Transcription Analysis of Arabidopsis Membrane Transporters and Hormone Pathways during Developmental and Induced Leaf Senescence

TPs

A key role of senescence in plant tissues is the ordered degradation of macromolecules and mobilization of the products, during which supposedly TPs are critically involved. However, until recently only few senescence-associated TPs were reported (Himelblau and Amasino, 2001; Quirino et al., 2001), and, only after the advent of large-scale expression profiling, more (putative) Arabidopsis TPs that are up-regulated during senescence were identified (Buchanan-Wollaston et al., 2003, 2005; Guo et al., 2004; Lin and Wu, 2004).

The Arabidopsis membrane proteins were systematically reclassified (see “Materials and Methods”), resulting in 2,234 predicted Arabidopsis proteins with at least three transmembrane (TM) spans (Supplemental Table I, column F). A categorization by the Transporter Classification system (TC; Busch and Saier, 2004) returned 994 proteins assigned to 84 TP families, including 32 proteins with less than three TM spans. In addition, 69 putative TPs are listed that could not be assigned to a TC family (Supplemental Table IV).

Of the 963 putative TP genes on the array, during NS 153 are up-regulated (173 during NS or DIS or DET) and 60 are down-regulated (72 during NS or DIS or DET; Supplemental Table V). Remarkably, the proportion of up-regulated TPs is about 1.5 times higher than that of all genes in the three senescence experiments, whereas the overall proportion of down-regulated TPs is similar to that of all genes (Fig. 2 ).

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Figure 2.

TP families with biased frequencies of significantly up- or down-regulated genes during senescence. Behind the TP family designation as defined in the TC system, the TC family code (in brackets) and the number of genes on the array assigned to this family (in parentheses) are indicated. Positive and negative values on the x axis indicate the fraction of up- and down-regulated genes, respectively.

In 27 of the 80 TC families, no significant regulation of genes is observed in any of the three senescence experiments, and 26 families are either too small to calculate a relative frequency or exhibit regulation of only a single gene in one experiment. In 21 of the remaining 27 families, a tendency toward up-regulation during senescence is apparent (Fig. 2). The only predominantly down-regulated families are the major intrinsic proteins, divalent anion:Na⁺ symporters, and auxin efflux carriers. The preponderance of up-regulated TPs corresponds well with the substrates known to be transported from senescent leaves to sink organs (amino acids, inorganic phosphorus, sugars, purines, pyrimidines, and metal ions; Himelblau and Amasino, 2001; Soudry et al., 2005). The up-regulation for amino acid and oligopeptide TPs correlates with the high amount of protein degradation taking place during senescence and the subsequent need to export the breakdown products to the sink organs (Hörtensteiner and Feller, 2002). Many TPs are commonly regulated in the three types of senescence experiments, indicating that the nutrient fluxes might be largely similar (Fig. 2; Supplemental Table V). However, during NS a higher fraction of amino acid and oligopeptide TPs are up-regulated. This probably reflects the slower and developmentally controlled progression of the senescence process during aging compared to DET and DIS, presumably enabling a more efficient and thorough nutrient mobilization during NS.

Subcellular localization predictions for the TPs were retrieved from the ARAMEMNON database and are indicated in Supplemental Tables IV and V. The genes encoding TPs with predicted localization in the plasma membrane (secretory pathway) exhibit a preferential up-regulation during NS and DET. Similar to the total set of regulated genes, the genes encoding (putative) plastidic localized TPs display a preferential down-regulation during all three senescence experiments. This might reflect the decline in photoassimilate export from plastids during the progression of senescence. Only 14 putative TPs with predicted plastidic localization are up-regulated during NS (Supplemental Table V). It is conceivable that these proteins are specifically involved in recycling of degradation products and energy allocation in the senescing plastids. The up-regulation of the putative aminophospholipid translocase AtALA3 later in senescence might indicate a role in exporting thylakoid membrane degradation products.

In a recent study, 74 putative TPs up-regulated during developmental senescence were identified (Buchanan-Wollaston et al., 2005). Four of these are not represented on the microarrays used here, and five are not TM proteins according to the ARAMEMNON database. Forty-eight of the remaining genes also show up in the NS experiment (Supplemental Table V, column G). The remaining 17 genes were not rated as up-regulated in our experiment because they did not pass the significance criteria.

Kinases and Receptor-Like Kinases

In Arabidopsis, nearly 1,000 genes are estimated to encode proteins belonging to the eukaryotic protein kinase superfamily. More than 60% of these are receptor-like kinases (RLKs) whose kinase domains belong to the same family as the mammalian interleukin receptor-associated kinases and the Drosophila melanogaster Pelle proteins (Shiu and Bleecker, 2001).

