Stomatal Density and Bio-water Saving

Joumal of Integratve Plant Biology 2007, 49 (10); 1435- 1444.Invited Review.Stomatal Density and Bio-water SavingYao Wang, Xi Chen and Cheng-Bin Xiang'(School o1 Life Sciences, Universty of Science and Technology of China, Hefei 230027, China)AbstractBio-water saving is to increase water use eficiency of crops or crop yield per unit of water input Plant water use efficiencyisdetermlned by photosynthesis and transpiration, for both of which stomata are crucial. Stomata are pores on leaf epidermisfor both water and carbon dioxide fuxes that are controlled by two major factors: stomatal behavior and density. Stomatalbehavlor has been the focus of intensive research, while less attention has been paid to stomatal density. Recently, anumber of genes controlling stomatal development have been ldentified. This review summarizes the recent progress onthe genes regulating stomatat density, and discusses the role of stomatal density in plant water use fficiency and thepossibility to increase plant water use eficiency, hence blo-water saving by genetically manipulating stomatal density.Key words: Arabidopsis; photosynthesis; stomata; stomatal density;: stomatal formation; transpiration; water use eficiency.Wang Y, Chen x, Xiang CB (2007). Stomatal density and bio-water saving. J Integr. Plant Biol, 49(10). 1435 1444.Available online at www. blackell-synergy.ocominstoc/jipb, www.jipb.netThere are two major ways to increase the water use eficiengy increase water use eficiency for the obvious reason of less inputin agriculture. One is the engineering-based cropping systemthan engineering-based methods.where modem irigation techniques play an important role. MuchBy definition, plant WUE is determined by both photosyn-progress has been made to increase water use eficiency by thesis and transpiration. Increasing photosynthesis and/or demanaging imigation (Kacira and Ling 2001; Jones 2004; Kangcreasing transpiration would elevate plant WUE. Stomata playand Zhang 2004; Bazzani 2005; Facchi et al. 2005; Sun et al.a crucial role in both processes and hold the keys to increase2005; Giordano et al. 2007). The other way is "biological waterplant WUE, and hence bio-water saving. The physiological andsaving" (bio-water saving) which was coined by Lun Shan inmolecular basis of bio-water saving is complicated and is highly1991 to emphasize the physiological and ecological bases of linked to drought tolerance mechanisms (Chaves and Oliveirawater saving by crops in agricuture (Shan 1991). The concept2004).of biowater saving was further developed by Yuanchun ShiStoma is one of the key factors affecting plant WUE. Stomaand defined as "more agricutural products output with the same as a specifc organ first appeared in trestrial land plants aboutor less water input by exploiting the physiological and genetic400 millin years ago to adapt new variable environmentspotential of organisms themselves" (Shi 1999). The core of(Edwards et al. 1998). Different plant species have evolvedbio water saving is to increase "water use eficiency' (WUE)diferent stomata with great variation of size, density (numberof plants, which is defined as a ratio of photosynthesis rateof stomata per unit area), and morphology. There are two typesto transpiration rate (Smith 1980) and used as an indicator forof stomata: dumb bllshaped and kidney-shaped stoma. Theplantwater saving capability. In contrast to the irigation method,former is often seen in monocotyledons, while the latter isbiological water saving is a more eficient and economic way tousually in dicots. It is believed that dumb-bell-shaped stomataare more evolutionarily advanced than the kidney-shaped onesRecered 29 May 2007 Acepted7 Jun. 2007because they can alter their width more easily when guardSupported by the Chinese Academy of Sciences (KSCX2- YW-N-012) andcells undergo a small change (Raschke 1979). This fast speedMOST (2003CB1 14305).opening and closing strategy can save energy and increase*Author for crorespondence.photosynthesis and water use eficiency (Granz and AssmannTel: +86 ()551 360 732;1991)中国煤化工also changed with aFax: +86 ()551 360 1443;millenstomatal densities be-E-mail: or .causefYHC N M H Gnd low cartbon dioxide◎2007 Institute of Botany, the Chinese Academy of Sciencesconcentration. Gymnosperm and angiosperm adapted to dryerdol: 111172 9072 205.5climnates have lower stomatal densities (Hil 1994).1436 Joumal of Integrative Plant