外源ALA缓解ABA抑制草莓根系伸长生长的机理研究

doi:10.16420/j.issn.0513-353x.2021-1215

摘要/Abstract

摘要：

以栽培草莓‘红颜’（Fragaria × ananassa Duch.‘Benihoppe’）为材料，探讨5-氨基乙酰丙酸（ALA）与脱落酸（ABA）以及生长素（IAA）之间的关系，以期为ALA在草莓生产上应用提供理论依据。结果显示，外源ABA处理抑制草莓根系伸长生长，而ALA缓解ABA的抑制效应。ABA处理降低草莓根尖内源生长素含量，ALA则促进内源ABA含量提高。ABA和（或）ALA处理对草莓根尖ABA生物合成关键基因NCED1、NECD2，以及ABA氧化代谢基因CYP707A的表达没有显著影响。但ABA处理诱导其受体基因PYL4和PYL8以及ABA信号通路关键蛋白激酶基因SnRK2.1、SnRK2.2、SnRK2.3、SnRK2.4、SnRK2.5和SnRK2.6表达上调，而ALA却没此效应，说明ALA-ABA调控草莓根系伸长生长效应不涉及ABA信号途径。另一方面，ABA和（或）ALA处理对IAA合成基因YUC1表达没有影响；ABA处理下调YUC2和YUC3以及IAA内向运输基因AUX1表达，但是这种效应不能被ALA逆转。值得关注的是，IAA外向运输蛋白编码基因PIN1在ABA处理后表达下调，而ALA可以逆转ABA效应，暗示该基因可能参与ALA-ABA调控草莓根系生长。利用携带绿色荧光蛋白（GFP）基因的转基因拟南芥植株研究发现，ABA抑制AtPIN1-GFP表达，而ALA逆转ABA的抑制效应。生物信息分析表明，栽培草莓PIN1氨基酸序列与其他蔷薇科植物的同源性较高，蛋白质两端存在多个跨膜区域。将带有GFP的FaPIN1转入本氏烟草，其荧光信号分布于细胞质膜。克隆FaPIN1全长，构建到雌二醇诱导表达载体上并转入拟南芥。这种超表达FaPIN1植株根系生长对ABA处理的敏感性下降，ALA缓解效应也下降。以上结果说明，ALA缓解ABA抑制草莓根系生长的关键点在于IAA极性运输蛋白基因PIN1的表达，即ALA通过促进IAA极性运输来缓解ABA抑制草莓根系生长。

关键词: 草莓, 根系, 生长, 5-氨基乙酰丙酸, 脱落酸, 生长素

Abstract:

The relationship among 5-aminolevulinic acid（ALA），abscisic acid（ABA）and auxin （IAA）in strawberry roots growth were discussed. It was found that exogenous ABA treatment significantly inhibited strawberry root growth，while ALA alleviated the inhibitory effect by ABA. ABA reduced the endogenous IAA content in the root tips of strawberry，while ALA promoted endogenous ABA content. The expressions of NCED1，NCED2 and CYP707A，referring to ABA biosynthesis and catabolism，respectively，were not different among treatments，whereas that of PYL4 and PYL8，both coding ABA receptors，and SnRK2.1，SnRK2.2，SnRK2.3，SnRK2.4，SnRK2.5 and SnRK2.6，which code the protein kinases in ABA signaling pathway were up-regulated by ABA but not mediated by ALA. These results suggested that the genes in ABA signaling route were not involved in ALA-ABA regulating root growth of strawberry. On the other hand，ABA down-regulated expressions of YUC2，YUC3（referring to IAA synthesis） and AUX1（coding auxin influx carrier）. However，the down-regulation of gene expressions by ABA were not reversed by ALA. This also means that these genes are not so important in ALA-ABA-regulated root growth of strawberry. Nevertheless，the expression of PIN1，which codes IAA exporter carrier，was down-regulated by ABA，and ALA reversed the down-regulation，suggesting that PIN1 may be involved in ALA-ABA regulating root growth of strawberry. In a transgenic Arabidopsis carrying the green fluorescent protein gene（GFP），it was found that AtPIN1-GFP expression was inhibited by ABA and reversed by ALA. Bioinformatic analysis showed that the amino acid sequence of FaPIN1 in the cultivated strawberry is high homology with many Rosaceous plants，with several transmembrane regions located at both ends of the polypeptide. When FaPIN1-GFP was transferred into tobacco （Nicotiana benthamiana），the fluorescence was distributed in the plasma membrane. When the cloned FaPIN1 was constructed into an estradiol-induced expression vector and transformed into Arabidopsis，the root growth of the transgenic plants was less sensitive to ABA treatment，and the ALA mitigation was also less significant. These results suggest that FaPIN1 is an important factor during ALA-ABA regulating root growth，and the alleviation of ALA on ABA-inhibiting root growth may be dependent on the promotion of IAA polar transport in the root tip of strawberry.

Key words: Fragaria × ananassa, root, growth, 5-aminolevulinic acid, ABA, auxin

中图分类号:

S668.4

饶智雄, 安玉艳, 曹荣祥, 唐泉, 汪良驹. 外源ALA缓解ABA抑制草莓根系伸长生长的机理研究[J]. 园艺学报, 2023, 50(3): 461-474.

RAO Zhixiong, AN Yuyan, CAO Rongxiang, TANG Quan, WANG Liangju. Studies on Mechanisms of ALA Alleviating ABA Inhibiting Root Growth of Strawberry[J]. Acta Horticulturae Sinica, 2023, 50(3): 461-474.

