Exploring the Physiological Multiplicity of Native Microalgae from the Ecuadorian Highland, Italian Lowland and Indoor Locations in Response to UV-B.

The differential effects of UV-B on the inhibition or activation of protective mechanisms to maintain cells photosynthetically active were investigated in native microalgae. Four strains were used, including two Chlorella sorokiniana strains, F4 and LG1, isolated from a Mediterranean inland swamp and a recycled cigarette butt's substrate, respectively, and two isolates from an Ecuadorian highland lake related to Pectinodesmus pectinatus (PEC) and Ettlia pseudoalveolaris (ETI). Monocultures were exposed to acute UV-B (1.7 W m-2) over 18 h under controlled conditions. UV-B-untreated microalgae were used as the control. Comparative physiological responses, including photosynthetic pigments, non-enzymatic antioxidants, and chlorophyll a fluorescence, were evaluated at specific time points. Results showed that UV-B significantly compromised all the physiological parameters in F4, thereby resulting in the most UV-B-sensitive strain. Contrarily, UV-B exposure did not lead to changes in the PEC physiological traits, resulting in the best UV-B-resistant strain. This could be attributed to the acclimation to high light habitat, where maintaining a constitutive phenotype (at the photosynthetic level) is strategically advantageous. Differently, LG1 and ETI at 12 h of UV-B exposure showed different UV-B responses, which is probably related to acclimation, where in LG1, the pigments were recovered, and the antioxidants were still functioning, while in ETI, the accumulation of pigments and antioxidants was increased to avoid further photodamage. Consequently, the prolonged exposure in LG1 and ETI resulted in species-specific metabolic regulation (e.g., non-enzymatic antioxidants) in order to constrain full photoinhibition under acute UV-B.

UV-B Irradiation Effect on Microalgae Performance in the Remediation of Effluent Derived from the Cigarette Butt Cleaning Process.

In this study, the potential of ultraviolet B (UV-B) radiation to alleviate the effects of pollutants in cigarette butt wastewater (CBW) was investigated using different Chlorella sorokiniana strains (F4, R1 and LG1). Microalgae were treated with UV-B (1.7 W m-2) for 3 days prior to their exposure to CBW and then incubated for 4 days in the absence or presence of UV-B. UV-B-untreated microalgae were used as the control. Comparative physiological responses, including photosynthetic pigments and non-enzymatic antioxidants, as well as nicotine and nicotyrine removal, were evaluated in 7-day cultures. UV-B treatments did not negatively impact algal chlorophyll or carotenoid production. UV-B acclimation was strain-dependent, correlating with native environment adaptations and genetic constitutions. UV-B as a pretreatment had long-term positive effects on non-enzymatic antioxidant capacity. However, LG1 needed more time to readjust the pro-oxidant/antioxidant balance, as it was the most UV-B-sensitive. Phenolic compounds played an important role in the antioxidant system response to UV-B, while flavonoids did not contribute to the total antioxidant capacity. Although cross-resistance between UV-B and CBW was observed in F4 and R1, only R1 showed nicotine/nicotyrine catabolism induction due to UV-B. Overall, the results suggest that UV-B activates defense pathways associated with resistance or tolerance to nicotine and nicotyrine.

Remediation Capacity of Different Microalgae in Effluents Derived from the Cigarette Butt Cleaning Process.

Microalgal-based remediation is an ecofriendly and cost-effective system for wastewater treatment. This study evaluated the capacity of microalgae in the remediation of wastewater from cleaning process of smoked cigarette butts (CB). At laboratory scale, six strains (one from the family Scenedesmaceae, two Chlamydomonas debaryana and three Chlorella sorokiniana) were exposed to different CB wastewater dilutions to identify toxicity levels reflected in the alteration of microalgal physiological status and to determine the optimal conditions for an effective removal of contaminants. CB wastewater could impact on microalgal chlorophyll and carotenoid production in a concentration-dependent manner. Moreover, the resistance and remediation capacity did not only depend on the microalgal strain, but also on the chemical characteristics of the organic pollutants. In detail, nicotine was the most resistant pollutant to removal by the microalgae tested and its low removal correlated with the inhibition of photosynthetic pigments affecting microalgal growth. Concerning the optimal conditions for an effective bioremediation, this study demonstrated that the Chlamydomonas strain named F2 showed the best removal capacity to organic pollutants at 5% CB wastewater (corresponding to 25 butts L−1 or 5 g CB L−1) maintaining its growth and photosynthetic pigments at control levels.

