Antisense suppression of phospholipase D alpha retards abscisic acid- and ethylene-promoted senescence of postharvest Arabidopsis leaves.

Membrane disruption has been proposed to be a key event in plant senescence, and phospholipase D (PLD; EC 3.1.4.4) has been thought to play an important role in membrane deterioration. We recently cloned and biochemically characterized three different PLDs from Arabidopsis. In this study, we investigated the role of the most prevalent phospholipid-hydrolyzing enzyme, PLD alpha, in membrane degradation and senescence in Arabidopsis. The expression of PLD alpha was suppressed by introducing a PLD alpha antisense cDNA fragment into Arabidopsis. When incubated with abscisic acid and ethylene, leaves detached from the PLD alpha-deficient transgenic plants showed a slower rate of senescence than did those from wild-type and transgenic control plants. The retardation of senescence was demonstrated by delayed leaf yellowing, lower ion leakage, greater photosynthetic activity, and higher content of chlorophyll and phospholipids in the PLD alpha antisense leaves than in those of the wild type. Treatment of detached leaves with abscisic acid and ethylene stimulated PLD alpha expression, as indicated by increases in PLD alpha mRNA, protein, and activity. In the absence of abscisic acid and ethylene, however, detached leaves from the PLD alpha-deficient and wild-type plants showed a similar rate of senescence. In addition, the suppression of PLD alpha did not alter natural plant growth and development. These data suggest that PLD alpha is an important mediator in phytohormone-promoted senescence in detached leaves but is not a direct promoter of natural senescence. The physiological relevance of these findings is discussed.     

Deferral of leaf senescence with calcium

In view of the possibility that senescence may be a consequence of the deterioration of membrane compartments in the cells of leaves, calcium was studied as a possible agent which might defer senescence. The senescence of corn leaf discs was deferred by added calcium, and the effect was additive to the cytokinin deferral of senescence. Likewise, the senescence of Rumex leaf discs was deferred by added calcium, and the effect was additive to the gibberellin deferral of senescence. Detailed experiments with corn leaf discs established that the increase in apparent free space associated with senescence was completely prevented by calcium. An increase in hydraulic permeability during senescence was likewise demonstrated, and this increase was deferred by calcium; calcium plus benzyladenine was even more effective. Each of the measured functions of leaf senescence (chlorophyll content, protein decrease, apparent free space increase, and hydraulic permeability increase) was suppressed by calcium, and the interpretation is offered that the effects are a consequence of the calcium function in maintaining cellular membranes.     

Lipid Profiling Demonstrates That Suppressing Arabidopsis Phospholipase Dδ Retards ABA-Promoted Leaf Senescence by Attenuating Lipid Degradation

Senescence is the last phase of the plant life cycle and has an important role in plant development. Degradation of membrane lipids is an essential process during leaf senescence. Several studies have reported fundamental changes in membrane lipids and phospholipase D (PLD) activity as leaves senesce. Suppression of phospholipase Dα1 (PLDα1) retards abscisic acid (ABA)-promoted senescence. However, given the absence of studies that have profiled changes in the compositions of membrane lipid molecules during leaf senescence, there is no direct evidence that PLD affects lipid composition during the process. Here, we show that application of n-butanol, an inhibitor of PLD, and N-Acylethanolamine (NAE) 12∶0, a specific inhibitor of PLDα1, retarded ABA-promoted senescence to different extents. Furthermore, phospholipase Dδ (PLDδ) was induced in leaves treated with ABA, and suppression of PLDδ retarded ABA-promoted senescence in Arabidopsis. Lipid profiling revealed that detachment-induced senescence had different effects on plastidic and extraplastidic lipids. The accelerated degradation of plastidic lipids during ABA-induced senescence in wild-type plants was attenuated in PLDδ-knockout (PLDδ-KO) plants. Dramatic increases in phosphatidic acid (PA) and decreases in phosphatidylcholine (PC) during ABA-induced senescence were also suppressed in PLDδ-KO plants. Our results suggest that PLDδ-mediated hydrolysis of PC to PA plays a positive role in ABA-promoted senescence. The attenuation of PA formation resulting from suppression of PLDδ blocks the degradation of membrane lipids, which retards ABA-promoted senescence.     

