组织透明法观察葡萄叶片生长过程中气孔与叶脉形态结构特征变化
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摘要
本文以‘摩尔多瓦’葡萄叶片为试验材料,采用组织透明法观察了葡萄叶片生长过程中叶片表皮细胞、气孔和叶脉形态结构的变化,测定与气孔和叶脉功能相关的生理指标变化,比较了组织透明法、指甲油法和撕取法在观察葡萄叶片气孔上的实验效果。结果表明:组织透明法能够清晰观察到葡萄叶片生长过程中叶片表皮细胞、气孔和叶脉形态结构的变化。相对于指甲油法、撕取法,组织透明法操作简单,且能保持组织完整性,提升观察叶片细胞结构的实验效果。从叶片生长过程中气孔形态结构变化规律来看,葡萄嫩叶齿尖存在大量的大气孔,而叶片中部只观察到中央大气孔和正在发育的气孔保卫细胞母细胞。随着叶片生长,叶片气孔保卫细胞母细胞逐渐发育形成成熟气孔,叶片末端叶脉也是随叶片生长而生长,叶脉密度逐渐增加,提高了树体往叶片供水的效率。从与气孔和叶脉功能相关的各项生理指标变化来看,气孔导度随着气孔逐渐形成和成熟而逐渐升高,但叶片含水量和水势下降,有利于拉升水分和养分从根系往地上树体各器官运输,满足整个植株生长发育的需要。
In this study, ‘Moldova' grapevine(Vitis vinifera) leaves were cleared to examine leaf epidermal cells, stomata, and minor vein system. These features were compared with other techniques such as leaf imprints using nail polish and tearing/peeling of leaf epidermis. Among these techniques, the tissue clearing technique emerged as the best way of studying leaf morphological features as it is easy and provides clear images of the highest resolution. For instance, it revealed numerous large stomates scattered throughout the abaxial surfaces of dentate margins of young leaves as opposed to few large stomates centrally localized along with guard mother cells in the lamina. As the leaves developed, the guard mother cells transformed into mature stomata and the minor vein system increased its vein density. These collectively increased stomatal conductance but the water content and water potential declined explaining the paradigm that plants use most water to transpire and only a small quantity to sustain growth.
In this study, ‘Moldova' grapevine(Vitis vinifera) leaves were cleared to examine leaf epidermal cells, stomata, and minor vein system. These features were compared with other techniques such as leaf imprints using nail polish and tearing/peeling of leaf epidermis. Among these techniques, the tissue clearing technique emerged as the best way of studying leaf morphological features as it is easy and provides clear images of the highest resolution. For instance, it revealed numerous large stomates scattered throughout the abaxial surfaces of dentate margins of young leaves as opposed to few large stomates centrally localized along with guard mother cells in the lamina. As the leaves developed, the guard mother cells transformed into mature stomata and the minor vein system increased its vein density. These collectively increased stomatal conductance but the water content and water potential declined explaining the paradigm that plants use most water to transpire and only a small quantity to sustain growth.
