Role of Na+ transporters HKT1;1 and HKT1;2 in tomato salt tolerance. I. Function loss of cheesmaniae alleles in roots and aerial parts

We wish to thank Elena Sanchez Romero (EEZ-CSIC) for her technical assistance, the Scientific Instrumentation Service at EEZ-CSIC, Granada, for their ICP-OES mineral analysis and Michael O'Shea for proofreading the manuscript. The study was funded by grant AGL201782452-C2-1R (A.B.) and grant AGL2017-82452-C2-2R (M.J.A.), both from the Spanish Ministerio de Ciencia e Innovacion/Agencia Estatal de Investigacion, MCIN/AEI/10.13039/501100011033, and FEDER "Una manera de hacer Europa", as well as grant ACCESP2018 (J.A.T.) from the University of Granada. J.E. was supported by a JAE Intro-CSIC grant, JAEINT_19_00566.We analyzed the physiological impact of function loss on cheesmaniae alleles at the HKT1;1 and HKT1;2 loci in the roots and aerial parts of tomato plants in order to determine the relative contributions of each locus in the different tissues to plant Na+/K+ homeostasis and subsequently to tomato salt tolerance. We generated different reciprocal rootstock/scion combinations with non-silenced, single RNAi-silenced lines for ScHKT1;1 and ScHKT1;2, as well as a silenced line at both loci from a near isogenic line (NIL14), homozygous for the Solanum cheesmaniae haplotype containing both HKT1 loci and subjected to salinity under natural greenhouse conditions. Our results show that salt treatment reduced vegetative growth and altered the Na+/K+ ratio in leaves and flowers; negatively affecting fruit production, particularly in graft combinations containing single silenced ScHKT1;2- and double silenced ScHKT1;1/ScHKT1;2 lines when used as scion. We concluded that the removal of Na+ from the xylem by ScHKT1;2 in the aerial part of the plant can have an even greater impact than that on Na+ homeostasis at the root level under saline conditions. Also, ScHKT1;1 function loss in rootstock greatly reduced the Na+/K+ ratio in leaf and flower tissues, minimized yield loss under salinity. Our results suggest that, in addition to xylem Na+ unloading, ScHKT1;2 could also be involved in Na+ uploading into the phloem, thus promoting Na+ recirculation from aerial parts to the roots. This recirculation of Na+ to the roots through the phloem could be further favoured by ScHKT1;1 silencing at these roots.Spanish Ministerio de Ciencia e Innovacion/Agencia Estatal de Investigacion AGL2017-82452-C2-1R AGL2017-82452-C2-2RFEDER "Una manera de hacer Europa" MCIN/AEI/10.13039/501100011033University of Granada ACCESP2018JAE Intro-CSIC grant JAEINT_19_0056

HKT1;1 and HKT1;2 Na+ Transporters from Solanum galapagense Play Different Roles in the Plant Na+ Distribution under Salinity

Salt tolerance is a target trait in plant science and tomato breeding programs. Wild tomato accessions have been often explored for this purpose. Since shoot Na+/K+ is a key component of salt tolerance, RNAi-mediated knockdown isogenic lines obtained for Solanum galapagense alleles encoding both class I Na+ transporters HKT1;1 and HKT1;2 were used to investigate the silencing effects on the Na and K contents of the xylem sap, and source and sink organs of the scion, and their contribution to salt tolerance in all 16 rootstock/scion combinations of non-silenced and silenced lines, under two salinity treatments. The results show that SgHKT1;1 is operating differently from SgHKT1;2 regarding Na circulation in the tomato vascular system under salinity. A model was built to show that using silenced SgHKT1;1 line as rootstock would improve salt tolerance and fruit quality of varieties carrying the wild type SgHKT1;2 allele. Moreover, this increasing effect on both yield and fruit soluble solids content of silencing SgHKT1;1 could explain that a low expressing HKT1;1 variant was fixed in S. lycopersicum during domestication, and the paradox of increasing agronomic salt tolerance through silencing the HKT1;1 allele from S. galapagense, a salt adapted species

Combining Genetic and Transcriptomic Approaches to Identify Transporter-Coding Genes as Likely Responsible for a Repeatable Salt Tolerance QTL in Citrus

