GH3 Gene Research Articles

The characterization of six auxin-induced tomato GH3 genes uncovers a member, SlGH3.4, strongly responsive to arbuscular mycorrhizal symbiosis.

In plants, the GH3 gene family is widely considered to be involved in a broad range of plant physiological processes, through modulation of hormonal homeostasis. Multiple GH3 genes have been functionally characterized in several plant species; however, to date, limited works to study the GH3 genes in tomato have been reported. Here, we characterize the expression and regulatory profiles of six tomato GH3 genes, SlGH3.2, SlGH3.3, SlGH3.4, SlGH3.7, SlGH3.9 and SlGH3.15, in response to different phytohormone applications and arbuscular mycorrhizal (AM) fungal colonization. All six GH3 genes showed inducible responses to external IAA, and three members were significantly up-regulated in response to AM symbiosis. In particular, SlGH3.4, the transcripts of which were barely detectable under normal growth conditions, was strongly activated in the IAA-treated and AM fungal-colonized roots. A comparison of the SlGH3.4 expression in wild-type plants and M161, a mutant with a defect in AM symbiosis, confirmed that SlGH3.4 expression is highly correlated to mycorrhizal colonization. Histochemical staining demonstrated that a 2,258 bp SlGH3.4 promoter fragment could drive β-glucuronidase (GUS) expression strongly in root tips, steles and cortical cells of IAA-treated roots, but predominantly in the fungal-colonized cells of mycorrhizal roots. A truncated 654 bp promoter failed to direct GUS expression in IAA-treated roots, but maintained the symbiosis-induced activity in mycorrhizal roots. In summary, our results suggest that a mycorrhizal signaling pathway that is at least partially independent of the auxin signaling pathway has evolved for the co-regulation of the auxin- and mycorrhiza-activated GH3 genes in plants.

Engineering cellulosic bioreactors by template assisted DNA shuffling and in vitro recombination (TADSir)

The current study focuses on development of a bioreactor engineering strategy based on exploitation of the Arabidopsis thaliana genome. Chimeric A. thaliana glycosyl hydrolase (GH) gene libraries were assembled using a novel directed evolution strategy (TADSir: template assisted DNA shuffling and in vitro recombination) that promotes DNA recombination by reassembly of DNA fragments on unique gene templates. TADSir was modeled using a set of algorithms designed to simulate DNA interactions based on nearest neighbor base stacking interactions and Gibb’s free energy differences between helical coil and folded DNA states. The algorithms allow for target gene prediction and for in silica analysis of chimeric gene library composition. Further, the study investigated utilization of A. thaliana GH sequence space for bioreactor design by evolving 20 A. thaliana genes representing the GH1, GH3, GH5, GH9 and GH10 gene families. Notably, TADSir achieved streamlined engineering of Saccharomyces cerevisiae and spinach mesophyll protoplast bioreactors capable of processing CM cellulose, Avicel and xylan.

Two New Xylanases with Different Substrate Specificities from the Human Gut Bacterium Bacteroides intestinalis DSM 17393

Xylan is an abundant plant cell wall polysaccharide and is a dominant component of dietary fiber. Bacteria in the distal human gastrointestinal tract produce xylanase enzymes to initiate the degradation of this complex heteropolymer. These xylanases typically derive from glycoside hydrolase (GH) families 10 and 11; however, analysis of the genome sequence of the xylan-degrading human gut bacterium Bacteroides intestinalis DSM 17393 revealed the presence of two putative GH8 xylanases. In the current study, we demonstrate that the two genes encode enzymes that differ in activity. The xyn8A gene encodes an endoxylanase (Xyn8A), and rex8A encodes a reducing-end xylose-releasing exo-oligoxylanase (Rex8A). Xyn8A hydrolyzed both xylopentaose (X5) and xylohexaose (X6) to a mixture of xylobiose (X2) and xylotriose (X3), while Rex8A hydrolyzed X3 through X6 to a mixture of xylose (X1) and X2. Moreover, rex8A is located downstream of a GH3 gene (xyl3A) that was demonstrated to exhibit β-xylosidase activity and would be able to further hydrolyze X2 to X1. Mutational analyses of putative active site residues of both Xyn8A and Rex8A confirm their importance in catalysis by these enzymes. Recent genome sequences of gut bacteria reveal an increase in GH8 Rex enzymes, especially among the Bacteroidetes, indicating that these genes contribute to xylan utilization in the human gut.

