摘要: 目的研究高温处理对银灰杨小孢子发生的影响，揭示高温处理导致杨树花粉败育的细胞学机理，旨在完善高温处理诱导配子染色体加倍、选育林木三倍体的技术、�/sec> 方法本研究以银灰杨为试验材料，利�?8 ℃和41 ℃的高温，对不同减数分裂时期的银灰杨花粉母细胞进�?�? h处理，研究处理温度、减数分裂时期以及持续处理时间对败育花粉比率的影响。在此基础上，通过比较未经高温处理和高温处理后银灰杨花粉母细胞减数分裂染色体行为、微管骨架动态变化以及减数后绒毡层细胞发育的差异，揭示高温处理诱导银灰杨花粉败育的细胞学机理、�/sec> 结果�?）减数分裂时期、处理温度、持续处理时间以及减数分裂时期与处理温度、减数分裂时期与持续处理时间的交互作用均对败育花粉比率具有极显著的影响�?1 ℃高温于中期Ⅰ持续处�? h的败育花粉比率最高，为（25.11 ± 4.28�?。（2）与对照组相比，银灰杨雄花芽经高温处理后，花粉母细胞减数分裂微管骨架表现出不同程度的解聚，中期Ⅰ和中期Ⅱ的部分纺锤体微管缺失，致使后期Ⅰ和后期Ⅱ的同源染色体或姊妹染色单体分离异常，产生大量的落后染色体。这些落后染色体被遗弃在细胞质中，引起微核的产生，四分体时期形成多分体而导致花粉败育。（3）高温处理可导致银灰杨花药绒毡层退化延迟，但仍能正常开裂，释放出花粉粒，因此绒毡层延迟退化不是高温处理诱导银灰杨花粉败育的原因、�/sec> 结论高温处理诱导银灰杨花粉母细胞中期Ⅰ和中期Ⅱ的部分纺锤体微管缺失，致使后期Ⅰ和后期Ⅱ产生大量的落后染色体，引起大量多分体的产生，是高温处理诱导银灰杨花粉败育的细胞学机理、�/sec>

关键诌�
• 银灰�?/a> /
• 高温/
• 败育花粉/
• 减数分裂/
• 微管骨架/
• 绒毡屁�/a>

Abstract: ObjectiveThe effects of high temperature on microsporogenisis and cytological mechanism of pollen abortion induced by high temperature are conducted in P. canescens, which aiming at improving the technology of forest triploid breeding though inducing gamete chromosme doubling by high temperature. MethodIn this study, the effects of high temperature, meiotic stage and duration on percentage of aborted pollen were conducted after male flower buds were exposed to 38 � for 3 or 6 h in P. canescens. Subsequently, the differences in chromosome behaviour, meiotic microtubule cytoskeleton and development of tapetum between untreated pollen mother cells (PMCs) and treated PMCs were studied to reveal the cytological mechanism of aborted pollen production induced by high temperature. Result(1) The results indicated that meiotic stages, temperature, duration, meiotic stage × temperature interactions and meiotic stage × duration interactions had significant effects on percentage of aborted pollen. The highest percentage of aborted pollen was 25.11 ± 4.28% when PMCs were treated with 41 � for 3 h at metaphase �? (2) Compared with PMCs in the control group, meiotic microtubule cytoskeleton was depolymerized and some spindles were lost within the PMCs at metaphase � and �? leading to the abnormal chromosome segregation at anaphase � and � and forming a great number of lagging chromosomes. These lagging chromosomes was retained within cytoplasm, causing some micronuclei to form. Therefore, aborted pollen formed due to the formation of polyads at tetrad stage. (3) Although high temperature could also delay the degradation of the tapetum, the anthers normally dehisced and pollen grains were released. Thus, the delayed degradation of the tapetum was not responsible for the formation of aborted pollen. ConclusionAfter male flower buds was treated by 38 � for 3 or 6 h, some spindles were lost within the PMCs at metaphase � and �? leading to form a great number of lagging chromosomes and polyads, which was the cytological mechanism of aborted pollen formation induced by high temperature.

Key words:
• Populus canescens/
• high temperature/
• aborted pollen/
• meiosis/
• microtubule cytoskeleton/
• tapetum

�?nbsp; 1银灰杨高温处理组（a）和对照组（b）的败育花粉

黑色箭头指向的为败育花粉。The black arrow shows aborted pollen.

