CfCHLM, from Cryptomeria fortunei, Promotes Chlorophyll Synthesis and Improves Tolerance to Abiotic Stresses in Transgenic Arabidopsis thaliana

We found that the CfCHLM protein showed a high similarity with CHLM proteins from the other species (Figure 1A). The phylogenetic analysis showed that the CHLM proteins of gymnosperms and angiosperms clustered separately; among them, CfCHLM was most closely related to TcCHLM (Figure 1B). Our prediction analysis showed that the CfCHLM protein is predominantly composed of alpha helices and random coils (Figure 1C), which is similar to the structural composition of the A. thaliana CHLM protein [17]. Our subcellular localization results showed that CfCHLM is located in chloroplasts (Figure 2A), and it has been reported that CHLM is located in both envelope and thylakoid chloroplast membranes [14], which is similar to what was found in our study. Therefore, we speculated that the CfCHLM protein may bind to the chloroplast membrane and promote chl synthesis.

The expression levels of CfCHLM varied in different tissues, with the highest expression found in the needles and the lowest in the roots and seeds (Figure 3A). In rice, the gene encoding the CHLM protein, YGL18, has the highest expression in the leaves and the lowest expression in the roots [16], and, even though the expression of this gene in seeds has not been detected, the tissues with the highest and lowest expression levels in rice are similar to those identified in our results. In addition, abiotic stress had an effect on the gene expression levels (Figure 3B–E). In this study, the expression of CfCHLM in C. fortunei was shown to be downregulated under abiotic stress, and the downregulation of the CHLM gene may have inhibited the synthesis of chl. Many studies have shown that the synthesis of chl is inhibited under various abiotic stresses [28,29,30,31], which demonstrates the downregulation of the CfCHLM gene in response to stress.

In our study, the chl levels of plants decreased under abiotic stress, but the chl levels of CfCHLM transgenic A. thaliana were higher than those of WT (Figure 4N–P, Figure 5N–P and Figure 6N–P). Many studies have shown that the chl content of plants can be affected by abiotic stresses. For example, salt stress affects the chl content of cowpeas (Vigna unguiculata) [32]; drought stress causes a decrease in the chl content in rice [33]; and cold stress specifically inhibits chl biosynthesis in young pakchoi (Brassica rapa ssp. chinensis) leaves [31]. The above studies are similar to our results, demonstrating that the CfCHLM gene promotes chl synthesis in A. thaliana.

Chl fluorescence parameters reflect the photochemical processes and efficiency of plants. F_v/F_m is a reliable indicator of photoinhibition—the lower the F_v/F_m is, the higher the degree of photoinhibition—and this indicator can be used to identify plant resistance to stress. Y(II) can reflect the actual photosynthetic efficiency of a plant, and qP can reflect the electron transfer activity of the PSII reaction center [20,34]. Plants produce large amounts of reactive oxygen species (ROS) when subjected to abiotic stress, and ROS promote the senescence of plant leaves [35], which reduces a plant’s ability to photosynthesize. The mechanism of photosynthesis involves various components, including photosystem activity, the electron transport system, the CO₂ transport level, and so on [36]. Chl fluorescence parameters are closely related to photosynthesis in plants and can reflect the photosynthetic capacity of plants [37]. Therefore, when plants are subjected to stress in a manner which results in a decrease in their photosynthetic capacity, their chl fluorescence parameters will also decrease accordingly. In our study, the F_v/F_m, Y(II), and qP values of A. thaliana showed a decreasing trend under the stress treatments compared to CK (Figure 4K–M, Figure 5K–M and Figure 6K–M). Arminian et al. [38] found that the chl fluorescence parameters of canola (Brassica napus L.) leaves decreased during cold. Li et al. [39] found that the F_v/F_m and qP of two different varieties of cucumber (Jinchun No.4, Zhongnong No.12) under high temperatures both decreased. Hou et al. [40] found that hypertonic salt stress reduced the F_v/F_m and qP values in A. thaliana. These studies showed that chl fluorescence parameters decrease under stress, similarly to our results, but, in our study, the F_v/F_m, Y(II), and qP values of transgenic A. thaliana were higher under each stress than those of the WT (Figure 4K–M, Figure 5K–M and Figure 6K–M), demonstrating that the CfCHLM gene can enhance photosynthesis in A. thaliana.

