2.1 Plant Materials, Growth Conditions, and Stress Treatment
Samples of the tea plant cultivar C. sinensis (L.) O. Kuntze cv.
Shuchazao were collected from the experimental garden of Anhui
Agricultural University and the tea plant germplasm resource garden in
Guohe Town. The tissue samples used for quantitative real-time
polymerase chain reaction (qRT-PCR) analysis were as follows: buds,
first leaves, second leaves, third leaves, fourth leaves, mature leaves,
old leaves, young stems, and young
roots. All samples were immediately frozen in liquid nitrogen and stored
at -80 °C.
A. thaliana ecotype Columbia-0 (Col-0) was grown under long-day
conditions 16 h of white light and 8 h of darkness at 22 ± 2 °C and a
light intensity of 100 mol m-2 s-1in the greenhouse at Anhui Agricultural University. Six-week-oldA. thaliana plants overexpressing CsHSP24.6 andCspTAC5 were selected, and wild-type (WT) A. thaliana was
used as a control. The plants were heat treated once for 6 hours at 45
°C, and were treated continuously at 40,000 lx light for three days, the
changes were investigated using a plant chlorophyll fluorometer. To
analyze the salt tolerance of A. thaliana , transgenic seeds were
sowed on Murashige and Skoog (MS) medium. After vernalization at 4 °C
for 3 d, the seedlings were grown in the greenhouse for 5 d, after which
they were transferred to MS medium containing 100 mM NaCl. After 7 d,
photographs were taken to record phenotypes and to measure related
physiological indicators. Data processing was done using SPSS. The NaCl
treatment method was as described by Johannes
Loubser39.
The expression patterns of CsHsp24.6 under biological and abiotic
stresses were also investigated. Tea branches having the same growth
conditions were picked from the tea garden and divided into five groups,
after which they were placed in Erlenmeyer flasks containing the same
amount of water. After 24 h of adaptation, the water was replaced with
200 mM NaCl solution for Group 1 and with 25% PEG4000 solution for
Group 2. Group 3 was treated by smearing 0.25% MeJA on each leaf.
Groups 4 and 5 were treated at 4 °C and 40 °C, respectively. Leaves
tissues were collected at seven time points: 0, 6, 12, 24, 36, 42, and
60 h for Group 1; 0, 4, 8, 16, 24, and 36 h for Group 2; at 0, 6, 12,
24, and 38 h for Group 3; 0, 6, 12, 24, 36, 72, and 96 h for Group 4;
and 0, 1, 2, 4, 8, 12, and 36 h for Group 5. At each time point, three
repeats were performed. Samples were immediately frozen in liquid
nitrogen and kept at −80 °C for RNA extraction.