A role for ABCB19‐mediated polar auxin transport in seedling photomorphogenesis mediated by cryptochrome 1 and phytochrome B

During seedling establishment, blue and red light suppress hypocotyl growth through the cryptochrome 1 (cry1) and phytochrome B (phyB) photosensory pathways, respectively. How these photosensory pathways integrate with growth control mechanisms to achieve the appropriate degree of stem elongation was investigated by combining cry1 and phyB photoreceptor mutations with genetic manipulations of a multidrug resistance‐like membrane protein known as ABCB19 that influenced auxin distribution within the plant, as evidenced by a combination of reporter gene assays and direct auxin measurements. Auxin signaling and ABCB19 protein levels, hypocotyl growth rates, and apical hook opening were measured in mutant and wild‐type seedlings exposed to a range of red and blue light conditions. Ectopic/overexpression of ABCB19 (B19OE) greatly increased auxin in the hypocotyl, which reduced the sensitivity of hypocotyl growth specifically to blue light in long‐term assays and red light in high‐resolution, short‐term assays. Loss of ABCB19 partially suppressed the cry1 hypocotyl growth phenotype in blue light. Hypocotyl growth of B19OE seedlings in red light was very similar to phyB mutants. Altered auxin distribution in B19OE seedlings also affected the opening of the apical hook. The cry1 and phyB photoreceptor mutations both increased ABCB19 protein levels at the plasma membrane, as measured by confocal microscopy. The B19OE plant proved to be a useful tool for determining aspects of the mechanism by which light, acting through cry1 or phyB, influences the auxin transport process to control hypocotyl growth during de‐etiolation.     

Arabidopsis thaliana TERMINAL FLOWER2 is involved in light-controlled signalling during seedling photomorphogenesis

Plants respond to changes in the environment by altering their growth pattern. Light is one of the most important environmental cues and affects plants throughout the life cycle. It is perceived by photoreceptors such as phytochromes that absorb light of red and far-red wavelengths and control, for example, seedling de-etiolation, chlorophyll biosynthesis and shade avoidance response. We report that the terminal flower2 (tfl2) mutant, carrying a mutation in the Arabidopsis thaliana HETEROCHROMATIN PROTEIN1 homolog, functions in negative regulation of phytochrome dependent light signalling. tfl2 shows defects in both hypocotyl elongation and shade avoidance response. Double mutant analysis indicates that mutants of the red/far-red light absorbing phytochrome family of plant photoreceptors, phyA and phyB, are epistatic to tfl2 in far-red and red light, respectively. An overlap between genes regulated by light and by auxin has earlier been reported and, in tfl2 plants light-dependent auxin-regulated genes are misexpressed. Further, we show that TFL2 binds to IAA5 and IAA19 suggesting that TFL2 might be involved in regulation of phytochrome-mediated light responses through auxin action.     

The growth of tomato (Lycopersicon esculentum Mill.) hypocotyls in the light and in darkness differentially involves auxin.

Light and auxin antagonistically regulate hypocotyl elongation. We have investigated the physiological interactions of light and auxin in the control of tomato (Lycopersicon esculentum Mill.) hypocotyl elongation by studying the auxin-insensitive mutant diageotropica (dgt). The length of the hypocotyls of the dgt mutant is significantly reduced when compared to the wild type line Ailsa Craig (AC) in the dark and under red light, but not under the other light conditions tested, indicating that auxin sensitivity is involved in the elongation of hypocotyls only in these conditions. Similarly, the auxin transport inhibitor naphthylphthalamic [correction of naphtylphtalamic] acid (NPA) differentially affects elongation of dark- or light-grown hypocotyls of the MoneyMaker (MM) tomato wild type. Using different photomorphogenic mutants, we demonstrate that at least phytochrome A, phytochrome B1 and, to a much lesser extent [correction of extend], cryptochrome 1, are necessary for a switch from an auxin transport-dependent elongation of hypocotyls in the dark to an auxin transport-independent elongation in the light. Interestingly, the dgt mutant and NPA-treated seedlings exhibit a looped phenotype only under red light, indicating that the negative gravitropism of hypocotyls also differentially involves auxin in the various light conditions.     

