Conserved expression of the Arabidopsis ACT1 and ACT 3 actin subclass in organ primordia and mature pollen.

Abstract

We have proposed that ancient and divergent classes of plant actin genes have been preserved throughout vascular plant evolution, because they have distinct patterns of gene regulation. The hypothesis was explored for AC7Y and ACT3, which represent one of the six ancient subclasses in the Arabidopsis actin gene family. Comparison of AC7Y and ACT3 cDNA and genomic sequences revealed highly divergent flanking and intron sequences, whereas they encoded nearly identical proteins. Quantification of their leve1 of divergence suggests that they have not shared a common ancestor for 30 to 60 million years. Gene-specific FINA gel blot hybridization and reverse transcriptase-polymerase chain reaction analyses demonstrated that the distribution of AC7Y and ACT3 mRNAs was very similar: both preferentially accumulated at high levels in mature pollen and at very low levels in the other major organs. The 5' flanking regions of both genes, including the promoter, leader exon and intron, and thefirst 19 codons, werefused to the P-giucuronidase (GUS) reporter gene. The expression of these reporter fusions was examined in a large number of transgenic Arabidopsis plants. Histochemical assays demonstrated that both ACTT-GUS and ACT3-GUS constructs were expressed preferentially in pollen, pollen tubes, and in all organ primordia, including those in roots, shoots, and the inflorescence. Comparison of the 5' flanking regions of AC7Y and ACT3 revealed a number of short conserved sequences, which may direct their common transcriptional and post-transcriptional regulation. The expression patterns observed were distinct from those of any other Arabidopsis actin subclass. The conservation of their expression pattern and amino acid sequences suggests that this actin subclass plays a distinct and required role in the plant cytoskeleton.

Distribution of actin gene isoforms in the Arabidopsis leaf measured in microsamples from intact individual cells

“…However, because ACT7 and 11 were detected neither in single-cell epidermal samples nor in mesophyll samples, we conclude that expression of these two species is specific to trichomes. We did not detect ACT1 and 3 in trichomes, in contrast to the results of An et al (1996a).…”

Strong, constitutive expression of the Arabidopsis ACT2/ACT8 actin subclass in vegetative tissues

“…ACT11, the next closest subclass, is expressed most strongly in the emerging inflorescence and in developing pollen and ovules (Huang et aL, 1996c). The ACT1/ACT3 (An et al, 1996) and ACT4/ACT12 subclasses are extremely divergent in sequence from ACT2/ACT8. Both of these subclasses are strongly and preferentially expressed in mature pollen, but each show distinct expression programs in organ primordia and root caps, respectively.…”

“…The ACT8 transcript had multiple 5' ends at 33, 37, and 42 nt downstream of the TATATA sequence. Multiple transcription start sites have been identified in several plant actin genes (An et al, 1996;Pearson and Meagher, 1990). Alignment of the 5' flanking sequences (Figure 2) revealed a 9 bp deletion/insertion difference between the TATA boxes and the transcriptional start sites of ACT2 and ACT8.…”

“…Total RNA from roots, stems, leaves, flowers, pollen, and siliques was resolved on a formaldehyde agarose gel, imprinted on a nylon membrane, and probed with the gene-specific 3' regions of ACT2 and ACT8 (An et al, 1996). Figure 4 shows the resulting autoradiograms.…”

“…Four independent samples containing 1 ~g of ACT2 ~,-genomic DNA and 1 i~g of pTZ19U vector plasmid (United States Biochemical Corporation, Cleveland, OH) were digested with BamHI, Sail, Bglll, and Hindlll, respectively. After inactivation of the restriction enzymes and ligation in 20 ~1, a 1 pl sample of the fused actinvector product was PCR-amplified for 25 cycles with the PLAC-12NBAM and M13-20 oligonucleotides as primers (Table 3) as described in An et al (1996). PLACT12N-BAM is an antisense primer with 24 bases complementary to codons 12-19 in all plant actin genes, an 11 base sequence containing a BamHI restriction site, and a 5 nt clamp at the 5' end.…”