KT/HAK/KUP家族基因在桃叶片发育过程中的表达及其对钾肥施放的响应

Expression of KT/HAK/KUP family genes during leaf development of peaches and their responses to potassium fertilizer applications

【目的】从转录水平分析KT/HAK/KUP家族基因在桃叶片发育不同时期的表达特征及对钾肥施用的响应情况,明确关键基因,旨在揭示果树K^+吸收与转运机制的分子基础,并为果树施肥及高效园艺作物的遗传改良与育种提供理论依据。【方法】以‘霞晖6号’桃树为研究对象,通过设置钾肥处理,分析其对桃叶片发育、光和性状及钾素营养状况的影响;通过HNO_3-HClO_4法消解叶片样品,利用ICP-AES设备测定叶片发育不同时期的钾离子含量;利用荧光实时定量qRT-PCR技术分析KT/HAK/KUP家族基因在叶片发育不同时期的动态表达及对钾肥施用的差异响应。【结果】钾肥施用显著提高了叶片中叶绿素含量和干鲜质量比,增强了钾素富集水平,并有效促进了叶片的光合作用(净光合速率Pn和气孔导度Gs均显著上升)和蒸腾作用(蒸腾速率Tr显著增加);KUP1-16在叶片发育不同时期的表达水平存在差异,KUP14在叶片整个发育时期的表达水平显著高于其他基因,其次是KUP3和KUP7,而KUP12和KUP13在叶片生长发育的整个过程中均没有检测到表达量;在桃叶片发育不同时期,KUP家族基因在转录水平对钾肥施用的响应不同:钾肥处理显著抑制了KUP14从施用钾肥至初秋时期(9月11日)的表达,表明该基因在桃叶片正常发育过程中起重要作用,KUP3对钾肥施放最为敏感,其表达水平在叶片发育不同时期是持续被诱导的,表明该基因更倾向于在钾素供应丰富的情况下发挥作用;尽管KUP11和KUP16在正常生长情况下的表达量相对较低,但在叶片发育不同时期是稳定不变的,且不受钾肥施放的影响,说明它们在叶片发育及钾素营养动态中持续发挥作用。【结论】钾肥施放促进桃叶片发育,改善叶片钾营养状况,并增强叶片的光合作用。KT/HAK/KUP家族基因在桃叶片发育不同时期的表达水平存在差异,并在转录水平对钾肥施放的响应是不同的。

