Guodong Ren

Identification of a Novel Chloroplast Protein AtNYE1 Regulating Chlorophyll Degradation during Leaf Senescence in Arabidopsis

A dramatic increase of chlorophyll (Chl) degradation occurs during senescence of vegetative plant organs and fruit ripening. Although the biochemical pathway of Chl degradation has long been proposed, little is known about its regulatory mechanism. Identification of Chl degradation-disturbed mutants and subsequently isolation of responsible genes would greatly facilitate the elucidation of the regulation of Chl degradation. Here, we describe a nonyellowing mutant of Arabidopsis (Arabidopsis thaliana), nye1-1, in which 50% Chl was retained, compared to less than 10% in the wild type (Columbia-0), at the end of a 6-d dark incubation. Nevertheless, neither photosynthesis- nor senescence-associated process was significantly affected in nye1-1. Characteristically, a significant reduction in pheophorbide a oxygenase activity was detected in nye1-1. However, no detectable accumulation of either chlorophyllide a or pheophorbide a was observed. Reciprocal crossings revealed that the mutant phenotype was caused by a monogenic semidominant nuclear mutation. We have identified AtNYE1 by positional cloning. Dozens of its putative orthologs, predominantly appearing in higher plant species, were identified, some of which have been associated with Chl degradation in a few crop species. Quantitative polymerase chain reaction analysis showed that AtNYE1 was drastically induced by senescence signals. Constitutive overexpression of AtNYE1 could result in either pale-yellow true leaves or even albino seedlings. These results collectively indicate that NYE1 plays an important regulatory role in Chl degradation during senescence by modulating pheophorbide a oxygenase activity.

EIN3 and ORE1 Accelerate Degreening during Ethylene-Mediated Leaf Senescence by Directly Activating Chlorophyll Catabolic Genes in Arabidopsis

Degreening, caused by chlorophyll degradation, is the most obvious symptom of senescing leaves. Chlorophyll degradation can be triggered by endogenous and environmental cues, and ethylene is one of the major inducers. ETHYLENE INSENSITIVE3 (EIN3) is a key transcription factor in the ethylene signaling pathway. It was previously reported that EIN3, miR164, and a NAC (NAM, ATAF, and CUC) transcription factor ORE1/NAC2 constitute a regulatory network mediating leaf senescence. However, how this network regulates chlorophyll degradation at molecular level is not yet elucidated. Here we report a feed-forward regulation of chlorophyll degradation that involves EIN3, ORE1, and chlorophyll catabolic genes (CCGs). Gene expression analysis showed that the induction of three major CCGs, NYE1, NYC1 and PAO, by ethylene was largely repressed in ein3 eil1 double mutant. Dual-luciferase assay revealed that EIN3 significantly enhanced the promoter activity of NYE1, NYC1 and PAO in Arabidopsis protoplasts. Furthermore, Electrophoretic mobility shift assay (EMSA) indicated that EIN3 could directly bind to NYE1, NYC1 and PAO promoters. These results reveal that EIN3 functions as a positive regulator of CCG expression during ethylene-mediated chlorophyll degradation. Interestingly, ORE1, a senescence regulator which is a downstream target of EIN3, could also activate the expression of NYE1, NYC1 and PAO by directly binding to their promoters in EMSA and chromatin immunoprecipitation (ChIP) assays. In addition, EIN3 and ORE1 promoted NYE1 and NYC1 transcriptions in an additive manner. These results suggest that ORE1 is also involved in the direct regulation of CCG transcription. Moreover, ORE1 activated the expression of ACS2, a major ethylene biosynthesis gene, and subsequently promoted ethylene production. Collectively, our work reveals that EIN3, ORE1 and CCGs constitute a coherent feed-forward loop involving in the robust regulation of ethylene-mediated chlorophyll degradation during leaf senescence in Arabidopsis.

