The ability to measure plant hormones quantitatively is important as plant hormones regulate plant growth, development and response to biotic and abiotic cues. In this protocol, we describe the quantitative analysis of major plant hormones from crude plant extracts. Plant hormones are determined using reverse-phase liquid chromatography-tandem mass spectrometry with multiple reaction monitoring. The method provides quantification of most major plant hormones in a single run from 50 mg of fresh plant tissue. Extraction and quantitative analysis of 40 samples takes 2-3 d.
A sensitive approach based on electrospray ionization tandem mass spectrometry has been employed to profile membrane lipid molecular species in Arabidopsis undergoing cold and freezing stresses. Freezing at a sublethal temperature induced a decline in many molecular species of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG) but induced an increase in phosphatidic acid (PA) and lysophospholipids. To probe the metabolic steps generating these changes, lipids of Arabidopsis deficient in the most abundant phospholipase D, PLD␣, were analyzed. The PC content dropped only half as much, and PA levels rose only half as high in the PLD␣-deficient plants as in wild-type plants. In contrast, neither PE nor PG levels decreased significantly more in wild-type plants than in PLD␣-deficient plants. These data suggest that PC, rather than PE and PG, is the major in vivo substrate of PLD␣. The action of PLD␣ during freezing is of special interest because Arabidopsis plants that are deficient in PLD␣ have improved tolerance to freezing. The greater loss of PC and increase in PA in wild-type plants as compared with PLD␣-deficient plants may be responsible for destabilizing membrane bilayer structure, resulting in a greater propensity toward membrane fusion and cell death in wild-type plants.Eukaryotic membranes contain diverse lipid molecular species, and the lipid composition changes in response to both internal and external cues. Knowing how lipid molecular species change and how the changes are generated is important to the understanding of membrane and cell functions. Detailed study of membrane lipid changes, however, has been technically challenging because of the complexity of lipid molecular species and analytical procedures. Recently, an approach based on electrospray ionization tandem mass spectrometry (ESI-MS/ MS) 1 has been developed to comprehensively analyze lipid composition in animal and yeast cells (1-9). It requires only simple sample preparation and small samples to identify and quantify lipid molecular species. Expansion of this approach to plants, which harbor unique lipids, such as galactosylglycerolipids, should greatly facilitate the understanding of lipid functions in plant growth, development, and stress responses.Plant stress caused by freezing has been an area of intensive research for many years, but the molecular and cellular mechanisms of freezing injury and tolerance are not well understood (10 -12). The best documented freezing injury occurs at the membrane level. One major form of freezing damage is due to the formation of lipid hexagonal II phase in regions where the plasma membrane and the chloroplast envelope are closely apposed (13,14). Changes in membrane lipid composition occur when plants are exposed to freezing temperatures (15). Lipid hydrolysis has been proposed to be mainly responsible for the change, but the role of lipid hydrolysis in freezing injury and tolerance is not clear.In plants, several lipolytic enzymatic activities have been described, ...
A bscisic acid (ABA) plays an important role in plant growth, development, and responses to environmental stresses, such as drought, salinity, and low temperature (1). Reversible protein phosphorylation is involved in the early events of ABA signal transduction (2, 3). Specific protein kinases are activated in response to ABA and have been proposed to play a positive role in ABA signaling (2, 4). On the other hand, protein phosphatases 2C (PP2C), such as ABI1, ABI2, and AtPP2CA, are negative regulators in ABA responses (5). The loss of ABI1 or ABI2 PP2C activity in the intragenic revertants of abi1-1 or abi2-1 leads to an enhanced response to ABA (6, 7), whereas overexpression of ABI1 or AtPP2CA blocks the expression of ABAinducible genes in Arabidopsis protoplasts (3). Antisense (AS) inhibition of AtPP2CA results in the enhancement of cold-and ABA-induced gene expression (8). Recently, ABI1 was shown to interact with ATHB6, a transcriptional regulator (9), and with PKS18, a SOS2-like protein kinase (10), whereas ABI2 and AtPP2CA were found to regulate SOS2 kinase and K ϩ channels, respectively (10-12). However, how the activity and function of PP2Cs are regulated in plant cells is still unclear.Recent studies indicate that phospholipase D (PLD) is involved in ABA responses. PLD activity has been implicated in mediating the ABA inhibition of ␣-amylase secretion (13), ABA-regulated stomatal movement (14), and ABA-induced gene expression (15,16). Arabidopsis has 12 PLDs, and multiple types of PLDs display distinctly different catalytic and regulatory properties (17). Molecular and genetic data have indicated that a specific PLD participates in signaling the ABA response. AS inhibition of PLD␣1 diminished stomatal closure induced by ABA or drought and increased water loss in Arabidopsis, whereas overexpression of PLD␣1 resulted in an increased sensitivity to ABA (18). In addition, specific PLDs have been shown to regulate many other plant functions, including cell patterning, programmed cell death, and stress tolerance (19-21). However, the direct target of PLD action is unknown. In this study, we present evidence for a direct link between PLD␣1 and PP2C in the ABA signaling response. Materials and Methods Plant Materials and Growth.A T-DNA-insertional mutant of PLD␣1 (PLD␣1-KO) was identified from SALK 053785 seeds obtained from the Arabidopsis Biological Resource Center (Columbus, OH). The site of T-DNA insertion was confirmed by DNA sequencing. The generation of PLD␣1-AS plants was described in ref. 22. Seeds of PLD␣1-KO, PLD␣1-AS, and wild type of Arabidopsis thaliana (ecotype Columbia) were sown in soil and kept at 4°C for 2 days. Plants were grown in a growth chamber with cool white fluorescent light of 100 mol m Ϫ2 ⅐s Ϫ1 under 14-h light͞10-h dark and 23°C͞18°C cycles.Water Loss and Stomatal Aperture Measurements. Detached leaves from 6-week-old plants were exposed to cool white light (125 mol m Ϫ2 ⅐s Ϫ1 ) at 23°C. Leaves were weighed at various time intervals, and the loss of fresh weight (%) was used to in...
