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found to cause defects in leaf development (Aoyama et al.,1995). This result could reflect the leaf phenotype observed in the prl1 mutant (Németh et al.,1998).

Root development is extremely impaired in the prl1 mutant. This is reflected in the tran-scription alteration of multiple genes associated to root development. For example, MYB77, an interacting partner of auxin response transcription factors (Shin et al.,2007) is down-regulated 2,5–2,7-fold in prl1 roots. As MYB77 plays a pivotal role in the control of auxin responses, in-cluding differentiation of lateral root and control of root elongation, its altered regulation may directly be related to the root developmental defects observed in prl1. ROOT HAIR DEFECTIVE 2 (RHD2) is up-regulated in roots during the time-course (2,5 fold) whereas is down-regulated in the RNA-Seq experiment (3,1 fold). The absence of RHD2 is reflected in a lack of root hairs due to a reduced O^•−2 production (Renew et al.,2005). This different behaviour of RHD2 in the dynamic (ER-PRL1/prl1) and static (prl1) situation could be a reflect of the adaptation changes taking place when PRL1 is depleted, whereas the down-regulation of RHD2 in the prl1 mutant has a direct impact in root development.

In summary, grasping complex alterations in gene regulation reflected by steady-state differ-ences and dynamic changes in transcript levels, gives an overview to infer the different regulation of developmental processes seen in the pleiotropic phenotype of prl1.

3.1.1.2 Altered transcription of genes in hormone regulation

By characterizing hormonal responses of the prl1 mutant,Németh et al.(1998) observed a hyper-sensitivity to cytokinins, ethylene, abscisic acid and auxin. Extensive changes in transcript levels of genes involved in these hormonal pathways have been observed by comparative transcript profiling of prl1 and wild type seedlings, as well as during the time-course of PRL1 depletion.

For example, CYTOKININ-RESPONSIVE GATA FACTOR 1 (CGA1) was identified as a gene with an early light-dependent response to cytokinin signalling (Naito et al.,2007). During the time-course, CGA1 is 2,66-fold down-regulated, suggesting an impaired mechanism of cytokinin signalling. Furthermore, CYTOKININ OXIDASE 2 (CKX2) is up-regulated (2,6 fold) in the RNA-Seq analysis. CKX2 over-expression reduces the active cytokinin content, producing smaller plants (Werner et al.,2003). A lower content of active cytokinins could be the cause of the semi-dwarf phenotype of prl1.

Several genes related to ethylene signalling were found to be miss-regulated in the tiling array dataset. For example, EIN3-BINDING F-BOX 1 and 2 (EBF1 and EBF2), were found to be up-regulated in the prl1 mutant (4,4 and 6,2 fold, respectively). EBF1 and EBF2 were shown to target EIN3 to degradation, repressing ethylene action (Gagne et al.,2004). During the time course, ACC OXIDASE 1 (ACO1), a key gene in ethylene production, was found to be 2,54-fold up-regulated in shoots growing in 0,1 % sucrose, whereas no change was observed in shoots growing in 3 % sucrose. This differential regulation suggest an activation of ethylene production when PRL1 is depleted, only in sucrose-limiting conditions. Contrarily, ACC SYNTHASE 7 (ACS7) was found to be 2-fold down-regulated in the sucrose-limiting conditions, whereas is up-regulated in the prl1 mutant (2,07 fold in the RNA-Seq experiment). ACS7 has been shown to have an increased expression upon treatment of GA3, ABA or salt (Wang et al.,2005). This difference in expression reflects the adapting situation the ER-PRL1/prl1 plants are undergoing, whereas in the static situation (prl1 plants), the increased regulation of ACS7 reflects a chronic stress.

Multiple responses related to abscisic acid (ABA) have been related to stress situations.

UDP-GLUCOSYL TRANSFERASE 71B6(UGT71B6) encodes for an ABA glucosyl-transferase. Over-expression of UGT71B6 was shown to produce an accumulation of glucosylated ABA (Priest et al., 2006). UGT71B6 up-regulation in the tiling array experiment suggests that activation of this gene is a consequence of an increased ABA content or ABA signalling. This up-regulation can lead to an accumulation of glucosylated ABA in the prl1 mutant due to an overproduction of bioactive ABA. In fact, ABA DEFICIENT 2 (ABA2), a gene encoding a key enzyme in ABA biosynthesis (Léon-Kloosterziel et al.,1996), is up-regulated (2 fold) when PRL1 content decreases. Similarly, ABSCISIC ACID RESPONSIVE ELEMENTS-BINGING FACTOR 3(ABF3) is also up-regulated during the time course. Interestingly, ABF3 over-expression is reported to confer tolerance to stresses including low and high temperature, oxidative stress and water deficiency (Kim et al.,2004). Contrarily, ABSCISIC ACID RESPONSIVE ELEMENTS-BINGING FACTOR 1(ABF1), a bZIP transcription factor, is 4,2-fold down-regulated, suggesting an ABA2-independent repression of ABF1 (Uno et al.,2000) during the time-course. These data suggest that ABA biosynthesis is constitutively activated in the prl1 mutant. Moreover, altered regulation of these genes correlates with an enhanced ABA sensitivity of prl1 during seed germination and seedling development (Németh et al.,1998).

