Senescence-Inducible Expression of Isopentenyl Transferase Extends Leaf Life, Increases Drought Stress Resistance and Alters Cytokinin Metabolism in Cassava

Cassava (Manihot esculenta Crantz) sheds its leaves during growth, especially within the tropical dry season. With the production of SAG12-IPT transgenic cassava we want to test the level of leaf retention and altered cytokinin metabolism of transgenic plants via the autoregulatory senescence inhibition system. After confirmation of transgene expression by molecular analysis and phenotype examination in greenhouse plants, two transgenic plant lines, 529-28 and 529-48, were chosen for further investigation. Detached mature leaves of 529-28 plants retained high levels of chlorophyll compared with wild-type leaves after dark-induced senescence treatment. Line 529-28 showed significant drought tolerance as indicated by stay-green capacity after drought stress treatment. Field experiments proved that leaf senescence syndrome was significantly delayed in 529-28 plants in comparison with wild-type and 529-48 plants. Physiological and agronomical characterizations of these plants also revealed that the induced expression of IPT had effects on photosynthesis, sugar allocation and nitrogen partitioning. Importantly, the 529-28 plants accumulated a high level of trans-zeatin-type cytokinins particularly of corresponding storage O-glucosides to maintain cytokinin homeostasis. Our study proves the feasibility of prolonging the leaf life of woody cassava and also sheds light on the control of cytokinin homeostasis in cassava leaves.     

转基因(SAG12-IPT)青菜的迟衰特性

利用根癌农杆菌感染方法将融合基因SAG12 IPT导入青菜 ,转基因植株明显表现出衰老延迟的生理现象。SAG12 IPT的抗衰老作用表现为 :在衰老过程中转基因青菜叶片中叶绿素含量高于未转基因的青菜 ,PCR分析结果表明该融合基因已经转入青菜中。激素检测结果表明转基因青菜叶片中细胞分裂素含量高于未转基因植株 ,说明抗衰老与叶片内细胞分裂素含量提高有关。另外 ,转基因植株不仅表现出活体植株衰老延迟 ,而且长在植株上的与离体的叶片滞绿时间延长。这些为蔬菜的耐储存育种提供了新的思路 ,同时为该融合基因在十字花科经济作物中的应用提供了理论依据。     

Functional significance of shade-induced leaf senescence in dense canopies: an experimental test using transgenic tobacco

Canopy photosynthesis models have predicted an optimal leaf area index (LAI; leaf area per unit surface area) and leaf nitrogen distribution at which whole-plant carbon gain per unit N is maximized. In this study we experimentally tested these models, using transgenic P(SAG12)-IPT tobacco (SAG; Nicotiana tabacum L.) plants with delayed leaf senescence and therefore a greater LAI and more uniform N distribution than the wild type (WT). In a competition experiment, the increased density of surrounding WT plants caused a greater reduction in dry mass of mature SAG target plants than in that of WT target plants, indicating negative effects of delayed leaf senescence on performance at high canopy density. Vegetative SAG plants achieved a lower calculated daily carbon gain than competing WT plants because the former retained leaves with a negative carbon gain in the shaded, lower part of the canopy. Sensitivity analyses showed that the carbon gain of SAG plants would increase if these lower leaves were shed and the N reallocated from these leaves were used to form additional leaf area at the canopy top. This strategy, which is adopted by the WT, is most advantageous because it results in the shading of competing neighbors.     

Mechanisms of the light-dependent induction of cell death in tobacco plants with delayed senescence

The relationship between leaf senescence and cell death was investigated using tobacco with delayed senescence due to auto-regulated production of cytokinin (SAG12-IPT). Although leaf senescence ultimately results in cell death, the results show that senescence and cell death can be uncoupled: in nutrient-deficient, but not in fertilized SAG12-IPT plants, necrotic lesions were detected in old, but otherwise green leaves. By contrast, wild-type leaves of the same age were yellow, but not necrotic. Chlorophyll fluorescence analysis revealed an over-reduction of the electron transport chain in old SAG12-IPT leaves, in combination with characteristic spatial patterns of minimum fluorescence (F0) quantum efficiency of open photosystem II centres (F(v)/F(m)) and non-photochemical quenching (NPQ), as determined by fluorescence imaging. The same patterns of F0, F(v)/F(m), and NPQ were induced by incubation of leaf discs from nutrient-deficient SAG12-IPT plants under illumination, but not in the dark, indicating that light-dependent reactions were responsible for the cell death. RT-PCR analysis showed that the pathogenesis-related (PR) genes PR-1b and PR-Q were strongly induced in old SAG12-IPT tobacco leaves with necrotic lesions. In addition, the ethylene-synthesis gene ACO was induced before lesions became visible in SAG12-IPT. It is proposed that over-reduction of the electron transport chain in combination with decreased electron consumption due to nutrient-deficiency led to oxidative stress, which, mediated by ethylene formation, can induce PR gene expression and hypersensitive cell death. Probably as a consequence of inefficient nutrient mobilization, flower development was prematurely aborted and reproduction thereby impaired in nutrient-deficient SAG12-IPT plants.     

