Multi-level analysis of the interactions between REVOLUTA and MORE AXILLARY BRANCHES 2 in controlling plant development reveals parallel, independent and antagonistic functions.

Class III homeodomain leucine zipper (HD-ZIPIII) transcription factors play fundamental roles in controlling plant development. The known HD-ZIPIII target genes encode proteins involved in the production and dissipation of the auxin signal, HD-ZIPII transcription factors and components that feedback to regulate HD-ZIPIII expression or protein activity. Here, we have investigated the regulatory hierarchies of the control of MORE AXILLARY BRANCHES2 (MAX2) by the HD-ZIPIII protein REVOLUTA (REV). We found that REV can interact with the promoter of MAX2 In agreement, rev10D gain-of-function mutants had increased levels of MAX2 expression, while rev loss-of-function mutants showed lower levels of MAX2 in some tissues. Like REV, MAX2 plays known roles in the control of plant architecture, photobiology and senescence, which prompted us to initiate a multi-level analysis of growth phenotypes of hd-zipIII, max2 and respective higher order mutants thereof. Our data suggest a complex relationship of synergistic and antagonistic activities between REV and MAX2; these interactions appear to depend on the developmental context and do not all involve the direct regulation of MAX2 by REV.

The Non-JAZ TIFY Protein TIFY8 of Arabidopsis thaliana Interacts with the HD-ZIP III Transcription Factor REVOLUTA and Regulates Leaf Senescence.

The HD-ZIP III transcription factor REVOLUTA (REV) is involved in early leaf development, as well as in leaf senescence. REV directly binds to the promoters of senescence-associated genes, including the central regulator WRKY53. As this direct regulation appears to be restricted to senescence, we aimed to characterize protein-interaction partners of REV which could mediate this senescence-specificity. The interaction between REV and the TIFY family member TIFY8 was confirmed by yeast two-hybrid assays, as well as by bimolecular fluorescence complementation in planta. This interaction inhibited REV's function as an activator of WRKY53 expression. Mutation or overexpression of TIFY8 accelerated or delayed senescence, respectively, but did not significantly alter early leaf development. Jasmonic acid (JA) had only a limited effect on TIFY8 expression or function; however, REV appears to be under the control of JA signaling. Accordingly, REV also interacted with many other members of the TIFY family, namely the PEAPODs and several JAZ proteins in the yeast system, which could potentially mediate the JA-response. Therefore, REV appears to be under the control of the TIFY family in two different ways: a JA-independent way through TIFY8, which controls REV function in senescence, and a JA-dependent way through PEAPODs and JAZ proteins.

Moderate DNA methylation changes associated with nitrogen remobilization and leaf senescence in Arabidopsis.

The lifespan of plants is restricted by environmental and genetic components. Following the transition to reproductive growth, leaf senescence ends cellular life in monocarpic plants to remobilize nutrients to storage organs. In Arabidopsis, we initially observed altered leaf to seed ratios, faster senescence progression, altered leaf nitrogen recovery after transient nitrogen removal, and ultimately enhanced nitrogen remobilization from the leaves in two methylation mutants (ros1 and the triple dmr1/2 cmt3 knockout). Analysis of the DNA methylome in wild type Col-0 leaves identified an initial moderate decline of cytosine methylation with progressing leaf senescence, predominantly in the CG context. Late senescence was associated with moderate de novo methylation of cytosines, primarily in the CHH context. Relatively few differentially methylated regions, including one in the ROS1 promoter linked to down-regulation of ROS1, were present, but these were unrelated to known senescence-associated genes. Differential methylation patterns were identified in transcription factor binding sites, such as the W-boxes that are targeted by WRKYs. Methylation in artificial binding sites impaired transcription factor binding in vitro. However, it remains unclear how moderate methylome changes during leaf senescence are linked with up-regulated genes during senescence.

Specificity of H2O2 signaling in leaf senescence: is the ratio of H2O2 contents in different cellular compartments sensed in Arabidopsis plants?

