Skip to main content
NCBI home page
Annals of Botany logoLink to Annals of Botany
. 2007 Jun 25;101(2):249–259. doi: 10.1093/aob/mcm115

Differential Effects of NAA and 2,4-D in Reducing Floret Abscission in Cestrum (Cestrum elegans) Cut Flowers are Associated with their Differential Activation of Aux/IAA Homologous Genes

Bekele Abebie 1,2, Amnon Lers 2, Sonia Philosoph-Hadas 2, Raphael Goren 1,*, Joseph Riov 1, Shimon Meir 2
PMCID: PMC2711013  PMID: 17591611

Abstract

Background and Aims

A previous study showed that the relative effectiveness of 2,4-dichlorophenoxyacetic acid (2,4-D) compared with that of 1-naphthaleneacetic acid (NAA) in reducing floret bud abscission in cestrum (Cestrum elegans) cut flowers was due to its acropetal transport. The aim of the present study was to examine if the differential effect of these auxins on floret abscission is reflected in the expression of Aux/IAA genes in the floret abscission zone (AZ).

Methods

cDNAs were isolated by PCR-based cloning from the floret AZ of auxin-treated cut flowers. The expression patterns of the cDNAs in various tissues and the effect of indole-3-acetic acid (IAA), applied with or without cycloheximide, on their expression in the floret AZ were examined by northern blot analysis. The regulation of transcript accumulation in the floret AZ in response to NAA or 2,4-D was measured by real-time PCR during auxin pulsing of cut flowers and vase life, concomitantly with floret abscission.

Key Results

Six isolated cDNAs were identified to represent Aux/IAA homologous genes, designated as Cestrum elegans (Ce)-IAA1 to Ce-IAA6. Four Ce-IAA genes were characterized as early auxin-responsive genes (ARGs), and two (Ce-IAA1 and Ce-IAA5) as late ARGs. Only Ce-IAA5 was AZ-specific in floret buds. A temporal regulation of Ce-IAA transcript levels in the floret AZ was found, with 2,4-D inducing higher expression levels than NAA in floret buds. These Ce-IAA expression levels were negatively correlated with floret abscission.

Conclusions

The differential transport characteristics of NAA and 2,4-D in cestrum cut flowers were reflected in differential activation of the Ce-IAA genes identified in the floret AZ. Therefore, Aux/IAA genes can be used as molecular markers to measure auxin activity, which reflects free auxin level in the AZ. Two of the identified genes, Ce-IAA1 and Ce-IAA5, may also have a regulatory role in abscission.

Key words: Abscission zone; auxin; Aux/IAA gene expression; Cestrum elegans; cut flowers; cycloheximide; 2,4-D; floret abscission; NAA

INTRODUCTION

The abscission process involves the interplay between indole-3-acetic acid (IAA) and ethylene (Abeles and Rubinstein, 1964; Sexton, 1997; Taylor and Whitelaw, 2001), which in turn affects the differentiation and function of target cells in the abscission zone (AZ) (Osborne, 1984). The generally accepted model is that the basipetal polar IAA flux through the AZ prevents abscission by rendering the AZ insensitive to ethylene. When the level of auxin decreases and the level of ethylene increases, the process of cell separation is initiated (Osborne, 1984). The direction of the auxin flux is also important for determining whether abscission will occur (Morris, 1993; Roberts et al., 2002). Thus, unlike various auxin-mediated physiological processes which are a result of transient and local changes in auxin levels (Woodward and Bartel, 2005), prevention of organ abscission has been found to require a continuous and constant polar supply of auxin to the AZ (Morris, 1993; Roberts et al., 2002).

In a previous study (Abebie et al., 2005) it has been demonstrated that the synthetic auxins, 1-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D), applied as pulse treatments to cestrum (Cestrum elegans) cut flowers, showed differential effects in reducing floret abscission. Floret abscission was significantly delayed by 2,4-D due to its significant acropetal transport, whereas NAA was much less effective in reducing abscission because of its low rate of acropetal transport. Consequently, more free 2,4-D than NAA accumulated in the floret AZ. As auxin activity is known to be mediated via activation of auxin-responsive genes, it is reasonable to assume that the differential accumulation of free NAA or 2,4-D level in the floret AZ may lead to differential activation of auxin-induced genes in this tissue.

Exogenous auxins induce several classes of genes known as early or late auxin-responsive genes (ARGs). Whereas the early ARGs are induced within minutes following auxin application, in the absence of de novo protein synthesis (Abel et al., 1995; Abel and Theologis, 1996), the late ARGs are probably triggered by events unleashed by the early-induced genes. The early ARGs include mainly members of the following gene families: Aux/IAA, small auxin up-regulated (SAUR) and Gretchen Hagen3 (GH3) (Abel and Theologis, 1996; Hagen and Guilfoyle, 2002). The most characterized gene products are Aux/IAA proteins, which are short-lived, nuclear localized, and have four conserved domains (Abel et al., 1995). Whereas domain I is an active repression domain that is transferable and dominant over activation domains (Tiwari et al., 2004), domain II plays a role in Aux/IAA stability (Worley et al., 2000). Domains III and IV function as homo- or hetero-dimerization domains of Aux/IAA-Aux/IAA or Aux/IAA-ARF (auxin-responsive factors) proteins. Although the function of Aux/IAA dimers is not clear, they may have a topology similar to that of prokaryotic transcriptional repressors, which allows them to bind DNA directly (Morgan et al., 1999). Thus, Aux/IAA proteins might also directly regulate gene transcription (Paciorek and Friml, 2006).

The function of Aux/IAA genes is well understood in terms of depression of ARF-induced transcription. However, downstream events and the function of Aux/IAA isoforms in various auxin-responsive pathways are less well understood. Differential expression of Aux/IAA genes in response to light (Liscum and Reed, 2002) as well as during tension wood formation (Moyle et al., 2002) and brassinosteroid-mediated growth responses (Nakamura et al., 2006) have been reported. Regulation of Aux/IAA-like mRNA level by ethylene was also observed in tomato fruit, but not in leaves, indicating a tissue-specific control (Jones et al., 2002). Aux/IAA proteins might both activate and inhibit auxin responses, depending on the particular Aux/IAA protein and/or the tissue (Reed, 2001).

It is becoming clear that the transport inhibitor response 1 (TIR1) and its three analogues, auxin-receptor F-box proteins (AFB1–AFB3), are functional auxin receptors that mediate the effect of auxin on gene expression (Dharmasiri et al., 2005a, b; Kepinski and Leyser, 2005). Generally, auxin regulates transcription by promoting the interaction between the Aux/IAA proteins, TIR1, and the closely related AFBs, and by targeting the Aux/IAA proteins for degradation (Gray et al., 2001; Zenser et al., 2001, 2003).

