Micro-scale chlorophyll analysis and developmental expression of a cytokinin oxidase/dehydrogenase gene during leaf development and senescence

Chlorophyll content is a baseline measurement in many plant studies. With the miniaturization of many molecular and biochemical assays the sample size needed for these assays has been greatly reduced. Chlorophyll is commonly determined by spectrophotometry. Traditionally this analysis has been carried out in 10 mm cuvettes with volumes of 0.2–2.0 ml. The NanoDrop™ spectrophotometer requires a sample volume of 1–2 μl which provides the opportunity to analyze far smaller samples. Chlorophyll analyses are a critical part of any study of senescence and the cytokinins are considered to play a key role in regulating senescence. The tissue specific levels of cytokinins are thought to be regulated, at least in part, by expression of the cytokinin degradation enzyme cytokinin oxidase/dehydrogenase (CKX). Here an assay is described for the spectral determination of chlorophyll using the NanoDrop™ spectrophotometer that allows the determination of chlorophyll from part samples of clover leaves while leaving sufficient tissue for quantitative real-time PCR study of CKX expression. The expression of TrCKX2 increased during leaf development along the stolon of Trifolium repens but was stable during the onset and progression of senescence suggesting that TrCKX2 was not an initiator of leaf senescence but may have facilitated the progression.     

EPR spin labeling measurements of thylakoid membrane fluidity during barley leaf senescence

Physical properties of thylakoid membranes isolated from barley were investigated by the electron paramagnetic resonance (EPR) spin labeling technique. EPR spectra of stearic acid spin labels 5-SASL and 16-SASL were measured as a function of temperature in secondary barley leaves during natural and dark-induced senescence. Oxygen transport parameter was determined from the power saturation curves of the spin labels obtained in the presence and absence of molecular oxygen at 25 °C. Parameters of EPR spectra of both spin labels showed an increase in the thylakoid membrane fluidity during senescence, in the headgroup area of the membrane, as well as in its interior. The oxygen transport parameter also increased with age of barley, indicating easier diffusion of oxygen within the membrane and its higher fluidity. The data are consistent with age-related changes of the spin label parameters obtained directly by EPR spectroscopy. Similar outcome was also observed when senescence was induced in mature secondary barley leaves by dark incubation. Such leaves showed higher membrane fluidity in comparison with leaves of the same age, grown under light conditions. Changes in the membrane fluidity of barley secondary leaves were compared with changes in the levels of carotenoids (car) and proteins, which are known to modify membrane fluidity. Determination of total car and proteins showed linear decrease in their level with senescence. The results indicate that thylakoid membrane fluidity of barley leaves increases with senescence; the changes are accompanied with a decrease in the content of car and proteins, which could be a contributing factor.     

Natural developmental variations in leaf and plant senescence in Arabidopsis thaliana

Leaf senescence is a developmentally regulated process that contributes to nutrient redistribution during reproductive growth and finally leads to tissue death. Manipulating leaf senescence through breeding or genetic engineering may help to improve important agronomic traits, such as crop yield and the storage life of harvested organs. Here, we studied natural variations in the regulation of plant senescence among 16 Arabidopsis thaliana accessions. Chlorophyll content and the proportion of yellow leaves were used as indicator parameters to determine leaf and plant senescence respectively. Our study indicated significant genotype effects on the onset and development of senescence. We selected three late- and five early-senescence accessions for further physiological studies. The relationship between leaf and plant senescence was accession-dependent. There was a significant correlation between plant senescence and the total number of leaves, siliques and plant bolting age. We monitored expression of two senescence marker genes, SAG12 and WRKY53 , to evaluate progression of senescence. Our data revealed that chlorophyll content does not fully reflect leaf age, because even fully green leaves had already commenced senescence at the molecular level. Integrating senescence parameters, such as the proportion of senescent leaves, at the whole plant level provided a better indication of the molecular status of the plant than single leaf senescence parameters.     

Hormonal regulation of leaf senescence through integration of developmental and stress signals

Leaf senescence is a genetically controlled dismantling programme that enables plants to efficiently remobilise nutrients to new growing sinks. It involves substantial metabolic reprogramming whose timing is affected by developmental and environmental signals. Plant hormones have long been known to affect the timing of leaf senescence, but they also affect plant development and stress responses. It has therefore been difficult to tease apart how the different hormones regulate the onset and progression of leaf senescence, i.e., whether they directly affect leaf senescence or affect it indirectly by altering the developmental programme or by altering plants’ response to stress. Here we review research on hormonal regulation of leaf senescence and propose that hormones affect senescence through differential responses to developmental and environmental signals. We suggest that leaf senescence strictly depends on developmental changes, after which senescence can be induced, depending on the type of hormonal and environmental cues.     