Urban conditions affect soil characteristics and physiological performance of three evergreen woody species.

Physiological studies conducted mainly in metropolitan areas demonstrated that urban environments generate stressful conditions for plants. However, less attention has been paid to plant response to urban conditions in small cities. Here, we evaluated to what extent the health and physiological functions of some Mediterranean urban species [Quercus ilex L., Nerium oleander L. and Pittosporum tobira (Thunb.) W.T. Aiton] were impacted by urban and peri-urban conditions in Pisa (Italy), a small medieval city with narrow streets that impede efficient public transport causing oversized private transport. Experimental period spanned from late-summer to winter in concomitance with the sharp increase in air pollutants. Climate and air quality, soil physical and chemical properties, and plant physiological traits including leaf gas exchanges, chlorophyll fluorescence and leaf pigments were assessed. In soil, the organic carbon affected aggregates and water stability and the concentrations of some micro-elements decreased in winter. Air pollutants impaired leaf gas exchanges and photochemical processes at photosystem II, depending on species, season, and urban conditions. Shrubs were more susceptible than the tree species, highlighting that the latter adapted better to pollutants along an urban-peri-urban transect in Mediterranean environments. This study gives information on the physiological adaptability of some of the most frequent Mediterranean urban species to stressful conditions and demonstrated that, even in a small city, urban conditions influence the physiology and development of vegetation, affecting the plant health status and its ability to provide key ecosystem services.

The TGN/EE SNARE protein SYP61 and the ubiquitin ligase ATL31 cooperatively regulate plant responses to carbon/nitrogen conditions in Arabidopsis.

Ubiquitination is a post-translational modification involving the reversible attachment of the small protein ubiquitin to a target protein. Ubiquitination is involved in numerous cellular processes, including the membrane trafficking of cargo proteins. However, the ubiquitination of the trafficking machinery components and their involvement in environmental responses are not well understood. Here, we report that the Arabidopsis thaliana trans-Golgi network/early endosome localized SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein SYP61 interacts with the transmembrane ubiquitin ligase ATL31, a key regulator of resistance to disrupted carbon (C)/nitrogen/(N)-nutrient conditions. SYP61 is a key component of membrane trafficking in Arabidopsis. The subcellular localization of ATL31 was disrupted in knockdown mutants of SYP61, and the insensitivity of ATL31-overexpressing plants to high C/low N-stress was repressed in these mutants, suggesting that SYP61 and ATL31 cooperatively function in plant responses to nutrient stress. SYP61 is ubiquitinated in plants, and its ubiquitination level is upregulated under low C/high N-nutrient conditions. These findings provide important insights into the ubiquitin signaling and membrane trafficking machinery in plants.

Hormonal Regulation in Different Varieties of Chenopodium quinoa Willd. Exposed to Short Acute UV-B Irradiation.

Increased ultraviolet-B (UV-B) due to global change can affect plant development and metabolism. Quinoa tolerates extreme conditions including high UV levels. However, the physiological mechanisms behind its abiotic stress tolerance are unclear, especially those related to UV-B. We previously demonstrated that 9.12 kJ m-2 d-1 may induce UV-B-specific signaling while 18.24 kJ m-2 d-1 promotes a UV-B-independent response. Here, we explored the effects of these UV-B doses on hormonal regulation linked to plant morphology and defense among diverse varieties. Changes in fluorescence parameters of photosystem II, flavonoids and hormones (indoleacetic acid (IAA), jasmonic acid (JA), abscisic acid (ABA) and salicylic acid (SA)) were surveyed under controlled conditions. Here, we showed that the sensitivity to short acute UV-B doses in varieties from different habitats is influenced by their parental lines and breeding time. UV-B sensitivity does not necessarily correlate with quinoa's geographical distribution. The role of flavonoids in the UV-B response seems to be different depending on varieties. Moreover, we found that the extent of changes in JA and SA correlate with UV-B tolerance, while the increase of ABA was mainly related to UV-B stress.

Photosynthetic performance of five cool-season turfgrasses under UV-B exposure.

