Temperature-induced phase shifting of circadian rhythms in cotton seedlings as related to variations in chilling resistance

The relationship between the degree of chilling resistance and phase shifting caused by low-temperature pulses was examined in two circadian rhythms in cotton (Gossypium hirsutum L. cv. Deltapine 50) seedlings grown under light-dark cycles of 1212 h at 33° C. The seedlings showed a circadian rhythm of chilling resistance and of cotyledon movement. A pulse of 19° C for 12 h during the chilling-sensitive phase (light period) caused a phase delay of 6 h, while a similar temperature pulse during the chilling-resistant phase (dark period) did not cause any phase shift. Exposure to 19° C, 85% RH (relative humidity) for 12 h during the dark period induced chilling resistance in the following otherwise chilling-sensitive light period. In this light period a 12-h 19° C pulse did not cause a phase shift of chilling resistance. Pulses of low temperatures (5–19° C) were more effective in causing phase delays in the rhythm of cotyledon movement when given during the chilling-sensitive phase than when given during the chilling-resistant phase. A 12-h pulse of 5° C, 100% RH during the light period caused a phase delay of cotyledon movement of 12 h. However, when that pulse had been preceded by a chill-acclimating exposure to 19° C, 85% RH for 12 h during the dark period the phase delay was shortened to 6 h. The correlation between higher degree of chilling resistance and the prevention or shortening of the phase delay caused by low temperatures indicates that the mechanism that increases chilling resistance directly or indirectly confers greater ability for prevention of phase shifting by low temperatures in circadian rhythms.Abbreviations CT circadian time - LDC light-dark cycle of 24 h - RH relative humidity     

In vitro assay and light regulation of nitrate reductase in red alga Gracilaria chilensis

Nitrate reductase (NR) is the first enzyme in the nitrogen assimilation pathway. The in vitro NR activity of Gracilaria chilensis was assayed under different conditions to reveal its stability and biochemical characteristics, and an optimized in vitro assay is described. Maximal NR activities were observed at pH 8.0 and 15 degrees C. The apparent Km value for NADH was 8 microM and for nitrate 680 microM. Crude extracts of G. chilensis stored at 4 degrees C showed a 50% decrease of NR activity after 24 h. The highest NR activity value (253.20+/-2.60 x 10(-3) U g(-1)) was obtained when 100% von Stosch medium (500 microM NO3-) was added before extraction of apical parts. Algae under light:dark cycles of 12:12h exhibited circadian fluctuation of NR activity and photosynthesis with more than 2 times higher levels in the light phase. No evidence of endogenous diel rhythm controlling NR activity or photosynthesis was observed. Light pulses lasting 10 or 60 min during the darkness increased the NR activity by 30% and 45%, respectively. The results indicate that NR and photosynthesis are regulated mainly by light and not by a biological clock.     

Nitrate assimilation in the forage legume Lotus japonicus L.

Nitrate assimilation in the model legume, Lotus japonicus, has been investigated using a variety of approaches. A gene encoding a nitrate-inducible nitrate reductase (NR) has been cloned and appears to be the only NR gene present in the genome. Most of the nitrate reductase activity (NRA) is found in the roots and the plant assimilates the bulk of its nitrogen in that tissue. We calculate that the observed rates of nitrate reduction are compatible with the growth requirement for reduced nitrogen. The NR mRNA, NRA and the nitrate content do not show a strong diurnal rhythm in the roots and assimilation continues during the dark period although export of assimilated N to the shoot is lower during this time. In shoots, the previous low NR activity may be further inactivated during the dark either by a phosphorylation mechanism or due to reduced nitrate flux coincident with a decreased delivery through the transpiration stream. From nitrate-sufficient conditions, the removal of nitrate from the external medium causes a rapid drop in hydraulic conductivity and a decline in nitrate and reduced-N export. Root nitrate content, NR and nitrate transporter (NRT2) mRNA decline over a period of 2 days to barely detectable levels. On resupply, a coordinated increase of NR and NRT2 mRNA, and NRA is seen within hours.     

植物中硝酸还原酶和亚硝酸还原酶的作用

植物通过硝酸盐同化途径以硝酸盐和氨的形式吸收氮元素。硝酸盐的同化是一个受到严格控制的过程,其中两个先后参加反应的酶——硝酸还原酶(NR)和亚硝酸还原酶(NiR)对初级氮的同化起主要调控。在高等植物中,NR和NiR基因的转录及转录后加工受到各种内在和外在因素的影响,翻译后调控是消除亚硝酸盐积累的重要机制。随着分子生物学技术的发展,可以更容易地通过突变体和转基因方式来研究NR和NiR基因的调控。     

Nitrate reductase and glutamate dehydrogenase activities in Skeletonema costatum as measures of nitrogen assimilation rates

