Abstract. We previously isolated and sequenced the major trypsin inhibitor from Amaranthus hypochondriacus seeds. This amaranth trypsin inhibitor (AmTI) is a ...
Plant Molecular Biology 41: 15–23, 1999. © 1999 Kluwer Academic Publishers. Printed in the Netherlands.
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Cloning and characterization of a trypsin inhibitor cDNA from amaranth (Amaranthus hypochondriacus) seeds Silvia Vald´es-Rodr´ıguez1,∗ , Alejandro Blanco-Labra1 , Glenda Anatoli Boradenenko2 , Alfredo Herrera-Estrella1 and June Simpson1
Guti´errez-Benicio1 ,
1 Unidad
de Biotecnolog´ıa e Ingenier´ıa Gen´etica de Plantas, Centro de Investigaci´on y de Estudios Avanzados del IPN, Apdo postal 629, C.P. 36500, Irapuato, Guanajuato, M´exico (∗ author for correspondence); 2 Instituto de Ciencias Agr´ıcolas, Universidad de Guanajuato, Apdo postal 311, C.P. 36500 and Irapuato, Guanajuato, M´exico Received 12 February 1999; accepted in revised form 9 June 1999
Key words: Amaranthus hypochondriacus), gene expression, trypsin inhibitor
Abstract We previously isolated and sequenced the major trypsin inhibitor from Amaranthus hypochondriacus seeds. This amaranth trypsin inhibitor (AmTI) is a 69 amino acid protein with high homology to members of the potato-1 inhibitor family. This paper describes the cloning and expression of a cDNA encoding this trypsin inhibitor in various vegetative tissues of the amaranth plant during seed development and imbibition, and investigates the possible induction of AmTI expression by wounding. We obtained a 393 bp cDNA sequence with an open reading frame corresponding to a polypeptide with 76 amino acid residues. With the exception of one residue (Ser-41), the polypeptide agrees with the amino acid sequence previously reported, plus 7 more residues at the N-terminus. These N-terminal residues are thought to be part of the signal used for intracellular sorting. The organ specificity of AmTI gene expression was investigated by northern analysis, showing that mRNA corresponding to AmTI genes was present in stems of plants growing under normal conditions. The kinetics of accumulation of the AmTI-mRNA, protein, and inhibitory activity during seed development and imbibition was determined. AmTI-mRNA accumulation reached a maximum at 14 days after anthesis (daa) and then gradually decreased, being barely detectable 36 daa. The AmTI protein accumulation followed the same profile as the inhibitory activity, both were delayed with respect to the mRNA. The maximum level was observed 22 daa, and then gradually decreased until a steady state was reached as seed maturation proceeded. Upon imbibition, a gradual decrease in AmTI protein and inhibitory activity was shown; however, an AmTI transcript was detected 24 h after imbibition. In contrast to representative members of the potato I family, this inhibitor was not inducible by wounding of leaves.
Introduction Proteinase inhibitors have long been proposed to play an important role as defense proteins in plants against the attack of insects (Ryan, 1990). This has been demonstrated by the transfer of proteinase inhibitor genes from different sources to several plants of ecoThe nucleotide sequence data reported will appear in the EMBL and GenBank Nucleotide Sequence Databases under the accession number AJ132473.
nomic interest, resulting in transgenic plants more resistant to predation (Boulter, 1993; Altpeter et al., 1999). However, another important and less understood role of these inhibitors is related to the regulation of the activity of endogenous proteases, particularly during the stage of protein deposition in storage tissues (Baumgartner and Chrispeels, 1977). In addition, inhibitor proteins themselves have also been suggested to act as storage proteins (Richardson, 1991).
