تاثیر بیش ‏بیان ژن‌های اسیدفسفاتاز Atpap17 و Atpap26 بر عملکرد و اجزاء عملکرد دانه تحت تنش شوری در گیاه Arabidopsis Thaliana

تنش شوری با تداخل در واکنش‌های فیزیولوژیک و اختلال در جذب عناصر غذایی کلیدی نظیر فسفر، منجر به کاهش شدید عملکرد دانه‌ ‌گیاهان می‌گردد. فعالیت فسفاتازها از جمله سازوکارهای گیاهان به منظور تامین و حفظ هومئوستازی فسفر درون سلولی است. از مجموع 29 اسیدفسفاتاز ارغوانی شناخته شده در گیاه arabidopsis thaliana، افزایش سطح رونوشت ژن دو اسیدفسفاتاز atpap17 و atpap26 در شرایط تنش شوری اثبات شده است. در پژوهش حاضر از ژنوتیپ‏ های‌ بیش‌بیان (oe17 و oe26)، جهش ‏یافته‌ ساده (mu17 و mu26) و جهش ‏یافته دوگانه (dm) ژن‌های مذکور به منظور مطالعه اثر atpap17 و atpap26 بر عملکرد و اجزاء عملکرد دانه گیاه آرابیدوپسیس، استفاده شد. براساس آستانه تحمل به شوری در این گیاه (mm100 ‌کلریدسدیم) و ژنوتیپ‌های مورد مطالعه، غلظت‌های 0، 50، 100 و mm150 ‌کلریدسدیم (nacl) بررسی شد. طی تنش، درصد گل‌دهی، تعداد غلاف در بوته، تعداد بذر در غلاف، وزن هزار دانه، عملکرد کل دانه و همچنین محتوای فسفر کل و آزاد اندازه‌گیری شد. نتایج حاکی از افزایش 1.7 و 0.89 برابری عملکرد کل دانه گیاهان‌oe17 وoe26 در مقایسه باwt در غلظتmm 150 بود، در حالی بود که گیاهان جهش‌یافته فاقد عملکرد دانه‌‌‌ در آن غلظت‌ بودند. از طرف دیگر، محتوای فسفات کل در گیاهان ‌oe17 وoe26 به‌ترتیب 1.7 و 1.5 برابر بیش‌تر از wt در غلظتmm150 بود. با این وجود گیاهان جهش‌یافته mu17، mu26 و dm به‌ترتیب کاهش معنی‌دار 39%، 21% و 52% در محتوای فسفات کل در مقایسه با wt و در غلظت‌ مشابه‌ نشان دادند. تغییر در عملکرد دانه ژنوتیپ‏های مورد مطالعه در تحقیق حاضر تحت شرایط تنش شوری شدید، به روشنی حاکی از تاثیر ژن‌هایpap17 وpap26 جهت فراهم نمودن فسفر کافی در دسترس و بهبود عملکرد دانه این گیاهان بود. با توجه به نتایج حاصل از مطالعه گیاه آرابیدوپسیس، می‌توان تاثیر دو اسید فسفاتا atpap17 وatpap26 را به‌عنوان کاندیدای موثر در بهبود عملکرد دانه در شرایط تنش شوری در گیاهان زراعی مطالعه نمود.

Effect of AtPAP17 and AtPAP26 genes overexpression on yield and yield components under salt stress in Arabidopsis thaliana plant

