Cytokinin stress changes the developmental regulation of several defence-related genes in tobacco

Tobacco shoots exposed to elevated endogenous or exogenous cytokinin levels are unable to develop roots and lack apical dominance. We have isolated cDNA copies of five mRNA species that accumulate to elevated levels in such cytokinin-stressed shoots via differential screening of a cDNA library of transgenic shoots which contain an active T-DNA cytokinin gene (T-cyt gene) from Agrobacterium tumefaciens. Four of the cDNA clones were found to correspond to plant defence-related mRNAs, encoding extensin, chitinase, PR-1 and a PR-1-like protein, respectively. In normal tobacco plants PR-1 mRNA is relatively rare in all organs. The other four mRNAs occur at relatively low levels in shoots, especially in leaves, but are very prevalent in roots. Extensin mRNA, for example, is not detectable in leaves, while it is an abundant mRNA in roots and stems. In normal shoots cultured on cytokinin-containing medium all five mRNAs accumulate to elevated levels, similar to those found in transgenic T-cyt shoots. We conclude that the imposed cytokinin stress causes changes in the tissue-specific control of the levels of several defence-related mRNA species in tobacco.     

Carbon partitioning in leaves and tubers of transgenic potato plants with reduced activity of fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase

The role of fructose-2,6-bisphosphate (Fru-2,6-P₂) in regulation of carbon metabolism was investigated in transgenic potato plants ( Solanum tuberosum L. cv Dianella) transformed with a vector containing a cDNA-sequence encoding fructose-6-phosphate,2-kinase (F6P,2-K, EC 2.7.1.105)/fructose-2,6-bisphosphatase (F26BPase, EC 3.1.3.46) in sense or antisense direction behind a CaMV 35S promoter. The activity of F6P,2-K in leaves was reduced to 5% of wild-type (WT) activity, and the level of Fru-2,6-P₂ was reduced both in leaves (10% of the WT level) and in tubers (40% of the WT level). Analysis of photosynthetic ¹⁴CO₂ metabolism, showed that in plant lines with reduced Fru-2,6-P₂ level the carbon partitioning in the leaves was changed in favour of sucrose biosynthesis, and the soluble sugars-to-starch labelling ratio was doubled. The levels of soluble sugars and hexose phosphates also increased in leaves of the transgenic plants. Most notably, the levels of hexoses were four- to six-fold increased in the transgenic plants. In tubers with reduced levels of Fru-2,6-P₂ only minor effects on carbohydrate levels were observed. Furthermore, carbon assimilation in tuber discs supplied with [U-¹⁴C]-sucrose showed only a moderate increase in labelling of hexoses and a decreased labelling of starch. Similar results were obtained using [U-¹⁴C]-glucose. No differences in growth of the transgenic lines and the WT were observed. Our data provide evidences that Fru-2,6-P₂ is an important factor in the regulation of photosynthetic carbon metabolism in potato leaves, whereas the direct influence of Fru-2,6-P₂ on tuber metabolism was limited.     

Development of necrosis and activation of disease resistance in transgenic tobacco plants with severely reduced catalase levels

Numerous studies argue that salicylic acid (SA) is an important component of the plant signal transduction pathway(s) leading to disease resistance. The discovery that the SA-binding protein is a catalase, whose activity is blocked by SA, led to the proposal that one of SA's modes of action is to inhibit this H₂O₂-degrading enzyme and thus elevate H₂O₂ levels. To test this model, an attempt was made to mimic the action of SA by reducing the synthesis of catalase using antisense RNA technology. Analyses of transgenic tobacco plants that expressed the tobacco catalase 1 ( cat1 ) or catalase 2 ( cat2 ) gene in an antisense orientation indicate that there is no correlation between modest to high levels of reduction in catalase activity and activation of plant defenses such as pathogenesis-related (PR)-1 protein synthesis. However, three independent antisense catalase transgenic plants (ASCAT1 Nos 16, 17, and 28), which exhibited the most severe reduction in catalase activity (∼90% or more), developed chlorosis or necrosis on some of their lower leaves. These same leaves accumulated very high levels of PR-1 proteins and showed enhanced resistance to tobacco mosaic virus. Necrosis and elevated SA, which appear to result from severe depression of catalase levels, may be responsible for the induction of these defense responses.     