16 Amaranth (Amaranthus hypochondriacus) is an ancient crop in America, whose grains possess a high nutritional quality protein, due to its relatively high content of Lys and sulfur amino acids (Segura-Nieto et al., 1992; Bressani, 1994). Considering that amaranth growth has been restricted to a few specific areas, it represents a potential source of genes for insect control. We have previously reported the presence of proteinase inhibitors in amaranth seeds which recognize insect enzymes (Valdés-Rodríguez et al., 1993; Chagolla-López et al., 1994). In particular, the amaranth trypsin inhibitor (AmTI) is a serine proteinase inhibitor that recognizes trypsin, chymotrypsin and trypsin-like proteinases extracted from the insect Prostephanus truncatus, an important pest of storage maize grains. This inhibitor belongs to the potato Iinhibitor family, showing the closest homology (67%) with Fagopyrum esculentum trypsin inhibitor (Belozersky et al., 1995), with Lycopersicon peruvianum trypsin inhibitor (Wingate and Ryan, 1991) and 51% with the proteinase inhibitor V extracted from the seeds of Cucurbita maxima (Krishnamoorty et al., 1990). There are many classes of inhibitor proteins in plants, and even within a class, it is not clear whether the same or related genes in any one plant species are involved in all of the reported responses (Richardson, 1991). To date, in many species containing proteinase inhibitor I homologues, functional variation has been deduced from genomic DNA sequence differences in both coding and noncoding regions. Variations in specificity, timing and site of expression support the idea of their possible involvement in different plant functions (Krishnamoorty et al., 1990; Beuning et al., 1994). The physiological roles of the amaranth trypsin inhibitor have not been fully investigated; however, such roles need to be considered for the rational manipulation of trypsin inhibitor proteins. Expression analysis of these genes will provide fundamental information to facilitate their manipulation as a source of insect resistance in the development of transgenic plants. In this study we have isolated an amaranth trypsin inhibitor cDNA and analyzed its expression in vegetative organs during development and imbibition of seeds, and examined the possibility of induction by wounding.
Materials and methods Plant and seed material Amaranth (Amaranthus hypochondriacus cv. Nutrisol) plants were grown in the field for 90 days. Considering that embryogenesis and seed maturation occur within seven weeks after anthesis, seeds at different stages of development were harvested at 9, 14, 22, 29, 36, 41 and 48 daa. Seeds at each time point were collected from different plants which were selected by height and size of their panicles. Samples were frozen in liquid nitrogen and stored at −80 ◦ C until processing. Mature amaranth seeds were surface-sterilized by shaking them in a 1% diluted commercial hypochlorite solution for 10 min, followed by rinsing in sterile deionized water. The seeds were placed on a wet sponge covered with filter paper and incubated in the dark at 28 ◦ C. Samples were collected at 3 h intervals throughout a 24 h period, freeze-dried and stored at −80 ◦ C. Experiments to obtain mRNA in vegetative tissues were performed on 10-day old seedlings and 26-day old plants (three- to four-leaf stage). These were grown at 25 ◦ C under 12 h light (185 µmol m−2 s−1 ) and 12 h dark. Dissected seedlings and plants were stored at −80 ◦ C. Wound treatment Twenty-six day old plants were used for wound induction experiments. Each plant was wounded by crushing twice with pliers the lower leaf in the middle, perpendicular to the mid vein. Wounded and unwounded leaves were sampled at 0, 2, 4 and 6 h, frozen in liquid nitrogen, and stored at −80 ◦ C before analysis. Preparation of protein extracts from developing and germinating seeds Seeds were ground to a fine powder and suspended in water (1:5 w/v) containing proteinase inhibitors (0.1 mM iodoacetic acid, 0.001 mM pepstatin, 1 mM p-methylsulfonate fluoride and 10 mM ethylenediamine tetraacetic acid). They were homogenized three times for 30 s in a tissue homogenizer ultra turrax (Hankel & Jenkel). The resulting extracts were centrifuged twice at 10 000×g for 10 min. All steps were carried out at 4 ◦ C. These extracts were freeze-dried and kept at −80 ◦ C until use in western analysis. To determine trypsin inhibitor activities, fresh extracts
