Responses of leaf stomatal and mesophyll conductance to abiotic stress factors

Ll Sheng-lan ,TAN Ting-ting ,FAN Yuan-fang ,Muhammad Ali RAZA ,WANG Zhong-lin ,WANG Bei-bei ,ZHANG Jia-wei,TAN Xian-ming,CHEN Ping,lram SHAFlQ,YANG Wen-yu,YANG Feng

1 College of Agronomy,Sichuan Agricultural University,Chengdu 611130,P.R.China

2 Sichuan Engineering Research Center for Crop Strip Intercropping,Chengdu 611130,P.R.China

3 Key Laboratory of Crop Ecophysiology and Farming System in Southwest,Ministry of Agriculture,Chengdu 611130,P.R.China

Abstract Plant photosynthesis assimilates CO2 from the atmosphere,and CO2 diffusion efficiency is mainly constrained by stomatal and mesophyll resistance.The stomatal and mesophyll conductance of plants are sensitive to abiotic stress factors,which affect the CO2 concentrations at carboxylation sites to control photosynthetic rates.Early studies conducted relevant reviews on the responses of stomatal conductance to the environment and the limitations of mesophyll conductance by internal structure and biochemical factors.However,reviews on the abiotic stress factors that systematically regulate plant CO2 diffusion are rare.Therefore,in this review,the rapid and long-term responses of stomatal and mesophyll conductance to abiotic stress factors (such as light intensity,drought,CO2 concentration and temperature) and their physiological mechanisms are summarized.Finally,future research trends are also investigated.

Keywords: CO2 diffusion,abiotic stress factors,stomatal conductance,mesophyll conductance

1.lntroduction

Photosynthesis is the basis of crop yield formation,and sunlight triggers the process of photosynthesis that induces gaseous exchange in the leaves through stomata(Makino 2010).Consequently,CO2from the surrounding environment is diffused into the leaves,followed by its fixation at the carboxylation sites of Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in the chloroplast stroma (Harrisonet al.2019).The CO2supply determines the photosynthetic rate to a large extent (in C3plants) (Xionget al.2015).However,CO2must overcome multiple obstacles to reach the chloroplast stroma (Xionget al.2015).Initially,concentration gradients allow the CO2to conquer the boundary layer (rb) and stomatal (rs)resistance and reach the substomatal cavities.Then,CO2continues to diffuse through the intercellular space to the cell wall,where carbonic anhydrase (CA) catalyzes its dissolution.Subsequently,CO2must overcome mesophyll resistance (rm) to arrive at the chloroplast stroma.These CO2molecules eventually approach the carboxylation sites of Rubisco to participate in the Calvin-Benson cycle (Fig.1).Notably,the reciprocals ofrb,rsandrmare boundary layer conductance (gb),stomatal conductance(gs) and mesophyll conductance (gm),respectively (Evanset al.2009;Harrisonet al.2019).Thegsandgmmainly regulate photosynthesis by limiting CO2diffusion (Xionget al.2017).

Stomata are small pores on the surface of leaves and stems that are mostly dumbbell-shaped in monocotyledons and kidney-shaped in dicotyledons (Lawson and Matthews 2020).The development and patterning of stomata determine stomatal density,size and distribution.All these anatomical characteristics,together with stomatal aperture,determinegsto a large extent (Xiong 2016).Abiotic environmental signals usually optimize gas exchange by altering stomatal development and stomatal aperture,which helps plants adapt to changing environments (Casson and Hetherington 2010).Thegsis mostly affected by light intensity,CO2concentration,water and temperature (Gaoet al.2016).

Similarly,gmis also sensitive to several conditions(Hanet al.2017).Thegmis generally constrained by leaf structure (mesophyll cell wall surface area exposed to intercellular airspace per leaf area,Sm;surface area of chloroplasts exposed to inter-cellular airspaces,Sc;cell wall) and biochemical factors (the content and activity of CA and aquaporins,AQPs) (Flexaset al.2008).Importantly,gsandgmlimit photosynthetic rate (mostly in C3plants),because any decrease in CO2diffusion or utilization would directly lead to a photosynthesis rate decrease (Xionget al.2017;Ubiernaet al.2018).

Thegsandgmform a complex response mechanism to optimize plant growth in various stress conditions.Reviews ongsresponses to the environment and their physiological mechanisms are available (Murataet al.2015;Lee and Bergmann 2019).Papers about internal limiting factors ofgmin leaves have been published in recent years as well (Evans 2020).However,reviews on the systematic regulation of plant CO2diffusion by abiotic stress factors are limited.With the growing world population (Simkinet al.2015),frequent drought disasters(Luoet al.2019),increasing global CO2concentration(Engineeret al.2015) and climate warming (Li Yet al.2019),clarifying the responses of CO2diffusion efficiency to abiotic stress factors in different crops is of considerable significance for ensuring high and stable food production.Consequently,the responses ofgsandgmto changes in light intensity,drought,CO2concentration and temperature are reviewed here.Moreover,the related physiological mechanism is explained briefly.

2.Stomatal conductance regulation in plants under variable environmental factors

Under the given environmental conditions,gsis mainly affected by stomatal morphological characteristics,including stomatal density,size,and aperture (Xiong 2016).Stomatal aperture and density modulate different plant responses to the environment.For instance,changes in the stomatal aperture represent the rapid response of plant leaves to the environment,while a change in stomatal density represents the long-term response (Dowet al.2014).

