,, 骆永丽, 李文倩, 王元元, 张秋霞, 陈金, 金敏, 李勇, 王振林山东农业大学农学院/作物生物学国家重点实验室,山东泰安 271018Effects of Different Spring Nitrogen Topdressing Modes on Lodging Resistance and Lignin Accumulation of Winter Wheat
DONG HeHe,, LUO YongLi, LI WenQian, WANG YuanYuan, ZHANG QiuXia, CHEN Jin, JIN Min, LI Yong, WANG ZhenLinCollege of Agronomy, Shandong Agricultural University/State Key Laboratory of Crop Biology, Tai’an 271018, Shandong责任编辑: 杨鑫浩
收稿日期:2020-05-14接受日期:2020-07-29网络出版日期:2020-11-01
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Received:2020-05-14Accepted:2020-07-29Online:2020-11-01
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董荷荷, 骆永丽, 李文倩, 王元元, 张秋霞, 陈金, 金敏, 李勇, 王振林. 不同春季追氮模式对小麦茎秆抗倒性能及木质素积累的影响[J]. 中国农业科学, 2020, 53(21): 4399-4414 doi:10.3864/j.issn.0578-1752.2020.21.009
DONG HeHe, LUO YongLi, LI WenQian, WANG YuanYuan, ZHANG QiuXia, CHEN Jin, JIN Min, LI Yong, WANG ZhenLin.
0 引言
【研究意义】倒伏是小麦高产的限制因子,黄淮海麦区每年约有10%的面积发生倒伏,严重的年份高达20%以上,甚至出现绝收[1,2]。合理的氮肥调控可以有效缓解个体与群体的矛盾,改善群体结构,不合理追肥易造成茎秆质量降低,群体结构变差[3,4,5]。氮肥合理后移能够增加基部节间壁厚、充实度及木质素积累量,从而提升茎秆的抗倒性能[4,6]。目前在生产上仍存在高产农田过量施用氮肥的问题[7,8]。因此,试验在高施氮量300 kg·hm-2基施1/3条件下设置不同的追肥时期,探究高施氮量条件下不同春季追氮模式对茎秆各节间生育后期抗倒伏能力的影响,以期为小麦高产稳产抗逆应变栽培提供理论依据。【前人研究进展】氮肥调控主要包括合理的氮肥施用量和合适的基追比两方面,是作物增产的重要途径[9]。前人研究表明,在小麦和玉米上,施足底肥,分次施肥有助于提高作物籽粒产量及茎秆强度,孕穗期追肥可以提高小麦茎秆中木质素的积累量,提高茎秆的抗折力,进而提高茎秆的抗倒伏能力[10,11,12]。茎秆的抗倒伏性能与植株高度、茎粗、壁厚、抗折力等生理指标密切相关,还与茎秆的解剖结构以及木质素代谢有关[13,14,15,16,17,18,19,20,21]。合理的氮肥运筹可以提高茎秆基部第二节间木质素积累量以及木质素合成途径关键酶的活性,且合理的氮肥施用量可以提高茎秆各节间中木质素单体的含量[4,15]。过量施氮会导致水稻茎秆基部节间中木质素、纤维素、半纤维素的含量降低,使茎秆抗折力下降,茎秆质量劣化。水稻中穗肥过量降低了木质素合成基因PAL、COMT、4CL3、CCR、CAD2、CAD7在茎秆发育早期的表达量[22,23]。木质素是植物体中重要的苯酚类代谢产物,和纤维素、半纤维素约占植物体干重的15%—40%,存在于细胞壁的多聚糖框架中以增强植物的机械强度[24,25,26]。木质素是由松柏醇、芥子醇和对香豆醇聚合形成的物质,在相关酶的催化作用下对应生成愈创木基木质素（G型木质素）、紫丁香基木质素（S型木质素）和对羟基苯基木质素（H型木质素）[27,28,29],木质素单体的含量及比例与茎秆的机械强度有密切关系,其中起主要机械支撑作用的是S型木质素[13,21,30-32]。关于木质素合成途径的分子调控前人已经进行了较多的研究,木质素合成途径呈网络状,表现在中间产物及木质素的侧基修饰类型丰富,许多产物的生成不止一条合成途径,且这些木质素合成途径相关基因均表现为多基因家族[32]。木质素中3种单体含量和比例受物种、品种、生育时期、环境因素、木质素合成相关酶基因上调、下调以及相互作用的影响[33,34,35,36,37,38]。PAL是木质素合成途径的关键起始酶,其表达丰度影响着整个木质素代谢过程,PAL基因下调时,植物体内木质素含量降低[39]。F5H或COMT基因调控均可严重影响S型木质素的生物合成[40,41],水稻中过量表达F5H增加了S型单体的含量,减少了G型单体的含量,影响水稻的抗倒伏能力[34]。【本研究切入点】关于氮肥对小麦茎秆抗倒伏能力的研究多集中于氮肥施用量,对不同抗倒伏品种小麦茎秆性能影响的研究也多集中于基部节间。生育后期受外界天气及茎秆自身变化的影响,上部节间也容易发生倒伏[42,43],不同春季追氮模式对茎秆基部第二节间茎秆抗倒伏能力的调控已有报道,但对茎秆各节间抗折力、木质素单体积累以及木质素合成相关酶基因的表达量尚无深入研究。【拟解决的关键问题】本试验以抗倒伏性能不同的2个小麦品种为材料,在总施氮量300 kg·hm-2的条件下,研究不同春季追氮模式对茎秆各节间木质素积累规律的影响及抗折力的影响,为提高小麦各节间抗倒伏能力及小麦高产稳产抗逆应变栽培提供理论依据。1 材料与方法
1.1 试验设计
2017—2018和2018—2019年在山东农业大学农学实验站进行大田试验,试验选用倒伏敏感型品种山农16和抗倒伏品种济麦22。试验田土壤为棕壤土,前茬作物为玉米,秸秆全部还田。小麦全生育期施用纯N 300 kg·hm-2（尿素）,P2O5 90 kg·hm-2（过磷酸钙）,K2O 100 kg·hm-2（氯化钾）,种植密度300万株/hm2。钾肥、磷肥和1/3的氮肥作为基肥均匀施入田中,翻耕入土。剩余氮肥后期开沟追施,设置4个追肥时期,分别为等量二次性追氮和剩余一次性追氮,即起身期﹕孕穗期1/3﹕1/3（T1）、拔节期﹕开花期1/3﹕1/3（T2）、孕穗期一次性追施2/3（T3）、拔节期一次性追施2/3（CK）。小区面积9 m2（3 m×3 m）,每个处理设置3次重复,共24个小区,人工开沟播种,行距25 cm,其他管理同一般大田。1.2 测定项目和方法
