The physiological role and mechanism of nutrient storage within vacuoles of specific cell types is poorly understood. Transcript profiles from Arabidopsis thaliana leaf cells differing in calcium concentration ([Ca], epidermis <10 mM versus mesophyll >60 mM) were compared using a microarray screen and single-cell quantitative PCR. Three tonoplast-localized Ca 2+ transporters, CAX1 (Ca 2+ /H + -antiporter), ACA4, and ACA11 (Ca 2+ -ATPases), were identified as preferentially expressed in Ca-rich mesophyll. Analysis of respective loss-of-function mutants demonstrated that only a mutant that lacked expression of both CAX1 and CAX3, a gene ectopically expressed in leaves upon knockout of CAX1, had reduced mesophyll [Ca]. Reduced capacity for mesophyll Ca accumulation resulted in reduced cell wall extensibility, stomatal aperture, transpiration, CO 2 assimilation, and leaf growth rate; increased transcript abundance of other Ca 2+ transporter genes; altered expression of cell wall-modifying proteins, including members of the pectinmethylesterase, expansin, cellulose synthase, and polygalacturonase families; and higher pectin concentrations and thicker cell walls. We demonstrate that these phenotypes result from altered apoplastic free [Ca 2+ ], which is threefold greater in cax1/cax3 than in wild-type plants. We establish CAX1 as a key regulator of apoplastic [Ca 2+ ] through compartmentation into mesophyll vacuoles, a mechanism essential for optimal plant function and productivity.
Circadian clocks are 24-h timing devices that phase cellular responses; coordinate growth, physiology, and metabolism; and anticipate the day-night cycle. Here we report sensitivity of the Arabidopsis thaliana circadian oscillator to sucrose, providing evidence that plant metabolism can regulate circadian function. We found that the Arabidopsis circadian system is particularly sensitive to sucrose in the dark. These data suggest that there is a feedback between the molecular components that comprise the circadian oscillator and plant metabolism, with the circadian clock both regulating and being regulated by metabolism. We used also simulations within a three-loop mathematical model of the Arabidopsis circadian oscillator to identify components of the circadian clock sensitive to sucrose. The mathematical studies identified GIGANTEA (GI) as being associated with sucrose sensing. Experimental validation of this prediction demonstrated that GI is required for the full response of the circadian clock to sucrose. We demonstrate that GI acts as part of the sucrose-signaling network and propose this role permits metabolic input into circadian timing in Arabidopsis.he Arabidopsis thaliana circadian clock confers growth and competitive advantage (1). The phase of circadian rhythms in plants is adjusted by light signals to entrain the clock to dawn and dusk (2). Additionally, it has been proposed that the Arabidopsis circadian clock is sensitive to nitrogen acting as a nutritional cue (3) and phytohormones, possibly as an input from stress signaling and growth pathways (4). In Arabidopsis, circadian oscillations are generated and maintained by interlocking transcriptionaltranslational feedback loops and posttranslational regulation (1,(5)(6)(7)(8). In the morning, light activates the expression of two Myblike transcription factors, CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), leading to expression of PSEUDO RESPONSE REGULATORS (PRR) 7 and 9, which in turn repress CCA1 and LHY expression. This "morning" loop of the oscillator is connected to "evening" expressed genes through direct repression of the expression of TIMING OF CAB EXPRESSION 1 (TOC1/PRR1) by binding of LHY/CCA1 to the TOC1 promoter. TOC1 is expressed in the evening and feeds back to activate LHY/CCA1 through an unknown pathway. TOC1 physically interacts with and antagonizes CCA1 HIKING EXPEDITION (CHE), a transcriptional repressor of CCA1 (9). CCA1 and LHY are also coupled with TOC1 through GIGANTEA (GI). LHY/CCA1 repress GI and GI forms a loop with TOC1, GI activating TOC1 and TOC1 repressing GI. GI encodes a protein of unknown biochemical function but in blue light, GI physically interacts with and stabilizes ZEITLUPE (10), an F-box protein that targets TOC1 for degradation (11). To entrain the circadian oscillator, signals are incorporated from light, second messengers, and metabolites to alter circadian clock gene expression and modulate circadian function (7,(12)(13)(14)(15).Carbohydrates are a major energy store in most forms ...