Delayed Leaf Senescence in Tobacco Plants Transformed with tmr,a Gene for Cytokinin Production in Agrobacterium

The aim of this study was to investigate whether enhanced levels of endogenous cytokinins could influence plant development, particularly leaf senescence. Tobacco plants were transformed with the Agrobacterium tumefaciens gene tmr, under the control of the soybean heat shock promoter HS6871. This gene encodes the enzyme isopentenyl transferase, which catalyzes the initial step in cytokinin biosynthesis. After heat shock, the cytokinin level increased greatly and the level of tmr mRNA, undetectable at 20[deg]C, rose and remained high for up to 8 hours. The levels of cytokinin and tmr mRNA were substantially lower by 24 hours. Transformed plants grown at 20[deg]C were shorter, had larger side shoots, and remained green for longer than untransformed plants. The differences were more pronounced after several heat shocks of whole plants or defined areas of leaves. Our results demonstrated that plant morphology and leaf senescence can be manipulated by changing the endogenous level of cytokinins.     

Studies on the mechanisms of ozone tolerance: Cytokinin-like activity of N-[2-(2-oxo-1-imidazolidinyl)ethyl]-N'-phenylurea, a compound protecting against ozone injury

The cytokinin-like activity of the growth regulating chemical EDU, N-[2-(2-oxo-1-imidazolidinyl)ethyl]-N'-phenylurea, was determined and compared with the actitivity of kinetin using the tobacco callus bioassay. EDU has a pronounced stimulatory effect on callus growth at concentrations of 5 × 10⁻⁴ and 1 × 10⁻³ M but was 5 000 times less potent than the synthetic cytokinin, kinetin. Senescence regulation and oxidant resistance induced by EDU and kinetin were also studied. EDU retarded the breakdown of chlorophyll, protein and RNA in 0₃-sensitive tobacco leaf discs during senescence. EDU was much more effective in arresting senescence and in protecting against 0₃ injury than kinetin. Results indicate the EDU-induced plant tolerance to 0₃ phytotoxicity may be indirect through enzyme induction regulation.     

Arabidopsis KNOXI proteins activate cytokinin biosynthesis

Plant architecture is shaped through the continuous formation of organs by meristems. Class I KNOTTED1-like homeobox (KNOXI) genes are expressed in the shoot apical meristem (SAM) and are required for SAM maintenance. KNOXI proteins and cytokinin, a plant hormone intimately associated with the regulation of cell division, share overlapping roles, such as meristem maintenance and repression of senescence, but their mechanistic and hierarchical relationship have yet to be defined. Here, we show that activation of three different KNOXI proteins using an inducible system resulted in a rapid increase in mRNA levels of the cytokinin biosynthesis gene isopentenyl transferase 7 (AtIPT7) and in the activation of ARR5, a cytokinin response factor. We further demonstrate a rapid and dramatic increase in cytokinin levels following activation of the KNOXI protein SHOOT MERISTEMLESS (STM). Application of exogenous cytokinin or expression of a cytokinin biosynthesis gene through the STM promoter partially rescued the stm mutant. We conclude that activation of cytokinin biosynthesis mediates KNOXI function in meristem maintenance. KNOXI proteins emerge as central regulators of hormone levels in plant meristems.     

Effects of cytokinin and senescence-inducing factors on expression of P ARR5 -GUS gene construct during leaf senescence in transgenic Arabidopsis thaliana plants

