In autumn and spring, citrus leaves with a Ponkan (Citrus reticulata Blanco cv. Ponkan) genetic background (Harumi, Daya, etc.) are prone to abnormal physiological chlorosis. The effects of different degrees of chlorosis (normal, mild, moderate and severe) on photosynthesis and the chlorophyll metabolism of leaves of Citrus cultivar (Harumi) were studied via field experiment. Compared with severe chlorotic leaves, the results showed that chlorosis could break leaf metabolism balance, including reduced chlorophyll content, photosynthetic parameters, antioxidant enzyme activity and enzyme activity related to chlorophyll synthesis, increased catalase and decreased enzyme activity. In addition, the content of chlorophyll synthesis precursors showed an overall downward trend expected for uroporphyrinogen III. Furthermore, the relative expression of genes for chlorophyll synthesis (HEMA1, HEME2, HEMG1 and CHLH) was down-regulated to some extent and chlorophyll degradation (CAO, CLH, PPH, PAO and SGR) showed the opposite trend with increased chlorosis. Changes in degradation were more significant. In general, the chlorosis of Harumi leaves might be related to the blocked transformation of uroporphyrinogen III (Urogen III) to coproporphyrinogen III (Coprogen III), the weakening of antioxidant enzyme system activity, the weakening of chlorophyll synthesis and the enhancement in degradation.

The GLK gene family is of great significance in regulating chloroplast development and participating in chlorophyll synthesis. However, the mechanism of GLK involvement in Citrus chlorophyll synthesis remains unclear. In this study, bioinformatics methods were used to analyze the gene structure, protein evolution, chromosome distribution, promoter elements and expression profile of GLK gene family in Citrus. Overall, 27 CsGLK TFs were identified from Citrus genome and divided into three subgroups according to the conserved domains. All members were distributed on nine chromosomes. The tandem replication events (ka/ks < 1) indicated that CsGLK TFs underwent a purification selection evolutionary process. The intron variation might be a vital configuration for the evolution of CsGLK genes. The expression pattern of CsGLKs showed that family members had higher expression levels in different tissues and at different growth stages and could actively respond to dark stress. CsGLK TFs of the same group had similar structures, but their expression patterns were quite different, indicating that they may have different functions and not be redundant. Correlation analysis showed that CsGLK2, CsGLK9, CsGLK10, CsGLK11, CsGLK20 and CsGLK24 were significantly positive correlations with Chl a and Chl b contents. In addition, CsGLK2, CsGLK5, CsGLK10, CsGLK11, CsGLK12, CsGLK15, CsGLK20 and CsGLK24 were significantly positive related to Mg-Proto IX, Proto IX and Pchl.