Maximal biomass production can occur in corn (Zea mays) in the absence of NO3 accumulation in either leaves or roots

In order to investigate effects of limited NO₃ availability in corn ( Zea mays L. cv. Brulouis) 17-day-old plants were grown for a further 25 days on sand in a growth chamber. The plants received frequent irrigation with a complete nutrient solution containing 0.2, 0.6, 1.5 or 3.0 mM NO₃. With 0.2 mM NO; nitrate levels in both roots and leaves diminished rapidly and were almost zero after 10 days treatment. Concurrently, as signs of nitrogen deficiency appeared, shoot growth was restricted, whereas root growth was enhanced. In addition, the concentration of reduced nitrogen and malate in the leaves declined, and in vitro nitrate reductase activity (NRA. EC 1.6.6.1), soluble protein and chlorophyll levels of leaf tissue were depressed and starch concentration was enhanced. With 0.6 mM NO₃ in the nutrient solution, the decrease in NO₃ levels in the tissues and the increase in root development were similar to those observed with 0.2 mM NO₃. However, shoot growth, reduced nitrogen concentration in leaves, and the above-mentioned biochemical characteristics were almost identical to those obtained at 1.5 and 3.0 mM NO₃. This indicates that when supplied with 0.6 mM NO₃, corn plants were able to absorb sufficient NO3 to support maximal biomass production without appreciable NO₃ accumulation in roots or shoot. It is, thus, suggested that the plants responded to low NO₃, availability in medium by enhancing root growth and by maximizing NO₃ reduction relative to NO₃ accumulation.     

The ectopic overexpression of a citrus gibberellin 20-oxidase enhances the non-13-hydroxylation pathway of gibberellin biosynthesis and induces an extremely elongated phenotype in tobacco

Transgenic plants of Nicotiana tabacum overexpressing a gibberellin (GA) 20-oxidase cDNA ( CcGA20ox1 ) from citrus, under the control of the 35S promoter, were taller (up to twice) and had larger inflorescences and longer flower peduncles than those of control plants. Hypocotyls of transgenic seedlings were also longer (up to 4 times), and neither the seedlings nor the growing plants elongated further after application of GA₃. Hypocotyl and stem lengths were reduced by application of paclobutrazol, and this inhibition was reversed by exogenous GA₃. The ectopic overexpression of CcGA20ox1 enhanced the non-13-hydroxylation pathway of GA biosynthesis leading to GA₄, apparently at the expense of the early-13-hydroxylation pathway. The level of GA₄ (the active GA from the non-13-hydroxylation pathway) in the shoot of transgenic plants was 3–4 times higher than in control plants, whereas that of GA₁, formed via the early-13-hydroxylation pathway (the main GA biosynthesis pathway in tobacco), decreased or was not affected. GA₄ applied to the culture medium or to the expanding leaves was found to be at least equally active as GA₁ on stimulating hypocotyl and stem elongation of tobacco plants. The results suggest that the tall phenotype of tobacco transgenic plants was due to their higher content of GA₄, and that the GA response was saturated by the presence of the transgene.     

