Analysis of Vascular Development in the hydra Sterol Biosynthetic Mutants of Arabidopsis

Background

The control of vascular tissue development in plants is influenced by diverse hormonal signals, but their interactions during this process are not well understood. Wild-type sterol profiles are essential for growth, tissue patterning and signalling processes in plant development, and are required for regulated vascular patterning.

Methodology/Principal Findings

Here we investigate the roles of sterols in vascular tissue development, through an analysis of the Arabidopsis mutants hydra1 and fackel/hydra2, which are defective in the enzymes sterol isomerase and sterol C-14 reductase respectively. We show that defective vascular patterning in the shoot is associated with ectopic cell divisions. Expression of the auxin-regulated AtHB8 homeobox gene is disrupted in mutant embryos and seedlings, associated with variably incomplete vascular strand formation and duplication of the longitudinal axis. Misexpression of the auxin reporter proIAA2∶GUS and mislocalization of PIN proteins occurs in the mutants. Introduction of the ethylene-insensitive ein2 mutation partially rescues defective cell division, localization of PIN proteins, and vascular strand development.

Conclusions

The results support a model in which sterols are required for correct auxin and ethylene crosstalk to regulate PIN localization, auxin distribution and AtHB8 expression, necessary for correct vascular development.     

Cadmium interferes with maintenance of auxin homeostasis in Arabidopsis seedlings

Auxin and its homeostasis play key roles in many aspects of plant growth and development. Cadmium (Cd) is a phytotoxic heavy metal and its inhibitory effects on plant growth and development have been extensively studied. However, the underlying molecular mechanism of the effects of Cd stress on auxin homeostasis is still unclear. In the present study, we found that the root elongation, shoot weight, hypocotyl length and chlorophyll content in wild-type (WT) Arabidopsis seedlings were significantly reduced after exposure to Cd stress. However, the lateral root (LR) formation was markedly promoted by Cd stress. The level and distribution of auxin were both greatly altered in primary root tips and cotyledons of Cd-treated plants. The results also showed that after Cd treatment, the IAA content was significantly decreased, which was accompanied by increases in the activity of the IAA oxidase and alteration in the expression of several putative auxin biosynthetic and catabolic genes. Application of the auxin transport inhibitor, 1-naphthylphthalamic acid (NPA) and 1-naphthoxyacetic acid (1-NOA), reversed the effects of Cd on LR formation. Additionally, there was less promotion of LR formation by Cd treatment in aux1-7 and pin2 mutants than that in the WT. Meanwhile, Cd stress also altered the expression of PINs and AUX1 in Arabidopsis roots, implying that the auxin transport pathway is required for Cd-modulated LR development. Taken together, these findings suggest that Cd stress disturbs auxin homeostasis through affecting auxin level, distribution, metabolism, and transport in Arabidopsis seedling.     

Auxin Control of Embryo Patterning

Plants start their life as a single cell, which, during the process of embryogenesis, is transformed into a mature embryo with all organs necessary to support further growth and development. Therefore, each basic cell type is first specified in the early embryo, making this stage of development excellently suited to study mechanisms of coordinated cell specification—pattern formation. In recent years, it has emerged that the plant hormone auxin plays a prominent role in embryo development. Most pattern formation steps in the early Arabidopsis embryo depend on auxin biosynthesis, transport, and response. In this article, we describe those embryo patterning steps that involve auxin activity, and we review recent data that shed light on the molecular mechanisms of auxin action during this phase of plant development.     

Gene expression profile of zeitlupe/lov kelch protein1 T-DNA insertion mutants in Arabidopsis thaliana: Downregulation of auxin-inducible genes in hypocotyls

Elongation of hypocotyl cells has been studied as a model for elucidating the contribution of cellular expansion to plant organ growth. ZEITLUPE (ZTL) or LOV KELCH PROTEIN1 (LKP1) is a positive regulator of warmth-induced hypocotyl elongation under white light in Arabidopsis, although the molecular mechanisms by which it promotes hypocotyl cell elongation remain unknown. Microarray analysis showed that 134 genes were upregulated and 204 genes including 15 auxin-inducible genes were downregulated in the seedlings of 2 ztl T-DNA insertion mutants grown under warm conditions with continuous white light. Application of a polar auxin transport inhibitor, an auxin antagonist or an auxin biosynthesis inhibitor inhibited hypocotyl elongation of control seedlings to the level observed with the ztl mutant. Our data suggest the involvement of auxin and auxin-inducible genes in ZTL-mediated hypocotyl elongation.     

