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Making new molecules — evolution of structures for novelmetabolites in plantsDaniel J Kliebenstein

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Secondary metabolites are essential plant fitness within the

natural environment by providing defense against attacking

and competing organisms including bacteria, fungi, insects,

animals and other plants. These compounds’ defensive

function is frequently intertwined with specific accumulation in

novel developmental structures. While, the biochemical

community is making great strides in identifying the genetic and

biochemical mechanisms that allow these chemicals to be

synthesized there is vastly less progress on understanding the

developmental mechanisms that is equally key to their

defensive function. In this review, I briefly delve into several

novel developmental structures and provide evolutionary

hypothesis for how they may have evolved and how they could

be unique systems for studying key developmental processes

that have heretofore been recalcitrant to study.

Address

Department of Plant Sciences, University of California, Davis, CA 95616,

USA

Corresponding author: Kliebenstein, Daniel J ([email protected])

Current Opinion in Plant Biology 2013, 16:112–117

This review comes from a themed issue on Growth and development

Edited by Michael Scanlon and Marja Timmermans

For a complete overview see the Issue and the Editorial

Available online 5th January 2013

1369-5266/$ – see front matter, # 2012 Elsevier Ltd. All rights

reserved.

http://dx.doi.org/10.1016/j.pbi.2012.12.004

IntroductionA general discussion of plant metabolism is often cen-

tered around two major classes. Primary metabolism is the

metabolism which allows a plant to utilize water, carbon

dioxide and minerals to create metabolites required to

make and maintain cells (sugars, fatty acids, amino acids

and nucleic acids) [1]. These chemicals were slotted into

a biological function early in the evolution of life as

reflected by their synthetic genes being largely conserved

across all known plants. Secondary metabolites (also

referred to as natural products, phytochemicals, defense

chemicals, defense metabolites, specialized metabolites,

among others) are the chemicals required for plant inter-

actions with the environment (e.g., pest and pathogen

defense compounds, ultraviolet-B sunscreens) [1–4]. This

relationship of secondary metabolites with an ever-fluctu-

ating biotic environment imparts an evolutionary pressure


upon plants to constantly create new secondary metab-

olites causing most secondary metabolites to be lineage-

specific. Interestingly this lineage novelty does not mean

that any given secondary chemical is any less essential to

the survival of a plant species within its natural environ-

ment than primary metabolites, plants lacking the appro-

priate secondary metabolism frequently have dramatically

reduced fitness if any residual fitness in the field [5]. Where

primary and secondary chemicals differ is that in the

artificial laboratory environment secondary metabolite

deficient plants have sufficient viability to be easily

manipulated in any genetic, biochemical or cytological

study. This makes systems associated with secondary

metabolites frequently easier to study and manipulate than

those affiliated with primary metabolites.

While secondary metabolites are essential to a plant’s

survival, this is only true under certain conditions when

the appropriate pathogen, herbivore or competitor is

present [6–8]. In the absence of the appropriate pathogen,

herbivore or competitor the compounds are highly

expensive to synthesize. In Arabidopsis thaliana, the syn-

thesis of the glucosinolate defense compounds imparts a

significant cost in utilizing more than 10% of the energy

available in the metabolic network with an associated cost

of decreased growth [9–11]. This conditionality of benefit

imparts a very complex cost/benefit calculation that the

plant species must solve to optimize the benefit of the

compound while diminishing its associated costs. Numer-

ous models for this optimization focus on the induction of

defense compounds in response to the pathogen or insect

[12,13]. Another optimization approach is to developmen-

tally or ontogenically regulate the secondary metabolite

so that it is present only in the tissues or life stages when

the specific attacking organism occurs [14–18]. Together

these regulatory systems can form a complex interaction

of genotype, ontogeny, tissue development and environ-

ment [19,20]. In addition to these macrolevel concepts,

another theorized mechanism for the plant to optimize

the cost/benefit calculation is to restrict the localization

of secondary metabolites to sites/tissues of optimal

activity. This idea is supported by the fact that the

accumulation of key secondary metabolites is often lim-

ited to specialized tissues such as glandular trichomes,

laticifers, secretory cavities, secondary phloem, resin

ducts or specialized cell compartments like the cell wall,

vacuole or cuticular wax layer [21]. This sequestration

from the broader tissues allows the defense chemical to

be generated in highly localized pockets of higher con-

centration, simultaneously increasing the immediate

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Making new molecules — evolution of structures for novel metabolites in plants Kliebenstein 113

Glossary

Definitions of specialized tissues discussed within the text. This is not

meant as an exhaustive list of all specialized tissues associated with

secondary metabolism in plants.

