Sporamin Gene Research Articles

ACRE, a class of AP2/ERF transcription factors, activates the expression of sweet potato ß-amylase and sporamin genes through the sugar-responsible element CMSRE-1

Sugars, synthesized by photosynthesis in source organs, are loaded and utilized as an energy source and carbon skeleton in sink organs, and also known to be important signal molecules regulating gene expression in higher plants. The expression of genes coding for sporamin and β-amylase, the two most abundant proteins in storage roots of sweet potato, is coordinately induced by sugars. We previously reported on the identification of the carbohydrate metabolic signal-responsible element-1 (CMSRE-1) essential for the sugar-responsible expression of two genes. However, transcription factors that bind to this sequence have not been identified. In this study, we performed yeast one-hybrid screening using the sugar-responsible minimal promoter region of the ß-amylase gene as bait and a library composed only transcription factor cDNAs of Arabidopsis. Two clones, named Activator protein binding to CMSRE-1 (ACRE), encoding AP2/ERF transcription factors were isolated. ACRE showed transactivation activity of the sugar-responsible minimal promoter in a CMSRE-1-dependent manner in Arabidopsis protoplasts. Electric mobility shift assay (EMSA) using recombinant proteins and transient co-expression assay in Arabidopsis protoplasts revealed that ACRE could actually act to the CMSRE-1. Among the DEHYDRATION -RESPONSIVE ELEMENT BINDING FACTOR (DREB) subfamily, almost all homologs including ACRE, could act on the DRE, while only three ACREs could act to the CMSRE-1. Moreover, ACRE-homologs of Japanese morning glory also have the same property of DNA-binding preference and transactivation activity through the CMSRE-1. These findings suggested that ACRE plays an important role in the mechanism regulating the sugar-responsible gene expression through the CMSRE-1 conserved across plant species.

High-level expression of sucrose inducible sweet potato sporamin gene promoter: β-glucuronidase fusion gene in transgenic Nicotiana plumbaginifolia hairy roots

High-level expression of sucrose inducible sweet potato sporamin gene promoter: β-glucuronidase fusion gene in transgenic Nicotiana plumbaginifolia hairy roots

Isolation and characterization of cDNA clones encoding a novel subfamily sporamin B in sweet potato

Abstract. Effendy J, Labonte DR, Efendi D. 2019. Isolation and characterization of cDNA clones encoding a novel subfamily sporamin B in sweet potato. Biodiversitas 20: 3033-3041. Three cDNAs (IbH23, IbH41, and IbH74) encoding sporamin of sweet potato, were isolated from a skinning injury cDNA library. The open reading frame consisted of 288 nucleotides and the deduced polypeptide sequences consist of 96 amino acids with missing 127 amino acid residues at 5′. The sequences shared identity [42-62% (IbH23 and IbH41) and 40-61% (IbH74)] to sporamins A and B previously identified in other sweet potato species. The IbSpors contained three of four signature cysteine residues of sporamin family (Cys135, Cys198, Cys209). In addition to similarity to sporamins, these cDNA clones also showed similarities to miraculin (29-35%); Kunitz trypsin inhibitor (29-36%); factor Xa inhibitor (32-38%); subtilisin inhibitor (26-37%); seed protein (32%); tumor-related protein (36% in IbH23 and IbH41); and [wound-responsive protein (30%), sexual organ responsive protein (29%), and proteinase inhibitor (26%) can only be found in IbH74]. The three partial mRNA transcripts showed induced and transient expression in response to skinning injury. The phylogenetic analysis revealed that they belonged to a new member of sporamin gene subfamily B and may play key roles in plant defense and wound repair in response to skinning injury in storage roots of sweet potato.

