Author: Troy Miller
Miller, Troy, 2024 Novel insights into the nitrogen assimilation pathways in chickpea (Cicer arietinum) nodules, Flinders University, College of Science and Engineering
Terms of Use: This electronic version is (or will be) made publicly available by Flinders University in accordance with its open access policy for student theses. Copyright in this thesis remains with the author. You may use this material for uses permitted under the Copyright Act 1968. If you are the owner of any included third party copyright material and/or you believe that any material has been made available without permission of the copyright owner please contact copyright@flinders.edu.au with the details.
Nitrogen is an integral component for effective plant growth, crucial to the synthesis of amino acids,
proteins, and chlorophyll, thereby directly influencing crop yield. Modern farming practices rely heavily on
synthetic nitrogen fertilisers, which are not only expensive but wasteful due to inefficient nitrogen
utilization by plants leading to environmental contamination. Leguminous plants like chickpea (Cicer
arietinum) possess a symbiotic relationship with soil bacteria, collectively known as rhizobia, with the ability
to fix atmospheric nitrogen into a bioavailable form usable by the plant, offering an alternative to fertilisers.
This symbiosis takes place in nodules, specialised organs located on roots that house the rhizobia. Within
the nodule cells rhizobia differentiate into the symbiotic bacteroids contained in symbiosomes, organelle
like structures surrounded by a plant derived membrane known as the symbiosome membrane.
Atmospheric nitrogen is reduced to ammonia in bacteroids and exported through the symbiosome as
ammonium, then further metabolised into amides glutamine (Gln) & asparagine (Asn) or ureides allantoin
& allantoic acid. These serve as a means to store and remobilise nitrogen to support plant growth. The
biosynthesis of amides or ureides differs between legumes and it is unclear in the literature which pathway
occurs in chickpea. Previous studies have shown that improving the export of amides or ureides from the
nodules of other legumes can significantly improve the rate of nitrogen fixation. However, before this can
be accomplished in chickpea, the pathway of nitrogen fixation and transporters responsible in amide or
ureide export from the nodules must be known.
A colorimetric assay was employed to quantify total ureides in leaf, root, and nodule tissue under low N
conditions (0.5 mM KNO3). Chickpea exhibited significantly reduced levels of ureides across all tissues
compared to soybean, a typical ureide producing legume. Moreover, qRT-PCR showed an apparent
upregulation of amide biosynthesis and negligible expression of the ureide biosynthesis pathway in
nodules, particularly the enzyme Pur1, a rate limiting step of this pathway.
To back up these observations a model of nitrogen fixation in chickpea was developed via RNA sequencing
comparing gene expression from nodule tissue preceding and during nitrogen fixation. A significant
upregulation of the entire amide biosynthesis pathway was observed, in contrast to non-differential
expression of the ureide pathway. Further analysis identified several (7) promising amide transporters
exhibiting significant upregulation during nitrogen fixation. Based on single cell RNA (scRNA) performed in
Medicago truncatula nodules, these transporters were predictively localised within nodule compartments
such as the parenchyma and were likely involved in fixed nitrogen export. Saccharomyces cerevisiae
complementation assays employing a mutant strain deficient in amino acid transport showed broad amino
acid transport properties by the proteins encoded by these genes, particularly the amides, Gln and Asn.
Data suggests that chickpea predominantly synthesise amides over ureides for nitrogen export from the
nodules. This export is likely facilitated by the transporters CaAAP6, CaAVT6A, CaUmamiT9, CaUmamiT18, CaUmamiT20, predictively localised to the nodule parenchyma and shown to transport a broad range of amino acids including the amides Asn and Gln. These transporters present promising targets for future genetic manipulation experiments to improve nitrogen fixation in chickpea.
Keywords: Ckickpea, Cicer arietinum, Nitrogen Fixation, Nodules, Rhizobia, Amino Acids, Amides, Ureides
Subject: Biotechnology thesis
Thesis type: Doctor of Philosophy
Completed: 2024
School: College of Science and Engineering
Supervisor: Sunita Ramesh