A rapid turnover of Aux/IAA proteins, which is known to occur via ubiquitin-mediated auxin signalling and which depends on auxin concentration (Gray et al., 1999, 2001; Gray and Estelle, 2000; Kepinski and Leyser, 2002), is essential for normal auxin-induced responses (Leyser, 2002). Therefore, the level of free auxin and Aux/IAA turnover in the cells seem to play an important role in auxin responses. According to the commonly accepted model (Hagen and Guilfoyle, 2002), Aux/IAA-ARF heterodimers are formed at low auxin concentrations and repress the expression of ARGs, while at high auxin concentrations Aux/IAA repressors dissociate from ARFs and degrade rapidly (Gray and Estelle, 2000; Worley et al., 2000). This implies that a certain critical threshold level of free auxin should be available in specific cells for normal plant growth and developmental processes.

Based on the accumulated evidence in the literature and our previous study (Abebie et al., 2005), we have hypothesized that the differential accumulation of NAA and 2,4-D in cestrum florets might induce differential activation of auxin-induced genes in the AZ, thereby causing differences in the reduction of floret abscission in response to the two auxins. In order to test this hypothesis, six cDNAs of Aux/IAA homologous genes were identified in the floret AZ of cestrum cut flowers and used as molecular markers to study auxin activity. The identified cDNAs, designated as Cestrum elegans (Ce)-IAAs, were further characterized and their expression levels, in parallel to the kinetics of floret abscission, were measured in response to NAA and 2,4-D. The results demonstrate differential expression patterns of the Ce-IAA genes in response to NAA or 2,4-D during auxin pulsing and vase life of the cut flowers. Floret bud abscission was negatively correlated with Ce-IAA gene expression in the AZ. This indicates the need for a continuous supply of free auxin to the AZ, for maintaining a constant level of Ce-IAA gene expression and reduced floret abscission.

MATERIALS AND METHODS

Plant system and treatments

Commercial size cut flowers of cestrum (Cestrum elegans Schlecht ‘Smithi’) were obtained from a local commercial plantation or from plants grown in a net-house. Unless otherwise stated, 30-cm-long cut flowers bearing a few open florets at the apex were used for the different experiments. The leaves were removed up to 12 cm from the base of the cut flowers, and each cut flower was placed in a 50-mL Falcon tube containing 7 mL of pulsing solution composed of 0·2 mm NAA or 2,4-D and 0·02 % 8-hydroxyquinoline citrate (8-HQC) as a preservative. Except when otherwise mentioned, the cut flowers were pulsed for 4 h at 20 °C under light at an intensity of 14 µmol m−2 s−1 and then transferred to 4 °C for an additional 20 h of pulsing under darkness. After pulsing, the lower 2 cm of each cut flower was trimmed off, and the pulsing solution was replaced with 20 mL of a bactericide solution containing 50 µL L−1 sodium dichloroisocyanureate (TOG-6, Milchan Brothers Ltd, Israel). Cut flowers placed in TOG-6 solution were incubated for 4 d in an observation room maintained at 20 °C, 60–70 % relative humidity and 12-h photoperiod (light intensity of 14 µmol m−2 s−1) to follow their vase life. This light intensity was used in all treatments. Additional TOG-6 solution was added when necessary during vase life to replace the amount lost by transpiration. For monitoring abscission of floret buds or open florets during vase life, individual inflorescences from the cut flowers were tapped gently at daily intervals, and the abscised floret buds or open florets were counted. At the end of the vase life incubation, floret buds or open florets that did not abscise were also counted and summed up with the number of abscised buds or florets to determine the accumulated percentage of the abscised organs during and at the end of the experiment. Unless otherwise mentioned, each experiment was conducted twice with five or six replicates, and data from one representative experiment are presented.

For studying gene expression, tissue sections, 3–4 mm long (approx. 1 g), which included the floret AZ and 1·5–2 mm of tissue from both sides of the separation layer, were excised from control or auxin-treated cut flowers at different time intervals during pulsing and vase life. The samples were collected into 2·1-mL Eppendorf tubes pre-cooled at –20 °C. For studying tissue specificity of Aux/IAA homologous genes, cut flowers were pulsed with 2,4-D for 24 h at 20 °C under light. Tissue samples were then excised from control and treated cut flowers, including florets (sepals of buds and open florets, perianthes, pedicels), leaves (leaf AZ, young and old leaf blades) and stems. The tissue samples were frozen in liquid N2 and stored at –80 °C until RNA isolation.

For studying gene induction in the floret AZ, various in vitro treatments were applied according to Moyle et al. (2002), with some modifications. Tissue samples of the floret AZ were excised from cut flowers as described above, placed in 1 % agar containing 50 µm MES buffer, pH 6·5, and incubated overnight at room temperature to remove endogenous auxin. Samples were then incubated for 1 h in either half-strength Murashige and Skoog (1/2 MS), 1/2 MS plus 10 µm IAA or 50 µm cycloheximide (CHX), or were pre-incubated for 30 min in 1/2 MS with 50 µm CHX and then transferred for an additional 1 h to 1/2 MS containing 10 µm IAA and 50 µm CHX. In all treatments, the 1/2 MS medium contained 0·5 % agarose. Untreated control samples were incubated for 1 h on moist Whatman No. 1 paper. At the end of incubation, the tissue samples were frozen in liquid N2 and stored at –80 °C until RNA isolation.

RNA extraction

Up to 1 g of frozen tissue was ground to a fine powder in liquid N2 with a pre-cooled pestle and mortar, and total RNA was extracted with cetyltrimethylammonium bromide (CTAB) according to the extraction procedures described by Liao et al. (2004). Briefly, after an overnight LiCl precipitation of the total RNA at 4 °C and a subsequent centrifugation (20 000g for 40 min at 4 °C), the pellet was re-suspended in 0·5 mL of sterile water and transferred to sterile Eppendorf tubes. Absolute ethanol (1·8:1, v/v) and sodium acetate, pH 5·5, (0·3 m final concentration) were then added, and the RNA was precipitated by an overnight incubation at a–20 °C, pelleted by centrifugation (20 000g for 20 min at 4 °C), washed twice with 75 % ethanol, and re-suspended in an appropriate amount of sterile water. The RNA was quantified by NanoDrop® ND-1000 spectrophotometer (NanoDrop Technologies, Inc., Rockland, DE, USA), and stored at –80 °C for further use.

