Identification and Analysis of NAC Related to Petal Senescence and Stress Responses of Petunia
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摘要:
目的 NAC（NAM, ATAF and CUC）参与植物生长发育和多种逆境胁迫响应过程的调控。本文旨在鉴定和研究对矮牵牛生长发育和逆境胁迫响应的关键NAC成员，为优质抗逆矮牵牛育种提供基因资源。 方法 以腋生矮牵牛（Petunia axillaris）基因组为参考基因组，利用矮牵牛花器官衰老过程、烟草脆裂病毒（Tobacco rattle virus, TRV）侵染、低磷、低温、NaCl、铜离子和干旱胁迫处理后的转录组数据，分析矮牵牛NAC基因（PaNACs）差异表达情况，并对差异表达PaNACs的启动子顺式作用元件及转录因子结合位点进行分析。利用实时荧光定量PCR验证了部分差异表达PaNACs在矮牵牛花衰老过程中的表达情况，并预测了差异表达PaNACs编码蛋白的潜在靶基因。 结果 鉴定的131个PaNAC基因中，59个（45.04%）被鉴定为花器官衰老和逆境胁迫响应过程中的差异表达基因。PaNAC72、PaNAC22、PaNAC29、PaNAC40、PaNAC2、PaNAC90、PaNAC83、PaNAC56、PaNAC36和PaNAC35在至少3个生物学过程响应中差异表达显著，其中拟南芥衰老关键基因AtNAP的直系同源基因PaNAC29在花器官衰老过程和低温、低磷、铜离子胁迫逆境处理中显著上调表达；PaNAC72在除受铜离子胁迫外的所有6种处理中表达差异显著；PaNAC22在花器官衰老过程和低温和低磷胁迫中上调表达，在铜离子和干旱逆境下调表达。启动子分析结果显示这10个PaNAC启动子区域存在多种逆境胁迫响应相关元件，且大量响应衰老和逆境胁迫的差异表达基因的启动子区域存在NAC的结合位点。 结论 PaNACs广泛参与矮牵牛生长发育及逆境胁迫响应，其中PaNAC29可能是花衰老关键的正调控因子，PaNAC72广泛响应多种逆境胁迫。 Abstract:Objective NACs in petunia responsible for the growth, floral senescence, and stress response were identified and analyzed. Method Based on the Petunia axillaris genome, transcriptomes on the flower in senescence as well as some other organs under the stress of inoculated tobacco rattle virus (TRV), low phosphorus, low temperature, NaCl, copper ion, or drought were obtained. Expression under stress, cis-acting elements, and transcription factor binding sites in promoters of differentially expressed PaNACs were analyzed. The expression in flower senescence was determined using qRT-PCR, and putative target genes of proteins encoded by them predicted. Result Of the 131 PaNACs, 59 (i.e., 45.04% of all) were identified as differentially expressed genes (DEGs) during flower senescence and in response to stresses. PaNAC72, PaNAC22, PaNAC29, PaNAC40, PaNAC2, PaNAC90, PaNAC83, PaNAC56, PaNAC36, and PaNAC35 exhibited significant differential expressions in response to at least 3 stress treatments. Among them, PaNAC29, an orthologue of the Arabidopsis key senescence-related gene AtNAP, was highly upregulated during flower senescence and in response to low temperature, low phosphorus, or copper ion treatment. PaNAC72 was significantly affected by all except copper ion treatment. PaNAC22 was upregulated during flower senescence and in responses to low temperature and low phosphorus treatments but downregulated in the presence of copper ion or under drought condition. Multiple stress responsive elements presented in the promoters of the 10 PaNACs, and many senescence and stress responsive DEGs containing NAC binding sites in their promoters. Conclusion NAC (NAM, ATAF and CUC) involved widely in the growth, development, and stress responses of plants. PaNACs in petunia, such as PaNAC29 appeared to be a key positive regulator of floral senescence, and PaNAC72 responsive to a wide variety of stresses. -
Key words:
- Petunia hybrida /
- NAC /
- transcriptome /
- senescence /
- stress /
- bioinformatics
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图 1 拟南芥AtNAC和矮牵牛PaNAC系统发育树
Figure 1. Phylogenetic tree for Arabidopsis AtNACs and petunia PaNACs
图 2 矮牵牛花衰老过程中PaNACs表达情况
A：火山图展示矮牵牛NAC家族成员在花衰老过程中的差异表达基因；B：热图展示矮牵牛花衰老过程中表达显著变化的NAC基因；C：D0和D4时期矮牵牛花表型；D：PaNAC29和PaNAC72的RT-qPCR验证结果；***： P＜ 0.001。
Figure 2. Expressions of PaNACs during petunia flower senescence
A: Volcano diagram showing differentially expressed PaNACs in flower senescence; B: heatmap showing NACs with significant changes in expression during petunia flower senescence; C: petunia flower phenotypes on D0 and D4; D: RT-qPCR validation results of PaNAC29 and PaNAC72, respectively; ***: P＜0.001.