The excessive accumulation of chloride (Cl−) in leaves due to salinity is frequently related to decreased yield in citrus. Two salt tolerance experiments to detect quantitative trait loci (QTLs) for leaf concentrations of Cl−, Na+, and other traits using the same reference progeny derived from the salt-tolerant Cleopatra mandarin (Citrus reshni) and the disease-resistant donor Poncirus trifoliata were performed with the aim to identify repeatable QTLs that regulate leaf Cl− (and/or Na+) exclusion across independent experiments in citrus, as well as potential candidate genes involved. A repeatable QTL controlling leaf Cl− was detected in chromosome 6 (LCl-6), where 23 potential candidate genes coding for transporters were identified using the C. clementina genome as reference. Transcriptomic analysis revealed two important candidate genes coding for a member of the nitrate transporter 1/peptide transporter family (NPF5.9) and a major facilitator superfamily (MFS) protein. Cell wall biosynthesis- and secondary metabolism-related processes appeared to play a significant role in differential gene expression in LCl-6. Six likely gene candidates were mapped in LCl-6, showing conserved synteny in C. reshni. In conclusion, markers to select beneficial Cleopatra mandarin alleles of likely candidate genes in LCl-6 to improve salt tolerance in citrus rootstock breeding programs are provided

Genetic dissection of tomato rootstock effects on scion traits under moderate salinity

The present study approaches the QTL dissection of rootstock effects on a commercial hybrid variety grafted on a population of RILs derived from Solanum pimpinellifolium, genotyped for 4370 segregating SNPs from the SolCAP tomato panel and grown under moderate salinity. Results are compared to those previously obtained under high salinity. The most likely functional candidate genes controlling the scion [Na+] were rootstock HKT1;1 and HKT1;2 as it was previously reported for non-grafted genotypes. The higher fruit [Na+] found when rootstock genotype was homozygote for SpHKT1 supports the thesis that scion HKT1 is loading Na+ into the phloem sap in leaves and unloading it in sink organs. A significant increment of small, mostly seedless, fruits was found associated with SlHKT1 homozygous rootstocks. Just grafting increased the incidence of blossom end rot and delayed fruit maturation but there were rootstock RILs that increased commercial fruit yield under moderate salinity. The heritability and number of QTLs involved were lower and different than those found under high salinity. Four large contributing (>17 %) rootstock QTLs, controlling the leaf concentrations of B, K, Mg and Mo were detected whose 2 Mbp physical intervals contained B, K, Mg and Mo transporter-coding genes, respectively. Since a minimum of 3 QTLs (two of them coincident with leaf K and Ca QTLs) were also found governing rootstock-mediated soluble-solids content of the fruit under moderate salinity, grafting desirable crop varieties on stress-tolerant rootstocks tenders an opportunity to increase both salt tolerance and quality

Two closely linked tomato HKT coding genes are positional candidates for the major tomato QTL involved in Na+/K+ homeostasis

The location of major quantitative trait loci (QTL) contributing to stem and leaf [Na+] and [K+] was previously reported in chromosome 7 using two connected populations of recombinant inbred lines (RILs) of tomato. HKT1;1 and HKT1;2, two tomato Na+-selective class I-HKT transporters, were found to be closely linked, where the maximum logarithm of odds (LOD) score for these QTLs located. When a chromosome 7 linkage map based on 278 single-nucleotide polymorphisms (SNPs) was used, the maximum LOD score position was only 35kb from HKT1;1 and HKT1;2. Their expression patterns and phenotypic effects were further investigated in two near-isogenic lines (NILs): 157-14 (double homozygote for the cheesmaniae alleles) and 157-17 (double homozygote for the lycopersicum alleles). The expression pattern for the HKT1;1 and HKT1;2 alleles was complex, possibly because of differences in their promoter sequences. High salinity had very little effect on root dry and fresh weight and consequently on the plant dry weight of NIL 157-14 in comparison with 157-17. A significant difference between NILs was also found for [K+] and the [Na+]/[K+] ratio in leaf and stem but not for [Na+] arising a disagreement with the corresponding RIL population. Their association with leaf [Na+] and salt tolerance in tomato is also discussed