A citrate efflux transporter important for manganese distribution and phosphorus uptake in rice.

The plant citrate transporters, functional in mineral nutrient uptake and homeostasis, usually belong to the multidrug and toxic compound extrusion transporter family. We identified and functionally characterized a rice (Oryza sativa) citrate transporter, OsCT1, which differs from known plant citrate transporters and is structurally close to rice silicon transporters. Domain analysis depicted that OsCT1 carries a bacterial citrate-metal transporter domain, CitMHS. OsCT1 showed citrate efflux activity when expressed in Xenopus laevis oocytes and is localized to the cell plasma membrane. It is highly expressed in the shoot and reproductive tissues of rice, and its promoter activity was visible in cells surrounding the vasculature. The OsCT1 knockout (KO) lines showed a reduced citrate content in the shoots and the root exudates, whereas overexpression (OE) line showed higher citrate exudation from their roots. Further, the KO and OE lines showed variations in the manganese (Mn) distribution leading to changes in their agronomical traits. Under deficient conditions (Mn-sufficient conditions followed by 8 days of 0 µm MnCl2 · 4H2 O treatment), the supply of manganese towards the newer leaf was found to be obstructed in the KO line. There were no significant differences in phosphorus (P) distribution; however, P uptake was reduced in the KO and increased in OE lines at the vegetative stage. Further, experiments in Xenopus oocytes revealed that OsCT1 could efflux citrate with Mn. In this way, we provide insights into a mechanism of citrate-metal transport in plants and its role in mineral homeostasis, which remains conserved with their bacterial counterparts.

Root-Expressed Rice PAP3b Enhances Secreted APase Activity and Helps Utilize Organic Phosphate.

Phosphate (Pi) deficiency leads to the induction of purple acid phosphatases (PAPs) in plants, which dephosphorylate organic phosphorus (P) 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 resupply. 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 (APase) with broad substrate specificity. The overexpression (OE) of OsPAP3b in rice led to increased secreted APase activity and improved mineralization of organic P sources, which resulted in better growth of transgenics compared to the wild type when grown on organic P as an exogenous P substrate. Under Pi deprivation, OsPAP3b knock-down and knock-out lines showed no significant changes in total P content and dry biomass. However, the expression of other phosphate starvation-induced genes and the levels of metabolites were found to be altered in the OE and knock-down 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 isoform 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.

Monogalactosyl diacylglycerol synthase 3 affects phosphate utilization and acquisition in rice.

Galactolipids are essential to compensate for the loss of phospholipids by 'membrane lipid remodelling' in plants under phosphorus (P) deficiency conditions. Monogalactosyl diacylglycerol (MGDG) synthases catalyse 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 3 gene (OsMGD3) was identified and functionally characterized. We showed that the plant phosphate (Pi) status and the transcription factor PHOSPHATE STARVATION RESPONSE 2 (OsPHR2) are involved in the transcriptional regulation of OsMGD3. CRISPR/Cas9 knockout and overexpression lines of OsMGD3 were generated to explore its potential role in rice adaptation to Pi deficiency. Compared with the wild type, OsMGD3 knockout lines displayed a reduced Pi acquisition and utilization while overexpression lines showed an enhancement of the same. Further, OsMGD3 showed a predominant role in roots, altering lateral root growth. Our comprehensive lipidomic analysis revealed a role of OsMGD3 in membrane lipid remodelling, in addition to a role in regulating diacylglycerol and phosphatidic acid contents 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.

Purple acid phosphatases: roles in phosphate utilization and new emerging functions.

Plants strive for phosphorus (P), which is an essential mineral for their life. Since P availability is limiting in most of the world's soils, plants have evolved with a complex network of genes and their regulatory mechanisms to cope with soil P deficiency. Among them, purple acid phosphatases (PAPs) are predominantly associated with P remobilization within the plant and acquisition from the soil by hydrolyzing organic P compounds. P in such compounds remains otherwise unavailable to plants for assimilation. PAPs are ubiquitous in plants, and similar enzymes exist in bacteria, fungi, mammals, and unicellular eukaryotes, but having some differences in their catalytic center. In the recent past, PAPs' roles have been extended to multiple plant processes like flowering, seed development, senescence, carbon metabolism, response to biotic and abiotic stresses, signaling, and root development. While new functions have been assigned to PAPs, the underlying mechanisms remained understood poorly. Here, we review the known functions of PAPs, the regulatory mechanisms, and their relevance in crop improvement for P-use-efficiency. We then discuss the mechanisms behind their functions and propose areas worthy of future research. Finally, we argue that PAPs could be a potential target for improving P utilization in crops. In turn, this is essential for sustainable agriculture.

Identification of Purple Acid Phosphatases in Chickpea and Potential Roles of CaPAP7 in Seed Phytate Accumulation.

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.

OsHAD1, a Haloacid Dehalogenase-Like APase, Enhances Phosphate Accumulation.

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, OsHAD1 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 (Oryza sativa) seedlings. Furthermore, overexpression of OsHAD1 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.

Assessment of Arabidopsis thaliana CENH3 promoter in Brassica juncea for development of haploid inducer lines.

Centromeres are epigenetically specified by the centromeric histone H3 protein (CENH3). The timing and level of expression of CENH3 is tightly regulated to match the demands of the host cell. So far in plants, only CENH3 promoter of Arabidopsis thaliana (L.) Heynh. has been characterized. However, whether CENH3 promoters retain their characteristic mode of regulation in other species remains to be established. In the present study, activity of AtCENH3 promoter was investigated using reporter gene assay in Brassica juncea (L.) Czem. A 1156 bp promoter fragment of AtCENH3 gene (At1g01370) including the first 111 nucleotides of the coding sequence was amplified and cloned into the pORE-R2 binary vector to ensure translation fusion with the uidA coding sequences. The Agrobacteriun tiunefaciens strain GV3101 harbouring the recombinant construct was used to transform B. juncea cv. RLM198 hypocotyl explants. Histochemical assay of To and T, transgenics showed GUS expression in shoot apical meristem, leaf, sepal, flower pedicel and root tip. Intense GUS expression was observed in meristematic tissues, particularly at shoot and root apices. However, mature leaves, flowers, pollen and ovules exhibited very low or no GUS expression. Our results showed that AtCENH3 promoter regulates cognate gene expression in Brassica juncea as it does in A. thaliana, and hence a suitable candidate for developing haploid inducer line in B. juncea.

Brassica juncea Lines with Substituted Chimeric GFP-CENH3 Give Haploid and Aneuploid Progenies on Crossing with Other Lines.

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.