ARR5-gene expression was studied in the course of natural leaf senescence and detached leaf senescence in the dark using Arabidopsis thaliana plants transformed with the P _ARR5 -GUS gene construct. GUS-activity was measured as a marker of ARR5-gene expression. Chlorophyll and total protein amounts were also estimated to evaluate leaf senescence. Natural leaf senescence was accompanied by the progressive decline in the GUS-activity in leaves of the 2nd and 3rd nodes studied, and this shift of GUS-activity was more pronounced than the loss of chlorophyll content. The ability of the ARR5-gene promoter to respond to cytokinin was not eliminated during natural leaf senescence, as was demonstrated by a cytokinin-induced increase in GUS activity in leaves after their detachment and incubation on benzyladenine (BA, 5 × 10⁻⁶ M) in the dark. Leaf senescence in the dark was associated with the further decrease in the GUS-activity. The ARR5-gene promoter response to cytokinin was enhanced with the increase of the age of plants, taken as a source of leaves for cytokinin treatments. Hence, although the expression of the ARR5 gene reduces during natural and dark/detached leaf senescence, the ARR5-gene sensitivity to cytokinin was maintained in both cases and even increased with the leaf age. This data suggest that the ARR5 gene, which belongs to the type-A negative regulators of plant response to cytokinin, could be a feedback regulator able to prevent retardation by cytokinin of leaf senescence when it is important for plant life. Growth regulators either reduced ARR5 gene response to cytokinin during senescence of mature detached leaves in the dark (SA, meJA, ABA, SP) or increased it (IAA), thus modifying the resulting rate of its expression.     

植物磷脂酶D基因表达与衰老的关系

磷脂酶D (PLD)是一种重要的磷脂水解酶,在植物细胞中普遍存在。磷脂酶D能激活许多重要的细胞生理功能,包括调控细胞膜的重建、跨膜信号传导及细胞内调控、细胞骨架组装、防御反应以及种子萌发和植物的衰老等。对磷脂酶D的基本特性、磷脂酶D基因特异性表达模式及其活性抑制与植物衰老的关系进行了综述,并探讨和展望了今后植物磷脂酶D基因的研究方向。     

Exogenous Jasmonic Acid and Cytokinin Antagonistically Regulate Rice Flag Leaf Senescence by Mediating Chlorophyll Degradation,Membrane Deterioration,and Senescence-Associated Genes Expression

Although it is well known that jasmonic acid (JA) and cytokinin (CK) are involved in regulating leaf senescence, the antagonistic mechanisms of JA and CK on leaf senescence are still unknown. To explore the antagonistic effects of JA and CK on leaf senescence, we treated detached rice flag leaves with JA and CK under dark conditions, and evaluated their chlorophyll contents, membrane deterioration, and expression levels of chlorophyll-degradation-related genes (CDRGs) and senescence-associated genes (SAGs). Our results demonstrated that exogenous application of JA promoted chlorophyll degradation by enhancing the expression levels of CDRGs, promoted membrane deterioration by accelerating the increases in lipid peroxidation and membrane permeability, enhanced the expression levels of SAGs, and consequently accelerated rice flag leaf senescence. On the other hand, exogenous application of CK retarded chlorophyll degradation by down-regulating the expression levels of CDRGs, retarded membrane deterioration by retarding the increases in lipid peroxidation and membrane permeability, down-regulated the expression levels of SAGs, and consequently delayed rice flag leaf senescence. Furthermore, the senescence-accelerating effect of a certain concentration of JA was nullified by the senescence-retarding effect of a certain concentration of CK. These results suggested that exogenous applications of JA and CK were able to antagonistically regulate flag leaf senescence by mediating chlorophyll degradation, membrane deterioration, and SAGs expression. In addition, our results suggested that the progression of flag leaf senescence might not only depend on the level of JA or CK but also depend on the balance between JA and CK.     

Root-synthesized cytokinins improve shoot growth and fruit yield in salinized tomato (Solanum lycopersicum L.) plants

Salinity limits crop productivity, in part by decreasing shoot concentrations of the growth-promoting and senescence-delaying hormones cytokinins. Since constitutive cytokinin overproduction may have pleiotropic effects on plant development, two approaches assessed whether specific root-localized transgenic IPT (a key enzyme for cytokinin biosynthesis) gene expression could substantially improve tomato plant growth and yield under salinity: transient root IPT induction (HSP70::IPT) and grafting wild-type (WT) shoots onto a constitutive IPT-expressing rootstock (WT/35S::IPT). Transient root IPT induction increased root, xylem sap, and leaf bioactive cytokinin concentrations 2- to 3-fold without shoot IPT gene expression. Although IPT induction reduced root biomass (by 15%) in control (non-salinized) plants, in salinized plants (100?mM NaCl for 22?d), increased cytokinin concentrations delayed stomatal closure and leaf senescence and almost doubled shoot growth (compared with WT plants), with concomitant increases in the essential nutrient K(+) (20%) and decreases in the toxic ion Na(+) (by 30%) and abscisic acid (by 20-40%) concentrations in transpiring mature leaves. Similarly, WT/35S::IPT plants (scion/rootstock) grown with 75?mM NaCl for 90?d had higher fruit trans-zeatin concentrations (1.5- to 2-fold) and yielded 30% more than WT/non-transformed plants. Enhancing root cytokinin synthesis modified both shoot hormonal and ionic status, thus ameliorating salinity-induced decreases in growth and yield.     

