A transcriptome‐wide study on the microRNA‐ and the Argonaute 1‐enriched small RNA‐mediated regulatory networks involved in plant leaf senescence

Leaf senescence is an important physiological process during the plant life cycle. However, systemic studies on the impact of microRNAs (miRNAs) on the expression of senescence‐associated genes (SAGs) are lacking. Besides, whether other Argonaute 1 (AGO1)‐enriched small RNAs (sRNAs) play regulatory roles in leaf senescence remains unclear. In this study, a total of 5,123 and 1,399 AGO1‐enriched sRNAs, excluding miRNAs, were identified in Arabidopsis thaliana and rice (Oryza sativa), respectively. After retrieving SAGs from the Leaf Senescence Database, all of the AGO1‐enriched sRNAs and the miRBase‐registered miRNAs of these two plants were included for target identification. Supported by degradome signatures, 200 regulatory pairs involving 120 AGO1‐enriched sRNAs and 40 SAGs, and 266 regulatory pairs involving 64 miRNAs and 42 SAGs were discovered in Arabidopsis. Moreover, 13 genes predicted to interact with some of the above‐identified target genes at protein level were validated as regulated by 17 AGO1‐enriched sRNAs and ten miRNAs in Arabidopsis. In rice, only one SAG was targeted by three AGO1‐enriched sRNAs, and one SAG was targeted by miR395. However, five AGO1‐enriched sRNAs were conserved between Arabidopsis and rice. Target genes conserved between the two plants were identified for three of the above five sRNAs, pointing to the conserved roles of these regulatory pairs in leaf senescence or other developmental procedures. Novel targets were discovered for three of the five AGO1‐enriched sRNAs in rice, indicating species‐specific functions of these sRNA–target pairs. These results could advance our understanding of the sRNA‐involved molecular processes modulating leaf senescence.     

Ammonium accumulation is associated with senescence of rice leaves

The relationship between ammonium accumulation and senescence of detached rice leaves was investigated. Ammonium accumulation in detached rice leaves coincided closely with dark-induced senescence. Exogenous NH4Cl and methionine sulfoximine, which caused an accumulation of ammonium in detached rice leaves, promoted senescence. Treatments such as light and benzyladenine, which retarded senescence, decreased ammonium level in detached rice leaves. Abscisic acid, which promoted senescence, increased ammonium level in detached rice leaves. The current results suggest that ammonium accumulation may be involved in regulating senescence. Evidence was presented to show that ammonium accumulated in detached rice leaves increases tissue sensitivity to ethylene. The accumulation of ammonium in detached rice leaves during dark-induced senescence is attributed to a decrease in glutamine synthetase activity and an increase in reduction of nitrate.     

Ethylene regulates the timing of leaf senescence in Arabidopsis

The plant hormone ethylene influences many aspects of plant growth and development, including some specialized forms of programmed senescence such as fruit ripening and flower petal senescence. To study the relationship between ethylene and leaf senescence, etr1-1, an ethylene-insensitive mutant in Arabidopsis, was used. Comparative analysis of rosette leaf senescence between etr1-1 and wild-type plants revealed that etr1-1 leaves live approximately 30% longer than the wild-type leaves. Delayed leaf senescence in etr1-1 coincided with delayed induction of senescence-associated genes (SAGs) and higher expression levels of photosynthesis-associated genes (PAGs). In wild-type plants, exogenous ethylene was able to further accelerate induction of SAGs and decrease expression of PAGs. The extended period of leaf longevity in etr1-1 was associated with low levels of photosynthetic activity. Therefore, the leaves in etr1-1 functionally senesced even though the apparent life span of the leaf was prolonged.     

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 .     

