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矿质营养是植物生长发育的物质基础。氮素作为植物生长发育过程中需求量较大、影响较大的矿质元素之一,被称为生命元素[1-2]。氮不仅是蛋白质的重要组成成分,也是核酸、磷脂、光合色素等多种生物大分子的重要组分[3]。由于氮素在植物生命活动中所处的特殊地位,探究氮素营养对植物生长的影响一直是农业领域的研究热点。JIANG C Q等[4]的研究结果表明,烟草在高氮和低氮处理下的生物量均低于对照,增加供氮量可在一定程度上提高烟草叶片的叶绿素含量和光合能力。邢倩等[5]在对冬小麦的研究中发现,氮、磷、钾3种大量元素中,氮素的缺乏对冬小麦净光合速率的影响较大。BOUSSADIA O等[6]发现,较低的供氮水平使不同品种橄榄的生物量、叶片氮含量、叶绿素含量、光合速率均显著下降。相似的研究结果在对玉米、棉花、咖啡、木薯等的研究中也得以体现[7-10]。上述研究结果表明,缺氮胁迫影响作物的生长,从而影响作物的产量,因此说明及时监测和诊断植物生长发育中氮素营养的供应水平,对指导作物增产生产有积极意义。通过叶绿素荧光仪可测定植物叶片的光合能力,借此可判断作物的供氮水平。叶绿素荧光仪在测定植物叶片光合能力中具有快捷、灵敏、无损伤等特点,而被广泛应用于植物营养供应的监测中[11]。
棕榈科植物广泛分布于热带及亚热带地区,在热带经济作物中占据着十分重要的地位[12]。槟榔(Areca catechu L.)是棕榈科槟榔属植物,在我国有着一千多年的栽培历史,是海南省重要的特色经济作物之一[13]。近些年随着槟榔在海南的大面积推广种植,很多地区形成了栽种密度大、物种单一的槟榔种植体系,且不注重水肥管理及土壤改良,造成土壤耕性下降和肥力不足,导致槟榔植株无法获得充足养分,使得槟榔生长受到抑制、抗性降低、收获期变短、产量品质下降[14]。这给槟榔种植、加工等行业的健康发展造成障碍。优化水肥管理、提高土壤肥力、培育健壮种苗是提高槟榔生产水平的有效措施。氮素作为大量元素之一,氮肥的施用是提高土壤肥力、培育壮苗中的重要一环。目前,氮素对槟榔生长发育影响的相关研究鲜有报道。笔者利用叶绿素荧光仪和光合仪测定缺氮条件下槟榔苗叶片的光合特性,并依据测定结果探索针对槟榔缺氮的便捷、有效的诊断方法,旨在为槟榔育苗中合理施用氮肥提供理论依据。
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供试槟榔苗为‘热研一号’槟榔,由中国热带农业科学院椰子研究所刘立云研究员与黄丽云博士提供。选取长势一致的1~2叶龄槟榔幼苗,经2周水培适应和平衡后,分别培养于全素营养液(CK)(表1)和缺氮营养液(-N)中。全素营养液配方见表1。每日保持通气泵充气2次(30 min·次)。培养地点:海南大学海甸校区温室大棚(自然光照)。培养3个月后,观测CK、-N处理的槟榔苗的生物量,和自新叶向下依次3片叶片(叶序号:L1,L2,L3)的光合指标、叶绿素荧光参数、叶绿素含量和叶片组织显微结构。1个处理取样3株苗,重复3次。
表 1 全素营养液配方
Table 1. Formula of total nutrient solution
母液 组分 摩尔质量/(g·mol−1) 母液浓度/1000×(mmol·L−1) 使用浓度/(mmol·L−1) 母液含量/1000×(g·L−1) 1 KNO3 101.1 1500 1.5 151.65 Ca(NO3)2·4H2O 236.15 1200 1.2 283.38 MgCl2 95.21 25 0.025 2.38 NH4NO3 80.04 400 0.4 32.02 K2SO4 174.27 300 0.3 52.28 MnSO4·H2O 169.01 1.5 1.5×10−3 0.254 ZnSO4·7H2O 287.55 1.5 1.5×10−3 0.431 2 CuSO4·5H2O 249.71 0.5 0.5×10−3 0.125 H2MoO4 161.95 0.14 0.14×10−3 0.02 MgSO4·7H2O 246.48 500 0.5 123.24 (NH4)2SO4 132.4 300 0.3 39.72 3 Fe-EDTA(Na2) 367.1 40 0.04 14.68 4 NaB4O7·10H2O 381.37 2.5 2.5×10−3 0.95 5 KH2PO4 136.09 250 0.25 34.02 -
将新鲜槟榔苗分别按地上部、地下部拍照记录,并在称取鲜质量后,放入烘箱(40 ℃, 12 h)烘干至恒重后,称干质量。
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称取新鲜叶片0.1 g,放入干净的研钵中,加入1 mL丙酮以及适量石英砂和碳酸钙粉末帮助研磨(研磨过程在冰上进行),研磨过程中,再加入3 mL 80%丙酮研磨至样品变为白色,将白色样品放入黑暗环境下静置5 min,然后用滤纸过滤,最后将滤液用80%丙酮定容至5 mL。使用UV721分光光度计分别测量645,663 nm处的吸光度,每个样品重复3次。按照Arnon(1949)的方法计算叶绿素含量。数据分析采用DunCan检测。
叶绿素a = 12.7A663−2.69A645;
叶绿素b = 22.9 A645−4.68 A663。
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使用CIRAS-3 型便携式光合仪,于2019−12−15(晴天)上午10:00−11:00,测定叶片净光合速率、胞间CO2浓度、气孔导度等光合参数。将槟榔苗暗适应30 min后,使用Dual-Pam-100测定叶片PSⅡ的最大光化学效率Fv/Fm和实际光化学效率 Y(Ⅱ)、光化学淬灭参数qP、非光化学淬灭参数NPQ等。采用IBM SPSS Statistics 20中的单因素方差分析选项对数值进行统计分析。
