Sanjay K. Gupta

Investigations on the role of acid invertase and UDP-glucose pyrophosphorylase in potato clones with varying resistance to cold-induced sweetening

Fifteen potato (Solanum tuberosum L.) clones with varying resistance to cold-induced sweetening were analysed for vacuolar acid invertase (AcInv, EC 3.2.1.26) and UDP-Glc pyrophosphorylase (UGPase; EC 2.7.7.9) activities related to their ability to accumulate sugars following cold storage (4 C). The UGPase isozyme profiles for each clone were also determined. Immunoblot analysis demonstrated a 55 kD protein, in seven of the 15 clones, that reacted with UGPase antisera in addition to the 53 kD UGPase subunit previously reported. The UGPase activity of these clones was significantly lower than that of the “single subunit” clones. Basal AcInv activity showed a positive correlation to the Glc:Suc ratio across the clones that was moderately significant before and after cold temperature storage. The activity of UGPase, which limits the rate of Suc formation, was of secondary importance in limiting the rate of hexogenesis when vacuolar AcInv activity was excessive. It is suggested that with the potato clones from this breeding program that AcInv (and its inhibitor) plays a dominant role in the hexogenic pathway by regulating the hexose:Suc ratio. The finding of a new protein that is reactive with UGPase antisera may prove to play an important function in the regulation of Suc formation in potatoes.ResumenQuince clones de papa (Solanum tuberosum L.) con varios grados de resistencia al endulzamiento inducida por el frío, fueron analizados para invertasa ácida (AcInv; EC 3.2.1.26) vacuolar y actividades de UDP-Glc fosforilasa (UGPasa; EC 2.7.7.9), relacionadas con su habilidad para acumular azucares después de su almacenamiento en frío (4 C). También se determinaron para cada clon los perfiles de la isoenzima UGPasa. El análisis inmunoblot demostró en siete de los 15 clones una proteína kD que reaccionó con el antisuero UGPasa cuando se agregó a la subunidad 53 kD UGPasa previamente reportadas. La actividad de UGPasa en estos clones fue significativamente mas baja que la de los clones de la “subunidad simple.” La actividad basal del AcInv demostró una correlación positiva con la proporción Glc: Sac en los clones que fueron moderadamente significativos antes y después del almacenamiento a baja temperatura. La actividad de la UGPasa la cual imita la tasa de formación de sacarosa, fue de importancia secundaria en limitar la tasa de hexogénesis cuando la actividad vacuolar del AcInv fue excesiva. Se sugiere que con los clones de papa de este programa, el AcInv (y su inhibidor) juega un rol dominante en la vía hexogénica regulando la proorción hexosa: sacarosa. El hallazgo de una nueva proteína que reacciona con el antisuero UGPasa, puede evidenciar que esta juega una función importante en regular la formación de sacarosa en papa.

Physicochemical and kinetic properties of unique isozymes of UDP-Glc pyrophosphorylase that are associated with resistance to sweetening in cold-stored potato tubers.

Isozymes of UGPase with unique catalytic properties were purified from the cold-induced-sweetening (CIS) resistant cultivar Snowden (Solanum tuberosum). Two distinct peaks of UGPase activity were obtained when protein extracts were subjected to anion-exchange chromatography on DEAE-Sephacel. Polypeptides in the first eluted fraction (A-I) were ionically similar to the UGPase isozyme UGP3 previously purified and characterized from the cold-sweetening sensitive cultivar Norchip (Sowokinos et al. 1993, Plant Physiol 101: 1073-1080). Seventy-two percent of the total endogenous UGPase activity in Snowden (cv.) tubers, however, was found in a more basic protein fraction (A-II) that is not found in the Norchip cultivar. This study reports on the physicochemical and kinetic properties of these new polypeptides that demonstrate UGPase activity. The reaction in the direction of UDP-Glc synthesis was specific for the substrates Glc-1-P and UTP and there was an absolute requirement for Mg2+ ions. The catalytic properties of UGP5 were markedly different from UGPase isozymes previously described in terms of (1) affinity for the substrate Glc-1-P, (2) pH optimum, (3) maximum reaction velocity and (4) sensitivity to product inhibition with UDP-Glc. Chi-square analysis of fifty-four genetically diverse potato lines revealed that resistance to CIS was highly correlated with the presence of the A-II isozymes of UGPase. The kinetic properties of these unique forms of UGPase may underlie, in part, a tubers ability to resist sweetening in the cold.

