Francesca Secchi

Sensing embolism in xylem vessels: the role of sucrose as a trigger for refilling

Refilling of embolized vessels requires a source of water and the release of energy stored in xylem parenchyma cells. Past evidence suggests that embolism presence can trigger a biological response that is switched off upon successful vessel refilling. As embolism formation is a purely physical process and most biological triggers rely on chemical sensors, we hypothesized that accumulation of osmotic compounds in walls of embolized vessels are involved in the embolism sensing mechanism. Analysis of Populus trichocarpas response to infiltration of sucrose, monosaccharides, polyethylene glycol and potassium chloride into the xylem revealed that only presence of sucrose resulted in a simultaneous physiological and molecular response similar to that induced by embolism. This response included reduction of the starch pool in xylem parenchyma cells and significant correlation of gene expression from aquaporins, amylases and sugar transporter families. The work provides evidence of the ability of plants to sense embolism and suggests that sucrose concentration is the stimulus that allows plants to trigger a biological response to embolism.

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

Embolism and the refilling of xylem vessels are intrinsic to the ability of plants to handle the transport of water under tension. Although the formation of an embolized vessel is an abiotic process, refilling against the pressure gradient requires biological activity to provide both the energy and the water needed to restore xylem transport capacity. Here, we present an analysis of the dynamics of embolism and refilling in Populus trichocarpa and follow temporal dynamics of co-occurring changes in expression level of aquaporins. Under mesic conditions, we found that the percent loss of conductance (PLC) varied diurnally by as much as 20%, suggesting a continuous embolism/refilling cycle. An increase in water stress tilted the balance between the two processes and increased the PLC to as much as 80%. Subsequent re-watering resulted in the reversal of water stress and recovery of PLC to pre-stress levels. Stem parenchyma cells responded to drought stress with considerable up-regulation of the PIP1 subfamily of water channels but not the PIP2 subfamily. Even more significant was the finding that PoptrPIP1.1 and PoptrPIP1.3 genes were up-regulated in response to embolism, but not to water stress, and were down-regulated after embolism removal, suggesting a local ability of plants to sense an embolism presence.

Adjustments of water use efficiency by stomatal regulation during drought and recovery in the drought-adapted Vitis hybrid Richter-110 (V. berlandieri x V. rupestris).

The hybrid Richter-110 (Vitis berlandieri x Vitis rupestris) (R-110) has the reputation of being a genotype strongly adapted to drought. A study was performed with plants of R-110 subjected to water withholding followed by re-watering. The goal was to analyze how stomatal conductance (g(s)) is regulated with respect to different physiological variables under water stress and recovery, as well as how water stress affects adjustments of water use efficiency (WUE) at the leaf level. Water stress induced a substantial stomatal closure and an increase in WUE, which persisted many days after re-watering. The g(s) during water stress was mainly related to the content of ABA in the xylem and partly related to plant hydraulic conductivity but not to leaf water potential. By contrast, low g(s) during re-watering did not correlate with ABA contents and was only related to a sustained decreased hydraulic conductivity. In addition to a complex physiological regulation of stomatal closure, g(s) and rate of transpiration (E) were strongly affected by leaf-to-air vapor pressure deficit (VPD) in a way dependent of the treatment. Interestingly, E increased with increasing VPD in control plants, but decreased with increasing VPD in severely stressed plants. All together, the fine stomatal regulation in R-110 resulted in very high WUE at the leaf level. This genotype is revealed to be very interesting for further studies on the physiological mechanisms leading to regulation of stomatal responsiveness and WUE in response to drought.

