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Cell-type-specific metabolism in plants

March 2023
The Plant Journal 114(5)

March 2023
114(5)

DOI:10.1111/tpj.16214

Authors:

Danilo de Menezes Daloso

Universidade Federal do Ceará

Thomas Christopher Rhys Williams

University of Brasília

Every plant organ contains tens of different cell types, each with a specialized function. These functions are intrinsically associated with specific metabolic flux distributions that permit the synthesis of the ATP, reducing equivalents and biosynthetic precursors demanded by the cell. Investigating such cell-type-specific metabolism is complicated by the mosaic of different cells within each tissue combined with the relative scarcity of certain types. However, techniques for the isolation of specific cells, their analysis in situ by microscopy, or modelling of their function in silico have permitted insight into cell-type-specific metabolism. In this review we present some of the methods used in the analysis of cell-type-specific metabolism before describing what we know about metabolism in several cell types that have been studied in depth; (1) leaf source and sink cells, (2) glandular trichomes which are capable of rapid synthesis of specialised metabolites, (3) guard cells that must accumulate large quantities of the osmolytes needed for stomatal opening, (4) cells of seeds involved in storage of reserves and (5) the mesophyll and bundle sheath cells of C4 plants that participate in a CO2 concentrating cycle. Metabolism is discussed in terms of its principal features, connection to cell function and what factors affect the flux distribution. Demand for precursors and energy, availability of substrates and suppression of deleterious processes are identified as key factors in shaping cell-type specific metabolism.

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SPECIAL ISSUE ARTICLE

Cell-type-speciﬁc metabolism in plants

Danilo de Menezes Daloso

, Eva Gomes Morais

, Karen Fernanda Oliveira e Silva

and

Thomas Christopher Rhys Williams

2,*

Lab Plant, Departamento de Bioqu

ımica e Biologia Molecular, Universidade Federal do Cear

a, Fortaleza-CA 60451-970,

Brazil, and

Departamento de Bot^

anica, Instituto de Ci^

encias Biol 

ogicas, Universidade de Bras

ılia, Asa Norte, Bras

ılia-DF 70910-900,

Brazil

Received 14 December 2022; revised 20 March 2023; accepted 25 March 2023.

*For correspondence (e-mail tcrwilliams@unb.br).

SUMMARY

Every plant organ contains tens of different cell types, each with a specialized function. These functions are

intrinsically associated with speciﬁc metabolic ﬂux distributions that permit the synthesis of the ATP, reduc-

ing equivalents and biosynthetic precursors demanded by the cell. Investigating such cell-type-speciﬁc

metabolism is complicated by the mosaic of different cells within each tissue combined with the relative

scarcity of certain types. However, techniques for the isolation of speciﬁc cells, their analysis in situ by

microscopy, or modeling of their function in silico have permitted insight into cell-type-speciﬁc metabolism.

In this review we present some of the methods used in the analysis of cell-type-speciﬁc metabolism before

describing what we know about metabolism in several cell types that have been studied in depth; (i) leaf

source and sink cells; (ii) glandular trichomes that are capable of rapid synthesis of specialized metabolites;

(iii) guard cells that must accumulate large quantities of the osmolytes needed for stomatal opening; (iv)

cells of seeds involved in storage of reserves; and (v) the mesophyll and bundle sheath cells of C4 plants

that participate in a CO

concentrating cycle. Metabolism is discussed in terms of its principal features, con-

nection to cell function and what factors affect the ﬂux distribution. Demand for precursors and energy,

availability of substrates and suppression of deleterious processes are identiﬁed as key factors in shaping

cell-type-speciﬁc metabolism.

Keywords: cell-types, guard cellsglandular trichomesC4 photosynthesisreserve accumulation.

INTRODUCTION

Multicellular plants are characterized by the presence of

tissues and organs containing a variety of different cell

types; for example, the model plant Arabidopsis thaliana

contains at least seven different types of cell in its leaf

veins (Kim et al., 2021) and 15 cell types in its roots (Brady

et al., 2007), and 56 cell types were identiﬁed over six

organs during construction of a single cell cis-regulatory

atlas for maize (Marand et al., 2021). These cell types per-

form myriad specialized functions, ranging from control-

ling gas exchange to storing the reserves that will be used

during germination and seedling growth, and from ﬁxing

in the leaves to assimilating nitrogen in the roots.

The differences in function of these cells are inevitably

reﬂected in metabolism that is speciﬁc to each cell type.

However, many studies of plant metabolism obscure these

differences, as biomolecules are typically extracted from

entire organs for analysis. To circumvent this problem,

experimental methods including cell isolation and puriﬁca-

tion, imaging techniques and computational modeling

have been developed that allow us to access information

on cell-type-speciﬁc metabolic ﬂuxes. In this review we

describe some of the techniques developed for the study

of cell-type-speciﬁc metabolism, before detailing several

selected cases where we have information about how

metabolism differs in these cells, why such differences are

important for function, and what deﬁnes them at a cellular

and molecular level.

METHODS FOR STUDYING CELL-TYPE-SPECIFIC

METABOLISM

Studies of cell-type-speciﬁc metabolism are complicated

by the fact that the target cells are usually sparsely

Ó2023 Society for Experimental Biology and John Wiley & Sons Ltd. 1

The Plant Journal (2023) doi: 10.1111/tpj.16214

distributed or present at low abundance. Different tech-

niques have been developed to allow sorting, isolation and

puriﬁcation of different cell types, and modern bioimaging

technologies, together with the use of genetically encoded

biosensors, can provide precious information regarding

cell-type-speciﬁc metabolism in situ. Methods that involve

physical separation vary in their speed, yield and the purity

of the material produced. Thus, some methods may be

appropriate for analysis of slowly turning over macro-

molecules, but not for metabolites that are rapidly pro-

duced and consumed. In certain cases, complete puriﬁca-

tion of a cell type may not be possible, and therefore

methods to assess the degree of enrichment and potential

presence of artifacts resulting from the puriﬁcation proce-

dure are necessary and should be reported. In the follow-

ing sections, we discuss the most commonly used

strategies for visualization, isolation and metabolic analy-

sis of speciﬁc cell types.

Isolation and puriﬁcation of speciﬁc cell types

Isolation of a small number of type-speciﬁc cells with lim-

ited contamination from surrounding tissue

In this section, we will brieﬂy discuss some of the most

common methods used to isolate speciﬁc cell types, such

as production of plant cell suspension cultures, protoplast-

ing, ﬂuorescence-activated cell sorting (FACS), laser-

assisted microdissection (LAM) and micromanipulation

systems (Liu et al., 2022; Misra et al., 2014). A plant cell

suspension culture is a population of undifferentiated cells

growing in a liquid medium. Despite its high biomass

yield, it is not advisable to use such material for study of

speciﬁc cell types as the undifferentiated state of the cells

means that their metabolism does not reﬂect a speciﬁc cell

type and their metabolome may not represent that of the

cells from which the culture is derived (Misra et al., 2014).

In contrast, plant protoplasts are cells devoid of cell wall

commonly obtained in high yields (around hundreds of

thousands of cells) by enzymatic digestion of differentiated

cells, meaning that they represent speciﬁc cell types.

Because plant tissues can contain many different speciﬁc

cell types, plant protoplasting may be subjected to contam-

ination with non-target cells (de Souza et al., 2020).

In this context, protoplasts from different cell types

can be sorted out and isolated by FACS, a specialized type

of ﬂow cytometry that is capable of sorting cells based on

their size, granularity and ﬂuorescence (Hu et al., 2016).

Isolation of speciﬁc cell types by FACS requires samples to

be in the form of a suspension of ﬂuorescently labeled

cells, which must be circularly shaped in order to not dis-

turb the laminar ﬂow (Dole

zel et al., 2007) Considering that

protoplasts are spherical, represent speciﬁc cell types and

can yield a great quantity of material, protoplasts are a

suitable plant-derived starting material for FACS. Metabo-

lite turnover, however, is extremely fast and prone to occur

due to the slightest perturbations, consequently even the

most careful sample isolation is likely to result in alter-

ations in the metabolome. Untargeted metabolic proﬁling

of ﬁve speciﬁc cell types in Arabidopsis roots has been

used to investigate how combining protoplasting and sort-

ing by FACS might interfere in the metabolome (Mous-

saieff et al., 2013). This study showed that 10% of the

robust masses were more abundant due to interference of

the isolation procedure, and these metabolites were there-

fore excluded from the dataset.

Nonetheless, some metabolome alterations on

account of the isolation process are intrinsic to the

method. For instance, cell wall invertase is a key enzyme in

sucrose metabolism (Nishanth et al., 2018), and metabo-

lites related to pathogen protection are secreted and

deposited in the extracellular space (de Souza et al., 2020).

Therefore, metabolic changes related to such physiological

processes are lost during protoplasting. Cell-wall-related

information can be recovered by LAM, which is a general

term referring to high spatial resolution techniques that

efﬁciently isolate single cells, or groups of cells, from a

solid tissue sample. Brieﬂy, LAM requires samples to be

ﬁxed, or even frozen, and embedded in parafﬁn or tissue-

freezing media and cut into thin microsections. Sample

microsections are then observed under a microscope to

visualize and target cells, which are microdissected using a

laser and retrieved (Day et al., 2005; Gross et al., 2015;Hu

et al., 2016). The number of cells extracted using LAM can

be adjusted in accordance with the analytical methods that

will be employed. Efﬁcient and rapid target cell identiﬁca-

tion depends not only on the capability of the operator or

software to recognizing the cells, but also depends on

sample preparation and on how the target cells are distrib-

uted in the tissue (Nelson et al., 2006). The ever-increasing

advances in computational technologies can result in faster

and more precise target cell identiﬁcation.

Target isolation by LAM occurs by capture, excision or

by a combination of these methods (Day et al., 2005; Ohtsu

et al., 2007). In laser capture microdissection, a thin trans-

parent thermoplastic cap is placed over the sample and an

infrared laser melts only the capped region located over

target cells. As a result, only target cells adhere to the cap

and are removed from the tissue when the cap is lifted

(Day et al., 2005). In laser microdissection by excision, an

ultraviolet laser ablates the region surrounding target cells

and effectively separates them from the adjacent contami-

nating tissue (Datta et al., 2015). After the cutting, target

cells are retrieved by diverse strategies. If the sample stage

is inversely oriented, then after the cutting the target sam-

ple simply falls out into a collection vessel. In laser micro-

dissection and pressure catapulting, a defocused UV laser

propels the cut target cells upward into a collection vessel

containing whichever solution is required in the analysis

steps (Nelson et al., 2006).

Ó2023 Society for Experimental Biology and John Wiley & Sons Ltd.,

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The LAM has great advantages for cell type metabo-

lism studies, including a below micrometer spatial resolu-

tion and the possibility of collecting tens of thousands of

cells in a few minutes to hours (Nelson et al., 2006). LAM

is thus a fast, accurate and versatile method that has been

applied to transcriptomics investigations of diverse cell

types, including trichomes (Olofsson et al., 2012), guard

cells (Simeoni et al., 2022), embryos (Florez-Rueda

et al., 2020), and speciﬁc cells from epidermal, mesophyll

and vascular tissues of leaves (Berkowitz et al., 2021).

Despite these advantages, LAM is not yet a widespread

speciﬁc cell type isolation procedure for metabolomics

studies because sample preparation steps can affect the

metabolome. For instance, some metabolites can be solu-

ble in the reagents used during sample ﬁxation and

parafﬁn-embedding. Although cryosectioning is a viable

alternative for plant sample preparation for LAM, it may

also render identiﬁcation of target cells more challenging

(Fang & Schneider, 2014; Nelson et al., 2006). Notwith-

standing this problem, LAM has been used for metabolic

analysis of trichomes (Qin et al., 2022), resin duct epithelial

cells (Turner et al., 2019), vascular bundles (Fang & Schnei-

der, 2014) and tissues of developing seeds (Schiebold

et al., 2011).

