Both wound-inducible and tuber-specific expression are mediated by the promoter of a single member of the potato proteinase inhibitor II gene family

A chimeric gene consisting of 1.3 kb of the 5' regulatory region of a member of the potato proteinase inhibitor II gene family, the coding region of the bacterial β-glucuronidase (GUS) gene and 260 bp of the proteinase inhibitor II 3'-untranslated region containing the poly(A) addition site was introduced into potato and tobacco by Agrobacterium tumefaciens mediated transformation. Analysis of transgenic plants demonstrates systemic, wound-inducible expression of this gene in stem and leaves of potato and tobacco. Constitutive expression was found in stolons and tubers of non-wounded potato plants. Histochemical experiments based on the enzymatic activity of the GUS protein indicate an association of the proteinase inhibitor II promoter activity with vascular tissue in wounded as well as in systemically induced non-wounded leaves, petioles, potato stems and in developing tubers. These data prove that one single member of the proteinase inhibitor II gene family contains cis-active elements, which are able to respond to both developmental and environmental signals. Furthermore they support the hypothesis of an inducing signal (previously called proteinase inhibitor inducing factor), which is released at the wound site and subsequently transported to non-wounded parts of the plant via the vascular system from where it is released to the surrounding tissue.     

Systemic induction of proteinase-inhibitor-II gene expression in potato plants by wounding

The systemic induction of expression of the gene for proteinase inhibitor II after wounding different parts of potato (Solanum tuberosum L.) plants was analysed at the RNA level. Wounding of either leaves or tubers led to an induction of expression of this gene in non-wounded upper and lower leaves as well as in the upper stem segment, whereas no expression was observed in nonwounded roots or in the lower stem segment. The signal mediating the systemic induction in nonwounded tissue must therefore be able to move both acropetally and basipetally. The systemic wound response is specific for the expression of the proteinase-inhibitor-II gene as no influence was observed for the expression of genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase and the tuber storage protein patatin which were examined in parallel with the proteinase-inhibitor-II gene.Abbreviation ssRubisco small subunit of ribulose-1,5-bis-phosphate carboxylase     

Nucleotide sequence of proteinase inhibitor II encoding cDNA of potato (Solanum tuberosum) and its mode of expression

Summary Two cDNA clones containing the complete coding region of a developmentally controlled (tuber-specific) as well as environmentally inducible (wound-inducible) gene from potato (Solanum tuberosum) have been sequenced. The open reading frame codes for 154 amino acids. Its sequence is highly homologous to the proteinase inhibitor II from tomato, indicating that the cDNA's encode the corresponding proteinase inhibitor II of potato. In addition the putative potato proteinase inhibitor II contains a sequence which is completely homologous with that of another small peptide proteinase inhibitor from potato, called PCI-I. Evidence is presented that this small peptide is probably derived from the proteinase inhibitor II by posttranslational processing.Northern type experiments using RNA from wounded and nonwounded leaves demonstrate that RNA homologous to the putative proteinase inhibitor II cDNA's accumulates in leaves as a consequence of wounding, whereas normally the expression of this gene is under strict developmental control, since it is detected only in tubers of potato (Rosahl et al. 1986). In addition the induction of this gene in leaves can also be achieved by the addition of different polysaccharides such as poly galacturonic acid or chitosan. In contrast to the induction of its expression by wounding in leaves, wounding of tubers results in a disappearance of the proteinase II inhibitor m-RNA from these organs.     

Signal Peptide of Potato PinII Enhances the Expression of Cry1Ac in Transgenic Tobacco

Insect-resistant plants have been developed throughexpression of insecticidal proteins from Bacillusthuringiensis (Bt) in the early 1980s [1,2]. However, forcontrol of insect pests, it is necessary to increase theexpression of Bt protein overall or in specific plant tissues.To increase the expression level, synthetic Bt genes havebeen developed and used to produce transgenic plants[2–5]. A number of approaches have been taken to increasethe expression level of foreign proteins in transgeni…     

Posttranslational modification of an isoinhibitor from the potato proteinase inhibitor II gene family in transgenic tobacco yields a peptide with homology to potato chymotrypsin inhibitor I.

