JXB Advance Access first published online on August 30, 2007
This version published online on September 4, 2007
Journal of Experimental Botany, doi:10.1093/jxb/erm165
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
© 2007 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER |
Senescence-associated genes induced during compatible viral interactions with grapevine and Arabidopsis
1Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Alameda 340, Santiago de Chile, Casilla 114-D, Chile
2Carnegie Institution, Department of Plant Biology, 260 Panama Street, Stanford, CA 94305, USA
* To whom correspondence should be addressed. E-mail: parce{at}bio.puc.cl
Received 16 May 2007; Revised 19 June 2007 Accepted 25 June 2007
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionSupplementary materialReferences |
|---|
The senescence process is the last stage in leaf development and is characterized by dramatic changes in cellular metabolism and the degeneration of cellular structures. Several reports of senescence-associated genes (SAGs) have appeared, and an overlap in some of the genes induced during senescence and pathogen infections has been observed. For example, the enhanced expression of SAGs in response to diseases caused by fungi, bacteria, and viruses that trigger the hypersensitive response (HR) or during infections induced by virulent fungi and bacteria that elicit necrotic symptoms has been observed. The present work broadens the search for SAGs induced during compatible viral interactions with both the model plant Arabidopsis thaliana and a commercially important grapevine cultivar. The transcript profiles of Arabidopsis ecotype Uk-4 infected with tobacco mosaic virus strain Cg (TMV-Cg) and Vitis vinifera cv. Carménère infected with grapevine leafroll-associated virus strain 3 (GLRaV-3) were analysed using microarray slides of the reference species Arabidopsis. A large number of SAGs exhibited altered expression during these two compatible interactions. Among the SAGs were genes that encode proteins such as proteases, lipases, proteins involved in the mobilization of nutrients and minerals, transporters, transcription factors, proteins related to translation and antioxidant enzymes, among others. Thus, part of the plant's response to virus infection appears to be the activation of the senescence programme. Finally, it was demonstrated that several virus-induced genes are also expressed at elevated levels during natural senescence in healthy plants.
Key words: Arabidopsis, compatible viral interactions, grapevine, senescence, transcript profiling, viral disease
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary materialReferences |
|---|
Leaf senescence is a regulated process that corresponds to the last stage in leaf development. It is characterized by dramatic changes in cellular metabolism and the sequential degeneration of cellular structures. At this stage, nutrients accumulated in leaves during the growing season are mobilized and recycled in new leaves or storage organs. The most visible change in senescent leaves is chlorophyll degradation, which is accompanied by losses in photosynthetic activity along with decreases in total RNA and protein. In addition to ageing, multiple developmental and environmental signals are able to trigger senescence. Drought, darkness, leaf detachment, and the hormones abscisic acid (ABA) and ethylene induce leaf yellowing (Lim et al., 2005). Among the attempts to clarify the molecular mechanisms that control this dynamic process, a number of senescence-associated genes (SAGs) have been identified (Lim et al., 2005). These SAGs encode diverse proteins including RNases, proteases, lipases, proteins involved in the mobilization of nutrients and minerals, transporters, transcription factors, proteins related to translation, and antioxidant enzymes. In contrast, senescence-down-regulated genes (SDGs) mainly encode proteins involved in photosynthesis (Buchanan-Wollaston et al., 2003; Gan, 2003).
A relationship between senescence and disease development is suggested by the induction of a common set of genes by these two processes. Some SAGs code for proteins related to defence-associated processes [e.g. pathogenesis-related (PR) proteins] that are induced during the hypersensitive response (HR) against avirulent pathogens (Quirino et al., 1999; Gepstein et al., 2003). Infection of plants by bacterial or fungal pathogens also induces genes that are expressed at high levels during senescence. For instance, in Arabidopsis thaliana, the expression of the gene LSC54, a putative metallothionein associated with senescence, is detected during the HR induced by incompatible bacteria and fungi and against virulent pathogens that induce necrotic symptoms (Butt et al., 1998). In tobacco, the expression of a cysteine protease (SAG12) is induced during the HR against viruses and bacteria (Pontier et al., 1999). Interestingly, the Arabidopsis mutant hys1/cpr5 shows accelerated senescence and constitutively high levels of defence responses, supporting a link between these processes (Yoshida et al., 2002). Thus, the link between defence response and senescence could involve programmed cell death. Recently, a WRKY transcription factor, a negative regulator of leaf senescence, has been implicated in the defence response (Ulker et al., 2007). We are interested in the effect of viral disease development on plant physiology at the molecular level. The present work broadens the search for SAGs induced by compatible ampelovirus grapevine leafroll-associated virus-3 (GLRaV-3) and crucifer-infecting tobacco mosaic virus (TMV-Cg) infections of a commercially important grapevine cultivar and Arabidopsis ecotype Uk-4, respectively.
The infection caused by TMV-Cg in Arabidopsis is an informative model for compatible interactions with viruses. TMV-Cg replicates and spreads both cell-to-cell and long distance in several Arabidopsis ecotypes without inducing plant death (Arce-Johnson et al., 2003). Viral proteins accumulate in all plant tissues, but symptoms are usually not observed, with only some leaf curling and premature ageing occurring in a small number of ecotypes. Grapevine plants are the subject of diverse viral infections that survive in plants for several annual cycles. GLRaV-3 causes an economically important disease in grapevines, affecting several aspects of plant physiology without triggering resistance responses (Espinoza et al., 2007). Different symptoms have been associated with GLRaV-3, including leaf deformation, interveinal mosaics, deficiency in grape cluster development, decrease in total levels of carotenoids, anthocyanin, and chlorophyll, chlorosis, and an important inhibition of the electron transport chain in photosystem II (Bertamini et al., 2004). These phenotypical alterations are probably caused by changes in sugar levels since GLRaV-3 spreads through the phloem, where it may affect the sink–source balance, as described for other viruses with similar tissue specificity (Olesinski et al., 1996; Herbers et al., 1997). Modification in sugar distribution and reduced photosynthetic activity occurring in infected grapevine plants could induce the expression of SAGs, as has been described for other species (Wingler et al., 2006).
DNA microarray techniques permit the simultaneous analysis of a large number of genes and have been widely used to determine transcriptional responses to pathogen attack (Wan et al., 2002). Studies of compatible interactions in Arabidopsis have shown that different viruses induce plant gene expression changes in a wide array of cellular processes, including the induction of genes associated with defence and stress, signal transduction, and metabolism (Golem et al., 2003; Whitham et al., 2003). In addition, several host genes are down-regulated during compatible interactions in several species (Maule et al., 2002; Golem et al., 2003; Whitham et al., 2003).