Figure 1.Aborted pollen in the treatment (a) and control group (b) ofP. canescens

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�?nbsp; 2银灰杨花粉母细胞对照组和38 ℃高温处理组的染色体行为

a. 对照组后期Ⅰ；b. 对照组后期Ⅱ；c. 对照组的四分体；d. 38 ℃高温持续处�? h后期Ⅰ的落后染色体；e. 38 ℃高温持续处�? h后期Ⅱ的落后染色体；f. 38 ℃高温持续处�? h后四分体时期产生的多分体；g. 38 ℃高温持续处�? h后期Ⅰ的落后染色体；h. 38 ℃高温持续处�? h后期Ⅱ的落后染色体；i. 38 ℃高温持续处�? h后四分体时期产生的多分体。黑色箭头指向的为落后染色体。标尺为10.0 μm。a, anaphase � in control group; b, lagging chromosomes at anaphase � in control group; c, normal tetrad in control group; d, lagging chromosomes in anaphase � derived from the treatment with 38 � for 3 h; e, lagging chromosomes in anaphase � derived from the treatment with 38 � for 3 h; f, polyad derived from the treatment with 38 � for 3 h; g, lagging chromosomes in anaphase I derived from the treatment with 38 � for 6 h; h, lagging chromosomes in anaphase � derived from the treatment with 38 � for 6 h; I, polyad derived from the treatment with 38 � for 6 h. The black arrow shows the lagging chromosomes. Bars = 10.0 μm.

Figure 2.Meiotic chromosome behaviours of PMCs in the control group and the treated groups by 38 � high temperature for 3 or 6 hr inP. canescens.

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�?nbsp; 3银灰杨花粉母细胞对照�?8 ℃高温处理组的微管骨枵�/p>

a. 对照组中期Ⅰ；b. 对照组后期Ⅰ；c. 对照组中期Ⅱ；d. 对照组后期Ⅱ；e. 对照组末期Ⅱ；f. 处理组（38 ℃高温处�? h）的中期Ⅰ；g. 处理组（38 ℃高温处�? h）的后期Ⅰ；h. 处理组（38 ℃高温处�? h）的中期Ⅱ；i. 处理组（38 ℃高温处�? h）的后期Ⅱ；j. 处理组（38 ℃高温处�? h）的末期Ⅱ；k. 处理组（38 ℃高温处�? h）中期Ⅰ；l. 处理组（38 ℃高温处�? h）的后期Ⅰ；m. 处理组（38 ℃高温处�? h）的中期Ⅱ；n. 处理组（38 ℃高温处�? h）的后期Ⅱ；o. 处理组（38℃高温处�? h）的末期Ⅱ。标尺为10.0 μm。a, metaphase � in the control group; b, anaphase � in the control group; c, metaphase � in the control group with; d, anaphase � in the control group; e, telophase � in the control group; f, metaphase � in the treatment group with 38 � for 3 h; g, anaphase � in the treatment group with 38 � for 3 h; h, metaphase � in the treatment group with 38 � for 3 h; i, anahase � in the treatment group with 38 � for 3 h; j, telophase � in the treatment group with 38 � for 3 h; k, metaphase � in the treatment group with 38 � for 6 h; l, anaphase � in the treatment group with 38 � for 6 h; m, metahase � in the treatment group with 38 � for 6 h; n, anahase � in the treatment group with 38 � for 6 h; o, telophase � in the treatment group with 38 � for 6 h. Scale bars = 10.0 μm.

Figure 3.The meiotic microtubular cytoskeletons of PMCs in the control group and the treated groups by 38 � inP. canescens.

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�?nbsp; 4银灰杨对照组�?8 ℃高温处理组的花药绒毡层发育进程