The Agilent chip carries probes for 376 kinase and 610 RLK genes that fall into 62 (sub)families (Supplemental Table VI). More than 470 RLK/Pelle proteins are predicted by the ARAMEMNON database to contain a single α-helical TM domain.

Overall, 55 (9) kinase and 91 (42) RLK genes are up-regulated (down-regulated) during NS or DIS or DET (Supplemental Table VII). Thus, the same proportion (15%) of kinases and RLKs are up-regulated, whereas the proportion of down-regulated kinases is lower. Thirteen of the 61 kinase and RLK families are not regulated during senescence, and 32 families are either too small to calculate a relative frequency or exhibit regulation of only a single gene in one experiment. From 27 kinase and RLK families with more conspicuous patterns, 13 are shown in Figure 3 . In contrast to the TP genes, the differences in expression patterns between NS, DIS, and DET are striking in some subfamilies. Some genes are up-regulated in all three senescence types; however, more frequently up-regulation is observed in NS, but not in DIS and DET. For example, 11 members of the DUF26 subfamily are rapidly up-regulated during the 5-week and 6-week NS stages, but not in DIS and DET. AtCRK10 is even down-regulated during DIS and DET (Supplemental Table VII). AtCRK7 and AtCRK37 are salicylic acid (SA) responsive (Supplemental Fig. 5, G and W; Buchanan-Wollaston et al., 2005), and several of these CRKs (for Ca²⁺-dependent protein kinase-related kinases) were shown to be involved in pathogen defense responses and hypersensitive response-like cell death (Chen et al., 2003, 2004). Leu-rich repeat-Ia is another RLK subfamily with several NS up-regulated genes. Here, four of the five genes are also up-regulated during DET, but not during DIS. This group includes AtSIRK, which also is SA induced (Supplemental Fig. 5L; Buchanan-Wollaston et al., 2005). The senescence-associated expression of AtSIRK and a close relative in bean, SARK, was noted before (Hajouj et al., 2000; Robatzek and Somssich, 2002).

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Figure 3.

RLK families with biased frequencies of significantly up- or down-regulated genes during senescence. Behind the kinase family designations, the number of genes on the array assigned to this family is indicated in parentheses. Positive and negative values on the x axis indicate the fraction of up- and down-regulated genes, respectively.

Plant-Specific Genes and Senescence

Leaf senescence is a unique plant-specific (PS) developmental program. We therefore examined a possible correlation between senescence-associated expression and PS genes. In Arabidopsis, 3,848 genes were identified that encode PS proteins and 2,436 genes with relatives in all other organisms (AO; Gutierrez et al., 2004a, 2004b). Of the 21,500 genes on the array, 2,996 are PS, whereas 2,391 have relatives in AO (Supplemental Table VIII). Relative to all genes on the array, the frequency of PS genes regulated during senescence is only slightly higher than the frequency of regulated AO genes (Table IV ).

Of the 1,931 TM genes on the array, comprising TPs and other proteins with more than three TM spans (Supplemental Table I, column F), 10% are in the PS and 15% in the AO groups. The frequency of senescence-up-regulated TM genes in the AO group is approximately 3 times higher than that of the up- or down-regulated PS TM genes and the down-regulated TM genes common to all organisms, suggesting that the majority of TP functions involved in leaf senescence are common to all taxa and only few senescence-specific activities are required.

Differences become apparent in the two other protein categories. Of the 1,985 putative transcription factors (TFs) on the array, 32% are in the PS and only 1.6% in the AO groups. During developmental senescence, 133 (21%) of all PS TFs are either up- or down-regulated, whereas only four TFs in the AO group (12.5% of all AO TFs) are regulated. Even when taking into account that Arabidopsis has 2 to 3 times more TFs than Caenorhabditis elegans or D. melanogaster and only 8% to 23% of Arabidopsis TFs show similarity to TFs in non-plant eukaryotes (Arabidopsis Genome Initiative, 2000), there remains a disparity that suggests that PS TFs are key players in the regulatory pathways leading to and coordinating leaf senescence. An opposite bias is observed in the kinase and RLK categories. Apparently, there exist almost no strictly PS (receptor-like) kinases in Arabidopsis. All these proteins have relatives in other taxa, 65% of them even in AO (Table IV).