Expression of the beta-oxidation gene 3-ketoacyl-CoA thiolase 2 (KAT2) is required for the timely onset of natural and dark-induced leaf senescence in Arabidopsis

The onset of leaf senescence is regulated by a complex mechanism involving positive and negative regulators. Among positive regulators, jasmonic acid (JA) accumulates in senescing leaves and the JA-insensitive coi1-1 mutant displays delayed leaf senescence in Arabidopsis. A strong activated expression of the gene coding for the JA-biosynthetic beta-oxidation enzyme 3-ketoacyl-CoA thiolase 2 (KAT2) in natural and dark-induced senescing leaves of Arabidopsis thaliana is reported here. By using KAT2::GUS and KAT2::LUC transgenic plants, it was observed that dark-induced KAT2 activation occurred both in excised leaves as well as in whole darkened plants. The KAT2 activation associated with dark-induced senescence occurred soon after a move to darkness, and it preceded the detection of symptoms and the expression of senescence-associated gene (SAG) markers. Transgenic plants with reduced expression of the KAT2 gene showed a significant delayed senescence both in natural and dark-induced processes. The rapid induction of the KAT2 gene in senescence-promoting conditions as well as the delayed senescence phenotype and the reduced SAG expression in KAT2 antisense transgenic plants, point to KAT2 as an essential component for the timely onset of leaf senescence in Arabidopsis.     

Strigolactone signaling regulates rice leaf senescence in response to a phosphate deficiency

Strigolactones (SLs) act as plant hormones that inhibit shoot branching and stimulate secondary growth of the stem, primary root growth, and root hair elongation. In the moss Physcomitrella patens, SLs regulate branching of chloronemata and colony extension. In addition, SL-deficient and SL-insensitive mutants show delayed leaf senescence. To explore the effects of SLs on leaf senescence in rice (Oryza sativa L.), we treated leaf segments of rice dwarf mutants with a synthetic SL analogue, GR24, and evaluated their chlorophyll contents, ion leakage, and expression levels of senescence-associated genes. Exogenously applied GR24 restored normal leaf senescence in SL-deficient mutants, but not in SL-insensitive mutants. Most plants highly produce endogenous SLs in response to phosphate deficiency. Thus, we evaluated effects of GR24 under phosphate deficiency. Chlorophyll levels did not differ of in the wild-type between the sufficient and deficient phosphate conditions, but increased in the SL-deficient mutants under phosphate deficiency, leading in the strong promotion of leaf senescence by GR24 treatment. These results indicate that the mutants exhibited increased responsiveness to GR24 under phosphate deficiency. In addition, GR24 accelerated leaf senescence in the intact SL-deficient mutants under phosphate deficiency as well as dark-induced leaf senescence. The effects of GR24 were stronger in d10 compared to d17. Based on these results, we suggest that SLs regulate leaf senescence in response to phosphate deficiency.     

Convergence and divergence in gene expression profiles induced by leaf senescence and 27 senescence-promoting hormonal, pathological and environmental stress treatments

In addition to age and developmental progress, leaf senescence and senescence-associated genes (SAGs) can be induced by other factors such as plant hormones, pathogen infection and environmental stresses. The relationship is not clear, however, between these induced senescence processes and developmental leaf senescence, and to what extent these senescence-promoting signals mimic age and developmental senescence in terms of gene expression profiles. By analysing microarray expression data from 27 different treatments (that are known to promote senescence) and comparing them with that from developmental leaf senescence, we were able to show that at early stages of treatments, different hormones and stresses showed limited similarity in the induction of gene expression to that of developmental leaf senescence. Once the senescence process is initiated, as evidenced by visible yellowing, generally after a prolonged period of treatments, a great proportion of SAGs of developmental leaf senescence are shared by gene expression profiles in response to different treatments. This indicates that although different signals that lead to initiation of senescence may do so through distinct signal transduction pathways, senescence processes induced either developmentally or by different senescence-promoting treatments may share common execution events.     

Hormonal regulation of leaf senescence in Lilium

In addition to floral senescence and longevity, the control of leaf senescence is a major factor determining the quality of several cut flowers, including Lilium, in the commercial market. To better understand the physiological process underlying leaf senescence in this species, we evaluated: (i) endogenous variation in the levels of phytohormones during leaf senescence, (ii) the effects of leaf darkening in senescence and associated changes in phytohormones, and (iii) the effects of spray applications of abscisic acid (ABA) and pyrabactin on leaf senescence. Results showed that while gibberellin 4 (GA(4)) and salicylic acid (SA) contents decreased, that of ABA increased during the progression of leaf senescence. However, dark-induced senescence increased ABA levels, but did not affect GA(4) and SA levels, which appeared to correlate more with changes in air temperature and/or photoperiod than with the induction of leaf senescence. Furthermore, spray applications of pyrabactin delayed the progression of leaf senescence in cut flowers. Thus, we conclude that (i) ABA plays a major role in the regulation of leaf senescence in Lilium, (ii) darkness promotes leaf senescence and increases ABA levels, and (iii) exogenous applications of pyrabactin inhibit leaf senescence in Lilium, therefore suggesting that it acts as an antagonist of ABA in senescing leaves of cut lily flowers.     