Biology Vol. 49 No. 10 2007Stomata play pivotal roles in global water and carbon cyclesof plants in response to environmental changes (Httherington(Hetherington and Woodward 2003). Stomatal movement, den-and Woodward 2003). Due to dfferent species and livingsity, and distribution determine plant water and COz exchange,environment, the stomatal size and density vary to a great extenthence photosynthesis and transpiration. Both processes are(Willmer 1996). Plants with high stomatal density tend to haveessential for higher plants. Transpiration is required for nutrientsmall-sized stomata (Beering and Woodward 1997). However,absorption as well as maintaining the body temperature of plantsthe selection mechanisms of diferent densty-size pattemingwhile photosynthesis assimilates carbon dioxide into photosyn-are not wel understood. The stomatal size is highly correlated tothates. Through stomata on leaf epidemis, water and carbonthe drought senstivity of plants. Large stomata lead to a slowerdioxide exchange between air and plants. Therefore, watermovement, thus a slower response to dehydration (Hetheringtonloss is an unavoidable occurrence. When plants are lackingand Woodward 2003).water, stomata will close to minimize the water loss. ClosingCarbon dioxide concentration is another factor infuencingstomata reduces transpiration and saves water but restrictsstomatal density. Increasing CO2 concentration can reduceCO2 exchange, adversely afcting photosynthesis. This confictstomatal index and density (Woodward 1987; Woodward andbetween photosynthesis and transpiration on stomata appearsKelly 1995). The stomatal conductance is also reduced butto be unsolvable. But through evolution plants have developedthe photosynthesis rate rises when CO2 is enriched (Beeringsophisticated strategies and mechanisms to compromise be-and Woodward 1997; Woodward 1998; Woodward et al.2002).tween photosynthesis and transpiration, Among these, stomatalBut exceptions do exist. For instance, Arabidopsis ecotypebehavior, density, and size play central roles in contoling waterC24 shows no response of stomatal density to high CO2and CO2 fluxes through stomata in the compromise.concentration (Woodward and Kelly 1995; Gray et al. 2000).Stomatal behavior or movement has been the focus of inten-The signal for stomatal patterning might be transmited from thesive research aiming at how stomatal movement is controlledold leaves to the new ones (Lake et al. 2001; Coupe et al.2006;by various environmental factors and how to genetically maDriscoll et al. 2006; Miyazawa et al. 2006). There is a strongnipulate plants to increase WUE by enhancing its sensitvity.correlation between CO2 concentration and stomatal density soMuch progress has been made in understanding the regulatorythat the CO2 content in ancient atmosphere from stomata fossilnetwork of stomatal movement (Hetherington and Woodwardcan be estimated (McElwain and Chaloner 1995; Hetherington2003; Lake and Gray 2007; Nilson and Assmann 2007). Inand Woodward 2003). CO2 signaling pathway is a typical long-contrast, less attention has been paid to the effects of stomatalrange one. It was found that exposing mature leaves to highCO2density and size on photosynthesis and transpiration. At lastforconcentration could reduce the stomatal density of developingone reason, the lack of natually occurring mutants of varyingleaves (Brownlee 2001; Lake et al. 2002; Woodward 2002). Thestomatal densities and sizes in the same genetic backgroundsignaling that links environments and stomata is not so clear.hampered the analysis of the roles of stomatal density inOne gene named HIC in Arabidopsis is known as a key nodeplant water use effciency. Recently, a number of genes werein the pathway of CO2 elevation leading to decreased stomataiidentifed affecting stomatal density and might provide new waysdensity (Gray et al.2000). Light intensity can also afect stomatalto answer these questions.fomation (Gay and Hurd 1975; Lake et al. 2002; Thomas et al.Stomatal density and movement have huge effects on photo-2004). High light intensity leads to a higher stomatal densitysynthesis and transpiration. However, stomatal density or dis-(Ticha 1982).tribution could not be accurately correlated with photosynthesisStomatal movement is a response to environmental changes,rate or transpirationrate. Thus, the tem "stomatalindex* definedand it is contolld by environmental factors. Stomatal openingas a proportion of