Benke Kuai

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.

Regulation of leaf senescence and crop genetic improvement.

Leaf senescence can impact crop production by either changing photosynthesis duration, or by modifying the nutrient remobilization efficiency and harvest index. The doubling of the grain yield in major cereals in the last 50 years was primarily achieved through the extension of photosynthesis duration and the increase in crop biomass partitioning, two things that are intrinsically coupled with leaf senescence. In this review, we consider the functionality of a leaf as a function of leaf age, and divide a leafs life into three phases: the functionality increasing phase at the early growth stage, the full functionality phase, and the senescence and functionality decreasing phase. A genetic framework is proposed to describe gene actions at various checkpoints to regulate leaf development and senescence. Four categories of genes contribute to crop production: those which regulate (I) the speed and transition of early leaf growth, (II) photosynthesis rate, (III) the onset and (IV) the progression of leaf senescence. Current advances in isolating and characterizing senescence regulatory genes are discussed in the leaf aging and crop production context. We argue that the breeding of crops with leaf senescence ideotypes should be an essential part of further crop genetic improvement.

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.

Ectopic overexpression of AtHDG11 in tall fescue resulted in enhanced tolerance to drought and salt stress.

Tall fescue (Festuca arundinacea Schreb.) is a cool-season perennial grass, which has been conventionally grown in the temperate area. However, as a major type of cool-season turf grass, its growth has been extended to the sub-tropical climate or even to the transitional climate between the sub-tropical and the tropical, and, in some cases, to heavily salinized lands. The extended growth imposes a serious challenge to its tolerance to the abiotic stress, particularly to drought, salt and high temperature. Here, we report a successful introduction of Arabidopsis AtHDG11 into the tall fescue via Agrobacterium-mediated transformation. The ectopic overexpression of AtHDG11 under the control of CaMV 35S promoter with four enhancers resulted in significantly enhanced tolerance to drought and salt stress. No obvious adverse effects on growth and development were observed in the transgenic plants. The enhanced stress tolerance was associated with a more extensive root system, a lower level of malondialdehyde, a nearly normal Na+/K+ ratio, a higher level of proline and a kinetically accelerated induction of SOD and CAT activities observed in the transgenic plants during drought and/or salt stress, indicating that an enhanced ROS scavenging capability might play a significant role in the acquired tolerance to the abiotic stress.

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.

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.

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 Key Role of Chlorocatechol 1,2-Dioxygenase in Phytoremoval and Degradation of Catechol by Transgenic Arabidopsis

Transgenic exploitation of bacterial degradative genes in plants has been considered a favorable strategy for degrading organic pollutants in the environment. The aromatic ring characteristic of these pollutants is mainly responsible for their recalcitrance to degradation. In this study, a Plesiomonas-derived chlorocatechol 1,2-dioxygenase (TfdC) gene (tfdC), capable of cleaving the aromatic ring, was introduced into Arabidopsis (Arabidopsis thaliana). Morphology and growth of transgenic plants are indistinguishable from those of wild-type plants. In contrast, they show significantly enhanced tolerances to catechol. Transgenic plants also exhibit strikingly higher capabilities of removing catechol from their media and high efficiencies of converting catechol to cis,cis-muconic acid. As far-less-than-calculated amounts of cis,cis-muconic acid were accumulated within the transgenic plants, existence of endogenous TfdD- and TfdE-like activities was postulated and, subsequently, putative orthologs of bacterial tfdD and tfdE were detected in Arabidopsis. However, no TfdC activity and no putative orthologs of either tfdC or tfdF were identified. This work indicates that the TfdC activity, conferred by tfdC in transgenic Arabidopsis, is a key requirement for phytoremoval and degradation of catechol, and also suggests that microbial degradative genes may be transgenically exploited in plants for bioremediation of aromatic pollutants in the environment.

Characterization of a novel developmentally retarded mutant (drm1) associated with the autonomous flowering pathway in Arabidopsis

ABSTRACTA developmentally retarded mutant (drm1) was identified from ethyl methanesulfonate (EMS)-mutagenized M2 seeds in Columbia (Col-0) genetic background. The drm1 flowers 109 d after sowing, with a whole life cycle of about 160 d. It also shows a pleiotropic phenotype, e.g., slow germination and lower germination rate, lower growth rate, curling leaves and abnormal floral organs. The drm1 mutation was a single recessive nuclear mutation, which was mapped to the bottom of chromosome 5 and located within a region of 20-30 kb around MXK3.1. There have been no mutants with similar phenotypes reported in the literature, suggesting that DRM1 is a novel flowering promoting locus. The findings that the drm1 flowered lately under all photoperiod conditions and its late flowering phenotype was significantly restored by vernalization treatment suggest that the drm1 is a typical late flowering mutant and most likely associated with the autonomous flowering pathway. The conclusion was further confirmed by the revelation that the transcript level of FLC was constantly upregulated in the drm1 at all the developmental phases examined, except for a very early stage. Moreover, the transcript levels of two other important repressors, EMF and TFL1, were also upregulated in the drm1, implying that the two repressors, along with FLC, seems to act in parallel pathways in the drm1 to regulate flowering as well as other aspects of floral development in a negatively additive way. This helps to explain why the drm1 exhibits a much more severe late-flowering phenotype than most late-flowering mutants reported. It also implies that the DRM1 might act upstream of these repressors.