Ribonuclease and Chlorophyllase Activities in Senescing Leaves

The activities of two enzymes, ribonuclease and chlorophyllase were investigated during the senescence of leaves. Ribonuclease activities were measured in primary leaves of Phaseolus vulgaris, and related to the levels of nucleic acid, protein and chlorophyll. Similarly, changes in chlorophyllase activity during senescence of leaves of Raphanus sativus were measured and related to chlorophyll. During senescence the levels of each enzyme as well as its respective substrate declined. Retardation of senescence, by excision of young tissue from intact plants or by treatment of detached leaves with cytokinins resulted in a maintainace of both the substrate and enzyme levels. It was concluded that high levels of ribonuclease and chlorophyllase activity are not linked directly with the degradation of RNA and chlorophyll during leaf senescence.     

Induction and suppression of RNase activity by growth regulators in senescing ragi leaves

Activity of RNase was studied in attached and detached leaves of 7-day-old ragi ( Eleusine coracana Gaertn. cv PR 202) plants during senescence using crude enzyme extracts. The RNase activity was relatively constant in attached leaves. In excised leaves incubated in the dark there was a rapid rise in enzyme activity up to 48 h, followed by a decline. No such decrease was observed in the light. Benzimidazole and gibberellic acid suppressed the activity of RNase up to 48 h in the dark and 96 h in the light. Both the growth regulators also prevented the post-48 h decline in RNase activity of dark incubated excised leaves. Decline in the levels of chlorophyll and RNA in the illuminated excised leaves was not affected by 3-(3,4-dichlorophenyl)-1,1-dimethyIurea, but the inhibitor prevented the photo-induced rise in RNase activity. Cycloheximide and actinomycin D could completely prevent both detachment (increase in enzyme activity after the leaf is excised) and photo-induced rise in RNase activity. Benzimidazole and gibberellic acid prevented the rise in the activity of RNase on one hand and maintained it on the other by their influence on its biosynthesis. Photoinduction of RNase and photo-induced retardation of senescence are concluded to be two different processes.     

Structural and functional similarities between MRP and RNase P

RNase P, the enzyme responsible for 5-end processing of tRNAs and 4.5S RNA, has been extensively characterized fromE. coli. The RNA component ofE. coli RNase P, without the protein, has the enzymatic activity and is the first true RNA enzyme to be characterized. RNase P and MRP are two distinct nuclear ribonucleoprotein (RNP) particles characterized in many eukaryotic cells including human, yeast and plant cells. There are many similarities between RNase P and MRP. These include: (1) sequence specific endonuclease activity; (2) homology at the primary and secondary structure levels; and (3) common proteins in both the RNPs. It is likely that RNase P and MRP originated from a common ancestor.     

Expression of Three RNase Activities during Natural and Dark-Induced Senescence of Wheat Leaves

We have monitored the activities of RNases WL_A, WL_B, and WL_C (A Blank, TA McKeon [1991] Plant Physiol 97: 1402-1408) during leaf senescence in wheat (Triticum aestivum L. cv Chinese Spring). When seedlings were induced to senesce in darkness, protein loss from primary leaves began immediately. RNase WL_B activity was unchanged for 2 days and then rose linearly, reaching a sixfold elevation in 7 days. RNase WL_C activity declined for 2 days and then rose linearly, reaching a twofold elevation in 7 days. RNase WL_A activity declined in the first 2 days and was unchanged thereafter. Although differentially expressed, these RNase activities may respond to a common regulatory mechanism(s) which, at 2 days of darkness, signals progression into a more advanced stage of senescence. The RNase activities were also differentially expressed during light-induced recovery, returning to normal levels in dissimilar patterns. In flag leaves of greenhouse-grown wheat, the three RNase activities increased during the early postanthesis period when protein content was stable and underwent further, accelerated accumulation during senescence. RNase WL_B activity showed the largest overall senescence-associated elevation (sixfold), followed by RNase WL_C (fourfold) and RNase WL_A (threefold).     

