The systemin precursor gene regulates both defensive and developmental genes in Solanum tuberosum

Javier Narva´ ez-Va´ squez and Clarence A. Ryan, Jr.*

Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340

Contributed by Clarence A. Ryan, Jr., October 1, 2002 Transformation of Solanum tuberosum, cv. Desiree, with the to- tubers for reproduction, wound-inducible defense-signaling mato prosystemin gene, regulated by the 35S cauliflower mosaic pathway components in leaves and stems were recruited to virus promoter, resulted in constitutive increase in defensive pro- provide a mechanism to store proteins in tuber cells for vege- teins in potato leaves, similar to its effects in tomato plants, but tative propagation. also resulted in a dramatic increase in storage protein levels in potato tubers. Tubers from selected transformed lines contained 4- Materials and Methods to 5-fold increases in proteinase inhibitor I and II proteins, >50% Potato Transformation. A previously constructed chimeric cauli- more soluble and dry weight protein, and >50% more total flower mosaic virus (CaMV)–tomato prosystemin gene was used nitrogen and total free amino acids than found in wild-type tubers. that contained a cDNA fragment encoding the complete tomato These results suggest that the prosystemin gene plays a dual role prosystemin ORF fused in the sense orientation with the 35S in potato plants in regulating proteinase inhibitor synthesis in CaMV promoter within the binary vector pGA 643. The physical leaves in response to wounding and in regulating storage protein map of this chimeric gene and details on its construction have synthesis in potato tubers in response to developmental cues. The been reported (11). This construct was introduced into Agrobac- results indicated that components of the systemin signaling path- terium tumefaciens strain LA 4404 and was used to transform way normally found in leaves have been recruited by potato plants potato leaf and tuber tissues. Both leaf and potato tuber discs to be developmentally regulated to synthesize and accumulate were surface sterilized and preconditioned by incubating for 2 large quantities of storage proteins in tubers. days in tobacco feeder plates and were then soaked for 20–30 min in 10–20 ml of Murashige and Skoog (MS) medium (MS tomato ͉ systemic wound signaling ͉ storage proteins ͉ defense response salts͞B5 vitamins͞100 mg͞liter m-inositol͞3% sucrose, pH 5.9), containing 107 Agrobacterium cells per ml. The explants were roteinase inhibitors I and I (Inh I and II) proteins were blotted dry with sterile filter paper and incubated again on Pinitially found as major storage proteins in potato tubers tobacco feeder plates for another 2 days and then transferred to (1–3) and later as wound-inducible antinutrient defensive pro- potato cell culture medium (MS medium͞5 ␮M zeatin͞3 ␮M teins in leaves of several Solanaceae species (4, 5). The two indoleacetic acid͞0.7% agarose), containing 250 mg͞ml proteins can account for up to 10% of the total soluble proteins Cefotaxime. The small calli that grew from the explants after of mature potato tubers (6, 7) and up to 2% of the soluble 2–3 weeks were transferred to fresh potato medium containing proteins in leaves of tomato plants in response to mechanical 100 mg͞liter kanamycin. The explants were transferred to fresh wounding or herbivore attacks. The wound-inducible expression medium every 2 weeks (growing calli were separated from the of Inh I and II in tomato and potato leaves has been extensively rest of the explant) until the first well-differentiated shoots investigated (5, 8), and their synthesis was shown to be initiated appeared (after 4–6 weeks). The level of kanamycin in the by the release of a mobile 18-aa polypeptide signaling molecule medium was gradually reduced to allow rooting, first to 50 called systemin (9). Systemin is released from damaged cells at mg͞liter and then to 20 mg͞liter. Regenerated plants were the site of wounding and systemically activates the expression of transferred to soil and grown to maturity in a greenhouse. over 20 defense-related genes, including Inh I and II, in leaves throughout the plants (5). Systemin is synthesized as part of a Proteinase Inhibitor Assays. Inh I and II proteins were quantified larger 200-aa protein called prosystemin (10), whose cDNA has by immunoradial diffusion (12, 13) by using pure potato inhib- been overexpressed in transgenic tomato plants in both the itors I and II as standards. Juice was expressed from leaves and antisense and sense orientations. In the antisense plants, sys- tubers of control (wild-type) and transgenic plants. A transverse temic wound signaling does not occur (10), whereas in the sense section (about one-quarter of an inch in width) was excised from plants, the leaves constitutively express high levels of the wound- the center of each tuber, including cortical and pith tissue. The inducible defensive proteins in the absence of wounding, acting section was diced into small pieces and crushed with mortar and as if the plants were in a permanent wounded state (11). pestle to express the juice. The juice was collected in a microfuge Although Inh I and II were first discovered in potato tubers tube kept on ice and centrifuged at 10,500 ϫ g to clarify. (1), systemin had not been considered previously to be involved Centrifugation had no effect on the levels of Inh I and II in the in the regulation of tuber storage proteins. We have now juice. Inhibitor concentration was expressed in microgram per constitutively overexpressed the tomato prosystemin gene in milliliter of extracted juice. potato plants in its sense orientation, similar to previous exper- iments with tomato plants (11). The transgenic plants overex- RNA Extraction and Analysis. Total RNA was extracted from leaves pressing the prosystemin gene were found to regulate not only and tubers of wild-type and transgenic plants. The leaves were the synthesis and accumulation of proteinase inhibitors in leaves immersed in liquid nitrogen, ground to a fine powder, and stored but also the synthesis of major storage proteins in potato tubers. at Ϫ80°C until used. Total leaf RNA was extracted by using the Tubers from selected transgenic potato lines were found to contain highly elevated levels of soluble proteins, including Inh I and II and patatin, over tubers from untransformed wild-type Abbreviation: Inh I and II, proteinase inhibitors I and II. plants. The evidence suggests that as potato plants evolved *To whom correspondence should be addressed. E-mail: [email protected].