Laticifer: Elongated cells with altered cytoplasm that accumulate latex

and frequently also accumulate defense compounds.

S-cell: Cells within the Brassicaceae that have developmental properties

similar to the laticifers but accumulate glucosinolates and not latex.

Extrafascicular phloem: An additional phloem network within the

cucurbits that appears to function for the transport of defense

compounds.

Glandular trichomes: Specialized multi-cellular epidermal protrudance

that is often optimized as a metabolic factory to produce and store volatile

oils and other potential defense compounds.

Secretory cavity: Subdermal structures that produce and accumulate

defense compounds. Typically the structure has a storage lumen

surrounded by secretory/synthesis cells and an outer layer of

parenchymatous cells.

Resin duct: A duct within woody tissues that is lined with glandular

epithelium to secrete resin and other defense chemicals.

dose to herbivores and pathogens and decreasing the

overall synthetic cost of making the chemical in every

cell. Another potential benefit to localized accumulation

of secondary metabolites is that this limited distribution

can decrease the ability of herbivores and pathogens to

evolve resistance to the defense chemistry [22,23].

Thus, an essential component of any secondary metab-

olite defense is the generation and proper function of

the specialized accumulation tissue. Yet very little is

known about how these specialized tissues (glandular

trichomes, secondary phloem, laticifers, secretory

cavities, resin ducts, among others) develop or are

regulated. The nescience of these specialized devel-

opmental systems has long been ascribed to the com-

mon knowledge that these tissues are not present in

key model systems and as such not genetically amen-

able. The advent of new genomics technologies is

beginning to erode the model systems technical

advantage [24]. However, this antimodel system argu-

ment also has an underlying presumption that each

specialized developmental system is novel and thus, a

unique, independent and different evolutionary event

with unique genes, among others. At one point, there

was a similar argument about floral development and

the generation of novelty but the past two decades

have shown that the ABC modular model of floral

development simultaneously allows for a common

set of gene functions to create an impressive range

of novelty using gene duplications, gene losses, neo-

functionalization and subfunctionalization events

[25,26]. In this review, I would like to posit the

argument that this reiteration and novelization of

common gene regulatory models for fundamental tis-

sues like vasculature and trichomes could also explain

the vast majority of plant secondary metabolite

specialized tissues. The key difference being that

the complete underlying central developmental

models have yet to be found and described in a way


to allow for novelization of the tissue for nonfloral tissues.

In fact it would be easier to identify these underlying gene

regulatory models using the specialized novelty develop-

mental structure that was derived from the original struc-

ture rather than the original structure.

Vasculature specialization and modificationfor defense metabolism?A key specialized tissue for plant secondary metabolism is

the laticifer, specialized cell types dominantly defined as

having latex [27]. Other key characteristics to laticifers are

being highly elongated vascular associated cells with

altered cytoplasm [27]. More critically to this review,

laticifers are highly polyphyletic throughout the plant

kingdom which has suggested that most laticifers represent

independent evolutions with an implicit assumption of

independent genes for each event. However, the fact that

nearly every major plant group has laticifers suggests that

there must be a central module of genes providing the

potential to repeatedly evolve laticifers. The question then

becomes what is this central module of genes enabling

repeated laticifer evolution?