Leaf senescence is an integral part of plant development and is driven by endogenous cues such as leaf or plant age. Developmental senescence aims to maximize the usage of carbon, nitrogen and mineral resources for growth and/or for the sake of the next generation. This requires efficient reallocation of the resources out of the senescing tissue into developing parts of the plant such as new leaves, fruits and seeds. However, premature senescence can be induced by severe and long-lasting biotic or abiotic stress conditions. It serves as an exit strategy to guarantee offspring in an unfavorable environment but is often combined with a trade-off in seed number and quality. In order to coordinate the very complex process of developmental senescence with environmental signals, highly organized networks and regulatory cues have to be in place. Reactive oxygen species, especially hydrogen peroxide (H2O2), are involved in senescence as well as in stress signaling. Here, we want to summarize the role of H2O2 as a signaling molecule in leaf senescence and shed more light on how specificity in signaling might be achieved. Altered hydrogen peroxide contents in specific compartments revealed a differential impact of H2O2 produced in different compartments. Arabidopsis lines with lower H2O2 levels in chloroplasts and cytoplasm point to the possibility that not the actual contents but the ratio between the two different compartments is sensed by the plant cells.

A guideline for leaf senescence analyses: from quantification to physiological and molecular investigations.

Leaf senescence is not a chaotic breakdown but a dynamic process following a precise timetable. It enables plants to economize with their resources and control their own viability and integrity. The onset as well as the progression of leaf senescence are co-ordinated by a complex genetic network that continuously integrates developmental and environmental signals such as biotic and abiotic stresses. Therefore, studying senescence requires an integrative and multi-scale analysis of the dynamic changes occurring in plant physiology and metabolism. In addition to providing an automated and standardized method to quantify leaf senescence at the macroscopic scale, we also propose an analytic framework to investigate senescence at physiological, biochemical, and molecular levels throughout the plant life cycle. We have developed protocols and suggested methods for studying different key processes involved in senescence, including photosynthetic capacities, membrane degradation, redox status, and genetic regulation. All methods presented in this review were conducted on Arabidopsis thaliana Columbia-0 and results are compared with senescence-related mutants. This guideline includes experimental design, protocols, recommendations, and the automated tools for leaf senescence analyses that could also be applied to other species.

REVOLUTA and WRKY53 connect early and late leaf development in Arabidopsis.

As sessile organisms, plants have to continuously adjust growth and development to ever-changing environmental conditions. At the end of the growing season, annual plants induce leaf senescence to reallocate nutrients and energy-rich substances from the leaves to the maturing seeds. Thus, leaf senescence is a means with which to increase reproductive success and is therefore tightly coupled to the developmental age of the plant. However, senescence can also be induced in response to sub-optimal growth conditions as an exit strategy, which is accompanied by severely reduced yield. Here, we show that class III homeodomain leucine zipper (HD-ZIPIII) transcription factors, which are known to be involved in basic pattern formation, have an additional role in controlling the onset of leaf senescence in Arabidopsis. Several potential direct downstream genes of the HD-ZIPIII protein REVOLUTA (REV) have known roles in environment-controlled physiological processes. We report that REV acts as a redox-sensitive transcription factor, and directly and positively regulates the expression of WRKY53, a master regulator of age-induced leaf senescence. HD-ZIPIII proteins are required for the full induction of WRKY53 in response to oxidative stress, and mutations in HD-ZIPIII genes strongly delay the onset of senescence. Thus, a crosstalk between early and late stages of leaf development appears to contribute to reproductive success.

Complex Formation between the Transcription Factor WRKY53 and Antioxidative Enzymes Leads to Reciprocal Inhibition.