a. 对照组四分体时期；b. 对照组四分体形成�? d；c. 对照组四分体形成�? d；d. 对照组四分体形成�? d；e. 对照组四分体形成�? d；f. 38 ℃的高温持续处理3 h后四分体时期；g. 38 ℃的高温持续处理3 h，四分体形成�? d；h. 38 ℃的高温持续处理3 h，四分体形成�? d；i. 38 ℃的高温持续处理3 h四分体形成后6 d� j. 38 ℃的高温持续处理3 h，四分体形成�? d；k. 38 ℃的高温持续处理6 h后四分体时期；l. 38 ℃的高温持续处理6 h，四分体形成�? d� m. 38 ℃的高温持续处理6 h，四分体形成�? d；n. 38 ℃的高温持续处理6 h，四分体形成�? d� o. 38 ℃的高温持续处理6 h，四分体形成�? d。黑色箭头指向的为绒毡层。标尺为5.0 μm。a, tapetum of anthers from the control group at the tetrad stage. b, tapetum of anthers from the control group 2 days after the tetrad stage. c, tapetum of anthers from the control group 4 days after the tetrad stage. d, tapetum of anthers from the control group 6 days after the tetrad stage. e, tapetum of anthers from the control group 8 days after the tetrad stage. f, tapetum of anthers in the treatment with 38 � for 3 h at the tetrad stage. g, tapetum of anthers in the treatment with 38 � for 3 h 2 days after the tetrad stage. h, tapetum of anthers in the treatment with 38 � for 3 h 4 days after the tetrad stage. i, tapetum of anthers in the treatment with 38 � for 3 h 6 days after the tetrad stage. j, tapetum of anthers in the treatment with 38 � for 3 h 8 days after the tetrad stage. k, tapetum of anthers in the treatment with 38 � for 6 h at the tetrad stage. l, tapetum of anthers in the treatment with 38 � for 6 h 2 days after the tetrad stage. m, tapetum of anthers in the treatment with 38 � for 6 h 4 days after the tetrad stage. n, tapetum of anthers in the treatment with 38 � for 6 h 6 days after the tetrad stage. o, tapetum of anthers in the treatment with 38 � for 6 h 8 days after the tetrad stage. The black arrow points to the tapetum. Scale bars = 5.0 μm.

Figure 4.Tapetum development process of anthers in the control group and the treated groups by 38 � high temperature inP. canescens.

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�?nbsp; 1不同高温处理组合的败育花粉比玆�/p>

Table 1.Percentage of aborted pollen derived from different high temperature treatment combinations ofP. canescens

减数分裂时期 Meiotic stage	处理温度 Treating temperature/ℂ�/td>	持续处理时间 Duration/h	败育花粉比率 Percentage of aborted pollen/%
细线朞�br/>Leptotene	38	3	9.45 ± 0.92
		6	17.15 ± 2.56
	41	3	11.29 ± 1.77
		6	17.92 ± 2.55
偶线朞�br/>Zygotene	38	3	12.78 ± 5.06
		6	22.45 ± 2.21
	41	3	17.39 ± 5.65
		6	20.37 ± 0.83
粗线朞�br/>Pachytene	38	3	15.72 ± 2.02
		6	15.35 ± 4.33
	41	3	18.61 ± 5.20
		6	16.16 ± 1.35
双线朞�br/>Diplotene	38	3	19.90 ± 3.03
		6	19.33 ± 0.96
	41	3	20.97 ± 4.10
		6	20.93 ± 1.70
终变朞�br/>Diakinesis	38	3	14.17 ± 1.80
		6	13.83 ± 1.73
	41	3	16.95 ± 1.82
		6	20.62 ± 3.49
中期⅟�br/>Metaphase ⅟�/td>	38	3	10.82 ± 3.41
		6	13.76 ± 2.06
	41	3	25.11 ± 4.28
		6	24.13 ± 4.05
对照 Control			7.58 ± 1.32
注：对照组为不进行高温处理的雄花芽。下同。Note: The untreated male buds by high temperature serves as the control group. The same below.

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�?nbsp; 238 ℃处�? h�? h后出现落后染色体的花粉母细胞比率

Table 2.Percentage of lagging chromosomes induced by 38 � for 3 h and 6 h

持续处理时间 Duration/h	落后染色体的花粉母细胞比玆�br/>Percentage of PMCs with lagging chromosomes/%
	后期� Anaphase ⅟�/td>	后期� Anaphase Ⅰ�/td>
3	40.67 ± 3.06 b	33.33 ± 8.00 b
6	55.33 ± 4.16 b	48.67 ± 3.06 b
对照 Control	22.67 ± 4.16 a	13.33 ± 5.03 a
注：不同小写字母表示�?i>ɑ< 0.05水平显著。Note: Different lowercase represents significance atɑ< 0.05 level.