In addition to LAM, micromanipulation systems also

allow study of speciﬁc cell types with minimal contamina-

tion. These systems comprise microcapillaries with an

aperture of a few micrometers coupled to a micromanipu-

lator, which either captures speciﬁc cells in a solution, col-

lects content from inside the cells or infuses cells with

working solutions. For instance, micromanipulation can be

used to isolate living cells from Arabidopsis female game-

tophytes (Englhart et al., 2017), to collect the inside content

of trichomes and other epidermal cell types (Ebert

et al., 2010), or to infuse stomatal cavity with working solu-

tions to investigate stomatal closure (Guzel Deger

et al., 2015). Micromanipulation systems are more easily

applied in cases where cells of interest are found in the sur-

face of the tissue, for example, trichomes and guard cells.

In cases where the target cells are embedded in a tissue, it

is necessary to ﬁrst liberate such cells in a solution. In the

isolation of living two-cell Arabidopsis proembryos, seeds

were ﬁrst treated with enzymatic solution to degrade cell

wall from the seed coat and facilitate the later dissection

with the aid of the microneedles (Zhou et al., 2019). Micro-

manipulation has the advantage of isolating living cells;

however, it presents a low throughput and requires highly

skilled operators to not only avoid unintentionally piercing

the cells but also to perform isolation in short enough time

for metabolic studies (Ebert et al., 2010; Hu et al., 2016).

Trichome isolation

Trichomes are uni- or multicellular structures found as

extensions of the plant epidermis (Wang et al., 2021), and

can be removed from frozen tissues with the aid of instru-

ments such as precooled ﬁne-scaled forceps (Ebert

et al., 2010), razor blades (Roka et al., 2018), brushes

(Balcke et al., 2017), bead-beaters (Johnson et al., 2017), or

by simply hand agitating a tube containing frozen tissue

and ﬁne powdered dry ice (Rodziewicz et al., 2019). In this

latter study, cannabis ﬂowers were snap-frozen in liquid

nitrogen and transferred to a falcon tube with dry ice.

Then, the tube was capped with a 140-lm nylon mesh and

hand shaken into a cooled beaker. The fast mechanical agi-

tation in dry ice detaches trichomes from the tissue while

maintaining them in a cold environment, and the mesh

facilitates removal of the leaf-contaminating tissue. A tri-

chome isolation method well suited for metabolic studies

consists of using a frozen brush to scrape off trichomes

from the leaf surface into a container with liquid nitrogen

(Balcke et al., 2014). This technique snap freezes only the

trichome, while the rest of the leaf continues ﬂexible. A fur-

ther ﬁltration step through a nylon mesh can also increase

purity of the sample. The trichome isolation methods

described above are efﬁcient, fast and simple to perform.

However, they do not consider that a tissue can simulta-

neously present different types of trichomes. In this sce-

nario, isolation by micromanipulation is a more suitable

method for studying a speciﬁc type of trichome (Livingston

et al., 2020). Optimized protocols can produce sufﬁcient

quantities of trichomes for analysis of gene expression

(Zager & Lange, 2018), proﬁling of primary and secondary

metabolites, and even analysis of

C incorporation follow-

ing labeling experiments (Balcke et al., 2017).

Guard cell isolation

Guard cells are specialized cell types that delimit the sto-

matal pore. These cells are mainly found in the leaf epider-

mis, but in certain species they can also be found in stems

and/or reproductive structures. Guard cell metabolism has

mainly been studied using three different starting materials

(Figure 1): isolated epidermal peels, guard cell enriched

epidermal fragments and guard cell protoplasts (GCPs;

Yao et al., 2018).

Epidermal peels can be obtained using instruments

such as sharp tweezers, forceps, liquid medical adhesive

or adhesive tape (Geilfus et al., 2018; Zimmerli et al., 2012).

Isolated epidermal peels are an excellent material for mon-

itoring stomatal movements as microscope image quality

is not reduced by the presence of mesophyll cells (MCs;

Zhu, Jeon, et al., 2016). However, these peels also contain

viable trichomes and pavement cells; an additional cell

lysis procedure can be used to improve purity, making

guard cell enriched epidermal peels suitable for further

analysis. Methods of cell lysis for improving purity include

use of cellulotytic enzymes, such as Macerozyme R-10, Cel-

lulase R-10 and Cellulysin, and physical methods such as

sonication. It is not possible to obtain epidermal peels

Ó2023 Society for Experimental Biology and John Wiley & Sons Ltd.,

The Plant Journal, (2023), doi: 10.1111/tpj.16214

Cell-type-speciﬁc metabolism in plants 3

from all species, and the analyses that can be performed

will depend on how much material can readily be isolated.

Epidermal fragments are obtained by homogenizing

leaves in a waring blender followed by extensive washing

to produce clean fragment samples (Kopka et al., 1997;

Kruse et al., 1989). The blending process breaks MCs, tri-

chomes, vascular tissue and pavement cells, while most

guard cells remain viable in the epidermal fragments

(Antunes et al., 2017). The increased sample yield com-

pared with epidermal peels enables mass spectrometry

(MS)-based metabolomics analysis and even isotope label-

ing experiments (Daloso, Antunes, et al., 2015; Misra

et al., 2015). Moreover, because non-target cells are found

lysed and non-functional, epidermal fragments are also

found to be more pure in comparison to epidermal peels.

Due to the prolonged and vigorous extraction processes

fragments are generally ﬁrst prepared, and the guard cells

are then subjected to a new condition (light, dark, exoge-

nous application of abscisic acid, etc.), which will be the

objective of the study.

The GCPs represent the purest material available for

investigation of guard cell metabolism. Methods for GCP

isolation typically yield samples with purity above 98%,

though this comes at the cost of low yield and long prepa-

ration time (Pandey et al., 2002). Such a high degree of

purity makes GCPs well suited to omics analysis (Zhu,

Jeon, et al., 2016). Moreover, GCPs display signal

responses that are usually observed in guard cells in

planta, and thus can be used for signal perception and

transduction studies (Fan et al., 2004).

Seed embryo isolation

Seed embryonic cells are embedded into layers of parental

tissue, making embryo isolation potentially even more

challenging than the isolation of epidermal cells, especially

in small sized seeds. After seed collection, embryos can be

isolated by dissection, enzymatic digestion, grinding or a

combination of these methods. Embryo dissection in

siliques is usually performed with a pair tungsten or ﬁne

glass needles coupled to a micromanipulation system

under a microscope. Glass microcapillaries are used to col-

lect and transfer isolated embryos to another well contain-

ing a washing solution (Kao & Nodine, 2020; Zhou

et al., 2019). Dissection of seed embryonic cells in bigger

seeds should consider the use of the appropriate size

apparatus. Bulk rupture methods can be more efﬁcient

regarding embryo collection (Kao & Nodine, 2020). Isolated

seeds can be directly placed into an enzyme solution con-

taining cellulolytic enzymes. Enzyme digestion breaks

down the cell wall of the seed coat cells facilitating later

stages of seed hand dissection (Zhou et al., 2019) or grind-

ing steps (Zhao et al., 2020) and separation of the embryo

from the embryo sac. Extensive washing using microcapil-

laries is essential to remove endosperm and seed coat

contaminants.

Analysis of purity and viability of cell preparations

Purity is generally assessed by microscopy and involves

the quantiﬁcation of target cells compared with contami-

nant cells. For instance, tobacco guard cell enriched

Figure 1. Schematic representation of the commonly used methods for guard cells isolation and cell viability analysis.

(a) Isolated epidermal peels (IEPs) are obtained by peeling off epidermis using tweezers or adhesive tape.

(b) Guard cell enriched epidermal fragments (GCEFs) are obtained by blending and ﬁltrating leaves.

(c) IEP are converted to guard cell enriched epidermal peels (GCEPs) either by sonication or by a weak enzymatic digestion. Note that in GCEPs obtained by

weak enzymatic digestion, outlines from pavement cells are not seen as a result from cell wall digestion by cellulolytic enzymes.

(d) Guard cell protoplasts (GCPs) are obtained by submitting GCEFs/GCEPs to strong enzymatic solution. Fluorescein diacetate (FDA) and neutral red are only

capable of staining viable and functional cells.

Ó2023 Society for Experimental Biology and John Wiley & Sons Ltd.,

The Plant Journal, (2023), doi: 10.1111/tpj.16214

4Danilo de Menezes Daloso et al.

epidermal fragments display about 98% guard cell purity

(Antunes et al., 2017; Daloso, Antunes, et al., 2015; Kopka

et al., 1997; Kruse et al., 1989), while protocols for GCPs

usually range between 98% and 99.5% in purity (Zhu, Jeon,

et al., 2016). Purity can also be assessed by analyzing the

expression of cell-speciﬁc marker genes (Leonhardt

et al., 2004) that are highly expressed in a speciﬁc cell type,

and usually have low expression in other cell types (Qiu

et al., 2021). For instance, NtRbcS-T1 and NtMALD1 are

speciﬁcally expressed in tobacco glandular trichomes

(GTs) and can therefore be used as markers (Pottier

et al., 2020). In the same way, guard cell purity in a sample

can be analyzed by expression of At1g22690 and AtMYB60

(Galbiati et al., 2011; Yang et al., 2008). The use of marker

genes to identify speciﬁc leaf cell types has received signif-

icant attention and has been reviewed in depth (Liu

et al., 2022).

Cell viability is usually assessed with the aid of ﬂuo-

rescent probes such as ﬂuorescein diacetate (FDA), propi-

dium iodide (PI) and neutral red (Figure 1). FDA hydrolysis

in the cytosol of viable cells results in emission of green

ﬂuorescence (Huang et al., 1986). PI emits red ﬂuorescence

but can only penetrate dead cells. Finally, neutral red,

which also emits red ﬂuorescence, accumulates in the vac-

uole of viable cells (Timmers et al., 1995). The combination

of these probes can therefore indicate the relative propor-

tions of living and dead cells. For example, the FDA–PI

combination showed that the blending process to extract

guard cell enriched epidermal fragments results in viable

guard cells while mesophyll, epidermal and trichomes are

predominantly non-viable (Antunes et al., 2017; Daloso,

Antunes, et al., 2015).

Methods for studying cell-type-speciﬁc metabolism

Mass spectrometry (MS)

Following cell separation, metabolism can be investigated

using transcriptomics and proteomics; however, tech-

niques for analysis of the metabolome of speciﬁc cell types

have also been developed. Whilst nuclear magnetic reso-

nance (NMR) spectroscopy and x-ray diffraction can be

employed, MS, coupled to capillary (CE), gas (GC) or liquid

(LC) chromatography, is the most used, due to its high sen-

sitivity (Misra et al., 2014). Adaptations of well-established

protocols (Lisec et al., 2006) have enabled metabolomics

analysis of protoplasts (Robaina-Est

evez et al., 2017) or iso-

lated guard cells via GC–MS (Daloso, Antunes, et al., 2015).

Similarly, GC–MS and/or LC–MS have also been used to

perform metabolomics analysis in guard cell preparations

(Geng et al., 2016; Jin et al., 2013; Misra et al., 2015; Zhu

et al., 2020; Zhu & Assmann, 2017), seed embryos

(Schwender & Ohlrogge, 2002), epidermal bladder cells

(Barkla & Vera-Estrella, 2015), trichomes (Livingston

et al., 2020) and other cell types (Misra et al., 2014). High-

sensitivity MS and methods for separation and

identiﬁcation of metabolites in complex biological samples

have also created the possibility of single-cell metabolo-

mics, though these techniques offer the additional chal-

lenge of harvesting sufﬁcient cells to meet the sensitivity

threshold of the analytical platform (Guo et al., 2021).

