Effects of senescence-induced alteration in cytokinin metabolism on source-sink relationships and ontogenic and stress-induced transitions in tobacco

Senescence and reserve mobilization are integral components of plant development, are basic strategles in stress mitigation, and regulated at least in part by cytokinin. In the present study the effect of altered cytokinin metabolism caused by senescence-specific autoregulated expression of the Agrobacterium tumefaciens IPT gene under control of the P_SAG12 promoter (P_SAG12-IPT) on seed germination and the response to a water-deficit stress was studied in tobacco (Nicotiana tabacum L.). Cytokinin levels, sugar content and composition of the leaf strata within the canopy of wild-type and P_SAG12-IPT plants confirmed the reported altered source–sink relations. No measurable difference in sugar and pigment content of discs harvested from apical and basal leaves was evident 72 h after incubation with (+)-ABA or in darkness, indicating that expression of the transgene was not restricted to senescing leaves. No difference in quantum efficiency, photosynthetic activity, accumulation of ABA, and stomatal conductance was apparent in apical, middle and basal leaves of either wild-type or P_SAG12-IPT plants after imposition of a mild water stress. However, compared to wild-type plants, P_SAG12-IPT plants were slower to adjust biomass allocation. A stress-induced increase in root:shoot ratio and specific leaf area (SLA) occurred more rapidly in wild-type than in P_SAG12-IPT plants reflecting delayed remobilization of leaf reserves to sink organs in the transformant. P_SAG12-IPT seeds germinated more slowly even though abscisic acid (ABA) content was 50% that of the wild-type seeds confirming cytokinin-induced alterations in reserve remobilization. Thus, senescence is integral to plant growth and development and an increased endogenous cytokinin content impacts source–sink relations to delay ontogenic transitions wherein senescence in a necessary process.     

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.     

Effects of P(SAG12)-IPT gene expression on development and senescence in transgenic lettuce

An ipt gene under control of the senescence-specific SAG12 promoter from Arabidopsis (P(SAG12)-IPT) significantly delayed developmental and postharvest leaf senescence in mature heads of transgenic lettuce (Lactuca sativa L. cv Evola) homozygous for the transgene. Apart from retardation of leaf senescence, mature, 60-d-old plants exhibited normal morphology with no significant differences in head diameter or fresh weight of leaves and roots. Induction of senescence by nitrogen starvation rapidly reduced total nitrogen, nitrate, and growth of transgenic and azygous (control) plants, but chlorophyll was retained in the lower (outer) leaves of transgenic plants. Harvested P(SAG12)-IPT heads also retained chlorophyll in their lower leaves. During later development (bolting and preflowering) of transgenic plants, the decrease in chlorophyll, total protein, and Rubisco content in leaves was abolished, resulting in a uniform distribution of these components throughout the plants. Homozygous P(SAG12)-IPT lettuce plants showed a slight delay in bolting (4-6 d), a severe delay in flowering (4-8 weeks), and premature senescence of their upper leaves. These changes correlated with significantly elevated concentrations of cytokinin and hexoses in the upper leaves of transgenic plants during later stages of development, implicating a relationship between cytokinin and hexose concentrations in senescence.     

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.     

Involvement of nitrogen and cytokinins in photosynthetic acclimation to elevated CO2 of spring wheat