引文
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Carins MR,Jordan GJ,Brodribb TJ(2012).Differential leaf expansion can enable hydraulic acclimation to sun and shade.Plant Cell Environ,35(8):1407-1418
Carins MR,Jordan GJ,Brodribb TJ(2014).Acclimation to humidity modifies the link between leaf size and the density of veins and stomata.Plant Cell Environ,37(1):124-131
Casson SA,Hetherington AM(2010).Environmental regulation of stomatal development.Curr Opin Plant Biol,13(1):90-95
Chung K,Wallace J,Kin SY,et al(2013).Structural and molecular interrogation of intact biological systems.Nature,497(7449):332
Cui HA,Liu YQ,Bai HX(2007).A new method to observe stomas.Acta Agri Boreali-Occident Sin,16(6):305-306(in Chinese with English abstract)[崔宏安,柳延琴,白红霞(2007).观察气孔的一种新方法.西北农业学报,16(6):305-306]
Du Z,Zhao SX,Zhou HY,et al(1995).The use of differential interference contrast microscope and whole-clearing of ovries for observation on embryo sacs structure of rice and wheat.J Agri Biotechnol,3(4):94-97(in Chinese with English abstract)[杜中,赵世绪,周海鹰等(1995).用微分干涉差显微镜和子房整体透明法观察水稻和小麦的胚囊结构.农业生物技术学报,3(4):94-97]
Gong R,Gao Q(2015).Research progress in the effects of leaf hydraulic characteristics on plant physiological functions.Chin J Plant Ecol,39(3):300-308(in English with Chinese abstract)[龚容,高琼(2015).叶片结构的水力学特性对植物生理功能影响的研究进展.植物生态学报,39(3):300-308]
Guo HB,Lu YG,Feng JH,et al(2006).Further observation on the formation and development of autotetraploid rice embryo sac using laser scanning confocal microscopy.Acta Laser Biol Sin,15(2):111-117(in Chinese with English abstract)[郭海滨,卢永根,冯九焕等(2006).利用激光扫描共聚焦显微术对同源四倍体水稻胚囊形成与发育的进一步观察.激光生物学报,15(2):111-117]
Hao JH,Qiang S(2007).The examples and aalysis of wholemount clearing techniques in plant biology.Chin Bull Bot,24(4):490-497(in Chinese with English abstract)[郝建华,强胜(2007).整体透明技术在植物生物学中的应用实例及其剖析.植物学通报,24(4):490-497]
Huang CL,Zhang DP(1996).Studies on physiological and environmental factors inducing diurnal photosynthetic depression in grapevine leaves.Acta Hort Sin,23(2):128-132(in Chinese with English abstract)[黄丛林,张大鹏(1996).葡萄叶片光合速率日间降低内外因调控的研究.园艺学报,23(2):128-132]
Kondo T,Kajita R,Miyazaki A,et al(2010).Stomatal density is controlled by a mesophyll-derived signaling molecule.Plant Cell Physiol,51(1):1-8
Li A,Gong H,Zhang B,et al(2010).Micro-optical sectioning tomography to obtain a high-resolution atlas of the mouse brain.Science,330(6009):1404-1408
Li GP,Huang QC,Qin GY(2006).The observation of pollen and pollen tube of Cedrus deodara with laser scanning confocalm icroscope.Acta Laser Biol Sin,15(1):1-8(in Chinese with English abstract)[李国平,黄群策,秦广雍(2006).用激光扫描共聚焦显微镜观察雪松花粉和花粉管.激光生物学报,15(1):1-8]
Li JH,Luo GG(1996).Dynamics of the leaf growth and photosynthetic characteristics for kyoho grapevines.Acta Horti Sin,23(3):213-217(in Chinese with English abstract)[李建华,罗国光(1996).巨峰葡萄叶片生长动态与光合特性的研究.园艺学报,23(3):213-217]