In the study presented here, microarrays were used to determine if SAGs are expressed at elevated levels in grapevine plants infected with a virulent viral pathogen. The transcript profile of the model plant Arabidopsis ecotype Uk-4 infected with TMV-Cg and Vitis vinifera cv. Carménère infected with GLRaV-3 was compared using microarrays slides of the reference species Arabidopsis.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary materialReferences |
|---|
Plant material
Arabidopsis thaliana (L.) Heynh ecotype Uk-4 plants were grown in vitro in Gamborg's B5 solid medium. The growth chamber was adjusted to 23 °C with a 16/8 h light/dark photoperiod. Three rosette leaves per plant from 4-week-old plants were inoculated after rosette growth is complete and before inflorescence emergence [stage 5.10 according to Boyes et al. (2001)]. Virus inoculation was done using cotton swabs dusted with carborundum and imbibed in a solution of 10 ng µl–1 of TMV-Cg in 20 mM sodium phosphate buffer pH 7.4. Control plants were inoculated in the same manner with buffer in place of the viral solution. A pool of four plants growing in the same vessel was considered as one biological replicate. Plants of V. vinifera cultivar Carménère, both healthy and GLRaV-3-infected, were obtained from the nursery of Agronomy Faculty, Pontificia Universidad Católica de Chile. Healthy plants were checked by enzyme-linked immunosorbent assay (ELISA) against the most common grapevine viruses (grapevine virus A, grapevine virus B, arabis mosaic virus, grapevine fanleaf virus, grapevine fleck virus, tomato ringspot virus, and grapevine leafroll-associated viruses 1, 2, 3, and 7) and all tested negative for all viruses. Healthy and virus-infected plants were maintained in separate greenhouses at 25–28 °C with a 16/8 h light/dark photoperiod. Medial leaves approximately 6 cm in diameter were collected on the same day during the growing season to ensure collected leaves were of the same age. Individual grapevine plants were considered as separate biological replicates; samples were collected from five replicates.
Evaluation of systemic viral disease
In Arabidopsis plants, the TMV-Cg systemic infection was confirmed by western blot analysis of cauline leaves with a specific polyclonal antibody against the TMV-Cg coat protein (Pereda et al., 2000). In grapevine Carménère plants, the presence of GLRaV-3 was confirmed by RT-PCR. Total RNA was extracted from leaves (Goes da Silva et al., 2005) and cDNA synthesis was performed with SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions. A 945 bp fragment encoding part of the GLRaV-3 coat protein was amplified by PCR using the primers C50 and H49 (Minafra and Hadidi, 1994). 18S rRNA was amplified as a control. All primers used in this work are listed in Table 1.
|
Microarray hybridization
Total RNA was extracted from rosette and cauline leaves of TMV-Cg-infected and control Arabidopsis plants using the Trizol reagent (Invitrogen) following the supplier's protocols. Total RNA from grapevine plants was isolated using a modified TRIS-LiCl protocol (Goes da Silva et al., 2005). RNA was quantified in a spectrophotometer and the integrity was checked in agarose gels run under denaturing conditions.
Microarray experiments were performed following the protocols described by the Arabidopsis Functional Genomics Consortium (AFGC) (Ramonell et al., 2002). Total RNA (30 µg) was used to synthesize single-stranded cDNA using SuperScript II Reverse Transcriptase according to the manufacturer's protocols (Invitrogen). The second strand synthesis and labelling was achieved using Klenow fragment and incorporating Cy3-dUTP or Cy5-dUTP for cDNA labelling from healthy control plants and virus-infected plants, respectively (Amersham Pharmacia, Piscataway, NJ, USA). Fluorescently labelled cDNAs were purified using a PCR clean. Up kit (Qiagen, Hilden, Germany), and dye incorporation was quantified by measuring the sample absorbance at 260, 550, and 650 nm. The frequency of dye incorporation is calculated as A550x58.47/A260 and A650x35.08/A260 for Cy3 and Cy5, respectively. Samples with a frequency of incorporation >10 were used for hybridization.
The Y2001 AFGC DNA microarrays slides were used in this study. These slides were designed from 11 500 Arabidopsis cDNA clones collected from public sources and 3000 gene-specific amplicons (Wu et al., 2001). Clones on the array represent genes expressed in different tissues, including leaves, steams, roots, flowers, siliques, and also several developmental stages. For hybridization to the microarray slides, labelled samples were dissolved in hybridization buffer containing yeast tRNA, poly(A), poly(T), SDS, and SSC as described in the AFGC microarray protocol. Heat-denaturated probes were applied to the microarray slide, hybridized at 65 °C for 16 h, and washed. Slides were scanned using a GenePix Personal 4100A scanner, with 10 µm resolution. Image analysis and quantification of spot or feature intensities were performed using GENEPIX 3.0 software (Axon, CA, USA).
Microarray data analysis
Normalization of spot intensity values was performed with the Vector Xpression 3.1 software (Invitrogen) by fitting the Lowess curve to the background-corrected data, with f=30% (Yang et al., 2002). Normalization was applied to each slide and to each block, to avoid mistakes associated with each independent slide and with the position of each spot on the slide. Spots with a detectable signal (i.e. with a signal intensity > background value ±2 SDs) were selected (Schenk et al., 2000). To identify genes with statistically significant changes in expression level, the statistical method significance analysis of microarrays (SAM) was used (Tusher et al., 2001). For Arabidopsis experiments, SAM analysis was done considering genes with hybridization data for all the five biological replicates used. For grapevine experiments, genes with reproducible data in at least four of the five replicates were considered. Genes were ranked by the magnitude of their score values, which are calculated for each gene considering their change of expression relative to the standard deviation of repeated data for that gene. Thirty-two balanced permutations were done and, for each one, score values were calculated and genes ranked. A 
=1.2 value with a false discovery rate of 0.6% was used for the Arabidopsis-TMV-Cg experiment and a less stringent =1.04 value with a false discovery rate of 5.9% was used for the grapevine-GLRaV-3 experiment, considering that a heterologous hybridization was performed in the last case. (All raw data are available at http://www.bio.puc.cl/labs/arce/index.htm.)