Auxin, ethylene and brassinosteroids: tripartite control of growth in the Arabidopsis hypocotyl

Dark-grown Arabidopsis seedlings develop an apical hook by differential cell elongation and division, a process driven by cross-talk between multiple hormones. Auxins, ethylene and gibberellins interact in the formation of the apical hook. In the light, a similar complexity of hormonal regulation has been revealed at the level of hypocotyl elongation. Here, we describe the involvement of brassinosteroids (BRs) in auxin- and ethylene-controlled processes in the hypocotyls of both light- and dark-grown seedlings. We show that BR biosynthesis is necessary for the formation of an exaggerated apical hook and that either application of BRs or disruption of BR synthesis alters auxin response, presumably by affecting auxin transport, eventually resulting in the disappearance of the apical hook. Furthermore, we demonstrate that ethylene-stimulated hypocotyl elongation in the light is largely controlled by the same mechanisms as those governing formation of the apical hook in darkness. However, in the light, BRs appear to compensate for the insensitivity to ethylene in hls mutants, supporting a downstream action of BRs. Hence, our results indicate that HLS1, SUR1/HLS3/RTY1/ALF1 and AMP1/HPT/COP2/HLS2/PT act on the auxin-ethylene interaction, rather than at the level of BRs. A model for the tripartite hormone interactions is presented.     

Inhibition of brassinosteroid biosynthesis by either a dwarf4 mutation or a brassinosteroid biosynthesis inhibitor rescues defects in tropic responses of hypocotyls in the arabidopsis mutant nonphototropic hypocotyl 4

The nonphototropic hypocotyl 4 (nph4)/auxin response factor 7 (arf7) mutant of Arabidopsis (Arabidopsis thaliana) is insensitive to auxin and has defects in hypocotyl tropism, hook formation, differential leaf growth, and lateral root formation. To understand an auxin-signaling pathway through NPH4, we carried out screening of suppressor mutants of nph4-103 and obtained a dwarf suppressor mutant, suppressor of nph4 (snp2). snp2 had short hypocotyls in the dark condition and dark green and round leaves, short petioles, and more lateral shoots than the wild type in the light condition. The snp2 phenotypes were rescued by adding brassinolide to the growth medium in both light and dark conditions. Genetic mapping, sequence analysis, and a complementation test indicated that snp2 was a weak allele of DWARF4 (DWF4), which functions in brassinosteroid (BR) biosynthesis. snp2, which was renamed dwf4-101, exhibited photo- and gravitropisms of hypocotyls similar to those of the wild type with a slightly faster response in gravitropism. dwf4-101 almost completely suppressed defects in both tropisms of nph4-103 hypocotyls and completely suppressed hyponastic growth of nph4-103 leaves. Treatment with brassinazole, an inhibitor of BR biosynthesis, also partially rescued the tropic defects in nph4-103. Hypocotyls of nph4-103 were auxin insensitive, whereas hypocotyls of dwf4-101 were more sensitive than those of the wild type. dwf4-101 nph4-103 hypocotyls were as sensitive as those of dwf4-101. Auxin inducibility of massugu 2 (MSG2)/IAA19 gene expression was reduced in nph4-103. mRNA level of MSG2 was reduced in dwf4-101 and dwf4-101 nph4-103, but both mutants exhibited greater auxin inducibility of MSG2 than the wild type. Taken together, dwf4-101 was epistatic to nph4-103. These results strongly suggest that BR deficiency suppresses nph4-103 defects in tropic responses of hypocotyls and differential growth of leaves and that BR negatively regulates tropic responses.     