Cloning and sequencing of Ce-IAA cDNA fragments

For cloning Ce-IAA genes, we used total RNA extracted from the floret AZ of cut flowers pulsed with either NAA or 2,4-D, which was mixed in equal proportions. First-strand cDNA was synthesized from 1 µg of total RNA with the EZ-First strand synthesis kit for RT-PCR (Biological Industries, Beit-Haemek Ltd, Israel), according to the manufacturer's instructions. Based on comparison of the amino acid sequences encoded by Aux/IAA genes in pea (Pisum sativum), soybean (Glycine max), mung bean (Vigna radiata) and Arabidopsis (Arabidopsis thaliana), degenerate forward and reverse primers were designed from the conserved domains II (5′-RTIGTIGGITGGCCICCIRT-3′) and IV (5′-ACRTCICCIRCIARCATCCA-3′), respectively (Oeller et al., 1993). PCR cloning of the Aux/IAA cDNA fragments was performed with a high-fidelity DNA polymerase. The PCR protocol consisted of an initial denaturation at 94 °C for 2 min followed by 30 cycles of 20 s at 94 °C, 57 °C for 20 s and 72 °C for 30 s, and a final extension at 72 °C for 5 min on an automated gradient thermal cycler (Model 9600, Perkin-Elmer Japan Co.). The resulting 290- and 370-bp PCR products were gel-purified using a QIAQUICK gel purification kit (Qiagen), TA-cloned into the pGEM-T Easy vector system II (Promega) and transformed into JM109 bacteria (Promega). The nucleotide sequence of the cloned Aux/IAA inserts was determined for both strands and used in search of homologous sequences in the GeneBank database using the BLASTX algorithm (Altschul et al., 1990). The sequences were aligned using ClustalW software (Thompson et al., 1994). The 3′ cDNA sequences were cloned using the 5′/3′-RACE second-generation kit (Roche Applied Sciences). The first-strand cDNA for 3′-RACE was synthesized from 1 µg of total RNA using the oligo dT-anchor primer supplied with the kit, according to the manufacturer's instructions. Gene-specific forward and/or nested primers were used to amplify the 3′ end with FastStart Taq DNA polymerase (Roche Applied Sciences) and the PCR anchor primer provided with the kit. The PCR protocols were adjusted in accordance with the melting temperature of each primer. The determined cDNA sequences were deposited in the data bank and their accession numbers are indicated in Table 1.

Table 1.

Nucleotide sequences of primers used for the quantitative real-time PCR reaction

Clone/accession no. Forward primer (F) Reverse primer (R)
Ce-IAA1 (DQ900819) 5′-CACCAACATATGAAGACAAGG-3′ 5′-GCTTCAGAACCCTTCATG-3′
Ce-IAA2 (DQ900820) 5′-GCTTCTTGTTCCCTAACATATG-3′ 5′-GAAACGTGATGAGCCAATAG-3′
Ce-IAA3 (DQ900821) 5′-GTGTTCAATGCTATCCATCC-3′ 5′-GTAAAGCATCTACCCATGTTG-3′
Ce-IAA4 (DQ900822) 5′-GTGAGATGGATCAGAATATGTTG-3′ 5′-GTCCAAATTAAAATACCACTTC- 3′
Ce-IAA5 (DQ900823) 5′-GAATAGGTGTGAGCTGTGTG-3′ 5′-GTAGCTAGGGAGCAGCTG-3′
Ce-IAA6 (DQ900824) 5′-GTTTTCGCATGCTTCTTAAG-3′ 5′-GGTCGTGGTAGCTGTAAGTAG-3′
18S rRNA 5′-GCGACGCATCATTCAAATTTC 5′-TCCGGAATCGAACCCTAATTC
Open in a new tab

Northern blot analysis

Twenty micrograms of total RNA was denatured and separated on a 1·0% agarose gel containing 0·66 m formaldehyde and then transferred to Hybond N+ (Amersham Pharmacia Biotech) membranes by standard capillary transfer methods (Ausubel et al., 1998). Transferred RNA was fixed to membranes by UV cross-linker (ULTRA LUM-UVC-508, Ultra Lum Inc., Paramount, CA, USA) for 2 min. The membranes with immobilized RNA were pre-hybridized in hybridization tubes for 2 h at 42 °C in a hybridization buffer containing 5× sodium chloride/sodium citrate (SSC), 5× Denhardt's reagents, 50 mm phosphate buffer, pH 6·5, 50 % formamide, 0·5 % sodium dodecyl sulfate (SDS) and 100 µg mL−1 salmon sperm DNA. The hybridization buffer was replaced with fresh buffer, and gene-specific denatured random-primed 32P-labelled Ce-IAA probes were added to the hybridization tubes. Hybridization was carried out overnight at 42 °C with constant rotation. The membranes were washed sequentially twice with 2× SSC for 5 min at room temperature, twice with 2× SSC at 65 °C for 15 min, and twice with 0·1× SSC at 65 °C for 15 min. All SSC solutions contained 0·1 % SDS. The membranes were then exposed to Imaging Plate BAS III (Fuji Film Co. Ltd, Japan) for 24 h, analysed using a Bio Image Analyser (BAS-5000, Fuji Film Co. Ltd), and processed with Photoshop 7·0.

Real-time PCR

Transcript levels of the Aux/IAA cDNAs were measured by quantitative real-time PCR (qRT-PCR) as described by Meir et al. (2006), with some modifications. Briefly, a pair of forward and reverse primer sequences were selected from the 3′ untranslated region (UTR) of each clone (Table 1) and checked with the Primer Express 2·0 software (Applied Biosystems). Each pair of primers was examined for its specificity to the relevant Aux/IAA gene and compared with genomic sequences available in the NCBI database to ensure that the primers amplify a unique and desired cDNA segment. cDNA was synthesized from 2 mg of total RNA using Moloney murine leukaemia virus reverse transcriptase (Promega) and a mixture of an oligo-(dT)24 primer and random primers (Promega).

RESULTS

Molecular cloning of auxin-induced cDNAs from floret AZ and the structure of their corresponding proteins

Aux/IAA cDNA clones were isolated using a mixture of total RNA extracted from the floret AZ excised from cut flowers pulsed with either NAA or 2,4-D. Two different size PCR products were obtained, which were subsequently TA-cloned. Following screening and sequence analysis of the different Aux/IAA cDNA clones, six different homologues of Aux/IAA genes designated as Ce-IAA1 to Ce-IAA6 were identified. The 3′ region was cloned with 3′RACE, and all cDNAs clones, except for Ce-IAA1, included sequences up to the transcript poly-A tail site.

The deduced partial amino acid sequences of the cloned Ce-IAA genes included about 68–75 % of their expected full-length coding sequences, based on the full-length sequences of Aux/IAA genes of representative plants (Fig. 1). The deduced partial amino acid sequences were aligned with the Aux/IAA protein sequences from various angiosperms, including A. thaliana, Nicotiana tabaccum and Populus tremula × Populus tremuloides. A high degree of homology was observed, similar to that observed among the representative plant sequences. The Ce-IAA polypeptides contained the highly conserved domains II (partial), III and IV, which are characteristic of the Aux/IAA protein family in angiosperms (Fig. 1). All six Ce-IAA genes also contained a part of the bipartite nuclear localization signal (NLS) in domains II and IV.

Fig. 1.

Fig. 1.

