Density- and moisture-dependent effects of arbuscular mycorrhizal fungus on drought acclimation in wheat.

Arbuscular mycorrhizal fungus (AMF) is widely viewed as an ecosystem engineer to help plants adapt to adverse environments. However, a majority of the previous studies regarding AMF's eco-physiological effects are mutually inconsistent. To clarify this fundamental issue, we conducted an experiment focused on wheat (Triticum aestivum L.) plants with or without AMF (Funneliformis mosseae) inoculation. Two water regimes (80% and 40% field water capacity, FWC80 (CK) and FWC40 (drought stress) and four planting densities (6 or 12 plants per pot as low densities, 24 or 48 plants per pot as high densities) were designed. AMF inoculation did not show significant effects on shoot biomass, grain yield, and water use efficiency (WUE) under the low densities, regardless of water regimes. However, under the high densities, AMF inoculation significantly decreased shoot biomass, grain yield and WUE in FWC80, while it significantly increased these parameters in FWC40, showing density and/or moisture-dependent effects of AMF on wheat performance. In FWC40, the relationships between reproductive biomass (y-axis) vs. vegetative biomass (x-axis) (R-V), and between grain biomass (y-axis, sink) vs. leaf biomass (x-axis, source) fell into a typical allometric pattern (α > 1, P < 0.001), and the AMF inoculation significantly increased the values of α. Yet in FWC80, they were in an isometric pattern (α ≈ 1, P < 0.001) and AMF addition had no significant effects on α. Similarly, AMF did not significantly change the isometric relationship between leaf biomass (i.e., metabolic rate) and shoot biomass (body size) in FWC80, while it significantly decreased the α of allometric relationship between both of them in FWC40 (α > 1, P < 0.001). We therefore, sketched a generalized model of R-V and sink-source relationships as affected by AMF, in which AMF inoculation might enhance the capabilities of sink acquisition and utilization under drought stress, while having no significant effect under the well watered conditions. Our findings demonstrate dual density- and moisture-dependent effects of AMF on plant development and provide new insights into current ecological applications of AMF as an ecosystem engineer.

Atg5-independent autophagy regulates mitochondrial clearance and is essential for iPSC reprogramming.

Successful generation of induced pluripotent stem cells entails a major metabolic switch from mitochondrial oxidative phosphorylation to glycolysis during the reprogramming process. The mechanism of this metabolic reprogramming, however, remains elusive. Here, our results suggest that an Atg5-independent autophagic process mediates mitochondrial clearance, a characteristic event involved in the metabolic switch. We found that blocking such autophagy, but not canonical autophagy, inhibits mitochondrial clearance, in turn, preventing iPSC induction. Furthermore, AMPK seems to be upstream of this autophagic pathway and can be targeted by small molecules to modulate mitochondrial clearance during metabolic reprogramming. Our work not only reveals that the Atg5-independent autophagy is crucial for establishing pluripotency, but it also suggests that iPSC generation and tumorigenesis share a similar metabolic switch.

Arabidopsis sucrose transporter SUT4 interacts with cytochrome b5-2 to regulate seed germination in response to sucrose and glucose.

It remains unknown whether a sucrose transporter mediates sugar signaling. Here, we report that the Arabidopsis (Arabidopsis thaliana) sucrose transporter SUT4 interacts with five members of the Arabidopsis cytochrome b5 (Cyb5) family, and sucrose represses the interaction between SUT4 and a Cyb5 member Cyb5-2/A. We observed that down-regulation of SUT4 and three cytochrome b5 members (Cyb5-2, Cyb5-4, and Cyb5-6) confers the sucrose- and glucose-insensitive phenotypes in the sucrose/glucose-induced inhibition of seed germination. The sut4 cyb5-2 double mutant displays slightly stronger sucrose/glucose-insensitive phenotypes than either the sut4 or cyb5-2 single mutant. We showed that the SUT4/Cyb5-2-mediated signaling in the sucrose/glucose-induced inhibition of seed germination does not require ABA or the currently known ABI2/ABI4/ABI5-mediated signaling pathway(s). These data provide evidence that the sucrose transporter SUT4 interacts with Cyb5 to positively mediate sucrose and glucose signaling in the sucrose/glucose-induced inhibition of seed germination.