Regulation of reactive oxygen species and cytosolic free calcium ([Ca 2+ ] cyt ) is central to plant function. Annexins are small proteins capable of Ca 2+ -dependent membrane binding or membrane insertion. They possess structural motifs that could support both peroxidase activity and calcium transport. Here, a Zea mays annexin preparation caused increases in [Ca 2+ ] cyt when added to protoplasts of Arabidopsis thaliana roots expressing aequorin. The pharmacological profile was consistent with annexin activation (at the extracellular plasma membrane face) of Arabidopsis Ca 2+ -permeable nonselective cation channels. Secreted annexins could therefore modulate Ca 2+ influx. As maize annexins occur in the cytosol and plasma membrane, they were incorporated at the intracellular face of lipid bilayers designed to mimic the plasma membrane. Here, they generated an instantaneously activating Ca 2+ -permeable conductance at mildly acidic pH that was sensitive to verapamil and Gd 3+ and had a Ca 2+ -to-K + permeability ratio of 0.36. These results suggest that cytosolic annexins create a Ca 2+ influx pathway directly, particularly during stress responses involving acidosis. A maize annexin preparation also demonstrated in vitro peroxidase activity that appeared independent of heme association. In conclusion, this study has demonstrated that plant annexins create Ca 2+ -permeable transport pathways, regulate [Ca 2+ ] cyt , and may function as peroxidases in vitro.
Cucumber mosaic virus (CMV) encodes the 2b protein, which plays a role in local and systemic virus movement, symptom induction and suppression of RNA silencing. It also disrupts signalling regulated by salicylic acid and jasmonic acid. CMV induced an increase in tolerance to drought in Arabidopsis thaliana. This was caused by the 2b protein, as transgenic plants expressing this viral factor showed increased drought tolerance, but plants infected with CMVΔ2b, a viral mutant lacking the 2b gene, did not. The silencing effector ARGONAUTE1 (AGO1) controls a microRNA-mediated drought tolerance mechanism and, in this study, we noted that plants (dcl2/3/4 triple mutants) lacking functional short-interfering RNA-mediated silencing were also drought tolerant. However, drought tolerance engendered by CMV may be independent of the silencing suppressor activity of the 2b protein. Although CMV infection did not alter the accumulation of the drought response hormone abscisic acid (ABA), 2b-transgenic and ago1-mutant seeds were hypersensitive to ABA-mediated inhibition of germination. However, the induction of ABA-regulated genes in 2b-transgenic and CMV-infected plants was inhibited more strongly than in ago1-mutant plants. The virus engenders drought tolerance by altering the characteristics of the roots and not of the aerial tissues as, compared with the leaves of silencing mutants, leaves excised from CMV-infected or 2b-transgenic plants showed greater stomatal permeability and lost water more rapidly. This further indicates that CMV-induced drought tolerance is not mediated via a change in the silencing-regulated drought response mechanism. Under natural conditions, virus-induced drought tolerance may serve viruses by aiding susceptible hosts to survive periods of environmental stress.
Appropriate stimulus-response coupling requires that each signal induces a characteristic response, distinct from that induced by other signals, and that there is the potential for individual signals to initiate different downstream responses dependent on cell type. How such specificity is encoded in plant signaling is not known. One possibility is that information is encoded in signal transduction pathways to ensure stimulus-and cell type-specific responses. The calcium ion acts as a second messenger in response to mechanical stimulation, hydrogen peroxide, NaCl, and cold in plants and also in circadian timing. We use GAL4 transactivation of aequorin in enhancer trap lines of Arabidopsis (Arabidopsis thaliana) to test the hypothesis that stimulus-and cell-specific information can be encoded in the pattern of dynamic alterations in the concentration of intracellular free Ca 2+ ( ] i dynamics in response to touch, cold, and hydrogen peroxide, which in the case of the latter two signals are common to all cell types tested. GAL4 transactivation of aequorin in specific leaf cell types has allowed us to bypass the technical limitations associated with fluorescent Ca 2+ reporter dyes in chlorophyll-containing tissues to identify the cell-and stimulus-specific complexity of [Ca 2+ ] i dynamics in leaves of Arabidopsis and to determine from which tissues stress-and circadian-regulated [Ca 2+ ] i signals arise. Ca2+ is an important second messenger involved in a wide range of responses in plants (Dodd et al., 2010 (Dodd et al., 2010). However, the utility of fluorescent Ca 2+ indicators in plants has been usually restricted to a few specific cell types, including the guard cells, root hairs, and pollen tubes, because these tissues are low in chlorophyll. In other tissues, chlorophyll fluorescence reduces the signal-to-noise ratio to unacceptable levels. Furthermore, in situ measurements of cytosolic-free Ca 2+ concentration ([Ca 2+
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