Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (3): 461-473.doi: 10.3864/j.issn.0578-1752.2020.03.001

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Functional Characterization of a Maize Plasma Membrane Intrinsic Protein ZmPIP2;6 Responses to Osmotic, Salt and Drought Stress

ZHOU Lian,XIONG YuHan,HONG XiangDe,ZHOU Jing,LIU ChaoXian,WANG JiuGuang,WANG GuoQiang,CAI YiLin()   

  1. Maize Research Institute, College of Agronomy and Biotechnology, Southwest University, Chongqing 400715
  • Received:2019-07-03 Accepted:2019-08-02 Online:2020-02-01 Published:2020-02-13
  • Contact: YiLin CAI E-mail:caiyilin@163.com

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Abstract:

【Objective】Plasma membrane Intrinsic Proteins (PIPs) exist widely in the membrane system of plant cells, which are essential to water transport and water balance in plant. The aim of this study is to explore the function of ZmPIP2;6 in plant water stress tolerance, and provide potential gene resources for new varieties of stress tolerance maize breeding. 【Method】Amino acid sequences of ZmPIP2;6 was analyzed and compared with other PIPs that involved in water stress tolerance. To verify the subcellular localization of ZmPIP2;6, ZmPIP2;6-GFP infusion vector was constructed and assessed using maize protoplasts isolated from leaves of maize seedlings. Tissues from root, stem, mature leaf, immature tassel, immature ear, endosperm and embryo of maize were isolated. Samples from root and leaf of maize were collected at different time after PEG or NaCl treatment. Total RNA were extracted, and expression pattern of ZmPIP2;6 in different tissues or under water stress condition was investigated by qRT-PCR. Transgenic Arabidopsis plants that overexpressed ZmPIP2;6 were generated and identified. The phenotype of ZmPIP2;6 overexpression transgenic Arabidopsis that tolerated to osmotic, salt or drought stress were monitored and primary root length and leaf water loss rate were measured. A number of stress responsive genes in ZmPIP2;6 overexpression Arabidopsis were detected under drought or salt condition.【Result】Analysis and comparison of the amino acid sequences showed that ZmPIP2;6 shared the same conserved structural domains and had a high degree of sequence similarity with other PIPs. The subcellular localization was assessed using maize protoplasts indicated ZmPIP2;6 was located on the plasma membrane. qRT-PCR result showed that ZmPIP2;6 was highly expressed in tassel. Treatment with PEG or NaCl resulted in induced expression of ZmPIP2;6 in root and leaf of maize. Overexpression of ZmPIP2;6 in transgenic Arabidopsis showed enhanced osmotic and salt stress tolerance in MS media plate and improved drought stress tolerance in soil condition compared to wild type. Expressions of related genes in the stress signaling pathway were changed in ZmPIP2;6 overexpression Arabidopsis under drought or salt condition. 【Conclusion】 Expression of ZmPIP2;6 was up-regulated under osmotic or salt stress condition. Overexpression of ZmPIP2;6 in Arabidopsis enhanced osmotic, salt and drought stress tolerance. A number of stress responsive genes in ZmPIP2;6 overexpression Arabidopsis were affected under salt or drought stress condition. These results indicated that ZmPIP2;6 might be involved in plant water stress responsive pathway.

Key words: maize (Zea mays L.), plasma membrane intrinsic proteins, osmotic stress, drought stress, salt stress

Fig. 1

Comparison of the amino acid sequences of PIPs"

Fig. 2

Subcellular localization of ZmPIP2;6 in maize protoplasts ZmPIP2;6-GFP was colocalized with a mCherry-labeled plasma membrane marker (mCherry-PM; CD3-1007). Bars=20 μm"

Fig. 3

Expression pattern of ZmPIP2;6 under normal and stress conditions A: Relative expression levels of ZmPIP2;6 in root, stem, mature leaf, immature tassel, immature ear, endosperm and embryo; B: Trifoliolate maize seedlings were treated with 10% PEG in nutrient solution; C: Trifoliolate maize seedlings were treated with 100 mmol·L-1 NaCl in nutrient solution"

Fig. 4

Generation of ZmPIP2;6 overexpression transgenic lines A: Schematic illustration of T-DNA sequence of ZmPIP2;6 overexpression vector; B: PCR detection of T1 generation of ZmPIP2;6 overexpression transgenic lines; 1-5: Five independent transgenic lines; MK: DNA Marker, PC: Positive control, NC: WT negative control; C: qRT-PCR analysis of three representative ZmPIP2;6 overexpression transgenic lines"

Fig. 5

Salt and osmotic stress tolerance of ZmPIP2;6 overexpression transgenic Arabidopsis"

Fig. 6

Phenotypes of ZmPIP2;6 overexpression transgenic lines under drought treatment and water loss rate A. Phenotype of WT and ZmPIP2;6 overexpression transgenic Arabidopsis seedlings under normal and dehydration conditions for 14 days and re-watered for another 3 days. B. Determination of water loss from detached leaves of WT and ZmPIP2;6 overexpression transgenic lines"

Fig. 7

Expression analysis of Arabidopsis stress responsive genes"

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