Arf2 Mutants Research Articles

The Knockdown of AUXIN RESPONSE FACTOR 2 Confers Enhanced Tolerance to Salt and Drought Stresses in Tomato (Solanum lycopersicum L.).

Auxin response factors (ARFs) act as key elements of the auxin-signaling pathway and play important roles in the process of a plant's growth, development, and response to environmental conditions. We studied the implication of the SlARF2 gene in the tomato response to salt (150 mM of NaCl) and drought (15% PEG 20000) stresses. The functional characterization of SlARF2 knockdown tomato mutants revealed that the downregulation of this gene enhanced primary root length and root branching and reduced plant wilting. At the physiological level, the arf2 mutant line displayed higher chlorophyll, soluble sugars, proline, and relative water contents as well as lower stomatal conductance and a decreased malondialdehyde content. Moreover, SlARF2 knockdown tomato mutants demonstrated higher activities of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) under salt and drought stresses than the wild type. Indeed, the stress tolerance of the arf2 mutant was also reflected by the upregulation of stress-related genes involved in ROS scavenging and plant defense, including SOD, CAT, dehydration-responsive element-binding protein, and early responsive to dehydration, which can ultimately result in a better resistance to salt and drought stresses. Furthermore, the transcriptional levels of the Δ1-pyrroline-5-carboxylate synthase (P5CS) gene were upregulated in the arf2 mutant after stress, in correlation with the higher levels of proline. Taken together, our findings reveal that SlARF2 is implicated in salt and drought tolerance in tomato and provides some considerable elements for improving the abiotic stress tolerance and increasing the crop yields of tomato.

ARF2 positively regulates flavonols and proanthocyanidins biosynthesis in Arabidopsis thaliana.

Auxin response factor 2 acts as a positive regulator to fine-tune the spatial and temporal accumulation of flavonoid compounds, mainly flavonols and proanthocyanidins in Arabidopsis. Auxin response factor (ARF) proteins are reported to involve in auxin-mediated regulation of flavonoid biosynthesis. However, the detailed regulation mechanism of ARF remains still unknown. Here, we provide genetic and molecular evidence that one of the twenty-three ARF members-ARF2-positively regulates flavonoid biosynthesis at multi-level in tissue-specific manner in Arabidopsis thaliana. Loss-of-function mutation of ARF2 led to significant reduction in flavonoid content (e.g., flavonols and proanthocyanidins) in the seedlings and seeds of the Arabidopsis arf2 mutants. Over-expression of ARF2 increased flavonols and proanthocyanidins content in Arabidopsis. Additionally, the changes of flavonoid content correlate well with the transcript abundance of several regulatory genes (e.g., MYB11, MYB12, MYB111, TT2, and GL3), and key biosynthetic genes (e.g., CHS, F3'H, FLS, ANS, ANR, TT12, TT19, and TT15), in the arf2 mutant and ARF2 over-expression lines. Transient transactivation assays with site-directed mutagenesis confirmed that ARF2 directly regulates the expression of MYB12 and FLS genes in the flavonol pathway and ANR in the proanthocyanidin pathway, and indirectly regulates MYB11 and MYB111 genes in the flavonol pathway, and ANS, TT12, TT19 and TT15 genes in the proanthocyanidin pathway. Further genetic results indicated that ARF2 acts upstream of MYB12 to regulate flavonol accumulation, and of TT2 to regulate proanthocyanidins accumulation. In particular, yeast two-hybrid assays revealed that ARF2 physically interacts with TT2, a master regulator of proanthocyanidins biosynthesis. Combined together, these results indicated that ARF2 functions as a positive regulator for the fine-tuned spatial and temporal regulation of flavonoids (mainly flavonols and proanthocyanidins) accumulation in Arabidopsis.

Two TPL-Binding Motifs of ARF2 Are Involved in Repression of Auxin Responses.

