Isolation and characterization of a myo-inositol-1-phosphate synthase gene from yellow passion fruit (Passiflora edulis f. flavicarpa) expressed during seed development and environmental stress

Abstract

Background and aims: Myo-inositol-1l-phosphate synthase (MIPS) catalyses the conversion of d-glucose 6-phosphate to 1-l-myo-inositol-1-phosphate, the first and rate-limiting step in the biosynthesis of all inositol-containing compounds. Inositol phospholipids play a vital role in membrane trafficking and signalling pathways, auxin storage and transport, phytic acid biosynthesis, cell wall biosynthesis and production of stress-related molecules. In the present study, an MIPS cDNA from developing Passiflora edulis f. flavicarpa seeds was characterized and an investigation made into its spatial and differential expression, as well as changes in its transcription during exposure of growing plants to cold and heat stresses.

Methods: The MIPS-encoding gene was isolated by polymerase chain reaction (PCR) methods, and transcript levels were examined using semi-quantitative reverse transcription-PCR (RT-PCR) during seed development and in response to heat and cold stress. In addition, the copy number of the cloned PeMIPS1 gene in the genome of Passiflora edulis, P. eichleriana, P. caerulea, P. nitida and P. coccinea was determined by Southern blot analyses.

Key results: A full-length cDNA clone of the PeMIPS1 from P. edulis was isolated and characterized. Southern blot analyses indicated that the genomic DNA might have diverse sequences of MIPS-encoding genes and one copy of the cloned PeMIPS1 gene in the genomes of P. edulis, P. eichleriana, P. caerulea, P. nitida and P. coccinea. RT-PCR expression analyses revealed the presence of PeMIPS1 transcripts in ovules, pollen grains and leaves, and during the seed developmental stages, where it peaked at 9 d after pollination. The PeMIPS1 gene is differentially regulated under cold and heat stress, presenting a light-responsive transcription.

Conclusions: Experimental data suggest that PeMIPS1 transcription plays an important role in the establishment of developmental programmes and during the response of plants to environmental changes. The PeMIPS1 is differentially transcribed during cold and heat stress, presenting a light response pattern, suggesting that it is important for environmental stress response.

Fig. 1.

Bootstrap consensus phylogenetic tree of myo-inositol-1-phosphate synthase (MIPS) genes from plant species. The tree includes sequences of MIPS available in GenBank and TIGR databases. Passiflora edulis is highlight in bold, as Pe. Abbreviation of the species and accession number of each sequence are: Aa, Actinidia arguta (AY005128); Ac, Allium cepa (TC248); Afp, Aquilegia formosa × pubescens (TC14836); At, Arabidopsis thaliana (U04876); As, Avena sativa (AB059557); Am, Avicennia marina (AY028259); Bn, Brassica napus (U66307); Cp, Citrus paradise (Z32632); Gm, Glycine max (AY382834); Gh, Gorssypium hirsutum (TC27410); Hv, Hordeum vulgare (AF056325); Lj, Lotus japonicus (TC8275); Le, Lycopersicon esculentum (TC154132); Mt, Medicago trunculata (TC93972); Mc, Mesembryanthemum crystallinum (U32511); Nt, Nicotiana tabacum (AB059557); Os, Oryza sativa (AB012107); Pv, Phaseolus vulgaris (AM048843); Pisp, Pinus sp. (TC64990); Posp, Populus sp. (TC19238); Pc, Porteresia coarctata (AF412340); So, Saccharum officinarum (TC65413); Si, Sesamum indicum (AF284065); St, Solanum tuberosum (TC112573); Sp, Spirodela polyrrhiza (Z11693); Sm, Suaeda maritima (AF433879); Tp, Tripolium pratense (AB236831); Ta, Triticum aestivum (AF120148); Vv, Vitis vinifera (TC45187); Xv, Xerophyta viscosa (AY323824); Zm, Zea mays (AF56326). Bootstrap values >50 % are displayed on the nodes.

Fig. 2.

Southern blot analysis of the MIPS gene in the genome of Passiflora species. The genomes of P. edulis (Pe), P. eichleriana (Pei), P. caerulea (Pca), P. nitida (Pn) and P. coccinea (Pco) were digested with EcoRI (E), HindIII (H) and XhoI (X). The membrane was probed with the 693 bp internal fragment from the PeMIPS1 gene at low (A) and high stringency (B) of washing conditions.

Fig. 3.

Differential transcription of the PeMIPS1 gene from passion fruit (P. edulis) in different organs (A) and developing seeds (B) 3, 9, 15, 21 and 27 days after pollination (DAP). Ov, ovules; PG, pollen grains; St, stem; L, leaves; Pt, petals; LG, leaf gland. The upper bands are consistent with the expected fragment amplified from the PeMIPS1 gene and the lower band corresponds to transcripts from the PeEFα gene (elongation factor EF-1α; internal control).

Fig. 4.

Effects of temperature and light on transcription of the PeMIPS1 gene in leaves of passion fruit plants 8 weeks after germination. Plants were exposed for 8 and 16 h at different temperatures in the dark (A) and under a continuous light intensity of 200 µmol m⁻² s⁻¹ (B). A plant before treatment (maintained at room temperature and a light intensity of 10 µmol m⁻² s⁻¹) is indicated by ‘Ct’. The upper bands are consistent with the expected fragment amplified from the PeMIPS1 gene and the lower band corresponds to transcripts from the PeEFα gene (elongation factor EF-1α; internal control).