Li YK,Zhang GG,Zhao LN,et al(2011).The study on the embryos development of Boehmeria tricuspis by whole clearing techniques.Plant Fiber Sci China,33(3):142-146(in Chinese with English abstract)[李彦坤,臧巩固,赵立宁等(2011).整体透明技术观察悬铃叶苎麻胚胎发育的方法研究.中国麻业科学,33(3):142-146]
Liberato JR,Barreto RW,Shivas RG(2005).Leaf-clearing and staining techniques for the observation of conidiophores in the Phyllactinioideae(Erysiphaceae).Australas Plant Path,34(3):401-404
Liu HB,Bai YG,Zhang JH,et al(2011).Study on the rule of hydraulic of grape in SPAC system in extreme arid region.Res Soil Water Conserv,18(6):185-189(in Chinese with English abstract)[刘洪波,白云岗,张江辉等(2011).极端干旱区葡萄spac系统水流阻力规律研究.水土保持研究,18(6):185-189]
Ma HQ,Luo GG(1999).Morphology and development of adaxial stomata on tooth-tips of grape leaves.J China Agri Uni,4(5):118-124(in Chinese with English abstract)[马会勤,罗国光(1999).葡萄叶片齿尖上表皮气孔形态及发育特征.中国农业大学学报,4(5):118-124]
Schulte PJ,Brooks JR(2003).Branch junctions and the flow of water through xylem in Douglas-fir and ponderosa pine stems.J Exp Bot,54(387):1597-1605
Song LQ,Hu CM,Hou XL,et al(2015).Relationship between photosynthetic characteristics and leaf vein density in Sorghum bicolor and Perilla frutescens.Chin Bull Bot,50(1):100-106(in Chinese with English abstract)[宋丽清,胡春梅,侯喜林等(2015).高梁、紫苏叶脉密度与光合特性的关系.植物学报,50(1):100-106]
Villani TS,Koroch AR,Simon JE(2013).An improved clearing and mounting solution to replace chloral hydrate in microscopic applications.Appl Plant Sci,1(5):429-438
Xie ZS,Bondada B,Li B,et al(2016).Visualization of axial water transport pathways in grapevines using dye-tracing technique.Plant Physiol J,52(8):1159-1168(in Chinese with English abstract)[谢兆森,Bhaskar Bondada,李勃等(2016).采用染料示踪法可视化研究葡萄新梢水分运输途径.植物生理学报,52(8):1159-1168]
Yang B,Treweek,JB,Kulkami RP,et al(2014).Single-cell phenotyping within transparent intact tissue through whole-body clearing.Cell,158(4):945-958
Yang HY(1986).Observations of embryo sacs,embryos,endosperms and embryoids by whole staining with transparent technique.J Integrative Plant Biol,28(6):575-581(in English with Chinese abstract)[杨弘远(1986).用整体染色与透明技术观察胚囊、胚、胚乳和胚状体.植物生态学报,28(6):575-581]
Yang HY(1988).Application of whole clearing technique in plant embryology.Chin Bull Bot,5(2):114-116(in Chinese with English abstract)[杨弘远(1988).植物胚胎学中的整体透明技术.植物学通报,5(2):114-116]
Yang LS,Li GP(2004).Procedures for observation of embryo sac structure of angiosperms.Res Exp Lab,23(2):9-11(in Chinese with English abstract)[杨鹭生,李国平(2004).被子植物胚囊结构的观察方法.实验室研究与探索,23(2):9-11]
Young BA,Sherwood RT,Bashaw EC(1979).Cleared pistil and thick-sectioning techniques for detecting aposporous apomixis in grasses.Can J Bot,57:1668-1672
Zhang Y,Yang SJ,Sun M,et al(2014).Stomatal traits are evolutionarily associated with vein density in basal angiosperms.Plant Sci J,32(4):320-328(in English with Chinese abstract)[张亚,杨石建,孙梅等(2014).基部被子植物气孔性状与叶脉密度的关联进化.植物科学学报,32(4):320-328]
Zhao LS,Liu LX,Gu JY,et al(2014).A method to improve sample preparation for observing stomata guard cells in Triticum aestivum.Chin Bull Bot,49(1):120-126(in Chinese with English abstract)[赵林姝,刘录祥,古佳玉等(2014).一种小麦叶片气孔保卫细胞观察样品的制备方法.植物学报,49(1):120-126]
