Expression of a CO2-permeable aquaporin enhances mesophyll conductance in the C4 species Setaria viridis.

A fundamental limitation of photosynthetic carbon fixation is the availability of CO2. In C4 plants, primary carboxylation occurs in mesophyll cytosol, and little is known about the role of CO2 diffusion in facilitating C4 photosynthesis. We have examined the expression, localization, and functional role of selected plasma membrane intrinsic aquaporins (PIPs) from Setaria italica (foxtail millet) and discovered that SiPIP2;7 is CO2-permeable. When ectopically expressed in mesophyll cells of Setaria viridis (green foxtail), SiPIP2;7 was localized to the plasma membrane and caused no marked changes in leaf biochemistry. Gas exchange and C18O16O discrimination measurements revealed that targeted expression of SiPIP2;7 enhanced the conductance to CO2 diffusion from the intercellular airspace to the mesophyll cytosol. Our results demonstrate that mesophyll conductance limits C4 photosynthesis at low pCO2 and that SiPIP2;7 is a functional CO2 permeable aquaporin that can improve CO2 diffusion at the airspace/mesophyll interface and enhance C4 photosynthesis.

Evolution of chloroplast retrograde signaling facilitates green plant adaptation to land.

Chloroplast retrograde signaling networks are vital for chloroplast biogenesis, operation, and signaling, including excess light and drought stress signaling. To date, retrograde signaling has been considered in the context of land plant adaptation, but not regarding the origin and evolution of signaling cascades linking chloroplast function to stomatal regulation. We show that key elements of the chloroplast retrograde signaling process, the nucleotide phosphatase (SAL1) and 3'-phosphoadenosine-5'-phosphate (PAP) metabolism, evolved in streptophyte algae-the algal ancestors of land plants. We discover an early evolution of SAL1-PAP chloroplast retrograde signaling in stomatal regulation based on conserved gene and protein structure, function, and enzyme activity and transit peptides of SAL1s in species including flowering plants, the fern Ceratopteris richardii, and the moss Physcomitrella patens Moreover, we demonstrate that PAP regulates stomatal closure via secondary messengers and ion transport in guard cells of these diverse lineages. The origin of stomata facilitated gas exchange in the earliest land plants. Our findings suggest that the conquest of land by plants was enabled by rapid response to drought stress through the deployment of an ancestral SAL1-PAP signaling pathway, intersecting with the core abscisic acid signaling in stomatal guard cells.

Non-selective cation channel activity of aquaporin AtPIP2;1 regulated by Ca2+ and pH.

The aquaporin AtPIP2;1 is an abundant plasma membrane intrinsic protein in Arabidopsis thaliana that is implicated in stomatal closure, and is highly expressed in plasma membranes of root epidermal cells. When expressed in Xenopus laevis oocytes, AtPIP2;1 increased water permeability and induced a non-selective cation conductance mainly associated with Na+ . A mutation in the water pore, G103W, prevented both the ionic conductance and water permeability of PIP2;1. Co-expression of AtPIP2;1 with AtPIP1;2 increased water permeability but abolished the ionic conductance. AtPIP2;2 (93% identical to AtPIP2;1) similarly increased water permeability but not ionic conductance. The ionic conductance was inhibited by the application of extracellular Ca2+ and Cd2+ , with Ca2+ giving a biphasic dose-response with a prominent IC50 of 0.32 mм comparable with a previous report of Ca2+ sensitivity of a non-selective cation channel (NSCC) in Arabidopsis root protoplasts. Low external pH also inhibited ionic conductance (IC50 pH 6.8). Xenopus oocytes and Saccharomyces cerevisiae expressing AtPIP2;1 accumulated more Na+ than controls. Establishing whether AtPIP2;1 has dual ion and water permeability in planta will be important in understanding the roles of this aquaporin and if AtPIP2;1 is a candidate for a previously reported NSCC responsible for Ca2+ and pH sensitive Na+ entry into roots.

VitiCanopy: A Free Computer App to Estimate Canopy Vigor and Porosity for Grapevine.

Leaf area index (LAI) and plant area index (PAI) are common and important biophysical parameters used to estimate agronomical variables such as canopy growth, light interception and water requirements of plants and trees. LAI can be either measured directly using destructive methods or indirectly using dedicated and expensive instrumentation, both of which require a high level of know-how to operate equipment, handle data and interpret results. Recently, a novel smartphone and tablet PC application, VitiCanopy, has been developed by a group of researchers from the University of Adelaide and the University of Melbourne, to estimate grapevine canopy size (LAI and PAI), canopy porosity, canopy cover and clumping index. VitiCanopy uses the front in-built camera and GPS capabilities of smartphones and tablet PCs to automatically implement image analysis algorithms on upward-looking digital images of canopies and calculates relevant canopy architecture parameters. Results from the use of VitiCanopy on grapevines correlated well with traditional methods to measure/estimate LAI and PAI. Like other indirect methods, VitiCanopy does not distinguish between leaf and non-leaf material but it was demonstrated that the non-leaf material could be extracted from the results, if needed, to increase accuracy. VitiCanopy is an accurate, user-friendly and free alternative to current techniques used by scientists and viticultural practitioners to assess the dynamics of LAI, PAI and canopy architecture in vineyards, and has the potential to be adapted for use on other plants.

