Spad 502 chlorophyll meter

Shoot fresh (FW) and dry (DW) weight were measured, and relative water content calculated as RWC = (FW-DW)/FW. Before harvesting, leaf chlorophyll content was measured as a SPAD index using a Minolta Chlorophyll Meter SPAD-502 (Konica Minolta, Osaka, Japan) on the third true leaf, at a position about one quarter of the length of the leaf from the leaf tip. Stomatal conductance (Gs) was also measured on the same leaf using a Decagon leaf porometer (Decagon Devices Inc., WA, Australia), under constant light conditions (artificial light of 150 μmol m⁻² s-¹). The number of necrotic leaves was also counted at harvest.

Photosynthetic rate, stomatal conductance and transpiration rate in the leaves of the salt-tolerant transgenic plants and wild-type plants grown in pots for 2 weeks under 200 mM NaCl stress were measured according to the methods of Liu et al. [18] . Relative chlorophyll content (SPAD value in fresh leaves) was measured as described by Fernández-Falcón et al. [28] with Chlorophyll Meter SPAD-502 (Minolta, Japan). The experiments were conducted at 9-11 a.m. of sunny days.

The parameters measured at the beginning of flowering included: leaf greenness index SPAD correlated with chlorophyll content using a Chlorophyll Meter SPAD-502 (Konica Minolta, Osaca, Japan), stomatal conductance with an SC1 porometer (Dekagon Devices, Pullman, WA, USA), net intensity of photosynthesis and leaf transpiration using a LI-COR gas analyzer (Portable Photosynthesis System, Lincoln, NE, USA). The measurements included five plants per treatment. Three well developed leaves from the central section of each plant were selected, four readings per leaf were performed and average values were calculated. The measurements were conducted between 10.00 a.m. and noon, on a cloudless day, at natural CO₂ level and average PAR 1101 μmol m⁻² s⁻¹ read with a Radiometer-Photometer RF-100 (Snopan, Białystok, Poland).

Six leaves per plant for 12L vines and three leaves per plant for 3L vines were measured using the portable Chlorophyll Meter SPAD 502 (Minolta Corp., Ramsey, NJ, USA). On each leaf, five SPAD readings were taken at each leaf lobe and then averaged.

The rice seeds were grown in the growth chambers with 12 h-dark/12 h-light at four temperatures (20, 24, 28, 32 °C) and the light intensity of 120 μmol of photons m⁻² s⁻¹. The extracts were obtained from 100 mg of the third fresh leaves at the 3-leaf stage seedlings and homogenised in 10 ml of 100 % acetone. Spectrophotometric quantification was carried out in BECKMANCOULTER-DU720. Total Chl, Chla, Chlb and carotenoid contents were determined according to the methods of Arnon (1949 (link)) and Alan (1994 (link)). In 2010, WT and tcm5 plants were grown in experimental rice fields of Shanghai Normal University by conventional methods. Leaf chlorophyll SPAD values (Additional file 1: Figure S1) by means of CHLOROPHYLL METER (SPAD-502, Minolta Co., Ltd, Japan), which can provide a simple, quick portable and non-destructive method for estimating leaf Chl content (Peng et al. 1993 (link); Turner and Jund 1991 (link); Dwyer et al. 1991 (link)) were measured every week from transplanting (summer) to heading (autumn). Finally, agronomic traits of rice plants (Additional file 1: Figure S2) were measured at maturity.

A final observation on chlorophyll content and gas exchange was done at 4 months after sowing. All measurements were carried out between 09:00 until 11:00 with the relative humidity of the air, ambient temperature and ambient CO₂ concentration were about 70%, 33–36°C and 370–390 μmol.mol^-1, respectively. The rate of 1000 μmol CO₂ m^-2 s^-1 was used as the light-saturated rate of photosynthesis.
The net photosynthetic rate (P_n), stomatal conductance (G_s), and concentration of intercellular CO₂ (C_i) were measured on fully expanded leaves seedlings using CIRAS-3 Portable Photosynthesis System (Amesbury, MA, USA). Chlorophyll content was determined with the Chlorophyll Meter, SPAD-502, Konica Minolta (Tokyo, Japan). These measurements were made on five randomly selected seedlings for each treatment with three replicates.

Seeds of three independent transgenic lines (Ubi:OsHSP20-L5, -L16 and -L20) and wild type (WT) were surface sterilized and plated on 1/2 MS medium for stress assays. For heat stress, the plates were treated at 50 °C for 12 h and then transferred to 25 °C for 10 d. For salt stress, the seeds were shifted to 1/2 MS medium with 100 mM NaCl for 14 d after germinated on 1/2 MS medium for 3 d. The germination percentages were scored and the lengths of the roots were measured for estimating the stress tolerances. The chlorophyll contents of leaves were measured with a chlorophyll meter SPAD-502 (Konica-Minolta, Japan) and recorded as SPAD values. All experiments were done at least three repeats and the data for each repeat were measured from more than 30 seeds or seedlings.