17 were prepared in the same way, but the use of proteinase inhibitors was avoided. Determination of total protein content was performed by the Bradford protein assay (BioRad) using BSA as a standard. Proteinase inhibition assay The inhibitory activity of seed extracts against trypsin was determined spectrophotometrically as described by Schwert and Takenaka (1955). Trypsin and seed extracts were preincubated for 3 min at 30 ◦ C and the residual proteolytic activity was determined by measuring the hydrolysis of BAEE (N-benzoyl-L-arginine ethyl ester; Sigma Chemical) at pH 8.1. Gel electrophoresis and immunoblotting Extracted seed proteins were separated by electrophoresis in 13% acrylamide by the method of Schagger and von Jagow (1987). Proteins were transferred electrophoretically onto Immun-Blot PVDF membrane (BioRad) with a BioRad Mini Trans Blot Module. Immunoblots were processed as described (Towbin et al., 1979). Antibodies against amaranth trypsin inhibitor were obtained by ammonium sulfate precipitation from antiserum of immunized rabbits and dialyzed against PBS. Detection of the bound antibody (diluted 1:3000) was done using Goat Anti-Rabbit IgG alkaline phosphatase conjugate (Gibco) and visualized with 5-bromo-4-chloro-3-indolylphosphate/nitroblue tetrazolium chloride according to the manufacturer’s instructions (Gibco). Isolation of AmTI cDNA The seed cDNA library used in this work was reported by Barba de la Rosa et al. (1996). The λgt22 cDNA library made from developing seeds of Amaranthus hypochondriacus was propagated in Y1090 Escherichia coli and λDNA was purified as described (Sambrook et al., 1989). A DNA probe for screening the cDNA library was produced by polymerase chain reaction (PCR). Based on the amino acid sequence previously reported (Valdés-Rodríguez et al., 1993), two degenerate oligonucleotide pools were designed using the DNASIS program (Hitachi). The oligonucleotides were the following: 50 -AARCARGARTGGCCNGARCT-30 and 50 -ACNACCCANACNCGRTCRCA-30 (where N is T, C, G, A and R is A, G). The first corresponds to amino acids 7 to 13 and has a sense orientation, whereas the second corresponds to amino acids 50 to
56 and has an antisense orientation. The PCR was performed with Taq polymerase (0.5 U) under standard conditions using the above oligonucleotides (0.5 µM) as primers and 25 ng of DNA isolated from the seed cDNA library. The amplification program included 10 cycles of 1 min at 94 ◦ C, 1.5 min at 50 ◦ C, 1 min at 72 ◦ C followed by 40 cycles of 1 min at 94 ◦ C, 1.5 min at 50 ◦ C, 1 min at 72 ◦ C and a final step at 72 ◦ C for 7 min. The 147 bp PCR amplification product was cloned into pCR II according to the instructions of the supplier (Invitrogen) and sequenced. Sequencing was performed with an automatic sequencer (ABI Prism 377) based on the dideoxynucleotide chain termination method (Sanger, 1977). This PCR product was labeled by random priming with 32 P-α-dCTP as described by the supplier (Boehringer Mannheim) and used as a probe. About 30 000 plaque-forming units of the λgt22 seed cDNA library were screened with the PCR product by in situ plaque hybridization. Membrane hybridization was performed by standard protocols (Sambrook et al., 1989). Stringency washes were performed in 0.1× SSC, 0.1% SDS at 60 ◦ C. Membranes were exposed overnight to X-Omat film. The EcoRI/Asp718 inserts of the positive clones were subcloned into pBS (Stratagene). Positive clones were purified by the ethidium bromide method (Stemmer, 1991) and sequenced from EcoRI with a reverse universal primer. The fragment NotI which included the cDNAAmTI clone plus 45 bp (37 bp from pBS and 18 bp upstream from the start of the open reading frame of the cDNA clone) was radiolabeled and used as a probe in the Southern and northern analysis. Southern blot analysis Amaranth nuclear DNA was isolated as described by Hernández et al. (1995) from the aerial tissues of 26day old plants which were maintained in the dark for 8 days before harvesting. The DNA was digested with EcoRI, EcoRV and HindIII restriction enzymes and electrophoresed on a 0.8% agarose gel and blotted onto Hybond N+ nylon membrane (Amersham). The membranes were hybridized at 60 ◦ C for at least 18 h in 0.5 M phosphate buffer pH 7.4, 7% SDS, 0.1 mg/ml calf thymus DNA and 1% BSA. The final wash was carried out in 0.1× SSC, 0.1% SDS for 10 min at 60 ◦ C.
