The Plant Vascular System: Evolution, Development and FunctionsF

2010; Schreiber et al. 2011). The next decade should
bring rapid progress as molecular biology continues to
merge with comparative and evolutionary whole plant
physiology.
Long-distance Signaling Through
the Phloem
Over the past several decades, considerable attention has
been paid to unraveling the mechanics of phloem loading.
Genetic and molecular studies have identified the major players
that mediate in the loading of sugars, predominantly sucrose,
into the CC-SE complex. Interestingly, in terms of the apoplasmic
loaders, the recent identification of the permease that
controls release of sucrose from the phloem parenchyma cells
into the CC apoplasm (Figure 13B, I) served to complete the
molecular characterization of this important pathway (Chen
et al. 2012b). Based on such studies and extensive physiological
experiments, the nature of the photosynthates (sugars
and amino acids) loaded into the phloem translocation stream
is well established.
The phloem has also been shown to carry additional cargo,
including the phytohormones auxin, gibberellins, cytokines and
abscisic acid (Hoad 1995), signaling agents involved in plant
defense (discussed later in the review), as well as certain
proteins and various forms of RNA (Lough and Lucas 2006;
Buhtz et al. 2010). That specific proteins are present in the
phloem has been recognized for some time (Fisher et al.
1992; Bostwick et al. 1992), and furthermore, some such
proteins have been shown to move within the translocation
stream (Golecki et al. 1998, 1999; Xoconostle-Cázares et al.
1999).
Phloem proteins: Potential roles in enucleate SE
maintenance and long-distance signaling
Phloem exudate can be collected from a number of plant
species, and this feature has been used to develop proteomic
databases for these species (Barnes et al. 2004; Giavalisco
et al. 2006; Lin et al. 2009; Rodriguez-Medina et al. 2011).
This collection process requires that an incision be made
into the petiole or stem in order to allow the phloem to
“bleed.” Thus, due care is required to minimize the level
of protein contamination from surrounding (CCs and phloem
parenchyma) tissues. As excision results in an abrupt pressure
drop between the sieve tube system and the surrounding cells,
it is generally appreciated that some level of contamination
is unavoidable (Atkins et al. 2011). Here, use of molecular
markers such as Rubisco (Doering-Saad et al. 2006; Giavalisco
et al. 2006; Lin et al. 2009), can help in assessing the extent
to which contamination may have occurred. Generally,
Insights into Plant Vascular Biology 339
contamination does not appear to be an important issue,
especially for the prominent proteins, but proteins present in
very low abundance need to be viewed with a degree of
caution.
Other methods, including cutting aphid stylets (Aki et al.
2008; Gaupels et al. 2008a), EDTA-induced phloem exudation
(Gaupels et al. 2008b; Batailler et al. 2012) and laser microdissection
of phloem tissues (Deeken et al. 2008) have also
been employed to develop phloem databases. Collectively,
these studies have established phloem proteome databases
containing more than 1,000 proteins, with activities encompassing
a very broad range of activities, including enzymes involved
in metabolic networks, amino acid synthesis, protein turnover,
RNA binding, transcriptional regulation, stress responses, defense,
and more.
The next step will be to partition these proteins into those
involved in local maintenance of the functional enucleate sieve
tube system and long-distance signaling. For these studies,
a combination of hetero-grafting experiments conducted between
species from different genera or families, and advanced
mass spectroscopy methods, will prove most useful. The cucurbits,
such as pumpkin, cucumber, melon and watermelon,
from which analytical quantities of phloem exudate can generally
be collected, may prove ideal for this purpose. The
recent completion of annotated genomes for three of these
cucurbits (Huang et al. 2009; Garcia-Mas et al. 2012; Guo
et al. 2012) adds to the utility of these species for such critical
experiments.
The complexity of the phloem proteome raises the question
as to the stability of these proteins and the mechanism by
which they might be turned over within the sieve tube system.
The large population of proteinase inhibitors probably
prevents turnover by simple proteolysis (Dinant and Lucas
2012). However, identification in the phloem sap of ubiquitin
and numerous enzymes involved in protein ubiquitination and
turnover, including all the components of the 26S proteasome
(Figure 19), indicates that enucleate SEs likely have retained the
ability to engage in protein sorting and turnover (Lin et al. 2009).
Thus, once they have performed their function(s), phloem
proteins can be degraded either through export into neighboring
CCs, or in loco via the ubiquitin-26S proteasome pathway.
The mature, enucleate sieve tube system also has been
shown to contain all the enzymes and associated activities
required for a complete antioxidant defense system (Walz et al.
2002; Lin et al. 2009; Batailler et al. 2012). Interestingly, these
enzyme activities appear to increase in response to imposed
drought stress (Walz et al. 2002). This complement of enzymes
would appear to function, locally, to afford protection against
oxidative stresses, thereby preventing damage to essential
components of the SEs. Such local maintenance functions will
likely be performed by a specific subset of the proteins detected
in phloem exudates.