Deciphering Kunitz proteinase inhibitors in white clover (Trifolium repens L.) : a transcriptional study : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Plant Molecular Biology at Massey University, Palmerston North, New Zealand

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This thesis investigates the Kunitz Proteinase Inhibitors (KPI) gene family in white clover (Trifolium repens L.) as this family of inhibitors is one of the most abundant among the serine proteinase inhibitor families in legume species. In other studies, these proteins have mainly been shown to serve as storage proteins and to also act as potent defensive factors against insect herbivory. As they are involved in regulating proteolytic activity, the question arises as to how much they are also involved with regulating plant growth and development and how they respond to different stresses other than insect herbivory? Here, in this thesis effort has been under taken to answer these questions using the perennial legume white clover which is a major contributor to pasture productivity in New Zealand. However, as yet, very little is known about the occurrence of Kunitz proteinase inhibitor (Tr‐KPI) genes or the functions of these genes in white clover. In this study, therefore, the spectrum of Tr‐KPI genes is characterized, and the regulation of expression at the transcriptional level of different members of the gene family is examined. To obtain KPI genes from white clover, degenerate primers were designed based on known legume KPI sequences. Four full length cDNA were obtained using degenerate and later gene‐specific primers. Blast searching of the JCVI and NCBI database showed that they encode for proteins fall into the soybean trypsin inhibitor super‐family (STI) and were named Tr‐KPI1, Tr‐KPI2, Tr‐KPI4 and Tr‐KPI5. The expression in the transcript level of these four genes showed that Tr‐KPI1, Tr‐KPI2, Tr‐KPI5 are constitutively expressed in vegetative and reproductive parts whereas Tr‐KPI4 is more organ‐specific such that it is expressed in the root and mature seed. A leaf and root developmental study showed that Tr‐KPI2 and Tr‐KPI5 are more developmentally regulated and transcript abundance during a germination time course study also suggests the involvement of Tr‐KPI1 and Tr‐KPI5 during seedling establishment. To explore the function of these genes further, different forms of biotic and abiotic stress were applied to white clover. A mechanical wounding study revealed the possible involvement of Tr‐KPI1, Tr‐KPI2 and Tr‐KPI5 in plant defense in both local and systemic tissues, and Tr‐KPI4 in the systemic tissue. A shoot herbivore (Spodoptera litura) and root herbivore (root knot nematode Meloidogyne trifoliophila and cyst nematode Heterodera trifolii) were also used to characterize the involvement of the Tr‐KPI genes in plant defense response. Expression of the Tr‐KPI genes against the generalist herbivore S. litura further supported the view that the Tr‐KPIs in white clover are involved in plant defense responses where local (leaf), basipetal (root) and acropetal i (apical tissue) tissues were compared. The expression results suggest that Tr‐KPI1, Tr‐KPI2 and Tr‐KPI5 are induced by herbivore attack and Tr‐KPI1 was found to be most involved (1600‐fold at 24 h) followed by Tr‐KPI2 and Tr‐KPI5. In the nematode experiment, inoculation by a cyst nematode was able to trigger the expression of Tr‐KPI1, Tr‐KPI4 and Tr‐KPI5 in the root tissue at day 4 and a systemic response of nematode feeding was also observed in the leaf tissue for these genes at day 8. Invasion by the root‐knot nematode did not result in any significant up‐regulation for Tr‐KPI genes at day 4 and day 8. This finding suggests that Tr‐KPIs might be involved in defense against cyst nematode invasion but not by root‐knot nematodes. To further elucidate the involvement of Tr‐KPI genes under cyst nematode attack, a resistant line 17R and a susceptible line 23S were used. In the resistant line 17R, all four Tr‐KPI genes were significantly expressed by day 4 and day 8, and in the susceptible line 23S, high transcript abundance was observed only at day 4. Therefore, it can be proposed that Tr‐KPIs in white clover are important in defense against white clover cyst nematode in combination with other defense genes. For an abiotic stress study, water deficiency and limited phosphorus (Pi) treatments were employed to examine the expression of the Tr‐KPI genes in white clover. For the water deficiency trials, two treatments were imposed: a pre‐stressed (PS) treatment in which plants were subjected to a water deficit for 7 days, followed by watering for a further 7 days before the experimental water deficit was applied, or a non‐pre stressed (NPS) treatment in which plants were subjected immediately to a water deficit. The level of Tr‐NCED1 (9‐cis‐epoxycarotenoid dioxygenase) expression, coding for an enzyme involved in ABA biosynthesis, was also investigated to prove that a water deficit is perceived by the plants. The Tr‐NCED1 level was found to be up‐regulated in the NPS treatment when compared with the level observed in fully hydrated tissue. Under the NPS and PS treatments, the transcription level of Tr‐KPI1 and Tr‐KPI5 were induced significantly in the leaf tissue when compared with the control. Interestingly, the pre‐stressed treatment triggered the expression of all three genes studied which were significantly higher compared to the expression level under the NPS treatment. To further characterize the role of Tr‐KPIs under water stress, a drought tolerant ecotype Tienshan and drought susceptible cultivar, Kopu was used. A clear upregulation of Tr‐KPI1 in Tienshan and Tr‐KPI5 in Kopu was observed under the PS treatment when compared with the initial moisture content and NPS treatments indicating some selective pressure on the Tr‐KPIs under water stress in susceptible and resistant plants. In a macro‐nutrient (Pi) limitation experiment, where the growing root is divided in different developmental regions comprising the elongation zone (EZ), the visible lateral root zone (VL) and the mature zone, a higher level of Tr‐KPIs expression was observed in the growing zone rather than mature root zone. Although expression of all four Tr‐KPIs was up‐regulated in the EZ region, only Tr‐KPI2 and Tr‐KPI4 showed an extended level of expression in the visible lateral root zone indicating a possible involvement in lateral root formation as Pi limitation does induce a higher number of lateral root primordia. In leaf tissue, the down regulation of Tr‐KPIs was observed up to 12 h of the Pi starved treatment and the transcript level started to increase from 24 h onward indicating that Tr‐KPIs are not early response genes in leaf tissue. Finally, the cis‐binding elements in the promoter regions of four Tr‐KPI genes indicate that this gene family in white clover is controlled by different transcription factors. A number of growth and development‐related transcription factor binding sites such as AREF, ASRC, LFY, MADS and biotic and abiotic stress responsive transcription factors binding sites such as EINL, MYBL, MYBS, MYCL, and WNAC have been identified in all the four promoter sequences, although differences in the pattern and frequency were observed across the four Tr‐KPI genes were observed. This further highlights that this gene family is regulated by a complex network of hormonal and other stress induced cues.
White clover, Proteinase, Inhibitors, White clover