stomatal cells to total epidermal cells andand cdosing are mainly controlled by guard cllturgor (Schroeder"stomatal conductance" as the water and CO2 exchange abilityet al.2001). Guard cell turgor change is caused by massive ionof leaves are often used. Elevation of stomatal index or stomatalfuxes and can be infuenced by many factors such as light,conductance will significantly increase the photosynthesis rate,phytohormone, potassium ion, calcium ion, malate, sucrose,but meanwhil, the transpiration rate will rise too. The besttc. (Assmann and Wang 2001; Assmann 2003). Guard cellcompromise between photosynthesis and transpiration wouldsignaling is ikely a scale-free network rather than a stand-alonemaximize CO2 uptake and minimize water loss, and ultimatelypathway (Hetherington and Woodward 2003). Hence the guardachieve the possible maximal water use eficincy.cell signaling is very robust. Missing a few components in thescal-free network will not interfere with the stomata movement(Hetherington and Woodward 2003). These mechanisms en-Environment Factors Affecting Stomatalsure中国煤化工ocess can tlerate andDensity and MovementadaptLiglHC N M H Gnovement (Eckert andEnvironment factors can affect plant stomatal density and disti-Kaldenhoff 2000), especially blue light (io et al, 1985;bution during the development of plants, rffecting the plasticity Matsuoka and Tokutomi 2005). Arabidopsis perceives blueStomatal Density and Bio water Saving 1437light signals via photreceptors (Gorton et al. 1993). There areto inibitions from some cues. Neighbor cll also has divisionfive major photoreceptors of blue light in Arabidopsis: CRY1,capacity and it may divide into meristemoid, which is calledCRY2, CRY3, PHOT1 and PHOT2 (Kinoshita et al. 2001; Maosatelite meristemoid (SM). Several asymmetric divisions seemet al. 2005). Most of them have been proved to take partrandom. In fact, the patterns are highly controlled (Geislerin the stomatal opening. Recenty, a protein kinase HT1 thatet al. 1998). In every division, the cell plate adopts specialtransduces a stomatal opening signal from low CO2 and blueorientation according to the cell plate in the last division. Serialslight has been identifed (Hashimoto et al.2006).of asymmetric divisions appear to form an inward spiral (TelferDrought is the most frequent abiotic stress encounteredand Poethig 1994). When a cell enters into the symmetricby plants. Water deficit is first sensed by plant roots anddivision stage, guard mother cells have no chance to contactphytohomone ABA synthesis is activated in the roots. Thewith other GMCs. A satlite meristemoid can also diferentiateroot-derived ABA is transported to leaves (Sauter et al. 2001;into a new stomatal complex that prevents contact with the pre-Wilkinson and Davies 2002; Jia and Davies 2007) to induceexisting stoma. A meristemoid follows the stomatal formationstomatal closing and prevent stomatal opening through dfferentpathway, whether dividing into a new M or convering to a GMCpathways (Mishra et al. 2006).is largely dependent on the near stoma. During these events,there must be cell to cell communication to regulate nomalpattem formations and to correct occasional mistakes in timeGenetic Control of Stomatal Density(Von Groll et al. 2002).Although stomatal density is affected by environmental factors,Genes afecting stomatal densityits genetic control is evident (Nadeau and Sack 2002a, 2003;Hetherington and Woodward 2003). For most of the twentiethERECTA was first identified more than a decade ago as acentury, the regulation of stomatal movement was the focusregulator in many aspects of plant development such as inflo-of intensive research (Hetherington and Brownlee 2004; Nilsonrescence development and architecture (Hanzawa et al.2000;and Assmann 2007; Shimazaki et al. 2007). However, someDouglas et al. 2002), organ growth, and flower developmentmutants with abnormal surface morphology were isolated and(Shpak et al. 2005). A linkage between ERECTA and stomatacharacterized (Schiefelbein and Somerille 1990; Hulskampowes to a quantitative trait loci (QTL) study about transpirationet al. 1994; Masucci and Schiefelbein 1994). In some of theseeficiency. Carbon isotope discrimination was exploited as amutants, the unusual stomatal density and distribution were bselection marker to screen mutants of transpiration effciencyserved. Although such mutants with abnormal stomatal patterms(Masle et al. 2005). The mutant erecta, a