Diurnal and circadian regulation of putative potassium channels in a leaf moving organ

Abstract

In a search for potassium channels involved in light- and clock-regulated leaf movements, we cloned four putative K channel genes from the leaf-moving organs, pulvini, of the legume Samanea saman. The S. saman SPOCK1 is homologous to KCO1, an Arabidopsis two-pore-domain K channel, the S. saman SPORK1 is similar to SKOR and GORK, Arabidopsis outward-rectifying Shaker-like K channels, and the S. saman SPICK1 and SPICK2 are homologous to AKT2, a weakly-inward-rectifying Shaker-like Arabidopsis K channel. All four S. saman sequences possess the universal K-channel-specific pore signature, TXXTXGYG, strongly suggesting a role in transmembrane K(+) transport. The four S. saman genes had different expression patterns within four leaf parts: "extensor" and "flexor" (the motor tissues), the leaf blades (mainly mesophyll), and the vascular bundle ("rachis"). Based on northern blot analysis, their transcript level was correlated with the rhythmic leaf movements: (a) all four genes were regulated diurnally (Spick2, Spork1, and Spock1 in extensor and flexor, Spick1 in extensor and rachis); (b) Spork1 and Spock1 rhythms were inverted upon the inversion of the day-night cycle; and (c) in extensor and/or flexor, the expression of Spork1, Spick1, and Spick2 was also under a circadian control. These findings parallel the circadian rhythm shown to govern the resting membrane K(+) permeability in extensor and flexor protoplasts and the susceptibility of this permeability to light stimulation (Kim et al., 1993). Thus, Samanea pulvinar motor cells are the first described system combining light and circadian regulation of K channels at the level of transcript and membrane transport.

Figure 1

Schematic representation of the top part of the leaf of S. saman, displaying movement (semicircular arrow) between the open and the folded positions (dark gray and light gray, respectively). P_II and P_III, Secondary and tertiary pulvini, respectively; L (blackened areas), leaf blades with the larger veins excluded; R, rachis; ra, rachilla; E, F, and vb, extensor, flexor, and the vascular bundle regions of the secondary pulvinus shown schematically, enlarged. The dotted lines indicate planes of E and F excision.

Figure 2

Sequence analysis of the S. saman putative K channels. A, Hydropathy plot of the predicted amino acid sequences of the S. saman K channels. Lines, Kyte and Doolittle hydrophobicity values using an 11-amino acid window. Black, Predicted transmembrane domains. Gray, Predicted pore domains. B, Alignment of predicted amino acid sequences of the most conserved parts in the S. saman—the pore region (P) and its flanking transmembrane segments (S)—of S. saman K channels with their respective homologs from Arabidopsis (SPICK1, SPICK2 versus AKT2; SPORK1 versus SKOR and GORK; and SPOCK1 versus KCO1). Identical and highly similar amino acids are labeled with black boxes, and less similar ones are labeled with gray boxes (see “Materials and Methods”). TXXTVGYGD represents the core of the pore domain and is the most conserved sequence among the plant K channels.

Figure 3

Rhythmic variation of S. saman K channels expression levels in the motor tissues. All panels, except Spick1 and Spick2: D/L, phosphor-imager scans of total RNA northern blots from E or F parts of the secondary pulvini, using the channel cDNA as a homologous probe and a probe to ribosomal RNA 18S (rRNA). Spick1 and Spick2: D/L, scans of autoradiograms of mRNA northern blots, probed with the channel cDNA (top) and poly-deoxy-Thymidine (pdT; bottom). Numbers at the bottom are abbreviations of the time of sampling: noon (13), evening (19), night (01), morning (07). D/L, Diurnal alternations of dark and light (on: 5 am; off: 9 pm); D/L-INV, dark-light illumination inverted (on: 5 pm; off: 9 am); leaves were harvested 7 d after the inversion. D/D, continuous darkness; measurements between h 39 and 58 after lights went off at the end of a normal day. White bars, light; gray bars, dark; hatched bars, subjective day; hatched gray bars, subjective night. Note that, while the mRNA levels of all four channels fluctuated in E and/or F during D/L, and, in Spork1 and Spock1, also after D/L inversion, Spock1 mRNA level did not fluctuate during continuous darkness (D/D).

Figure 4

Temporal pattern of expression of S. saman K channel genes. A, top, An angle between rachis and terminal rachilla in an intact, tree-attached leaf (see Fig. 1). D/L, dark/light alternations; D/D, continuous darkness. Inset, Pulvinus angles illustrated. All other panels show normalized transcript levels of the individual genes (indicated) in the different tissues (indicated, as in Fig. 1) during D/L or during D/D. Symbols (±se; number of repeats are in parentheses) are mean transcript levels in various leaf parts. Where not seen, the error is smaller than the symbol (except in Spick2 leaf, 07 h: a single sample). *, Transcript levels significantly higher than in (at least one) other sample(s) in the same tissue. See also a summary in Table II. Horizontal bars: white, day; black, night; hatched, subjective day; hatched gray, subjective night. Abscissa, Abbreviated hours (as in Fig. 3) and, beneath, in the 2nd line, time count in D/D starting with the last lights off signal at the end of a normal day. B, Whole pulvinus: an additional (single) series of normalized mRNA signals obtained from a whole pulvinus total RNA probed sequentially with three channel probes (see “Materials and Methods” for details).