Phytochrome A, phytochrome B and HY4 are involved in hypocotyl growth responses to natural radiation in Arabidopsis: weak de-etiolation of the phyA mutant under dense canopies

The roles of phytochrome A (phyA), phytochrome B (phyB) and a putative blue-light (BL) photoreceptor (HY4) in the control of hypocotyl growth by natural radiation were investigated using phyA, phyB and hy4 mutants of Arabidopsis thaliana. Full sunlight inhibited hypocotyl growth to a larger extent in wild-type (WT) than in phyA, phyB and, particularly, hy4 seedlings. In WT seedlings, hypocotyl growth was promoted by selectively lowering BL irradiance, lowering red-light (R) plus far-red-light (FR) irradiance or lowering the R/FR ratio (which was achieved either by increasing FR or by reducing R). The effects of lowering BL were reduced in hy4 and exaggerated in phyA seedlings. The effects of lowering R+FR were reduced in phyA and exaggerated in hy4 seedlings. Neither phyB nor hy4 mutants responded to low R/FR ratios. Neighbouring plants reflecting FR without shading caused subtle reductions of the R/FR ratio. This signal promoted hypocotyl growth in WT but not in phyA, phyB or hy4 seedlings. Intermediate canopy shade produced similar effects in all genotypes. Under deep shade, de-etiolation was severely impaired in phyA seedlings, which died prematurely. Thus, the FR ‘high-irradiance reaction’ mediated by phyA could be important for seedling survival under dense canopies.     

The Shade Avoidance Syndrome in Arabidopsis: The Antagonistic Role of Phytochrome A and B Differentiates Vegetation Proximity and Canopy Shade

Light limitation caused by dense vegetation is one of the greatest threats to plant survival in natural environments. Plants detect such neighboring vegetation as a reduction in the red to far-red ratio (R:FR) of the incoming light. The low R:FR signal, perceived by phytochromes, initiates a set of responses collectively known as the shade avoidance syndrome, intended to reduce the degree of current or future shade from neighbors by overtopping such competitors or inducing flowering to ensure seed production. At the seedling stage these responses include increased hypocotyl elongation. We have systematically analyzed the Arabidopsis seedling response and the contribution of phyA and phyB to perception of decreased R:FR, at three different levels of photosynthetically active radiation. Our results show that the shade avoidance syndrome, induced by phyB deactivation, is gradually antagonized by phyA, operating through the so-called FR-High Irradiance Response, in response to high FR levels in a range that simulates plant canopy shade. The data indicate that the R:FR signal distinguishes between the presence of proximal, but non-shading, neighbors and direct foliar shade, via a intrafamily photosensory attenuation mechanism that acts to suppress excessive reversion toward skotomorphogenic development under prolonged direct vegetation shade.     

A genome-wide association study identifies variants underlying the Arabidopsis thaliana shade avoidance response

Shade avoidance is an ecologically and molecularly well-understood set of plant developmental responses that occur when the ratio of red to far-red light (R:FR) is reduced as a result of foliar shade. Here, a genome-wide association study (GWAS) in Arabidopsis thaliana was used to identify variants underlying one of these responses: increased hypocotyl elongation. Four hypocotyl phenotypes were included in the study, including height in high R:FR conditions (simulated sun), height in low R:FR conditions (simulated shade), and two different indices of the response of height to low R:FR. GWAS results showed that variation in these traits is controlled by many loci of small to moderate effect. A known PHYC variant contributing to hypocotyl height variation was identified and lists of significantly associated genes were enriched in a priori candidates, suggesting that this GWAS was capable of generating meaningful results. Using metadata such as expression data, GO terms, and other annotation, we were also able to identify variants in candidate de novo genes. Patterns of significance among our four phenotypes allowed us to categorize associations into three groups: those that affected hypocotyl height without influencing shade avoidance, those that affected shade avoidance in a height-dependent fashion, and those that exerted specific control over shade avoidance. This grouping allowed for the development of explicit hypotheses about the genetics underlying shade avoidance variation. Additionally, the response to shade did not exhibit any marked geographic distribution, suggesting that variation in low R:FR-induced hypocotyl elongation may represent a response to local conditions.     