15818–15821 ͉ PNAS ͉ November 26, 2002 ͉ vol. 99 ͉ no. 24 www.pnas.org͞cgi͞doi͞10.1073͞pnas.232591199 Downloaded by guest on September 29, 2021 Table 1. Levels of Inh I and II in juice from leaves of first- generation transgenic potato plants overexpressing the tomato prosystemin gene Inh I, ␮g͞ml Inh II, ␮g͞ml Total, ␮g͞ml Plant leaf juice leaf juice leaf juice

Wild-type control 34 15 49 Fig. 1. Constitutive expression of the tomato prosystemin gene in leaves of Transgenic control* 23 20 43 transgenic potato plants. WT, wild-type control plant; T0, transgenic control T2 75 77 152 plant transformed with a plasmid lacking the prosystemin gene; T2, T3, and T6, T3 90 87 177 transgenic plants. An 18S ribosomal RNA gene probe was used as a loading T6 110 100 210 control. Inh I and II proteins were quantified as described in Materials and Methods. Values are the average values of two measurements per plant; each measure- Trizol reagent (GIBCO͞BRL) following the manufacturer’s ment was made by using pooled juice from at least two leaves. recommendations. Potato tubers were chopped in small pieces, *Control plants transformed with a plasmid lacking the prosystemin gene. frozen with liquid nitrogen, ground to a fine powder with a Ϫ mortar and pestle, and stored at 80°C. Total RNA was then identified T2, T3, and T6 transformants as having high levels of extracted according to Singh et al. (14), with some modifications. proteinase inhibitors (Table 1). The levels of proteinase inhib- Briefly, Ϸ100 mg of ground tuber tissue was extracted with a mix ␮ ⅐ ͞ itors in leaves of these three transformants ranged from 4- to of 400 l of 0.1 M Tris HCl, pH 7.4, containing 1% (wt vol) 5-fold higher than levels in leaves of wild-type plants or in leaves sodium sulfite and 500 ␮l of water-saturated phenol, and reex- ͞ of the transgenic control plants lacking the prosystemin trans- tracted with an equal volume of acid–phenol chloroform (5:1, gene. The constitutive expression of the two inhibitors was Ambion, Austin, TX). The RNA was precipitated with equal similar to their expression in leaves of tomato plants that were volumes of isopropanol containing 0.1 volume of 3 M sodium transformed with the prosystemin gene (11), where the overex- acetate (pH 5.2) at Ϫ20°C overnight. RNA quality was deter- pression of the prosystemin gene caused the constitutive expres- mined by gel fractionation in agarose formaldehyde followed by sion of several wound- and systemin-inducible genes, as if the ethidium bromide staining and UV light visualization before plants were under constant herbivore attacks (11, 17). The effect analyzing for specific mRNAs. A tomato prosystemin cDNA, Inh appears to be caused by the abnormal elevated synthesis and I and II cDNAs, and an RT-PCR-amplified patatin cDNA from processing of prosystemin, resulting in the release of systemin potato tissue were used as probes in gel blot analyses as described throughout the plants. (15). An 18S ribosomal RNA gene probe was used as a loading Assays for Inh I and II proteins in the juice from the transgenic control. Membranes were washed once with 2ϫ SSPE (1 ϫ 0.115 tubers also revealed that the proteins were present at strikingly M NaCl͞10 mM NaH PO ͞mM EDTA, pH 7.4) for 20 min at 4 high levels, averaging about a 2.5-fold increase for both inhibi- room temperature, two to three times with 2ϫ SSPE͞1% SDS, tors