Modification of tobacco plant development by sense and antisense expression of the tomato viroid-induced AGC VIIIa protein kinase PKV suggests involvement in gibberellin signaling
© Hammond and Zhao; licensee BioMed Central Ltd. 2009
Received: 9 April 2009
Accepted: 18 August 2009
Published: 18 August 2009
The serine-threonine protein kinase gene, designated pkv (protein kinase- viroid induced) was previously found to be transcriptionally activated in tomato plants infected with the plant pathogen Potato spindle tuber viroid (PSTVd). These plants exhibited symptoms of stunting, and abnormal development of leaf, root, and vascular tissues. The encoded protein, PKV, is a novel member of the AGC VIIIa group of signal-transducing protein kinases; however, the role of PKV in plant development is unknown. In this communication, we report the phenotypic results of over expression and silencing of pkv in transgenic tobacco.
Over expression of pkv in Nicotiana tabacum cv. Xanthi (tobacco) resulted in stunting, reduced root formation, and delay in flowering, phenotypes similar to symptoms of PSTVd infection of tomato. In addition, homozygous T2 tobacco plants over expressing PKV were male sterile. Antisense expression of pkv, on the other hand, resulted in plants that were taller than non-transformed plants, produced an increased number of flowers, and were fertile. Exogenous application of GA3 stimulated stem elongation in the stunted, sense-expressing plants. PKV sense and antisense expression altered transcript levels of GA biosynthetic genes and genes involved in developmental and signaling pathways, but not genes involved in salicylic acid- or jasmonic acid-dependent pathways. Our data provide evidence suggesting that PKV plays an important role in a GA signaling pathway that controls plant height and fertility.
We have found that the over expression of the tomato protein kinase PKV resulted in stunting, modified vascular tissue development, reduced root formation, and male sterility in tobacco, and we propose that PKV regulates plant development by functioning in critical signaling pathways involved in gibberellic acid metabolism.
Viroids, the smallest known pathogenic agents of plants, are distinguished from plant viruses by their lack of a protein coat and absence of protein coding capacity . Covalently-closed, circular RNA molecules ranging in size from 239-401 nucleotides, viroids replicate and move from cell to cell and throughout the plant without a helper virus, but with the aid of host cell components . Potato spindle tuber viroid (PSTVd) and the pospiviroids replicate and accumulate in the nucleus [3, 4]. PSTVd causes a serious disease of tomato characterized by stunting, abnormal development of root and vascular tissues, leaf epinasty and deformation; the severity of symptoms ranging from mild to lethal depending on the viroid strain . Many of the symptoms caused by viroid infection suggest an imbalance in growth hormones. A significant decrease in endogenous gibberellins (GA3 and/or GA1) has been observed in viroid-infected plants .
The molecular basis of symptom formation in viroid-infected plants is unknown, although the nuclear location of PSTVd suggests interactions with the host genome and/or transcription machinery. Viroid infection of tomato results in increased transcription of defense-related genes [7–14]. Using macroarray technology, Itaya et al.  also observed induction or suppression of genes encoding proteins involved in stress responses, cell wall structure, chloroplast function, and protein metabolism. Growth reduction in citrus caused by infection with Citrus exocortis viroid (CEVd) (a pospiviroid) was correlated with reduced levels of gibberellin 20-oxidase (GA20ox) mRNA , and Qi and Ding  reported that LeExp2 expansin gene expression is down-regulated in viroid-infected tomato plants, suggesting that stunting results from restricted cell expansion.
Although gene expression in viroid-infected plants is altered, little is known of the link between the viroid infection process and gene activation or suppression and resulting symptoms. Single or multiple nucleotide substitutions in the pathogenicity region [18–21] or the central conserved region  of PSTVd can dramatically alter symptoms in infected tomato plants. Surprisingly, constitutive expression a secondary hairpin derived from the pathogenicity region of the virulent RG1 strain of PSTVd resulted in viroid symptoms in transformed tomato . RNA-induced silencing of host genes was proposed as the mechanism of pathogenesis and a 19-20 nucleotide sequence similarity between the pathogenicity region of PSTVd and a putative transcription factor (GenBank BI969092) suggested that PSTVd may silence regulatory genes . Enhanced accumulation of viroid-specific siRNAs [23–27], and a bifunctional nuclease and RNase that may be involved in the regulation of plant development  were found to be associated with viroid infection in PSTVd-infected tomato plants.
