Peroxisomal enzyme activities in attached senescing ... - Springer Link

Planta

Planta (1991)184:151-153

9 Springer-Verlag1991

Peroxisomal enzyme activities in attached senescing leaves Laura Pistelli, Luigi De Bellis, and Amedeo Alpi Dipartimento di Biologia delle Piante Agrarie, UniversitA di Pisa, Viale delle Piagge 23, 1-56124 Pisa, Italy Received 27 July; accepted 9 November 1990

Abstract. Recently it has been demonstrated that detached leaves show glyoxysomal enzyme activities when incubated in darkness for several days. In this report glyoxylate-cycle enzymes have been detected in leaves of rice (Oryza sativa L.) and wheat (Triticum durum L.) from either naturally senescing or dark-treated plants. Isolated peroxisomes of rice and wheat show isocitrate lyase (EC 4.1.3.1), malate synthase (EC 4.1.3.2) and/3oxidation activities. L e a f peroxisomes f r o m dark-induced senescing leaves show glyoxylic-acid-cycle enzyme activities two to four times higher than naturally senescing leaves. The glyoxysomal activities detected in leaf peroxisomes during natural foliar senescence m a y represent a reverse transition of the peroxisomes into glyoxysomes.

Key words: Isocitrate lyase - L e a f senescence - Malate synthase - Oryza - Peroxisome - Senescence (natural, i n d u c e d ) - Triticum

The role in higher-plant cells of peroxisomes with tissuespecific functions has been well established (Huang et al. 1983), and recently the /3-oxidation of fatty acids has been characterized as a c o m m o n function for all classes of peroxisomes (Gerhardt 1986; Pistelli et al. 1989). G u t and Matile (1988) have shown that barley leaf segments contain glyoxylate-cycle enzymes after induction of senescence. Furthermore, De Bellis et al. (1990) demonstrated the occurrence of isocitrate lyase and malate synthase in peroxisomes of dark-treated detached rice and leaf beet leaves and p u m p k i n cotyledons. During the initial stages of dark-induced leaf senescence, chloroplasts show evident signs of degeneration (KuraH o t t a et al. 1990); by contrast peroxisomes maintain their integrity and undergo surprising variations in their enzyme complements. In the present study, evidence has been gathered a b o u t the occurrence of peroxisomal glyoxylate-cycle enzymes in naturally senescing leaves of rice and wheat. Plants of rice (Oryza sativa L. cv. Arborio) and wheat (Tritieum durum cv. Creso) were grown in the greenhouse. Third leaves from

rice and wheat plants were harvested when clear symptoms of senescence appeared, 90 and 120 d for rice and wheat, respectively, after sowing. Other experiments were carried out using 60-d-old rice plants and 35-d-old wheat plants transferred to complete darkness for 11 and 7 d, respectively. Third leaves were collected for the experiments. Five grams fresh weight (FW) of rice and wheat leaves were used to isolate organelles as described previously (De Bellis et al. 1990). Enzyme activities were assayed according to the cited references: catalase (EC 1.11.1.6), Aebi (1974); fl-hydroxypyruvate reductase (EC 1.1.1.81), Huang and Beevers (1972); triosephosphate isomerase (EC 5.3.1.1), Feierabend (1975); cytoehrome-c oxidase (EC 1.9.3.1), Hodges and Leonard (1974) modified by Pistelli et al. (1989); isocitrate lyase (EC 4.1.3.1), Franzisket and Gerhardt (1980); malate synthase (EC 4.1.3.2), Hock and Beevers (1966); overall fl-oxidation (palmitoyl-CoA-dependent NAD reduction) Cooper and Beevers (1969). Proteins were determined using a Bio-Rad (Watford, UK) protein assay kit with a bovine serum albumin as standard. Total chlorophyll was determined according to Inskeep and Bloom (1985) using N,N-dimethylformamide as solvent. Sucrose concentrations were determined refractometrically. Figure 1 shows the separation by sucrose density gradients of organelles f r o m leaves of rice plants grown for 11 d in the dark or f r o m naturally senescing leaves. The behaviour of organelles and m a r k e r enzymes was similar to that reported in previous papers (Pistelli et al. 1989; De Bellis et al. 1990). Key enzymes of the glyoxylate cycle and overall /3oxidation activity were tested along the gradient fractions. Isocitrate lyase and malate synthase activities were found under both experimental conditions; their profile followed that of catalase./?-Oxidation activity was also observed in naturally senescing and in dark-grown rice leaves. Table 1 shows, for rice and wheat, the specific activities of the various peroxisomal enzymes in their characteristic fractions under the examined conditions. The activity of hydroxypyruvate reductase was relatively higher in naturally senescent leaves than in dark-treated leaves, whereas isocitrate lyase and malate synthase showed greater activity in permanently darkened leaves. The higher activities of isocitrate lyase and malate synthase

L. Pistelli et al. : Glyoxylate-cycle enzymes in senescing leaves

152 RICE-DARK

1:i

R I C E - NATURALLY

INCUBATED

o oc.L HPR

i

CHL

o--o

o - - o HPR & - - & B-OX

o--o

--

SENESCENT

&--AB-OX

90.8 -E

!

90.6

v

E

.J

-0.4

i1. ' o 90.2 rv O -I '

L-..-_ 14-

:

~.

ooooood ~>.

CYT OX

CYT OX ICL PROT

0--0

~o

r n - - n ICL

> F-- 1 2 -

D--n o--o

O - - O PROT

12T

lO E

> 10" F-r

\

8