Open in a new tab

Sequence alignment of the primary protein sequence encoded by six cestrum Aux/IAA genes (Ce-IAA1–Ce-IAA6) and representative Aux/IAA protein sequences from Arabidopsis (At-IAA6, Q38824; At-IAA7, CAB46059; At-IAA8, AAC49047), tobacco (Nt-iaa2·3, AAD32142; Nt-iaa4·3, AAD32144; Nt-iaa4·5, AAD32145; Nt-iaa28, AAD32146) and hybrid aspen (Ptt-IAA8, CAC84712). Alignment was limited to the partial available sequence of Ce-IAA proteins and full-length sequences of the representative proteins. The sequences were aligned using ClustalW software (Thompson et al., 1994). The black and grey shaded areas represent at least 50% amino acid identity or similarity, respectively. The conserved basic residues that comprise the NLS sequence in domains II and IV, and the conserved amino acids of the protein destabilization element in domain II (box), which are the basic features of Aux/IAA proteins, are indicated.

Auxin induction of Ce-IAA genes

To examine whether the identified Ce-IAA genes are auxin-inducible and whether this induction is dependent on protein synthesis, AZ explants excised from nearly open and open florets were treated with IAA with or without CHX and their expression levels were measured. Transcript levels of Ce-IAA4 were below the detection limit by northern blot analysis. The data presented in Fig. 2 show that treatment with IAA for 1 h resulted in up-regulation of all five Ce-IAA genes at different degrees, thus confirming that these genes are auxin-inducible. CHX treatment increased the levels of four Ce-IAA genes, except for Ce-IAA5, compared with the untreated controls, to a lesser or greater degree than IAA. CHX applied together with IAA decreased the IAA-induced expression level of Ce-IAA1, increased the expression level of Ce-IAA2 above the level of its IAA-induced transcript, and abolished the IAA-induced expression level of Ce-IAA5. However, the combined CHX and IAA treatment did not affect transcript levels of the Ce-IAA3 and Ce-IAA6 genes compared with their levels in explants treated with IAA alone. These results indicate that only the response of Ce-IAA1 and Ce-IAA5 genes to IAA seems to depend on de novo protein synthesis.

Fig. 2.

Fig. 2.

Open in a new tab

Effect of IAA and/or CHX on induction of five Ce-IAA genes in cestrum floret AZ. Explants (3–4 mm long) were excised from the floret AZ and incubated overnight on 1 % agar plates for depletion of the endogenous auxin. The samples were then incubated for 1 h in either 1/2 MS, 1/2 MS containing 10 µm IAA or 50 µm CHX, or were pre-incubated for 30 min in 1/2 MS with 50 µm CHX and then transferred for an additional 1 h to 1/2 MS containing 10 µm IAA and 50 µm CHX. In all the treatments the 1/2 MS medium contained 0·5 % agarose. Untreated control samples were incubated for 1 h on moist Whatman No. 1 paper. Total RNA (20 mg) was denatured, loaded on each lane, blotted onto Hybond N+ membranes and hybridized with gene-specific Ce-IAA probes amplified from the 3′UTR of the clone.

Organ/tissue-specific expression of Ce-IAA genes

Organ- or tissue-specific expression patterns of the Ce-IAA genes was measured by northern blot analysis. Total RNA was isolated from different parts of untreated or 2,4-D-treated cut flowers. 2,4-D was selected for pulse treatment due to its increased acropetal movement in cestrum cut flowers (Abebie et al., 2005). Except for Ce-IAA4 and Ce-IAA6, expression of which was too low to be detected by northern blot analysis, the other four Ce-IAA genes showed differential expression levels in different tissues and in response to 2,4-D (Fig. 3). Ce-IAA1 was expressed in all the examined untreated tissues/organs, except for young and old leaves (Fig. 3A), and its expression level increased significantly in response to 2,4-D (Fig. 3B). The expression of Ce-IAA1 in leaves was limited only to the AZ. No expression of the Ce-IAA2 gene could be detected in any of the examined untreated tissues (Fig. 3A), but it was induced in floret bud organs, open floret AZ, pedicels and leaf AZ following 2,4-D treatment (Fig. 3B). Ce-IAA3 was expressed in all the examined untreated tissues, except for leaves and stems (Fig. 3A), and its expression level slightly increased following 2,4-D treatment (Fig. 3B). Ce-IAA5 was expressed only in the untreated floret bud AZ (Fig. 3A), but following 2,4-D treatment its expression was detected also in floret bud sepals, open floret AZ, pedicels and leaf AZ (Fig. 3B). These data demonstrate that the Ce-IAA genes vary in their expression levels in various tissues and in their degree of response to 2,4-D.

Fig. 3.

Fig. 3.

Open in a new tab

Expression patterns of four Ce-IAA genes in various organs or tissues excised from untreated (A) or 2,4-D-treated (B) cestrum cut flowers. The shoots (30 cm long) were pulsed for 24 h with 0·2 mm 2,4-D at 20 °C in the observation room. Samples were collected from the different organs or tissues of control and 2,4-D-treated shoots. Total RNA (20 mg) was denatured, loaded per each lane, blotted onto Hybond N+ membranes and hybridized with gene-specific Ce-IAA probes amplified from the 3′UTR of the clone.

Effect of NAA and 2,4-D pulsing on expression of Ce-IAA genes in the floret AZ

Experiments were conducted to compare the effect of NAA and 2,4-D pulsing of cut flowers on induction of the identified Ce-IAA genes in the floret AZ. Cut flowers were pulsed with either 0·2 mm NAA or 2,4-D, and the pulsing solution was then replaced with a bactericide solution (TOG-6) during vase life. The results of the qRT-PCR analysis show that all six Ce-IAA genes exhibited a differential expression pattern in the AZ of both floret buds (Fig. 4) and open florets (Fig. 5) during and after NAA or 2,4-D pulsing. The expression of these genes increased in the floret bud AZ of cut flowers in response to auxin treatment, particularly in the 2,4-D-treated flowers, after 4 and 24 h of pulsing, compared with their expression levels in the floret bud AZ of control cut flowers (Fig. 4). The highest increase in the transcript levels of the Ce-IAA1, 2, 5 and 6 genes was obtained after 48 h of incubation in response to 2,4-D pulsing (Fig. 4A, B, E, F). By contrast, the induction of the Ce-IAA3 gene in response to both auxins was similar throughout the incubation period (Fig. 4C). The expression level of the Ce-IAA4 gene was not affected by the two auxins throughout the incubation period (Fig. 4D). A sharp decline in the transcript level of five of the induced genes was observed after 48 h of incubation (Fig. 4A–C, E, F).

Fig. 4.

Fig. 4.

Open in a new tab

Effect of NAA or 2,4-D pulsing of cestrum cut flowers on expression of six Ce-IAA genes in the AZ of floret buds during pulsing and subsequent vase life. The cut flowers were pulsed with 0·2 mm of the different auxin solutions in 0·02 % 8-HQC for 24 h and then transferred to a TOG-6 solution during vase life. Total RNA was extracted from the AZ of floret buds during pulsing and subsequent vase life. The level of gene expression was determined by RT-PCR. The transcript level was comparatively quantified vs. ribosomal 18S cDNA, which served as an internal reference (housekeeping gene) to normalize gene expression. The primer pairs used for each gene and for the 18S gene are presented in Table 1. The relative amount of each specific transcript is in reference to its own level in untreated controls (0 time = 1·0). The results represent average values of duplicate RT-PCR reactions for the same cDNA in one representative experiment.

