Effects of NaHCO3 stress on growth, photosynthesis and chlorophyll fluorescence characteristics in Populus davidiana × P. bolleana overexpressed TaLEA
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摘要: 为了筛选耐苏打盐渍土的转基因杨树优良株系, 以苏打盐渍土的主要成分NaHCO3胁迫处理转TaLEA基因的山新杨各株系与对照(NT)，测定不同株系的苗高和地径等生长性状，测定净光合速率(Pn)、胞间CO2浓度(Ci)、气孔导度(Gs)、蒸腾速率(Tr)等光合参数以及最大光化学效率(Fv/Fm)、光化学猝灭系数(qP)、非光化学猝灭系数(NPQ)、实际光化学电子产量(ΦPSII)等叶绿素荧光动力学参数，利用染色法分别比较各株系的超氧离子和过氧化氢的累积情况，进而综合评价不同株系对NaHCO3胁迫的响应。结果表明，NaHCO3胁迫处理前、后各株系的苗高和地径生长量发生明显分化，由处理前的差异不显著，到处理20 d后的显著，转基因株系的苗高、地径普遍较对照高，表现突出的是转基因株系SL2号，其平均苗高和地径分别较对照提高了17.4%、15.7%；对照的盐害指数是转基因株系的3.3倍，并且转基因株系的平均存活率是对照的2.8倍。各转基因株系的光合参数和荧光参数也较对照增强，Pn、Tr在胁迫20 d后转基因株系较对照分别高了237.38%、649.02%，Gs、Ci在胁迫15 d时转基因株系较对照分别高了119.05%、24.56%，其中SL2号转基因株系较对照分别提高150%、25.81%。各转基因株系的Fv/Fm平均为0.68，对照只有0.45，前者的qP和ΦPSII只略高于后者，后者的NPQ却是前者的6倍。转基因株系的超氧离子和过氧化氢累积量均少于对照。在相同的碱胁迫环境下，转基因山新杨，尤其是SL2，能保持较快的生长量、较强的光合能力和较低的ROS水平，初步选择其为耐苏打盐渍土的杨树转基因优良株系。Abstract: In order to find the transgenic poplar which can tolerate soda-saline soil, the lines overexpressed TaLEA gene and the control (NT) were treated with NaHCO3, the main components of soda-saline soil. And to evaluate salt-tolerant properties, we measured some parameters of different lines, including height and ground diameter, net photosynthetic rate(Pn), intercellular CO2 concentration(Ci), stomatal conductance(Gs), transpiration rate(Tr), maximal photochemical efficiency(Fv/Fm), photochemical quenching coefficient (qP), non-photochemical quenching coefficient (NPQ), and the actual photochemical electron yield (ΦPSII). Moreover, the accumulation of superoxide anion and hydrogen peroxide in each line was compared by staining method. The results showed that obvious changes had taken place in height and ground diameter among different lines after NaHCO3 stress. There was no significant difference before treatment, but after treating for 20 days, significant differences happened. Compared with control, all transgenic lines increased in height and ground diameter, especially the SL2 transgenic line, whose average height and ground diameter were increased by 17.4% and 15.7%, respectively. Furthermore, the salt damage index of wild type was 3.3 times of transgenic lines, and the survival rate of transgenic lines was 2.8 times of wild type. The photosynthetic parameters and fluorescence parameters of the transgenic lines after 20 days stress were also higher than wild type, Pn and Tr were increased by 237.38% and 649.02%, respectively compared with the wild type. Compared with control, Gs and Ci of transgenic lines were increased by 119.05% and 24.56% under 15 days stress, and Gs, Ci of SL2 transgenic line were increased by 150% and 25.81%. Besides, the average Fv/Fm of each transgenic line was 0.68, the wild type was 0.45. The qP and PSII of transgenic plants were only slightly higher than that of the wild type, but the NPQ of wild type was 6 times of wild type.The accumulation of superoxide anion and hydrogen peroxide in transgenic lines was less than that in wild type. All in all, transgenic lines, especially SL2, could maintain rapid growth, strong photosynthetic capacity and lower level of ROS under alkali stress environment, so SL2 was initially selected as an excellent transgenic line of poplar because of salinity tolerance.
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Keywords:
- transgene /
- NaHCO3 stress /
- growth /
- photosynthesis /
- chlorophyll fluorescence /
- TaLEA
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图 1 碱性盐害级值
A.0级无盐害症状；B.1级轻度盐害(有部分叶尖、叶缘变黄)；C.2级中度盐害(有大约1/2的叶片、叶缘焦枯)；D.3级重度盐害(大部分叶片、叶缘焦枯或脱落)；E.4级极重度盐害(复水后植株死亡)。
Figure 1. Level of alkaline salt stress
A, symptom of 0 salt injury; B, level 1 for mild salt damage (the tip of leaf or leaf margin turned yellow); C, level 2 for moderate salt damage (about 1/2 blade or leaf margin turned withered); D, level 3 for severe salt damage (most of the leaf, leaf margin turned withered or was off); E, level 4 for extremely severe salt damage (plant dead after rewatering).