2.1.Rapid response of the stomatal aperture to environmental factors

Stomatal opening in the rapid response ofgsto abiotic environmental factors is caused by the plasma membrane hyperpolarization of guard cells,which needs the activation of H+-ATPases (Kimet al.2010;Papanatsiouet al.2019).Membrane hyperpolarization induces K+uptake into guard cellsviavoltage-gated inward K+inchannels (KAT).Water is then absorbed into the guard cells,and the stomata are opened (Kimet al.2010;Kinoshita and Hayashi 2011).

Stomatal closure is primarily triggered by the release of anions and K+from guard cells (Geigeret al.2011).The plasma membrane of guard cells has two main kinds of anion channels.The first kind is the slow-activating sustained (S-type) anion channel,which produces a slow and continuous anion outflow (Kimet al.2010).S-type channels,mostly SLOWANION CHANNEL-ASSOCIATED(SLAC1) and SLAC1 homolog 3 (SLAH3),are permeable to Cl-and NO3-(Geigeret al.2011).Another kind is the rapid-transient (R-type) anion channel,which is instantaneously activated within 50 ms.ALUMINUMACTIVATED MALATE TRANSPORTER 12/QUICKLY ACTIVATING ANION CHANNEL 1 (ALMT12/QUAC1)is an important R-type anion channel.R-type anion channels mediate malate transport in stomatal closure(Meyeret al.2010).The activation of S-type anion channels has been identified as a key step in stomatal closure (Kimet al.2010).Subsequently,the outflow of anions through channels induces depolarization of the plasma membrane and activates voltage-gated outward K+channels,triggering stomatal closure (Munemasaet al.2015).

Light intensityStomata,as one of the key structures of plant photosynthesis,has strong light adaptability (Gaoet al.2016).Thegsunder fluctuating light rapidly varies with light intensity (Matthewset al.2018;Zhanget al.2019).However,the speed of the stomatal response to light intensity is determined by genotype and growth conditions (Durandet al.2020).For example,stomata in open habitats are more sensitive to changing light intensity compared with plants grown in shade (Xionget al.2014).Overall,this phenomenon may be correlated withPATROL1,which facilitates the direction of H+-ATPases for plasma membrane in guard cells (Hashimoto-Sugimotoet al.2013).Plant Rho-type GTPase 2 (ROP2)-ROP interactive binding motif-containing protein 7 (RIC7)negatively regulates light-induced stomatal opening inArabidopsisby interacting with and inhibiting the function of exocyst subunit Exo70 family protein B1 (Exo70B1)(Honget al.2016).OsKAT2 (T235R)-overexpressing rice showed delayed stomatal opening when the plant was transferred from darkness to light (Moonet al.2017).Similarly,rice plants with knockout geneSLAC1show highergsthan normal plants under fluctuating light (Yamoriet al.2020).Nonetheless,the high temperature caused by strong light intensity will increase the transpiration rate,reduce the leaf water potential,and ultimately lead to a decrease in the stomatal aperture (Gaoet al.2016).

DroughtUnder drought conditions,small peptide CLAV ATA3/EMBRYO-SURROUNDING REGION-RELATED 25(CLE25) transmits the stress signal felt byArabidopsisroots to the leaves through vascular tissue,which is received by BARELY ANY MERISTEM (BAM) (Takahashiet al.2018).Drought induces an increase in abscisic acid (ABA) levels in roots and leaves (Geet al.2017),thus closing stomata to reduce water loss (Takahashiet al.2018;Luoet al.2019).In addition to ABA,plants also produce other factors that regulate stomatal closure under drought stress,such as other plant hormones(auxins,methyl jasmonate,etc.),microbial elicitors and polyamines.Among these factors,ABA is considered to be the crucial factor,and the mechanism of ABA-induced stomatal closure under drought has been extensively studied (Lvet al.2017;Agurlaet al.2018;Meeterenet al.2019).

The transportation of ABA to guard cells may be carried out by some members of the ATP-binding cassette G(ABCG) subfamily (Meriloet al.2015).For instance,ABCG40 is a plasma membrane transporter for active ABA uptake into guard cells (Kanget al.2010).ABCG25,which is mainly expressed in vascular tissues,has also been reported to be involved in ABA transportation(Kuromoriet al.2010).RCN1/OsABCG5,which is located in the plasma membrane of guard cells,has proven to be indispensable during drought-triggered stomatal closure in rice (Matsudaet al.2016).Additionally,an unidentified transporter is involved in root ABA transport but it cannot transport ABA against the concentration gradient (Joneset al.2014).Importantly,the rapid ABA response may not need to import the ABA produced outside of the guard cells (Meriloet al.2015) because autonomous ABA synthesis in the guard cells is sufficient during droughtinduced stomatal closure (Fig.2) (Baueret al.2013).