1.2.1 茎秆抗折力的测定 于小麦开花期、灌浆期、成熟期取15株生长均匀一致的植株,分别测量各节间的抗折力,使用的仪器为浙江托普仪器有限公司生产的茎秆强度测定仪（YYD-1）,测量参照PENG等[18]和ZHENG等[13]的方法。将小麦从顶部向下依次分为I5、I4、I3、I2、I1,把每一节间中间部分置于茎秆强度测定仪的凹槽中,支撑长度5 cm,此时茎秆与压力器探头垂直,手持压力传感器手柄匀速下压,茎秆被折断时屏幕显示的数值（单位为N）表示为该节间的抗折力。1.2.2 木质素含量的测定 木质素含量的测定参照SYROS等[27]的方法略有改动。取鲜样置于研钵中,加液氮研磨成粉状。称取0.1 g于10 mL离心管中,加入8 mL 95%乙醇过夜浸提后离心去上清。沉淀中加入8 mL正己烷﹕乙醇=2﹕1的混合溶液浸提,离心弃上清,重复2次,50℃烘干至恒重。干燥物用2.5 mL 25%乙溴酰冰醋酸（体积比）溶解,70℃水浴保温30 min,加热期间摇晃2次混匀,冷水中快速冷却至室温,然后加入0.9 mL 2 mol·L-1 NaOH溶液终止反应,加0.1 mL 7.5 mol·L-1盐酸羟胺和4 mL冰乙酸,混匀后离心5 min,吸取0.1 mL上清液,加7.9 mL冰乙酸（前2次取样含量较少,可取上清液0.2 mL,加3.8 mL冰乙酸稀释20倍）稀释后测定A280nm。以单位质量鲜样在A280nm处的吸光值表示木质素含量。
1.2.3 木质素单体含量的测定 参照ZHENG等[17]的方法,茎秆样品研磨过筛,经NaCl溶液、无水乙醇、95%乙醇、丙酮、色谱氯仿﹕色谱甲醇1﹕1萃取后50℃烘干备用。准确称取0.02 g 上述烘干备用的样品,置于50 mL白色消解管中（微波消解仪配套白色50 mL消解管）,加入3 mL 2 mol·L-1的NaOH和0.5 mL的硝基苯,混匀后放入微波消解仪中（MULTIWAVE 3000）,150℃保温1 h,然后将样品转移到10 mL离心管中并加入0.5 mL 2 mol·L-1 NaOH冲洗白管,冲洗后的液体转移到10 mL离心管中,5 000×g离心5 min。吸取2.7 mL上清液转移到新的10 mL离心管中,加入4 mL乙酸乙酯（CNW）,用振荡器漩涡混匀5 000×g离心后,吸取3 mL上清转移到新的离心管中。重复加入4 mL乙酸乙酯,漩涡混匀吸取4 mL上清液,与上一步骤得到的3 mL乙酸乙酯合并。将7 mL有机相利用真空离心浓缩仪（Thermo Fisher）蒸干,蒸干后加6 mL 50%的乙腈水复溶样品,用振荡器涡旋并于超声仪中进行超声,使样品充分溶解混匀。将样品用50%的乙腈水稀释后过0.22 μm有机滤器,利用超高效液相-三重四级杆质谱仪（Xevo TQ-S,Waters,Milford,MA,USA）进行样品定性及定量的测定。
1.2.4 小麦茎秆木质素合成关键酶基因表达丰度的测定 将按照节间分好的鲜样用液氮研磨至粉末状,然后迅速转移到无RNA酶、无DNA酶、无菌的2 mL离心管中,利用全式金试剂盒提取RNA,然后用全式金反转录试剂盒反转录为cDNA。配制96孔板所需体系,每孔配制20 μL,引物序列如表1所示。将20 μL体系分别加入96孔板相应孔内（操作要缓慢,避免产生气泡）,将96孔板放入ABI 7500型实时荧光定量PCR仪（赛默飞）中反应,进行目的基因的表达分析。
Table 1
表1
表1木质素合成途径中关键基因引物及探针的序列
Table 1
| 名称 | 序列-F (5′-3′) | 序列-R (5′-3′) |
|---|---|---|
| ACTIN | GGGACCTCACGGATAATCTAATG | CGTAAGCGAGCTTCTCCTTTAT |
| PAL | CATCTTGGAGGGAAGCTCATAC | GACTTGGTGGCAAATCGAATAAC |
| C4H | GCCGAGAGCAAGATCCTCGT | CGTGCTTCTCCTCCTCCAGG |
| HCT | TGTCAGCATTGCTCCGTGGA | CCAGGAGCCATGAACACCGG |
| C3H | CCCATCCTCGGCATCACCAT | CGCCCTTCTCAGTCGTGTCA |
| CCR | CGTGATGGTGCTGAAGAAAC | CGATCATCGAAGCCGATACA |
| CAD | GAGGTCGTCAAGATGGAC | CTAGCTCTTTCTCCCTCTG |
| 4CL | TGCACACTGGAGACATTGGC | TTCGAGTTCCGCAGGAGGTA |
| F5H | AGCTCCCCTCTCTCAAGTGC | GACACAGTCCTCGGCGTTCT |
| COMT | GATCCATGACAACGAGTCTACC | CGAATCAATCGACGACACAAAC |
| CSE | GCCAGCAAGGACAAGACCATCA | GCTGAGCCAGGCGAGGATGT |
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Table 2
表2
表2不同春季追氮模式对不同抗倒伏型小麦品种各节间抗折力的影响
Table 2
| 年份 Year | 节间 Internode | 处理 Treatment | SN16 | JM22 | ||||
|---|---|---|---|---|---|---|---|---|
| 开花期 Anthesis stage | 灌浆期 Filling stage | 成熟期 Maturity stage | 开花期 Anthesis stage | 灌浆期 Filling stage | 成熟期 Maturity stage | |||
| 2017-2018 | I1 | CK | 5.99ab | 6.65c | 3.82bcd | 7.54a | 7.79cd | 4.36ef |
| T1 | 5.92bc | 7.75a | 4.76a | 7.52a | 9.38a | 5.38a | ||
| T2 | 4.85ef | 7.03bc | 4.00bc | 6.83b | 8.05cd | 4.74de | ||
| T3 | 5.24de | 7.28ab | 4.83a | 5.89d | 8.62b | 4.83bcd | ||
| I2 | CK | 6.47a | 5.59ef | 3.64cd | 7.17ab | 6.39fg | 4.20fg | |
| T1 | 5.62bcd | 6.97bc | 4.80a | 7.03b | 8.11c | 5.26ab | ||
| T2 | 4.97e | 5.81de | 3.95bc | 6.29c | 7.03e | 4.77de | ||
| T3 | 5.48cd | 6.16d | 4.06b | 5.78de | 7.66d | 4.78cd | ||
| I3 | CK | 5.19de | 4.61g | 3.15fg | 5.47efg | 5.83hi | 4.04fgh | |