Given that plant metabolism is spatially more com-

plex than that of animals and microorganisms (Sweetlove

& Fernie, 2013), the resolution of metabolomics can be fur-

ther improved by the use of in situ MS imaging

approaches. In MS imaging, the ionization method that

should be used depends on the dimensions and properties

of the targeted samples as the spatial resolution of the ion-

ization method will determine the pixel dimension. The

most common ionization methods include matrix-assisted

laser deionization (MALDI; 1–100 lm resolution), second-

ary ion MS (0.1–1lm resolution) and desorption electro-

spray ionization (approx. 200 lm resolution; Dong

et al., 2016). Each pixel will be analyzed for the intensity of

each metabolite fragment identiﬁed in the mass spectra,

and the data used to produce a map showing the localiza-

tion and accumulation of each metabolite (Yin et al., 2022).

MALDI-MS imaging has been successfully used to identify

the localizations of alkaloids and other metabolites in idio-

blasts, laticifer structures, and different cells from leaves,

stems, ﬂower and rhizomes (H€

olscher et al., 2009;Li

et al., 2014; Yamamoto et al., 2016; Yamamoto et al., 2019).

Fluorescent probes

Recent advances in spectroscopy and the use of probes

and genetically encoded ﬂuorescent biosensors allow in

vivo analysis of metabolism at the cell-type level. Different

sensors have been created to investigate the spatial–tem-

poral dynamics of different metabolites (e.g. sucrose, cit-

rate), dinucleotides [NAD(P)(H)] and ATP, amongst others

(De Col et al., 2017; Kroll et al., 2021; Smith et al., 2021).

These approaches have also been used to understand local

and systemic propagation of calcium (Ca) and reactive oxy-

gen species waves throughout the plant (Fichman & Mit-

tler, 2020; Grenzi et al., 2021; Resentini et al., 2021). Most

of these biosensors are used via microscopy and can even

be targeted to a speciﬁc subcellular localization during

plant transformation. Therefore, these approaches offer

great potential for investigation of not only cell-type-

speciﬁc metabolism but also metabolic and ionic dynamics

within organelles of different cell types (Schwarzlader &

Zurbriggen, 2021).

Isotope labeling experiments

Experiments where isolated cells, organs or tissues are

incubated with substrates containing stable isotopes, such

C and

N, followed by quantiﬁcation of incorporation

of label into metabolic intermediates by MS or NMR spec-

troscopy can be used to identify active metabolic pro-

cesses (Silva et al., 2016) or new pathways (Schwender

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The Plant Journal, (2023), doi: 10.1111/tpj.16214

Cell-type-speciﬁc metabolism in plants 5

et al., 2004). These methods have been applied to guard

cell enriched epidermal fragments (Daloso, Antunes,

et al., 2015), tomato trichomes (Balcke et al., 2017), and the

M and BS cells of the C4 plant maize (Arrivault et al., 2017;

Medeiros et al., 2022) amongst others. Radioisotopes may

also be used as labels, taking advantage of highly sensitive

methods for their detection in cell types that can often only

be isolated in relatively low abundance such as trichomes

(Johnson et al., 2017). For certain systems, carefully

designed labeling experiments may allow quantiﬁcation of

metabolic ﬂuxes. In these metabolic ﬂux analysis (MFA)

experiments, a computational model is used to simulate

label distribution within a metabolic network; free ﬂux

parameters within the model are then iteratively adjusted

to maximize agreement between the simulated label distri-

bution and that measured experimentally (Kruger & Rat-

cliffe, 2021). MFA has been used successfully to quantify

ﬂuxes during seed reserve accumulation in Brassica napus,

A. thaliana, sunﬂower and soybean embryos (Allen &

Young, 2013; Alonso et al., 2007; Lonien & Schwen-

der, 2009; Schwender et al., 2006). Whilst this method pro-

vides robust quantiﬁcation of ﬂuxes, the number of types

of tissue to which it can be applied is relatively limited due

to the need for metabolic, and typically isotopic, steady

state. Advances in analytical techniques and the use of

cell-speciﬁc marker proteins may allow MFA to be used to

quantify ﬂuxes in additional cell types (Rossi et al., 2017).

In silico modeling

Cell-type-speciﬁc metabolic models can be used to

investigate the particularities of each cell type that composes

a plant, and how they respond to changes in the surround-

ing micro- and macro-environments (Marshall-Colon

et al., 2017). Flux balance analysis (FBA) simulations using

plant stochiometric metabolic models, especially those cre-

ated at the genome-scale level, can provide detailed predic-

tions of cell-type-speciﬁc metabolic ﬂux with greater spatial

resolution than experimental metabolomics approaches

(Clark et al., 2020; Moreira, Lima, et al., 2019; Shi & Schwen-

der, 2016). For this reason, different models have been cre-

ated to understand the metabolic ﬂuxes of particular cell

types, such as MCs (the predominant cell type in leaf

models), guard cells, embryos, trichomes and seedlings

(Arnold & Nikoloski, 2014; Cheung et al., 2014; Christopher

et al., 2019; Johnson et al., 2017; Moreira, Shaw, et al., 2019;

Robaina-Est

evez et al., 2017; Schwender et al., 2006;Sham-

eer et al., 2018; Shameer et al., 2019; Shameer et al., 2020;

Tan & Cheung, 2020;T

€

opfer et al., 2020). These models may

incorporate information from single-cell transcriptomics

analysis, which are often more readily available than meta-

bolic data. Cell-type-speciﬁc models can then be incorpo-

rated into multi-scale models (Broddrick et al., 2022;Chew

et al., 2016; Smith et al., 2019), contributing to better under-

stand the functioning and plasticity of the plant system, with

clear implications for systems-based breeding and metabolic

engineering (Amthor et al., 2019; Zhu, Lynch, et al., 2016).

Because these models are underdetermined, they produce

predictions of metabolic network function under different

conditions, making FBA and MFA complementary

approaches.

CELL-TYPE-SPECIFIC METABOLIC PHENOTYPES

The methods described above have provided detailed

information regarding the metabolism of a wide range of

different cell types. In the following sections we focus on

several contrasting cell types and discuss the factors that

contribute to the establishment of their metabolic pheno-

types, including the inﬂuence of substrate supply, demand

for biosynthetic precursors and cofactors, and the need to

avoid deleterious processes.

Source and sink cells

A deﬁning characteristic of multicellular plants is the pres-

ence of both source cells, which are net exporters of

photoassimilates, and sink cells, which are net importers

(Fettke & Fernie, 2015). Whilst there are many different

types of source and sink cells in a single plant, and indeed

within every organ, here we focus on the MCs of fully

developed leaves as an example of a source cell type and

compare their metabolism principally with sink leaves (Fig-

ure 2). In addition to providing a point of contrast, the dif-

ferences in metabolism between source and sink cells are

relevant for plant growth. Whilst one might think that

source cells are the most important determinants of plant

growth rate, given that they are responsible for the distri-

bution of photoassimilates to the entire plant, it seems

likely that source and sink tissues co-limit plant growth

(Burnett et al., 2016; Fernie et al., 2020; Jonik et al., 2012).

It is therefore important to understand the particularities of

both cell types to obtain information that may be used to

engineer plants with increased growth and yield. Indeed, a

recent systems-based metabolic engineering study has

modiﬁed up to 20 genes in both source and sink tissues of

tomato, resulting in plants with up to 23% increased fruit

yield (Vallarino et al., 2020). Although similar increases in

fruit yield have been achieved by single gene transforma-

tion (Nunes-Nesi et al., 2005), this study highlights the

need to considering manipulating both source and sink

cells to improve the capacity to produce, export and trans-

port carbon and nitrogen to the harvested organ (Vallarino

et al., 2020). Studies of this nature are important in break-

ing down the paradigm that improved yield is mainly (or

solely) obtained by improving either the photosynthetic

capacity of source cells or the capacity of sink cells to

import sugars and/or synthesize starch (Sonnewald &

Fernie, 2018).

Source cells have the highest photosynthetic capacity

amongst the cells of a plant, whilst sink organs are

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The Plant Journal, (2023), doi: 10.1111/tpj.16214

6Danilo de Menezes Daloso et al.

characterized by high activity of catabolic enzymes and

high expression of sugar transporters that favor the import

of sugars (K€

uhn & Grof, 2010) (Figure 2). Source and sink

leaves also differ in the manner in which they use sucrose,

with glycolytic ﬂuxes toward the tricarboxylic acid (TCA)

cycle and associated pathways higher in sink than in

source leaves (Dethloff et al., 2017). Sink and source cells

not only differ in their capacity to produce and import/

export sugars, but also on how they degrade and use

(respire) them (Farrar, 1985; Gordon et al., 1985; Ho, 1988;

Walker & Ho, 1977). For example, higher

C-enrichment in

phenylalanine and shikimate was observed in sink than in

source leaves of

C-sucrose-fed Arabidopsis plants (Fig-

ure 2; Dethloff et al., 2017). This is consistent with the

higher concentrations of secondary metabolites found in

sink leaves (Gobbo-Neto et al., 2017). The metabolism of

malate and the pathways associated with the TCA cycle

also seem to be regulated differently in sink and source

cells (Centeno et al., 2011). Sink leaves had higher

C-

enrichment in metabolites of, or associated with, the TCA

cycle, including glutamate and glutamine, than source

leaves of

C-sucrose-fed Arabidopsis plants (Dethloff

et al., 2017). This is in close agreement with

C-labeling

experiments MFA showing that photosynthetic ﬂuxes are

barely directed toward the synthesis of TCA cycle-

associated metabolites in source leaves (Abadie

et al., 2017; Gauthier et al., 2010; Ma et al., 2014; Szecowka

et al., 2013; Tcherkez et al., 2005). This is because the TCA

cycle of source leaves is negatively regulated by light at

the transcriptional and post-translational levels; TCA cycle

genes are downregulated in the light (Eprintsev, Fedorin,

Karabutova, & Igamberdiev, 2016; Eprintsev, Fedorin,

Sazonova, & Igamberdiev, 2016; Igamberdiev et al., 2014),

pyruvate dehydrogenase (PDH) is inhibited by phosphory-

lation (Tovar-M

endez et al., 2003), and succinate dehydro-

genase (SDH) and fumarase are deactivated by

thioredoxin-mediated redox regulation (Daloso, M€

uller,

et al., 2015). Indeed, positional

C-labeling analysis indi-

cates that the ﬂuxes from glycolysis and phosphoenolpyr-

uvate carboxylase (PEPc) toward glutamate are restricted

by thioredoxin (TRX) o1 in Arabidopsis rosettes, which are

mostly composed of source leaves (Lima et al., 2021). It

remains unclear, however, whether these regulatory mech-

anisms are also restricting TCA cycle activity in sink leaves.

Alternatively, the differential regulation of the TCA cycle

between sink and source cells may be due to differential

substrate availability.

Beyond carbon and nitrogen metabolism, lipid metab-

olism has also been extensively studied in sink cells, given

their potential for biofuel production. Key regulators for

the synthesis of fatty acids and triacylglycerols, for the sta-

bility of lipid droplets and for the degradation of lipids

through b-oxidation have been revealed and genetically

manipulated to improve oil content in sink or source cells

Figure 2. Schematic representation of the metabolic ﬂuxes in sink and source Arabidopsis leaves.

Thicker arrows indicate metabolic reactions with higher ﬂuxes. Source leaves are characterized by their higher photosynthetic capacity, indicated by the thicker

arrows in the Calvin–Benson cycle (CBC) and sucrose synthesis, which is preferentially used for either starch synthesis in the source leaf or exported to sink tis-

sues.

C-labeling kinetic analysis indicates that the ﬂuxes derived from RuBisCO-mediated CO

assimilation is marginally used for the synthesis of metabolites

associated to the TCA cycle (Ma et al., 2014; Szecowka et al., 2013). This is associated with the light-inhibition of respiratory metabolism mediated by transcrip-

tional and posttranslational mechanisms (for review, see Fonseca-Pereira et al., 2021). By contrast, the

C-enrichment in metabolites of, or associated with, the

TCA cycle are higher in sink than source leaves when fed with

C-sucrose (Dethloff et al., 2017). These ﬁndings collectively suggest that the ﬂuxes throughout

the TCA cycle and Glu/Gln pathway are higher in sink than source leaves. Higher

C-enrichment in shikimate and Phe was also observed in sink leaves fed with

C-sucrose, when compared with source leaves (Dethloff et al., 2017), in agreement with the higher accumulation of secondary metabolites in these leaves.