Identification of BFN1, a bifunctional nuclease induced during leaf and stem senescence in Arabidopsis

Nuclease I enzymes are responsible for the degradation of RNA and single-stranded DNA during several plant growth and developmental processes, including senescence. However, in the case of senescence the corresponding genes have not been reported. We describe the identification and characterization of BFN1 of Arabidopsis, and demonstrate that it is a senescence-associated nuclease I gene. BFN1 nuclease shows high similarity to the sequence of a barley nuclease induced during germination and a zinnia (Zinnia elegans) nuclease induced during xylogenesis. In transgenic plants overexpressing the BFN1 cDNA, a nuclease activity of about 38 kD was detected on both RNase and DNase activity gels. Levels of BFN1 mRNA were extremely low or undetectable in roots, leaves, and stems. In contrast, relatively high BFN1 mRNA levels were detected in flowers and during leaf and stem senescence. BFN1 nuclease activity was also induced during leaf and stem senescence. The strong response of the BFN1 gene to senescence indicated that it would be an excellent tool with which to study the mechanisms of senescence induction, as well as the role of the BFN1 enzyme in senescence using reverse genetic approaches in Arabidopsis.     

Indoleacetic Acid-Induced Decrease of the Ribomiclease Activity in vivo

A study has been made on the influence of indole-3-acetic acid (IAA) on the ribonuclease (RNase) activity in wheat coleoptile sections and green pea stem sections. The hormonal effects on the enzyme activity, ribonncleic acid (RNA) metabolism and growth have been compared. Addition of 10^?5M IAA to the plant sections causes their RNase activity to decrease and their elongation to increase. Removal of the added IAA results in increasing enzyme activity and decreasing growth. The altered enzyme activities are paralleled by opposite changes in the RNA net synthesis. Administration of crystalline RNase to the plant tissue depresses growth. There is thus evidence that the in vivo effect of IAA on the RNase activity is of importance for the hormonal regulation of RNA metabolism and growth. The IAA-induced reduction in the enzyme activity involves cellular metabolism. The effect can be suspended by means of p-chloromercuribenzoate. A possible mechanism for the reduction is discussed.     

Suppression of LX ribonuclease in tomato results in a delay of leaf senescence and abscission

Although present in different organisms and conserved in their protein sequence, the biological functions of T2 ribonucleases (RNase) are generally unknown. Tomato (Lycopersicon esculentum) LX is a T2/S-like RNase and its expression is known to be associated with phosphate starvation, ethylene responses, and senescence and programmed cell death. In this study, LX function was investigated using antisense tomato plants in which the LX protein level was reduced. LX protein levels normally become elevated when leaves senesce and antisense inhibition of LX retarded the progression of senescence. Moreover, we observed a marked delay of leaf abscission in LX-deficient plants. This correlated with specific induction of LX protein in the tomato mature abscission zone tissue. LX RNase gene regulation and the consequences of antisense inhibition indicate that LX has an important functional role in both abscission and senescence.     

Identification and functional characterization of a leucine-rich repeat receptor-like kinase gene that is involved in regulation of soybean leaf senescence

We report here the cloning and characterization of a soybean receptor-like kinase (RLK) gene, designated GmSARK (Glycine max senescence-associated receptor-like kinase), which is involved in regulating leaf senescence. The conceptual protein product of GmSARK contains typical domains of LRR receptor-like kinases: a cytoplasmic domain with all the 11 kinase subdomains, a transmembrane domain and an extracelullar domain containing 9 Leucine-Rich Repeat (LRR) units that may act as a receptor. The expression of GmSARK in soybean leaves was up-regulated in all the three tested senescence systems: senescing cotyledons, dark-induced primary leaf senescence and the natural leaf senescence process after florescence. Furthermore, the RNA interference (RNAi)-mediated knocking-down of GmSARK dramatically retarded soybean leaf senescence. A more complex thylakoid membrane system, higher foliar level of chlorophyll content and a very remarkable delay of senescence-induced disintegration of chloroplast structure were observed in GmSARK-RNAi transgenic leaves. A homolog of maize lethal leaf-spot 1 gene, which has been suggested to encode a key enzyme catalyzing chlorophyll breakdown, was isolated and nominated Gmlls1. The expression level of Gmgtr1 gene, which encodes a key enzyme of chlorophyll synthesis, was also analyzed. It was found that Gmlls1 was up-regulated and Gmgtr1 was down-regulated during senescence in wild-type soybean leaves. However, both of the up-regulation of Gmlls1 and down-regulation of Gmgtr1 were retarded during senescence of GmSARK-RNAi transgenic leaves. In addition, over-expression of the GmSARK gene greatly accelerated the senescence progression of CaMV 35S:GmSARK transgenic plants. Taken together, these results strongly suggested the involvement of this LRR-RLK in regulation of soybean leaf senescence, maybe via regulating chloroplast development and chlorophyll accumulation. Multiple functions of GmSARK besides its regulation of leaf senescence were also discussed. Electronic Supplementary Material Supplementary material is available for this article at Rui Gan, Peng-Li Li and Yuan-Yuan Ma contributed equally to this work.     