Receptors,such as G-PROTEIN COUPLED RECEPTOR (GPCR)-TYPEGPROTEINS 1(GTG1),GTG2 and PYRABACTIN RESISTANCE(PYR)/PYRABACTIN RESISTANCE 1 LIKE (PYL)/REGULATORY COMPONENT OF ABA RECEPTOR(RCAR) (Fig.2),recognize ABA levels in guard cells to regulate stomatal response (Lianget al.2018).Among these receptors,GTG1 and GTG2 are targeted to the plasma membrane of guard cells and interact with G PROTEINLPHA SUBUNIT (GPA1) (Wanget al.2001).GPA1 then functions in the inhibition of K+inchannels in guard cells,such as POTASSIUM CHANNEL INARABIDOPSISTHALIANA1 (KAT1) (Fig.2) (Wanget al.2001;Sutteret al.2007).Similarly,several proteins of the PYR/PYL/RCAR family are also known to interact with and inhibit type 2Cs protein phosphatase (PP2Cs),which mainly include ABA-INSENSITIVE 1 (ABI1),ABI2 and HOMOLOGY TO ABI1 (HAB1) (Santiagoet al.2009).Furthermore,ABA ligand-PYR/PYL/RCAR-PP2C complex activates Ca2+-independent protein kinases SUCROSE NON-FERMENTING 1-RELATED PROTEIN KINASE 2s(SnRK2s),especially OPEN STOMATA 1 (OST1/SnRK2.6)(Fig.2) (Weineret al.2010).BRASSINOSTEROIDINSENSITIVE 1 ASSOCIATED RECEPTOR KINASE 1(BAK1) can also promote the phosphorylation of OST1,thereby positively regulating ABA signaling in stomatal closure (Shanget al.2016).Antagonistically,phosphatase ABI1 interacts with BAK1 and suppresses its promoting effect on OST1invivo(Fig.2) (Shanget al.2016).Moreover,ABA-triggered stomatal closure requires OST1-dependent Plasma membrane Intrinsic Protein2;1 (PIP2;1) phosphorylation to increase guard cell permeability to water and hydrogen peroxide (H2O2)(Grondinet al.2015).

Fig.2 Mechanism of the rapid regulation of stomatal closure by abscisic acid (ABA).GTG1,G-protein coupled receptor (GPCR)-type G proteins 1;GTG2,G-protein coupled receptor (GPCR)-type G proteins 2;PYR/PYL/RCAR,pyrabactin resistance/pyrabactin resistance 1 like/regulatory component of ABA receptor;GPA1,G proteinlpha subunit;KAT1,potassium channel in Arabidopsis thaliana 1;PP2Cs,type 2Cs protein phosphatase;ABI1,ABA-insensitive 1;ABI2,ABA-insensitive 2;HAB1,homology to ABI1;BAK1,brassinosteroid-insensitive 1 associated receptor kinase 1;OST1,open stomata 1;SnRK2s,sucrose non-fermenting 1-related protein kinase 2s;GCA2,growth controlled by abscisic acid 2;CPKs,calcium-dependent protein kinase;CIPK23 CBLinteracting protein kinase 23;ROS,reactive oxygen species;MAPKs,mitogen-activated protein kinases;NO,nitric oxide;GHR1,guard cell hydrogen peroxide-resistant 1;ICa,Ca2+-permeable non-selective cation;[Ca2+]cyt,Ca2+ in cytosolic;GORK,guard cell outward rectifying K+ channel.

Any decrease in PP2Cs activity directly enhances the activity of hyperpolarization-dependent Ca2+-permeable non-selective cation (ICa) channels in the plasma membrane of guard cells,increasing the concentration of Ca2+in cytosolic ([Ca2+]cyt) (Murataet al.2001).The inhibition of phosphatase ABI2 by ABA releases GUARD CELL HYDROGEN PEROXIDE-RESISTANT1 (GHR1),which is involved in initiating ICaand resides in the guard cell plasma membrane (Fig.2) (Munemasaet al.2015).Alternatively,Ca2+-independent protein kinase OST1 directly interacts with and initiates both RESPIRATORY BURST OXIDASE HOMOLOGUE F (AtRBOHF) and AtRBOHD NADPH oxidase (Sirichandraet al.2009),which function in ABA-mediated reactive oxygen species (ROS,mainly H2O2) elevation (Bharathet al.2021).Subsequently,these ROS regulate ABA signaling through a feedback cycle by directly down-regulating the phosphatase activities of ABI1 and ABI2 (Meinhard and Grill 2001;Raghavendraet al.2010).Mitogenactivated protein kinases (MAPKs) regulate stomatal closure downstream of H2O2under drought stress.For instance,MAP3Kθ1promotes the accumulation of ABA under drought (Jiaet al.2020).However,the specific mechanism should still be studied further due to the complexity and diversity of MAPK family members (Axelet al.2016;Duet al.2019).Furthermore,ROS promotes nitric oxide (NO) production,which inhibits K+inchannels and initiates K+outand ICachannels (Agurlaet al.2018).Nevertheless,NO may not be the crucial factor in the rapid stomatal closure induced by ABA (Meeterenet al.2019).NAD kinase2 (NADK2) positively regulates ABAinduced stomatal closure by increasing the production of H2O2,Ca2+and NO in guard cells inArabidopsis(Sunet al.2017),while RIC7 performs negative regulation by inhibiting H2O2generation (Zhuet al.2021).

ROS also activate ICachannels (Fig.2) (Kwaket al.2003).[Ca2+]cytcan be increased by Ca2+release from intracellular Ca2+stores,but this mechanism should still be comprehensively clarified (Munemasaet al.2015).Importantly,cytosolic Ca2+has been shown to act as a second messenger in ABA signaling in guard cells(Huanget al.2019).In the absence of a [Ca2+]cyttransient signal in guard cells,ABA-induced stomatal closure accounts for only 30% of the normal level,and the closure speed is considerably slowed down (Siegelet al.2009).Interestingly,ABA enhances/primes the capability of guard cells to respond to increased [Ca2+]cytlevels (Younget al.2006;Siegelet al.2009).Notably,the ABA-insensitive mutantgrowthcontrolledbyabscisicacid2(gca2) shows a different pattern of [Ca2+]cytchange induced by ABA (Allenet al.2001).In the absence of ABA,Ca2+responsiveness was inhibited by PP2Cs;thus,PP2Cs mediated the Ca2+regulation specifically induced by ABA (Brandtet al.2015).In the presence of ABA,the change in [Ca2+]cytactivates S-type anion channels (SLAC1) and suppresses K+inchannels (Fig.2) (Siegelet al.2009;Agurlaet al.2018).Furthermore,ABC protein MULTIDRUGRESISTANCE PROTEIN 5 (AtMRP5) plays an essential transduction role in the activation of S-type anion channels by ABA and Ca2+(Suhet al.2007).

