Hong-Wei Xue

Control of Root Cap Formation by MicroRNA-Targeted Auxin Response Factors in Arabidopsis

The plant root cap mediates the direction of root tip growth and protects internal cells. Root cap cells are continuously produced from distal stem cells, and the phytohormone auxin provides position information for root distal organization. Here, we identify the Arabidopsis thaliana auxin response factors ARF10 and ARF16, targeted by microRNA160 (miR160), as the controller of root cap cell formation. The Pro35S:MIR160 plants, in which the expression of ARF10 and ARF16 is repressed, and the arf10-2 arf16-2 double mutants display the same root tip defect, with uncontrolled cell division and blocked cell differentiation in the root distal region and show a tumor-like root apex and loss of gravity-sensing. ARF10 and ARF16 play a role in restricting stem cell niche and promoting columella cell differentiation; although functionally redundant, the two ARFs are indispensable for root cap development, and the auxin signal cannot bypass them to initiate columella cell production. In root, auxin and miR160 regulate the expression of ARF10 and ARF16 genes independently, generating a pattern consistent with root cap development. We further demonstrate that miR160-uncoupled production of ARF16 exerts pleiotropic effects on plant phenotypes, and miR160 plays an essential role in regulating Arabidopsis development and growth.

Coexpression Analysis Identifies Rice Starch Regulator1, a Rice AP2/EREBP Family Transcription Factor, as a Novel Rice Starch Biosynthesis Regulator

Starch biosynthesis is important for plant development and is a critical factor in crop quality and nutrition. As a complex metabolic pathway, the regulation of starch biosynthesis is still poorly understood. We here present the identification of candidate regulators for starch biosynthesis by gene coexpression analysis in rice (Oryza sativa). Starch synthesis genes can be grouped into type I (in seeds; sink tissues) and type II (in vegetative tissues; source tissues), and 307 and 621 coexpressed genes are putatively involved in the regulation of starch biosynthesis in rice seeds and vegetative tissues, respectively. Among these genes, Rice Starch Regulator1 (RSR1), an APETALA2/ethylene-responsive element binding protein family transcription factor, was found to negatively regulate the expression of type I starch synthesis genes, and RSR1 deficiency results in the enhanced expression of starch synthesis genes in seeds. Seeds of the knockout mutant rsr1 consistently show the increased amylose content and altered fine structure of amylopectin and consequently form the round and loosely packed starch granules, resulting in decreased gelatinization temperature. In addition, rsr1 mutants have a larger seed size and increased seed mass and yield. In contrast, RSR1 overexpression suppresses the expression of starch synthesis genes, resulting in altered amylopectin structure and increased gelatinization temperature. Interestingly, a decreased proportion of A chains in rsr1 results in abnormal starch granules but reduced gelatinization temperature, whereas an increased proportion of A chains in RSR1-overexpressing plants leads to higher gelatinization temperatures, which is novel and different from previous reports, further indicating the complicated regulation of starch synthesis and determination of the physicochemical properties of starch. These results demonstrate the potential of coexpression analysis for studying rice starch biosynthesis and the regulation of a complex metabolic pathway and provide informative clues, including the characterization of RSR1, to facilitate the improvement of rice quality and nutrition.

Characterization and expression profiles of miRNAs in rice seeds

Small RNAs (sRNAs) are common and effective modulators of gene expression in eukaryotic organisms. To characterize the sRNAs expressed during rice seed development, massively parallel signature sequencing (MPSS) was performed, resulting in the obtainment of 797 399 22-nt sequence signatures, of which 111 161 are distinct ones. Analysis on the distributions of sRNAs on chromosomes showed that most sRNAs originate from interspersed repeats that mainly consist of transposable elements, suggesting the major function of sRNAs in rice seeds is transposon silencing. Through integrative analysis, 26 novel miRNAs and 12 miRNA candidates were identified. Further analysis on the expression profiles of the known and novel miRNAs through hybridizing the generated chips revealed that most miRNAs were expressed preferentially in one or two rice tissues. Detailed comparison of the expression patterns of miRNAs and corresponding target genes revealed the negative correlation between them, while few of them are positively correlated. In addition, differential accumulations of miRNAs and corresponding miRNA*s suggest the functions of miRNA*s other than being passenger strands of mature miRNAs, and in regulating the miRNA functions.