| T1 | 4.42fg | 5.72def | 4.12b | 5.75de | 6.60f | 4.61de | ||
| T2 | 4.40fg | 4.68g | 3.18fg | 5.27fg | 6.23fgh | 3.82hij | ||
| T3 | 3.76ij | 5.49ef | 3.50def | 4.22j | 6.49f | 5.22abc | ||
| I4 | CK | 4.08ghi | 4.02ij | 2.70hij | 5.54def | 4.55k | 3.26kl | |
| T1 | 4.35fgh | 5.31f | 3.55de | 5.45efg | 5.98ghi | 4.82bcd | ||
| T2 | 3.89hi | 4.52gh | 2.93ghi | 5.07gh | 5.63ij | 3.97gh | ||
| T3 | 3.35jk | 5.30f | 3.20efg | 4.71hi | 5.33j | 3.92ghi | ||
| I5 | CK | 3.20kl | 3.46k | 1.84k | 4.33ij | 3.48l | 2.68m | |
| T1 | 3.40jk | 4.47ghi | 2.97gh | 4.68hi | 4.60k | 3.53jk | ||
| T2 | 2.54m | 3.74jk | 2.47j | 4.08jk | 4.38k | 3.04lm | ||
| T3 | 2.73lm | 4.09hij | 2.59ij | 3.61k | 4.35k | 3.56ijk | ||
| 2018-2019 | I1 | CK | 6.46a | 6.93cd | 3.92cde | 8.07a | 7.70bc | 4.25de |
| T1 | 6.48a | 7.94a | 4.99a | 7.80a | 8.54a | 5.31a | ||
| T2 | 5.36bc | 7.31bc | 4.28bc | 6.85b | 8.07ab | 4.89b | ||
| T3 | 5.76b | 7.78ab | 4.49ab | 6.64bc | 8.20ab | 4.84b | ||
| I2 | CK | 5.05cd | 5.62ef | 3.36fgh | 5.58e | 6.65d | 4.13de | |
| T1 | 5.45bc | 6.67d | 4.74ab | 5.69e | 8.16ab | 4.75bc | ||
| T2 | 4.80de | 5.87e | 3.93cde | 5.04f | 7.39c | 3.86e | ||
| T3 | 4.29efghi | 6.97cd | 3.97cde | 4.40gh | 7.33c | 4.33d | ||
| I3 | CK | 4.44efg | 4.75gh | 2.91hi | 5.62e | 5.45gh | 3.88e | |
| T1 | 4.64def | 5.55ef | 4.28bcd | 5.55e | 6.52de | 4.76bc | ||
| T2 | 3.79ijk | 5.15fg | 3.60efg | 4.48gh | 6.07ef | 3.94de | ||
| T3 | 4.11ghi | 5.20fg | 3.68ef | 5.06f | 6.12ef | 4.36cd | ||
| I4 | CK | 4.06ghi | 4.04ij | 2.28j | 5.82de | 4.65ij | 3.30gh | |
| T1 | 4.35efgh | 5.68ef | 3.77def | 6.22cd | 6.19def | 3.82ef | ||
| T2 | 3.55jk | 4.35hi | 3.15hi | 5.66e | 5.45gh | 3.33fgh | ||
| T3 | 3.51k | 5.45ef | 2.93gh | 4.75fg | 5.89fg | 3.86e | ||
| I5 | CK | 3.92hij | 4.04ij | 1.24k | 4.63fg | 4.07k | 3.07gh | |
| T1 | 4.22fghi | 4.57h | 3.13gh | 4.71fg | 5.13hi | 3.33gh | ||
| T2 | 3.40k | 3.62j | 3.05h | 3.59i | 4.45jk | 2.95h | ||
| T3 | 2.58l | 4.49hi | 2.39ij | 4.09hi | 5.05hi | 3.41fg | ||
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比较2种类型品种不同节间的抗折力,从I1—I5呈现逐渐下降的趋势。以2017—2018年灌浆期T1处理的5个节间为例,与I1相比,山农16 I2、I3、I4、I5抗折力分别下降了11.19%、35.49%、45.95%、73.38%,济麦22抗折力分别降低了15.66%、42.12%、56.86%、103.91%。不同节间之间抗折力差异显著,上部节间抗折力显著小于下部节间,在生育后期受恶劣天气的影响更容易发生倒伏,因此上部节发生倒伏的可能性同样不可忽视。
2.2 不同春季追氮模式对小麦茎秆木质素积累量的影响
不同春季追氮模式对小麦茎秆木质素的积累具有调控作用。抗倒伏品种济麦22各时期木质素积累量高于山农16,在不同生育时期,木质素的积累量呈现显著差异。随着生育进程推进,木质素总积累量增加,各生育阶段木质素积累量呈逐渐减小的趋势（图1）。图1
新窗口打开|下载原图ZIP|生成PPT图1不同春季追氮模式对小麦茎秆木质素含量的影响
SN16表示山农16,JM22表示济麦22。J表示拔节期,A表示开花期,F表示灌浆期,M表示成熟期。下同
Fig. 1Effect of different spring nitrogen topdressing modes on lignin content of the five internodes
SN16 and JM22 represent Shannong16 and Jimai22. J represent jointing stage, A represent anthesis stage, F represent filling stage, M represent maturity stage. The same as below