A member of the potato proteinase inhibitor II (PPI-II) gene family under the control of the cauliflower mosaic virus 35S promoter has been introduced into tobacco (Nicotiana tabacum). Purification of the PPI-II protein that accumulates in transgenic tobacco has confirmed that the N-terminal signal sequence is removed and that the inhibitor accumulates as a protein of the expected size (21 kD). However, a smaller peptide of approximately 5.4 kD has also been identified as a foreign gene product in transgenic tobacco plants. This peptide is recognized by an anti-PPI-II antibody, inhibits the serine proteinase chymotrypsin, and is not observed in nontransgenic tobacco. Furthermore, amino acid sequencing demonstrates that the peptide is identical to a lower molecular weight chymotrypsin inhibitor found in potato tubers and designated as potato chymotrypsin inhibitor I (PCI-I). Together, these data confirm that, as postulated to occur in potato, PCI-I does arise from the full-length PPI-II protein by posttranslational processing. The use of transgenic tobacco represents an ideal system with which to determine the precise mechanism by which this protein modification occurs.     

Expression of tobacco class II catalase gene activates the endogenous homologous gene and is associated with disease resistance in transgenic potato plants

We have previously shown that healthy potato plants respond poorly to salicylic acid (SA) for activating disease resistance against the late blight fungal pathogen Phytophthora infestans. However, SA is essential for the establishment of potato systemic acquired resistance (SAR) against P. infestans after treatment with the fungal elicitor arachidonic acid (AA). To understand the molecular mechanisms through which AA induces SA-dependent SAR in potato, we have recently studied the expression of potato class II catalase (Cat2St) in comparison with its tobacco homologue, Cat2Nt, which has previously been shown to bind SA. In the present study, we show that tobacco Cat2Nt is expressed at high levels and accounts for almost half of total SA-binding activity detected in tobacco leaves. In contrast, potato Cat2St is not expressed in healthy leaves, which is associated with the low SA responsiveness of potato plants for activation of disease resistance mechanisms. Upon treatment with AA, expression of potato Cat2St is induced not only in AA-treated leaves, but also in the upper untreated parts of the plants, concomitant with the establishment of SA -dependent SAR to P. infestans. Moreover, expression of the tobacco Cat2Nt gene in transgenic potato plants leads to constitutive expression of the endogenous potato Cat2St gene and is associated with enhanced resistance to P. infestans. These results collectively indicate that plant SA-binding class II catalases may play an important role in the development of disease resistance, possibly by serving as biological targets of SA.     

Signal peptide of potato PinII enhances the expression of Cry1Ac in transgenic tobacco

The modified Cry l Ac was expressed in transgenic tobacco plants. To allow secretion of the CrylAc protein into the intercellular space, the signal peptide sequence of potato proteinase inhibitor II (pinII) was N-terminally fused to the CrylAc encoding region. Expression of Cry 1 Ac in transgenic tobacco plants was assayed with ELISA. The results showed that pinII signal peptide sequence enhanced the expression of Cry lAc protein and led to the secretion of the Cry 1 Ac protein in transgenic tobacco plants. GFP gene was also fused to the signal peptide sequence and transformed to tobacco. The results of fluorescent detection showed that GFP had localized in the apoplast of transgenic plants.     