Neomycin: An Effective Inhibitor of Jasmonate-Induced Reactions in Plants

Jasmonates are important phytohormones involved in both plant developmental processes as well as defense reactions. Many JA-mediated plant defense responses have been studied in model plants using mutants of the jasmonate signaling pathway. However, in plant species where JA-signaling mutants are not accessible, the availability of a tool targeting JA signaling is crucial to investigate jasmonate-dependent processes. Neomycin is a poly-cationic aminoglycoside antibiotic that blocks the release of Ca2+ from internal stores. We examined the inhibitory activities of neomycin on different jasmonate-inducible responses in eight different plant species: Intracellular calcium measurements in Nicotiana tabacum cell culture, Sporamin gene induction in Ipomoea batatas, PDF2.2 gene expression in Triticum aestivum, Nepenthesin protease activity measurement in Nepenthes alata, extrafloral nectar production in Phaseolus lunatus, nectary formation in Populus trichocarpa, terpene accumulation in Picea abies, and secondary metabolite generation in Nicotiana attenuata. We are able to show that neomycin, an easily manageable and commercially available compound, inhibits JA-mediated responses across the plant kingdom.

Effects of nitrogen forms on carbohydrate metabolism and storage‐root formation of sweet potato

AbstractIn this study, we examined the effects of different forms of nitrogen (N) fertilizer on carbohydrate metabolism and storage root formation in sweet potato [Ipomoea batatas L. (Lam.) cv. Shangshu 19 and cv. Jixu 23] in 2015–2016. Two fertilizer treatments, ammonium nitrogen (AN) and amide nitrogen (XN), were applied at 60 kg ha−1 in a two‐factor split‐plot design. The effects of nitrogen form on the morphology of adventitious roots, carbohydrate metabolism in potential storage roots, and number of storage roots per plant in sweet potato were investigated. The results show that during the early growth phase, the AN treatment significantly increased the number of adventitious roots, root tips, root length density, and fresh weight of roots (in pot trials). This treatment also significantly decreased the sucrose concentration of potential storage roots and increased the activities of cell wall, vacuolar, and cytoplasmic invertases. However, XN‐treated potential storage roots showed a relatively high starch concentration, activities of sucrose synthase and ADP‐glucose pyrophosphorylase, and transcription of sporamin genes. At the canopy closure period, the AN treatment significantly increased the number of storage roots of 0.5–5.0 cm in diameter and decreased the number of those > 5 cm in diameter compared to the control. The XN treatment induced the opposite effects. In the harvesting period, the AN treatment produced the highest storage root yield and number of storage roots per plant. Thus, in field trials the AN treatment induced a greater increase in production by increasing the number of storage roots.

Overexpression of ADP-glucose pyrophosphorylase (IbAGPaseS) affects expression of carbohydrate regulated genes in sweet potato [Ipomoea batatas (L.) Lam. cv. Yulmi

ADP-glucose pyrophosphorylase is crucial in starch metabolism. The open reading frame of IbAGPaseS gene is comprised of 1539-base pairs and encodes a polypeptide of 512 amino acid residues. The deduced IbAGPaseS protein sequence was homologous with that of other AGPase proteins of various plant species. Also, the IbAGPaseS gene appears to be a member of multiple gene families and further, the differences in the copy numbers among the ten cultivars were considered to lead to differences in starch contents. IbAGPaseS was expressed in whole tissues and revealed high activity in thick-pigmented roots and developing tuberous roots. RT-PCR analysis indicated that the main role of IbAGPaseS is starch biosynthesis accumulating sink tissues during sweet potato root development. The IbAGPaseS cDNA was introduced to sweet potato under the control of the CaMV 35S promoter using Agrobacterium-mediated gene transfer techniques, thereby affecting the expression levels of carbohydrate regulated genes and sporamin gene. These results suggest that IbAGPaseS affects carbohydrate gene regulation.

Differential activation of sporamin expression in response to abiotic mechanical wounding and biotic herbivore attack in the sweet potato.