Acclimation of photosynthetic capacity to elevated CO₂ involves a decrease of the leaf Rubisco content. In the present study, it was hypothesized that nitrogen uptake and partitioning within the leaf and among different aboveground organs affects the down-regulation of Rubisco. Given the interdependence of nitrogen and cytokinin signals at the whole plant level, it is also proposed that cytokinins affect the nitrogen economy of plants under elevated CO₂, and therefore the acclimatory responses. Spring wheat received varying levels of nitrogen and cytokinin in field chambers with ambient (370 μmol mol⁻¹) or elevated (700 μmol mol⁻¹) atmospheric CO₂. Gas exchange, Rubisco, soluble protein and nitrogen contents were determined in the top three leaves in the canopy, together with total nitrogen contents per shoot. Growth in elevated CO₂ induced decreases in photosynthetic capacity only when nitrogen supply was low. However, the leaf contents of Rubisco, soluble protein and total nitrogen on an area basis declined in elevated CO₂ regardless of nitrogen supply. Total nitrogen in the shoot was no lower in elevated than ambient CO₂, but the fraction of this nitrogen located in flag and penultimate leaves was lower in elevated CO₂. Decreased Rubisco: chlorophyll ratios accompanied losses of leaf Rubisco with CO₂ enrichment. Cytokinin applications increased nitrogen content in all leaves and nitrogen allocation to senescing leaves, but decreased Rubisco contents in flag leaves at anthesis and in all leaves 20 days later, together with the amount of Rubisco relative to soluble protein in all leaves at both growth stages. The results suggest that down regulation of Rubisco in leaves at elevated CO₂ is linked with decreased allocation of nitrogen to the younger leaves and that cytokinins cause a fractional decrease of Rubisco and therefore do not alleviate acclimation to elevated CO₂.     

Leaf senescence of Quercus myrtifolia as affected by long-term CO2 enrichment in its native environment

The long‐term effects of elevated (ambient plus 350 μmol mol^?1) atmospheric CO₂ concentration (C_a) on the leaf senescence of Quercus myrtifolia Willd was studied in a scrub‐oak community during the transition from autumn (December 1997) to spring (April 1998). Plants were grown in large open‐top chambers at the Smithsonian CO₂ Research Site, Merritt Island Wildlife Refuge, Cape Canaveral, Florida. Chlorophyll (a + b) concentration, Rubisco activity and N concentration decreased by 75%, 82%, and 52%, respectively, from December (1997) to April (1998) in the leaves grown at ambient C_a. In contrast, the leaves of plants grown at elevated C_a showed no significant decrease in chlorophyll (a + b) concentration or Rubisco activity, and only a 25% reduction in nitrogen. These results indicate that leaf senescence was delayed during this period at elevated C_a. Delayed leaf senescence in elevated C_a had important consequences for leaf photosynthesis. In elevated C_a the net photosynthetic rate of leaves that flushed in Spring 1997 (last year's leaves) and were 13 months old was not different from fully‐expanded leaves that flushed in 1998, and were approximately 1 month old (current year's leaves). In ambient C_a the net photosynthetic rate of last year's leaves was 54% lower than for current year's leaves. When leaves were fully senesced, nitrogen concentration decreased to about 40% of the concentration in non‐senesced leaves, in both CO₂ treatments. In April, net photosynthesis was 97% greater in leaves grown in elevated C_a than in those grown at ambient. During the period when elevated C_a delayed leaf senescence, more leaves operating at higher photosynthetic rate would allow the ecosystem dominated by Q. myrtifolia to gain more carbon at elevated C_a than at ambient C_a.     

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.     

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.     

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.     

Light Restriction Delays Leaf Senescence in Winter Oilseed Rape (Brassica napus L.)

Oilseed rape (Brassica napus L.) is a crop with a complex aerial architecture that can cause self-shading leading to a vertical light gradient over the foliage. Mutual shading between neighboring plants at a high sowing density also results in an alteration of photosynthetically active radiation (PAR) absorption by lower leaves. The aim of this study was to analyze the impact that light restriction on lower leaves has on shoot architecture, biomass production and allocation, nitrogen (N) fluxes, and progression of sequential senescence. Field-grown plants were collected at the end of the vegetative rest period and grown in hydroponic conditions until pod maturity. A shading treatment corresponding to a 43.4 % reduction of PAR was applied at the early flowering stage. N uptake and fluxes of N allocation and remobilization were determined by supplying K¹⁵NO₃ in the nutrient solution. Photosynthesis and expression of SAG12 and Cab genes (indicators of leaf senescence progression) were also analyzed on different leaf ranks. The results showed that shading enhanced leaf development on the main stem and ramifications to optimize light capture. The expression pattern of the SAG12/Cab molecular indicator suggested a delay in leaf senescence that allowed leaf life span to be extended resulting in a more efficient leaf compound remobilization, with lower N residual contents in fallen leaves under shading. N uptake increased and N remobilization fluxes were enhanced from source organs (leaves and stem) toward sink organs (flowers). Profuse branching and late senescing varieties would be of interest for further selection programs under high sowing densities.     

