REGULATION OF GENES INVOLVED IN LIPID CATABOLISM IN CaMVINFECTED ARABIDOPSIS
Pietro Giuntini1,3, Luigi De Bellis2, Amedeo Alpi3 and Joel J. Milner1
Plant Molecular Science Group, IBLS, Glasgow University, Glasgow, UK
University of Lecce, Lecce, Italy 3 Dep. Crop Biology, University of Pisa, Pisa, Italy
1
2
Di.S.Te.B.A.,
Fatty acids play an essential role in all living cells, either as components of membrane
lipids or as important forms of high energy storage compounds. For this reason, fatty acid
catabolism is an important process found in germinating oilseeds but also present in a
basal level throughout the plant life-cycle and in a wide variety of tissue. In mature plants,
levels of peroxysomal -oxidation gene expression increase during foliar senescence,
carbohydrate starvation and abiotic stress (wounding).
ACX is the first enzyme of the -oxidation cycle and is believed to act as a control point for
all the cycle. In Arabidopsis the ACX family is composed of at least four isozymes with
distinct but partially overlapping substrate specificities. AtACX1 is particularly active
medium- to long-chain saturated acyl-CoA; AtACX2 prefers long-chain unsaturated acylCoAs; AtACX3 preferred substrates are medium-chain acyl-CoAs, whereas AtACX4 is
active only on short-chain acyl-CoAs.During virus infection, the need to support
replication imposes an increased metabolic load on the plant. We therefore examined the
expression of -oxidation genes during infection by cauliflower mosaic virus (CaMV; a
compatible pathogen of Arabidopsis). Gene expression was analyzed in infected wild-type
(Col-0) Arabidopsis and in NahG transgenic plants (which are unable to accumulate
salicylic acid and which accumulate three times as much virus as wild-type plants).
Arabidopsis plants were infected on the first two true leaves with CaMV (isolate: Cabb BJI) approximately 10-13 days after germination and plant material were collected 21 days
post infection. RNA was extracted and gene expression was analyzed in Northern blots,
using probes for six genes that encode proteins involved in lipid catabolism. Virus
replication was estimated using a probe for CaMV Gene VI. PR-1 is a marker for SAsignalled defence. In Northern blots, levels of transcripts from four genes, AtACX1,
AtACX2, AtACX4 and THI (3,3-ketoacyl-CoA thiolase) were greatly increased at 21 dpi,
whereas the levels of AtACS3 and AIM1 (multi functional protein: hydratase
/dehydrogenase) showed moderate increases. Expression in infected NahG plants was
similar to wild-type, although virus levels were three times greater.
Real-Time Quantitative PCR was used to obtain a more precise estimate of mRNA levels.
Data were normalized using reference dye (ROX) in the PCR mix and normalized to ACT2
mRNA as an internal standard. At 21 dpi, the time at which virus accumulation reaches a
maximum, levels of ACX1 and ACX2 mRNA were 5-6 times greater than in uninfected
controls; levels of THI and AIM1 mRNAs were 3-4 times greater than in uninfected
controls. We observed no significant differences in transcript levels of any of the these
genes between either infected or uninfected NahG and wild-type.
We conclude that during CaMV infection several genes encoding peroxysomal enzymes
are very significantly up-regulated. However, these changes in gene activity do not
correlate directly with virus replication (and hence the apparent increase in metabolic
load), because transcript levels in NahG (which supports higher levels of replicating virus)
were similar to wild-type.