Proceedings of the XLV Italian Society of Agricultural Genetics - SIGA Annual Congress

Salsomaggiore Terme, Italy - 26/29 September, 2001

ISBN 88-900622-1-5

Oral Communication Abstract

EXPRESSION OF DIFFERENT PCD-RELATED NUCLEASES DURING MALE GAMETOGENESIS IN BARLEY (HORDEUM VULGARE L.)

ZAINA G.*, DE AMICIS F.*, MORASSUTTI C.*, FOGHER C.**, MARCHETTI S.*

* Dipartimento di Produzione Vegetale e Tecnologie Agrarie, Università di Udine, Via delle Scienze 208, 33100 Udine

stefano.marchetti@dpvta.uniud.it

** Istituto di Botanica e Genetica Vegetale, Università Cattolica del S. Cuore, Via Emilia Parmense 84, 29100 Piacenza

programmed cell death, nuclease, male gametogenesis, microspore, barley

Production of functional pollen grains relies significantly on deterioration and death of the anther tapetum, a tissue that releases structural and nutritional components for microspore eterotrophic growth. It was demonstrated that the selective killing of tapetal cells occurs by the programmed cell death (PCD) process. Since most observations on cell death in reproductive tissues have been on the histological and cytological levels, we focused our research on the biochemical and molecular mechanisms that underline this process.

PCD involves hydrolysis of genomic DNA, which must be catalysed by endonucleases active towards dsDNA. In barley the association to PCD was proposed for the S1-type nucleases Bnuc1 and BEN1, expressed during leaf senescence and seed germination respectively. Our research group demonstrated the co-expression of Bnuc1 and BEN1 in immature barley anthers containing microspores at the uninucleate stage. Afterthat, a full-length cDNA was isolated from a cDNA library prepared from immature anther mRNA. The corresponding CDS shows a high similarity with that of Bnuc1 (88%) and BEN1 (89%), whereas a large discrepancy can be noted in both the 5’- and 3’-UTR. The clone represents therefore a different gene coding for a novel putative nuclease (Bnuc2: barley nuclease 2; GenBank: AJ311603). To understand their possible association to PCD occuring in developing anthers, the expression pattern of these nucleases was analysed at different developmental stages of androgenesis using RT-PCR and Northern blot techniques.

Previously, our group demonstrated that a significant Mg²⁺-dependent nuclease activity was released in diffusates of barley uninucleate microspores. The endonuclease responsible for this activity (BMN: barley microspore nuclease) was purified, biochemically characterized and its N-terminal aminoacidic sequence determined.

Since the deduced aminoacid sequence of Bnuc1, Bnuc2 and BEN1 does not include the N-terminal sequence of BMN, it can also be concluded that a swarm of nucleases are expressed during male gametogenesis. Furthermore, sequence and expression analyses suggest that Bnuc1, Bnuc2 and BEN1 constitute a PCD-associated nuclease pool on the barley genome.