Identification of an important site for function of the type 2C protein phosphatase ABI2 in abscisic acid signalling in Arabidopsis.

It is known that the clade A protein phosphatase 2Cs (PP2Cs), including ABI1 and ABI2 and other PP2C members, are key players that function directly downstream of the PYR/PYL/RCAR abscisic acid (ABA) receptors. Here, identification of a crucial site for function of ABI2 protein phosphatase in ABA signalling is reported. It was observed that a calcium-dependent protein kinase (CDPK) phosphorylation site-like motif (CPL) in the ABI2 molecule is required for the interactions of ABI2 with the two members of the ABA receptors PYL5 and PYL9 and with a downstream protein kinase SnRK2.6, and for the catalytic activity of ABI2 in vitro, as well as for the response of ABI2 to the ABA receptors PYL5/PYL9 in relation to the ABA receptor-induced inhibition of the ABI2 phosphatase activity. Further, genetic evidence was provided to demonstrate that this CPL is required for the function of ABI2 to mediate ABA signalling. These data reveal that this CPL is an important site necessary for both the phosphatase activity of ABI2 and the functional interaction between ABI2 and PYL5/9 ABA receptors, providing new information to understand primary events of ABA signal transduction.

Two coupled components of the mitogen-activated protein kinase cascade MdMPK1 and MdMKK1 from apple function in ABA signal transduction.

Plant mitogen-activated protein kinase (MAPK) cascades are involved in a range of biotic and abiotic stress responses, but many members of the MAPK family involved in signal transduction of the stress-related hormone ABA remain to be identified and how they regulate ABA signaling is still unclear. Here we characterized biochemically an apple MAPK signaling cascade MdMKK1-MdMPK1, which is transiently activated by ABA. Expression of MdMKK1 or MdMPK1 in the reference plant Arabidopsis (Arabidopsis thaliana) confers ABA hypersensitivity in both seed germination and seedling growth, showing that MdMKK1 and MdMPK1 are positively involved in ABA signaling. Expression of MdMKK1 or MdMPK1 up-regulates expression of several ABA-responsive transcription factor-encoding genes including ABI5. Furthermore, MdMPK1 phosphorylates the Arabidopsis ABI5 protein through the unique residue Ser314, showing that ABI5 is a potential direct downstream component of MAPK in ABA signaling. These findings indicate that the apple MdMKK1-MdMPK1-coupled signaling cascade may function in ABA signaling by regulating both expression and the phosphorylation status of the important ABA signaling component ABI5 or ABI5-like transcription factors.

Two calcium-dependent protein kinases, CPK4 and CPK11, regulate abscisic acid signal transduction in Arabidopsis.

Many biochemical approaches show functions of calcium-dependent protein kinases (CDPKs) in abscisic acid (ABA) signal transduction, but molecular genetic evidence linking defined CDPK genes with ABA-regulated biological functions at the whole-plant level has been lacking. Here, we report that ABA stimulated two homologous CDPKs in Arabidopsis thaliana, CPK4 and CPK11. Loss-of-function mutations of CPK4 and CPK11 resulted in pleiotropic ABA-insensitive phenotypes in seed germination, seedling growth, and stomatal movement and led to salt insensitivity in seed germination and decreased tolerance of seedlings to salt stress. Double mutants of the two CDPK genes had stronger ABA- and salt-responsive phenotypes than the single mutants. CPK4- or CPK11-overexpressing plants generally showed inverse ABA-related phenotypes relative to those of the loss-of-function mutants. Expression levels of many ABA-responsive genes were altered in the loss-of-function mutants and overexpression lines. The CPK4 and CPK11 kinases both phosphorylated two ABA-responsive transcription factors, ABF1 and ABF4, in vitro, suggesting that the two kinases may regulate ABA signaling through these transcription factors. These data provide in planta genetic evidence for the involvement of CDPK/calcium in ABA signaling at the whole-plant level and show that CPK4 and CPK11 are two important positive regulators in CDPK/calcium-mediated ABA signaling pathways.

