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Land plants have evolved adaptive regulatory mechanisms enabling the survival of environmental stresses associated with terrestrial life. Here, we focus on the evolution of the regulatory CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) component of the ethylene signaling pathway that modulates stress-related changes in plant growth and development. First, we compare CTR1-like proteins from a bryophyte, Physcomitrella patens (representative of early divergent land plants), with those of more recently diverged lycophyte and angiosperm species (including Arabidopsis [Arabidopsis thaliana]) and identify a monophyletic CTR1 family. The fully sequenced P. patens genome encodes only a single member of this family (PpCTR1L). Next, we compare the functions of PpCTR1L with that of related angiosperm proteins. We show that, like angiosperm CTR1 proteins (e.g. AtCTR1 of Arabidopsis), PpCTR1L modulates downstream ethylene signaling via direct interaction with ethylene receptors. These functions, therefore, likely predate the divergence of the bryophytes from the land-plant lineage. However, we also show that PpCTR1L unexpectedly has dual functions and additionally modulates abscisic acid (ABA) signaling. In contrast, while AtCTR1 lacks detectable ABA signaling functions, Arabidopsis has during evolution acquired another homolog that is functionally distinct from AtCTR1. In conclusion, the roles of CTR1-related proteins appear to have functionally diversified during land-plant evolution, and angiosperm CTR1-related proteins appear to have lost an ancestral ABA signaling function. Our study provides new insights into how molecular events such as gene duplication and functional differentiation may have contributed to the adaptive evolution of regulatory mechanisms in plants., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

Phosphate (Pi) is a macronutrient that is essential for plant growth and development. However, the low mobility of Pi impedes uptake, thus reducing availability. Accordingly, plants have developed physiological strategies to cope with low Pi availability. Here, we report that the characteristic Arabidopsis thaliana Pi starvation responses are in part dependent on the activity of the nuclear growth-repressing DELLA proteins (DELLAs), core components of the gibberellin (GA)-signaling pathway. We first show that multiple shoot and root Pi starvation responses can be repressed by exogenous GA or by mutations conferring a substantial reduction in DELLA function. In contrast, mutants having enhanced DELLA function exhibit enhanced Pi starvation responses. We also show that Pi deficiency promotes the accumulation of a green fluorescent protein-tagged DELLA (GFP-RGA [repressor of ga1-3]) in root cell nuclei. In further experiments, we show that Pi starvation causes a decrease in the level of bioactive GA and associated changes in the levels of gene transcripts encoding enzymes of GA metabolism. Finally, we show that the GA-DELLA system regulates the increased root hair length that is characteristic of Pi starvation. In conclusion, our results indicate that DELLA-mediated signaling contributes to the anthocyanin accumulation and root architecture changes characteristic of Pi starvation responses, but do not regulate Pi starvation-induced changes in Pi uptake efficiency or the accumulation of selected Pi starvation-responsive gene transcripts. Pi starvation causes a reduction in bioactive GA level, which, in turn, causes DELLA accumulation, thus modulating several adaptively significant plant Pi starvation responses.

Plant morphogenesis is profoundly influenced by light (a phenomenon known as photomorphogenesis). For example, light inhibits seedling hypocotyl growth via activation of phytochromes and additional photoreceptors. Subsequently, information is transmitted through photoreceptor-linked signal transduction pathways and used (via previously unknown mechanisms) to control hypocotyl growth. Here we show that light inhibition of Arabidopsis (Arabidopsis thaliana) hypocotyl growth is in part dependent on the DELLAs (a family of nuclear growth-restraining proteins that mediate the effect of the phytohormone gibberellin [GA] on growth). We show that light inhibition of growth is reduced in DELLA-deficient mutant hypocotyls. We also show that light activation of phytochromes promotes the accumulation of DELLAs. A green fluorescent protein (GFP)-tagged DELLA (GFP-RGA) accumulates in elongating cells of light-grown, but not dark-grown, transgenic wild-type hypocotyls. Furthermore, transfer of seedlings from light to dark (or vice versa) results in rapid changes in hypocotyl GFP-RGA accumulation, changes that are paralleled by rapid alterations in the abundance in hypocotyls of transcripts encoding enzymes of GA metabolism. These observations suggest that light-dependent changes in hypocotyl GFP-RGA accumulation are a consequence of light-dependent changes in bioactive GA level. Finally, we show that GFP accumulation and quantitative modulation of hypocotyl growth is proportionate with light energy dose (the product of exposure duration and fluence rate). Hence, DELLAs inhibit hypocotyl growth during the light phase of the day-night cycle via a mechanism that is quantitatively responsive to natural light variability. We conclude that DELLAs are a major component of the adaptively significant mechanism via which light regulates plant growth during photomorphogenesis.

