The Development of Student Research Skills in Second Year Plant Biology

In 2011, students in Agricultural Sciences and Viticulture & Oenology were first provided with opportunities to develop research skills in plant biology through the course Foundations in Plant Science II. Students worked in small groups and completed an open-ended research project under the guidance of an academic mentor. Each group of students were given the freedom to plan and manage an experiment; collect, analyse and interpret data independently and to present their results both orally and in writing. Students reported that the group project was a positive experience where they were able to develop skills in scientific report writing. In 2012, students were challenged by aspects of the research project including experimental design and identifying published papers to support their hypotheses. In 2013, when we provided more support and structure using on-line and in-class tutorials, students were better able to work in groups, source appropriate literature and analyse data using statistics as their confidence in research and questioning ideas had improved

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 S. 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 Ca 2+

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. Coexpression 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

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. Coexpression 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

Characterisation of the TaALMT1 protein as an Al(3+)-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.Wen-Hao Zhang, Peter R. Ryan, Takayuki Sasaki, Yoko Yamamoto, Wendy Sullivan and Steve D. Tyerma

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.Chuang Wang, Wei Huang, Yinghui Ying, Shuai Li, David Secco, Steve Tyerman, James Whelan and Huixia Sho