For P acquisition, the PHT gene family controlling P transport provides several candidate genes. In Arabidopsis, the PHTgenes that encode phosphate transporters responsible for transport of P anions are well characterised and are grouped into four families . PHT proteins other than PHT1 are involved in the uptake, distribution and remobilisation of P within the plant, however, PHT1 in the plasma membrane is the most important . Phosphate stress induces expression of these genes. However, the use of PHT transporters in plant breeding has been limited by P toxicity and other side effects of unbalanced P regulation associated with the over expression of some transporter genes. For example, OsPHT1;9 and OsPHT1;10 over expressing rice plants have reduced biomass under high phosphate compared to wild-type plants. Accessory proteins, encoded for by genes like PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR , are also important for proper functioning of P transporter genes. Homologs of PHT1 transporter genes, transcriptional factors including the SPX gene family, and genes involved in RSA determination such as PDR2 and LPR1/2 could be candidate genes for improving phosphate acquisition in watercress, but these genes have yet to be identified in aquatic crops.The transcriptional regulation of PUE is complex: there is some overlap with genes involved in both phosphate acquisition and phosphate utilisation, such as the global transcriptional regulation by PHR1 and PHL1. Here we target genes primarily involved in utilisation,square planter pots including those responsible for P transport within the plant, alternate metabolic pathways, and internal Pscavenging.
Using an Arabidopsis Affymetrix gene chip, changes in global gene expression have been analysed in response to P deprivation. The expression of 612 genes was induced and 254 genes suppressed including upregulation of phosphate transporters such as PHT1 genes and PHO1;H1. Genes involved in protein biosynthesis were down regulated during deficiency, likely representing P recycling strategies. PHT transporters also play a role in P utilisation through re-translocation of P within the plant. Five of the 13 maize PHT1 genes are induced in other tissues such as leaves, anthers, pollen and seeds, suggesting PHT1 involvement in diverse processes such as root to-shoot distribution. PHO1 is another central element responsible for P homeostasis and transport. Pho1 mutants exhibit P deficiency in the shoots due to lack of P loading into the xylem vessels. Phosphatases are important for remobilisation of fixed P. Eleven genes encoding different purple acid phosphatases were reported to be upregulated under P starvation in Arabidopsi. The Arabidopsis genome encodes 29 purple acid phosphatases, some of which are excreted into the soil, such as PAP12 and PAP26. Only phosphatase activity within the plant is relevant to watercress breeding: in a f lowing water system, any P made available around the roots by secreted phosphatases would rapidly wash away. As well as being a major secreted phosphatase, PAP26 is regarded as the predominant intracellular acid phosphatase in Arabidopsis and is upregulated two-fold under Pdeficiency. PAP26 functions in PUE by scavenging P from intracellular and extracellular P-ester pools, to increase P availability in the plant. Homologs of PAP26 should be investigated in watercress.
Genes involved in lipid metabolism and biosynthesis represent the largest group of core PSI genes in Arabidopsis, demonstrating their importance in PUE under P starvation. Three genes encode “plant-type” PEPC enzymes in Arabidopsis . PPC1 is expressed in roots and f lowers, PPC2 in all organs and PPC3 in only roots. PEPC activity is affected by phosphorylation by PPCK , thus PPCK1 and PPCK2 genes are additional important components for P-bypassing. Membrane phospholipids constitute approximately 20% of the total P in the leaves of P-sufficient plants. This represents a large pool of P that can be remobilised. 7% of the P-responsive genes found by Misson et al.were involved in lipid biosynthesis pathways in Arabidopsis. This includes the genes MGD2 and MGD3 whose expression changed 11-fold and 48- fold under P deficiency, respectively. These genes encode major enzymes for galactolipid biosynthesis and are involved in replacing phospholipids in cell membranes with non-phosphorus lipids such as galactolipid digalactosyldiacylglycerol. PECP1 is involved in the liberation of P from phospholipids and is upregulated under P deprivation, with up to 1785-fold increases in expression reported in roots. PSR2 encodes a phosphatase involved in galactolipid biosynthesis and whose expression increases 174-fold in P deprived seedlings. Both PECP1 and PSR2 have similar roles in the dephosphorylation of phosphocholine in the galactolipid synthesis pathway. However, despite their massive upregulation, it has been observed that inactivation of PECP1 and PSR2 does not alter plant growth or plant P content under P-deprivation so PCho is not likely a major source of P under limiting conditions.