Wheat catalase expressed in transgenic rice can improve tolerance against low temperature stress

We examined whether the expression of wheat catalase (EC 1.11.1.6) cDNA in transgenic rice ( Oryza sativa L.) could enhance tolerance against low temperature injury. Transgenic rice plants expressing wheat CAT protein showed an increase of activities in leaves at 25°C, 2- to 5-fold that in non-transgenic rice. At 5°C, catalase activities were about 4–15 times higher than those in non-transgenic rice were. A comparison of damage observed in leaves as they withered due to chilling at 5°C showed that transgenic rice displayed an increased capability to resist low temperature stress. The exposure of these plants to low temperature at 5°C for 8 days resulted in decreased catalase activities in leaves at 25°C, but the transgenic plants indicated 4 times higher residual catalase activities than those of non-transgenic ones. The concentration of H₂O₂ in leaves was kept lower in transgenic rice than that of the control plants during the 8 days chilling. These results suggest that the improved tolerance against low temperature stress in genetically engineered rice plants be attributed to the effective detoxification of H₂O₂ by the enhanced catalase activities.     

Influence of nitrate and ammonium on the growth and 2,4-diaminobutyric acid composition of flatpea (Lathyrus sylvestris L.)

Abstract. Presence of 2.4-diaminobutyric acid (A₂bu), a neurotoxin, in tissues of flatpea ( Lathyrus sylvestris L.) necessitates a thorough understanding of the regulation of this nonprotein amino acid before the species can be recommended to livestock producers for forage applications. To determine how different concentrations and ratios of NO₃ and NH⁺₄ in growth media influence the levels of A₂bu and other free amino acids in the 'Lathco'flatpea cultivar, plants were grown hydroponically in controlled environments. The concentration of A₂bu was highest in tissues when the NO₃ to NH⁺₄ ratio in the nutrient solution was low. Responses of amides and other nonprotein amino acids, especially in the roots, followed a similar trend. Free protein amino acids in leaves and stems were generally unaffected by changes in NO₃ to NH⁺₄ ratios. In roots, protein amino acids increased as the NO₃ to NH⁺₄ ratio in the growth medium increased. Ammonium inhibited shoot and root growth; NO₃ alleviated the toxic effects of NH⁺₄. Soluble protein concentrations were higher in the shoots of NO₃-fed plants and in the roots of plants supplied with NH⁺₄. These results suggest that accumulation of A₂bu and other nonprotein amino acids, as well as asparagine and glutamine, plays a role in detoxification of NH⁺₄ and storage of N.     

Up-regulation of an N-terminal truncated 3-hydroxy-3-methylglutaryl CoA reductase enhances production of essential oils and sterols in transgenic Lavandula latifolia

Spike lavender ( Lavandula latifolia ) essential oil is widely used in the perfume, cosmetic, flavouring and pharmaceutical industries. Thus, modifications of yield and composition of this essential oil by genetic engineering should have important scientific and commercial applications. We generated transgenic spike lavender plants expressing the Arabidopsis thaliana HMG1 cDNA, encoding the catalytic domain of 3-hydroxy-3-methylglutaryl CoA reductase (HMGR1S), a key enzyme of the mevalonic acid (MVA) pathway. Transgenic T₀ plants accumulated significantly more essential oil constituents as compared to controls (up to 2.1- and 1.8-fold in leaves and flowers, respectively). Enhanced expression of HMGR1S also increased the amount of the end-product sterols, β-sitosterol and stigmasterol (average differences of 1.8- and 1.9-fold, respectively), but did not affect the accumulation of carotenoids or chlorophylls. We also analysed T₁ plants derived from self-pollinated seeds of T₀ lines that flowered after growing for 2 years in the greenhouse. The increased levels of essential oil and sterols observed in the transgenic T₀ plants were maintained in the progeny that inherited the HMG1 transgene. Our results demonstrate that genetic manipulation of the MVA pathway increases essential oil yield in spike lavender, suggesting a contribution for this cytosolic pathway to monoterpene and sesquiterpene biosynthesis in leaves and flowers of the species.     