Autophagy Genes and Senescence

During senescence, different pathways contribute to degradation of proteins and other macromolecules, one of them being autophagy (ATG). In Arabidopsis, some ATG genes are encoded by small gene families, e.g. there are nine orthologs of the yeast ATG8 and five orthologs of the ATG18 protein. Knockout or RNAi mutants of AtATG4a/b, AtATG5, AtATG7, AtATG9, and AtATG18a display accelerated senescence and hypersensitivity to nutrient starvation (Doelling et al., 2002; Hanaoka et al., 2002; Yoshimoto et al., 2004; Thompson et al., 2005; Xiong et al., 2005), indicating that autophagy is not essential for the senescence program but plays an important role in nutrient remobilization. Consistently, in Arabidopsis suspension-cultured cells, several ATG genes are up-regulated at the onset of carbon starvation (Contento et al., 2004; Rose et al., 2006).

Remarkably, nineteen of the 21 Arabidopsis ATG genes (Supplemental Table IX) on the microarray are transcriptionally activated (Supplemental Fig. 2). It appears most ATG genes are coordinately up-regulated at a stage in developmental senescence when chlorophyll degradation starts to get visible, i.e. between the 6-week and 25%-bleaching stages. Figure 4 shows the expression profiles of the most dramatically up-regulated ATG genes, 7, 8a, 8e, and 9. The prolonged observation time of induced and developmental senescence progression enabled the recognition of senescence-associated expression patterns of several ATG genes that had not been recognized in other studies (Buchanan-Wollaston et al., 2005; Xiong et al., 2005). It has been proposed that the Arabidopsis ATG system is ubiquitously present and not restricted to a certain developmental phase or nutritional state (for review, see Thompson et al., 2005). Consistent with this assumption is the significant background expression level of many ATG genes in 4-week-old plants (e.g. AtATG4b, 8c–f, 8i, and 18g; Supplemental Table IX). On the other hand, the massive up-regulation of many ATG genes during all three senescence conditions suggests that autophagy is important for the efficient remobilization and recycling of nutrients.

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Figure 4.

Autophagy. The expression profiles of four senescence-associated ATG genes are shown. Additional ATG gene expression profiles are shown in Supplemental Figure 2. The averages of the normalized signal values (see “Materials and Methods”) were plotted against the sampling time points. The time scale of the x axis is not linear and different for the NS, DIS, and DET senescence experiments. For the developmental senescence (NS) curve, the signal ratio between the last sampling time point (75y for 75% yellow surface; see Table I) and the 4-week-old reference sample (4w/0 d) is indicated.

Cytokinin

Cytokinins (CKs) regulate cell division and various metabolic and developmental processes, including senescence. CK has a regulatory function early in senescence as CK levels are reduced in senescing leaves, whereas the exogenous application or endogenous overexpression of CKs delays senescence (Smart et al., 1991; Gan and Amasino, 1995).

For this study, 137 genes associated with various aspects of CK homeostasis were compiled (Supplemental Table X). The eight (putative) Arabidopsis adenylate isopentenyltransferase enzymes and two CK trans-hydroxylases that are possibly involved in CK biosynthesis are very weakly expressed in the three senescence experiments (“not well above background”), and none is up-regulated.

For the CK-degrading CK oxidase/dehydrogenases (CKX; Schmülling et al., 2003) and the CK-inactivating N- and O-glucosylases, the situation is different. Of the six CKX genes on the array, only AtCKX5 is expressed at a high confidence level and increases weakly (2.1- to 2.9-fold) but continuously during NS, DIS, and DET. For AtCKX2 and AtCKX4, similar patterns at lower levels are apparent. AtCKX6 appears to be down-regulated. The role of CKX proteins in senescence is as yet unclear, which is illustrated by the observation that, unexpectedly, overexpression of CKX genes may result in retardation of leaf senescence (Werner et al., 2003). Four of the five active glycosyltransferases from Arabidopsis (Hou et al., 2004) are weakly expressed and barely regulated. However, the O-glycosyltransferase AtUGT85A1 shows a remarkable activation in all three senescence conditions (Fig. 5A ). It is thus conceivable that this protein is involved in senescence-specific inactivation of CKs by conjugation.

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Figure 5.

Cytokinin. The expression profiles of six senescence-associated CK genes are shown. The data were processed as described for Figure 4.

CK levels could also be reduced during senescence by translocation in the vascular system. Two members of the purine TP family, AtPUP1 and AtPUP2, can mediate CK uptake in yeast and thus are likely candidates for CK TPs (Bürkle et al., 2003). AtPUP1 and three other family members are up-regulated (Fig. 5, B and C; Supplemental Table X) and none of the remaining AtPUPs is down-regulated (AtPUP2 is not represented on the array), consistent with a role of purine permeases in exporting CKs during senescence.