Polar auxin transport is required for the inhibition by blue light of the elongation-related LeEXT tomato gene

Hypocotyl elongation is an early developmental process regulated antagonistically by light and auxin. To highlight the interaction between both signals, we studied the photoregulation of the auxin-induced tomato (Lycopersicon esculentum Mill.) gene LeEXT involved in this process. RNA gel blot analysis indicated that this gene is down-regulated in response to blue light. We demonstrate that this response is principally mediated by the blue light photoreceptor cry1, but an interaction with the red/far-red light photoreceptors phyA, phyB1 and phyB2 has also been established. Furthermore, the polar auxin transport inhibitor NPA reverts the blue light inhibition of Lycopersicon esculentum gene encoding xyloglucan endotransglycosylase (LeEXT) expression, when it has the opposite effect in the dark or under red light. These results provide strong support for a specific interaction between auxin and blue light transduction pathways in the control of LeEXT expression, and therefore, of hypocotyl elongation in tomato.     

The Arabidopsis mutant alh1 illustrates a cross talk between ethylene and auxin

Ethylene or its precursor 1-aminocyclopropane-1-carboxylic acid (ACC) can stimulate hypocotyl elongation in light-grown Arabidopsis seedlings. A mutant, designated ACC-related long hypocotyl 1 (alh1), that displayed a long hypocotyl in the light in the absence of the hormone was characterized. Etiolated alh1 seedlings overproduced ethylene and had an exaggerated apical hook and a thicker hypocotyl, although no difference in hypocotyl length was observed when compared with wild type. Alh1 plants were less sensitive to ethylene, as reflected by reduction of ACC-mediated inhibition of hypocotyl growth in the dark and delay in flowering and leaf senescence. Alh1 also had an altered response to auxin, whereas auxin levels in whole alh1 seedlings remained unaffected. In contrast to wild type, alh1 seedlings showed a limited hypocotyl elongation when treated with indole-3-acetic acid. Alh1 roots had a faster response to gravity. Furthermore, the hypocotyl elongation of alh1 and of ACC-treated wild type was reverted by auxin transport inhibitors. In addition, auxin up-regulated genes were ectopically expressed in hypocotyls upon ACC treatment, suggesting that the ethylene response is mediated by auxins. Together, these data indicate that alh1 is altered in the cross talk between ethylene and auxins, probably at the level of auxin transport.     

Auxin and red light in the control of hypocotyl hook opening in beans

Evidence is presented to support the suggestion that endogenous auxinlike substances participate in controlling the unbending of the hypocotyl hook of Phaseolus vulgaris L. (cv. Black Valentine). An acidic indole was detected in hook diffusates by fluorometry; triiodobenzoic acid, an inhibitor of auxin transport, prevented red light-induced unbending, and indoleacetic acid can be substituted for tissue just above the elbow region as an inhibitor of opening. Indoleacetic acid also stimulated growth of shank cells, and red light increased the sensitivity of this tissue to the hormone. A small red light-induced stimulation of auxin transport through the inside half of the hypocotyl shank was observed and may be related to light-induced unbending of the hook.     

Genetic dissection of blue-light sensing in tomato using mutants deficient in cryptochrome 1 and phytochromes A, B1 and B2

Several novel allelic groups of tomato (Solanum lycopersicum L.) mutants with impaired photomorphogenesis have been identified after gamma-ray mutagenesis of phyA phyB1 double-mutant seed. Recessive mutants in one allelic group are characterized by retarded hook opening, increased hypocotyl elongation and reduced hypocotyl chlorophyll content under white light (WL). These mutants showed a specific impairment in response to blue light (BL) resulting from lesions in the gene encoding the BL receptor cryptochrome 1 (cry1). Phytochrome A and cry1 are identified as the major photoreceptors mediating BL-induced de-etiolation in tomato, and act under low and high irradiances, respectively. Phytochromes B1 and B2 also contribute to BL sensing, and the relative contribution of each of these four photoreceptors differs according to the light conditions and the specific process examined. Development of the phyA phyB1 phyB2 cry1 quadruple mutant under WL is severely impaired, and seedlings die before flowering. The quadruple mutant is essentially blind to BL, but experiments employing simultaneous irradiation with BL and red light suggest that an additional non-phytochrome photoreceptor may be active under short daily BL exposures. In addition to effects on de-etiolation, cry1 is active in older, WL-grown plants, and influences stem elongation, apical dominance, and the chlorophyll content of leaves and fruit. These results provide the first mutant-based characterization of cry1 in a plant species other than Arabidopsis.     

