Altered Architecture and Enhanced Drought Tolerance in Rice via the Down-Regulation of Indole-3-Acetic Acid by TLD1/OsGH3.13 Activation

Plant architecture is determined by genetic and developmental programs as well as by environmental factors. Sessile plants have evolved a subtle adaptive mechanism that allows them to alter their growth and development during periods of stress. Phytohormones play a central role in this process; however, the molecules responsible for integrating growth- and stress-related signals are unknown. Here, we report a gain-of-function rice (Oryza sativa) mutant, tld1-D, characterized by (and named for) an increased number of tillers, enlarged leaf angles, and dwarfism. TLD1 is a rice GH3.13 gene that encodes indole-3-acetic acid (IAA)-amido synthetase, which is suppressed in aboveground tissues under normal conditions but which is dramatically induced by drought stress. The activation of TLD1 reduced the IAA maxima at the lamina joint, shoot base, and nodes, resulting in subsequent alterations in plant architecture and tissue patterning but enhancing drought tolerance. Accordingly, the decreased level of free IAA in tld1-D due to the conjugation of IAA with amino acids greatly facilitated the accumulation of late-embryogenesis abundant mRNA compared with the wild type. The direct regulation of such drought-inducible genes by changes in the concentration of IAA provides a model for changes in plant architecture via the process of drought adaptation, which occurs frequently in nature.Plant architecture is vitally important for rice (Oryza sativa), as it is closely related to yield potential. Plants with a desirable structural form are capable of increased grain production in resource-limited fields compared with plants with less desirable builds. The three fundamental determinants of rice architecture, tiller number, leaf/tiller angle, and plant height, which are also important agronomic traits, are largely under the control of genetic and developmental programs; however, they are also influenced by environmental factors.The effects of environmental conditions on plant architecture are derived from the fact that increased planting density inhibits the production of vegetative branches by grasses due to shade and nutrient deficiencies (Doust, 2007). In addition, sessile plants are frequently subjected to abiotic stresses, such as water deficiency, that can decrease the number of tillers and plant height.The genetic and environmental interactions involved in determining plant architecture are thought to be mediated by plant hormones. The fact that some of the genes cloned from morphologically defective mutants encode components involved in hormone biosynthesis, perception, and signaling suggests that the phytohormone orchestra plays a crucial role in the creation of plant architecture, and auxin appears to be the central player. As seen in the maintenance of apical dominance, basipetal polar auxin transport in the shoot suppresses axillary bud outgrowth at the shoot base, likely through interplay with other messengers, including cytokinin and strigolactone, a newly discovered branching hormone (for review, see McSteen, 2009).The establishment of an auxin gradient is prerequisite for its functions in plant morphogenesis, which include effects on root patterning, vascular tissue differentiation, axillary bud formation, flower organ development, and tropistic growth (Benková et al., 2003; Friml et al., 2003; Esmon et al., 2006). The auxin maxima in the plant body determine the location of primordia outgrowth, which involves the creation of distinct cell types that will give rise to various organs (Reinhardt et al., 2000; Grieneisen et al., 2007). Thus, the presence of stable local auxin maxima is a deciding factor in the ability of a plant to adopt an appropriate structural design. The asymmetric accumulation of auxin in certain cells results mainly from directional transport as well as from the dynamic biosynthesis, degradation, and conjugation of free indole-3-acetic acid (IAA), the main form of active auxin in plants (Woodward and Bartel, 2005; Paciorek and Friml, 2006). In rice, the perturbation of auxin homeostasis causes pleiotropic abnormalities leading to dramatic changes in plant architecture. For example, enhanced or reduced IAA biosynthesis via the overexpression or repression of OsYUCCA1 results in fluctuations in the level of IAA. These fluctuations produce severe abnormalities in shoot, root, and stem development, leading to dwarfism in transgenic rice plants (Yamamoto et al., 2007).The maintenance of IAA homeostasis through the conversion of free IAA to a conjugated form is a conserved mechanism in monocots and dicots. Several gene families have been identified that are involved in the conjugation of free IAA with sugars, amino acids, or methyl groups (Qin et al., 2005; Woodward and Bartel, 2005). Proteins belonging to the GH3 family are responsible for converting active IAA to its inactive form via the conjugation of IAA with amino acids (Staswick et al., 2005). GH3 was first identified in Glycine max as an early auxin-responsive gene (Hagen and Guilfoyle, 1985). GH3 functions in the negative feedback regulation of IAA concentration, in that excess IAA up-regulates GH3 expression, causing the IAA conjugated to amino acids to be either stored or degraded. It has been shown that members of this gene family in Arabidopsis (Arabidopsis thaliana) are also regulated by hormones and environmental factors, including salicylic acid (SA), abscisic acid (ABA), pathogen infection, and light (Takase et al., 2004; Park et al., 2007a, 2007b; Zhang et al., 2007). Therefore, in addition to functioning in growth and development under normal conditions, GH3 genes also participate in plant resistance to biotic and abiotic stress. Similarly, OsGH3.8 and OsGH3.1 in rice reportedly play dual roles in development and bacterial resistance through the regulation of auxin signaling (Ding et al., 2008; Domingo et al., 2009). However, no other OsGH3 members have been reported that function in abiotic stress adaptation in rice.Drought stress triggers the production of ABA and induces the expression of numerous genes via ABA-dependent and -independent pathways. Synchronized changes in plant architecture during drought-stress adaptation have been observed; however, no molecular mechanism has been reported. Here, we describe the cloning of OsGH3.13, a new member of the GH3 gene family, from a gain-of-function mutant, tld1-D (for increased number of tillers, enlarged leaf angles, and dwarfism). TLD1/OsGH3.13 is suppressed in aboveground tissues in rice under normal growth conditions in order to maintain a reasonable structural design; however, it is strongly induced in rice seedlings subjected to drought stress. The activation of TLD1/OsGH3.13 in tld1-D mutant rice results in IAA deficiency and dramatic changes in architecture; however, it also enhances drought tolerance. The loss-of-function mutant tld1 does not show visible differences from wild-type plants in normal growth and drought conditions. Here, we provide evidence that the down-regulation of IAA facilitates the accumulation of late-embryogenesis abundant (LEA) mRNA and switches the focus of rice plants from growth to stress adaptation at the expense of plant architecture and tissue patterning. Our results demonstrate the crucial role of IAA in integrating diverse developmental and environmental cues.     