A potential clue comes from the identification of S-cells

within A. thaliana [28,29�]. S-cells are inflorescence

phloem associated elongated cells with altered cytoplasm

and high levels of the glucosinolate secondary metab-

olites. As such, S-cells have been proposed to be identical

developmental structures to laticifers. Removing the

latex requirement for laticifer definition could allow for

more plant species to have laticifer-like structures than

has originally been described. While Arabidopsis leaves

do not have classical S-cells they do have unique single

cells scattered along the vascular bundle that can be seen

when using glucosinolate-specific markers [30]. This

positioning is identical to where an S-cell/laticifer should

be located but these glucosinolate idioblasts do not con-

nect to form a contiguous network. The repeated posi-

tioning near the vasculature for glucosinolate idioblasts,

S-cells and laticifers suggests a hypothesis that laticifers/

S-cells are evolutionarily derived from a vascular devel-

opmental module. This specialized cell type is often

phloem associated suggesting that it may represent a

novelization of the phloem developmental program.

Additionally, laticifers can show tip development which

is also true of xylem during secondary growth [31]. Thus,

if this novel vascular hypothesis is true, it would require

the combination of components from the xylem and

phloem modules. Unfortunately, the absolute require-

ment for an intact vasculature to create a viable plant

has inhibited our understanding of the underlying genetic

modules. However, if this novelization of a vascular

module hypothesis is true it suggests that identifying

the genetic module controlling laticifer/S-cell develop-

ment could be an easier avenue to understanding the

genetics of vascular development. An interesting test of

this model would be to analyze all known Arabidopsis


114 Growth and development

Figure 1

Epidermis

Peppermint ArtemesiaCurrent Opinion in Plant Biology

Patterning of cell division in glandular trichome formation in Peppermint

and Artemesia. The timing, orientation and patterning of cell divisions

from the epidermal bulge stage are shown for both Peppermint and

Artemesia. Lines represent nucleated cells separated by intact cell walls.

vascular or auxin mutants for alterations in S-cell de-

velopment.

Another potential novel modification of a plant vascular

developmental program for the specific purpose of sec-

ondary metabolism is present within the cucurbits. The

cucurbits have two phloems, one within the vascular bun-

dle, the fascicular phloem, and an extrafascicular phloem

that is scattered through the stem. Recent work has shown

that the extrafascicular phloem is largely transporting

defense-related compounds like secondary metabolites

while the fascicular phloem is facilitating the traditional

phloem transport functions [32�]. Interestingly, extrafasci-

cular phloem proteins have a completely different pro-

teome and metabolome suggesting that this is the

evolution of a complete new transport system derived from

the phloem, almost as if the phloem duplicated and then

subfunctionalized. Very little is understood about the

evolution of complicated new systems, networks or tissues

and this system could provide a key system to investigate

how a complex structure like the phloem can be retooled to

create a completely new vascular component.

Asymmetric cell division and glandulartrichomesAnother specialized developmental structure that is

critical to the function of many plant defense compounds

is glandular trichomes. These structures arise via a com-

plex arrangement of precise cell divisions. For instance,

the mint glandular trichomes first arise as protruding

epidermal cells that then undergo a precise series of cell

divisions including symmetric and asymmetric periclinal

and anticlinal divisions to produce the precise glandular

trichome structure [33] (Figure 1). Interestingly, gland-

ular trichome development in Artemesia which also

begins as an epidermal protrudance proceeds by a com-

pletely different timing and patterning of the cell div-

isions leading to a different glandular trichome structure

[34] (Figure 1). Thus, both species have developed

fundamentally the same structure using precise coordi-

nated cell divisions but each species utilizing different

patterns. The question then becomes have these two

species utilized the same cellular regulatory genes to

evolve these different sequence of cell division patterns.

Further, is the final structure the optimal glandular tri-

chome for each species or is the final structure a con-

sequence of the stochastic nature of evolution and how

and when the two species co-opted cellular division genes

to the process of trichome formation? Intriguingly, some

species have multiple types of glandular trichomes raising

the possibility that each type has a different cellular

program [35�,36,37].

In spite of this fascinating developmental program with

specific and differential cell division, almost nothing is

known about the genetics underlying the development of

glandular trichomes. The main trichome model is the


Arabidopsis hair trichome which is a single cell with endo-

reduplication in contrast to the multi-cellular glandular

trichome [38,39]. While it is tempting to postulate that

hair trichomes and glandular trichomes utilize the same

genetic program with hair trichomes eliminating the

cellularization involved in forming glandular trichomes

this may not be the case. In tomato that has both hair and

glandular trichomes it is possible to genetically abolish

glandular trichome formation without concomitant

effects on hair trichomes suggesting that in tomato the

hair and glandular trichomes use different genetic

machinery [40]. Thus glandular trichomes and the

specific cell division patterning are an underutilized study

system that should provide enticing insights with signifi-

cant implications both for plant defense and the control of

cell division within plants.