Phosphorylation/dephosphorylation of host proteins most likely also plays a role in viroid pathogenicity. CEVd infection of tomato plants was found to alter the general pattern of leaf protein phosphorylation . PSTVd infection of tomato induced the phosphorylation of a host-encoded 68,000 Mr protein immunologically related to the mammalian ds-RNA dependent protein kinase (PKR) [29–31]; however, the plant ortholog of PKR has not been isolated. PSTVd strains varying in pathogenicity, on the other hand, resulted in differential activation of the mammalian PKR in vitro, suggesting a possible triggering event in viroid pathogenesis .
PKV is a novel member of the AGC VIIIa protein kinase superfamily , members of which have significant homologies to cyclic nucleotide-dependent protein kinases, however, little is known about the role of AGC VIIIa kinases in plants. Within subgroup AGC VIIIa, only PINOID  and Adi3  have been genetically characterized, and have been shown to play fundamental roles in auxin signalling and plant cell death, respectively.
To better understand the biological role of PKV in plant development, we introduced sense and antisense copies of the full-length pkv gene into experimental Nicotiana tabacum cv. Xanthi tobacco plants by stable transformation. N. tabacum encodes a homolog of PKV, with 99% sequence identity to the tomato gene (GenBank EU196240), which is also transcriptionally activated to a low level in PSTVd-infected tobacco plants (tobacco is not a symptomatic host of PSTVd; data not shown). We demonstrate that over expression of PKV in tobacco results in dwarfing and reduced root formation, similar to symptoms of PSTVd infection of tomato plants. Results from hormone supplement and gene expression studies suggest that gibberellic acid biosynthetic and/or signalling pathways are regulated by PKV.
Molecular analysis of transgenic plants
To determine the role of pkv gene expression in plant development, a full-length copy of the open reading frame  was cloned in the sense and antisense orientations into the binary vector, pBI121, where transcription of the inserted gene is under control of the CaMV35S promoter. Several independently transformed lines for each construct were obtained with similar phenotypes, and three lines of each construct was selected for further investigations. Southern blot analysis revealed that plants contained a single insert and segregated with a 3:1 ratio (data not shown).
Phenotypic changes in tobacco in response to transgenic expression of pkv
Morphometric analysis of fully-grown untransformed (X), PKVS and PKVAS plants.
Plant size (mm)
989.3 ± 16.67
1219.33 ± 9.94a
700.67 ± 52.51a
71.67 ± 1.76
91.33 ± 0.8a
44.6 ± 0.8a
Size of 10th internode (cm)
3.9 ± 0.15
5.0 ± 0.03a
1.86 ± 0.1a
42 ± 1.45
65.6 ± 3.8a
17.6 ± 1.8a
Flower size (cm)
4.46 ± 0.03
4.43 ± .06
3.67 ± .16a
3.5 ± 0.1
3.5 ± 0.2
1.63 ± .13a
3.72 ± .02
3.75 ± .1
2.2 ± .1a
Northern blot analysis of salicylic acid- and jasmonic acid-signaling pathways in transgenic plants
Exogenous application of gibberellic acid reverses stunting in plants over expressing PKV
Transcript levels of genes involved in GA biosynthesis and leaf expansion
Endogenous GA levels have also reported to be modulated by the tobacco knotted-1 homeobox gene NTH (Nicotiana tabacum homeobox gene 15)  and the tobacco bZIP transcriptional activator gene RSG (repression of shoot growth) , which regulates the transcript levels of GA biosynthetic enzymes. Over expression of the NTH15 gene in tobacco has been found to lead to decreased transcript levels of GA20ox and to abnormal leaf morphology with little or no effect on GA3β transcript levels . In our study, NTH transcript levels were down-regulated in XS plants compared to non-transformed control, and were up-regulated in XAS plants (Figure 7). The dominant-negative form of RSG represses the expression of the ent-kaurene oxidase gene in the GA biosynthetic pathway, reducing GA levels, and resulting in a dwarfing phenotype in tobacco . In our studies, RSG levels in X, XS, and XAS plants appeared to be similar to one another (Figure 7).
The change in plant height in XS or XAS plants could be due to changes in cell number or cell size. Transcript levels of LeExp2 (EXP), the major expansin gene expressed in stems and hypocotyls of tomato and regulated by auxin, have been shown to correlate with growth rate in rapidly expanding hypocotyl tissue due to increased cell size . In our analysis of EXP transcript levels in tobacco leaf tissue using real time RT-PCR (experiment was repeated several times), there was a positive correlation between plant and leaf size and EXP expression in XS and XAS plants, with EXP transcripts expressed at 0.28 fold in XS plants as compared to non-transgenic plants (X), while EXP transcripts were up-regulated and in XAS plants with respect to non-transgenic plants (Figure 7). This result confirms what was reported by Qi and Ding  in dwarfed, viroid-infected plants.