Turfgrasses are monocotyledonous plants from the family Poaceae. They are widely used in green spaces and are considered one of the most economically important horticultural crops in the world. Turfgrass quality is affected by several environmental factors including light, which is involved in the quality decline of transplanted sod. Ultraviolet-B (UV-B) is an important regulator of plant growth and development. Plants growing and/or stored in protected systems, such as in sod production, may be more vulnerable to UV-B damage than those growing in the field due to acclimation. Few studies on the effects of UV-B on turfgrass physiology have been published. Therefore, the aim of this study was to evaluate the influence of UV-B irradiation on the photosynthetic performance of five cool-season turfgrasses, namely Agrostis stolonifera L., Festuca arundinacea Schreb., Poa supina Schrad., Poa pratensis L. and Lolium perenne L. Turfgrasses were exposed to 18.25 kJ m-2 d-1 biologically effective UV-B in growth chambers under controlled conditions. Measurements included photosynthetic pigments, chlorophyll fluorescence and gas exchanges monitored for 16 d-UV-B treatment and after recovery. Content of pigments decreased with UV-B exposure with significant differences among the species. UV-B also affected the photosystem II (PSII) efficiency depending on the exposure period and species. Similarly, gas exchange parameters showed different effects among species after UV-B exposure compromising the assimilation of CO2. Multivariate analysis highlighted three main clusters of species confirming their different UV-B tolerance and ability to restore PSII photochemistry after recovery, from which Festuca arundinacea resulted to be the most tolerant.

Physiological responses of maca (Lepidium meyenii Walp.) plants to UV radiation in its high-altitude mountain ecosystem.

Ultraviolet (UV) radiation is a small fraction of the solar spectrum, which acts as a key environmental modulator of plant function affecting metabolic regulation and growth. Plant species endemic to the Andes are well adapted to the harsh features of high-altitude climate, including high UV radiation. Maca (Lepidium meyenii Walpers) is a member of Brassicaceae family native to the central Andes of Peru, which grows between 3500 and 4500 m of altitude, where only highland grasses and few hardy bushes can survive. Even though maca has been the focus of recent researches, mainly due to its nutraceutical properties, knowledge regarding its adaptation mechanisms to these particular natural environmental conditions is scarce. In this study, we manipulated solar UV radiation by using UV-transmitting (Control) or blocking (UV-block) filters under field conditions (4138 m above the sea level) in order to understand the impact of UV on morphological and physiological parameters of maca crops over a complete growing season. Compared to the UV-blocking filter, under control condition a significant increase of hypocotyl weight was observed during the vegetative phase together with a marked leaf turnover. Although parameters conferring photosynthetic performance were not altered by UV, carbohydrate allocation between above and underground organs was affected. Control condition did not influence the content of secondary metabolites such as glucosinolates and phenolic compounds in hypocotyls, while some differences were observed in the rosettes. These differences were mainly related to leaf turnover and the protection of new young leaves in control plants. Altogether, the data suggest that maca plants respond to strong UV radiation at high altitudes by a coordinated remobilization and relocation of metabolites between source and sink organs via a possible UV signaling pathway.

Physiological responses of Lepidium meyenii plants to ultraviolet-B radiation challenge.

BACKGROUND: Ultraviolet-B (UV-B) radiation can affect several aspects ranging from plant growth to metabolic regulation. Maca is a Brassicaceae crop native to the Andes growing in above 3500 m of altitude. Although maca has been the focus mainly due to its nutraceutical properties, it remains unknown how maca plants tolerate to harsh environments, such as strong UV-B. Here, we present the first study that reports the physiological responses of maca plants to counteract and recover to repeated acute UV-B irradiation. RESULTS: In detail, plants were daily exposed to acute UV-B irradiation followed by a recovery period under controlled conditions. The results showed that repeated acute UV-B exposures reduced biomass and photosynthetic parameters, with gradual senescence induction in exposed leaves, reduction of young leaves expansion and root growth inhibition. Negative correlation between increased UV-B and recovery was observed, with marked production of new biomass in plants treated one week or more. CONCLUSIONS: A differential UV-B response was observed: stress response was mainly controlled by a coordinated source-sink carbon allocation, while acclimation process may require UV-B-specific systemic defense response reflected on the phenotypic plasticity of maca plants. Moreover, these differential UV-B responses were also suggested by multifactorial analysis based on biometric and physiological data.

Photosynthetic and Growth Responses of Arundo donax L. Plantlets Under Different Oxygen Deficiency Stresses and Reoxygenation.

