Yunrong Wu

OsPTF1, a Novel Transcription Factor Involved in Tolerance to Phosphate Starvation in Rice

We report here on a novel transcription factor with a basic helix-loop-helix domain for tolerance to inorganic phosphate (Pi) starvation in rice (Oryza sativa). The gene is designated OsPTF1. The expression of OsPTF1 is Pi starvation induced in roots while constitutively expressed in shoots, as shown by northern-blot analysis. Overexpression of OsPTF1 enhanced tolerance to Pi starvation in transgenic rice. Tillering ability, root and shoot biomass, and phosphorus content of transgenic rice plants were about 30% higher than those of the wild-type plants in Pi-deficient conditions in hydroponic experiments. In soil pot and field experiments, more than 20% increase in tiller number, panicle weight, and phosphorus content was observed in transgenic plants compared to wild-type plants at low-Pi levels. In Pi-deficient conditions, transgenic rice plants showed significantly higher total root length and root surface area, which results in a higher instantaneous Pi uptake rate over their wild-type counterparts. Microarray analysis for transgenic plants overexpressing OsPTF1 has been performed to investigate the downstream regulation of OsPTF1.

OsARF12, a transcription activator on auxin response gene, regulates root elongation and affects iron accumulation in rice (Oryza sativa)

• Auxin has an important role in maintaining optimal root system architecture (RSA) that can cope with growth reductions of crops caused by water or nutrient shortages. However, the mechanism of controlling RSA remains largely unclear. Here, we found a limiting factor of RSA--OsARF12--an auxin response factor whose knockout led to decreased primary root length in rice (Oryza sativa). • OsARF12 as a transcription activator can facilitate the expression of the auxin response element DR5::GFP, and OsARF12 was inhibited by osa-miRNA167d by transient expression in tobacco and rice callus. • The root elongation zones of osarf12 and osarf12/25, which had lower auxin concentrations, were distinctly shorter than for the wild-type, possibly as a result of decreased expression of auxin synthesis genes OsYUCCAs and auxin efflux carriers OsPINs and OsPGPs. The knockout of OsARF12 also altered the abundance of mitochondrial iron-regulated (OsMIR), iron (Fe)-regulated transporter1 (OsIRT1) and short postembryonic root1 (OsSPR1) in roots of rice, and resulted in lower Fe content. • The data provide evidence for the biological function of OsARF12, which is implicated in regulating root elongation. Our investigation contributes a novel insight for uncovering regulation of RSA and the relationship between auxin response and Fe acquisition.

OsCYT-INV1 for alkaline/neutral invertase is involved in root cell development and reproductivity in rice (Oryza sativa L.)

A short root mutant was isolated from an EMS-generated rice mutant library. Under normal growth conditions, the mutant exhibited short root, delayed flowering, and partial sterility. Some sections of the roots revealed that the cell length along the longitudinal axis was reduced and the cell shape in the root elongation zone shrank. Genetic analysis indicated that the short root phenotype was controlled by a recessive gene. Map-based cloning revealed that a nucleotide substitution causing an amino acid change from Gly to Arg occurred in the predicted rice gene (Os02g0550600). It coded an alkaline/neutral invertase and was homologous to Arabidopsis gene AtCyt-inv1. This gene was designated as OsCyt-inv1. The results of carbohydrate analysis showed an accumulation of sucrose and reduction of hexose in the Oscyt-inv1 mutant. Exogenously supplying glucose could rescue the root growth defects of the Oscyt-inv1 mutant. These results indicated that OsCyt-inv1 played important roles in root cell development and reproductivity in rice.

A Gain-of-Function Mutation in OsIAA11 Affects Lateral Root Development in Rice

Lateral roots are important to plants for the uptake of nutrients and water. Several members of the Aux/IAA family have been shown to play crucial roles in lateral root development. Here, a member of the rice Aux/IAA family genes, OsIAA11 (LOC_Os03g43400), was isolated from a rice mutant defective in lateral root development. The gain-of-function mutation in OsIAA11 strictly blocks the initiation of lateral root primordia, but it does not affect crown root development. The expression of OsIAA11 is defined in root tips, lateral root caps, steles, and lateral root primordia. The auxin reporter DR5-GUS (β-glucuronidase) was expressed at lower levels in the mutant than in wild-type, indicating that OsIAA11 is involved in auxin signaling in root caps. The transcript abundance of both OsPIN1b and OsPIN10a was diminished in root tips of the Osiaa11 mutant. Taken together, the results indicate that the gain-of-function mutation in OsIAA11 caused the inhibition of lateral root development in rice.