The Arabidopsis CK-signaling pathway consists of CK perception by His protein kinases (AHKs) and activation of phosphorelay carriers, of B-type and eventually of A-type nuclear response regulators (ARRs), which control the primary CK-responsive genes by a negative feedback mechanism (for review, see Hwang et al., 2002). AtAHK3 is the only CK receptor that is detectably expressed, but it is not (or only very weakly: 2.0- to 2.6-fold) up-regulated. Of two phosphorelay mediators and 11 B-type ARRs on the array, only AtARR1 seems to be weakly (2.6- to 3.4-fold) up-regulated. Several A-type ARRs are rapidly induced by CK (Rashotte et al., 2003; Brenner et al., 2005; Kiba et al., 2005). Consistent with a depletion of CK during senescence progression, four of those are down-regulated in NS, DIS, and DET (Fig. 5, D–G). Down-regulation of these genes also was observed by Buchanan-Wollaston et al. (2005). A remarkable exception is AtARR16, which is strongly up-regulated upon CK treatment (D'Agostino et al., 2000; Rashotte et al., 2003; Kiba et al., 2005). This gene is also up-regulated during NS and DIS (but not DET; Fig. 5H), suggesting a special function in CK signaling that deviates from the other A-type ARRs.

Recently 71 up-regulated and 11 down-regulated immediate CK-response genes of Arabidopsis were identified (Brenner et al., 2005). From a simplistic viewpoint, one would expect that these genes are not regulated or down-regulated during senescence, if the CK level decreases. Indeed, 80% of the 69 CK-response genes on the array are not regulated (43 genes) or down-regulated (12 genes) during senescence, whereas the remaining 14 genes are up-regulated (Supplemental Table X). This suggests that one-fifth of the immediate CK-response genes are under positive control of other regulators during senescence.

Jasmonic Acid

Jasmonic acid (JA) is critically involved in senescence (for review, see He and Gan, 2003). JA application induces premature senescence and its level increases in senescing leaves (He et al., 2002). Eleven of 19 JA-biosynthesis and six of 11 JA-signaling or -response genes are up- or down-regulated, and some exhibit strikingly different regulation in NS, DIS, and DET (Supplemental Table XI; Supplemental Fig. 3, A–K). Eight JA-biosynthesis genes are specifically and transiently up-regulated in NS. These include the highly expressed lipoxygenase AtLOX2 (Supplemental Fig. 3C), allene oxide synthase AtAOS1 (Supplemental Fig. 3F), and 12-oxophytodienoate reductase AtOPR3 (Fig. 6A ; Supplemental Fig. 3B) that catalyze sequential reactions in the JA-biosynthesis pathway. Their coordinate expression during NS, but not DIS or DET, suggests that this pathway is in leaves specifically activated by developmental stimuli. AtOPR3 is the isoenzyme also predominantly responsible for JA biosynthesis during pollen development, and, like AtAOS1, it is essential for male gametophyte development and anther elongation. AtOPR1 and AtOPR2 are also up-regulated during senescence (Supplemental Fig. 3, A and H), but the roles of these less efficient isoenzymes are not clear. Only one gene, the uncharacterized putative allene oxide cyclase family protein AtAOC4 (Supplemental Fig. 3I), is weakly down-regulated. In striking contrast, two other genes, AtαDOX1 encoding a fatty acid α-dioxygenase (Supplemental Fig. 3J) and AtLOX1 (Fig. 6B; Supplemental Fig. 3K), are up-regulated to very high levels in detached leaves but not in NS and DIS, indicating that these genes are predominantly responding to wounding. Taken together, the expression profiles indicate that, during developmental senescence and upon wounding, different isozymes are active in the JA synthesis pathway and that in dark-induced senescence JA possibly does not play major role.

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Figure 6.

JA. The expression profiles of four senescence-associated JA genes are shown. Additional JA gene expression profiles are shown in Supplemental Figure 3. The data were processed as described for Figure 4.

Several genes that are supposedly involved in JA signaling or response are up- or down-regulated during senescence (Supplemental Fig. 3, L–Q). The ribonuclease 1 gene AtRNS1 is strongly up-regulated in later stages during NS, but not in DIS and DET (Fig. 6C; Supplemental Fig. 3L). AtRNS1 also is very rapidly and transiently induced upon wounding but declines to almost noninduced level within 48 h (LeBrasseur et al., 2002), explaining that it is not detected in the DET experiment. The late expression peak of RNS1 would be consistent with a participation in nucleic acid degradation late in senescence. The only JA-signaling gene that is strictly down-regulated in all senescence conditions is AtCOS1 (Fig. 6D; Supplemental Fig. 3Q). It encodes lumazine synthase, which catalyzes the penultimate step in riboflavin synthesis. It is suggested that AtCOS1 acts in the riboflavin pathway as a suppressor of certain JA responses, including senescence (attenuated by the SCF/COI1 complex; Xiao et al., 2004). The strong down-regulation of AtCOS1 during senescence corroborates this model.