OBPC Symposium: Maize 2004 &; beyond-developmental and molecular genetics of embryogenesis in plants

Summary Embryo development is a very key phase in the life cycle of seed plants. At maturity, the embryo contains the complete machinery to elaborate the entire plant body. While the embryogenic process is an innate feature of the zygote, gametic and somatic cells can undergo embryogenesis under the appropriate culture conditions. Embryogenesis is a highly regulated process and the use of mutants, especially in Arabidopsis, has allowed the identification of genes regulating pattern formation during this process. The use of such mutants has revealed the eritical roles of auxin levels and transport in the establishment of embryo axis. Root and shoot apical meristem function and integrity, have been defined by examination of genes involved in their identity and function. Further knowledge of the molecular and biochemical aspects of zygotic embryogenesis should contribute to our understanding of the underlying regulatory pathways and networks and also provide critical insights into unique totipotent features of the plant cell.     

Auxin-induced Fruit Set in Capsicum annuum L. Requires Downstream Gibberellin Biosynthesis

A hierarchical scheme for the central role of the plant hormones auxin and gibberellins in fruit set and development has been established for the model plants Arabidopsis and tomato. In the fruit crop Capsicum annuum, the importance of auxin as an early signal in fruit set has also been recognized; however, the effect of gibberellins and their interaction with auxin has not yet been studied. The aim of this study was to determine the role of gibberellin and the hierarchy between auxin and gibberellin. We applied gibberellin alone or in combination with auxin or with the gibberellin biosynthesis inhibitor paclobutrazol on stigmas of emasculated flowers. Gibberellin application enhanced fruit set, whereas application of paclobutrazol reduced fruit set. The effect of paclobutrazol treatment could be counteracted by coapplication of gibberellin but not by auxin_. These results indicate that in C. annuum, like in Arabidopsis and tomato, auxin is the major inducer of fruit set that acts in part by inducing gibberellin biosynthesis. Interestingly, gibberellin does not significantly contribute to the final fruit size but seems to play an important role in preventing flower and fruit abscission, a major determinant of production loss in C. annuum. At the same time, gibberellin together with auxin seems to balance cell division and cell expansion during fruit growth.     

Two homologous INDOLE-3-ACETAMIDE (IAM) HYDROLASE genes are required for the auxin effects of IAM in Arabidopsis

Indole-3-acetamide (IAM) is the first confirmed auxin biosynthetic intermediate in some plant pathogenic bacteria. Exogenously applied IAM or production of IAM by overexpressing the bacterial iaaM gene in Arabidopsis causes auxin overproduction phenotypes. However, it is still inconclusive whether plants use IAM as a key precursor for auxin biosynthesis. Herein, we reported the isolation IAM HYDROLASE 1 (IAMH1) gene in Arabidopsis from a forward genetic screen for IAM-insensitive mutants that display normal auxin sensitivities. IAMH1 has a close homolog named IAMH2 that is located right next to IAMH1 on chromosome IV in Arabidopsis. We generated iamh1 iamh2 double mutants using our CRISPR/Cas9 gene editing technology. We showed that disruption of the IAMH genes rendered Arabidopsis plants resistant to IAM treatments and also suppressed the iaaM overexpression phenotypes, suggesting that IAMH1 and IAMH2 are the main enzymes responsible for converting IAM into indole-3-acetic acid (IAA) in Arabidopsis. The iamh double mutants did not display obvious developmental defects, indicating that IAM does not play a major role in auxin biosynthesis under normal growth conditions. Our findings provide a solid foundation for clarifying the roles of IAM in auxin biosynthesis and plant development.     