Semi-quantitative RT-PCR
For semi-quantitative RT-PCR (sqRT-PCR), 2.5 µg of DNase-treated total RNA was used to synthesize cDNA, and PCRs were done with 1 µl of each cDNA sample. A master mix was prepared for each gene and all sqRT-PCRs were performed with three biological replicates using an Eppendorf Mastercycler Gradient. In order to establish the kinetics of amplification for each gene, PCRs with 19–44 cycles were performed. For all genes evaluated, logarithmic amplification was observed with 33 cycles, and this point was chosen to compare gene expression levels. The following thermal cycle conditions were used: 94 °C for 3 min, followed by 94 °C for 50 s, 50 s at the Tm of each specific pairs of primers (Table 1), and 72 °C for 1 min for 33 cycles. Genes evaluated and primers used are listed in Table 1. All PCR products had an average length of 300–560 bp. Relative expression levels of genes in each sample were normalized to the expression of glyceraldehyde-3-phosphate dehydrogenase (GPDH), which was expressed at a constant level in the present experimental conditions. Quantification was done using Scion Image beta 4.0.2 software (Scion Corporation, USA).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary materialReferences |
|---|
Viral infection of Arabidopsis and grapevine plants
The infection of Arabidopsis ecotype Uk-4 plants with TMV-Cg was evaluated by western blot analysis of the viral coat protein. Ten days after inoculation of rosette leaves, Arabidopsis plants were asymptomatic and TMV-Cg coat protein was detected in uninoculated cauline leaves, showing that TMV-Cg had spread systemically by this time (Fig. 1). Samples for subsequent microarrays studies were collected at this time, when flower buds were visible and no symptoms of leaf senescence are present. The presence of GLRaV-3 in grapevine plants was determined by RT-PCR. The expected amplified fragment was observed exclusively in infected plants, and no amplification was observed from healthy plants (Fig. 1). The presence of GLRaV-3 in grapevine plants correlated with plant symptoms, such as red coloration of leaves and chlorosis. These plants were used for microarray analysis.
|
Transcript profile in Arabidopsis thaliana and Vitis vinifera during compatible viral disease
From microarray data of the Arabidopsis response to TMV-Cg infection, it was estimated that about 71–78% of features (genes) on the Arabidopsis microarrays exhibited fluorescent signals higher than background plus 2 SDs when hybridized with leaf samples. Therefore, about 10 000–11 000 features could be evaluated. SAM identified 1561 Arabidopsis genes that were differentially expressed between healthy and TMV-Cg-infected plants. Among these genes were 1167 induced and 394 repressed genes (see Supplementary Table S1 at JXB online). A survey of these genes provides an overview of the metabolic and cellular changes occurring during viral infection of Arabidopsis. The Gene Ontology (GO) categories for biological process, molecular function, and cellular component were assigned to each gene (Berardini et al., 2004). A wide range of biological processes were affected by viral infection of Arabidopsis. About 25% of the induced genes fell into the categories translation, proteolysis, protein modifications, and targeting and folding of proteins (Fig. 2). These categories, in addition to genes with unknown functions and genes of metabolism, constituted 50% of all induced genes. Ubiquitin-related genes are also induced by viral disease in Arabidopsis. Functions involving cell rescue, defence, death, and ageing included both induced and repressed genes. Based on cellular localization, most of the repressed genes in infected Arabidopsis plants were associated with the endomembrane system. Interestingly, the abundance of chloroplastic localized genes among the repressed genes reflects the lower photosynthetic rate accompanying compatible interactions.
|
The changes in gene expression in V. vinifera cv. Carménère infected by GLRaV-3 were analysed at the genomic level using the Arabidopsis microarray platform, which has provided reliable data in several cases (Girke et al., 2000; Huang et al., 2000; Enard et al., 2002; Prendergast et al., 2002; Gu and Gu, 2003; Horvath et al., 2003; Becher et al., 2004; Lachance et al., 2004; Weber et al., 2004). About 50–60% of the total features on the array had a detectable fluorescent signal when hybridized with grapevine leaf samples, meaning that 7000–8000 grapevine genes could be analysed. Statistical analysis of the grapevine data set identified 477 differentially expressed genes. Among these genes were 184 induced and 293 repressed genes (Supplementary Table S2 available at JXB online). Genes modified by viral disease in grapevine plants were classified by GO according to biological process and cellular localization (Fig. 3). This analysis showed that a wide range of cellular processes were affected in V. vinifera cv. Carménère during the GLRaV-3 infections. It is noteworthy that about 8% of induced genes were classified in the category cell rescue, defence, death, ageing, and redox status. Similar to the Arabidopsis response to TMV-Cg, the ubiquitin–proteasome pathway is represented exclusively in the group of viral-induced genes. Other relevant processes affected during the infection included transport, transcription, and RNA processing. The main biological processes repressed during viral infection were translation, proteolysis, protein modifications, and targeting and folding of proteins, reaching 17% of the total. Processes associated with metabolism constituted 18% of repressed genes, but this group also included induced genes, reaching 10% in this case. The biosynthesis of different types of macromolecules was also affected, indicating that the lower metabolic rate observed in other plant–virus interactions also occurs in viral infections of grapevine. The chromatin and DNA modifications and metabolism categories were most affected in the repressed group of genes compared with the induced genes. Chloroplastic genes are greatly repressed in infected Carménère plants. Transcript profiling of virus-infected grapevine studied with cDNA microarrays of the model plant Arabidopsis are in good agreement with a previous analysis done with the V. vinifera GeneChip® from Affymetrix (Espinoza et al., 2007). Both analyses showed a similar group of biological processes affected by viral disease, validating the use of heterologous microarrays for gene expression in grapevine plants.
|
Association of compatible viral disease and leaf senescence
It has been proposed that an overlap exists between leaf senescence and pathogen defence programmes in plants (Lim et al., 2005). With the aim of identifying genes affected by viral diseases in Arabidopsis and grapevine plants, which have also been described as SAGs, a comparison between the data set generated in the present work and previously published large-scale transcript profiling in senescent tissues was done (Buchanan-Wollaston et al., 2003, 2005; Gepstein et al., 2003; Andersson et al., 2004; Guo et al., 2004; Zimmermann et al., 2004; Lim et al., 2005).
