American Journal of Botany 99(7): 1249–1254. 2012.
BRIEF COMMUNICATION
CELLULAR LOCALIZATION OF AQUAPORIN MRNA IN HYBRID
POPLAR STEMS1
ADRIANA M. ALMEIDA-RODRIGUEZ2 AND UWE G. HACKE3
Department of Renewable Resources, University of Alberta, Edmonton, AB, T6G 2E3, Canada
• Premise of the study: Aquaporins (AQPs) are channel proteins, and their function is mostly associated with transmembrane
water transport. While aquaporin genes are known to be expressed in woody poplar stems, little is known about AQP expression at the cellular level. Localization of AQP expression to particular cell and tissue types is a necessary prerequisite in understanding the biological role of these genes.
• Methods: Subsets of plants were subjected to 6 wk of high nitrogen fertilization (high N plants) or to a controlled drought.
Experimental treatments affected cambial activity and wood anatomy. RNA in situ hybridization was used to characterize
spatial expression of three AQP genes in stem cross sections.
• Key results: The strongest labeling consistently occurred in the cambial region and in adjacent xylem and phloem cells. Expression was also detected in rays. Contact cells exhibited high expression, while expression in other ray cells was more variable.
High N plants exhibited a broader band of expression in the cambial region than plants receiving only adequate N fertilization
(control plants) and plants subjected to drought.
• Conclusions: Water channels in stems were expressed in a manner that allows hydraulic coupling between xylem and other
tissues that may serve as water reservoirs, including phloem and pith parenchyma. Expression of AQPs in rays may increase
radial flow of water from xylem and phloem to the cambial region where AQPs may help sustain rapid cell division and expansion of developing vessel elements.
Key words: aquaporins; cambium; in situ hybridization; Populus; secondary growth; xylogenesis.
Aquaporins (AQPs) are proteins that facilitate the movement
of water or other small solutes through membranes. Compared
with other organisms, plants have a large number of AQP genes
(Gomes et al., 2009). In poplar, 56 AQP genes have been identified (Gupta and Sankararamakrishnan, 2009; Almeida-Rodriguez
et al., 2010). AQPs are classified into several subfamilies,
including plasma membrane intrinsic proteins (PIPs; further
divided into PIP1 and PIP2 groups) and tonoplast intrinsic proteins (TIPs).
Unraveling the function of individual AQP genes in different
plant organs is an active area of research. Most studies on AQPs
have focused on roots and leaves of herbaceous plants, while
stems of trees have received less attention. Gene expression
studies showed that AQPs are expressed in woody poplar stems
(e.g., Secchi and Zwieniecki, 2010), but to better understand
the biological role of these genes, tissue and cellular level localization studies are required. Despite the putative importance of
AQPs for radial water transport in stems, to our knowledge only
one study has focused on localizing AQPs in the secondary xylem of stems (Sakr et al., 2003). In their study on walnut stems,
1 Manuscript received 27 February 2011; revision accepted 13 June 2012.
The authors thank B. Thomas (Alberta-Pacific Forest Industries Inc.,
Boyle, Alberta) for providing the plant material and greenhouse space.
Funding (to U.H.) was provided by the Canada Research Chair program,
an Alberta Ingenuity New Faculty Award, and NSERC.
2 Present address: Department of Biological Sciences, University of
Montreal, Montreal, QC, H1X 2B2, Canada
3 Author for correspondence (e-mail: [email protected])
doi:10.3732/ajb.1200088
PIP2 aquaporins were localized in paratracheal parenchyma
cells, suggesting that PIP2s could play a role in water transport
between these cells and adjacent vessels.
AQPs in woody stems may facilitate radial water exchange
between phloem and xylem and are widely believed to play a
role in xylem refilling (Nardini et al., 2011). On the basis of
these putative functions, we hypothesized that AQPs would be
expressed in rays, although expression in ray cells may not be
homogeneous. Ray cells that are connected with vessels through
pits (contact cells) control the exchange of solutes and water
between parenchyma and vessels (Sauter et al., 1973; Evert,
2006). Ray cells that have no contact with vessels (isolation
cells) function as storage cells (Evert, 2006). Considering that it
is primarily the contact cells that facilitate water exchange
between the xylem sap and the symplastic continuum, we expected to find stronger expression of AQPs in contact cells than
in isolation cells.