18 Northern blot analysis Total RNA was isolated from seeds, seedling and plant tissues as described (Schuler and Zielinski, 1989). RNA (30–40 µg/lane) was separated on a denaturing 1.3% agarose gel containing 2.1 M formaldehyde, and transferred onto Hybond N+ nylon membrane. The membranes were hybridized as described above. Hybridization with a labeled human ribosomal probe containing the 28S sequence was carried out as a control to demonstrate consistent loading of RNA in each well.
Results Isolation of an AmTI-cDNA clone With the oligonucleotides described in Materials and methods as primers in a PCR reaction, a DNA fragment was amplified from the seed cDNA library. The 147 bp product obtained was the size expected for the region spanned by the primers and was cloned into the vector pCR II for subsequent sequencing. The amino acid sequence deduced from the nucleotide sequence compared with the two reported AmTI amino acid sequences coincides completely with the sequence of the inhibitor isolated from Amaranthus caudatus (Hejgaard et al., 1994), whereas it differs in one amino acid (Ser-41) with the sequence reported from our group for an AmTI isolated from A. hypochondriacus (Valdés-Rodríguez et al., 1993). After screening the amaranth seed λgt22 cDNA library by plaque hybridization using the PCR product as a probe, we obtained 4 positive cDNA clones. The sizes of the restriction fragments (EcoRI/Asp718) within the lambda clones were the same. After subcloning, a near full-length sequence was determined. The AmTI-cDNA sequence and deduced amino acid sequence are shown in Figure 1. The cDNA is 393 bp in length. It contains a 228 bp open reading frame from 19 to 246 and has a 147 bp untranslated 30 end. Two polyadenylation signals (AATAAA and AATATA) are present. The deduced amino acid sequence was identical to the sequenced protein in Amaranthus hypochondriacus with the exception of the Ser residue mentioned above. In addition, the cDNA clone predicted an extra 7 amino acid sequence at the N-terminus including two methionines. The hydropathic profile of this short peptide (Figure 2) indicates a highly hydrophilic nature.
Figure 1. Nucleotide sequence of the cDNA for trypsin inhibitor from amaranth (AmTI) (Amaranthus hypochondriacus). The deduced amino acid sequence is shown below the nucleotide sequence. The vertical arrow indicates the site of processing to give the mature protein. Stop signals are indicated with asterisks. The black bars indicate the amino acids used to design primers. Polyadenylation signals are underlined.
Figure 2. Hydropathy plot of the deduced AmTI cDNA amino acid sequence. This profile was predicted by the method of Kyte and Doolittle (1982). The hydropathy values were calculated using an 11 residue window.
Southern blot analysis A Southern blot of restricted A. hypochondriacus genomic DNA, probed with 32 P-labeled cDNA (Figure 3), identified two strongly labeled fragments with EcoRI, a strong and a weak fragment with EcoRV and a single fragment with HindIII at a stringency of 0.1× SSC, 0.1% SDS. This suggests the existence of 1 or 2 copies of the AmTI gene in the genome of amaranth, in contrast to the multicopy families found for most proteinase inhibitor genes in other plants.
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Figure 3. Southern blot analysis of amaranth genomic DNA. DNA was digested with EcoRI (E), EcoRV (V) and HindIII (H), subjected to electrophoresis on a 0.8% w/v agarose gel, blotted and probed with a 32 P-labeled cDNA fragment obtained by NotI restriction from pBS. Molecular weights were calculated by using the program RFLP scan (Scanalytics, a division of CsP, Inc., 1996).