OsCYP2, a chaperone involved in degradation of auxin‐responsive proteins, plays crucial roles in rice lateral root initiation

Auxin plays a pivotal role in many facets of plant development. It acts by inducing the interaction between auxin-responsive [auxin (AUX)/indole-3-acetic acid (IAA)] proteins and the ubiquitin protein ligase SCF(TIR) to promote the degradation of the AUX/IAA proteins. Other cofactors and chaperones that participate in auxin signaling remain to be identified. Here, we characterized rice (Oryza sativa) plants with mutations in a cyclophilin gene (OsCYP2). cyp2 mutants showed defects in auxin responses and exhibited a variety of auxin-related growth defects in the root. In cyp2 mutants, lateral root initiation was blocked after nuclear migration but before the first anticlinal division of the pericycle cell. Yeast two-hybrid and in vitro pull-down results revealed an association between OsCYP2 and the co-chaperone Suppressor of G2 allele of skp1 (OsSGT1). Luciferase complementation imaging assays further supported this interaction. Similar to previous findings in an Arabidopsis thaliana SGT1 mutant (atsgt1b), degradation of AUX/IAA proteins was retarded in cyp2 mutants treated with exogenous 1-naphthylacetic acid. Our results suggest that OsCYP2 participates in auxin signal transduction by interacting with OsSGT1.

cDNA-AFLP analysis of inducible gene expression in rice seminal root tips under a water deficit.

The seminal roots of an upland rice variety, Azucena, showed accelerated elongation in response to a water deficit. The elongation of cortical cells in the elongation zone is rapidly stimulated within 16 h by the water deficit. cDNA-AFLP analysis was used to examine gene expression in seminal root tips at four time points (4, 16, 48 and 72 h) during the water deficit. One hundred and six unique genes induced by the water deficit were obtained. The expression patterns of these genes were confirmed by Northern blot analysis based on 21 selected genes representing different patterns. The 106 upregulated genes were composed of 60 genes of known function, 28 genes of unknown function and 18 novel genes. Sixty genes of known functions were involved in transport facilitation, metabolism and energy, stress- and defense-related proteins, cellular organization and cell-wall biogenesis, signal transduction, expression regulator and transposable element, suggesting that seminal root tips undergo a complex adaptive process in response to the water deficit. Expression of 22 genes reached a maximum within 16 h of water deficit treatment. These included aquaporin (PIP2a), 9-cis-epoxycarotenoid dioxygenase (NCED1) and a negative regulator of gibberellin signal transduction (SPY); eight other genes participated in cell wall loosening or vesicle traffic.

A novel short-root gene encodes a glucosamine-6-phosphate acetyltransferase required for maintaining normal root cell shape in rice.

Glycosylation is a posttranslational modification occurring in many secreted and membrane-associated proteins in eukaryotes. It plays important roles in both physiological and pathological processes. Most of these protein modifications depend on UDP-N-acetylglucosamine. In this study, a T-DNA insertional rice (Oryza sativa) mutant exhibiting a temperature-sensitive defect in root elongation was isolated. Genetic and molecular analysis indicated that the mutated phenotype was caused by loss of function of a gene encoding a glucosamine-6-P acetyltransferase (designated OsGNA1), which is involved in de novo UDP-N-acetylglucosamine biosynthesis. The aberrant root morphology of the gna1 mutant includes shortening of roots, disruption of microtubules, and shrinkage of cells in the root elongation zone. Our observations support the idea that protein glycosylation plays a key role in cell metabolism, microtubule stabilization, and cell shape in rice roots.