BackgroundPlants respond differently to mechanical wounding and herbivore attack, using distinct pathways for defense. The versatile sweet potato sporamin possesses multiple biological functions in response to stress. However, the regulation of sporamin gene expression that is activated upon mechanical damage or herbivore attack has not been well studied.ResultsBiochemical analysis revealed that different patterns of Reactive oxygen species (ROS) and antioxidant mechanism exist between mechanical wounding (MW) and herbivore attack (HA) in the sweet potato leaf. Using LC-ESI-MS (Liquid chromatography electrospray ionization mass spectrometry analysis), only the endogenous JA (jasmonic acid) level was found to increase dramatically after MW in a time-dependent manner, whereas both endogenous JA and SA (salicylic acid) increase in parallel after HA. Through yeast one-hybrid screening, two transcription factors IbNAC1 (no apical meristem (NAM), Arabidopsis transcription activation factor (ATAF), and cup-shaped cotyledon (CUC)) and IbWRKY1 were isolated, which interact with the sporamin promoter fragment of SWRE (sporamin wounding-responsive element) regulatory sequences. Exogenous application of MeJA (methyl jasmonate), SA and DIECA (diethyldithiocarbamic acid, JAs biosynthesis inhibitor) on sweet potato leaves was employed, and the results revealed that IbNAC1 mediated the expression of sporamin through a JA-dependent signaling pathway upon MW, whereas both IbNAC1 and IbWRKY1 coordinately regulated sporamin expression through JA- and SA-dependent pathways upon HA. Transcriptome analysis identified MYC2/4 and JAZ2/TIFY10A (jasmonate ZIM/tify-domain), the repressor and activator of JA and SA signaling among others, as the genes that play an intermediate role in the JA and SA pathways, and these results were further validated by qRT-PCR (quantitative real-time polymerase chain reaction).ConclusionThis work has improved our understanding of the differential regulatory mechanism of sporamin expression. Our study illustrates that sweet potato sporamin expression is differentially induced upon abiotic MW and biotic HA that involves IbNAC1 and IbWRKY1 and is dependent on the JA and SA signaling pathways. Thus, we established a model to address the plant-wounding response upon physical and biotic damage.

High-level fructooligosaccharide production in transgenic tobacco plants

β-fructofuranosidase (FFase) of Aspergillus niger ATCC 20611 can transfer fructosyl residues from one sucrose to another for the synthesis of glucose and fructooligosaccharides composed of 1-kestose (GF2), nystose (GF3), and β-fructofuranosylnystose (GF4). The FFase gene, under the control of the sporamin gene promoter from sweet potato, was introduced into tobacco plants. Sporamin promoter activity is induced by sugar and exhibits preferential expression in stem and root tissues. Thin-layer and high performance liquid chromatographic analyses showed that soluble extracts from the transgenic plants contained considerable amounts of fructooligosaccharides such as GF2 and GF3. The conversion of sucrose into fructooligosaccharides did not affect plant growth or development. Our results indicate that the transgenic plants could be utilized as bioreactors, and this opens up the possibility for efficient production of fructooligosaccharides in sucroseproducing plants such as sugar beet and sugarcane.

Systemin, Hydroxyproline-Rich Systemin and the Induction of Protease Inhibitors

Systemin, an 18-amino acid signaling peptide isolated from tomato leaves, has been found to be an integral component of the jasmine acid signaling pathway, leading to the synthesis of protease inhibitors (PIs). The discovery of systemin has led to a search for other peptide signals involved in defense in the Solanaceae and in other plant families. A new class of peptides having similar signaling properties but little sequence homology to systemin have been found and termed hydroxyproline-rich glycopeptide systemins (HypSys). These small (18-20 amino acids) glycopeptides, like systemin, are derived from larger precursor proteins (proHypSys) and until recently were thought to function only in protection from herbivore attack. However, HypSys peptides isolated from petunia induced the defensin gene, known for its involvement in pathogen defense. More recently, a HypSys glycopeptide was isolated from sweet potato, a member of the Convolvulaceae family and found to induce the sporamin gene which codes for the major storage protein in tubers with trypsin inhibitor activity. These recent discoveries expand the function and range of the HypSys family of glycopeptides and establish these unique inducible signaling molecules as potential components of defense pathways throughout the Eudicots. Herein we review the signaling and structural properties of systemin and the HypSys glycopeptides and their roles in the induction of PIs.