[Objective]As one of the most abundant cation elements in plant cells,potassium（K^＋） contributes to many physiological and metabolic processes,such as maintenance of cellular osmolarity,photosynthetic apparatus and the control of the closing and opening of stomata.K^＋ deficiency negatively affect plant growth,especially of photosynthesis,chlorophyll content and chloroplast ultrastructure.In orchards,K^＋ is also closely related to flower formation,fruit quality and fruit yield.K^＋ deficiency is inevitably one of the major challenges to fruit productivity and quality.However,molecular mechanisms toward K^＋ nutrition in fruit trees are very rare.To maintain normal growth and development,an optimal amount of K^＋ must be effectively absorbed from the soil via the roots,and then subtly transported and allocated into different organs or tissues throughout the whole plant.Notably,K^＋ transporters are known for acquiring K^＋,catalyzing K^＋ uptake across a wide range of external concentrations,mediating K^＋ movement within the plant as well as its efflux into the environment and possibly maintaining cation homeostasis in plants.Notably,individual members of the KT/HAK/KUP family（the largest K^＋ transporter family in plants）,have been found in diverse species,including Arabidopsis,barley,rice,tomato,and alligator weed.Several members of the KT/HAK/KUP family have been characterized in plants,either by overexpression in Arabidopsis suspension cells or in the analyses of T- DNA insertion mutants.The ubiquitous presence of these KT/HAK/KUP members in plants implies that they play a key role in enhancing plant tolerance to adverse environments,including drought,salt and cold.The knowledge of the K^＋ uptake and transport mechanism in model plants directly provides insights for the maintaining of fruit crops,i.e.breeding for higher output,and better quality or more stress tolerant cultivars.As one of the most economically important fruit crops,the peach（Prunus persica L.Batch） has been genetically well-characterized.In previous studies,we have identified 16 KUP（K^＋ uptake transporter） family genes in the ＇Xiahui6＇ peach,and analyzed the physiological response of ＇Xiahui6＇ seedlings and expression profiles of each KUP gene in response to abiotic stresses,including K^＋ deficiency,drought and heavy metal stresses.In this present study,we analyze leaf development,K^＋ accumulation status,photosynthesis and expression profiles of KUP genes during different leaf development stages,especially in response to K fertilizer treatment.This study not only directly reveals a close relationship between K^＋ nutritional status and fruit tree growth（especially of leaf development）,but also provides the theoretical basis for K fertilization in natural orchards or installation horticulture and genetic improvement and breeding of efficient horticulture crops.[Methods]＇Xiahui6＇ peach trees were used throughout our study.The K fertilizer treatment experiment was carried out via applying834 g KC1（K2O 60%） to each peach tree on the 40 th day after full bloom.We also analyzed the effects of K2 O fertilizer applications on leaf development,K^＋ nutritional status,and photosynthesis.After being digested via the HNO3-HClO4 method,the K^＋ concentration of peach leaves were determined on ICP-AES apparatus during different leaf developmental stages.Photosynthetic analysis was carried out on a portable photosynthetic system LI- 6400 to determine the net photosynthetic rate（Pn）,stomatal conductance（Gs）,and transpiration rate（Tr） at the terminal leaflet of fully grown second peach leaves.Using quantitative real-time PCR,we analyzed expression profiles of the KT/HAK/KUP family genes during whole leaf development,and identified key K^＋ transporter genes.We further analyzed the response of KT/HAK/KUP family genes subjected to K fertilizer application during different leaf developmental stages.[Results]The leaf chlorophyll content was relatively high during the whole fruit development stage,especially from the peach fruitlet formation（early May） to full fruit ripening（mid-July）.The highest leaf chlorophyll content occurred in mid-June.After the peaches were harvested,the leaf chlorophyll content began to reduce,and reached its lowest level during late October.However,the K fertilizer application significantly enhanced the leaf chlorophyll content,compared to the control,especially during early May to mid-July.The ratio of dry and fresh leaf mass was higher after fruit harvest than that during the whole fruit development stage,and the K fertilizer application significantly enhanced the ratio of dry and fresh leaf mass.Similar to that of chlorophyll content,the photosynthesis in the leaves were more active during the phase from early May to September（including the whole fruit development and ripening stages）,as evidenced by the higher net photosynthetic rate,higher stomatal conductance,and higher transpiration rate.The K fertilizer application favorably strengthened leaf photosynthesis,as evidenced by the enhanced net photosynthetic rate,stomatal conductance and transpiration rate,as compared to the control.In particular,the highest K^＋ accumulation occurred in mid-June,which was the second exponential growth phase of peach fruits,while the K fertilizer application significantly enhanced the K^＋ nutritional status,with an increase of 21.30%（May7th）,19.44%（June 17th）,18.81%（July 29th） and 19.12%（October 21st） for the K^＋ concentrations,respectively.In addition,genes of KUP1-16 were distinctly expressed during different leaf development stages.KUP11 was the most expressed gene,followed by KUP3 and KUP7,whereas KUP12 and KUP13 were not detected in the leaves throughout the whole development process.Moreover,the KUP family genes were differentially regulated by the K application,i.e.,KUP14 was the most expressed gene throughout the whole development process in the control leaves,which was significantly reduced by the K application,indicating that this gene may play an important role in normal leaf growth and development.KUP3 was most sensitive to the K application,whose expression was consistently induced,but was induced throughout the whole development stage,implying that KUP3 is more prone to be active within a rich K^＋environment.Although expression of KUP11 and KUP16 were relatively low in the control leaves,they were consistently steady during the whole leaf development process and had little effect from the K application.[Conclusion]The K fertilizer application favorably improves the leaf growth status in peaches,enhances the leaf K^＋ nutritional status,promotes leaf photosynthesis,and differentially regulates the KT/HAK/KUP family genes during different leaf development stages.This study directly reveals a molecular basis to study K^＋ uptake and transport mechanisms in fruit trees,which allows for better understanding of the correlation between the K^＋ nutrition level and leaf growth status,especially in perennial woody plants.