Nitrate reductase (NADH-NR) and glutamate dehydrogenase (NADPH-GDH)activities were measured in Skeletonema costatum (Grev.) Clevein ammonium and nitrate limited continuous cultures before andafter additions of nitrate and/or ammonium. Comparisons of enzymicactivity with nitrogen uptake and assimilation rates, externaland internal nitrate concentrations, and external ammonium concentrationswere made in order to assess the roles of NR and GDH in nitrogenassimilation and to determine their suitability as measuresof nitrogen assimilation rates. NR activity appeared to be inducedby internal rather than external nitrate concentrations. Ammoniumin the medium reduced NR activity under some environmental conditions,but not others. However, ammonium acted indirectly, perhapsby causing the accumulation of an internal pool of an intermediateof ammonium assimilation. NR activity was found to approximatenitrate assimilation rates during growth limited by the nitratesupply and undeT some conditions in the presence of high nitrateand ammonium concentrations in the medium. Under other environmentalconditions, NR activity did not agree with nitrate assimilationrates; a second nitrate reducing mechanism may operate whenthese conditions prevail. GDH activities were consistently low,representing less than 5% of the ammonium uptake and assimilationrates. Consequently, it is proposed that ODH is not the primaryammonium assimilating enzyme under most environmental conditionsand cannot be used as a measure of ammonium assimilation. ¹ Contribution number 1095 from the Department of Oceanography,University of Washington     

A nitrate-induced frq-less oscillator in Neurospora crassa

When nitrate is the only nitrogen source, Neurospora crassa's nitrate reductase (NR) shows endogenous oscillations in its nitrate reductase activity (NRA) on a circadian time scale. These NRA oscillations can be observed in darkness or continuous light conditions and also in a frq(9) mutant in which no functional FRQ protein is formed. Even in a white-collar-1 knockout mutant, NRA oscillations have been observed, although with a highly reduced amplitude. This indicates that the NRA oscillations are not a simple output rhythm of the white-collar-driven frq oscillator but may be generated by another oscillator that contains the nit-3 autoregulatory negative feedback loop as a part. In this negative feedback loop, a product in the reaction chain catalyzed by nitrate reductase, probably glutamine, induces repression of the nitrate reductase gene and thus downregulates its own production. This is the first example of an endogenous, nutritionally induced daily rhythm with known molecular components that is observed in the absence of an intact FRQ protein.     

Rapid and slow modulation of nitrate reductase activity in the red macroalga <Emphasis Type="Italic">Gracilaria chilensis</Emphasis> (Gracilariales,Rhodophyta): influence of different nitrogen sources

Nitrate is one of the most important stimuli in nitrate reductase (NR) induction, while ammonium is usually an inhibitor. We evaluated the influence of nitrate, ammonium or urea as nitrogen sources on NR activity of the agarophyte Gracilaria chilensis. The addition of nitrate rapidly (2 min) induced NR activity, suggesting a fast post-translational regulation. In contrast, nitrate addition to starved algae stimulated rapid nitrate uptake without a concomitant induction of NR activity. These results show that in the absence of nitrate, NR activity is negatively affected, while the nitrate uptake system is active and ready to operate as soon as nitrate is available in the external medium, indicating that nitrate uptake and assimilation are differentially regulated. The addition of ammonium or urea as nitrogen sources stimulated NR activity after 24 h, different from that observed for other algae. However, a decrease in NR activity was observed after the third day under ammonium or urea. During the dark phase, G. chilensis NR activity was low when compared to the light phase. A light pulse of 15 min during the dark phase induced NR activity 1.5-fold suggesting also fast post-translational regulation. Nitrate reductase regulation by phosphorylation and dephosphorylation, and by protein synthesis and degradation, were evaluated using inhibitors. The results obtained for G. chilensis show a post-translational regulation as a rapid response mechanism by phosphorylation and dephosphorylation, and a slower mechanism by regulation of RNA synthesis coupled to de novo NR protein synthesis.     

An overnight chill induces a delayed inhibition of photosynthesis at midday in mango (Mangifera indica L.)

The effect of a cold night on photosynthesis in herbaceous chilling-sensitive crops, like tomato, has been extensively studied and is well characterized. This investigation examined the behaviour of the sub-tropical fruit tree, mango, to enable comparison with these well-studied systems. Unlike tomato, chilling between 5 degrees C and 7 degrees C overnight produced no significant inhibition of light-saturated CO(2) assimilation (A:) during the first hours following rewarming, measured either under controlled environment conditions or in the field. By midday, however, there was a substantial decline in A:, which could not be attributed to photoinhibition of PSII, but rather was associated with an increase in stomatal limitation of A: and lower Rubisco activity. Overnight chilling of tomato can cause severe disruption in the circadian regulation of key photosynthetic enzymes and is considered to be a major factor underlying the dysfunction of photosynthesis in chilling-sensitive herbaceous plants. Examination of the gas exchange of mango leaves maintained under constant conditions for 2 d, demonstrated that large depressions in A: during the subjective night were primarily the result of stomatal closure. Chilling did not disrupt the ability of mango leaves to produce a circadian rhythm in stomatal conductance. Rather, the midday increase in stomatal limitation of A: appeared to be the result of altered guard cell sensitivity to CO(2) following the dark chill.     

