Abstract
To understand the early signaling steps in the response of plant cells to increased environmental temperature, 2-D difference gel electrophoresis was used to study the proteins in microsomes of Arabidopsis seedlings that are regulated early during heat stress. Using mass spectrometry, 19 microsomal proteins that showed an altered expression level within 5 min after heat treatment were identified. Among these proteins, annexin 1 (AtANN1) was one of those up-regulated rapidly after heat-shock treatment. Functional studies show loss-of-function mutants for AtANN1 and its close homolog AtANN2 were more sensitive to heat-shock treatment, whereas plants overexpressing AtANN1 showed more resistance to this treatment. Correspondingly, the heat-induced expression of heat-shock proteins and heat-shock factors is inhibited in ann1/ann2 double mutant, and the heat-activated increase in cytoplasmic calcium concentration ([Ca(2+)]cyt) is greatly impaired in the ann1 mutant and almost undetectable in ann1/ann2 double mutant. Taken together these results suggest that AtANN1 is important in regulating the heat-induced increase in [Ca(2+)]cyt and in the response of Arabidopsis seedlings to heat stress.
Highlights
Temperatures above the optimum are sensed as heat stress (HS)1 by all living organisms
Prior reports showed this increase is regulated by plasma membrane-localized cyclic nucleotide-gated channels (CNGCs) [8, 9] and phospholipase C9 (PLC9) [26], but there may be other calcium channels or membrane-localized proteins involved in regulating this rapid and dynamic change in [Ca2ϩ]cyt in response to HS treatment
Because of prior studies showing that Among these proteins was annexin 1 (AtANN1) promoted the Ca2ϩ permeability of plasma membranes, we selected it for further study from the microsomal proteins in Arabidopsis seedlings whose level changed rapidly after heat shock
Summary
Plant Materials and Heat Shock Treatment—Arabidopsis thaliana seeds were surface sterilized using 75% (v/v) ethanol and sowed in glass plates containing 25 ml growth medium containing half-strength Murashige and Skoog (1/2 MS) salt, 1.5% (w/v) sucrose, and 0.8% (w/v) agar. To prepare the seedlings for 2D-DIGE protein sample extraction, Arabidopsis seedlings were grown in liquid medium containing 1/2 MS salt and 1.5% (w/v) sucrose under long day at 22 °C for 1 week. The transcript abundance of AtANN1 and AtANN2 in wild type and mutant seedlings was determined by semi-quantitative Reverse transcription PCR using 5Јatggcgactcttaaggtttc-3Ј (left) and 5Ј-agcatcatcttcaccgagaag-3Ј (right) for AtANN1 and gene specific genotyping primer set for SALK_ 054223 and SALK_112492. Expression Analysis of HSFs and HSPs by RT-PCR and Western Blot—Wild type and ann1–2/ann double mutant seedlings were grown in the same glass plates containing 15 ml growth medium (1/2 MS salt, 1.5% sucrose, and 0.8% agar) for 1 week at 22 °C under long-day condition. Determination of Total Ca2ϩ in Plant Tissues—Fifty seven-day-old Arabidopsis seedlings grown in 1/2 MS medium supplied with 1% sucrose were grouped as one sample and oven dried at 65 °C overnight. Total plant Ca2ϩ concentration was determined with a flame atomic absorption spectrophotomer (Model AA240Z, Agilent Technologies, Santa Clara, CA)
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