Functional characterization of the rice SPX-MFS family reveals a key role of OsSPX-MFS1 in controlling phosphate homeostasis in leaves.

• Proteins possessing the SPX domain are found in several proteins involved in inorganic phosphate (Pi) transport and signalling in yeast and plants. Although the functions of several SPX-domain protein subfamilies have recently been uncovered, the role of the SPX-MFS subfamily is still unclear. • Using quantitative RT-PCR analysis, we studied the regulation of SPX-MFS gene expression by the central regulator, OsPHR2 and Pi starvation. The function of OsSPX-MFS1 in Pi homeostasis was analysed using an OsSPX-MFS1 mutant (mfs1) and osa-miR827 overexpression line (miR827-Oe). Finally, heterologous complementation of a yeast mutant impaired in Pi transporter was used to assess the capacity of OsSPX-MFS1 to transport Pi. • Transcript analyses revealed that members of the SPX-MFS family were mainly expressed in the shoots, with OsSPX-MFS1 and OsSPX-MFS3 being suppressed by Pi deficiency, while OsSPX-MFS2 was induced. Mutation in OsSPX-MFS1 (mfs1) and overexpression of the upstream miR827 (miR827-Oe) plants impaired Pi homeostasis in the leaves. In addition, studies in yeast revealed that OsSPX-MFS1 may be involved in Pi transport. • The results suggest that OsSPX-MFS1 is a key player in maintaining Pi homeostasis in the leaves, potentially acting as a Pi transporter.

Characterization of the TaALMT1 protein as an Al3+-activated anion channel in transformed tobacco (Nicotiana tabacum L.) cells.

TaALMT1 encodes a putative transport protein associated with Al(3+)-activated efflux of malate from wheat root apices. We expressed TaALMT1 in Nicotiana tabacum L. suspension cells and conducted a detailed functional analysis. Protoplasts were isolated for patch-clamping from cells expressing TaALMT1 and from control cells (empty vector transformed). With malate(2-) as the permeant anion in the protoplast, an inward current (anion efflux) that reversed at positive potentials was observed in protoplasts expressing TaALMT1 in the absence of Al(3+). This current was sensitive to the anion channel antagonist niflumate, but insensitive to Gd(3+). External AlCl(3) (50 microM), but not La(3+) and Gd(3+), increased the inward current in TaALMT1-transformed protoplasts. The inward current was highly selective to malate over nitrate and chloride (P(mal) >> P(NO3) >or= P(Cl), P(mal)/P(Cl) >or=18, +/-Al(3+)), under conditions with higher anion concentration internally than externally. The anion currents displayed a voltage and time dependent deactivation at negative voltages. Voltage ramps revealed that inward rectification was caused by the imposed anion gradients. Single channels with conductances between 10 and 17 pS were associated with the deactivation of the current at negative voltages, agreeing with estimates from voltage ramps. This study of the electrophysiological function of the TaALMT1 protein in a plant heterologous expression system provides the first direct evidence that TaALMT1 functions as an Al(3+)-activated malate(2-) channel. We show that the Al(3+)-activated currents measured in TaALMT1-transformed tobacco cells are identical to the Al(3+)-activated currents observed in the root cells of wheat, indicating that TaALMT1 alone is likely to be responsible for those endogenous currents.

Sources of water used by riparian Eucalyptus camaldulensis overlying highly saline groundwater.

Water sources of Eucalyptus camaldulensis Dehn. trees were investigated on a semiarid floodplain in south-eastern Australia. The trees investigated ranged in distance from 0.5 to 40 m from a stream, with electrical conductivity 0.8 dSm-1, and grew over groundwater with electrical conductivity ranging from 30 to 50 dSm-1. The sources of water being used by the trees were investigated using the naturally occurring stable isotopes of water and measurements of soil water potential. Xylem water potential and leaf conductance were also examined to identify the trees' response to using these sources of water. Trees at distances greater than about 15 m from the stream used no stream water. The trees used groundwater in summer and a combination of groundwater and rain-derived surface-soil water (0.05-0.15 m depth) in winter. In doing so they suffered water stress at electrical conductivities higher than approximately 40 dSm-1 (equivalent to approximately -1.4 MPa). Trees adjacent to the stream used stream water directly in summer, but may have used stream water from the soil profile in winter, after the stream had risen and recharged the soil water. E. camaldulensis appeared to be partially opportunistic in the sources of water they used.