The transcription factor WRKY53 of the model plant Arabidopsis thaliana is an important regulator of leaf senescence. Its expression, activity and degradation are tightly controlled by various mechanisms and feedback loops. Hydrogen peroxide is one of the inducing agents for WRKY53 expression, and a long-lasting intracellular increase in H2O2 content accompanies the upregulation of WRKY53 at the onset of leaf senescence. We have identified different antioxidative enzymes, including catalases (CATs), superoxide dismutases (SODs) and ascorbate peroxidases (APXs), as protein interaction partners of WRKY53 in a WRKY53-pulldown experiment at different developmental stages. The interaction of WRKY53 with these enzymes was confirmed in vivo by bimolecular fluorescence complementation assays (BiFC) in Arabidopsis protoplasts and transiently transformed tobacco leaves. The interaction with WRKY53 inhibited the activity of the enzyme isoforms CAT2, CAT3, APX1, Cu/ZuSOD1 and FeSOD1 (and vice versa), while the function of WRKY53 as a transcription factor was also inhibited by these complex formations. Other WRKY factors like WRKY18 or WRKY25 had no or only mild inhibitory effects on the enzyme activities, indicating that WRKY53 has a central position in this crosstalk. Taken together, we identified a new additional and unexpected feedback regulation between H2O2, the antioxidative enzymes and the transcription factor WRKY53.

Determination of the in vivo redox potential by one-wavelength spectro-microscopy of roGFP.

For the quantitative analysis of molecular processes in living (plant) cells, such as the perception and processing of environmental and endogenous signals, new combinatorial approaches in optical and spectroscopic technologies are required and partly already became established in many fields of the life sciences. One hallmark of the in vivo analysis of cell biological processes is the use of visible fluorescent proteins to create fluorescent fusion proteins. Recent progress has been made in generating a redox-sensitive mutant of green fluorescent proteins (roGFP), which exhibits alterations in its spectral properties in response to changes in the redox state of the surrounding medium. An established method to probe the local redox potential using roGFP is based on a ratiometric protocol. This readout modality requires two excitation wavelengths, which makes the technique less suited for in vivo studies of e.g. dynamic samples. We clarify the origin of the redox sensitivity of roGFP by ab initio calculations, which reveal a changed protonation equilibrium of the chromophore in dependence on the redox potential. Based on this finding, we test and compare different spectroscopic readout modalities with single wavelength excitation to determine the local redox potential and apply these techniques to live cell analytics.

Senescence-specific alteration of hydrogen peroxide levels in Arabidopsis thaliana and oilseed rape spring variety Brassica napus L. cv. Mozart.

In order to analyze the signaling function of hydrogen peroxide (H(2)O(2)) production in senescence in more detail, we manipulated intracellular H(2)O(2) levels in Arabidopsis thaliala (L.) Heynh by using the hydrogen-peroxide-sensitive part of the Escherichia coli transcription regulator OxyR, which was directed to the cytoplasm as well as into the peroxisomes. H(2)O(2) levels were lowered and senescence was delayed in both transgenic lines, but OxyR was found to be more effective in the cytoplasm. To transfer this knowledge to crop plants, we analyzed oilseed rape plants Brassica napus L. cv. Mozart for H(2)O(2) and its scavenging enzymes catalase (CAT) and ascorbate peroxidase (APX) during leaf and plant development. H(2)O(2) levels were found to increase during bolting and flowering time, but no increase could be observed in the very late stages of senescence. With increasing H(2)O(2) levels, CAT and APX activities declined, so it is likely that similar mechanisms are used in oilseed rape and Arabidopsis to control H(2)O(2) levels. Under elevated CO(2) conditions, oilseed rape senescence was accelerated and coincided with an earlier increase in H(2)O(2) levels, indicating that H(2)O(2) may be one of the signals to inducing senescence in a broader range of Brassicaceae.

The genetic interaction of REVOLUTA and WRKY53 links plant development, senescence, and immune responses.