Responses to auxin are also enhanced in the prl1 mutant. Intriguingly, while MYB77 shows down-regulation in roots during the time-course, a set of IAA and ARF factors (including Shy2/IAA3, IAA6, IAA7, IAA14, AXR3/IAA17, IAA29, ARF10, ARF11), as well as PINOID and ENHANCER OF PINOID, which act as central regulators of auxin responses, are up-regulated in shoots during the time-course. Alteration of transcript levels of genes encoding components of auxin signalling may thus correlate with the enhanced auxin sensitivity trait of prl1.

During the time course, PIN-FORMED 1 and 3 (PIN1 and PIN3) were found to be up-regulated in shoots growing in 0,1 % sucrose. Alteration of PIN expression has been shown to have effects in root growth and embryo patterning (Blilou et al.,2005). These genes do not appear as altered in the tiling or RNA-Seq analysis, indicating that the PIN-dependent auxin homeostasis could be differently controlled in the prl1 mutant than in the PRL1 depleting situation. Other example of altered expression of auxin-related genes during the time-course is AUXIN-REGULATED GENE INVOLVED IN ORGAN SIZE(ARGOS), a gene induced by auxin, which is 2,14-fold up-regulated after PRL1 depletion. Interestingly, ARGOS over-expression has been related to an over-growth of Arabidopsisorgans (Hu et al.,2003). The up-regulation of ARGOS after PRL1 depletion suggests the activation of a compensatory mechanism to keep the growing rate of the ER-PRL1/prl1 plants.

ARABIDOPSIS THALIANA HOMEOBOX PROTEIN 2(ATHB-2) is involved in auxin perception.

When auxin is applied to the athb-2 mutant, the phenotype is restored (Steindler et al.,1999).

Furthermore, ATHB-2 expression has been shown to be repressed by rich far-red light (Carabelli et al.,1993) and involved in mediated shade-avoidance responses (Steindler et al.,1999). These results suggest a deficient auxin response in prl1, since ATHB-2 is 2-fold down-regulated in the RNA-Seq sample, whereas is 3,23-fold up-regulated during the time course, which could be explained as an adaptation mechanism to control auxin homeostasis during PRL1 depletion.

Remarkably, depletion of PRL1 in the time-course experiment resulted in 2 to 6-fold coordi-nated up-regulation of transcript levels of several genes involved in the biosynthesis of tryptophan (ANTHRANILATE SYNTHASE ALPHA SUBUNIT 1, α and β subunits of TRYPTOPHAN SYNTHASE TRYPTOPHAN AMINOTRANSFERASE) and tryptophan-derived indolacetic acid derivatives, includ-ing indol-3-acetaldoxime (IAOx), a precursor of glucosynolates, such as the antifungal camalexin.

In correlation with the activation of this pathway, genes encoding signalling components of

3.1. TRANSCRIPTIONAL REGULATORY EFFECTS OF PRL1 3. DISCUSSION

the salicylic acid-triggered camalexin-based pathogencity responses, such as PAD3 and EPS1 are up-regulated during the time-course.

Jasmonic acid biosynthesis is also modified in the prl1 mutant and this is reflected in the transcription profile. ALLENE OXIDE CYCLASE 4 (AOC4) and LIPOXYGENASE 1 (LOX1) encode for enzymes which catalyse essential steps in jasmonic acid biosynthesis (Ziegler et al.,2000).

Both are up-regulated in the time-course. In addition, jasmonate signalling is also impaired in the prl1 mutant. This is shown by the up-regulation of JASMONATE-ZIM-DOMAIN PROTEIN 6 and 7 (JAZ6 and JAZ7) in the tiling arrays (1,8 and 2,3 fold, respectively). JAZ6 has been shown to rapidly increase its expression upon wounding-induced stress (Chung et al.,2008).