Characterization of shade avoidance responses in Lotus japonicus

Sessile plants must continuously adjust their growth and development to optimize photosynthetic activity under ever-fluctuating light conditions. Among such light responses in plants, one of the best-characterized events is the so-called shade avoidance, for which a low ratio of the red (R):far-red (FR) light intensities is the most prominent stimulus. Such shade avoidance responses enable plants to overtop their neighbors, thereby enhancing fitness and competitiveness in their natural habitat. Considerable progress has been achieved during the last decade in understanding the molecular mechanisms underlying the shade avoidance responses in the model rosette plant, Arabidopsis thaliana. We characterize here the fundamental aspects of the shade avoidance responses in the model legume, Lotus japonicus, based on the fact that its phyllotaxis (or morphological architecture) is quite different from that of A. thaliana. It was found that L. japonicus displays the characteristic shade avoidance syndrome (SAS) under defined laboratory conditions (a low R:FR ratio, low light intensity, and low blue light intensity) that mimic the natural canopy. In particular, the outgrowth of axillary buds (i.e., both aerial and cotyledonary shoot branching) was severely inhibited in L. japonicus grown in the shade. These results are discussed with special emphasis on the unique aspects of SAS observed with this legume.     

Interaction of red to far red light ratio and ethylene in regulating stem elongation of <Emphasis Type="Italic">Helianthus annuus</Emphasis>

Sunflower (Helianthus annuus L.) stems showed increased elongation under two types of vegetative shade: canopy shade (low red to far red [R/FR] ratio) and neighbouring proximity shade (FR enrichment). Hypocotyls also elongated more under narrow-band FR light than under narrow-band R light. Ethylene levels were determined in actively elongating 7-day-old hypocotyls and 17-day-old internodes under three R/FR ratios. Ethylene levels were lower in both sunflower hypocotyls and internodes when the R/FR ratio was reduced. Both FR enrichment of normal R/FR ratio and narrow-band FR light with very low light irradiance resulted in reduction in ethylene levels in 7-day-old hypocotyls. Further, in application experiments, sunflower stems grown under low R/FR ratio were more sensitive to ethephon and less sensitive to aminoethoxyvinylglycine (AVG) than stems grown under high R/FR ratio. Low R/FR ratio appears to initiate reduction in ethylene levels in sunflower seedlings, allowing maximum stem elongation. These results, and findings of other authors, suggest that various plant species may have developed different ways of regulating stem elongation and ethylene levels in response to low R/FR ratio.     

Impaired phytochrome-mediated shade-avoidance responses in the aurea mutant of tomato

Internode extension-growth responses to neighbouring plants and to red to far-red ratios (R:FR) were investigated in wild-type (WT) and aurea (au)-mutant seedlings of tomato grown under natural radiation. The genomic location of the au mutant is not known, but one of its consequences is the reduced phytochrome level. In WT seedlings, internode growth was promoted by the presence of non-shading neighbours reflecting far-red light (FR), the shade of a tall canopy, FR provided as a supplement during the photoperiod, and FR pulses either provided at the end of the day or delayed into the dark period. Supplementary FR during the photoperiod also promoted growth in herbicide-treated partially bleached WT seedlings. The au mutant showed higher background extension-growth rates, but only responded to the most severe treatments: deep shade light and very low R:FR at the end of the day, i.e. au-mutant seedlings were less sensitive than WT seedlings to R:FR signals. Wild-type seedlings were transferred from the glasshouse to a growth room and exposed to white light with two levels of phytochrome-absorbable radiation but similar phytochrome photoequilibria and radiation for photosynthesis. The plants exposed to the lowest level showed a transient increase of internode extension growth rate and a simultaneous reduction of response to FR pulses, i.e. reproduced some of the features of au-mutant seedlings. Phytochrome itself could set the degree of response to Pfr during neighbour detection.     