Calcium-dependent protein kinases (CDPKs;inArabidopsis,CPKs) act as a Ca2+sensor to transfer the calcium signal.CPK3/4/5/6/11/21/23 have been characterized as the positive sensors in guard cell ABA signaling (Fig.2) (Moriet al.2006;Zhuet al.2007;Geigeret al.2011;Meriloet al.2013;Murataet al.2015).Another calcium signal sensor,namely CALCINEURIN-B LIKE PROTEINS (CBLs),interacts with CBL-INTERACTING PROTEIN KINASES (CIPKs) and modulates CIPK activity (D’Angeloet al.2007).CIPK23,as an interacting protein of CBL1 and CBL9,enhances the activities of SLACI and SLAH3 (Fig.2) (Maierhoferet al.2014).

OST1,GHR1 and CPKs all can phosphorylate S-type anion channels SLAC1 or SLAH3 (Geigeret al.2011;Huaet al.2012).By contrast,PP2Cs directly down-regulate the activity of SLAC1 (Brandtet al.2015).Additionally,OST1/SnRK2.6 phosphorylates the R-type anion channel ALMT12/QUAC1 as well (Imeset al.2013).Overall,ABA-induced stomatal closure depends on two pathways:one is dependent on the [Ca2+]cyt,while the other is Ca2+-independent.However,complete ABA-induced stomatal rapid movement requires cross-regulation of Ca2+-independent and Ca2+-dependent responses (Fig.2)(Brandtet al.2015).Ultimately,the activation of S-and R-type anion channels leads to the efflux of K+through voltage-dependent outward potassium ion channel GUARD CELL OUTWARD RECTIFYING K+CHANNEL(GORK) (Hosyet al.2003) with the participation of K+UPTAKE TRANSPORTERs (KUPs) (Fig.2) (Osakabeet al.2013).

In addition to regulating anion loss in guard cell stroma,ABA also adjusts solute loss in the guard cell vacuole (Eisenachet al.2017).For instance,in ABAinduced stomatal closure,K+channel two pore K+1(TPK1) in the vacuolar membrane can be activated by the change in [Ca2+]cytand mediate K+eラux (Gobertet al.2007).Subsequently,the outflow of K+leads to vacuolar membrane depolarization and activates ALUMINUM ACTIVATED MALATE TRANSPORTER4 (AlMT4).In return,AlMT4 mediates the outflow of malate from the vacuole,which is required for ABA-triggered stomatal closure (Eisenachet al.2017).Finally,the loss of anions and K+results in a decrease in guard cell turgor and stomatal closure (Munemasaet al.2015).

CO2 concentrationLow concentrations of CO2rapidly open stomata,while high concentrations decrease stomatal aperture andgsto ensure efficient CO2influx(Klein and Ramon 2019;Tanget al.2020).Stomatal opening caused by low CO2concentrations is probably regulated byPATROL1,which promotes H+-ATPases localization in the plasma membrane of guard cells(Hashimoto-Sugimotoet al.2013).Facing high concentrations of CO2,BETA CARBONIC ANHYDRASE 1(βCA1) and βCA4 directly mediate the early transduction of CO2signals inArabidopsisguard cells,thereby increasing bicarbonate ions (HCO3-) (Fig.3) (Huet al.2011).In such conditions,HIGH LEAF TEMPERATURE 1(HT1) is a crucial negative regulator for the signaling pathway of stomatal closure (Hashimotoet al.2006).The activation of HT1 is suppressed by MPK4 and MPK12in vitro(Fig.3) (H?raket al.2016).The regulation of HT1 by MPK4/12 may also function in low CO2-induced stomatal opening (Chateret al.2015).The MATE-type transporter protein RESISTANT TO HIGH CO2(RHC1)also inhibits HT1 activity in a bicarbonate-dependent manner (Fig.3) (Tianet al.2015).

The sensitivity of guard cells to ABA might increase in CO2enrichment;therefore,ABA is an essential factor in CO2-induced stomatal closure as well (Chateret al.2015;Shiet al.2015).As mentioned above,ABA will form complexes with PYR/RCAR receptors and PP2Cs,releasing OST1 and GHR1 from PP2Cs’ inhibition and activating SLAC1 (Fig.3) (Fujiiet al.2009;Huaet al.2012).Analogously,HT1 inhibits the activation of SLAC1 by depressing GHR1 and OST1 inXenopus laevisoocytes (Fig.3) (H?raket al.2016).SLAC1 is an essential S-type anion channel in the stomatal response of plants to CO2(Negiet al.2008).Additionally,evidence regarding the involvement of R-type anion channel AtALMT12 in the stomatal closure response to elevated CO2is available (Meyeret al.2010).Elevated HCO3-enhances the sensitivity of SLAC1 to intracellular free Ca2+(Xueet al.2011).ABA and CO2-induced stomatal closure of ABA-insensitive mutantgca2are inhibited in leaf epidermis and intact leaves.Therefore,GCA2may act downstream of the convergence of the two signaling transduction networks (Israelssonet al.2007;Tanget al.2020).In addition,CO2-mediated stomatal opening and closure incpk3/5/6/11/23quintuple mutants are defective(Schulzeet al.2020).