The Clubroot Pathogen (Plasmodiophora brassicae) Influences Auxin Signaling to Regulate Auxin Homeostasis in Arabidopsis.

The clubroot disease, caused by the obligate biotrophic protist Plasmodiophora brassicae, affects cruciferous crops worldwide. It is characterized by root swellings as symptoms, which are dependent on the alteration of auxin and cytokinin metabolism. Here, we describe that two different classes of auxin receptors, the TIR family and the auxin binding protein 1 (ABP1) in Arabidopsis thaliana are transcriptionally upregulated upon gall formation. Mutations in the TIR family resulted in more susceptible reactions to the root pathogen. As target genes for the different pathways we have investigated the transcriptional regulation of selected transcriptional repressors (Aux/IAA) and transcription factors (ARF). As the TIR pathway controls auxin homeostasis via the upregulation of some auxin conjugate synthetases (GH3), the expression of selected GH3 genes was also investigated, showing in most cases upregulation. A double gh3 mutant showed also slightly higher susceptibility to P. brassicae infection, while all tested single mutants did not show any alteration in the clubroot phenotype. As targets for the ABP1-induced cell elongation the effect of potassium channel blockers on clubroot formation was investigated. Treatment with tetraethylammonium (TEA) resulted in less severe clubroot symptoms. This research provides evidence for the involvement of two auxin signaling pathways in Arabidopsis needed for the establishment of the root galls by P. brassicae.

옥신 반응 SMALL AUXIN UP RNA 유전자의 최근 연구 동향 및 작물 개량을 위한 적용

Auxin is a key plant hormone which regulates overall plant growth development. A number of researches to investigate auxin signaling identified three major classes of early auxin response genes: AUX/IAA, GH3 and SMALL AUXIN UP RNA (SAUR). Among these genes, in planta functions of SAUR gene family are largely ambiguous, while both AUX/IAA and GH3 genes are analyzed to mediate negative feedback on auxin response. SAUR genes encode small plant-specific proteins. SAUR gene products are highly unstable and transiently expressed in the tissue- and developmental-specific manners in response to auxin and various environmental stimuli. In the decades, molecular and genetic approaches to elucidate in planta functions of SAURs have been hampered by several factors such as the unstable molecular features and functional redundancy among them. However, a series of recent studies focusing on several subgroups of SAUR gene family made significant progress in our understanding of its biochemical and physiological functions. These works suggest that many SAUR proteins mainly regulate auxin-related cell expansion and auxin transport. In this review, the recent progress in SAUR research and prospects for crop improvement through its genetic manipulation are discussed.

Distribution of indole-3-acetic acid in Petunia hybrida shoot tip cuttings and relationship between auxin transport, carbohydrate metabolism and adventitious root formation

To determine the contribution of polar auxin transport (PAT) to auxin accumulation and to adventitious root (AR) formation in the stem base of Petunia hybrida shoot tip cuttings, the level of indole-3-acetic acid (IAA) was monitored in non-treated cuttings and cuttings treated with the auxin transport blocker naphthylphthalamic acid (NPA) and was complemented with precise anatomical studies. The temporal course of carbohydrates, amino acids and activities of controlling enzymes was also investigated. Analysis of initial spatial IAA distribution in the cuttings revealed that approximately 40 and 10 % of the total IAA pool was present in the leaves and the stem base as rooting zone, respectively. A negative correlation existed between leaf size and IAA concentration. After excision of cuttings, IAA showed an early increase in the stem base with two peaks at 2 and 24 h post excision and, thereafter, a decline to low levels. This was mirrored by the expression pattern of the auxin-responsive GH3 gene. NPA treatment completely suppressed the 24-h peak of IAA and severely inhibited root formation. It also reduced activities of cell wall and vacuolar invertases in the early phase of AR formation and inhibited the rise of activities of glucose-6-phosphate dehydrogenase and phosphofructokinase during later stages. We propose a model in which spontaneous AR formation in Petunia cuttings is dependent on PAT and on the resulting 24-h peak of IAA in the rooting zone, where it induces early cellular events and also stimulates sink establishment. Subsequent root development stimulates glycolysis and the pentose phosphate pathway.Electronic supplementary materialThe online version of this article (doi:10.1007/s00425-013-1907-z) contains supplementary material, which is available to authorized users.

