Journal of Integrative Agriculture
Advanced Online Publication: 2013
Doi: 10.1016/S2095-3119(13)60607-3
Phytochrome D is Involved in Red Light-Induced Negative Gravitropism in
Arabidopsis thaliana
*
LI Jian-ping1, 2 , HOU Pei1, ZHEN Xu1, SONG Mei-fang1, SU Liang1 and YANG Jian-ping1
1
Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R.China
2
Institute of Nuclear and Biotechnology, Xinjiang Academy of Agricultural Sciences. Urumqi 830091, P.R.China
Abstract
The phytochrome gene family, which in most dicot plants consists of phytochromes A-E (phyA-phyE),
regulates plant responses to ambient light environments.
PhyA and phyB have been characterized in detail, but
studies on phyC-phyE have reported discrepant functions.
In this study, we show that phyD regulates the
gravitropic response of Arabidopsis thaliana by inhibiting gravitropic growth of hypocotyls grown under red light.
PhyD had only a limited effect on the gravitropic growth of roots.
PhyD also enhanced phyB-regulated
gravitropic responses in hypocotyls. Moreover, the regulation of hypocotyl gravitropic responses by phyD was
dependent upon the red light fluence rate.
Key words: phytochrome D, gravitropism, Arabidopsis thaliana1
INTRODUCTION
In response to the detection of various environmental signals such as light, gravity, touch, and humidity, the
absorption of water and nutrients and the rate of photosynthesis are altered in plants, resulting in a change in plant
growth (Miyo 2010).
Light is an important environmental influence on plant growth and development; plant
photoreceptors detect the quality and direction of light, and plant growth is adjusted in response. Phytochromes
are the primary photoreceptors involved in the regulation of red/far-red light-induced responses. Arabidopsis has
five phytochromes, phyA to phyE.
Phylogenetic analysis suggests that these phytochromes can be clustered into
four subfamilies: phyA, phyB/D, phyC, and phyE (Sharrock and Clack 2003). PhyA, a light-liable phytochrome,
mediates responses to far-red light; phyB is light stable and is dominant in responses to red light (Franklin et al.
2003; Monte et al. 2003). Within the light-stable-phytochrome family, phyB, phyD, and phyE act redundantly to
control multiple physiological responses, including leaf expansion, hypocotyl development, and flowering time
(Franklin et al. 2003).
Gravity is an important environmental cue for modulating the directional growth of plants. Generally, plant
shoots grow upward (i.e., negative gravitropism), and roots grow downward (i.e., positive gravitropism) (Briggs
and Christie 2002; Blancaflor and Masson 2003) tropic responses. Both gravity and light can affect the gravitropic
growth of plants. For example, dark-grown hypocotyls of Arabidopsis grow upward, but seedlings grown in red
or far-red light grow in random directions (Liscum and Hangarter 1993; Poppe et al. 1996; Robson and Smith,
LI Jian-ping, E-mail: [email protected]; Correspondence YANG Jian-ping, Tel/Fax: +86-10-82105859, E-mail:
[email protected]
1996; Hennig et al. 2002). Red and far-red light can also influence gravitropic responses in roots (Lu et al. 1997,
Takano et al. 2001).
Thus, gravitropic responses in plants are modulated by signals from light-activated
photoreceptors (Whippo and Hangarter 2003; Blakeslee et al. 2004), and roles in light-regulated gravitropism
have been identified for several phytochromes.
PhyA is the primary photoreceptor involved in far-red
light-regulated gravitropic responses of roots and hypocotyls in Arabidopsis (Parks and Spalding 1996; Hennig et
al. 2002). Both phyA and phyB influence the gravitropic responses of maize roots and Arabidopsis hypocotyls
(Liscum and Hangarter 1993; Lu et al. 1996; Poppe et al. 1996; Robson and Smith 1996; Parks and Spalding
1999). PhyC participates in the regulation of negative gravitropic and phototropic responses of hypocotyls
(Kumar et al. 2008).
