| Potato(Solanum tuberosum L.)is the most important non-grain food crop in the world and plays a role in food security.Significant amounts of potatoes are used for processed products like chips,French fries and others.In order to ensure a continuous supply and reduce sprouting,water loss and pathogenesis,potato tubers are often stored at low temperature(normally less than 10°C).However,cold storage often leads to an accumulation of reducing sugars(RS)in tubers,which is known as cold-induced sweetening(CIS).RS can react with ?-amino acid groups of nitrogenous compounds in a non-enzymatic Maillard reaction during frying,resulting in undesired browning of processed products.Starch degradation has been reported to be one of the main pathways involed in potato CIS,however,the functional proof for specific amylases genes responsible for potato CIS is still not available.In the present research,specific amylases,their fuctions and possible regulatory mechanism underlying potato CIS associated with starch degredation were dissected.The main results are as follows:1.Two ?-amylases and seven ?-amylases were identified in the potato genome sequence consortium database which are unevenly distributed on the potato chromosomes.The phylogenetic analysis of ?-amylase and ?-amylase proteins from diverse plant species suggest that amylases may be evolutionarily conserved in planta,?-amylases could be grouped into three subfamilies and ?-amylases were clustered in four subfamilies.Consistent with the phylogenetic analysis,?-amylase genes in the same subfamily possess similar gene structure in most cases.The structure of conserved glucosyl hydrolase domains of ?-amylase proteins predicted that StBAM9 may be an inactive ?-amylase.2.Based on the expression analyses of ?-amylase and ?-amylase gene families in different organs of CIS-sensitive potato cultivars E-Potato 3(E3)and CIS-resistant clone AC030-06 at different storage periods,StAmy23,StBAM1 and StBAM9 were found to have higher transcripts and to be strongly induced by low temperature in tubers among other amylase genes.3.To clarify the subcellular locatization of the amylases,StAmy23,StBAM1 and StBAM9 were fused with GFP and the starch granule marker StGBSS was fused with RFP.The free RFP and chlorophyll autofluorescence were taken as indicators of the cytosolic and chloroplast stroma localizations,respectively.StAmy23-GFP,StBAM1-GFP and StBAM9-GFP were co-expressed with StGBSS-RFP and the free RFP in Nicotiana benthamiana leaf cells.The results showed that StAmy23 was located in the cytoplasm,StBAM1 localized in the plastidic stroma while StBAM9 was located on the surface of starch granules.Moreover,St BAM1 and StBAM9 chloroplast transit peptides were identified to locate on the starch granules,whereas StBAM1 and StBAM9 without chloroplast transit peptides were located in the cytosol and nucleus.Therefore,we speculate that the chloroplast transit peptide might be prerequisite for subcellular location of StBAM1 and StBAM9.4.To explore the roles of StAmy23,StBAM1 and StBAM9 in potato CIS,the expression vectors for individually suppressing St BAM1 and StBAM9 and collectively suppressing these two genes were transformed into a CIS-sensitive cultivar E3 by Agrobacteriummediated RNA interference.The transgenic plants of CIS-sensitive potato cultivar Solara with RNAi-StAmy23 have been previously obtained.The results showed that StBAM1 and StBAM9 were essential for leaf starch breakdown during the day but only StBAM9 might be the primary contributor at night.Total ?-amylase activity was reduced in cold-stored tubers of RNAi-StBAM1 and RNAi-StBAM1+StBAM9 but not in RNAi-StBAM9 tubers.A higher starch content and lower RS levels were observed in cold-stored RNAi-St BAM1 and RNAi-StBAM9 tubers compared with control.Interestingly,collective suppression of both StBAM1 and St BAM9 had a more severe phenotype than single repression,suggesting a functional overlap between them.Furthermore,the soluble starch showed an increase in cold-stored RNAi-StBAM1 tubers but an obvious decrease in RNAi-StBAM9 tubers.These suggest that StBAM1 may regulate potato CIS by acting on soluble starch,while StBAM9 may directly attack starch granules.In addition,StAmy23 suppression resulted in higher soluble phytoglycogen accompanied by a lower RS content in cold-stored tubers,demonstrating that StAmy23 may impact on potato CIS by breaking down soluble phytoglycogen.These findings suggest that StAmy23,StBAM1 and StBAM9 play critical and distinct roles in potato CIS by attacking different substrates in different locations.To our knowledge,this is the first report to dissect the starch degradation pathway in potato CIS.5.The investigation of paired interactions between amylases(StAmy23,St BAM1 and StBAM9)and enzymes associated with starch metabolism(StGWD,StLSF1,StLSF2 and StGBSS)in the Y2 H system was performed.The results showed that StBAM9 might interact with StLSF2,however,recombinant StBAM9 or StBAM1 had no effects on dephosphorylation of StLSF2,suggesting that StLSF2 may be not a main factor influencing StBAM9-mediated starch degradation.6.The yeast-two hybrid(Y2H)library was constructed for the mixture of potato CISresistant clones 10908-06 and CW2-1 and CIS-sensitive E3 tubers stored at 4°C for 5 days.The StBAM9 and StBAM9 without chloroplast transit peptide were employed as the baits for the library screening.There were 63 potential interaction proteins obtained.Noticeably,?-amylase StBAM1 was captured by using StBAM9 without chloroplast transit peptide as bait.Moreover,Y2 H and BiFC assay proved that StBAM9 and StBAM1 interacted on the starch granules.This is the first report for existence of the interaction between ?-amylases in planta.Therefore,we speculate that StBAM9 may recruit StBAM1 to starch granules from plastidic stroma and thereby to facilitate starch breakdown. |
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