Biological Function of Gene MiISU1 for Fe-S Cluster Assembly in Mangifera indica

doi:10.13560/j.cnki.biotech.bull.1985.2022-0616

Abstract

Abstract:

Iron-sulfur（Fe-S）cluster assembly genes play important roles in plant growth and development. We focused on the cloning and functional determination of MiISU1 involved in mitochondrial Fe-S cluster assembly in mango（Mangifera indica）and analyzed its biological functions, which may provide gene resources and theoretical foundation for the molecular mechanisms of Fe-S cluster assembly in tropical crops. The MiISU1 was isolated by homology cloning technology from mango. The tissue-specific expression characteristics and their responses to different Fe supplies were detected by quantitative real-time PCR. Functional verification of MiISU1 was carried out via creating over-expressed transgenic Arabidopsis seedlings. Results showed that MiISU1 was isolated from diploid mango ‘Guire 82’, whose expression was the highest in young fruits, followed by mature fruits, full-bloom flowers and new leaves, but was low in new roots and new phloem. In addition, Fe depletion significantly reduced the expression of MiISU1 in the roots of 1-year-old grafted seedlings, while high Fe toxicity significantly enhanced the expression of MiISU1 in the whole tested seedlings. Moreover, over-expression of MiISU1 in Arabidopsis significantly enhanced plant growth, accompanied with enhanced Fe accumulation, leaf total chlorophyll, nitrite reductase（NIR）, aconitase（ACO）and succinate dehydrogenase（SDH）activities in transgenic lines, and improved plant resistance to Fe depletion via enhancing the root growth and development. MiISU1 plays a key role in Fe nutrition and metabolism of mango, which is regulated by external Fe status.

Key words: Mangifera indica, Fe metabolism, Fe-S cluster assembly machinery, ISU1, Fe deficiency stress, transgenic Arabidopsis

CUI Ji-jie, CAI Wen-bo, ZHUANG Qing-hui, GAO Ai-ping, HUANG Jian-feng, CHEN Ya-hui, SONG Zhi-zhong. Biological Function of Gene MiISU1 for Fe-S Cluster Assembly in Mangifera indica[J]. Biotechnology Bulletin, 2023, 39(2): 139-146.

Figures/Tables 7

Fig. 1 Amino acid sequence identity analysis of ISU1 homologs from 13 plant species At: Arabidopsis thaliana; Bd: Brachypodium distachyon; Cs: Citrus sinensis; Es: Thellungiella salsuginea; Fv: Fragaria vesca; Gm: Glycine max; Mi: Mangifera indica; Md: Malus domestica; Os: Oryza sativa; Pp: Prunus persica; Pt: Populus trichocarpa; Sl: Solanum lycopersicum; Vi: Vitis vinifera. The same below

Fig. 2 Phylogenetic tree of ISU homologs from 13 plant species

Fig. 3 Tissue-specific expression analysis of MiISU1 gene in mango

Fig. 4 Differential responses of MiISU1 gene to different Fe supplies in mango tissue culture seedlings * indicates significant difference at the P < 0.05 level, ** indicates extremely significant difference at the P < 0.01 level

Fig. 5 Generation and verification of MiISU1 over-expres-sion transgenic Arabidopsis seedlings A: Schematic diagram of recombinant expression vector construction; B: PCR verification of T1 transgenic lines

Fig. 6 Phenotype analysis of MiISU1 over-expression tran-sgenic Arabidopsis seedlings A: Control conditions; B: Fe deficiency treatment

Table 1 Physiological analysis of transgenic Arabidopsis seedlings under Fe depletion for 5 d

处理Treatment	对照Control		缺铁Fe depletion
处理Treatment	野生型Wild type	T₃转基因株系 T₃ transgenic lines	野生型 Wild type	T₃转基因株系 T₃ transgenic lines
整株生物鲜重Fresh weight of whole seedling/g	60.62 ± 5.01	78.24 ± 5.29**	21.75 ± 2.53	28.39 ± 3.21**
地上部生物鲜重Fresh weight of shoots/g	49.42 ± 3.16	62.52 ± 3.44**	16.08 ± 1.92	15.73 ± 1.68
根部生物鲜重Fresh weight of roots/g	10.58 ± 1.44	15.72 ± 1.51**	5.67 ± 0.65	12.66 ± 1.47**
整株铁含量 Fe concentration/（g·Kg^-1 DW）	0.32 ± 0.02	0.47 ± 0.04**	0.08 ± 0.01	0.13 ± 0.01**
叶片叶绿素含量Total leaf chlorophyll/（g·kg^-1 FW）	0.46 ± 0.03	0.51 ± 0.05	0.17 ± 0.02	0.23 ± 0.02**
NiR活性NiR activity/（U·mg^-1·protein^-1）	1.23 ± 0.11	1.51 ± 0.12*	0.32 ± 0.02	0.34 ± 0.02
ACO活性 ACO activity/（U·mg^-1·protein^-1）	0.43 ± 0.02	0.53 ± 0.03*	0.17 ± 0.01	0.23 ± 0.01**
SDH活性SDH activity/（U·mg^-1·protein^-1）	6.97 ± 0.41	9.31 ± 0.46**	2.17 ± 0.22	2.83 ± 0.25**

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