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.     

Delayed leaf senescence in ethylene-deficient ACC-oxidase antisense tomato plants: molecular and physiological analysis

To determine the role of ethylene during tomato (Lycopersicon esculentum Mill. cv. Alisa Craig) leaf senescence, transgenic ACC oxidase antisense plants were analysed. Northern analysis of wild-type plants indicated that ACC oxidase mRNA accumulation normally begins in pre-senescent green leaves but was severely reduced in the antisense plants. Although the levels of ethylene evolved by wild-type and transgenic leaves increased during the progression of senescence, levels were extremely low in transgenic leaves. Leaf senescence, as assessed by colour change from green to yellow, was clearly delayed by 10–14 days in the antisense plants when compared with wild-type plants. Northern analysis of the photosynthesis-associated genes, cab and rbcS, indicated that levels of the corresponding mRNAs were higher in transgenic leaves which were not yet senescing compared with senescing wild-type leaves of exactly the same age. Northern analysis using probes for tomato fruit ripening-related genes expressed during leaf senescence indicated that once senescence was initiated the expression pattern of these mRNAs was similar in transgenic and wild-type leaves. In the antisense plants chlorophyll levels, photosynthetic capacity and chlorophyll fluorescence were higher when compared with senescing wild-type plants of the same age. Photosynthetic capacity and the quantum efficiency of photosystem II were maintained for longer in the transformed plants at values close to those observed in wild-type leaves prior to the visible onset of senescence. These results indicate that inhibiting ACC oxidase expression and ethylene synthesis results in delayed leaf senescence, rather than inducing a stay-green phenotype. Once senescence begins, it progresses normally. Onset of senescence is not, therefore, related to a critical level of ethylene. The correlation between higher levels prior to senescence and early onset, however, suggests that ethylene experienced by the plant may be a significant contributing factor in the timing of senescence.     

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.     

Consequences of CO2 and light interactions for leaf phenology, growth, and senescence in Quercus rubra

We investigated how light and CO₂ levels interact to influence growth, phenology, and the physiological processes involved in leaf senescence in red oak (Quercus rubra) seedlings. We grew plants in high and low light and in elevated and ambient CO₂. At the end of three years of growth, shade plants showed greater biomass enhancement under elevated CO₂ than sun plants. We attribute this difference to an increase in leaf area ratio (LAR) in shade plants relative to sun plants, as well as to an ontogenetic effect: as plants increased in size, the LAR declined concomitant with a decline in biomass enhancement under elevated CO₂ Elevated CO₂ prolonged the carbon gain capacity of shade‐grown plants during autumnal senescence, thus increasing their functional leaf lifespan. The prolongation of carbon assimilation, however, did not account for the increased growth enhancement in shade plants under elevated CO₂. Elevated CO₂ did not significantly alter leaf phenology. Nitrogen concentrations in both green and senesced leaves were lower under elevated CO₂ and declined more rapidly in sun leaves than in shade leaves. Similar to nitrogen concentration, the initial slope of A/C_i curves indicated that Rubisco activity declined more rapidly in sun plants than in shade plants, particularly under elevated CO₂. Absolute levels of chlorophyll were affected by the interaction of CO₂ and light, and chlorophyll content declined to a minimal level in sun plants sooner than in shade plants. These declines in N concentration, in the initial slope of A/C_i curves, and in chlorophyll content were consistent with declining photosynthesis, such that elevated CO₂ accelerated senescence in sun plants and prolonged leaf function in shade plants. These results have implications for the carbon economy of seedlings and the regeneration of red oak under global change conditions.     