Long-term effects of low levels of SO2 on bean plants (Phaseolus vulgaris). II. Immission-response effects on biomass production: quantity and quality

Bean plants ( Phaseolus vulgaris L. cv. Processer) were grown in water culture with separate air supply to roots for four to five weeks at five levels of SO₂ ranging from 10 μg m⁻³ to 950 μg m⁻³. At harvest the plant material was divided into six fractions: root, stem, fruit and leaves of three age groups.
Plants were mainly affected at and above approx. 250 μg m⁻³ SO₂. Fresh weight was reduced in mature and old leaves, and roots and fruit. Dry weight was also reduced in mature and old leaves, and roots and stem. A reduction was found in chlorophyll a and chlorophyll b in mature and old leaves, and also starch was reduced in the leaves. Sulfur content of leaves and fruit increased with exposure time and concentration, while Br, Ca, Cl, K, Mn, P and Zn increased at the highest SO₂ level only. Total (but not specific) peroxidase activity increased in all aerial fractions, i.e. soluble protein increased just like peroxidase activity. Seventeen studied amino acids all increased on the average by 38% in mature bean pods.
The observed effects may be parts of a reaction for survival and propagation of the plant, as fruit quality was not affected, indeed, it sometimes improved slightly. The latter observation is of commercial interest.     

大气CO2浓度升高对玉米非结构性碳水化合物和籽粒品质的影响

本试验利用改进的开顶式气室(OTC)在黄土高原长武农业生态试验站田间模拟大气CO₂浓度升高环境,设置3个处理:CK(田间环境,自然大气CO₂浓度)、OTC(OTC气室,自然大气CO₂浓度)、OTC_e(OTC气室,CO₂浓度700 μmol·mol^-1),探讨春玉米在不同生育期各器官非结构性碳水化合物(NSC)及籽粒品质(可溶性糖、淀粉和粗蛋白)对大气CO₂浓度升高的响应,为揭示旱作区春玉米对大气CO₂浓度升高的适应机理提供科学依据。结果表明: 大气CO₂浓度升高对玉米NSC含量、积累量的影响因器官和生育期不同而异。与CK和OTC相比,OTC_e促进了生殖生长阶段叶、茎和根NSC的活化再分配,提高了叶片、茎秆和根系NSC转运到籽粒的量(ATM_NSC)、向籽粒的转运率(AR_NSC)以及对籽粒的贡献率(AC_NSC);与CK相比,OTC带来的增温效应抑制了茎和根NSC的活化再分配,促进了叶NSC的活化再分配,显著提高了玉米叶ATM_NSC、AR_NSC、AC_NSC。在两年试验中,大气CO₂浓度升高对玉米籽粒可溶性糖、淀粉和粗蛋白含量无显著影响。     

Elevated pyruvate,orthophosphate dikinase (PPDK) activity alters carbon metabolism in C3 transgenic potatoes with a C4 maize PPDK gene

Changes in carbon metabolism and δ¹³C value of transgenic potato plants with a maize pyruvate,orthophosphate dikinase (PPDK; EC 2.7.9.1) gene are reported. PPDK catalyzes the formation of phospho enol pyruvate (PEP), the initial acceptor of CO₂ in the C₄ photosynthetic pathway. PPDK activities in the leases of transgenic potatoes were up to 5.4‐fold higher than those of control potato plants (wild‐type and treated control plants). In the transgenic potato plants, PPDK activity in leaves was negatively correlated with pyruvate content (r²= 0.81), and was positively correlated with malate content (r²= 0.88). A significant increase in the δ¹³C value was observed in the transgenic potato plants, suggesting a certain contribution of PEP carboxylase as the initial acceptor of atmospheric CO₂. These data suggest that elevated PPDK activity may alter carbon metabolism and lead to a partial operation of C₄‐type carbon metabolism. However, since parameters associated with CO₂ gas exchange were not affected, the altered carbon metabolism had only a small effect on the total photosynthetic characteristics of the transgenic plants.     