Influence of ozone on ribonuclease activity in wheat (Triticum aestivum) leaves

Ribonucleases (RNases) degrade RNA and exert a major influence on gene expression during development and in response to biotic and abiotic stresses. RNase activity typically increases in response to pathogen attack, wounding and phosphate (P(i)) deficiency. Activity also increases during senescence and other programmed cell death processes. The air pollutant ozone (O(3)) often induces injury and accelerated senescence in many plants, but the biochemical mechanisms involved in these responses remain unclear. The objective of this study was to determine whether RNase activity and isozyme expression was stimulated in wheat (Triticum aestivum L.) flag leaves following treatment with O(3). Plants were treated in open-top chambers with charcoal-filtered air (27 nmol O(3) mol(-1)) (control) or non-filtered air plus O(3) (90 nmol O(3) mol(-1)) (O(3)) from seedling to reproductive stage. After exposure for 56 days, RNase activity was 2.1 times higher in flag leaf tissues from an O(3)-sensitive cultivar in the O(3) treatment compared with the control, which generally coincided with foliar injury and lower soluble protein concentration, but not soluble leaf [P(i)]. Soluble [P(i)] in leaf tissue extracts from the O(3) and control treatments was not significantly different. RNase activity gels indicated the presence of three major RNases and two nucleases, and their expression was enhanced by the O(3) treatment. Isozymes stimulated in the O(3) treatment were also stimulated in naturally senescent flag leaf tissues from plants in the control. However, soluble [P(i)] in extracts from naturally senescent flag leaves was 50% lower than that found in green flag leaves in the control treatment. Thus, senescence-like pathological responses induced by O(3) were accompanied by increased RNase and nuclease activities that also were observed in naturally senescent leaves. However, [P(i)] in the leaf tissue samples suggested that O(3)-induced injury and accelerated senescence was atypical of normal senescence processes in that P(i) export was not observed in O(3)-treated plants.     

Changes in the metabolism of nucleic acids during the growth and senescence of tobacco callus

Changes in DNA and RNA metabolism, DNA composition and RNA species in callus of tobacco ( Nicotiana rustica L. cv. Gansu Yellow Flower) were investigated during the growth and senescence. DNA and RNA contents remained almost unchanged during the callus growth period, but started to decrease synchronously at the time that callus senescence was initiated. Synthesis of DNA and RNA, as measured by incorporation of [³H]-labelled precursor, increased during the growth period and did not decrease until late in senescence. The activities of DNase and RNase (pH 4.5) increased during the early senescence period in accordance with the decrease in the levels of DNA and RNA, but appeared to decrease during late senescence. These results suggest that the decrease in the levels of DNA and RNA in senescing tobacco callus may stem from the increase in the hydrolytic activities of DNase and RNase (pH 4.5) in the early stage of senescence, and that the slowdown of synthesis in the late senescence period may also be a cause. DNA and RNA electrophoresis showed that a low-molecular-weight satellite DNA band disappeared after the onset of senescence and that the nuclear main band DNA gradually decreased, whereas the high-molecular-weight satellite DNA seemed to undergo no significant changes during the senescence period tested. Of the RNA species, 4–5S RNA was far more susceptible to damage during senescence than 25S and 18S rRNA. This suggests different susceptibilities of different DNA and RNA components to damage during the senescence of tobacco callus or alternatively a highly sequenced degradation of DNA and RNA molecules.     