In this study we compared the gravitropic responses of phytochrome mutants and wild-type (WT) Arabidopsis
thaliana plants to explore the role of phyD in root and hypocotyl gravitropic responses and to discover how
phytochromes inhibit hypocotyl negative gravitropism.
RESULTS
Phytochrome D mediates red light-regulated hypocotyl gravitropism
To determine the role of phyD in gravitropism, we conducted a time-course analysis of root and hypocotyl growth.
We compared the gravitropic curvatures of roots and hypocotyls from phyD single mutant seedlings with those of
roots and hypocotyls from WT plants (Landsberg erecta (Ler) background) by re-orientating Petri dishes from the
vertical to the horizontal position under red light (20.0 μE.m-2.S-1).
There were no significant differences in
gravitropism between the roots of the phyD mutant and the WT plant roots, with the exception of the 15-h time
point (Fig. 1-A).
In contrast, the gravitropic curvature of hypocotyls was inhibited in red light-grown phyD
seedlings relative to WT seedlings, especially at 3 and 24 h (Fig. 1-B).
Fig. 1 Curvature of roots and hypocotyls in wild-type (Ler) and phyD mutant seedlings of Arabidopsis thaliana
grown under red light (20.0 μE m-2 S-1).
C, hypocotyls.
A, analysis of RT-PCR and Western blot of phyD mutant.
Time-course studies following 90° re-orientation of the Petri dishes.
standard deviations.
B, roots.
Error bars indicate
Time points of mutants that show a significant difference (P<0.05) relative to the WT at the
corresponding time points are indicated *.
The same as below.
PhyB and phyD synergistically regulate the hypocotyl gravitropic response under red light
Previous studies have demonstrated interactions between phytochrome signalling and gravitropic responses and
have suggested that phyB plays a predominant role in the regulation of gravitropic growth (Poppe et al. 1996;
Robson and Smith 1996; Kumar et al. 2008). Given that phyB and phyD are closely related phylogenetically,
we speculated that they interact in controlling gravitropic growth of Arabidopsis. First, we investigated the
gravitropic response of WT plants and phyB, phyD, and phyB phyD mutant plants grown under red light by
determining the growth orientation of roots and hypocotyls as the degree from vertical (Fig. 2-A-E). The growth
angles from vertical were markedly less for the roots of the phyB and phyB phyD mutants compared with the
angles for the phyD mutant and WT roots (Fig. 2-B, C, D, E; dark), and the root angles did not differ between WT
and phyD (Fig. 2-B, C; dark).
These results are consistent with previous studies (Liscum and Hangarter 1993;
Correll et al. 2003, 2005) and suggest an important role of phyB in root gravitropic responses. Hypocotyl
gravitropism showed similar results, except there was also a difference between the angles of phyD and WT
hypocotyls (Fig. 2-B and C; grey).
Moreover, a slight yet significant decrease was observed in the angle from
vertical for the hypocotyls of phyB phyD seedlings compared with phyB seedlings (Fig. 2-D and E; grey).
Fig. 2 Scatter diagram of hypocotyl and root orientation with respect to the gravity vector for wild-type (Ler)
and mutant Arabidopsis thaliana seedlings (n=160) that were grown vertically under red light (20.0 μE m-2 S-1).
To confirm these results and further understand the kinetics of hypocotyl gravitropism, we performed
time-course studies by determining the change in curvature of light-grown seedlings stimulated by re-orientating
the Petri dishes by 90°in red light.
As expected, there was no difference in root curvature between phyD and
WT, whereas the roots of phyB and phyB phyD had less curvature than those of phyD and WT (Fig. 3-A).
The
curvature of the hypocotyls was significantly less in both phyB and phyB phyD compared with WT, with
significant reductions in curvature at 9, 12, 15, and 24 h in phyB and at 3, 9, 12, 15, and 24 h in phyB phyD (Fig.
