Unanticipated regulatory roles for Arabidopsis phytochromes revealed by null mutant analysis

Abstract

In view of the extensive literature on phytochrome mutants in the Ler accession of Arabidopsis, we sought to secure a phytochrome-null line in the same genetic background for comparative studies. Here we report the isolation and phenotypic characterization of phyABCDE quintuple and phyABDE quadruple mutants in the Ler background. Unlike earlier studies, these lines possess a functional allele of FT permitting measurements of photoperiod-dependent flowering behavior. Comparative studies of both classes of mutants establish that phytochromes are dispensable for completion of the Arabidopsis life cycle under red light, despite the lack of a transcriptomic response, and also indicate that phyC is nonfunctional in the absence of other phytochromes. Phytochrome-less plants can produce chlorophyll for photosynthesis under continuous red light, yet require elevated fluence rates for survival. Unexpectedly, our analyses reveal both light-dependent and -independent roles for phytochromes to regulate the Arabidopsis circadian clock. The rapid transition of these mutants from vegetative to reproductive growth, as well as their insensitivity to photoperiod, establish a dual role for phytochromes to arrest and to promote progression of plant development in response to the prevailing light environment.

Fig. 1.

phyABCDE and phyABDE mutants are photomorphogenically similar. (A) Immunoblot analysis confirms the identities of phyAB(C)DE mutants; the weak phyC band of phyABDE is detected after long exposure (bottom blot). (B) White light-grown adult plants on soil under short-day conditions for 6 wk, from left to right: WT (Ler), phyB, phyAB, phyBCDE, phyABDE, and phyABCDE. (Scale bar, 2 cm.) (C) Rc50-grown, 5-wk-old adult plants on soil, the plant order is same as B (Scale bar, 1 cm.) (D) Hypocotyl lengths of 4-d-old seedlings grown in darkness or under 50 µmol⋅m⁻²⋅s⁻¹ fluence rate of continuous red (Rc), white (Wc), or blue (Bc) light (mean ± SEM, n = 30∼50). (E) Germination of phyAB(C)DE mutants vary and are promoted more effectively by GA₄₊₇ than by GA₃. Seeds were sown on phytagar plates with (MS+) or without MS salts (MS−) and supplied with or without 100 µM GA, stratified for 4 d, and then grown under Rc50 for 4 d before germination scoring. All mutant lines tested were independently grown and harvested; the two phyABDE lines are plotted in blue and the three phyABCDE lines in red.

Fig. 2.

phyAB(C)DE mutants can synthesize a low level of chlorophyll under red light. (A) Rc50-grown, 5-d-old phyAB(C)DE seedlings have a nearly etiolated phenotype with marginal greening; some seedlings have cotyledons fully enclosed by a seed coat. (B) Five-day-old phyAB(C)DE seedlings accumulate very low levels of chlorophyll (n = 3, SD is given for the peak value). (C) Dark-grown phyAB(C)DE seedlings can efficiently photoconvert dark-accumulated protochlorophyllide into chlorophyll(ide) after exposure to Rc50 for 15 min, similar to WT and phyAB (n = 3).

Fig. 3.

Transcriptomic analysis of phyAB(C)DE response to red light. (A) Venn diagram of red light responsive genes in 4-d-old WT, phyABDE, and phyABCDE (D, dark; Rc = 50 µmol⋅m⁻²⋅s⁻¹ red light). (B) Expression patterns (Rc vs. D) of the nine Rc responsive genes in phyABDE; white dots denote significantly differential expression, and asterisk indicates stress-responsive genes. (C) Expression levels of the two Rc-inducible genes in phyABCDE, At5g53710 and At5g15960/70(KIN1/2). Expression levels are normalized to WT-D of each gene; DR2 = 4-d darkness followed by 2 h of Rc50 exposure. *Statistical significance (adjusted P < 0.05) from the same genotype grown in the dark.

Fig. 4.

Flowering of phyAB(C)DE mutants is insensitive to photoperiod. The data are presented as mean with SEM (n = 20). LD, long-day conditions (16-h L/8-h D); SD, short-day conditions (8-h L/16-h D).

Fig. 5.

Expression response of ATHB2 (A) and PIL1 (B) to various light treatments. Three-week-old plants grown on soil under SD conditions (8-h L/16-h D) at 16 °C were transferred to darkness for 4 or 6 h, 4 h followed by 2 h of red light (30 µmol⋅m⁻²⋅s⁻¹), or 2 h of red plus far-red light (R:FR = 0.2) treatments. Expression levels are the means from three biological replicates ± SD.

Fig. 6.

Circadian rhythms in the phyAB(C)DE mutants. (A) Normalized bioluminescence of seedlings containing a pCCA1:LUC2 reporter construct. Plants were entrained to 12 L:12 D cycles for 6 d before being moved to 27 µmol⋅m⁻²⋅s⁻¹ Rc. Data presented for each line were normalized to the average bioluminescence over 72 h following background subtraction. (B) phyAB(C)DE mutants have a shorter period in comparison with WT at low fluence rates, but a longer period at higher red light fluence rates. Seedlings were entrained as in A before being moved to Rc at the indicated fluence rate. Error bars indicate SEM (n ≥ 6).