Genes expressed at elevated levels in Arabidopsis leaf tissue infected by TMV-Cg and in V. vinifera cv. Carménère leaves infected by GLRaV-3 that have been previously described as SAGs are shown in Table 2. These genes encode proteins involved in different functions, including genes that code for transcription factors, proteins with roles in perception and signal transduction, macromolecule degradation, stress, transport, and hormone-related proteins, among others. Genes usually described as senescence-associated, such as cysteine and aspartyl proteases, components of the ubiquitin–proteasome pathway, hydrolases, and autophagy pathway components, were also induced during compatible viral interactions with grapevine and Arabidopsis. Several genes encode transporters of sugar, peptides, amino acids, and ABC transporters, which could participate in the substrate and nutrient mobilization that occurs as part of the senescence programme. Some transcripts were associated with plant hormones, such as auxin, ethylene, and ABA. Although ethylene has been recognized as a senescence-promoting hormone, the roles of auxin and ABA during leaf senescence are not well known. Another important component of the overlap between senescence and plant responses to viral infections is the induction of defence-related genes. These include genes encoding several proteins that detoxify reactive oxygen species (ROS), as well as lipid transfer protein (LTP) and harpin-induced family protein 1 (HIN1) genes, which were previously described as part of the defence response against bacteria and fungi, or during the HR response induced by viruses (Sohal et al., 1999; Lee et al., 2001; Park et al., 2002; Takahashi et al., 2004). Also, LTP and HIN1 genes were previously shown to be induced in GLRaV-3-infected grapevine, confirming results obtained in this work (Espinoza et al., 2007). SAG proteins with unknown functions were affected by viral disease in Arabidopsis and grapevine plants, including a CBS domain protein and several expressed proteins. Although in this work a function for these genes was not established, they constitute another example of the overlap between the defence and senescence pathways, and these genes are potential targets to study.
|
Expression of senescence-associated genes during viral disease in grapevine and Arabidopsis plants
SAGs that were up- or down-regulated during the infections by TMV-Cg and GLRaV-3 in Arabidopsis and grapevine plants, respectively, were further evaluated by sqRT-PCR. The expression of the following genes was analysed in healthy and TMV-Cg-infected Arabidopsis plants: catalase 3 (AT1G20620), aspartyl protease (Asp Prot; AT2G17760), glutamine-dependent asparagine synthase (ASN1; AT3G47340), metallothionein protein 2B (MT-2B; AT5G02380), senescence-associated protein (Sen Assoc; AT5G46700), and a senescence-induced receptor kinase (SIRKb; AT5G48380). In healthy and GLRaV-3-infected grapevine plants, the following genes were evaluated: a CBS domain-containing protein (CBS prot; TC45123), peroxidase 20 (PER20; NP842538), expressed protein similar to vanilloid-receptor (Exp prot; TC44784), Asp Prot (TC49155), sugar transporter family protein 1 (SFP1; TC43544), the senescence-associated gene SAG102 (TC39250), dehydration-responsive gene (RD21A; TC38379), ribosomal protein L20 (RIBL20; TC44286), and copper chaperone (CuCHAP; TC45583). All transcript levels were normalized to the expression of the GPDH gene from Arabidopsis (At-GPDH; AT1G13440) or from V. vinifera (Vv-GPDH; TC44984). It has been shown that GPDH behaves as a housekeeping gene in a broad repertory of biotic and abiotic treatments (Mahalingam et al., 2003). For both plant species, GPDH expression was not significantly different between viral-infected and control plants in the present experimental conditions.
Transcripts corresponding to MT-2B, catalase 3, and Asp Prot were expressed at low levels in Arabidopsis plants and their expression increased as a consequence of infection with TMV-Cg virus (Fig. 4). Transcripts for SIRKb, Sen Assoc, and ASN1 are not expressed at a detectable level in healthy Arabidopsis leaves. However, these transcripts clearly accumulated to a much higher levels in TMV-Cg-infected leaves. In grapevine plants (Fig. 5), transcripts corresponding to SAG102 and the expressed protein (TC44784) are present in GLRaV-3-infected plants but not in non-infected tissues. Asp Prot and SFP1 transcripts were hardly detected in healthy tissues but showed a marked increase in infected leaves. Transcripts of a CBS domain-containing protein and peroxidase 20 are present in healthy plants, but they accumulated to higher levels in virus-infected grapevine plants. On the other hand, transcripts corresponding to RD21A, RIBL20, and CuCHAP genes are clearly repressed in grapevine plants in response to GLRaV-3 disease. These sqRT-PCR results validate the results obtained from the microarray analysis, both for the homologous hybridization with Arabidopsis samples and for the heterologous analysis in grapevines.
|
|
Expression of virus-induced genes during natural leaf senescence in Vitis vinifera
To establish if genes that are induced as part of the grapevine response to viral disease are also induced during senescence in this species, the expression of some viral-induced genes was evaluated in Carménère grapevine leaves undergoing natural senescence. Leaves at three developmental stages, ranging from mature green leaves (S1), leaves with early yellowing (S2), and fully senescent leaves (S3), were sampled. Genes induced by viral infection in grapevine plants analysed by sqRT-PCR were HIN1 (TC40876), NAM (no apical meristem) protein (TC43527), tropinone reductase, described as the senescence-associated gene SAG13 (TC40352), and an LTP (TC46011) (Fig. 6). The expression of the cysteine protease SAG12 (TC38379) was included as a control for a previously described SAG (Noh et al., 1999). SAG12 and HIN1 were expressed in the three evaluated stages, with a modest increase as senescence progressed from the S1 to S3 stage. The NAM transcript was detected at a low level in S1, but its expression increased in parallel with the increase of senescence. On the other hand, SAG13 and LTP transcripts were undetectable at S1; however, their transcripts were present late in senescence. Thus, the expression of genes induced during viral disease in Carménère grapevine plants was also induced during leaf senescence triggered by natural factors, showing a clear correspondence between natural senescence and plant response during viral compatible disease.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary materialReferences |
|---|
The transcriptome changes associated with viral disease in grapevine were evaluated previously using the homologue platform from Affymetrix (Espinoza et al., 2007). However, the availability of this technology is limited, thus an attempt was made to determine if it is possible to apply a heterologous microarray system to study changes in grapevine gene expression. In this study, samples from V. vinifera were hybridized to an Arabidopsis cDNA microarray. Several studies have demonstrated that cross-species hybridization (hybridizing samples from one species to DNA of a different but similar species on a microarray) provides reliable and informative data (Girke et al., 2000; Huang et al., 2000; Enard et al., 2002; Prendergast et al., 2002; Gu and Gu, 2003; Horvath et al., 2003; Becher et al., 2004; Lachance et al., 2004; Weber et al., 2004). Fulton et al. (2002) have identified a set of >1000 conserved orthologous marker genes that have remained relatively stable in sequence since the divergence of dicotyledonous families (i.e. tomato, Arabidopsis, and Medicago). Thus, there can be significant sequence conservation among homologous genes in different plant families, sufficient to permit heterologous hybridization on cDNA microarrays. Over 50% of non-control features in the V. vinifera treatment channel (Cy5) had a signal intensity greater than background value ±2 SDs. The differential expression of V. vinifera genes during GLRaV-3 infection was validated by sqRT-PCR, confirming the results obtained with the heterologous microarray.