Genome‐wide identification,expression analysis of auxin‐responsive GH3 family genes in maize (Zea mays L.) under abiotic stresses

Auxin is involved in different aspects of plant growth and development by regulating the expression of auxinresponsive family genes. As one of the three major auxinresponsive families, GH3(Gretchen Hagen3) genes participate in auxin homeostasis by catalyzing auxin conjugation and bounding free indole-3-acetic acid(IAA) to amino acids.However, how GH3 genes function in responses to abiotic stresses and various hormones in maize is largely unknown.Here, the latest updated maize(Zea mays L.) reference genome sequence was used to characterize and analyze the Zm GH3 family genes from maize. The results showed that 13 Zm GH3 genes were mapped on fi ve maize chromosomes(total10 chromosomes). Highly diversi fi ed gene structures and tissue-speci fi c expression patterns suggested the possibility of function diversi fi cation for these genes in response to environmental stresses and hormone stimuli. The expression patterns of Zm GH3 genes are responsive to several abiotic stresses(salt, drought and cadmium) and major stress-relatedhormones(abscisic acid, salicylic acid and jasmonic acid)Various environmental factors suppress auxin free IAA contents in maize roots suggesting that these abiotic stresses and hormones might alter GH3-mediated auxin levels. The respon siveness of Zm GH3 genes to a wide range of abiotic stresses and stress-related hormones suggested that Zm GH3 s are involved in maize tolerance to environmental stresses.     

