A sweetpotato SRD1 promoter confers strong root-, taproot-, and tuber-specific expression in Arabidopsis, carrot, and potato.

Harvestable, starch-storing organs of plants, such as fleshy taproots and tubers, are important agronomic products that are also suitable target organs for use in the molecular farming of recombinant proteins due to their strong sink strength. To exploit a promoter directing strong expression restricted to these storage organs, we isolated the promoter region (3.0 kb) of SRD1 from sweetpotato (Ipomoea batatas cv. 'White Star') and characterized its activity in transgenic Arabidopsis, carrot, and potato using the ß-glucuronidase (GUS) gene (uidA) as a reporter gene. The SRD1 promoter conferred root-specific expression in transgenic Arabidopsis, with SRD1 promoter activity increasing in response to exogenous IAA. A time-course study of the effect of IAA (50 µM) revealed a maximum increase in SRD1 promoter activity at 24 h post-treatment initiation. A serial 5' deletion analysis of the SRD1 promoter identified regions related to IAA-inducible expression as well as regions containing positive and negative elements, respectively, controlling the expression level. In transgenic carrot, the SRD1 promoter mediated strong taproot-specific expression, as evidenced by GUS staining being strong in almost the entire taproot, including secondary phloem, secondary xylem and vascular cambium. The activity of the SRD1 promoter gradually increased with increasing diameter of the taproot in the transgenic carrot and was 10.71-fold higher than that of the CaMV35S promoter. The SRD1 promoter also directed strong tuber-specific expression in transgenic potato. Taken together, these results demonstrate that the SRD1 promoter directs strong expression restricted to the underground storage organs, such as fleshy taproots and tubers, as well as fibrous root tissues.

Dammarenediol-II production confers TMV tolerance in transgenic tobacco expressing Panax ginseng dammarenediol-II synthase.

Panax ginseng is one of the famous medicinal plants. Ginsenosides, a class of tetracyclic triterpene saponins, are mainly responsible for its pharmacological activity. Most ginsenosides are composed of dammarenediol-II aglycone with various sugar moieties. Dammarenediol-II synthase is the first enzyme in the biosynthesis of ginsenosides. Here, we report that transgenic tobacco expressing the P. ginseng dammarenediol-II synthase gene (PgDDS) produced dammarenediol-II, and conferred resistance to Tobacco mosaic virus (TMV). Upon infection with TMV, lesions developed more rapidly in transgenic tobacco plants, and their size was smaller than those of wild-type plants. Transgenic tobacco plants showed a low level of both the viral titer and mRNA accumulation of TMV coat protein (CP) compared with the wild type. The production of dammarenediol-II in transgenic tobacco stimulated the expression of tobacco pathogenesis-related genes (PR1 and PR2) under both virus-untreated and -treated conditions. When the leaves of wild-type plants were inoculated with a mixture of TMV and dammarenediol-II, the leaves exhibited a reduced viral concentration and TMV-CP expression than those receiving TMV treatment alone. When the leaves of P. ginseng were infected with TMV, transcription of PgDDS was significantly increased. Transgenic P. ginseng plants harboring a ß-glucuronidase (GUS) gene driven by the PgDDS promoter were constructed. The GUS expression was activated when the transgenic ginseng plants were treated with TMV. These results indicate that the medicinally important dammarenediol-II can be ectopically produced in tobacco, and the production of dammarenediol-II in tobacco plants allows them to adopt a viral defense system.

Expression and functional characterization of three squalene synthase genes associated with saponin biosynthesis in Panax ginseng.