Expression during seed development To examine AmTI expression during seed development, we monitored the mRNA, the AmTI protein and AmTI inhibitory activity from seeds during maturation. The level of AmTI mRNA (Figure 4, panel C, top) 9 daa was low, reaching its maximum 14 daa, and then decreased. A gradual decrease after day 14 was observed in two different experiments (data not shown); however, data in Figure 4C show a more pronounced decrease at day 29. This probably indicates variations in the expression level among the different tested plants. The pattern of accumulation of AmTI (Figure 4, panel B) did not correspond directly to that of the mRNA. The presence of AmTI was observed from 9 daa, whereas the maximum level of protein was reached 22 daa, which means it appeared later than the maximum mRNA level. After this point, AmTI protein levels slightly decreased to a steady-state level until seed maturation. Both inhibitory activity (Figure 4, panel A) and the level of AmTI protein showed the same pattern during seed development. Although inhibitory activity was not measured 9 daa, an increase from 14 to 22 daa was observed, where maximum inhibitory activity was reached. After this time, the activity decreased to 83% of the maximum value and from there on, no significant changes were detected. The fact that maximum values for mRNA and protein did not coincide indicates that there might be a post-
Figure 4. Differential expression of amaranth trypsin inhibitor during seed development at 9, 14, 22, 29, 36, 41 and 48 daa. A. Inhibitory activity (TIU/g dry weight) of AmTI against trypsin was measured using BAEE as sustrate. Residual proteolytic activity was determined after preincubating seed extracts and trypsin for 3 min at 30 ◦ C. B. Western blot analysis. Proteins (5 µg) isolated from developing seeds were electrophoresed onto a 13% acrylamide gel by the Schägger and von Jagow (1987) method and blotted. AmTI was detected by using an alkaline phosphatase immunoassay. C is a control of purified AmTI. C. Northern blot analysis. A 30 µg portion of total RNA from developing seeds were fractionated by electrophoresis on a 1.2 M formaldehyde agarose gel, blotted and probed with AmTI-cDNA. C is a NotI fragment containing the cDNA sequence. The bottom panel shows the results of hybridization with a human gene for 28S ribosomal RNA used as a control.
transcriptional process controlling the expression of this gene. AmTI expression during seed imbibition In order to determine whether AmTI was utilized during seed imbibition as would be expected for a seed storage protein, mature amaranth seeds were imbibed in dark at 28 ◦ C for 24 h. Under these conditions, 6% of the seeds germinated (radicle protrusion) 9 h after inhibition (hai), 25% 12 hai and 90% 15 hai, reaching a value of 92% 24 hai. Since most of the seed germinated 15 hai, this time was considered as the germination time. Levels of mRNA, protein and inhibitory activity were monitored in samples collected every 3 h. No mRNA transcript was detected dur-
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Figure 6. Gel blot analysis of RNA from seedlings and mature amaranth plants. Total RNA (40 µg) extracted from radicle (Rd), hypocotyls (H), cotyledons (Ct) from 10-day old seedlings and roots (R), stems (S) and leaves (L) of 26-day old plants hybridized with AmTI-cDNA; C, probe used as hybridization control. The bottom panel shows the results of hybridization with human 28S ribosomal DNA, used as a loading control.
plants are shown in Figure 6. The results showed that the AmTI cDNA hybridized exclusively to a 450 bp RNA from stem. No detectable hybridization signals were obtained from any other plant organs. Induction of AmTI by wounding Figure 5. Differential expression of amaranth trypsin inhibitor during seed imbibition. Amaranth seeds were imbibed in the dark at 28 ◦ C and samples were collected at 3, 6, 9, 12, 15, 18, 21 and 24 hai. A. Inhibitory activity. B. Western blot analysis. Protein (5 µg) isolated from germinating seeds. C. Northern blot analysis achieved as described in Figure 4.
ing the first 21 hai (Figure 5, panel C, top), but a clear signal was detected 24 hai. Western analysis showed a gradual decrease of AmTI protein during seed imbibition (Figure 5, panel B). This agrees with the decline observed in inhibitory activity (Figure 5, panel A). Both results suggest that AmTI protein level decreased, probably as a result of its use to support the synthesis of new proteins. The low level of inhibitory activity (TIU/g dry weight) found during the imbibition experiment was most likely due to the use of an old lot of seeds harvested 3 years before. This was confirmed using a fresh lot of seeds, which showed higher levels of inhibitory activity (6 times more). However, the decrease in inhibitory activity during imbibition occurred in a similar way. Expression of AmTI in different organs The expression pattern of AmTI was determined by northern blot hybridization experiments. AmTI mRNA levels in different organs of 10-day old seedlings and 26-day old fully developed amaranth
Wound induction of AmTI mRNA was investigated in 26-day old amaranth plants. In order to follow the time course of appearance of AmTI mRNA upon wounding, the lower leaves of young amaranth plants were damaged by applying pressure and the mRNA was isolated at 2 h intervals for a period of 6 h. Northern blot analysis failed to detect AmTI mRNA in either wounded or unwounded leaves (data not shown).