Jasmonic acid promotes degreening via MYC2/3/4- and ANAC019/055/072-mediated regulation of major chlorophyll catabolic genes

Degreening caused by rapid chlorophyll (Chl) degradation is a characteristic event during green organ senescence or maturation. Pheophorbidexa0a oxygenase gene (PAO) encodes a key enzyme of Chl degradation, yet its transcriptional regulation remains largely unknown. Using yeast one-hybrid screening, coupled with inxa0vitro and inxa0vivo assays, we revealed that Arabidopsis MYC2/3/4 basic helix-loop-helix proteins directly bind to PAO promoter. Overexpression of the MYCs significantly enhanced the transcriptional activity of PAO promoter in Arabidopsis protoplasts, and methyl jasmonate (MeJA) treatment greatly induced PAO expression in wild-type Arabidopsis plants, but the induction was abolished in myc2xa0myc3xa0myc4. In addition, MYC2/3/4 proteins could promote the expression of another Chl catabolic enzyme gene, NYC1, as well as a key regulatory gene of Chl degradation, NYE1/SGR1, by directly binding to their promoters. More importantly, the myc2xa0myc3xa0myc4 triple mutant showed a severe stay-green phenotype, whereas the lines overexpressing the MYCs showed accelerated leaf yellowing upon MeJA treatment. These results suggest that MYC2/3/4 proteins may mediate jasmonic acid (JA)-induced Chl degradation by directly activating these Chl catabolic genes (CCGs). Three NAC family proteins, ANAC019/055/072, downstream from MYC2/3/4 proteins, could also directly promote the expression of a similar set of CCGs (NYE1/SGR1, NYE2/SGR2 and NYC1) during Chl degradation. In particular, anac019xa0anac055xa0anac072 triple mutant displayed a severe stay-green phenotype after MeJA treatment. Finally, we revealed that MYC2 and ANAC019 may interact with each other and synergistically enhance NYE1 expression. Together, our study reveals a hierarchical and coordinated regulatory network of JA-induced Chl degradation.

Reverse Genetic Identification of CRN1 and its Distinctive Role in Chlorophyll Degradation in Arabidopsis

Recent identification of NYE1/SGR1 brought up a new era for the exploration of the regulatory mechanism of Chlorophyll (Chl) degradation. Cluster analysis of senescence associated genes with putative chloroplast targeting sequences revealed several genes sharing a similar expression pattern with NYE1. Further characterization of available T-DNA insertion lines led to the discovery of a novel stay-green gene CRN1 (Co-regulated with NYE1). Chl breakdown was significantly restrained in crn1-1 under diversified senescence scenarios, which is comparable with that in acd1-20, but much more severe than that in nye1-1. Notably, various Chl binding proteins, especially trimeric LHCP II, were markedly retained in crn1-1 four days after dark-treatment, possibly due to a lesion in disassociation of protein-pigment complex. Nevertheless, the photochemical efficiency of PSII in crn1-1 declined, even more rapidly, two days after dark-treatment, compared to those in Col-0 and nye1-1. Our results suggest that CRN1 plays a crucial role in Chl degradation, and that loss of its function produces various side-effects, including those on the breakdown of Ch-protein complex and the maintenance of the residual photosynthetic capability during leaf senescence.

ABF2, ABF3, and ABF4 Promote ABA-Mediated Chlorophyll Degradation and Leaf Senescence by Transcriptional Activation of Chlorophyll Catabolic Genes and Senescence-Associated Genes in Arabidopsis.

Chlorophyll (Chl) degradation is an integral process of leaf senescence, and NYE1/SGR1xa0has been demonstrated as a key regulator of Chl catabolism in diverse plant species. In this study, using yeast one-hybrid screening, we identified three abscisic acid (ABA)-responsive element (ABRE)-binding transcription factors, ABF2 (AREB1), ABF3, and ABF4 (AREB2), as the putative binding proteins of the NYE1 promoter. Through the transactivation analysis, electrophoretic mobility shift assay, and chromatin immunoprecipitation, we demonstrated that ABF2, ABF3, and ABF4 directly bound to and activated the NYE1 promoter inxa0vitro and inxa0vivo. ABA is a positive regulator of leaf senescence, and exogenously applied ABA can accelerate Chl degradation. The triple mutant of the ABFs, abf2abf3abf4, as well as two ABA-insensitive mutants, abi1-1 and snrk2.2/2.3/2.6, exhibited stay-green phenotypes after ABA treatment, along with decreased induction of NYE1 and NYE2 expression. In contrast, overexpression of ABF4 accelerated Chl degradation upon ABA treatment. Interestingly, ABF2/3/4 could also activate the expression of two Chl catabolic enzyme genes, PAO and NYC1, by directly binding to their promoters. In addition, abf2abf3abf4 exhibited a functional stay-green phenotype, and senescence-associated genes (SAGs), such as SAG29 (SWEET15), might be directly regulated by the ABFs. Taken together, our results suggest that ABF2, ABF3, and ABF4 likely act as key regulators in mediating ABA-triggered Chl degradation and leaf senescence in general in Arabidopsis.

