The dependence of leaf senescence on the balance between 1‐aminocyclopropane‐1‐carboxylate acid synthase 1 (ACS1)‐catalysed ACC generation and nitric oxide‐associated 1 (NOS1)‐dependent NO accumulation in Arabidopsis

• Ethylene and nitric oxide (NO) act as endogenous regulators during leaf senescence. Levels of ethylene or its precursor 1‐aminocyclopropane‐1‐carboxylate acid (ACC) depend on the activity of ACC synthases (ACS), and NO production is controlled by NO‐associated 1 (NOA1). However, the integration mechanisms of ACS and NOA1 activity still need to be explored during leaf senescence.
• Here, using experimental techniques, such as physiological and molecular detection, liquid chromatography‐tandem mass spectrometry and fluorescence measurement, we investigated the relevant mechanisms.
• Our observations showed that the loss‐of‐function acs1‐1 mutant ameliorated age‐ or dark‐induced leaf senescence syndrome, such as yellowing and loss of chlorophyll, that acs1‐1 reduced ACC accumulation mainly in mature leaves and that acs1‐1‐promoted NOA1 expression and NO accumulation mainly in juvenile leaves, when compared with the wild type (WT). But the leaf senescence promoted by the NO‐deficient noa1 mutant was not involved in ACS1 expression. There was a similar sharp reduction of ACS1 and NOA1 expression with the increase in WT leaf age, and this inflection point appeared in mature leaves and coincided with the onset of leaf senescence.
• These findings suggest that NOA1‐dependent NO accumulation blocked the ACS1‐induced onset of leaf senescence, and that ACS1 activity corresponds to the onset of leaf senescence in Arabidopsis.

     

Regulation of plant disease resistance, stress responses, cell death, and ethylene signaling in Arabidopsis by the EDR1 protein kinase

ENHANCED DISEASE RESISTANCE 1 (EDR1) encodes a CTR1-like kinase and was previously reported to function as a negative regulator of disease resistance and ethylene-induced senescence. Here, we report that the edr1 mutant displays enhanced stress responses and spontaneous necrotic lesions under drought conditions in the absence of pathogen, suggesting that EDR1 is also involved in stress response signaling and cell death regulation. Double mutant analysis revealed that these drought-induced phenotypes require salicylic acid but not ethylene signaling pathways. In addition, the edr1-mediated ethylene-induced senescence phenotype was suppressed by mutations in EIN2, but not by mutations in SID2, PAD4, EDS1, or NPR1, suggesting that EDR1 functions at a point of cross talk between ethylene and salicylic acid signaling that impinges on senescence and cell death. Two edr1-associated phenotypes, drought-induced growth inhibition and ethylene-induced senescence, were suppressed by mutations in ORE9, implicating ubiquitin-mediated protein degradation in the regulation of these phenotypes. However, the ore9 mutation did not suppress edr1-mediated enhanced disease resistance to powdery mildew or spontaneous lesions, indicating that these phenotypes are controlled by separate signaling pathways. To investigate the function of the EDR1 kinase domain, we expressed the C-terminal third of EDR1 in wild-type Columbia and edr1 backgrounds under the control of a dexamethasone-inducible promoter. Overexpression of the EDR1 kinase domain in an edr1 background had no obvious effect on edr1-associated phenotypes. However, overexpression of the EDR1 kinase domain in a wild-type Columbia background caused dominant negative phenotypes, including enhanced disease resistance to powdery mildew and enhanced ethylene-induced senescence; thus, the overexpressed EDR1 kinase domain alone does not exert EDR1 function, but rather negatively affects the function of native EDR1 protein.     

Possible role of light in the maintenance of EIN3/EIL1 stability in Arabidopsis seedlings

To examine the mechanism of EIN3-mediated gene expression by ethylene, the expression patterns of ethylene-inducible genes by ethylene were monitored in Col-0 and ethylene signaling mutants. In Col-0, the inducibility of ACC oxidase by ethylene in light-grown seedlings was much higher than in dark-grown seedlings. While the expression of ACC oxidase was highly increased by ethylene not only in Col-0 but in ein3-1 under light treatment, this pattern was completely abrogated in etiolated ein3-1 seedlings, suggesting the expression of EIN3-mediated ACC oxidase genes could be affected by light. To check if the level of EIN3 and EIL1 was regulated by light, cell-free degradation assays were performed. This resulted in the rapid degradation of these proteins within 1h after adding dark-grown cell extracts and this degradation was retarded by light-grown extracts. Here, we propose that light may act as a negative regulator in the destabilization of EIN3/EIL1.     

