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

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 [33] 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).

Transcript and protein levels of PKV in control and transgenic lines were examined by northern and western blot analyses, respectively (Figure 2). pkv transcripts were readily apparent in sense (XS) plants as opposed to untransformed (X) plants (lane 2 and 1, respectively, Figure 2A). By contrast, there was evidence of reduced accumulation and corresponding degradation of the introduced pkv transcripts in the antisense (XAS) plants (lane 3, Figure 2A), an indication of gene silencing of the tobacco homolog of PKV and resulting from formation of double-stranded RNAs of target gene transcripts and the introduced antisense gene transcripts. Western blot analysis of PKV protein synthesis revealed the presence of a protein band of approximately 52 kDa reacting to PKV-specific antiserum in XS plants (Figure 2B, lane 5). Although tobacco contains a PKV homolog, no detectable PKV-related protein was evident in the non transgenic control (X) or XAS plants, indicating very low levels of basal expression, similar to what was found in healthy tomato plants [33].

Phenotypic changes in tobacco in response to transgenic expression of pkv

Tobacco plants transformed with the XS and antisense XAS constructs exhibited dramatic alterations in appearance and development compared to non-transformed control plants (X) (Figure 3). XS plants were dwarfed and had noticeably reduced root systems in tissue culture and as young plants (Figure 3A and 3B), phenotypes that are similar to symptoms observed in tomato plants infected by PSTVd. XAS transformants, on the other hand, were taller than non-transformed tobacco plants and had a more extensive root system (not shown in potted plants). The phenotypic responses of the transformants were most pronounced in mature tobacco plants, where XS plants were stunted, possessed thick, dark leaves, and exhibited reduced flowering and root formation, and XAS plants were taller and had increased numbers of inflorescences and flowers (Figure 3C). Morphometric analysis of the mature plants (Table 1) revealed that internode number and stem length were decreased in XS plants and increased in XAS plants. The internode length contributed to the overall difference in height from the non-transformed control. Visualization of the vascular tissue in cross-sections of leaf petioles of control and transgenic plants revealed increased numbers of lignified xylem elements (darker cells) in XAS petioles as compared to petioles derived from similarly positioned leaves of X plants, whereas there was a decreased number of lignified elements in XS plants (Figure 3D).

	X	XAS	XS
Plant size (mm)	989.3 ± 16.67	1219.33 ± 9.94a	700.67 ± 52.51a
Internode #	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
# flowers	42 ± 1.45	65.6 ± 3.8a	17.6 ± 1.8a
Flower size (cm)	4.46 ± 0.03	4.43 ± .06	3.67 ± .16a
Stigma (cm)	3.5 ± 0.1	3.5 ± 0.2	1.63 ± .13a
Style (cm)	3.72 ± .02	3.75 ± .1	2.2 ± .1a
Pollen (T1)	viable	viable	not viable

^aValues are significantly different compared to the X control (P < 0.05) as measured using the

unpaired t test. Six plants of each line were used for the analysis with three lines per construct.

Total height and number of internodes was measured from the base of the plant to the base of the inflorescence when all of the flowers were developed.

Flower size was smaller in XS plants (Figure 4A and Table 1) with resulting shorter style and stigma. In addition, XS plants had greatly reduced fertility, while seed set in XAS plants was not affected. When pollen viability was examined by staining with acetocarmine, the cytoplasm of non-transgenic and XAS pollen stained red, indicating viability, while the majority of the XS pollen of T2, homozygous, selfed plants had collapsed and was nonviable (arrows, Figure 4B). When the XS flowers were pollinated with X pollen, however, the plants produced more seeds than the XS parent, indicating that the XS plants were male sterile but female fertile. Fifty per cent of the seeds resulting from the X × XS cross germinated.