Metabolite fingerprinting in transgenic lettuce

Metabolite fingerprinting has been achieved using direct atmospheric pressure chemical ionization-mass spectrometry (APCI-MS) and linked gas chromatography (GC-APCI/EI-MS) for transgenic lettuce (Lactuca sativa L. cv. Evola) plants expressing an IPT gene under the control of the senescence-specific SAG12 promoter from Arabidopsis thaliana (P(SAG12)-IPT). Mature heads of transgenic lettuce and their azygous controls were maintained under defined conditions to assess their shelf life. Transgenic lettuce plants exhibited delayed senescence and significant increases (up to a maximum of threefold) in the concentrations of three volatile organic compounds (VOCs), corresponding to molecular masses of 45, 47 and 63, when compared with heads from azygous plants. These VOCs were identified as acetaldehyde (45), ethanol (47) and dimethyl sulphide (63). The increase in dimethyl sulphide was paralleled by an accumulation of reactive oxygen species (ROS) in the heads of transgenic plants. These results demonstrate the applicability of metabolic fingerprinting techniques to elucidate the underlying pleiotropic responses of plants to transgene expression.     

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.     

Senescence-induced ectopic expression of the A. tumefaciens ipt gene in wheat delays leaf senescence, increases cytokinin content, nitrate influx, and nitrate reductase activity, but does not affect grain yield

The manipulation of cytokinin levels by senescence-regulated expression of the Agrobacterium tumefaciens ipt gene through its control by the Arabidopsis SAG12 (senescence-associated gene 12) promoter is an efficient tool for the prolongation of leaf photosynthetic activity which potentially can affect plant productivity. In the present study, the efficiency of this approach was tested on wheat (Triticum aestivum L.)-a monocarpic plant characterized by a fast switch from vegetative to reproductive growth, and rapid translocation of metabolites from leaves to developing grains after anthesis. When compared with the wild-type (WT) control plants, the SAG12::ipt wheat plants exhibited delayed chlorophyll degradation only when grown under limited nitrogen (N) supply. Ten days after anthesis the content of chlorophyll and bioactive cytokinins of the first (flag) leaf of the transgenic plants was 32% and 65% higher, respectively, than that of the control. There was a progressive increase in nitrate influx and nitrate reductase activity. However, the SAG12::ipt and the WT plants did not show differences in yield-related parameters including number of grains and grain weight. These results suggest that the delay of leaf senescence in wheat also delays the translocation of metabolites from leaves to developing grains, as indicated by higher accumulation of ((15)N-labelled) N in spikes of control compared with transgenic plants prior to anthesis. This delay interferes with the wheat reproductive strategy that is based on a fast programmed translocation of metabolites from the senescing leaves to the reproductive sinks shortly after anthesis.     

转ipt和反义ACO基因番茄的叶片衰老相关特性

以ipt和反义ACO转化的两类转基因番茄纯系为材料,研究在植株不同生长发育阶段,不同叶位中,与叶片衰老相关的生理生化指标.结果表明:两类基因导入番茄后,均可增强内源iPA和IAA表达水平,增加或保持番茄叶片的叶绿素含量、提高光合效率,进而明显地延缓植株的叶片衰老,提高单株果实产量.但它们调控叶片衰老的途径不同,ipt主要通过提高CTK的水平延缓叶片衰老,而反义ACO则主要是通过抑制乙烯生成,间接提高IAA的水平来实现.     

Increased cytokinin levels in transgenic PSAG12–IPT tobacco plants have large direct and indirect effects on leaf senescence, photosynthesis and N partitioning

We studied the impact of delayed leaf senescence on the functioning of plants growing under conditions of nitrogen remobilization. Interactions between cytokinin metabolism, Rubisco and protein levels, photosynthesis and plant nitrogen partitioning were studied in transgenic tobacco (Nicotiana tabacum L.) plants showing delayed leaf senescence through a novel type of enhanced cytokinin syn‐thesis, i.e. targeted to senescing leaves and negatively auto‐regulated (P_SAG12–IPT), thus preventing developmental abnormalities. Plants were grown with growth‐limiting nitrogen supply. Compared to the wild‐type, endogenous levels of free zeatin (Z)‐ and Z riboside (ZR)‐type cytokinins were increased up to 15‐fold (total ZR up to 100‐fold) in senescing leaves, and twofold in younger leaves of P_SAG12–IPT. In these plants, the senescence‐associated declines in N, protein and Rubisco levels and photosynthesis rates were delayed. Senescing leaves accumulated more (¹⁵N‐labelled) N than younger leaves, associated with reduced shoot N accumulation (–60%) and a partially inverted canopy N profile in P_SAG12–IPT plants. While root N accumulation was not affected, N translocation to non‐senescing leaves was progressively reduced. We discuss potential consequences of these modified sink–source relations, associated with delayed leaf senescence, for plant productivity and the efficiency of utilization of light and minerals.     

