Density and size of stomata in the leaves of different hybrids (Vitis sp.) and Vitis vinifera varieties

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1 Vitis 55, (2016) DOI: Density nd size of stomt in the leves of different hybrids (Vitis sp.) nd Vitis vinifer vrieties S. Boso, P. Ggo, V. Alonso-Villverde, J. L. Sntigo nd M. C. Mrtinez Misión Biológic de Glici, Consejo Superior de Investigciones Científics (CSIC), Slcedo, Pontevedr, Spin Summry A number of studies hve highlighted differences in the density of stomt between Vitis species, but few hve exmined differences between vrieties of V. vinifer. The density nd size of the stomt in the lower epidermis of leves belonging to 12 grpevine vrieties, direct producer hybrid (DPH) involving V. vinifer nd non-vinifer prent, nd the non-vinifer rootstocks 'SO4' nd '110-Richter', were therefore exmined. Trnsprent nil polish peel prints of the re between the min nd right lterl veins were produced for 10 leves per vriety. These prints were then exmined under light microscope nd the number of stomt in unit re of mm 2 counted. Imge nlysis softwre ws then used to mesure the length nd width of ll those counted. Rootstock 'SO4', 'Chssels Dorée', 'Albriño' nd 'Cbernet Suvignon' hd the highest stomtl densities (ll > 34 stomt per unit re), while 'Cstñl', 'Torrontés' nd 'Ciño Blnco' nd 'Jcquez' (DPH), hd the smllest (ll < stomt per unit re). 'Treixdur' nd 'Ciño Blnco' hd significntly longer nd wider stomt thn ll the other vrieties exmined; the DPH 'Jcquez' hd mong the shortest nd nrrowest. No reltionship ws seen, however, between men vrietl lef size nd the stomtl density or stomtl size; nor ws ny seen between the vribles exmined nd the condition of belonging to V. vinifer or not. Key words: Vitis vinifer; HPD; rootstocks; number of stomt; nil polish prints; stom size. Introduction Stomt re smll pores through which plnts exchnge gses nd must regulte wter loss (Bernrd 1978, Düring 1980, Ren et l. 2003). Unfortuntely, they lso provide route vi which fungi nd other pthogens cn enter the plnt. The leves of grpevines (Vitis vinifer L.) hve stomt only on the lower epidermis, where they re rrnged in no pprent order. All stomt re hve two kidney-shped gurd cells tht surround the pore or ostiole (Bernrd 1978, Düring 1980, Alonso-Villverde et l. 2011, Boso et l. 2010), lthough some uthors report differences in stomtl morphology between vrieties within different Vitis species (Swnepoel nd Villiers 1987, Codrenu 2006), s well s in their opening nd closing mechnisms, nd their involvement in physiologicl processes (Liu et l. 1978, Teixeir et l. 2009, Rogiers et l. 2011). A reltionship hs lso been reported between the density nd size of stomt nd susceptibility to downy mildew (Lu et l. 2010). Studying grpevine stomt cn, however, be difficult since the results obtined my differ depending on number of fctors. Indeed, single plnt of the 'Crignn' vriety my show differences in leves exmined t budding, flowering, budset, verison, nd fruit ripening (Bernrd 1978), nd Düring (1980) hs reported the sme for other Vitis species, e.g. 'Silvner' nd 'Müller-Thurgu'. Results my lso vry depending on the position of lef on shoot (Bernrd 1978, Mrtínez nd Grenn 1999) or the type of shoot exmined, e.g. fruiting shoot (i.e. derived from the previous yer's wood), wter shoot (which derives from the trunk), or xillry shoot (derived from lef xill on green shoot) (Plliotti et l. 2000). Thus, plnts of the sme vriety my return very different results if they hve not been rised under exctly the sme climtic conditions nd following the sme trining nd pruning prctices, etc. (Kr nd Özeker 1999, Gómez del Cmpo et l. 2004, Ben Slem-Fnyou et l. 2005). The type of rootstock used my lso influence the results (Fregoni et l. 1978, Kr nd Özeker 1999), s cn the vegettive stte of the plnt. Rigorous smpling is therefore key to success in studies on grpevine stomt. The im of the present work ws to compre the density nd size of the stomt of different V. vinifer vrieties, nd other non-vinifer members of Vitis, including direct producer hybrid (DPH). Mteril nd Methods Plnt mteril nd plot chrcterist i c s : The studied plnt mteril included 12 vrieties of V. vinifer, some of interntionl winemking importnce ('Albriño', 'Alicnte Henri Bouschet', 'Cbernet Suvignon' nd 'Chssels Dorée'), nd others little known outside their trditionl Spnish growing regions but beginning to wken interest ('Albrin Blnco', 'Ciño Blnco', Correspondence to: Dr. M. C. Mrtínez Rodriguez, Misión Biológic de Glici, Consejo Superior de Investigciones Científics (CSIC), Crblleir 8, Slcedo, Pontevedr, Spin. Fx: E-mil: crmenmrtinez@mbg.csic.es The uthor(s). This is n Open Access rticle distributed under the terms of the Cretive Commons Attribution Shre-Alike License (