Pn, Gs, and Tr can also be used as important indicators of plant chloroplast function. In response to external stress, plants lower their Gs to reduce water loss, forming a self-protection mechanism. Many studies have shown that Pn is positively correlated with Gs in plants under normal growth conditions, but, under stress conditions, influenced by other factors, Pn is not as well correlated with Gs [41,42,43]. Zhang et al. [44] found that the Pn, Gs, and Tr of wild apricot (Prunus armeniaca L. var. ansu) decreased with decreasing soil moisture. Ying et al. [45] found that the Pn, Gs, and Tr values of happy tree (Camptotheca acuminata) decreased with increasing levels of water stress. In this study, the A. thaliana lines (WT and transgenic lines) showed a decreasing trend for Pn, Gs, and Tr under the stress treatments compared to CK (Figure 4Q–S, Figure 5Q–S and Figure 6Q–S). Under heat stress, drought stress, and salt stress, the Gs and Tr of transgenic A. thaliana were lower than those of WT (Figure 4R–S, Figure 5R–S and Figure 6R–S), demonstrating that transgenic A. thaliana was more capable of self-protection than WT. Notably, both Gs and Tr decreased under 300 mM salt stress but rebounded under 400 mM salt stress in the A. thaliana lines (WT and transgenic lines) (Figure 6R–S). We hypothesized that, under 300 mM salt stress, the A. thaliana plants adapted to the stress and, thus, reduced their Gs, which led to a reduction in Tr and Pn, but, under 400 mM salt stress, cell damage led to an increase in Gs and Tr but a reduction in Pn. These results indicated that CfCHLM enhanced chloroplast function in A. thaliana.

A phenotype is the most intuitive manifestation of plants under stress. In our study, A. thaliana leaves showed different degrees of damage under various stresses (Figure 4A–I, Figure 5A–I and Figure 6A–I). Notably, in our study, A. thaliana under the 10% PEG treatment was greener than CK (Figure 5D–F). Ma et al. [46] found that PEG pretreatment increased plants’ chl content, chl fluorescence, and photosynthetic parameters, promoted rice seedling growth, and regulated the tolerance of rice seedlings to drought. We, therefore, hypothesized that, because 10% PEG was a moderate concentration or the treatment period was short, this treatment had a positive effect instead of acting as a stress. This could be improved in the future by increasing the concentration or extending the treatment time.

SOD, POD, and CAT are important antioxidant enzymes that scavenge ROS in plants. SOD is the first line of defense against plant antioxidants, scavenging excess superoxide anions from cells, while POD and CAT scavenge disproportionate amounts of H₂O₂ into water and oxygen molecules [47]. The MDA content can be used as an indicator of oxidative damage to plant cell membranes [48]. Our results indicated that the SOD, POD, and CAT activities and MDA content showed an increasing trend in A. thaliana under abiotic stress, but, compared to WT, CfCHLM transgenic A. thaliana generally possessed a higher antioxidant enzyme activity and a lower MDA content (Figure 4T–W, Figure 5T–W and Figure 6T–W). Zhang et al. [49] found that the antioxidant enzyme activities and MDA content of Limonium sinense Kuntze increased with increasing time under NaCl stress. Wang et al. [50] found that cold stress increased antioxidant enzyme activities and MDA concentrations in maize (Zea mays) seedlings. Khanna-Chopra and Chauhan [51] found that, under heat stress, antioxidant enzyme activities were higher in the heat-tolerant wheat (Triticum aestivum) line Hindi62 than in the heat-sensitive wheat line PBW343, and this increased antioxidant capacity contributed to an improved heat tolerance. Huang et al. [17] found that mutant plants of A. thaliana chlm-4 accumulated excessive ROS and had a defective enzyme system for scavenging ROS. Our results were similar to those of these reports, showing that transgenic A. thaliana had a higher antioxidant capacity and could effectively reduce membrane lipid peroxidation in leaves, demonstrating that the CfCHLM gene enhances plant stress tolerance.