Fig. 5.

Fig. 5.

Open in a new tab

Effect of NAA or 2,4-D pulsing of cestrum cut flowers on expression of six CeIAA genes in the AZ of open florets during pulsing and subsequent vase life. The cut flowers were pulsed with the auxins, and total RNA was extracted from the AZ of open florets and assayed as detailed in Fig. 4. The results represent average values of duplicate RT-PCR reactions for the same cDNA in one representative experiment.

The relative expression level of all Ce-IAA genes in the AZ of open florets in control cut flowers remained almost unchanged during pulsing and vase life, but it increased in a similar manner in response to both auxins (Fig. 5), including also Ce-IAA2, Ce-IAA5 and Ce-IAA6, which showed only a very weak response to NAA in the floret bud AZ (Fig. 4). Similar to floret buds, the maximum level of transcript accumulation in the AZ of open florets in response to both auxins was observed after 48 h, and this was followed by a sharp decline thereafter. Generally, after 72 or 96 h the transcript levels of all Ce-IAA genes in the AZ of both floret buds (Fig. 4) and open florets (Fig. 5) excised from NAA- or 2,4-D-treated cut flowers decreased nearly to the basal levels obtained in control flowers.

Effect of NAA and 2,4-D on floret abscission

Following a 24-h pulse treatment of cut flowers, NAA and 2,4-D showed differential effects in reducing the abscission of floret buds (Fig. 6A) or open florets (Fig. 6B) during vase life. The kinetics of floret bud abscission (Fig. 6A) showed that 48 h after pulsing, more than 20 % of the floret buds abscised in the control cut flowers, whereas no floret bud abscission occurred in the auxin-treated flowers. Whereas about 90 % of the floret buds abscised in control cut flowers 72 h after pulsing, the percentage of abscised floret buds in response to NAA or 2,4-D was 50 and 5 %, respectively. Ninety-six hours after pulsing, more than 98 and 95 % of the floret buds abscised in control and NAA-treated cut flowers, respectively. However, at this time point, only 50 % of the floret buds abscised in 2,4-D-treated cut flowers.

Fig. 6.

Fig. 6.

Open in a new tab

Effect of NAA or 2,4-D pulsing of cestrum cut flowers on abscission of floret buds (A) and open florets (B) during vase life. The cut flowers (30 cm long) bearing mainly floret buds (A) or open florets (B) were pulsed with 0·2 mm of the different auxin solutions in 0·02 % 8-HQC for 24 h and then transferred to a TOG-6 solution during vase life. At the indicated time intervals, the inflorescence heads were placed into polyethylene bags and tapped gently. Abscised floret buds or open florets were then counted and their accumulated number is presented as percentage of total floret buds or open florets per inflorescence. Data represent the mean values of six shoots ± s.e.

The effect of the two auxins on abscission of open florets was examined in a parallel experiment. The data presented in Fig. 6B show that generally the overall abscission of open florets was significantly delayed compared with that of floret buds. In the control and NAA-treated cut flowers, open florets started to abscise 96 h after pulsing, without much difference between them, whereas in 2,4-D-treated flowers almost no florets abscised at this time point (Fig. 6B). In summary, the data show that 2,4-D significantly delayed floret bud abscission (Fig. 6A) and completely prevented the abscission of open florets (Fig. 6B) during 96 h of vase life compared with NAA.

DISCUSSION

It has previously been shown that the differential effects of NAA and 2,4-D in reducing floret bud and open floret abscission in cestrum cut flowers seem to be associated with the failure of NAA to move acropetally and to accumulate in the floret AZ at a sufficient level required for continuous auxin signalling (Abebie at al., 2005). In contrast, 2,4-D exhibited a significant acropetal transport. These phenomena might in turn create a differential activation of auxin-induced genes in the floret AZ. Thus, the differential effect of the two auxins on abscission may be reflected in the regulation of ARGs such as Aux/IAA family members. To examine this possibility, cDNAs corresponding to six Aux/IAA homologous genes, designated as Ce-IAA1 to Ce-IAA6, were cloned from the floret AZ of cestrum cut flowers following auxin treatment. These Ce-IAA genes were characterized as early or late ARGs, exhibiting variable spatial expression patterns in various tissues/organs. The temporal expression patterns of these Ce-IAA genes in the floret AZ following a pulse treatment of cut flowers with NAA or 2,4-D was characterized, in parallel to the kinetics of floret abscission. In this way, the identified Ce-IAA genes could be used as potential molecular markers to measure auxin activity, which reflects the levels of free auxin in the floret AZ following auxin pulsing.

Based on their deduced partial amino acid sequences, all the identified six Ce-IAA genes showed homology to other members of the Aux/IAA gene family of angiosperms (Fig. 1). The presence of a part of the basic NLS sequence in domains II and IV indicates that all the Ce-IAA genes are possibly located in the nucleus. The expression of five of the identified six Ce-IAA genes, except for Ce-IAA4, was induced by IAA (Fig. 2), NAA and 2,4-D (Figs 3–5). A slight induced expression of Ce-IAA4 in response to NAA and 2,4-D was detected by RT-PCR analysis in the AZ of open florets (Fig. 5D), even in tissues where no expression was detected without exogenous auxin (Fig. 3). These results further confirm that all the identified six Ce-IAA genes are auxin-inducible.

To exclude any possible effect of wound- and auxin-induced ethylene on Aux/IAA gene expression, explants were either treated with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) or were exposed to exogenous ethylene. In addition, auxin pulsing of cut flowers was performed in the presence of the ethylene action inhibitor silver thiosulfate (STS). As no effect on the transcript levels of the Ce-IAA genes was observed following these treatments (data not shown), it can be concluded that the identified Ce-IAA genes in the floret AZ are not induced by ethylene.

Apart of their fast induction by auxin, Aux/IAA genes are generally characterized by the pattern of their mRNA accumulation when de novo protein synthesis is blocked. Thus, Aux/IAA genes that are induced, super-induced or unaffected by CHX are suggested to be early ARGs, as their induction is not repressed by inhibition of protein synthesis (Abel et al., 1995; Dargeviciute et al., 1998). Pre-treatment of cestrum AZ explants with CHX reduced the response of Ce-IAA1 and Ce-IAA5 to IAA (Fig. 2), indicating that de novo protein synthesis is required for the auxin induction of these genes. Therefore, these two genes can be classified as late ARGs. Given that IAA induction of the other Ce-IAA genes was either slightly increased (Ce-IAA2) or unaffected (Ce-IAA3 and Ce-IAA6) by the CHX pre-treatment, they may be classified as early ARGs. The inductive effect of CHX on the expression of four Ce-IAA genes may be ascribed to its stabilizing effect on mRNA (Koshiba et al., 1995), or to the involvement of a repressor in the regulation of some Aux/IAA genes.