Viral infection caused by GLRaV-3 in grapevine plants, as well as the infection of Arabidopsis by TMV-Cg, are compatible interactions that do not trigger effective resistance against the pathogen. Therefore, the viruses spread to all tissues via the phloem, altering nutrient distribution in the plant and also changing the sink–source balance. Viral diseases can also alter nitrogen metabolism, similar to the changes described in senescent leaves (Pageau et al., 2006). In addition, sugar distribution can be affected in viral-infected tissues, and processes that utilize sugar as a signal molecule, like leaf senescence, could be altered.
Several components of the senescence process are also elicited during the HR or in plant defence responses against pathogens that trigger necrotic symptoms. The aim of this work was to evaluate whether the senescence pathway might also be elicited in compatible viral interactions, in which no necrosis or HR occurs. Arabidopsis and grapevine genes that were differentially expressed in response to viral infection were exhaustively compared with previously described SAGs. An extensive overlap between plant responses to these two processes was observed. SAGs induced by viral compatible disease in both Arabidopsis and grapevine plants are discussed below.
Genes with regulatory roles
Leaf senescence is an active and highly regulated process. Putative regulators include transcription factors, as well as proteins involved in signal perception and transduction. Different families of transcription factors, such as NAC, MyB, WRKY, AP2, bZIP, and zinc finger types C2H2 and C3H, reported previously to play a role in senescence (Guo et al., 2004), were induced during viral infection in the present experimental conditions, making these interesting candidates for future studies (Table 2). Among these transcription factors, Myb, AP2, bZIP, and WRKY have been associated with defence responses (Eulgem, 2005), reflecting the overlap between these processes. NAC transcription factors are present exclusively in plants and have been implicated in different developmental processes (Guo et al., 2004). The overexpression of NAC transcription factor leads to a phenotype of premature senescence, and T-DNA insertional null mutants exhibited a delay in senescence (Guo and Gan, 2006). In species such as Arabidopsis and wheat, the expression of NAC occurs when the first senescence symptoms are visible (Guo and Gan, 2006; Uauy et al., 2006), as was observed for the grapevine homologue gene (Fig. 6). Moreover, the orthologous NAC genes from rice and kidney bean restore the Arabidopsis null to a wild-type phenotype (Guo and Gan, 2006). Thus, a conserved role for NAC transcription factors during leaf senescence could be suggested. WRKY transcription factors are also present exclusively in plants and have been implicated in a variety of processes (i.e. defence, development, and senescence). Although several WRKY transcription factors are expressed at elevated levels in senescent leaves, only a few reports have demonstrated direct regulation of SAG expression by WRKY transcription factors (Robatzek et al., 2002; Miao et al., 2004). Recently it has been proposed that WRKY70, a negative regulator of the senescence response, is involved in signal transduction in plant defence responses (Ulker et al., 2007). Potential components of the senescence signalling pathway were induced by viral disease in Arabidopsis and grapevine plants, including kinases and calcium-binding proteins (Table 2). Some of these proteins may be related to signal perception or transduction acting both in plant responses to virulent viruses and in senescence.
Macromolecule degradation
Protein degradation is the main biochemical event that occurs in senescent leaves (Lim et al., 2005). Transcripts corresponding to aspartyl and cysteine proteases are induced during senescence and were also induced as part of plant responses during compatible viral interactions (Table 2). In addition, genes belonging to the ubiquitin–proteasome pathway appeared to be highly induced during compatible interactions (Figs 2, 3; Table 2). The ubiquitin–proteasome pathway contributes to the active protein degradation that occurs in Arabidopsis senescent leaves and probably has a regulatory role for specific proteins (Gepstein et al., 2003). Genes encoding F-box proteins were also induced during the compatible viral–plant interactions (Table 2). These proteins are part of the Skp/Cullin/F-box complex that controls the selective degradation of target proteins. Furthermore, it has been shown that a mutation in the ORE9 gene, which codes for an F-box protein, causes a delay in senescence (Woo et al., 2001). Indeed, it has been proposed that the ubiquitin pathway could be involved in the specific degradation of a putative negative regulator of leaf senescence.
The transcript corresponding to the ATG5 gene, a gene implicated in the autophagy pathway, was induced by viral disease in grapevine plants. Autophagy has an essential role in plant nutrient recycling and is involved in seed nutrient storage, in responses to nutrient deficiencies and in defence responses by limiting cell death during HR. Mutants in different components of the pathway display early senescence and are hypersensitive to nitrogen or carbon starvation, accompanied by a faster loss of organellar and cytoplasmic proteins (Thompson and Vierstra, 2005; Thompson et al., 2005; Seay et al., 2006). The induction of transcripts associated with autophagy could be involved in the nutrient recycling related to senescence that is occurring in plants during compatible viral disease.
Lipid degradation is another catabolic process present in senescent leaves (Lim et al., 2005) and was represented in this study by the induction of genes for hydrolases during the compatible viral infections (Table 2). Lipase and acyl hydrolase (such as the SAG101 gene) are responsible for fatty acid degradation affecting membrane integrity. Fatty acids released could contribute to the energetic demands of senescent tissues through ß-oxidation.