PLDζ1 and PLDζ2 encode phospholipases D zeta 1 and 2 that hydrolyse major phospholipids such as phosphatidylcholine which yields phosphatidic acid and PA phosphatase and releases DAG and P. Phospholipids can also be replaced by sulfolipids. SQD2 is the primary gene in this pathway and encodes an enzyme that catalyses the final step in the sulfolipid biosynthesis. GDPD1 is involved in the formation of glycerol-3- phosphate from phospholipid products , that can be dephosphorylated to release P. Homologs of PHT1 genes responsible for P redistribution, within the plant, genes involved in P scavenging , genes implemented in metabolic pathways that bypass P use including galactolipid biosynthetic pathways and those involved in sulfolipid biosynthesis could be candidate genes for improving phosphate utilisation in watercress.Watercress root research is virtually completely absent in the literature, with no studies on root responses to phosphate availability. Nevertheless, the finite nature of rock phosphate and the fact that watercress cultivation methods have the potential to result in environmental damage , are clear drivers, as with soil-grown crops, to breed for watercress with improved PUE. Uncovering phenotypic traits and the molecular basis for PUE are important early steps in breeding for phosphate use efficiency. No commercial breeding programs exist for watercress worldwide, but germplasm collections are emerging. Development of new watercress varieties is also no doubt limited by the lack of genomic information for this crop. Payne screened a germplasm collection under indoor and outdoor conditions and identified significant variation in several traits including stem length, stem diameter and antioxidant concentrations. Voutsina used RNA-seq to analyse the first watercress transcriptome and identified differences in antioxidant capacity and glucosinolate biosynthesis across a germplasm collection of watercress, with 71% of the watercress transcripts annotated based on orthology to Arabidopsis. Jeon et al. independently used RNA-seq approaches to assemble the watercress transcriptome de novo. They identified 33 candidate genes related to glucosinolate biosynthetic pathways using Arabidopsis glucosinolate genes to search for homologous sequences in the watercress transcriptome. Additionally, a watercress mapping population comprising 259 F2 individuals was established by Voutsina using parents with contrasting nutrient and growth phenotypes. Genotype-by-sequencing of this mapping population enabled the construction of first genetic linkage map for watercress and identified 17 QTL for morphological traits of interest, growing blackberries in containers antioxidant capacity and cytotoxicity against human cancer cells. However, no root traits were assessed in this work. Screening this mapping population and wider germplasm for root traits may reveal individuals with extreme PUE phenotypes, which could allow the development of markers and QTL associated with this complex trait. RNA-seq approaches could ultimately lead to the identification of candidate genes. Together these findings will assist in developing commercial cultivars with a reduced need for phosphate, and a reduced negative environmental impact.