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剪取叶片中部约3 cm×5 cm见方,置于FAA固定液中固定12 h,采用树脂半薄切片法制片。用leica 2235切片机切片,切片厚度为5 μm。切片经甲苯胺蓝-O(TBO)染色后,置于Nikon生物光学显微镜下观察并拍照。每个处理挑选3张完整切片,每张切片随机选取10个视野,用ImageJ1.43软件分别测定叶片厚度、叶片上下表皮细胞厚度、叶肉细胞间隙面积、单位面积视野内叶肉细胞数量(叶肉细胞密度)。采用ImageJ1.43对细胞大小等参数进行测量,并使用IBM SPSS Statistics 20中的单因素方差分析及独立样本t检测进行统计分析。
Effects of Nitrogen Deficiency on the Photosynthetic Characteristics of Areca catechu L. Seedling Leaves
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摘要: 以‘热研一号’槟榔幼苗为试验材料,通过缺氮处理,观测槟榔苗生长状态;测定槟榔苗的生物量、叶绿素含量、叶绿素荧光参数,并结合气体交换指标,分析缺氮胁迫对槟榔幼苗叶片光合特性的影响。结果显示,与对照(CK)相比,缺氮胁迫下,槟榔苗地上部矮小,叶片发黄,生物量降低但差异不显著;而地下部根系发达,根冠比显著升高。缺氮处理的槟榔苗各叶序叶片叶绿素a、b含量均显著低于对照,且叶绿素b含量降低幅度更大。缺氮胁迫还导致槟榔苗各叶序叶片的光合速率、蒸腾速率、气孔导度、最大光化学效率(Fv/Fm)、实际光化学效率Y(Ⅱ)、光化学淬灭参数qP等均显著下降,而细胞间CO2浓度、非光化学淬灭参数NPQ显著上升。缺氮胁迫还导致槟榔苗各叶序叶片的叶肉细胞密度显著低于对照。结果表明,缺氮胁迫使槟榔苗叶片叶绿素合成受到抑制,PSⅡ反应中心活性下降,光合效率降低,导致整株生物量下降;在缺氮条件下,槟榔苗会通过调整地上部与地下部的生物量的分配以维持根系与地上部的生长平衡,同时槟榔苗可通过采取降低叶绿素b在光合色素中的比例以及提高叶片厚度和上表皮细胞厚度等策略来增强热耗散机制,从而适应热带高温、强光的生存环境。Abstract: The growth and leaf microstructure of seedlings of arecanut (Areca catechu L.) under nitrogen deficiency were observed, and the biomass, chlorophyll content, chlorophyll fluorescence parameters and gas exchange index of the arecanut seedling leaves were determined to analyze the effect of nitrogen deficiency stress on photosynthetic characteristics of arecanut seedling leaves. The results showed that compared with the control the arecanut seedlings were shorter in the aboveground part, yellow in the leaves and lower in the biomass but without significant difference, while they were well developed in the root system in the underground part and had a significantly high ratio of root to crown. The contents of chlorophyll a, b in each leaf of the first three leaves from the top of the arecanut seedlings was significantly lower under the nitrogen deficiency stress than in the control, and the content of chlorophyll b decreased more significantly. The nitrogen deficiency stress reduced significantly leaf chlorophyll content, photosynthetic rate, transpiration rate, stomatal conductance, the maximal photochemical efficiency (Fv/Fm), actual photochemical efficiency Y (Ⅱ) and photochemical quenching coefficient qP but increased the intercellular CO2 concentration and the non-photochemical quenching coefficient NPQ significantly. The density of mesophyll cells in all the first three leaves of the arecanut seedlings was significantly lower in the nitrogen