may delay senescence of the whole plant. This fact has been interpreted in diverse ways, the most ac-
cepted idea being that hormonal factors produced by flowers and fruits affect the levels of signals re-
sponsible for senescence in the rest of the plant [2]. There is also evidence of roots playing a role as a
source of growth substances during senescence of fruiting plants. Thus, simultaneous senescence of all
plant parts is probably a result of interorgan signaling.
III. EXPERIMENTAL SYSTEMS TO STUDY SENESCENCE
The importance of having appropriate experimental systems and controls deserves special attention. Sys-
tems to study senescence have to be well defined and as close as possible to the natural environmental
conditions of the plants under study. In this sense, intact plants are the best experimental systems and
should be used whenever possible. Moreover, environmental conditions need to be under careful control.
Usually, plant senescence experiments are carried out under optimal greenhouse or growth chamber con-
ditions. However, stresses due to field suboptimal supplies of water and mineral nutrients, extreme tem-
peratures, salt, ultraviolet (UV) light, and wounding may heavily modify the natural senescence behavior
and should be taken into account. Light conditions are also crucial. For example, chlorophyll breakdown
is strongly retarded by continuous illumination when compared with leaves kept in the dark. Even light
quality (in relation to red or far-red components) is relevant because of the participation of phytochrome
in light-mediated responses during senescence [29,30].
A. Nonintact Plants
Senescence studies of bulky plants, such as trees, under controlled laboratory conditions may require the
use of simpler systems. The use of excised plant parts often produces important physiological and bio-
chemical changes, especially in the detached organ but also in the rest of the plant. If the use of a nonin-
tact plant system is unavoidable, precautions should be taken in analyzing the results because of the cor-
relative nature of many plant processes. As information grows, there is more and more evidence of
interrelation between all plant parts during senescence processes, probably through hormonal crosstalk
[31]. Thus, it is always necessary to contrast the observations with the changes undergone by intact or-
gans.
On the other hand, in testing the influence of some plant parts on the senescence of the remaining
ones, a common procedure consists of the surgical elimination of these parts. Usually, the replacement of
the excised organ with an agar block containing the putative hormonal effector or another type of exoge-
nous application completes the experiment [32]. The conclusions that can be extracted from this class of
work are limited. Exogenous application of any biological material is subjected to strong limitations such
as uptake, sequestration, transport, and metabolism of the active material and the difficulty of quantitat-
ing the amount of it within the target tissue [33].
B. Mutants and Transgenic Plants
Currently, the use of intact (but genetically modified) plants is making it possible to dissect senescence
without interferences from other processes. Therefore, mutants and transgenic plants are becoming
widely accepted as the best tools for this purpose.
Once a natural or induced mutation affecting senescence has been spotted, the next goal is usually to
identify the mutated gene and to characterize the phenotype at the molecular level in order to evaluate its
functional role in the senescence process. In this regard, current protocols that use map-based cloning,
large-scale complementary DNA (cDNA) sequencing, and polymerase chain reaction (PCR)-based meth-
ods for screening DNA insertion tagged populations of plants genetically engineered with transposons
[34] or T-DNA from Agrobacterium tumefaciens[35,36] facilitate the rescue of the mutated gene (for a
review see Ref. 37). Besides, in Arabidopsis thaliana, DNA microarrays (chips) to detect changes in gene
expression [38] and seed collections of plants carrying deletions of single genes (knockouts) [39] are com-
mercially available for identifying relevant genes and characterizing their roles in plant senescence. For
convenience, most work on characterization of senescence-related genes by mutant analysis has been
done in the small-genome weed A. thaliana, but this first step usually leads (or will lead in due time) to
the characterization and eventual manipulation of homologous genes from crop plants.
184 PEÑARRUBIA AND MORENO