Plant hormone-responsive genes
Transcripts encoding proteins involved in the synthesis or perception and signal transduction of hormones such as ethylene, auxin, and ABA were induced during both viral diseases studied in this work (Table 2). Ethylene is a senescence-promoting hormone in plants and could have a role in co-ordinating the time of senescence onset rather than in the progression of senescence. Plants overproducing ethylene do not show early senescent phenotypes, suggesting that factors such as leaf age may be required for ethylene-mediated regulation of senescence (Gan, 2003). The participation of the other hormones in senescence is less clear. It is known that Arabidopsis mutants with deficiencies in ABA biosynthesis or signalling, or in auxin responses exhibit altered or delayed senescence, respectively (Gan, 2003; Lim et al., 2005). The specific role of these hormones in plant senescence has not been established. However, some auxin- and ABA-responsive genes are expressed at higher levels in both senescent and virus-infected leaves (Table 2).
It has been demonstrated that salicylic acid (SA) and methyl jasmonate (MeJ) influence the expression of SAGs (Lim et al., 2005), and several SA- and MeJ-dependent genes are expressed during compatible viral disease (Table 2 see Supplementary Tables S1 and S2 at JXB online). Experimental data have shown the role of these molecules during the plant defence response. Endogenous levels of SA increase during the resistance response and mutant plants with higher or lower levels of SA are associated with more resistant or more susceptible phenotypes, respectively (O'Donnell et al., 2003). However, it has been demonstrated that during viral compatible interactions, SA synthesis is not induced and plants with deficiencies in SA signalling pathways, MeJ, or ethylene are not more susceptible to the infection caused by compatible viruses (Huang et al., 2005; Love et al., 2005). Thus, SA basal levels present in leaf tissues would be able to allow expression of SA-dependent genes.
Expression of defence genes and senescence
Numerous transcripts specifically associated with resistance and defence responses that have been described in senescence were also induced during the compatible viral plant interactions studied here. These include genes that code for proteins involved in ROS detoxification, HIN1 and LTP, among others (Figs 2, 3; Table 2). ROS production is observed during senescence as well as in incompatible plant–pathogen interactions that lead to the HR. These highly reactive molecules cause extensive cellular damage if they are not actively removed. Particularly in senescence, it is important to retard cell death until nutrient mobilization occurs, which could explain the induction of ROS detoxification genes. LTPs have been described in animals, fungi, plants, and bacteria. Although LTPs are able to exchange membrane lipids in vitro, their role in plant defence is unclear. LTPs are induced in response to abiotic stress and during defence responses against bacteria and fungi. LTP transcript levels show an increase during infections with viruses that trigger the HR (Park et al., 2002) and also in Arabidopsis infected with cauliflower mosaic virus, a virus that induces local lesions in inoculated leaves and systemic symptoms (Sohal et al., 1999). Also, the expression of various LTPs has been consistently observed during plant senescence (Yoshida et al., 2001; Gepstein et al., 2003). On the other hand, the HIN1 transcript has been detected in tobacco during the HR in response to viruses and bacteria, where a role in cell death has been proposed (Lee et al., 2001; Takahashi et al., 2004). HIN1 expression has also been detected during senescence in Arabidopsis (Pontier et al., 1999; Yoshida et al., 2001; Buchanan-Wollaston et al., 2003). In the present work, the expression of LTP and HIN1 in susceptible plant species infected with viruses and during natural senescence in grapevine plants was demonstrated.
Overlap between plant leaf responses during viral compatible interactions and senescence
The relationship between plant defence and senescence has been suggested by the elevated expression of SAGs during HR or in systemic diseases trigged by fungi or bacteria that produce necrotic symptoms. In these cases, cell death appears to be the connection between plant responses to pathogens and senescence. In this work, the relationship of senescence to viral disease development was studied for viruses that do not elicit necrosis. The work clearly shows that plant–virus compatible interactions elicit the expression of several senescence-associated genes (Table 2; Fig. 5). Moreover, the expression of virus-induced genes occurs during the natural senescence in grapevine. A portion of SAGs encode proteins also involved in pathogen defence. The expression of defence genes during leaf senescence could imply that cells of senescent leaves are stressed, and the expression of some defence genes could have a protective effect in those cells, such as protecting them from oxidative damage or against attack by opportunistic pathogens, and thereby allowing the completion of the senescence programme. The generation of ROS could be responsible for the partial activation of the senescence programme during viral diseases. ROS are necessary for the expression of defence-related genes and also act as promoters of senescence (Hung et al., 2004). This relationship may represent a strategy by plants for adapting to viral pathogens, recycling nutrients from infected leaves and mobilizing them to distant tissues, allowing a plant–pathogen relationship to be established, even for long periods of time.
|
Supplementary material |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary materialReferences |
|---|
Supplementary material is available at JXB online.
Table S1. A list of differentially expressed genes in Arabidopsis thaliana ecotype Uk-4 during infection with TMV-Cg.
Table S2. A list of differentially expressed genes in Vitis vinifera cv. Carménère during infection with GLRaV-3.
|
Acknowledgements |
|---|
This work was supported by the Chilean Genome Initiative FONDEF G02S1001 and the FONDECYT 1040789 of the Chilean National Council of Sciences and Technology (CONICYT) to PAJ. We thank Bi-Huei Hou (Carnegie Institute) for her assistance with preparing and hybridizing the microarrays. This work is also supported in part by funds from the National Science Foundation, the US Department of Energy, and the Carnegie Institution to S.S.
|
Footnotes |
|---|
This version of the paper is now Open Access.
|
Abbreviations |
|---|
ABA, abscisic acid; AFGC, Arabidopsis Functional Genomics Consortium; ELISA, enzyme-linked inmunoabsorbent assay; FDR, false discovery rate; GLRaV-3, grapevine leafroll-associated virus strain 3; GPDH, glyceraldehyde-3-phosphate dehydrogenase; HIN1, harpin-induced 1; HR, hypersensitive response; LTP, lipid transfer protein; NAM, no apical meristem protein; PR, pathogenesis-related protein; ROS, reactive oxygen species; SAG, senescence-associated gene; SAG12, senescence-associated cysteine protease; SAG13, senescence-associated tropinone reductase; SAM, significance analysis of microarrays; SDG, senescence-down-regulated gene; TMV-Cg, tobacco mosaic virus strain Cg.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary materialReferences |
|---|
Andersson A, Keskitalo J, Sjodin A, et al. A transcriptional timetable of autumn senescence. Genome Biology (2004) 5:R24.[CrossRef][Medline]
Arce-Johnson P, Medina C, Padgett H, Huanca W, Espinoza C, Beachy RN. Analysis of local and systemic spread of the crucifer-infecting TMV-Cg virus in tobacco and several Arabidopsis thaliana ecotypes. Functional Plant Biology (2003) 30:401–408.[CrossRef][ISI]
Becher M, Talke IN, Krall L, Kramer U. Cross-species microarray transcript profiling reveals high constitutive expression of metal homeostasis genes in shoots of the zinc hyperaccumulator Arabidopsis halleri. The Plant Journal (2004) 37:251–268.[ISI][Medline]
Berardini TZ, Mundodi S, Reiser L, et al. Functional annotation of the Arabidopsis genome using controlled vocabularies. Plant Physiology (2004) 135:745–755.