over levels found in wild-type tubers (Table 2). To deter- for 15–30 min at 65°C and then exposed 15–32 h to x-ray film or mine whether levels of other proteins present in the tuber juice to a PhosphorImager (Bio-Rad). of transformants T2, T3, and T6 were also elevated, the juice was analyzed by SDS͞PAGE. The protein profiles of juice from these Determination of Tuber Percent Nitrogen. Fresh tubers (100–200 g tubers are compared in Fig. 2 with proteins from the expressed each) from second-generation-propagated wild-type and trans- juice from wild-type tubers. The levels of Inh I, Inh II, and other genic potato plants grown in a greenhouse were selected for total storage proteins, including patatin and a group of proteinase nitrogen analysis. Tubers were diced into small pieces and freeze-dried. Once dried, tuber pieces were ground to a fine inhibitors of molecular masses near 20 kDa (18), showed pro-

powder, weighed, and analyzed for percent nitrogen, dry weight, nounced increases over the proteins of the control plants. PLANT BIOLOGY by using a LECO combustion analyzer CNS-2000 (LECO, St. Differences in the levels of individual proteins were found Joseph, MI) (16), at the Analytical Sciences Laboratory, Uni- among transgenic lines, indicating that a differential regulation versity of Idaho Holm Research Center, Moscow, ID. of storage proteins by prosystemin may be occurring, perhaps resulting from insertional effects during transformation and͞or Statistical Analysis. One-way analysis of variance followed by somaclonal variations that occurred during regeneration of Dunnett’s multiple comparison test was performed to compare plants (19). mean values of inhibitor protein levels and nitrogen content in Small tubers from plants of transgenic lines T2, T3, and T6 the tubers from wild-type and transgenic plants. were used to propagate second generations in larger pots in the greenhouse, supplemented with lights. Five tubers (50–120 g) Results and Discussion The constitutive overexpression of the prosystemin gene in the Table 2. Inh I and II and soluble protein levels in juice expressed leaves of transgenic plants was confirmed in gel blot analyses of from wild-type and transgenic potato tubers prosystemin mRNA by using the tomato prosystemin cDNA as a probe. In 18 transformants that were regenerated, various Inh I Inh II Soluble protein levels of expression of the prosystemin gene were found. Pro- Plant ␮g͞ml % ␮g͞ml % ␮g͞ml % systemin mRNA blots from leaves of the three transformants T2, T3, and T6 that expressed the highest levels of the prosystemin Wild-type 234 100 351 100 2.8 100 mRNA are shown in Fig. 1. Prosystemin mRNA was not detected T2 538 230 970 277 5.2 185 in leaves of either the wild-type or in the transformed control T3 626 267 851 242 5.4 193 plants, but in assays at lower stringency, low levels of prosystemin T6 590 252 930 265 4.7 168 mRNA could be detected, similar to levels found in leaves of Inh I and II proteins were quantified as described in Materials and Methods. wild-type tomato plants (10). Total water-soluble proteins in the clarified juice were assayed by using the Immunological assays of the levels of Inh I and II proteins in Bradford assay, with bovine serum albumin as the standard. Shown are the expressed juice of leaves of the young transgenic plants also average values from six independent assays.

