Proteomic analysis of different mutant genotypes of Arabidopsis led to the identification of 11 proteins correlating with adventitious root development

A lack of competence to form adventitious roots by cuttings or explants in vitro occurs routinely and is an obstacle for the clonal propagation and rapid fixation of elite genotypes. Adventitious rooting is known to be a quantitative genetic trait. We performed a proteomic analysis of Arabidopsis (Arabidopsis thaliana) mutants affected in their ability to develop adventitious roots in order to identify associated molecular markers that could be used to select genotypes for their rooting ability and/or to get further insight into the molecular mechanisms controlling adventitious rooting. Comparison of two-dimensional gel electrophoresis protein profiles resulted in the identification of 11 proteins whose abundance could be either positively or negatively correlated with endogenous auxin content, the number of adventitious root primordia, and/or the number of mature adventitious roots. One protein was negatively correlated only to the number of root primordia and two were negatively correlated to the number of mature adventitious roots. Two putative chaperone proteins were positively correlated only to the number of primordia, and, interestingly, three auxin-inducible GH3-like proteins were positively correlated with the number of mature adventitious roots. The others were correlated with more than one parameter. The 11 proteins are predicted to be involved in different biological processes, including the regulation of auxin homeostasis and light-associated metabolic pathways. The results identify regulatory pathways associated with adventitious root formation and represent valuable markers that might be used for the future identification of genotypes with better rooting abilities.     

生长素、乙烯和一氧化氮对拟南芥下胚轴插条形成不定根的调节

研究生长素、乙烯和一氧化氮（NO）对拟南芥下胚轴插条形成不定根的调节,以及生长素和乙烯信号转导成员在IAA促进不定根形成中的作用的结果表明：拟南芥切条以IAA和硝普钠（N0供体）单独处理7d后的不定根形成均受到促进,其中以50μmol·L^-1 IAAμmol·L^-1 SNP的促进作用为最强,乙烯的促进作用不明显;生长素运输和信号转导以及乙烯信号转导相关突变体对IAA促进生根作用的敏感性比野生型有所下降,特别是IAA14功能获得型的突变体。IAA和NO在促进不定根形成中有协同效应。     

Strigolactones suppress adventitious rooting in Arabidopsis and pea

Adventitious root formation is essential for the propagation of many commercially important plant species and involves the formation of roots from nonroot tissues such as stems or leaves. Here, we demonstrate that the plant hormone strigolactone suppresses adventitious root formation in Arabidopsis (Arabidopsis thaliana) and pea (Pisum sativum). Strigolactone-deficient and response mutants of both species have enhanced adventitious rooting. CYCLIN B1 expression, an early marker for the initiation of adventitious root primordia in Arabidopsis, is enhanced in more axillary growth2 (max2), a strigolactone response mutant, suggesting that strigolactones restrain the number of adventitious roots by inhibiting the very first formative divisions of the founder cells. Strigolactones and cytokinins appear to act independently to suppress adventitious rooting, as cytokinin mutants are strigolactone responsive and strigolactone mutants are cytokinin responsive. In contrast, the interaction between the strigolactone and auxin signaling pathways in regulating adventitious rooting appears to be more complex. Strigolactone can at least partially revert the stimulatory effect of auxin on adventitious rooting, and auxin can further increase the number of adventitious roots in max mutants. We present a model depicting the interaction of strigolactones, cytokinins, and auxin in regulating adventitious root formation.     

A mitochondrial mutator system in maize

Terfestatin A (TrfA), terphenyl-beta-glucoside, was isolated from Streptomyces sp. F40 in a forward screen for compounds that inhibit the expression of auxin-inducible genes in Arabidopsis (Arabidopsis thaliana). TrfA specifically and competitively inhibited the expression of primary auxin-inducible genes in Arabidopsis roots, but did not affect the expression of genes regulated by other plant hormones such as abscisic acid and cytokinin. TrfA also blocked the auxin-enhanced degradation of auxin/indole-3-acetic acid (Aux/IAA) repressor proteins without affecting the auxin-stimulated interaction between Aux/IAAs and the F-box protein TIR1. TrfA treatment antagonized auxin responses in roots, including primary root inhibition, lateral root initiation, root hair promotion, and root gravitropism, but had only limited effects on shoot auxin responses. Taken together, these results indicate that TrfA acts as a modulator of Aux/IAA stability and thus provides a new tool for dissecting auxin signaling.     