RCN1-regulated phosphatase activity and EIN2 modulate hypocotyl gravitropism by a mechanism that does not require ethylene signaling

The roots curl in naphthylphthalamic acid1 (rcn1) mutant of Arabidopsis (Arabidopsis thaliana) has altered auxin transport, gravitropism, and ethylene response, providing an opportunity to analyze the interplay between ethylene and auxin in control of seedling growth. Roots of rcn1 seedlings were previously shown to have altered auxin transport, growth, and gravitropism, while rcn1 hypocotyl elongation exhibited enhanced ethylene response. We have characterized auxin transport and gravitropism phenotypes of rcn1 hypocotyls and have explored the roles of auxin and ethylene in controlling these phenotypes. As in roots, auxin transport is increased in etiolated rcn1 hypocotyls. Hypocotyl gravity response is accelerated, although overall elongation is reduced, in etiolated rcn1 hypocotyls. Etiolated, but not light grown, rcn1 seedlings also overproduce ethylene, and mutations conferring ethylene insensitivity restore normal hypocotyl elongation to rcn1. Auxin transport is unaffected by treatment with the ethylene precursor 1-aminocyclopropane carboxylic acid in etiolated hypocotyls of wild-type and rcn1 seedlings. Surprisingly, the ethylene insensitive2-1 (ein2-1) and ein2-5 mutations dramatically reduce gravitropic bending in hypocotyls. However, the ethylene resistant1-3 (etr1-3) mutation does not significantly affect hypocotyl gravity response. Furthermore, neither the etr1 nor the ein2 mutation abrogates the accelerated gravitropism observed in rcn1 hypocotyls, indicating that both wild-type gravity response and enhanced gravity response in rcn1 do not require an intact ethylene-signaling pathway. We therefore conclude that the RCN1 protein affects overall hypocotyl elongation via negative regulation of ethylene synthesis in etiolated seedlings, and that RCN1 and EIN2 modulate hypocotyl gravitropism and ethylene responses through independent pathways.     

Phytochromes and cryptochromes regulate the differential growth of Arabidopsis hypocotyls in both a PGP19-dependent and a PGP19-independent manner

Photoreceptors, phytochromes and cryptochromes regulate hypocotyl growth under specific conditions, by suppressing negative gravitropism, modulating phototropism and inhibiting elongation. Although these effects seem to be partially caused via the regulation of the phytohormone auxin, the molecular mechanisms underlying this process are still poorly understood. In our present study, we demonstrate that the flabby mutation enhances both phytochrome- and cryptochrome-inducible hypocotyl bending in Arabidopsis. The FLABBY gene encodes the ABC-type auxin transporter, PGP19, and its expression is suppressed by the activation of phytochromes and cryptochromes. Our current results therefore indicate that the phytochromes and cryptochromes have at least two effects upon the tropic responses of the hypocotyls in Arabidopsis: the enhancement of hypocotyl bending through the suppression of PGP19, and a PGP19-independent mechanism that induces hypocotyl bending. By the using an auxin polar transport assay and DR5:GUS expression analysis, we further find that the phytochromes inhibit basipetal auxin transport, and induce the asymmetric distribution of auxin in the hypocotyls. These data suggest that the control of auxin transport by phytochromes and cryptochromes is a critical regulatory component of hypocotyl growth in response to light.     