Constitutive sense and antisense expression of PKV affects plant development
Our results suggest that PKV regulates plant development by modulating levels of GA and the enzymes involved in GA biosynthesis. Constitutive over expression of PKV resulted in visually obvious phenotypes of stunting and reduced root formation. In addition, the plants were male infertile which may be attributed to defective pollen. Accumulation of PKV transcripts or protein in flowers may disrupt pollen production by affecting development of stamens and microspore mother cells, resulting in nonviable pollen. Antisense expression of pkv (XAS), on the other hand, resulted in plants that were taller than non-transformed plants, with enhanced root development and greater numbers of flowers in the inflorescence, but with no obvious influence on seed set.
The abnormal development induced by constitutive over expression of PKV resembles that found in viroid-infected plants. For example, reduced flower size was evident in Hop stunt viroid (HSVd)-infected cucumber  and in chrysanthemum infected with Chrysanthemum stunt viroid . In addition to stunting, infection of tomato by severe strains of PSTVd reduced pollen viability . Symptoms of viroid infection in tissue culture are varied but include specific inhibition of root formation by HSVd in cucumber  and root and shoot formation by CEVd in tomato . Finally, vascular system development was arrested in CEVd-infected tomato plants .
Changes in pkv expression result in altered expression levels of GA biosynthetic genes
Many factors are responsible for dwarfing phenotypes of plants, but the roles of GA, auxins, and brassinosteroid hormones are the most studied . These hormones control separate processes that contribute to stem elongation, and a deficiency due to reduced synthesis of the hormones or the lack of response to the hormones may lead to a dwarf phenotype. The elongation response of XS seedlings to exogenous GA suggests that they contain lower levels of active GA and are not impaired in their response to the hormone (Figure 4). Altered GA levels impact lignin biosynthesis and development of xylem vessels in tobacco plants . The reduced number of xylem vessels in the petioles of XS plants and increased number in XAS plants also suggests decreased and increased levels of active GA's, respectively (Figure 2D).
GA20ox1 and GA3β hydroxylase are key biosynthetic enzymes in the pathways to active GA4 and GA1 and are subject to feedback regulation . The transcripts of these genes are up-regulated in GA-deficient XS plants, as might be expected (Figure 7). Positive and negative signaling components in GA signal transduction have been characterized, and include the DELLA proteins and bZIP transcriptional activators and repressors [37, 49]. Further investigations of transcriptional activation of enzymes and activators/repressors involved in GA biosynthesis and catabolism will help unravel the biochemical pathways responsible for the dwarfing phenotype in XS plants.
Because of the reduced height of XS plants and increased height of XAS plants, we would expect lower and higher levels, respectively, of LeExp2 (EXP) transcripts in these plants. As shown in Figure 7, EXP transcript levels as evaluated are reduced in leaves of XS plants compared to control and XAS plants. As the LeExp2 gene is expressed primarily in stems and hypocotyls of plants , the reduced leaf size in XS and enlarged leaf size in XAS also result from altered LeExp2 gene expression.
NTH15, a knotted-1 homeobox gene, and the bZIP transcriptional activator gene RSG modulate endogenous GA levels by regulating the expression of GA biosynthetic enzymes. For example, over expression of NTH15 decreases the expression of GA20ox1, leading to abnormal leaf and flower morphology. Following that reasoning, the results in Figure 7 showing that down regulation of NTH expression levels corresponds to an increase in GA20ox1 transcripts in XS plants. These results suggest the NTH may be involved in PKV signaling.
The RSG protein binds and activates the promoter of GA3, encoding ent-kaurene oxidase, another key enzyme in the GA biosynthetic pathway. Expression of a dominant-negative form of RSG gene in tobacco plants severely inhibits stem internode growth . In our study, RSG transcript levels are not repressed in XS plants, suggesting that RSG may not play a role in PKV signaling However, as RSG is activated through phosphorylation , the lack of regulation at the transcriptional level does not eliminate its involvement in the regulation of endogenous GA levels in XS and XAS plants. Further investigations of transcriptional activation of enzymes and activators/repressors involved in GA biosynthesis and catabolism will help unravel the biochemical pathways responsible for the dwarfing phenotype in XS plants.