Discussion One of the main goals in the study of proteinase inhibitors is the identification of their corresponding genes that would allow their use to obtain transgenic plants with higher resistance to specific insect attack. A rational manipulation of plant proteins such as the trypsin inhibitors for crop improvement depends to a large extent on the knowledge of their specific functions. As an initial step towards this goal, it is first necessary to determine the localization and specific expression patterns of these proteins during different stages of plant development. Here, we report the isolation and characterization of an amaranth trypsin inhibitor cDNA. Based on the knowledge of the amino acid sequence, it was possible to design pools of oligonucleotides for a given sequence of amino acids and use them as primers in a PCR reaction. The amplified fragment predicted an amino acid sequence which agrees with
21 the amino acid sequence of AmTI previously reported by our group, with the exception of one residue (Ser-41 instead of Asp). This finding would support the possibility of 2 AmTI genes in Amaranthus hypochondriacus as suggested by Southern analysis. The sequence reported here agrees 100% with the sequence of a trypsin inhibitor isolated from A. caudatus (Hejgaard et al., 1994). The isolated AmTI-cDNA predicts a larger precursor with at least 7 additional residues at the N-terminus. This agrees with data from several other seed storage proteins whose nucleotide sequence predicts an N-terminal signal sequence extension. It is well known that sorting and targeting of seed storage proteins begin during their biosynthesis on membrane-bound polysomes, where an N-terminal signal peptide mediates their segregation into the lumen of the endoplasmic reticulum. After cleavage of the signal peptide, the proteins are subjected to different modifications such as glycosylation, disulfide bridge formation and oligomerization. These types of processing finally produce the protein with the proper structure to be deposited in endoplasmic reticulumderived protein bodies or else to be further transferred into protein storage vacuoles, which are later on transformed in protein bodies. Several storage proteins are also processed after arriving into the protein storage vacuoles. Some of their precursors have short N- or C-terminal targeting sequences which are detached after arrival into the protein storage vacuoles (Muntz, 1998). Within the short AmTI N-terminal sequence, two methionine residues are included which represent two potential sites of protein translation initiation. However, neither one of the two methionines were within the optimal context for the initiation of the translation process considered in eukaryotes (Kozak, 1991). Considering the hydropathic profile of this peptide, it lacks the characteristics of a typical signal peptide (Bednarek and Raikel, 1992). But, taking into account that this inhibitor is to be stored within the protein bodies, it could be part of a larger signal peptide. On the other hand, when the mature AmTI protein and its deduced amino acid precursor sequence are aligned, it is clear that the precursor is processed after an asparagine residue. This type of processing has been suggested to occur in various seed proteins stored in protein bodies. For processing to occur at this amino acid, it is necessary that the residue is exposed on the surface of the protein (Hara-Nishimura et al., 1995). This is highly probable, considering the hydropathic
profile of the N-terminal segment (Figure 2), which shows a hydrophilic character. Most proteinase inhibitors are encoded by multigene families that are expressed differentially (Jofuku and Goldberg, 1989). In the case of AmTI, the results indicate that this inhibitor belongs to a small (probably 2 members) gene family and that there is a high degree of homology between the genes, considering the stringency of the washes used in the Southern blotting and the single amino acid difference in the deduced and determined protein sequences. Data presented here show that the AmTI genes are expressed in developing seeds. Its synthesis occurs during the first three weeks of seed development, after which the mRNA almost disappears. AmTI protein and the trypsin inhibitory activity reach their maximum level one week later than the mRNA, and then decrease and remain steady until maturation. This indicates that once synthesized, the active protein is stably accumulated during seed maturation. The delay in maximum AmTI protein accumulation with respect to maximum mRNA synthesis, as well as the significant increase in AmTI protein but not in inhibitory activity, suggest that post-transcriptional and post-translational processes are probably involved