lsosof-functionwere isolated, only a limited number of genes were identifed.mutant of ERECTA was isolated. The mutant displayed aBased on their function, these genes can be categorized intodwarf body statue and compact rosette (Masle et al. 2005).three groups: stomatal density, stomatal patteming, and cell di-The transpiration efciency was lower in erecta than that invisin. Some genes may have more than one single fnctionIn the wild type. Transfoming CaMV35S ERECTA into erectaaddition, polyploidy, a common phenomenon in plant evolutioncould complement and restore the wild type phenotype. Thus,is associated with changes in stomatal density and size (ChenERECTA is a good candidate gene for improving transpiration2007).effciency in Arabidopsis (Masle et al. 2005). The leaf anatomywas also dfferent in that erecta had fewer mesophyl tssuesand a higher stomatal density. But the stomatal index of erectaStomatal formation pathway In Arabidopsisis the same as that of the wild type, implcating that ERECTAIn Arabidopsis, a stoma is composed of a pore fanked by twowould probably function through increasing epidemal cell size,guard clls. In the wild bype, there is at least one cell between but not regulating stomatal development (Masle et al.2005).different stomata, which means that stomata are not adjacentAt the same time, another research group also found an(Sachs 1991). The stomatal formation pathway ensures this unusual stomatal patterming in the erecta mutant. They extendone-ell-rule. When a stem coll decides to diferentiate the guardtheir sights to the other two ERECTA family genes: ERL1 andcell fate, it first becomes a meristemoid mother cell (MMC)ERL2 (Shpak et al. 2005). These three genes synegisically(Nadeau and Sack 2002a). Then through one asymmetric control the stomatal patteming. Double or triple mutants showeddivision, MMC splits into a small cell meristemoid (M) and aa more severe disruption of stomatal patterns than erectalarge cell neighbor cell (NC). An M is a 8elf-renewable cell. It (Shpak et al. 2005). ERECTA and its two paralogous genescould divide into a new M and another NC asymetrcally. Afterencod中国煤化工kinases (LRR-RLKs)no more than three asymmetric divisions (Sema and Fenoll(ToriYHC N M H G.only prtaly epistatic:d that TMM apared2002), M converts to a guard mother cell (GMC), which can to be edivide equally into two guard cells (GCs) (Larkin et al. 1997).to ERL1. Interestingly, ERECTA, ERL1 and ERL2 together areA guard cell retains its dividing ability but cannot divide due epistatic to TMM (Shpak et al. 2005). Thus, the relationship1438 Jourmal of ltegrative Plant Biology Vol. 49 No. 102007of these genes is not simply upstream or downstream, Oneplane in the wrong orientation and thus fails to avoid contacthypothesis assumed that ERL1 could interact with T00 MANYbetween different stomata. The stomata in the same custermOUTH (TMM) when SDD1 ligand is present (Ingram 2005).are likely to be derived from one precursor cell. This view isThe binding of ERL1, SDD1 ligand and TMM would disruptsupported by analyzing promoter actity for cell division com-ERECTA, ERL1 and ERL2 complex and thus spoil the MAPKKKpetence and mitotic actity (Sema and Fenoll 1997). Besidescascade.the morphology changes, the stomatal density on cotyledons inSTOMATAL DENSITY AND DISTRIBUTION1 (SDD1) wastmm is higher than that in the wild type (Yang and Sack 1995).isolated from the lsso-unction mutant sdd1 with a higherSo TMM functions in the control of not only plane orientationstomatal density (Berger and Atmann 2000). Compared to tmm,but also stomatal density. TMM defect results in an increasedsdd1 had fewer stomata clusters, suggesting that SDD1 doesprecursor cell formation and actity (Yang and Sack 1995).not interupt the patterm fomation but the density and distribu-Combined with incorrect division orientation, the tmm mutanttion of stomata (Berger and Altmann 2000). Overexpression ofshows a high-denslty-cluster pattem. In general, TMM limitsSDD1 decreases the stomatal density and forms some arestedthe precursor cell formation and keeps a low meristemoid cellstomata (Von Groll et al.2002). The stomatal density elevationinitiation. When this meristemoid cell adopts its stomatal cellis due to increased satelite stomata fomation in sdd1 mutantdestination, TMM ensures the correct cell division orientation(Berger and Atmann 2000). There are no obvious phenotypesby retrieving the formation of the adjacent cell (Geisler et al.other than the above mentioned stomatal density such as2000). TMM is expressed primarily in stomatal precursor cellsplant growth, leaf anatomy structure, cell number, and cell sizeand some neighbor cells. During the stomatal lineage, the(Berger and Altmann 2000). But altered light conditions maydifrentiation stage is earier, TMM expression level is highincrease the carbon dioxide assimilation rate in sdd1 (Schluterin meristemoid, medium in guard mother cell and immatureet al. 2003). SDD1 encodes a subisin-like serine proteaseguard cell, and low in mature guard cell (Nadeau and Sack(Berger and Altmann 2000) and is localized in the plasma2002a). These results suggest that TMM may promote themembrane (Von Groll et al. 2002). Therefore, it may act tocell divisions. TMM encodes a leucine-ich repeat receptor-likemodify a substrate such as a receptor (Berger and Altmannprotein (LRR-RLP) (Nadeau and Sack 2002a). A large LRR2000; Von Groll and Altmann 2001; Sema and Fenoll 2002; family exists in Arabidopsis and is thought to be importantVon Groll et al. 2002).in protein to protein interaction (Kobe and Kajava 2001). TheCarbon dioxide partial pressure is an important factorfirst identified LRR-RLP is Cf-9 in tomato, which is a disease-that regulates stomatal density (Beering and Chaloner 1992;resistant gene (Jones et al. 1994). But TMM functions mainly inHetherington and Woodward 2003). Mutant hic was isolateddevelopment. LRR-RLPS contain a leucine-rich repeat outsidefrom the C24 ecotype (Gray et al. 2000). It was first identfedcell surface, a short C-terminus in cell, and a transmembranein a promoter trapping screen project instead of a stomataldomain (Fritz-l aylin et al. 2005). TMM seems to be localized inmutant screening. Although hic gives the same morphologythe plasma membrane (Nadeau and Sack 2002b). Unusually,as the parental line C24 under normal conditions, when Covery diferent phenotypes of stomatal density can be observedconcentration elevates, hic shows a greatly increased stomatalin the tmm mutant. Excess stomata are found in leaves, whiledensity, while C24 shows a lttle change (Gray et al. 2000),no stomata are found in the inforescence stem (Yang and SackWith map-based cloning, HIC (HIGH CARBON DIOXIDE) gene1995). Interestingly, there is an apex-to-base gradient stomatalwas identified as a putative 3-keto acyl coenzyme A synthasedensity from excess to no stomata in flower stalks (Geisler et al.(KCS), which is involved in the synthesis of vey-long-chain1998)_ This region-specifc patterm suggests that TMM has bothfalty acids (Gray et al. 2000). Why HIC mutation results inpositive and negative efecs in the same plant.such a phenotype is intriguing. It is speculated that at leastFOUR LIPS (FLP) functions simiary to TMM in some as-one of the substrates of the HIC enzyme is a component of the pects. The loss-of- FLP mutants show an increased stomatalsignal pathway linking CO2 and stomatal density (Gray et al.density as tmm mutants. But unlike tmm, fp has fewer clusters.2000). HICis the frst gene that directly reflects how environmentThe typical phenotype of f册p is two linked stomata and unpairedsignals tigger stomata-related gene expression in plants.guard cell (Yang and Sack 1995). These two linking stomataphenotypes suggest FLP may function in the late difentiationstage, the symmetric division, through restrction to GMC divi-Genes responsible for stomatal pattemingsion capacity (Yang and Sack 1995). FLP encodes a two-repeatTMM is the first gene identified to regulate stomatal pat-(R2R3) MYB transcription factor (MYB124) (Lai et al. 2005).tems (Yang and Sack 1995). Lossof-function mutation showsR2R3中国煤化工domain (Ogata et al.stomatal dusters on leaf epidermis, espeilly on cotyledons.1992).s redundant functions.Refering t切o the stomatal formation steps mentioned above,MYB8MHCNMHGthewildtype.DoubleTMM appears to be required for correct orientation of the cellmutant of MYB88 and FLP shows a more severe intemuption ofplate in asymmetric divisions. The tmm mutant places the cell stomatal formation (Lai et al. 2005). Since tmm and fp shareStomatal Density and Bio water Saving 1439notype where all of the epidermal cells of cotyledons were tumedhierarchy in the development pathway was genetically analyzed. into stomata. But double mutants showed lethal phenotypes.The relationship between TMM and FLP seems complicated.To solve this problem, Wang and colleagues used an inducibleDouble