Brassinosteroid signaling regulates phosphate starvation-induced malate secretion in plants.

Inorganic phosphate (Pi) is often limited in soils due to precipitation with iron (Fe) and aluminum (Al). To scavenge heterogeneously distributed phosphorus (P) resources, plants have evolved a local Pi signaling pathway that induces malate secretion to solubilize the occluded Fe-P or Al-P oxides. In this study, we show that Pi limitation impaired brassinosteroid signaling and downregulated BRASSINAZOLE-RESISTANT 1 (BZR1) expression in Arabidopsis thaliana. Exogenous 2,4-epibrassinolide treatment or constitutive activation of BZR1 (in the bzr1-D mutant) significantly reduced primary root growth inhibition under Pi-starvation conditions by downregulating ALUMINUM-ACTIVATED MALATE TRANSPORTER 1 (ALMT1) expression and malate secretion. Furthermore, AtBZR1 competitively suppressed the activator effect of SENSITIVITY TO PROTON RHIZOTOXICITY 1 (STOP1) on ALMT1 expression and malate secretion in Nicotiana benthamiana leaves and Arabidopsis. The ratio of nuclear-localized STOP1 and BZR1 determined ALMT1 expression and malate secretion in Arabidopsis. In addition, BZR1-inhibited malate secretion is conserved in rice (Oryza sativa). Our findings provide insight into plant mechanisms for optimizing the secretion of malate, an important carbon resource, to adapt to Pi-deficiency stress.

Rice ACID PHOSPHATASE 1 regulates Pi stress adaptation by maintaining intracellular Pi homeostasis.

The concentration and homeostasis of intracellular phosphate (Pi) are crucial for sustaining cell metabolism and growth. During short-term Pi starvation, intracellular Pi is maintained relatively constant at the expense of vacuolar Pi. After the vacuolar stored Pi is exhausted, the plant cells induce the synthesis of intracellular acid phosphatase (APase) to recycle Pi from expendable organic phosphate (Po). In this study, the expression, enzymatic activity and subcellular localization of ACID PHOSPHATASE 1 (OsACP1) were determined. OsACP1 expression is specifically induced in almost all cell types of leaves and roots under Pi stress conditions. OsACP1 encodes an acid phosphatase with broad Po substrates and localizes in the endoplasmic reticulum (ER) and Golgi apparatus (GA). The phylogenic analysis demonstrates that OsACP1 has a similar structure with human acid phosphatase PHOSPHO1. Overexpression or mutation of OsACP1 affected Po degradation and utilization, which further influenced plant growth and productivity under both Pi-sufficient and Pi-deficient conditions. Moreover, overexpression of OsACP1 significantly affected intracellular Pi homeostasis and Pi starvation signalling. We concluded that OsACP1 is an active acid phosphatase that regulates rice growth under Pi stress conditions by recycling Pi from Po in the ER and GA.

Characterization of the PHOSPHATE RESPONSE 2-dependent and -independent Pi-starvation response secretome in rice.

Many proteins secreted from plant cells into the surrounding extracellular space help maintain cell structure and regulate stress responses in the external environment. In this study, under Pi-replete and depleted conditions, 652 high-confidence secreted proteins were quantified from wild-type (WT) and PHOSPHATE RESPONSE 2 (OsPHR2)-overexpressing suspension-cultured cells (SCCs). These proteins were functionally grouped as phosphatases, signal transduction proteins, pathogen-related (PR) proteins, cell wall-remodeling proteins, and reactive oxygen species (ROS) metabolism proteins. Although PHOSPHATE RESPONSE (PHR) transcription factors regulate two-thirds of Pi-responsive genes at the transcriptional level, only 30.6% of the Pi-starvation-regulated secreted proteins showed significant changes in OsPHR2-overexpressing SCCs. The OsPHR2-dependent systemic Pi signaling pathway mainly regulates phosphatases and PR proteins, which are involved in the utilization of organophosphate, pathogen resistance, and colonization by rhizosphere microorganisms. The OsPHR2-independent local Pi signaling pathway, on the other hand, largely regulated ROS metabolism proteins, cell wall-remodeling proteins, and signal transduction proteins, which are involved in modifying cell wall structure and root architecture. The functions of differentially expressed secreted proteins between WT and OsPHR2-overexpressing plants under Pi-sufficient and Pi-deficient conditions were further confirmed by analysis of the acid phosphatase activity, ROS content, and cell wall composition.

Inhibition of nitric oxide production under alkaline conditions regulates iron homeostasis in rice.

Rice is one of the most susceptible plants to iron (Fe) deficiency under neutral and alkaline conditions. Alkaline stress induces H2 O2 production and increases the deposition of Fe on the root surface, which causes leaf chlorosis and Fe deficiency in rice. Gene chip and qRT-PCR analysis indicated that the expression of the nitrate reductase (NR) genes were downregulated by alkaline treatment, which resulted in significantly decreased nitrate activity and nitric oxide (NO) production in the epidermis and stele, where H2 O2 accumulated. In contrast, treatment with sodium nitroprusside (SNP), a NO donor, strongly alleviated alkaline-induced Fe deficiency by limiting Fe plaque formation. Increasing the NO signal significantly reduced the accumulation of H2 O2 and the lignin barrier but enhanced phenolic acid secretion in the root epidermis and stele under alkaline conditions. The secreted phenolic acid effectively mobilized the apoplast Fe and increased Fe uptake in roots, thereby alleviating the Fe-deficiency response and downregulating the expressions of Fe-uptake genes under alkaline conditions. In conclusion, alkaline stress inhibits NR activity and NO production in the roots of rice, which play vital roles in the mobilization of the apoplast Fe by regulation of H2 O2 and phenolic acid concentrations.

Purple acid phosphatase 10c encodes a major acid phosphatase that regulates plant growth under phosphate-deficient conditions in rice.

Whilst constitutive overexpression of particular acid phosphatases (APases) can increase utilization of extracellular organic phosphate, negative effects are frequently observed in these transgenic plants under conditions of inorganic phosphate (Pi) sufficiency. In this study, we identified rice purple acid phosphatase 10c (OsPAP10c) as being a novel and major APase that exhibits activities associated both with the root surface and with secretion. Two constructs were used to generate the OsPAP10c-overexpression plants by driving its coding sequence with either a ubiquitin promoter (UP) or the OsPAP10c-native promoter (NP). Compared with the UP transgenic plants, lower expression levels and APase activities were observed in the NP plants. However, the UP and NP plants both showed a similar ability to degrade extracellular ATP and both promoted root growth. The growth performance and yield of the NP transgenic plants were better than the wild-type and UP plants in both hydroponic and field experiments irrespective of the level of Pi supply. Overexpression of APase by its native promoter therefore provides a potential way to improve crop production that might avoid increased APase activity in untargeted tissues and its inhibition of the growth of transgenic plants.

Genome-wide analysis of haloacid dehalogenase genes reveals their function in phosphate starvation responses in rice.

The HAD superfamily is named after the halogenated acid dehalogenase found in bacteria, which hydrolyses a diverse range of organic phosphate substrates. Although certain studies have shown the involvement of HAD genes in Pi starvation responses, systematic classification and bioinformatics analysis of the HAD superfamily in plants is lacking. In this study, 41 and 40 HAD genes were identified by genomic searching in rice and Arabidopsis, respectively. According to sequence similarity, these proteins are divided into three major groups and seven subgroups. Conserved motif analysis indicates that the majority of the identified HAD proteins contain phosphatase domains. A further structural analysis showed that HAD proteins have four conserved motifs and specified cap domains. Fewer HAD genes show collinearity relationships in both rice and Arabidopsis, which is consistent with the large variations in the HAD genes. Among the 41 HAD genes of rice, the promoters of 28 genes contain Pi-responsive cis-elements. Mining of transcriptome data and qRT-PCR results showed that at least the expression of 17 HAD genes was induced by Pi starvation in shoots or roots. These HAD proteins are predicted to be involved in intracellular or extracellular Po recycling under Pi stress conditions in plants.

Improved the Activity of Phosphite Dehydrogenase and its Application in Plant Biotechnology.

Phosphorus (P) is a nonrenewable resource, which is one of the major challenges for sustainable agriculture. Although phosphite (Phi) can be absorbed by the plant cells through the Pi transporters, it cannot be metabolized by plant and unable to use as P fertilizers for crops. However, transgenic plants that overexpressed phosphite dehydrogenase (PtxD) from bacteria can utilize phosphite as the sole P source. In this study, we aimed to improve the catalytic efficiency of PtxD from Ralstonia sp.4506 (PtxDR4506), by directed evolution. Five mutations were generated by saturation mutagenesis at the 139th site of PtxD R4506 and showed higher catalytic efficiency than native PtxDR4506. The PtxDQ showed the highest catalytic efficiency (5.83-fold as compared to PtxDR4506) contributed by the 41.1% decrease in the K m and 2.5-fold increase in the k cat values. Overexpression of PtxDQ in Arabidopsis and rice showed increased efficiency of phosphite utilization and excellent development when phosphite was used as the primary source of P. High-efficiency PtxD transgenic plant is an essential prerequisite for future agricultural production using phosphite as P fertilizers.

A Na2CO3-Responsive Chitinase Gene From Leymus chinensis Improve Pathogen Resistance and Saline-Alkali Stress Tolerance in Transgenic Tobacco and Maize.

Salinity and microbial pathogens are the major limiting factors for crop production. Although the manipulation of many genes could improve plant performance under either of these stresses, few genes have reported that could improve both pathogen resistance and saline-alkali stress tolerance. In this study, we identified a new chitinase gene CHITINASE 2 (LcCHI2) that encodes a class II chitinase from Leymus chinensis, which grows naturally on alkaline-sodic soil. Overexpression of LcCHI2 increased chitinase activity in transgenic plants. The transgenic tobacco and maize exhibited improved pathogen resistance and enhanced both neutral salt and alkaline salt stress tolerance. Overexpression of LcCHI2 reduced sodium (Na+) accumulation, malondialdehyde content and relative electrical conductivity in transgenic tobacco under salt stress. In addition, the transgenic tobacco showed diminished lesion against bacterial and fungal pathogen challenge, suggesting an improved disease resistance. Similar improved performance was also observed in LcCHI2-overexpressed maize under both pathogen and salt stresses. It is worth noting that this genetic manipulation does not impair the growth and yield of transgenic tobacco and maize under normal cultivation condition. Apparently, application of LcCHI2 provides a new train of thought for genetically engineering saline-alkali and pathogen resistant crops of both dicots and monocots.