Phosphorus (P) deficiency limits plant growth and yield. Since plants can absorb only the inorganic form of P (Pi), a large portion of soil P (organic and inorganic P complexes) remains unused. Here, we identified and characterized a PHR2-regulated, novel low-Pi-responsive haloacid dehalogenase (HAD)-like hydrolase, While OsHAD1 is a functional HAD protein having both acid phosphatase and phytase activities, it showed little homology with other known low-Pi-responsive HAD superfamily members. Recombinant OsHAD1 is active at acidic pH and dephosphorylates a broad range of organic and inorganic P-containing substrates, including phosphorylated serine and sodium phytate. Exogenous application of recombinant OsHAD1 protein in growth medium supplemented with phytate led to marked increases in growth and total P content of Pi-deficient wild-type rice () seedlings. Furthermore, overexpression of in rice resulted in enhanced phosphatase activity, biomass, and total and soluble P contents in Pi-deficient transgenic seedlings treated with phytate as a restricted Pi source. Gene expression and metabolite profiling revealed enhanced Pi starvation responses, such as up-regulation of multiple genes involved in Pi uptake and solubilization, accumulation of organic acids, enhanced secretory phosphatase activity, and depletion of ATP in overexpression lines as compared with the wild type. To elucidate the underlying regulatory mechanisms of OsHAD1, we performed in vitro pull-down assays, which revealed the association of OsHAD1 with protein kinases such as OsNDPKs. We conclude that, besides dephosphorylation of cellular organic P, OsHAD1 in coordination with kinases may regulate the phosphorylation status of downstream targets to accomplish Pi homeostasis under limited Pi supply.

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Purple acid phosphatases (PAPs) play important roles in phosphate (Pi) acquisition and utilization. These PAPs hydrolyze organic Phosphorus (P) containing compounds in rhizosphere as well as inside the plant cell. However, roles of PAPs in one of the most widely cultivated legumes, chickpea (Cicer arietnum L.), have not been unraveled so far. In the present study, we identified 25 putative PAPs in chickpea (CaPAPs) which possess functional PAP motifs and domains. Differential regulation of CaPAPs under different nutrient deficiencies revealed their roles under multiple nutrient stresses including Pi deficiency. Interestingly, most of the CaPAPs were prominently expressed in flowers and young pods indicating their roles in flower and seed development. Association mapping of SNPs underlying CaPAPs with seed traits revealed significant association of low Pi inducible CaPAP7 with seed weight and phytate content. Biochemical characterization of recombinant CaPAP7 established it to be a functional acid phosphatase with highest activity on most abundant organic-P substrate, phytate. Exogenous application of recombinant CaPAP7 enhanced biomass and Pi content of Arabidopsis seedlings supplemented with phytate as sole P source. Taken together, our results uncover the PAPs in chickpea and potential roles of CaPAP7 in seed phytate accumulation.

Haploids and doubled haploids are invaluable for basic genetic studies and in crop improvement. A novel method of haploid induction through genetic engineering of the Centromere Histone Protein gene, CENH3, has been demonstrated in Arabidopsis. The present study was undertaken to develop haploid inducer (HI) lines of Brassica juncea based on the principles elaborated in Arabidopsis. B. juncea was found to carry three copies of CENH3 which generated five different transcripts, of which three transcripts resulted from alternative splicing. Unlike Arabidopsis thaliana where native CENH3 gene was knocked out for constructing HI lines, we used RNAi approach to knockdown the native CENH3 genes. Further, to rescue CENH3 silenced cells, a GFP-CENH3-tailswap construct having N terminal GFP fused to H3.3 tail sequences and synthetic CENH3 histone fold domain sequences was devised. A total 38 transgenic B. juncea plants were regenerated following co-transformation with both silencing and rescue cassettes and transgenics carrying either or both the constructs were obtained. Transgenic status was confirmed through PCR, Southern and qRT-PCR analyses. Co-transformed lines were crossed to untransformed B. juncea or a line expressing only GFP-tailswap. FACS and cytological analyses of progenies revealed partial or complete elimination of B. juncea chromosomes thereby giving rise to aneuploids and haploid. This is the first report in a polyploid crop demonstrating that CENH3 engineering could be used to develop HI lines.

Phosphate (Pi) deficiency leads to the induction of purple acid phosphatases (PAPs) in plants, which dephosphorylates organic phosphorus complexes in the rhizosphere and intracellular compartments to release Pi. In this study, we demonstrate that OsPAP3b belongs to group III low molecular weight PAP, and is low Pi responsive, preferentially in roots. The expression of OsPAP3b is negatively regulated with Pi re-supply. Interestingly, OsPAP3b was found to be dual localized to the nucleus and secretome. Furthermore, OsPAP3b is transcriptionally regulated by OsPHR2 as substantiated by DNA-protein binding assay. Through in-vitro biochemical assays, we further demonstrate that OsPAP3b is a functional acid phosphatase with broad substrate specificity. Overexpression of OsPAP3b in rice led to increased secreted APase activity and improved mineralization of organic P sources, reflected in better growth of transgenics compared to wild type when grown on organic P as exogenous P substrate. Under Pi deprivation, OsPAP3b knockdown and knockout lines showed no significant changes in total P content and dry biomass. However, the expression of other phosphate starvation-induced (PSI) genes and the levels of metabolites were found to be altered in the overexpression and knockdown lines. In addition, in-vitro pull-down assay revealed multiple putative interacting proteins of OsPAP3b. Our data collectively suggest that OsPAP3b can aid in organic P utilization in rice. The APase isoforms behavior and nuclear localization indicate its additional role, possibly in stress signaling. Considering its important roles, OsPAP3b could be a potential target for improving low Pi adaptation in rice.

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Galactolipids are essential for compensating for the loss of phospholipids by “membrane lipid remodeling” in plants under phosphorus (P) deficiency. Monogalactosyl diacylglycerol (MGDG) synthases catalyze the synthesis of MGDG which is further converted into digalactosyl diacylglycerol (DGDG), later replacing phospholipids in the extraplastidial membranes. However, the roles of these enzymes are not well explored in rice. In this study, the rice MGDG synthase gene, OsMGD3 was identified and functionally characterized. We showed that plant phosphate (Pi) status and transcription factor OsPHR2 are involved in the transcriptional regulation of OsMGD3. CRISPR/Cas9 knockout (KO) and overexpression (OE) lines of OsMGD3 were generated to explore its potential role in rice adaptation to Pi deficiency. Compared to WT, OsMGD3 KO lines displayed a reduction while OE lines showed an enhanced, Pi acquisition and utilization. Further, OsMGD3 showed a predominant role in roots, altering lateral root growth. Our comprehensive lipidomic analysis revealed the role of OsMGD3 in membrane lipid remodeling in addition to a role in regulating diacylglycerol and phosphatidic acid levels that affected the expression of Pi transporters. Our study highlights the role of OsMGD3 in affecting both internal P utilization and P acquisition in rice.

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