Effects of kinetin on phosphorylation of leaf membrane proteins.

Isolated Chinese cabbage leaf membranes were phosphorylated by membrane-associated protein kinase(s) in the presence or [gamma-32P]ATP. Membrane-associated 32P radioactivity appeared to be bound to membrane proteins. Both smooth cell membranes and chloroplast lamellae reacted with ATP. Phosphorylation of the membranes was inhibited by Ca2+ and partially inhibited by kinetin or 6-benzyladenine. The possibility that cytokinin effects on membrane phosphorylation might increase ion availability was investigated in vivo. It was found that Ca2+ could substitute for kinetin in the leaf disc expansion assay.     

Interrelations between Gibberellic Acid, Cytokinins and Abscisic Acid in Retarding Leaf Senescence

The interrelation between the effects of abscisic acid (ABA) and the effects of cytokinins and gibberellic acid in retarding leaf senescence was investigated. Leaf discs from plants of Taraxacum megallorrhizon, Rumex pulcber and Tropaeolum majus were floated on solutions of cytokinin or GA to which given amounts of ABA were added. After five days, chlorophyll was extracted and the amount estimated spectrophoto-metrically. The interrelation between the effects of abscisic acid and cytokinins differed from that between the effects of ABA and gibberellic acid. Abscisic acid reduced the senescence retarding effect of GA more than that of cytokinins. A high concentration of cytokinins nullified the senescence enhancing effect of low concentrations of ABA. GA did not reverse the effects of ABA.     

Cytokinins in plant senescence: From spray and pray to clone and play

Three approaches have been used to investigate the inhibitory role of the cytokinin class of phytohormones in plant senescence: external application of cytokinins, measurement of endogenous cytokinin levels before and during senescence, and manipulation of endogenous cytokinin production in transgenic plants. In transgenic plant studies, endogenous cytokinin levels are manipulated by expression of IPT, a gene encoding isopentenyl transferase. Transgenic plants expressing IPT from a variety of promoters exhibit developmental and morphological alterations and often display retarded leaf senescence. A recently developed autoregulatory senescence-inhibition system targets cytokinin production quantitatively, spatially and temporally, and results in transgenic plants that exhibit significantly delayed senescence without abnormalities. These transgenic studies not only confirm the regulatory role of cytokinins in plant senescence, but also provide a way to manipulate senescence for potential agricultural applications.     

Contrasting patterns of cytokinins between years in senescing aspen leaves

Cytokinins are plant hormones that typically block or delay leaf senescence. We profiled 34 different cytokinins/cytokinin metabolites (including precursors, conjugates and degradation products) in leaves of a free‐growing mature aspen (Populus tremula) before and after the initiation of autumnal senescence over three consecutive years. The levels and profiles of individual cytokinin species, or classes/groups, varied greatly between years, despite the fact that the onset of autumn senescence was at the same time each year, and senescence was not associated with depletion of either active or total cytokinin levels. Levels of aromatic cytokinins (topolins) were low and changed little over the autumn period. Diurnal variations and weather‐dependent variations in cytokinin content were relatively limited. We also followed the expression patterns of all aspen genes implicated as having roles in cytokinin metabolism or signalling, but neither the pattern of regulation of any group of genes nor the expression of any particular gene supported the notion that decreased cytokinin signalling could explain the onset of senescence. Based on the results from this tree, we therefore suggest that cytokinin depletion is unlikely to explain the onset of autumn leaf senescence in aspen.     

