DEVELOPMENTAL CHANGES OF DENDROBIUM CRUMENATUM SEEDS DURING EARLY STAGES OF GERMINATION

Abstract

Germination of D. crumenatum seeds on Vacin and Went medium containing sucrose showed that hydrolysis of storage granules is initiated after about two weeks in culture and that the root and shoot initials were formed after about 4 weeks. Seeds sown on culture medium without sucrose did not form any shoots or roots and the seedlings remained at the protocorm stage until they senesced.

In vivo protein synthesis experiments indicated that protein synthesis increased gradually from 6 days postgermination, reached maximal level at 18 days and thereafter declined. Sodium dodecyl sulfate gel electrophoresis of buffer-soluble proteins showed several developmental patterns, including: (a) disappearance of the 23 and 33 kDa polypeptides within 6 days of germination which could be the storage proteins present initially; (b) early appearance and progressive increase of the 10, 11 and 18 kDa polypeptides, presumably of chloroplast and peroxisomal origin. Protein microsequencing of a 10 kDa polypeptide band revealed 60% identity to the peroxisomal biosynthesis protein PASS of Pichia pastoris (yeast).

W c have analyzed the structure of genes encoding the glyoxylate cycle key enzyme isocitratc lyase (ICL) from this orchid. Restriction mapping, nucleotide sequence, and DNA gel blot hybridization analyses of cDNA clones indicated that there are two ICL genes in the n. cm111e11al11111 genome based upon their divergence in the 5 ' - and 3 ' - untranslated regions. The deduced amino acid sequence of the ICL1 1-3 cDNA clone, (2.1 kb), revealed more than 70% identity to ICL of other higher plants. Gene-specific primers were used to study expression of ICL gene in both embryos and seedlings. ICL gene transcripts were already present in the non- germinated seeds, increased in amout to a peak at day 3 after germination, and thereafter declined. Comparison of the sequence with ICL of other dicot species shows a conserved C-terminal tripeptide, Ser-Ag-Leu, which resembles peroxisomal targetting sequence.

We have also analyzed one other gene in this orchid, MSl-2.(1.2 kb), showing 40% similarity to malate synthase at the DNA level and 37% similarity to the 33 kDa oxygenevolving protein of photosystem II at the protein level. However, the nucleotide sequence showed a great possibility of it being a pseudo gene. The N-terminal region of this gene resembles that of the transit peptide present in most chloroplast proteins. Further investigation of this gene shows a probable cytochrome-b-heme signature. Surprisingly, gene-specific primers showed that the transcripts for this gene were present not only in the dry non-germinated seeds and the seedlings but also in the immature seeds.