Integrative Comparison of the Role of the PHOSPHATE RESPONSE1 Subfamily in Phosphate Signaling and Homeostasis in Rice

A subfamily of phosphate-responsive genes is functionally diverse in the regulation of phosphate signaling and homeostasis. Phosphorus (P), an essential macronutrient for all living cells, is indispensable for agricultural production. Although Arabidopsis (Arabidopsis thaliana) PHOSPHATE RESPONSE1 (PHR1) and its orthologs in other species have been shown to function in transcriptional regulation of phosphate (Pi) signaling and Pi homeostasis, an integrative comparison of PHR1-related proteins in rice (Oryza sativa) has not previously been reported. Here, we identified functional redundancy among three PHR1 orthologs in rice (OsPHR1, OsPHR2, and OsPHR3) using phylogenetic and mutation analysis. OsPHR3 in conjunction with OsPHR1 and OsPHR2 function in transcriptional activation of most Pi starvation-induced genes. Loss-of-function mutations in any one of these transcription factors (TFs) impaired root hair growth (primarily root hair elongation). However, these three TFs showed differences in DNA binding affinities and messenger RNA expression patterns in different tissues and growth stages, and transcriptomic analysis revealed differential effects on Pi starvation-induced gene expression of single mutants of the three TFs, indicating some degree of functional diversification. Overexpression of genes encoding any of these TFs resulted in partial constitutive activation of Pi starvation response and led to Pi accumulation in the shoot. Furthermore, unlike OsPHR2-overexpressing lines, which exhibited growth retardation under normal or Pi-deficient conditions, OsPHR3-overexpressing plants exhibited significant tolerance to low-Pi stress but normal growth rates under normal Pi conditions, suggesting that OsPHR3 would be useful for molecular breeding to improve Pi uptake/use efficiency under Pi-deficient conditions. We propose that OsPHR1, OsPHR2, and OsPHR3 form a network and play diverse roles in regulating Pi signaling and homeostasis in rice.

OsGLU3, a Putative Membrane-Bound Endo-1,4-Beta-Glucanase, Is Required for Root Cell Elongation and Division in Rice (Oryza sativa L.)

Plant roots move through the soil by elongation. This is vital to their ability to anchor the plant and acquire water and minerals from the soil. In order to identify new genes involved in root elongation in rice, we screened an ethyl methane sulfonate (EMS)-mutagenized rice library, and isolated a short root mutant, Osglu3-1. The map-based cloning results showed that the mutant was due to a point mutation in OsGLU3, which encodes a putative membrane-bound endo-1,4-β-glucanase. Osglu3-1 displayed less crystalline cellulose content in its root cell wall, shorter root cell length, and a slightly smaller root meristem as visualized by restricted expression of OsCYCB1,1:GUS. Exogenous application of glucose can suppress both the lower root cell wall cellulose content and short root phenotypes of Osglu3-1. Consistently, OsGLU3 is ubiquitously expressed in various tissues with strong expression in root tip, lateral root, and crown root primodia. The fully functional OsGLU3-GFP was detected in plasma membrane, and FM4-64-labeled compartments in the root meristem and elongation zones. We also found that phosphate starvation, an environmental stress, altered cell wall cellulose content to modulate root elongation in a OsGLU3-dependant way.

Identification of a novel mitochondrial protein, short postembryonic roots 1 (SPR1), involved in root development and iron homeostasis in Oryza sativa

• A rice mutant, Oryza sativa short postembryonic roots 1 (Osspr1), has been characterized. It has short postembryonic roots, including adventitious and lateral roots, and a lower iron content in its leaves. • OsSPR1 was identified by map-based cloning. It encodes a novel mitochondrial protein with the Armadillo-like repeat domain. • Osspr1 mutants exhibited decreased root cell elongation. The iron content of the mutant shoots was significantly altered compared with that of wild-type shoots. A similar pattern of alteration of manganese and zinc concentrations in shoots was also observed. Complementation of the mutant confirmed that OsSPR1 is involved in post-embryonic root elongation and iron homeostasis in rice. OsSPR1 was found to be ubiquitously expressed in various tissues throughout the plant. The transcript abundance of various genes involved in iron uptake and signaling via both strategies I and II was similar in roots of wild-type and mutant plants, but was higher in the leaves of mutant plants. • Thus, a novel mitochondrial protein that is involved in root elongation and plays a role in metal ion homeostasis has been identified.