GH3 expression and IAA-amide synthetase activity in pea (Pisum sativum L.) seedlings are regulated by light, plant hormones and auxinic herbicides

The formation of auxin conjugates is one of the important regulatory mechanisms for modulating IAA action. Several auxin-responsive GH3 genes encode IAA-amide synthetases that are involved in the maintenance of hormonal homeostasis by conjugating excess IAA to amino acids. Recently, the data have revealed novel regulatory functions of several GH3 proteins in plant growth, organ development, fruit ripening, light signaling, abiotic stress tolerance and plant defense responses. Indole-3-acetyl-aspartate (IAA-Asp) synthetase catalyzing IAA conjugation to aspartic acid in immature seeds of pea (Pisum sativum L.) was purified and characterized during our previous investigations. In this study, we examined the effect of auxin and other plant hormones (ABA, GA, kinetin, JA, MeJA, SA), different light conditions (red, far-red, blue, white light), and auxinic herbicides (2,4-D, Dicamba, Picloram) on the expression of a putative GH3 gene and IAA-amide synthesizing activity in 10-d-old pea seedlings. Quantitative RT-PCR analysis indicated that the PsGH3-5 gene, weakly expressed in control sample, was visibly induced in response to all plant hormones, different light wavelengths and the auxinic herbicides tested. Protein A immunoprecipitation/gel blot analysis using anti-AtGH3.5 antibodies revealed a similar pattern of changes on the protein levels in response to all treatments. IAA-amide synthetase activity determined with aspartate as a substrate, not detectable in control seedlings, was positively affected by a majority of treatments. Based on these results, we suggest that PsGH3-5 may control the growth and development of pea plants in a way similar to the known GH3 genes from other plant species.

A genomics approach to understanding the role of auxin in apple (Malus x domestica)fruit size control

BackgroundAuxin is an important phytohormone for fleshy fruit development, having been shown to be involved in the initial signal for fertilisation, fruit size through the control of cell division and cell expansion, and ripening related events. There is considerable knowledge of auxin-related genes, mostly from work in model species. With the apple genome now available, it is possible to carry out genomics studies on auxin-related genes to identify genes that may play roles in specific stages of apple fruit development.ResultsHigh amounts of auxin in the seed compared with the fruit cortex were observed in 'Royal Gala' apples, with amounts increasing through fruit development. Injection of exogenous auxin into developing apples at the start of cell expansion caused an increase in cell size. An expression analysis screen of auxin-related genes involved in auxin reception, homeostasis, and transcriptional regulation showed complex patterns of expression in each class of gene. Two mapping populations were phenotyped for fruit size over multiple seasons, and multiple quantitative trait loci (QTLs) were observed. One QTL mapped to a region containing an Auxin Response Factor (ARF106). This gene is expressed during cell division and cell expansion stages, consistent with a potential role in the control of fruit size.ConclusionsThe application of exogenous auxin to apples increased cell expansion, suggesting that endogenous auxin concentrations are at least one of the limiting factors controlling fruit size. The expression analysis of ARF106 linked to a strong QTL for fruit weight suggests that the auxin signal regulating fruit size could partially be modulated through the function of this gene. One class of gene (GH3) removes free auxin by conjugation to amino acids. The lower expression of these GH3 genes during rapid fruit expansion is consistent with the apple maximising auxin concentrations at this point.