Bertamini M, Muthuchelian K, Nedunchezhian N. Effect of grapevine leafroll on the photosynthesis of field grown grapevine plants (Vitis vinifera L. cv. Lagrein). Journal of Phytopathology (2004) 152:145–152.[CrossRef][ISI]
Boyes DC, Zayed AM, Ascenzi R, McCaskill AJ, Hoffman NE, Davis KR, Gorlach J. Growth stage-based phenotypic analysis of Arabidopsis: a model for high throughput functional genomics in plants. The Plant Cell (2001) 13:1499–1510.
Buchanan-Wollaston V, Earl S, Harrison E, Mathas E, Navabpour S, Page T, Pink D. The molecular analysis of leaf senescence—a genomics approach. Plant Biotechnology Journal (2003) 1:3–22.[CrossRef][ISI][Medline]
Buchanan-Wollaston V, Page T, Harrison E, et al. Comparative transcriptome analysis reveals significant differences in gene expression and signalling pathways between developmental and dark/starvation-induced senescence in Arabidopsis. The Plant Journal (2005) 42:567–585.[CrossRef][ISI][Medline]
Butt A, Mousley C, Morris K, Beynon J, Can C, Holub E, Greenberg JT, Buchanan-Wollaston V. Differential expression of a senescence-enhanced metallothionein gene in Arabidopsis in response to isolates of Peronospora parasitica and Pseudomonas syringae. The Plant Journal (1998) 16:209–221.[CrossRef][ISI][Medline]
Enard W, Khaitovich P, Klose J, et al. Intra- and interspecific variation in primate gene expression patterns. Science (2002) 296:340–343.
Espinoza C, Vega A, Medina C, Schlauch K, Cramer G, Arce-Johnson P. Gene expression associated with compatible viral diseases in grapevine cultivars. Functional and Integrative Genomics (2007) 7:95–110.[CrossRef]
Eulgem T. Regulation of the Arabidopsis defense transcriptome. Trends in Plant Science (2005) 10:71–78.[CrossRef][ISI][Medline]
Fulton TM, Van der Hoeven R, Eannetta NT, Tanksley SD. Identification, analysis, and utilization of conserved ortholog set markers for comparative genomics in higher plants. The Plant Cell (2002) 14:1457–1467.
Gan S. Mitotic and postmitotic senescence in plants. Sci of Aging Knowledge Environment (2003) 2003:RE7.
Garcia-Hernandez M, Berardini TZ, Chen G, et al. TAIR: a resource for integrated Arabidopsis data. Functional and Integrative Genomics (2002) 2:239–253.[CrossRef]
Gepstein S, Sabehi G, Carp MJ, Hajouj T, Nesher MF, Yariv I, Dor C, Bassani M. Large-scale identification of leaf senescence-associated genes. The Plant Journal (2003) 36:629–642.[CrossRef][ISI][Medline]
Girke T, Todd J, Ruuska S, White J, Benning C, Ohlrogge J. Microarray analysis of developing Arabidopsis seeds. Plant Physiology (2000) 124:1570–1581.
Goes da Silva F, Iandolino A, Al-Kayal F, et al. Characterizing the grape transcriptome. Analysis of expressed sequence tags from multiple Vitis species and development of a compendium of gene expression during berry development. Plant Physiology (2005) 139:574–597.
Golem S, Culver JN. Tobacco mosaic virus induced alterations in the gene expression profile of Arabidopsis thaliana. Molecular Plant–Microbe Interactions (2003) 16:681–688.[CrossRef]
Gu JG, Gu X. Induced gene expression in human brain after the split from chimpanzee. Trends in Genetics (2003) 19:63–65.[CrossRef][ISI][Medline]
Guo Y, Cai Z, Gan S. Transcriptome of Arabidopsis leaf senescence. Plant, Cell and Environment (2004) 27:521–549.[CrossRef]
Guo Y, Gan S. AtNAP, a NAC family transcription factor, has an important role in leaf senescence. The Plant Journal (2006) 46:601–612.[CrossRef][ISI][Medline]
Herbers K, Tacke E, Hazirezaei M, Krause KP, Melzer M, Rohde W, Sonnewald U. Expression of a luteoviral movement protein in transgenic plants leads to carbohydrate accumulation and reduced photosynthetic capacity in source leaves. The Plant Journal (1997) 12:1045–1056.[CrossRef][ISI][Medline]
Horvath DP, Schaffer R, West M, Wisman E. Arabidopsis microarrays identify conserved and differentially expressed genes involved in shoot growth and development from distantly related plant species. The Plant Journal (2003) 34:125–134.[CrossRef][ISI][Medline]
Huang GS, Yang SM, Hong MY, Yang PC, Liu YC. Differential gene expression of livers from ApoE deficient mice. Life Science (2000) 68:19–28.[CrossRef][ISI][Medline]
Huang Z, Yeakley JM, Garcia EW, Holdridge JD, Fan JB, Whitham SA. Salicylic acid-dependent expression of host genes in compatible Arabidopsis–virus interactions. Plant Physiology (2005) 137:1147–1159.
Hung KT, Kao CH. Hydrogen peroxide is necessary for abscisic acid-induced senescence of rice leaves. Journal of Plant Physiology (2004) 161:1347–1357.[CrossRef][ISI][Medline]
Lachance PE, Chaudhuri A. Microarray analysis of developmental plasticity in monkey primary visual cortex. Journal of Neurochemistry (2004) 88:1455–1469.[ISI][Medline]
Lee J, Klessig DF, Nurnberger T. A harpin binding site in tobacco plasma membranes mediates activation of the pathogenesis-related gene HIN1 independent of extracellular calcium but dependent on mitogen-activated protein kinase activity. The Plant Cell (2001) 13:1079–1093.