Differential expression of the potato proteinase inhibitor II promoter in transgenic tobacco

We studied temporal and spatial expression patterns of the potato proteinase inhibitor II (PI-II) promoter, using transgenic tobacco (Nkotiana tabacum L cv. Xanthi) plants that carried a fusion between the PI-II promoter and the chloramphenicol acetyltransferase (cat) gene. Pl-ll promoter activity was low when plants were young, but increased as plants grew. In 8-week-old plants, old leaves showed higher activity than young leaves. At flowering stage (ca. 15 weeks), the overall promoter activity was reduced to a lower level except in the petals. Compared with stems or petioles at the flowering stage, the roots and floral organs showed minimal activity for the Pl-ll promoter. We used several environmental stimuli to examine the induction of the Pl-ll promoter in different organs. Promoter induction was effected by wounding or methyl jasmonate in stems, petioles, sepals, and leaves. The induction was highest in leaves, as was sucrose-enhanced wound induction. These results suggest that the Pl-ll gene is temporally and spatially regulated. We also established a transient assay system in tobacco BY2 suspension cells to elucidate the upstream regulatory region of the Pl-ll promoter. A field strength of 0.75 kV/cm and 400 μF capacitance were optimal electroporation conditions for our transient assay.     

Expression of a tuber-specific storage protein in transgenic tobacco plants: demonstration of an esterase activity

A chimaeric gene composed of the 5' upstream region of STLS1, a leaf/stem specifically expressed gene from Solanum tuberosum, and the RNA-coding as well as the 3' downstream region of patatin, the major storage protein of potato tubers, has been transferred into tobacco plants using the Agrobacterium system. The introduction of this gene led to a leaf/stem specific expression of a 42-kd large protein which immunocrossreacts with patatin antiserum. Only low amounts of immunoreacting protein of smaller size could be detected in transgenic tobacco leaves indicating that the patatin protein is fairly stable in this heterologous environment. The size of the protein as well as the size of the RNA detected in transgenic tobacco leaves using a patatin-specific probe indicates that the patatin RNA was accurately processed in both leaf and stem tissue of tobacco. The expression of the patatin gene led to the appearance of a new esterase activity in the transformed tobacco which co-migrated with a protein immunoreacting with patatin antiserum. These data therefore demonstrate that patatin in addition to serving as a storage protein displays an enzymatic activity.     

Expression of E. coli inorganic pyrophosphatase in transgenic plants alters photoassimilate partitioning

Transgenic plants were constructed expressing a novel cytosolic inorganic pyrophosphatase in order to reduce the cytosolic pyrophosphate content. To this end the Escherichia coli gene ppa encoding inorganic pyrophosphatase was cloned between the 35S CaMV promoter and the poly(A) site of the octopine synthase gene and transferred into tobacco and potato plants by Agrobacterium-mediated gene transfer. Regenerated plants were tested for the expression of the ppa gene by Northern blots and activity gels. Plants expressing active inorganic pyrophosphatase showed a dramatic change in photoassimilate partitioning. In both transgenic tobacco and potato plants the ratio between soluble sugars and starch was increased by about 3-4-fold in source leaves as compared with the wild-type. However, whereas source leaves of transgenic tobacco plants accumulated much higher levels of glucose (up to 68-fold), fructose (up to 24-fold), sucrose (up to 12-fold) and starch (up to 8-fold) this was not observed in potato plants where the change in assimilate partitioning in source leaves was due to an increase of about 2-fold in sucrose and a reduction in starch content. Expression of the cytosolic inorganic pyrophosphatase in tobacco results in stunted growth of vegetatively growing plants due to a reduced internode distance. Upon flowering the transgenic plants increase their growth rate, reaching almost the same height as control plants at the end of the growth period. Old source leaves accumulate up to 100-fold more soluble sugars than control leaves. This increase in soluble sugars is accompanied by a reduction in chlorophyll content (up to 85%). Transgenic potato plants showed a less dramatic change in their growth behaviour. Plants were slightly reduced in size, with stems more highly branched. Tuber number increased 2-3-fold, but tuber weight was lower resulting in no net increase in fresh weight.     