Proposed functional role of PKV in plant development and viroid pathology
PKV is a member of the AGC protein kinases (specifically Group AGC VIIIa), members of which have been shown to play roles in growth signaling pathways, regulation of transcription, and programmed cell death . Key features of the AGCVIIIa family, and shared by PKV and related kinases, include the DFD motif in the activation loop, a 50-80 amino acid variable insertion (T-loop extension) in subdomain VII of the conserved catalytic domain, and a C-terminal hydrophobic domain known as PIF (PDK1 [3-phosphoinositide-dependent protein kinase 1]-interacting fragment), characterized by the amino acid motif FxxFs/TF/Y (represented by FEYF in PKV) (Figure 1). Two members of the AGC VIIIa family, PINOID, which regulates organ development by enhancing polar auxin transport in plants [34, 51], and Adi3, which negatively regulates plant cell death  are activated by PDK1 phosphorylation [52–55]. PKV activity may also be regulated by PDK1 phosphorylation. PDK1 interaction with PKV through the PIF domain at the carboxy terminus of PKV (Figure 1) would result in phosphorylation of the serine at position 303 in the T-loop extension of PKV (Figure 1), thereby transactivating PKV and leading to autophosphorylation at additional sites. Phosphorylated and activated PKV would subsequently transphosphorylate downstream transcriptional activators or repressors, or genes encoding enzymes involved in GA biosynthesis or degradation, resulting in stunted plants exhibiting morphogenic abnormalities that characterize viroid-induced disease.
Unlike viroid- or virus-infected tomato plants, transcripts of pathogenesis-related proteins were not induced in pkv sense-transformed tobacco plants, suggesting that pkv downstream signaling is not through salicylic acid- or jasmonic acid-dependent pathways. The putative promoter region of pkv contains G- (CACGTG) and H-box (GGTAGG) cis elements  which are known to interact with, and be transcriptionally activated by, bZIP transcriptional factors. These cis elements are speculated to be responsible for early responses to pathogen attack , but they also function in the regulation of genes by developmental stimuli . The genes encoding PKV and the PR proteins may be transcriptionally activated by the same bZIP transcription factors but diverge into separate signaling pathways in tomato. Further studies are needed to determine the extent to which PSTVd infection regulates the transcription of pkv and to dissect the signaling pathways and downstream substrates of PKV phosphorylation. PSTVd RNA may bind to the pkv promoter directly and enhance transcription. Alternatively, because of its partially double-stranded structure , viroid RNA could trigger posttranscriptional gene silencing of plant transcriptional activators or repressors, as proposed by Wang et al. , or viroid RNAs could act as transcriptional activators by targeting the noncoding regulatory regions in gene promoters. Alternatively, PSTVd RNA may bind to the pkv promoter directly and enhance transcription. Although the exact mechanism of transcriptional activation by short dsRNAs is unknown, Li et al.  demonstrated this mechanism in human cells. Further studies are needed to determine the extent to which PSTVd infection regulates the transcription of pkv and to dissect the upstream signaling components, including PDK1, and and downstream substrates of PKV phosphorylation to dissect the role of PKV in plant development.
In this study, we have found that the over expression of the tomato serine-threonine protein kinase gene PKV resulted in stunting, modified vascular tissue development, reduced root formation, and male sterility in tobacco, while silencing of the endogenous tobacco PKV gene by antisense expression resulted in a taller stature and increased numbers of influorescences in transgenic plants. The combined results of reversal of stunting in plants over expressing PKV by exogenous application of GA and the altered transcript levels of GA biosynthetic genes suggests that PKV regulates plant development by functioning in critical signaling pathways involved in GA metabolism and GA-regulated transcriptional networks. Our findings establish a foundation to further investigate the molecular mechanisms of how PKV interacts with critical cellular factors, including transcriptional regulators such as DELLA-domain proteins , that lead to altered plant development. Ultimately, it may also lead to a greater understanding of the signaling pathways functioning in viroid pathogenicity.
PKV sense (GenBank accession no. AY849915; 33) and PKV antisense sequences were cloned into the binary vector pBI121 (Clontech, Inc., Palo Alto, CA) at the BamHI/SstI sites, releasing the GUS gene. For preparation of the sense insert, construct pGEXKG/PKV  was doubly digested with BamHI and SstI, and the PKV sense fragments were recovered. To prepare the antisense insert, construct pET28a/PKV  was first digested with EcoRI to release the PKV insert, which was then ligated back into the pET28a vector at the EcoRI site, generating clones with opposite orientations. Antisense clones were selected and one of the clones was doubly digested with BamHI and SstI, giving rise the antisense insert, which contained cohesive ends compatible to the above prepared pBI121 vector. The pBI121 constructs were transformed into Agrobacterium tumefaciens LBA4404.
Plant growth conditions
Tobacco plants were grown under greenhouse conditions under natural daylight with a temperature of 20-25°C.
