Scientia Agricultura Sinica ›› 2023, Vol. 56 ›› Issue (9): 1633-1645.doi: 10.3864/j.issn.0578-1752.2023.09.002

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

Overexpression of Wheat TaCYP78A5 Increases Flower Organ Size

PENG HaiXia1(), KA DeYan2, ZHANG TianXing3, ZHOU MengDie2, WU LinNan2, XIN ZhuanXia1, ZHAO HuiXian2, MA Meng2()   

  1. 1 College of Landscape Architecture and Art, Northwest A&F University, Yangling 712100, Shaanxi
    2 College of Life Sciences, Northwest A & F University, Yangling 712100, Shaanxi
    3 College of Agronomy, Northwest A&F University, Yangling 712100, Shaanxi
  • Received:2022-11-28 Accepted:2023-02-22 Online:2023-05-01 Published:2023-05-10

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Abstract:

【Objective】The function and mechanism of TaCYP78A5 regulating flower organ size was preliminarily analyzed by means of expression pattern analysis, transgenic overexpression and cytological observation. The results provide genetic resources and theoretical basis for crop genetic improvement. 【Method】According to the sequence information of CYP78A family members of different species in EnsemblePlants genome database, sequence alignment and evolutionary analysis were carried out for the homologous genes of TaCYP78A5 in wheat and other species. The gene and protein structures and the expression patterns of different organs of wheat TaCYP78A5 were analyzed by bioinformatics. Through the strategy of constitutive overexpression and local specific overexpression in reproductive organs of TaCYP78A5 in Arabidopsis, it is clear that TaCYP78A5 has the function of regulating flower organ size. The cytological characteristics of flower organs of different transgenic Arabidopsis were observed under microscope, and the cytological mechanism of TaCYP78A5 regulating flower organ size was analyzed. The function of TaCYP78A5 in regulating wheat spike size and other spike traits was clarified by using the strategy of transgenic overexpression in wheat. The correlation analysis of haplotype data and spike phenotype data of 323 wheat accessions was used to explore the effect of TaCYP78A5 expression on spike size and other spike traits of different wheat accessions. 【Result】The gene and protein sequence similarity of wheat TaCYP78A5 and Arabidopsis AtCYP78A5 is low, but the gene and protein structure similarity is high. Wheat TaCYP78A5 and Arabidopsis AtCYP78A5 are widely expressed in many organs, but highly expressed in flower organs. Compared with wild type, the constitutive overexpression of TaCYP78A5 in Arabidopsis could lead to the enlargement of flower organs and a significant increase in petal area of 13.5%-35.4%. Moreover, the specific overexpression of TaCYP78A5 only in the ovule was enough to cause the enlargement of the flower organ of Arabidopsis, and the petal area increased significantly by 9%-22.1%. On the contrary, the flower organ of Arabidopsis cyp78a5 mutant was significantly smaller than that of wild type, and the petal area was significantly reduced by 27%. The constitutive overexpression of TaCYP78A5 in Arabidopsis resulted in a significant increase in the size of petal epidermal cells by 49%-54% compared with wild type, and a significant decrease in the number of cells by 11%-19% compared with wild type. Locally specific overexpression of TaCYP78A5 in Arabidopsis also resulted in a significant increase in the size of petal epidermal cells by 20%-49% compared with wild type, and a significant decrease in the number of cells by 8%-24% compared with wild type. The constitutive overexpression of TaCYP78A5 in wheat resulted in the increase of wheat spike length by 7.9%-8.9%, glume area by 9.6%-14.7%, and grain number per spike by 12.4%-23.8%. The spikelet number per spike and grain number per spikelet showed different degrees of change. The results of haplotype analysis showed that among 323 wheat accessions, wheat accessions with higher TaCYP78A5-A expression level had longer spike length, more grains per spikelet and fewer spikelets per spike than wheat accessions with lower TaCYP78A5-A expression level, but there was no significant difference in grain number per spike. 【Conclusion】TaCYP78A5 promoted the growth of flower organs in a non cellular self-made mode. The overexpression of TaCYP78A5 in wheat and Arabidopsis could lead to the enlargement of flower organs.

Key words: wheat, TaCYP78A5, overexpression, flower organ size, cell expansion

Fig. 1

Structure, expression pattern and evolutionary analysis of TaCYP78A5 A: Phylogenetic tree analysis of TaCYP78A5 (deduced protein sequence from TaCYP78A5-A) and other CYP78A family members; B: Exon and intron architecture of TaCYP78A5-A, TaCYP78A5-B, and TaCYP78A5-D, the black box represents the coding region of the protein, and the cleaved part represents the region missing from TaCYP78A5-A and TaCYP78A5-D compared with TaCYP78A5-B, the sequences encoding the hydrophobic region, the oxygen-binding site and the heme-binding site are denoted by yellow, blue and red box representation; C: Organ expression patterns of Arabidopsis AtCYP78A5 and wheat TaCYP78A5"

Fig. 2

Sequence alignment and structure analysis of wheat TaCYP78A5 and Arabidopsis AtCYP78A5 proteins A: Multiple sequence alignment analysis of wheat TaCYP78A5 and Arabidopsis AtCYP78A5 proteins, the hydrophobic region, oxygen binding domain and heme binding domain are marked with yellow, blue and red respectively; B: Tertiary structure prediction of wheat TaCYP78A5 and Arabidopsis AtCYP78A5 proteins"

Fig. 3

Effects of the overexpression of TaCYP78A5 on flower organs size in Arabidopsis A-H: Morphology of flower organs; I-J: Statistical analysis of the petal area (n>36), take the maximum projected area of petals as the petal area; * and ** indicate significant difference levels at P<0.05 and P<0.01. The same as below. The scale in A-D is 0.5 cm; Scale in E-H is 500 μm"

Fig. 4

Effects of the overexpression of TaCYP78A5 on epidermal cells of petals in Arabidopsis A: Morphology of petals; B-E: Morphology of epidermal cells in the middle of the petals; F-G: Statistical analysis of the area of epidermal cells in the middle of the petals (n>50); H-I: Statistical analysis of the number of epidermal cells of the petals (n>50). The scale in A is 500 μm; The scale in B-E is 50 μm"

Fig. 5

Effects of the overexpression of TaCYP78A5 on spike traits in wheat A-C: Phenotypes of spikes, glumes and spikelets at 12 days after flowering; D: Phenotypes of grains at 30 days after flowering; E-I: Statistical analysis of spike length, glume area, spikelet numbers per spike, grain numbers per spikelet and grain numbers per spike at 12 days after flowering (n>8), all plants were planted under the same culture conditions in the greenhouse. The scale in A-D is 1 cm"

Fig. 6

Effect of TaCYP78A5 expression on spike traits in different wheat accessions A: Phenotypes of spikes of wheat accessions with Ap-HapⅠ and Ap-HapⅡ; B-E: Statistical analysis of spike length, spikelet numbers per spike, grain numbers per spikelet and grain numbers per spike of wheat accessions with Ap-HapⅠ (n=265) and Ap-HapⅡ (n=58). Phenotypic data are derived from the mean value of 323 wheat accessions at five environmental points"

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