Auxin signaling is finalized by activator auxin response factors (aARFs) that are released from Auxin/Indole-3-Acetic Acid (Aux/IAA) repressors and directly activate auxin-responsive genes. However, it remains to be answered how repressor ARFs (rARFs) exert their repression function. In this study, we assessed the molecular and biological functions of two putative co-repressor-binding motifs (EAR and RLFGI) of ARF2 (a rARF) in Arabidopsis thaliana. In the yeast two-hybrid assay, the EAR mutation moderately and the RLFGI mutation, or both motifs, almost completely disrupted the interaction between the co-repressor TOPLESS (TPL) and the repressive motifs-containing middle domain (MD) of ARF2. The ARF2-MD interacted not only with TPL but also with TPL homologs (TPRs). Root hair-specific overexpression of rARFs (ARF1–4, 9–11, and 16) considerably inhibited root hair growth, suggesting that rARFs generally function as repressors in the auxin-responsive root hair single cell. Individual mutation of the ARF2 EAR or RLFGI motif slightly and both mutations greatly compromised ARF2-mediated inhibition of root hair growth and auxin-responsive gene expression. In addition, flowering time and seed size, two representative arf2 mutant phenotypes, were examined to assess the function of the repressive motifs in mutant-complementation experiments. ARF2-mediated inhibition of flowering and seed growth was suppressed considerably by the individual mutation of EAR or RLFGI and almost completely by both mutations. These results suggest that EAR and RLFGI work together as major repressive motifs for ARF2 to recruit TPL/TPR co-repressors and to exhibit its repressive biological functions.

ARF2 coordinates with PLETHORAs and PINs to orchestrate ABA-mediated root meristem activity in Arabidopsis .

Multiple hormones, including abscisic acid (ABA) and auxin, regulate cell division and differentiation of Arabidopsis root meristems. AUXIN RESPONSE FACTOR 2 (ARF2) functions as a negative regulator of ABA responses, as seed germination and primary root growth of arf2 mutants are hypersensitive to ABA. In this study, we found that ABA treatment reduced the expression levels of the PIN-FORMEDs (PIN) auxin efflux carriers, PIN1, PIN3, PIN4, and PIN7, to a greater extent in the root meristems of arf2-101 mutant than in the wild type. Also, arf2-101 pin1 and arf2-101 pin4 double mutants show less ABA-induced inhibition of root meristem activity than the arf2-101 mutants. Furthermore, ARF2 positively mediates the transcripts of transcription factor PLETHORA 1 (PLT1) gene but negatively mediates PLT2 at protein level in root meristems. Using a dexamethasone (DEX)-inducible transgenic line, Pro35S:PLT2-GR, we showed that PLT2 greatly promotes cell division and completely inhibits cell differentiation in root meristems of the arf2-101 mutant once PLT2 is induced by DEX, which can be partially reversed by ABA treatment, suggesting that ABA regulates root meristem activity in both ARF2-dependent and independent pathways. Our results uncover a complex regulatory architecture in which ARF2 coordinates with PLTs and PINs to orchestrate ABA-mediated regulation of root meristem activity in Arabidopsis.

Phosphorylation of ARF2 Relieves Its Repression of Transcription of the K+ Transporter Gene HAK5 in Response to Low Potassium Stress.

Potassium (K+) plays crucial roles in plant growth and development. In natural environments, K+ availability in soils is relatively low and fluctuating. Transcriptional regulation of K+ transporter genes is one of the most important mechanisms in the plant's response to K+ deficiency. In this study, we demonstrated that the transcription factor ARF2 (Auxin Response Factor 2) modulates the expression of the K+ transporter gene HAK5 (High Affinity K+ transporter 5) in Arabidopsis thaliana The arf2 mutant plants showed a tolerant phenotype similar to the HAK5-overexpressing lines on low-K+ medium, whose primary root lengths were longer than those of wild-type plants. High-affinity K+ uptake was significantly increased in these plants. ARF2-overexpressing lines and the hak5 mutant were both sensitive to low-K+ stress. Disruption of HAK5 in the arf2 mutant abolished the low-K+-tolerant phenotype of arf2 As a transcriptional repressor, ARF2 directly bound to the HAK5 promoter and repressed HAK5 expression under K+ sufficient conditions. ARF2 can be phosphorylated after low-K+ treatment, which abolished its DNA binding activity to the HAK5 promoter and relieved the inhibition on HAK5 transcription. Therefore, HAK5 transcript could be induced, and HAK5-mediated high-affinity K+ uptake was enhanced under K+ deficient conditions. The presented results demonstrate that ARF2 plays important roles in the response to external K+ supply in Arabidopsis and regulates HAK5 transcription accordingly.