Squalene synthase (SQS) catalyzes the biosynthesis of squalene by condensing two molecules of farnesyl pyrophosphate (FPP), a key precursor in sterol and triterpene biosynthesis. Previously, we reported that PgSS1 overexpression results in the enhanced biosynthesis of both phytosterols and triterpene saponins in Panax ginseng. Here, cDNAs encoding two new SQS homologs (PgSS2 and PgSS3) from a P. ginseng expressed sequence tag (EST) library are described. Functional complementation analysis revealed that ectopic expression of PgSS1, PgSS2 and PgSS3 in the yeast erg9 mutant strain 2C1 lacking SQS activity restored ergosterol prototrophy. The recombinant mutant yeast produced squalene, squalene epoxide and ergosterol. PgSS1 (mRNA) was highly transcribed in all organs, whereas PgSS2 and PgSS3 (mRNAs) were only transcribed in specific organs. All three genes were activated positively by an elicitor (methyl jasmonate), but their transcriptional patterns were different. In situ hybridization analysis revealed that both PgSS1 and PgSS3 transcripts were preferentially accumulated near conducting tissue in the petiole. The PgSS1 and PgSS3 promoters were isolated, and the tissue- and organ-specific regulation of PgSS genes was examined. Transgenic ginseng was constructed by introducing PgSS1 and PgSS3 promoters fused to the ß-glucuronidase (GUS) gene. GUS expression driven by the PgSS1 promoter was found in both roots and shoots, but PgSS3-driven GUS was only found in shoots. These results suggest that the three SQS genes are differently expressed and that all three SQS enzymes are involved in squalene production in P. ginseng.

Transcriptional regulation of the cinnamyl alcohol dehydrogenase gene from sweet potato in response to plant developmental stage and environmental stress.

Cinnamyl alcohol dehydrogenase (CAD) is a key enzyme in the biosynthesis of lignin. We have isolated full length of a cDNA encoding CAD (IbCAD1) that was previously identified as the most abundant gene in an EST library of sweetpotato suspension cells. Phylogenetic analysis revealed that IbCAD1 belongs to the family of defense-related CADs. High levels of IbCAD1 mRNA were found in the roots of sweetpotato, but not in the leaves and petioles. The IbCAD1 gene transcripts were highly induced by cold, wounding, and reactive oxygen species. Analyses of transcriptional regulation of the IbCAD1 gene in transgenic tobacco plants carrying the IbCAD1 promoter-GUS revealed that IbCAD1 promoter expression was strong in the roots, but barely detectable in the cotyledons. IbCAD1 promoter activity increased with increasing root age, and strong promoter expression was observed in the lateral root emergence sites and in root tips. Weak GUS expression was observed in lignified tissues of vascular system of mature leaves and stems. IbCAD1 promoter activity was strongly induced in response to the biotic and abiotic stresses, with the strongest inducer being wounding, and was also induced by salicylic acid (SA) and jasmonic acid (JA) as well as by abscisic acid (ABA) and 6-benzylaminopurine. Taken together, our data suggest that IbCAD1 can be involved in JA- and SA-mediated wounding response and ABA-mediated cold response, respectively. The IbCAD1 gene may play a role in the resistance mechanism to biotic and abiotic stresses as well as in tissue-specific developmental lignification.

SRD1 is involved in the auxin-mediated initial thickening growth of storage root by enhancing proliferation of metaxylem and cambium cells in sweetpotato (Ipomoea batatas).

A sweetpotato (Ipomoea batatas cv. 'Jinhongmi') MADS-box protein cDNA (SRD1) has been isolated from an early stage storage root cDNA library. The role of the SRD1 gene in the formation of the storage root in sweetpotato was investigated by an expression pattern analysis and characterization of SRD1-overexpressing (ox) transgenic sweetpotato plants. Transcripts of SRD1 were detected only in root tissues, with the fibrous root having low levels of the transcript and the young storage root showing relatively higher transcript levels. SRD1 mRNA was mainly found in the actively dividing cells, including the vascular and cambium cells of the young storage root. The transcript level of SRD1 in the fibrous roots increased in response to 1000 muM indole-3-acetic acid (IAA) applied exogenously. During the early stage of storage root development, the endogenous IAA content and SRD1 transcript level increased concomitantly, suggesting an involvement of SRD1 during the early stage of the auxin-dependent development of the storage root. SRD1-ox sweetpotato plants cultured in vitro produced thicker and shorter fibrous roots than wild-type plants. The metaxylem and cambium cells of the fibrous roots of SRD1-ox plants showed markedly enhanced proliferation, resulting in the fibrous roots of these plants showing an earlier thickening growth than those of wild-type plants. Taken together, these results demonstrate that SRD1 plays a role in the formation of storage roots by activating the proliferation of cambium and metaxylem cells to induce the initial thickening growth of storage roots in an auxin-dependent manner.