Introduction Seedyield as an important and quantitative trait for grain crops is determined by yield components, although it could also be adversely influenced by genotype and environment. Salinity limits seed yield via interfering with major physiological functions, disrupting ion homeostasis and diminishing nutrient uptake (such as phosphorus) in plant cells. Phosphorus plays an important role in photosynthesis, respiration, and regulation of a number of enzymes as well as signaling pathways. Due to the vital roles of phosphorus in cells, plant growth and productivity are frequently limited by low phosphorus availability. One of the adaptive changes of plants under phosphate deficient condition is the increase in phosphatase activity which is one of the primary plant responses to Pi releasing and recycling from both internal and external resources. Purple acid phosphatases (PAPs) are a group of APases that catalyze the hydrolysis of a wide range of phosphate esters and anhydrides in plants. The ultimate aim of salinity tolerance research is to increase the ability of plants to maintain growth and productivity in saline soils through the identification genes associated with responding to salt stress. Our current knowledge making AtPAP17 and AtPAP26 genes promising candidate for biotechnological strategies to improve Pi acquisition and utilization, and enhance yield components under NaCl stress condition.   Materials and methods The Arabidopsis thaliana seeds, ecotype Columbia0 (Col0), atpap17 and atpap26 homozygous TDNA insertion mutant lines (Mu17 and Mu26), double mutant of atpap17/26 (DM), AtPAP17 and AtPAP26 overexpressing lines (OE17 and OE26) were used. After seeds stratified at 4°C for 48 h, the plants were cultivated (1peat moss: 1perlite: 1cocopeat) in growth chambers with a 16 h light (1000 Lux), 8 h dark photoperiod at 25°C. Plants grown on this condition fertilized 48 hourly by subirrigation with similar Hoagland’s solution containing 1.25 mM KH2PO4 for 28 days. Subsequently, the seedlings were subjected to salt stress by applying 50, 100, and 150 mM NaCl with the same Hoagland’s solution containing 1.25 mM KH2PO4 for 16 days (longterm). The control plants were grown without addition of NaCl.Trend of flowering (for eight days after salt stress), pods number per plant, seeds number per each pod, and l000seed weight obtained during the salt stress period. Finally, the total seed yield (total seed yield obtained from pods N. per plant × seed N. per each pod × l000seed weight) was also calculated for all plants. Total phosphorus (TP) and free phosphorus inorganic (Pi) contents were also measured. There were three replications (with 15 plants on each replication) of each treatment. Least significant differences were used for means separation at the 0.01 probability levels.   Results and discussion Results from the study revealed that the yieldrelated parameters and total yield as well as PT and Pi contents were gradually decreased in the genotypes with increase salt stress to 100 and 150 mM NaCl. However, no similar amount of decrease was observed among them under same growth conditions. Results showed that increase of NaCl concentration was associated with decreases in phosphorus accumulation in plants, and alternatively, phosphorus deficiency stress in plants caused to decline in the seed yield. The PT content of OE17 and OE26 were significantly higher than that in WT, although Pi content of OE17 was nosignificantly higher, and Pi content of OE26 was significantly higher in compared with WT at 150 mM NaCl. These findings showed that AtPAP26 beyond AtPAP17 plays functional role in internal Pirecycling or increasing the availability of Pi for plant by releasing Pi from external organophosphates when seedlings were deprived of the Pi supply. DM plants did not have any pod at 150 mM NaCl, the DM plants (at both 100 and 150 mM NaCl) and Mu26 (at 150 mM NaCl) lacked any seed per pod, and also the number of seed per pod of Mu17 were significantly less than that of WT at 150 mM NaCl. In addition to these genotypes that did not have any seed, and subsequently no onethousandseed weight, the seeds of Mu17 showed lack viability, with increasing level of salt concentration and period of salt stress. Thus, Mu17 and Mu26 (at 150 mM NaCl), and DM (at both 100 and 150 mM NaCl) genotypes could not obtain total seed yield; However, OE17 and OE26 produced the highest total seed yield, under both 100 and 150 mM NaCl. These results indicated that the plants responded to salinity depending on severity, duration of the stress and potential of them. Our results clearly demonstrated that overexpression of AtPAP17 and AtPAP26 genes is an effective approach to improve P acquisition. In addition, since AtPAP17 and AtPAP26 have both acid phosphatase and alkalineperoxidase activity, they could be involved in phosphate scavenging and recycling as well as the metabolism of reactive oxygen species. These results could suggest that the physiological roles of AtPAP17 and AtPAP26 might be related to the adaptation of Arabidopsis to NaCl stress, possibly through its involvement in reactive oxygen species forming, scavenging and stressresponding signal transduction pathways.   Conclusions It was clear that enhancing yield production was associated with Pi homeostasis in plants, and homeostasis of Pi for yield enhancement was related with the potential of the genotypes to recycle and scavenge Pi from intracellular and extracellular, and translocate Pi, under salt stress. Overall, the results suggest that AtPAP17 and AtPAP26 genes to supply of homeostasis of Pi could be used for the increase the ability to maintain of yield, under salt stress in Arabidopsis plants. Hence, the study of the positive effect of two AtPAP17 and AtPAP26 phosphatases on seed yield and seed yield components will be useful in generating of salttolerant crops.