Salicylic Acid

Salicylic acid (SA) plays a key role as mediator of plant stress responses, including disease and systemic acquired resistance. Recently, it was reported that the SA-signaling pathway is also active in the control of gene expression during developmental senescence (Morris et al., 2000). A transcriptome analysis in senescing Arabidopsis leaves from wild-type plants and SA-deficient NahG mutants revealed that many SAGs are dependent on the SA-signaling pathway (Buchanan-Wollaston et al., 2005).

Four of six genes that are (possibly) involved in SA biosynthesis are regulated during NS (Supplemental Fig. 4, A–D; Supplemental Table XII). Among them is the gene encoding isochorismate synthase (AtICS1), a central enzyme in the pathway. It is conspicuous that this gene is only induced during NS, but not after shading or detachment. In contrast, the ICS1 homolog At1g18870 is also induced by shading, but like ICS1 not after detachment of leaves. The two Phe ammonia-lyase genes are only weakly regulated. Eight of 12 SA-signaling genes are regulated during NS, and also in this group of genes no or less regulation during DIS and DET is observed (Supplemental Fig. 4, E–L). Differential up-regulation is most prominent for AtEDS4, which encodes an orphan MATE-related efflux carrier. AtEDS4 is induced by SA, UV light, and after pathogen infection, depending on the two other SA-signaling proteins (AtEDS1 and AtPAD4; Nawrath et al., 2002). These also are up-regulated in NS, but not in DIS and DET (Supplemental Fig. 4, H and I). AtEDS1 and AtPAD4 interact with each other, and both genes are transducers of redox signals and important activators of SA signaling (for review, see Wiermer et al., 2005). Only one SA-signaling gene, AtAGD2, is down-regulated during senescence.

The expression profiles of 44 SA-responsive genes (Supplemental Table XII), including 30 of the 32 senescence-enhanced and SA-dependent genes identified by Buchanan-Wollaston et al. (2005), reveal an interesting trend. Twenty-six genes are up-regulated (Supplemental Figs. 5 and 10A), none is down-regulated, and all except one are genes identified in the NahG mutant. It is somewhat surprising that of the other 14 SA-responsive genes reported in the literature, only one (AtAF2; Supplemental Fig. 5P) is up-regulated during senescence. Remarkably, the majority of the senescence-associated SA-responsive genes are only up-regulated during NS, but not (or only weakly) during DIS or DET, resembling the expression profiles of the SA-biosynthesis and -signaling genes mentioned above. In summary, these data provide additional evidence for SA as a modulator of gene expression in naturally senescing leaves.

Ethylene

The plant hormone ethylene (ET) is well known as an accelerator of senescence. The importance of ET signaling during senescence is illuminated by a delayed senescence phenotype of ET-insensitive mutants like ein2 in Arabidopsis (Oh et al., 1997). However, ET is not essential for the onset and progression of senescence (Grbic and Bleecker, 1995; Jing et al., 2002).

Of 69 genes (proposed to be) involved in ET biosynthesis or signaling (Supplemental Table XIII), 18 are up- or down-regulated (Supplemental Fig. 6). The 1-aminocyclopropane-1-carboxylic acid (ACC) synthases (AtACS) catalyze the rate-limiting step in the ET-biosynthesis pathway. Arabidopsis encodes nine ACS proteins, of which eight form functional homodimers, that underly a complex spatial and temporal regulation (Tsuchisaka and Theologis, 2004). Only three of the functional ACC synthases (AtACS2, AtACS5, and AtACS6) are significantly expressed and up-regulated during NS (Supplemental Fig. 6, A–C). In the subsequent step, ACC is converted to ET by the ACC oxidase (AtEAT1), which is also up-regulated (Supplemental Fig. 6D). In addition, a nitrilase (AtNIT4) that is thought to play a role in cyanide detoxification during ET biosynthesis (Piotrowski et al., 2001) is up-regulated (Supplemental Fig. 6E). Possibly, this set of proteins is responsible for an increase in ET production in senescing leaves.

