Yasuhito Sakuraba

STAY-GREEN and Chlorophyll Catabolic Enzymes Interact at Light-Harvesting Complex II for Chlorophyll Detoxification during Leaf Senescence in Arabidopsis

This work shows that the chloroplast-localized components of the chlorophyll catabolic pathway dynamically interact with each other, possibly forming a multiprotein complex specifically localizing to light-harvesting complex II. This interaction likely channels chlorophyll breakdown intermediates and thereby prevents potential chlorophyll-derived phototoxicity during leaf senescence. During leaf senescence, plants degrade chlorophyll to colorless linear tetrapyrroles that are stored in the vacuole of senescing cells. The early steps of chlorophyll breakdown occur in plastids. To date, five chlorophyll catabolic enzymes (CCEs), NONYELLOW COLORING1 (NYC1), NYC1-LIKE, pheophytinase, pheophorbide a oxygenase (PAO), and red chlorophyll catabolite reductase, have been identified; these enzymes catalyze the stepwise degradation of chlorophyll to a fluorescent intermediate, pFCC, which is then exported from the plastid. In addition, STAY-GREEN (SGR), Mendel’s green cotyledon gene encoding a chloroplast protein, is required for the initiation of chlorophyll breakdown in plastids. Senescence-induced SGR binds to light-harvesting complex II (LHCII), but its exact role remains elusive. Here, we show that all five CCEs also specifically interact with LHCII. In addition, SGR and CCEs interact directly or indirectly with each other at LHCII, and SGR is essential for recruiting CCEs in senescing chloroplasts. PAO, which had been attributed to the inner envelope, is found to localize in the thylakoid membrane. These data indicate a predominant role for the SGR-CCE-LHCII protein interaction in the breakdown of LHCII-located chlorophyll, likely to allow metabolic channeling of phototoxic chlorophyll breakdown intermediates upstream of nontoxic pFCC.

Phytochrome-interacting transcription factors PIF4 and PIF5 induce leaf senescence in Arabidopsis

Plants initiate senescence to shed photosynthetically inefficient leaves. Light deprivation induces leaf senescence, which involves massive transcriptional reprogramming to dismantle cellular components and remobilize nutrients. In darkness, intermittent pulses of red light can inhibit senescence, likely via phytochromes. However, the precise molecular mechanisms transducing the signals from light perception to the inhibition of senescence remain elusive. Here, we show that in Arabidopsis, dark-induced senescence requires phytochrome-interacting transcription factors PIF4 and PIF5 (PIF4/PIF5). ELF3 and phytochrome B inhibit senescence by repressing PIF4/PIF5 at the transcriptional and post-translational levels, respectively. PIF4/PIF5 act in the signalling pathways of two senescence-promoting hormones, ethylene and abscisic acid, by directly activating expression of EIN3, ABI5 and EEL. In turn, PIF4, PIF5, EIN3, ABI5 and EEL directly activate the expression of the major senescence-promoting NAC transcription factor ORESARA1, thus forming multiple, coherent feed-forward loops. Our results reveal how classical light signalling connects to senescence in Arabidopsis.

The Arabidopsis Transcription Factor NAC016 Promotes Drought Stress Responses by Repressing AREB1 Transcription through a Trifurcate Feed-Forward Regulatory Loop Involving NAP

The Arabidopsis transcription factor NAC016 activates drought stress responses by inducing NAP transcription and repressing AREB1 transcription by binding to different regions of the AREB1 promoter. Drought and other abiotic stresses negatively affect plant growth and development and thus reduce productivity. The plant-specific NAM/ATAF1/2/CUC2 (NAC) transcription factors have important roles in abiotic stress-responsive signaling. Here, we show that Arabidopsis thaliana NAC016 is involved in drought stress responses; nac016 mutants have high drought tolerance, and NAC016-overexpressing (NAC016-OX) plants have low drought tolerance. Using genome-wide gene expression microarray analysis and MEME motif searches, we identified the NAC016-specific binding motif (NAC16BM), GATTGGAT[AT]CA, in the promoters of genes downregulated in nac016-1 mutants. The NAC16BM sequence does not contain the core NAC binding motif CACG (or its reverse complement CGTG). NAC016 directly binds to the NAC16BM in the promoter of ABSCISIC ACID-RESPONSIVE ELEMENT BINDING PROTEIN1 (AREB1), which encodes a central transcription factor in the stress-responsive abscisic acid signaling pathway and represses AREB1 transcription. We found that knockout mutants of the NAC016 target gene NAC-LIKE, ACTIVATED BY AP3/PI (NAP) also exhibited strong drought tolerance; moreover, NAP binds to the AREB1 promoter and suppresses AREB1 transcription. Taking these results together, we propose that a trifurcate feed-forward pathway involving NAC016, NAP, and AREB1 functions in the drought stress response, in addition to affecting leaf senescence in Arabidopsis.