Genome-wide investigation and expression analysis suggest diverse roles of auxin-responsive GH3 genes during development and response to different stimuli in tomato (Solanum lycopersicum)

In plants, auxin-mediated responses are regulated by diverse proteins. One such class of proteins, i.e. GH3, is involved in the conjugation of IAA to amino acids and provides a negative feedback loop to control auxin homoeostasis. In order to have a better understanding of the mechanism of the auxin action, 15 genes encoding GH3 members were identified using existing EST databases of tomato. Their orthologs were identified from tobacco, potato, N. benthemiana, pepper, and petunia. Phylogenetic analysis of AtGH3, SlGH3, and their Solanaceae orthologs provided insights into various orthologous relationships among these proteins. These genes were found to be responsive to a variety of signals including, phytohormones and environmental stresses. Analysis of AuxRE elements in their promoters showed variability in the sequence as well as number of this element. Up-regulation of only 11 SlGH3 genes, in response to exogenous auxin, suggested possible relationship between the diversity in the sequence and number of AuxRE element with the auxin inducibility. Expression analysis of SlGH3 genes in different vegetative and reproductive tissues/stages suggested limited or no role for most of the SlGH3 genes at the initiation of fruit ripening. However, up-regulation of SlGH3-1 and -2 at the onset of fruit ripening indicates that these genes could have a role in fruit ripening. The present study characterizes GH3 gene family of tomato and its evolutionary relationship with members of this family from other Solanaceae species and Arabidopsis. It could help in the identification of GH3 genes and revelation of their function during vegetative/reproductive development stages from other Solanaceae members.

Deficiency in riboflavin biosynthesis affects tetrapyrrole biosynthesis in etiolated Arabidopsis tissue

Tetrapyrrole biosynthesis is controlled by multiple environmental and endogenous cues. Etiolated T-DNA insertion mutants were screened for red fluorescence as result of elevated levels of protochlorophyllide and four red fluorescent in the dark (rfd) mutants were isolated and identified. rfd3 and rfd4 belong to the group of photomorphogenic cop/det/fus mutants. rfd1 and rfd2 had genetic lesions in RIBA1 and FLU encoding the dual-functional protein GTP cyclohydrolase II/3,4-dihydroxy-2-butanone-4-phosphate synthase and a negative regulator of tetrapyrrole biosynthesis, respectively. RIBA1 catalyses the initial reaction of the metabolic pathway of riboflavin biosynthesis and rfd1 contains reduced contents of riboflavin and the flavo-coenzymes FMN and FAD. Transcriptome analysis of rfd1 revealed up-regulated genes encoding nucleus-localized factors involved in cytokinin signalling and numerous down-regulated LEA genes as well as an auxin-inducible GH3 gene. Alteration of cytokinin metabolism of rfd1was confirmed by elevated contents of active forms of cytokinin and stimulated expression of an ARR6::GUS reporter construct. An etiolated quadruple ckx (cytokinin oxidase) mutant with impaired cytokinin degradation as well as different knockout mutants for the negative AUX/IAA regulators shy2-101 (iaa3), axr2-1 (iaa7) and slr-1 (iaa14) showed also excessive protochlorophyllide accumulation. The transcript levels of CHLH and HEMA1 encoding Mg chelatase and glutamyl-tRNA reductase were increased in rfd1 and the AUX/IAA loss-of-function mutants. It is proposed that reduced riboflavin synthesis impairs the activity of the flavin-containing cytokinin oxidase, increases cytokinin contents and de-represses synthesis of 5-aminolevulinic acid of tetrapyrrole metabolism in darkness. As result of the mutant analyses, the antagonistic cytokinin and auxin signalling is required for a balanced tetrapyrrole biosynthesis in the dark.