Analysis of Xylem Sap from Functional (Nonembolized) and Nonfunctional (Embolized) Vessels of Populus nigra : Chemistry of Refilling

It is assumed that the refilling of drought-induced embolism requires the creation of an osmotic gradient between xylem parenchyma cells and vessel lumens to generate the water efflux needed to fill the void. To assess the mechanism of embolism repair, it is crucial to determine if plants can up-regulate the efflux of osmotically active substances into embolized vessels and identify the major components of the released osmoticum. Here, we introduce a new approach of sap collection designed to separate water from nonembolized (functional) and embolized (nonfunctional) vessels. This new approach made possible the chemical analysis of liquid collected from both types of vessels in plants subjected to different levels of water stress. The technique also allowed us to determine the water volumes in nonfunctional vessels as a function of stress level. Overall, with the increase of water stress in plants, the osmotic potential of liquid collected from nonfunctional vessels increased while its volume decreased. These results revealed the presence of both sugars and ions in nonfunctional vessels at elevated levels in comparison with liquid collected from functional vessels, in which only traces of sugars were found. The increased sugar concentration was accompanied by decreased xylem sap pH. These results provide new insight into the biology of refilling, underlining the role of sugar and sugar transporters, and imply that a large degree of hydraulic compartmentalization must exist in the xylem during the refilling process.

Isolation and functional characterization of three aquaporins from olive (Olea europaea L.)

To study the molecular bases of water transport in olive we characterized cDNAs from Olea europaea cv “Leccino” related to the aquaporin (AQP) gene family. A phylogenetic analysis of the corresponding polypeptides confirmed that they were part of water channel proteins localized in the plasma membrane and in the tonoplast. The full-length sequences were obtained by RACE-PCR and were named OePIP1.1, OePIP2.1 and OeTIP1.1. The OePIP2.1 and OeTIP1.1 encode functional water channel proteins, as indicated by expression assays in Xenopus laevis oocytes. OePIP1.1 and OePIP2.1 expression levels are high in roots and twigs and low in leaves. The highest hybridization signal of OeTIP1.1 was detected in twigs, while in roots and leaves the expression was low. To investigate the effect of abiotic stress on the transcript level of olive AQP genes, olive trees were subjected to drought treatment and the expression levels of the genes were measured by Northern-blot analysis. The transcript levels of each gene diminished strongly in plants submitted to drought stress, when soil moisture, twig water potential and twig hydraulic conductivity progressively decreased. The downregulation of AQP genes may result in reduced membrane water permeability and may limit loss of cellular water during periods of water stress. A possible role for AQPs on shoot embolism repair is discussed.

Transcriptome Response to Embolism Formation in Stems of Populus trichocarpa Provides Insight into Signaling and the Biology of Refilling

The mechanism of embolism repair in transpiring plants is still not understood, despite significant scientific effort. The refilling process is crucial to maintaining stem transport capacity and ensuring survival for plants experiencing dynamic changes in water stress. Refilling air-filled xylem vessels requires an energy and water source that can only be provided by adjacent living parenchyma cells. Here, we report an analysis of the transcriptome response of xylem parenchyma cells after embolism formation in Populus trichocarpa trees. Genes encoding aquaporins, ion transporters, and carbohydrate metabolic pathways were up-regulated, and there was a significant reduction in the expression of genes responding to oxidative stress. Thus, a novel view of the plant response to embolism emerges that suggests a role for oxygen in embolized vessels as a signal triggering xylem refilling and for the activity of cation transport as having a significant role in the generation of the energy gradient necessary to heal embolized vessels. These findings redefine current hypotheses surrounding the refilling phenomenon and provide insight into the complexity of the biological response to the seemingly simple physical event of xylem embolism formation.

Down-Regulation of Plasma Intrinsic Protein1 Aquaporin in Poplar Trees Is Detrimental to Recovery from Embolism

Aquaporins are involved in the maintenance of xylem transport system capacity, they promote recovery from stress, and they contribute to the control of stomatal conductance under water stress. During their lifecycles, trees encounter multiple events of water stress that often result in embolism formation and temporal decreases in xylem transport capacity. The restoration of xylem transport capacity requires changes in cell metabolic activity and gene expression. Specifically, in poplar (Populus spp.), the formation of xylem embolisms leads to a clear up-regulation of plasma membrane protein1 (PIP1) aquaporin genes. To determine their role in poplar response to water stress, transgenic Populus tremula × Populus alba plants characterized by the strong down-regulation of multiple isoforms belonging to the PIP1 subfamily were used. Transgenic lines showed that they are more vulnerable to embolism, with 50% percent loss of conductance occurring 0.3 MPa earlier than in wild-type plants, and that they also have a reduced capacity to restore xylem conductance during recovery. Transgenic plants also show symptoms of a reduced capacity to control percent loss of conductance through stomatal conductance in response to drought, because they have a much narrower vulnerability safety margin. Finally, a delay in stomatal conductance recovery during the period of stress relief was observed. The presented results suggest that PIP1 genes are involved in the maintenance of xylem transport system capacity, in the promotion of recovery from stress, and in contribution to a plant’s control of stomatal conductance under water stress.