AQPs in woody stems could also play a role in xylogenesis
by increasing radial movement of water from xylem and phloem
to the cambial region. In this region, water channels may help
sustain rapid cell division and expansion of developing xylem
and phloem cells. Developing vessel elements, in particular,
undergo rapid expansion. Since this process is mainly driven by
water uptake, expansion of tracheary elements may be facilitated by water channels (Groover et al., 2010).
A role of AQPs in xylogenesis is consistent with a recent
study on hybrid poplar stems (Hacke et al., 2010). In that study,
it was found that saplings receiving high nitrogen (N) fertilization (high N plants) exhibited enhanced secondary growth compared with plants receiving only adequate N fertilization.
Influences of N on cambial activity were paralleled by changes
American Journal of Botany 99(7): 1249–1254, 2012; http://www.amjbot.org/ © 2012 Botanical Society of America
1249
1250
[Vol. 99
AMERICAN JOURNAL OF BOTANY
in AQP expression. Of the 11 genes that were examined, five
PIP2s and two TIPs showed higher transcript abundance in
stem xylem of high N plants. However, in this study RNA was
collected from the bulk xylem; a cellular-level localization of
transcripts was not performed.
The main objectives of this present study were (1) to visualize
the tissue- and cell-specific expression of three AQPs previously
found to be expressed in the secondary xylem of hybrid poplar
(Hacke et al., 2010) and (2) to determine if and how transcript
localization varies in response to experimental treatments. We
reasoned that if particular AQPs are involved in wood development, they will exhibit strong localized expression in the cambial
region, and transcript localization patterns will differ in response
to environmental conditions that affect cambial activity.
Besides N, water availability is also known to affect wood
cell development in poplar stems. Drought-stressed poplars had
narrower vessels in early summer and had fewer enlarging cambial
cell derivatives in the wood-forming zone than well-watered
control trees (Arend and Fromm, 2007). Hence, the effect of
drought on vessel element expansion is opposite to the effect of
high N availability.
In this present study, hybrid poplars were grown in a controlled environment under adequate N (control plants) vs. high
N conditions (high N plants) and were well watered. A third set
of plants received adequate N but was subjected to a controlled
drought. We hypothesized that the fast growth of high N plants
would be associated with strong labeling of the cambial region
and that drought-stressed plants would exhibit a weaker signal
in the cambial region. We also explored how AQP expression
in ray cells responded to growing conditions.
MATERIALS AND METHODS
Plant material—Shoot cuttings of the clone Populus בOkanese’ were provided from a single tree by Alberta-Pacific Forest Industries. ‘Okanese’ is a cross
between Populus בWalker’ and Populus ×petrowskyana. ‘Walker’ is a cross
between Populus deltoides and P. ×petrowskyana (Kalcsits et al., 2009). Rooted
cuttings were produced and grown in a greenhouse under semicontrolled conditions (22°/20°C day night, 18 h/6 h light/dark, 60% relative humidity). The soil
consisted of peat moss, vermiculite, and Turface calcined clay. Plants received
2 g·L−1 NPK 15-30-15 fertilizer once weekly over 6 wk. Plants were then randomly assigned to three treatments. High N plants received 10 mmol/L NH4NO3
in 0.5× Hocking’s solution (Hocking, 1971), while control and drought-stressed
plants received 1 mmol/L NH4NO3 in 0.5× Hocking’s solution. Plants were
fertilized three times a week. High N and control plants were watered to field
capacity daily, while drought-stressed plants received 200 mL of fertilizer solution three times a week as the only source of water.
Plant growth measurements and tissue collection for microscopy—After 6 wk
of treatments, the growth of six plants per treatment was measured, and tissues
were sampled. Growth measurements included plant height, stem diameter
(10 cm above soil surface) and the cumulative area of leaves between leaf plastochron index (LPI; Larson and Isebrands, 1971) 7 to 9. Leaf area was measured using a LI-3100 leaf area meter (Li-Cor, Lincoln, Nebraska, USA). Tissue
samples were taken from stem segments that had developed secondary growth
(located between LPI 18 and 19), and secondary leaf veins from young leaves
(LPI 4). Samples were immediately submerged in FAA (10% formaldehyde,
5% glacial acetic acid, and 50% ethanol), recut in the laboratory, and fixed
overnight in FAA solution at 4°C. Samples were dehydrated and embedded in
paraffin as described previously (Brewer et al., 2006).