In annual plants, tight coordination of successive developmental events is of primary importance to optimize performance under fluctuating environmental conditions. The recent finding of the genetic interaction of WRKY53, a key senescence-related gene with REVOLUTA, a master regulator of early leaf patterning, raises the question of how early and late developmental events are connected. Here, we investigated the developmental and metabolic consequences of an alteration of the REVOLUTA and WRKY53 gene expression, from seedling to fruiting. Our results show that REVOLUTA critically controls late developmental phases and reproduction while inversely WRKY53 determines vegetative growth at early developmental stages. We further show that these regulators of distinct developmental phases frequently, but not continuously, interact throughout ontogeny and demonstrated that their genetic interaction is mediated by the salicylic acid (SA). Moreover, we showed that REVOLUTA and WRKY53 are keys regulatory nodes of development and plant immunity thought their role in SA metabolic pathways, which also highlights the role of REV in pathogen defence. Together, our findings demonstrate how late and early developmental events are tightly intertwined by molecular hubs. These hubs interact with each other throughout ontogeny, and participate in the interplay between plant development and immunity.

A HECT E3 ubiquitin ligase negatively regulates Arabidopsis leaf senescence through degradation of the transcription factor WRKY53.

WRKY transcription factors play a central role in controlling leaf senescence in Arabidopsis. One important member, WRKY53, is tightly regulated by various mechanisms, and is a convergence node between senescence and pathogen responses. Using WRKY53 in a yeast two-hybrid screen, we isolated the HECT domain E3 ubiquitin ligase UPL5. In contrast to mammals, Arabidopsis contains only seven HECT E3 ubiquitin ligases, whose targets and functions are largely unknown. In yeast cells, UPL5 interacts with WRKY53 via its leucine zipper domain, and this interaction was confirmed in the cytoplasm of plant cells by a bimolecular fluorescence complementation assay. UPL5 was able to use the WRKY53 protein as a substrate for polyubiquitination in an in vitro system, and induction of UPL5 expression by an ethanol-inducible system in upl5 plants led to degradation of the WRKY53 protein. Expression of both genes is regulated antagonistically in response to hydrogen peroxide, jasmonic acid and plant development. Two T-DNA insertion lines (upl5-1 and upl5-2) showed the same senescence phenotype as WRKY53 over-expressers. Over-expression of WRKY53 in the upl5 background enhanced the accelerated senescence phenotype of WRKY53 over-expressers. Therefore, we conclude that UPL5 regulates leaf senescence in Arabidopsis through degradation of WRKY53 and ensures that senescence is executed in the correct time frame.

G-Box binding factor1 reduces CATALASE2 expression and regulates the onset of leaf senescence in Arabidopsis.

Hydrogen peroxide (H(2)O(2)) is discussed as being a signaling molecule in Arabidopsis (Arabidopsis thaliana) leaf senescence. Intracellular H(2)O(2) levels are controlled by the H(2)O(2)-scavenging enzyme catalase in concert with other scavenging and producing systems. Catalases are encoded by a small gene family, and the expression of all three Arabidopsis catalase genes is regulated in a senescence-associated manner. CATALASE2 (CAT2) expression is down-regulated during bolting time at the onset of leaf senescence and appears to be involved in the elevation of the H(2)O(2) level at this time point. To understand the role of CAT2 in senescence regulation in more detail, we used CAT2 promoter fragments in a yeast one-hybrid screen to isolate upstream regulatory factors. Among others, we could identify G-Box Binding Factor1 (GBF1) as a DNA-binding protein of the CAT2 promoter. Transient overexpression of GBF1 together with a CAT2:beta-glucuronidase construct in tobacco (Nicotiana benthamiana) plants and Arabidopsis protoplasts revealed a negative effect of GBF1 on CAT2 expression. In gbf1 mutant plants, the CAT2 decrease in expression and activity at bolting time and the increase in H(2)O(2) could no longer be observed. Consequently, the onset of leaf senescence and the expression of senescence-associated genes were delayed in gbf1 plants, clearly indicating a regulatory function of GBF1 in leaf senescence, most likely via regulation of the intracellular H(2)O(2) content.

Editorial for Special Issue "Leaf Senescence" in Plants.

Senescence in plants is often described as the last step in the life history of a plant [...].

Disparate Dynamics of Gene Body and cis-Regulatory Element Evolution Illustrated for the Senescence-Associated Cysteine Protease Gene SAG12 of Plants.