Gibberellin (GA) biosynthesis is activated during the time course, this is observed in the up-regulation (2,51 fold) of GIBBERELLIN 3-OXIDASE 1 (GA3OX1) and the down-up-regulation (2,97 fold) of GIBBERELLIN 2-OXIDASE 6 (GA2OX6). Both gene products have antagonistic effects in the biosynthesis of active GAs (Hedden and Kamiya,1997), leading to an increased amount of bioactive GAs during the time-course. A similar situation was observed in the tiling results: a gibberellin 2-oxidase was found to be down-regulated (1,8 fold) in prl1, suggesting that gibberellin inactivation is negatively regulated in the prl1 mutant. Gibberellin signalling could also be modified during the time-course. Genes encoding two members of the RGA-LIKE DELLA protein family, which act as redundant negative regulators of GA responses, showed a 5,5 and 1,7-fold up-regulation in shoots analysed in the time-course experiment.

The above-mentioned regulatory changes in various hormone response pathways suggest to be the cause of the observed hormone hypersensitivity traits in the prl1 mutant. However, only genetic analysis can confirm the exact implication.

3.1.1.3 DNA and RNA-related processes are modified

In the tiling array dataset, ARABIDOPSIS MEI2-LIKE PROTEIN 5 (AML5) was found to be 3 fold up-regulated. AML5 was shown to be required for a proper meiosis in Arabidopsis (Kaur et al., 2006). An over-expression of AML5 in prl1 could be an effect of a compensating mechanism targeted at maintaining DNA stability. Contrarily, ROOT AND POLLEN ARFGAP (RPA), a protein that binds to single-stranded DNA at stalled replication forks, was found to be down-regulated in the RNA-Seq experiment (3,6 fold). RPA was shown to perform an important role during pollen development (Boavida et al.,2009), since its absence produces semi-sterility. The down-regulation of this gene in prl1 would suggest DNA instability, and especially during pollen development.

RPAdown-regulation could be reflected in the lower seed production of prl1.

During PRL1 depletion, several genes related to DNA stability have an altered expression. For example, DNA-DAMAGE REPAIR/TOLERATION 100 (DRT100), which was reported to increase E. colitolerance to mutagenic conditions (Pang et al.,1992), is 3,4-fold up-regulated. CYTIDINE DEAMINASE 1 (CDA1) is 2,67-fold up-regulated during the time-course in shoots growing in presence of 0,1 % sucrose. CDA1 was found to deaminate cytidine and deoxycytidine and shares sequence and structure homology with known prokaryotic cytidine deaminases involved in DNA repairing (Faivre-Nitschke et al.,1999). Conversely, UV REPAIR DEFECTIVE 3 (UVR3) is 2,58-fold down-regulated in prl1. UVR3 was found to encode a photolyase that repairs the cyclobutane pyrimidine dimer caused by UV light (Nakajima et al.,1998). Together this suggests that DNA is destabilized during PRL1 depletion.

Chromatin remodelling is also affected during PRL1 depletion. REPRESSOR OF SILENCING 1 (ROS1) is 1,95-fold up-regulated. ROS1 mediates an active DNA de-methylation by removing 5-methylcytosine (Morales-Ruiz et al.,2006). This implies that during PRL1 depletion, an active de-methylation is taking place. Furthermore, METHYLATED DNA-BINDING DOMAIN 11 (MBD11) is 1,73-fold up-regulated. MDB11 belongs to the same family of MBD7 (Scebba et al.,2003) which indirectly interacts with PRL1 via PRMT11 (Salchert,1997;Scebba et al.,2007). Overall, these results suggest that changes in DNA methylation in a genome-wide scale are taking place during PRL1 depletion.

Interestingly, a substantial number of genes, related to DNA stability, were found in the PRL1-depleting situation, whereas less were found in the tiling and RNA-Seq experiments. This difference suggests that the transcriptional regulation of genes leading to DNA stability is com-pensated in the prl1 mutant.

DNA stability could have a direct impact on RNA-dependent processes such as transcription and pre-mRNA splicing. Recent QPCR profiling of TF mRNAs revealed hundreds of regulatory genes that show differential expression in the prl1 mutant (Baruah et al.,2009). The transcript profiling experiments presented here also indicate differential regulation of several genes involved in post-transcriptional gene silencing. Specifically, ARGONAUTE7 (AGO7) is 2,73-fold up-regulated in shoots growing in 0,1 % sucrose. AGO7 is involved in defence against virus and its transcript is up-regulated by the presence of specific viral proteins, whereas the siRNA biogenesis is reduced due to virus-dependent mechanisms (Shivaprasad et al.,2008). This up-regulation of AGO7 suggests that during PRL1 depletion, siRNA biogenesis is constitutively activated.