Cloning and characterization of tomato leaf senescence-related cDNAs

Senescence-related cDNA clones designated SENU1, 4, 5 (senescence up-regulated) and SEND32, 33, 34, 35 and 36 (senescence down-regulated) isolated from a tomato leaf cDNA library [9] were characterized. Southern analysis showed that SEND32 is encoded by a single-copy gene while SEND33, 34, 35, 36 and SENU1 and SENU5 are members of small gene families. DNA and protein database searches revealed that SEND32, SEND35, SENU1 and SENU5 are novel cDNAs of unknown function. SEND33 encodes ferredoxin, SEND34 encodes a photosystem II 10 kDa polypeptide and SEND36 encodes catalase. The SENU4 sequence is identical to the P6 tomato protein previously reported to be pathogenesis-related [46]. The mRNA levels of SENU1, 4 and 5 increased during leaf senescence and SENU1 and SENU5 were also expressed at high levels during leaf development and in other plant organs. The SENU4 mRNA was associated more specifically with leaf senescence, although low expression was also detected in green fruit. The mRNAs for all SEND clones decreased during tomato leaf development and senescence and all except SEND32 were expressed at low levels in other plant organs. The accumulation of mRNA homologous to SENU4 and the decrease in abundance of SEND32 provide good molecular markers for leaf senescence.     

The effect of plant cytokinin hormones on the production of ethylene, nitric oxide, and protein nitrotyrosine in ageing tobacco leaves

Transgenic plants with genetically increased or decreased levels of cytokinins were used to investigate the effect of cytokinin level on the production of ethylene, a plant hormone with suggested role in senescence, and the production of nitric oxide, potentially important signalling and regulatory molecule. The production of these gases was followed during the course of leaf development and senescence. The production of ethylene and nitric oxide is under genetic control of genes other than those involved in regulation of senescence. The difference in basic ethylene and NO levels in different tobacco cultivars was higher than their changes in senescence. The results of this study did not indicate a direct link between ethylene production and cytokinin levels. However, there was a decreased production of NO in senescent leaves. Low cytokinins level was associated with increased NO production during leaf development. Protein nitrotyrosine proved to be a better indicator of the reactive nitrogen species than measuring of the NO production. Higher nitrotyrosine concentrations were found in insoluble proteins than in the soluble ones, pointing to membrane proteins as the primary targets of the reactive nitrogen species. In plants with elevated cytokinin levels the content of nitrated proteins decreased both in soluble and insoluble fractions. This finding indicates an antioxidative function of cytokinins against reactive nitrogen species.     

Leaf senescence is delayed in tobacco plants expressing the maize homeobox gene knotted1 under the control of a senescence-activated promoter.

Leaf senescence is an active process involving remobilization of nutrients from senescing leaves to other parts of the plant. Whereas senescence is accompanied by a decline in leaf cytokinin content, supplemental cytokinin delays senescence. Plants that overexpress isopentenyl transferase (ipt), a cytokinin-producing gene, or knotted1 (kn1), a homeobox gene, have many phenotypes in common. Many of these phenotypes are characteristic of altered cytokinin physiology. The effect of kn1 on leaf senescence was tested by driving its expression using the promoter of the senescence-associated gene SAG12. SAG:kn1 tobacco plants showed a marked delay in leaf senescence but otherwise developed normally. The delay in senescence was revealed by an increase in chlorophyll content in SAG:kn1 leaves relative to leaves of the control plants and by a decrease in the number of dead leaves. Senescence was also delayed in detached leaves of SAG:kn1 plants. Delayed senescence was accompanied by increased leaf cytokinin content in older leaves expressing kn1. These experiments extend the current understanding of kn1 function and suggest that in addition to mediating meristem maintenance, kn1 is capable of regulating the onset of senescence in leaves.     

Characterisation of a cysteine protease cDNA from Lolium multiflorum leaves and its expression during senescence and cytokinin treatment

A cysteine protease cDNA clone (See1) highly homologous to barley aleurain was isolated from Lolium multiflorum leaves. During leaf senescence, expression of the See1 mRNA and protein was strongly enhanced. In dark-incubated leaf segments, cytokinin delayed senescence and reduced expression of both See1 mRNA and protein.