Gene regulation networks precisely orchestrate the expression of genes that are closely associated with defined physiological and developmental processes such as leaf senescence in plants. The Arabidopsis thaliana senescence-associated gene 12 (AtSAG12) encodes a cysteine protease that is (i) involved in the degradation of chloroplast proteins and (ii) almost exclusively expressed during senescence. Transcription factors, such as WRKY53 and WRKY45, bind to W-boxes in the promoter region of AtSAG12 and play key roles in its activation. Other transcription factors, such as bZIPs, might have accessory functions in their gene regulation, as several A-boxes have been identified and appear to be highly overrepresented in the promoter region compared to the whole genome distribution but are not localized within the regulatory regions driving senescence-associated expression. To address whether these two regulatory elements exhibiting these different properties are conserved in other closely related species, we constructed phylogenetic trees of the coding sequences of orthologs of AtSAG12 and screened their respective 2000 bp promoter regions for the presence of conserved cis-regulatory elements, such as bZIP and WRKY binding sites. Interestingly, the functional relevant upstream located W-boxes were absent in plant species as closely related as Arabidopsis lyrata, whereas an A-box cluster appeared to be conserved in the Arabidopsis species but disappeared in Brassica napus. Several orthologs were present in other species, possibly because of local or whole genome duplication events, but with distinct cis-regulatory sites in different locations. However, at least one gene copy in each family analyzed carried one W-box and one A-box in its promoter. These gene differences in SAG12 orthologs are discussed in the framework of cis- and trans-regulatory factors, of promoter and gene evolution, of genetic variation, and of the enhancement of the adaptability of plants to changing environmental conditions.

Arabidopsis WRKY53, a Node of Multi-Layer Regulation in the Network of Senescence.

Leaf senescence is an integral part of plant development aiming at the remobilization of nutrients and minerals out of the senescing tissue into developing parts of the plant. Sequential as well as monocarpic senescence maximize the usage of nitrogen, mineral, and carbon resources for plant growth and the sake of the next generation. However, stress-induced premature senescence functions as an exit strategy to guarantee offspring under long-lasting unfavorable conditions. In order to coordinate this complex developmental program with all kinds of environmental input signals, complex regulatory cues have to be in place. Major changes in the transcriptome imply important roles for transcription factors. Among all transcription factor families in plants, the NAC and WRKY factors appear to play central roles in senescence regulation. In this review, we summarize the current knowledge on the role of WRKY factors with a special focus on WRKY53. In contrast to a holistic multi-omics view we want to exemplify the complexity of the network structure by summarizing the multilayer regulation of WRKY53 of Arabidopsis.

Impact of Alternatively Polyadenylated Isoforms of ETHYLENE RESPONSE FACTOR4 with Activator and Repressor Function on Senescence in Arabidopsis thaliana L.

Leaf senescence is highly regulated by transcriptional reprogramming, implying an important role for transcriptional regulators. ETHYLENE RESPONSE FACTOR4 (ERF4) was shown to be involved in senescence regulation and to exist in two different isoforms due to alternative polyadenylation of its pre-mRNA. One of these isoforms, ERF4-R, contains an ERF-associated amphiphilic repression (EAR) motif and acts as repressor, whereas the other form, ERF4-A, is lacking this motif and acts as activator. Here, we analyzed the impact of these isoforms on senescence. Both isoforms were able to complement the delayed senescence phenotype of the erf4 mutant with a tendency of ERF4-A for a slightly better complementation. However, overexpression led to accelerated senescence of 35S:ERF4-R plants but not of 35S:ERF4-A plants. We identified CATALASE3 (CAT3) as direct target gene of ERF4 in a yeast-one-hybrid screen. Both isoforms directly bind to the CAT3 promoter but have antagonistic effects on gene expression. The ratio of ERF4-A to ERF4-R mRNA changed during development, leading to a complex age-dependent regulation of CAT3 activity. The RNA-binding protein FPA shifted the R/A-ratio and fpa mutants are pointing towards a role of alternative polyadenylation regulators in senescence.

Nitrogen Supply Drives Senescence-Related Seed Storage Protein Expression in Rapeseed Leaves.