Ó2023 Society for Experimental Biology and John Wiley & Sons Ltd.,

The Plant Journal, (2023), doi: 10.1111/tpj.16214

Cell-type-speciﬁc metabolism in plants 7

(Yang et al., 2022). However, whilst the spatial characteriza-

tion of lipids in sink cells, especially in seeds, has been

investigated by isolating embryos or using magnetic reso-

nance imaging (MRI) analysis, information on lipid metab-

olism in isolated source cells is less abundant. The

possibility of producing biodiesel from C4 leaves (Luo

et al., 2022; Zale et al., 2016) creates a demand to under-

stand the spatial resolution and regulation of lipid metabo-

lism in photosynthetic cells. For instance, histochemical

and MS-based lipidomics analysis have revealed the

dynamic of lipid droplet degradation in guard cells and

their role in stomatal opening (McLachlan et al., 2016).

These and other approaches typically used in seeds can

then be applied to improve our understanding of the regu-

lation of lipid metabolism in source cells.

Glandular trichomes

The GTs are epidermal structures present in about 30% of

vascular plants that are specialized in the production of

secondary metabolites (Huchelmann et al., 2017). GTs are

generally multi-cellular and formed from apical, stalk and

basal cells, and the metabolites they produce may accumu-

late intra- or extracellularly. For example, in trichomes that

accumulate volatile compounds these may be stored in

subcuticular spaces, as occurs in peppermint, or between

the secretory cells as in tomato (Schuurink & Tissier, 2020).

The metabolites accumulated in GTs typically have anti-

herbivory activity, making GTs an important part of plant

defenses against biotic stress, but they also produce com-

pounds with pharmaceutical applications such as the anti-

malarial artemisinin that accumulates in the GTs of

Artemisia annua (Chen et al., 2021), meaning that their

metabolism has also been studied with a view to genetic

engineering (Huchelmann et al., 2017).

The GTs are capable of high rates of secondary

metabolite production with the secretory cells of pepper-

mint peltate GTs able to ﬁll the subcuticular cavity with

monoterpene essential oil in a single day (Turner

et al., 2000). Cell puriﬁcation experiments have indicated

that the biosynthetic process, from sugars to secondary

metabolites, is entirely contained within the cells of the

GTs themselves (McCaskill et al., 1992), meaning that these

rates of production are largely due to their specialized

metabolism. Flux balance analysis of a stoichiometric

model of mint (Johnson et al., 2017) predicted a high rate

of ATP recycling and production of reduced ferredoxin;

large amounts of ATP are required for monoterpene pre-

cursor synthesis and reduced ferredoxin is required for

reductive enzymes of the methylerythritol 4-phosphate

(MEP) pathway, the plastidic route for terpene precursor

production, which is highly active in these cells. Radio-

labeling experiments performed on GTs isolated using

beat-beating also provide evidence for fermentation in

these cells as the ATP synthase inhibitor oligomycin

impacts terpene production when they are incubated with

moderate concentrations of sucrose but not high concen-

trations, and alcohol dehydrogenase activity and tran-

scripts can also be detected. It may be that the waxy

cuticle of the GTs and high activity of oxygen-consuming

cytochrome p450 enzymes results in low internal oxygen

and the need to use fermentation to maintain ATP synthe-

sis (Johnson et al., 2017).

A metanalysis of metabolic ﬂuxes suggests similar

ﬂux distributions in the GTs of different species (Figure 3),

including the presence of fermentation and ferredoxin

reduction by NADPH (Zager & Lange, 2018). Sugar uptake

in the form of sucrose is highly important, as even when

the secretory cells have photosynthetic activity these cells

are carbon sinks as demonstrated by

C-labeling experi-

ments and high expression of sucrose-degrading enzymes

in tomato GTs isolated by cryo-brushing (Balcke

et al., 2017). Light energy absorbed by GTs with photosyn-

thetic capacity is therefore likely important in the genera-

tion of ATP and NADPH for biosynthetic processes as well

as the reﬁxation of CO

released by respiration (Balcke

et al., 2017). Indeed, tobacco GTs express a speciﬁc form

of the ribulose-1,5-bisphosphate carboxylase/oxygenase

(RubisCO) small subunit that has a relatively high turnover

but low CO

afﬁnity, and which may therefore be suited to

the high-CO

concentration conditions of GTs (Laterre

et al., 2017). Further study is required though as this small

subunit isoform is not present in some species where

reﬁxation of CO

released by respiration is known to occur,

such as B. napus (Brand & Tissier, 2022). Interestingly, epi-

thelial duct cells isolated from loblolly pine (Pinus taeda)

needles using LAM that produce diterpenes, sesquiter-

penes and monoterpenes as part of their oleoresin appear

to have a similar metabolism to cells of GTs of herbaceous

species, showing high MEP pathway activity, ATP synthe-

sis, heterotrophic regeneration of ferredoxin and fermenta-

tion (Turner et al., 2019). Thus, despite their distant

evolutionary relationship, the cells of both species have a

similar ﬂux distribution imposed by their specialization in

the production and secretion of terpenes.

High levels of expression of enzymes involved in sec-

ondary metabolite synthesis appear to be a deﬁning char-

acteristic of the secretory cells of GTs and a determinant of

their specialized metabolism. There is generally a strong

correlation between the class of metabolite synthesized

and the abundance of transcripts coding for enzymes that

produce this class (Aziz et al., 2005; Zager & Lange, 2018),

and transcript abundance may also point to which pathway

is used for metabolite production (Jin et al., 2014). This pat-

tern may also extend beyond a single pathway. Cannabis

produces a complex mixture of cannabinoids, monoter-

penes and sesquiterpenes in the capitate-stalked GTs of

female plants during ﬂowering. Cannabinoids, in particular,

require GPP from the MEP pathway as well as olivetonic

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The Plant Journal, (2023), doi: 10.1111/tpj.16214

8Danilo de Menezes Daloso et al.

acid from the polyketide pathway; for all these metabolites

strong correlations between their abundance and the abun-

dance of transcripts coding for enzymes in the relevant bio-

synthetic pathways were detected (Zager et al., 2019). GTs

may also show highly specialized gene expression patterns

with more than 90% of metabolism-associated transcripts

coding for enzymes involved in bitter acid and prenylﬂava-

noid biosynthesis in hop for example (Zager & Lange, 2018).

Secondary metabolism withdraws intermediates from pri-

mary metabolic pathways, and enzymes involved in the

production of these precursors are also strongly expressed

in GTs. For example, enzymes such as ATP-citrate lyase,

which produces the cytosolic acetyl-CoA needed for the

mevalonic acid pathway, and plastidic isoforms of glyco-

lytic enzymes involved in production of pyruvate and GAP

for the MEP pathway are enriched in tomato GTs (Balcke

et al., 2017). Transcription factors that drive high expres-

sion of enzymes involved in both primary and secondary

metabolism in GTs have been identiﬁed. Many of these,

however, also have effects on trichome development mak-

ing separating their direct roles in enzyme expression from

indirect developmental effects challenging (Brand & Tis-

sier, 2022). Gene duplication also appears to be important

as it permits the evolution of enzyme isoforms associated

with the specialized metabolism of GT cells (Brand & Tis-

sier, 2022). For example, type II isoforms of DXS, the ﬁrst

enzyme of the MEP pathway, tend to be associated with

isoprene-producing tissues. Similarly, the ferredoxin iso-

form identiﬁed in heterotrophic mint GTs has a different

redox potential to the isoform involved in photosynthetic

electron transport that allows it to receive electrons from

NADPH (Johnson et al., 2017).

Figure 3. Metabolic ﬂuxes observed in glandular trichomes (GTs) producing terpenes.

In this example, based on a model proposed for tomato type VI GTs (Balcke et al., 2017), IPP/DMAPP are produced via the plastidic methylerythritol 4-phosphate

(MEP) pathway from pyruvate and triose-phosphate as well as via the cytosolic MVA pathway using acetyl-CoA produced by ATP-citrate lyase. In addition to gly-

colysis, pyruvate is also produced by a plastidic NADP-malic enzyme. Terpene precursor production results in release of CO

that can be reﬁxed via Calvin–Ben-

son cycle activity. Despite this photosynthetic activity, metabolism depends on the uptake of sugars. Ethanolic fermentation has been detected in GTs (Zager &

Lange, 2018) and is also represented here.

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The Plant Journal, (2023), doi: 10.1111/tpj.16214

Cell-type-speciﬁc metabolism in plants 9

There is also evidence for interaction between path-

ways of secondary metabolism in GTs. Tomato type VI

GTs produce both ﬂavonoids and volatile terpenes;

mutants lacking chalcone synthase, an enzyme necessary

for ﬂavonoid synthesis, not only lack anthocyanins but are

also affected in terpene production (Kang et al., 2014). Ter-

pene synthesis is reduced to 10% of normal with downre-

gulation of transcripts coding for enzymes necessary for

their synthesis speciﬁcally in the type VI GTs of plants lack-

ing the CH1 gene (Sugimoto et al., 2022). The mechanism

of co-regulation of these pathways is unclear, but appears

unlikely to be due to accumulation of a speciﬁc metabolic

precursor as lack of other enzymes in the same pathway

has a similar affect.

The specialized metabolism of GT cells is also depen-

dent on the trichomes having a structure that allows for

the release or accumulation of compounds such as ter-

penes. Generally, capitate GTs, which have a stalk that is

longer than half the head height, exude resinous sub-

stances, whilst peltate GTs, which have a large head and

unicellular or bicellular stalk, accumulate volatile com-

pounds in intercellular or subcuticular cavities (Huchel-

mann et al., 2017; Tissier et al., 2017). For example, volatile

secondary metabolites are stored in a cavity in tomato type

IV GTs (intercellular), cannabis GTs and mint peltate GTs

(subcuticular), which allows these substances to accumu-

late to high levels without potentially negative effects on

cell physiology. It seems likely that both the active secre-

tory pathway as well as membrane transporter proteins

are important in exporting volatile compounds to these

cavities (Tissier et al., 2017).

Guard cells

Guard cells are specialized cells found in the leaf epidermis

surrounding the stomatal pore, an aperture in the leaf epi-

dermis that allows the entrance of CO

for photosynthesis

and the efﬂux of water vapor through transpiration.

Growth and stress tolerance are therefore actively regu-

lated by stomatal movements (Lawson, 2009). Changes in

guard cell metabolism ultimately control the opening and

closure of the stomatal pore. Whilst stomatal opening is

induced by the accumulation of osmolytes within guard

cells, their efﬂux or consumption causes the stomatal pore

to close. The homeostasis of ions and metabolites in guard

cells, mediated by their transport and turnover, is thus key

in regulating the rate of stomatal movements. This process

is highly dynamic, responding to different endogenous

and environmental signals, in which MCs and subsidiary

cells act as important sources of metabolites for guard

cells (Figure 4) (Fl€

utsch & Santelia, 2021; Wang

et al., 2019). A guard cell is therefore a highly responsive

cell, acting as a hub for sensing environmental signals and

memorizing stress conditions (Auler et al., 2022). Investiga-

tions of guard cell metabolism have mainly focussed on

how these cells respond to environmental conditions, such

as CO

, dark, light, phytohormones and stress (Daloso,

Antunes, et al., 2015; Daloso, Williams, et al., 2016; Fran-

zisky et al., 2021; Freire et al., 2021; Geng et al., 2017;

Hedrich & Marten, 1993; Jin et al., 2013; Medeiros

et al., 2018; Misra et al., 2015; Outlaw & Lowry, 1977; Tal-

bott & Zeiger, 1996; Zhu et al., 2020). These studies have

been carried out using epidermal peels, guard cell

enriched epidermal fragments and protoplasts. Several

unique characteristics of guard cell metabolism (Figure 4)

have been revealed thanks to these and other guard cell

metabolic studies.

