Caspase-like,ptvpe And Ptcdd In Relation To Programmed Cell Death In Xylem Cell Of Poplar

The formation of wood (secondary xylem) involves sequential developmental events encompassing differentiation of xylem mother cells from the vascular cambium, cell division, cell expansion, cell wall thickening, programmed cell death (PCD), and formation of heart wood. To understand the regulation of programmed cell death in wood formation, several candidate factors, including caspase-like proteases (CLPs), PtVPE (vacuolar processing enzyme) and calcium dependent DNase (PtCDD), were investigated in Populus tomentosa Carr. and Populus nigra L. The major findings are described as followings:1. Caspase-like activities in protein extracts of immature xylem from Populus nigra L. were measured by using AMC substrates. The results showed that the substrates Ac-DEVD-AMC (caspase-3) and Ac-VEID-AMC (caspase-6) are cleaved by proteases activated in wood formation. The DEVDase exhibited 63.1% higher activity than the VEIDase. Ac-LEVD-AMC (caspase-4) and Ac-YVAD-AMC (caspase-1) were also cleaved but at much lower levels. Setting the untreated control as 100% activity, Ac-DEVD-CHO significantly inhibited the DEVDase activity by 82.7%. Because pH can affect enzyme activity, we investigated the optimal pH for the caspase-like activities, which revealed that the pH range for the DEVDase is very narrow, with the peak at pH4.0 and the range at acidic pH3.5-4.5. In addition, caspase-3 inhibitors Ac-DEVD-CHO or Ac-DEVD-CMK were used to treat biennial branches from Populus nigra L. Cambium area was obviously thickened by the treatment with the mixture of Ac-DEVD-CHO and Ac-DEVD-CMK. DNA fragmentation was reduced in developing vascular tissues as revealed using the TdT-mediated dUTP nick-end labeling (TUNEL) method. Collectedly, we showed that caspase-3 inhibitors can affect the normal programmed cell death of xylem cells during wood formation, and caspase-3-like/DEVDase might involve in programmed cell death in wood formation.2. In the present study, we cloned NAC068 and NAC154 promoters from P. tomentosa genomic DNA with the length at 901 bp and 668 bp respectively, constructed plant transformation vectors using theβ-glucuronidase (GUS) reporter gene system and detected the expression patterns of PtNAC068 and PtNAC154 promoter in transgenic poplars (P. alba×P. glandulosa). Histochemical GUS assay showed GUS activity driven by PtNAC068 promoter was mainly in vascular tissues of stems, leaves, petioles and roots, while that driven by PtNAC154 promoter was confined to the developing secondary xylem of stems, petioles and veins of leaves. GUS expression in internodes 3-8 of ProNAC068::GUS transgenic plants was 30-fold higher than that in ProNAC154::GUS transgenic plants. The differences in expression pattern indicate that PtNAC068 and PtNAC154 may be involved in two distinct aspects of vascular tissue development. The characterization of these promoters will lay the foundation to control exogenous gene specific expression in vascular system of transgenic poplars.3. A full-length cDNA designated asγ-PtVPE encoding a vacuolar processing enzyme gene was cloned from immature xylem of P. tomentosa. Theγ-PtVPE cDNA is 1609bp long and contains an open reading frame of 1482bp encoding 493 amino acids. To explore the role of PtVPE in secondary vascular development, we constructed the expression vectors, including pBIRNAi-PtVPE, pBI121-CaMV35S-sensePtVPE, pBI121-ProNAC068-sensePtVPE and pBI121-ProNAC068-antisensePtVPE, with sense and antisense orientation driven by CaMV35S or ProNAC068 promoters. The four expression vectors were transformed into hybrid poplar 84K via the Agrobactria-mediated leaf-disc transformation method, and the two sense expression vectors were transformed into Arabidopsis by the floral dip method. The putative transgenic poplar and Arabidopsis plants were screened out by genomic PCR amplification of the NPTII gene and PtVPE. Overexpression of PtVPE in Arabidopsis delayed growth, reduced number of trichomes and the total length of leaves, and thickened secondary xylem of stem and hypocotyl. In ProNAC068-driven PtVPE transgenic Arabidopsis, no trichome was found on the leaves, and secondary xylem of stem and hypocotyl were also thickened. These results showed that PtVPE is possibly involved in plant growth and development, especially in the regulation of secondary growth of xylem.4. To study the role of PtCDD in wood formation, we constructed the expression vectors of PtCDD, including pBI121-CaMV35S-sensePtCDD, pBI121-ProNAC068-sensePtCDD and pBI121-ProNAC068-antisensePtCDD, driven by CaMV35S or ProNAC068 promoters. Another expression vector PX6-PtCDD was constructed using inducible plant expression vector PX6. The expression vectors were transformed into hybrid poplar 84K, tobacco and Arabidopsis. The putative transgenic plants were screened out by genomic PCR amplification of the NPTII gene and PtCDD. In ProNAC068-driven PtCDD transgenic Arabidopsis, shoot apical meristem was destroyed during vegetative growth phase, while axillary meristem was promoted. The bolting was delayed for 12-14 days compared with the wild type. The CaMV35S-driven PtCDD transgenic tobacco plants also slowed growth, due to the apical dominance was damaged, and new lateral buds produced from leaf axils. In CaMV35S-driven PtCDD transgenic poplar plants lacked main stem and showed 3-5 lateral branches. These results showed that PtCDD is possibly involved in cell death, and possibly affected the organization of apical meristem thus to hinder the stem development.