Chlorophyll Degradation: The Tocopherol Biosynthesis-Related Phytol Hydrolase in Arabidopsis Seeds Is Still Missing

Two known chlorophyll-related types of phytol hydrolases may only play limited roles in tocopherol biosynthesis in Arabidopsis seeds, implicating another unknown dephytylase that may exist. Phytyl diphosphate (PDP) is the prenyl precursor for tocopherol biosynthesis. Based on recent genetic evidence, PDP is supplied to the tocopherol biosynthetic pathway primarily by chlorophyll degradation and sequential phytol phosphorylation. Three enzymes of Arabidopsis (Arabidopsis thaliana) are known to be capable of removing the phytol chain from chlorophyll in vitro: chlorophyllase1 (CLH1), CLH2, and pheophytin pheophorbide hydrolase (PPH), which specifically hydrolyzes pheophytin. While PPH, but not chlorophyllases, is required for in vivo chlorophyll breakdown during Arabidopsis leaf senescence, little is known about the involvement of these phytol-releasing enzymes in tocopherol biosynthesis. To explore the origin of PDP for tocopherol synthesis, seed tocopherol concentrations were determined in Arabidopsis lines engineered for seed-specific overexpression of PPH and in single and multiple mutants in the three genes encoding known dephytylating enzymes. Except for modestly increasing tocopherol content observed in the PPH overexpressor, none of the remaining lines exhibited significantly reduced tocopherol concentrations, suggesting that the known chlorophyll-derived phytol-releasing enzymes do not play major roles in tocopherol biosynthesis. Tocopherol content of seeds from double mutants in NONYELLOWING1 (NYE1) and NYE2, regulators of chlorophyll degradation, had modest reduction compared with wild-type seeds, although mature seeds of the double mutant retained significantly higher chlorophyll levels. These findings suggest that NYEs may play limited roles in regulating an unknown tocopherol biosynthesis-related phytol hydrolase. Meanwhile, seeds of wild-type over-expressing NYE1 had lower tocopherol levels, suggesting that phytol derived from NYE1-dependent chlorophyll degradation probably doesn’t enter tocopherol biosynthesis. Potential routes of chlorophyll degradation are discussed in relation to tocopherol biosynthesis.

NON-YELLOWING2 (NYE2), a Close Paralog of NYE1, Plays a Positive Role in Chlorophyll Degradation in Arabidopsis

Plant senescence is an integral part of plant development, with old organs senescing in association with the emergence and development of nascent ones. Rapid chlorophyll (Chl) degradation, the most prominent event during green organ senescence, is proposed to be a key detoxification process to facilitate massive nutrient remobilization (Lim et al., 2007). Biochemistry of Chl degradation has been extensively elucidated, but its regulation remains largely unknown. A significant step in the elucidation of Chl degradation regulation is the identification of NON-YELLOWING1/STAY-GREEN1 (NYE1/SGR1) gene in diverse species, which is responsible for the green cotyledon trait of Mendels pea (for a review, see Hortensteiner, 2009).

The role of ANAC072 in the regulation of chlorophyll degradation during age- and dark-induced leaf senescence

Key messageANAC072 positively regulates both age- and dark-induced leaf senescence through activating the transcription ofNYE1.AbstractLeaf senescence is integral to plant development, which is age-dependent and strictly regulated by internal and environmental signals. Although a number of senescence-related mutants and senescence-associated genes (SAGs) have been identified and characterized in the past decades, the general regulatory network of leaf senescence is still far from being elucidated. Here, we report the role of ANAC072, an SAG identified through bioinformatics analysis, in the regulation of chlorophyll degradation during natural and dark-induced leaf senescence. The expression of ANAC072 was increased with advancing leaf senescence in Arabidopsis. Leaf degreening was significantly delayed under normal or dark-induced conditions in anac072-1, a knockout mutant of ANAC072, with a higher chlorophyll level detected. In contrast, an overexpression mutant, anac072-2, with ANAC072 transcription markedly upregulated, showed an early leaf-yellowing phenotype. Consistently, senescent leaves of the loss-of-function mutant anac072-1 exhibited delays in the decrease of photosynthesis efficiency of photosystem II (Fv/Fm ratio) and the increase of plasma membrane ion leakage rate as compared with corresponding leaves of wild-type Col-0 plants, whereas the overexpression mutant anac072-2 showed opposite changes. Our data suggest that ANAC072 plays a positive role during natural and dark-induced leaf senescence. In addition, the transcript level of NYE1, a key regulatory gene in chlorophyll degradation, relied on the function of ANAC072. Combining these analyses with electrophoretic mobility shift assay and chromatin immunoprecipitation, we demonstrated that ANAC072 directly bound to the NYE1 promoter in vitro and in vivo, so ANAC072 may promote chlorophyll degradation by directly upregulating the expression of NYE1.