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图 2 NaHCO3胁迫对叶片光合参数的影响
A.叶片的净光合速率(Pn)；B.胞间CO2浓度(Ci)；C.蒸腾速率(Tr)；D.气孔导度(Gs)。A, net photosynthetic rate (Pn); B, intercellular CO2 concentration (Ci); C, transpiration rate (Tr); D, stomatal conductance (Gs).
Figure 2. Effects of NaHCO3 stress on photosynthetic parameters of leaves
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图 3 NaCHO3胁迫对叶片叶绿素荧光动力学参数的影响
A.最大光化学效率(Fv/Fm)；B.光化学猝灭系数(qP)；C.非光化学猝灭系数(NPQ)；D.实际光化学电子产量(ΦPSII)。
Figure 3. Effects of NaCHO3 stress on chlorophyll fluorescence kinetics parameters of leaves
A, maximum photochemical efficiency (Fv/Fm); B, photochemical quenching coefficient (qP); C, non-photochemical quenching coefficient (NPQ); D, actual photochemical electron yield (ΦPSII).
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图 4 NaHCO3胁迫下转基因山新杨与NT细胞内超氧离子累积比较
Figure 4. Comparison of superoxide anion accumulation in the cells of transgenic lines and NT under NaHCO3 stress
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图 5 NaHCO3胁迫下转基因山新杨与NT细胞内过氧化氢累积比较
Figure 5. Comparison of hydrogen peroxide accumulation in transgenic poplar and NT cells under NaHCO3 stress
表 1 NaHCO3胁迫前后高生长比较
Table 1 Comparison in height growth before and after NaHCO3 stress
株系
Line胁迫前、后平均苗高
Average height before and after stress/cm非胁迫条件下
平均苗高
Average height
without stress/cmNaHCO3对高生长的
影响Effect of
NaHCO3 on height
growth/%胁迫前
Before stress胁迫后
After stress绝对高生长
Absolute height growthNT 85.55±3.67 122.11±5.02 c 36.55±5.40 b 147.78±4.42 17.37±2.07ab SL1 84.33±2.36 130.17±2.79 bc 46.00±8.38 ab 147.17±1.01 11.55±1.53ab SL2 88.67±3.21 143.33±7.23 a 54.44±6.16 a 149.67±8.21 4.21±1.75d SL3 85.33±2.60 128.89±6.50 bc 43.56±5.54 ab 145.00±4.58 10.96±7.31bc SL4 85.56±2.14 122.19±2.72 c 37.08±3.83 b 150.67±5.69 18.80±4.36 a SL5 86.30±4.39 132.89±6.44 b 46.92±7.87 ab 139.22±5.42 4.57±1.20 cd SL6 87.05±1.25 127.22±3.53 bc 40.17±2.42 b 143.17±2.56 11.10±3.59ab 平均Mean 86.11±2.80 129.54±4.89 43.53±5.66 146.09±4.55 11.21±2.73 注：不同小写字母表示在P < 0.05水平上差异显著。下同。Notes:different lowercase letters mean significant differences at P < 0.05 level. Same as below. 
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表 2 NaHCO3胁迫前后地径生长比较
Table 2 Comparison in ground diameter growth before and after NaHCO3 stress
株系
Line胁迫前、后平均地径
Average ground diameter before and after stress/mm非胁迫条件下
平均地径Mean
ground diameter
without stress/mmNaHCO3对地径生长
的影响Effect of
NaHCO3 on ground
diameter growth/%胁迫前
Before stress胁迫后
After stress绝对地径生长
Absolute ground diameter growthNT 2.18±0.017 6.48±0.133 c 4.30±0.51 7.53±0.59 13.57±7.71 SL1 2.16±0.10 7.22±0.18 ab 5.06±0.15 8.18±0.63 11.68±8.57 SL2 2.23±0.036 7.50±0.055 a 5.27±0.38 8.49±0.42 11.60±4.02 SL3 2.04±0.058 6.80±0.084 bc 4.77±0.26 7.88±0.63 13.24±7.11 SL4 2.18±0.079 6.92±0.38 bc 4.68±0.35 8.04±0.46 13.87±5.64 SL5 2.18±0.085 6.71±0.36 c 4.53±0.78 7.99±0.47 15.64±8.71 SL6 2.20±0.046 6.79±0.26 bc 4.58±0.35 7.96±0.49 14.50±6.86 平均Mean 2.17±0.063 6.92±0.23 4.74±0.40 8.01±0.52 13.44±6.94
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表 3 碱性盐害指数及存活率
Table 3 Index of salt-alkaline stress and survival rate
株系
Line碱害指数
Index of salt-alkaline stress存活率
Survival rate/%NT 0.79 30 SL1 0.13 87 SL2 0.28 100 SL3 0.30 80 SL4 0.18 90 SL5 0.22 90 SL6 0.34 62
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