ET signal transduction supposedly follows a “linear” pathway, with membrane-bound receptors at the beginning, TFs at the end of the chain, and multiple positive and negative regulators in between. The five ET receptors or the next downstream protein, AtCTR1, are not regulated in NS, DIS, or DET, but several other components of ET signal transduction are. The TP-like AtRAN1 protein, which might be required to form functional ET receptors (Woeste and Kieber, 2000), and the downstream component AtEIN3 are significantly up-regulated in all three senescence experiments, most rapidly in DIS and DET (Supplemental Fig. 6, H and I). AtEIN2, located between AtCTR1 and AtEIN3, is not represented on the array. The AtEIN3 and AtEIL TFs regulate gene expression by binding directly to primary ET-response elements in the promoters of various genes. One immediate target of AtEIN3 is AtERF1a (Solano et al., 1998) that appears to be transiently up-regulated (Supplemental Fig. 6). AtERF1a is a TF whose transcriptional activation requires simultaneously the JA- and ET-signaling pathways (Lorenzo et al., 2003). Five other ERF proteins are also up-regulated but with different profiles (Supplemental Fig. 6, K–O). AtERF1b, AtERF2, and AtEBP1 are ET-responsive activators of GCC-box genes (Büttner and Singh, 1997; Fujimoto et al., 2000).The weakly up-regulated AtRCD1 modulates ET, abscisic acid (ABA), and methyl-JA responses (Ahlfors et al., 2004). Two uncharacterized putative TFs, At5g07580 and At5g25190, annotated as possibly involved in ET signaling, are down-regulated (Supplemental Fig. 6, Q and R).

Buchanan-Wollaston et al. (2005) recently identified 21 SAGs that show reduced expression in ET- and JA-deficient plants. Of the 19 genes represented on the Agilent microarray (Supplemental Table XIII), one yields no significant signal. All others are strongly induced during NS (Supplemental Fig. 7) and four of them are known to be involved in JA signaling (AtRNS1; Supplemental Fig. 3L; AtNAC3; Supplemental Fig. 3O), JA biosynthesis (AtαDOX1; Supplemental Fig. 3J), and to be SA responsive (At1g19250; Supplemental Fig. 5M). However, as their expression patterns during DIS and DET differ, at least some of these genes are additionally regulated by other signaling pathways. A subgroup of six genes appears to be specific for developmental senescence as they do not respond at all to shading or leaf detachment, including the S-like RNAse AtRNS1, the exopolygalacturonases and endopolygalacturonases AtPGA3 and AtADPG1, and the ABC-TP AtPDR12. Another subgroup of genes is most intensely and apparently transiently up-regulated in detached leaves and thus possibly responding to wounding (Supplemental Fig. 7, J–N).

In summary, these data indicate a coordinated up-regulation of ET-biosynthesis genes during leaf senescence in Arabidopsis that is accompanied by changes in expression of several ET-signaling components. It appears that some downstream response factors are NS-specifically and -transiently transcribed at later stages in senescence development.

Auxin

Although auxin effects on abscission and senescence were first reported more than 50 years ago (for review, see Noodén, 1988), the involvement of this hormone in the senescence development is much less understood than that of ET, JA, or CK. In Arabidopsis senescing leaves, the indole-3-acetic acid (IAA) concentration is 2-fold higher than in nonsenescing leaves (Quirino et al., 1999). However, auxin treatment leads to a transient decrease in SAG12 expression (Noh and Amasino, 1999). Recently, it was described that the AUXIN RESPONSE FACTORs 1 and 2 (AtARF1 and AtARF2) are involved in regulation of abscission and senescence (Ellis et al., 2005; Okushima et al., 2005). More than 110 proteins implicated in auxin biosynthesis, metabolism, transport, and signaling were compiled from Woodward and Bartel (2005), Remington et al. (2004), The Arabidopsis Information Resource Gene Ontologies, and gene descriptions (Supplemental Table XIV). The expression profiles of the regulated genes are shown in Supplemental Figure 8.

The observation that eight of the 15 (putative) Arabidopsis IAA-biosynthesis genes are significantly regulated suggests that leaf senescence is associated with changes in IAA synthesis. A moderate transcriptional increase of Trp synthase a (AtTSA1; Supplemental Fig. 8A) is observed in NS and DET, but not DIS. Downstream of Trp, the Arabidopsis amidohydrolase AtAMI1 converts in vitro indole-3-acetamide to IAA (Pollmann et al., 2003). As AtAMI1 transcription decreases during NS, DIS, and DET (Supplemental Fig. 8B), the indole-3-acetamide pathway is presumably not active in senescence. However, six genes encoding proteins that are supposedly acting in the IPA and/or IAOx pathway are heavily up-regulated (Supplemental Fig. 8, C–H). Most intriguingly, the IAA1d oxidase (AtAO1) and nitrilases AtNIT1 to 3 that directly convert indole-3-acetaldehyde and indole-3-acetonitrile, respectively, into IAA are transcriptionally up-regulated. Quirino et al. (1999) have also reported the senescence-associated induction of AtNIT1 to 3 and found that it is accompanied by an increase in IAA and decrease in indole-3-acetonitrile. Suspiciously, transcription of AtAO1, AtTSA1, AtSUR2, and AtCYP92B2 remains almost constant in dark-induced senescence.