During stomatal opening, guard cells accumulate

potassium (K

) and its counterions chloride (Cl



), nitrate

(NO

) and malate. The uptake of potassium through spe-

ciﬁc channels is a consequence of membrane hyperpolari-

zation through the action of H

-ATPases, which in turn

demand large quantities of ATP. Starch is an important

substrate for ATP production and malate synthesis during

stomatal opening, and its metabolism in guard cells there-

fore shows certain particularities (Figure 4). For example,

starch from Arabidopsis guard cells is degraded in the ﬁrst

hours of light (Antunes et al., 2017;Fl

€

utsch et al., 2020;

Horrer et al., 2016), likely as mechanism to support the

energetic and osmotic demands of the stomatal opening

process (Santelia & Lunn, 2017). In contrast, starch synthe-

sis is nearly linear in the light and degradation is linear

during the night in MCs (Martins et al., 2013). Sucrose also

plays a role in stomatal opening (Daloso, Anjos, & Fer-

nie, 2016); increasing sucrose hydrolysis in guard cells

through expression of acid invertase in potato or sucrose

synthase (SuSy) in tobacco increased stomatal opening

(Antunes et al., 2012; Daloso, Williams, et al., 2016), whilst

decreases in SuSy2 expression in tobacco guard cells

reduced the rate of stomatal opening (Freire et al., 2021).

C-labeling experiments performed using epidermal frag-

ments suggest that sucrose breakdown contributes to

energy and organic acid counterion production (Daloso,

Antunes, et al., 2015). Sucrose in guard cells may be

derived from photosynthesis and starch, and can form a

sucrose futile cycle (SFC; Robaina-Est

evez et al., 2017).

However, the transcriptomes of these cells are also

enriched in membrane sucrose transporters (Daloso,

Anjos, & Fernie, 2016), and knockdown of the SUT1 trans-

porter resulted in decreased stomatal opening, indicating

that at least part of guard cell sucrose is taken up from the

apoplast (Figure 4; Antunes et al., 2017).

Guard cells are photosynthetically active (Lawson

et al., 2003), and photosynthesis could therefore potentially

contribute to energy or osmolyte production. Whilst car-

bon ﬁxation via the Calvin–Benson cycle can be detected

together with that catalyzed by PEPc (Daloso, Antunes,

et al., 2015; Gotow et al., 1988), it remains unclear how

much ﬁxation via RuBisCO contributes to carbon balance

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The Plant Journal, (2023), doi: 10.1111/tpj.16214

10 Danilo de Menezes Daloso et al.

and osmolyte accumulation (Lawson, 2009). Photosyn-

thetic electron transport appears to make an important

contribution to production of the ATP and reducing equiva-

lents needed for osmolyte accumulation (Suetsugu

et al., 2014).

Despite their photosynthetic nature, guard cells also

present several characteristics of sink cells (Daloso,

Antunes, et al., 2015;Fl

€

utsch et al., 2022; Hite et al., 1993),

as compared with MCs they have higher ﬂuxes toward the

TCA cycle and glutamate/glutamine metabolic pathways

(Lima et al., 2021). It is therefore not clear whether guard

cell metabolism more closely resembles that of sink or

source cells. Furthermore, guard cells from C3 plants have

high expression of genes associated with C4 metabolism

(Daloso, Anjos, & Fernie, 2016), higher assimilation of CO

mediated by PEPc (Daloso et al., 2017; Robaina-Est

evez

et al., 2017) and high CO

assimilation in the dark (Gotow

et al., 1988; Lima et al., 2021), resembling C4 and CAM.

Several subcellular particularities are also observed such

as the formation of a SFC in the cytosol and greater trans-

port of CO

from the chloroplast to the cytosol and of

malate from the cytosol to the chloroplasts(Robaina-

Est

evez et al., 2017). A four-phase ﬂux balance analysis of

guard cells reinforced the ﬂexibility of their metabolism,

highlighting how different pathways may operate as a

function of light intensity and sucrose supply as well as

the energetic equivalence of Cl



and malate as K

counter-

ions (Tan & Cheung, 2020).

Given the important role of guard cells for the regula-

tion of photosynthesis and transpiration, a full understand-

ing of guard cell metabolism may aid obtaining plants with

improved water use efﬁciency through metabolic engineer-

ing (Lawson & Vialet-Chabrand, 2018). However, the lack

of a protocol to isolate these cells from frozen leaves

makes it harder to understand the dynamics of guard cell

metabolism in planta. Separation could theoretically be

done by LAM, but an enormous amount of guard cells

would be required for metabolomic analysis. The

Figure 4. Schematic representation of the metabolic ﬂuxes in guard cells during light- or dark-induced stomatal opening/closure conditions.

Thicker arrows indicate metabolic processes with high ﬂuxes in each condition. Guard cells are characterized by having characteristics of both sink and source

cells, but with limited CO

assimilation capacity mediated by ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). Thus, these cells seem to import

sucrose and other sugars during both stomatal opening and closure conditions (for review, see Fl€

utsch & Santelia, 2021). During the dark-to-light transition (left

ﬁgure), starch and sucrose are degraded likely to meet the energetic and osmotic demand for stomatal opening, in which a close connection between carbohy-

drates, tricarboxylic acid (TCA) cycle activity and Gln metabolism has been observed in different studies (for review, see Lima et al., 2018). Modeling and

C-

labeling analyses suggest that guard cells have high phosphoenolpyruvate carboxylase (PEPc) activity (Robaina-Est

evez et al., 2017), including in the dark

(Gotow et al., 1988), as indicated by the thick arrows between PEP and OAA in both ﬁgures. In contrast to the light, it has been hypothesized that stomatal clo-

sure conditions (e.g. dark, high CO

) would favor gluconeogenesis toward starch synthesis rather than glycolysis, as indicated by the reverse arrow from PEP to

hexoses. However, the speed of stomatal closure cannot be solely explained by the activation of gluconeogenesis. The efﬂux of organic osmolytes (e.g. malate)

from the symplast to the apoplast of guard cells seem thus required to enable a rapid stomatal closure (Hedrich & Marten, 1993; Raschke & Dittrich, 1977). Fur-

ther evidence has shown that Glu can induce stomatal closure, but the mechanism behind such a result remains to be identiﬁed.

Ó2023 Society for Experimental Biology and John Wiley & Sons Ltd.,

The Plant Journal, (2023), doi: 10.1111/tpj.16214

Cell-type-speciﬁc metabolism in plants 11

establishment of a protocol for separating MCs and guard

cells from the same frozen leaf material assumes therefore

a paramount importance to improve our understanding of

guard cell metabolism.

Seeds and embryos

Seeds contain signiﬁcant quantities of reserves that are

used for maintenance and growth of the embryo during

germination and the seedling phase. Following histodiffer-

entiation of the embryo, these substances, including carbo-

hydrates, lipids and proteins, accumulate in reserve tissues

of the cotyledons or endosperm during seed maturation.

The cells of these tissues therefore exhibit specialized ﬂux

distributions associated with the production of precursors,

their assembly into reserves and the generation of the ATP

and reducing equivalents needed for these processes. In

addition to the demands resulting from reserve accumula-

tion, metabolism is also affected by substrate availability,

whether organic compounds supplied by the mother plant

or through gas exchange, the photosynthetic capacity of

the embryo and even shape, size and space available for

its growth (Muller-Landau, 2010).

Flux distribution in these cells varies with reserve

composition and has been extensively studied using MFA.

Oilseeds are generally characterized by high ﬂuxes through

glycolysis and plastidic pyruvate dehydrogenase to pro-

duce acetyl-CoA for fatty acid synthesis (Figure 5), with

malic enzyme supplementing pyruvate production (Allen

et al., 2009; Schwender et al., 2006; Sweetlove et al., 2013).

On the other hand, metabolism in the endosperm of bar-

ley, which is about 60% starch, 8% protein and 7% lipid by

mass, is mainly directed toward the production of starch

from imported sugars with energy generated through TCA

cycle activity and oxidative phosphorylation (Rolletschek

et al., 2015).

The ﬂux distribution is also determined by which sub-

strates are available, which in turn is a consequence of

supply by the endosperm or maternal tissue. In soybean

cotyledons, for example, carbon from glutamine and

asparagine are fed into the TCA cycle, with part of the car-

bon then withdrawn for the synthesis of other amino acids

or fatty acids (Allen et al., 2009). Substrate supply not only

affects ﬂux distribution but also the reserves that are accu-

mulated; as the ratio of carbon to nitrogen fed to soybean

embryos increases the concentration of protein decreases

from 47% to 14% of dry mass but with no change in

embryo growth rate (Allen & Young, 2013). Sophisticated

MRI techniques are now beginning to be used to probe

endosperm metabolism, which has an important effect on

substrate supply to the embryo in B. napus and other spe-

cies (Rolletschek, Mayer, et al., 2021).

The space that the embryo has available for growth

also affects reserve accumulation and hence metabolism;

B. napus embryos grown in vitro accumulate more starch,

which is less energy dense, as their size is less constrained

than embryos developing in planta within a seed coat, and

mechanical restriction appears to induce a switch from

embryo growth to maturation (Borisjuk et al., 2013; Roll-

etschek, Muszynska, & Borisjuk, 2021).

Depending on the species, signiﬁcant quantities of

light may reach the embryo, meaning that photosynthesis

can potentially contribute to carbon and energy balance

during reserve accumulation. In A. thaliana,Glycine max

and B. napus, lipid storage, and consequently seed yield,

is greatly affected by light (Li et al., 2006; Ruuska

et al., 2004). The cells of cotyledons accumulating oil

release CO

as part of the plastidic pyruvate dehydroge-

nase reaction that produces acetyl-CoA for fatty acid syn-

thesis. In green embryos this CO

can be reﬁxed by

RuBisCO operating without the Calvin cycle, and this

RuBisCO bypass has been detected in B. napus (Schwen-

der et al., 2004), soybean (Allen et al., 2009) and Arabidop-

sis (Lonien & Schwender, 2009). CO

reﬁxation in these

cotyledon cells results in increased carbon conversion efﬁ-

ciency into fatty acids when compared with heterotrophic

oilseed cotyledons such as those of sunﬂower (Alonso

et al., 2007). Light energy is not only used for CO

ﬁxation

with the ATP and NADPH produced by chloroplast electron

transport also used for reserve synthesis, reducing the

need for ﬂux through the TCA cycle (Allen et al., 2009;

Lonien & Schwender, 2009; Schwender et al., 2006).

Embryos of different species will receive different

amounts of light as a result of plant morphology and

growth conditions, and the amount of light reaching the

embryos is likely to affect ﬂux through the RuBisCO bypass

(Borisjuk et al., 2013) with greater ﬂux in B. napus, where

pods are directly exposed to sun, compared with soybean

where pods are below the leaf canopy (Allen et al., 2009;

Lonien & Schwender, 2009). Even within a single embryo,

cells in different cotyledons may receive different quanti-

ties of light and hence exhibit different metabolic ﬂuxes. In

B. napus embryos, one cotyledon is folded over the other

and the resulting shading produces differences in metabo-

lism; cells of the outer cotyledon and the hypocotyl/radicle

axis have greater photosynthetic activity, in contrast to

those of the inner cotyledon that grows essentially hetero-

trophically (Borisjuk et al., 2013). Alterations in ﬂux

between the cotyledons reﬂect the embryo’s adjustment to

ATP/NADPH production, which is related to the level of

light exposure.

The effects of light on seed metabolism are not lim-

ited to those with green embryos. For example, in silico

modeling of the endosperm and embryos of barley (Hor-

deum vulgare) and wheat (Triticum aestivum) revealed an

adjustment in metabolic ﬂux due to light-induced changes

in solute uptake. In the green pericarp that surrounds these

tissues there were changes in photosynthetic and TCA

cycle ﬂuxes that allowed reﬁxation of CO

released by the

Ó2023 Society for Experimental Biology and John Wiley & Sons Ltd.,

The Plant Journal, (2023), doi: 10.1111/tpj.16214

12 Danilo de Menezes Daloso et al.

endosperm and guaranteed greater efﬁciency in the con-

version of carbon into seed reserves (Rolletschek

et al., 2015).

