MATERIALS AND METHODS
Plant
materials and growth conditions
Two cowpea genotypes, Yacine and 58-77, generously provided by Drs
Philip Roberts and Bao-Huhyn Lam, from University of California,
Riverside were used for all experiments. These lines exhibited apparent
tolerance and sensitivity respectively to HL stress. One seed was sown
in 1 L pot filled with Suremix potting medium (Michigan Grower Products
Inc, USA) and watered with half-strength Hoagland’s nutrient solution.
Plants were staged in a reach-in growth chamber with the following
conditions: RH, 50%; temperature, 30/20 °C day/night; light intensity,
up to 400 μmol m-2 s-1 and
photoperiod of 10/14 h light/dark. Seedlings were moved to a Dynamic
Environment Photosynthesis Imager (DEPI) chamber (Cruz et al., 2016) for
treatments 3–4 d or 12–14 d after germination for the young leaves or
mature leaves respectively for treatments and assessment of
photosynthetic parameters. Where necessary, older plants were used but
leaves of similar maturity were used for all measurements. The relative
humidity (RH) varied from 20–40% for the HT treatments whereas it was
kept at 50% for the Control treatment.
High
temperature and high-light treatments
Various HT and HL treatments
are described in detail within the corresponding figure descriptions.
Briefly, HT treatments were imposed by heating the air temperature in
the chamber using the native environmental control embedded in the
growth chamber to 45 °C. For the temperature response curves, the
temperature was changed by 5 °C every 2 h (starting from GT of 30 °C)
but measurements were made after exposure for at least 1 h. Illumination
in the DEPI chamber was provided by LED lights with wavelength in
photosynthetically active radiation (PAR) range. The light intensity
treatments consisted of low light (LL — 300 μmol m-2s-1) and high-light (HL — either 1000 or 1500 μmol
m-2 s-1).
Chlorophyll
fluorescence and absorbance change measurements
the maximal PSII efficiency (FV/FM),
plants were dark adapted for 20 min before measurements were made. Most
chlorophyll fluorescence measurements were made using either the
MultispeQ V1 linked to the PhotosynQ platform
[www.photosynq.org , (Kuhlgert et al., 2016)] or the
image-based DEPI chamber (Cruz et al., 2016). Some fluorescence
measurements were also performed simultaneously with gas exchange
measurements using the LI-COR 6800 (Lincoln, Nebraska, USA).
The electrochromic shift was assessed simultaneously with chlorophyll
fluorescence measurements using the MultispeQ V1, thus enabling us to
connect PSII function with downstream reactions such as the ATP synthase
activity. The thylakoid conductivity to protons
(g H+) was determined by the
dark interval relaxation kinetics (DIRK) of the electrochromic shift
(ECS) at 520 nm (Thomas J Avenson, Cruz, Kanazawa, & Kramer, 2005;
Takizawa, Cruz, Kanazawa, & Kramer, 2007) and was utilized as a measure
for the chloroplast ATP synthase activity. The proton motive force
(pmf ) was assessed as the amplitude of the first-order decay
kinetics of the ECS trace in the first 300 ms [ECSt, (Kanazawa &
Kramer, 2002; Kanazawa et al., 2017)].
Photoprotection (the rapidly reversible form of NPQ,q E) and photo-inhibitory quenching (the slowly
relaxing form of NPQ, q I) were assessed as
described in (Tietz, Hall, Cruz, & Kramer, 2017), using the MultispeQ
V1 or DEPI platform. Briefly, following initial pulses to determine
FO’ and
FM’, leaves were dark adapted for 2
min after which q E would have relaxed, leavingq I, enabling us to determine
FM”.