Arabidopsis seedling growth response and recovery to ethylene. A kinetic analysis

Responses to the plant hormone ethylene are mediated by a family of five receptors in Arabidopsis that act in the absence of ethylene as negative regulators of response pathways. In this study, we examined the rapid kinetics of growth inhibition by ethylene and growth recovery after ethylene withdrawal in hypocotyls of etiolated seedlings of wild-type and ethylene receptor-deficient Arabidopsis lines. This analysis revealed that there are two phases to growth inhibition by ethylene in wild type: a rapid phase followed by a prolonged, slower phase. Full recovery of growth occurs approximately 90 min after ethylene removal. None of the receptor null mutations tested had a measurable effect on the two phases of growth inhibition. However, loss-of-function mutations in ETR1, ETR2, and EIN4 significantly prolonged the time for recovery of growth rate after ethylene was removed. Plants with an etr1-6;etr2-3;ein4-4 triple loss-of-function mutation took longer to recover than any of the single mutants, while the ers1;ers2 double mutant had no effect on recovery rate, suggesting that receiver domains play a role in recovery. Transformation of the ers1-2;etr1-7 double mutant with wild-type genomic ETR1 rescued the slow recovery phenotype, while a His kinase-inactivated ETR1 construct did not. To account for the rapid recovery from growth inhibition, a model in which clustered receptors act cooperatively is proposed.     

Ethylene response pathway is essential for ARABIDOPSIS A-FIFTEEN function in floral induction and leaf senescence

ARABIDOPSIS A-FIFTEEN (AAF) encodes a plastid protein and was originally identified as a SENESCENCE-ASSOCIATED GENE. Previously, we found that overexpression of AAF (AAF-OX) in Arabidopsis led to accumulated reactive oxygen species and promoted leaf senescence induced by oxidative stress, which was suppressed by a null mutant, ein2-5, in ethylene response pathway. Whether AAF function is involved in ethylene biosynthesis and/or the response pathway remained unknown. Here we show that neither overexpression (AAF-OX) nor a null mutant (aaf-KO) of AAF generates a higher level of ethylene than the wild type and display a typical triple-response phenotype in etiolated seedlings treated with 1-aminocyclopropane-1-carboxylic acid (ACC). Nevertheless, ein2-5 suppresses the phenotypes of early flowering and age-dependent leaf senescence in AAF-OX plants. We reveal that a functional ethylene response is essential for AAF function in leaf senescence and floral induction, but AAF is unlikely a regulatory component integral to the ethylene pathway.     

Suppression of edr2-mediated powdery mildew resistance, cell death and ethylene-induced senescence by mutations in ALD1 in Arabidopsis

EDR2 is a negative regulator of the defense response and cell death in Arabidopsis. Loss-of-function of EDR2 leads to enhanced resistance to powdery mildew. To identify new components in the EDR2 signal transduction pathway, mutations that suppress edr2 resistant phenotypes were screened. Three mutants, edts5-1, edts5-2 and edts5-3 (edr (t)wo (s)uppressor 5), were identified. The EDTS5 gene was identified by map-based cloning and previously was shown to encode an aminotransferase (ALD1). Therefore we renamed these three alleles ald1-10, ald1-11 and ald1-12, respectively. Mutations in ALD1 suppressed all edr2-mediated phenotypes, including powdery mildew resistance, programmed cell death and ethylene-induced senescence. Accumulation of hydrogen peroxide in edr2 was also suppressed by ald1 mutation. The expression of defense-related genes was up-regulated in the edr2 mutant, and the up-regulation of those genes in edr2 was suppressed in the edr2/ald1 double mutant. The ald1 single mutant displayed delayed ethylene-induced senescence. In addition, ald1 mutation suppressed edr1-mediated powdery mildew resistance, but could not suppress the edr1/edr2 double-mutant phenotype. These data demonstrate that ALD1 plays important roles in edr2-mediated defense responses and senescence, and revealed a crosstalk between ethylene and salicylic acid signaling mediated by ALD1 and EDR2.     

Distinct modulations of the hexokinase1-mediated glucose response and hexokinase1-independent processes by HYS1/CPR5 in Arabidopsis

The Arabidopsis mutant hypersenescence 1 (hys1), that is allelic to constitutive expresser of pathogenesis-related genes 5 (cpr5), displays phenotypes related to glucose signalling and defence responses. In the present study, it is shown that the hys1 mutation boosts the inhibitory effects of glucose upon the greening of seedlings and reduces the antagonistic activities of ethylene and cytokinin toward this inhibition. Neither the glucose content nor the sensitivities to ethylene, cytokinin, and abscisic acid were found to differ between wild-type and hys1 seedlings. However, disruption of the gene encoding hexokinase1 (HXK1), which acts as a glucose sensor, partially suppressed the glucose hypersensitive phenotype of the hys1 mutant. These results thus suggest that the hys1 mutation promotes a process associated with the HXK1-mediated glucose response during greening. By contrast, additional hys1 phenotypes, including an increase in salicylic acid (SA), production of abnormal trichomes, and early senescence, were not suppressed by the loss of HXK1. Surprisingly, the hxk1 and hys1 mutations acted synergistically towards an increased SA accumulation. Hence, HYS1/CPR5 appears to be a versatile protein that modulates both the HXK1-mediated glucose response and various HXK1-indepndent processes that are involved in growth control. A possible role for HYS1/CPR5 as a component of the networks that regulate growth control is discussed.     

