Morphological and transcriptomic responses/acclimations of erect-type submerged macrophyte Hydrilla verticillata both at low-light exposure and light recovery phases

Light intensity is a determinant for submerged macrophytes. Little is known about their molecular responses to low-light exposure, despite being more informative and responsive than morphological traits. For erect-type submerged macrophytes, the stem is more crucial relative to the leaf in acclimation to low-light stress, but receives less attention. We determined morphological and stem transcriptomic responses/acclimations of Hydrilla verticillata to extremely and mildly low light (7.2 and 36 μmol photons m–2 s–1, respectively), i.e. EL and ML, with the radiation intensity of 180 μmol photons m–2 s–1 as the control. Low-light exposure continued for nine days, followed by a seven-day recovery phase (180 μmol photons m–2 s–1). At the exposure phase, the low-light treatments, in particular the EL, decreased relative growth ratio, but induced greater height and longer stem internode distance and epidermal cell. Such responses/acclimations continued into the recovery phase, despite more or less changes in the magnitude. Transcriptome showed that the photosynthetic system was inhibited at the exposure phase, but the macrophyte adjusted hormone synthesis relating to cell division and elongation. Moreover, the EL activated cell stress responses such as DNA repair. Following light recovery, the macrophyte exhibited strong-light response, although energy metabolism enhanced. Especially, the EL-enriched pathways relating to anthocyanin synthesis at such phase, indicating an activation of photoprotective mechanism. Our findings suggest that negative influences of low light occur at both low-light exposure and recovery phases, but submerged macrophytes would acclimate to light environments. Transcriptome can show the molecular basis of plant responses/acclimations, including but not limited to morphology. This study establishes a bridge connecting morphological and molecular responses/acclimations.

Methods
At the end of both low-light exposure and light recovery phases, we chose three replicated plastic bottles from each growth chamber and determined morphological parameters of H. verticillata. Two healthy individuals from each plastic bottle were harvested, washed, and dried with filter paper to determine the total biomass, the number of stem internodes, and the average distance of stem internodes. In addition, plant height and stem diameter (at 5 cm down the tip) were measured with a ruler (0.1 cm precision) and a micrometer (0.02 mm precision), respectively. After the measurement, the H. verticillata individuals in each plastic bottle were separated into leaves, stems and roots, oven-dried at 80°C to a constant weight, and then weighed to obtain the dry weight. The stem mass ratio was defined as the ratio of stem dry weight to total dry weight. Relative growth rate (RGR) was calculated as:

                                                                                                                    RGR=W1-W2/∆T

where W1 is the initial fresh weight (mg), W2 is the final fresh weight (mg), and ∆T is the time (d). In addition, a scan with the electron microscope (JSM-5610LV, Thermo Fisher Scientific, USA) was used to determine stem epidermal cell length (at least 15 cells per sample) at 5 cm down the tip.