Jasmonate-Dependent and COI1-Independent Defense Responses Against Sclerotinia sclerotiorum in Arabidopsis thaliana: Auxin is Part of COI1-Independent Defense Signaling

The jasmonate receptor COI1 is known to facilitate plant defense responses against necrotrophic pathogens, including the ascomycete Sclerotinia sclerotiorum. However, it is not known to what extent jasmonates contribute to defense nor have COI1-independent defense pathways been sufficiently characterized. Here we show that the susceptibility to S. sclerotiorum of the aos mutant, deficient in biosynthesis of jasmonic acid (JA) and its precursor 12-oxophytadienoic acid, was elevated to a level reminiscent of that of hypersusceptible coi1 mutants. In contrast, susceptibility of the JA-deficient opr3 mutant was comparable with that of the wild type. A set of 99 genes responded similarly to infection with S. sclerotiorum in wild-type and coi1 mutant leaves. Expression of this COI1-independent gene set correlated with known differences in gene expression between wild-type plants and a mutant in the transcriptional repressor auxin response factor 2 (arf2). Susceptibility to S. sclerotiorum was reduced in two arf2 mutants early during infection, implicating ARF2 as a negative regulator of defense responses against this pathogen. Hypersusceptibility of an axr1 mutant to S. sclerotiorum confirmed the contribution of auxin action to defense responses against this fungal pathogen.

Auxin Response Factor2 (ARF2) and Its Regulated Homeodomain Gene HB33 Mediate Abscisic Acid Response in Arabidopsis

The phytohormone abscisic acid (ABA) is an important regulator of plant development and response to environmental stresses. In this study, we identified two ABA overly sensitive mutant alleles in a gene encoding Auxin Response Factor2 (ARF2). The expression of ARF2 was induced by ABA treatment. The arf2 mutants showed enhanced ABA sensitivity in seed germination and primary root growth. In contrast, the primary root growth and seed germination of transgenic plants over-expressing ARF2 are less inhibited by ABA than that of the wild type. ARF2 negatively regulates the expression of a homeodomain gene HB33, the expression of which is reduced by ABA. Transgenic plants over-expressing HB33 are more sensitive, while transgenic plants reducing HB33 by RNAi are more resistant to ABA in the seed germination and primary root growth than the wild type. ABA treatment altered auxin distribution in the primary root tips and made the relative, but not absolute, auxin accumulation or auxin signal around quiescent centre cells and their surrounding columella stem cells to other cells stronger in arf2-101 than in the wild type. These results indicate that ARF2 and HB33 are novel regulators in the ABA signal pathway, which has crosstalk with auxin signal pathway in regulating plant growth.

Yield assessment of integument‐led seed growth following targeted repair of auxin response factor 2

It is becoming increasingly vital to improve the yield of seed crops to feed an expanding population and, more recently, for biofuel production. One strategy to increase the yield is to increase the seed size, provided that there is not a concomitant decrease in seed number. In a previous study, we described a mutant in the auxin response factor 2 (ARF2) gene which produced extra cells in the seed coat and, subsequently, enlarged seeds. However, arf2 mutant plants also show severely reduced self-fertility caused, in part, by over-elongated sepals that prevent flower opening. As a low seed set increases individual seed size, a meaningful comparison of the yield in arf2 and wild-type plants could not be conducted. In this study, we show that targeted expression of wild-type ARF2 in the sepals and petals of arf2-9 mutant flowers restores flower opening and dramatically increases seed set. The restored plants retain both enlarged integuments and increased seed size, reinforcing previous evidence that arf2 mutations increase seed weight through their effect on integuments and not only via reduced fertility. We also show that the measurement of the harvest index in Arabidopsis is useful in assessing the impact of introduced traits on the yield.

AUXIN RESPONSE FACTOR1andAUXIN RESPONSE FACTOR2regulate senescence and floral organ abscission inArabidopsis thaliana

In plants, both endogenous mechanisms and environmental signals regulate developmental transitions such as seed germination, induction of flowering, leaf senescence and shedding of senescent organs. Auxin response factors (ARFs) are transcription factors that mediate responses to the plant hormone auxin. We have examined Arabidopsis lines carrying T-DNA insertions in AUXIN RESPONSE FACTOR1 (ARF1) and ARF2 genes. We found that ARF2 promotes transitions between multiple stages of Arabidopsis development. arf2 mutant plants exhibited delays in several processes related to plant aging, including initiation of flowering, rosette leaf senescence, floral organ abscission and silique ripening. ARF2 expression was induced in senescing leaves. ARF2 regulated leaf senescence and floral organ abscission independently of the ethylene and cytokinin response pathways. arf1 mutations enhanced many arf2 phenotypes, indicating that ARF1 acts in a partially redundant manner with ARF2. However, unlike arf2 mutations, an arf1 mutation increased transcription of Aux/IAA genes in Arabidopsis flowers, supporting previous biochemical studies that indicated that ARF1 is a transcriptional repressor. Two other ARF genes, NPH4/ARF7 and ARF19, were also induced by senescence, and mutations in these genes enhanced arf2 phenotypes. NPH4/ARF7 and ARF19 function as transcriptional activators, suggesting that auxin may control senescence in part by activating gene expression.