Lim PO, Nam HG. The molecular and genetic control of leaf senescence and longevity in Arabidopsis. Current Topics in Developmental Biology (2005) 67:49–83.[ISI][Medline]
Love AJ, Yun BW, Laval V, Loake GJ, Milner JJ. Cauliflower mosaic virus, a compatible pathogen of Arabidopsis, engages three distinct defense-signaling pathways and activates rapid systemic generation of reactive oxygen species. Plant Physiology (2005) 139:935–948.
Mahalingam R, Gomez-Buitrago A, Eckardt N, Shah N, Guevara-Garcia A, Day P, Raina R, Fedoroff NV. Characterizing the stress/defense transcriptome of Arabidopsis. Genome Biology (2003) 4:R20.[CrossRef][Medline]
Maule A, Leh V, Lederer C. The dialogue between viruses and hosts in compatible interactions. Current Opinion in Plant Biology (2002) 5:279–284.[CrossRef][ISI][Medline]
Miao Y, Laun T, Zimmermann P, Zentgraf U. Targets of the WRKY53 transcription factor and its role during leaf senescence in Arabidopsis. Plant Molecular Biology (2004) 55:853–867.[ISI][Medline]
Minafra A, Hadidi A. Sensitive detection of grapevine virus A, B, or leafroll-associated III from viruliferous mealybugs and infected tissue by cDNA amplification. Journal of Virological Methods (1994) 47:175–188.[CrossRef][ISI][Medline]
Noh YS, Amasino RM. Identification of a promoter region responsible for the senescence-specific expression of SAG12. Plant Molecular Biology (1999) 41:181–194.[CrossRef][ISI][Medline]
O'Donnell PJ, Schmelz EA, Moussatche P, Lund ST, Jones JB, Klee HJ. Susceptible to intolerance—a range of hormonal actions in a susceptible Arabidopsis pathogen response. The Plant Journal (2003) 33:245–257.[CrossRef][ISI][Medline]
Olesinski AA, Almon E, Navot N, Perl A, Galun E, Lucas WJ, Wolf S. Tissue-specific expression of the tobacco mosaic virus movement protein in transgenic potato plants alters plasmodesmal function and carbohydrate partitioning. Plant Physiology (1996) 111:541–550.[Abstract]
Pageau K, Reisdorf-Cren M, Morot-Gaudry JF, Masclaux-Daubresse C. The two senescence-related markers, GS1 (cytosolic glutamine synthetase) and GDH (glutamate dehydrogenase), involved in nitrogen mobilization, are differentially regulated during pathogen attack and by stress hormones and reactive oxygen species in Nicotiana tabacum L. leaves. Journal of Experimental Botany (2006) 57:547–557.
Park CJ, Shin R, Park JM, Lee GJ, You JS, Paek KH. Induction of pepper cDNA encoding a lipid transfer protein during the resistance response to tobacco mosaic virus. Plant Molecular Biology (2002) 48:243–254.[CrossRef][ISI][Medline]
Pereda S, Ehrenfeld N, Medina C, Delgado J, Arce-Johnson P. Comparative analysis of TMV-Cg and TMV-U1 detection methods in infected Arabidopsis thaliana. Journal of Virological Methods (2000) 90:135–142.[CrossRef][ISI][Medline]
Pontier D, Gan S, Amasino RM, Roby D, Lam E. Markers for hypersensitive response and senescence show distinct patterns of expression. Plant Molecular Biology (1999) 39:1243–1255.[CrossRef][ISI][Medline]
Prendergast BJ, Mosinger B Jr, Kolattukudy PE, Nelson RJ. Hypothalamic gene expression in reproductively photoresponsive and photorefractory Siberian hamsters. Proceedings of the National Academy of Sciences, USA (2002) 99:16291–16296.
Quackenbush J, Cho J, Lee D, Liang F, Holt I, Karamycheva S, Parvizi B, Pertea G, Sultana R, White J. The TIGR gene indices: analysis of gene transcript sequences in highly sampled eukaryotic species. Nucleic Acids Research (2001) 29:159–164.
Quirino BF, Normanly J, Amasino RM. Diverse range of gene activity during Arabidopsis thaliana leaf senescence includes pathogen-independent induction of defense-related genes. Plant Molecular Biology (1999) 40:267–278.[CrossRef][ISI][Medline]
Ramonell K, Zhang B, Ewing R, Chen Y, Xu D, Stacey G, Somerville S. Microarray analysis of chitin elicitation in Arabidopsis thaliana. Molecular Plant Pathology (2002) 3:301–311.[CrossRef][ISI]
Robatzek S, Somssich IE. Targets of AtWRKY6 regulation during plant senescence and pathogen defense. Genes and Development (2002) 16:1139–1149.
Schenk PM, Kazan K, Wilson I, Anderson JP, Richmond T, Somerville SC, Manners JM. Coordinated plant defense responses in Arabidopsis revealed by microarray analysis. Proceedings of the National Academy of Sciences, USA (2000) 7:11655–11660.
Seay M, Patel S, Dinesh-Kumar SP. Autophagy and plant innate immunity. Cell Microbiology (2006) 8:899–906.[CrossRef]
Sohal AK, Pallas JA, Jenkins GI. The promoter of a Brassica napus lipid transfer protein gene is active in a range of tissues and stimulated by light and viral infection in transgenic Arabidopsis. Plant Molecular Biology (1999) 41:75–87.[CrossRef][ISI][Medline]
Takahashi Y, Berberich T, Yamashita K, Uehara Y, Miyazaki A, Kusano T. Identification of tobacco HIN1 and two closely related genes as spermine-responsive genes and their differential expression during the tobacco mosaic virus-induced hypersensitive response and during leaf- and flower-senescence. Plant Molecular Biology (2004) 54:613–622.[CrossRef][ISI][Medline]
Thompson AR, Doelling JH, Suttangkakul A, Vierstra RD. Autophagic nutrient recycling in Arabidopsis directed by the ATG8 and ATG12 conjugation pathways. Plant Physiology (2005) 138:2097–2110.
Thompson AR, Vierstra RD. Autophagic recycling: lessons from yeast help define the process in plants. Current Opinion in Plant Biology (2005) 8:165–173.[CrossRef][ISI][Medline]
Tusher VG, Tibshirani R, Chu G. Significance analysis of microarrays applied to the ionizing radiation response. Proceedings of the National Academy of Sciences, USA (2001) 98:5116–5121.
Uauy C, Distelfeld A, Fahima T, Blechl A, Dubcovsky J. A NAC gene regulat