Promotion of nonfood species production to marginal, degraded lands abandoned by mainstream agriculture is affected by extremes of water availability (droughts and floods), which have increased in frequency and intensity and account for severe yield reduction. Arundo donax L., known as giant cane or giant reed, spontaneously grows in different kinds of environments with limitation to low temperature and is thus widespread in temperate and hot areas around the world. Moreover, this perennial rhizomatous grass has been recognized as a leading candidate crop in the Mediterranean for lignocellulosic feedstock due to its high C3 photosynthetic capacity, positive energy balance and low agroecological management demand. In this study, the photosynthetic performance and growth response of A. donax to waterlogging and submergence stress following a time course as well as their respective re-oxygenation were analyzed under reproducible and controlled environment conditions. Results of growth response showed that biomass production was strongly conditioned by the availability of oxygen. In fact, only waterlogged plants showed similar growth capacity to those under control conditions, while plants under submergence resulted in a dramatic reduction of this trait. The simultaneous measurements of both gas exchanges and chlorophyll fluorescence highlighted an alteration of both stomatal and non-stomatal photosynthetic behaviors during a short/medium period of oxygen deprivation and re-oxygenation. Photosynthetic CO2 uptake was strictly related to a combination of stomatal and mesophyll diffusional constrains, depending on the severity of the treatment and exposure time. Conditions of waterlogging and hypoxia revealed a slight growth plasticity of the species in response to prolonged stress conditions, followed by a fast recovery upon reoxygenation. Moreover, the rapid restoration of physiological functions after O2 deprivation testifies to the environmental plasticity of this species, although prolonged O2 shortage proved detrimental to A. donax by hampering growth and photosynthetic CO2 uptake.

Nitrate Reductase Modulation in Response to Changes in C/N Balance and Nitrogen Source in Arabidopsis.

Environmental cues modulate the balance of carbon (C) and nitrogen (N) which are essential elements for plant metabolism and growth. In Arabidopsis, photochemical efficiency of PSII, phosphorylation status and localization of many enzymes, and the level of total soluble sugars were affected by an unbalanced C/N ratio. Since differences in C/N affect these parameters, here we checked whether different sources of N have different effects when a high C/N ratio is imposed. NO3- and NH4+ were separately provided in C/N medium. We investigated the effects on photochemical efficiency of PSII, the level of total soluble sugars and nitrate reductase activity under stressful C/N conditions compared with control conditions. We found that treated plants accumulated more total soluble sugars when compared with control. Photochemical efficiency of PSII did not show significant differences between the two sources of nitrogen after 24 h. The actual nitrate reductase activity was the result of a combination of activity, activation state and protein level. This activity constantly decreased starting from time zero in control conditions; in contrast, the actual nitrate reductase activity showed a peak at 2 h after treatment with NO3-, and at 30 min with NH4+. This, according to the level of total soluble sugars, can be explained by the existence of a cross-talk between the sugars in excess and low nitrate in the medium that blocks the activity of nitrate reductase in stressful sugar conditions until the plant is adapted to the stress.

Physiological effects of short acute UVB treatments in Chenopodium quinoa Willd.

Increased ultraviolet B (UVB) radiation due to global change can affect plant growth and metabolism. Here, we evaluated the capacity of quinoa to resist under short acute UVB irradiation. Quinoa was daily exposed for 30 or 60 min to 1.69 W m-2 UVB. The results showed that 30 min exposure in 9 d-course did not cause severe alterations on photosynthetic pigments and flavonoids, but a significant increase of antioxidant capacity was observed. Otherwise, 60 min UVB in 5 d-course reduced almost all these parameters except for an increase in the de-epoxidation of xanthophyll cycle pigments and led to the death of the plants. Further studies of gas exchange and fluorescence measurements showed that 30 min UVB dramatically decrease stomatal conductance, probably associated to reactive oxygen species (ROS) production. Inhibition of photosynthetic electron transport was also observed, which could be a response to reduce ROS. Otherwise, irreversible damage to the photosynthetic apparatus was found with 60 min UVB probably due to severe ROS overproduction that decompensates the redox balance inducing UVB non-specific signaling. Moreover, 60 min UVB compromised Rubisco carboxylase activity and photosynthetic electron transport. Overall, these data suggest that quinoa modulates different response mechanisms depending on the UVB irradiation dosage.

