Cloning, expression, and molecular characterization of a small pea gene family regulated by low levels of ultraviolet B radiation and other stresses

Abstract

A pea (Pisum sativum) DNA fragment (termed MB3) was isolated by differential display of cDNAs obtained from total leaf RNA of ultraviolet B (UV-B) radiation-treated plants. Longer cDNAs were cloned by rapid amplification of cDNA ends in the 3' to 5' direction. Three different, but very similar, cDNAs were cloned, sadA, sadB, and sadC, the major difference between them being a 36-bp deletion in the coding region of sadB. Southern blotting confirmed the occurrence of at least three genes in the pea genome. Database comparisons of the SAD protein sequences revealed high identity (46%) and similarity (77%) with a putative tomato (Lycopersicon esculentum) short-chain alcohol dehydrogenase. Very low levels of UV-B radiation (the biologically effective radiation normalized to 300 nm = 0.08 W m(-2)) was shown to up-regulate expression, a dose considerably lower than that needed to induce expression of the well-known UV-B defensive chalcone synthase and phenylalanine ammonia lyase genes. RNase protection assay revealed that primarily sadA and sadC mRNA accumulation was enhanced by UV-B. In addition to UV-B irradiation, ozone fumigation, wounding, aluminum stress, and salt stress induced increased transcript levels of the sad genes in pea.

Figure 1

Sequences of the sad genes and the corresponding proteins. a, DNA sequences of the sadA, sadB, sadC, and MB3 cDNAs from pea cv Greenfeast. The start and stop codons are boxed, the MB3 primer sequences are underlined, and the nucleotides of the first primer matching the sad sequences are double underlined. Possible poly(A) signals (Hunt, 1994) are marked with a shaded area. b, Protein sequence alignment of the pea sadA, sadB, and sadC gene products with the translated sequences of the tomato gad3 gene (GenBank accession no. U21801; Jacobsen and Olszewski, 1996), the Arabidopsis Atsad2 gene product (GenBank accession no. AC004411; the second of four sad genes in the same genomic DNA region and by us named AtSad2), and Streptomyces exfoliatus (Marekov et al., 1990; SwissProt accession no. P19992). The catalytic YXXSK region is boxed, as well as the nucleotide binding site (Ghosh et al., 1991).

Figure 2

Southern blot probed with sadA cDNA, showing the restriction pattern of genomic pea DNA cleaved with the enzymes BamHI (B), EcoRI (E), HindIII (H), and XbaI (X), hybridized with [³²P]dCTP-labeled cDNA of the sadA gene. The positions of M_r standards are shown to the right and are expressed in numbers of bases.

Figure 3

Northern blot showing the presence of mRNA hybridizing to sadA cDNA under different types of stresses. a, Pea leaves exposed (U) or not exposed (C) to UV-B (UV-B_BE,300 = 1.6 W m⁻²) for 0, 3, 6, or 12 h; b, pea leaves exposed (O) or not exposed (C) to ozone, approximately 100 nL L⁻¹ for 12 h each 24-h experimental period. The experiment was carried out for 0, 24, or 48 h. c, Nonwounded control leaves (C), wounded leaves (W), and neighbors to wounded leaves (N) 0, 3, 6, or 24 h after wounding; d, pea roots exposed (A) or not exposed (C) to aluminum in hydroponic cultures, as described in the “Materials and Methods,” for 0, 3, 6, or 24 h; e, pea roots exposed (S) or not exposed (C) to NaCl in hydroponic cultures, as described in “Materials and Methods,” for 0, 3, 6, or 24 h. The same Northern blots hybridized to cDNA for 18S rRNA are shown below the corresponding sadA-hybridized northern blot to compare the loading of the total RNA samples on the gels and the transfer of the RNA to the membrane.

Figure 4

Northern blot showing the presence of mRNA hybridizing to [³²P]dCTP-labeled cDNA of the sadA, chs, and pal genes after exposure to low dose UV-B (UV-B_BE,300 = 0.08 W m⁻²) for 3, 6, or 12 h. See Figure 3 for abbreviations.

Figure 5

RNase protection assay performed with total RNA from UV-B-exposed or control plants. Probes specific for sadA (lanes 1–4), sadB (lanes 5–8), or sadC (lanes 9–12) were used. Equal amounts of ³²P-labeled probe (6 × 10⁴ cpm) and total RNA (10 μg) were hybridized in all samples. The samples are as follows: lanes 1, 5, and 9, probe hybridized with total RNA from control plants before digestion; lanes 2, 6, and 10, probe hybridized with total RNA from UV-B-exposed plants (12 h of UV-B_BE,300 = 1.6 W m⁻²) before digestion; lanes 3, 7, and 11, probe hybridized with total RNA from yeast before digestion; lanes 4, 8, and 12, probe hybridized with total RNA from yeast, no digestion (only 5% of the total reaction volume was added in each of these three lanes due to the high specific acivity of these radioactive probes). R denotes the radiolabeled RNA ladder with the distance between each band corresponding to even 100 bases.

Figure 6

Northern blot showing the presence of mRNA hybridizing to sadA cDNA in different types of tissue. In lanes 1 to 16 the tissue is from cv Greenfeast, whereas in lanes 17 and 18 the tissue is from the mutant argenteum. The tissue types are as follows: 1, Dry seeds; 2, seeds after 16 h of soaking in tap water; 3, shoots 9 d after sowing; 4, cotyledons 14 d after sowing; 5, leaf buds 14 d after sowing; 6, cotyledons 21 d after sowing; 7, second leaf pairs 21 d after sowing; 8, third leaf pairs 21 d after sowing; 9, fourth leaf pairs 21 d after sowing; 10, fifth leaf pairs 21 d after sowing; 11, roots from plants grown in vermiculite 14 d after sowing; 12, roots from plants grown in vermiculite 21 d after sowing; 13, roots from plants grown in vermiculite 42 d after sowing; 14, stems 21 d after sowing; 15, tendrils; 16, flowers (white); 17, flowers (purplish); and 18, pods.