Plant phosphatidylinositol (PI) metabolic pathway involves in many signaling pathways and developmental processes, including gene expression, defense, auxin function, light control, ion regulation, seed germination, rearrangement of cytoskeleton, and leaf senescence, etc. Using Arabidopsis as material, we focused on the physiological roles of PI pathways in plant development and cell response to environmental factors, with the purpose to explain the functional mechanisms and interaction with other signaling pathways. cDNAs encoding various key enzymes involved in the PI pathway, i.e. PI 4-kinase (PI4-K) and isoforms, PI-4-phosphate 5-kinase (PIP-K), phospholipase D (PLD), inositol 1,4,5-trisphosphate kinase (IPK), and inositol polyphosphate 5-phosphatase were isolated. Expression pattern analysis and functional characterizations were performed.

Based on the analysis of Arabidopsis genome, 82 proteins were involved in PI pathways, and expression pattern of the relevant coding genes were analyzed via cDNA chips under different conditions, including plant hormones (auxin, cytokinin, ABA, GA and brassinosteroid, ethylene) and environmental factors (drought, low-temperature, adversity and disease resistance), which provide comprehensive information on the functional analysis. Relevant knock-out mutants with T-DNA insertion were identified and functional analysis showed that phospholipase D mediated auxin sensitivity through regulation assembly of vesicle, phosphatidylinositol-4-phosphate 5-kinase regulated rice flowering through modification of flowering time genes, or regulated root development through modification of sugar metabolism, inositol polyphosphate kinase effected growth of root hair and pollen tube, and its antisense transgenic lines showed insensitive to EGTA and hypersensitive to Ca2+, 5PTase involved the cotyledon vascular tissue development through regulating auxin homeostasis

 

 

1. Membrane targeting capacities of special domains presented in PI4K and PIPK

MONR motif of PIP-5-kinase, as well as PPC (a highly charged domain) region of PI4-kinase, have the affinity to membrane. These conclusions have been gained from experimental results of transient experssion GFP fusion protein in onion epidermal cells and yeast-based SMET system. Both of them could bind different sort of phospholipids in vitro, respectively providing new regulatory mechanisms of PI signaling pathway.

Fig. A: Hydropathy plots deduced from the OsPI4K2 amino acid sequences were generated with the Kyte-Doolittle algorithm (Kyte and Doolittle, 1982), where increased hydrophobicity is denoted by positive values. Amino acids regions 227-601 of OsPI4K2 are strongly negative, representing a novel charged region, called PPC, which is present only in higher plantβtype PI4Ks at present.

Fig. B-F: Detection of green fluorescence from fusions of GFP with the PPC region of OsPI4K2 [pA7-PPC(OsPI4K2)] (D), as well as the region containing repeat motifs pA7-OsPI4K2-227:401 (E), and pA7-OsPI4K2-402:601 (F), indicated plasma membrane localizations after plasmolysis. The empty vector pA7-GFP was used as negative control (B). The vector pA7-AtMT2 (Sohlenkamp et al., 2002) was used as positive control (C). Cell plasmolysis was achieved by incubating onion epidermal strips in 1 M sucrose solution for 10 min. Bar=20 µm. To facilitate the observation, section Z-series of images at different interval through the specimens was presented.

 

2. PIPK involved in flower timing determination and root development process

OsPIPK1（OsPIPK1, a rice phosphatidylinositol phosphate kinase, regulates rice heading by modifying the expression of floral induction genes. Plant Molecular Biology. 2004）。

The analysis of antisense transgenic lines showed that OsPIPK could regulate rice flowering through modification the transcription of flowering time-related genes (OsPIPK1, a rice phosphatidylinositol monophosphate kinase, regulates rice heading by modifying the expression of floral induction genes. Plant Molecular Biology. 2004).

Fig. A&B: Earlier flowering of T2 and T3 generations of OsPIPK1-deficient plants. The date of flowering was defined as the heading date at which panicles emerged from the flag leaf. T2, generation, A；T3 generation B。

Fig. C: RT-PCR analysis of transcripts of selected floral initiation genes in OsPIPK1-deficient plants. Transcripts of the floral initiation genes OsGI, Hd1, OsCKA2, RFT1 and OsMADS1 were analyzed via RT-PCR. Total RNAs were isolated from rice meristems at the heading stage (40, 45, 50, 55 and 60 days) and RT-PCR was performed for 36 cycles with rice actin amplied as a positive internal control.

An Arabidopsis phosphatidylinositol phosphate-5-kinase 9 (AtPIP5K9) was isolated.The subcellular localization of AtPIP5K was examined by transient expression of AtPIPK9::GFP in onion epidermal cells. Western blot analysis showed the recombinant protein, expressed in E. coli, has the expected molecular weight, 90kDa. Observation of the promoter::GUS transgenic plants revealed that AtPIPK9 was transcribed in all tissues. Analysis of the T-DNA insertion mutant line indicates that AtPIPK9 regulates root cell elongation.

Fig. left: T-DNA insertion mutant resulting in enhanced AtPIP5K9 showed shortened primary roots.

Fig. right: The subcellular localization of AtPIP5K9 was identified by transient expression of GFP fusion proteins in epidermal onion cells.

 

3.PLDζ 2 modified auxin sensitivity through regulating vesicle transportation

Arabidopsis PLDζ2 was isolated by screening cDNA library using PCR strategy and recombinant protein was expressed by E. coliin vitro. After protein expression and purification, a single fragment of protein in expected molecular weight, 120kD, was obtained. The expression of PLDζ2 was accumulated on vascular tissues and stomata of cotyledons and elongation zone of seedling root. It was also detected in pollen grain and embryo of young seeds. The overespression of PLDζ2 lead to hypersensitivity to IAA. The primary root of PLD ζ2 overexpressing transgenic plants was restrained with 10-7 and 10-8 M adscititious auxin. Further analysis showed that this hypersensitivity was due to the modification of vesicle transport. (The Plant Cell, 2007)

Fig. left: Effect of 1-butanol, the specific inhibitor of PLD, on auxin induction of DR5::GUS expression. DR5::GUS seedlings were grown in Murashige and Skoog medium for 7 d and were treated with 0.1 µM IAA, with a different concentration 1-butanol or 2-butanol for 3 h before histochemical GUS staining . A-E treated with a concentration of: 0 (A), 0.2% (B), 0.4% (C), 0.6% (D), 0.8% (E) 1-butanol, the auxin induced GUS stain in elongation region of root were obviously shorted and weaken. F, treated with 0.8% 2-butnaol, without effect on auxin induced GUS staining.

Fig. right: PLDζ2 overexpression lines show altered BFA sensitive. In wild type, when treated with 50 µM BFA 2 hours, BFA compartments were appeared in all the cell; while in PLDζ2 overexpression lines only partially cell appeared BFA compartment.

 

4. IPK involved the germination of pollen tube and the growth of root hair through Ca2+-dependent manner

Inositol 1,4,5-trisphosphate 6/3-kinase involved in the regulation of internal calcium store in plant cells, and play critical roles in pollen grain germination and pollen tube growth. Promoter–reporter gene studies revealed that Arabidopsis IPK was highly expressed in floral tissues including pollen grain, pollen tube and pistil. Transgenic approaches employing antisense strategy showed that transgenic plants harboured enhanced pollen tube and root hair growth comparing to control plants under Ca2+ deficient conditions (A role of Arabidopsis inositol polyphosphate kinase, AtIPK2, in pollen germination and root growth. Plant Physiology. 2005).

Transgenic plants under IPK deficiency showed enhanced growth of root and pollen tubes under different Ca2+ deficient conditions. Further studies through cross of IPK-deficiency plants and Ca2+-marker lines, and overexpressing of IPK will facilitate the physiological characterizations.

Fig. upper: Root hairs formed in the presence of supplemented EGTA at concentrations of 0.1 and 0.5 mM. Images of joint position of hypocotyls and roots and root were obtained by SEM. Note, that the AtIPK2aantisense line produced longer root hairs at both concentrations of EGTA.

Fig. bottom: Visual analysis of pollen grains and pollen tube growth. Pollen sampled from the Arabidopsis wild type (A, C and E), and AtIPK2aantisense line A8 (B, D and F) was germinated in the presence of varying concentrations of supplemented Ca2+(0, 5, or 50 mM), or in the presence of 0.1 mM EGTA. Pictures were taken 6 h after pollen germination. Bar=20 mm.

 

5. 5PTase involved auxin-related cotyledon vein development

Analysis of inositol polyphosphate phosphatase gene family on genome level, including the expression pattern analysis, structural analysis revealed the presence of 15 isoforms, which can be subgrouped into two subfamilies. All isoforms have high conserved catalytic domain but only members of subfamily II have WD40 repeats. The expression pattern analysis revealed differential expressions of individuals. FRA3 expressed in seedling, root, leaf and flower. 5PTase8 expressed in shoot meristem and ovule. 5PTase12 transcribed in seedlings and mature pollen grain. 5PTase13 expressed in seedlings and the apex of inflorescence. 5PTase14 transcribed in the lamina, vascular tissues and hydratodes of cotyledon and leaf hydratodes. The transcription level has been induced by blue light, red light and far-red light. We have isolated 5 full cDNA of 5PTase by screening library using isotope and PCR strategy and done the protein expression, purification and western-blot confirmation in vitro about 5PTase2, 13 and FRA3 using E. coli and insect cell system. Enzymatic assay results showed that they have 5-phosphatase activity against IP3 and IP4. Among them, 5PTase13 aims at against IP4 mostly.

One isoform, named 5PTase13, belong to subfamily II, was specially expressed in cotyledons, hypocotyls and roots of seedlings ，as well as the apex of inflorescence stem including hydratodes of last leaf, petal, sepal and pollen grain. Its transcription seemed to be variational during seedling development and similar to auxin distributing. 5PTase was also induced by blue light and darkness and decreased by IAA, GA, KT and BR. The At5PTase13 loss-of-function mutant was identified and confirmed. Phenotypic analysis showed the abnormal cotyledon vein pattern, root development, gravity sensitivity and shorter hypocotyls under blue light, which could be rescued or partly resumed by exogenous applied auxin. Transcription of some genes encoding proteins critical for multiple auxin biosynthesis and polar transport have been altered, suggesting the roles of At5PTase13 in hormone (auxin)-dependant cotyledon vein and root development. Yeast one-hybrid results indicated At5PTase13 involved in auxin-related Arabidopsis cotyledon vascular tissues development by effecting CYP83B1 transcription level through its WD40 domain. The transcription level of PIN2 and PIN4 suggested that the At5PTase13 involves root development and gravity response through effecting auxin polar transport. Yeast two hybrid results showed that At5PTase13 involved in blue light signaling through a novel way without interaction with CRY1 and COP1 directly (At5PTase13 modulates cotyledon vein development through regulating auxin homeostasis. Plant Physiology. 2005).

Fig. A: Comparing to the cotyledon veins of wild type plants (a, 4-days, mid-vein and distal secondary vein developed), those of At5pt13 showed various abnormal patterns (b-i), including altered numbers of veins (b, c), improper orientation (d, e), additional loops (f, g), and coarse (i, comparing to that of wild type plants, h). The abnormalities are highlighted by arrows. Vascular tissues were observed using differential interference contrast (DIC) microscopy.

Fig. B: Altered auxin accumulation and distribution in At5PTase13-deficient plants.

GUS activity in homozygous DR5-GUS/At5pt13 cross offspring showed altered remarkable auxin

levels and distribution in seedlings grown at 1 DAG (two independent homozygous lines, L1 and 2, -NAA, are shown) after germination. Adscititious NAA in low concentration could enhance auxin levels in both DR5-GUS/At5pt13 and control plants. Bar=2mm.

Fig C: The hypothesized model of how At5PTase13 involves in the cotyledon vein development through regulating auxin homeostasis and Ins(1,3,4,5)P4 related Ca2+. In normal conditions, At5PTase13 suppresses CYP83B1 and keeps higher IAA/IAN homeostasis. In At5pt13,

release of CYP83B1 leads to more IAN in vivo and lower IAA/IAN homeostasis. Ins(1,3,4,5)P4 related Ca2+ may interact with auxin homeostasis to modulate the vascular development.


6. Arabidopsis phosphatidylinositol monophosphate 5-kinase 2 is involved in root gravitropism through regulation of polar auxin transport by affecting the cycling of PIN proteins

Phosphatidylinositol monophosphate 5-kinase (PIP5K) catalyzes the synthesis of PI-4,5-bisphosphate (PtdIns(4,5)P2) by phosphorylation of PI-4-phosphate at the 5 position of the inositol ring, and is involved in regulating multiple developmental processes and stress responses. We present the functional characterization of Arabidopsis PIP5K2, which is expressed during lateral root initiation and elongation, and whose expression is enhanced by exogenous auxin. The knockout mutant pip5k2 shows reduced lateral root formation, which could be recovered with exogenous auxin, and interestingly, delayed root gravity response that could not be recovered with exogenous auxin. Crossing with the DR5-GUS marker line and measurement of free IAA content confirmed the reduced auxin accumulation in pip5k2. In addition, analysis using the membrane-selective dye FM4-64 revealed the decelerated vesicle trafficking caused by PtdIns(4,5)P2 reduction, which hence results in suppressed cycling of PIN proteins (PIN2 and 3), and delayed redistribution of PIN2 and auxin under gravistimulation in pip5k2 roots. On the contrary, PtdIns(4,5)P2 significantly enhanced the vesicle trafficking and cycling of PIN proteins. These results demonstrate that PIP5K2 is involved in regulating lateral root formation and root gravity response, and reveal a critical role of PIP5K2/PtdIns(4,5)P2 in root development through regulation of PIN proteins, providing direct evidence of crosstalk between the phosphatidylinositol signaling pathway and auxin response, and new insights into the control of polar auxin transport. (Cell Research, 2011)