3-B).
There was a slight difference in hypocotyl curvature between phyD and WT at 3, 9 and 24 h (Fig. 3-B).
Fig. 3 Curvature of roots and hypocotyls of wild-type (Ler) and phyB, phyD, and phyB phyD mutant seedlings
of Arabidopsis thaliana grown under red light (20.0 μE m-2 S-1).
re-orientation of Petri dishes (n≥20 for each time point).
A, time-course of root curvature after 90°
B, time-course of hypocotyl curvature after 90°
re-orientation of Petri dishes (n≥20 for each time point).
Light fluence rates affect the gravitropic response in hypocotyls
Light has been proven to be an important factor in gravitropic growth through interactions with phytochrome
signalling. In previous studies, gravitropism was shown to be impaired by red light (Britz and Galston 1982;
Woitzik and Mohr 1988). To characterize the effect of light, we measured a fluence rate response curve.
Arabidopsis WT and phyB, phyD and phyB phyD mutants were grown in continuous red light at fluence rates of 0
(darkness), 5.0, 20.0, 50.0, 100.0, and 200.0 μE m-2 S-1 for 24 h. With increases in the red light fluence rate from
5.0 to 20.0 to 50.0 μE m-2 S-1, the curvature of the hypocotyls accelerated.
curvature was observed at 100 and 200 μE m-2 S-1.
However, reduced acceleration of the
The loss of phyB in Arabidopsis resulted in continuous
inhibition of gravitropism in the hypocotyl at all fluence rates, and phyD acted synergistically with phyB at a
fluence rate of 20.0 μE m-2 S-1.
Interestingly, a significant difference in gravitropic growth between the phyD
mutant and WT was found only at a fluence rate of 20.0 μE m-2 S-1.
These results indicate that the effect of red
light on hypocotyl gravitropism is fluence rate-dependent.
Fig. 4 Effect of fluence rate on red light-regulated gravitropic growth of hypocotyls. Wild-type (Ler) and phyB,
phyD, and phyB phyD mutant seedlings of Arabidopsis thaliana were grown on vertically orientated plates under
red light of different fluence rates (0, 5.0, 20.0, 50.0, 100.0, and 250 μE m-2 S-1) for 24 h. The hypocotyl curvature
was measured and plotted versus the fluence rate.
DISCUSSION
Phytochromes have been shown to play a role in gravitropism (Liscum and Hangarter 1993). Both phyA and
phyB are involved in controlling the gravitropic response of maize roots and the gravitropic orientation and
inhibition of elongation of Arabidopsis hypocotyls (Poppe et al. 1996; Robson and Smith 1996; Lu and Feldman
1997; Parks and Spalding 1999).
Most of these previous studies were based on observations of mutant
phenotypes. The present study used a single phyD mutant and a double phyB phyD mutant.
The results for the
curvature of both roots and hypocotyls in these mutants revealed that phyD attenuated negative gravitropic growth
in hypocotyls under red light and that phyB and phyD may act synergistically to regulate this response. Recent
studies have also shown that both phyC and phyD mediate positive phototropism in hypocotyls and stems under
blue light (Correll et al. 2003) and inhibit red light-based positive phototropism in roots (Correll et al. 2005).
PhyB and phyE have been reported to attenuate gravitropism in inflorescence and stems in Arabidopsis (Kumar
and Kiss 2006). Thus, the entire phytochrome family appears to be involved the regulation of tropic responses,
and phytochromes may interact with each other or with other photoreceptors such as cryptochromes to regulate
tropic responses.
Evidences have shown that phyD plays appurtenant role in the inhibition of hypocotyl elongation (Franklin et al.