Arabidopsis PLDζ2 Regulates Vesicle Trafficking and Is Required for Auxin Response

Phospholipase D (PLD) and its product, phosphatidic acid (PA), play key roles in cellular processes, including stress and hormonal responses, vesicle trafficking, and cytoskeletal rearrangements. We isolated and functionally characterized Arabidopsis thaliana PLDζ2, which is expressed in various tissues and enhanced by auxin. A PLDζ2-defective mutant, pldζ2, and transgenic plants deficient in PLDζ2 were less sensitive to auxin, had reduced root gravitropism, and suppressed auxin-dependent hypocotyl elongation at 29°C, whereas transgenic seedlings overexpressing PLDζ2 showed opposite phenotypes, suggesting that PLDζ2 positively mediates auxin responses. Studies on the expression of auxin-responsive genes and observation of the β-glucuronidase (GUS) expression in crosses between pldζ2 and lines containing DR5-GUS indicated that PLDζ2, or PA, stimulated auxin responses. Observations of the membrane-selective dye FM4-64 showed suppressed vesicle trafficking under PLDζ2 deficiency or by treatment with 1-butanol, a PLD-specific inhibitor. By contrast, vesicle trafficking was enhanced by PA or PLDζ2 overexpression. Analyses of crosses between pldζ2 and lines containing PIN-FORMED2 (PIN2)–enhanced green fluorescent protein showed that PLDζ2 deficiency had no effect on the localization of PIN2 but blocked the inhibition of brefeldin A on PIN2 cycling. These results suggest that PLDζ2 and PA are required for the normal cycling of PIN2-containing vesicles as well as for function in auxin transport and distribution, and hence auxin responses.

Brassinosteroids stimulate plant tropisms through modulation of polar auxin transport in Brassica and Arabidopsis.

Brassinosteroids (BRs) are important plant growth regulators in multiple developmental processes. Previous studies have indicated that BR treatment enhanced auxin-related responses, but the underlying mechanisms remain unknown. Using 14C-labeled indole-3-acetic acid and Arabidopsis thaliana plants harboring an auxin-responsive reporter construct, we show that the BR brassinolide (BL) stimulates polar auxin transport capacities and modifies the distribution of endogenous auxin. In plants treated with BL or defective in BR biosynthesis or signaling, the transcription of PIN genes, which facilitate functional auxin transport in plants, was differentially regulated. In addition, BL enhanced plant tropistic responses by promoting the accumulation of the PIN2 protein from the root tip to the elongation zone and stimulating the expression and dispersed localization of ROP2 during tropistic responses. Constitutive overexpression of ROP2 results in enhanced polar accumulation of PIN2 protein in the root elongation region and increased gravitropism, which is significantly affected by latrunculin B, an inhibitor of F-actin assembly. The ROP2 dominant negative mutants (35S-ROP2-DA/DN) show delayed tropistic responses, and this delay cannot be reversed by BL addition, strongly supporting the idea that ROP2 modulates the functional localization of PIN2 through regulation of the assembly/reassembly of F-actins, thereby mediating the BR effects on polar auxin transport and tropistic responses.

PIP5K9, an Arabidopsis Phosphatidylinositol Monophosphate Kinase, Interacts with a Cytosolic Invertase to Negatively Regulate Sugar-Mediated Root Growth

Phosphatidylinositol monophosphate 5-kinase (PIP5K) plays an essential role in coordinating plant growth, especially in response to environmental factors. To explore the physiological function of PIP5K, we characterized Arabidopsis thaliana PIP5K9, which is constitutively expressed. We found that a T-DNA insertion mutant, pip5k9-d, which showed enhanced PIP5K9 transcript levels, had shortened primary roots owing to reduced cell elongation. Transgenic plants overexpressing PIP5K9 displayed a similar root phenotype. Yeast two-hybrid assays identified a cytosolic invertase, CINV1, that interacted with PIP5K9, and the physiological relevance of this interaction was confirmed by coimmunoprecipitation studies using plant extracts. CINV1-deficient plants, cinv1, had reduced activities of both neutral and acid invertases as well as shortened roots. Invertase activities in pip5k9-d seedlings were also reduced, suggesting a negative regulation of CINV1 by PIP5K9. In vitro studies showed that PIP5K9 interaction indeed repressed CINV1 activities. Genome-wide expression studies revealed that genes involved in sugar metabolism and multiple developmental processes were altered in pip5k9-d and cinv1, and the altered sugar metabolism in these mutants was confirmed by metabolite profiling. Together, our results indicate that PIP5K9 interacts with CINV1 to negatively regulate sugar-mediated root cell elongation.