Expression of a grape calcium-dependent protein kinase ACPK1 in Arabidopsis thaliana promotes plant growth and confers abscisic acid-hypersensitivity in germination, postgermination growth, and stomatal movement.

Calcium is an important second messenger involved in abscisic acid (ABA) signal transduction. Calcium-dependent protein kinases (CDPKs) are the best characterized calcium sensor in plants and are believed to be important components in plant hormone signaling. However, in planta genetic evidence has been lacking to link CDPK with ABA-regulated biological functions. We previously identified an ABA-stimulated CDPK from grape berry, which is potentially involved in ABA signaling. Here we report that heterologous overexpression of ACPK1 in Arabidopsis promotes significantly plant growth and enhances ABA-sensitivity in seed germination, early seedling growth and stomatal movement, providing evidence that ACPK1 is involved in ABA signal transduction as a positive regulator, and suggesting that the ACPK1 gene may be potentially used for elevating plant biomass production.

The Mg-chelatase H subunit is an abscisic acid receptor.

Abscisic acid (ABA) is a vital phytohormone that regulates mainly stomatal aperture and seed development, but ABA receptors involved in these processes have yet to be determined. We previously identified from broad bean an ABA-binding protein (ABAR) potentially involved in stomatal signalling, the gene for which encodes the H subunit of Mg-chelatase (CHLH), which is a key component in both chlorophyll biosynthesis and plastid-to-nucleus signalling. Here we show that Arabidopsis ABAR/CHLH specifically binds ABA, and mediates ABA signalling as a positive regulator in seed germination, post-germination growth and stomatal movement, showing that ABAR/CHLH is an ABA receptor. We show also that ABAR/CHLH is a ubiquitous protein expressed in both green and non-green tissues, indicating that it might be able to perceive the ABA signal at the whole-plant level.

Abscisic acid stimulates a calcium-dependent protein kinase in grape berry.

It has been demonstrated that calcium plays a central role in mediating abscisic acid (ABA) signaling, but many of the Ca2+-binding sensory proteins as the components of the ABA-signaling pathway remain to be elucidated. Here we identified, characterized, and purified a 58-kD ABA-stimulated calcium-dependent protein kinase from the mesocarp of grape berries (Vitis vinifera x Vitis labrusca), designated ACPK1 (for ABA-stimulated calcium-dependent protein kinase1). ABA stimulates ACPK1 in a dose-dependent manner, and the ACPK1 expression and enzyme activities alter accordantly with the endogenous ABA concentrations during fruit development. The ABA-induced ACPK1 stimulation appears to be transient with a rapid effect in 15 min but also with a slow and steady state of induction after 60 min. ABA acts on ACPK1 indirectly and dependently on in vivo state of the tissues. Two inactive ABA isomers, (-)-2-cis, 4-trans-ABA and 2-trans, 4-trans-(+/-)-ABA, are ineffective for inducing ACPK1 stimulation, revealing that the ABA-induced effect is stereo specific to physiological active (+)-2-cis, 4-trans-ABA. The other phytohormones such as auxin indoleacetic acid, gibberellic acid, synthetic cytokinin N-benzyl-6-aminopurine, and brassinolide are also ineffective in this ACPK1 stimulation. Based on sequencing of the two-dimensional electrophoresis-purified ACPK1, we cloned the ACPK1 gene. The ACPK1 is expressed specifically in grape berry covering a fleshy portion and seeds, and in a developmental stage-dependent manner. We further showed that ACPK1 is localized in both plasma membranes and chloroplasts/plastids and positively regulates plasma membrane H+-ATPase in vitro, suggesting that ACPK1 may be involved in the ABA-signaling pathway.