Protein degradation and nitrogen remobilization during leaf senescence

Leaf senescence, a type of programmed cell death, is a complex and highly regulated process that involves the degradation of macromolecules, including proteins, nucleic acids, and lipids. Nutrients, especially nitrogen, are re-mobilized from senescing leaves to newly developing tissues or reserve organs. Our review focuses on three pathways for protein breakdown and the resorption of N during this process: the ubiquitin/proteosome system, the chloroplast degradation pathway, and the vacuolar and autophagic pathway. We propose that two relative biochemical cycles exist for amino acid recycling and N-export — the GS/GOGAT cycle and the PPDK-GS/GOGAT cycle.     

A senescence-associated gene of Arabidopsis thaliana is distinctively regulated during natural and artificially induced leaf senescence

We have characterized the structure and expression of a senescence-associated gene (sen1) of Arabidopsis thaliana. The protein-coding region of the gene consists of 5 exons encoding 182 amino acids. The encoded peptide shows noticeable similarity to the bacterial sulfide dehydrogenase and 81% identity to the peptide encoded by the radish din1 gene. The 5-upstream region contains sequence motifs resembling the heat-shock- and ABA-responsive elements and the TCA motif conserved among stress-inducible genes. Examination of the expression patterns of the sen1 gene under various senescing conditions along with measurements of photochemical efficiency and of chlorophyll content revealed that the sen1 gene expression is associated with Arabidopsis leaf senescence. During the normal growth phase, the gene is strongly induced in leaves at 25 days after germination when inflorescence stems are 2–3 cm high, and then the mRNA level is maintained at a comparable level in naturally senescing leaves. In addition, dark-induced senescence of detached leaves or of leaves in planta resulted in a high-level induction of the gene. Expression of the sen1 gene was also strongly induced in leaves subjected to senescence by 0.1 mM abscisic acid or 1 mM ethephon treatment. The induced expression of the gene by dark treatment was not significantly repressed by treatment with 0.1 mM cytokinin or 50 mM CaCl₂ which delayed loss of chlorophyll but not that of photochemical efficiency.     

Activation of latent phenolase during spinach leaf senescence

The observed increase of phenolase activity and of its rate of activation during spinach leaf senescence is due to reduced binding of latent phenolase to the thylakoid membranes and not to de novo synthesis. The same amount of phenolase which is active in isolated thylakoid membranes from senescent leaves can be found in the membranes of non-senescent leaves after activation of latent enzyme. Tracer experiments give evidence that one multiple form which is responsible for the bulk activity in senescent leaves, is synthesized before, but not after the onset of senescence, indicating that pre-existing latent phenolase is converted to easily activating forms.     

Molecular analysis of natural leaf senescence in Arabidopsis thaliana

Using artificial canopies, several authors have shown that horizontally propagated and overall propagated radiation beneath the canopy differ substantially in spectral distribution in the red (R) and far red (FR) wavelengths. Given the lack of information about light quality under real crop canopies, the R:FR ratio of vertical and horizontal radiation beneath field-grown maize, soybean and wheat was monitored until leaf area index (LAI) reached 4, 2.5 and 6.9, respectively.
A Li-Cor 1800 spectroradiometer with a remote cosine receptor fitted with a quartz fibre-optic light-guide was used. To isolate radiation coming from a given direction, a black coated tube was fitted to the cosine receptor. The viewing angle was 15°. In open conditions, the values of R:FR from the upper hemisphere were between 1.07 and 1.20. For vertically and horizontally-propagated light, average values were 1.22 and 0.75 respectively.
Beneath the canopy, both R:FR and photosynthetic photon flux density (PPFD) from the entire upper hemisphere decreased in relation to LAI and crop height. R:FR of the horizontal component were found to be generally much lower than the vertical, which decreased significantly only in the later measurements.
The lowest R:FR values were recorded under wheat and soybean canopies. Even the very low LAIs present at early development stages were enough to cause a sharp decrease of R:FR in the horizontal fluxes. Referring to the entire upper hemisphere, PPFD transmittance and R:FR as a percentage of the external references appeared well correlated.     

Regulation of ethylene biosynthesis during senescence of oat leaf segments

The evolution of endogenous ethylene, the conversion of 1-aminocylopropane-1-car-boxylic acid (ACC) to ethylene and the amounts of ACC (free and conjugated) have been followed during the senescence of oat ( Avena sativa L. cv. Victory) leaf segments. During the first three days of incubation of leaf segments in darkness, endogenous ethylene evolution and ACC-dependent ethylene production displayed a close relationship, both showing an increase followed by a decrease to the basal rate. However, unlike ethylene production, the level of ACC increased during the five days of incubation in the dark without any decline. It is concluded that ACC synthesis does not limit ethylene production, at least in the last stages of leaf senescence when ethylene production markedly decreased. The level of conjugated ACC increased and reached a plateau already at the first day of incubation. Yet, at the progressive stages of senescence, when the level af ACC gradually increased, no further conjugation of ACC could be detected. Thus, conjugation of ACC cannot account for ethylene drop at the last stages of oat leaf senescence.     