We compared the expression patterns of the Ce-IAA genes in different tissues excised from untreated and 2,4-D-pulsed cut flowers. Among the six examined Ce-IAA genes, the transcript levels of two genes, Ce-IAA4 and Ce-IAA6, were below the detection limit of the northern blot analysis even following 2,4-D treatment; two genes, Ce-IAA1 and Ce-IAA3, were expressed in most of the examined tissues and their expression level increased in response to 2,4-D; one gene, Ce-IAA2, was expressed only in response to 2,4-D in most of the tissues examined; and one gene, Ce-IAA5, was expressed only in the floret bud AZ, but 2,4-D treatment increased its expression also in other tissues (Fig. 3). These results indicate that the Ce-IAA genes cloned from the floret AZ are not specific to the AZ and they differ in their response to 2,4-D. Indeed, it was previously reported that many of the Aux/IAA genes are known to show differential expression patterns in different tissues in response to exogenous auxins, light stimuli and at different stages of development (Ainley et al., 1988; Yamamoto et al., 1992; Abel et al., 1995; Dargeviciute et al., 1998). However, most of the examined Ce-IAA genes were expressed in the flower tissues, and none of them was expressed in young or old/non-senescent leaves (Fig. 3), suggesting that other Ce-IAA genes might be expressed in the leaves.

The expression levels of the Ce-IAA genes in the floret AZ excised from auxin-treated or untreated cut flowers (Figs 4 and 5) were negatively correlated with the rate of floret abscission (Fig. 6). This suggests that the free auxin level in the floret AZ declined with time, leading to the induction of abscission. Thus, the general pattern of a decline in Ce-IAA expression levels, which occurred after 48 h in the AZ of floret buds (Fig. 4) or after 96 h in the AZ of open florets (Fig. 5), coincided with the rate of floret bud abscission (Fig. 6). At low concentrations of free auxin, Aux/IAA proteins are reported to hetero-dimerize with ARF proteins and repress transcription of other downstream ARGs (Hagen and Guilfoyle, 2002). This implies that a certain amount of free auxin should be available or supplied to the AZ cells for continuous maintenance of Aux/IAA gene expression in these cells, which is required for normal auxin signalling. For example, the decline in transcript levels of most of the Ce-IAA genes below the basal level (1·0) in the AZ of floret buds after 72 h (Fig. 4) or open florets after 96 h of the control (Fig. 5), which coincided with a significant abscission of floret buds or open florets at these time points (Fig. 6), probably reflected a decline in endogenous auxin levels in the AZ. Conversely, abscission of floret buds or open florets in cut flowers pulsed with either NAA or 2,4-D was low (Fig. 6) as long as the transcript level of most of the Ce-IAA genes was at least equal to or above the basal level (Figs 4 and 5).

The data obtained in the present study demonstrate that open florets differ from floret buds in several aspects. They exhibited the following patterns: (1) reduced abscission in response to all treatments (Fig. 6B), (2) prolonged expression of the Ce-IAA genes in control plants (Fig. 5) and (3) a similar induction of Ce-IAA genes by the two auxins (Fig. 5). These phenomena might result from an elevated auxin level in open florets compared with floret buds, which is probably derived from mature flower organs such as the anthers (Aloni et al., 2006; Yasuor et al., 2006). It is reasonable to assume that a high endogenous auxin level in open florets will reduce their sensitivity to abscission, induce a prolonged expression of the Ce-IAA genes, and increase the auxin activity of the relatively low amount of NAA that reaches the florets in inducing the Ce-IAA genes. The possibility that the differential activation of Ce-IAA genes by NAA and 2,4-D might have stemmed from their differential binding to the auxin receptor should also be considered. NAA has been shown to have a higher affinity for the auxin receptor, TIR1, than 2,4-D in Arabidopsis (Dharmasiri et al., 2005a, b; Kepinski and Leyser, 2005). However, our observations do not support this possibility. First, we have previously shown that NAA almost failed to reach the floret AZ owing to a lack of acropetal transport (Abebie et al., 2005). Secondly, 2,4-D exhibited a higher activity than NAA in the induction of the Ce-IAA genes in cestrum floret buds (Fig. 4). Hence, the issue of differential binding does not seem to be relevant in this system. It seems, therefore, that inhibition of floret bud abscission by 2,4-D depends mainly on its improved transport to the floret AZ from the site of its application (Abebie et al., 2005) rather than on its differential binding to the auxin receptor.

The results presented in Figs 4–6 indicate that as long as a continuous supply of free auxin to the AZ was maintained, as reflected in continuous expression of Aux/IAA genes above the basal level (1·0), floret abscission was reduced. It is now known that the Aux/IAA proteins are actually repressors of auxin-induced transcription, and that auxin promotes the degradation of this large family of transcriptional regulators, leading to diverse downstream auxin effects (Worley et al., 2000; Gray et al., 2001). This allows ARF proteins to bind to auxin responsive elements (AREs) within the promoters and either activate or repress target gene expression. Auxins are also known to repress transcription of some genes whose expression is correlated with senescence and/or abscission (Noh and Amasino, 1999; Hong et al., 2000; Tucker et al., 2002). Functional analysis of the ARF1, ARF2, ARF7 and ARF19 genes suggested that these transcription regulators act with partial redundancy to promote senescence and floral organ abscission (Ellis et al., 2005; Okushima et al., 2005). Thus, changes in auxin gradients across the AZ may either delay or promote abscission, possibly by modulating ARF activity, as observed in A. thaliana floral organs (Taylor and Whitelaw, 2001; Ellis et al., 2005).

Ce-IAA1 and Ce-IAA5 genes, which were characterized as late ARGs (Fig. 2), are of particular interest in view of the need for continuous auxin supply to the AZ to delay organ abscission, a system characterized by a slow response to auxin. Ce-IAA1 was specifically expressed in the leaf AZ, but not in the blade of old or young leaves, while Ce-IAA5 was specifically expressed only in the floret bud AZ (Fig. 3A). These data suggest that the Ce-IAA1 and Ce-IAA5 genes, apart from being molecular markers for auxin activity, may also have a regulatory role in the abscission process of leaves and flowers, respectively, and therefore deserve further attention. As in other plants, the Aux/IAA genes in cestrum were found to form a gene family with differential member expression. It is possible that only specific members of this Aux/IAA gene family, expression of which is more localized in the AZ, are involved in regulation of the abscission process. Recently, a more direct correlation between organ abscission and expression of Aux/IAA genes in the AZ was demonstrated in Mirabilis jalapa (Meir et al., 2006). The transcript levels of two Aux/IAA homologues cloned from the AZ of Mirabilis (Mj-Aux/IAA1 and 2) were repressed following removal of the auxin source by either leaf deblading or stem decapitation. The expression of these two clones was de-repressed by applying IAA to the cut end of the petiole or stump, concomitantly with its effect in preventing abscission of these organs (Meir et al., 2006).