Mutation of the Arabidopsis NAC016 Transcription Factor Delays Leaf Senescence

The highly ordered process of senescence forms the final stage of leaf development; a large set of senescence-associated genes (SAGs) execute this orderly dismantling of the photosynthetic apparatus and remobilization of cellular components. A number of transcription factors (TFs) modulate SAG expression to promote or delay senescence. Here we show that NAC016, the previously uncharacterized senescence-associated NAM/ATAF1/2/CUC2 (senNAC) TF in Arabidopsis thaliana, promotes senescence. Leaves of nac016 mutants remained green under senescence-inducing conditions, and leaves of NAC016-overexpressing (NAC016-OX) plants senesced early. Under dark-induced senescence (DIS) conditions, nac016 mutants had low ion leakage, and retained the proper balance of photosystem proteins and normal grana thylakoid shape much longer than wild-type plants, suggesting that nac016 acts as a functional stay-green type senescence mutant. Under DIS conditions, SAGs (NYC1, PPH, SGR1/NYE1 and WRKY22), including senNACs (JUB1, NAP, ORE1, ORS1 and VNI2), were down-regulated in nac016 mutants and up-regulated in NAC016-OX plants. In addition to its role in senescence, NAC016 also affects abiotic stress. Under salt and oxidative stress conditions, NAC016 expression rapidly increased in developing leaves, possibly to promote senescence. Indeed, under the stress conditions, nac016 mutants stayed green and NAC016-OX plants senesced rapidly. To identify direct targets of the NAC016 TF in the regulation of leaf senescence, we conducted yeast one-hybrid assays, which strongly suggested that NAC016 binds to the promoters of NAP and ORS1. Based on these results, we propose that NAC016 regulatory mechanisms promoting leaf senescence exhibit cross-talk with the salt and oxidative stress-responsive signaling pathways.

The rice faded green leaf locus encodes protochlorophyllide oxidoreductase B and is essential for chlorophyll synthesis under high light conditions

NADPH:protochlorophyllide oxidoreductase (POR) catalyzes photoreduction of protochlorophyllide (Pchlide) to chlorophyllide in chlorophyll (Chl) synthesis, and is required for prolamellar body (PLB) formation in etioplasts. Rice faded green leaf (fgl) mutants develop yellow/white leaf variegation and necrotic lesions during leaf elongation in field-grown plants. Map-based cloning revealed that FGL encodes OsPORB, one of two rice POR isoforms. In fgl, etiolated seedlings contained smaller PLBs in etioplasts, and lower levels of total and photoactive Pchlide. Under constant or high light (HL) conditions, newly emerging green leaves rapidly turned yellow and formed lesions. Increased levels of non-photoactive Pchlide, which acts as a photosensitizer, may cause reactive oxygen accumulation and lesion formation. OsPORA expression is repressed by light and OsPORB expression is regulated in a circadian rhythm in short-day conditions. OsPORA was expressed at high levels in developing leaves and decreased dramatically in fully mature leaves, whereas OsPORB expression was relatively constant throughout leaf development, similar to expression patterns of AtPORA and AtPORB in Arabidopsis. However, OsPORB expression is rapidly upregulated by HL treatment, similar to the fluence rate-dependent regulation of AtPORC. This suggests that OsPORB function is equivalent to both AtPORB and AtPORC functions. Our results demonstrate that OsPORB is essential for maintaining light-dependent Chl synthesis throughout leaf development, especially under HL conditions, whereas OsPORA mainly functions in the early stages of leaf development. Developmentally and physiologically distinct roles of monocot OsPORs are discussed by comparing with those of dicot AtPORs.

Mutation of Oryza sativa CORONATINE INSENSITIVE 1b (OsCOI1b) delays leaf senescence.

Jasmonic acid (JA) functions in plant development, including senescence and immunity. Arabidopsis thaliana CORONATINE INSENSITIVE 1 encodes a JA receptor and functions in the JA-responsive signaling pathway. The Arabidopsis genome harbors a single COI gene, but the rice (Oryza sativa) genome harbors three COI homologs, OsCOI1a, OsCOI1b, and OsCOI2. Thus, it remains unclear whether each OsCOI has distinct, additive, synergistic, or redundant functions in development. Here, we use the oscoi1b-1 knockout mutants to show that OsCOI1b mainly affects leaf senescence under senescence-promoting conditions. oscoi1b-1 mutants stayed green during dark-induced and natural senescence, with substantial retention of chlorophylls and photosynthetic capacity. Furthermore, several senescence-associated genes were downregulated in oscoi1b-1 mutants, including homologs of Arabidopsis thaliana ETHYLENE INSENSITIVE 3 and ORESARA 1, important regulators of leaf senescence. These results suggest that crosstalk between JA signaling and ethylene signaling affects leaf senescence. The Arabidopsis coi1-1 plants containing 35S:OsCOI1a or 35S:OsCOI1b rescued the delayed leaf senescence during dark incubation, suggesting that both OsCOI1a and OsCOI1b are required for promoting leaf senescence in rice. oscoi1b-1 mutants showed significant decreases in spikelet fertility and grain weight, leading to severe reduction of grain yield, indicating that OsCOI1-mediated JA signaling affects spikelet fertility and grain filling.

