Knock‐down of transcript abundance of a family ofKunitz proteinase inhibitorgenes in white clover (Trifolium repens) reveals a redundancy and diversity of gene function

Abstract

SummaryThe transcriptional regulation of four phylogenetically distinct members of a family of Kunitz proteinase inhibitor (KPI) genes isolated from white clover (Trifolium repens; designated Tr-KPI1, Tr-KPI2, Tr-KPI4 and Tr-KPI5) has been investigated to determine their wider functional role.The four genes displayed differential transcription during seed germination, and in different tissues of the mature plant, and transcription was also ontogenetically regulated. Heterologous over-expression of Tr-KPI1, Tr-KPI2, Tr-KPI4 and Tr-KPI5 in Nicotiana tabacum retarded larval growth of the herbivore Spodoptera litura, and an increase in the transcription of the pathogenesis-related genes PR1 and PR4 was observed in the Tr-KPI1 and Tr-KPI4 overexpressing lines.RNA interference (RNAi) knock-down lines in white clover displayed significantly altered vegetative growth phenotypes with inhibition of shoot growth and a stimulation of root growth, while knock-down of Tr-KPI1, Tr-KPI2 and Tr-KPI5 transcript abundance also retarded larval growth of S. litura. Examination of these RNAi lines revealed constitutive stress-associated phenotypes as well as altered transcription of cellular signalling genes.These results reveal a functional redundancy across members of the KPI gene family. Further, the regulation of transcription of at least one member of the family, Tr-KPI2, may occupy a central role in the maintenance of a cellular homeostasis.

Kunitz Proteinase Inhibitors Limit Water Stress Responses in White Clover (Trifolium repens L.) Plants

“…Two cultivars and one ecotype of white clover with different water requirements, namely, Kopu, Huia and Tienshan, were provided by AgResearch Grasslands, Palmerston North, New Zealand. The RNAi knock-down lines are described in Islam et al (2015a) and are in the Huia background. The small-leaved white clover ecotype Tienshan is considered to display some drought tolerance; cv.…”

“…While harvesting the leaf and root materials, the plants were selected randomly to eliminate any biasness. The development of RNAi knock-down lines for the four Tr-KPIs namely Tr-KPI1 , Tr-KPI2 , Tr-KPI4 , and Tr-KPI5 is described in detail in Islam et al (2015a) . The positive RNAi lines (T 0 ) and a number of non-expressing lines (designated as controls, C) were transferred to soil and maintained in a temperature-controlled containment glasshouse at 22°C, with ambient light and humidity.…”

“…Plant Kunitz proteinase inhibitors (KPIs) belong to the serine proteinase inhibitor (PI) group and numerous KPIs have now been identified from different plant species ( Rawlings et al, 2016 ). In most plant species, the KPIs comprise a multi-gene family: there are 13 members in soybean ( Jofuku and Goldberg, 1989 ), 21 in potato ( Heibges et al, 2003a , b ), 31 in poplar ( Philippe et al, 2009 ) and more than 11 in white clover ( Islam et al, 2015a ). The gene family generally encodes proteins of approximately 18–22 KDa in size with a single reactive site ( Laskowski and Kato, 1980 ).…”

“…The activity of PIs including KPIs against insect digestive proteinases has led to the promotion of their use as tolerance determinants in transgenic plants ( Green and Ryan, 1972 ; Hilder et al, 1987 ; Ryan, 1990 ; Jongsma and Bolter, 1997 ; Lee et al, 1999 ; McManus et al, 2005 ; Lima et al, 2011 ). Recent evidence also suggests that KPIs in plants are involved in programmed cell death, growth, and development ( Karrer et al, 1998 ; Yeu et al, 2007 ; Kim et al, 2009 ; Laluk and Mengiste, 2011 ; Pereira et al, 2011 ; Boex-Fontvieille et al, 2015 ; Islam et al, 2015a ). Moreover, changes in expression profile of KPIs in response to water limitation suggests a function for these proteins in abiotic stress, possibly by targeting specific proteinases and as such limiting their proteolytic activities ( Downing et al, 1992 ; Kang et al, 2001 ; Desclos et al, 2008 ; Kidric et al, 2014 ).…”

“…Previously we described four phylogenetically distinct members of the KPI gene family from white clover ( Trifolium repens L.; Tr-KPI ) and showed that a range of developmental and biotic stress-associated cues regulate their expression ( Islam et al, 2015a , b ). It was also shown that knock-down of these Tr-KPIs affected plant development, increased oxidative stress and revealed altered transcription of cellular signaling genes.…”