mutant of tmm and fp shows a mixed phenotype of aRNA interference (RNA) technique instead of T-DNA insertionhigh frequency of stomatal clusters with several unpaired guardlines (Wang et al. 2007). Aithough the lssof-function mutantscells (Yang and Sack 1995). But there are some discrepanciesceased continued growth after the cotyledons were open, theyin the details of dfferent organs. Generally, tmm is epistatic to still observed the exciting morphology on the cotyledon sur-face. Overexpression of MKK4/MKK5 or MPK3/MPK6 showedThe YODA gene was identifed as a regulator of extra-no stomatal diferentiation. Therefore, the YODA-MKK4/MKK5-embryonic cell fate (Lukowitz et al.2004). Later, it was shown toMPK3/MPK6 has been established as a complete MAPKKKalso be a regulator in guard cell fate detemination (Bergmanncascade. It could be inferred that such a MAPKKK signalinget al. 2004). Loss-of-YODA mutants produce more stomata,pathway functions not only in stomatal development but also inwhile YODA overexpression lines produce no stomata in leaves.similar stem cell dffentiation processes (Wang et al. 2007).Compared with tmm and sdd1, which could only partly disturbFAMA was first identifed in a yoda microarray experimentstomatal pattems, YODA disrupts the stomalal development (Bergmann et al. 2004). A more detailed characterization ofpathway more completely (Bergmann et al. 2004). YODA e肌FAMA was conducted (Ohashilto and Bergmann 2006). FAMAcodes a MAPKKK. In Arabidopsis, there is a large MAPKKK is a bHLH transcription factor. Mutant fama-1 is a lossof-family with at least 25 members (Tena et al. 2001). It tiggersfunction mutant but can stil transcribe FAMA, however thethe downstream phosphorylation cascades and thus affectsamount of FAMA transcript is too ltte to consider. Mutantthe expression of many other genes. A microaray analysisfama-1 has a normal morphology in seedling but very compactwas conducted for yoda and found that several hundreds ofrosette leaves and mufi-ifertile top flowers. There are nogenes were changed at the transcriptional level (Bergmannnomal stomata on leaf epidermis. Remnant guard cells areet al.2004). T-DNAinsetional mutants were used to functinallyformed as dusters of small and narow cells. Gain-of-functionanalyze some of these genes. One locus named FAMA encodesmutation of FAMA has an opposite phenotype to null mutation.a MYB transcription factor. Loss-of- FAMA leads to dwarf plant It is concluded that FAMA functions in GMC symmetric divisionand immature guard cell dusters similar to the double mutant ofstages (Ohashi-lto and Bergmann 2006). FAMA imits the GMCmyb88 and fp. Likely, YODA may act in the same pathway asdivision times and ensures that one GMC only produces twoTMM and SDD1.guard cells.A model of integrating action was proposed (Gray andAnother two FAMA bHLH family transcription factors haveHetherington 2004). SDD1 may modifty a substrate outsidebeen reported (Gray 2007; MacAlister et al. 2007; Pltteri et al.the membrane. The modifed substrate is then activated as2007). SPEECHLESS (SPCH) is necessary for the itiationa lIgand of TMM. TMM is a transmembrane protein with anof protodermal cell into stomatal cell lineage. Null mutationexrtraclular receptor domain, but it lacks kinase activity inside of SPCH leads to a small seedling. Only pavement cells andthe membrane. TMM passes the signal through a co-receptortrichomes existin spch (MacAlister et al.2007). This no-stomataand activates YODA to trigger phosphorylation cascade. Thisphenotype is a result of stem cell failure to enter the stomatalhypothesis is very similar to a known CL AVATA pathwaycell lineage. Constitutive expression of SPCH shows more{Jeong et al. 1999; Trotochaud et al. 1999). CLV1 and CLV2meristemoid initiation. So SPCH is suficient to direct stem cellsare both transmembrane proteins with extraellular receptor to difentiate into meristemoid. SPCH encodes a 364 aminoactivities. They fom a heterodimer to receive extermal signals.acid protein with a nuclear localization signal. It shares a veryThe diference between them is that CLV1 has itracellularhigh similarity with FAMA and another bHLH transcription factor,phosphorylation sites. CLV3 is a ligand of CLV1/CLV2 complex, MUTE. MUTE controls meristemoid to divide asymeicallybut CLV3 must be processed by a subtilase before it bindsto form a guard mother cell. In the MUTE knockout mutant,to CLV1/CLV2 receptor. There is also a hypothesis about a