CONCLUSIONS

The six cDNAs cloned from the floret AZ of cestrum cut flowers were found to represent six different members of the Aux/IAA gene family, characterized as auxin-inducible, early or late ARGs, which mostly are not specific to the AZ. The differential accumulation of NAA and 2,4-D in cestrum floret AZ, derived from their different transport characteristics, was reflected in differential activation of these identified six Ce-IAA genes in the floret AZ, and in their differential efficiency in reducing floret abscission. The ability of 2,4-D to move acropetally and to accumulate in the floret organs, which was correlated with higher expression levels of Ce-IAA genes and reduced floret abscission, indicates the importance of accumulation of free auxin in the AZ for reducing floret abscission. Therefore, the expression of Aux/IAA genes can be used as molecular markers for estimating changes in free auxin level and activity in the AZ. In addition, the data indicate that a certain critical threshold level of free auxin has to be supplied continuously to the AZ cells for effective and continuous expression of Aux/IAA genes. We suggest that the regulation of the abscission process might require expression of specific Aux/IAA genes, localized in the AZ, rather than an expression of all Aux/IAA genes. A possible candidate for this role in cestrum is the Ce-IAA5 gene, a late ARG, which is specifically expressed in floret bud AZ, and the transcript level of which increased significantly in response to 2,4-D.