Isolation of photorespiratory mutants from Lotus japonicus deficient in glutamine synthetase

A mutagenesis programme using ethyl methanesulphonate (EMS) was carried out on Lotus japonicus (Regel) Larsen cv. Gifu in order to isolate photorespiratory mutants in this model legume. These mutants were able to grow in a CO₂-enriched atmosphere [0.7% (v/v) CO₂] but showed stress symptoms when transferred to air. Among them, three mutants displayed low levels of glutamine synthetase (GS; EC 6.3.1.2) activity in leaves. The mutants accumulated ammonium in leaves upon transfer from 0.7% (v/v) CO₂ to air. F₁ plants of back crosses to wild type were viable in air and F₂ populations segregated 3 : 1 (viable in air : air-sensitive) indicative of a single Mendelian recessive trait. Complementation tests showed that the three mutants obtained were allelic. Chromatography on DEAE-Sephacel used to separate the cytosolic and plastidic GS isoenzymes together with immunological data showed that: (1) mutants were specifically affected in the plastidic GS isoform, and (2) in L. japonicus the plastidic GS isoform eluted at lower ionic strength than the cytosolic isoform, contrary to what happens in most plants. The plastidic GS isoform present in roots of wild type L. japonicus was also absent in roots of the mutants, indicating that this plastidic isoform from roots was encoded by the same gene than the GS isoform expressed in leaf tissue. Viability of mutant plants in high-CO₂ conditions indicates that plastidic GS is not essentially required for primary ammonium assimilation. Nevertheless, mutant plants did not grow as well as wild type plants in high-CO₂ conditions.     

Enhanced Accumulation of BiP in Transgenic Plants Confers Tolerance to Water Stress

The binding protein (BiP) is an important component of endoplasmic reticulum stress response of cells. Despite extensive studies in cultured cells, a protective function of BiP against stress has not yet been demonstrated in whole multicellular organisms. Here, we have obtained transgenic tobacco (Nicotiana tabacum L. cv Havana) plants constitutively expressing elevated levels of BiP or its antisense cDNA to analyze the protective role of this endoplasmic reticulum lumenal stress protein at the whole plant level. Elevated levels of BiP in transgenic sense lines conferred tolerance to the glycosylation inhibitor tunicamycin during germination and tolerance to water deficit during plant growth. Under progressive drought, the leaf BiP levels correlated with the maintenance of the shoot turgidity and water content. The protective effect of BiP overexpression against water stress was disrupted by expression of an antisense BiP cDNA construct. Although overexpression of BiP prevented cellular dehydration, the stomatal conductance and transpiration rate in droughted sense leaves were higher than in control and antisense leaves. The rate of photosynthesis under water deficit might have caused a degree of greater osmotic adjustment in sense leaves because it remained unaffected during water deprivation, which was in marked contrast with the severe drought-induced decrease in the CO(2) assimilation in control and antisense leaves. In antisense plants, the water stress stimulation of the antioxidative defenses was higher than in control plants, whereas in droughted sense leaves an induction of superoxide dismutase activity was not observed. These results suggest that overexpression of BiP in plants may prevent endogenous oxidative stress.     

Effect of gibberellic acid on K+ (Rb+) uptake and transport in sunflower roots

Four-week-old sunflower plants ( Helianthus annuus L. cv. Halcón), grown in different nutrient solutions, were used to study the effects of gibberellic acid (GA₃) on K⁺ (Rb⁺) uptake by roots or transport to the shoot. Gibberellic acid application to the nutrient solution did not affect the exudation process of excised roots. When GA₃ was sprayed on leaves 2 to 6 days before excising the roots, the rate of exudation and the K⁺ flux increased. When the exudation study was done keeping the roots in a nutrient solution in which Rb⁺ replaced K⁺, the GA₃ effects were evident also on Rb⁺ uptake and transport. In intact plants, GA₃ increased the Rb⁺ transported to the shoot but did not affect Rb⁺ accumulation in the root. It is suggested that these GA₃ effects can be explained if it is assumed that GA₃ acts on the transport of ions to the xylem vessels.