Involvement of HLS1 in sugar and auxin signaling in Arabidopsis leaves

Sugar regulates a variety of genes and controls plant growth and development similarly to phytohormones. As part of a screen for Arabidopsis mutants with defects in sugar-responsive gene expression, we identified a loss-of-function mutation in the HOOKLESS1 (HLS1) gene. HLS1 was originally identified to regulate apical hook formation of dark-grown seedlings (Lehman et al., 1996, Cell 85: 183-194). In hls1, sugar-induced gene expression in excised leaf petioles was more sensitive to exogenous sucrose than that in the wild type. Exogenous IAA partially repressed sugar-induced gene expression and concomitantly activated some auxin response genes such as AUR3 encoding GH3-like protein. The repression and the induction of gene expression by auxin were attenuated and enhanced, respectively, by the hls1 mutation. These results suggest that HLS1 plays a negative role in sugar and auxin signaling. Because AUR3 GH3-like protein conjugates free IAA to amino acids (Staswick et al., 2002, Plant Cell 14: 1405-1415; Staswick et al., 2005, Plant Cell 17: 616-627), enhanced expression of GH3-like genes would result in a decrease in the free IAA level. Indeed, hls1 leaves accumulated a reduced level of free IAA, suggesting that HLS1 may be involved in negative feedback regulation of IAA homeostasis through the control of GH3-like genes. We discuss the possible mechanisms by which HLS1 is involved in auxin signaling for sugar- and auxin-responsive gene expression and in IAA homeostasis.     

芒果生长素反应因子类蛋白的cDNA克隆和表达

通过SSH法获得了一个与不定根形成相关的差异表达的cDNA片段,其推导的氨基酸序列与拟南芥的生长素反应因子(ARF)类蛋白具有较大的同源性,因此将它命名为MiARF。用所设计的基因特异引物进行3′RACE扩增获得包含完整读码框架(ORF)的MiARF1（GenBank登录号为AY255705）和MiARF2（GenBank登录号为AY300808）。MiARF1全长为3272bp,其中,ORF含2523bp,5′非翻译区（5′UTR）含285bp, 3′非翻译区(3′UTR)含464bp。由该序列所推导的氨基酸序列与拟南芥ARF2（BAB10162）的ID值为64%, E值为0,在DNA结合区域（DBD）、III和IV区域的同源性更高,ID值均大于80%。MiARF2cDNA全长为1474bp,其中ORF含981bp,5′非翻译区含285bp, 3′非翻译区含208bp,由该序列所推导的氨基酸序列与拟南芥ARF2（BAB10162）的ID值为84%,E值为e-151。 MiARF2仅具有DBD保守区并与MiARF1的基本相同,但缺乏III和IV区域。Virtural Northern 杂交表明：MiARF2在生根的组织中表达水平高, 而在非生根的组织中未见表达;MiARF1在生根及非生根的组织中均有表达。     

Synergistic interaction between coumarin 1,2-benzopyrone and indole-3-butyric acid in stimulating adventitious root formation in Vigna radiata (L.) Wilczek cuttings: I. Endogenous free and conjugated IAA and basic isoperoxidases

Cuttings from 7-day-old Vigna radiata seedlings were treated for 24 h with various concentrations of coumarin and/or indole-3-butyric acid (IBA), applied either alone or in combination, in order to stimulate adventitious root formation (ARF). The effects of treatment on endogenous free and conjugated indole-3-acetic acid (IAA), basic peroxidase (basic PER) activity and its isoperoxidases analysis and their relation to ARF were then investigated at the potential rooting sites during the first 96 h after application. Simultaneously, combined treatments acted synergistically in inducing more adventitious roots in treated cuttings than in those treated with coumarin or IBA individually, as compared with the control. Endogenous free IAA increased transiently in treated cuttings as compared with the control and the maximum increase occurred with the combined treatment. This suggests that coumarin and IBA may act synergistically in increasing the endogenous free IAA level during the induction phase of rooting to initiate more roots. Likewise, higher level of conjugated IAA was also found in treated cuttings than in untreated ones, during the primary events of ARF, with the maximum level occurring in the combined treatment. Comparison of the dynamics of conjugated IAA and activity of basic PERs led to conclusion that the former but not the latter is responsible for downregulation of endogenous IAA levels significantly during the primary events of ARF. A sharp increases in basic PERs occurred during the secondary events of ARF, suggesting their role in root initiation and development rather than root induction.     