图/表 10

表1 草莓根系RT-qPCR分析用引物

Table 1 The primers for RT-qPCR analysis of strawberry roots

引物名称 Primer name	正向序列（5′-3′） Forward primer	反向序列（5′-3′） Reverse primer
ACTIN	F-TGGGTTTGCTGGAGATGAT	R-GAGTTAGGAGAACTGGGTGC
YUCCA1	F-GAAAGGTGAGCGTGGGTTA	R-TATGGCGACGACGATAGAG
YUCCA2	F-CTCCGTCTTCATCTCCCTAA	R-CAAACGTACTCAATCTCCTCC
YUCCA3	F-CTTGTCGTCGGTTGTGGAA	R-GCCTGTTGAGCCCGTATTT
AUX1	F-CTGGTTAGCTTCACGGTCTA	R-GAACTCTTAGCGATGTGGG
PIN1	F-GGGAAAGTGGAGGGTAGAAGA	R-TGCCACCTGTAGGATACGAGA
CYP707A	F-AGACGTGGAGTTTGAGGGTT	R-TAGTTGTGAGGTGGTGGAGG
NCED1	F-AGTCCGACGAGGTTGTTGTG	R-CCGAGTCTTTCTACCGAGCAG
NCED2	F-TTATCTCGCCATTGCTGAAC	R-GAAGGAAGAAAGAAAGGCTCAC
PYL4	F-CAACCCACAAGCCTACAAGCA	R-TCGTGACGGAGCGGTAGTTC
PYL8	F- CATACTACTGCCTGTCTGTC	R-GGATGAAACCTGCTACTT
SnRK2.1	F-ATTCACAACCCAAATCAACT	R-ACATCTGCTATCTTCCCATC
SnRK2.2	F-ATCCTTACTGTACGCTACGC	R-ACTTCCTCCTTCCTTCATTT
SnRK2.3	F-ACCACAATAATTTCACCACCAT	R-ACCTACCAAGATTCCGAGCT
SnRK2.4	F-TCTTGAATCGGGTTTCTTGT	R-CCTTCATTGGCTCATACCTC
SnRK2.5	F-CTACCGACTCAACTCACC	R-GTTCTCCGCTACACTTCT
SnRK2.6	F-CTTCCGCAAGACAATACA	R-AGATCAGATGACGGCACT
SnRK2.8	F-CCACAGATCACTCCGCCACC	R-TCGTCTTCGCTGAACCTCCC
SnRK2.9	F-TGCTCTTCCCGTCCAGTCAC	R-CCGGCCTTGCATATTCTCGT

表2 草莓FaPIN1亚细胞定位和FaPIN1超表达试验用引物

Table 2 The primers for subcellular localization and overexpression analysis of FaPIN1

引物名称 Primer name	正向序列（5′-3′） Forward primer	反向序列（5′-3′） Reverse primer
PIN1-ORF	ATGATCACATTATCAGACTTTTAC	TCATAGCCCCAGCAAAATG
PIN1 for GFP	GAGAACACGGGGGACTCTAGAATGATCACATTATCAG ACTTTTAC	CGCCCTTGCTCACCATGGATCCTAGCCCCAGCAAA ATGTAG
PIN1 for pER8	CTAGTCGACTCTAGCCTCGAGATGATCACATTATCAGA CTTTTAC	GGGAGGCCTGGATCGACTAGTTCATAGCCCCAGCA AAATG

图1 ABA抑制草莓根系生长的浓度效应（A）和ALA对ABA（2.5 μmol • L-1）抑制草莓根系生长的缓解效应（B）图中数据为30条以上根系的平均值。相同小写字母代表差异不显著。下同。

Fig. 1 The concentration effects of ABA inhibiting strawberry root growth（A）and the alleviation of exogenous ALA on the root growth of strawberry inhibited by ABA（B） The data in each figure are the means of more than 30 roots. The same small letters represent no significant difference. The same below.

图2 外源ABA和ALA处理对草莓根尖内源生长素和脱落酸量的影响

Fig. 2 Effects of exogenous ABA and ALA on the endogenous IAA and ABA in the root-tips of strawberry

图3 外源ABA和ALA处理对草莓根尖脱落酸信号相关基因表达水平的影响

Fig. 3 Effects of ABA and ALA treatments on the gene expressions related to ABA signal in the root-tips of strawberry

图4 外源ABA和ALA处理对草莓根尖生长素合成运输相关基因表达水平的影响

Fig. 4 Effects of ABA and ALA treatments on the gene expressions related to IAA biosynthesis and transport in the root-tips of strawberry

图5 ALA和（或）ABA处理对转基因拟南芥根尖IAA荧光信号强度的影响 A ~ C：携带绿色荧光蛋白的PIN1、PIN2和DR5转基因植株根尖荧光照片（上）和与明场合并后的照片（下）；D：相对荧光强度。

Fig. 5 Effects of ALA and（or）ABA on auxin fluorescence signal intensity in the root tips of Arabidopsis A-C are PIN1，PIN2 and DR5 transgenic plants carried with green fluorescence protein gene（GFP）.The upper half is the fluorescence image，while the down half is the merged image with bright field. D is the relative fluorescence intensity of GFP related with IAA in three transgenic plants after treatments.

图6 草莓FaPIN1跨膜区预测（A）和亚细胞定位（B） A中蓝色的长方形表示位于FaPIN1蛋白两端的跨膜结构域。

Fig. 6 Prediction of the transmembrane regions（A）and subcellular location（B）of FaPIN1 The blue rectangle indicates transmembrane domains located at either ends of the FaPIN1 protein in A.

图7 PIN1的系统进化分析

Fig. 7 Phylogenetic analysis of FaPIN1

图8 ABA和ALA处理对诱导超表达FaPIN1的转基因拟南芥根系生长的影响

Fig. 8 Effect of ABA and ALA on root growth of FaPIN1 transgenic Arabidopsis

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