meristemoid divides more than three times Pltteri et al.TMM inactivation (Shpak et al. 2005). When SDD1 ligands2007). The division foms a very small triangle meristemoidare absent, TMM could dissociate from its co-receptor, thusin the middle of the inward spiral. The centered meristemoidativating ERECTA family member RLKs and the downstream cannot dfferentiate into a guard mother celto form a stoma. TheMAPKKK pathway. But this is only a hypothesis that has notCaMV35S -MUTE transgenic plants display a no-pavement-cellbeen experimentaly proved.phenotype. AI of the epidermal cells convert into guard cells,Recently, the YODA downteam components were resolved.which中国煤化IitionfrommeristemoidThis cascade includes two MKKs and two MPKs (Wang et al.to gua=Id FAMA, three highly2007). Both MKKs and MPKs were reported before (Tena et al.conselC N M H Gcan tandemly control2001; Zhang and Klessig 2001; Nakagami et al.2005; Pedleythe stomatal development pathway as master regulators (Grayand Martin 2005). Loss of-function mutant gave an unusual phe- 2007). .1440 Joumal of Integrative Plant Biology Vol. 49 No. 10 2007very similar to that found in the plants overexpressing ERECTASome genes controlling call expanslon and cell division(Masle et al. 2005). We have identifed the gene that enoodescan affect stomatal densitya homeodomain transcripin factor. Overexpression of theSince stomatal density is inevitably affected by cell densityranscription factor in tobacco and nice can reduce stomatal(number of cells per unit area) which is in tum affected by celldensity (Figure 1) as well as improve drought tolerance andsize. Theretically, any factors affecting cell density will affectroot architecture. Interestingly, although the stomatal density isstomatal density. Cell division includes cytokinesis and karyoki-reduced, biomass is increased in the mutants and transgenicnesis. Many Arabidopsis mutants of abnormal cytokinesis haeve plants (CB Xiang, unpubl. data, 2007).been characterized. Some of these gene mutations can affectstomata density (Sollner et al. 2002; Inze and De Veylder 2006).STOMATAL CYTOKINESIS-DEFECTIVE 1 (SCD1) is a geneDegree of Ploidy can Change Stomatalinvolved in cell plane formation. The scd1 mutant of EMSDensity and Sizemutagenesis onigin was identifed to encode the SCD1 gene,a unique gene in Arabidopsis (Falbel et al.2003). The mutationPolyploidy is a common phenomenon in plants (Chen 2007).in the scd1 mutant was mapped at c∞odon 392, a single pointUnder certain conditions, being polyploidy is just a nomalmutation from C to T, corresponding amino acid changes fromnecessary process of growth, such as the trichome developmentserine to phenylalanine. The scd1 mutant shows smaller, darker(Melragno et al. 1993). In some cases, being polyploidy is forgreen and shorter roots than the wild type. The cell wall in thea certain purpose. For example, some plants will increase theirscd1 mutant is not complete. This defective cytokinesis leads toploidy levels to enlarge the cell volume, so that they can betterfom multinucleate cells and cell wall protrusions. Furthermore,suvive an unfavorable situation (Xiong et al. 2006).the decreased functional stomata in scd1 due to incompleteThere appears to be a general trend that the higher level thecell wall formation results in low CO2 assimilation. The scd1 isploidy, the lowerthe stomatal density and the larger the stomatala temperature-sensitive mutant. Two T-DNA inserion lines ofsize (Hetherington and Woodward 2003). Naturally occuringSCD1 show a more severe phenotype than scd1, suggestingtetraploid and hexaploid wheat varieties show increased cellthat T-DNA lines are completely loss offunction mutants. Inter-size associated with increased grain yield, reduced stomatalestingly, T-DNA lines lost temperature sensitivity. To manifestdensities, and increased stomatal sizes (Dunstone and Evansthe roles of SCD1 In stomatal development, T-DNA lines were1974; Xiong et al. 2006). The change in stomatal densityused because of complete loss of function. No GMC and lessand sizes in polyploid C4 grass Panicum vigatum is usuallycell expansion in the pavement cell were observed in the T-DNAaccompanied with increased photosynthesis (Wamer et al.lines. The most evident feature was fewer lobes of epidermnal1987). Tissue clture-derived rice polyploids are associatedcells in the mutant and thus to form a more rectangular shape. Inwith reduced stomatal density, increased size of