Inter- and intraspecific variability in physiological traits and post-anoxia recovery of photosynthetic efficiency in grasses under oxygen deprivation.

Low oxygen conditions occur in grass sites due to high and frequent precipitation, poor soil quality, and over-irrigation followed by slow drainage. Three warm-season and one cool-season grass were analyzed at metabolic level during a time-course experiment performed in a controlled anoxic environment. Prolonged oxygen depletion proved detrimental by leading to premature death to all the species, with the exception of seashore paspalum. Moreover, the anoxia tolerance observed in these grasses has been associated with slow use of carbohydrates, rather than with their relative abundance, which was more important than their antioxidant capacity. Further physiological characterization of eight seashore paspalum genotypes to anoxia was also performed, by examining the variation in photosystem II (PSII) efficiency and gas exchange during post-anoxia recovery. Multivariate analysis highlighted the presence of three main clusters of seashore paspalum genotypes, characterized by different ability to restore the PSII photochemistry during recovery after one day of anoxia. Taken together, our data demonstrate that the analysis of post-anoxia recovery of fluorescence and gas exchange parameters can represent a fast and reliable indicator for selecting species and cultivars more able to acclimate their photosynthetic apparatus.

Seedling Establishment of Tall Fescue Exposed to Long-Term Starvation Stress.

In germinating seeds under unfavorable environmental conditions, the mobilization of stores in the cotyledons is delayed, which may result in a different modulation of carbohydrates balance and a decrease in seedling vigor. Tall fescue (Festuca arundinacea Schreb.) caryopses grown at 4°C in the dark for an extended period in complete absence of nutrients, showed an unexpected ability to survive. Seedlings grown at 4°C for 210 days were morphologically identical to seedlings grown at 23°C for 21 days. After 400 days, seedlings grown at 4°C were able to differentiate plastids to chloroplast in just few days once transferred to the light and 23°C. Tall fescue exposed to prolonged period at 4°C showed marked anatomical changes: cell wall thickening, undifferentiated plastids, more root hairs and less xylem lignification. Physiological modifications were also observed, in particular related to sugar content, GA and ABA levels and amylolytic enzymes pattern. The phytohormones profiles exhibited at 4 and 23°C were comparable when normalized to the respective physiological states. Both the onset and the completion of germination were linked to GA and ABA levels, as well as to the ratio between these two hormones. All plants showed a sharp decline in carbohydrate content, with a consequent onset of gradual sugar starvation. This explained the slowed then full arrest in growth under both treatment regimes. The analysis of amylolytic activity showed that Ca2+ played a central role in the stabilization of several isoforms. Overall, convergence of starvation and hormone signals meet in crosstalk to regulate germination, growth and development in tall fescue.

The efficient physiological strategy of a tomato landrace in response to short-term salinity stress.

Landraces represent an important part of the biodiversity well-adapted under limiting environmental conditions. We investigated the response of two Southern Italy tomato landraces, the well-known San Marzano (our commercial standard) and a local accession called "Ciettaicale", to different levels of sodium chloride in water irrigation (from 0 up to 600 mM) for a short-time exposure (one week). The combination of the chlorophyll a fluorescence and gas exchange analyses suggested that Ciettaicale maintained a higher efficiency of photosystem II (PSII) photochemistry and CO2 utilization at high salinity concentrations than San Marzano. Stomatal and non-stomatal limitations occurred in San Marzano according to the reduction of maximum efficiency of PSII photochemistry and the increase of intercellular CO2 concentration. Higher Na+/K+ ratio and higher concentration of total soluble sugars contributed to non-stomatal limitations in San Marzano leaves. These effects were significantly less evident in Ciettaicale. We also observed changes in total antioxidant capacity and leaf pigment content that emphasized the occurrence of modifications in the photosynthetic apparatus according to salt gradient. The more efficient assimilates supply and an integrated root protection system provided by sugars and antioxidants can explain the significantly higher root/shoot ratio in Ciettaicale. Overall, our results suggest that a comprehensive assessment of salinity tolerance in a genotypes comparison could be also obtained evaluating plant response to high salinity levels at early vegetative stage. In addition, further studies will be carried out in order to evaluate the possibility of using Ciettaicale in tomato improvement programs.

Effect of carbon/nitrogen ratio on carbohydrate metabolism and light energy dissipation mechanisms in Arabidopsis thaliana.