In general, yield and fruit quality strongly rely on efficient nutrient remobilization during plant development and senescence. Transcriptome changes associated with senescence in spring oilseed rape grown under optimal nitrogen supply or mild nitrogen deficiency revealed differences in senescence and nutrient mobilization in old lower canopy leaves and younger higher canopy leaves [1]. Having a closer look at this transcriptome analyses, we identified the major classes of seed storage proteins (SSP) to be expressed in vegetative tissue, namely leaf and stem tissue. Expression of SSPs was not only dependent on the nitrogen supply but transcripts appeared to correlate with intracellular H2O2 contents, which functions as well-known signaling molecule in developmental senescence. The abundance of SSPs in leaf material transiently progressed from the oldest leaves to the youngest. Moreover, stems also exhibited short-term production of SSPs, which hints at an interim storage function. In order to decipher whether hydrogen peroxide also functions as a signaling molecule in nitrogen deficiency-induced senescence, we analyzed hydrogen peroxide contents after complete nitrogen depletion in oilseed rape and Arabidopsis plants. In both cases, hydrogen peroxide contents were lower in nitrogen deficient plants, indicating that at least parts of the developmental senescence program appear to be suppressed under nitrogen deficiency.

Arabidopsis thaliana WRKY25 Transcription Factor Mediates Oxidative Stress Tolerance and Regulates Senescence in a Redox-Dependent Manner.

Senescence is the last developmental step in plant life and is accompanied by a massive change in gene expression implying a strong participation of transcriptional regulators. In the past decade, the WRKY53 transcription factor was disclosed to be a central node of a complex regulatory network of leaf senescence and to underlie a tight multi-layer control of expression, activity and protein stability. Here, we identify WRKY25 as a redox-sensitive up-stream regulatory factor of WRKY53 expression. Under non-oxidizing conditions, WRKY25 binds to a specific W-box in the WRKY53 promoter and acts as a positive regulator of WRKY53 expression in a transient expression system using Arabidopsis protoplasts, whereas oxidizing conditions dampened the action of WRKY25. However, overexpression of WRKY25 did not accelerate senescence but increased lifespan of Arabidopsis plants, whereas the knock-out of the gene resulted in the opposite phenotype, indicating a more complex regulatory function of WRKY25 within the WRKY subnetwork of senescence regulation. In addition, overexpression of WRKY25 mediated higher tolerance to oxidative stress and the intracellular H2O2 level is lower in WRKY25 overexpressing plants and higher in wrky25 mutants compared to wildtype plants suggesting that WRKY25 is also involved in controlling intracellular redox conditions. Consistently, WRKY25 overexpressers had higher and wrky mutants lower H2O2 scavenging capacity. Like already shown for WRKY53, MEKK1 positively influenced the activation potential of WRKY25 on the WRKY53 promoter. Taken together, WRKY53, WRKY25, MEKK1 and H2O2 interplay with each other in a complex network. As H2O2 signaling molecule participates in many stress responses, WRKK25 acts most likely as integrators of environmental signals into senescence regulation.

Study of Hydrogen Peroxide as a Senescence-Inducing Signal.

In many plant species, leaf senescence correlates with an increase in intracellular levels of reactive oxygen species (ROS) as well as differential regulation of anti-oxidative systems. Due to their reactive nature, reactive oxygen species (ROS) were considered to have only detrimental effects for long time. However, ROS turned out to be more than just toxic by-products of aerobic metabolism but rather major components in different signaling pathways. Considering its relatively long half-life, comparably low reactivity, and its ability to cross membranes, especially hydrogen peroxide, has gained attention as a signaling molecule. In this article, a set of tools to study hydrogen peroxide contents and the activity of its scavenging enzymes in correlation with leaf senescence parameters is presented.

Correction: Phosphorylation Affects DNA-Binding of the Senescence-Regulating bZIP Transcription Factor GBF1. Plants 2015, 4, 691-709.

The authors wish to make the following corrections to their paper [1].[...].