Analyses of inheritance patterns and consistent expression of sporamin and chitinase PjChi-1 genes in Brassica napus

With 6 figures and 2 tables Abstract Stable and consistent expression of foreign genes in serial propagation is a crucial factor to induce effective and efficient resistance through genetic engineering in insects and fungi. To better understand the inheritance patterns and expression of transgenes, two genes (sporamin and chitinase PjChi-1) in T1 progeny of four single-copy T0 transformants were characterized. PCR detection revealed that the segregation ratio was 3 : 1 for tested plants of each T0 transformant, which agreed with the Mendelian rules. In RT-PCR analysis, 10 positive progeny plants of T0-11 line showed a stable expression of sporamin and PjChi-1 but not in wild type. Moreover, a higher expression level of sporamin gene in T1-11-3 was confirmed compared to the other progeny plants using Northern blot hybridization technique. Artificial inoculation of 10 plants (T1-11) with Lepidoptera larvae (Plutella xylostella) and fungus (Sclerotinia sclerotiorum) in in vitro bioassays showed significant resistance compared to the wild type. Progeny T1-11-3 showed the stronger resistance, which was consistent with the higher expression level of sporamin gene in Northern blot hybridization detection, whereas there was no difference in insect and fungi resistance among these positive T1-11 plants. These results indicate that both combined genes have been inherited from parental transformants to their progeny and showed a consistent resistance to insect and fungus in all positive T1 progeny.

Activation tagging of a gene for a protein with novel class of CCT‐domain activates expression of a subset of sugar‐inducible genes in Arabidopsis thaliana

In the current studies, we examined sugar-inducible gene expression using the Arabidopsis thaliana line sGsL, which carries luciferase (LUC) and beta-glucuronidase (GUS) reporter genes under the control of a 210-bp promoter derived from the sweet potato sporamin gene (Spo(min)). We isolated an enhancer activation-tagged mutant of this line that showed high-level expression of LUC and GUS under non-inducing low-sugar conditions. The Activator ofSpo(min)::LUC2 (ASML2) gene located close to the enhancer encodes a protein belonging to a previously uncharacterized class of CCT (CONSTANS, CONSTANS-like, TOC1) domain proteins. Overexpression of ASML2 cDNA in the sGsL line resulted in enhanced expression of not only LUC and GUS reporters but also several endogenous sugar-inducible genes, including Atbeta-Amy, ApL3, and VSP2. Transient co-expression of 35S::ASML2 with the Spo(min)::LUC or Atbeta-Amy::LUC reporter in protoplasts resulted in an approximately 2.4 or 5.6-fold transactivation of LUC expression, respectively. Expression of ASML2 was high in reproductive organs, and expression in seedlings was slightly enhanced by sugars, but not by abscisic acid. These results suggest that ASML2 functions as a transcriptional activator and regulates the expression of at least a subset of sugar-inducible genes.

Analysis of a Sugar Response Mutant of Arabidopsis Identified a Novel B3 Domain Protein That Functions as an Active Transcriptional Repressor