Auxin redistribution and shifts in PIN gene expression during Arabidopsis grafting

Auxin is important in the development of plant vascular tissues. Reconnection of vascular bundles between scion and stock is a primary aim of grafting, and polar auxin transport greatly affects the formation of a continuous vascular model. The role of auxin in the process of graft-union development was studied by grafting the seedlings of Arabidopsis thaliana (L.) Heynh. DR5:GUS marker plants, which exert the auxinspecific responses. Auxin induced the DR5:GUS expression in the vascular bundles around graft surface and stimulated the formation of multiple vascular bundle reconnections on the third day after grafting (DAG). DR5:GUS expression was delayed for one day in both scion and stock and dramatically declined by the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA). Vascular bundle reconnection was observed only on the 4th DAG. These results suggest that auxin stimulates the reconnection of the vascular bundles, whereas NPA inhibits it. We studied the role of PIN proteins in graft development by grafting seedlings of PIN:GUS plants. PIN had different expression patterns in the graft process. Expression levels of PIN genes were analyzed by real-time PCR. All PIN genes had the higher expression level at the third DAG. We conclude that auxin stimulates the development of graft unions, and the patterns of expressions of PIN family genes can affect the development of graft-union by controlling the auxin flow.     

Control of vein patterning by intracellular auxin transport

The vein networks of plant leaves are among the most spectacular expressions of biological pattern, and the principles controlling their formation have continually inspired artists and scientists. Control of vein patterning by the polar, cell-to-cell transport of the plant signaling molecule auxin—mediated in Arabidopsis primarily by the plasma-membrane-localized PIN1—has long been known. By contrast, the existence of intracellular auxin transport and its contribution to vein patterning are recent discoveries. The endoplasmic-reticulum-localized PIN5, PIN6, and PIN8 of Arabidopsis define an intracellular auxin-transport pathway whose functions in vein patterning overlap with those of PIN1-mediated intercellular auxin transport. The genetic interaction between the components of the intracellular auxin-transport pathway is far from having been resolved. The study of vein patterning provides experimental access to gain such a resolution—a resolution that in turn holds the promise to improve our understanding of one of the most fascinating examples of biological pattern formation.     

Narciclasine modulates polar auxin transport in Arabidopsis roots

Plant development displays an exceptional plasticity and adaptability that involves the dynamic, asymmetric distribution of the phytohormone auxin. Polar auxin flow, which requires transport facilitators of the PIN family, largely contributes to the establishment and maintenance of auxin gradients and mediates multiple developmental processes. Here, we report the effects of narciclasine (NCS), an Amaryllidaceae alkaloid isolated from Narcissus tazetta bulbs, on postembryonic development of Arabidopsis roots. Arabidopsis seedlings grown on NCS showed defects in root gravitropism which correlates with a reduction in auxin transport in roots. Expressions of auxin transport genes were affected and the polar localization of PIN2 protein was altered under NCS treatment. Taken together, we propose that NCS modulates auxin transport gene expression and PIN2 localization, and thus affects auxin transport and auxin distribution necessary for postembryonic development of Arabidopsis roots.     

Research progresses on <Emphasis Type="Italic">GH3s</Emphasis>, one family of primary auxin-responsive genes

Auxin plays a very important role in plant growth and development. Those genes that are specifically induced by auxin within minutes of exposure to the hormone are referred to as early/primary auxin-responsive genes, mainly including the auxin/indole-3-acetic acid (Aux/IAA), the small auxin-up RNA (SAUR), and the GH3 gene families. So far, GH3 genes have been identified in various plant species including soybean, Arabidopsis, rice, tobacco, pungent pepper, sweet orange, pine, and moss. Twenty members of GH3 family were identified in Arabidopsis and these genes were classified into three groups (Group I–III) based on their sequence similarities and substrate specificities. GH3s belong to acyl adenylate-forming firefly luciferase superfamily and can catalyze adenylation of specific substrates. Group I adenylates jasmonic acid (JA), and Group II adenylates indole-3-acetic acid (IAA) and salicylic acid (SA), respectively. Because of the presence of Auxin-Responsive Elements (AuxRE) in the GH3s’ promoter regions, Auxin Response Factors (ARFs) are able to bind to the AuxRE and regulate expression of some GH3s, which in turn modulate the auxin homeostasis. Identification of GH3 mutants in Arabidopsis reveals the function of GH3s in hypocotyl elongation under different light conditions, root growth, stress adaptation, sensitivity to MeJA, or susceptibility to P. syringae. Taken together, GH3s may be linkers among auxin, JA, SA and light signal transduction pathways.