deficiency treatment than in the control. The results showed that nitrogen deficiency stress inhibited the synthesis of chlorophyll, and reduced the activity of PSII reaction center and the photosynthetic efficiency, leading to lower production of the biomass of the arecanut seedlings. Under nitrogen deficiency the arecanut seedlings regulate their biomass distribution between the aboveground and underground parts to maintain their balanced growth between the aboveground part and the root system, and at the same time they may reduce the proportion of chlorophyll b in the photosynthetic pigments and improve the thickness of leaves and the cells of the upper epidermis of the leaves to enhance their heat dissipation mechanism for their adaptation to the environment of high temperature and strong sunlight in the tropics.
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图 4 不同供氮水平处理下槟榔苗叶片叶绿素荧光参数
不同小写字母表示P<0.05水平差异显著。Fv/Fm:PSⅡ的最大光化学效率;Y(Ⅱ):PSⅡ的实际光化学效率;qP:光化学淬灭参数;NPQ:非光化学淬灭参数。
Fig. 4 Chlorophyll fluorescence parameters of arecanut seedling leaves under different nitrogen levels
Different lowercase letters showed significant difference at P < 0.05 level. Fv/Fm: Maximal photochemical efficiency; Y(Ⅱ): Actual photochemical efficiency;qP: Photochemical quenching coefficient; NPQ: Non-photochemical quenching coefficient.
图 5 不同供氮水平处理下槟榔苗叶片显微结构
A:对照处理槟榔苗各叶序叶片显微图;B:缺氮处理槟榔苗各叶序叶片显微图;Ⅰ:上表皮细胞;Ⅱ:叶肉细胞;Ⅲ:下表皮细胞;柱状图上不同小写字母表示在P<0.05水平差异显著。
Fig. 5 Microstructure of arecanut seedling leaves under different nitrogen levels
A: Micrographs of the first three leaves of arecanut seedlings in the control ; B: Micrograph of the first three leaves of the arecanut seedling under nitrogen deficiency streess; Ⅰ: Upper epidermal cell; Ⅱ: Mesophyll cells; Ⅲ: Lower epidermal cell; Different lowercase letters on the bar chart indicate significant differences at a P<0.05 level.
表 1 全素营养液配方
Table 1 Formula of total nutrient solution
母液 组分 摩尔质量/(g·mol−1) 母液浓度/1000×(mmol·L−1) 使用浓度/(mmol·L−1) 母液含量/1000×(g·L−1) 1 KNO3 101.1 1500 1.5 151.65 Ca(NO3)2·4H2O 236.15 1200 1.2 283.38 MgCl2 95.21 25 0.025 2.38 NH4NO3 80.04 400 0.4 32.02 K2SO4 174.27 300 0.3 52.28 MnSO4·H2O 169.01 1.5 1.5×10−3 0.254 ZnSO4·7H2O 287.55 1.5 1.5×10−3 0.431 2 CuSO4·5H2O 249.71 0.5 0.5×10−3 0.125 H2MoO4 161.95 0.14 0.14×10−3 0.02 MgSO4·7H2O 246.48 500 0.5 123.24 (NH4)2SO4 132.4 300 0.3 39.72 3 Fe-EDTA(Na2) 367.1 40 0.04 14.68 4 NaB4O7·10H2O 381.37 2.5 2.5×10−3 0.95 5 KH2PO4 136.09 250 0.25 34.02 -
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