stomata, androots there are no such phenotypes, but it seems more sensitiveincreased photosynthesis. The leaf and the whole plant areto cytoskeleton and vesicular taffcking inhibitor in mutants.larger. However, these polyploids have frility problems (FuB-ype CDKs are unique in plants (Boudolf et al. 2004).et al.1999).CDKB1;1 expression is confined to stomatal precursor clls andguard clls. A dominant negative mutant CDKB1;1.N161 led tolower CDK activity and showed smaller leaf size caused byPerspectivecell expansion and restricted division aility. The stomatal indexis also lower in the mutant. There are about 58.5% abnormalTo cope with ever changing lving conditions. sessile plants havestomata in CDKB1;1.N161 plants, but the guard cell ident isdeveloped mechanisms to defend themselves and optimallyusenot atered. A number of additional genes involved in the cellthe resources in their surroundings such as water and nutrientscycle were found to change cell density and consequenty alterfor their growth and development Environmental cues havestomatal density (Inze and De Veylder 2006).great efects on plant growth and development as well as geneticThe ABA overtysensitive mutant abo1 mutant with adrought-controls.resistant phenotype also dfrertnally modulates the develop-Stomatal density and size are dominant factors determiningment and growth of adjacent guard cells and shows reducedleaf conductance, which is crucial for both photosynthesis andstomatal density. ABO1 is a new alele of ELO2, a subunit oftranspiration and∞onsequenty affect plant WUE. Traditinally,holo-Elongator (Chen et al. 2006).the studies on the relationship between stomatal density andWe have isolated a gain-of-function Arabidopsis mutant withplant Wlnlant species with varyingimproved drought tolerance (Yu et al. 2004). This mutant alsostoma中国煤化工ntgenetic backgroundtsshows improved root architecture and reduced stomatal density.of theCN M H Gudies would certainlyThe reduced stomatal density is associated with increasedcomplcante ue Thuerpreuauon ul unu rusults. Although a generalstomatal size. The leaf surface phenotype of this mutant istrend could be seen, it is hard to draw accurate conclusions.Stomatal Density and Bio water Saving 1441AWT35S-HS1Stomatal densityCell densityStomatal indexAve01020304050607080050 100 150 200 250 05 101520Stomata/mm2Cells/mm2Flgure 1. Decrease of stomatal density by overexpressing an AHS1 homeodomain protin in transgenic tbaco.(A) Comparson of stomatal density on the adaxial leaf sutace between the wild type (WT)and the ransgenic tobaco ovrexpressing HS1 (35S-HS1)(magifcation: 200x).(B) Comparisons of stomatal density, cell density, and stomatal index betwen the wld type and the ransgenic lines overexressing HS1. Ave,average of transgenic ines.Recent advances in stomatal development and the identifica-been created by overexpressing the homeodomain transcriptiontion of a number of genes regulating stomatal density make itfactor (Figure 1). These plants would provide a unique opportu-possible to generate transgenic plants with dfferent stomatal nity to precisely assess the role of stomatal density and size indensities. In the same genetic background, the transgenicplant WuEplants would alow accurate analyses of the effect of stomataltap |中国煤化Ilensily may be a moredensity on plant WUE. This has been demonstrated using theamena，CN M H Gstonatal be; stomatal behavior inhomeodomain transcription factor in our laboratory. Transgenic; polyploidtobacco and rice lines with a range of stomatal densities have plants also imply that reduced stomatal density with associated1442 Joumal of Integretive Plant Biology Vol. 49 No. 10 2007enlargement of stomata may be a general evolution trend forKNAT1 and ERECTA regulate inforescence architecture in Arahigher plantWUE. But unfortunately, few genes regulating stom-bidopsis. Plant Coll 14, 547- 558.atal density have been reported so far for use in either studyingDriscoll SP, Prins A, Olmos E, Kunen KJ, Foyer CH (2006). Spec-the role of stomatal density in plant WUE or applying to bio-ication of adaxial and abaxial stomata, epidermal stucure andwater saving in agrioulture. Several genes such as ERECTA,photosynthesis to CO2 enichment in maize leaves. J. Exp. Bot.SDD1, and the above mentioned homeodomain transcription57, 381 -390.factor have no apparent adverse effects on plant growth andDunstone RL, Evans LT (1974). 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