Another factor that shapes metabolism in cells of seed

tissues is oxygen balance, as there is a need for external

oxygen to ensure aerobic respiration (Borisjuk & Roll-

etschek, 2009; Rolletschek et al., 2002). Low capacity for

oxygen diffusion can cause hypoxia, which in turn results

in reduced ATP production as oxygen is the terminal elec-

tron acceptor in the mitochondrial electron transport chain,

ultimately decreasing ATP availability and resulting in

shifts in metabolism (Greenway & Jane, 2003; Klok et al.,

2002), including activation of fermentation and reduced

TCA cycle ﬂux as predicted by ﬂux balance analysis of bar-

ley embryos (Grafahrend-belau et al., 2009).

Oxygen supply to the inner part of the seed therefore

has great inﬂuence over the quantity and size of seeds

(Kuang et al., 1998), and seed structure and architecture

are relevant for understanding the metabolic processes

that occur during its development. Computerized tomogra-

phy has been used to evaluate the development of B.

napus seeds, seeking to understand the impact of empty

spaces that are found in the seed (Verboven et al., 2013).

The testa and hypocotyl showed several empty spaces,

while the cotyledons revealed small and poorly intercon-

nected spaces. It is likely that the size and interconnectivity

of these empty spaces are the main determinants of gas

exchange potential, which itself affects the local respiratory

activity within a developing seed, and allow high gas

exchange rates both in the seed coat and along the axial

direction of the hypocotyl (Verboven et al., 2013).

To understand the importance of oxygen availability

or restriction in seed and embryo development, and how

this can cause changes in metabolic patterns, it is essential

to elucidate the role of nitric oxide (NO) in oxygen signal-

ing. NO is a gaseous free radical that responds to the pres-

ence of molecular oxygen and is thought to mediate

important functions such as low oxygen detection,

Figure 5. The principal metabolic ﬂuxes observed in cotyledon cells of green embryos accumulating fatty acids based on a model for soybean (Allen

et al., 2009).

released during acetyl-CoA production is reﬁxed by ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) acting without a complete Calvin–Benson

cycle (Schwender et al., 2004). In addition to sucrose, carbon also enters metabolism as glutamine, which can be converted to 2-oxoglutarate and used for ana-

pleurotic replenishment of the tricarboxylic acid (TCA) cycle. TCA cycle intermediates are also withdrawn synthesis of amino acids or fatty acids via malic

enzyme; for clarity, ﬂuxes to amino acids are not shown.

Ó2023 Society for Experimental Biology and John Wiley & Sons Ltd.,

The Plant Journal, (2023), doi: 10.1111/tpj.16214

Cell-type-speciﬁc metabolism in plants 13

respiratory control, ATP availability and seed storage activ-

ity (Rolletschek et al., 2005). The availability of oxygen con-

trols the concentration of NO and, in turn, NO regulates

the consumption availability of oxygen in the seed. This

oxygen self-regulating mechanism prevents seed anoxia

and indicates a key role for NO in regulating storage activ-

ity, which can be observed in the form of changes in

steady-state metabolite concentrations and biosynthetic

ﬂuxes under NO treatment (Benamar et al., 2008; Linder-

mayr et al., 2005; Neill et al., 2003).

C4 metabolism

C4 photosynthesis is present in about 3% of vascular

plants, and is particularly common in those growing in hot

and dry regions with abundant light. This type of photo-

synthesis depends on the operation of a carboxylation-

decarboxylation cycle distributed between leaf MCs and

bundle sheath cells (BSCs). C4 photosynthesis is thought

to have evolved independently at least 61 times

(Sage, 2016), likely because the C4 cycle repurposes

enzymes that are present in all plants (Aubry et al., 2011)

together with the signiﬁcant advantages that C4 metabo-

lism offers in certain environmental conditions. Firstly, the

C4 cycle results in elevated concentrations of CO

in the

BSCs, which results in suppression of photorespiration

and increased rates of carbon ﬁxation by RubisCO. Whilst

variable between species and conditions,

C-labeling

experiments in maize suggested photorespiratory ﬂux was

5% of the rate of carboxylation, ﬁve times less than

expected for a C3 plant (Arrivault et al., 2017). The effective

pumping of CO

from mesophyll to bundle sheath also

tends to decrease leaf intercellular CO

concentrations,

which in turn favors the diffusion of CO

into the leaf; C4

plants can therefore maintain high rates of photosynthesis

with low stomatal conductance resulting in increased

water use efﬁciency (Way et al., 2014). These characteris-

tics are particularly advantageous in hot environments,

which favor photorespiration, with limited water and nitro-

gen availability and abundant light energy needed to drive

the C4 cycle (Schluter & Weber, 2020). Today, grasses in

savannahs such as the Brazilian Cerrado senso stricto are

predominately C4, and differences in the abundance of C3

and C4 species can even be detected between closely

located open savannah and gallery forests (Amaral

et al., 2021).

The C4 cycle is associated with speciﬁc metabolic phe-

notypes in the MCs and BSCs. Phosphoenolpyruvate (PEP)

is carboxylated by PEPc in the mesophyll using bicarbon-

ate produced by carbonic anhydrase to produce the C4

organic acid oxaloacetate (Figure 6). Oxaloacetate is con-

verted to malate or aspartate, which move to the BSC via

plasmodesmata. C4 organic acids are decarboxylated to

release CO

in the BSCs that can then be ﬁxed via the Cal-

vin cycle and a three-carbon molecule (pyruvate or alanine)

that will be returned to the MC where it can be converted

back to PEP to complete the cycle (Schluter & Weber, 2020).

Three different C4 pathways are typically identiﬁed, which

are named after the enzyme responsible for catalyzing

decarboxylation (plastidic NADP-ME, mitochondrial NAD-

ME and cytosolic PEPCK), but which also vary in the mole-

cules that move between the MCs and BSCs and cofactor

consumption and synthesis in the two cell types (Wang

et al., 2014). Whilst for some time it was thought that each

C4 species used only a speciﬁc C4 subtype, it now appears

that different decarboxylation pathways may operate

simultaneously, to varying extents, within the same spe-

cies (Pick et al., 2011; Wang et al., 2014). Moreover, the ﬂux

through each subtype pathway may vary based on envi-

ronmental conditions, such as light intensity (Medeiros

et al., 2022) or developmental stage (Pick et al., 2011).

Other metabolite exchanges between MCs and BSCs

are also important in C4 photosynthesis. Plants that pre-

dominantly use an NADP-ME C4 cycle such as maize, sug-

arcane and sorghum tend to exhibit low abundance of BSC

PSII (Meierhoff & Westhoff, 1993). NADPH is produced in

BSCs by NADP-ME itself but can also be produced in BSCs

using triose-phosphate imported from MCs via NADPH-

dependent GA3PDH, with the 3PGA produced being

returned to the MCs and recycled (Arrivault et al., 2017).

This cycle also appears important in proton balancing

between the cell types (Shaw & Maurice Cheung, 2019).

Exchange of the metabolites involved in these cycles

requires concentration gradients between the cell types to

drive diffusion, and metabolite proﬁling of BSCs and MCs

of maize obtained by leaf homogenization followed by ﬁl-

tration at low temperature have conﬁrmed the presence of

such gradients for malate, triose-phosphate and 3PGA

(Arrivault et al., 2017). The need to establish concentration

gradients may contribute to the simultaneous operation of

C4 subtype pathways; as rates of diffusion depend on the

size of a concentration gradient, multiple pathways would

reduce the need to maintain high concentrations of a sin-

gle metabolite (Wang et al., 2014). Other metabolic pro-

cesses show at least some degree of restriction to one of

the cell types; transient starch production in mature maize

leaves occurs in the BSCs (Weise et al., 2011) whilst nitrate

assimilation occurs in the MCs (Kopriva, 2011). Mecha-

nisms are also required to avoid leakage of carbon from

the C4 cycle into other processes; for example, elevated

organic acid concentrations in BSCs could potentially

result in carbon leaking into the TCA cycle. Although it is

not clear how this occurs,

C-labeling experiments in

maize indicate that such leakage is minimal (Arrivault

et al., 2017).

Due to the physiological advantages of C4 photosyn-

thesis under hot and dry conditions, the potential for its

introduction into C3 crops such as rice has been exten-

sively evaluated (Ermakova et al., 2020). As such, we now

Ó2023 Society for Experimental Biology and John Wiley & Sons Ltd.,

The Plant Journal, (2023), doi: 10.1111/tpj.16214

14 Danilo de Menezes Daloso et al.

have detailed information regarding the metabolism of

MCs and BSCs, as well as the anatomical and gene expres-

sion patterns that are necessary to establish a functioning

C4 cycle.

As might be expected given its complexity, C4 photo-

synthesis does not appear to have evolved in a single step.

Analysis of C3-C4 intermediate species, which are often

phylogenetically close to C4 species (Schluter &

Weber, 2020), indicates expression of glycine decarboxyl-

ase almost exclusively in BSCs as a likely ﬁrst step in C4

evolution (Borghi et al., 2022). Glycine decarboxylation,

releasing CO

, in BSCs is sufﬁcient to reduce the CO

com-

pensation point in C3-C4 intermediate species relative to

C3 species, but also requires establishment of metabolite

shuttles between the MCs and BSCs to balance nitrogen

and reduce equivalent metabolism. Metabolite proﬁling of

nine species from the Flaveria genus indicates high con-

centrations of serine in BSCs that could drive its diffusion

to the MCs (Borghi et al., 2022), and it could be that further

shuttles involving malate, aspartate, pyruvate and alanine

proposed in C3-C4 intermediate species represent the basis

for evolution of a full C4 cycle (Mallmann et al., 2014).

Clear patterns of transcript abundance are observed in

the BSCs and MCs of C4 species, with transcripts for Calvin

cycle, decarboxylation and photorespiratory enzymes

preferentially expressed in the BSCs, and those for PEPC,

PPDK and CA in the MCs (Schluter & Weber, 2020). As part

of efforts to produce a C4 rice, promoter elements that

drive speciﬁc expression in these cell types have been

identiﬁed (Ermakova et al., 2020). Gene duplication

appears to be important for C4 evolution as the additional

copies of genes may acquire speciﬁc functions associated

with MC or BSC metabolism, such as the C4 speciﬁc iso-

forms of PEPC (Chollet et al., 1996), or even affect leaf ana-

tomical traits such as vein spacing (Huang et al., 2021). C3

Flaveria species have a BSC speciﬁc and a ubiquitous iso-

form of the GLDP protein, one of the subunits of glycine

decarboxylase; in C4 species of the same genus the ubiqui-

tous form is a pseudogene leaving these plants with only

the BSC isoform that could participate in a photorespira-

tory pump (Schulze et al., 2013). A whole-genome duplica-

tion event also appears to have enabled the evolution of

C4 in Gynandropsis gynandra (Huang et al., 2021).

Differences in chloroplast development and function

between MCs and BSCs, and when compared with C3 spe-

cies, are also a key feature of C4 metabolism, with chloro-

plasts much more abundant in BSCs. Indeed, this is a

signiﬁcant barrier to C4 engineering as the plastids nor-

mally present in the BSCs of rice could not support

the photosynthetic activity necessary for a C4 cycle.

Figure 6. The principal metabolic ﬂuxes observed in the mesophyll cells (MCs) and bundle sheath cells (BSCs) of mature leaves of a C4 plant.