GH3-mediated auxin homeostasis links growth regulation with stress adaptation response in Arabidopsis

Plants constantly monitor environmental fluctuations to optimize their growth and metabolism. One example is adaptive growth occurring in response to biotic and abiotic stresses. Here, we demonstrate that GH3-mediated auxin homeostasis is an essential constituent of the complex network of auxin actions that regulates stress adaptation responses in Arabidopsis. Endogenous auxin pool is regulated, at least in part, through negative feedback by a group of auxin-inducible GH3 genes encoding auxin-conjugating enzymes. An Arabidopsis mutant, wes1-D, in which a GH3 gene WES1 is activated by nearby insertion of the (35)S enhancer, exhibited auxin-deficient traits, including reduced growth and altered leaf shape. Interestingly, WES1 is also induced by various stress conditions as well as by salicylic acid and abscisic acid. Accordingly, wes1-D was resistant to both biotic and abiotic stresses, and stress-responsive genes, such as pathogenesis-related genes and CBF genes, were upregulated in this mutant. In contrast, a T-DNA insertional mutant showed reduced stress resistance. We therefore propose that GH3-mediated growth suppression directs reallocation of metabolic resources to resistance establishment and represents the fitness costs of induced resistance.     

Distinct modes of adventitious rooting in Arabidopsis thaliana

The literature describes different rooting protocols for Arabidopsis thaliana as models to study adventitious rooting, and results are generally perceived as comparable. However, there is a lack of investigations focusing on the distinct features, advantages and limitations of each method in the study of adventitious rooting with both wild-type (WT) ecotypes and their respective mutants. This investigation was undertaken to evaluate the adventitious rooting process in three different experimental systems, all using A. thaliana, analysing the same rooting parameters after transient exposure to auxin (indole-3-acetic acid) and control conditions: excised leaves, de-rooted plants and etiolated seedlings. The founding tissues and sites of origin of roots differed depending on the system used, whereas all rooting patterns were of the direct type (i.e., without callus formation). None of the systems had an absolute requirement for exogenous auxin, although rooting was enhanced by this phytohormone, with the exception of de-rooted plants, which had adventitious rooting strongly inhibited by exogenous auxin. Root elongation was much favoured in isolated leaves. Auxin-overproducing mutants could not be used in the detached leaf system due to precocious senescence; in the de-rooted plant system, these mutants had a WT-like rooting response, whereas the expression of the 'rooty' phenotype was only evident in the etiolated seedling system. Adventitious rooting of etiolated WT seedlings in the presence of exogenous auxin was inhibited by exogenous flavonoids, which act as auxin transport inhibitors; surprisingly, the flavonoid-deficient mutant chs had a lower rooting response compared to WT. Although Arabidopsis is an excellent model system to study adventitious rooting, physiological and developmental responses differed significantly, underlining the importance of avoiding data generalisation on rooting responses derived from different experimental systems with this species.     

Mediators,Genes and Signaling in Adventitious Rooting

Adventitious roots are a post-embryonic root which arise from the stem and leaves and from non-pericycle tissues in old roots and it is one of the most important ways of vegetative propagation in plants. Many exogenous and endogenous factors regulate the formation of adventitious roots, such as Ca²⁺, sugars, auxin, polyamines, ethylene, nitric oxide, hydrogen peroxide, carbon monoxide, cGMP, MAPKs and peroxidase, etc. These mediators are thought to function as signaling and mediate auxin signal transduction during the formation of adventitious roots. To date, only a few genes have been identified that are associated with the general process of adventitious rooting, such as ARL1, VvPRP1, VvPRP2, HRGPnt3, LRP1 and RML, etc. Auxin has been shown to be intimately involved in the process of adventitious rooting and function as crucial role in adventitious rooting. Great progress has been made in elucidating the auxin-induced genes and auxin signaling pathway, especially in auxin response Aux/IAA and ARF genes family and the auxin receptor TIR1. Although, some of important aspects of adventitious rooting signaling have been revealed, the intricate signaling network remains poorly understood.     