Vessel density, vessel diameter, and histochemical staining—Cross sections (10 µm) of stems were cut with a microtome and prepared for microscopy.
After dewaxing in Histochoice (Sigma, St. Louis, Missouri, USA) and sequential rehydration, sections were rinsed with phosphate-buffered saline (PBS) and
exposed to phloroglucinol-HCl solution (1 g phloroglucinol, 2.7 N HCl, and
80% ethanol) until lignified cell walls turned red. Sections were observed with
a light microscope (DM3000, Leica, Wetzlar, Germany), and images were captured with a digital camera (DFC420C, Leica). Image analysis software
(ImagePro Plus version 6.1, Media Cybernetics, Silver Spring, Maryland,
USA) was used to measure the number of vessels per area (mm2) and the mean
diameter of lignified vessels (Dv) near the cambial region in plants from different treatments.
Another set of sections was stained with a standard safranin-fast green combination, which highlights nuclei and lignified cell walls in red, and cytoplasm and
cellulosic cell walls in blue. To assess the location of living cells in the cambial
region and developing xylem, we stained sections with DAPI (4′,6-diamidino2-phenylindole) and observed with an epifluorescence microscope (DM3000).
DAPI stains nuclear DNA in living fibers and ray cells.
mRNA in situ hybridization—Digoxigenin-labeled antisense and sense
probes for PtPIP2;3, PtPIP2;5, and PtTIP2;1 (terminology follows Gupta and
Sankararamakrishnan, 2009) were generated with a DIG RNA labeling kit
(Roche Diagnostics) according to the manufacturer’s protocol. Probes were 272,
346, and 349 bp for PIP2;5, PIP2;3, and TIP2;1, respectively. The probes were
designed to target part of the 3′-coding region and the 3′-untranslated regions
specific to each gene (Appendix S1, see Supplemental Data with the online version
of this article). Templates were synthesized by in vitro transcription of PCR
amplicons, cloned into pGEM T Easy vector (Promega, Madison, Wisconsin,
USA) and sequenced to determine orientation (Appendix S2, see online Supplemental Data). Cross-sections of stem segments were dewaxed and rehydrated
according to Brewer et al. (2006). Sense probe hybridizations were used as
controls. In situ hybridization was performed using an IsHyb In Situ Hybridization Kit (Biochain, Hayward, California, USA) following the manufacturer’s
instructions. An additional step for stopping the Proteinase K proteolysis was
included by washing the sections with 2 mg/mL glycine in 1× PBS RNase-free
buffer for 1 min. Sections were observed with a light microscope.
Statistics—All statistical analyses were carried out using the program
SigmaStat 3.5 (Systat, Point Richmond, California, USA). Differences due to
the effects of treatments were analyzed using a one-way ANOVA followed by
a Tukey’s test. Data are presented as means ± SE. Differences were considered
significantly different at P ≤ 0.05.
RESULTS
Differences in morphology and wood anatomy in response
to experimental treatments are summarized in Table 1. High
N plants produced thicker stems and more leaf area (as well as
larger leaves) than controls and drought-stressed plants; controls
were intermediate. High N plants also had the widest vessels;
vessel diameters were intermediate in controls and smallest in
drought plants. Vessel diameters were negatively correlated with
vessel density. Moreover, in drought plants, lignification was
observed in closer proximity to the cambial zone than in controls and high N plants (online Appendix S3). The contrasting
growing conditions not only affected cambial activity and wood
anatomy, but also resulted in treatment-specific differences in
the distribution of AQP mRNA (online Appendix S4).
In drought plants, all three AQPs were highly expressed in
the cambial region and in phloem (Fig. 1A–C, E). Expression of
TIP2;1 in the bark extended from the cambium to the periderm
(Fig. 1F); the PIP2s showed similar expression patterns in the
bark. In the xylem, transcripts of all three AQPs were detected
in developing vessel elements and fibers in the expansion zone
as well as in living fibers in the zone of secondary wall formation (Fig. 1E). A hallmark of AQP expression in drought plants
was relatively homogeneous labeling of contact and isolation
cells in rays (Fig. 1; online Appendix S4). Strong expression
was also detected at the interface between xylem rays and
parenchyma cells in the pith. Sense controls of the three genes
did not show a signal (Fig. 1G–I).
