Sunflower germin-like proteins: evolution, gene structure and functional characterization.

EHRENBOLGER F.; BERACOCHEA V.; PELUFFO L.; ALMASIA N.; VAZQUEZ ROVERE C.; HOPP H.E.; PANIEGO N.; HEINZ R.A.; LIA V.V.

Mar del Plata

Simposio; 18th International sunflower Conference; 2012

ASAGIR

  ·                Germin-like proteins (GLPs) are ubiquitous plant glycoproteins which belong to the cupin super family. They are encoded by a heterogeneous group of genes present in many land plants including monocots, dicots, gymnosperms and mosses. They were classified in a number of subfamilies that ranges from 6 to 12 depending on the species. Several studies have reported that GLP family members may function as complex QTL conferring broad-spectrum resistance to fungal and bacterial pathogens. Although overexpression of GLPs from different plant species has been shown to confer tolerance to Sclerotinia sclerotiorum, one of the main constraints of sunflower production worldwide, no data is available to date on the diversity and molecular characterization of these proteins in sunflower. Here we investigate the evolution, diversification, and function of sunflower GLPs (HaGLPs) with the aim of identifying new candidate genes for crop improvement. ·                Sixteen sunflower expressed sequence tags (ESTs) containing germin motifs were retrieved from publically available databases and proprietary tissue specific EST libraries. Phylogenetic analysis was conducted using 212 protein sequences including representatives from wheat, rice, barley, Arabidopsis, soybean and wild Helianthus species, among others. Intron/exon organization was examined by comparing ESTs to the corresponding genomic sequences obtained through PCR amplification. Expression patterns were studied by Northern blot and RT-PCR. Arabidopsis transgenic plants overexpressing HaGLP1 were obtained via Agrobacterium tumefaciens- mediated transformation. Infection experiments with the fungal pathogens Rhizoctonia solanii and S. sclerotiorum were assayed on agar plates using 5-10 day-old seedlings. ·                Phylogenetic reconstruction allowed identification of 9 putative subfamilies within HaGLPs. The HaGLP genes are divergent in terms of their primary sequence, the size of their encoded proteins and the length of the introns. Pairwise comparisons of the coding regions showed average sequence similarities of 51 and 44% at the nucleotide and amino acid levels, respectively. All HaGLPs analyzed possess N-terminal secretory signals. Expression studies showed that most HaGLPs are transcribed in major plant organs, albeit to varying degrees in different sunflower tissues (root, leaf, stem, flower, receptacle, and seed). Transgenic Arabidopsis lines overexpressing HaGLP1 showed enhanced tolerance to Rhizoctonia solani and 6% crude extracts (6%) of these transgenic lines inhibited the mycelial growth of S. sclerotiorum. ·                 The number of GLP subfamilies identified for sunflower is comparable to the number described for complete well-annotated angiosperm genomes, and relationships within these lineages are fairly congruent with established phylogenetic relationships among taxa. The presence of signal peptides in all HaGLPs suggests cell wall/extracellular matrix targeting. The antifungal properties of HaGLP1 will be discussed in the context of the generation of H2O2 produced by its enzymatic activity. ·                The results from infection and mycelial inhibition assays performed on Arabidopsis transgenic plants overexpressing HaGLP1, along with the affiliations of some members of the family to confirmed defense-related germins of other species, suggest that HaGLPs are valuable candidate genes for future breeding efforts.