Overexpression of the Arabidopsis Gene UPRIGHT ROSETTE Reveals a Homeostatic Control for Indole-3-Acetic Acid

Auxins are phytohormones that are essential for many aspects of plant growth and development. The main auxin produced by plants is indole-3-acetic acid (IAA). IAA exists in free and conjugated forms, corresponding to the bioactive and stored hormones, respectively. Free IAA levels, which are crucial for various physiological activities, are maintained through a complex network of environmentally and developmentally responsive pathways including IAA biosynthesis, transport, degradation, conjugation, and conjugate hydrolysis. Among conjugated IAA forms, ester- and amide-type conjugates are the most common. Here we identify a new gene, UPRIGHT ROSETTE (URO), the overexpression of which alters IAA homeostasis in Arabidopsis (Arabidopsis thaliana). We previously identified a semidominant mutant, uro, which had multiple auxin-related phenotypes. We show here that compared to wild-type plants, the uro plants contain increased levels of free and ester-conjugated IAA, and decreased levels of amino-conjugated IAA. uro plants carrying the pDR5:β-glucuronidase (GUS) construct have strong GUS staining in cotyledons and stem, and their cotyledons are able to generate roots on auxin-free medium, further confirming that this mutant contains higher levels of free IAA. The URO gene encodes a C2H2 zinc-finger protein that belongs to a plant-specific gene family. The response to URO overexpression is evolutionarily conserved among plants, as GUS activity that may reflect free IAA levels was increased markedly in transgenic p35S:URO/pGH3:GUS/Physcomitrella patens and pNOS:URO/pGH3:GUS/P. patens plants.Auxin is an important plant hormone that modulates numerous processes throughout plant growth and development. It has roles in tropic responses to light and gravity, general root and shoot architecture, organ initiation and patterning, and vascular development (Woodward and Bartel, 2005; Benjamins and Scheres, 2008; Vanneste and Friml, 2009). The major naturally occurring auxin in plants is indole-3-acetic acid (IAA). Local free IAA is bioactive and contributes to the regulation of plant growth and development. Free IAA can be converted into conjugated forms, which lose biological activity, through modification of the indole ring or side chain (Ljung et al., 2002; Bajguz and Piotrowska, 2009). Hydrolysis of IAA conjugates, which releases free IAA, is another important aspect in plant IAA homeostatic control. The major IAA conjugates, ester- and amide-type conjugated IAA, can be both hydrolyzed to free IAA (Ljung et al., 2002; Bajguz and Piotrowska, 2009).In higher plants, free IAA concentration is usually very low, often in the nanomolar range (Woodward and Bartel, 2005). In whole seedlings of Arabidopsis (Arabidopsis thaliana), the amide-type IAA conjugates constitute approximately 90% of the IAA pool, whereas the ester-type conjugates and free IAA account for approximately 10% and 1%, respectively (Tam et al., 2000). This generally maintained distribution of IAA forms indicates that an appropriate amount of free IAA in local plant tissues is crucial for specific physiological activities and must be strictly maintained.Recent studies have provided important information toward the understanding of IAA homeostasis. SUPERROOT1 (SUR1) and SUR2 encode a C-S lyase (Mikkelsen et al., 2004) and a cytochrome P450 protein (Barlier et al., 2000), respectively. Both of these genes are likely to be involved in glucosinolate biosynthesis. Since both glucosinolate and IAA biosyntheses require common precursors, a block in glucosinolate biosynthesis diverts the precursor compounds into IAA biosynthesis. Mutant seedlings of sur1 and sur2 have an elongated hypocotyl, epinastic cotyledons, and an increased number of lateral roots. The YUCCAs, which encode flavin monooxygenase-like proteins, constitute another group of genes involved in IAA biosynthesis (Zhao et al., 2001; Cheng et al., 2006, 2007; Kim et al., 2007). Dominant activation-tagged yucca mutants have elongated hypocotyls, epinastic leaves, and increased apical dominance (Zhao et al., 2001; Kim et al., 2007). The loss-of-function sur1 and 2 mutants and the gain-of-function yucca mutants contain increased levels of free IAA at specific developmental stages. The sur1 mutant also has increased levels of conjugated IAA on a whole-seedling basis. It has been proposed that the increased levels of IAA conjugates in sur1 may be due to an increased rate of conjugation, induced by the larger amount of free IAA (Ljung et al., 2002). TAA1 encodes an aminotransferase, which catalyzes the formation of indole-3-pyruvic acid from l-Trp in another IAA biosynthetic pathway (Tao et al., 2008). Loss-of-function taa1 mutants have a reduction in free IAA levels, demonstrating the importance of the indole-3-pyruvic acid-dependent IAA biosynthesis pathway.In addition to the enzymes that are involved in IAA biosynthesis, enzymes involved in auxin that conjugate formation and hydrolysis also affect IAA homeostasis. These include IAA glucosyl-transferase, auxin-conjugate hydrolases, and GH3 IAA-amino acid synthases (Bartel and Fink, 1995; Davies et al., 1999; LeClere et al., 2002; Rosamond et al., 2002; Rampey et al., 2004; Staswick et al., 2005; Ludwig-Müller et al., 2009). The IAR4 gene, which encodes a putative mitochondrial pyruvate dehydrogenase E1α-subunit, is required for maintenance of amino-type conjugate levels. In the iar4-3 mutant, the IAA amino-conjugate level was significantly increased (Quint et al., 2009). Several genes in the GH3 family, such as GH3.2, GH3.3, GH3.4, GH3.5, GH3.6, and GH3.17 in Arabidopsis (Staswick et al., 2005) or GH3-like genes in moss (Ludwig-Müller et al., 2009), encode IAA-amido synthetases that catalyze the formation of auxin-amino acid conjugates. In addition, several amidohydrolases, such as ILR1, ILL1, ILL2, and IAR3, are involved in cleaving IAA amino acid conjugates to release free IAA in Arabidopsis (Davies et al., 1999; LeClere et al., 2002). The triple mutant ilr1 iar3 ill2 has lower levels of free IAA, and higher levels of amino-type IAA conjugates (Rampey et al., 2004).The recent research has led to a clear picture showing that there are a number of components affecting IAA homeostasis in plants. However, little is known about how the coordination of these components is regulated to fulfill each corresponding function. Perhaps one limitation to this question is that most of the IAA homeostasis-affecting components identified thus far are metabolic enzymes, not putative regulatory factors. We previously reported characterizations of a semidominant Arabidopsis mutant, upright rosette (uro), which displayed multiple auxin-related phenotypes (Sun et al., 2000; Guo et al., 2004; Yuan et al., 2007). Our genetic analyses revealed that the uro phenotypes are closely linked to a T-DNA insertion and are caused by a single nuclear gene mutation (Sun et al., 2000). In this study, we report the identification of the URO gene, which encodes a putative C2H2 zinc-finger protein. We demonstrate that the uro auxin-related phenotypes are caused by URO overexpression. Our data also suggest that a potentially existing plant regulatory system(s) for IAA homeostasis control responds to the URO overexpression, as the uro mutation resulted in an altered distribution of the free, ester-, and amide-conjugated IAA levels in Arabidopsis. In addition, we show that the response to URO overexpression is consistent in Physcomitrella moss plants, suggesting a conserved homeostasis control of IAA in plants.     

