Gene expression patterns underlying changes in xylem structure and function in response to increased nitrogen availability in hybrid poplar.

Nitrogen availability has a strong influence on plant growth and development. In this study, we examined the effect of nitrogen availability on xylogenesis in hybrid poplar (Populus trichocarpa x deltoides H11-11). Saplings of hybrid poplar were fertilized for 33 d with either high or adequate levels of ammonium nitrate. We observed enhanced radial growth, wider vessels and fibres and thinner fibre walls in the secondary xylem of high N relative to adequate N plants. These anatomical differences translated into altered hydraulic properties with xylem being more transport efficient but also more vulnerable to drought-induced cavitation in high N plants. The changes in xylem structure and function were associated with differences in gene expression as revealed by the transcriptome analysis of the developing xylem region. We found 388 genes differentially expressed (fold change ±1.5, P-value ≤ 0.05), including a number of genes putatively involved in nitrogen and carbohydrate metabolism and various aspects of xylem cell differentiation. Several genes encoding known transcriptional regulators of secondary cell wall deposition were down-regulated in high N plants, corresponding with thinner secondary cell walls in these plants. The results of this study provide us with gene candidates potentially affecting xylem hydraulic and structural traits.

Cellular localization of aquaporin mRNA in hybrid poplar stems.

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.

Influence of evaporative demand on aquaporin expression and root hydraulics of hybrid poplar.

When light levels and evaporative demand increase, dynamic physiological changes in roots may be required to restore the water balance at the whole plant level. We hypothesized that a dynamic increase in root hydraulic conductance (L(P)) and aquaporin (AQP) expression could moderate the transpiration-induced drop in water potential (Ψ), allowing continued gas exchange in hybrid poplar (Populus trichocarpa × deltoides) saplings. Fifty-six AQPs have been identified in poplar, but little information about their expression patterns in roots is available, especially from a whole-plant water relations perspective. We measured AQP expression and L(P) in plants subjected to different levels of light and evaporative demand. Shaded plants had only one-tenth the root area of plants growing at higher light levels. Shade-grown saplings experiencing a sudden increase in light exhibited a threefold higher L(P) than plants remaining in shade. This dynamic increase in L(P) corresponded with increased transcript abundance of 15 AQPs out of a total of 33 genes simultaneously assessed by quantitative RT-PCR. The tissue-level localization of transcripts of four AQPs was studied with in situ hybridization. Comprehensive expression profiling in conjunction with physiological and morphological measurements is a valuable reference for future studies on AQP function in poplar.

Functional characterization of drought-responsive aquaporins in Populus balsamifera and Populus simonii×balsamifera clones with different drought resistance strategies.

We have characterized poplar aquaporins (AQPs) to investigate their possible functions in differential drought responses of Populus balsamifera and Populus simonii×balsamifera leaves. Plants were exposed to mild and severe levels of drought stress and to drought stress recovery treatment, and their responses were compared with well-watered controls. Compared with P. balsamifera, P. simonii×balsamifera used drought avoidance as the main drought resistance strategy, and rapidly reduced stomatal conductance in response to stress. This strategy is correlated with growth rate reductions. Eleven AQPs were transcriptionally profiled in leaves from these experiments and five were functionally characterized for water channel activity. PIP1;3 and PIP2;5 were among the most highly expressed leaf AQPs that were responsive to drought. Expression of PIP1;3 and five other AQPs increased in response to drought in the leaves of P. simonii×balsamifera but not in P. balsamifera, suggesting a possible role of these AQPs in water redistribution in the leaf tissues. PIP2;5 was upregulated in P. balsamifera, but not in P. simonii×balsamifera, suggesting that this AQP supports the transpiration-driven water flow. Functional characterization of five drought-responsive plasma membrane intrinsic proteins (PIPs) demonstrated that three PIP2 AQPs (PIP2;2, PIP2;5, PIP2;7) functioned as water transporters in Xenopus laevis oocytes, while the two PIP1 AQPs (PIP1;2 and PIP1;3) did not, consistent with the notion that they may be functional only as heterotetramers.

Influence of nitrogen fertilization on xylem traits and aquaporin expression in stems of hybrid poplar.

We studied the influence of nitrogen (N) on hydraulic traits and aquaporin (AQP) expression in the stem xylem of hybrid poplar saplings (Populus trichocarpa (Torr. & Gray) x deltoides Bartr. ex Marsh clone H11-11). Plants were grown in a controlled environment and were kept well watered throughout the experiments. Hydraulic measurements were done on basal and distal stem segments of plants receiving high N fertilization (high N plants) versus plants receiving only adequate N fertilization (adequate N plants). High N plants grew faster and exhibited more leaf area than adequate N controls. These morphological differences were paralleled by wider vessels and higher specific conductivities (K(S)) in high N plants. However, stems of high N plants were more vulnerable to xylem cavitation, at least in one of two experiments, and showed lower wood densities than stems of adequate N plants. Leaf area was strongly correlated with cross-sectional xylem area in both plant groups. Since higher K(S) in high N plants was accompanied by concomitant increases in leaf area, leaf-specific conductivities were similar in both plant groups. Influences of N on hydraulic traits were paralleled by changes in AQP expression. Seven AQPs were upregulated in the stem xylem of high N plants, five of which have been identified recently as water transporters. The enhanced growth of secondary xylem of high N plants has been shown to result from both increased cambial activity as well as increased cell size. We suggest that some of these water-transporting AQPs could play a role in xylogenesis, facilitating the influx of water into the zone of differentiating and maturing cells in secondary xylem, including expanding vessels.

Symbiotic association between Salix purpurea L. and Rhizophagus irregularis: modulation of plant responses under copper stress.

There are increasing concerns about trace metal levels such as copper (Cu) in industrial sites and the broader environment. Different studies have highlighted the role of mycorrhizal associations in plant tolerance to trace metals, modulating some of the plant metabolic and physiological responses. In this study, we investigated the role of the symbiotic association betweenRhizophagus irregularisandSalix purpureaL. in modulating plant responses under Cu stress. We measured Cu accumulation, oxidative stress-related, photosynthetic-related and hydraulic traits, for non-inoculated (non-arbuscular mycorrhizal fungi) and inoculated saplings exposed to different Cu concentrations. We found thatS. purpureais a suitable option for phytoremediation of Cu, acting as a phytostabilizer of this trace metal in its root system. We observed that the symbiotic association modulates a broad spectrum of metabolic and physiological responses inS. purpureaunder Cu conditions, including (i) a reduction in gas exchange associated with chlorophyll content changes and (ii) the sequestration of Cu into the cell walls, modifying vessels anatomy and impacting leaf specific conductivity (KL) and root hydraulic conductance (LP). UpholdingKLandLPunder Cu stress might be related to a dynamic Aquaporin gene regulation ofPIP1;2along with an up-regulation ofTIP2;2in the roots of inoculatedS. purpurea.