Furthermore, the stability of mRNA could be affected during the time-course. GENE WITH UNSTABLE TRANSCRIPT 15(GUT15) is 1,72-fold up-regulated in the low-sucrose medium. GUT15 was discovered as a short-lived transcript upon treatment with a transcription inhibitor (Taylor and Green,1995). GUT15 is actually a non-coding RNA with a mRNA structure. Alterations such the one observed for GUT15 in non-coding RNAs have been related to responses to stress, genomic imprinting and ribozyme activity (Rymarquis et al.,2008). These results strongly suggest that DNA and RNA stability are modified during PRL1 depletion in a genome-wide scale.

3.1.1.4 Specific effects of prl1 on biotic and abiotic stress reponses

A key to further dissect PRL1 effects on regulatory genes in abiotic stress response is the down-regulation of CBF1 and CBF2, as well as DREP1A, 2A and DREB2B during the time-course. CBF1/2 are key regulators of low temperature response pathways (Thomashow,1999). Down-regulation of CBF1/2 transcript levels during the time-course does not only correlate with enhanced cold sensitivity of the mutant, but also with a coordinated down-regulation in transcription of several low temperature-induced marker genes, such as COLD REGULATED B5 (CORB5) and COR47.

DREB1Abelongs to the cold-induced CBF class of AP2 transcription factors and transcription of the DREB2 family is stimulated by drought and osmotic stress (Shinozaki and Yamaguchi-Shinozaki, 2000). As for CBF1/2, down-regulation of DREB2A/B transcription during the time-course leads to a coordinated down-regulation of transcript levels of numerous genes involved in osmotic and drought stress responses. These genes include members of the EARLY RESPONSIVE TO DEHYDRATIONgene family (ERD4, 5,6,7, and 15), RESPONSIVE TO DEHYDRATION 19 (RD19), members of the ARABIDOPSIS ZINC-FINGER PROTEIN TF family (AZF1, 2, and 3), STZ (SALT TOLERANCE ZINC FINGER), SZF1 (SALT-INDUCIBLE ZINC FINGER 1) and others.

3.1. TRANSCRIPTIONAL REGULATORY EFFECTS OF PRL1 3. DISCUSSION

However, all osmotic stress marker genes, which are positively regulated by ABA or JA are remarkably up-regulated in the prl1 mutant. These include, members of the RESPONSIVE TO DESICCATIONfamily, such as RD2, RD20, RD22, RD26 and several dehydrin-encoding genes.

Activation of JA, ABA, glucosynolate, ethylene and salicylic acid-regulated pathways is indicated by the up-regulation of over hundred of known genes involved in pathogen-signalling pathways. Activation of SA biosynthesis –and signalling– during the time-course is suggested by the 2,24-fold up-regulation of ISOCHORISMATE SYNTHASE 2 (ICS2), involved in SA biosynthesis (Garcion et al.,2008).

This result correlates well with the high transcription of SA-stimulated classical genes, includ-ing the PATHOGENESIS-RELATED GENEs PR1 and PR4, which have been previously observed also byNémeth et al.(1998). Analogously, up-regulation of the JA signalling pathway is indicated by increased transcript levels of JA-induced genes, including PDF1.2, PDF1.2a and PDF1.4. Although not exposed to pathogen, the time-course also displays activation of several elicitor-induced genes, such as ELICITOR-ACTIVATED GENE 3-1 (ELI3-1) and ELI3-2 and AVRRPT2-INDUCED GENEs (AIG1 and AIG2). Activation of HR responses is indicated by an up-regulated transcription of 26 genes encoding various enzymes of phenylpropanoid pathway (including CHALCONE SYNTHASE and PHYLALANINE AMMONIA LYASEdescribed by Németh et al, 1998) in prl1.

Contrarily, during the time-course, specific genes that mediate pathogen recognition are down-regulated, including RECOGNITION OF PERONOSPORA PARASITICA 4 and 5 (RPP4 and RPP5, respectively) which confer resistance for downy mildew (van der Biezen et al.,2002;Parker et al.,1997, respectively). Another example is BOTRYTIS-INDUCED KINASE1 (BIK1), which is up-regulated upon Botrytis cinerea infection, shows a 3,87-fold down-regulation during the time course.