Adventitious rooting in hypocotyls of sunflower (Helianthus annuus) seedlings. IV. The role of changes in endogenous free and conjugated indole‐3‐acetic acid

We have studied the role of endogenous auxin on adventitious rooting in hypocotyls of derooted sunflower (Helianthus annuus L. var. Dahlgren 131) seedlings. Endogenous free and conjugated indole-3-acetic acid (IAA) were measured in three segments of hypocotyls of equal length (apical, middle, basal) by using gas chromatography-mass spectrometry with [¹³C₆]-IAA as an internal standard. At the time original roots were excised (0 h), the free IAA level in the hypocotyls showed an acropetally decreasing gradient, but conjugated IAA level increased acropetally; i.e. free to total IAA ratio was highest in the basal portion of hypocotyls. The basal portion is the region where most of root primordia were found. Some primordia were seen in this region within 24 h after the roots were excised. The quantity of free IAA in the middle portion of the hypocotyl increased up to 15 h after excision and then decreased. In this middle region there were fewer root primordia, and they could not be seen until 72 h. In the apical portion the amount of free IAA steadily increased and no root primordia were seen by 72 h. Surgical removal of various parts of the hypocotyl tissues caused adventitious root formation in the hypocotyl regions where basipetally transported IAA could accumulate. Reduction in the basipetal flow of auxin by N-1-naphthylphthalamic acid and 2,3,5-tri-iodobenzoic acid resulted in fewer adventitious roots. The fewest root primordia were seen if the major sources of endogenous auxin were removed by decapitation of the cotyledons and apical bud. Exogenous auxins promoted rooting and were able to completely overcome the inhibitory effect of 2,3,5-tri-iodobenzoic acid. Exogenous auxins were only partially able to overcome the inhibitory effect of decapitation. We conclude that in sunflower hypocotyls endogenously produced auxin is necessary for adventitious root formation. The higher concentrations of auxin in the basal portion may be partially responsible for that portion of the hypocotyl producing the greatest number of primordia. In addition to auxins, other factors such as wound ethylene and lowered cytokinin levels caused by excision of the original root system cuttings must also be important.     

Insensitivity of the diageotropica tomato mutant to auxin

The sensitivity of excised hypocotyl segments to indoleacetic acid (IAA) in two assays, ethylene production and elongation, was determined in the ethylene-requiring tomato (Lycopersicon esculentum Mill.) mutant, diageotropica (dgt), and its isogenic parent, cv VFN8. Endogenous (uninduced) ethylene synthesis rates were slightly lower in dgt hypocotyls than in VFN8 hypocotyls. Ethylene production was essentially unaffected by IAA in dgt, but was stimulated up to 10-fold by 10 micromolar IAA in VFN8. Elongation of dgt hypocotyls was also insensitive to concentrations of IAA as high as 100 micromolar, as compared to significant elongation of VFN8 hypocotyls in response to 0.1 micromolar IAA. A range of IAA analogs active in VFN8 was also ineffective in stimulating elongation of dgt hypocotyls, suggesting that the differences were not due to rapid metabolism of IAA by dgt tissues. Auxin-induced elongation of VFN8 hypocotyls was unaffected by 2,3,5-triiodobenzoic acid and naphthylphthalamic acid, indicating that polar auxin transport was not a factor in these experiments. Exogenous and auxin-induced ethylene had no effect on the elongation respone of either genotype, nor did exogenous ethylene restore the sensitivity of dgt hypocotyls to IAA. Despite their apparent insensitivity to auxin, dgt hypocotyls elongated dramatically and synthesized ethylene rapidly in response to 1.2 micromolar fusicoccin. These results suggest that the primary effect of the dgt mutation is to reduce the sensitivity of the tissue to auxin. As altered regulation of ethylene synthesis is only one symptom of this fundamental deficiency, dgt should more properly be considered to be the auxin-insensitive tomato mutant.     