2 18 S. Boso et l. 'Ciño Tinto', 'Cstñl', 'Godello', 'Menci', 'Torrontés' nd 'Treixdur'). Three vrieties of non-vinifer were lso exmined: the rootstocks '110-Richter' ( hybrid of V. berlndieri Resseguier nº2 nd Rupestris Mrtín) nd 'SO4' ( hybrid of V. berlndieri nd V. ripri [Oppenheim, selection of Teleki nº4]) (Glet 1995), nd the DHP known s Jcquez ( teinturier of uncertin origin) (Sntigo et l. 2008). Except for the two rootstocks, ll were grfted onto '110-Richter' in April 1994 (10 replics per vriety) nd grown on trellises t the Misión Biológic de Glici (CSIC) in northwestern Spin (42 25 N, 8 38 W; ltitude 20 m). All were subject to Sylvoz pruning. Climte dt: The men temperture for the re for the lst 50 yers ws 14.2 C, nd the men rinfll 1650 mm (with wide nnul vrition). Men temperture, mximum temperture, minimum temperture nd totl rinfll were lso recorded for the study period t the Slcedo wether sttion (nº1485) next to the experimentl plot. Smpling methods nd vribles m e s u r e d : Adult leves were smpled on the sme dy between budset nd verison in 2008 (July 18) nd 2009 (July 16). When the mjority of green shoots hd produced between 12 nd 14 internodes, the 8 th lef from the bse of fertile green shoot (sun exposed) ws tken from 10 plnts per vriety. These leves were lbelled nd plced in plstic bgs for trnsport to the lbortory. A trnsprent nil polish peel print ws then mde of n pproximtely 1 1 cm re of the underside of ech lef, close to the petiolr sinus between the min vein nd the right left lterl vein (D Ambrogio de Argüeso 1986). For this, fine lyer of nil vrnish ws pplied to the lower surfce nd llowed to dry. It ws then peeled off with the help of sclpel. All prints were preserved in luminium foil t room temperture until use. Ech print ws plced 25 on microscope slide onto which drop of distilled wter hd been plced, nd covered with coverslip. Observtions were mde using Nikon Eclipse E200 opticl microscope (objective 40 ). The number of stomt within visul field of mm 2 (herefter unit re) ws counted t three different points. Representtive photogrphs were tken for ech vriety (objective 40 ) nd the stomtl lengths nd widths recorded using NIS-Elements Bsic Reserch v.3.1 softwre. Sttisticl nlysis: Differences between the men stomtl density nd size for ech vriety were nlysed by ANOVA. An F test ws then performed, compring ech fixed fctor (vrieties) with its error. Significnt F vlues were subjected to comprison of mens using Fisher s protected lest significnt difference (LSD) test. All clcultions were performed using SAS System v. 9.1 softwre. Results nd Discussion Fig. 1 shows the wether dt over the two yers in which the study ws performed. The men nnul temperture for 2008 ws C nd the rinfll ws mm; for 2009 these figures were C nd mm. The yer 2009 ws therefore wetter nd colder. Indeed, in June 2009, the rinfll recorded ws mm compred to mm in 2008, nd with the exception of Mrch nd My, the tempertures were lwys cooler thn in Results collected over both yers for "stomtl density", "stomtl length" nd "stomtl width" were subjected to ANOVA. Significnt differences were detected bewteen the vrieties (p < 0.01) nd between yers for stomtl length (p < 0.05) nd density (p < 0.01). The interction vriety yer hd significnt (p < 0.01) effect on ll these 500,0 450, ,0 Men Temperture ( C) ,0 300,0 250,0 200,0 150,0 Rinfll totl (mm) 5 100,0 50,0 0 Jnury Februry Mrch April My June Month July August September October November December 0,0 Men Temperture 2008 Men Temperture 2009 Rinfll 2008 Rinfll 2009 Fig. 1: Temperture ( C) nd rinfll (mm), s recorded t the Slcedo nº1485 wether sttion (ner the plot) over the experimentl period (2008 nd 2009).