Flavonoid-related regulation of auxin accumulation in<Emphasis Type="Italic"> Agrobacterium tumefaciens</Emphasis>-induced plant tumors

Agrobacterium tumefaciens-induced plant tumors accumulate considerable concentrations of free auxin. To determine possible mechanisms by which high auxin concentrations are maintained, we examined the pattern of auxin and flavonoid distribution in plant tumors. Tumors were induced in transformants of Trifolium repens (L.), containing the beta-glucuronidase ( GUS)-fused auxin-responsive promoter ( GH3) or chalcone synthase ( CHS2) genes, and in transformants of Arabidopsis thaliana (L.) Heynh., containing the GUS-fused synthetic auxin response element DR5. Expression of GH3::GUS and DR5::GUS was strong in proliferating metabolically active tumors, thus suggesting high free-auxin concentrations. Immunolocalization of total auxin with indole-3-acetic acid antibodies was consistent with GH3::GUS expression indicating the highest auxin concentration in the tumor periphery. By in situ staining with diphenylboric acid 2-aminoethyl ester, by thin-layer chromatography, reverse-phase high-performance liquid chromatography, and two-photon laser-scanning microscopy spectrometry, tumor-specific flavones, isoflavones and pterocarpans were detected, namely 7,4'-dihydroxyflavone (DHF), formononetin, and medicarpin. DHF was the dominant flavone in high free-auxin-accumulating stipules of Arabidopsis leaf primordia. Flavonoids were localized at the sites of strongest auxin-inducible CHS2::GUS expression in the tumor that was differentially modulated by auxin in the vascular tissue. CHS mRNA expression changes corresponded to the previously analyzed auxin concentration profile in tumors and roots of tumorized Ricinus plants. Application of DHF to stems, apically pretreated with alpha-naphthaleneacetic acid, inhibited GH3::GUS expression in a fashion similar to 1-N-naphthyl-phthalamic acid. Tumor, root and shoot growth was poor in inoculated tt4(85) flavonoid-deficient CHS mutants of Arabidopsis. It is concluded that CHS-dependent flavonoid aglycones are possibly endogenous regulators of the basipetal auxin flux, thereby leading to free-auxin accumulation in A. tumefaciens-induced tumors. This, in turn, triggers vigorous proliferation and vascularization of the tumor tissues and suppresses their further differentiation.     

Deletion of MP/ARF5 domains III and IV reveals a requirement for Aux/IAA regulation in Arabidopsis leaf vascular patterning

Combinatorial interactions of AUXIN RESPONSE FACTORs (ARFs) and auxin/indole acetic acid (Aux/IAA) proteins through their common domains III and IV regulate auxin responses, but insight into the functions of individual proteins is still limited. As a new tool to explore this regulatory network, we generated a gain-of-function ARF genotype by eliminating domains III and IV from the functionally well-characterized ARF MONOPTEROS(MP)/ARF5. This truncated version of MP, termed MPΔ, conferred complementing MP activity, but also displayed a number of semi-dominant traits affecting auxin signaling and organ patterning. In MPΔ, the expression levels of many auxin-inducible genes, as well as rooting properties and vascular tissue abundance, were enhanced. Lateral organs were narrow, pointed and filled with parallel veins. This effect was epistatic over the vascular hypotrophy imposed by certain Aux/IAA mutations. Further, in MPΔ leaves, failure to turn off the procambium-selecting gene PIN1 led to the early establishment of oversized central procambial domains and very limited subsequent lateral growth of the leaf lamina. We conclude that MPΔ can selectively uncouple a single ARF from regulation by Aux/IAA proteins and can be used as a genetic tool to probe auxin pathways and explore leaf development.