Effects of cytokinin production under two SAG promoters on senescence and development of tomato plants

Two promoters of senescence-associated ARABIDOPSIS genes, SAG12 and SAG13, were used in tomato plants to express IPT that catalyzes the rate-limiting step in cytokinin biosynthesis. Expression of these heterologous promoters in tomato plants was analyzed using the reporter gene beta-glucuronidase. Both promoters are expressed in tomato leaves in a manner similar to their expression in ARABIDOPSIS plants. The SAG12 promoter is very specific to senescing leaves, whereas the SAG13 promoter is expressed in mature leaves prior to the onset of visible senescence and its expression increases in senescing leaves. Expression of both promoters in tomato tissues other than leaves was very low . IPT expressed under the control of SAG12 and SAG13 promoters ( PSAG12::IPT and PSAG13::IPT, respectively) resulted in suppression of leaf senescence and advanced flowering, as well as in a slight increase in fruit weight and fruit total soluble solids (TSS). However, expression of PSAG13::IPT also led to stem thickening, short internodal distances and loss of apical dominance. In contrast to the autoregulation of PSAG12::IPT, PSAG13::IPT is expressed at higher levels in mature leaves. This difference is likely due to PSAG13::IPT exhibiting two phases of expression - a senescence-independent expression prior to the onset of senescence that is not subjected to autoregulation by cytokinin, and enhanced expression throughout senescence which is autoregualted by cytokinin. This moderate different autoregulated behavior of PSAG12::IPT and PSAG13::IPT markedly influenced plant development, emphasizing the biological effects of cytokinin in addition to senescence inhibition.     

Relationship between hexokinase and cytokinin in the regulation of leaf senescence and seed germination

Arabidopsis hexokinase (AtHXK1), an enzyme that catalyses hexose phosphorylation, accelerates leaf senescence, whereas the plant hormone cytokinin inhibits senescence. Previous work in our laboratory has shown that isopentenyl transferase (IPT), a key gene in the biosynthesis of cytokinin, expressed under promoters of the senescence-associated genes SAG12 or SAG13 (P(SAG12)::IPT and P(SAG13)::IPT, respectively), inhibits leaf senescence in tomato plants. To study the relationship between hexokinase and cytokinin in the regulation of leaf senescence, we created and analysed double-transgenic tomato plants expressing both AtHXK1 and either P(SAG12)::IPT or P(SAG13)::IPT. We found that expression of IPT in the double-transgenic plants could not prevent the accelerated senescence induced by over-expression of AtHXK1. Since cytokinin inhibits senescence via an apoplastic invertase that produces extracellular hexoses, whereas AtHXK1 is an intracellular mitochondria-associated hexokinase, our results suggest that intracellular sugar sensing via AtHXK1 is dominant over extracellular sugar sensing with regard to leaf senescence. Interestingly, the heterologous SAG12 and SAG13 promoters are also expressed in germinating tomato seed, around the radicle penetration zone, suggesting that seed germination involves a senescence process that is probably necessary for radicle emergence. Indeed, seed expressing P(SAG12)::IPT and P(SAG13)::IPT exhibited delayed radicle emergence, possibly due to delayed endosperm senescence.     

Extracellular invertase is an essential component of cytokinin-mediated delay of senescence

Leaf senescence is the final stage of leaf development in which the nutrients invested in the leaf are remobilized to other parts of the plant. Whereas senescence is accompanied by a decline in leaf cytokinin content, exogenous application of cytokinins or an increase of the endogenous concentration delays senescence and causes nutrient mobilization. The finding that extracellular invertase and hexose transporters, as the functionally linked enzymes of an apolasmic phloem unloading pathway, are coinduced by cytokinins suggested that delay of senescence is mediated via an effect on source-sink relations. This hypothesis was further substantiated in this study by the finding that delay of senescence in transgenic tobacco (Nicotiana tabacum) plants with autoregulated cytokinin production correlates with an elevated extracellular invertase activity. The finding that the expression of an extracellular invertase under control of the senescence-induced SAG12 promoter results in a delay of senescence demonstrates that effect of cytokinins may be substituted by these metabolic enzymes. The observation that an increase in extracellular invertase is sufficient to delay leaf senescence was further verified by a complementing functional approach. Localized induction of an extracellular invertase under control of a chemically inducible promoter resulted in ectopic delay of senescence, resembling the naturally occurring green islands in autumn leaves. To establish a causal relationship between cytokinins and extracellular invertase for the delay of senescence, transgenic plants were generated that allowed inhibition of extracellular invertase in the presence of cytokinins. For this purpose, an invertase inhibitor was expressed under control of a cytokinin-inducible promoter. It has been shown that senescence is not any more delayed by cytokinin when the expression of the invertase inhibitor is elevated. This finding demonstrates that extracellular invertase is required for the delay of senescence by cytokinins and that it is a key element of the underlying molecular mechanism.     