Fig.3 Mechanism of the rapid regulation of S-type anion channels by a high concentration of CO2 mediated by guard cells.βCA1,β carbonic anhydrase 1;βCA4,β carbonic anhydrase 4;MPK4/12,MAP kinases 4/12;RHC1,resistant to high CO2;HT1,high leaf temperature 1;[Ca2+]cyt,Ca2+ in cytosolic;CPKs,calcium-dependent protein kinase;GCA2,growth controlled by abscisic acid 2;JA,jasmonic acid;PYR/RCAR,pyrabactin resistance/regulatory component of abscisic acid (ABA) receptor;PP2Cs,type 2Cs protein phosphatase;ABI1,ABA-insensitive 1;ABI2,ABA-insensitive 2;OST1,open stomata 1;ROS,reactive oxygen species;GHR1,guard cell hydrogen peroxide-resistant 1.

With the participation of ABA receptors,the increase in HCO3-can raise ROS levels,which is also required for CO2-triggered stomatal closure (Chateret al.2015).Elevated CO2promotes the synthesis of jasmonic acid (JA)through an unknown mechanism with the action of CAs(Genget al.2016).JA is reported to positively regulate stomatal closure by increasing ROS levels and changing[Ca2+]cytin guard cells (Genget al.2016).These ROS(H2O2,NO) are crucial signaling molecules between OST1 and SLAC1 in CO2-induced stomatal movements (Shiet al.2015).H2O2and NO production and transient [Ca2+]cytare considered convergence points of the ABA and CO2signals in response to CO2-mediated stomatal closure(Tianet al.2015).Simultaneously,the loss of anion across the guard cell plasma membrane initiates voltagegated K+transport channels,resulting in stomatal closure(Murataet al.2015).Overall,elevated CO2-induced stomatal closure also has two pathways: ABA-dependent and ABA-independent pathways in guard cells (Fig.3).

In addition to guard cells,mesophyll cells also mediate the response of stomata to CO2(Mottet al.2008).In response to elevated CO2,mesophyll cells release malate to activate guard cell R-type anion channels through the extracellular malate pathway,leading to anion (malate)loss and subsequent stomatal closure (Hedrichet al.2001).Simultaneously,the release of malate by guard cells into the cell wall further stimulates the R-type anion channels,providing positive feedback for stomatal closure (Leeet al.2008).However,a high intracellular malate concentration inhibits the activity of S-type anion channels,while low-millimolar cytosolic levels activate the Cl-current of the anion channels (Wang and Blatt 2011).The efflux of malate and the starch synthesis during stomatal closure may lead to low-millimolar cytosolic malate concentrations in guard cells (Wang Cet al.2018).The intracellular malate affects the activity of S-type anion channels indirectly by regulating the upstream SLAC1(Wang Cet al.2018).Moreover,ABC TRANSPORTER B FAMILY MEMBER 14 (ATABCB14) is a malate uptake transporter in guard cell plasma membrane,and mutantatabcb14is insensitive to the stomatal closure response to elevated CO2(Leeet al.2008).Thus,malate may be a messenger that integrates mesophyll cell photosynthesis and stomatal movement (Dalosoet al.2016).

TemperatureSome researchers previously reported thatgsincreased with temperature (von Caemmerer and Evans 2015;Urbanet al.2017),while some suggestedgshas no response to temperature (Cerasoliet al.2014;von Caemmerer and Evans 2015).By contrast,some studies also provided evidence that an increase in temperature promotes stomatal closure (Lahret al.2015).Therefore,the stomata can be opened,closed or remain unaffected as the temperature rises depending on species and growth conditions.However,the stomatal limitations are great at high temperatures regardless of the stoma response (Sage and Kubien 2007).

2.2.Long-term regulation of stomatal conductance by environmental factors

Stomatal density,which is determined by stomatal development and patterning,is regarded as one of the crucial adaptive strategies of plants through unpleasant environmental conditions (Dalosoet al.2016;Xiong 2016).Stomatal development is regulated by three basic helix-loop-helix (bHLH) transcription factors,including SPEECHLESS (SPCH),MUTE,FAMA and their heterodimerization partners INDUCER OF CBF1 (ICE1)/SCREAM1 (SCRM1) and/or SCRM2,which form a central transcriptional cascade that regulates it sequentially (Lee and Bergmann 2019).Additionally,two partially redundant paralogous R2R3 MYB transcription factors,namely FOUR LIPS (FLP) and MYB88,are positive regulators for stomatal development (Leeet al.2014).The upstream of bHLH transcription factors is the mitogen-activated protein(MAP) kinases cascade,which negatively regulates stomatal development.This cascade includes MAP kinase kinase kinase YDA,MAP kinase kinase MKK4/5 and MAP kinases MPK4/5/7/9 (MAPK) (Balcerowiczet al.2014).

Moreover,the stomatal distribution in leaves follows the one-cell spacing rule for optimal gaseous fluxes and efficient functioning (Lawson and Matthews 2020).Many stomata regulatory factors are involved in the process of correcting stomatal development and patterning.The negative regulators include subtilisin protease STOMATAL DENSITY AND DISTRIBUTION 1 (SDD1) (von Grollet al.2002) and small-molecule peptides EPIDERMAL PATTERNING FACTOR 1 (EPF1),EPF2 and CHALLAH(CHAL)/EPFL6.The EPFs are derived from the EPIDERMAL PATTERNING FACTOR family (EPFf).EPFf rely on leucine-rich repeat (LRR) receptor-like kinases of the ERECTA family (ERf) (comprised of ER,ERECTALIKE1 (ERL1) and ERL2) and the LRR receptor-like protein TOO MANY MOUTHS (TMM) (Weiet al.2020) for their proper operation.Importantly,the primary positive regulatory factor named EPF9/STOMAGEN competitively combines ER with EPF2 to positively regulate stomatal development (Leeet al.2015).

