Date Published: February 13, 2018
Publisher: Public Library of Science
Author(s): Sanjana Negi, Himanshu Tak, T. R. Ganapathi, Keqiang Wu.
Deposition of secondary cell wall in the xylem elements is controlled by a subgroup of NAC (NAM, ATAF, CUC) family, known as vascular-related NAC transcription factors (VNDs). In the present study, we analyzed the 5’ upstream regulatory region of two banana NAC transcription factors (MusaVND6 and MusaVND7) for tissue specific expression and presence of 19-bp secondary-wall NAC binding element (SNBE)-like motifs. Transgenic banana plants of Musa cultivar Rasthali harboring either PMusaVND7::GUS or PMusaVND6::GUS showed specific GUS (β-D-Glucuronidase) activity in cells of the xylem tissue. Approximately 1.2kb promoter region of either MusaVND6 or MusaVND7 showed presence of at least two SNBE-like motifs. This 1.2kb promoter region was retarded in a gel shift assay by three banana VND protein (VND1,VND2 and VND3). The banana VND1-VND3 could also retard the mobility of isolated SNBE-like motifs of MusaVND6 or MusaVND7 in a gel shift assay. Transcript levels of MusaVND6 and MusaVND7 were elevated in transgenic banana overexpressing either banana VND1, VND2 or VND3. Present study suggested a probable regulation of banana VND6 and VND7 expression through direct interaction of banana VND1- VND3 with SNBE-like motifs. Our study also indicated two promoter elements for possible utilization in cell wall modifications in plants especially banana, which is being recently considered as a potential biofuel crop.
Evolution of secondary cell wall deposition was a prominent feature which not only provided the mechanical strength required for the vertical growth of plants but also helped in the long distance transport of water and minerals. This secondary cell wall is laid in cells of xylem tissue and is composed of cellulose, hemicelluloses, lignin and xylan. Water transport in plants from roots to other organs is through the channels of xylem tissue composed of tracheids and vessel elements. These tracheids develop an array of secondary wall depositions in the form of reticulate, pitted, helical and annular thickenings. Secondary cell wall deposition is regulated and highly coordinated for systematic deposition of multiple components by many genes among which a subgroup of NAC transcription factors, VNDs (vascular related NAC transcription factors) are most important. Secondary cell wall deposition in tracheids is accompanied with programmed cell death, hence regulation of the activity of secondary cell wall associated genes in specialized cells is of prime importance for proper functioning and homeostasis of plants.
Modifications of secondary cell wall including reduction of lignin especially in plants important for biofuel and pulp production holds great importance. The efficient conversion of lignocellulosic biomass (chiefly made up of secondary cell wall) into ethanol through fermentation process is deterred by high lignin content . Hence reduction of lignin and increased deposition of hydrolysable cellulose through elevated deposition of secondary cell wall can be achieved byNAC transcription factors and other potential genetic factors. Moreover, many important crop plants like banana are prone to lodging during fruiting stage (due to heavy bunch weight), which can be improved through increased mechanical strength of pseudostem by elevatingsecondary cell wall deposition [19,20]. Information on regulation of secondary wall thickening especially in context of VNDs in plants like Arabidopsis, poplar and Brachypodium among otherhave been generated suggesting that these transcription factors regulate a number of downstream genes for coordinated regulation of secondary cell wall deposition [21–24]. In Arabidopsis seven members have been included in VND subgroup of NAC family and VND6 and VND7are suggested to be master regulators for secondary wall synthesis [5,6]. Attempts to overexpress these genes for secondary cell wall modification have often resulted in unwanted effect of biomass reduction probably through transdifferentiation of essential tissue into tracheids as these genes were overexpressed under constitutive promoter [5,7]. Hence, identification and characterization of potential xylem specific regulatory DNA element for regulated expression of VNDs is indispensable . In this study we have characterized two 5’upstream regulatory region (PMusaVND7 and PMusaVND6) with specific activation in xylem tissue element which can be utilized in future for biomass engineering. Some examples of utilization of xylem specific promoter element have been reported in the past. In Arabidopsis, the expression of C4H (cinnamate 4-hydroxylase), an important gene for lignin synthesis was restricted under the activation of VND6 promoter (a vessel specific promoter) which reduced the lignin content (without plant growth reduction), thus increasing the hydrolysis of cellulose for sugar release . Another study has reduced lignin content through expression of bacterial 3-dehydroshikimate dehydratase (QsuB) under the PC4Hand emphasized the need of fiber cell specific promoter for higher stringency in lignin reduction . Moreover, studies on genetic factors regulating secondary wall deposition in banana are important due to the fact that banana has recently being researched as a potential second generation biofuel crop due to high amount of lignocellulosic biomass being left out after the fruit harvest [26–28]. Quantitative RT-PCR analysis to determine the transcript levels of VND6 and VND7 in native banana indicated differential expression of MusaVND6 and MusaVND7 in different organs. Transcript levels of both VND6 and VND7was high in corm and pseudostem compared to other organs (S6 Fig). Though the transcript level of VND7was higher than VND6 in different organ, but it does not correlate with the relative GUS activity observed in transgenic banana harboringPMusaVND7::GUS and PMusaVND6::GUS. Lower GUS activity in transgenic banana harboring PMusaVND6::GUS than transgenic banana harboring PMusaVND7::GUS might be due to reasons such as integration of T-DNA in less active chromatin regions or requirement of additional upstream DNA element for achievement of full potential inexpression of VND6. In the present study we have successfully analyzed the xylem specific activity of PMusaVND6and PMusaVND7.