July 2012]
ALMEIDA-RODRIGUEZ AND HACKE—AQUAPORIN LOCALIZATION IN POPLAR STEMS
1251
Means (±SE) for growth and xylem characteristics of poplar saplings fertilized with high amounts of nitrogen, control plants receiving only
adequate amounts of nitrogen, and drought-stressed plants.
TABLE 1.
Treatment
High N
Control
Drought-stress
Height (cm), F2,15 = 21.82
Dstem (mm), F2,15 = 42.64
AL (cm2), F2,15 = 104.90
Dv (µm), F2,25 = 44.49
VD (no./mm2), F2,25 = 109.63
207.9 (4.3) A
191.2 (5.4) A
164.2 (4.5) B
10.8 (0.3) A
9.2 (0.2) B
8.2 (0.1) C
705.5 (31.1) A
381.0 (22.4) B
251.3 (9.7) C
54.3 (1.5) A
45.1 (2.4) B
34.3 (1.0) C
115.2 (7.4) A
155.9 (15.8) B
357.4 (17.2) C
Notes: Variables shown are plant height (height), stem diameter (Dstem) measured 10 cm above pots, leaf area (AL) measured between leaf plastochron
index 7–9, mean vessel diameter (Dv), and mean vessel density (VD). Different letters indicate significant differences (P < 0.01).
Expression in the cambial region of the control plants was
similar to the expression in the drought plants. However, compared
with drought-stressed plants, control plants tended to show weaker
labeling in rays and in the phloem (Appendix S4; images not
shown).
The fast growth and increased cambial activity exhibited by
high N plants was associated with strong expression of all
three genes in the cambial region and in developing phloem
and xylem. Transcripts of all three AQPs occurred in living
fibers located in older xylem with mature (and thus functional)
vessels, resulting in a broad band of expression in fibers adjacent to the cambial region (Fig. 2A–C). DAPI staining confirmed the presence of nuclei in living fibers in this region of
the xylem (online Appendix S5). High N plants also exhibited
strong expression of all three genes in contact cells (Fig. 2D–F)
and in regions where rays connected with the pith (Fig. 2D).
Fig. 1. In situ mRNA hybridization of three aquaporin genes in stem cross sections of Populus plants subjected to a controlled drought. Sections were
hybridized with DIG-labeled antisense (A, D, G) PIP2;3, (B, E, H) PIP2;5, and (C, F, I) TIP2;1 RNA probes. (G–I) Negative controls hybridized with
DIG-labeled sense probes. (G) PIP2;3, (H) PIP2;5, (I) TIP2;1. Regions of aquaporin expression are indicated by dark-purple staining. High expression
occurred in the cambial region and in ray cells. Ray cells adjacent to vessels showed particularly strong labeling (arrows in D). Expression extended into
the cell expansion zone (arrowheads in [E] point to expanding vessel elements). cz, cambial zone; p, phloem; pd, periderm; pi, pith; v, vessel; x, xylem.
Bars = 100 μm.
1252
AMERICAN JOURNAL OF BOTANY
[Vol. 99
Fig. 2. In situ mRNA hybridization of three aquaporin genes in stem cross sections of well-watered Populus plants receiving high nitrogen fertilization
(high N plants). Sections were hybridized with DIG-labeled antisense (A, D) PIP2;3, (B, E) PIP2;5, and (C, F) TIP2;1 RNA probes. Regions of aquaporin
expression are indicated by dark-purple staining. High N plants exhibited a broad band of expression in the cambial region, phloem, and developing xylem
(A–C). Strong expression of PIP2;3 occurred in ray cells near the pith (D). TIP2;1 was expressed in contact cells (arrows in F); expression in other ray
cells was weak or absent. cz, cambial zone; p, phloem; pi, pith; v, vessel; x, xylem. Bars = 100 μm.
Expression in isolation cells was heterogeneous, as shown for
TIP2;1 in Fig. 2F.
A strong signal was consistently observed in the bark (Fig. 3A);
phloem companion cells showed particularly strong labeling
(Fig. 3B, arrowheads). In striking contrast, no expression was
detected in sieve tube members. The absence of transcripts in
these cells is consistent with their lack of a nucleus.
DISCUSSION
which necessarily involves the crossing of cell membranes.
