184. Abstract

Phospholipase D (PLD) catalyzes the hydrolysis of glycerophospholipids to produce phosphatidic acid, a metabolite proposed to participate in a wide range of cellular events such as cell proliferation, vesicle trafficking, and membrane remodeling (Exton, 1997). In plants, activation of PLD has been suggested to play an important role in the plant responses to ABA, ethylene, and wounding (Ritchie and Gilroy, 1998). It has also been proposed that PLD is involved in senescence, nutrient starvation, and plant-pathogen interactions (Fan et al., 1999). PLD has been purified from a number of plants and studied extensively in aspects of its enzymatic properties. Isolation of PLD genes has been reported from castor bean (Wang et al., 1994), rice, maize (Ueki, et al.,1995), and cabbage (Kim et al., 1999), as well as yeast (Rose et al., 1995), mouse (Colley et al., 1997), and human (Hammond et al., 1995). In addition, three distinct PLDs, designated as PLDalpha, PLDbeta, and PLDgamma, have been identified from Arabidopsis (Qin et al., 1997). It was found that PLDalpha requires millimolar concentrations of Ca2+, whereas PLDbeta and PLDgamma are active in the presence of polyphosphoinositide and micromolar levels of Ca2+. Moreover, possible catalytic and regulatory domains of PLD have been revealed. Recently, it was demonstrated that the three PLDs in Arabidopsis have different patterns of subcellular distribution and tissue expression (Fan et al., 1999), which may suggest that these PLDs have respectively unique cellular functions. Here we describe the identification of a PLD gene from the cotton fiber.

In our effort to isolate genes activated during the secondary wall stage in cotton fiber development by differential screening of a cDNA library of 24 days postanthesis (DPA) fibers, we selected a phage clone which showed considerably stronger signal intensity in the autoradiograph of 24 DPA probes (representing the secondary wall stage) compared with 14 DPA probes (representing the primary wall stage). This plaque was then subjected to in vivo excision and DNA sequencing. The insert was subsequently found to be over 1.0 kb and displayed high homology to PLD at the derived amino acid level by searching the GenBank database. In order to obtain the corresponding full-length cDNA, 5' RACE was performed using the Marathon cDNA amplification kit (Clontech) and a gene-specific primer at the position of 500 bp from the cDNA 3' end. After the 3.5 kb RACE product was cloned into pPCR-Script vector (Stratagene), analysis of the DNA sequence indicated that a complete cotton fiber PLD gene was isolated.

The full-length cDNA, designated as cPLD, consists of 4058 bp with the longest open reading frame extending from an ATG initiation codon at position 1370 to a TAA stop codon at position 3857. The deduced polypeptide of 829 amino acids has a predicted molecular mass of 93,039 Da and a pI of 7.6. Comparison of cPLD with Arabidopsis PLDs showed that the potential cotton PLD shares 42%, 66%, and 64% amino acid identity with PLDalpha, -beta, and -gamma, respectively. The overall similarity is 64%, 76%, and 77%, respectively. Multiple sequence alignment indicated that cPLD possesses two HXKXXXXD motifs which exist in all plant PLDs and are critical for catalytic function (Qin et al., 1997). There are also two potential polyphophoinositide binding sites near the C terminus (Qin et al., 1997). However, only three of the four consensus basic residues are conserved in cPLD. Moreover, a Ca2+/phospholipid binding C2 domain is present near the N terminus with one of the consensus acid residues substituted by a neutral amino acid (Qin et al., 1997), suggesting presumably reduced affinity of cPLD for Ca2+. To further investigate the expression of cPLD during cotton fiber development, Northern blot analysis was conducted. It was found that cPLD had a higher expression level in the secondary wall stage than in the primary wall stage. Exactly how the function of this cPLD correlates to fiber growth remains to be determined.
 

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