Li 3000 area meter

Shoots and roots were harvested 21 days after the start of the different pH values and N forms treatment, frozen in liquid nitrogen, the stored at −80°C for physiological analysis, and dried partially at 105°C for 20 min and then 75°C for 24 h to obtain a constant weight. Root morphology was estimated from images of plants taken by a root scan machine (Epson 1,680, Indonesia), with WinRhizo Pro Vision 5.0 analysis program (Canada). A Li-3000 area meter (Li-Cor Inc., Lincoln, NE, United States) was used to measure leaf area. Free NH₄⁺ concentration in plant shoot and root was measured according to the method of Balkos et al. (2010) (link). Soluble sugars concentration in tissues were measured from the hydroalcoholic extracts using a colorimetric assay with an anthrone reagent (Wang et al., 2019 (link)). Free amino acids concentration in shoot and root was determined using the method of Moore and Stein (1954) (link). GS activity and nitrate reductase (NR) activity were determined, as described previously (Wang et al., 2016 (link)).

At the post-anthesis stage, the LI-3000 area meter (Li-Cor. Inc., Lincoln, NE, United States) was used to measure the green flag leaf area during drought stress treatment (WD). The specific leaf weight was calculated as follows:
where WL is the flag leaf weight and AL is the flag leaf area.
When half of the spikes under the treatment had reached grain maturity, the grain filling duration (GFD) (the number of days from blossoming to grain development) was recorded. At maturity, three pots were haphazardly chosen from every treatment to ascertain the number of tillers with spikes, the number of grains/spikes, spike length, the weight of 1,000 grains, grain yield/pot, plant biomass, and drought index (DI). The DI was determined as the proportion of grain yield under drought stress (YD) and grain yield under WW development (YW).

We also sampled 40 terminal branches of each sex for leaf and branch dimensions. To do this we removed all leaves between nodes from an apical branch per individual and noted leaf numbers and total leaf area per annual branch, as well as node length and stem diameter. We used these to determine mean leaf area and we dried leaves at room temperature to determine specific leaf area. Specific leaf area is correlated with many aspects of leaf physiology^{30 (link)}. We measured leaf area with a LI-3000 Area Meter (LICOR, Lincoln, NE, USA) and leaves were dried for > 48 h at 70 °C in a drying oven to obtain dry mass. To get a measure of percentage seed set, we counted numbers of florets per single randomly collected inflorescence and numbers of seeds in a single cone on the same individual in each of 50 female individuals. We then dissected through a sample of 300 seeds from a cone from 32 individuals and used the visual presence of endosperm as indicative of a viable seed.