in the control of the expression of this gene during seed development. During imbibition, a gradual decrease in the levels of AmTI occurred in the first 24 h. This decrease, however, is not fast enough to be considered as leaching from the seed as has been reported to occur with some inhibitors from legume seeds (Wilson, 1980). The decrease is more evident 15 hai at which time 90% of the seeds have germinated, indicating that AmTI is used once germination has already occurred. The pattern of AmTI synthesis during seed development and its decrease during imbibition would correspond to a typical pattern of a storage protein. The accumulation and subsequent liberation of amino acids from storage proteins require that endogenous proteolysis be tightly regulated. Expression of proteinase inhibitors by the same signals that regulate storage proteins may ensure that endogenous vacuolar proteases (Boller and Kenda, 1979) do not degrade newly synthesized storage proteins. The reserves might be subsequently mobilized by the de novo production of proteases. A way to prevent hydrolysis of the storage proteins is through the presence of specific proteinase inhibitors. It was reported that some storage proteins themselves have proteinase inhibitor activities (Kai-Wun et al., 1997).
22 In this work, the expression pattern of AmTI in vegetative organs from seedlings and in mature plants was studied. In these tissues, an appreciable amount of AmTI-mRNA was shown in stems. The transcript size was approximately 450 bp, suggesting that the isolated cDNA clone is nearly full-length. It has long been thought that seed protein genes are either expressed exclusively during embryogenesis or are expressed at very low levels in mature plant organs (Goldberg et al., 1989; Jofuku and Goldberg, 1989). However, in the case of proteinase inhibitors, some reports have indicated a great variability in their expression patterns, suggesting their participation in several distinct plant functions. Winged bean Kunitz chymotrypsin inhibitor (WCI) accumulates in seeds and tuberous roots and a small amount of the WCI protein and mRNA can also be detected in the sieve tubes of stems. Since endogenous proteases are presumed to be involved in the autophagy of the cytoplasm content of sieve tubes (Habu et al., 1996), it was suggested that this inhibitor could be involved in the regulation of the activities of these enzymes. Similarly, the presence of AmTI in stems might also be indicative of its participation in both defense systems against insects or bacteria that invade the stem and regulation of endogenous proteolytic activities during the development of these tissues. In addition to the roles previously suggested for AmTI within the seed, the fact that new mRNA was detected 24 hai is indicative of new requirements for this protein during the early stages of seedling growth. This requirement was specific for this phase in the developmental process, since no mRNA was detected in any tissue in 10-day old seedlings. However, 26-day old plants showed in stems the presence of this mRNA, indicating that a new requirement must be fulfilled again. Finally, data on wound induction showed no leaf AmTI synthesis 6 h after wounding. However, the time of response should be monitored over a longer timecourse if definitive conclusions are to be drawn. The isolation of the trypsin inhibitor cDNA has allowed the identification of the site where the precursor molecule is to be processed after synthesis. The expression pattern of this trypsin inhibitor in the amaranth plant was also determined, showing its expression not only in seeds, but also in low levels in stems. The synthesis and accumulation of this protein during seed formation and its later decrease after germination has been shown, indicating that this protein is probably used to support seedling growth. However, the fact that after germination some synthesis is detected
could indicate its possible involvement in some other unknown functions during seedling growth. Finally, the isolation of a genomic clone and the identification of the sequences that regulate its expression will be useful in further studies on the defense mechanism of the plant and the role of AmTI during development.
Acknowledgements The authors thank Drs N. Villegas and A. Guevara for expert assistance in obtaining the amaranth cDNA, and M. Sánchez for help in hybridization analysis. The skilful technical assistance by A.L. Ruíz, V. Zárate and A. Guerrero is gratefully acknowledged. S.V-R thanks CONACYT and CONCYTEG for a doctoral fellowship. Comparison of the nucleotides and protein sequence with sequences in the databases was performed at NCBI using the BLAST network service.
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