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The pea (Pisum sativum) gene Cyp15a encodes a protein with sequence similarity to cysteine proteases. Expression of Cyp15a was investigated during pea seedling development and in response to environmental stress. Cyp15a shows increased transcription and elevated mRNA levels in plant tissues that are partially dehydrated or treated with 0.6 M mannitol. Cyp15a mRNA levels also increase in seedlings treated with 0.2-0.25 M NaCl or KCl. During development, Cyp15a mRNA levels increase within 6 to 12 h in cotyledons and axes during germination and continue to increase for at least 96 h. Illumination of dark-grown seedlings increased Cyp15a mRNA abundance in elongating and non-elongating stem tissues. GA and ABA, which modulate the abundance of many seed-localized cysteine proteases, did not significantly modulate Cyp15a mRNA levels in stems. The protein encoded by Cyp15a contains a typical amino-terminal secretory targeting domain. This domain is followed by a pro-sequence containing ca. 110 amino acids that is found in other cysteine proteases. Polyclonal antibodies, directed against CYP15a, recognized both the larger pro-form and the cleaved mature form of CYP15a on western blots. Immunolocalization assays indicated that both forms of the protein are located in cell walls of stem cortical cells.

We previously reported that the pea (Pisum sativum) gene, Trg31, shows increased transcription and elevated mRNA levels in plant tissues which are dehydrated and lose turgor. The protein encoded by Trg31 is homologous to members of the MIP intrinsic membrane protein superfamily. Expression of Trg31 was characterized during pea seedling development and in transgenic tobacco using Trg31 promoter::Gus fusions. In pea, Trg31 mRNA abundance was highest in roots followed by flowers, stems and leaves. In roots, Trg31 mRNA levels were highest in non-elongating regions and low in root tips. In dark-grown seedlings, Trg31 mRNA levels were high in stems and illumination caused mRNA abundance in stems to decrease. Histochemical analysis of transgenic tobacco expressing Trg31 promoter::Gus constructs showed high GUS activity in root to shoot and hypocotyl to cotyledon junctions and cotyledons in germinating seedlings. High activity was also observed in the leaf marginal meristem and trichomes. In more mature seedlings, Trg31 promoter activity was observed in the non-elongating portion of the root and in stems especially in the vascular tissue. A gradient of expression was noted in leaf to stem junction zones with highest expression in the younger tissues. Very high expression was observed in stems of flowers and other floral tissues including the calyx, corolla, style, ovules, pods and pollen. This expression pattern suggests that the Trg31 gene product may facilitate transport from sources, through transmitting tissues to sinks.

Reduction of turgor in pea shoots caused the accumulation of several poly(A) RNAs. cDNA clones derived from three different poly(A) RNAs which accumulate in wilted pea shoots were isolated, sequenced and expression of the corresponding genes examined. Clone 7a encoded a 289 amino acid protein. The C-terminal 180 amino acids of this protein were homologous to soybean nodulin-26. RNA hybridizing to cDNA 7a was abundant in roots, and induced in shoots by dehydration, heat shock and to a small extent by ABA. Hydropathic plots indicate that the protein encoded by cDNA 7a contains six potential membrane spanning domains similar to proteins which form ion channels. Clone 15a encoded a 363 amino acid protein with high homology to cysteine proteases. RNA hybridizing to cDNA 15a was more abundant in roots than shoots of control plants. Dehydration of pea shoots induced cDNA 15a mRNA levels whereas heat shock or ABA treatment did not. Clone 26g encoded a 508 amino acid protein with 30% residue identity to several aldehyde dehydrogenases. RNA hybridizing to cDNA 26g was induced by dehydration of shoots but not roots and heat shock and ABA did not modulate RNA levels. Levels of the three poly(A) RNAs increased 4-6-fold by 4 h after wilting and this increase was not altered by pretreatment of shoots with cycloheximide. When wilted shoots were rehydrated, RNA hybridizing to cDNA 26g declined to pre-stress levels within 2 h. Run-on transcription experiments using nuclei from pea shoots showed that transcription of the genes which encode the three poly(A) RNAs was induced within 30 min following reduction of shoot turgor. One of the genes showed a further increase in transcription by 4 h after dehydration whereas transcription of the other 2 genes declined. These results indicate that plant cells respond to changes in cell turgor by rapidly increasing transcription of several genes. Furthermore, the expression of the turgor-responsive genes varies with respect to the time course of induction and reversibility of the wilting-induced changes.

A 6.25 kbp barley plastid DNA region located between psbA and psbD-psbC were sequenced and RNAs produced from this DNA were analyzed. TrnK(UUU), rps16 and trnQ(UUG) were located upstream of psbA. These genes were transcribed from the same DNA strand as psbA and multiple RNAs hybridized to them. TrnK and rsp16 contained introns; a 504 amino acid open reading frame (ORF504) was located within the trnK intron. Between trnQ and psbD-psbC was a 2.24 kbp region encoding psbK, psbI and trnS(GCU). PsbK and psbI are encoded on the same DNA strand as psbD-psbC whereas trnS(GCU) is transcribed from the opposite strand. Two large RNAs accumulate in barley etioplasts which contain psbK, psbI, anti-sense trnS(GCU) and psbD-psbC sequences. Other RNAs encode psbK and psbI only, or psbK only. The divergent trnS(GCU) located upstream of psbD-psbC and a second divergent trnS(UGA) located downstream of psbD-psbC were both expressed. Furthermore, RNA complementary to psbK and psbI mRNA was detected, suggesting that transcription from divergent overlapping transcription units may modulate expression from this DNA region.