Differences in plant canopy height in the natural community or agricultural intercropping systems (e.g.,a maize and soybean strip intercropping system) create long-term shading conditions for the lower leaves or plants (Valladares and Niinemets 2008;Du J Bet al.2018).Thegsin shaded leaves was lower than that of sun-exposed leaves (Fig.4),mainly due to the decrease in stomatal density (Wanget al.2020;Weiet al.2020;Shafiqet al.2021).Only a few mature stomata were observed on the cotyledons of seedlings under darkgrown conditions (Balcerowiczet al.2014).Plant mature leaves can sense the light intensity signal and transmit it to new ones,thereby regulating stomatal development(Vráblováet al.2018).The light induces the expression of EPF9/STOMAGEN,SPCH,MUTE,FAMA,EPF2 and TMM to promote the stomatal formation (Hronkováet al.2015).

Fig.4 Comparison of stomatal densities of two cultivated soybean cultivars (ND12,Nandou 12;GX7,Guixia 7) under normal light(left) and low light (right).

Light-regulated stomatal development is possibly mediated through crosstalk between the cryptochrome(CRY)-phy to chrome (phy)-CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1,E3 ubiquitin ligase)signaling system and the MAP kinase cascade signaling pathway (Kanget al.2009).COP1 is a key repressor of light signaling (Kanget al.2009).The activity of COP1 in darkness is enhanced,inhibiting stomatal differentiation and development (Lau and Deng 2012).Upstream of COP1,CRY,phyAandphyBpromote stomatal development by negatively regulating COP1 in the light (Balcerowiczet al.2014),while the downstream of COP1 is repressor YDA (Kanget al.2009).COP1-and TMM/ER/ERL-mediated signaling converge to YDA and inhibit stomata formation by promoting SPCH and ICE1 degradation (Zhaoet al.2017).Concurrently,COP1 also directly degrades ICE proteins by mediating their ubiquitination pathways (Leeet al.2017).Additionally,light promotes the transcription and protein abundance of ANGUSTIFOLIA3 (AN3),through the photoreceptormediated pathways,which then downregulates the expression ofCOP1(Menget al.2018) andYDA(Menget al.2015).Furthermore,PHYTOCHROME INTERACTING FACTOR 4 (PIF4) acts in aphyBdependent manner to regulate stomatal development in response to strong light (Boccalandroet al.2009;Cassonet al.2009).PIF4 also suppresses stomatal formation by directly repressing the expression of light-regulated genes,such asGATANITRATE-INDUCIBLECARBON METABOLISM-INVOLVED(GNC) andGNC-LIKE(GNL)in the upstream of theSPCH,MUTEandSCRM/2(Klermundet al.2016).

Plant stomatal density has various adaptation strategies due to drought stress of different degrees and duration times.The stomatal densities ofSchizonepeta tenuifolia(Li Ket al.2019),olive (Bosabalidis and Kofidis 2002) and the abaxial surface of eggplant leaves were increased in response to long-term water stress (Fuet al.2013).Alternatively,Arabidopsis(Hepworthet al.2015),rice (Ouyanget al.2010),tomato (Du Q Jet al.2018) and wheat (Liet al.2017) preferred to reduce stomatal density to improve drought tolerance.Similarly,moderate water deficits have shown a positive impact on the stomatal density of grass and jujube,although a severe drought led to a reduction in the stomatal density of their leaves (Liuet al.2006;Xu and Zhou 2008).

The increased stomatal density of plants in moderate water stress may be caused by epidermal cell expansion and leaf area reduction (Bosabalidis and Kofidis 2002).A high stomatal density helps in effectively controlling the gas exchange during mild drought (Fuet al.2013).However,a severe drought would inhibit the guard cell division associated with senescence,thereby reducing stomatal density (Xu and Zhou 2008).Moreover,miRNAPu-miR172dofPopulus ussuriensisis up-regulated by water stress,which down-regulatesGT-2LIKE1(GTL1) (Liuet al.2020).GTL1negatively regulatesSDD1,thus up-regulating the expression ofSDD1in response to drought (Yooet al.2010).Analogously,MADS-box transcriptional factor AGAMOUS-LIKE16 (AGL16) inArabidopsisguard cells is down-regulated by drought stress and plays a negative role inSDD1transcription (Xiang C Bet al.2020).Then the expression product ofSDD1decreases stomatal density by activating the MAP kinase cascade(Yooet al.2010).Meanwhile,the MAP kinase cascade is also activated by ABA (Tanakaet al.2013;Liet al.2016) and EPFs,which are accumulated during water deficits (Kumariet al.2014;Hugheset al.2017;Liuet al.2019).In addition,drought stress induces the expression of rice receptor-like kinase OsSIK1 (Ouyanget al.2010),ArabidopsisMedicagoTruncatulacoldacclimation specific protein 31 (MtCAS31) (Xieet al.2012) and maize NAC transcription factorZmNAC49(Xiang Yet al.2020) to reduce stomatal density.

