. A decrease in algae would soon result in a decrease in Daphnia and later a decrease in damsel flies.

Field experiments that compared peach (P. persica) trees with and without EFNs were conducted in at two sites (Fruit Research Station, Kearneysville, West Virginia, and University of Maryland Western Maryland Research and Education Center, Keedysville, Maryland, USA). P. persica has three leaf EFN phenotypes: Globose (small, circular EFN glands) Reniform (large, kidneyshaped EFN glands) No glands

Dormant peach seedlings were potted and held in the greenhouse at 18218C for 4 weeks to break dormancy Trees (320 total) of a consistent size range were selected after verifying leaf EFN phenotype (160 trees with EFN reniform glands, 160 trees without EFN glands)

All trees were treated with avermectin against arthropods infestations in greenhouse

and transplanted in eld plots.

Hybrid willow trees (1 m tall) were planted into buffer rows separating the plots of each site (1-m spacing).

Necessary agronomic practices (Fungicides, herbicides, Chlorpyrifos etc.) except

application of Insectides throughout the experimental period was observed. Arthropods community structure estimation The arthropods communities structure were characterized using

Pair rank abundance

Indices of diversity
Richness index The estimation was done randomly twice monthly on limbs in morning hrs by jarring method

Limbs were tapped trice with rubber hose in 0.58m2 canvas tray underneath to collect dislodged arthropods. The dislodged arthropods were identified in taxomical order on sight and then replaced to avoid disruption of the community. Then, grouping was done based on trophic levels as Herbivores and predators. Folivory Feeding assays in the laboratory showed that: Regularly Oedophrys hilleri (Coleoptera: Curculionidae) inhabited the peach tree canopies

Popillia japonica (Coleoptera: Scarabaeidae)

Diabrotica undecimpunctata howardi (Coleoptera: Chrysomelidae) But only, O. hilleri and P. japonica caused measurable foliage injury, and their feeding was easily separated in the eld.

Paired rankabundance plots comparing the arthropod communities on peach trees +EFN or -EFN.
Year I

This

releaved

that alter

EFNs

can

signicantly

arthropod

community structure.
92.8% Year II

The dominance of ants could be

crucial to plant establishment in the

first year of growth, when seedlings are more vulnerable to herbivory .

For each plot, the abundance of arthropods in each taxon on trees with EFNs is shown in rank order (right side), paired with the abundance of each corresponding taxon on trees lacking EFNs (left side). The total number of individuals (N), Simpsons diversity index(D), Margalef s richness index (R)

Effect of peach tree leaf EFN presence (+EFN) or absence (EFN) on predator and herbivore abundances
Year I

Year II

Other predators include Araneae, Asilidae, Cantharidae, and Coccinellidae; other herbivores include Aphididae, Chrysomelidae, Cicadellidae, and Scarabaeidae.

Ants colonization

Metabolic costs associated with production of EFN outweighed the folivory by herbivores. Thus,

Trees beneted from >50% less folivory than trees without

EFNs. Both terminal carbon assimilation (13C) and trunk growth were higher for trees producing EFNs. threefold increase in bud production compared to trees
Year I Abundance (by limb jarring) of (a) ants and (b) herbivores on peach trees with leaf extraoral nectaries present (+EFN) or absent (-EFN) in year I and year II.

without EFNs.

Year II

To determine if EFN-mediated plant defense is exclusively to ants as a functional group or if other predators play that defensive role for the peach tree.

Hence, Ants exclusion experiments were conducted on trees with and without EFNs

1. Trees were vigorously shaken to empty all ants present 2. Trangle traps (wide sticky barrier) were applied or use against visiting ants

3. The ground under the samples trees were hand weeded to prevent crawling of interference ants to vegetation canopies

Abundance of predator & herbivores during ants exclusion

The results indicate that ant exclusion from the trees with EFNs

triggered a shift that favored the chief defoliator, O. hilleri, which

became the dominant arthropod on those trees. The defoliator accounted for 60.8% of arthropods on trees with EFNs when ants were excluded, compared to 26.2% without ant exclusion

(a) Ants and (b) herbivores on peach trees with leaf extraoral nectaries present (+EFN) or absent (-EFN) and with or without ant exclusion treatment, year II.

Folivory effect by ants

Monthly folivory rates, measured as percentage of injured leaves

Findings show that folivory for trees with EFNs increased 5x if ants were excluded in May, a time when the trees bear young leaf material that is not chemically

protected .