Elevated CO2 alters tissue balance of nitrogen metabolism and downregulates nitrogen assimilation and signalling gene expression in wheat seedlings receiving high nitrate supply

Tissue and canopy-level evidence suggests that elevated carbon dioxide (EC) inhibits shoot nitrate assimilation in plants and thereby affects nitrogen (N) and protein content of the economic produce. It is speculated that species or genotypes relying more on root nitrate assimilation can adapt better under EC due to the improved/steady supply of reductants required for nitrate assimilation. A study was conducted to examine the effect of EC on N assimilation and associated gene expression in wheat seedlings. Wheat genotypes, BT-Schomburgk (BTS) with comparatively high leaf nitrate reductase (NR) activity and Gluyas Early (GE) with high root NR activity were grown in hydroponic culture for 30 days with two different nitrate levels (0.05 mM and 5 mM) in the climate controlled growth chambers maintained at either ambient (400 ± 10 μmol mol⁻¹) or EC (700 ± 10 μmol mol⁻¹) conditions. Exposure to EC downregulated the activity of enzyme NR and glutamate synthase (GOGAT) in leaf tissues, whereas in roots, activities of both the enzymes were upregulated by exposure to EC. In addition, EC downregulated N assimilation and signalling gene expression under high N availability. Root N assimilation was less affected in comparison with shoot N assimilation; thereby, the proportion of root contribution towards total assimilation was higher. The results suggest that EC could alter and re-programme N assimilation and signalling in wheat seedlings. The genotype and tissue-specific effects of EC on N assimilation also warrants the need for identification of suitable genotypes and revision of fertiliser regime for tapping the beneficial effects of EC conditions.

     

The nodulation and nyctinastic leaf movement is orchestrated by clock gene LHY in Medicago truncatula

As sessile organisms, plants perceive, respond, and adapt to the environmental changes for optimal growth and survival. The plant growth and fitness are enhanced by circadian clocks through coordination of numerous biological events. In legume species, nitrogen‐fixing root nodules were developed as the plant organs specialized for symbiotic transfer of nitrogen between microsymbiont and host. Here, we report that the endogenous circadian rhythm in nodules is regulated by MtLHY in legume species Medicago truncatula. Loss of function of MtLHY leads to a reduction in the number of nodules formed, resulting in a diminished ability to assimilate nitrogen. The operation of the 24‐h rhythm in shoot is further influenced by the availability of nitrogen produced by the nodules, leading to the irregulated nyctinastic leaf movement and reduced biomass in mtlhy mutants. These data shed new light on the roles of MtLHY in the orchestration of circadian oscillator in nodules and shoots, which provides a mechanistic link between nodulation, nitrogen assimilation, and clock function.     

DCMU inhibits in vivo nitrate reduction in illuminated barley (C(3)) leaves but not in maize (C(4)): a new mechanism for the role of light?

The leaves of C(4) plants possess a superior metabolic efficiency not only in terms of photosynthetic carbon assimilation, but also in terms of inorganic nitrogen assimilation, when compared to C(3)plants. In vivo nitrate assimilation efficiency of leaves is dependent on light, but the obligatory presence of light has been debated and its role remains confounded. This problem has not been addressed from the standpoint of the C(3) vs. C(4) nature of the species investigated, which may actually hold the key to resolve the controversy. Here, we present the first report providing evidence for differential photo-regulation of leaf nitrate reduction in barley ( Hordeum vulgare L.) vs. maize ( Zea mays L.) plants, which may help explain the superior nitrogen-use efficiency (and hence superior productivity) of maize plants. The novel finding that carbohydrate-depleted maize leaves were able to reduce nitrate when photosynthesis was inhibited by 3-(3',4'-dichlorophenyl)-1,1'-dimethylurea (DCMU) in the presence of light, raises a very important question about the possibilities of a new photo-regulatory mechanism for supporting nitrate reduction in maize leaves operating independently of photosynthetic carbon dioxide fixation. On the other hand, leaves of barley could not carry out any in vivo nitrate assimilation, whatsoever, under these conditions. We find another fundamental difference between the two species in terms of differential regulation of nitrate reductase (NR; EC 1.6.6.1). In barley leaves, NR activity and activation state remained unaffected due to DCMU, but in sharp contrast, both were appreciably upregulated in maize. Collectively, the results indicate that enzyme capacity is not limiting for nitrate reduction in leaves, as the NR activity was higher in barley than in maize. The maize leaves may have had a selective advantage due to C(4) morphology/metabolism in terms of maintaining a better reductant/carbon skeleton supply for nitrate reduction.