3 Density nd size of stomt in leves 19 vribles (Tb. 1). This grees with reports by other uthors (Tichá 1982, Wentworth et l. 2006) which indicte wether conditions (rinfll, tmospheric CO 2 concentrtion, light intensity, ir temperture), photoperiod nd crop mngement etc., to ffect these vribles. Thus, ANOVAs were performed seprtely for the different vribles nd yer. Agin, significnt differences (p < 0.01) were detected between vrieties (Tb. 1). Tb. 2 shows the men stomtl densities, lengths nd widths for ech yer In 2009, the vrieties hd lrger stomtl density thn in 2008 (35.63 stomt per unit re compred to per unit re). They were lso longer in 2009 t men 42.1 μm compred to μm in 2008, nd wider, t men 26.9 μm compred to μm (Tb. 2). At first, these findings might be the result of the greter rinfll in 2009 during erly-mid summer (157.8 nd 69 mm in June nd July respectively) compred to the sme months in 2008 (14.4 nd 33.4 mm) when lef development is gretest). However, Plliotti et l. (2001) demonstrted tht the increse of individul lef re in well wtered vines produced the decrese of stomtl density compred to low wtered vines. The sme nd other uthors (Plliotti et l. Tble 1 ANOVAs involving stomtl density, length nd width Vrible Vrieties (V) Yer (Y) Vriety Yer d Error Stomtl length (μm) g L -1 b C.M. c *** e * *** Stomtl width (μm) g L C.M *** 31.95ns *** g L Stomtl density C.M *** *** *** Stomtl length (μm) g L C.M *** Stomtl width (μm) g L C.M *** g L Stomtl density C.M *** Stomtl length (μm) g L C.M *** Stomtl width (μm) g L C.M *** g L Stomtl density C.M *** Number of stomt in unit re of mm 2 ; b Degrees of freedom; c Men squre; d Interction vriety x yer; *Yer; e ns, not significnt; *p < 0.05; ***p < Tble 2 Comprison of men stomtl densities, lengths nd widths in ech yer of the study Genotypes Stomtl length (µm) Stomtl width (µm) Stomtl density Albrín Blnco def fg d efg efgh bc Albriño bcd 44.42bc bc b bcd Alicnte Henri Bouschet bcd de c gh b cd Cbernet Suvignon f de c bc bc bc Ciño Blnco bc bc gh de Ciño Tinto bcd de c bcde cdef cd Cstñl b g b i fgh e Chssels Dorée bcd de d defg bcdef Godello ef fg c fg bcde de Jcquez g g d i efgh e Menci bcd bc c bcdef defg SO bcd de bc bcde Torrontés f d cdef h e Treixdur * c hi bcd de 110-Ritcher bc cd c bcd cdef bcd LSD (0.05) * Vlues with the sme letter re significntly different (p < 0.05).