A recessive mutation hsi2 of Arabidopsis (Arabidopsis thaliana) expressing luciferase (LUC) under control of a short promoter derived from a sweet potato (Ipomoea batatas) sporamin gene (Spo(min)LUC) caused enhanced LUC expression under both low- and high-sugar conditions, which was not due to increased level of abscisic acid. The hsi2 mutant contained a nonsense mutation in a gene encoding a protein with B3 DNA-binding domain. HSI2 and two other Arabidopsis proteins appear to constitute a novel subfamily of B3 domain proteins distinct from ABI3, FUS3, and LEC2, which are transcription activators involved in seed development. The C-terminal part of HSI2 subfamily proteins contained a sequence similar to the ERF-associated amphiphilic repression (EAR) motif. Deletion of the C-terminal portion of HSI2 lost in the hsi2 mutant caused reduced nuclear targeting of HSI2. Null allele of HSI2 showed even higher Spo(min)LUC expression than the hsi2 mutant, whereas overexpression of HSI2 reduced the LUC expression. Transient coexpression of 35SHSI2 with Spo(min)LUC in protoplasts repressed the expression of LUC activity, and deletion or mutation of the EAR motif significantly reduced the repression activity of HSI2. These results indicate that HSI2 and related proteins are B3 domain-EAR motif active transcription repressors.

ACTIVATOR of Spomin::LUC1/WRINKLED1 of Arabidopsis thaliana Transactivates Sugar-inducible Promoters

We isolated an enhancer activation-tagged mutant of Arabidopsis thaliana line sGsL carrying the luciferase (LUC) gene under control of a short sugar-inducible promoter derived from a sweet potato sporamin gene (Spomin) that showed high level expression of LUC under non-inducing conditions. The activator of Spomin::LUC1 (ASML1) gene located downstream of the enhancer encoded an APETALA2 (AP2)-type AP2 domain protein, and this gene was shown recently to be responsible for the wrinkled1 mutation which causes defective accumulation of seed storage oil. Overexpression of ASML1 cDNA in sGsL plants resulted in enhanced expression of not only the LUC reporter but also endogenous sugar-inducible genes including Atbeta-Amy encoding beta-amylase. Transient co-expression of 35S::ASML1 with Spomin::LUC or Atbeta-Amy::LUC reporters in protoplasts resulted in an approximately 10-fold transactivation of LUC expression. This transactivation was lost when the C-terminal acidic region of ASML1 was deleted. Expression of ASML1 was high in reproductive organs, and ASML1 mRNA showed transient accumulation in leaves after treatment with 6% sucrose, whereas it did not respond to abscisic acid. These results suggest that ASML1/WRI1 is a transcriptional activator involved in the activation of a subset of sugar-responsive genes and the control of carbon flow from sucrose import to oil accumulation in developing seeds.

Two cis-acting regulatory elements are involved in the sucrose-inducible expression of the sporamin gene promoter from sweet potato in transgenic tobacco

In this study, we generated transgenic tobacco plants that express the beta-glucuronidase (GUS) gene under the control of the 305-bp 5'-upstream region of a gene coding for sporamin A of sweet potato. Expression of GUS in excised tobacco leaves was induced by sucrose, mimicking the sugar-inducible expression of the endogenous sporamin genes in sweet potato. Deletion of the sequences extending from position -305 (relative to the transcription start site) to -283 and from -146 to -87 resulted in an approximately 40-fold enhancement in GUS reporter expression. Furthermore, the sequence from -282 to -165 conferred sucrose-inducibility on the -89 core promoter of the Cauliflower Mosaic Virus 35S RNA gene. Analysis of internal deletions, linker scanning and the introduction of base substitutions in the sequence between positions -282 and -165 indicated that sucrose-responsiveness conferred by this region was dependent on the presence of two cis-acting elements, termed CMSREs (carbohydrate metabolite signal responsive elements) 1 and 2, which are separated by a spacer. A sequence similar or identical to the core of CMSRE-1 (TGGACGG) is also present in the promoters of several other sugar-inducible genes, and sequences encopassing the TGGACGG-related motifs from two of these could functionally replace the CMSRE-1 in the truncated sporamin A promoter. These results suggest that the TGGACGG element plays an important role in the sucrose-inducible expression of a group of plant genes.