Carboxylation occurs in the MCs through phosphoenolpyruvate carboxylase (PEPc) activity with the malate produced from OAA moving to the BSCs through

plasmodesmata where it is decarboxylated by NADP-ME in the chloroplast. Released CO

is ﬁxed by the Calvin–Benson cycle resulting in sucrose and starch

synthesis. Pyruvate returns to the MCs and is converted to PEP to complete the cycle. For clarity, only the NADP-ME pathway of decarboxylation is shown, and

additional substrate cycles discussed in the text are omitted.

Ó2023 Society for Experimental Biology and John Wiley & Sons Ltd.,

The Plant Journal, (2023), doi: 10.1111/tpj.16214

Cell-type-speciﬁc metabolism in plants 15

A proto-Kranz anatomy, which involves increased organ-

elle volume, and abundance of RubisCO in chloroplasts

and GDC in mitochondria appears a likely step in C4 evolu-

tion (Sage et al., 2014), and can be recreated in the vascu-

lar sheath cells of rice through expression of golden-like 2

transcription factors (Wang et al., 2017). As mentioned,

PSII abundance in the BSCs of C4 species that mainly use

NADP-ME for decarboxylation is low, and this is typically

associated with the presence of few grana stacks (Meierh-

off & Westhoff, 1993) and greater cyclic electron ﬂow nec-

essary for ATP generation (Dal’Molin et al., 2010).

Cell-speciﬁc enzyme expression is necessary but not

sufﬁcient for C4 to function, as leaf anatomy also plays a

crucial role, with almost all C4 species having Kranz anat-

omy (Borghi et al., 2022). Firstly, C4 species typically have

minimal spacing between leaf veins, with each vein sepa-

rated by only four cells, whilst in C3 species this number

can be as high as 20 (Langdale, 2011). This organization is

necessary for operation of the C4 cycle between the two

cell types. BSCs and MCs are also connected by extensive

plasmodesmata that permit rapid metabolite exchange;

comparison of C3 and C4 species revealed the presence of

up to nine times more plasmodesmata for each MC-BSC

interface in C4 plants (Danila et al., 2016). The C4 cycle

therefore requires a combination of a cell-speciﬁc pattern

enzyme expression, together with developmental pro-

cesses that result in the correct spacing and connection of

cells and the activation of chloroplast development in the

BSCs.

CONCLUSIONS

Advances in isolation protocols and analytical techniques

have generated signiﬁcant insight into metabolism in spe-

ciﬁc cell types. Ultimately no single technique is appropri-

ate for all cell types, but between LAM and FACs a wide

range of cell types can be isolated, and MS-based analyti-

cal platforms are now sensitive to probe the metabolism of

cells isolated in this manner. Evaluation and reporting of

cell purity and potential artifacts caused by isolation is

highly important.

Different cell types in plants are associated with major

differences in the distribution of metabolic ﬂux. The in-

depth studies of cell-type-speciﬁc metabolism carried out

so far indicate that metabolic differences may have multi-

ple causes that are often found in combination. Firstly,

cells may be specialized in the production of certain mole-

cules, demanding ﬂux through speciﬁc metabolic pro-

cesses as occurs during fatty acid biosynthesis in B. napus

embryos or terpene synthesis in the GTs of mint. Synthesis

of such compounds, and their assembly into macromole-

cules, also requires ATP and reducing equivalents, and

their production will also shape the distribution of meta-

bolic ﬂux.

Metabolism may also differ between cell types due to

the substrates available to them. The clearest example of

this is the difference between source and sink cells; metab-

olism in one dominated by CO

ﬁxation and direct use of

light as an energy source, whilst in the other mitochondrial

respiration is typically required to meet demands for ATP

and NADPH. Subtler, but no less important, differences

may arise from the set of substrates that can be trans-

ported into the cell or released from internal reserves, as

occurs during a day–night cycle in guard cells, in embryo

cells receiving substrate from surrounding tissues, or the

availability of oxygen that will affect the extent to which

oxidative phosphorylation may function.

Cell-type-speciﬁc metabolism may also be necessary

to suppress potentially deleterious processes. One of the

consequences of the C4 photosynthetic cycle, for example,

is suppression of photorespiration, whilst the restriction of

production of certain secondary metabolites to specialized

cell types and the accumulation of these metabolites in

specialized structures avoids potential autotoxicity and

ﬁnely regulates the growth-stress tolerance trade-off.

The ﬂexibility of photosynthesis is also made clear by

the metabolic phenotypes of speciﬁc cell types; whilst

Calvin–Benson cycle function resulting in net CO

ﬁxation

and driven by predominantly linear electron transport is

present in the mesophyll of mature C3 leaves, these pro-

cesses can fulﬁll different roles in other metabolic con-

texts. For example, reﬁxation of CO

occurs in green

embryos and photosynthetic GTs without true Calvin–Ben-

son cycle operation, whilst the ATP and reducing power

generated by photosynthesis in these cells is needed for

other biosynthetic processes.

Further technical advances, including the develop-

ment of methods for cell isolation, techniques for in vivo

imaging, increased sensitivity of analytical methods and in

silico modeling, will allow us to obtain more detailed infor-

mation on cell-type-speciﬁc metabolism that both

increases our understanding of plant metabolic physiology

and reveals new opportunities for metabolic engineering.

ACKNOWLEDGEMENTS

DMD and TCRW thank the National Council for Scientiﬁc and

Technological Development (CNPq, grants 404817/2021-1 and

305288/2020) for ﬁnancial support. DMD thanks the National Insti-

tute of Science and Technology in Plant Physiology under Stress

Conditions (INCT Plant Stress Physiology –grant 406455/2022-8)

for ﬁnancial support. TCRW thanks the Fundac

ao de Apoio de Pes-

quisa do Distrito Federal (FAPDF, grant 00193-00001067/2021-48)

for ﬁnancial support. The authors also thank the research fellow-

ships granted by CNPq to DMD and TCRW, and the scholarship

granted by the Brazilian Federal Agency for Support and Evalua-

tion of Graduate Education (CAPES-Brazil) to EGM.

CONFLICT OF INTEREST

The authors declare that they have no conﬂicts of interest.

Ó2023 Society for Experimental Biology and John Wiley & Sons Ltd.,

The Plant Journal, (2023), doi: 10.1111/tpj.16214

16 Danilo de Menezes Daloso et al.

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13C labelling reveals details of the soybean (Glycine max (L.) Merrill) seedling metabolic network

Article

Apr 2024

Seedlings depend upon seed reserves for the prevision of carbon skeletons for growth and energy production. Post-germinative growth of soybean is therefore marked by the breakdown of carbohydrates, proteins, and lipids and the interconversion of the products of these catabolic processes. Here, we refined a method for 13C isotope labelling of heterotrophic soybean seedlings and used it to probe metabolism during this critical phase of plant development. We anticipated that 13C labelling would reveal differences in metabolism between the cotyledons (COT) and hypocotyl-root axis (HRA). Feeding with U-13C glucose followed by analysis of isotope incorporation indicated uptake and metabolism of this labelled precursor by both COT and HRA. Fractional enrichments were generally greater in the HRA reflecting the catabolism of unlabelled reserves of lipids and proteins in COT. Mass isotopomer distributions confirmed operation of the TCA cycle and glycolysis along with hexose-phosphate cycling in both organs, whilst amino acid synthesis was limited, as expected, given the significant protein reserves. COT differed from HRA in TCA cycle citrate and anapleurotic metabolism. Experiments with 13C glycine indicated that glycine decarboxylase and serine hydroxymethyltransferase enzymes may function in heterotrophic tissues as well as in photorespiration. Labelling of the majority of metabolites was constant over time, suggesting that the experimental system could be used for metabolic flux analysis. Overall stable isotope labelling provided significant insight into metabolism of soybean seedlings and could be used to investigate seedling metabolism in other genotypes or species.

A role for fermentation in aerobic conditions as revealed by computational analysis of maize root metabolism during growth by cell elongation

Article

Oct 2023

The root is a well‐studied example of cell specialisation, yet little is known about the metabolism that supports the transport functions and growth of different root cell types. To address this, we used computational modelling to study metabolism in the elongation zone of a maize lateral root. A functional‐structural model captured the cell‐anatomical features of the root and modelled how they changed as the root elongated. From these data, we derived constraints for a flux balance analysis model that predicted metabolic fluxes of the 11 concentric rings of cells in the root. We discovered a distinct metabolic flux pattern in the cortical cell rings, endodermis and pericycle (but absent in the epidermis) that involved a high rate of glycolysis and production of the fermentation end‐products lactate and ethanol. This aerobic fermentation was confirmed experimentally by metabolite analysis. The use of fermentation in the model was not obligatory but was the most efficient way to meet the specific demands for energy, reducing power and carbon skeletons of expanding cells. Cytosolic acidification was avoided in the fermentative mode due to the substantial consumption of protons by lipid synthesis. These results expand our understanding of fermentative metabolism beyond that of hypoxic niches and suggest that fermentation could play an important role in the metabolism of aerobic tissues.

Understanding plant pathogen interactions using spatial and single-cell technologies

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Full-text available

Aug 2023

Plants are in contact with diverse pathogens and microorganisms. Intense investigation over the last 30 years has resulted in the identification of multiple immune receptors in model and crop species as well as signaling overlap in surface-localized and intracellular immune receptors. However, scientists still have a limited understanding of how plants respond to diverse pathogens with spatial and cellular resolution. Recent advancements in single-cell, single-nucleus and spatial technologies can now be applied to plant – pathogen interactions. Here, we outline the current state of these technologies and highlight outstanding biological questions that can be addressed in the future.

Advancing Engineered Plant Living Materials through Tobacco BY-2 Cell Growth and Transfection within Tailored Granular Hydrogel Scaffolds

Article

May 2024

In this study, an innovative approach is presented in the field of engineered plant living materials (EPLMs), leveraging a sophisticated interplay between synthetic biology and engineering. We detail a 3D bioprinting technique for the precise spatial patterning and genetic transformation of the tobacco BY-2 cell line within custom-engineered granular hydrogel scaffolds. Our methodology involves the integration of biocompatible hydrogel microparticles (HMPs) primed for 3D bioprinting with Agrobacterium tumefaciens capable of plant cell transfection, serving as the backbone for the simultaneous growth and transformation of tobacco BY-2 cells. This system facilitates the concurrent growth and genetic modification of tobacco BY-2 cells within our specially designed scaffolds. These scaffolds enable the cells to develop into predefined patterns while remaining conducive to the uptake of exogenous DNA. We showcase the versatility of this technology by fabricating EPLMs with unique structural and functional properties, exemplified by EPLMs exhibiting distinct pigmentation patterns. These patterns are achieved through the integration of the betalain biosynthetic pathway into tobacco BY-2 cells. Overall, our study represents a groundbreaking shift in the convergence of materials science and plant synthetic biology, offering promising avenues for the evolution of sustainable, adaptive, and responsive living material systems.

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Article

Jan 2024

The tricarboxylic acid (TCA) cycle is an important metabolic pathway to underpin stomatal movements, given that respiration is thought to be the main energy source for guard cell (GC) metabolism. However, it is still unclear how the metabolic fluxes throughout the TCA cycle and associated pathways are regulated in GCs. Here we used a 13C-positional isotopomer approach and performed a multi-species/cell-types analysis based on previous 13C-labelling studies carried out using Arabidopsis rosettes, maize leaves, Arabidopsis source and sink leaves, and isolated GCs from Arabidopsis and tobacco. We aimed to compare flux modes through the TCA cycle and associated pathways in GCs and leaves, which are mostly composed by mesophyll cells (MCs). Mesophyll cells showed high 13C-enrichment into alanine and aspartate following provision of 13CO2, whilst GCs and sink MCs showed high 13C-incorporation into glutamate/glutamine following provision of 13C-sucrose. Only GCs showed high 13C-enrichment in the carbon 1 atom of glutamine, which is derived from phosphoenolpyruvate carboxylase (PEPc)-mediated CO2 assimilation. The PEPc-mediated 13C-incorporation into malate was similar between GCs and MCs, but GCs had higher 13C-enrichment and accumulation of fumarate than MCs. The metabolic fluxes throughout the TCA cycle of illuminated GCs resemble those of sink MCs, but with different contribution from PEPc, glycolysis and the TCA cycle to glutamate/glutamine synthesis. We further demonstrate that transamination reactions catalysed by alanine and aspartate amino transferases may support non-cyclic TCA flux modes in illuminated MCs.

