Role Of GhAGPS1 And GhAGPL1 Genes In Gladiolus Corm Quality And Cormel Numbers

Gladiolus is one of popular cut flowers in the word and it plays an important role in the cut flower market. Gladiolus cut flower production mainly depends on the high quality of new corm. Corm commercial propagation depends on cormel quality. For many reasons, such as integrated insect pest, long-term asexual reproduction, climatic conditions which induces serious corm degradation. At present, high-quality corm depends entirely on imports in our country. Starch is the main storage compound in underground organs like Gladiolus corms and cormels. The synthesis and accumulation of starch plays an important role in corm development. ADP-glucose pyrophosphorylase (AGPase) plays an important role in regulating starch biosynthesis in storage organs. Therefore, we studied the role of two ADP-glucose pyrophosphorylase genes (GhAGPS1 and GhAGPL1) in corm and cormels’ development. Therefore, the research of AGPase gene in the growth and development of Gladiolus is of great importance. The study provides theoretical basis and technical support for improving corm quality, cormel number to promote breeding programs and commercial propagation.Here, a full length cDNA of GhAGPSl and a full length cDNA of GhAGPL1 were isolated from G hybridus ’Rose Supreme’ by RT-PCR and rapid amplification of cDNA ends (RACE). The GhAGPSl-GFP and GhAGPLl-GFP plasmid were transformed into onion epidermal cells by particle bombardment method to detect subcellular localization of GhAGPS1-GFP and GhAGPL1-GFP fusion proteins. The relative expression of the two genes in different organs and different treatments were analyzed by quantitative real-time PCR (qRT-PCR). The leaves were stained with Lugol’s solution. Starch content was determined using anthrone-sulfonic acid method. The starch granules in leaf and corm were observed by transmission electron microscopy. The transformations of GhAGPS1 and GhAGPLl in Arabidopsis apsl and apll mutant plants were done by infiltration with Agrobacterium by the floral dip method. We silenced GhAGPS1 and GhAGPL1 in Gladiolus corms by virus-induced gene silencing (VIGS) method to investigate the role of GhAGPS1 and GhAGPLl genes in the development process of corm and cormel.The results showed that:(1) Full-length GhAGPS1 is 1801 bp with a 1581 bp ORF. The predicted molecular weight (MW) of GhAGPS1 is 58,008 Da. GhAGPSl gene encodes a polypeptide of 526 amino acids. Isoelectric point (pI) of GhAGPS1 is 7.87. Full-length GhAGPLl is 1919 bp with a 1554 bp ORF. GhAGPL1 encodes a polypeptide of 517 amino acids with a calculated molecular mass of 56,522 Da and a pI of 7.47. The two proteins share high similarity and contain four conserved subdomains (an ATP-binding motif, a catalytic motif, a Glc-1-P motif and an activator site). (2) The GhAGPSl protein is a plastid localization protein, and the GhAGPL1 protein is a cytoplasm protein. (3) The highest transcriptional levels of GhAGPS1 and GhAGPLl were both observed in cormels and corms. Their expressions were induced by glucose and sucrose, a lesser extent by mannitol. Exogenous application of ABA decreased the expression of both GhAGPSl and GhAGPLl in Gladiolus. (4) Heterologous transformation of GhAGPSl and GhAGPL1 respectively into Arabidopsis apsl and apll mutants partially recovered their ability of starch synthesis. (5) Silencing GhAGPS1 and GhAGPL1 in Gladiolus corms decreased the transcriptional levels of two genes by 3.47-fold and 1.14-fold, the AGPase enzyme activity by 54% and 25%. The starch content was reduced by 69% and 51.25% in TRV-GhAGPSl and TRV-GhAGPL1 plants. Transmission electron microscopy analyses of leaf and corm sections confirmed that the number of starch granules was reduced in the silenced plants. Corm weight and cormel number reduced significantly in the silenced plants. The number of cormels declined from 4.08 (in TRV control plants) to 0.42 (TRV-GhAGPSl) or 0.58 (TRV-GhAGPL1). The results indicate that inhibiting the expression of GhAGPS1 and GhAGPL1 genes could impair starch synthesis, which results in the significantly lowered corm quality and cormel yield in Gladiolus.Taken together, the expression of GhAGPase genes (GhAGPS1 and GhAGPL1) affects the starch synthesis of Gladiolus corm. Inhibiting the expression reduced quality of corm and quantity of cormel. These results indicate that GhAGPase genes (GhAGPS1 and GhAGPL1) positively regulate corm enlargement and growth of Gladiolus. This study provides theory basis for breeding programs and commercial propagation.