不同处理下,拔节期至开花期木质素积累量表现为T1>T3>CK>T2,开花期到灌浆期及灌浆期到成熟期木质素积累量表现为T1>T3>T2>CK,2年结果趋于一致。以2018—2019年第二节间为例,拔节期至开花期山农16和济麦22在T1处理下木质素积累量分别比CK、T2、T3处理高出13.43%、30.69%、12.88%和30.79%、38.33%、29.47%,开花期至灌浆期山农16和济麦22在T1处理下木质素积累量分别比CK、T2、T3处理高出28.58%、0.27%、4.71%和22.11%、15.97%、16.80%,灌浆期至成熟期山农16和济麦22在T1处理下木质素积累量分别比CK、T2、T3处理高出23.11%、15.38%、8.96%和22.66%、10.97%、1.23%。说明起身期﹕孕穗期等量二次性追肥提高了茎秆后期各生育阶段木质素积累量。
相同处理下,不同节间之间存在差异,基部节间木质素积累量要高于上部节间,在2018—2019年T1处理中,山农16第一节间3个生育时期均值较第二节间、第三节间、第四节间、第五节间分别高出18.46%、30.63%、32.10%、46.53%。JM22第一节间3个生育时期均值较其他节间高出7.86%、24.01%、42.14%、49.51%。不同处理之间节间变化趋势与T1处理变化规律一致。3个生育阶段各节间木质素的积累量随生育期呈现降低的趋势。
2.3 不同春季追氮模式对小麦茎秆木质素单体含量及积累规律的影响
木质素是小麦茎秆的重要组成成分,由愈创木基木质素（G型）、紫丁香基木质素（S型）和对羟基苯基木质素（H型）3种单体组成。由图2可知,在小麦中G型、S型单体的含量显著高于H型,抗倒伏品种济麦22 3种单体的总含量显著高于倒伏敏感型品种山农16。开花期2种类型品种不同处理间S型单体含量的变化趋势表现为T1>T3>CK>T2,灌浆期和成熟期表现为T1>T3>T2>CK。随着生育进程推进,3种单体含量的总和逐渐增加,S型/G型的比例逐渐减少,S型单体含量占3种单体含量总和的31.05%—49.80%,G型单体含量占3种单体含量总和的41.94%—53.21%,G型+S型单体含量占3种单体含量总和的78.05%—93.31%（以2018—2019年数据为例）。不同春季追氮模式对小麦茎秆木质素含量及其积累规律有一定影响,以2018—2019年成熟期第二节间为例,山农16 T1处理S型单体含量分别比CK、T2、T3处理增加了13.01%、25.62%、5.55%,G型单体含量分别比CK、T2、T3处理增加了17.18%、18.01%、1.24%,JM22 T1处理S型单体含量分别比CK、T2、T3处理增加了24.62%、11.34%、11.22%,G型单体含量分别比CK、T2、T3处理增加了14.38%、9.67%、3.03%。追肥模式以起身期﹕孕穗期1﹕1等量二次性追氮模式显著增加了茎秆生育后期各节间G型、S型、H型单体的含量,拔节期全部追施不利于生育后期木质素单体的积累。图2
新窗口打开|下载原图ZIP|生成PPT图2不同春季追氮模式对不同抗倒伏品种小麦各节间木质素单体含量及积累的影响
Fig. 2The content and accumulation regularity of lignin monomers in different internodes of two wheat cultivars under different spring nitrogen topdressing modes
各节间变化规律趋于一致,从开花期到成熟期G型、S型、H型单体的含量逐渐增大。从I1至I5 S型、G型单体的含量逐渐下降,3种单体的总含量也逐渐下降。2018—2019年山农16成熟期第一节间S型单体含量较第二节间、第三节间、第四节间、第五节间分别高出13.71%、39.56%、45.72%、61.26%,G型单体含量较其他节间分别高出2.73%、10.95%、28.67%、54.14%。不同处理间各节间变化趋势一致,说明起身期﹕孕穗期1﹕1等量二次性追施氮肥有利于提高高氮肥处理下小麦生育后期茎秆各节间木质素单体的积累量,孕穗期一次性追氮也有利于生育后期茎秆各节间木质素3种类型单体的积累。
茎秆抗折力与木质素积累量、S型单体含量以及S型+G型单体含量呈极显著正相关（表3）。2018—2019年成熟期抗折力与木质素积累量以及S型单体含量、S型+G型单体含量相关系数分别为0.85、0.70和0.66,结果表明抗折力高的品种茎秆中木质素积累量以及S型单体含量、S型+G型单体含量较高。本试验中,T1处理茎秆抗折力优于其他处理,说明在高施氮量300 kg·hm-2基施1/3条件下可以通过起身期﹕孕穗期1﹕1等量二次性追施提高茎秆中木质素积累量、S型单体含量及G型+S型单体的含量进而提高茎秆的抗倒伏能力。
Table 3
表3
表3抗折力与木质素积累的相关分析
Table 3
| 年份 Year | 生育时期 Growth stage | 木质素积累量 Lignin accumulation | 木质素单体含量 Lignin monomer content | |
|---|---|---|---|---|
| S型 | S型+G型 | |||
| 2017-2018 | 开花期Anthesis stage | 0.82** | 0.61** | 0.68** |
| 灌浆期Filling stage | 0.93** | 0.63** | 0.61** | |
| 成熟期Maturity stage | 0.90** | 0.90** | 0.84** | |
| 2018-2019 | 开花期Anthesis stage | 0.75** | 0.74** | 0.71** |
| 灌浆期Filling stage | 0.91** | 0.82** | 0.70** | |
| 成熟期Maturity stage | 0.85** | 0.70** | 0.66** | |
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2.4 不同春季追氮模式对小麦茎秆木质素合成相关酶基因表达丰度的影响
苯丙氨酸解氨酶PAL是木质素合成途径的关键起始酶。由图3可知,木质素合成相关酶基因苯丙氨酸解氨酶（PAL）、肉桂酸4羟化酶（C4H）、羟基肉桂酰辅酶A转移酶（HCT）、香豆酸-3-羟基氧化酶（C3H）、肉桂酰辅酶 A 还原酶（CCR）、肉桂醇脱氢酶（CAD）、香豆酸辅酶A连接酶（4CL）、阿魏酸 5 羟化酶（F5H）、咖啡酸3氧甲基转移酶（COMT）、咖啡酰莽草酸酯酶（CSE）在抗倒伏品种济麦22中基因表达丰度高于倒伏敏感型品种山农16。随着生育进程的推进,PAL、C3H、C4H、COMT、F5H等木质素合成相关酶基因的表达丰度呈逐渐降低的趋势。开花期第二节间不同处理间表现为T1>CK>T3>T2,开花后7 d及之后的时期,不同处理间表现为T1>T3>T2>CK。说明在本试验追肥条件下,将追肥时期改为起身肥﹕孕穗期1﹕1等量二次性追施可以提高开花期及之后时期茎秆木质素合成相关酶基因的表达丰度,拔节期一次性追施以及过晚施肥均不利于茎秆中木质素合成途径相关酶基因的表达,进而影响茎秆中木质素单体的含量和抗折力,最终影响茎秆质量。图3