Functional analysis of putative genes encoding the PIP2 water channel subfamily in Populus trichocarpa

We located fully sequenced putative genes of the plasma membrane intrinsic proteins (PIPs) family in the Populus trichocarpa (Torr. Gray), genome. Of 23 gene candidates, we assigned eight genes to the PIP2 subfamily. All eight putative genes were expressed in vegetative tissues (roots, leaves, bark and wood), and all of them showed water channel activity after being expressed in Xenopus oocytes. Six of eight proteins were affected by mercury ions. No proteins were affected by the presence of nickel or tungsten ions, or by lowering the pH of bathing external solution from 7.4 to 6.5. The presence of copper ions caused seven of eight PIP2 proteins to increase their water transport capacity by as much as 50%. This systematic study of the PIP2 subfamily of proteins in P. trichocarpa provides a basic overview of their activity as water channels and will be a useful reference for future physiological studies of plant water relations that use P. trichocarpa as a model system.

Down-regulation of PIP1 aquaporin in poplar trees is detrimental to recovery from embolism.

Aquaporins are involved in the maintenance of xylem transport system capacity, they promote recovery from stress, and they contribute to the control of stomatal conductance under water stress. During their lifecycles, trees encounter multiple events of water stress that often result in embolism formation and temporal decreases in xylem transport capacity. The restoration of xylem transport capacity requires changes in cell metabolic activity and gene expression. Specifically, in poplar (Populus spp.), the formation of xylem embolisms leads to a clear up-regulation of plasma membrane protein1 (PIP1) aquaporin genes. To determine their role in poplar response to water stress, transgenic Populus tremula × Populus alba plants characterized by the strong down-regulation of multiple isoforms belonging to the PIP1 subfamily were used. Transgenic lines showed that they are more vulnerable to embolism, with 50% percent loss of conductance occurring 0.3 MPa earlier than in wild-type plants, and that they also have a reduced capacity to restore xylem conductance during recovery. Transgenic plants also show symptoms of a reduced capacity to control percent loss of conductance through stomatal conductance in response to drought, because they have a much narrower vulnerability safety margin. Finally, a delay in stomatal conductance recovery during the period of stress relief was observed. The presented results suggest that PIP1 genes are involved in the maintenance of xylem transport system capacity, in the promotion of recovery from stress, and in contribution to a plant’s control of stomatal conductance under water stress.

The physiological response of Populus tremula x alba leaves to the down-regulation of PIP1 aquaporin gene expression under no water stress.

In order to study the role of PIP1 aquaporins in leaf water and CO2 transport, several lines of PIP1-deficient transgenic Populus tremula x alba were generated using a reverse genetic approach. These transgenic lines displayed no visible developmental or morphological phenotypes when grown under conditions of no water stress. Major photosynthetic parameters were also not affected by PIP1 down regulation. However, low levels of PIP1 expression resulted in greater leaf hydraulic resistance (an increase of 27%), which effectively implicated PIP1 role in water transport. Additionally, the expression level of PIP1 genes in the various transgenic lines was correlated with reductions in mesophyll conductance to CO2 (gm), suggesting that in poplar, these aquaporins influenced membrane permeability to CO2. Overall, although analysis showed that PIP1 genes contributed to the mass transfer of water and CO2 in poplar leaves, their down-regulation did not dramatically impair the physiological needs of this fast growing tree when cultivated under conditions of no stress.