Functional Study on ZmSTART1 Regulation of Maize Vascular Bundle Formation

doi:10.13560/j.cnki.biotech.bull.1985.2024-0981

Abstract

Abstract:

Objective The objective of this study is to analyze the structure of ZmSTART1, clarify the expressing characteristics of ZmSTART1, analyze the function of ZmSTART1 in the process of vascular bundle construction, and thus provide a theoretical basis for the genetic improvement of corn lodging resistance and yield traits. Method Bioinformatics method was applied to analyze the structure characteristics of ZmSTART1 protein. Real-time PCR was used to analyze the temporal and spatial expression of ZmSTART1. ZmSTART1-GFP fusion technology was to transform tobacco leaves and determine the subcellular location of ZmSTART1. The overexpressing vector of ZmSTART11 was constructed, and Agrobacterium infection flocculent method was to transform Col-0 Arabidopsis thaliana. Real-time PCR technology was applied to validate the ZmSTART1 overexpressed A.thaliana positive seedlings. The vascular bundle characteristics of A. thaliana overexpressing ZmSTART1 were observedand the biological function of ZmSTART1 in regulating vascular bundle formation was clarified. Result Bioinformatics analysis revealed that ZmSTART1 was a hydrophobic protein containing only one START domain. The spatiotemporal expression characteristics of ZmSTART1 via real-time PCR technology showed that ZmSTART1 had higher expressions in the anthers, leaves, and the first internode. Subcellular localization analysis revealed that ZmSTART1 was located on the cytoplasmic membrane. Creating A.thaliana plants ZmSTART1-OE overexpressing the ZmSTART1 gene, it was found that transgenic A.thaliana overexpressing ZmSTART1-OE had more secondary and tertiary leaf veins than wild-type ones, and the number of enclosed spaces formed by the leaf veins was also significantly higher than that of the wild-type control. When ZmSTART1-OE transgenic A.thaliana leaves showed fourth order leaf veins, the wild-type control leaves only had third order leaf veins. Observing the development of stem vascular bundles, it was found that the wild-type A. thaliana had 6 vascular bundles in its primary stem structure, while ZmSTART1-OE had 7 vascular bundles in its primary stem structure. The number of fiber cell layers between bundles decreased, the safranin staining became lighter, and the lignin content decreased. Conclusion ZmSTART1 belongs to the START family and is located on the cytoplasmic membrane, playing an important role in the process of vascular bundle formation in maize.

Key words: ZmSTART1, expression characteristics, vascular bundle formation, maize, Arabidopsis thaliana

LIU Tong-tong, LI Xiao-hui, YANG Jun-long, CHEN Wang, YU Meng, WANG Chao-fan, WANG Feng-ru, KE Shao-ying. Functional Study on ZmSTART1 Regulation of Maize Vascular Bundle Formation[J]. Biotechnology Bulletin, 2025, 41(4): 115-122.

Figures/Tables 10

Table 1 Statistical analysis of morphological indexes

类型 Type	真叶面积 True leaf area/mm²	子叶面积 Cotyledon area/mm²	叶柄长 Petiole length/cm	株高 Plant height/cm	茎面积 Stem area/mm²	茎粗 Stem circumference/mm
WT	6.15±1.06aa	8.35±0.64aa	0.25±0.04aa	11±1.00aa	0.62±0.01aa	2.79±0.03aa
ZmSTART1-OE1	9.4±0.28AA	6.15±0.47AA	0.35±0.05AA	26.7±1.00AA	0.67±0.11aa	2.89±0.05aa
ZmSTART1-OE2	8.8±0.11AA	5.87±0.24AA	0.31±0.08Aa	22.5±1.87AA	0.63±0.13aa	2.81±0.17aa
ZmSTART1-OE3	8.8±0.23AA	5.15±0.33AA	0.28±0.07aa	18.6±1.65AA	0.62±0.07aa	2.79±0.13aa

Fig. 1 Structure analysis of ZmSTART1A: Schematic diagram of the domain in ZmSTART1 protein. B: Schematic diagram of the exons and introns in ZmSTART1 gene. C: The tertiary structure of ZmSTART1

Fig. 2 Phylogenetic tree analysis of START genes of maize (Zea mays) and A. thalianaGRMZM: Zea mays; At: Arabidopsis thaliana

Fig. 3 Expressions of ZmSTART1 in different tissues of maizeV1: 1 true leaf development stage. V3: 3 true leaf development stage. V5: 5 true leaf development stage. V7: 7 true leaf development stage. R1: Silking stage of maize. Different lowercase letters indicate a significant difference ≤0.05. The same below

Fig. 4 The subcellular distribution of ZmSTART1-GFP observed under laser confocal microscopy (bar=50 μm)

Fig. 5 Screening and identification of ZmSTART1 positive seedlingsA: Hygromycin screening. B: PCR identification. C: Analysis of RT-qPCR expression

Fig. 6 Phenotypic observation of transgenic linesoverexpressed ZmSTART1 in A. thaliana

Table 2 Phenotype data statistics of superficial veins and vascular bundles

类型	维管束个数	子叶二级叶脉	真叶二级叶脉数	真叶三级叶脉数	真叶四级叶脉数	子叶脉间区域
Type	No. of vascular bundles	Cotyledon vein/Veins	Total number of secondary veins in true leaves	Total number of tertiary veins in true leaves	Total number of fourth-order veins of true leaves	Region between veins of cotyledons
WT	6aa	5±1aa	12±1aa	12±1aa	0aa	5±1aa
ZmSTART1-OE1	7±1Aa	7±1AA	17±2AA	37±7AA	4±1AA	7±1AA
ZmSTART1-OE2	7±1Aa	7±1AA	16±2AA	33±8AA	3±1AA	6±7Aa
ZmSTART1-OE3	6±1aa	6±1Aa	15±2Aa	30±8AA	2±1AA	5±7aa

Fig. 7 Phenotype of the of ZmSTART1-overexpressed transgenic A.thaliana leaf veinA and B: The cotyledon and true leaf veins of wild type (A) and ZmSTART1-overexpressed transgenic Arabidopsis (B) respectively. C and D: The true leaf veins at all levels of wild type (C) and ZmSTART1-overexpressed transgenic Arabidopsis (D) respectively

Fig. 8 Stem cross seetional microstructure of transgenie A. thaliana overexpressing ZmSTART1A and B: Stem cross seetional microstructure of wild type (A) and ZmSTART1-overexpressed transgenic Arabidopsis (B) respectively. C and D: The vascular bundle of wild type (C) and ZmSTART1-overexpressed transgenic Arabidopsis (D) respectively

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