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[2]康宁, 白凤�? 张平�? �? 高温诱导胚囊染色体加倍获得毛白杨杂种三倍体[J]. 北京林业大学学报, 2015, 37(2): 79�?6. doi:10.13332/j.cnki.jbfu.2015.02.021

Kang N, Bai F Y, Zhang P D, et al. Inducing chromosome doubling of embryo sac in Populus tomentosawith high temperature exposure for hybrid triploids[J]. Journal of Beijing Forestry University, 2015, 37(2): 79�?6. doi:10.13332/j.cnki.jbfu.2015.02.021 [3]王君. 青杨派树种多倍体诱导技术研究[D]. 北京: 北京林业大学, 2009.

Wang J. Techniques of polyploid induction in Populusspp .(Section tacamahaca)[D]. Beijing: Beijing Forestry University, 2009. [4]李云, 朱之�? 田砚�? �? 极端温度处理白杨雌花芽培育三倍体植株的研究[J]. 北京林业大学学报, 2000, 22(5): 7�?2. doi:10.3321/j.issn:1000-1522.2000.05.002

Li Y, Zhu Z T, Tian Y T, et al. Obtaining triploids by high and low temperature treating female flower buds of white poplar[J]. Journal of Beijing Forestry University, 2000, 22(5): 7�?2. doi:10.3321/j.issn:1000-1522.2000.05.002 [5]Li Y, Wang Y, Wang P Q, et al. Induction of unreduced megaspores in Eucommia ulmoidesby high temperature treatment during megasporogenesis[J]. Euphytica, 2016, 212(3): 515�?24. doi:10.1007/s10681-016-1781-4 [6]杨珺. 桉树生殖生物学基础与染色体加倍技术研究[D]. 北京: 北京林业大学, 2015.

Yang J. Reproductive biology and techniques of chromosome doubling in Eucalyptus[D] .Beijing: Beijing Forestry University, 2015. [7]康向�? 朱之�? 张志�? 高温诱导白杨2 n花粉有效处理时期的研究[J]. 北京林业大学学报, 2000, 22(3): 1�?. doi:10.3321/j.issn:1000-1522.2000.03.001

Kang X Y, Zhu Z T, Zhang Z Z. Suitable period of high temperature treatment for 2 npollen of Populus tomentosa × P. bolleana[J]. Journal of Beijing Forestry College, 2000, 22(3): 1�?. doi:10.3321/j.issn:1000-1522.2000.03.001 [8]鲁敏. 响叶杨三倍体和四倍体诱导技术研究[D]. 北京: 北京林业大学, 2013.

Lu M. Techniques of triploid and teraploid induction in Populus adenopodaMaxim[D]. Beijing: Beijing Forestry University, 2013. [9]张磊, 王君, 索玉�? �? 高温诱导银白杨花粉染色体加倍研究[J]. 核农学报, 2010, 24(6): 1158�?165. doi:10.11869/hnxb.2010.06.1158

Zhang L, Wang J, Suo Y J, et al. Pollen chromosome doubling under high temperature in Populus albaL.[J]. Journal of Nuclear Agricultural Sciences, 2010, 24(6): 1158�?165. doi:10.11869/hnxb.2010.06.1158 [10]田梦�? 李燕�? 张平�? �? 高温诱导银灰杨花粉染色体加倍创制杂种三倍体[J]. 林业科学, 2018, 54(3): 39�?7.

Tian M D, Li Y J, Zhang P D, et al. Pollen chromosome doubling induced by high temperature exposure to produce hybrid triploids in Populus canescens[J]. Scientia Silvae Sinicae, 2018, 54(3): 39�?7. [11]Guan J Z, Wang J J, Cheng Z H, et al. Cytomixis and meiotic abnormalities during microsporogenesis are responsible for male sterility and chromosome variations in Houttuynia Cordata[J]. Genetics and Molecular Research, 2012, 11: 121�?30. doi:10.4238/2012.January.17.2 [12]Liu L W, Huang H, Gong Y Q, et al. Cytological and ultra-structural study on microsporogenesis of cytoplasmic male sterility in Raphanus sativus[J]. Joural of Integrative Plant Biology, 2009, 64(9): 716�?22. [13]Xu C G, Liu Z T, Zhang L P, et al. Organization of actin cytoskeleton during meiosisⅠin a wheat thermo-sensitive genic male sterile line[J]. Protoplasma, 2013, 250: 415�?22. doi:10.1007/s00709-012-0386-6 [14]张文�? 曹媛, 常童�? �? 毛白杨花粉败育过程中胼胝质的异常分布变化[J]. 东北林业大学学报, 2013, 41(1): 68�?1. doi:10.3969/j.issn.1000-5382.2013.01.017

