Expression analysis of photosynthesis-related genes in albino Artocarpus heterophyllus seedlings leaves

Albino Artocarpus heterophyllus Seedlings (AAS) were found in the preliminary investigation by our group and were used as materials for researching. The phenotype of AAS leaves were observed and measured. In parallel, the photosynthetic physiological parameters were determined under different photosynthetically active radiations (PAR). The results suggested that the length, width, area and thickness of AAS leaves were less than normal seedings. Likewise, the net photosynthetic rate, intercellular CO2 concentration, stomatal conductance and transpiration rate of AAS leaves were not susceptible to PAR in contrast to normal individuals. Furthermore, the transcriptome sequencing technology was performed to clarify the expression of genes related to photosynthesis. It is as expected that numerous down-regulated genes were found in the synthesis of photosynthetic pigments, as well as the pathways of photosynthesis - antenna proteins, photoreaction and carbon fixation reaction of AAS leaves. Compared to other albino plants, AAS have a longer life span and more stable phenotypic traits with larger leaves, which could provide ideal materials for investigating photosynthesis of woody plants.

The development profiles of the third leaf of AAS and CK were 114 observed within 20 days from the moment when the seedings germinated.

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The length, width and area of the leaves obtained from at least six plants 116 were measured.

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The mature leaves of AAS and CK were taken to make paraffin 119 sections following the method described by Jagadish et al. [16]. After that, The content of Carotenoids: X Caro (mg/g) = (1000A 470 -3.27 C Chl a 145 -104 C Chl b )/229(mg/L) ×25(ml) ×10 -3 / weight of fresh sample (g) 146 Data measurement and statistical analysis 147 The length, width and area of AAS and CK leaves as well as the 148 thickness of the epidermis, sponge tissue and palisade tissue of the leaves 149 were all measured by Image J, for which six or more repetitions were set.

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The mean and standard deviation were calculated, and statistically 151 significant differences were analyzed by U-test between the CK and AAS

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Gene assembly, KEGG annotation and analysis 169 The raw reads were filtered by removing the reads with low quality, 170 connector pollution and the content of unknown base N more than 5%.

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The filtered clean reads (PRJNA661997) were compared to the aspect ratio was observed in AAS leaves in contrast to CK, and 201 significant differences were shown from the 12th day (Fig 1c-f). The 202 daily growths of the length, width and area of AAS leaves were all less 203 compared with CK during the whole growth period. The length and width 204 of the leaves increased rapidly in the first 9 days, but tended to be gentle 205 in 9-17 days. While in the days after the 17th day, the length, width and 10 206 area showed negative growth, which is due to the premature atrophy of 207 AAS leaves (Fig 1f-h).  was less than that in CK, which was 215.15 µm and 64.25% of that in CK, 228 with significant difference compared with CK (Fig 2g). Carotenoids in AAS leaves were 0.013 mg·g -1 , 0.014 mg·g -1 , 0.027 240 mg·g -1 and 0.0077 mg·g -1 , which decreased down to 1.07%, 1.67%, 241 1.32% and 1.28% of CK levels (1.21 mg·g -1 , 0.84 mg·g -1 , 2.05 mg·g -1 and 242 0.60 mg·g -1 ), respectively (Fig 3a). The respiratory rate of AAS leaves 243 was 0.916 µmol·m -2 ·s -1 , which was 2.18 times of that in CK with 244 statistical significance (Fig 3b). From 0 to 1600 µmol·m -2 ·s -1 PAR, only leaves as expected (Fig 3c-f). Increasing the PAR from 0 to 600 249 µmol·m -2 ·s -1 resulted in an enhance in the net photosynthetic rate, while 250 conducted a decline simultaneously in the intercellular CO 2 concentration 251 of CK leaves. With further increases in PAR, the net photosynthetic rate 252 of CK leaves essentially saturated, and little fluctuations were observed in 253 the intercellular CO 2 concentration (Fig 3c-d). With the increase of PAR, 254 the stomatal conductance of CK leaves increased gradually. The 255 observation is consistent with the trend of transpiration rate in CK which 256 varied from that in AAS significantly (Fig 3e-f). These results clearly 257 showed that AAS leaves were not susceptible to PAR compared with CK.  respectively. Furthermore, the GC content was 41.25% (Table 1).     down-regulated. Among these genes, HemA and DVR were the key 309 enzyme genes (S1 Table). The enzyme genes involved in 10 steps were  Table) in AAS. The enzyme genes associated with 10 324 steps were down-regulated out of the 16 steps of carotenoids biosynthesis 325 in AAS (Fig 6).   Table).  Table).  Table). The gene expression in 395 5 steps out of 14 steps in this process were lower than CK obviously (Fig   396   9). as that of RNA-Seq data (Fig 10), indicating the reliability of RNA-Seq 412 sequencing data in this study (S6 Table).