Nonetheless, relatively consistent AQP expression in isolation
cells of drought-stressed plants suggests that AQPs may modulate water movement in xylem rays under certain conditions.
A specific role of water channels in contact cells could be to
contribute to the refilling of embolized vessels (Sakr et al., 2003;
Secchi and Zwieniecki, 2010). AQPs in contact cells could also
facilitate the uptake of water from functional vessels. Water
taken up by ray cells may be transported to the cambial region
in which all three genes were prominently expressed.
In situ hybridization is a powerful technique for studying
gene expression in specific cells and tissues. It complements
standard quantitative techniques such as real-time PCR, which
allow the detection of altered gene expression but provide no
information about cellular localization of transcripts. Such information is often required to generate or test hypotheses about
gene function. In this brief communication, we provide information about the localization of mRNA coding for three AQPs
in woody stems.
The AQPs studied here were expressed in a symplastic continuum extending from the periderm to the pith. This pattern
suggests that AQPs may play an integral role in the radial transport
of water in stems. Most PIP2s and TIPs have been identified as
functional water transporters (Gomes et al., 2009; Secchi et al.,
2009; Almeida-Rodriguez et al., 2010). The fact that expression
was particularly high in contact cells (Fig. 2F) suggests that
AQPs in these cells increase water exchange between apoplast
and symplast. Since isolation cells are connected via plasmodesmata (Sauter and Kloth, 1986; Chaffey and Barlow, 2001),
water movement between ray cells may be less dependent on
AQPs than water exchange between vessels and contact cells,
Fig. 3. In situ mRNA hybridization of PIP2;3 and PIP2;5 in stem
cross sections of well-watered Populus plants receiving high nitrogen fertilization (high N plants). PIP2;3 was expressed in the cambial zone, in
phloem cells, and in bark parenchyma cells (A). PIP2;5 showed particularly strong expression in phloem companion cells (arrowheads in B), but
not in sieve tube members. cz, cambial zone; p, phloem; pf, phloem fibers;
s, sieve tube member. Bars = 100 µm (A); 50 µm (B).
July 2012]
ALMEIDA-RODRIGUEZ AND HACKE—AQUAPORIN LOCALIZATION IN POPLAR STEMS
It has been hypothesized that AQPs localizing to the cambium
promote cambial activity and secondary growth (Hacke et al.,
2010). AQPs generally appear to be abundant in zones of cell
division and elongation in root tips, leaves, and reproductive
organs (Chaumont et al., 1998; Tyerman et al., 2002). Prominent
expression of AQPs in the cambium has rarely been reported,
even though transcripts and protein of individual AQPs (including
PIP2;3 and TIP2;1) have been detected in the cambial region of
poplar (Schrader et al., 2004; Berta et al., 2010; Nilsson et al.,
2010; Song et al., 2011; Plavcová et al., in press). The expression of AQPs in fibers that remain alive until they are quite far
away from the cambium (Fig. 2A–C; online Appendix S5) is
consistent with a recent study (Bollhoner et al., 2012) that
showed that fibers disintegrate cellular contents at a slower
pace than vessels.
Strong expression was also observed in the phloem, particularly in companion cells. In leaves where companion cells may
contribute to phloem loading, high AQP expression in these
cells likely facilitates water uptake by sieve tube–companion
cell complexes (Hachez et al., 2008). In stems, phloem unloading will be required to support secondary growth. Moreover,
besides its role in sugar transport, phloem may also serve as a
water reserve for the transpiration stream (Sevanto et al., 2011).
We observed strong expression of water channels in companion
cells of the drought-stressed plants, which may be indicative of
water exit from sieve tube–companion cell complexes and may
contribute to hydraulic coupling between phloem and xylem. In
situ hybridization experiments on secondary leaf veins (online
Appendix S6) also revealed a broad band of expression in the
phloem of drought-stressed leaves.
Besides expression in phloem and bark parenchyma, AQPs
were enriched at the interface between xylem rays and pith
parenchyma. In young stems, the pith may also represent an
important reservoir for water, and AQPs may contribute to the
hydraulic coupling of pith and xylem.