Selected Components of the Shade-Avoidance Syndrome Are Displayed in a Normal Manner in Mutants of Arabidopsis thaliana and Brassica rapa Deficient in Phytochrome B

Several growth parameters associated with the phytochrome-mediated shade avoidance syndrome have been measured in seedlings and mature plants of a wild-type and a hy3 mutant of Arabidopsis thaliana deficient in phytochrome B. Growth parameters were compared in plants grown in either white light (high red:far-red [R:FR] ratio) or white light plus added far-red (FR) light (low R:FR ratio). Wild-type Arabidopsis exhibited increased hypocotyl and petiole extension under a low, compared with a high, R:FR ratio. The hy3 mutant did not respond to low R:FR ratio by increase in hypocotyl or petiole length. Extension growth of wild-type plants was stimulated by brief end-of-day FR pulses, but similar treatment had no effect on extension growth of hy3 mutant plants. However, some responses to low R:FR ratio seen in the wild-type plants were also evident in the hy3 mutants. The number of days to bolting, the developmental stage at bolting, the leaf area, and the specific stem weight (weight per unit of length) all decreased in the wild-type and hy3 seedlings in response to low R:FR ratio. Low R:FR ratio caused a larger decrease in leaf area and specific stem weight in the mutant seedlings than in wild-type seedlings. The effects of low R:FR ratio on leaf area and specific stem weight were opposite to those of the hy3 lesion, which resulted in increased leaf area and specific stem weight in comparison with the wild type. Both leaf area and specific stem weight responses to low R:FR ratio also were unchanged in the ein mutant of Brassica rapa, known to be deficient in phytochrome B. These responses represent components of the shade-avoidance syndrome, and, consequently, the results indicate that phytochrome B cannot be solely responsible for the perception of R:FR ratio and the induction of shade-avoidance responses. The hypothesis is proposed that different phytochromes may be responsible for the regulation of extension growth and the regulation of lateral or radial expansion.     

Nuclear localization and interaction with COP1 are required for STO/BBX24 function during photomorphogenesis

Arabidopsis (Arabidopsis thaliana) SALT TOLERANCE/B-BOX ZINC FINGER PROTEIN24 (STO/BBX24) is a negative regulator of the light signal transduction that localizes to the nucleus of plant cells and interacts with CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) in the yeast (Saccharomyces cerevisiae) two-hybrid system. The protein contains two B-box zinc-finger motives at the N terminus and a conserved motif at the C-terminal part required for the interaction with COP1. BBX24 accumulates during deetiolation of young seedlings in the first hours of exposure to light. However, this accumulation is transient and decreases after prolonged light irradiation. Here, we identified the amino acidic residues necessary for the nuclear import of the protein. In addition, we created mutated forms of the protein, and analyzed them by overexpression in the bbx24-1 mutant background. Our results indicate that the degradation of BBX24 occurs, or at least is initiated in the nucleus, and this nuclear localization is a prerequisite to fulfill its function in light signaling. Moreover, mutations in the region responsible for the interaction with COP1 revealed that a physical interaction of the proteins is also required for degradation of BBX24 in the light and for normal photomorphogenesis.     

Characterization of Shade Avoidance Responses in Lotus japonicus

Sessile plants must continuously adjust their growth and development to optimize photosynthetic activity under ever-fluctuating light conditions. Among such light responses in plants, one of the best-characterized events is the so-called shade avoidance, for which a low ratio of the red (R):far-red (FR) light intensities is the most prominent stimulus. Such shade avoidance responses enable plants to overtop their neighbors, thereby enhancing fitness and competitiveness in their natural habitat. Considerable progress has been achieved during the last decade in understanding the molecular mechanisms underlying the shade avoidance responses in the model rosette plant, Arabidopsis thaliana. We characterize here the fundamental aspects of the shade avoidance responses in the model legume, Lotus japonicus, based on the fact that its phyllotaxis (or morphological architecture) is quite different from that of A. thaliana. It was found that L. japonicus displays the characteristic shade avoidance syndrome (SAS) under defined laboratory conditions (a low R:FR ratio, low light intensity, and low blue light intensity) that mimic the natural canopy. In particular, the outgrowth of axillary buds (i.e., both aerial and cotyledonary shoot branching) was severely inhibited in L. japonicus grown in the shade. These results are discussed with special emphasis on the unique aspects of SAS observed with this legume.     