Functional characterization of cellulases identified from the cow rumen fungus Neocallimastix patriciarum W5 by transcriptomic and secretomic analyses

BackgroundNeocallimastix patriciarum is one of the common anaerobic fungi in the digestive tracts of ruminants that can actively digest cellulosic materials, and its cellulases have great potential for hydrolyzing cellulosic feedstocks. Due to the difficulty in culture and lack of a genome database, it is not easy to gain a global understanding of the glycosyl hydrolases (GHs) produced by this anaerobic fungus.ResultsWe have developed an efficient platform that uses a combination of transcriptomic and proteomic approaches to N. patriciarum to accelerate gene identification, enzyme classification and application in rice straw degradation. By conducting complementary studies of transcriptome (Roche 454 GS and Illumina GA IIx) and secretome (ESI-Trap LC-MS/MS), we identified 219 putative GH contigs and classified them into 25 GH families. The secretome analysis identified four major enzymes involved in rice straw degradation: β-glucosidase, endo-1,4-β-xylanase, xylanase B and Cel48A exoglucanase. From the sequences of assembled contigs, we cloned 19 putative cellulase genes, including the GH1, GH3, GH5, GH6, GH9, GH18, GH43 and GH48 gene families, which were highly expressed in N. patriciarum cultures grown on different feedstocks.ConclusionsThese GH genes were expressed in Pichia pastoris and/or Saccharomyces cerevisiae for functional characterization. At least five novel cellulases displayed cellulytic activity for glucose production. One β-glucosidase (W5-16143) and one exocellulase (W5-CAT26) showed strong activities and could potentially be developed into commercial enzymes.

Two GH3 genes from longan are differentially regulated during fruit growth and development

In the present work, two full length cDNAs of GH3 genes, named DlGH3.1 and DlGH3.2 were cloned from pericarp and aril tissues of the longan fruit, respectively. Three conserved motifs, SSGTSAGERK, YASSE and YRVGD, as a characteristic of the acyladenylate/thioester forming enzyme superfamily were observed in DlGH3.1 and DlGH3.2 proteins. DlGH3.1 mainly expressed in pericarp tissues while DlGH3.2 accumulated in both the pericarp and aril tissues during fruit growth and development. In addition, NAA treatment induced the expression of DlGH3.1 and DlGH3.2 in the pericarp tissues at 21 and 77 days after anthesis (DAA), while only DlGH3.2 in the aril tissues could be induced by NAA at 77 DAA. More importantly, ABA and ethrel treatments suppressed the accumulations of DlGH3.1 and DlGH3.2 in the pericarp tissues of longan fruit at 21 DAA (a rapid growth stage of pericarp), but enhanced DlGH3.2 expression in the aril tissues at 77 DAA (a fruit ripening stage). Furthermore, the expression patterns of DlGH3.1 and DlGH3.2 showed different tissue specificity. Thus, our results suggest that DlGH3.1 gene expression might be associated with pericarp growth, while DlGH3.2 accumulation is likely to be related to both pericarp growth and fruit ripening, and the responses of DlGH3s to plant growth hormones are different and dependent on fruit development stage and fruit tissue.

Evolutionary history of the GH3 family of acyl adenylases in rosids

GH3 amino acid conjugases have been identified in many plant and bacterial species. The evolution of GH3 genes in plant species is explored using the sequenced rosids Arabidopsis, papaya, poplar, and grape. Analysis of the sequenced non-rosid eudicots monkey flower and columbine, the monocots maize and rice, as well as spikemoss and moss is included to provide further insight into the origin of GH3 clades. Comparison of co-linear genes in regions surrounding GH3 genes between species helps reconstruct the evolutionary history of the family. Combining analysis of synteny with phylogenetics, gene expression and functional data redefines the Group III GH3 genes, of which AtGH3.12/PBS3, a regulator of stress-induced salicylic acid metabolism and plant defense, is a member. Contrary to previous reports that restrict PBS3 to Arabidopsis and its close relatives, PBS3 syntelogs are identified in poplar, grape, columbine, maize and rice suggesting descent from a common ancestral chromosome dating to before the eudicot/monocot split. In addition, the clade containing PBS3 has undergone a unique expansion in Arabidopsis, with expression patterns for these genes consistent with specialized and evolving stress-responsive functions.

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.

YUCCA6 over-expression demonstrates auxin function in delaying leaf senescence in Arabidopsis thaliana.