The Arabidopsis hypocotyl, together with hormone mutants and chemical inhibitors, was used to study the role of auxin in cell elongation and its possible interactions with ethylene and gibberellin. When wild-type Arabidopsis seedlings were grown on media containing a range of auxin concentrations, hypocotyl growth was inhibited. However, when axr1-12 and 35S-iaaL (which have reduced auxin response and levels, respectively) were grown in the same conditions, auxin was able to promote hypocotyl growth. In contrast, auxin does not promote hypocotyl growth of axr3-1, which has phenotypes that suggest an enhanced auxin response. These results are consistent with the hypothesis that auxin levels in the wild-type hypocotyl are optimal for elongation and that additional auxin is inhibitory. When ethylene responses were reduced using either the ethylene-resistant mutant etr1 or aminoethoxyvinylglycine, an inhibitor of ethylene synthesis, auxin responses were unchanged, indicating that auxin does not inhibit hypocotyl elongation through ethylene. To test for interactions between auxin and gibberellin, auxin mutants were grown on media containing gibberellin and gibberellin mutants were grown on media containing auxin. The responses were found to be the same as wild-type Arabidopsis seedlings in all cases. In addition, 1 microM of the auxin transport inhibitor 1-naphthylphthalmic acid does not alter the response of wild-type seedlings to gibberellin. Double mutants were made between gibberellin and auxin mutants and the phenotypes of these appear additive. These results indicate that auxin and gibberellin are acting independently in hypocotyl elongation. Thus auxin, ethylene, and gibberellin each regulate hypocotyl elongation independently.

Active gibberellins (GAs) are endogenous factors that regulate plant growth and development in a dose-dependent fashion. Mutant plants that are GA deficient, or exhibit reduced GA responses, display a characteristic dwarf phenotype. Extragenic suppressor analysis has resulted in the isolation of Arabidopsis mutations, which partially suppress the dwarf phenotype conferred by GA deficiency and reduced GA-response mutations. Here we describe detailed studies of the effects of two of these suppressors, spy-7 and gar2-1, on several different GA-responsive growth processes (seed germination, vegetative growth, stem elongation, chlorophyll accumulation, and flowering) and on the in planta amounts of active and inactive GA species. The results of these experiments show that spy-7 and gar2-1 affect the GA dose-response relationship for a wide range of GA responses and suggest that all GA-regulated processes are controlled through a negatively acting GA-signaling pathway.

The gibberellins (GAs) are a complex family of diterpenoid compounds, some of which are potent endogenous regulators of plant growth. As part of a feedback control of endogenous GA levels, active GAs negatively regulate the abundance of mRNA transcripts encoding GA biosynthesis enzymes. For example, Arabidopsis GA4 gene transcripts encode GA 3beta-hydroxylase, an enzyme that catalyzes the conversion of inactive to active GAs. Here we show that active GAs regulate GA4 transcript abundance in a dose-dependent manner, and that down-regulation of GA4 transcript abundance is effected by GA4 (the product of 3beta-hydroxylation) but not by its immediate precursor GA9 (the substrate). Comparison of several different GA structures showed that GAs active in promoting hypocotyl elongation were also active in regulating GA4 transcript abundance, suggesting that similar GA:receptor and subsequent signal transduction processes control these two responses. It is interesting that these activities were not restricted to 3beta-hydroxylated GAs, being also exhibited by structures that were not 3beta-hydroxylated but that had another electronegative group at C-3. We also show that GA-mediated control of GA4 transcript abundance is disrupted in the GA-response mutants gai and spy-5. These observations define a sensitive homeostatic mechanism whereby plants may regulate their endogenous GA levels.

Plant growth and development are regulated by numerous internal and external factors. Among these, gibberellin (GA) (an endogenous plant growth regulator) and phytochrome (a photoreceptor) often influence the same processes. For example, in plants grown in the light Arabidopsis thaliana hypocotyl elongation is reduced by GA deficiency and increased by phytochrome deficiency. Here we describe experiments in which the phenotypes of Arabidopsis plants doubly homozygous for GA-related and phytochrome-related mutations were examined. The double mutants were studied at various stages in the plant life cycle, including the seed germination, young seedling, adult, and reproductive phases of development. The results of these experiments are complex, but indicate that a fully functional GA system is necessary for full expression of the elongated phenotypes conferred by phytochrome deficiency.

Several aspects of the photophysiology of wild-type Arabidopsis thaliana seedlings were compared with those of a phytochrome A null mutant, phyA-1, and a mutant, fhy1, that is putatively involved in the transduction of light signals from phytochrome A. Although phyA seedlings display a near wild-type phenotype when grown in white light (W), they nevertheless display several photomorphogenic abnormalities. Thus, whereas the germination of wild-type and fhy1 seeds is almost fully promoted by a pulse of red light (R) or by continuous far-red light (FR), phyA seed germination is responsive only to R. Following growth under day/night cycles, but not under continuous W, the hypocotyls of light-grown phyA and fhy1 seedlings are more elongated than those of wild-type seedlings. For seedlings grown under low red/far-red (R/FR) ratio light conditions, phyA and fhy1 seedlings display a more marked promotion of hypocotyl elongation than wild-type seedlings. Similarly, seedlings that are doubly null for phytochrome A and phytochrome B(phyA phyB) also have more elongated hypocotyls under low R/FR ratio conditions than phyB seedlings. This indicates that phytochrome A action in light-grown seedlings is antagonistic to the action of phytochrome B. Although wild-type, fhy1, and phyA seedlings flower at essentially the same time under both short-day and long-day conditions, an obvious consequence of phytochrome A deficiency is a pronounced late flowering under conditions where a short day of 8 h of fluorescent W is extended by 8 h of low-fluence-rate incandescent light. The evidence thus indicates that phytochrome A plays a role in seed germination, in the control of elongation growth of light-grown seedlings, and in the perception of daylength.