Carbon (C) and nitrogen (N) nutrient sources are essential elements for metabolism, and their availability must be tightly coordinated for the optimal growth and development in plants. Plants are able to sense and respond to different C/N conditions via specific partitioning of C and N sources and the regulation of a complex cellular metabolic activity. We studied how the interaction between C and N signaling could affect carbohydrate metabolism, soluble sugar levels, photochemical efficiency of photosystem II (PSII) and the ability to drive the excess energy in Arabidopsis seedlings under moderated and disrupted C/N-nutrient conditions. Invertase and sucrose synthase activities were markedly affected by C/N-nutrient status depending on the phosphorylation status, suggesting that these enzymes may necessarily be modulated by their direct phosphorylation or phosphorylation of proteins that form complex with them in response to C/N stress. In addition, the enzymatic activity of these enzymes was also correlated with the amount of sugars, which not only act as substrate but also as signaling compounds. Analysis of chlorophyll fluorescence in plants under disrupted C/N condition suggested a reduction of electron transport rate at PSII level associated with a higher capacity for non-radiative energy dissipation in comparison with plants under moderated C/N condition. In conclusion, the tight coordination between C and N not only affects the carbohydrates metabolism and their concentration within plant tissues, but also the partitioning of the excitation energy at PSII level between radiative (electron transport) and non-radiative (heat) dissipation pathways.

Pale-green phenotype of atl31atl6 double mutant leaves is caused by disruption of 5-aminolevulinic acid biosynthesis in Arabidopsis thaliana.

Arabidopsis ubiquitin ligases ATL31 and homologue ATL6 control the carbon/nitrogen nutrient and pathogen responses. A mutant with the loss-of-function of both atl31 and atl6 developed light intensity-dependent pale-green true leaves, whereas the single knockout mutants did not. Plastid ultrastructure and Blue Native-PAGE analyses revealed that pale-green leaves contain abnormal plastid structure with highly reduced levels of thylakoid proteins. In contrast, the pale-green leaves of the atl31/atl6 mutant showed normal Fv/Fm. In the pale-green leaves of the atl31/atl6, the expression of HEMA1, which encodes the key enzyme for 5-aminolevulinic acid synthesis, the rate-limiting step in chlorophyll biosynthesis, was markedly down-regulated. The expression of key transcription factor GLK1, which directly promotes HEMA1 transcription, was also significantly decreased in atl31/atl6 mutant. Finally, application of 5-aminolevulinic acid to the atl31/atl6 mutants resulted in recovery to a green phenotype. Taken together, these findings indicate that the 5-aminolevulinic acid biosynthesis step was inhibited through the down-regulation of chlorophyll biosynthesis-related genes in the pale-green leaves of atl31/atl6 mutant.

Ubiquitin ligase ATL31 functions in leaf senescence in response to the balance between atmospheric CO2 and nitrogen availability in Arabidopsis.

Carbon (C) and nitrogen (N) are essential elements for metabolism, and their availability, called the C/N balance, must be tightly coordinated for optimal growth in plants. Previously, we have identified the ubiquitin ligase CNI1/ATL31 as a novel C/N regulator by screening plants grown on C/N stress medium containing excess sugar and limited N. To elucidate further the effect of C/N balance on plant growth and to determine the physiological function of ATL31, we performed C/N response analysis using an atmospheric CO2 manipulation system. Under conditions of elevated CO2 and sufficient N, plant biomass and total sugar and starch dramatically increased. In contrast, elevated CO2 with limited N did not increase plant biomass but promoted leaf chlorosis, with anthocyanin accumulation and increased senescence-associated gene expression. Similar results were obtained with plants grown in medium containing excess sugar and limited N, suggesting that disruption of the C/N balance affects senescence progression. In ATL31-overexpressing plants, promotion of senescence under disrupted CO2/N conditions was repressed, whereas in the loss-of-function mutant it was enhanced. The ATL31 gene was transcriptionally up-regulated under N deficiency and in senescent leaves, and ATL31 expression was highly correlated with WRKY53 expression, a key regulator of senescence. Furthermore, transient protoplast analysis implicated the direct activation of ATL31 expression by WRKY53, which was in accordance with the results of WRKY53 overexpression experiments. Together, these results demonstrate the importance of C/N balance in leaf senescence and the involvement of ubiquitin ligase ATL31 in the process of senescence in Arabidopsis.