Three drought-responsive members of the nonspecific lipid-transfer protein gene family in Lycopersicon pennellii show different developmental patterns of expression

Abstract

Genomic clones of two nonspecific lipid-transfer protein genes from a drought-tolerant wild species of tomato (Lycopersicon pennellii Corr.) were isolated using as a probe a drought- and abscisic acid (ABA)-induced cDNA clone (pLE16) from cultivated tomato (Lycopersicon esculentum Mill.). Both genes (LpLtp1 and LpLtp2) were sequenced and their corresponding mRNAs were characterized; they are both interrupted by a single intron at identical positions and predict basic proteins of 114 amino acid residues. Genomic Southern data indicated that these genes are members of a small gene family in Lycopersicon spp. The 3'-untranslated regions from LpLtp1 and LpLtp2, as well as a polymerase chain reaction-amplified 3'-untranslated region from pLE16 (cross-hybridizing to a third gene in L. pennellii, namely LpLtp3), were used as gene-specific probes to describe expression in L. pennellii through northern-blot analyses. All LpLtp genes were exclusively expressed in the aerial tissues of the plant and all were drought and ABA inducible. Each gene had a different pattern of expression in fruit, and LpLtp1 and LpLtp2, unlike LpLtp3, were both primarily developmentally regulated in leaf tissue. Putative ABA-responsive elements were found in the proximal promoter regions of LpLtp1 and LpLtp2.

Figure 2

Deduced amino acid sequence alignment of nsLTPs from wild tomato (L. pennellii), cultivated tomato (L. esculentum), and tobacco (Nicotiana tabacum). Tomato sequences are from genes le16 (accession no. U81996) and TSW12 (accession no. X56040); tobacco sequences are from genes TobLTP1 (accession no. D13952) and NTLTP1 (accession no. X62395). The alignment was performed using ClustalW (1.60). Positions of identity with respect to LpLTP1 are indicated by dots. The asterisks mark identical residues; the arrowheads indicate conservative substitutions in all six genes. Eight Cys and four Pro residues at highly conserved positions in all plant nsLTPs are underlined. The number of amino acid residues is indicated to the right of each sequence.

Figure 3

Genomic Southern blot of L. pennellii (P) and L. esculentum (E). Genomic DNA (8 μg) was digested with HindIII. Blots were probed with either an Ltp conserved-region probe (Cod250), or gene-specific probes for LpLtp1, LpLtp2, and LpLtp3. The sizes of LpLtp-hybridizing fragments are indicated in kb.

Figure 1

Restriction endonuclease map of a 9.1-kb DNA fragment from the L. pennellii genomic clone Pen16. The indicated restriction fragments were subcloned into pBluescript for sequence analysis. The SalI site is provided by the EMBL3 phage vector. The exons (open bars) and introns (filled bars) of LpLtp1 and LpLtp2 are shown, with the direction of transcription indicated by the arrows.

Figure 4

nsLtp transcript accumulation during late development of leaves from normal (N) or wilted (W) L. pennellii plants. RNA (7.5 μg) isolated from the smallest leaf (L1) to a fully expanded leaf (L5) was probed with either the nsLtp conserved-region probe (Cod250), or gene-specific probes for LpLtp1, LpLtp2, or LpLtp3. The relative intensity (RI) of hybridization to each probe is graphed to the right of each autoradiogram. A photograph of one of the ethidium-bromide-stained gels is shown at the bottom for load comparison.

Figure 5

nsLtp transcript accumulation in stem, flower, and fruit from normal (N) or wilted (W) L. pennellii plants. RNA (7.5 μg) isolated from stems (St), closed (Fc), and open (Fo) flowers and immature fruits (Fr) was probed and analyzed as in Figure 4.

Figure 6

nsLtp transcript accumulation in ABA-treated leaves from L. pennellii. RNA (7.5 μg) isolated from detached, fully expanded leaf petioles that had been immersed for 6 h in either water (H), 10 mm Mes buffer (B), or increasing concentrations of ABA in 10 mm Mes buffer; or from fully expanded leaves (L5) of normal (N) or wilted (W) plants, was probed and analyzed as in Figure 4.