新窗口打开|下载原图ZIP|生成PPT图3不同春季追氮模式对小麦茎秆木质素合成途径相关酶基因表达的影响
5月3日标为开花后0 d,7 DAA、14 DAA、21 DAA分别表示开花后7 d,14 d,21 d
Fig. 3Effects of lignin synthesis pathway related gene expression on lodging resistance of different wheat cultivars under different spring nitrogen topdressing modes
May 3 is marked as 0 d after anthesis, 7 DAA, 14 DAA and 21 DAA respectively represent 7 d, 14 d and 21 d after anthesis.
不同节间木质素合成途径相关酶基因的表达丰度呈现I5>I4>I3>I2>I1的趋势。相同生育时期其余节间木质素合成途径相关酶基因的表达丰度与第二节间开花期后木质素合成途径相关酶的基因表达丰度变化趋势基本一致。结果表明,不同春季追氮模式对茎秆各节间木质素合成相关酶基因的表达有调控作用,起身期﹕孕穗期1﹕1等量二次性追施可以提高各节间开花期及之后生育时期木质素合成相关酶基因的表达。
2.5 不同春季追氮模式对小麦籽粒产量和产量构成因素的影响
山农16籽粒产量呈现T1>T2>T3>CK的趋势,济麦22籽粒产量呈现出T1>T2>CK>T3的趋势,2种类型品种均以T1处理籽粒产量最高,山农16在T1处理下2年籽粒产量平均比CK、T2、T3高7.47%、4.37%、5.49%。济麦22在T1处理下2年籽粒产量平均比其他处理高9.97%、6.58%、12.59%（表4）。CK、T3处理产量差异不显著,说明过晚追肥、一次性追肥均不利于籽粒产量的提高。不同处理之间,以T3处理千粒重最高,说明孕穗期追肥可以显著提高籽粒的产量。虽然T3处理千粒重较高,但由于追肥时期过晚,植株成穗数少,导致T3籽粒产量低于T1。拔节期一次性追氮模式虽可保证足够穗数,但不利于提高茎秆抗折能力。Table 4
表4
表4不同春季追氮模式对不同抗倒伏能力小麦籽粒产量的影响
Table 4
| 年份 Year | 品种 Cultivar | 处理 Treatment | 穗数 Spike number (×104·hm-2) | 穗粒数 Grain number per spikes | 千粒重 1000-grain weight (g) | 籽粒产量 Grain yield (kg·hm-2) |
|---|---|---|---|---|---|---|
| 2017-2018 | SN16 | CK | 662ab | 37.11ab | 40.89b | 7833.4c |
| TI | 688a | 38.46a | 42.08ab | 8387.1a | ||
| T2 | 652ab | 35.47b | 41.26b | 8066.3b | ||
| T3 | 628b | 38.07ab | 44.89a | 7997.3bc | ||
| JM22 | CK | 691a | 36.10a | 41.78c | 8336.8bc | |
| TI | 710a | 37.79a | 45.02ab | 9136.4a | ||
| T2 | 678a | 35.47a | 43.82bc | 8602.8b | ||
| T3 | 659a | 35.88a | 45.67a | 8107.8c | ||
| 2018-2019 | SN16 | CK | 719b | 39.35a | 44.30b | 8035.0c |
| TI | 756a | 39.60a | 45.39a | 8667.5a | ||
| T2 | 690bc | 38.56a | 44.60b | 8273.9b | ||
| T3 | 671c | 39.40a | 45.55a | 8168.4bc | ||
| JM22 | CK | 747b | 42.45ab | 45.91b | 8630.6bc | |
| TI | 775a | 42.73a | 46.92a | 9523.7a | ||
| T2 | 716c | 37.79c | 46.06b | 8904.9b | ||
| T3 | 694c | 40.26b | 46.86a | 8466.8c |
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由表5可知,对2018—2019年开花期、灌浆期、成熟期茎秆基部第二节间茎秆抗折力、木质素积累量等抗倒伏指标与籽粒产量进行相关分析,籽粒产量与木质素积累量以及S型单体含量呈极显著正相关（籽粒产量与开花期S型单体含量呈显著正相关）,籽粒产量与成熟期茎秆抗折力、木质素积累量和S型单体的含量呈极显著正相关,相关指数分别为0.69、0.91和0.81,与G型单体含量、S型+G型单体的含量呈显著正相关,相关系数为0.75和0.77。说明合理的栽培措施不仅可以提高茎秆的质量,籽粒产量也会增加。
Table 5
表5
表5茎秆抗倒伏指标与籽粒产量指标的相关分析（2018—2019）
Table 5
| 生育时期 Growth stage | 抗折力 Breaking strength | 木质素积累量 Lignin accumulation | 木质素单体含量Lignin monomer content | |||
|---|---|---|---|---|---|---|
| S型 | G型 | S型+G型 | ||||
| 籽粒产量 Yield | 开花期Anthesis stage | 0.66 | 0.86** | 0.71* | 0.69* | 0.75* |
| 灌浆期Filling stage | 0.82** | 0.92** | 0.85** | 0.82** | 0.83** | |
| 成熟期Maturity stage | 0.69** | 0.91** | 0.81** | 0.75* | 0.77* | |
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3 讨论
3.1 不同春季追氮模式对小麦茎秆抗倒性能的影响
