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

Abstract

Background: 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.

Results: 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.

Conclusion: 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.

Figure 1

Schematic diagram showing the key features of PKV. The protein kinase catalytic domain, putative nuclear localization signal and transmembrane domains, and T-loop extension (in subdomain VII) are indicated by boxes. S₃₀₃ = location of putative phosphorylated serine in the extension loop; FEYF, location of the COOH-terminal FEYF PIF motif. (http://predictprotein.org, [64])

Figure 2

Characterization of transcript and protein expression in transgenic plants. (A) Northern blot analysis of the accumulation of pkv transcripts in transgenic plants as described in the Methods section. Left panel, 20 ug of total RNA extracted from control (1, X), XS (2) and XAS (3). Ethidium bromide staining of ribosomal RNA (rRNA) is shown to illustrate equal loading of RNAs. Arrows indicate location of mRNAs hybridizing to the PKV DIG-labeled probe. Asterisk indicates degraded PKV transcript in XAS plants. (B) Western blot analysis of PKV protein accumulation in mature leaves of control and transgenic plants using a PKV-specific polyclonal antibody as described in the Methods section. Equal amounts of total protein were loaded per sample lane. Numbers to the left of the figure represent the size of prestained protein markers. Lane 1, X; Lane 2, XS; Lane 3, XAS. The arrow designates the location of the 52 kDa PKV protein.

Figure 3

Growth of transgenic tobacco plants expressing the sense (XS) or antisense (XAS) copy of pkv. (A) Three week-old in vitro plantlets of control (X), XS and XAS showing the reduced (XS) or enhanced (XAS) root production (arrows). (B) Five-week old plants in soil showing the dwarfing phenotype of XS. (C) Fully mature greenhouse-grown plants showing the increased height and flowering of XAS and the dwarfing habit and reduced/delayed flowering of XS. (D) Transverse petiole sections from leaves of mature control (X), XS, and XAS tobacco plants. Sections were observed by light microscopy without staining. Petioles of the fifth leaf from the base were taken from each plant. Original magnification, 100 ×. Arrows designate the columns of lignified xylem elements.

Figure 4

Morphology of flowers and pollen from of transformants. (A) Over expression of pkv results in reduced flower size (XS) as compared to control (X) and XAS. (B) Light microscopy of acetocarmine-stained pollen from control (X), XS, and XAS anthers. The arrow in XS points to defective pollen grains which appear white and collapsed in the picture and are adjacent to a viable pollen grain which appears red in the picture. Bar = 20 μm.

Figure 5

Northern blot analysis of the expression pattern of salicylic acid- and jasmonic acid- regulated genes. (A) Northern blot analysis of the accumulation Pr1a, Pr1b, and LAP transcripts in 20 μg of total RNA extracted from healthy tomato (lane 1), PSTVd-infected tomato (Viroid-infected tomato, lane 2), non-transformed control (X, lane 3), transgenic plants XS (lane 4) and XAS (lane 5), and tomatoes infected with Potato virus X (PVX-infected tomato, lane 7). Ethidium bromide staining of ribosomal RNA (rRNA) is shown to illustrate equal loading of RNAs. SA, salicylic acid-inducible genes; JA-jasmonic acid-inducible gene. (B) Northern blot analysis of total RNA samples as in (A) using only the Pr1b probe. Lane 1, Healthy tomato; Lane 2, PSTVd-infected tomato; Lane 3, X; Lane 4, XS; Lane 5, XAS; Lane 6, PSTVd-infected Xanthi tobacco.

Figure 6

Growth effects of in vitro application of hormone to control and transgenic seedlings. Ethanol (EtOH), GA₃ (10^-5M), pachlobutrazol (10^-5M), or GA₃(10^-5M) plus pachlobutrazol (P) (10^-5M) were added to solidified media. Plants were evaluated at 20 days after seeding.

Figure 7

Real-time quantitative RT-PCR (QPCR) analysis of the effects of transgenes on the expression of host genes. Total RNA isolated from leaf tissue of X, XS, and XAS plants was subjected to QPCR analysis using primers specific for the indicated genes as described in the Methods section. Relative transcript expression levels of each target were normalized with respect to actin and the transgenic values reflect fold change expression compared to non-transgenic (X) controls. Six biological replications were used to calculate mean values and standard deviations. Bars mark the standard deviation of the average. The line across the figure indicates the normalized level of 1 in the non-transgenic control.