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Article

Dec 2023
PLANT J

Gwendolyn K Kirschner

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Jun 2023
PLANT J

Integration of physiologically relevant photosynthetic energy flows into whole genome models of light‐driven metabolism

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Full-text available

Sep 2022

Abstract Characterizing photosynthetic productivity is necessary to understand the ecological contributions and biotechnology potential of plants, algae, and cyanobacteria. Light capture efficiency and photophysiology have long been characterized by measurements of chlorophyll fluorescence dynamics. However, these investigations typically do not consider the metabolic network downstream of light harvesting. In contrast, genome‐scale metabolic models capture species‐specific metabolic capabilities but have yet to incorporate the rapid regulation of the light harvesting apparatus. Here we combine chlorophyll fluorescence parameters defining photosynthetic and non‐photosynthetic yield of absorbed light energy with a metabolic model of the pennate diatom Phaeodactylum tricornutum. This integration increases the model predictive accuracy regarding growth rate, intracellular oxygen production and consumption, and metabolic pathway usage. Through the quantification of Excess Electron Transport (EET), we uncover the sequential activation of non‐radiative energy dissipation processes, cross compartment electron shuttling, and non‐photochemical quenching as the rapid photoacclimation strategy in P. tricornutum. Interestingly, the photon absorption thresholds that trigger the transition between these mechanisms were consistent at low and high incident photon fluxes. We use this understanding to explore engineering strategies for rerouting cellular resources and excess light energy toward bioproducts in silico. Overall, we present a methodology for incorporating a common, informative data type into computational models of light‐driven metabolism and show its utilization within the design‐build‐test‐learn cycle for engineering of photosynthetic organisms.

Metabolic engineering of energycane to hyperaccumulate lipids in vegetative biomass

Article

Full-text available

Aug 2022
BMC BIOTECHNOL

Background The metabolic engineering of high-biomass crops for lipid production in their vegetative biomass has recently been proposed as a strategy to elevate energy density and lipid yields for biodiesel production. Energycane and sugarcane are highly polyploid, interspecific hybrids between Saccharum officinarum and Saccharum spontaneum that differ in the amount of ancestral contribution to their genomes. This results in greater biomass yield and persistence in energycane, which makes it the preferred target crop for biofuel production. Results Here, we report on the hyperaccumulation of triacylglycerol (TAG) in energycane following the overexpression of the lipogenic factors Diacylglycerol acyltransferase 1-2 ( DGAT 1-2) and Oleosin 1 ( OLE 1) in combination with RNAi suppression of SUGAR-DEPENDENT 1 ( SDP 1) and Trigalactosyl diacylglycerol 1 ( TGD 1). TAG accumulated up to 1.52% of leaf dry weight (DW,) a rate that was 30-fold that of non-modified energycane, in addition to almost doubling the total fatty acid content in leaves to 4.42% of its DW. Pearson’s correlation analysis showed that the accumulation of TAG had the highest correlation with the expression level of ZmDGAT 1-2, followed by the level of RNAi suppression for SDP 1. Conclusions This is the first report on the metabolic engineering of energycane and demonstrates that this resilient, high-biomass crop is an excellent target for the further optimization of the production of lipids from vegetative tissues.

A high-efficiency trichome collection system by laser capture microdissection

Article

Full-text available

Aug 2022

Trichomes, which are classified as glandular or non-glandular, are hair-like epidermal structures that are present on aerial parts of most plant species. Glandular secretory trichomes (GSTs) have the capacity to secrete and store specialized metabolites, which are widely used as natural pesticides, food additives, fragrance ingredients or pharmaceuticals. Isolating individual trichomes is an essential way for identifying trichome-specific gene functions and discovering novel metabolites. However, the isolation of trichomes is difficult and time-consuming. Here, we report a method to isolate the GSTs from leaf epidermis dispense with fixation using laser capture microdissection (LCM). In this study, 150 GSTs were captured efficiently from Artemisia annua leaves and enriched for artemisinin measurement. UPLC analysis of microdissected samples indicated specific accumulation of secondary metabolites could be detected from a small number of GSTs. In addition, qRT-PCR revealed that the GST-specific structural genes involved in artemisinin biosynthesis pathway were highly expressed in GSTs. Taken together, we developed an efficient method to collect comparatively pure GSTs from unfixed leaved, so that the metabolites were relatively obtained intact. This method can be implemented in metabolomics research of purely specific plant cell populations and has the potential to discover novel secondary metabolites.

Research strategies for single‐cell transcriptome analysis in plant leaves

Article

Full-text available

Jul 2022

The recent and continuous improvement in single‐cell RNA sequencing (scRNA‐seq) technology has led to its emergence as an efficient experimental approach in plant research. However, compared with single‐cell research in animals and humans, the application of scRNA‐seq in plant research is limited by several challenges, including cell separation, cell type annotation, cellular function analysis, and cell–cell communication networks. In addition, the unavailability of corresponding reliable and stable analysis methods and standards has resulted in the relative decentralization of plant single‐cell research. Considering these shortcomings, this review summarizes the research progress in plant leaf using scRNA‐seq. In addition, it describes the corresponding feasible analytical methods and associated difficulties and problems encountered in the current research. In the end, we provide a speculative overview of the development of plant single‐cell transcriptome research in the future.

13CO2 Labelling Kinetics in Maize Reveal Impaired Efficiency of C4 Photosynthesis under Low Irradiance

Article

Full-text available

Jun 2022

C4 photosynthesis allows faster photosynthetic rates and higher water and nitrogen use efficiency than C3 photosynthesis, but at the cost of lower quantum yield due to the energy requirement of its biochemical carbon concentration mechanism. It has also been suspected that its operation may be impaired in low irradiance. To investigate fluxes under moderate and low irradiance, maize (Zea mays) was grown at 550 µmol photons m−2 s-l and 13CO2 pulse-labelling was performed at growth irradiance or several hours after transfer to 160 µmol photons m−2 s−1. Analysis by liquid chromatography/tandem mass spectrometry or gas chromatography/mass spectrometry provided information about pool size and labelling kinetics for 32 metabolites and allowed estimation of flux at many steps in C4 photosynthesis. The results highlighted several sources of inefficiency in low light. These included excess flux at phosphoenolpyruvate carboxylase, restriction of decarboxylation by NADP-malic enzyme, and a shift to increased CO2 incorporation into aspartate, less effective use of metabolite pools to drive intercellular shuttles, and higher relative and absolute rates of photorespiration. The latter provides evidence for a lower bundle sheath CO2 concentration in low irradiance, implying that operation of the CO2 concentration mechanism is impaired in this condition. The analyses also revealed rapid exchange of carbon between the Calvin-Benson cycle and the CO2-concentration shuttle, which allows rapid adjustment of the balance between CO2 concentration and assimilation, and accumulation of large amounts of photorespiratory intermediates in low light that provides a major carbon reservoir to build up C4 metabolite pools when irradiance increases.

On the role of guard cells in sensing environmental signals and memorising stress periods

Article

Full-text available

Jun 2022

The capacity to perceive and memorise adverse environmental conditions is pivotal for the survival of any biological system. Despite being sessile, plants do it with maestri. Plants can rapidly respond to changes in environmental cues and memorise stress conditions, as a consequence of their modularity and the presence of highly complex cell types, such as the guard cells. These cells integrate endogenous and environmental signals to regulate the opening of the stomatal pore, mainly found at leaf epidermis. Whilst the stomatal opening enables the influx of CO2 for photosynthesis, stomatal closure is important to reduce water loss during drought stress. Guard cell is thus crucial for the perception of environmental signals and a master regulator of water use efficiency (WUE). Furthermore, recent results indicate that guard cell gene expression precedes those observed in mesophyll cells when plants are subjected to drought and that guard cell is an important hub for stress memory. Here, we highlight these recent findings and provide an updated overview regarding the intrinsic complexity of guard cell structure and the signalling networks related to the perception and response to different environmental signals. We explored recently published guard cell transcriptomics data from plants under drought and discussed their implication for plant stress acclimation and metabolic engineer toward plant drought tolerance improvement. We further highlight the possible interplay among the mechanisms that regulate stress memory and stomatal speediness and how they can be used to improve WUE and/or stress tolerance in plants.

Transcriptional regulation of oil biosynthesis in seed plants: Current understanding, applications, and perspectives

Article

Full-text available

Apr 2022

Plants produce and accumulate triacylglycerol (TAG) in their seeds as an energy reservoir to support the processes of seed germination and seedling development. Plant seed oils are vital not only for human diets but also as renewable feedstock for industrial uses. TAG biosynthesis consists of two major steps: de novo fatty acid biosynthesis in the plastids and TAG assembly in the endoplasmic reticulum. The latest advances in unraveling transcriptional regulation have shed light on the molecular mechanisms of plant oil biosynthesis. This review summarizes the recent progress in understanding the regulatory mechanisms of well-characterized and newly discovered transcription factors and other types of regulators that control plant fatty acid biosynthesis. The emerging picture shows that plant oil biosynthesis responds to developmental and environmental cues that stimulate a network of interacting transcriptional activators and repressors, which, in turn, fine-tune the spatiotemporal regulation of the pathway genes.

Expression of the VvMYB60 Transcription Factor Is Restricted to Guard Cells and Correlates with the Stomatal Conductance of the Grape Leaf

Article

Full-text available

Mar 2022

Giulia Castorina

The modulation of stomatal activity is a relevant trait in grapes, as it defines the isohydric/anysohydric behavior of different cultivars and directly affects water-use efficiency and drought resistance of vineyards. The grape transcription factor VvMYB60 has been proposed as a transcriptional regulator of stomatal responses based on its ectopic expression in heterologous systems. Here, we directly addressed the cellular specificity of VvMYB60 expression in grape leaves by integrating independent approaches, including the qPCR analysis of purified stomata and the transient expression of a VvMYB60 promoter: GFP fusion. We also investigated changes in the VvMYB60 expression in different rootstocks in response to declining water availability. Our results indicate that VvMYB60 is specifically expressed in guard cells and that its expression tightly correlates with the level of stomatal conductance (gs) of the grape leaf. As a whole, these findings highlight the relevance of the VvMYB60 regulatory network in mediating stomatal activity in grapes.

Starch biosynthesis in guard cells has features of both autotrophic and heterotrophic tissues

Article

Full-text available

Mar 2022

The pathway of starch synthesis in guard cells (GCs), despite the crucial role starch plays in stomatal movements, is not well understood. Here, we characterized starch dynamics in GCs of Arabidopsis (Arabidopsis thaliana) mutants lacking enzymes of the Phosphoglucose isomerase (PGI)-Phosphoglucose mutase (PGM)-ADP-Glucose pyrophosphorylase (AGPase) starch synthesis pathway in leaf mesophyll chloroplasts or sugar transporters at the plastid membrane, such as Glucose-6-phosphate/Phosphate Translocators (GPTs), which are active in heterotrophic tissues. We demonstrate that GCs have metabolic features of both photoautotrophic and heterotrophic cells. GCs make starch using different carbon precursors depending on the time of day, which can originate both from GC photosynthesis and/or sugars imported from the leaf mesophyll. Furthermore, we unravel the major enzymes involved in GC starch synthesis and demonstrate that they act in a temporal manner according to the fluctuations of stomatal aperture, which is unique for GCs. Our work substantially enhances our knowledge on GC starch metabolism and uncovers targets for manipulating GC starch dynamics to improve stomatal behaviour, directly affecting plant productivity.

Mass spectrometry imaging techniques: a versatile toolbox for plant metabolomics

Article

Nov 2022
TRENDS PLANT SCI

Cell-type-specific metabolism in plants

Abstract

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