Dual regulation role of GH3.5 in salicylic acid and auxin signaling during Arabidopsis-Pseudomonas syringae interaction

Salicylic acid (SA) plays a central role in plant disease resistance, and emerging evidence indicates that auxin, an essential plant hormone in regulating plant growth and development, is involved in plant disease susceptibility. GH3.5, a member of the GH3 family of early auxin-responsive genes in Arabidopsis (Arabidopsis thaliana), encodes a protein possessing in vitro adenylation activity on both indole-3-acetic acid (IAA) and SA. Here, we show that GH3.5 acts as a bifunctional modulator in both SA and auxin signaling during pathogen infection. Overexpression of the GH3.5 gene in an activation-tagged mutant gh3.5-1D led to elevated accumulation of SA and increased expression of PR-1 in local and systemic tissues in response to avirulent pathogens. In contrast, two T-DNA insertional mutations of GH3.5 partially compromised the systemic acquired resistance associated with diminished PR-1 expression in systemic tissues. The gh3.5-1D mutant also accumulated high levels of free IAA after pathogen infection and impaired different resistance-gene-mediated resistance, which was also observed in the GH3.6 activation-tagged mutant dfl1-D that impacted the auxin pathway, indicating an important role of GH3.5/GH3.6 in disease susceptibility. Furthermore, microarray analysis showed that the SA and auxin pathways were simultaneously augmented in gh3.5-1D after infection with an avirulent pathogen. The SA pathway was amplified by GH3.5 through inducing SA-responsive genes and basal defense components, whereas the auxin pathway was derepressed through up-regulating IAA biosynthesis and down-regulating auxin repressor genes. Taken together, our data reveal novel regulatory functions of GH3.5 in the plant-pathogen interaction.     