Northern blot analysis of salicylic acid- and jasmonic acid-signaling pathways in transgenic plants

Induced defense and/or pathogenesis-related (PR) gene expression occurs in PSTVd-infected, dwarfed tomato plants [15]. To determine if PR expression is associated with stunting caused by over expression of PKV in tobacco, we probed Northern blots containing total RNA isolated from leaf tissue for transcripts of Pr1a1 and Pr1b, whose expression is salicylic acid-mediated. These genes were transcriptionally activated in viroid-infected tomato plants, as previously reported, but not in XS or XAS transformants, nor in PSTVd-infected tobacco plants, indicating that neither PSTVd infection nor PKV over expression induce a general pathogenesis response in tobacco (compare lane 2 with lanes 4 and 5 in Figure 5A and lane 2 and lanes 4-6 in Figure 5B). In addition, transcripts of the leucine aminopeptidase gene (LAP) (jasmonic acid-mediated; [36]) did not increase in transgenic tobacco plants, nor in viroid-infected tomato (whereas such transcripts did accumulate in Potato virus X-infected tomato), indicating that neither the dwarfing caused by PKV over expression in tobacco nor PSTVd pathogenesis in tomato involve jasmonic acid signaling (Figure 5A).

Exogenous application of gibberellic acid reverses stunting in plants over expressing PKV

To investigate whether the reduced stature of XS plants was due to a lower content of GA or to reduced response to GA, exogenous hormone applications were made to seedlings grown in vitro and seedling growth was monitored. The hypocotyl length of XS seedlings increased significantly in medium containing 10^-5 M GA₃, where it was not as greatly affected in control (X) or XAS seedlings (Figure 6, panel C). In medium containing 10^-5 pachlobutrazol, an inhibitor of GA biosynthesis, the height of both control and transgenic seedlings was reduced, and this inhibition was fully reversed by addition of 10^-5 M GA₃ (Figure 6, panel D). Therefore the plants respond normally to GA. This suggests that the dwarf phenotype of XS plants is due to a lower content of active GAs. The phenotype of mature XAS plants suggests that these plants may be producing more GA than the control, non-transformed plants

Transcript levels of genes involved in GA biosynthesis and leaf expansion

The dwarfing phenotype of XS plants and its reversal by exogenous GA₃ treatment suggested defects in GA biosynthesis [37]. The accumulation of mRNAs of genes involved in GA biosynthesis, GA20ox1 (GA20ox) and GA3β-hydroxylase (GA3β), in non-transformed (X), XS, and XAS tobacco plants was evaluated by real-time quantitative RT-PCR (Figure 7). GA20ox and GA3β transcript levels were 1.6-1.8 fold higher in XS plants compared to X or XAS. In XAS plants, GA3β transcript levels were reduced significantly compared to the non-transformed control. It has been reported that, as key enzymes in the last catalytic steps in the synthesis of active GA's, GA3β and GA20ox are subject to feedback regulation by GA [37]. Therefore, in XS plants, low levels of GA could result in induction of these enzymes, whereas their gene expression would be repressed in plants containing higher levels of GA.

Endogenous GA levels have also reported to be modulated by the tobacco knotted-1 homeobox gene NTH (Nicotiana tabacum homeobox gene 15) [38] and the tobacco bZIP transcriptional activator gene RSG (repression of shoot growth) [39], 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 [38]. 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 [39]. 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 [40]. 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 [17] 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 [41] and in chrysanthemum infected with Chrysanthemum stunt viroid [42]. In addition to stunting, infection of tomato by severe strains of PSTVd reduced pollen viability [41]. Symptoms of viroid infection in tissue culture are varied but include specific inhibition of root formation by HSVd in cucumber [43] and root and shoot formation by CEVd in tomato [44]. Finally, vascular system development was arrested in CEVd-infected tomato plants [45].

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 [46]. 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 [47]. 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 [48]. 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 [40], 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 [39]. 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 [39], 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 [50]. 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 [35] 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 [33] 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 [56], but they also function in the regulation of genes by developmental stimuli [57]. 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 [1], viroid RNA could trigger posttranscriptional gene silencing of plant transcriptional activators or repressors, as proposed by Wang et al. [22], 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. [58] 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.

DNA manipulations

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 [33] was doubly digested with BamHI and SstI, and the PKV sense fragments were recovered. To prepare the antisense insert, construct pET28a/PKV [33] 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. [60]. 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

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 [33].

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 [33] (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 [63] 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.

Microscopy

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.