Arabidopsis STAY-GREEN2 Is a Negative Regulator of Chlorophyll Degradation during Leaf Senescence

Chlorophyll (Chl) degradation causes leaf yellowing during senescence or under stress conditions. For Chl breakdown, STAY-GREEN1 (SGR1) interacts with Chl catabolic enzymes (CCEs) and light-harvesting complex II (LHCII) at the thylakoid membrane, possibly to allow metabolic channeling of potentially phototoxic Chl breakdown intermediates. Among these Chl catabolic components, SGR1 acts as a key regulator of leaf yellowing. In addition to SGR1 (At4g22920), the Arabidopsis thaliana genome contains an additional homolog, SGR2 (At4g11910), whose biological function remains elusive. Under senescence-inducing conditions, SGR2 expression is highly up-regulated, similarly to SGR1 expression. Here we show that SGR2 function counteracts SGR1 activity in leaf Chl degradation; SGR2-overexpressing plants stayed green and the sgr2-1 knockout mutant exhibited early leaf yellowing under age-, dark-, and stress-induced senescence conditions. Like SGR1, SGR2 interacted with LHCII but, in contrast to SGR1, SGR2 interactions with CCEs were very limited. Furthermore, SGR1 and SGR2 formed homo- or heterodimers, strongly suggesting a role for SGR2 in negatively regulating Chl degradation by possibly interfering with the proposed CCE-recruiting function of SGR1. Our data indicate an antagonistic evolution of the functions of SGR1 and SGR2 in Arabidopsis to balance Chl catabolism in chloroplasts with the dismantling and remobilizing of other cellular components in senescing leaf cells.

Delayed degradation of chlorophylls and photosynthetic proteins in Arabidopsis autophagy mutants during stress-induced leaf yellowing

Summary Under mild abiotic-stress conditions, Arabidopsis atg mutants showed a functional stay-green phenotype which is probably caused by the lack of chloroplastic autophagy and the retrograde regulation of senescence-associated gene expression.

7-Hydroxymethyl chlorophyll a reductase functions in metabolic channeling of chlorophyll breakdown intermediates during leaf senescence.

During natural or dark-induced senescence, chlorophyll degradation causes leaf yellowing. Recent evidence indicates that chlorophyll catabolic enzymes (CCEs) interact with the photosynthetic apparatus; for example, five CCEs (NYC1, NOL, PPH, PAO and RCCR) interact with LHCII. STAY-GREEN (SGR) and CCEs interact with one another in senescing chloroplasts; this interaction may allow metabolic channeling of potentially phototoxic chlorophyll breakdown intermediates. 7-Hydroxymethyl chlorophyll a reductase (HCAR) also acts as a CCE, but HCAR functions during leaf senescence remain unclear. Here we show that in Arabidopsis, HCAR-overexpressing plants exhibited accelerated leaf yellowing and, conversely, hcar mutants stayed green during dark-induced senescence. Moreover, HCAR interacted with LHCII in in vivo pull-down assays, and with SGR, NYC1, NOL and RCCR in yeast two-hybrid assays, indicating that HCAR is a component of the proposed SGR-CCE-LHCII complex, which acts in chlorophyll breakdown. Notably, HCAR and NOL are expressed throughout leaf development and are drastically down-regulated during dark-induced senescence, in contrast with SGR, NYC1, PPH and PAO, which are up-regulated during dark-induced senescence. Moreover, HCAR and NOL are highly up-regulated during greening of etiolated seedlings, strongly suggesting a major role for NOL and HCAR in the chlorophyll cycle during vegetative stages, possibly in chlorophyll turnover.

The Divergent Roles of STAYGREEN (SGR) Homologs in Chlorophyll Degradation

Degradation of chlorophyll (Chl) by Chl catabolic enzymes (CCEs) causes the loss of green color that typically occurs during senescence of leaves. In addition to CCEs, STAYGREEN1 (SGR1) functions as a key regulator of Chl degradation. Although sgr1 mutants in many plant species exhibit a stay-green phenotype, the biochemical function of the SGR1 protein remains elusive. Many recent studies have examined the physiological and molecular roles of SGR1 and its homologs (SGR2 and SGR-LIKE) in Chl metabolism, finding that these proteins have different roles in different species. In this review, we summarize the recent studies on SGR and discuss the most likely functions of SGR homologs.