Plant transformation and hormone treatments
Leaf pieces of Nicotiana tabacum cv. Xanthi were transformed with a suspension of A. tumefaciens LBA4404 bacterial cells containing the pBI121 PKV constructs as described by Horsch et al. . Stable incorporation of the genes was verified by either Southern blot hybridization or PCR analysis. Plant height, internode length, number of flowers, and flower size were measured from three plants of each category and the data were compared using the unpaired t test. Seeds of control and transgenic tobacco plants were germinated and grown in Petri dishes containing solidified sterile 1/2 MS media containing 3% sucrose and either 10-5 M GA3, 5 μM indolebutyric acid (Sigma Chemical Co., St Louis, MO) or 10-5 M pachlorbutrazol (PhytoTechnology Laboratories, Shawnee Mission, KS), alone or in combination, or ethanol as a control was added at the same concentration as used to solubilize the hormones.
Northern, Southern, and western blot analyses
Oligonucleotide primers used in experimental analyses.
GA20 oxidase 1
Northern blot hybridizations were carried out in a DIG Easy Hybridization buffer (Roche Molecular Biochemicals) at 47°C overnight. The blots were washed for 2 × 15 minutes in 2 × SSC (1 × SSC is 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0), 0.1% SDS at room temperature, followed by 2 × 15 minutes washed in 0.5 SSC, 0.1% SDS at 68°C. Colorimetric detection was made using the DIG wash and block buffer set (Roche Molecular Biochemicals) and NBT/BCIP according to manufacturer's instructions (Kirkegaard and Perry, Gaithersburg, MD). For Southern blot analysis, RT-PCR products were electrophoresed through a 1% agarose/TBE gel, blotted onto a nitrocellulose membrane, and hybridized using the same conditions used for Northern blot analyses .
Total proteins were extracted from plant tissues using a 2:1 ratio (buffer: tissue) of Cell- Lytic P Plant Cell Lysis/Extraction Reagent (Sigma Chemical Company) containing a 1:100 dilution of Plant Protease Inhibitor cocktail (Sigma Chemical Company) by grinding in an eppendorf tube using a disposable pestle. Samples were centrifuged for ten minutes at 16,000 × g in a microfuge. Laemmli sample buffer (Bio-Rad Laboratories, Hercules, CA) was added (2:1, sample/dye ratio), the sample was boiled for 10 minutes, and a 20 μl aliquot was electrophoresed under denaturing conditions through a 10-20% Tris-glycine gel (Invitrogen) and blotted according to manufacturers instructions onto a nitrocellulose membrane (Invitrogen). The size marker was the Precision Plus Kaledioscope prestained markers (Bio-Rad Laboratories). Western blot analysis was carried out using a polyclonal antibody raised against a PKV-GST fusion protein  (Cocalico Inc, Reamstown, PA) and a secondary goat anti-rabbit polyclonal antibody (Kirkegaard and Perry). Colorimetric detection was performed using NBT/BCIP according to manufacturers' instructions (Kirkegaard and Perry).
Quantitative real-time RT-PCR analysis (QPCR)
Total RNA was extracted from plant tissues using TriReagent. First strand cDNA was prepared using an oligo(dT) primer from 1 μg total RNA using the Advantage RT-for-PCR kit (Clontech). The cDNA reaction was diluted to 100 μl with sterile water. QPCR was performed using the first-strand cDNA generated above, selected primer sets designed using the Primer3 program  and listed in Table 2 as RQ primers, and the Brilliant® SYBR® Green QPCR Master Mix (Stratagene, La Jolla, CA). Each QPCR reaction contained 12.5 μl of 2× Brilliant® SYBR® Green QPCR Master Mix, 2 ul of the diluted cDNA, and 2.5 pM of each gene-specific primer. The conditions used for the QPCR reactions were: 95 C for 10 min, followed by 40 cycles of 95 C, 30 sec, 50 C, for 1 min, and 72 C for 30 sec in a Mx3000P QPCR System (Stratagene). Data were analyzed using the MxPro software (Stratagene) and relative transcript expression levels of each target were normalized with respect to the tobacco ACTIN gene (Accession no. EU938079). Six biological replications were used to calculate mean values and standard deviations.
Pollen grains were stained with acetocarmine and examined under light microscopy in a Zeiss Axioskop 2 microscope (Carl Zeiss, Inc., Thornwood, NY). Hand-cut sections of leaf petioles were examined using light microscopy.
We acknowledge the technical assistance of Dr. Nancy Kreger and Sharon Jhingory who participated in various aspects of the project, and Dr. Wei Wei for assistance with figures and critical reading of the manuscript. We also acknowledge Linda Walling at University of California, Riverside for the gift of a leucine aminopeptidase clone.
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