HAMBURG, GERMANY
University of Hamburg

The ZmJR2 gene encodes a putative GDSL-like lipolytic enzyme and is preferentially expressed during early maize (Zea mays L.) kernel development

— Lorbiecke, R; Paul, C; Wienand, U

In most plants, fatty acids are incorporated into both membrane and storage lipids. Hydrolysis and synthesis of lipids are tightly regulated during plant development. Whereas in vegetative tissues fatty acids primarily support membrane biosynthesis, developing seeds often accumulate lipids as storage compounds. During seed germination, fatty acids are released from lipids by lipolysis to provide energy e.g. for embryonic growth and development. While germination associated lipases have been well characterized in the past (Huang, A., In: Moore, T. S. (Ed.), Lipid metabolism in plants. Academic Press Inc., Florida, USA, pp. 569–589, 1993), information about the importance of lipolytic enzymes during kernel and embryo development is limited.

Here we report on cloning and expression of a maize gene, ZmJR2, which encodes a novel putative lipolytic enzyme that is preferentially and transiently expressed during early maize kernel development.

A partial cDNA clone of ZmJR2 was initially identified in a subtractive hybridization approach aimed at identifying genes that are differentially expressed during early kernel development (Lorbiecke, R. et al. Maize Gen. Coop. Newsletter 75: 20–21, 2001). Screening of a λ-cDNA library from developing kernels led to the identification of a 1.4 kbp cDNA clone that encodes a protein of 372 amino acids. Sequence comparison revealed a significant similarity between ZmJR2 and members of the prokaryotic GDSL family of secreted lipolytic enzymes (PROSITE PDOC00842). In prokaryotes, proteins of this family are characterized by five conserved sequence domains (Block I to V) and a Ser-Asp-His catalytic triad (Brumlik, M., Buckley, T. J. Bacteriol. 178: 2060–2064, 1996). In ZmJR2, both, the five conserved domains and the positions of the three amino acids of the catalytic triad are highly conserved. Further in silico analysis of ZmJR2 indicated the presence of an N-terminal signal peptide and suggested that ZmJR2 encodes a functional and secreted lipolytic enzyme. Several other putative GDSL-like plant proteins have already been predicted based on EST search (Brick, D. J. et al. FEBS Let. 377: 475–480, 1995). However, no detailed data concerning their expression profiles or functions have been described.

Northern analysis indicated that ZmJR2 is predominately expressed during early kernel development (Fig. 1). Transcripts could also be detected in unfertilized ovaries (Fig. 1, lane I) and their amounts increased until 10 days after pollination (dap). Afterwards, ZmJR2 transcript amounts dropped steadily. When analyzing embryos and remaining kernel tissues of dissected 20 dap kernels, ZmJR2 expression was found to be stronger in the remaining kernel tissues (Fig. 1: 20 dap, kernel, embryo).

Maize kernel development is divided into four phases a) mitosis, b) cell enlargement and differentiation, c) storage material accumulation and d) desiccation and maturation ((Lur, H. S. and Setter, T. L. Plant Physiol. 103: 273–280, 1993). A comparison between cytological data of kernel development and the ZmJR2 expression profile indicated that ZmJR2 transcript abundance paralleled the time courses of cell division and endoreduplication rates in the developing endosperm (Lur, H. S. and Setter, T. L. Plant Physiol. 103: 273–280, 1993; Lorbiecke et al. unpublished). Therefore, it could be hypothesized that ZmJR2 acts most likely in processes which provide the energy for mitotic activity and rapid expansion of the maize endosperm which take place between 4 dap and 15 dap (Olsen, O.A. Annu. Rev. Plant Physiol. Plant Mol. Biol. 52: 233–267, 2001). However, significant amounts of ZmJR2 transcripts were also detected in coleoptiles and primary roots (Fig. 1) suggesting that a putative ZmJR2 lipolytic activity might also be necessary for energy supply in other non-green tissue types of the maize plant.

 

Figure 1. Northern analysis of ZmJR2 in various tissues at different developmental stages of maize. Gene expression was analyzed during kernel development in unfertilized pistillate spikelets that were split into ovary (I) and glumes (II), in intact unfertilized pistillate spikelets (III), in developing kernels 6 dap, 8, dap, 10 dap, 12 dap, 14 dap and 20 dap, in silks, tassel, in stems and leaves from mature flowering plants, in shoots of 16 day-old plants (seedling 16 d), in coleoptiles and roots from 3 day-old dark grown seedlings, and in embryo and remaining kernel tissues of dissected 20 dap kernels (20 dap: kernel, embryo). Expression of the constitutively expressed maize gene ZmJR27 is given as a control for RNA integrity and loading.


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