In conclusion, we used in situ hybridization to locate AQP
mRNA in stems of hybrid poplar. The three genes included in
this study showed similar expression patterns. Strong expression occurred in various regions of cross sections, including the
cambial region, phloem, and rays. Expression patterns varied
depending on experimental treatments. Our findings complement recent studies on changes in radial conductance between
the xylem and the phloem (Sevanto et al., 2011; Steppe et al.,
2012), support models of embolism repair that involve xylem–
phloem interactions (Zwieniecki and Holbrook, 2009; Nardini
et al., 2011), and demonstrate that water channels in stems are
expressed in a manner that allows hydraulic coupling between
xylem and other tissues that may serve as water reservoirs, i.e.,
bark parenchyma, phloem, and pith parenchyma. Functional
experimentation is required to evaluate whether high AQP transcript levels in the cambial region help sustain high rates of cell
division and cell expansion.
LITERATURE CITED
ALMEIDA-RODRIGUEZ, A. M., J. E. K. COOKE, F. YEH, AND J. J. ZWIAZEK. 2010.
Functional characterization of drought-responsive aquaporins in Populus
balsamifera and Populus simonii ×balsamifera clones with different
drought resistance strategies. Physiologia Plantarum 140: 321–333.
AREND, M., AND J. FROMM. 2007. Seasonal change in the drought response
of wood cell development in poplar. Tree Physiology 27: 985–992.
BERTA, M., A. GIOVANNELLI, F. SEBASTIANI, A. CAMUSSI, AND M. L. RACCHI.
2010. Transcriptome changes in the cambial region of poplar (Populus
alba L.) in response to water deficit. Plant Biology 12: 341–354.
1253
BOLLHONER, B., J. PRESTELE, AND H. TUOMINEN. 2012. Xylem cell death:
Emerging understanding of regulation and function. Journal of
Experimental Botany 63: 1081–1094.
BREWER, P. B., M. G. HEISLER, J. HEJATKO, J. FRIML, AND E. BENKOVA. 2006.
In situ hybridization for mRNA detection in Arabidopsis tissue sections. Nature Protocols 1: 1462–1467.
CHAFFEY, N., AND P. BARLOW. 2001. The cytoskeleton facilitates a threedimensional symplasmic continuum in the long-lived ray and axial
parenchyma cells of angiosperm trees. Planta 213: 811–823.
CHAUMONT, F., F. BARRIEU, E. M. HERMAN, AND M. J. CHRISPEELS. 1998.
Characterization of a maize tonoplast aquaporin expressed in zones of
cell division and elongation. Plant Physiology 117: 1143–1152.
EVERT, R. F. 2006. Esau’s plant anatomy: Meristems, cells, and tissues
of the plant body: Their structure, function, and development, 3rd ed.
Wiley-Interscience, Hoboken, New Jersey, USA.
GOMES, D., A. AGASSE, P. THIÉBAUD, S. DELROT, H. GERÓS, AND F. CHAUMONT.
2009. Aquaporins are multifunctional water and solute transporters
highly divergent in living organisms. Biochimica et Biophysica Acta,
Biomembranes 1788: 1213–1228.
GROOVER, A. T., K. NIEMINEN, Y. HELARIUTTA, AND S. D. MANSFIELD. 2010. Wood
formation in Populus. In S. Jansson, R. P. Bhalerao, and A. T. Groover [eds.],
Genetics and genomics of Populus, vol. 8, Plant genetics and genomics:
Crops and models, 201–224. Springer, New York, New York, USA.
GUPTA, A. B., AND R. SANKARARAMAKRISHNAN. 2009. Genome-wide analysis of major intrinsic proteins in the tree plant Populus trichocarpa:
Characterization of XIP subfamily of aquaporins from evolutionary
perspective. BMC Plant Biology 9: 134. doi:10.1186/1471-2229-9134
HACHEZ, C., R. B. HEINEN, X. DRAYE, AND F. CHAUMONT. 2008. The expression pattern of plasma membrane aquaporins in maize leaf highlights their role in hydraulic regulation. Plant Molecular Biology 68:
337–353.
HACKE, U. G., L. PLAVCOVÁ, A. ALMEIDA-RODRIGUEZ, S. KING-JONES, W.
ZHOU, AND J. E. K. COOKE. 2010. Influence of nitrogen fertilization on
xylem traits and aquaporin expression in stems of hybrid poplar. Tree
Physiology 30: 1016–1025.
HOCKING, D. 1971. Preparation and use of a nutrient solution for culturing
seedlings of lodgepole pine and white spruce with selected bibliography.