Rice tillering dwarf mutant dwarf3 has increased leaf longevity during darkness-induced senescence or hydrogen peroxide-induced cell death

Senescence or cell death in plant leaves is known to be inducible by darkness or H(2)O(2). When the Arabidopsis gene MAX2/ORE9 is disrupted, leaf senescence or cell death in response to the above stimuli is delayed. Because the rice (Oryza sativa L.) gene DWARF3 (D3) is orthologous to MAX2/ORE9, we wished to know whether disruption of D3 also results in increased longevity in leaves. We found that darkness-induced senescence or H(2)O(2)-induced cell death in the third leaf [as measured by chlorophyll degradation, membrane ion leakage and expression of senescence-associated genes (SAGs)] in a d3 rice mutant was delayed by 1-3 d compared to that in its reference line Shiokari. Moreover, the mRNA levels of D3, HTD1 and D10, which are orthologs of Arabidopsis MAX2/ORE9, MAX3 and MAX4, respectively, increased during cell death. These results suggest that D3 protein in rice, like MAX2/ORE9 in Arabidopsis, is involved in leaf senescence or cell death.     

Quantitative trait loci mapping of dark-induced senescence in winter wheat (Triticum aestivum)

In order to explore the genetics of dark-induced senescence in winter wheat(Triticum aestivum L.),a quantitative trait loci(QTL)analysis was carried out in a doubled haploid population developed from a cross between the varieties Hanxuan 10(HX)and Lumai 14(LM).The senescence parameters chlorophyll content(Chl a+b,Chl a,and Chl b),original fluorescence(Fo),maximum fluorescence level(Fm),maximum photochemical efficiency(Fv/Fm),and ratio of variable fluorescence to original fluorescence(Fv/Fo)were evaluated in the second leaf of whole three-leaf seedlings subjected to 7 d of darkness.A total of 43 QTLs were identified that were associated with dark-induced senescence using composite interval mapping.These QTLs were mapped to 20 loci distributed on 11 chromosomes:1B,1D,2A,2B,3B,3D,5D,6A,6B,7A,and 7B.The phenotypic variation explained by each QTL ranged from 7.5% to 19.4%.Eleven loci coincided with two or more of the analyzed parameters.In addition,14 loci co-located or were linked with previously reported QTLs regulating flag leaf senescence,tolerance to high light stress,and grain protein content(Gpc),separately.     

Expression of a nitric oxide degrading enzyme induces a senescence programme in Arabidopsis

Nitric oxide (NO) has been proposed to act as a factor delaying leaf senescence and fruit maturation in plants. Here we show that expression of a NO degrading dioxygenase (NOD) in Arabidopsis thaliana initiates a senescence-like phenotype, an effect that proved to be more pronounced in older than in younger leaves. This senescence phenotype was preceded by a massive switch in gene expression in which photosynthetic genes were down-regulated, whereas many senescence-associated genes (SAGs) and the 1-aminocyclopropane-1-carboxylic acid (ACC) synthase gene ACS6 involved in ethylene synthesis were up-regulated. External fumigation of NOD plants with NO as well as environmental conditions known to stimulate endogenous NO production attenuated the induced senescence programme. For instance, both high light conditions and nitrate feeding reduced the senescence phenotype and attenuated the down-regulation of photosynthetic genes as well as the up-regulation of SAGs. Treatment of plants with the cytokinin 6-benzylaminopurin (BAP) reduced the down-regulation of photosynthesis, although it had no consistent effect on SAG expression. Metabolic changes during NOD-induced senescence comprehended increases in salicylic acid (SA) levels, accumulation of the phytoalexin camalexin and elevation of leaf gamma-tocopherol contents, all of which occurred during natural senescence in Arabidopsis leaves as well. Moreover, NO fumigation delayed the senescence process induced by darkening individual Arabidopsis Columbia-0 (Col-0) leaves. Our data thus support the notion that NO acts as a negative regulator of leaf senescence.     

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.