AUXIN RESPONSE FACTOR 2 (ARF2): a pleiotropic developmental regulator

AUXIN RESPONSE FACTORS (ARFs) regulate auxin-mediated transcriptional activation/repression. They are encoded by a gene family in Arabidopsis, and each member is thought to play a central role in various auxin-mediated developmental processes. We have characterized three arf2 mutant alleles, arf2-6, arf2-7 and arf2-8. The mutants exhibit pleiotropic developmental phenotypes, including large, dark green rosette leaves, delayed flowering, thick and long inflorescence, abnormal flower morphology and sterility in early formed flowers, large organ size and delayed senescence and abscission, compared with wild-type plants. In addition, arf2 mutant seedlings have elongated hypocotyls with enlarged cotyledons under various light conditions. The transcription of ACS2, ACS6 and ACS8 genes is impaired in the developing siliques of arf2-6. The phenotypes of all three alleles are similar to those of the loss-of-function mutants obtained by RNA interference or co-suppression. There is no significant effect of the mutation on global auxin-regulated gene expression in young seedlings, suggesting that ARF2 does not participate in auxin signaling at that particular developmental stage of the plant life cycle. Because ARF2 is thought to function as a transcriptional repressor, the prospect arises that its pleiotropic effects may be mediated by negatively modulating the transcription of downstream genes in signaling pathways that are involved in cell growth and senescence.

Arf1 GTPase plays roles in the protein traffic between the endoplasmic reticulum and the Golgi apparatus in tobacco and Arabidopsis cultured cells.

Arf GTPases are known to be key regulators of vesicle budding in various steps of membrane traffic in yeast and animal cells. We cloned the Arabidopsis Arf1 homologue, AtArf1, and examined its function. AtArf1 complements yeast arf1 arf2 mutants and its GFP-fusion is localized to the Golgi apparatus in plant cells like its animal counterpart. The expression of dominant negative mutants of AtArf1 in tobacco and Arabidopsis cultured cells affected the localization of co-expressed GFP-tagged proteins in a variety of ways. AtArf1 Q71L and AtArf1 T31N, GTP- and GDP-fixed mutants, respectively, changed the localization of a cis-Golgi marker, AtErd2-GFP, from the Golgi apparatus to the endoplasmic reticulum but not that of GFP-AtRer1B or GFP-AtSed5. GFP-AtRer1B and GFP-AtSed5 were accumulated in aberrant structures of the Golgi by AtArf1 Q71L. A soluble vacuolar protein, sporamin-GFP, was also located to the ER by AtArf1 Q71L. These results indicate that AtArf1 play roles in the vesicular transport between the ER and the Golgi and in the maintenance of the normal Golgi organization in plant cells.

ADP-Ribosylation factors do not activate yeast phospholipase Ds but are required for sporulation.

ADP-ribosylation factor (ARF) proteins in Saccharomyces cerevisiae are encoded by two genes, ARF1 and ARF2. The addition of the c-myc epitope at the C terminus of Arf1 resulted in a mutant (arf1-myc arf2) that supported vegetative growth and rescued cells from supersensitivity to fluoride, but homozygous diploids failed to sporulate. arf1-myc arf2 mutants completed both meiotic divisions but were unable to form spores. The SPO14 gene encodes a phospholipase D (PLD), whose activity is essential for mediating the formation of the prospore membrane, a prerequisite event for spore formation. Spo14 localized normally to the developing prospore membrane in arf1-myc arf2 mutants; however, the synthesis of the membrane was attenuated. This was not a consequence of reduced PLD catalytic activity, because the enzymatic activity of Spo14 was unaffected in meiotic arf1-myc arf2 mutants. Although potent activators of mammalian PLD1, Arf1 proteins did not influence the catalytic activities of either Spo14 or ScPld2, a second yeast PLD. These results demonstrate that ARF1 is required for sporulation, and the mitotic and meiotic functions of Arf proteins are not mediated by the activation of any known yeast PLD activities. The implications of these results are discussed with respect to current models of Arf signaling.