Jiong Gao

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.

TCP transcription factors are critical for the coordinated regulation of ISOCHORISMATE SYNTHASE 1 expression in Arabidopsis thaliana

Salicylic acid (SA) plays an important role in various aspects of plant development and responses to stresses. To elucidate the sophisticated regulatory mechanism of SA synthesis and signaling, we used a yeast one-hybrid system to screen for regulators of isochorismate synthase 1 (ICS1), a gene encoding the key enzyme in SA biosynthesis in Arabidopsis thaliana. A TCP family transcription factor AtTCP8 was initially identified as a candidate regulator of ICS1. The regulation of ICS1 by TCP proteins is supported by the presence of a typical TCP binding site in the ICS1 promoter. The binding of TCP8 to this site was confirmed by in vitro and in vivo assays. Expression patterns of TCP8 and its corresponding gene TCP9 largely overlapped with ICS1 under pathogen attack. A significant reduction in the expression of ICS1 during immune responses was observed in the tcp8 tcp9 double mutant. We also detected strong interactions between TCP8 and SAR deficient 1 (SARD1), WRKY family transcription factor 28 (WRKY28), NAC (NAM/ATAF1,ATAF2/CUC2) family transcription factor 019 (NAC019), as well as among TCP8, TCP9 and TCP20, suggesting a complex coordinated regulatory mechanism underlying ICS1 expression. Our results collectively demonstrate that TCP proteins are involved in the orchestrated regulation of ICS1 expression, with TCP8 and TCP9 being verified as major representatives.

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.

The Pathway and Regulation of Salicylic Acid Biosynthesis in Probenazole-Treated Arabidopsis

Probenazole (PBZ; 3-allyloxy-1,2-benzisothiazole-1,1-dioxide) is a highly effective chemical inducer of systemic-acquired resistance (SAR). It has been used widely to protect rice plants against the rice blast fungus Magnaporthe grisea. Previous studies have shown that PBZ induces SAR through enhanced accumulation of salicylic acid (SA). Plants synthesize SA by either a pathway that uses phenylalanine as substrate or another that involves isochorismate. To clarify how SA is produced in PBZ-treated Arabidopsis, we examined the expression patterns and enzyme activities of phenylalanine ammonia lyase (PAL) and isochorismate synthase (ICS), which are the main components of the phenylalanine and isochorismate pathways, respectively. PBZ exposure significantly improved the accumulation of SA and increased ICS activity. In the sid2–2 mutant, which has a defect in ICS1, PBZ had no effect on the level of endogenous SA or activity of ICS. In contrast, PAL activity and the expression of most PAL genes were down-regulated by such treatment in wild-type plants. These results suggest that SA is mainly synthesized via the ICS-mediated pathway in Arabidopsis.

Suppressor of Overexpression of CO 1 Negatively Regulates Dark-Induced Leaf Degreening and Senescence by Directly Repressing Pheophytinase and Other Senescence-Associated Genes in Arabidopsis

SOC1 inhibits dark-induced leaf degreening and senescence by directly binding to the CArG box of PPH, NYE1, and SAG113 promoters and inhibiting their expression at the transcriptional level in Arabidopsis. Although the biochemical pathway of chlorophyll (Chl) degradation has been largely elucidated, how Chl is rapidly yet coordinately degraded during leaf senescence remains elusive. Pheophytinase (PPH) is the enzyme for catalyzing the removal of the phytol group from pheophytin a, and PPH expression is significantly induced during leaf senescence. To elucidate the transcriptional regulation of PPH, we used a yeast (Saccharomyces cerevisiae) one-hybrid system to screen for its trans-regulators. SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1), a key flowering pathway integrator, was initially identified as one of the putative trans-regulators of PPH. After dark treatment, leaves of an SOC1 knockdown mutant (soc1-6) showed an accelerated yellowing phenotype, whereas those of SOC1-overexpressing lines exhibited a partial stay-green phenotype. SOC1 and PPH expression showed a negative correlation during leaf senescence. Substantially, SOC1 protein could bind specifically to the CArG box of the PPH promoter in vitro and in vivo, and overexpression of SOC1 significantly inhibited the transcriptional activity of the PPH promoter in Arabidopsis (Arabidopsis thaliana) protoplasts. Importantly, soc1-6 pph-1 (a PPH knockout mutant) double mutant displayed a stay-green phenotype similar to that of pph-1 during dark treatment. These results demonstrated that SOC1 inhibits Chl degradation via negatively regulating PPH expression. In addition, measurement of the Chl content and the maximum photochemical efficiency of photosystem II of soc1-6 and SOC1-OE leaves after dark treatment suggested that SOC1 also negatively regulates the general senescence process. Seven SENESCENCE-ASSOCIATED GENES (SAGs) were thereafter identified as its potential target genes, and NONYELLOWING1 and SAG113 were experimentally confirmed. Together, we reveal that SOC1 represses dark-induced leaf Chl degradation and senescence in general in Arabidopsis.