Comparison of phosphorus mobilization during monocarpic senescence in rice cultivars with sequential and non-sequential leaf senescence

The senescence pattern of the three uppermost leaves of four rice (Oryza sativa L.) cultivars viz. Ratna, Jaya, Masuri and Kalojira was analysed in terms of decline of chlorophyll and by measuring [³²P]-phosphate retention and export from leaf to grains during the reproductive development. With the advancement of reproductive development, the cultivars Masuri and Kalojira showed a sequential mode of senescence, but the cultivars Ratna and Jaya showed a non-sequential mode of leaf senescence where the flag leaf senesced earlier than the older second leaf. Foliar spraying with benzyladenine (0.5 mM) significantly delayed, and abscisic acid (0.1 mM) accelerated, leaf senescence. In untreated control plants, the second leaf had the highest export of labelled phosphate among the leaves at the grain formation stage (0–7 days) in Masuri and Kalojira. This was compensated by the flag leaf at the grain development stage (7–14 days), whereas export of [³²P]-phosphate was highest from the flag leaf of Ratna and Jaya at the grain development stage. Compared with the control, benzyladenine treatment caused higher retention of [³²P]-phosphate in the leaves and also export to the grains, but abscisic acid treatment gave lower retention and export of [³²P]-phosphate to the grains. The amount of [³²P]-phosphate export from a mother to a daughter shoot developed in the axil of the second leaf of plants with the panicle removed, was less than that to panicles remaining on control plants of all cultivars. When the panicle had been excised, the greatest export of [³²P]-phosphate took place from the second leaf to the daughter shoot in all cultivars. Excision of the panicle delayed leaf senescence as compared with intact controls and maintained an age-related leaf senescence pattern in all the four cultivars. The results presented here demonstrate that mobilization of phosphorus from leaf to grains, regardless of cultivar or age and position of the leaf, correlates well with the senescence of that leaf.     

Predictive capability of a leaf optical meter for determining leaf pigment status during senescence

We conducted an experiment to assess the predictive capability of a leaf optical meter for determining leaf pigment status of Acer mono Maxim., A. ginnala Maxim., Quercus mongolica Fisch., and Cornus alba displaying a range of visually different leaf colors during senescence. Concentrations of chlorophyll (Chl) a, Chl b, and total Chl [i.e., Chl (a+b)] decreased while the concentration of carotenoids (Car) remained relatively static for all species as leaf development continued from maturity to senescence. C. alba exhibited the lowest average concentration of Chl (a+b), Chl a, and Car, but the highest relative anthocyanin concentration, while Q. mongolica exhibited the highest Chl (a+b), Chl b, and the lowest relative anthocyanin concentration. A. mono exhibited the highest Chl a and Car concentrations. The relationships between leaf pigments and the values measured by the optical meter generally followed an exponential function. The strongest relationships between leaf pigments and optical measurements were for A. mono, A. ginnala, and Q. mongolica (R ² ranged from 0.64 to 0.95), and the weakest relationships were for C. alba (R ² ranged from 0.13 to 0.67). Moreover, optical measurements were more strongly related to Chl a than to Chl b or Chl (a+b). Optical measurements were not related to Car or relative anthocyanin concentrations. We predicted that weak relationships between leaf pigments and optical measurements would occur under very low Chl concentrations or under very high anthocyanin concentrations; however, these factors could not explain the weak relationship between Chl and optical measurements observed in C. alba. Overall, our results indicated that an optical meter can accurately estimate leaf pigment concentrations during leaf senescence — a time when pigment concentrations are dynamically changing — but that the accuracy of the estimate varies across species. Future research should investigate how species-specific leaf traits may influence the accuracy of pigment estimates derived from optical meters.     

The correlation between oxidative stress and leaf senescence during plant development

In plants, besides being the final step leading to the death of the whole organism, senescence has a developmental function involving the coordinated degradation of macromolecules and the mobilization of nutrients out of senescing tissues into developing parts of the plant. Free radicals are thought to play an essential role in senescence, especially those derived from oxygen. Since these molecules are extremely toxic, the levels of the different reactive oxygen species have to be tightly regulated. However, at low concentrations, hydrogen peroxide may also serve as a signalling molecule. Therefore, a coordinated regulation of the free radical scavenging system, which comprises enzymatic components such as catalase, superoxide dismutase and ascorbate peroxidase, and non-enzymatic molecules such as ascorbate and glutathione is essential. The increased radical levels displayed during senescence are not only caused by the elevated production of radicals but also by a loss in antioxidant capacity.