优化的氮肥施用模式（基肥﹕拔节肥﹕孕穗肥4﹕4﹕2）可以提高茎秆中木质素合成关键酶PAL、TAL、POD的活性以及木质素积累量,提高茎秆抗折力,最终提高茎秆抗倒伏能力[4]。施氮量过高导致茎秆壁厚、充实度降低,大小维管束数目及木质素积累量减少,茎秆抗折力降低,在生长发育后期易引发倒伏[15,23]。本试验探讨了高施氮量300 kg·hm-2且基施1/3条件下不同春季追氮模式对茎秆各节间抗折力的影响,不同处理间茎秆抗折力以起身肥﹕孕穗肥等量二次性追施茎秆抗折力优于其他处理,且2种类型品种抗折力随生育时期均呈现先增高后降低的趋势,在灌浆期达到最大值。S型单体主要为茎秆提供机械强度支撑[13,15]。在本试验条件下,S型单体的含量在2年生长季的不同生育时期均表现为起身期﹕孕穗期1﹕1等量二次性追施显著高于其他处理。开花期木质素各生育阶段积累量、单体的含量呈现T1>T3>CK>T2的规律,灌浆期和成熟期呈现T1>T3>T2>CK的趋势。起身期﹕孕穗期等量二次性追施（T1）提高了高氮肥种植条件下小麦茎秆各节间木质素合成相关酶基因的表达量、各生育阶段木质素积累量及单体的含量,说明起身肥﹕孕穗肥等量二次性追施通过提高各节间木质素合成途径关键酶的表达丰度,进而提高了茎秆各节间木质素的积累量,最终提高了各节间茎秆抗折力。茎秆质量和木质素代谢研究表明,木质素的含量与茎秆的抗倒伏能力密切相关,抗倒伏强的品种木质素含量较高[4,17]。在本研究中,相同处理间抗倒伏品种济麦22抗折力、木质素的积累量、单体的含量较倒伏敏感型品种山农16高。倒伏一般发生在小麦生长发育的中后期[5]。本研究探究了从开花期到成熟期,茎秆各节间木质素单体的含量、木质素总量等指标随生育时期的变化。木质素合成相关酶基因的表达量随着生育时期呈现降低趋势,木质素总积累量随生育时期增加,但积累速率逐渐降低。3种木质素单体S型、G型、H型的含量随生育时期逐渐增加,S型/G型单体的值减小,这与抗折力随生育时期进程的变化规律不一致。本研究还发现,2个生长季茎秆各节间抗折力与木质素积累量、S型单体含量以及S型+G型单体含量均呈显著正相关。结合前人研究结果,高施氮量300 kg·hm-2且基施1/3条件下,在小麦不同生育时期可以通过合理追肥定向调控茎秆的机械强度,将追肥时期改为起身期﹕孕穗期1﹕1等量二次性追施,提高茎秆各节间木质素合成关键酶基因的表达以及木质素单体的积累,以达到提高茎秆抗倒伏能力的目的。不同节间之间,茎秆基部节间短而充实,上部节间中空细长,且上部节间拔节时间晚,木质素积累量小于下部节间。从I1—I5茎秆抗折力、木质素积累量及单体的含量逐渐减少,木质素合成相关酶各节间表达规律与上述指标相反,从I1—I5相关酶基因的表达量逐渐增加,推测是由于上部节间拔节较晚,茎秆组织幼嫩,相关酶基因的表达量较高。
3.2 不同春季追氮模式对冬小麦籽粒产量的影响
在实际大田生产中,兼顾籽粒产量和茎秆质量一直是研究的重点[14-15,18]。小麦籽粒产量及构成因素受多种条件的影响,包括氮肥施入量、肥料利用率、施用时期以及氮肥基追比例[4,6,9-10]。合适的栽培措施可以保证茎秆质量,增加籽粒产量,且适宜的追肥时期可显著增加茎秆中的单糖、多糖的含量,提高茎秆充实度,促使纤维素及木质素的形成,提高抗折力,改善其抗倒伏性能[44,45,46]。本研究表明,起身期、拔节期追肥有助于提高小麦分蘖成穗率,孕穗肥有助于显著提高千粒重,减缓后期木质素积累速率的降低,增加木质素单体的含量。本研究还发现,从开花期到成熟期,籽粒产量与木质素积累量以及S型单体含量呈极显著正相关（籽粒产量与开花期S型单体含量呈显著正相关）,说明在增强茎秆抗倒伏能力的同时,籽粒产量也能得到提高。结合前人分析可得,在高施氮量300 kg·hm-2基施1/3情况下,在小麦生育后期以起身期﹕孕穗期1﹕1等量二次性追氮可以提高茎秆中木质素总积累量及木质素单体的积累,有助于提高茎秆抗折力,增加籽粒产量。4 结论
本试验条件下,基肥﹕起身肥﹕孕穗肥为1﹕1﹕1施用时较其他处理显著提高了小麦生育后期茎秆各节间的抗折力、木质素积累量、单体含量、木质素合成途径相关酶基因的表达以及产量。开花期、灌浆期、成熟期茎秆抗折力与木质素积累量、S型单体含量、S型+G型单体含量存在极显著正相关关系,说明抗倒伏品种具有更高的木质素合成相关基因的表达量、木质素积累量和木质素单体含量。起身期：孕穗期1﹕1等量二次性追施既可以保证单位面积成穗数,又能提高粒重,进而获得高产。拔节期一次性追肥虽然可以保证足够的穗数,但不利于茎秆各节间抗折力和产量的提高。因此,在高施氮量300 kg·hm-2且基施1/3条件下,起身期与孕穗期1﹕1等量二次性追氮模式可作为黄淮海麦区高施氮量300 kg·hm-2基施1/3条件下既能保证高产又能提高生育后期茎秆质量的春季适宜追氮模式。参考文献 原文顺序
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DOI:10.1126/science.1246843URLPMID:24833396 [本文引用: 2]
Research and development activities directed toward commercial production of cellulosic ethanol have created the opportunity to dramatically increase the transformation of lignin to value-added products. Here, we highlight recent advances in this lignin valorization effort. Discovery of genetic variants in native populations of bioenergy crops and direct manipulation of biosynthesis pathways have produced lignin feedstocks with favorable properties for recovery and downstream conversion. Advances in analytical chemistry and computational modeling detail the structure of the modified lignin and direct bioengineering strategies for future targeted properties. Refinement of biomass pretreatment technologies has further facilitated lignin recovery, and this coupled with genetic engineering will enable new uses for this biopolymer, including low-cost carbon fibers, engineered plastics and thermoplastic elastomers, polymeric foams, fungible fuels, and commodity chemicals.