Carins MR,Jordan GJ,Brodribb TJ(2012).Differential leaf expansion can enable hydraulic acclimation to sun and shade.Plant Cell Environ,35(8):1407-1418
Carins MR,Jordan GJ,Brodribb TJ(2014).Acclimation to humidity modifies the link between leaf size and the density of veins and stomata.Plant Cell Environ,37(1):124-131
Casson SA,Hetherington AM(2010).Environmental regulation of stomatal development.Curr Opin Plant Biol,13(1):90-95
Chung K,Wallace J,Kin SY,et al(2013).Structural and molecular interrogation of intact biological systems.Nature,497(7449):332
Cui HA,Liu YQ,Bai HX(2007).A new method to observe stomas.Acta Agri Boreali-Occident Sin,16(6):305-306(in Chinese with English abstract)[崔宏安,柳延琴,白红霞(2007).观察气孔的一种新方法.西北农业学报,16(6):305-306]
Du Z,Zhao SX,Zhou HY,et al(1995).The use of differential interference contrast microscope and whole-clearing of ovries for observation on embryo sacs structure of rice and wheat.J Agri Biotechnol,3(4):94-97(in Chinese with English abstract)[杜中,赵世绪,周海鹰等(1995).用微分干涉差显微镜和子房整体透明法观察水稻和小麦的胚囊结构.农业生物技术学报,3(4):94-97]
Gong R,Gao Q(2015).Research progress in the effects of leaf hydraulic characteristics on plant physiological functions.Chin J Plant Ecol,39(3):300-308(in English with Chinese abstract)[龚容,高琼(2015).叶片结构的水力学特性对植物生理功能影响的研究进展.植物生态学报,39(3):300-308]
Guo HB,Lu YG,Feng JH,et al(2006).Further observation on the formation and development of autotetraploid rice embryo sac using laser scanning confocal microscopy.Acta Laser Biol Sin,15(2):111-117(in Chinese with English abstract)[郭海滨,卢永根,冯九焕等(2006).利用激光扫描共聚焦显微术对同源四倍体水稻胚囊形成与发育的进一步观察.激光生物学报,15(2):111-117]
Hao JH,Qiang S(2007).The examples and aalysis of wholemount clearing techniques in plant biology.Chin Bull Bot,24(4):490-497(in Chinese with English abstract)[郝建华,强胜(2007).整体透明技术在植物生物学中的应用实例及其剖析.植物学通报,24(4):490-497]
Huang CL,Zhang DP(1996).Studies on physiological and environmental factors inducing diurnal photosynthetic depression in grapevine leaves.Acta Hort Sin,23(2):128-132(in Chinese with English abstract)[黄丛林,张大鹏(1996).葡萄叶片光合速率日间降低内外因调控的研究.园艺学报,23(2):128-132]
Kondo T,Kajita R,Miyazaki A,et al(2010).Stomatal density is controlled by a mesophyll-derived signaling molecule.Plant Cell Physiol,51(1):1-8
Li A,Gong H,Zhang B,et al(2010).Micro-optical sectioning tomography to obtain a high-resolution atlas of the mouse brain.Science,330(6009):1404-1408
Li GP,Huang QC,Qin GY(2006).The observation of pollen and pollen tube of Cedrus deodara with laser scanning confocalm icroscope.Acta Laser Biol Sin,15(1):1-8(in Chinese with English abstract)[李国平,黄群策,秦广雍(2006).用激光扫描共聚焦显微镜观察雪松花粉和花粉管.激光生物学报,15(1):1-8]
Li JH,Luo GG(1996).Dynamics of the leaf growth and photosynthetic characteristics for kyoho grapevines.Acta Horti Sin,23(3):213-217(in Chinese with English abstract)[李建华,罗国光(1996).巨峰葡萄叶片生长动态与光合特性的研究.园艺学报,23(3):213-217]
Li YK,Zhang GG,Zhao LN,et al(2011).The study on the embryos development of Boehmeria tricuspis by whole clearing techniques.Plant Fiber Sci China,33(3):142-146(in Chinese with English abstract)[李彦坤,臧巩固,赵立宁等(2011).整体透明技术观察悬铃叶苎麻胚胎发育的方法研究.中国麻业科学,33(3):142-146]