LITERATURE CITED

  1. Abebie B, Goren R, Huberman M, Meir S, Philosoph-Hadas S, Riov J. Prevention of bud and floret abscission in Cestrum cut flowers is related to the mode of transport and metabolism of synthetic auxins. Acta Horticulturae. 2005;682:789–794. [Google Scholar]
  2. Abel S, Theologis A. Early genes and auxin action. Plant Physiology. 1996;111:9–17. doi: 10.1104/pp.111.1.9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Abel S, Nguyen MD, Theologis A. The Ps-Iaa4/5-like family of early auxin-inducible messenger RNAs in Arabidopsis thaliana. Journal of Molecular Biology. 1995;251:533–549. doi: 10.1006/jmbi.1995.0454. [DOI] [PubMed] [Google Scholar]
  4. Abeles FB, Rubinstein B. Regulation of ethylene evolution and leaf abscission by auxin. Plant Physiology. 1964;39:963–969. doi: 10.1104/pp.39.6.963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Ainley WM, Walker JC, Nagao RT, Key JL. Sequence and characterization of 2 auxin-regulated genes from soybean. Journal of Biological Chemistry. 1988;263:10658–10666. [PubMed] [Google Scholar]
  6. Aloni R, Aloni E, Langhans M, Ullrich CI. Role of auxin in regulating Arabidopsis flower development. Planta. 2006;223:315–328. doi: 10.1007/s00425-005-0088-9. [DOI] [PubMed] [Google Scholar]
  7. Altschul SF, Gish W, Miller W, Myers E, Lipman DJ. Basic local alignment search tool. Journal of Molecular Biology. 1990;215:403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  8. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K. Current Protocols in Molecular Biology. New York: John Wiley & Sons; 1998. [Google Scholar]
  9. Dargeviciute A, Roux C, Decreux A, Sitbon F, Perrot-Rechenmann C. Molecular cloning and expression of the early auxin-responsive Aux/IAA gene family in Nicotiana tabacum. Plant and Cell Physiology. 1998;39:993–1002. doi: 10.1093/oxfordjournals.pcp.a029311. [DOI] [PubMed] [Google Scholar]
  10. Dharmasiri N, Dharmasiri S, Estelle M. The F-box protein TIR1 is an auxin receptor. Nature. 2005;a 435:441–445. doi: 10.1038/nature03543. [DOI] [PubMed] [Google Scholar]
  11. Dharmasiri N, Dharmasiri S, Weijers D, Lechner E, Yamada M, Hobbie L, et al. Plant development is regulated by a family of auxin receptor F-box proteins. Developmental Cell. 2005;b 9:109–119. doi: 10.1016/j.devcel.2005.05.014. [DOI] [PubMed] [Google Scholar]
  12. Ellis CM, Nagpal P, Young JC, Hagen G, Guilfoyle TJ, Reed JW. AUXIN RESPONSE FACTOR1 and AUXIN RESPONSE FACTOR2 regulate senescence and floral organ abscission in Arabidopsis thaliana. Genes and Development. 2005;132:4563–4574. doi: 10.1242/dev.02012. [DOI] [PubMed] [Google Scholar]
  13. Gray WM, Estelle M. Function of the ubiquitin-proteasome pathway in auxin response. Trends in Biochemical Sciences. 2000;25:133–138. doi: 10.1016/s0968-0004(00)01544-9. [DOI] [PubMed] [Google Scholar]
  14. Gray WM, del Pozo JC, Walker L, Hobbie L, Risseeuw E, Banks T, et al. Identification of an SCF ubiquitin-ligase complex required for auxin response in Arabidopsis thaliana. Genes and Development. 1999;13:1678–1691. doi: 10.1101/gad.13.13.1678. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gray WM, Kepinski S, Rouse D, Leyser O, Estelle M. Auxin regulates SCFTIR1-dependent degradation of AUX/IAA proteins. Nature. 2001;414:271–276. doi: 10.1038/35104500. [DOI] [PubMed] [Google Scholar]
  16. Hagen G, Guilfoyle T. Auxin-responsive gene expression: genes, promoters and regulatory factors. Plant Molecular Biology. 2002;49:373–385. [PubMed] [Google Scholar]
  17. Hong SB, Sexton R, Tucker ML. Analysis of gene promoters for two tomato polygalacturonases expressed in abscission zones and the stigma. Plant Physiology. 2000;123:869–881. doi: 10.1104/pp.123.3.869. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jones B, Frasse P, Olmos E, Zegzouti H, Li ZG, Latche A, et al. Down-regulation of DR12, an auxin-response-factor homolog, in the tomato results in a pleiotropic phenotype including dark green and blotchy ripening fruit. Plant Journal. 2002;32:603–613. doi: 10.1046/j.1365-313x.2002.01450.x. [DOI] [PubMed] [Google Scholar]
  19. Kepinski S, Leyser O. Ubiquitination and auxin signalling: a degrading story. Plant Cell. 2002;14:S81–S95. doi: 10.1105/tpc.010447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kepinski S, Leyser O. The Arabidopsis F-box protein TIR1 is an auxin receptor. Nature. 2005;435:446–451. doi: 10.1038/nature03542. [DOI] [PubMed] [Google Scholar]
  21. Koshiba T, Ballas N, Wong LM, Theologis A. Transcriptional regulation of Ps-Iaa4/5 and Ps-Iaa6 early gene expression by indoleacetic acid and protein synthesis inhibitors in pea (Pisum sativum) Journal of Molecular Biology. 1995;253:396–413. doi: 10.1006/jmbi.1995.0562. [DOI] [PubMed] [Google Scholar]
  22. Leyser O. Molecular genetics of auxin signalling. Annual Review of Plant Biology. 2002;53:377–398. doi: 10.1146/annurev.arplant.53.100301.135227. [DOI] [PubMed] [Google Scholar]
  23. Liao ZH, Chen M, Guo L, Gong YF, Tang F, Sun XF, Tang KX. Rapid isolation of high-quality total RNA from Taxus and Ginkgo. Preparative Biochemistry and Biotechnology. 2004;34:209–214. doi: 10.1081/PB-200026790. [DOI] [PubMed] [Google Scholar]
  24. Liscum E, Reed JW. Genetics of Aux/IAA and ARF action in plant growth and development. Plant Molecular Biology. 2002;49:387–400. [PubMed] [Google Scholar]
  25. Meir S, Hunter DA, Chen JC, Halaly V, Reid MS. Molecular changes occurring during acquisition of abscission competence following auxin depletion in Mirabilis jalapa. Plant Physiology. 2006;141:1604–1616. doi: 10.1104/pp.106.079277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Morgan KE, Zarembinski TI, Theologis A, Abel S. Biochemical characterization of recombinant polypeptides corresponding to the predicted beta alpha fold in Aux/IAA proteins. FEBS Letters. 1999;454:283–287. doi: 10.1016/s0014-5793(99)00819-4. [DOI] [PubMed] [Google Scholar]
  27. Morris DA. The role of auxin in the apical regulation of leaf abscission in cotton (Gossypium hirsutum L) Journal of Experimental Botany. 1993;44:807–814. [Google Scholar]
  28. Moyle R, Schrader J, Stenberg A, Olsson O, Saxena S, Sandberg G, Bhalerao RP. Environmental and auxin regulation of wood formation involves members of the Aux/IAA gene family in hybrid aspen. Plant Journal. 2002;31:675–685. doi: 10.1046/j.1365-313x.2002.01386.x. [DOI] [PubMed] [Google Scholar]
  29. Nakamura A, Nakajima N, Goda H, Shimada Y, Hayashi K, Nozaki H, et al. Arabidopsis Aux/IAA genes are involved in brassinosteroid-mediated growth responses in a manner dependent on organ type. Plant Journal. 2006;45:193–205. doi: 10.1111/j.1365-313X.2005.02582.x. [DOI] [PubMed] [Google Scholar]
  30. Noh YS, Amasino RM. Identification of a promoter region responsible for the senescence-specific expression of SAG12. Plant Molecular Biology. 1999;41:181–194. doi: 10.1023/a:1006342412688. [DOI] [PubMed] [Google Scholar]
  31. Oeller PW, Keller JA, Parks JE, Silbert JE, Theologis A. Structural characterization of the early indoleacetic acid-inducible genes, Ps-Iaa4/5 and Ps-Iaa6, of pea (Pisum sativum L) Journal of Molecular Biology. 1993;233:789–798. doi: 10.1006/jmbi.1993.1555. [DOI] [PubMed] [Google Scholar]
  32. Okushima Y, Overvoorde PJ, Arima K, Alonso JM, Chan A, Chang C, et al. Functional genomic analysis of the AUXIN RESPONSE FACTOR gene family members in Arabidopsis thaliana: unique and overlapping functions of ARF7 and ARF19. Plant Cell. 2005;17:444–463. doi: 10.1105/tpc.104.028316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Osborne DJ. Concepts of target cells in plant differentiation: review. Cell Differentiation. 1984;14:161–169. doi: 10.1016/0045-6039(84)90042-3. [DOI] [PubMed] [Google Scholar]
  34. Paciorek T, Friml J. Auxin signalling. Journal of Cell Science. 2006;119:1199–1202. doi: 10.1242/jcs.02910. [DOI] [PubMed] [Google Scholar]
  35. Reed JW. Roles and activities of Aux/IAA proteins in Arabidopsis. Trends in Plant Science. 2001;6:420–425. doi: 10.1016/s1360-1385(01)02042-8. [DOI] [PubMed] [Google Scholar]
  36. Roberts JA, Elliott KA, Gonzalez-Carranza ZH. Abscission, dehiscence, and other cell separation processes. Annual Review of Plant Biology. 2002;53:131–158. doi: 10.1146/annurev.arplant.53.092701.180236. [DOI] [PubMed] [Google Scholar]
  37. Sexton R. The role of ethylene and auxin in abscission. Acta Horticulturae. 1997;463:435–444. [Google Scholar]
  38. Taylor JE, Whitelaw CA. Signals in abscission. New Phytologist. 2001;151:323–339. [Google Scholar]
  39. Thompson JD, Higgins DG, Gibson TJ. CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research. 1994;22:4673–4680. doi: 10.1093/nar/22.22.4673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Tiwari SB, Hagen G, Guilfoyle TJ. Aux/IAA proteins contain a potent transcriptional repression domain. Plant Cell. 2004;16:533–543. doi: 10.1105/tpc.017384. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Tucker ML, Whitelaw CA, Lyssenko NN, Nath P. Functional analysis of regulatory elements in the gene promoter for an abscission-specific cellulase from bean and isolation, expression, and binding affinity of three TGA-type basic leucine zipper transcription factors. Plant Physiology. 2002;130:1487–1496. doi: 10.1104/pp.007971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Woodward AW, Bartel B. Auxin: regulation, action, and interaction. Annals of Botany. 2005;95:707–735. doi: 10.1093/aob/mci083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Worley CK, Zenser N, Ramos J, Rouse D, Leyser O, Theologis A, Callis J. Degradation of Aux/IAA proteins is essential for normal auxin signalling. Plant Journal. 2000;21:553–562. doi: 10.1046/j.1365-313x.2000.00703.x. [DOI] [PubMed] [Google Scholar]
  44. Yamamoto KT, Mori H, Imaseki H. cDNA cloning of indole-3-acetic acid-regulated genes – Aux22 and Saur from mung bean (Vigna radiata) hypocotyl tissue. Plant and Cell Physiology. 1992;33:93–97. [Google Scholar]
  45. Yasuor H, Abu-Abied M, Belausov E, Madmony A, Sadot E, Riov J, Rubin B. Glyphosate-induced anther indehiscence in cotton is partially temperature dependent and involves cytoskeleton and secondary wall modifications and auxin accumulation. Plant Physiology. 2006;141:1306–1315. doi: 10.1104/pp.106.081943. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Zenser N, Ellsmore A, Leasure C, Callis J. Auxin modulates the degradation rate of Aux/IAA proteins. Proceedings of the National Academy of Sciences of the United States of America. 2001;98:11795–11800. doi: 10.1073/pnas.211312798. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Zenser N, Dreher KA, Edwards SR, Callis J. Acceleration of Aux/IAA proteolysis is specific for auxin and independent of AXR1. Plant Journal. 2003;35:285–294. doi: 10.1046/j.1365-313x.2003.01801.x. [DOI] [PubMed] [Google Scholar]

Articles from Annals of Botany are provided here courtesy of Oxford University Press

RESOURCES