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DOI:10.1186/s12284-016-0103-8URLPMID:27369289 [本文引用: 2]
BACKGROUND: Lodging in rice production often limits grain yield and quality by breaking or bending stems. Excessive nitrogen (N) fertilizer rates are the cause of poor lodging resistance in rice, but little is known about the effect of top-dressing N application rates on the mechanical strength of japonica rice plants, especially how the anatomical structure in culms is affected by N. In this study, field experiments on two japonica rice varieties with three top-dressing N application rates, 0 kg N ha(-1) (LN), 135 kg N ha(-1) (MN), and 270 kg N ha(-1) (HN) as urea, were conducted. Wuyunjing23, a lodging-resistant japonica rice cultivar and W3668, a lodging-susceptible japonica rice cultivar were used. The lodging index, breaking strength, morphological and anatomical traits in culms were measured in this study. RESULTS: The visual lodging rate in japonica rice differed remarkably between genotypes and top-dressing N treatments. The higher lodging index of rice plants was primarily attributed to the weak breaking strength of the lower internodes. The longer elongated basal internodes were responsible for higher plant height and a higher lodging index. Correlation analysis showed that breaking strength was significantly and positively correlated with the thickness of the mechanical tissue but was significantly and negatively correlated with the inner diameter of the major axis (b2). With increasing top-dressing N rates, the sclerenchyma cells of the mechanical tissues and the vascular bundles of the Wuyunjing23 cultivar varied little. The plant height, inner diameter of the minor axis (a2) and b2 increased significantly, but the area of the large vascular bundle (ALVB) and the area of the small vascular bundle (ASVB) decreased significantly and resulted in lower stem strength and a higher lodging index under higher top-dressing N conditions. The culm diameter of the W3668 cultivar increased slightly with no significant difference, and the sclerenchyma cells in the mechanical tissues and vascular bundles showed deficient lignifications under high top-dressing N conditions. Moreover, the ALVB and the ASVB decreased significantly, while the area of air chambers (AAC) increased rapidly. CONCLUSIONS: An improvement in the lodging resistance of japonica rice plants could be achieved by reducing the length of the lower internodes, decreasing the inner culm diameter and developing a thicker mechanical tissue. Top-dressing N application increased the plant height and inner culm diameter and decreased the ALVB and the ASVB of the Wuyunjing23 cultivar and caused deficient lignified sclerenchyma cells, lowered the ALVB and the ASVB, and increased the AAC of the W3668 cultivar resulting in weaker stem strength and a higher lodging index.