The Arabidopsis thaliana YUCCA family of flavin monooxygenase proteins catalyses a rate-limiting step in de novo auxin biosynthesis. A YUCCA6 activation mutant, yuc6-1D, has been shown to contain an elevated free IAA level and to display typical high-auxin phenotypes. It is reported here that Arabidopsis plants over-expressing YUCCA6, such as the yuc6-1D activation mutant and 35S:YUC6 transgenic plants, displayed dramatic longevity. In addition, plants over-expressing YUCCA6 exhibited classical, delayed dark-induced and hormone-induced senescence in assays using detached rosette leaves. However, plants over-expressing an allele of YUCCA6, that carries mutations in the NADPH cofactor binding site, exhibited neither delayed leaf senescence phenotypes nor phenotypes typical of auxin overproduction. When the level of free IAA was reduced in yuc6-1D by conjugation to lysine, yuc6-1D leaves senesced at a rate similar to the wild-type leaves. Dark-induced senescence in detached leaves was accompanied by a decrease in their free IAA content, by the reduced expression of auxin biosynthesis enzymes such as YUCCA1 and YUCCA6 that increase cellular free IAA levels, and by the increased expression of auxin-conjugating enzymes encoded by the GH3 genes that reduce the cellular free auxin levels. Reduced transcript abundances of SAG12, NAC1, and NAC6 during senescence in yuc6-1D compared with the wild type suggested that auxin delays senescence by directly or indirectly regulating the expression of senescence-associated genes.

Time-Course Analysis of Levels of Indole-3-Acetic Acid and Expression of Auxin-Responsive GH3 Genes in Betula platyphylla

Indole-3-acetic acid (IAA) is a natural hormone that plays an important role in the essential physiological processes of plant growth, development, and environmental response. To maintain IAA homeostasis in plants, some members of the GH3 gene family may mediate the conjugation of amino acids to indole-3-acetic acid. In this study, nine birch BpGH3 genes were cloned, including three BpGH3.5 genes, three BpGH3.6 genes, two BpGH3.9 genes, and a single BpGH3.3 gene, that were likely to be auxin-responsive genes. Time-course expression analysis of these nine BpGH3 genes using real-time reverse transcriptase–polymerase chain reaction (RT-PCR) was conducted using birch plant tissues collected during a 1-year growth cycle. Further, we determined the endogenous free IAA content in birch during this period by using liquid chromatography tandem mass spectrometry. The results indicated that the IAA levels changed remarkably throughout the study period; the level remained relatively stable during the developmental stage from June 1 to July 1, and IAA was present in the birch leaves throughout the study period. The correlation between the expression of BpGH3 genes and IAA levels was analyzed using Pearson correlation analysis. The results revealed a significant negative correlation between the expressions of four BpGH3 genes, namely, BpGH3.3 (p < 0.05), BpGH3.5a (p < 0.01), BpGH3.5b (p < 0.01), and BpGH3.9a (p < 0.01) and the free IAA level, thereby suggesting that these genes might play roles in IAA regulation.

Rice GH3 gene family

Auxin is an indispensable hormone throughout the lifetime of nearly all plant species. Several aspects of plant growth and development are rigidly governed by auxin, from micro to macro hierarchies; auxin also has a close relationship with plant-pathogen interactions. Undoubtedly, precise auxin levels are vitally important to plants, which have many effective mechanisms to maintain auxin homeostasis. One mechanism is conjugating amino acid to excessive indole-3-acetic acid (IAA; main form of auxin) through some GH3 family proteins to inactivate it. Our previous study demonstrated that GH3-2 mediated broad-spectrum resistance in rice (Oryza sativa L.) by suppressing pathogen-induced IAA accumulation and downregulating auxin signaling. Here, we further investigated the expression pattern of GH3-2 and other GH3 family paralogues in the life cycle of rice and presented the possible function of GH3-2 on rice root development by histochemical analysis of GH3-2 promoter:GUS reporter transgenic plants.