An ABA-regulated and Golgi-localized protein phosphatase controls water loss during leaf senescence in Arabidopsis

It is known that a senescing leaf loses water faster than a non-senescing leaf and that ABA has an important role in promoting leaf senescence. However, questions such as why water loss is faster, how water loss is regulated, and how ABA functions in leaf senescence are not well understood. Here we report on the identification and functional analysis of a leaf senescence associated gene called SAG113. The RNA blot and GUS reporter analyses all show that SAG113 is expressed in senescing leaves and is induced by ABA in Arabidopsis. The SAG113 expression levels are significantly reduced in aba2 and abi4 mutants. A GFP fusion protein analysis revealed that SAG113 protein is localized in the Golgi apparatus. SAG113 encodes a protein phosphatase that belongs to the PP2C family and is able to functionally complement a yeast PP2C-deficient mutant TM126 (ptc1Δ). Leaf senescence is delayed in the SAG113 knockout mutant compared with that in the wild type, stomatal movement in the senescing leaves of SAG113 knockouts is more sensitive to ABA than that of the wild type, and the rate of water loss in senescing leaves of SAG113 knockouts is significantly reduced. In contrast, inducible over-expression of SAG113 results in a lower sensitivity of stomatal movement to ABA treatment, more rapid water loss, and precocious leaf senescence. No other aspects of growth and development, including seed germination, were observed. These findings suggest that SAG113, a negative regulator of ABA signal transduction, is specifically involved in the control of water loss during leaf senescence.     

Evidence for contribution of autophagy to Rubisco degradation during leaf senescence in Arabidopsis thaliana

During leaf senescence, Rubisco is gradually degraded and its components are recycled within the plant. Although Rubisco can be mobilized to the vacuole by autophagy via specific autophagic bodies, the importance of this process in Rubisco degradation has not been shown directly. Here, we monitored Rubisco autophagy during leaf senescence by fusing synthetic green fluorescent protein (sGFP) or monomeric red fluorescent protein (mRFP) with Rubisco in Arabidopsis (Arabidopsis thaliana). When attached leaves were individually exposed to darkness to promote their senescence, the fluorescence of Rubisco‐sGFP was observed in the vacuolar lumen as well as chloroplasts. In addition, release of free‐sGFP due to the processing of Rubisco‐sGFP was observed in the vacuole of individually darkened leaves. This vacuolar transfer and processing of Rubisco‐sGFP was not observed in autophagy‐deficient atg5 mutants. Unlike sGFP, mRFP was resistant to proteolysis in the leaf vacuole of light‐grown plants. The vacuolar transfer and processing of Rubisco‐mRFP was observed at an early stage of natural leaf senescence and was also obvious in leaves naturally covered by other leaves. These results indicate that autophagy contributes substantially to Rubisco degradation during natural leaf senescence as well as dark‐promoted senescence.     

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.     

Suboptimal nitrogen status sensitizes the photosynthetic apparatus in willow leaves to long term but not short term water stress

The response to drought was compared for willow plants of optimal leaf nitrogen content (100 N) and those of 86% of this content (86 N). Gas exchange measurements revealed that the carboxylation efficiency (CE) of photosynthesis was more sensitive to drought than the photosynthetic capacity in both N regimes. Since the leaf content of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was found to be much more resistant it is suggested that a decreased specific activity of Rubisco underlies the decreased CE. Although the rate of water consumption was the same for 86 N and 100 N plants the photosynthetic apparatus responded much more rapidly in the 86 N leaves. This increased sensitivity of 86 N leaves was not due to accelerated senescence as judged by comparison with parallel plants subjected to discontinued fertilization; the two categories of treatments resulted in the same loss of leaf nitrogen and Rubisco but drought induced a much more rapid photosynthetic depression. In contrast to the drought situation, 86 N and 100 N plants behaved similarly when compared under short term water stress. First, when single attached leaves were exposed to a sudden drop in air humidity the capacity of CO₂ uptake in both N regimes decreased about 20% over 10 min while the leaf water potential remained high. Second, in freely transpiring leaf discs cut from 86 N and 100 N leaves the same relationship between capacity of O₂ evolution and extent of dehydration was observed. The possible mechanisms underlying the increased susceptibility of 86 N leaves to drought is discussed; the water status of the roots not the leaves is suggested to be the determining factor.Abbreviations CE carboxylation efficiency - 100 N optimal nitrogen regime - 86 N suboptimal nitrogen regime with 86% of the optimal leaf nitrogen content, Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase