Plant hormones are critical in plant development. We focused on the effects of plant hormones on development of specific tissue or cells, the relevant functional mechanisms, interactions with other signaling pathways, with the emphasis on hormone-responsive genes, receptor, binding proteins and related signal transduction pathways.

 

Polar auxin transport, which is critical in plant embryo development, vascular tissuedevelopment and so on, plays significant roles on rice root development especially on lateral roots. Application of HFCA or TIBA, inhibitor of polar auxin transport, resulted in the shortened primary roots, less numbers of lateral and secondary roots. However, presence of primordial of lateral roots under inhibition of polar auxin transport revealed different functional mechanism comparing to that in dicots and monocots.

Treatment with polar auxin transport inhibitors, such as HFCA, NPA and TIBA, results in lignification of vascular tissues, altered development of vessel, expanding of motor cells, and alteration of transverse vein numbers. In addition, the differences of primary and secondary veins were getting non-obvious.

2.Studies of OsPIN family, the efflux carrier of auxin

Studies of rice genome identified of 8 putative OsPIN genes. Three full-length cDNAs were amplified (OsPIN3, 4, and 5). The promoter regions of OsPIN1, 2, 3, 4 were fused with reporter gene (GUS) and transformed to rice genome. Analysis of the transgenic lines showed that OsPIN1 was constitutively expressed, OsPIN2 was mainly expressed in leaf, OsPIN3 was expressed in leaf lateral veins and flowers, and OsPIN4 was expressed in leaf main veins and vascular bundle of the other organs. Physiological roles of them were studied through analyzing the transgenic plants with overexpressed or deficient genes. The deficiency of OsPIN3 leads to the abnormal pollen development.

Fig. A: Affection of PAT inhibitor (NPA) on rice seeding growth.

Fig. B: Morphological change of PAT inhibitor (NPA) treated leaves. The 1st leaf of rice becomes longer and narrower.

Fig. C: Ospin4 is expressed in vascular tissues of rice stem.

3. Auxin flow in anther filaments defect pollen grain development through affecting pollen mitosis

Auxin flow in filaments is important for pollen grain development. Through analyzing auxin responsible marker line DR5, we show that auxin is accumulated in anther (floral stages 10 to 12) and sepal (floral stages 12 to 15) during floral development. Studies employing the indoleacetic-acid-lysine synthetase (iaaL) coding gene, which could decline IAA biosynthesis, under control of an Arabidopsis promoter of phosphatidylinositol monophosphate 5-kinase 1 (AtPIP5K1), with anther filament-specific expression showed that block of auxin flow of filaments resulted in shortened filaments, and more interestingly, defected pollen grains. Similar phenotype was observed in tobacco plants transformed with the same construct. Detailed studies further show that the meiosis process of pollen grain is normal while the mitosis at later stage is significantly defected, revealing the effects of auxin flow in filaments on mitosis process of pollen grain. Analysis employing [14C]-IAA, as well the expression of AtPIN1, coding for auxin efflux carrier, demonstrated the presence of polar auxin transport in filaments.

Fig. A: AtPIP5K-iaaL transgenic plant (Arabidopsis or tobacco) exhibits shorter filament than that of wild type.

Fig. B: Pollen grains of AtPIP5K-iaaL transgenic plant are mainly vacuolated microspores but those of wild type are three-type mature pollen grains, indicating that mitosis process of pollen grains is heavily blocked at flower stage 12.

4. Interaction of auxin and brassinosteroids (BR): BR plays physiological roles on plant tropisms through regulation of polar auxin transport

Our studies showed that polar auxin transportation was altered by BR treatment and in BR-related mutants. Analysis of the auxin marker line DR5 treated with BR shows the altered distribution of auxin, indicating that BR affects the polar transport and distribution of auxin. The expressions of PIN gene family were studied by Real-time PCR in detail and the results showed that PIN genes were regulated under BR treatment, indicating that effects of BR on plant development such as hypocotyl elongation, root growth or tropistic responses, may be mediated by a general impact of BR on the expression of PIN genes. Studies on the localization of PIN2 from the root tip to the elongation zone, as defined by the analysis of PIN2-GFP fusion, indicating the BR regulation on its accumulation. In addition, analysis on the expression and localization of ROP2 (ROP2-YFP fusion) in response to gravity and BR stimulation, suggests that BR first affect ROP2 expression, localization and activity, thereby modulating expression and localization of PIN2, the latter being modulated by F-actin-dependent processes.

BR-caused altered distribution of endogenous auxin, revealed by detection of GUS activities of DR5-GUS-harbouring A. thaliana seedlings. 4-day-old seedlings growing in the medium supplemented with series concentrations of 24-eBL (0-0.2 μM) were analyzed. GUS activities were highly strengthened at leaves, the elongation zone of roots, and the junctions. Brown frames highlighted the significantly enhanced GUS expressions. Regions squared with black frames were enlarged in the flanking images. Bar=1 mm.

5. Membrane Steroid Binding Protein 1 (MSBP1) negatively controls cell elongation.

Four putative Steroid Binding Proteins (SBPs) were identified by searching Arabidopsis genome using homolog from other species. One membrane localization SBP (MSBP1) and one nucleus localization SBP (NSBP1) were functionally studied. MSBP1 was characterized as a negative regulator of cell elongation. Expression of MSBP1 in hypocotyl is suppressed by darkness and activated by light, suggesting that MSBP1, as a negative regulator of cell elongation, plays a role in plant photomorphogenesis. EMSA and ChIP assay confirmed MSBP1 as the target of HY5 and HYH, to mediate the blue light signaling and BR signaling for a complex network in photomorphogensis.

MSBP1 can bind to progesterone, 5-dihydrotestosterone, 24-epi-brassinolide (24-eBL), and stigmasterol with different affinities in vitro. Transgenic plants overexpressing MSBP1 showed short hypocotyl phenotype and increased steroid binding capacity in membrane fractions, whereas antisense MSBP1 transgenic plants showed long hypocotyl phenotypes and reduced steroid binding capacity, indicating that MSBP1 negatively regulates hypocotyl elongation (Arabidopsis membrane steroid-binding protein 1 (MSBP1) is involved in inhibition of cell elongation. As a cofactor of BAK1, MSBP1 could negatively regulate brassinosteroid signaling by enhancing the endocytosis of BAK1(Plant Cell.2005; Cell Research, 2009; Molecular Plant, 2011).

Fig. A: Predicted stereo structure of MSBP 1. Arrow indicates the putative binding pocket.

Fig. B: Growth of wild-type and transgenic plants in darkness for 4 DAG. T test analysis showed the significant changes of the shortened hypocotyls of MSBP1-overexpressing plants (P < 0.01).

Fig. C: A functional model for MSBP1. Transcription of MSBP1 is activated by light signaling. The MSBP1 protein is localized to the plasma membrane. MSBP1 may perceive a growth-inhibiting steroid signal.

We will emphasize on the signal mechanism of MSBP1. With the help of yeast two-hybrid and Co-IP strategies, we have proved the direct interaction of MSBP1 and the extracellular domain of BAK1. As BAK1 participates in BR signal transduction, we will focus on whether the MSBP1 involved in the BR signaling pathway.

 

6.OsMDP1 is a key negative regulator in BR signaling pathway

OsMDP1 (Oryza sativa MADS-domain-containing protein 1) encode a rice AG-like MADS-box protein. Expression pattern analyses indicated that OsMDP1 is mainly transcribed in vegetative tissues, including the mature leaf, coleoptile, root elongation zone, culm internode, and especially the joint region between the leaf blade and sheath. Further studies revealed that transcription of OsMDP1 is suppressed by brassinolide treatment. OsMDP1 deficiency induced by antisense technology resulted in shortened primary roots, elongated coleoptiles and enhanced lamina joint inclinations. Moreover, transgenic plants showed hypersensitivities to exogenous brassinolide in terms of lamina joint inclination and coleoptile elongation. OsMDP1-deficient plants were found to overexpress OsXTR1, which encodes xyloglucan endotransglycosylase, the cell-wall loosing enzyme necessary for cell elongation. Together, these results indicate that OsMDP1 plays a key negative regulatory role in BR signaling through suppression of OsXTR1.

 

OsMDP1 deficiency results in the enhanced inclination of etiolated leaf lamina joint following 24-eBL treatments.

7.Rice ABI5-like1 Regulates ABA and Auxin Responses by Affecting the Expression of ABRE-Containing Genes

Abscisic acid (ABA) regulates plant development and is crucial for plant responses to biotic and abiotic stresses. Studies have identified the key components of ABA signaling in Arabidopsis thaliana, some of which regulate the ABA responses by the transcriptional regulation of downstream genes. We present the functional identification of rice (Oryza sativa) ABI5-like1 (ABL1), which is a bZIP transcription factor. ABL1 is expressed in various tissues and is induced by the hormones ABA and IAA and stress conditions including salinity, drought and osmotic pressure. The ABL1 deficiency mutant, abl1, shows suppressed ABA responses, and ABL1 expression in the Arabidopsis abi5 mutant rescued the ABA sensitivity. The ABL1 protein is localized to the nucleus and can directly bind ABRE (G-box) elements in vitro. A gene expression analysis by DNA chip hybridization confirms that the large proportion of down-regulated genes of abl1 is involved in stress responses, being consistent with the transcriptional activating effects of ABL1. Further studies indicate that ABL1 regulates the plant stress responses by regulating a series of ABRE-containing WRKY family genes. In addition, the abl1 mutant is hypersensitive to exogenous IAA, and some ABRE-containing genes related to auxin metabolism or signaling are altered under ABL1 deficiency, suggesting that ABL1 modulates ABA and auxin responses by directly regulating the ABRE-containing genes. (Plant Physiology, 2011)