K tsta 100a

The proline, glycine-betaine (GB) and total soluble sugar contents of the samples were determined photometrically using a UV-Visible spectrophotometer (160A, Shimadzu Corp, Kyoto, Japan). The proline content was determined on the basis of its reaction with ninhydrin, according to the Bates method [25 ] as described in [26 ]. The GB content was determined using the periodide method [27 ] as described in [28 ] and the soluble sugars were estimated by the anthrone reagent method, as described in [26 ].
The megazyme total starch assay protocol was used to determine the starch content of the samples (K-TSTA-100A, Megazyme International Ireland, Wicklow, Ireland). The method is based on the enzymatic hydrolysis of starch to glucose using thermostable α-amylase and amyloglucosidase. The starch content was determined from the twice-washed pellet remaining after the extraction of total soluble sugars.
For each method, 5 x 0.2 g fresh weight (FW) samples were collected at the end of the experiments. Standards ranging from 0–50 μg/mL for the proline calibration curve, 0–400 μg/mL for GB and 0–100 μg/mL for glucose calibration curves were used to determine the metabolite contents of the samples, expressed in μg/g FW.

Freeze-dried samples from five full knockout events were homogenized in a Retsch Mixer Mill MM400 (Retsch, Germany), at 30 Hz, for 30 s, and 50 mg of homogenized material was utilized to determine total starch content using Total Starch Assay Kit (K-TSTA-100A, Megazyme, Wicklow, Ireland) following the manufacturer’s instructions. Briefly, total starch in the samples was converted into maltodextrins by thermostable α-amylase (100°C, 15 min), which were subsequently quantitatively hydrolyzed into D-glucose by amyloglucosidase (50°C, 30 min). The resulting D-glucose was measured in a colorimetric reaction employing glucose oxidase/peroxidase (GOPOD) reagent, and absorbance was measured at 510 nm (Multiskan GO, Thermo Fisher Scientific, USA). All analysis were performed in triplicates; total starch concentration was calculated using Mega-Calc™ (Megazyme, Ireland) and reported as mean values of percentage on dry weight (DW) basis.

Moisture, protein, fat and ash contents were determined using AACC official methods (AACC 44-15A, AACC 46–13, AACC 30–10 and 08–03, respectively) [19 ]. Starch and phytic acid contents were measured using the enzymatic kits K-TSTA-100A and K-PHYT, respectively, from Megazyme (Wicklow, Ireland). [20 (link)]. Samples were analyzed in duplicate and results were expressed in g/100 g of dry weight (dw).

Samples from all the diet components and feces were milled at 1 and 0.5 mm (Retsch cutting mill, model SM1, Retsch GmbH, Haan, Germany) and analyzed through AOAC [22 ] procedures for dry matter (DM) (934.01), organic matter (OM) (942.05), ether extract (EE) (920.39), crude protein (CP) (954.01), and starch (996.11). A Velp Scientifica apparatus (Velp Scientifica srl, Usmate, Italy) was used to determine Kjedhal N, which was then multiplied by a conversion factor of 6.25 to determine CP content. A solvent recovery extractor for oils and fats (JP Selecta, Det-Gras N, Barcelona, Spain) was used to determine EE content. To determine starch content, an enzymatic kit K-TSTA-100A (Megazyme, Enterprise Ireland, Dublin, Ireland) and a spectrometer UVmin-1240 (Shimadzu, Quioto, Japan) were used. Neutral detergent fiber (NDF) and acid detergent fiber (ADF) were obtained through the Robertson and Van Soest and Van Soest et al. [23 ,24 (link)] methodologies. Acid detergent lignin (ADL) was determined by solubilization of cellulose with sulfuric acid [23 ]. Soluble sugar content was determined using a UV-VIS spectrophotometry UVmin-1240 (Shimadzu, Quioto, Japan; 625 nm) through the anthrone methodology [25 (link)].

Total soluble sugars were extracted from leaves with 80% ethanol and employing a modified method [9 (link),54 (link)]. Fresh leaves, each containing 0.1 g of plant tissue, were taken in triplicate. The total soluble sugar content was measured at 620 nm by a Biospectrometer (Eppendorf, Hamburg, Germany) using glucose as the standard. The contents of soluble sugars are expressed as mg g⁻¹ FW.
Glucose, fructose and sucrose were extracted from the leaves using the method in Lu and Sharkey [55 (link)]. Fresh leaves, each containing 0.1 g of plant tissue, were taken in triplicate. The sugar concentrations were measured as described Stitt et al., 1989 and a Biospectrometer [56 ] (Eppendorf, Hamburg, Germany). To analyze starch content, the resulting sediments from aqueous ethanol extractions were autoclaved for 3 h in distilled H₂O and enzymatically digested to glucose according to the method described by Walters et al. [57 (link)]. α-amylase and amyloglucosidase from the Total Starch Kit were used to digest amylose and amylopectin into glucose. (Megazyme International Ireland Ltd.,Wichlow, Ireland, K-TSTA-100A). The sugar concentrations were determined enzymatically with a method described by Stitt et al., 1989 using a Biospectrometer [56 ] (Eppendorf, Hamburg, Germany).

Total starch (K-TSTA-100A), amylose/amylopectin assay (K-AMYL), and dietary fibre (K-TDFR-200A) kits were purchased from Megazyme Ltd. (Bray, Ireland), and the analyses were performed according to the manufacturer’s protocols.

To isolate starch from leaves, three biological replicates of 2 cm middle leaf segments were collected from fourth fully expanded true leaves into screw cap tubes (USA Scientific, 1420-9700) with a grinding bead (Advanced Materials, 4039GM-S050) and immediately frozen in liquid nitrogen and stored at −80 °C. Frozen samples were homogenized using a paint shaker. For starch quantification, leaves decolorized by 80% ethanol and starch concentration was subsequently measured using a starch assay kit (Megazyme, K-TSTA-100A).