To assess whether homologs of the candidate PUE genes identified in this study exist in watercress, available watercress transcriptome data was mined for 13 key PUE genes selected from Table 2. This included genes whose expression was induced more than tenfold in at least 2 independent studies , plus PHR1, the global regulator of P starvation responses. Annotated transcripts were obtained from transcriptomic studies of watercress by Voutsina et al., 2016; Jeon et al., 2017; Müller et al., 2021 and matches for these candidate genes were assessed by searching for genes using AGI . Across all studies, strong matches were found for PHT1;4, SPX1, PHR1, MDG3, PEPC1, PLDζ1/2, PSR2 and SQD2, with all corresponding e-values ranging from 0 to 3.00E-32. Additionally, Müller et al. identified homologous transcripts for MDG2. Voutsina et al. had transcripts corresponding to PHT1;3, and MDG2, and Jeon et al. had hits for PHT1;2 and PHT1;3. No matching transcripts were found for PHT1;1 in any of the three studies. Where FDR values were < 0.05, changes to expression patterns were noted. Interestingly, Müller et al. also observed varying levels of upregulation of PHT1;4 and PHR1 following submergence, which may suggest a link between phosphate starvation and submergence responses.Being a high-energy phosphate compound, PPi can serve as a phosphate donor and energy source, but it can, at high levels, become inhibitory to cellular metabolism. To maintain an optimal PPi level in the cytoplasm, timely degradation of excessive PPi is carried out by two major types of enzymes: soluble inorganic pyrophosphatases and proton-translocating membrane-bound pyrophosphatases. The importance of maintaining an optimal cellular PPi level has been demonstrated in several different organisms. Genetic mutations that lead to the absence of sPPase activity affects cell proliferation in Escherichia coli. In yeast, inorganic pyrophosphatase is indispensable for cell viability because loss of its function results in cell cycle arrest and autophagic cell death associated with impaired NAD+ depletion. In Arabidopsis, a tonoplast-localized proton-pumping pyrophosphatase AVP1 was shown to be the key enzyme for cytosolic PPi metabolism in different cell types of various plants. This enzyme activity has been correlated with the important function that AVP1 plays in many physiological processes. Arabidopsis fugu5 mutants lacking functional AVP1 show elevated levels of cytosolic PPi and display heterotrophic growth defects resulting from the inhibition of gluconeogenesis. This important role in controlling PPi level in plant cells is reinforced by a recent study showing that higher-order mutants defective in both tonoplast and cytosolic pyrophosphatases display much severe phenotypes including plant dwarfism, ectopic starch accumulation, decreased cellulose and callose levels, and structural cell wall defects. Moreover, the tonoplast-localized H+ -PPase AVP1 appears to be a predominant contributor to the regulation of cellular PPi levels because the quadruple knockout mutant lacking cytosolic PPase isoforms ppa1 ppa2 ppa4 ppa5 showed no obvious phenotypes. Interestingly, in companion cells of the phloem, AVP1 was also shown to be localized to the plasma membrane and function as a PPi synthase that contribute to phloem loading, photosynthate partitioning, and energy metabolism. On the other hand, AVP1 is also believed to contribute to the establishment of electrochemical potential across the vacuole membrane, which is important for subsequent vacuolar secondary transport and ion sequestration. Constitutive over expression of AVP1 improves the growth and yield of diverse transgenic plants under various abiotic stress conditions—including drought, salinity, as well as phosphorus and nitrogen deficiency—although the mechanism remains to be fully understood. Taken together, AVP1 serves as a multi-functional protein involved a variety of physiological processes in plants, some of which await to be fully understood. Magnesium is an essential macro-nutrient for plant growth and development, functioning in numerous biological processes and cellular functions, including chlorophyll biosynthesis and carbon fixation. Either deficiency or excess of Mg in the soil could be detrimental to plant growth and therefore plants have evolved multiple adaptive mechanisms to maintain cellular Mg concentration within an optimal range. In higher plants, the most well-documented Mg2+ transporters belong to homologues of bacterial CorA super family and are also called “MRS2” based on their similarity to yeast Mitocondrial RNA splicing 2 protein. Several members of the MGT family mediate Mg2+ transport in bacteria or yeast as indicated by functional complementation as well as 63Ni tracer assay. In plants, they have been shown to play vital roles in Mg2+ uptake, translocation, and homeostasis associated with their different sub-cellular localizations and diverse tissue-specific expression patterns. For instance, MGT2 and MGT3 are tonoplast localized and possibly involved in Mg2+ partitioning into mesophyll vacuoles; MGT4, MGT5, and MGT9 are strongly expressed in mature anthers and play a crucial role in pollen development and male fertility. MGT6 and MGT7 are shown to be most directly involved in Mg homeostasis because knocking-down or knocking-out either of the genes leads to hypersensitivity to low Mg conditions.