Transcription factors for sugar-inducible genes

Expression of a variety of sink-related genes for the synthesis of storage compounds are up-regulated in response to increased levels of sugars, while expression of many genes involved in photosynthesis and the breakdown of storage compounds is repressed by sugars. Despite of extensive studies on sugar signaling in plants, only few transcription factors involved in the expression of sugar-inducible genes have been identified so far. A transgenic Arabidopsis thaliana line carrying luciferase gene under the control of a short sugar-inducible promoter derived from a sweet potato sporamin gene was used to screen for loss-of-function type as well as gain-of-function type mutants. These genetic screens resulted in the identification of novel transcription factors that are involved in the expression of at least a subset of sugar-inducible genes in Arabidopsis.

Sporamin-mediated resistance to beet cyst nematodes (Heterodera schachtii Schm.) is dependent on trypsin inhibitory activity in sugar beet (Beta vulgaris L.) hairy roots.

Sporamin, a sweet potato tuberous storage protein, is a Kunitz-type trypsin inhibitor. Its capability of conferring insect-resistance on transgenic tobacco and cauliflower has been confirmed. To test its potential as an anti-feedant for the beet cyst nematode (Heterodera schachtii Schm.), the sporamin gene SpTI-1 was introduced into sugar beet (Beta vulgaris L.) by Agrobacterium rhizogenes-mediated transformation. Twelve different hairy root clones expressing sporamin were selected for studying nematode development. Of these, 8 hairy root clones were found to show significant efficiency in inhibiting the growth and development of the female nematodes whereas 4 root clones did not show any inhibitory effects even though the SpTI-1 gene was regularly expressed in all of the tested hairy roots as revealed by northern and western analyses. Inhibition of nematode development correlated with trypsin inhibitor activity but not with the amount of sporamin expressed in hairy roots. These data demonstrate that the trypsin inhibitor activity is the critical factor for inhibiting growth and development of cyst nematodes in sugar beet hairy roots expressing the sporamin gene. Hence, the sweet potato sporamin can be used as a new and effective anti-feedant for controlling cyst nematodes offering an alternative strategy for establishing nematode resistance in crops.

Wound-response regulation of the sweet potato sporamin gene promoter region.

Sporamin, a tuberous storage protein of sweet potato, was systemically expressed in leaves and stems by wound stimulation. In an effort to demonstrate the regulatory mechanism of wound response on the sporamin gene, a 1.25 kb sporamin promoter was isolated for studying the wound-induced signal transduction. Two wound response-like elements, a G box-like element and a GCC core-like sequence were found in this promoter. A construct containing the sporamin promoter fused to a beta-glucuronidase (GUS) gene was transferred into tobacco plants by Agrobacterium-mediated transformation. The wound-induced high level of GUS activity was observed in stems and leaves of transgenic tobacco, but not in roots. This expression pattern was similar to that of the sporamin gene in sweet potatoes. Exogenous application of methyl jasmonate (MeJA) activated the sporamin promoter in leaves and stems of sweet potato and transgenic tobacco plants. A competitive inhibitor of ethylene (2,5-norbornadiene; NBD) down-regulated the effect of MeJA on sporamin gene expression. In contrast, salicylic acid (SA), an inhibitor of the octadecanoid pathway, strongly suppressed the sporamin promoter function that was stimulated by wound and MeJA treatments. In conclusion, wound-response expression of the sporamin gene in aerial parts of plants is regulated by the octadecanoid signal pathway.

Sugar-responsible elements in the promoter of a gene for beta-amylase of sweet potato.

