PMC

Endogenous levels of 13-hydroperoxide derivatives. Volatiles present in the leaves of nontransformed potato plants (WT) and a transgenic antisense line showing the largest depletion in HPL activity (H57) were compared by head-space GLC analysis. All HPL-derived compounds [hexanal, (E)-2-hexenal; and (Z)-3 and (E)-2-hexenols] are reduced in the HPL-antisense H57 line, whereas levels of ethyl vinyl ketone (EVK) and (Z)-2-pentenal and pentenols (derived of LOX activity on 13-hydroperoxides) are increased. The identity of the peaks was determined by comparison of retention times with authentic standards and GC/MS determination of their structures (not shown).

HPL gene expression in HPL-antisense plants. A radioactive cDNA probe was used to detect simultaneously sense (s) and antisense (as) HPL transcripts in total RNA extracted from second (L2) and sixth (L6) leaves (as in Fig. ) of nontransformed potato plants (WT), and from three independent transgenic potato lines (H1, H4, and H57) expressing HPL-antisense RNA. The smaller hybridizing bands that can be observed in the lanes corresponding to the transgenic lines comigrate with the rRNAs, and are likely a result of trapped HPL-antisense transcripts.

Defense gene expression in nontransformed and HPL-depleted plants. Second and third leaves of nontransformed (WT) and HPL-depleted (H57) potato plants were wounded and harvested at different times after wounding (indicated in hours above the lanes). Small differences in the levels of sense HPL RNA (HPL) were observed on wounding, which may be related to slight differences in the ages of the leaves used. The accumulation of transcripts from wound-induced genes, allene oxide synthase (AOS), leucine aminopeptidase (LAP), lipoxygenases H1 (LOXH1) and H3 (LOXH3), proteinase inhibitor II (PIN2), and prosystemin (SYS), also was analyzed by Northern blotting. Equal RNA loading was verified by ethidium bromide staining of rRNA.

HPL gene expression in potato plants. HPL mRNA accumulation was determined by Northern blotting techniques, using an internal cDNA fragment as probe (HPL). Hybridization to the proteinase inhibitor II gene (PIN2) was used to monitor wound-induced gene expression. Ethidium bromide staining of rRNA was used to verify even loading of the gel. (A) Total RNA was isolated from the leaves of a healthy potato plant, starting from the top (lane 1, immature leaf) and sequentially following to the bottom (lane 7, approaching senescence). (B) Second (L2) and fifth (L5) leaves (from top to bottom) were wounded and harvested at different times thereafter (indicated in hours above the corresponding lanes). Nonwounded leaves (0) were collected as controls. (C) Total RNA was isolated from second leaves (L), and flowers at different developmental stages: small (lane 1), green (lane 2), and colored (lane 3) buds; and open (lane 4) and senescent (lane 5) flowers. RNA from potato berries (B) was included in the analysis also.