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DOI:10.1111/j.1469-8137.2010.03572.xURLPMID:21158867 [本文引用: 1]
* Reduced lignin content in perennial crops has been sought as a means to improve biomass processability for paper and biofuels production, but it is unclear how this could affect wood properties and tree form. * Here, we studied a nontransgenic control and 14 transgenic events containing an antisense 4-coumarate:coenzyme A ligase (4CL) to discern the consequences of lignin reduction in poplar (Populus sp.). During the second year of growth, trees were grown either free-standing in a field trial or affixed to stakes in a glasshouse. * Reductions in lignin of up to 40% gave comparable losses in wood strength and stiffness. This occurred despite the fact that low-lignin trees had a similar wood density and up to three-fold more tension wood. In free-standing and staked trees, the control line had twice the height for a given diameter as did low-lignin trees. Staked trees had twice the height for a given diameter as free-standing trees in the field, but did not differ in wood stiffness. * Variation in tree morphogenesis appears to be governed by lignin x environment interactions mediated by stresses exerted on developing cells. Therefore our results underline the importance of field studies for assessing the performance of transgenic trees with modified wood properties.
DOI:10.1007/s00425-017-2692-xURLPMID:28421330 [本文引用: 2]
MAIN CONCLUSION: Regulation of a gene encoding coniferaldehyde 5-hydroxylase leads to substantial alterations in lignin structure in rice cell walls, identifying a promising genetic engineering target for improving grass biomass utilization. The aromatic composition of lignin greatly affects utilization characteristics of lignocellulosic biomass and, therefore, has been one of the primary targets of cell wall engineering studies. Limited information is, however, available regarding lignin modifications in monocotyledonous grasses, despite the fact that grass lignocelluloses have a great potential for feedstocks of biofuel production and various biorefinery applications. Here, we report that manipulation of a gene encoding coniferaldehyde 5-hydroxylase (CAld5H, or ferulate 5-hydroxylase, F5H) leads to substantial alterations in syringyl (S)/guaiacyl (G) lignin aromatic composition in rice (Oryza sativa), a major model grass and commercially important crop. Among three CAld5H genes identified in rice, OsCAld5H1 (CYP84A5) appeared to be predominantly expressed in lignin-producing rice vegetative tissues. Down-regulation of OsCAld5H1 produced altered lignins largely enriched in G units, whereas up-regulation of OsCAld5H1 resulted in lignins enriched in S units, as revealed by a series of wet-chemical and NMR structural analyses. Our data collectively demonstrate that OsCAld5H1 expression is a major factor controlling S/G lignin composition in rice cell walls. Given that S/G lignin composition affects various biomass properties, we contemplate that manipulation of CAld5H gene expression represents a promising strategy to upgrade grass biomass for biorefinery applications.
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DOI:10.1007/s11103-015-0385-zURLPMID:26453352 [本文引用: 1]
Artificial microRNAs (amiRNA) provide a new feature in the gene silencing era. Concomitantly, reducing the amount of lignin in fiber-yielding plants such as jute holds significant commercial and environmental potential, since this amount is inversely proportional to the quality of the fiber. The present study aimed at reducing the lignin content in jute, by introducing amiRNA based vectors for down-regulation of two monolignoid biosynthetic genes of jute, coumarate 3-hydroxylase (C3H) and ferulate 5-hydroxylase (F5H). The transgenic lines of F5H-amiRNA and C3H-amiRNA showed a reduced level of gene expression, which resulted in about 25% reduction in acid insoluble lignin content for whole stem and 12-15% reduction in fiber lignin as compared to the non-transgenic plants. The results indicate successful F5H-amiRNA and C3H-amiRNA transgenesis for lignin reduction in jute. This is likely to have far-reaching commercial implications and economic acceleration for jute producing countries.
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Artificial microRNAs (amiRNAs) are similar to microRNAs (miRNAs) in that they are able to reduce the abundance of specific transcripts in plants by RNA-Induced Silencing Complex (RISC)-mediated cleavage and degradation, but differ in that they are designed for specific targets. The long generation times of forest trees have limited the discovery of mutations by conventional genetics. AmiRNAs can create gene-specific transcript reduction in transgenic trees in a single generation and may have broad application for functional genomics of trees. In this paper, we describe the specific down-regulation of multiple genes in the phenylalanine ammonia-lyase (PAL) gene family of Populus trichocarpa using amiRNA sequences incorporated in a P. trichocarpa miRNA-producing precursor, ptc-MIR408. Two different amiRNA constructs were designed to specifically down-regulate two different subsets of PAL genes, revealing differential regulation within the gene family. Down-regulation of subset A (PAL2, PAL4 and PAL5) by amiRNA-palA led to an increase in transcript abundance of subset B (PAL1 and PAL3). The reciprocal effect was not observed.
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We demonstrated the new target for lodging resistance in rice (Oryza sativa L.) by the analysis of physiological function of a locus for lodging resistance in a typhoon (lrt5) with the near isogenic line under rice
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