图 3 矮牵牛叶在烟草脆裂病毒（TRV）侵染过程中PaNACs表达情况
A：矮牵牛NAC家族成员在TRV病毒侵染过程中的差异表达基因火山图；B：矮牵牛在TRV病毒侵染过程中差异表达NAC基因热图；S0、S3、S6分别为病毒侵染后第0、3、6天。
Figure 3. Expressions of PaNACs in petunia leaves under TRV infestation
A: Volcano diagram showing differentially expressed PaNACs in TRV-infested petunia; B: heatmap showing NACs with significant changes in expression during TRV infestation in petunia. S0, S3, and S6: 0, 3, and 6 d after viral inoculation, respectively.
图 4 多种处理条件下矮牵牛PaNACs表达情况
A～E：气泡图展示矮牵牛NAC家族成员在低温（A）、低磷（B）、铜离子（C）、NaCl（D）和干旱（E）等非生物胁迫下的表达情况；F：韦恩图展示矮牵牛NAC家族成员在花衰老和非生物胁迫过程中的差异表达基因；G：矮牵牛花衰老和非生物胁迫中参与多个过程的PaNAC。
Figure 4. PaNACs expressions in petunia under various conditions
A–E: bubble plots demonstrating expressions of PaNACs under abiotic stresses, such as cold (A), low phosphorus (B), copper ions (C), NaCl (D), and drought (E); F: Wayne diagram showing differentially expressed PaNACs during flower senescence and under abiotic stresses; G: PaNACs involved in multiple processes in petunia flower senescence and abiotic stresses.
图 5 10个在花衰老和胁迫差异表达的PaNACs的启动子顺式作用元件及转录因子结合位点预测结果
A：10个在花衰老过程和胁迫处理下差异表达的PaNACs启动子顺式作用元件预测结果；B：10个在花衰老和胁迫差异表达的PaNAC启动子的转录因子结合位点预测。
Figure 5. Cis-acting elements and transcription factor binding sites in promoters of 10 PaNACs with differential expressions during flower senescence and under stresses
A: Cis-acting elements in promoters of 10 PaNACs showing differential expression during flower senescence and in response to stresses; B: transcription factor binding site prediction for promoters of 10 differentially expressed PaNACs during flower senescence and in response to stresses.
图 6 荧光定量PCR测定PaNAC在花衰老过程中的表达量
ns表示P＞ 0.05，*表示P ＜ 0.05 ，**表示P ＜ 0.01 ，***表示P ＜ 0.001。
Figure 6. Expressions of PaNACs during flower senescence by qRT-PCR
ns: P＞0.05; *: P＜0.05; **: P＜0.01; ***: P＜0.001.