Liberato JR,Barreto RW,Shivas RG(2005).Leaf-clearing and staining techniques for the observation of conidiophores in the Phyllactinioideae(Erysiphaceae).Australas Plant Path,34(3):401-404
Liu HB,Bai YG,Zhang JH,et al(2011).Study on the rule of hydraulic of grape in SPAC system in extreme arid region.Res Soil Water Conserv,18(6):185-189(in Chinese with English abstract)[刘洪波,白云岗,张江辉等(2011).极端干旱区葡萄spac系统水流阻力规律研究.水土保持研究,18(6):185-189]
Ma HQ,Luo GG(1999).Morphology and development of adaxial stomata on tooth-tips of grape leaves.J China Agri Uni,4(5):118-124(in Chinese with English abstract)[马会勤,罗国光(1999).葡萄叶片齿尖上表皮气孔形态及发育特征.中国农业大学学报,4(5):118-124]
Schulte PJ,Brooks JR(2003).Branch junctions and the flow of water through xylem in Douglas-fir and ponderosa pine stems.J Exp Bot,54(387):1597-1605
Song LQ,Hu CM,Hou XL,et al(2015).Relationship between photosynthetic characteristics and leaf vein density in Sorghum bicolor and Perilla frutescens.Chin Bull Bot,50(1):100-106(in Chinese with English abstract)[宋丽清,胡春梅,侯喜林等(2015).高梁、紫苏叶脉密度与光合特性的关系.植物学报,50(1):100-106]
Villani TS,Koroch AR,Simon JE(2013).An improved clearing and mounting solution to replace chloral hydrate in microscopic applications.Appl Plant Sci,1(5):429-438
Xie ZS,Bondada B,Li B,et al(2016).Visualization of axial water transport pathways in grapevines using dye-tracing technique.Plant Physiol J,52(8):1159-1168(in Chinese with English abstract)[谢兆森,Bhaskar Bondada,李勃等(2016).采用染料示踪法可视化研究葡萄新梢水分运输途径.植物生理学报,52(8):1159-1168]
Yang B,Treweek,JB,Kulkami RP,et al(2014).Single-cell phenotyping within transparent intact tissue through whole-body clearing.Cell,158(4):945-958
Yang HY(1986).Observations of embryo sacs,embryos,endosperms and embryoids by whole staining with transparent technique.J Integrative Plant Biol,28(6):575-581(in English with Chinese abstract)[杨弘远(1986).用整体染色与透明技术观察胚囊、胚、胚乳和胚状体.植物生态学报,28(6):575-581]
Yang HY(1988).Application of whole clearing technique in plant embryology.Chin Bull Bot,5(2):114-116(in Chinese with English abstract)[杨弘远(1988).植物胚胎学中的整体透明技术.植物学通报,5(2):114-116]
Yang LS,Li GP(2004).Procedures for observation of embryo sac structure of angiosperms.Res Exp Lab,23(2):9-11(in Chinese with English abstract)[杨鹭生,李国平(2004).被子植物胚囊结构的观察方法.实验室研究与探索,23(2):9-11]
Young BA,Sherwood RT,Bashaw EC(1979).Cleared pistil and thick-sectioning techniques for detecting aposporous apomixis in grasses.Can J Bot,57:1668-1672
Zhang Y,Yang SJ,Sun M,et al(2014).Stomatal traits are evolutionarily associated with vein density in basal angiosperms.Plant Sci J,32(4):320-328(in English with Chinese abstract)[张亚,杨石建,孙梅等(2014).基部被子植物气孔性状与叶脉密度的关联进化.植物科学学报,32(4):320-328]
Zhao LS,Liu LX,Gu JY,et al(2014).A method to improve sample preparation for observing stomata guard cells in Triticum aestivum.Chin Bull Bot,49(1):120-126(in Chinese with English abstract)[赵林姝,刘录祥,古佳玉等(2014).一种小麦叶片气孔保卫细胞观察样品的制备方法.植物学报,49(1):120-126]