SHALLOT-LIKE1 Is a KANADI Transcription Factor That Modulates Rice Leaf Rolling by Regulating Leaf Abaxial Cell Development

As an important agronomic trait, rice (Oryza sativa L.) leaf rolling has attracted much attention from plant biologists and breeders. Moderate leaf rolling increases the photosynthesis of cultivars and hence raises grain yield. However, the relevant molecular mechanism remains unclear. Here, we show the isolation and functional characterization of SHALLOT-LIKE1 (SLL1), a key gene controlling rice leaf rolling. sll1 mutant plants have extremely incurved leaves due to the defective development of sclerenchymatous cells on the abaxial side. Defective development can be functionally rescued by expression of SLL1. SLL1 is transcribed in various tissues and accumulates in the abaxial epidermis throughout leaf development. SLL1 encodes a SHAQKYF class MYB family transcription factor belonging to the KANADI family. SLL1 deficiency leads to defective programmed cell death of abaxial mesophyll cells and suppresses the development of abaxial features. By contrast, enhanced SLL1 expression stimulates phloem development on the abaxial side and suppresses bulliform cell and sclerenchyma development on the adaxial side. Additionally, SLL1 deficiency results in increased chlorophyll and photosynthesis. Our findings identify the role of SLL1 in the modulation of leaf abaxial cell development and in sustaining abaxial characteristics during leaf development. These results should facilitate attempts to use molecular breeding to increase the photosynthetic capacity of rice, as well as other crops, by modulating leaf development and rolling.

Arabidopsis Membrane Steroid Binding Protein 1 Is Involved in Inhibition of Cell Elongation

A putative Membrane Steroid Binding Protein (designated MSBP1) was identified and functionally characterized as a negative regulator of cell elongation in Arabidopsis thaliana. The MSBP1 gene encodes a 220–amino acid protein that 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. The reduced cell elongation of MSBP1-overexpressing plants was correlated with altered expression of genes involved in cell elongation, such as expansins and extensins, indicating that enhanced MSBP1 affected a regulatory pathway for cell elongation. Suppression or overexpression of MSBP1 resulted in enhanced or reduced sensitivities, respectively, to exogenous progesterone and 24-eBL, suggesting a negative role of MSBP1 in steroid signaling. Expression of MSBP1 in hypocotyls is suppressed by darkness and activated by light, suggesting that MSBP1, as a negative regulator of cell elongation, plays a role in plant photomorphogenesis. This study demonstrates the functional roles of a steroid binding protein in growth regulation in higher plants.

Rice ABI5-Like1 Regulates Abscisic Acid 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 (Arabidopsis thaliana), some of which regulate ABA responses by the transcriptional regulation of downstream genes. Here, we report the functional identification of rice (Oryza sativa) ABI5-Like1 (ABL1), which is a basic region/leucine zipper motif transcription factor. ABL1 is expressed in various tissues and is induced by the hormones ABA and indole-3-acetic acid 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 ABA-responsive elements (ABREs; G-box) in vitro. A gene expression analysis by DNA chip hybridization confirms that a large proportion of down-regulated genes of abl1 are involved in stress responses, 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 indole-3-acetic acid, 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.

Inositol trisphosphate-induced Ca2+ signaling modulates auxin transport and PIN polarity

The phytohormone auxin is an important determinant of plant development. Directional auxin flow within tissues depends on polar localization of PIN auxin transporters. To explore regulation of PIN-mediated auxin transport, we screened for suppressors of PIN1 overexpression (supo) and identified an inositol polyphosphate 1-phosphatase mutant (supo1), with elevated inositol trisphosphate (InsP(3)) and cytosolic Ca(2+) levels. Pharmacological and genetic increases in InsP(3) or Ca(2+) levels also suppressed the PIN1 gain-of-function phenotypes and caused defects in basal PIN localization, auxin transport and auxin-mediated development. In contrast, the reductions in InsP(3) levels and Ca(2+) signaling antagonized the effects of the supo1 mutation and disrupted preferentially apical PIN localization. InsP(3) and Ca(2+) are evolutionarily conserved second messengers involved in various cellular functions, particularly stress responses. Our findings implicate them as modifiers of cell polarity and polar auxin transport, and highlight a potential integration point through which Ca(2+) signaling-related stimuli could influence auxin-mediated development.