4 20 S. Boso et l. 2000, Gómez del Cmpo et l. 2007) dd tht rinfll is not the only limiting fctor; light intensity nd high tempertures (the mximum reched in July 2008 ws 32.5 C) lso pper to be involved. Dt on the individul lef re of the studied vines re being recorded t present in order to verify this behviour. The V. vinifer vrieties 'Treixdur' nd 'Ciño Blnco' lwys hd the longest stomt, while the DHP 'Jcquez' lwys hd mong the shortest (Fig. 2, Tb. 2). Those of 'Jcquez' were lso the nrrowest, while the vrieties 'Cstñl', 'Ciño Blnco', 'Albriño' nd the rootstock 'SO4' hd the widest. 'Cstñl' ctully showed different behviours in the different yers. In 2008 it returned the highest vlues for both vribles, while in 2009 it returned the lowest of ll. Torrontés showed the opposite behviour: in 2008 it returned the lowest vlues for both vribles, nd in 2009 it returned mong the highest. Tb. 2 shows the rootstock 'SO4' nd the V. vinifer vrieties 'Chssels Dorée', 'Albriño' nd 'Cbernet Suvignon' hd the lrgest stomtl densities (ll > 34 stomt per unit re), while 'Cstñl', 'Torrontés' nd 'Ciño Blnco' plus the DHP 'Jcquez' hd the smllest (ll < 26.5 per unit re). Swnepoel nd Villiers (1987), who studied the stomtl density nd stomtl index (number of stomt/ number of epiderml cell) of different hybrids (including 'Jcquez'), V. Vinifer cultivrs ('Pinot noir', 'Musct d'alexndrie') nd Americn roostocks ('99R', '143B Mgt', etc.), reported tht Americn spp. lwys hd highest vlues. In the present work, SO4 returned the highest stomtl density, but 'Jcquez' fell within the vrieties with the smllest vlues. No direct reltionship ws seen between number of stomt nd ny lef chrcteristic (such s size or the depth of the lterl sinuses). For exmple, 'Cstñl' nd 'Jcquez' hd mong the lowest stomtl densities but hve very lrge leves (Sntigo et l. 2008, Ggo et l. 2009; while Ciño Blnco hd low stomtl density yet hs smll leves (Sntigo et l. 2007). No cler reltionship ws seen between the stomtl density nd either stomtl length or width. For exmple, 'Jcquez' hd low stomtl density nd its stomt were smll, while 'Ciño Blnco' hd low stomtl density but its stomt were lrge, nd 'Cstñl' hd both lrge nd smll stomt on the sme lef in the sme yer. Neither ws ny cler reltionship seen between stomtl density nd known susceptibility to downy mildew (Boso et l. 2010, Boso nd Kssemeyer 2008); this grees with tht reported by other uthors (Mendgen 1996, Allègre et l. 2007, Jürges et l. 2009). Work performed by our group hs shown tht 'Cbernet Suvignon' nd the rootstocks '110-Richter' nd 'SO4' re more resistnt to this disese thn others (Boso nd Kssemeyer 2008), yet they hd high stomtl densities, while 'Ciño Blnco' nd 'Treixdur', which hd low stomtl densities, hve been shown rther susceptible (Boso et l. 2010). Both these results re the opposite of wht one might expect. Similrly, Polocci et l. (2014) reported the downy mildew-resistnt vriety Solris to hve high stomtl density. According to Wilkinson et l. (1995), the stomtl index vries little between vrieties, while the stomtl density differs more strongly. However, controversy surrounds whether the stomtl density is geneticlly predetermined or whether it cn vry ccording to conditions such s light intensity, humidity, ir temperture or soil temperture, etc. Plliotti et l. (2000) rgue the former cse, while others (Lke et l. 2001, Csson nd Gry 2008, Rogiers et l. 2011) rgue the ltter. Some uthors (Gómez del-cmpo et l. 2003, Rogiers et l. 2011), suggest tht while the ltter my be true there re genetic differences s well. According to Meisel et l. (2011), plnts hve "specilised vision system", tht llows them to perceive light signls vi specilised photoreceptors. These initite biochemicl events tht regulte the ctivity of the genome, thus llow Length of stom 2008 (ɥm) Width of stomt 2008 (ɥm) Length of stom 2009 (ɥm) Width of stomt 2009 (ɥm) 50.0 Size of stomt (ɥm) Albriño Albrin Binco Alicnte HB Cbernet Suvignon Ciño Binco Ciño Tinto Fig. 2: Size of stomt (length nd width) in 2008 nd Cstñl Chssels Dorée Grpevines Godello Jcquez Menci Torrontes Treixdur 110R-Rootstock SO4-Rootstock

5 Density nd size of stomt in leves 21 ing responses to environmentl stimuli to be mde. Sugno et l. (2010) recently reported tht the mesophyll cells of immture Arbidopsis leves express n intercellulr signlling fctor (stomgen) tht intercts with epiderml cell fctors to influence stomtl density. It my be, therefore, tht these photosynthetic cells, nd not the epiderml cells, ultimtely regulte this vrible. The differences detected in the present work cnnot be due to hving tken the exmined leves t different stges of development or from different plces on the shoot; ll were tken t the sme time from node 8, which, ccording to vrious uthors (Mrtinez nd Grenn 1999) is tht t which grpevine leves re structurlly nd morphologiclly most stble. Conclusion In conclusion, no reltionship ws seen between lef size nd stomtl size or density, nor between stomtl density nd size. Neither did belonging to V. vinifer or not pper to influence ny of the vribles exmined. 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