We determined the role of Phospholipase Da1 (PLDa1) and its lipid product phosphatidic acid (PA) in abscisic acid (ABA)-induced production of reactive oxygen species (ROS) in Arabidopsis thaliana guard cells. The plda1 mutant failed to produce ROS in guard cells in response to ABA. ABA stimulated NADPH oxidase activity in wild-type guard cells but not in plda1 cells, whereas PA stimulated NADPH oxidase activity in both genotypes. PA bound to recombinant Arabidopsis NADPH oxidase RbohD (respiratory burst oxidase homolog D) and RbohF. The PA binding motifs were identified, and mutation of the Arg residues 149, 150, 156, and 157 in RbohD resulted in the loss of PA binding and the loss of PA activation of RbohD. The rbohD mutant expressing non-PA-binding RbohD was compromised in ABA-mediated ROS production and stomatal closure. Furthermore, ABA-induced production of nitric oxide (NO) was impaired in plda1 guard cells. Disruption of PA binding to ABI1 protein phosphatase 2C did not affect ABA-induced production of ROS or NO, but the PA-ABI1 interaction was required for stomatal closure induced by ABA, H 2 O 2 , or NO. Thus, PA is as a central lipid signaling molecule that links different components in the ABA signaling network in guard cells.
Terrestrial plants lose water primarily through stomata, pores on the leaves. The hormone abscisic acid (ABA) decreases water loss by regulating opening and closing of stomata. Here, we show that phospholipase Dalpha1 (PLDalpha1) mediates the ABA effects on stomata through interaction with a protein phosphatase 2C (PP2C) and a heterotrimeric GTP-binding protein (G protein) in Arabidopsis. PLDalpha1-produced phosphatidic acid (PA) binds to the ABI1 PP2C to signal ABA-promoted stomatal closure, whereas PLDalpha1 and PA interact with the Galpha subunit of heterotrimeric G protein to mediate ABA inhibition of stomatal opening. The results reveal a bifurcating signaling pathway that regulates plant water loss.
Four types of phospholipase D (PLD), PLD␣, , ␥, and ␦, have been characterized in Arabidopsis, and they display different requirements for Ca 2ϩ , phosphatidylinositol 4,5-bisphosphate (PIP 2), substrate vesicle composition, and/or free fatty acids. However, all previously cloned plant PLDs contain a Ca 2ϩ-dependent phospholipid-binding C2 domain and require Ca 2ϩ for activity. This study documents a new type of PLD, PLD1, which is distinctively different from previously characterized PLDs. It contains at the N terminus a Phox homology domain and a pleckstrin homology domain, but not the C2 domain. A full-length cDNA for Arabidopsis PLD1 has been identified and used to express catalytically active PLD in Escherichia coli. PLD1 does not require Ca 2ϩ or any other divalent cation for activity. In addition, it selectively hydrolyzes phosphatidylcholine, whereas the other Arabidopsis PLDs use several phospholipids as substrates. PLD1 requires PIP 2 for activity, but unlike the PIP 2-requiring PLD or ␥, phosphatidylethanolamine is not needed in substrate vesicles. These differences are described, together with a genomic analysis of 12 putative Arabidopsis PLD genes that are grouped into ␣, , ␦, ␥, and based on their gene architectures, sequence similarities, domain structures, and biochemical properties.
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