Narva´ez-Va´squez and Ryan PNAS ͉ November 26, 2002 ͉ vol. 99 ͉ no. 24 ͉ 15819 Downloaded by guest on September 29, 2021 Fig. 4. Gel blot analyses of mRNAs coding for Inh I, Inh II, and patatin, in second-generation tubers from wild-type (WT) and transgenic potato plants Fig. 2. SDS͞PAGE analysis of soluble proteins in tubers from transgenic T2, T3, and T6. Et-Br, ethidium bromide-stained ribosomal RNA used as load- potato plants T2, T3, and T6, transformed with the tomato prosystemin gene, ing control. and from tubers from wild-type plants (WT). Clarified juice from tubers (30 ␮l) was mixed with 10 ␮lof4ϫ Laemmli’s sample buffer, boiled for 5 min, and loaded into 12.5% polyacrylamide gels. After electrophoresis, the protein profiles were visualized with Coomassie blue. lines. Free amino acids in the transgenic tubers ranged 5.3–6.8 ␮mol͞100 mg of dry weight compared with 4.2 ␮mol͞100 mg of dry weight in wild-type tubers, with glutamine exhibiting in- from each transgenic line and from wild-type control plants were creases of 2.5- to 2.9-fold (Table 3). This indicated that the assayed for prosystemin expression (Fig. 3). The assays indicated increased synthesis of storage proteins in the tubers was not that the prosystemin transgene was strongly expressed in the limited by amino acid availability, and that the expression of the tubers, similar to its expression in the leaves. prosystemin gene had caused the amino acid pools to increase to The increased expression of Inh I and II and patatin genes in accommodate the increased synthesis of storage proteins. the tubers was confirmed by gel blot analyses of their respective The mechanism by which prosystemin regulates protein syn- mRNAs (Fig. 4). All three genes were expressed at substantially thesis in tubers has not been established, but it is likely that higher levels than in wild-type tubers. This observation suggested signaling occurs through the octadecanoid pathway, as found in that the expression of the prosystemin gene in the tubers resulted leaves, by producing oxylipin signals from linolenic acid (21) and in the transcriptional regulation of the storage protein genes. active oxygen species (22). In leaves, systemin is processed from The increases in the levels of storage proteins in the tubers prosystemin and released on wounding, so that a developmen- were reflected in increases in total soluble proteins. In transgenic tally regulated mechanism for systemin processing and release ͞ tubers, the levels of soluble proteins were 4.7–5.4 mg ml, must be present in tubers. This might be accomplished by the Ϯ ͞ compared with 2.8 0.9 mg ml in tubers from wild-type plants synthesis of tuber-specific proteinases that are expressed con- (Table 2). Increases in total soluble protein were accompanied stitutively. The prosystemin processing proteinases in leaves has by an increase in percent total nitrogen. The average percent dry weight nitrogen in the tubers from transgenic plants was 1.7– 1.9%, compared with Ϸ1.3 Ϯ 0.1% in tubers from wild-type plants (Fig. 5). Mean values of inhibitor levels and percent nitrogen in tubers of each of the transgenic plants were signif- icantly higher (P Ͻ 0.001) than levels found in the tubers of wild-type plants (Fig. 5). Because free amino acids can represent a substantial contribution to potato tuber nitrogen (20), the free amino acid pool was analyzed in the tubers of the transgenic

