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Acta Agronomica Sinica ›› 2020, Vol. 46 ›› Issue (9): 1368-1379.doi: 10.3724/SP.J.1006.2020.94188


Mechanism of GhPIF4 regulating anther abortion under high temperature stress in cotton

CHEN Miao(), XIE Sai, WANG Chao-Zhi, LI Yan-Long, ZHANG Xian-Long, MIN Ling*()   

  1. National Key Laboratory of Crop Genetic Improvement / College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, Hubei, China
  • Received:2019-11-30 Accepted:2020-03-24 Online:2020-09-12 Published:2020-04-10
  • Contact: Ling MIN E-mail:2857520681@qq.com;lingmin@mail.hzau.edu.cn
  • Supported by:
    Funding from Fundamental Research Funds for the Central Universities(140419001);National Key Research and Development Program of China(2018YFD0100403)


PIF4, which belongs to the phytochrome interacting factor family, is a key transcription factor in response to temperature changes, and is widely involved in plant heat signaling transduction and hormonal signaling pathways. In this study, the GhPIF4 gene was cloned from ‘YZ1’ (Gossypium hirsutum). Subcellular localization results indicated that GhPIF4 is a nuclear located protein. qRT-PCR analysis and GUS staining of proGhPIF4:GUS transgenic cotton plants showed that the GhPIF4 gene was highly expressed in the anther, and its expression was significantly induced by high temperature (HT) in the ‘H05’ (HT sensitive line). With the overexpression of GhPIF4, different cotton transgenic lines showed different male fertility performances. Three transgenic lines with high GhPIF4 expression, OE5, OE7, and OE19, showed indehiscent anthers in the flower on the day of flowering and lower pollen activity than wild type ‘YZ1’. However, the transgenic line OE10 with slighter overexpression showed normal anther dehiscence and pollen activity. These results indicated that GhPIF4 causes anther abortion in an expression level-dependent manner. Furthermore, the IAA content was measured in OE7 and OE19 anthers at the tapetum degradation stage (9-14 mm buds) and mitotic stage (14-19 mm buds). As a result, the IAA content in OE7 and OE19 decreased compared to that in ‘YZ1’. Then, we analyzed the expression of auxin biosynthesis genes, including GhTAA1, GhYUC2, and GhCYP71A13, showing that the expression of these genes was down-regulated in OE7 and OE19, which was consistent with the decrease in IAA content. We hypothesize that overexpression of GhPIF4 might partially simulate the response of late stage anthers to HT, while constitutive overexpression of GhPIF4 may alter the auxin content in vegetative organs or early stage anthers, or the decline of IAA may lead to the anther abortion. These results provide a reference for further analysis of the function of GhPIF4 gene and understanding the mechanism of cotton anther abortion under HT.

Key words: cotton, high temperature, PIF4, auxin, anther/pollen abortion

Table 1

Vectors and strains used in the experiment"

载体/菌株 Vector/strain 用途 Purpose
pGEM-T Easy载体 pGEM-T Easy vector TA克隆 TA clone
pDONOR221载体 pDONOR221 vector BP反应 BP reaction
pGBW409载体 pGBW409 vector 35S:GhPIF4:His载体的构建 Construction of 35S:GhPIF4:His vector
pMDC43载体 pMDC43 vector GhPIF4亚细胞定位 Subcelluar localization of GhPIF4
pGWB433载体 pGWB433 vector GhPIF4启动子表达模式分析 Analysis of GhPIF4 promoter expression pattern
大肠杆菌TOP10 E. coli TOP10 载体转化 Vector transformation
农杆菌GV3101 Agrobacterium GV3101 棉花下胚轴的转化及烟草的侵染 Transformation of cotton hypocotyl and tobacco infection

Table 2

Sequence and purpose of the primers used in this study"

引物名称Primer name 引物序列Primer sequence (5°-3°) 用途Purpose
BP reaction
BP reaction
proGhPIF4-F TTTTTGTCTCCCATTACAGTATC Gene promoter amplification
proGhPIF4-R GGGTCATAAACTGGAAATTCAGA Gene promoter amplification

Fig. 1

Phylogenetic tree analysis of GhPIFs and GhPIL1s in Gossypium hirsutum and structural characteristics of GhPIF4 gene A: the phylogenetic tree analysis of GhPIFs and GhPIL1s in Gossypium hirsutum; B: comparison of amino acid sequences between GhPIF4, AtPIF4 and AtPIF5. Red line indicates bHLH domain; C: gene structure of GhPIF4. Red box and black line indicate exon and intron, respectively."

Fig. 2

Subcelluar localization of GhPIF4 35S:GFP: subcellular localization of the GFP in Nicotiana benthamiana; 35S:GhPIF4:GFP: subcellular localization of the GhPIF4:GFP fusion protein in Nicotiana benthamiana. Bar = 20 μm."

Fig. 3

Expression of GhPIF4 in different tissue and induced by high temperature in Gossypium hirsutum A and B: the expression analysis of GhPIF4 in H05 (A) and YZ1 (B), respectively. C: the expression pattern of proGhPIF4:GUS transgenic cotton; a: whole anther from the blooming flower; b: single anther from the blooming flower; c: sepal; d: petal; e: young leaf. D: the heat-induced expression analysis of GhPIF4 in ‘H05’ anthers at different development stages. Figures a, c, d, e, bar = 1 cm; figure b, bar = 100 μm; HN and HH refer to ‘H05’ under normal temperature and high temperature stress conditions, respectively; < 7 mm represents a mixture of flower buds less than 7 mm in length; **P < 0.01."

Fig. 4

Expression of GhPIF4 and Southern blotting in 35S:GhPIF4:His transgenic cottons A: the expression level of GhPIF4 in both T0 fertility plants and T0 sterility plants; B: southern blotting of T0 transgenic plants."

Fig. 5

Phenotype of 35S:GhPIF4:His transgenic cotton lines A: the whole anthers, bar = 1 cm; B: the single anther stained by TTC and observed under stereo microscope, bar = 500 μm."

Fig. 6

Content of IAA and the expression level of auxin synthesis genes in sterile anthers of GhPIF4 overexpression transgenic cottons A: the content of IAA was decreased in sterile anthers of GhPIF4 overexpression transgenic cottons; B-D: the expression level of auxin synthesis genes in sterile GhPIF4 overexpression transgenic cottons; GhTAA1 (B), GhYUC2 (C), GhCYP71A13 (D); **P < 0.01."

Fig. 7

Comparison of amino acid sequences between GhirPIF4 and GhPIF4 Red box indicates APB motif."

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