Photosynthesis and rhizome carbohydrate concentrations of switchgrass grown from reserve-depleted rhizomes

A long-standing question in perennial grass breeding and physiology is whether yield improvement strategies could compromise winter survival. Perennial grasses rely on the pool of carbohydrates accumulated in storage organs from the previous growing season for winter maintenance and spring regrowth. Yield improvement strategies could reduce winter survival if they increase biomass and grain yields at the expense of carbon allocation to storage. Therefore, it is crucial to better understand the dependence of regrowth on storage reserves. We experimentally depleted switchgrass (Panicum virgatum L.) rhizome reserves by storing rhizomes for two weeks at 5 °C (control treatment) and 25 °C (reserve-depleted treatment). During the storage period rhizome respiration was 5.3x higher at 25 °C (0.010 μmol CO2 g-1 min-1 at 5 °C vs. 0.054 μmolCO2 g-1 min-1 at 25 °C; P < 0.0001) and the starch content was depleted by 30% by the end of storage. Surprisingly, reserve-depleted switchgrass had 60 % larger leaf area, and produced ~40% more aboveground biomass than control plants. In addition, it restored its rhizome starch reserves to pre-storage levels. Switchgrass showed a large plasticity amongst its source-sink components to buffer the imposed reserve depletion. It increased plant photosynthesis by increasing the photosynthetic leaf area while keeping photosynthesis constant on a leaf area basis and readjusted the timing and activity of sink organs to maintain a constant allocation of carbon to storage that was greater than the control treatment. These results suggest that switchgrass, and potentially other perennial grasses, largely over-invest in storage reserves, therefore, current breeding strategies in perennial grasses aimed to extend the growing season should not compromise crop persistence. Our study also has implications on long-term yield dynamics as it highlights sink-limitations as potential driver of the yield decline commonly observed in perennial grasses 5+ years after cultivation.

Methods
Rhizomes used in this experiment were harvested from a 12-year-old switchgrass stand in the Michigan State University Agronomy Farm (42.713 N, -84.467 W) in East Lansing, Michigan, USA. Rhizomes were harvested during winter, in early January, and stored at 5 °C for two weeks before the start of the reserve-depletion treatment. Reserve-depletion was imposed by storing switchgrass rhizomes at 25 °C for 14 days, control treatment rhizomes remained at 5 °C for the same period.

Immediately after the storage period, rhizomes were planted individually in 2.54 L pots filled with a peat and perlite mix (SureMixTM; Michigan Grower Products, Galesburg, MI, USA) and kept in a greenhouse at 27 °C and 16hr photoperiod supplemented with artificial lights for the length of the growth cycle (~180 days). Rhizome respiration, development, and photosynthesis were measured repeatedly over the course of the experiment. These non-destructive measurements were taken on randomly assigned rhizomes weekly or biweekly. Leaf net CO2 assimilation rate and stomatal conductance to water were measured biweekly in the middle portion of the youngest fully expanded leaf with a Li-6800.

Aboveground biomass, rhizome and root biomass, and total leaf area were sampled 5 times during the experiment. Rhizome biomass was also sampled three times during the storage period on the first, 8th and 14th day of storage. These destructive measurements were taken on 10 randomly preassigned rhizomes per treatment every 30 – 40 days.

To harvest belowground tissues, soil was rinsed using tap water and roots were removed from rhizomes. Roots were dried and weighted separately as with aboveground biomass. All rhizome samples were weighed fresh, placed in an aluminum pouch and flash frozen in liquid nitrogen for glucose, sucrose, and starch content analyses. Total belowground biomass was estimated as the sum of root and rhizome biomass.

Leaf and rhizome samples were stored at -80°C until further processing. Leaf samples were ground to a fine powder with a mortar and pestle. Rhizome samples were ground with a spice mixer (Cuisinart; SG-10). All samples stayed in contact with liquid nitrogen during grinding and were then freeze-dried for at least 48 hours in a lyophilizer. Leaf and rhizome starch, sucrose and free glucose were measured at the Biomass Analytics Facility at Michigan State University.