2003) while root elongation from phyD were similar to WT for both darkand light-grown seedlings (Correll et al
2005). In our studies, we also demonstrated that phyD plays a role in hypocotyl gravitropic responses but not in
root. Although the inhibition of gravitropism by light is one of the less-understood light responses and it is not
also clear how phytochromes inhibit gravitropism, based on previous studies (Correll et al. 2005, Kumar et al.
2008), phytochromes (phyA and phyB especially) may directly be involved in regulating the inhibition of
hypocotyl elongation and gravitropism but may have not a direct effect of modulating the root gravity signaling
cascade through the elongation process. In addition, the crosstalk of the various auxins and phytochormes also
determine the elongation processes and tropisms (Marchant et al. 1999, Nagashima et al. 2008, Tajagasgu et al.
2009).
CONCLUSION
By analysing the phenotypes of phyB, phyD, and phyB phyD mutants of Arabidopsis, we demonstrated that phyD
is involved in the inhibition of hypocotyl gravitropic responses regulated by red light. Under red light, root growth
in response to gravity did not differ significantly between WT and phyD, whereas the curvature of phyD
hypocotyls was less than that of WT. Comparisons of the angles from vertical among the roots and hypocotyls of
the phyB, phyD, and phyB phyD mutants highlighted the role of phyD in red light-regulated gravitropic growth.
PhyD also enhanced phyB-regulated gravitropic responses in hypocotyls, which was confirmed by the time course
of hypocotyl curvature. The present results show that phytochromes regulate gravitropic responses in hypocotyls
under red light in a fluence rate-dependent manner.
MATERIALS AND METHODS
Plant materials and growth conditions
The Landsberg erecta ecotype of Arabidopsis thaliana was used in these studies. To generate the wild-type and
phyD mutant near-isogenic lines used here, the Ws (phyD-1) line was crossed to the La-er wild type.
And a
heterozygous phyD-1/+ backcross one (BC1) F1 plant was identified by PCR. A heterozygous phyD-1/+ BC7 F1
plant was selfed and F2 plants were screened by PCR to identify homozygous lines.
Experiments were
performed using two independent BC7 F3 or F4 seeds. BC7 F2 plants with homozygous genotype phyB phyD
were identified and used in these experiments.
Seeds were surface sterilized in 30% bleach with 0.01% (v/v) Triton X-100 for 15 min, rinsed five times in
sterile double-distilled water, and stored in water at 4°C for 3 d.
Then the seeds were sown on Murashige and
Skoog medium with 1% (w/v) sucrose, 0.05% MES (pH 5.7), and 0.9% (w/v) agar in square Petri dishes (100×15
mm) and irradiated with white light for 24 h to promote seed germination.
Gravitropism experiments
To test the gravitropic responses of the seedlings, the germination-induced plates were placed vertically in red
light (20.0 μE m-2 S-1) and seedlings grew along the surface of the agar for 96 h at 22°C.
Seedlings were
photographed and the angle of growth away from gravity was measured relative to the vertical position for
hypocotyls and roots using Image J program (Fukaki H et al. 1996). To measure the effect of light on hypocotyl
and root gravitropic curvature, the germination-induced dishes were grown vertically under darkness for 4 d, and
then the seedlings were transferred to new assay plates, which were placed in darkness or red light and rotated 90°.
The seedlings were photographed at 0, 1, 2, 3, 6, 9, 12, 15, and 24 h of unilateral light, respectively.
Images were digitally captured and analyzed using the image analysis program ImageJ (ver. 1.41). Curvature
was defined as the change in angle from the starting point. Organs that curved in the direction of the gravity
vector were assigned positive angles, and organs that curved away from the direction of the gravity vector were
assigned negative angles (Kiss et al. 1996). Experiments were repeated three times and values are reported as
the mean±SE.
Acknowledgements
This work was financially supported by funds from the Genetically Modified Organisms Breeding Major Projects
of China (2011ZX08010-002), the National Natural Science Foundation of China (30871438 and 31170267) and
the Natural Science Foundation of Xinjiang (2012211B49).
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