In high atmospheric CO2concentrations,the plantgsis generally decreased by the reduction in stomatal density (Kirschbaum and McMillan 2018;Habermannet al.2019).CO2enrichment upregulates the expression ofHIGHCARBONDIOXIDE(HIC) (Casson and Hetherington 2010).HICis expressed in guard cells and encodes a 3-ketoacyl coenzyme A (CoA) synthase(KCS) inArabidopsis,regulating stomatal density and index (Casson and Hetherington 2010).KCS is involved in the production of the very-long-chain fatty acid components of waxes,glycerolipids,sphingolipids and cutin.Therefore,changes in the structure and properties of the waxy cuticle may interfere with the diffusion of the endogenous inhibitors controlling stomatal development(Grayet al.2000).Major lipoxygenase (LOX)-controlled jasmonate cascades,which control the synthesis of jasmonates,are also related to the long-term responses of stomata to CO2levels (Lakeet al.2002).The enzyme CO2RESPONSE SECRETED PROTEASE (CRSP),has also been identified as a negative mediator for stomatal development (Engineeret al.2014).CRSP is regulated by elevated CO2and initiates the negative regulatory activity of EPF2 in the presence of CA1 and CA4 (Engineeret al.2014).In addition,ABA and ROS regulate the stomatal density in CO2enrichment (Chateret al.2015).However,research on the regulatory network of drought and elevated CO2with respect to stomatal development is lacking.

3.Environmental factors in the regulation of mesophyll conductance in plants

Mesophyll resistance significantly reduces the accumulation of CO2from the intercellular space to the chloroplast.Thegmsubstantially limits the CO2fixation rate and increases the water costs of carbon acquisition(Evans 2020).Plants can rapidly respond to the changing circumstances by adjustinggm,which is achieved by modulating cell wall properties (reflected by the nature of chemical interactions inside cell wall pores between CO2and cell wall components),membrane permeability (mainly reflected by the content and/or activity of CA and AQPs)and chloroplast morphology and distribution (reflected bySc) (Flexas and Diaz-Espejo 2014).Furthermore,plants can also respond to long-term abiotic stress conditions by regulatinggm,among which the effects of light intensity,CO2concentration,drought and temperature ongmhave been widely studied (Flexaset al.2008, 2012).In longterm environmental regulation,gmis mainly controlled by cell wall thickness,Sm,Sc,and the contents and activities of CA and PIPs (Hanet al.2017).

3.1.Light intensity

The rapid responses of leafgmto rising light intensity among different species show diverse trends.Some studies proved that plants increasegmto varying degrees over a range of rising irradiance (Doutheet al.2011;Caiet al.2017;Xionget al.2017).By contrast,other studies observed the absence of agmresponse to changing light intensity (Tazoeet al.2009;Yamoriet al.2009;Yanget al.2020).Interestingly,gmof rice growing with a high nitrogen supply increased with the light intensity,while that growing with a low nitrogen supply had no response to light intensity (Xionget al.2015).

Fluctuating light intensity may regulategmby changing chloroplast distribution (Sc) inArabidopsisleaves (Boex-Fontvieilleet al.2014).Light-mediated up-regulation ofPIP2expression also regulatesgminJuglansregia(Cochardet al.2007).Appropriately increasing the light intensity may promote the pumping of protons from the cytoplasm and initiate the activity of AQPs inQuercus macrocarpa.However,the transcription factors of AQPs and their activity are down-regulated in strong light,reducing thegmof bur oak (Voicuet al.2009).Furthermore,compared with sun leaves,gmof shade leaves rises with increasing light intensity more rapidly inEucalyptustereticornis,thus facilitating the rapid response of shade leaves to sunflecks (Campanyet al.2016).However,the modeling of layer-based photosynthesis within the leaf revealed that thegmof leaves with saturated photosynthesis under low light is less responsive to increasing light intensity,while those with unsaturated photosynthesis may be responsive to changing light(Theroux-Rancourt and Gilbert 2017).As mentioned above,thegmis changed byScand/or the content and activity of AQPs in fluctuating light intensity,which is dependent on the anatomical characters of species or leaves (Doutheet al.2011;Theroux-Rancourt and Gilbert 2017).

In the natural community,thegmof shade leaves is remarkably lower than that of sun leaves (Campanyet al.2016).The decrease ingmin shade leaves may be due to the reduction ofScand the considerable changes in structure and organization (Warrenet al.2007).However,the relationships betweengmand leaf anatomical characteristics are diverse in leaves from different species,ages and growth conditions (Piel 2003).

3.2.Drought

Plant photosynthesis is also limited bygmin long-term drought stress (Theroux-Rancourtet al.2014;Wang X Xet al.2018).Compared with stomatal limitation,nonstomatal limitation of photosynthesis (gmor Rubisco carboxylation capability) is dominant in chronic water stress (Olsovskaet al.2016;Zaitet al.2018).In drought,cell wall thickness is changed depend on plant species,timing and degree of stress (Saito and Terashima 2010;Liet al.2013;Ouyanget al.2017;Hanet al.2018).Chloroplast atrophy in drought stress results inScdecrease,and thusgmdecline (Ouyanget al.2017;Hanet al.2018).However,grapevine cultivars do not show any correlation betweengmvariability and anatomical structure in drought (Tomàset al.2014).Moreover,McMIPB,an AQPs gene in tobacco,functions in CO2transport and regulatesgmin drought stress (Kawaseet al.2013).Interestingly,gmis reduced by ABA in a dose-dependent manner in drought,which is considered to be crucial (Mizokamiet al.2015,2018).Partly,the decreases in CA content and activity also cause the decline ingmunder drought stress (Flexaset al.2012,2018).Therefore,the effects and magnitude of drought stress on plantgmvaries within plant species.