IAA homeostasis also is affected by formation and degradation of IAA conjugates and indole-3-butyric acid. About one-third of the genes implicated in these processes are transcriptionally induced during leaf senescence. Three IAA-amino acid synthetases are massively up-regulated in NS, DIS, and DET (Supplemental Fig. 8, I–K). These enzymes presumably help to maintain auxin homeostasis by inactivating excess IAA (Staswick et al., 2005). On the other hand, the two IAA amidohydrolases AtILR1 and AtIAR3 also are induced (Supplemental Fig. 8, L and M) that release free IAA from IAA-Leu, -Phe, and -Ala and thus counteract the IAA-amino acid synthetases. Moreover, the three genes AtACX1, AtACX4, and AtPED1 encoding acyl-CoA oxidases involved in conversion of indole-3-butyric acid into bioactive IAA (Zolman et al., 2000; Adham et al., 2005) are also up-regulated (Supplemental Fig. 8, N, P, and R).

Experiments with bean leaves suggested that changes in auxin gradients rather than the auxin concentration itself may modulate abscission and senescence development (Addicott et al., 1955). Of 13 TPs (suspected to be) involved in auxin transport, six are massively down-regulated in NS, DIS, and DET (Supplemental Fig. 8, S–X). The putative IAA permease AtAUX1 is localized asymmetrically in the plasma membrane of certain cells and facilitates IAA loading into the leaf vascular transport system and IAA unloading in the primary root apex. AtPIN3 is one of three down-regulated proteins from the PIN family involved in auxin efflux and expressed in gravity-sensing tissues like the columella, where it quickly relocates in the cell plasma membrane to the side oriented toward gravity when the gravity vector changes. ABC TPs of the PGP subfamily were recently identified as auxin TPs (for review, see Geisler and Murphy, 2006). AtPGP1 and AtPGP19 function in conjunction with TWISTED DWARF 1, supposedly as auxin exporters, and are probably involved in the proper polar distribution of auxin during plant development. AtPGP19 is strongly down-regulated during senescence (Supplemental Fig. 8W), and AtPGP1 also appears to be weakly down-regulated, but its expression level remains subsignificant (Supplemental Table XIV). Interestingly, AtPGP4, which presumably functions as auxin importer in root caps (Terasaka et al., 2005), is up-regulated in senescing leaves (Supplemental Fig. 8Y). These changes in expression of auxin TPs may contribute to the increased auxin concentration in senescing leaves that was observed by Quirino et al. (1999).

Auxin causes changes in the expression of many genes, and crucial factors in the response pathway are the ARF and Aux/IAA proteins. The Arabidopsis AtARF and AtIAA gene families consist of 23 and 29 members, respectively (Remington et al., 2004). Expression of most AtARF genes is below the confidence level in our experiments and does not change in NS, DIS, or DET. The only exception is MONOPTEROS (AtARF5/AtMP), which is induced late in natural senescence (Supplemental Fig. 9A). AtARF5 has not been assigned a role in senescence yet. Recently, regulation of leaf senescence by AtARF1 and 2 and a weak transcriptional repression/induction of AtARF1/AtARF2 during senescence have been observed (Lin and Wu, 2004; Ellis et al., 2005). In our experiments also, a 2- to 3-fold decrease/increase in AtARF1/AtARF2 expression can be seen, albeit below the significance threshold. In contrast to the ARFs, almost one-half of the 28 Aux/IAA genes are down-regulated during senescence (Supplemental Fig. 9, B–N). A function of Aux/IAA proteins in senescence has not yet been described, but the overall decline in AtIAA gene transcription suggests that the quality of auxin responses in leaves alters as a result of changes in signaling proteins.

A Role of GA and Brassinosteroids in Senescence?

Reports that demonstrate a link between GA activity and senescence are rare. In pea (Pisum sativum), exogenous GA3 can delay apical senescence, and endogenous GA concentration is lower in senescing than in flowering shoots (Zhu and Davies, 1997). In detached Arabidopsis leaves, CK and to a lower extent GA delay chlorophyll degradation. However, upon combining the two hormones, GA partially inhibits the CK effect, and it is suggested that the SPINDLY protein regulates this cross talk between the two signaling pathways (Greenboim-Wainberg et al., 2005).

Fifteen of the 18 genes on the array that are (putatively) involved in GA biosynthesis are expressed at subsignificant levels, and none is up-regulated (Supplemental Table XV). On the other hand, the functional and GA-inducible GA 2-oxidase 2 (AtGA2OX2) that deactivates GA (Thomas et al., 1999) is 18-fold up-regulated (Fig. 7A ). These results suggest that during senescence at least some gibberellins are deactivated.

