*4.1. Effect of Photoperiod Extension on Leaf Physiology*

Extending the natural solar photoperiod via the implementation of supplemental lighting has proven to be a beneficial lighting strategy in terms of plant growth and yield during greenhouse production of tomatoes [1]. However, decades of research have shown that photoperiod extension beyond a critical length causes photoperiod-related injury characterized by leaf chlorosis [9,34–36]. Indeed, this negative result observed under photoperiods greater than 17 h has nullified any theoretical advantages they may have [8].

The increased use of wavelength-specific LED fixtures during controlled environment plant production has the potential to play a pivotal role in reducing injury related to photoperiod extension. During CL, Matsuda et al. [14] indicated that young tomato plants (23 days after planting and 13 days into the treatment) grown under white light during the day and either red or orange light during the subjective nighttime period produced a lower degree of injury than plants grown under white light during the day and either white or blue light at night. A recent study by Lanoue et al. [7] produced injury-free tomatoes during a 6-month production period grown under CL by using an alternating spectrum of red during the day and blue during the subjective nighttime period. However, a reduction in the light level also occurred during the spectral shift from 200 µmol m−<sup>2</sup> s <sup>−</sup><sup>1</sup> of red light to 50 µmol m−<sup>2</sup> s <sup>−</sup><sup>1</sup> of blue light (close to light compensation point), which may have confounded any effects of the spectral shift [9]. In this study, the light intensity was maintained at the same level for the two lighting treatments, allowing for the comparison of spectral compositions/quality without any confounding effects.

During the initial stages of plant growth, plants grown under all lighting treatments were observed to be injury-free (Figures 3A and 4). However, as determined by measurements between 54 and 62 DIT (8–16 January 2019), plants grown under either red 23 h or mix 23 h treatments developed photoperiod-related injury, characterized by leaf chlorosis, consistent with previous research (Figures 3B and 4) [6,35–37]. At this period (around 2–4 weeks before the starting of fruit harvest—78 DIT; it usually takes 2 weeks for a cluster of fruit from mature green (full size) to reach harvesting stage—4 red fruits out of the 5 fruits in each cluster), the clusters of fruits were in the fastest growing stage (fastest increase in size). The plants had heavy fruit sink and weak leaf source due to low natural sunlight (Figure 2, 8–16 January 2019). During this period of measurements, parameters related to photosynthesis, such as LCP, QY, Pnmax, Vcmax, and Jmax, were all reduced from leaves that were exposed to both 23 h lighting treatments compared to those exposed to either 17 h lighting treatment (Tables 2 and 3). The downregulation in parameters related to photosynthesis may be related to either excess carbohydrate accumulation or a downregulation of *CAB-13*, affecting PSII function [9,38]. Interestingly, during this period of production, which was characterized by photoperiod-related injury and a downregulation in parameters related to photosynthesis, both stomatal conductance and transpiration rates were unaffected (Figure 5). It should be noted that Lanoue et al. [7] did not observe issues with carbon metabolism during CL. Therefore, the major driver of injury during extended photoperiods cannot simply be attributed to improper stomatal function or overaccumulation of carbohydrates. Furthermore, as a decrease in the chlorophyll content was observed (Table 5), photoperiod-related injury seems to be more closely related to light capture.

From 54 DIT (8 January 2019) onwards, plants and leaves grown under both 23 h treatments were observed to recover from the photoperiod-related injury (Figures 3C and 6C). The photosynthetic capacity and Fv/F<sup>m</sup> values from leaves under 23 h treatments returned to levels similar to the 17 h treatments, and fruit yield during the months of April and May also increased (Table 6). However, no changes to the experimental settings had occurred to invoke such changes. What did change was the peak intensity of natural solar radiation (Figure 2) and the natural photoperiod from January to April. In fact, from the period of peak photoperiod injury (62 DIT, 16 January 2019) to when photoperiod injury was completely alleviated (138 DIT, 2 April 2019), the solar radiation nearly doubled in average daily intensity from 88 W m−<sup>2</sup> to 165 W m−<sup>2</sup> (Figure 2). Moreover, during this period, the plant growth was more balanced—less fruit load due to fruit harvesting and more leaf growth due to strong sunlight. The changes in both solar radiation and plant growth balance (generative vs. vegetative or source vs. sink) may, in some way, account for the observed recovery of plants under both 23 h lighting treatments. Further specifically designed studies will be needed to separate the light intensity effect from that of fruit load/plant growth stage. Potentially, as the natural light intensity increased later in the study, the strong exogenous signaling (i.e., high peak light intensity) was able to

override the endogenous signal causing injury [34,37,39]. However, an in-depth look at this hypothesis was beyond the scope of our study.