Delay of leaf senescence in <Emphasis Type="Italic">Medicago sativa</Emphasis> transformed with the <Emphasis Type="Italic">ipt</Emphasis> gene controlled by the senescence-specific promoter SAG12

We report the successfull delay of leaf senescence in Medicago sativa. A highly regenerable clone of alfalfa was transformed with the construct SAG12-IPT, an approach that has already proved efficient in other crops. Several independent transformants were obtained as determined by Southern analysis and all the transformants expressed the transgene as measured by RT-PCR. In vitro and in vivo analyses showed that SAG12-IPT plants exhibited a stay-green phenotype that has the potential to greatly improve the quantity and quality of alfalfa forage.     

Regulated Expression of a Cytokinin Biosynthesis Gene IPT Delays Leaf Senescence and Improves Yield under Rainfed and Irrigated Conditions in Canola (Brassica napus L.)

Delay of leaf senescence through genetic modification can potentially improve crop yield, through maintenance of photosynthetically active leaves for a longer period. Plant growth hormones such as cytokinin regulate and delay leaf senescence. Here, the structural gene (IPT) encoding the cytokinin biosynthetic enzyme isopentenyltransferase was fused to a functionally active fragment of the AtMYB32 promoter and was transformed into canola plants. Expression of the AtMYB32xs::IPT gene cassette delayed the leaf senescence in transgenic plants grown under controlled environment conditions and field experiments conducted for a single season at two geographic locations. The transgenic canola plants retained higher chlorophyll levels for an extended period and produced significantly higher seed yield with similar growth and phenology compared to wild type and null control plants under rainfed and irrigated treatments. The yield increase in transgenic plants was in the range of 16% to 23% and 7% to 16% under rainfed and irrigated conditions, respectively, compared to control plants. Most of the seed quality parameters in transgenic plants were similar, and with elevated oleic acid content in all transgenic lines and higher oil content and lower glucosinolate content in one specific transgenic line as compared to control plants. The results suggest that by delaying leaf senescence using the AtMYB32xs::IPT technology, productivity in crop plants can be improved under water stress and well-watered conditions.     

PSAG12-ipt嵌合基因转化樱桃番茄的研究

利用根癌农杆菌(Agrobacterium tumefacienst)介导法将嵌合基因P SAG12-ipt导人樱桃番茄,经双重PCR检测和基因组总DNA Southern杂交测验,共获得17株转基因植株。田间生长实验观察结果显示,这17株转基因植株生长发育及形态正常,但其中的14株长势明显好于对照。对14株中的2株进行叶片叶绿素、细胞分裂素检测结果表明,转基因植株基本定型叶下部,不同叶位叶片叶绿素、细胞分裂素含量明显高于对照,基本定型叶上部叶片与对照基本一致,同一叶位叶片比对照延缓衰老15～20d。     

Antioxidant protection during ageing and senescence in chloroplasts of tobacco with modulated life span

We studied changes in antioxidant protection during ageing and senescence in chloroplasts of tobacco (Nicotiana tabacum L., cv. Wisconsin) with introduced SAG(12) promoter fused with ipt gene for cytokinin synthesis (transgenic plants with increased levels of cytokinins, SAG) or without it (control). Old leaves of SAG plants as well as their chloroplasts maintained higher physiological parameters compared to controls; accordingly, we concluded that their ageing was diverted due to increased cytokinin content. The chloroplast antioxidant protection did not decrease as well. Although antioxidant protection usually decreased in whole leaves of senescing control plants, ascorbate peroxidase (APX) and dehydroascorbate reductase (DHAR) activity, which maintained the high redox state of ascorbate, increased in chloroplasts of old control leaves.     

A gene encoding an acyl hydrolase is involved in leaf senescence in Arabidopsis

SAG101, a leaf senescence-associated gene, was cloned from an Arabidopsis leaf senescence enhancer trap line and functionally characterized. Reporter gene and RNA gel blot analyses revealed that SAG101 was not expressed until the onset of senescence in leaves. A recombinant SAG101 fusion protein overexpressed in Escherichia coli displayed acyl hydrolase activity. Antisense RNA interference in transgenic plants delayed the onset of leaf senescence for approximately 4 days. Chemically induced overexpression of SAG101 caused precocious senescence in both attached and detached leaves of transgenic Arabidopsis plants. These data suggest that SAG101 plays a significant role in leaf senescence.