Northern Forestry Research Centre Information Report Nor-XI. Canadian
Forest Service, Edmonton, Alberta, Canada.
KALCSITS, L., S. SILIM, AND K. TANINO. 2009. Warm temperature accelerates short photoperiod-induced growth cessation and dormancy
induction in hybrid poplar (Populus × spp.). Trees—Structure and
Function 23: 971–979.
LARSON, P. R., AND J. G. ISEBRANDS. 1971. The plastochron index as applied
to developmental studies of cottonwood. Canadian Journal of Forest
Research 1: 1–11.
NARDINI, A., M. A. LO GULLO, AND S. SALLEO. 2011. Refilling embolized
xylem conduits: Is it a matter of phloem unloading? Plant Science
180: 604–611.
NILSSON, R., K. BERNFUR, N. GUSTAVSSON, J. BYGDELL, G. WINGSLE, AND C.
LARSSON. 2010. Proteomics of plasma membranes from poplar trees
reveals tissue distribution of transporters, receptors, and proteins in
cell wall formation. Molecular & Cellular Proteomics 9: 368–387.
PLAVCOVÁ, L., U. G. HACKE, A. M. ALMEIDA-RODRIGUEZ, E. LI, AND C. J.
DOUGLAS. In press. Gene expression patterns underlying changes in
xylem structure and function in response to increased nitrogen availability in hybrid poplar. Plant, Cell and Environment.
SAKR, S., G. ALVES, R. L. MORILLON, K. MAUREL, M. DECOURTEIX, A.
GUILLIOT, P. FLEURAT-LESSARD, ET AL. 2003. Plasma membrane aquaporins are involved in winter embolism recovery in walnut tree. Plant
Physiology 133: 630–641.
SAUTER, J. J., W. ITEN, AND M. H. ZIMMERMANN. 1973. Studies on the
release of sugar into the vessels of sugar maple (Acer saccharum).
Canadian Journal of Botany 51: 1–8.
SAUTER, J. J., AND S. KLOTH. 1986. Plasmodesmatal frequency and radial translocation rates in ray cells of poplar (Populus × canadensis
Moench ‘robusta’). Planta 168: 377–380.
SCHRADER, J., J. NILSSON, E. MELLEROWICZ, A. BERGLUND, P. NILSSON, M.
HERTZBERG, AND G. SANDBERG. 2004. A high-resolution transcript profile
1254
AMERICAN JOURNAL OF BOTANY
across the wood-forming meristem of poplar identifies potential regulators of cambial stem cell identity. Plant Cell 16: 2278–2292.
SECCHI, F., B. MACIVER, M. L. ZEIDEL, AND M. A. ZWIENIECKI. 2009.
Functional analysis of putative genes encoding the PIP2 water
channel subfamily in Populus trichocarpa. Tree Physiology 29:
1467–1477.
SECCHI, F., AND M. A. ZWIENIECKI. 2010. Patterns of PIP gene expression
in Populus trichocarpa during recovery from xylem embolism suggest a major role for the PIP1 aquaporin subfamily as moderators of
refilling process. Plant, Cell & Environment 33: 1285–1297.
SEVANTO, S., T. HOLTTA, AND N. M. HOLBROOK. 2011. Effects of the hydraulic coupling between xylem and phloem on diurnal phloem diameter variation. Plant, Cell & Environment 34: 690–703.
SONG, D. L., W. XI, J. H. SHEN, T. BI, AND L. G. LI. 2011. Characterization
of the plasma membrane proteins and receptor-like kinases associated
with secondary vascular differentiation in poplar. Plant Molecular
Biology 76: 97–115.
STEPPE, K., H. COCHARD, A. LACOINTE, AND T. AMEGLIO. 2012. Could rapid
diameter changes be facilitated by a variable hydraulic conductance?
Plant, Cell & Environment 35: 150–157.
TYERMAN, S. D., C. M. NIEMIETZ, AND H. BRAMLEY. 2002. Plant aquaporins: Multifunctional water and solute channels with expanding
roles. Plant, Cell & Environment 25: 173–194.
ZWIENIECKI, M. A., AND N. M. HOLBROOK. 2009. Confronting Maxwell’s
demon: Biophysics of xylem embolism repair. Trends in Plant Science
14: 530–534.