BBX32, an Arabidopsis B-Box protein, functions in light signaling by suppressing HY5-regulated gene expression and interacting with STH2/BBX21

A B-box zinc finger protein, B-BOX32 (BBX32), was identified as playing a role in determining hypocotyl length during a large-scale functional genomics study in Arabidopsis (Arabidopsis thaliana). Further analysis revealed that seedlings overexpressing BBX32 display elongated hypocotyls in red, far-red, and blue light, along with reduced cotyledon expansion in red light. Through comparative analysis of mutant and overexpression line phenotypes, including global expression profiling and growth curve studies, we demonstrate that BBX32 acts antagonistically to ELONGATED HYPOCOTYL5 (HY5). We further show that BBX32 interacts with SALT TOLERANCE HOMOLOG2/BBX21, another B-box protein previously shown to interact with HY5. Based on these data, we propose that BBX32 functions downstream of multiple photoreceptors as a modulator of light responses. As such, BBX32 potentially has a native role in mediating gene repression to maintain dark adaptation.     

The regulation of cell wall extensibility during shade avoidance: a study using two contrasting ecotypes of Stellaria longipes

Shade avoidance in plants involves rapid shoot elongation to grow toward the light. Cell wall-modifying mechanisms are vital regulatory points for control of these elongation responses. Two protein families involved in cell wall modification are expansins and xyloglucan endotransglucosylase/hydrolases. We used an alpine and a prairie ecotype of Stellaria longipes differing in their response to shade to study the regulation of cell wall extensibility in response to low red to far-red ratio (R/FR), an early neighbor detection signal, and dense canopy shade (green shade: low R/FR, blue, and total light intensity). Alpine plants were nonresponsive to low R/FR, while prairie plants elongated rapidly. These responses reflect adaptation to the dense vegetation of the prairie habitat, unlike the alpine plants, which almost never encounter shade. Under green shade, both ecotypes rapidly elongate, showing that alpine plants can react only to a deep shade treatment. Xyloglucan endotransglucosylase/hydrolase activity was strongly regulated by green shade and low blue light conditions but not by low R/FR. Expansin activity, expressed as acid-induced extension, correlated with growth responses to all light changes. Expansin genes cloned from the internodes of the two ecotypes showed differential regulation in response to the light manipulations. This regulation was ecotype and light signal specific and correlated with the growth responses. Our results imply that elongation responses to shade require the regulation of cell wall extensibility via the control of expansin gene expression. Ecotypic differences demonstrate how responses to environmental stimuli are differently regulated to survive a particular habitat.     

The interaction of light quality and irradiance with gibberellins, cytokinins and auxin in regulating growth of Helianthus annuus hypocotyls

A reduced red to far-red (R/FR) light ratio and low photosynthetically active radiation (PAR) irradiance are both strong signals for inducing etiolation growth of plant stems. Under natural field conditions, plants can be exposed to either a reduced R/FR ratio or lower PAR, or to a combination of both. We used Helianthus annuus L., the sunflower, to study the effect of reduced R/FR ratio, low PAR or their combination on hypocotyl elongation. To accomplish this, we attempted to uncouple light quality from light irradiance as factors controlling hypocotyl elongation. We measured alterations in the levels of endogenous gibberellins (GAs), cytokinins (CKs) and the auxin indole-3-acetic acid (IAA), and the effect of exogenous hormones on hypocotyl growth. As expected, both reduced R/FR ratio and lower PAR can significantly promote sunflower hypocotyl elongation when given separately. However, providing the reduced R/FR ratio at a low PAR resulted in the greatest hypocotyl growth, and this was accompanied by significantly higher levels of endogenous IAA, GA1, GA8, GA20 and of a wide range of CKs. Providing a reduced R/FR ratio under normal PAR also significantly increased growth and again gave significantly higher levels of endogenous IAA, GAs and CKs. However, only under the de-etiolating influence of a normal R/FR ratio did lowering PAR significantly increase levels of GA1, GA8 and GA20. We thus conclude that light quality (e.g. the R/FR ratio) is the most important component of shade for controlling hypocotyl growth and elevated growth hormone content.