Chlorophyll Synthase under Epigenetic Surveillance Is Critical for Vitamin E Synthesis, and Altered Expression Affects Tocopherol Levels in Arabidopsis

Epigenetic analysis of chlorophyll synthase expression demonstrates its effects on chlorophyll and tocopherol synthesis. Chlorophyll synthase catalyzes the final step in chlorophyll biosynthesis: the esterification of chlorophyllide with either geranylgeranyl diphosphate or phytyl diphosphate (PDP). Recent studies have pointed to the involvement of chlorophyll-linked reduction of geranylgeranyl by geranylgeranyl reductase as a major pathway for the synthesis of the PDP precursor of tocopherols. This indirect pathway of PDP synthesis suggests a key role of chlorophyll synthase in tocopherol production to generate the geranylgeranyl-chlorophyll substrate for geranylgeranyl reductase. In this study, contributions of chlorophyll synthase to tocopherol formation in Arabidopsis (Arabidopsis thaliana) were explored by disrupting and altering expression of the corresponding gene CHLOROPHYLL SYNTHASE (CHLSYN; At3g51820). Leaves from the homozygous chlysyn1-1 null mutant were nearly devoid of tocopherols, whereas seeds contained only approximately 25% of wild-type tocopherol levels. Leaves of RNA interference lines with partial suppression of CHLSYN displayed marked reductions in chlorophyll but up to a 2-fold increase in tocopherol concentrations. Cauliflower mosaic virus35S-mediated overexpression of CHLSYN unexpectedly caused a cosuppression phenotype at high frequencies accompanied by strongly reduced chlorophyll content and increased tocopherol levels. This phenotype and the associated detection of CHLSYN-derived small interfering RNAs were reversed with CHLSYN overexpression in rna-directed rna polymerase6 (rdr6), which is defective in RNA-dependent RNA polymerase6, a key enzyme in sense transgene-induced small interfering RNA production. CHLSYN overexpression in rdr6 had little effect on chlorophyll content but resulted in up to a 30% reduction in tocopherol levels in leaves. These findings show that altered CHLSYN expression impacts tocopherol levels and also, show a strong epigenetic surveillance of CHLSYN to control chlorophyll and tocopherol synthesis.

NYEs/SGRs‐mediated chlorophyll degradation is critical for detoxification during seed maturation in Arabidopsis

In the seed industry, chlorophyll (Chl) fluorescence is often used as a major non-invasive reporter of seed maturation and quality. Breakdown of Chl is a proactive process during the late stage of seed maturation, as well as during leaf senescence and fruit ripening. However, the biological significance of this process is still unclear. NYE1 and NYE2 are Mg-dechelatases, catalyzing the first rate-limiting step of Chl a degradation. Loss-of-function of both NYE1 and NYE2 not only results in a nearly complete retention of Chl during leaf senescence, but also produces green seeds in Arabidopsis. In this study, we showed that Chl retention in the nye1 nye2 double-mutant caused severe photo-damage to maturing seeds. Upon prolonged light exposure, green seeds of nye1 nye2 gradually bleached out and eventually lost their germination capacity. This organ-specific photosensitive phenotype is likely due to an over-accumulation of free Chl, which possesses photosensitizing properties and causes a burst of reactive oxygen species upon light exposure. As expected, a similar, albeit much milder, photosensitive phenotype was observed in the seeds of d1 d2, a green-seed mutant defective in NYE/SGR orthologous genes in soybean. Taken together, our data suggest that efficient NYEs-mediated Chl degradation is critical for detoxification during seed maturation.