Cloning and functional expression of the gene encoding an inhibitor against Aspergillus flavus alpha-amylase, a novel seed lectin from Lablab purpureus (Dolichos lablab).

Maize is one of the more important agricultural crops in the world and, under certain conditions, prone to attack from pathogenic fungi. One of these, Aspergillus flavus, produces toxic and carcinogenic metabolites, called aflatoxins, as byproducts of its infection of maize kernels. The alpha-amylase of A. flavus is known to promote aflatoxin production in the endosperm of these infected kernels, and a 36-kDa protein from the Lablab purpureus, denoted AILP, has been shown to inhibit alpha-amylase production and the growth of A. flavus. Here, we report the isolation of six full-length labAI genes encoding AILP and a detailed analysis of the activities of the encoded proteins. Each of the six labAI genes encoded sequences of 274 amino acids, with the deduced amino acid sequences showing approximately 95-99% identity. The sequences are similar to those of lectin members of a legume lectin-arcelin-alpha-amylase inhibitor family reported to function in plant resistance to insect pests. The labAI genes did not show any of the structures characteristic of conserved structures identified in alpha-amylase inhibitors to date. The recombinant proteins of labAI-1 and labAI-2 agglutinated human red blood cells and inhibited A. flavus alpha-amylase in a manner similar to that shown by AILP. These data indicate that labAI genes are a new class of lectin members in legume seeds and that their proteins have both lectin and alpha-amylase inhibitor activity. These results are a valuable contribution to our knowledge of plant-pathogen interactions and will be applicable for developing protocols aimed at controlling A. flavus infection.

Two sweetpotato ADP-glucose pyrophosphorylase isoforms are regulated antagonistically in response to sucrose content in storage roots.

The transcriptional regulation of ADP-glucose pyrophosphorylase (AGPase) genes in detached leaves in response to exogenous sucrose has been investigated earlier; however the effects of endogenous sucrose on AGPase gene transcription in leaves or starch-accumulating tissues have not yet been determined. We therefore have investigated the relationship between endogenous sucrose content in the storage tissues of sweetpotato (Ipomoea batatas cv. Yulmi) and the rate of transcription of the two sweetpotato AGPase isoforms, ibAGP1 and ibAGP2, by means of transient expression analysis of their promoters. Sequence analysis of the two promoters identified putative sucrose-responsive elements on the ibAGP1 promoter and, conversely, putative sucrose-starvation elements on the ibAGP2 promoter. Transient expression analyses on transverse storage root sections revealed that the ibAGP1 and ibAGP2 promoters directed strong expression in the sweetpotato storage roots (diameter: 1.5 cm). Sucrose contents of the sweetpotato storage roots were positively correlated with growth of the storage root. In the storage roots, ibAGP1 promoter activity became stronger with increasing endogenous sucrose levels, while ibAGP2 promoter activity became markedly weaker. Consequently, ibAGP2 was expressed primarily during the early stages of storage root development, whereas ibAGP1 was abundantly expressed in the later stages, during which a profound level of starch accumulation occurs. The antagonistic regulation of the two promoters in response to endogenous sucrose levels was also confirmed in carrot (Daucus carota L. cv. Hapa-ochon) taproots.

Genomic organizations of two small subunit ADP-glucose pyrophosphorylase genes from sweetpotato.

