Functional Identification Of AbHemA2 In Chimeric Leaf Formation Of Ananas Comosus Var.bracteatus

Ananas comosus var.bracteatus has become an important new ornamental plant and the leaf flowers and fruits are appreciated,which can be used as potted ornamental,plant landscaping,and cut flowers due to its chimeric properties.Ananas comosus var.bracteatus chimeric leaves are composed of marginal albino tissue and central green tissue,which is an ideal material for studying the mechanism of leaf bleach mutation,and leaf chimeras are also excellent materials for studying chlorophyll synthesis accumulation,chloroplast development,photosynthesis and plant development.Further study of the formation causes of chimeric leaf is of great significance to revealing the formation mechanism of leaf color chimeras and cultivating new varieties of leaf color chimeras.Chlorophyll and heme are two competing branches of the anabolic pathway of plant tetrapyrrole compounds,which play an important role in photosynthesis,morphological construction,growth and development of plants.In this study,the anabolic differences between green and albino compounds in green and albino tissues were analyzed using the central green tissue and marginal albino tissues of the chimeric leaves of Ananas comosus var.bracteatus as materials,and combined with transcriptomics and proteomics sequencing results,it was screened that AbHemA2 gene is the key gene for bleaching and loss of green in the chimeric leaves of Ananas comosus var.bracteatus and the maintenance of the anabolic stability of tetrapyrrole compounds in leaves.The Abhem A2 gene sequence was cloned and functionally verified by RNAi genetic transformation,and the effect of exogenous ALA feeding on the synthesis pathway of Ananas comosus var.bracteatus tetrapyrrole compound was combined to investigate the role of AbHemA2 gene in Ananas comosus var.bracteatus mosaic leaf cell degreas and chlorophyll and heme anabolic regulation.The results of the resulting study are as follows:1 Ananas comosus var.bracteatus chimeric leaves have a significant difference in chlorophyll content between green and white tissues,with a difference of about 25times.There was no significant difference in ALA content,and the green tissue was slightly higher than the white tissue.On the contrary,the heme content in albino tissue is significantly higher than that in green tissue,and the content of Mg²⁺and Fe²⁺is also significantly higher than that in green tissue.Enzyme activity tests showed that glutamyl t RNA reductase（GluTR）activity in albino tissues was upregulated,but the difference was not significant.The analysis of reverse transcription quantitative real-time PCR（RT-q PCR）at the three developmental stages（S1,S2,S3）of chimeric leaves showed that the expression of AbHemA2 gene in the white tissue was upregulated,which was consistent with the results of the early transcriptome and proteomic sequencing of the research group.The high levels of AbHemA2 expression,GluTR enzyme activity and Fe²⁺concentration in the white tissues of the clear leaves promoted the synthesis and accumulation of heme,while the high levels of heme accumulation inhibited the synthesis of chlorophyll,thereby promoting the formation of albino leaves.2 ALA is the first precursor to the chlorophyll synthesis pathway,obtained by Glutamyl-t RNA which is catalyzed by Glutamyl-t RNA reductase encoded by the AbHemA2 gene.The endogenous ALA content of Ananas comosus var.bracteatus leaf significantly increased with the increase of exogenous ALA concentration（0,100,200 mg/L）Moderate level（100 mg/L）exogenous ALA feeding can significantly increase AbHemA2 gene expression,thereby increasing GluTR enzyme activity and promoting heme synthesis,but chlorophyll synthesis is blocked,which is consistent with the case of albino tissue of Ananas comosus var.bracteatus chimeric leaf;and excessively high levels（200 mg/L）foreign ALA feeding inhibits the expression of Abhem A2 gene,reduces GluTR enzyme activity,inhibits heme synthesis,and thus promotes chlorophyll synthesis,which is consistent with the green tissue of Ananas comosus var.bracteatus chimera leaves.It is shown that the ALA content can regulate the expression of AbHemA2 gene and the branch orientation of the anabolic flow of tetrapyrrole compounds to chlorophyll and heme,thereby determining the content and relative proportion of chlorophyll and heme,and affecting the leaf color.3 Identified a HemA gene in the Ananas comosus var.bracteatus genome,cloned to obtain a full c DNA length of 1981 bp,containing an open reading window（ORF）of 1617 bp,encoding 538 amino acids,named AbHemA2 according to the gene annotation.AbHemA2 has the N＿terminal domain,C＿terminal domain and NADPH binding domain common to the HemA family,and has the highest homology with Ananas comosus As HemA2 and more than 75%homology to the HemA2 protein of other species,which is a gene with high conservatity between species.4 Construct pTCK303-AbHemA2-RNAi interference vector and transform Ananas comosus var.bracteatus albino callus and tobacco leaves mediated by GV3101,and obtain Hyg-resistant tobacco and Ananas comosus var.bracteatus plants after screening hygromycin（Hygromycin B）medium.After PCR identification and testing,7 transgenic tobacco plants were obtained,and no transgenic plants were detected in Ananas comosus var.bracteatus resistant seedlings.The relative expression of AbHemA2 gene in the transgenic tobacco plants detected by q RT-PCR was significantly lower than that of wild-type tobacco,and the GluTR enzyme activity,chlorophyll content and heme content of the transgenic plants were significantly downregulated.Transgenic tobacco leaves are yellowed,sagging,weaker,and have significantly higher ALA levels than wild-type tobacco.This shows that when AbHemA2 expression is disturbed,GluTR enzyme activity decreases,but ALA is relatively accumulated due to inhibition of chlorophyll and heme synthesis.Moreover due to the lack of chlorophyll and heme,the plant develops a yellowing phenotype.