Using defense chemistry to find new cell typesDevelopmental structures are almost always defined by a

visual characteristic either directly observable like tri-

chomes or flowers or enhanced by chemical staining like

the casparian strip. Thus, our understanding of plant

development is limited by these very developmental

assays and visualization approaches. This limitation in

visualization raises the potential that the application of

new techniques to developmental investigations may

identify previously unknown cell types. One such tech-

nology being applied to developmental questions is mass-

spectrometry to map the patterning of secondary



metabolites within specific tissues. Mapping flavonoid

accumulation in Arabidopsis flowers showed that the

petal actually has two different developmental zones

that can only be separated by the differential accumu-

lation of two flavonoids [41]. A similar mapping of glu-

cosinolate accumulation within the Arabidopsis leaf

identified three sites of accumulation, near the vascula-

ture, at the leaf margin and distributed cells within the

leaf [42]. While the vasculature sites are probably unrec-

ognized vascular idioblasts based on gene-fusion studies

[30,43,44] and the leaf margin is a relatively unstudied

tissue, the potentially most interesting accumulation site

are the distributed cells within the leaf. These appear to

be single cells that are previously unrecognized meso-

phyll idioblasts and show a stochastic distribution within

the mesophyll. These glucosinolate mesophyll idioblasts

are solely recognizable by the accumulation of glucosi-

nolates and the expression of specific promoters for

glucosinolate pathway genes [45�,46]. This glucosinolate

mesophyll idioblast would only have been found using

molecular approaches targeted specifically to the gluco-

sinolate pathway. The finding of a new potential tissue

with a single metabolic pathway raises the intriguing

question of how many other unrecognized cell types

may be found when researchers begin targeting each

specific secondary metabolic pathway and its tissue pat-

terning in intact tissues.

Defense chemistry modifying developmentWhile development is usually considered to be the frame-

work upon which secondary metabolism is organized,

there is beginning to be intriguing evidence that plant

secondary metabolites may be perceived by the plant to

control developmental processes. In tomato, a tomatine

derivative can lead to the induction of programmed cell

death in what appears to be a direct recognition rather

than a generic toxicity mechanism [47,48]. Similarly,

glucosinolates have been linked to controlling develop-

mental processes like flowering time in Arabidopsis via

what also appears to be a specific recognition event [49�].This ability of plants to specifically utilize their secondary

metabolites to regulate development is best illustrated in

Raphanus sativa where a specific glucosinolate has taken

over the role of auxin in controlling hypocotyl responses

to light by interacting with the TIR1 receptor [50,51]. In

this system, the plant has evolved a replacement for auxin

in a key developmental process using a chemical that is

specific to R. sativa. Thus, it is possible to rapidly evolve

developmental programs both in terms of their con-

sequence and their cause. The impact of plant secondary

metabolites on plant development is typically not inves-

tigated so it remains to be seen how frequently secondary

metabolites may influence development.

Conclusions and future perspectivesThis review has focused on how secondary metabolites

and their accumulation sites can provide unique


opportunities for the study of plant development and

cellular patterning. The very fact that secondary metab-

olites are not conserved may actually simplify these

studies by allowing these developmental systems to be

genetically manipulated. Further, the developmental

structures while apparently independently evolved like

laticifers are probably facilitated by the use of a common

developmental genetic program possibly from a more

essential tissue like the central vasculature. This will

allow for rapid application of information from one lati-

cifer system to another or one glandular trichome to

another. Finally, the development of better metabolite

visualization platforms should greatly help our under-

standing of how many cell types and tissues actually occur

within a plant. Secondary metabolism and developmental

studies have the potential for great future synergism that

will hopefully be quickly tapped.

AcknowledgementsDaniel J Kliebenstein acknowledges funding from NSF DBI grant 0820580and NSF IOS grant 1021861. I apologize to colleagues whose work was notcited due to space constraints.

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