EIN2 regulates salt stress response and interacts with a MA3 domain-containing protein ECIP1 in Arabidopsis

Ethylene signalling regulates plant growth and development. However, its roles in salt stress response are less known. Here we studied functions of EIN2, a central membrane protein of ethylene signalling, and its interacting protein ECIP1 in salt stress responses. Mutation of EIN2 led to extreme salt sensitivity as revealed by phenotypic and physiological changes, and overexpression of C-terminus of EIN2 suppressed salt sensitivity in ein2-5, indicating that EIN2 is required for salt tolerance. Downstream components EIN3 and EIL1 are also essential for salt tolerance because ein3-1eil1-1 double mutant showed extreme salt-sensitive phenotype. A MA3 domain-containing protein ECIP1 was further identified to interact with EIN2 in yeast two-hybrid assay and GST pull-down assay. Loss-of-function of ECIP1 resulted in enhanced ethylene response but altered salt response during seed germination and plant growth. Double mutant analysis revealed that ein2-1 was epistatic to ecip1, and ecip1 mutation partially suppressed ethylene-insensitivity of etr2-1 and ein4-1. These studies strengthen that interactions between ECIP1 and EIN2 or ethylene receptors regulate ethylene response and stress response.     

Ethylene-induced stomatal closure is mediated via MKK1/3–MPK3/6 cascade to EIN2 and EIN3

Mitogen-activated protein kinases (MPKs) play essential roles in guard cell signaling, but whether MPK cascades participate in guard cell ethylene signaling and interact with hydrogen peroxide (H₂O₂), nitric oxide (NO), and ethylene-signaling components remain unclear. Here, we report that ethylene activated MPK3 and MPK6 in the leaves of wild-type Arabidopsis thaliana as well as ethylene insensitive2 (ein2), ein3, nitrate reductase1 (nia1), and nia2 mutants, but this effect was impaired in ethylene response1 (etr1), nicotinamide adenine dinucleotide phosphate oxidase AtrbohF, mpk kinase1 (mkk1), and mkk3 mutants. By contrast, the constitutive triple response1 (ctr1) mutant had constitutively active MPK3 and MPK6. Yeast two-hybrid, bimolecular fluorescence complementation, and pull-down assays indicated that MPK3 and MPK6 physically interacted with MKK1, MKK3, and the C-terminal region of EIN2 (EIN2 CEND). mkk1, mkk3, mpk3, and mpk6 mutants had typical levels of ethylene-induced H₂O₂ generation but impaired ethylene-induced EIN2 CEND cleavage and nuclear translocation, EIN3 protein accumulation, NO production in guard cells, and stomatal closure. These results show that the MKK1/3–MPK3/6 cascade mediates ethylene-induced stomatal closure by functioning downstream of ETR1, CTR1, and H₂O₂ to interact with EIN2, thereby promoting EIN3 accumulation and EIN3-dependent NO production in guard cells.     

Identification of three genetic loci controlling leaf senescence in Arabidopsis thaliana

Four mutants that show the delayed leaf senescence phenotype were isolated from Arabidopsis thaliana . Genetic analyses revealed that they are all monogenic recessive mutations and fall into three complementation groups, identifying three genetic loci controlling leaf senescence in Arabidopsis . Mutations in these loci cause delay in all senescence parameters examined, including chlorophyll content, photochemical efficiency of photosystem II, relative amount of the large subunit of Rubisco, and RNase and peroxidase activity. Delay of the senescence symptoms was observed during both age-dependent in planta senescence and dark-induced artificial senescence in all of the mutant plants. The results indicate that the three genes defined by the mutations are key genetic elements controlling functional leaf senescence and provide decisive genetic evidence that leaf senescence is a genetically programmed phenomenon controlled by several monogenic loci in Arabidopsis . The results further suggest that the three genes function at a common step of age-dependent and dark-induced senescence processes. It is further shown that one of the mutations is allelic to ein2-1 , an ethylene-insensitive mutation, confirming the role of ethylene signal transduction pathway in leaf senescence of Arabidopsis .