Acyl substrate preferences of an IAA-amido synthetase account for variations in grape (Vitis vinifera L.) berry ripening caused by different auxinic compounds indicating the importance of auxin conjugation in plant development

Nine Gretchen Hagen (GH3) genes were identified in grapevine (Vitis vinifera L.) and six of these were predicted on the basis of protein sequence similarity to act as indole-3-acetic acid (IAA)-amido synthetases. The activity of these enzymes is thought to be important in controlling free IAA levels and one auxin-inducible grapevine GH3 protein, GH3-1, has previously been implicated in the berry ripening process. Ex planta assays showed that the expression of only one other GH3 gene, GH3-2, increased following the treatment of grape berries with auxinic compounds. One of these was the naturally occurring IAA and the other two were synthetic, α-naphthalene acetic acid (NAA) and benzothiazole-2-oxyacetic acid (BTOA). The determination of steady-state kinetic parameters for the recombinant GH3-1 and GH3-2 proteins revealed that both enzymes efficiently conjugated aspartic acid (Asp) to IAA and less well to NAA, while BTOA was a poor substrate. GH3-2 gene expression was induced by IAA treatment of pre-ripening berries with an associated increase in levels of IAA-Asp and a decrease in free IAA levels. This indicates that GH3-2 responded to excess auxin to maintain low levels of free IAA. Grape berry ripening was not affected by IAA application prior to veraison (ripening onset) but was considerably delayed by NAA and even more so by BTOA. The differential effects of the three auxinic compounds on berry ripening can therefore be explained by the induction and acyl substrate specificity of GH3-2. These results further indicate an important role for GH3 proteins in controlling auxin-related plant developmental processes.     

Expression of two HOOKLESS genes in peas (Pisum sativum L.)

The apical hook of dark-grown dicotyledonous plants results from asymmetric growth of its inner and outer sides. It is a protective structure that prevents damage to the shoot apical meristem and the young leaves as the seedling pushes through the soil. Two phytohormones, ethylene and auxin, are thought to be involved in regulating apical hook formation. HOOKLESS1 (HLS1) of Arabidopsis was recognized as an ethylene-response gene whose product is required for hook formation. We cloned two cDNAs from peas, Ps-HLS1 and Ps-HLS2, whose products are functional homologs of HLS1. Both Ps-HLS1 and Ps-HLS2 complement the hls1 mutation in Arabidopsis. Expression of Ps-HLS1 is enhanced by ethylene and by IAA. Because the effect of ethylene is counteracted by 2,5-norbornadiene, an inhibitor of ethylene action, it appears that the primary factor in apical hook formation in peas is ethylene.     

Auxin and light control of adventitious rooting in Arabidopsis require ARGONAUTE1

Adventitious rooting is a quantitative genetic trait regulated by both environmental and endogenous factors. To better understand the physiological and molecular basis of adventitious rooting, we took advantage of two classes of Arabidopsis thaliana mutants altered in adventitious root formation: the superroot mutants, which spontaneously make adventitious roots, and the argonaute1 (ago1) mutants, which unlike superroot are barely able to form adventitious roots. The defect in adventitious rooting observed in ago1 correlated with light hypersensitivity and the deregulation of auxin homeostasis specifically in the apical part of the seedlings. In particular, a clear reduction in endogenous levels of free indoleacetic acid (IAA) and IAA conjugates was shown. This was correlated with a downregulation of the expression of several auxin-inducible GH3 genes in the hypocotyl of the ago1-3 mutant. We also found that the Auxin Response Factor17 (ARF17) gene, a potential repressor of auxin-inducible genes, was overexpressed in ago1-3 hypocotyls. The characterization of an ARF17-overexpressing line showed that it produced fewer adventitious roots than the wild type and retained a lower expression of GH3 genes. Thus, we suggest that ARF17 negatively regulates adventitious root formation in ago1 mutants by repressing GH3 genes and therefore perturbing auxin homeostasis in a light-dependent manner. These results suggest that ARF17 could be a major regulator of adventitious rooting in Arabidopsis.     