3.3.CO2 concentration

Wheatgmremarkably increases within 24 h under the induction of a low CO2concentration,which is regulated by increased CA activity (Bj?rkbackaet al.1999).However,the instantaneous increase of CO2reduces thegmof plants to varying degrees,and the extent of the decrease is related to plant species and increases in CO2(Flexaset al.2007,2014).To avoid a considerable drop in cell pH or an increase in cell leakage in high CO2partial pressure,the plant has to decrease its leafgmand keep the balance ofCiandCc(Flexaset al.2012).However,the adjustments ofgmmentioned above may be restricted by mesophyll or chloroplast structure,only allowinggmto vary within a relatively narrow range (Flexaset al.2012),such as the changes inSccaused by chloroplast behaviours (deformation,movement) (Tholenet al.2008;Tanget al.2017).Elevated CO2also regulates the expression ofPIP2and/or the activation state of PIPs to changegm(Alguacilet al.2009;Mizokamiet al.2018).

Nevertheless,no uniform variation trend ofgmamong plants growing under long-term high CO2concentration is observed (Flexaset al.2008).After growth in elevated CO2for several years,the sweetgumgmsignificantly increases,while thegmof spinach,cucumber and linden bean markedly decrease,and aspengmremains unchanged (Singsaaset al.2003).Similar to aspen,thegmof birch grown for several months andArabidopsisfor several weeks in elevated CO2did not change significantly (Eichelmannet al.2004;Mizokamiet al.2018).Therefore,CO2enrichment during growth may have different effects on various limiting factors ofgm,and such impacts are diverse based on species,CO2concentration and duration time (Shiet al.2010).

3.4.Temperature

With a short but lasting rise in temperature,gmcan be gradually increased,unchanged,or firstly increased and then decreased (von Caemmerer and Evans 2015;Li Yet al.2019;Shresthaet al.2019;Evans 2020).Bernacchi provided evidence that the response ofgmto temperature may be closely related to enzymatic reactions (Bernacchiet al.2002).CA and AQPs are proteins,and temperatures may affect their activities,which might cause an immediate effect ongm(Bernacchiet al.2002;Flexaset al.2008).However,Warren and Dreyer (2006) suggested that the rapid response ofgmto temperature may not be a simple protein-promoting process but a complex response of multiple processes with different temperature sensitivities.Evans and von Caemmerer developed a model that dividesrminto liquid phases (mainly determined by the cell wall,cytoplasm and chloroplast stroma) and membrane phases (mainly determined by the plasma membrane and chloroplast envelopes) to explain the differences ingmresponses to temperature in various species (Evans and von Caemmerer 2012;von Caemmerer and Evans 2015).In this model,the conductance of the liquid phase (gliq) is assumed to be negatively correlated with temperature,while that of membrane phases (gmem) has a positive exponential relationship with temperature (Evans and von Caemmerer 2012;von Caemmerer and Evans 2015).This model also reveals that,among different species,the effective activation path length of liquid phase diffusion and the energy of membrane permeability contribute differently togm;thus,gmresponds differently to the temperature (von Caemmerer and Evans 2015).However,a recent study suggested that leaf water potential should also be considered in this model (Li Yet al.2019).

4.Conclusion and outlook

Plant photosynthesis is affected by leafgsandgm,which are sensitive to abiotic environmental signals.Short-term environmental changes rapidly regulate CO2diffusion by modulating the stomatal aperture,cell wall properties,membrane permeability and chloroplast behavior in leaves.Simultaneously,guard cells rapidly convert environmental input signals into the activation of plasma membrane ion channels that quickly alter stomatal aperture andgs.These processes involve the responses of hormones (ABA,JA,etc.) and secondary messengers(Ca2+,ROS,NO,HCO3-,etc.).Long-term abiotic stress factors affect stomatal density andgsby modifying bHLH transcription factors by adjusting the activity and/or content of the MAPK cascade system,EPFs,ERf and SDD1.In addition,gmin long-term abiotic stress factors is dependent on species or different genotypes within a species and controlled by mesophyll anatomy,CA and AQPs.Therefore,leaf CO2diffusion adapts to the environmental changes through the short-and long-term physiological regulation mechanisms.

Although significant progress has been made in our knowledge of the regulation of CO2diffusion by abiotic stress,studies on the molecular mechanisms and networks ofgsandgmresponses to the changing environment are insufficient.In particular,the sensors of different environmental signals in plants,the biochemical protein basis on which signal transmission depends,and the transcription factors that regulate stomatal development all need further research and improvement.The overlapping or interactive effects of different abiotic signals on CO2diffusion efficiency and how signals from conflicting stresses are integrated to optimize plant gas exchange are also worth studying.Moreover,the mutually independent or co-regulatory relationships betweengsandgmare still unclear.A systematic study of the regulatory mechanism of abiotic stress on CO2diffusion will help modulategsandgmto increase water use efficiency without a penalty in carbon fixation and contribute to improving the photosynthesis rate by breeding selection and genetic engineering.

Acknowledgements

This work was supported by National Natural Science Foundation of China (32071963),the Chengdu Science and Technology Project,China (2020-YF09-00033-SN),a grant from the International S &T Cooperation Projects of Sichuan Province,China (2020YFH0126),and the China Agriculture Research System of MOF and MARA (CARS-04-PS19).

Declaration of competing interest

The authors declare that they have no conflict of interest.