Fig. 5. A comparison of the levels of Inh I and II proteins with the percent dry weight nitrogen in second-generation tubers from wild-type (WT) and trans- Fig. 3. Gel blot analyses of tomato prosystemin mRNA in tubers from genic potato plants T2, T3, and T6. Data are means Ϯ SD (n ϭ 6). Mean values wild-type (WT) and transgenic potato plants T2, T3, and T6. Et-Br, ethidium for the transgenic plants were statistically different from the wild-type plants bromide-stained ribosomal RNA used as loading control. (Dunnett’s test, P Ͻ 0.001).

15820 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.232591199 Narva´ ez-Va´ squez and Ryan Downloaded by guest on September 29, 2021 Table 3. Levels of free amino acids in potato tubers from constitutively expressed endogenous prosystemin gene, may wild-type and transgenic plants provide a new source of inexpensive high-quality protein for Plant Free amino acids* % increase Glutamine* % increase animal and human consumption. Studies of the systemin signaling system have revealed a new Wild type 4.2 100 0.57 100 level of complexity in plants. Systemin signaling in tomato leaves T2 5.7 137 1.53 268 has been shown to be mediated by a 160-kDa transmembrane T3 5.3 126 1.64 288 leucine-rich repeat (LRR) receptor kinase that exhibits a high T6 6.8 161 1.41 247 percentage of identity to the Arabidopsis brassinolide receptor Free amino acids were extracted from lyophilized tuber tissue with acidi- BRI1 (28), indicating a possible dual function for this receptor fied ethanol (80% ethanol͞0.25 M HCl) at 40°C for 1 h and analyzed with a in regulating both defense- and development-related genes. The Beckman 6300 analyzer (GMI, Albertville, MN) using pure amino acids as relationship of systemin signaling with brassinolide signaling has standards. become an intriguing question. Because of its ubiquity in plants *␮mol͞100 mg of lyophilized tuber tissue. (29), brassinolide signaling appears to have evolved much earlier than systemin signaling. Systemin signaling has been found in some species of the Solanaceae family but not others and is not not been identified, although several proteinases are wound- and found in other plant families. A role for systemin in tuberization systemin-inducible (5, 17) and may have roles in processing. in some potato species must have evolved at an even later time. Inh I and II were initially isolated from potato tubers (1–3) Thus, it appears that the brassinolide receptor evolved initially and, because of their high concentrations, they were considered and then was later recruited during the early evolution of the to be major storage proteins (23). A defensive role for the Solanaceae family to facilitate systemin signaling. As solana- inhibitors in potato tubers has not been established but, as in ceous species evolved, and tuberization from stems developed, leaves, they may have roles in defense against predators and some components of the systemin signaling pathway appear to pathogens. When denatured (cooked), potato tuber proteins are have been adopted by cells of developing tubers as a means of among the most nutritious plant proteins available to humans storing proteins. Systemin signaling appears to be an interesting (24–26). They are a major source of dietary protein world-wide, case of exaptation (30), the process of specific functions having with nearly 300 million tons of potatoes currently being produced evolved to a level where the process(es) is coopted to serve an on over 18 million hectares, representing over 6 trillion tons of entirely novel function in the organism. protein (20, 27). Transformation of potatoes has become rou- tine, even in developing countries. Thus, the potential for We thank S. Vogtman and C. Whitney for growing plants and G. Munske increasing the protein quantity of potato tubers of varieties for amino acid analyses. This research was supported by the College of grown in different countries, by the simple introduction of a Agriculture and Home Economics and the National Science Foundation.

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Narva´ ez-Va´ squez and Ryan PNAS ͉ November 26, 2002 ͉ vol. 99 ͉ no. 24 ͉ 15821 Downloaded by guest on September 29, 2021