Expression of genes coding for sporamin and beta-amylase, the two most abundant proteins in storage roots of sweet potato, is coordinately inducible in atypical vegetative tissues by sugars. A sweet potato gene for beta-amylase (beta-Amy) with introns as well as a beta-Amy::GUS fusion gene composed of the beta-Amy promoter and the GUS coding sequence, both showed sugar-inducible expression in leaves of transgenic tobacco which occurred via a hexokinase-independent pathway. Analyses using various 5'-terminal and internal deletions of the beta-Amy promoter indicated that truncated promoters of beta-Amy containing a sequence between -901 and -820, relative to the transcription start site, and the basic promoter region can confer sugar-inducible expression. This 82 bp region contained the TGGACGG sequence that plays an essential role in the sugar-inducible expression of the truncated promoter of the sporamin gene. Deletion or base substitutions of this element in the truncated beta-Amy promoter abolished the sugar-inducible expression, the results suggesting that the TGGACGG element plays an important role in the coordinate induction of expression of genes for beta-amylase and sporamin by sugars.

Sugar-Dependent Expression of the CHS-A Gene for Chalcone Synthase from Petunia in Transgenic Arabidopsis

Transgenic Arabidopsis thaliana plants were constructed by introduction of a fusion of the gene for beta-glucuronidase (GUS) to the CHS-A gene, which is one of the two genes for chalcone synthase that are actively expressed in the floral organs of petunia. The expression of the fusion gene CHS-A::GUS was low in transgenic Arabidopsis plantlets, but it was enhanced when plantlets or detached leaves were transferred to a medium that contained 0.3 molar sucrose, glucose, or fructose. No enhancement was observed when plantlets were transferred to a medium that contained 0.3 molar mannitol. Measurements of cellular levels of sugars revealed a tight linkage between the level of expression of the CHS-A::GUS gene and the level of accumulation of exogenously supplied sugars, in particular sucrose. The parallelism between the organ-specific accumulation of sugar and the organ-specific expression of the CHS-A::GUS gene was also observed in petunia and A. thaliana plants grown under normal conditions in soil. The consensus sequences for sugar responses, such as boxes II and III in members of the family of sporamin genes from the sweet potato, were found in the promoter region of the CHS-A gene that was used for fusion to the GUS gene. It is suggested that the expression of the CHS-A gene is regulated by sugars, as is the expression of other sugar-responsive genes, such as the genes for sporamin. A putative common mechanism for the control of expression of "sugar-related" genes, including the CHS-A gene, is discussed.

High-level expression of a sweet potato sporamin gene promoter: β-glucuroidase (GUS) fusion gene in the stems of transgenic tobacco plants is conferred by multiple cell type-specific regulatory elements

Genes coding for sporamin, the most abundant protein of the tuberous root of the sweet potato, are expressed at a high levels in the stems of plantlets cultured axenically on sucrose-containing medium. Their expression is also induced in leaf-petiole explants by high concentrations of sucrose. A fusion gene comprising of the 1 kb 5' upstream region of the gSPO-A1 gene coding for the A-type sporamin and the coding sequence of bacterial beta-glucuronidase (GUS) was introduced into the tobacco genome by Agrobacterium-mediated transformation. Transgenic tobacco plants cultured axenically on sucrose-containing medium expressed GUS activity predominantly in their stems. Histochemical examination of GUS activity using a chromogenic substrate showed a distinct spatial pattern of GUS staining in the stem. Strong GUS activity was detected in the internal phloem of the vascular system and at the node, especially at the base of the axillary bud. Relatively weaker GUS activity was also detected in pith parenchyma. A 5' deletion of the promoter to nucleotide -305, relative to the transcription start site, did not alter significantly the level of GUS activity or the spatial pattern of GUS staining in the stem. However, further deletions to -237 and -192 resulted in a decrease in the level of GUS activity in the stem that occurred simultaneously with the loss of GUS staining in both the internal phloem and at the base of the axillary bud. However, plants with these deletion constructs still exhibited the predominant expression pattern of GUS activity in the stem and GUS staining in the pith parenchyma cells. Deletion to -94 completely abolished the expression of GUS activity. These results indicate that a sequence between -305 and -237 contains a cis-regulatory element(s) that is required for expression of the GUS reporter gene in both the internal phloem and at the base of the axillary bud, while a sequence between -192 and -94 contains a cis-acting element(s) that is required for expression in pith parenchyma cells.