Miss-regulation of pathogen-induced genes during PRL1 depletion could affect the expression of genes involved in responses to reactive oxygen species (ROS). Several putative peroxidases, which reduce peroxides, were found to be up-regulated in the tiling and RNA-Seq dataset, whereas none was found to be down-regulated. The time-course experiment also revealed extensive changes in ROS signalling. For example, l-ASPARTATE OXIDASE (AO), is involved in the biosynthesis of NAD. The early steps of NAD biosynthesis occur in the plastid with the conversion of l-aspartate by AO (Katoh et al.,2006). This result suggests that the production of NAD is highly up-regulated during PRL1 depletion. Furthermore, RESPIRATORY BURST OXIDASE HOMOLOGUE D (RBOHD) is also up-regulated (2,29 fold), suggesting that the pathogen- and NADP-dependent systemic signal of oxidative burst in which RBOHD is involved (Miller et al., 2009), could be up-regulated during the time-course. Interestingly, several glutaredoxins, which transfer the ROS to glutathione, are down-regulated. This suggest that the glutathion-dependent mechanism of ROS scavenging is inactivated during PRL1 depletion.

Effects of ROS can lead to cell death, and during the time-course, ACCELERATED CELL DEATH 6 (ACD6), a gene involved in cell death (Dong,2004), is highly up-regulated (2,81 fold in roots growing in the sucrose-limiting situation). Absence of ACD6 provokes an enhanced resistance to Pseudomonas syringae (Rate et al.,1999) whereas its mRNA is over-expressed in uninfected tissues of infected plants by P. syringae (Lu et al.,2003). ACD6 up-regulation during the time-course reinforces the hypothesis that the systemic signal against pathogen is activated during PRL1 depletion. Furthermore, ARABIDOPSIS NAC DOMAIN CONTAINING PROTEIN 6 (ATNAC6), a positive regulator of senescence in leaves (Kim et al.,2009), is up-regulated not only in the sucrose-limiting conditions of the time-course, but also in the 3 % sucrose-containing medium.

Nevertheless, SUGAR TRANSPORT PROTEIN 13 (STP13), whose induction has also been related to programmed cell death (Nørholm et al.,2006), is 6,73-fold down-regulated during the time-course.

STP13could counter-act for ACD6 and ATNAC6 in the inactivation of the cell death programme during the time-course. These observations suggest an altered response in programmed cell death that could be reflected in the slower growth of the prl1 mutant.

3.1.2 RNA-Seq is a powerful tool for revealing alternative splicing

3.1.2.1 Annotation-dependent

The transcript profile analysis of the RNA-Seq data, carried out with Cufflinks is able to discern expression levels of alternative-spliced isoforms from the same locus. For example, the locus AT5G58720 shows a 3-fold over-expression in prl1 of the third isoform exclusively, whereas isoforms one and two do not show any changes in expression. Expression of AT5G58720 was not found to be altered in prl1 with the transcript profiling performed using the tiling arrays.

AT5G58720encodes a protein that has been shown to interact with the N-terminal part of PRL1 (Salchert,1997) and it is predicted to function in DNA binding and mismatch repair. AT5G58720 shares 44 % similarity with SILENCING DEFECTIVE 5 (SDE5), a gene proposed to be involved in the transport of trans-acting short interfering RNAs (tasiRNA,Hernández-Pinzón et al.,2007).

Specifically, the PAM2 motif, a motif for interaction with polyA binding proteins, is highly conserved between the two proteins. Interestingly, only the predicted product of AT5G58720.3 contains the sequence of the last intron, whereas the last exon of the representative isoform (AT5G58720.1) is missing. This difference in sequences can have an impact on the function, localization, stability or interactions of AT5G58720.3. Moreover, the sequence of AT5G58720 found to interact with PRL1 by Y2H (Salchert,1997) corresponds to AT5G58720.1, the only isoform containing the last exon. This suggests that the isoform that interacts with PRL1 is AT5G58720.1, whereas the product of AT5G58720.3, although is up-regulated in prl1, could have different implications in the phenotype.

A correct evaluation of expression levels of different isoforms of the same gene model has been explored only recently. For instance,Bohnert et al.(2009) commented on quantification strategies dealing with de novo transcripts, whereas,Richard et al.(2010) presented a method for prediction and quantification of alternative-spliced isoforms based on exon expression levels. This last quantification method is more efficient when an accurate annotation of the different isoforms

A correct evaluation of expression levels of different isoforms of the same gene model has been explored only recently. For instance,Bohnert et al.(2009) commented on quantification strategies dealing with de novo transcripts, whereas,Richard et al.(2010) presented a method for prediction and quantification of alternative-spliced isoforms based on exon expression levels. This last quantification method is more efficient when an accurate annotation of the different isoforms