Zhang W C, Cao Y, Chang T J, et al. Abnormal changes of callose distribution during pollen abortion in Populus tomentosaCarr.[J]. Journal of North-east Forestry University, 2013, 41(1): 68�?1. doi:10.3969/j.issn.1000-5382.2013.01.017 [15]李燕�? 高温诱导银灰杨产�? n花粉的细胞及分子机制[D]. 北京: 北京林业大学, 2017.

Li Y J. Cytological and molecular mechanism of 2 npollen production induced by high temperature in Populus canescens[D]. Beijing: Beijing Forestry University, 2017. [16]张媛. ‘抱头毛白杨’花粉败育的细胞学机理研究[D]. 北京: 北京林业大学, 2019.

Zhang Y. Cytological mechanism of pollen abortion in Populus tomentosa‘baotoubai’[D]. Beijing: Beijing Forestry University, 2019. [17]Koti S, Reddy K R, Reddy V R, et al. Interactive effects of carbon dioxide, temperature, and ultraviolet-B radiation on soybean ( Glycine maxL.) flower and pollen morphology, pollen production, germination, and tube lengths[J]. Journal Experiment botany, 2005, 56(412): 725�?36. doi:10.1093/jxb/eri044 [18]Sato S, Kamiyama M, Iwata N, et al. Moderate increase of mean daily temperature adversely affects fruit set of Lycopersicon esculentumby disrupting specific physiological processes in male reproductive development[J]. Annals of Botany, 2006, 97(5): 731�?38. doi:10.1093/aob/mcl037 [19]Porch T G, Jahn M. Effects of high-temperature stress on microsporogenesis in heat-sensitive and heat-tolerant genotypes of Phaseolus vulgaris[J]. Plant Cell & Environment, 2001, 24(7): 723�?31. [20]Peet M M, Sato S, Gardner R G. Comparing heat stress effects on male-fertile and male-sterile tomatoes[J]. Plant Cell & Environment, 1998, 21: 225�?31. [21]Young L W, Wilen R W, Bonham-Smith P C. High temperature stress of Brassica napusduring flowering reduces micro- and megagametophyte fertility, induces fruit abortion, and disrupts seed production[J]. Journal of Experiment botany, 2004, 396: 485�?95. [22]Cross R H, McKay S A B, McHughen A G, et al. Heat-stress effects on reproduction and seed set in Linum usitatissimumL. (flax)[J]. Plant Cell & Environment, 2003, 26(7): 1013�?020. [23]Johnsen Ø, Dæhlen O G, Østreng G, et al. Daylength and temperature during seed production interactively affect adaptive performance of Picea abiesprogenies[J]. New Phytologist, 2010, 168: 589�?96. [24]Johnsen Ø, Fossdal C G, Nagy N, et al. Climatic adaptation in Picea abiesprogenies is affected by the temperature during zygotic embryogenesis and seed maturation[J]. Plant Cell & Environment, 2010, 28(9): 1090�?102. [25]Lacey E P, Herr D. Parental effects in Plantago lanceolataL. �? Measuring parental temperature effects in the field[J]. Evolution, 2010, 54(4): 1207�?217. [26]Erickson A N, Markhart A H. Flower developmental stage and organ sensitivity of bell pepper ( Capsicum annuumL.) to elevated temperature[J]. Plant Cell & Environment, 2002, 25: 123�?30. [27]Cao Y Y, Duan H, Yang L N, et al. Effect of heat stress during meiosis on grain yield of rice cultivars differing in heat tolerance and its physiological mechanism[J]. Acta Agronomica Sinica, 2008, 34(12): 2134�?142. doi:10.1016/S1875-2780(09)60022-5 [28]Bomblies K, Higgins J D, Yant L. Meiosis evolves: adaptation to external and internal environments[J]. New phytologist, 2015, 208: 306�?23. doi:10.1111/nph.13499 [29]Wang J, Kang X Y. Distribution of microtubular cytoskeletons and organelle nucleoids during microsporogenesis in a 2 npollen producer of hybrid Populus[J]. Silvae Genetica, 2009, 58: 220�?26. doi:10.1515/sg-2009-0028 [30]李燕�? 田梦�? 刘燕, �? 银灰杨花粉母细胞减数分裂与微管骨架变化[J]. 北京林业大学学报, 2017, 39(5): 9�?6. doi:10.13332/j.1000-1522.20160417