Convergence of signaling pathways in the control of differential cell growth in Arabidopsis

Seedling apical hook development involves a complex interplay of hormones and light in the regulation of differential cell growth. However, the underlying molecular mechanisms that integrate these diverse signals to control bending of the embryonic stem are poorly understood. The Arabidopsis ethylene-regulated HOOKLESS1 (HLS1) gene is essential for apical hook formation. Herein, we identify two auxin response regulators that act downstream of HLS1 to control cell elongation in the hypocotyl. Extragenic suppressors of hls1 were identified as mutations in AUXIN RESPONSE FACTOR 2 (ARF2). The level of ARF2 protein was decreased by ethylene, and this response required HLS1. Exposure to light decreased HLS1 protein levels and evoked a concomitant increase in ARF2 accumulation. These studies demonstrate that both ethylene and light signals affect differential cell growth by acting through HLS1 to modulate the auxin response factors, pinpointing HLS1 as a key integrator of the signaling pathways that control hypocotyl bending.     

Activation of the indole-3-acetic acid-amido synthetase GH3-8 suppresses expansin expression and promotes salicylate- and jasmonate-independent basal immunity in rice

New evidence suggests a role for the plant growth hormone auxin in pathogenesis and disease resistance. Bacterial infection induces the accumulation of indole-3-acetic acid (IAA), the major type of auxin, in rice (Oryza sativa). IAA induces the expression of expansins, proteins that loosen the cell wall. Loosening the cell wall is key for plant growth but may also make the plant vulnerable to biotic intruders. Here, we report that rice GH3-8, an auxin-responsive gene functioning in auxin-dependent development, activates disease resistance in a salicylic acid signaling- and jasmonic acid signaling-independent pathway. GH3-8 encodes an IAA-amino synthetase that prevents free IAA accumulation. Overexpression of GH3-8 results in enhanced disease resistance to the rice pathogen Xanthomonas oryzae pv oryzae. This resistance is independent of jasmonic acid and salicylic acid signaling. Overexpression of GH3-8 also causes abnormal plant morphology and retarded growth and development. Both enhanced resistance and abnormal development may be caused by inhibition of the expression of expansins via suppressed auxin signaling.     

Auxin-related gene families in abiotic stress response in Sorghum bicolor

Sorghum, a C4 model plant, has been studied to develop an understanding of the molecular mechanism of resistance to stress. The auxin-response genes, auxin/indole-3-acetic acid (Aux/IAA), auxin-response factor (ARF), Gretchen Hagen3 (GH3), small auxin-up RNAs, and lateral organ boundaries (LBD), are involved in growth/development and stress/defense responses in Arabidopsis and rice, but they have not been studied in sorghum. In the present paper, the chromosome distribution, gene duplication, promoters, intron/exon, and phylogenic relationships of Aux/IAA, ARF, GH3, and LBD genes in sorghum are presented. Furthermore, real-time PCR analysis demonstrated these genes are differently expressed in leaf/root of sorghum and indicated the expression profile of these gene families under IAA, brassinosteroid (BR), salt, and drought treatments. The SbGH3 and SbLBD genes, expressed in low level under natural condition, were highly induced by salt and drought stress consistent with their products being involved in both abiotic stresses. Three genes, SbIAA1, SbGH3-13, and SbLBD32, were highly induced under all the four treatments, IAA, BR, salt, and drought. The analysis provided new evidence for role of auxin in stress response, implied there are cross talk between auxin, BR and abiotic stress signaling pathways.