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Figure 7.

GA and BRs. The expression profiles of four senescence-associated GA (A–D) and two BR genes (E and F) are shown. The data were processed as described for Figure 4.

None of the 16 genes that encode (putative) GA-signaling proteins are up-regulated, and AtGASA4 and AtGASA5, two members of the GASA family of short proteins of unknown function, are down-regulated (Fig. 7, B and C). Some GA-responsive genes display senescence-associated regulation. A well-known example is the GA-induced aquaporine AtTIP1.1 (Phillips and Huttly, 1994) that is down-regulated, as many other intrinsic proteins too (Fig. 2). Intriguingly, the GA-responsive xyloglucan endotransglycosylase AtMERI5 is massively up-regulated during senescence (Fig. 7D). AtMERI5 and other xyloglucan endotransglycosylases are suggested to be involved in cell wall weakening that is necessary for cell expansion (Xu et al., 1996). Studies on AtTIP1.1 and AtMERI5 revealed that there is cross talk between the GA and brassinosteroid (BR) pathways. AtTIP1.1 is antagonistically regulated by GA and BR, whereas transcription of AtMERI5 was shown to be positively and additively regulated by either GA or BR treatment (Kauschmann et al., 1996). As during leaf senescence GA activity appears to be reduced, the senescence-associated induction of AtMERI5 must be effected by a different regulator, possibly BR.

BRs appear to promote developmental senescence, as mutants deficient in BR biosynthesis (e.g. det2) or the BR receptor BRI1 have a retarded senescence progression (for review, see Clouse and Sasse, 1998). However, from a set of 99 BR-related genes on the array (14 biosynthesis and inactivation genes, 11 receptor and signaling genes, 52 BR-induced genes, and 29 BR-repressed genes; compiled from Müssig and Altmann [2003] and Lisso et al. [2005]), no clear picture emerges (Supplemental Table XVI). Of the BR-induced genes, 15 and 14 were significantly expressed and up- or down-regulated during NS, respectively. Of the BR-repressed genes, 13 were up- and 5 were down-regulated. Two of the 14 (putative) biosynthesis genes are down-regulated (AtCPD/At5g05690 and At3g55360; data not shown) and none is up-regulated, suggesting that BR biosynthesis does not significantly increase during senescence. Central transmitters of the BR signal from the receptor(s) to the target genes are the AtBIN2 kinase and the two nearly identical transcriptional modulators and regulators of BR signaling, AtBES1 and AtBZR1. AtBIN2 and AtBZR1 show no significant change in expression, whereas AtBES1 is down-regulated (Fig. 7E).

The BR receptors AtBRL1 and AtBRL3 supposedly function specifically in provascular differentiation (Cano-Delgado et al., 2004). As AtBRL3 is strongly up-regulated in later stages of NS, DIS, and DET (Fig. 7F), one could speculate that BR modulates the properties of the vascular tissue in senescing leaves toward efficient export of metabolites.

ABA

As treatment with ABA can induce leaf senescence, ABA is considered a promoter of senescence (Noodén, 1988). In Arabidopsis, ABA induces expression of several SAGs. Vice versa, during senescence several genes are up-regulated that are involved in ABA synthesis, metabolism, or signaling (Oh et al., 1996; Weaver et al., 1998; He et al., 2001; Buchanan-Wollaston et al., 2005). We investigated the expression profiles of 58 ABA-biosynthesis, -metabolism, and -signaling genes (Supplemental Table XVII). Four of 11 ABA-biosynthesis genes are up-regulated, including the 9-cis-epoxycarotenoid dioxygenases AtNCED2 and 3 that catalyze the cleavage of neoxanthin and the abscisic aldehyde oxidases AtAAO3 and 4 (Supplemental Fig. 10, A–D). On the other hand, the ABA-inactivating glucosylase UGT71B6 (Priest et al., 2005) is also up-regulated during NS (Supplemental Fig. 10E).

Of 30 ABA-signaling genes, eight are up-regulated and only two are down-regulated (Supplemental Fig. 10, F–O). Of 12 negative regulators of ABA responses, five protein phosphatases 2C are up-regulated (Supplemental Fig. 10, P–T). The ABA-responsive AtSEN1 gene is up-regulated during senescence (Supplemental Fig. 10U) as described by Oh et al. (1996). The ABA-repressible AtPIP1.5 and AtPIP2.6 genes (Jang et al., 2004), but also the ABA-inducible AtPIP2.2, are down-regulated during senescence (Supplemental Fig. 10, V–X). These complex and, for several ABA-related genes, opposite regulation patterns during natural and induced senescence support the notion that ABA is a modulator of senescence-associated physiological changes.