The Plant Vascular System: Evolution, Development and FunctionsF

in metaxylem cells and subsequently turns off again prior
to secondary cell wall differentiation. MYB46 is one of the
transcription factors partially required for synthesis of various
components of the secondary cell wall in the protoxylem and
subsequent metaxylem (Lee et al. 2006; Zhong et al. 2008).
This gene is expressed towards the end of the elongation
zone in protoxylem cells and then later in metaxylem cells.
Together, these findings suggest that, although there are no
morphological markers of protoxylem cell specification early
in developmental time, there are indeed molecular markers
and two distinct developmental states for protoxylem and
metaxylem cells: an “early” state and a “late” state. Gene
expression data support this observation, as there are distinct
gene expression profiles in cells marked by TMO5 as compared
to MYB46 (Brady et al. 2007).
As mentioned earlier, PHB, PHV, REV, COR and ATHB-
8 act redundantly to regulate xylem cell fate differentiation
and are sufficient to regulate xylem patterning. In a dominant
mutant background of PHB, phb-1d, which its mRNA is resistant
to miRNA degradation, ectopic protoxylem is observed
(Carlsbecker et al. 2010). PHB is therefore sufficient to specify
protoxylem differentiation. The developmental time point at
which the patterning of xylem cells is first regulated by these
transcription factors remains unknown. Upstream regulators
of SHR have yet to be identified, although SHR is expressed
throughout the vascular cylinder during vascular development.
Class III HD-ZIP transcription factors are also expressed in
various cell types of the vasculature, primarily early in root
developmental time in the meristematic zone; miR165a and
miR166b are also expressed with a peak in endodermis cells in
this same zone (Carlsbecker et al. 2010). Based solely on the
temporal nature of these expression patterns, the patterning
of protoxylem and metaxylem cells appears to occur prior to
the action of VND6 and VND7, although validation of this
hypothesis requires further experimentation.
Phloem cell patterning and differentiation
Only a few factors are known to regulate phloem cell patterning
and differentiation, despite protophloem being histologically
evident quite early in development relative to xylem cell types
(Mähönen et al. 2000). Mutations in OPS, ops-1 and ops-2,
result in irregular phloem differentiation within the root. In
WT roots, SE and CC differentiation is first marked by cell
elongation followed by callose deposition and subsequent cell
wall thickening in the longitudinal dimension. In the ops-1
mutant, cell elongation, callose deposition and cell wall thickening
fail to occur within the phloem cell lineage (Truernit
et al. 2012). In addition, the ops-1 mutant has impaired longdistance
phloem transport, likely because of gaps in phloem SE
continuity. However, OPS is not sufficient to specify phloem cell
differentiation. Overexpression of OPS results in precocious
Insights into Plant Vascular Biology 317
phloem cell differentiation within the root, but only within already
specified protophloem and metaphloem lineages (Truernit et al.
2012).
In roots, APL is expressed later than OPS, that is, at a
distance from the QC (Bonke et al. 2003; Truernit et al.
2012) (Figure 11B). In an apl mutant, protophloem cells are
misspecified as protoxylem cells, and there is a short root
phenotype (Bonke et al. 2003). Contrary to an ops mutant
phenotype, in an apl mutant background, no protophloem,
metaphloem, or CCs are present anywhere in the phloem pole
position within the vascular cylinder. APL plays a multifaceted
role in phloem development. First, APL regulates the timing
of the asymmetric cell divisions that would normally give rise
to the SE and CC lineages. Second, APL is required for
protophloem and metaphloem SE differentiation. Third, APL
represses protoxylem differentiation within cells in the phloem
pole position. However, despite all these roles, APL is not
sufficient to specify phloem cell specification and differentiation.
Clearly, additional factors remain to be identified in phloem
cell development. The presence of a master regulator for
phloem development like VND6/7 and the Class III HD-ZIP
family in xylem development that is both necessary and
sufficient has not been identified. Protophloem cells differentiate
earlier than metaphloem cells and CCs. APL protein
localization reflects this temporal difference in differentiation,
but the factor(s) that determines this temporal delay has yet to
be isolated. Finally, factors that determine the spatial patterning
of protophloem, metaphloem and CCs are similarly unknown.
Pericycle cell specification and differentiation
Pericycle cells have been divided into two populations based
on gene expression and function. Only xylem pole pericyle cells
are competent to become lateral root primordia. One marker of
xylem pole pericycle cell differentiation is the J0121 enhancer
trap that marks xylem pole pericycle cells after they exit from the
meristematic zone and pass through the elongation zone (Parizot
et al. 2008). A marker of intervening cells within the pericycle
tissue layer helped identify phloem pole pericycle cells. These
cells are marked by expression of S17, a basic leucine zipper
transcription factor. The function of phloem pole pericycle cells
has not been determined, nor are there histological markers of
phloem pole pericycle differentiation. However, phloem pole
pericycle cells have a very distinct expression pattern and
underlying transcriptional signature from that of xylem pole
pericycle cells, as determined by expression profiling of marked
populations of both of these cells relative to other cell types
in the Arabidopsis root (Brady et al. 2007). Interestingly, the
expression pattern of phloem pole pericycle cells more closely
reflects that of cells in the developing phloem cell lineage.
No early markers of either phloem or xylem pole pericycle
cells have been identified, nor have regulatory factors been