图 7 矮牵牛衰老和逆境胁迫中的差异表达基因及PaNAC的潜在靶基因的GO富集
A：矮牵牛花衰老和逆境胁迫中的差异表达基因数量；B：差异表达基因中PaNAC的潜在靶基因的GO富集。
Figure 7. Differentially expressed genes in petunia senescence and under stresses and GO enrichment of potential target genes of PaNAC
A: number of differentially expressed genes in petunia flower senescence and stresses; B: GO enrichment of differentially expressed genes for potential target genes of PaNAC.
表 1 矮牵牛花衰老及各种胁迫处理
Table 1. Petunia flower senescence and various stress treatments
胁迫
Stress处理方法
Treatment品种/组织
Cultivar/tissue数据来源
Data source花衰老
Flower senescence采样时间点：第0天（D0），花朵开放但在花药裂开之前；第4天（D4），花冠在尖端边缘显示枯萎迹象。 ‘米切尔二倍体’腋生矮牵牛/花瓣 PRJNA417209[12] 病毒胁迫
Virus stress用100 mmol·L−1酸盐缓冲液均质化的TRV (PPK20) 感染性汁液侵染，在侵染后第0天（S0）、3天（S3）和6天（S6）取样。 ‘蓝色好时’腋生矮牵牛/叶 PRJNA693880[13] 冷胁迫
Cold stress分别于4 °C低温处理后1、3、6、12 h收集叶片。 ‘超越’腋生矮牵牛/叶 PRJNA640832[14] 低磷胁迫
Low phosphorus切除来自节点2（侧芽）和7（顶芽）的腋芽；两种处理：正常磷（250 μmol·L−1）和低磷（5 µmol·L−1）。 ‘V26自交系’腋生矮牵牛/腋芽 PRJNA997338[15] NaCl胁迫
NaCl stress正常植株用Hoagland溶液进行培养；NaCl处理组用含150 mmol·L−1 NaCl的改良Hoagland溶液进行处理，在处理后0、6、24 h取叶片。 ‘米切尔二倍体’腋生矮牵牛/叶 PRJNA381775[16] 铜离子胁迫
Cu stress正常营养液培养28 d后，将植物转移至含有40 μmol·L−1 CuSO4(+Cu)的营养液中直至开花；正常植株始终用正常营养液培养。收集第7阶段（花药裂开）花瓣。 ‘米切尔二倍体’腋生矮牵牛/花瓣 PRJNA774370[17] 干旱胁迫
Drought stress对照组每天用100 mg·L−1氮灌溉直至试验结束，胁迫组5 d不浇水。并在第5天收集叶。 ‘米切尔二倍体’腋生矮牵牛/叶 PRJNA680631[18] 
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表 2 实时荧光定量PCR引物信息
Table 2. Oligonucleotide primers used for qRT-PCR analysis
基因名称
Gene上游引物
Forward primer下游引物
Reverse primer退火温度
Annealing temperature/ ℃PaNAC2 CTAATGTCGACCGCTCTGCT CATCGATTGTGGCCTTGGTG 57.34/57.03 PaNAC22 ATAGCCAACGTGACCGGAAG AGAGTAGTGAGGGTCGGTCC 57.65/58.56 PaNAC29 TCGGACCTTCCTCCAGGATT TATCGGTGCCTGTAGCCTTC 57/58 PaNAC35 GGATGACAGAAGCAGCAACG GTTCCCAAGGGTCATAGCGA 56.89/57.23 PaNAC36 TGGCAACAATTGGCGAGAGA ACCCAATCAGTCTTGGAGCC 57.23/57.56 PaNAC40 GTCTCCAGTGGGCCTGAATC TCAACCAGCTTGCTGAACCA 58.49/57.19 PaNAC72 TGTGTCACAGGGTACTCAAGC ACCGAATACCAAACGGGTCA 57/56.4 Actin7 TGCTGATCGTATGAGCAAGGAA GGTGGAGCAACAACCTTAATCTTC 56/56
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