Increased production of lateral roots, adventitious roots and root hairs all increase root surface area, and thus will increase P acquisition from the water and sediment and are important traits for PUE in watercress. In addition, the root cap can account for 20% of the phosphate absorbed by the roots of Arabidopsis. Therefore, increasing the number of roots increases the number of root tips and the number of these “hot spots” for phosphate acquisition.Plants are reliant on phosphate transporters to acquire P from the environment and transport P between tissues, and this includes for aquatic plants. The PHT1 family is the most widely studied group of P transporters and is primarily responsible for P uptake but also has a role for P transport between tissues. A broad range of expression patterns are associated with different PHT1 genes but generally, higher expression of PHT1 genes is associated with improved shoot biomass accumulation and P tolerance . Watercress with higher PHT1 expression may result in improved biomass accumulation in P deficient water, but this has yet to be tested. Additional traits that are important in other crops are organic acid exudation and phosphatase activity. Since these control release of P from organic forms in the soil, they are less relevant to watercress cultivation where P released from bound sources would be rapidly lost to the watercourse. However, phosphatases that remobilise P from intracellular sources have been identified in Arabidopsis so similar phosphatases could enhance internal P utilisation in watercress.Alongside phosphate acquisition, PUE also refers to more efficient P utilisation associated with re-translocation and recycling of stored P,indoor grow facility that relies on effective P transportation within the plant, P scavenging, and use of alternate biochemical pathways that bypass P use.
Re-translocation between plant tissues is governed by transporters such as PHT transporters and PHO transporters. Unlike, PHT transporters which regulate P acquisition too, PHO transporters are solely responsible for P transport into vascular tissues and cells. Alternative P use strategies includes substituting phospholipids in cell walls with sulfolipids and galactolipids. Several enzymes in the glycolytic pathway depend on P so bypass enzymes such as pyrophosphatedependent phosphofructokinase , phosphoenolpyruvate carboxylase and pyruvate phosphate dikinase can be recruited to use pyrophosphate for a P donor and conserve limited ATP pools. Several studies have reported increased PEPC activity under P deprivation. The mitochrondrial electron transport chain responds by utilising non-phosphorylative pathways. Acid phosphatases in intracellular spaces or present in the apoplast can increase P availability by remobilising P from senescent tissues and the extracellular matrix. Both aspects of PUE rely on accurate sensing of the P state within the plant and external environment to alter global gene expression and ensure appropriate responses to upregulate P uptake and P use pathways.QTL for overall PUE metrics as well as QTL for more specific architectural root traits associated with low P tolerance have been identified in several economically important crops including soybean, soybean , rice , maize and common bean. RSA is extremely plastic, subject to effects of hormone signalling, environmental stimuli and under the control of several genes so elucidating these QTL is challenging. Studies on other Brassicaceae species are likely of most genetic relevance for QTL mapping in watercress, however QTL associated with other species such as soybean, rice, sorghum and wheat are summarised in Table 1. P-starved Arabidopsis exhibit longer root hairs and higher root hair density, decreased primary root length and increased lateral root density.
Three QTL, were identified which explained 52% of the variance in primary root length. In rapeseed primary root length decreases, lateral root length and density increases with declining P concentration. Several QTL are associated with these changes and many co-locate with QTL for root traits in Arabidopsis. A more recent study used over 13 000 SNP markers to construct a genetic linkage map in rapeseed, where 131 QTL were identified in total across different growth systems and P availabilities. However, only four QTL were common to all conditions, demonstrating strong environmental effects determining these QTL. To date, there is no published literature on QTL associated with aerenchyma formation under low P in any plant species and no studies exist on QTL mapping for root traits in watercress. Identification of QTL and markers associated with PUE could accelerate breeding for nutrient use and reduce the environmental impact associated with watercress cultivation.Genes involved in transcriptional control are multifunctional under P deprivation; some have overlapping roles in RSA development, P signalling and P utilisation. They are discussed together here despite partial involvement in P utilisation. PHR1 and PHL1 code for transcription factors that play critical roles in the control of P starvation responses. PHR1 mediates expression of the microRNA miR399 which modulates the PHO2 gene, responsible for P allocation between roots and shoots and affects expression of other PSR genes such as PHT transporters . SPX transcription factors are important negative regulators of PSR via repression of PHR . The roles of several other transcription factor genes on RSA and other regulatory elements are summarised in Table 2 and Figure 3. Auxin, sugars and other hormones such as cytokinins, ethylene, abscisic acid , giberellins and strigolactones are implicated in phosphate-induced determination of RSA so genes involved in these pathways may be significant candidates. Under low P, auxin levels increase in root hair zones and root tips.
Auxin mutants such as taa1 and aux1 have impaired root hair growth in low P. Expression of the Arabidopsis auxin receptor gene TIR1 increases under low P availability which results in increased sensitivity to auxin and production of lateral roots. Mutants in auxin-inducible transcription factors also have disrupted root hair responses under low P. ROOT HAIR DEFECTIVE 6- LIKE-2 and ROOT HAIR DEFECTIVE 6-LIKE-4 are responsive to P deficiency and promote root hair initiation and elongation. ARF19 is a key transcription factor promoting auxin-dependent root hair elongation in response to low P . HPS1 is involved in regulating the sucrose transporter SUC2 and hps1 mutants exhibit significant P-starvation responses under P-sufficient conditions. Plants with impaired cytokinin receptors CRE1 and AHK3 show increased sugar sensitivity and increased expression of P-starvation genes. ETHYLENE RESPONSE FACTOR070 is a transcription factor critical for root development under P starvation. Though no studies exist for P-associated gene expression changes in watercress, Müller et al. used RNA sequencing approaches to identify responses to submergence in watercress and found several ABA biosynthesis and catabolism genes associated with stem elongation. This study provides a model for using transcriptomic approaches to explore hormone-induced morphological changes in watercress. For P acquisition, the PHT gene family controlling P transport provides several candidate genes. In Arabidopsis, the PHT genes 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,indoor grow rack 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, 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 downregulated 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 rootto-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 Arabidopsis . 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. Plants also respond to P starvation by utilising alternative metabolic pathways. 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.