Predictive Markers for Cold-Induced Sweetening Resistance in Cold Stored Potatoes (Solanum tuberosum L.)

An approach has been developed to screen a large number of potato clones for cold induced sweetening (CIS) resistance in breeding programs. Two key enzymes responsible for reducing sugar accumulation during cold storage were identified. Clones with the A-II isozymes of UDP-glucose pyrophosphorylase coupled with low activity of vacuolar acid invertase enzyme had increased resistance to CIS by forming less suc, which is subsequently hydrolyzed to the undesirable reducing sugars, glc and fru. Six named cultivars and 192 genetically diverse clones from various breeding programs in USA were analyzed over two years for the two key enzymes and sugar concentration in cold stored tubers. The predictability for CIS resistance during cold storage was 94% both years. Clones classified as class A accumulated low concentration of reducing sugar glc during cold storage. It is suggested that these two predictor enzymes can be used for screening parents and selections in potato breeding program.ResumenSe ha desarrollado una estrategia para evaluar un gran número de clones de papa para resistencia al endulzamiento inducido por frío (CIS) en programas de mejoramiento. Se identificaron dos enzimas clave como responsables de la acumulación de azúcar reductor durante el almacenamiento en frío. Los clones con las isoenzimas A-II de UDP-glucosa pirofosforilasa, acopladas con la baja actividad de la enzima vacuolar ácido invertasa, han aumentado la resistencia a CIS mediante la formación de menos sac, que es subsecuentemente hidrolizada a los azúcares reductores indeseables glc y fru. Se analizaron seis variedades con nombre y 192 clones genéticamente diversos de varios programas de mejoramiento en los EUA, durante dos años para las dos enzimas clave y concentración de azúcar en tubérculos almacenados en frío. La predicción de la resistencia a CIS durante el almacenamiento en frío fue de 94% en ambos años. Los clones clasificados como clase A acumularon baja concentración de los azucares reductores glc durante el almacenamiento en frío. Se sugiere que estas dos enzimas de predicción pueden usarse para analizar progenitores y selecciones en programas de mejoramiento en papa.

Linkage analysis and QTL mapping in a tetraploid russet mapping population of potato

BackgroundGenome-wide single nucleotide polymorphism (SNP) markers coupled with allele dosage information has emerged as a powerful tool for studying complex traits in cultivated autotetraploid potato (Solanum tuberosum L., 2n = 4× = 48). To date, this approach has been effectively applied to the identification of quantitative trait loci (QTLs) underlying highly heritable traits such as disease resistance, but largely unexplored for traits with complex patterns of inheritance.ResultsIn this study, an F1 tetraploid russet mapping population (162 individuals) was evaluated for multiple quantitative traits over two years and two locations to identify QTLs associated with tuber sugar concentration, processing quality, vine maturity, and other high-value agronomic traits. We report the linkage maps for the 12 potato chromosomes and the QTL location with corresponding genetic models and candidate SNPs explaining the highest phenotypic variation for tuber quality and maturity related traits. Significant QTLs for tuber glucose concentration and tuber fry color were detected on chromosomes 4, 5, 6, 10, and 11. Collectively, these QTLs explained between 24 and 46% of the total phenotypic variation for tuber glucose and fry color, respectively. The QTL on chromosome 10 was associated with apoplastic invertases, with ‘Premier Russet’ contributing the favorable allele for fry processing quality. On chromosome 5, minor-effect QTLs for tuber glucose concentration and fry color co-localized with various major-effect QTLs, including vine maturity, growth habit, tuber shape, early blight (Altenaria tenuis), and Verticillium wilt (Verticillium spp.).ConclusionsLinkage analysis and QTL mapping in a russet mapping population (A05141) using SNP dosage information successfully identified favorable alleles and candidate SNPs for resistance to the accumulation of tuber reducing sugars. These novel markers have a high potential for the improvement of tuber processing quality. Moreover, the discovery of different genetic models for traits with overlapping QTLs at the maturity locus clearly suggests an independent genetic control.