Effects of Oxidative Stress Caused by Oxygen and Hydrogen Peroxide on Energy Metabolism and Senescence in Oat Leaves

In attached oat leaves the levels of adenine nucleotides decreasedduring leaf development and senescence. However, the energycharge (EC) only decreased from 0.90 in 4-cm leaves to 0.80in senescent leaves. In detached leaves the levels of adeninenucleotides increased for 48 h, in association with an increasein RNase activity and a decrease in levels of RNA. The EC remainedhigh until late senescence when levels of adenine nucleotidesfell to about 30% of initial values. A decrease in energy parametersinduced by transfer from light to darkness and from high (21%)to low (0.5% and anoxia) concentrations of oxygen resulted inan increase in membrane permeability. Oxidative stress (above 0.5% O₂ induced an increase in levelsof malondialdehyde (MDA) and then in permeability, associatedwith a decrease in levels of adenine nucleotides. Oxidativestress provoked by 0.05 and 0.10 M H₂O₂ caused a more rapiddecrease in energy parameters than O₂. Under oxidative stress(above 0.5% O₂) there is, first of all, an increase in membranepermeability and then a decrease in energy parameters, whichin turn are involved with senescence via increases in oxidationof membranes and degradation of energy-producing systems. (Received October 6, 1987; Accepted October 19, 1988)     

Changes occurring during aging and senescence in a submerged aquatic angiosperm (Potamogeton pectinatus)

Changes occurring during aging and senescence of leaves of a submerged aquatic angiosperm ( Potamogeton pectinatus L.) were studied. Total chlorophyll and chlorophylls a and b were maximal in mature, and minimal in old leaves. The chlorophyll a to b ratio was highest in mature leaves. During senescence, the chlorophyll content and the ratio of chlorophyll a to b decreased. The content of DNA, RNA, protein and dry weight, and the activity of alkaline pyrophosphatase decreased while free amino acids, the activity of protease, RNase and acid pyrophosphatase, and the ratio of acid to alkaline pyrophosphatase activity increased during aging and senescence. Kinetin (0.23 m M ) deferred leaf senescence by delaying the loss of chlorophyll, protein, nucleic acids and dry weight, and reducing the rise in free amino acids, the activity of protease, RNase and acid pyrophosphatase and the ratio of acid to alkaline pyrophosphatase activity; while both 0.69 m M ethrel and 0.075 m M ABA hastened senescence. Kinetin pretreatment for an optimum period (12 h) followed by ethrel or ABA treatment partially erased the senescence-promoting effect of the latter. But treatments in a reverse order markedly reduced the delaying effect of kinetin on senescence.     

Retardation of leaf senescence by benzyladenine in intact bean plants

Summary Benzyladenine (BA) applied to primary leaves of intact bean plants delayed the senescence of both the leaves and the entire shoot. The retardation of senescence was manifested in higher levels of chlorophyll, protein, RNA and ribonuclease activity at all stages of development. Also, the levels of incorporation of labelled precursors into protein and RNA were enhanced. The effect of BA was largely independent of light intensity and the compound did not act merely as a nitrogen source.     

Biochemical Changes in Excised Leaves of Oryza sativa Subjected to Water Stress

Excised rice (Oryza sativa L. cv. Ratna) leaves were used to compare the changes in the levels of various biochemical intermediates and enzyme activities during senescence in turgid and water-stressed conditions. Chlorophyll, total protein and soluble protein content decreased but α-amino nitrogen content increased during the senescence of turgid leaves. In the leaves subjected to water stress, these changes were accelerated, the acceleration being greater with higher degree of water stress. Starch, soluble sugars, total carbohydrates and non-reducing sugar content decreased during senescence of turgid leaves. Water stress accelerated the changes in the levels of starch and non-reducing sugar, but the changes in the levels of soluble sugars and total carbohydrates were retarded. Reducing sugar content increased at first and then decreased in the turgid leaves, and water stress accelerated the change. The decline in the catalase activity and the increase in the peroxidase activity with time was faster in the water-stressed leaves than in the turgid leaves. Acid inorganic pyrophosphatase activity increased, but alkaline inorganic pyrophosphatase activity decreased during the senescence of turgid leaves, and such changes were accelerated by water stress. The results suggest that water stress does not accelerate all the processes connected with leaf senescence.     

RNase MRP and rRNA processing

RNase MRP is a ribonucleoprotein enzyme with a structure similar to RNase P. It is required for normal processing of precursor rRNA, cleaving it in the Internal Transcribed Spacer 1. Abbreviations: RNase MRP RNase for mitochondrial RNA processing; also involved in pre-rRNA processing; RNase P - RNase for pre-tRNA processing; snoRNA - small nucleolar RNA; RNP - RNA-protein particle; snoRNP - small nucleolar RNA-protein particle.