The genomic features of the small subunit ADP-glucose pyrophosphorylase (AGPase) isoforms are different in barley and maize. The two isoforms found in barley originated from one single gene through alternative splicing, while two independent genes encode the two isoforms in maize. To ascertain the genomic organizations of two small subunit AGPase isoforms in sweetpotato (ibAGP1 and ibAGP2), we isolated genomic DNAs containing the entire coding regions of two genes. Complete genomic structures of ibAGP1 and ibAGP2 were ascertained by the sequencing of these genomic regions. The transcribed regions of ibAGP1 and ibAGP2, comprising nine exons and eight introns, were distributed over 3.9 and 4.0 kb, respectively. The eight introns differed in length, from 76 to 946 bp in ibAGP1, and from 76 to 811 bp in ibAGP2, while the locations of introns in ibAGP1 and ibAGP2 were identical. There was 46-58% sequence identity between the intron sequences of the two genes. Intron sequence analyses suggested that either duplication in each intron, or gene conversion between introns of two isoforms, might cause major intron size differences between the two genes. Altogether, these results indicate that two small subunit AGPase isoforms in sweetpotato are encoded by two independent genes, in a fashion similar to that of maize small subunit AGPase genes.

Alterations in intracellular and extracellular activities of antioxidant enzymes during suspension culture of sweetpotato.

Cultured plant cells are a good system for the study of antioxidant mechanisms and for the mass production of antioxidants, because they can be grown under conditions of high oxidative stress. Alterations in the intracellular and extracellular activities of three antioxidant enzymes, superoxide dismutase (SOD), guaiacol-type peroxidase (POD), and glutathione peroxidase (GPX), were investigated in suspension cultures of sweetpotato (Ipomoea batatas) during cell growth. Intracellular SOD activities (units/mg protein) at 15 days after subculture (DAS) and 30 DAS were 10 and 20 times higher, respectively, compared with the SOD activity at 1 DAS, whereas intracellular specific POD and GPX activities did not significantly increase until after 15 DAS, when they rapidly increased. The extracellular activities of the three enzymes in culture medium were much higher than were the intracellular activities. The change in extracellular SOD activity was similar to that of extracellular GPX during cell growth. Those activities showed high levels until 5 DAS and then significantly decreased. Extracellular POD activity had an almost constant level regardless of the cell growth stage. In addition, intracellular SOD and POD isozymes were quite different from those isozymes in the culture medium. The changes in SOD and POD isozymes observed here suggest that different isozymes might modulate the levels of reactive oxygen intermediates during cell growth. Characterization of extracellular antioxidant enzymes discovered here would provide a new understanding for defense mechanism in plants.

Salt causes ion disequilibrium-induced programmed cell death in yeast and plants.

Programmed cell death (PCD) is a fundamental cellular process conserved in metazoans, plants and yeast. Evidence is presented that salt induces PCD in yeast and plants because of an ionic, rather than osmotic, etiology. In yeast, NaCl inhibited growth and caused a time-dependent reduction in viability that was preceded by DNA fragmentation. NaCl also induced the cytological hallmarks of lysigenous-type PCD, including nuclear fragmentation, vacuolation and lysis. The human anti-apoptotic protein Bcl-2 increased salt tolerance of wild-type yeast strain and calcineurin-deficient yeast mutant (cnb1Delta) that is defective for ion homeostasis, but had no effect on the NaCl or sorbitol sensitivity of the osmotic hypersensitive hog1Delta mutant -- results that further link PCD in the response to the ion disequilibrium under salt stress. Bcl-2 suppression of cnb1Delta salt sensitivity was ENA1 (P-type ATPase gene)-dependent, due in part to transcriptional activation. Salt-induced PCD (TUNEL staining and DNA laddering) in primary roots of both Arabidopsis thaliana wild type (Col-1 gl1) and sos1 (salt overly sensitive) mutant seedlings correlated positively with treatment lethality. Wild-type plants survived salt stress levels that were lethal to sos1 plants because secondary roots were produced from the shoot/root transition zone. PCD-mediated elimination of the primary root in response to salt shock appears to be an adaptive mechanism that facilitates the production of roots more able to cope with a saline environment. Both salt-sensitive mutants of yeast (cnb1Delta) and Arabidopsis (sos1) exhibit substantially more profound PCD symptoms, indicating that salt-induced PCD is mediated by ion disequilibrium.