Differential Protein Analysis of Pecan Hardwood Cuttings

in HortScience

Pecan cuttings are difficult for rooting. This study describes the pecan hardwood rooting process based on anatomic characteristics to understand root formation mechanisms of pecan cuttings. The expressed proteins of different periods during the adventitious rooting process of pecan seedling hardwood cuttings were identified and analyzed to evaluate the rooting mechanism. The expressed proteins of pecan cutting seedlings were also compared with other cultivar cuttings during the rooting period. Pecan seedling cuttings were developed at different air and substrate temperatures to induce root formation. Adventitious root formation of pecan hardwood cuttings was described, and the phloem at the base of the prepared cuttings was selected as the sample for the differential protein analysis. The results showed that adventitious root formation of pecan hardwood cuttings was the only product of callus differentiation, which originated from the cells of the cambium or vascular ray parenchyma. Such adventitious root primordia were developed from those calluses that formed the regenerative structure, and the expressed proteins during the adventitious rooting of pecan hardwood cutting were identified and analyzed by matrix-assisted laser desorption ionization–time of flight–mass spectrometry (MALDI-TOF-MS) to evaluate the rooting mechanism. Eight differentially expressed proteins were found in the rooting periods, and 15 differential proteins were found by comparing pecan cutting types, which were analyzed by peptide mass fingerprinting homology. The results show that the primordial cells were differentiated from the meristematic cells. Furthermore, the differentially expressed proteins contained energy metabolism proteins, adversity stress proteins, and signal transmission proteins. The energy metabolism-related proteins were adenosine triphosphate (ATP) synthase, photosynthesis-related proteins, and enolase. The adversity-stress proteins containing heat shock-related proteins and signal transmission proteins were mainly cytochrome enzymes and heme-binding proteins. Adventitious root formation of pecan cultivar hardwood cuttings was difficult. More trials should be performed from the potential aspects of high defensive protection and phloem morphologic structure.

Contributor Notes

This project was supported by Jiangsu Forestry Science and Technology Innovation and Promotion Project (LYKJ[2018]05-1) and Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

Corresponding author. E-mail: frpeng@njfu.edu.cn.

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    Pecan hardwood cuttings at key rooting period (20 d after cutting). It was the key period for the induction of adventitious roots. The basal phloem parts of both groups were selected for differential protein analysis. (A) Cutting materials from ‘Pawnee’. (B) Cutting materials from ‘Stuart’ seedling.

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    A field plot of transplanted rooted pecan seedling cuttings. The pecan seedling hardwood cuttings were treated with 500 mg·L–1 1-naphthalene acetic acid 6 h and planted onto a fixed nursery bed containing peat and perlite substrate with bottom artificial heating. (A) More than 90% of the pecan seedling cuttings show expanded leaves at 40 d. (B) About 80% of the pecan seedling cuttings developed adventitious roots at 50 d. (C) Most of the cuttings grew only one adventitious root at the beginning, around 40 d. (D) The root fibrils began to grow when three to five adventitious roots appeared or when the adventitious roots elongated to 2 to 3 cm at 50 d. (E) After 60 d, with more adventitious roots growing, the branches and leaves on the top of each cutting began to extend and expand.

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    Stem transection microstructure at different periods after the pecan hardwood cuttings of seedlings were treated with 1-naphthalene acetic acid. (A) Cross-section on day 10. (B) Cross-section on day 20. (C) Diagonal section on day 30. (D) Diagonal section on day 40. Cam, cambium; Cal, callus; Cor, cortex; En, endodermis; Ep, epidermis; Pd, periderm; Ph, phloem; Pi, pith; Pz, perimedullary zone; r, secondary ray (S, single secondary ray; D, double secondary ray); rh, root hair; Ve, vessel; and Xy, xylem.

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    Representative two-dimensional electrophoresis (2-DE) gel maps of proteins extracted from the pecan seedling hardwood cuttings at different growing periods. 2-DE of protein extracted from rooting region in the hardwood cuttings of Carya illinoinensis at different periods. (A) Samples from day 1. (B) Samples from day 20. (C) Samples from day 40. The most significant difference expressed protein spots (S1–S10), which marked in the maps, were selected for the protein homology analysis.

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    Representative two-dimensional electrophoresis (2-DE) gel maps of proteins extracted from the pecan ‘Pawnee’ and ‘Stuart’ seedling hardwood cuttings on day 20 of the callus formation period. (A) 2-DE gel map of proteins extracted from the pecan hardwood cuttings of ‘Pawnee’. L1 through L5 protein spots were greater than 10× expressed in this map. (B) 2-DE gel map of proteins extracted from the pecan hardwood cuttings of ‘Stuart’ seedlings. L6 through L15 protein spots were less than 0.1× expressed in this map. All 15 protein spots (L1–L15) were selected for homology analysis.

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