Impact of grafting on cold responsive gene expression in Satsuma mandarin (Citrus unshiu)

The major limitation preventing the expansion of Satsuma mandarin (Citrus unshiu) production in the Gulf Coast regions of the US is the risk of freeze damage. Poncirus trifoliata (L.) Raf has been widely used as a cold hardy rootstock for citrus production in freeze-prone environments. In this study, relative quantitative real-time RT–PCR was used to study cold responsive genes in Satsuma mandarin (C. unshiu), C. unshiu grafted on P. trifoliata rootstock and P. trifoliata following exposure to cold shock or cold acclimation treatments. Major changes in gene expression occurred in the different species during cold stress especially with respect to nitrate transporter, auxin responsive GH3, UDP-glycosyltransferase (UGT) and chalcone-flavanone isomerase. Very high levels of nitrate transporter were reached early in Poncirus following 60 h of cold shock treatment at 4°C, and in C. unshiu after 3 weeks of gradual cold acclimation. Also, high levels of auxin responsive GH3 were reached in Poncirus following both cold shock and cold acclimation, while levels in C. unshiu were low or reduced. Poncirus rootstock significantly affected gene expression in C. unshiu scion, resulting in a reduction of nitrate transporter, UGT and trehalose 6-phosphate synthase expression levels, and an increase in auxin responsive GH3 gene during cold acclimation. The impact of rootstock on citrus scion during cold stress might be associated with partitioning of nutrients and hormones.

Sequestration of auxin by the indole-3-acetic acid-amido synthetase GH3-1 in grape berry (Vitis vinifera L.) and the proposed role of auxin conjugation during ripening

In fleshy fruit, levels of indole-3-acetic acid (IAA), the most abundant auxin, decline towards the onset of ripening. The application of auxins to immature fruit can delay the ripening processes. However, the mechanisms by which the decrease in endogenous IAA concentrations and the maintenance of low auxin levels in maturing fruit are achieved remain elusive. The transcript of a GH3 gene (GH3-1), encoding for an IAA-amido synthetase which conjugates IAA to amino acids, was detected in grape berries (Vitis vinifera L.). GH3-1 expression increased at the onset of ripening (veraison), suggesting that it might be involved in the establishment and maintenance of low IAA concentrations in ripening berries. Furthermore, this grapevine GH3 gene, responded positively to the combined application of abscisic acid and sucrose and to ethylene, linking it to the control of ripening processes. Levels of IAA-aspartic acid (IAA-Asp), an in vitro product of recombinant GH3-1, rose after veraison and remained high during the following weeks of the ripening phase when levels of free IAA were low. A similar pattern of changes in free IAA and IAA-Asp levels was detected in developing tomatoes (Solanum lycopersicum Mill.), where low concentrations of IAA and an increase in IAA-Asp concentrations coincided with the onset of ripening in this climacteric fruit. Since IAA-Asp might be involved in IAA degradation, the GH3 catalysed formation of this conjugate at, and after, the onset of ripening could represent a common IAA inactivation mechanism in climacteric and non-climacteric fruit which enables ripening.

Auxin homeostasis during lateral root development under drought condition

Lateral root formation is a critical agronomic trait in plant architecture that determines crop productivity and environmental stress adaptability. It is therefore tightly regulated both by intrinsic developmental cues, such as abscisic acid (ABA) and auxin, and by diverse environmental growth conditions, including water deficit and high salinity in the soil. We have recently reported that an Arabidopsis R2R3-type MYB transcription factor, MYB96, regulates lateral root meristem activation under drought conditions via an ABA-auxin signaling crosstalk. In this signaling scheme, the MYB96-mediated ABA signals are incorporated into an auxin signaling pathway that involves a subset of GH3 gene encoding auxin-conjugating enzymes. The MYB96-overexpressing, activation tagging mutant, which is featured by having dwarfed growth and reduced lateral root formation, exhibits an enhanced drought resistance. In the mutant, expression of the GH3 genes was significantly elevated, which is consistent with the reduced lateral root formation. In contrast, the MYB96-deficient knockout mutant produced more lateral roots and was more susceptible to drought stress. Our observations strongly support that MYB96 is a molecular link that integrates ABA and auxin signals in modulating auxin homeostasis during lateral root development, particularly under water deficit conditions. It is also envisioned that the MYB96-mediated signals are related with pathogen resistance response, which is also profoundly affected by water content in plant cells.