The use of cysteine proteinase inhibitors to engineer resistance against potyviruses in transgenic tobacco plants

As the processing mechanism of all known potyviruses involves the activity of cysteine proteinases, we asked whether constitutive expression of a rice cysteine proteinase inhibitor gene could induce resistance against two important potyviruses, tobacco etch virus (TEV) and potato virus Y (PVY), in transgenic tobacco plants. Tobacco lines expressing the foreign gene at varying levels were examined for resistance against TEV and PVY infection. There was a clear, direct correlation between the level of oryzacystatin message, inhibition of papain (a cysteine proteinase), and resistance to TEV and PVY in all lines tested. The inhibitor was ineffective against tobacco mosaic virus (TMV) infection because processing of this virus does not involve cysteine proteinases. These results show that plant cystatins can be used against different potyviruses and potentially also against other viruses, whose replication involves cysteine proteinase activity.     

Colorado potato beetle larvae on potato plants expressing a locust proteinase inhibitor

The natural defence system of plants often involves inhibitors of digestive enzymes of their pests. Modem and environmental-friendly methods try to increase this plant resistance by expressing heterologous protease inhibitors in crops. Here we report the effects of expressing a gene from desert locust (Schistocerca gregaria) encoding two serine protease inhibitors in potato on Colorado potato beetle (Leptinotarsa decemlineata) larvae. The gene encoding both peptides on a single chain was used for Agrobacterium-mediated transformation of potato plants. The presence of the active inhibitor protein in the leaves was verified. The feeding bioassays in the laboratory showed that despite the low level of the peptide in leaves, CPB larvae on transgenic plants have grown slightly but significantly more slowly than those on control potato plants. The results support the notion that expression of multifunctional proteinase inhibitors of insect origin in plants might be a good strategy to improve insect resistance.     

Expression of a yeast-derived invertase in the cell wall of tobacco and Arabidopsis plants leads to accumulation of carbohydrate and inhibition of photosynthesis and strongly influences growth and phenotype of transgenic tobacco plants.

Chimeric genes consisting of the coding sequence of the yeast invertase gene suc 2 and different N-terminal portions of the potato-derived vacuolar protein proteinase inhibitor II fused to the 35S CaMV promoter and the poly-A site of the octopine synthase gene were transferred into tobacco and Arabidopsis thaliana plants using Agrobacterium based systems. Regenerated transgenic plants display a 50- to 500-fold higher invertase activity compared to non-transformed control plants. This invertase is N-glycosylated and efficiently secreted from the plant cell leading to its apoplastic location. Whereas expression of the invertase does not lead to drastic changes in transgenic Arabidopsis thaliana plants, transgenic tobacco plants show dramatic changes with respect to development and phenotype. Expression of the invertase leads to stunted growth due to reduction of internodal distances, to development of bleached and/or necrotic regions in older leaves and to suppressed root formation. In mature leaves, high levels of soluble sugars and starch accumulate. These carbohydrates do not show a diurnal turnover. The accumulation of carbohydrate is accompanied by an inhibition of photosynthesis, and in tobacco, by an increase in the rate of respiration. Measurements in bleached versus green areas of the same leaf show that the bleached section contains high levels of carbohydrates and has lower photosynthesis and higher respiration than green sections. It is concluded that expression of invertase in the cell wall interrupts export and leads to an accumulation of carbohydrates and inhibition of photosynthesis.     

Overexpression of jasmonic acid carboxyl methyltransferase increases tuber yield and size in transgenic potato

Jasmonates control diverse plant developmental processes, such as seed germination, flower, fruit and seed development, senescence and tuberization in potato. To understand the role of methyl jasmonate (MeJA) in potato tuberization, the Arabidopsis JMT gene encoding jasmonic acid carboxyl methyltransferase was constitutively overexpressed in transgenic potato plants. Increases in tuber yield and size as well as in vitro tuberization frequency were observed in transgenic plants. These were correlated with JMT mRNA level––the higher expression level, the higher the tuber yield and size. The levels of jasmonic acid (JA), MeJA and tuberonic acid (TA) were also higher than those in control plants. Transgenic plants also exhibited higher expression of jasmonate-responsive genes such as those for allene oxide cyclase (AOC) and proteinase inhibitor II (PINII). These results indicate that JMT overexpression induces jasmonate biosynthesis genes and thus JA and TA pools in transgenic potatoes. This results in enhanced tuber yield and size in transgenic potato plants.