Synthetic promoters consisting of defined cis-acting elements link multiple signaling pathways to probenazole-inducible system

Probenazole (3-allyloxy-1,2-benzisothiazole-1,1-dioxide, PBZ), the active component of Oryzemate, could induce systemic acquired resistance (SAR) in plants through the induction of salicylic acid (SA) biosynthesis. As a widely used chemical inducer, PBZ is a good prospect for establishing a new chemical-inducible system. We first designed artificially synthetic promoters with tandem copies of a single type of cis-element (SARE, JERE, GCC, GST1, HSRE, and W-box) that could mediate the expression of the β-glucuronidase (GUS) reporter gene in plants upon PBZ treatment. Then we combined different types of elements in order to improve inducibility in the PBZ-inducible system. On the other hand, we were surprised to find that the cis-elements, which are responsive to jasmonic acid (JA) and ethylene, also responded to PBZ, implying that SA, JA, and ethylene pathways also would play important roles in PBZ’s action. Further analysis demonstrated that PBZ also induced early events of innate immunity via a signaling pathway in which Ca2+ influx and mitogen-activated protein kinase (MAPK) activity were involved. We constructed synthesized artificial promoters to establish a PBZ chemical-inducible system, and preliminarily explored SA, JA, ethylene, calcium, and MAPK signaling pathways via PBZ-inducible system, which could provide an insight for in-depth study.摘要目的构建有效响应烯丙异噻唑 (PBZ) 诱导的人工合成启动子, 了解植物体内受PBZ诱导系统触发的信号途径。创新点通过分析包含已知顺式元件的人工合成启动子对PBZ 的响应性, 为构建一种基于PBZ诱导系统的新型化学诱导启动子提供了可能性, 并初步揭示了除水杨酸 (SA) 外, 茉莉酸 (JA) 和乙烯等多条信号途径可能共同参与了PBZ诱导的植物免疫反应过程。方法利用已知的响应相关信号通路的顺式作用元件构建人工合成启动子, 融合GUS报告基因后, 稳定转化拟南芥。通过检测PBZ处理过程中GUS酶活性的变化, 了解人工合成启动子对PBZ 的响应性, 分析PBZ诱导系统可能触发的信号途径。结论除了SA响应元件SARE 可以有效响应PBZ诱导外, 利用JA和乙烯响应元件JERE和GCC, 超敏反应 (HR) 相关的顺式元件HSRE和GST1, 以及植物抗病反应中重要顺式作用元件W-box构建的人工合成启动子也均可有效响应PBZ。另外, 通过人工合成启动子响应性分析的手段, 初步揭示了包括SA、JA、乙烯、钙离子和丝裂原活化蛋白激酶 (MAPKs) 在内的多条信号通路可能共同参与了PBZ诱导植物免疫反应的过程。

Association of the molecular regulation of ear leaf senescence/stress response and photosynthesis/metabolism with heterosis at the reproductive stage in maize

Maize exhibits a wide range of heterotic traits, but the molecular basis of heterosis at the reproductive stage has seldom been exploited. Leaf senescence is a degenerative process which affects crop yield and quality. In this study, we observed significantly delayed ear leaf senescence in the reciprocal hybrids of B73/Mo17 and Zheng58/Chang7-2 after silking, and all the hybrids displayed larger leaf areas and higher stems with higher yields. Our time-course transcriptome analysis identified 2,826 differentially expressed genes (DEGs) between two parental lines (PP-DEGs) and 2,328 DEGs between parental lines and the hybrid (PH-DEGs) after silking. Notably, several senescence promoting genes (ZmNYE1, ZmORE1, ZmWRKY53 and ZmPIFs) exhibited underdominant expression patterns in the hybrid, whereas putative photosynthesis and carbon-fixation (ZmPEPC)-associated, starch biosynthetic (ZmAPS1, ZmAPL), gibberellin biosynthetic genes (ZmGA20OX, ZmGA3OX) expressed overdominantly. We also identified 86 transcription factors from PH-DEGs, some of which were known to regulate senescence, stress and metabolic processes. Collectively, we demonstrate a molecular association of the regulations of both ear leaf senescence/stress response and photosynthesis/metabolism with heterosis at the late developmental stage. This finding not only extends our understanding to the molecular basis of maize heterosis but also provides basic information for molecular breeding.

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.

REF6 promotes lateral root formation through de-repression of PIN1/3/7 genes: H3K27 demethylation promotes lateral root formation

The H3K27 methyltransferase CLF inhibits lateral root (LR) formation through depositing the repressive H3K27me3 mark to the chromatin of PIN1, a key polar auxin transporter gene. Here, we show that the H3K27me3 demethylase REF6 promotes lateral root primordium initiation and LR emergence. REF6 directly binds to the chromatin of PIN1/3/7. Dysfunction in REF6 results in increased levels of H3K27me3 on PIN1/3/7 and suppressed expression of PIN genes. Genetic analysis of the clf ref6 double mutant revealed an antagonistic action between CLF and REF6, in terms of LR formation. Our findings indicate that H3K27 methylation and demethylation activities are likely coordinated to ensure proper LR organogenesis.