Li Y J, Tian M D, Liu Y, et al. Meiosis and organization of microtubule of pollen mother cells in Populus canescens[J]. Journal of Beijing Forestry University, 2017, 39(5): 9�?6. doi:10.13332/j.1000-1522.20160417 [31]Scott R J, Spielman M, Dickinson H G. Stamen structure and function[J]. The Plant Cell, 2004, 16: 46�?0. doi:10.1105/tpc.017012 [32]张虹, 梁婉�? 张大�? 花药绒毡层细胞程序性死亡研究进展[J]. 上海交通大学学�? 农业科学�? 2008, 26(1): 86�?0.

Zhang H, Liang W Q, Zhang D B. Research progress on tapetum programmed cell death[J]. Journal of Shanghai Jiao Tong University: Agricultural Science, 2008, 26(1): 86�?0. [33]Wu H M, Cheung A Y. Programmed cell death in plant reproduction. Plant Molecular Biology, 2000, 44: 267-281. [34]Sanders P M, Lee P, Biesgen C, et al. The arabidopsis DELAYED DEHISCENCE 1 gene encodes an enzyme in the jasmonic acid synthesis pathway[J]. The Plant Cell, 2000, 12: 1041�?061. doi:10.1105/tpc.12.7.1041 [35]Jin W, Horner H T, Palmer R G. Genetics and cytology of a new genic male-sterile soybean [ Glycine max(L.) Merr.][J]. Sex Plant Reproductive, 1997, 10: 13�?1. doi:10.1007/s004970050062 [36]Worrall D, Hird D L, Hodge R, et al. Premature dissolution of the microsporocyte callose wall causes male sterility in transgenic tobacco[J]. The Plant Cell, 1992, 4: 759�?71. [37]张鹏�? 小麦雄性不育系绒毡层异常代谢与小孢子败育的关系[J]. 中国农业科学, 2014, 47(9): 1670�?680. doi:10.3864/j.issn.0578-1752.2014.09.002

Zhang P F. Relationship between microspore abortion of CMS lines associated with nutrient metabolism disorder in tapetal of anther in wheat ( Triticum aestivumL.)[J]. Scientia Agricultura Sinica, 2014, 47(9): 1670�?680. doi:10.3864/j.issn.0578-1752.2014.09.002 [38]Li B, Chen X P, Wu Y R, et al. Gene characterization and molecular pathway analysis of reverse thermosensitive genic male sterility in eggplant ( Solanum melongenaL.)[J/OL]. Horticulture Research, 2019, 6(1): 16. DOI: 10.1038/s41438-019-0201-z. [39]Endo M, Tsuchiya T, Hamada K, et al. High temperatures cause male sterility in rice plants with transcriptional alterations during pollen development[J]. Plant & cell physiology, 2009, 11: 1911�?922. [40]Abiko M, Akibayashi K, Sakata T, et al. High-temperature induction of male sterility during barley ( Hordeum vulgareL.) anther development is mediated by transcriptional inhibition[J]. Sexual Plant Reproductive, 2005, 18(2): 91�?00. doi:10.1007/s00497-005-0004-2 [41]Oshino T, Abiko M, Saito R, et al. Premature progression of anther early developmental programs accompanied by comprehensive alterations in transcription during high-temperature injury in barley plants[J]. Mollecular Genetics and Genomics, 2007, 278(1): 31�?2. doi:10.1007/s00438-007-0229-x [42]Papini A, Mosti S, Brighigna L. Programmed-cell-death events during tapetum development of angiosperms[J]. Protoplasma, 1999, 207: 213�?21. doi:10.1007/BF01283002 [43]Varnier A L, Mazeyrat-Gourbeyre F, Sangwan R S, et al. Programmed cell death progressively models the development of anther sporophytic tissues from the tapetum and is triggered in pollen grains during maturation[J]. Journal of Structural Biology, 2005, 152(2): 118�?28. doi:10.1016/j.jsb.2005.07.011