Genetic dissection of the role of ethylene in regulating auxin-dependent lateral and adventitious root formation in tomato

In this study we investigated the role of ethylene in the formation of lateral and adventitious roots in tomato ( Solanum lycopersicum ) using mutants isolated for altered ethylene signaling and fruit ripening. Mutations that block ethylene responses and delay ripening – Nr ( Never ripe ), gr ( green ripe ), nor ( non ripening ), and rin ( ripening inhibitor ) – have enhanced lateral root formation. In contrast, the epi ( epinastic ) mutant, which has elevated ethylene and constitutive ethylene signaling in some tissues, or treatment with the ethylene precursor 1-aminocyclopropane carboxylic acid (ACC), reduces lateral root formation. Treatment with ACC inhibits the initiation and elongation of lateral roots, except in the Nr genotype. Root basipetal and acropetal indole-3-acetic acid (IAA) transport increase with ACC treatments or in the epi mutant, while in the Nr mutant there is less auxin transport than in the wild type and transport is insensitive to ACC. In contrast, the process of adventitious root formation shows the opposite response to ethylene, with ACC treatment and the epi mutation increasing adventitious root formation and the Nr mutation reducing the number of adventitious roots. In hypocotyls, ACC treatment negatively regulated IAA transport while the Nr mutant showed increased IAA transport in hypocotyls. Ethylene significantly reduces free IAA content in roots, but only subtly changes free IAA content in tomato hypocotyls. These results indicate a negative role for ethylene in lateral root formation and a positive role in adventitious root formation with modulation of auxin transport as a central point of ethylene–auxin crosstalk.     

The interaction of light irradiance with auxin in regulating growth of <Emphasis Type="Italic">Helianthus annuus</Emphasis> shoots

Plants growing under canopy shade or in near-neighboring proximity of taller vegetation are the receivers of shade light conditions. The effect of light irradiance (photosynthetically active radiation [PAR]), one of the main components of shade light, on the growth of various tissues of sunflower seedlings and the possible role of auxin were investigated. Gradual reductions in PAR irradiance level from near-normal to low and very low result in significant and gradual increases in sunflower hypocotyl growth and endogenous auxin content. Similar reductions in PAR level resulted in significant and gradual decreases in sunflower cotyledon and leaf growth, and endogenous auxin content. Exogenously applied auxin increased hypocotyl elongation under near-normal PAR, where IAA levels are below optimum, but decreased elongation under very low PAR, where IAA levels are already at optimum. These results suggests that auxin acts as positive growth regulator of sunflower hypocotyls subjected to low light irradiance stress. This is further supported by the transfer experiments where seedlings transferred, for example, from near-normal PAR to very low PAR showed increased elongation associated with increased IAA levels. Therefore, it is reasonable to conclude that light irradiance-mediated changes in hypocotyl elongation of young sunflower seedlings are regulated by endogenous auxin levels.     

Transgene-mediated auxin overproduction in Arabidopsis: hypocotyl elongation phenotype and interactions with the hy6-1 hypocotyl elongation and axr1 auxin-resistant mutants

Transgenic Arabidopsis thaliana plants constitutively expressing Agrobacterium tumefaciens tryptophan monooxygenase (iaaM) were obtained and characterized. Arabidopsis plants expressing iaaM have up to 4-fold higher levels of free indole-3-acetic acid (IAA) and display increased hypocotyl elongation in the light. This result clearly demonstrates that excess endogenous auxin can promote cell elongation in a whole plant. Interactions of the auxin-overproducing transgenic plants with the phytochrome-deficient hy6-1 and auxin-resistant axrl-3 mutations were also studied. The effects of auxin overproduction on hypocotyl elongation were not additive to the effects of phytochrome deficiency in the hy6-1 mutant, indicating that excess auxin does not counteract factors that limit hypocotyl elongation in hy6-1 seedlings. Auxin-overproducing seedlings are also qualitatively indistinguishable from wild-type controls in their response to red, far-red, and blue light treatments, demonstrating that the effect of excess auxin on hypocotyl elongation is independent of red and blue light-mediated effects. All phenotypic effects of iaaM-mediated auxin overproduction (i.e. increased hypocotyl elongation in the light, severe rosette leaf epinasty, and increased apical dominance) are suppressed by the auxin-resistant axr1-3 mutation. The axr1-3 mutation apparently blocks auxin signal transduction since it does not reduce auxin levels when combined with the auxin-overproducing transgene.