Drought stress responses of the Medicago truncatula - Ensifer meliloti symbiosis on nodule senescence and nitrogen fixation : a thesis presented in fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Science at Massey University, Palmerston North, New Zealand

Abstract

While the use of nitrogen (N) fertilizer has provided many benefits to agriculture, incessant use of it can reduce soil organic matter and fertility resulting in lower crop yields. Legume plants can fix its own N2 through symbiotic nitrogen fixation (SNF) to promote plant growth by developing facultative root organs called nodules. Therefore, increasing the rate of SNF to reduce dependence on N fertilizer is a promising strategy for sustainable legume production. In legume cultivation this symbiotic process confronts two major challenges. First, SNF is suppressed by readily available N in the soil and second, the early senescence of N-fixing nodules can limit any further fixation to occur. Previous reports show that N fertilizer supresses SNF activity in many legumes and supports plant growth better than SNF. Moreover, suppression of SNF can also be induced by drought stress which causes early nodule senescence and subsequent reduced rates of plant growth. This thesis addresses the process of SNF suppression in two 𝘔𝘦𝘥𝘪𝘤𝘢𝘨𝘰 𝘵𝘳𝘶𝘯𝘤𝘢𝘵𝘶𝘭𝘢 selected genotypes in response to external N treatment and also delivers a molecular view on the regulation of drought-induced nodule senescence processes.

I first hypothesized that Jemalong A17 and R108 may respond differently to external N treatment and drought stress, which both lead to SNF suppression. To determine this, 𝘔. 𝘵𝘳𝘶𝘯𝘤𝘢𝘵𝘶𝘭𝘢 plants were grown with the N₂-fixing symbiont 𝘌𝘯𝘴𝘪𝘧𝘦𝘳 𝘮𝘦𝘭𝘪𝘭𝘰𝘵𝘪 and ¹⁵N-labelled N fertilizer under well-watered and drought conditions. Plants were then harvested at different time points. N partitioned by SNF and N fertilizer were measured using isotope ratio mass spectroscopy. Results show that under well-watered conditions, N fertilized R108 plants used SNF for N uptake (upto 23% of total shoot N), when inoculated with 𝘌. 𝘮𝘦𝘭𝘪𝘭𝘰𝘵𝘪, and reduced N fertilizer uptake to balance total N uptake. Under drought stress, both Jemalong A17 and R108 plants derived assimilated N from SNF (upto 45% of total shoot N) while they significantly reduced the N uptake from N fertilizer. Moreover I found that SNF in association with 𝘌. 𝘮𝘦𝘭𝘪𝘭𝘰𝘵𝘪 not only benefitted the host by increasing the N supply but also primed the host plant to better tolerate drought stress by controlling the expression of drought-associated genes.

In legumes, nodules are the first organs to be responsive to drought stress. Once drought conditions are perceived, the host plant induces early nodule senescence in order to reduce the carbon investment in the nodules. Therefore, nodule senescence can be part of a drought-survival strategy. However, as SNF becomes limited due to early nodule senescence, nutrient stress can develop in the legume in addition to the drought stress. Here, I then hypothesized that the intrinsically destructive senescence process must be tightly regulated to function as a part of drought-survival strategy. 𝘔. 𝘵𝘳𝘶𝘯𝘤𝘢𝘵𝘶𝘭𝘢 protease inhibitor and iron scavenging genes, possibly involved in controlling nodule senescence, were identified. RNAi lines were constructed in which expression of a serpin or ferritins were knocked down. Both wild-type and RNAi lines were subjected to drought stress and the subsequent nodule activity and plant physiological responses were measured. Drought caused 𝘔. 𝘵𝘳𝘶𝘯𝘤𝘢𝘵𝘶𝘭𝘢 to initiate nodule senescence before plant growth was affected and before an increase in papain-like proteolytic activity and free iron levels were detected. Knock-down expression of serpin6 and ferritins caused increased protease activity, free iron levels, early nodule senescence and reduced plant growth. These results suggest that 𝘔. 𝘵𝘳𝘶𝘯𝘤𝘢𝘵𝘶𝘭𝘢 expresses serpin6 and ferritins in nodules to mediate ordered drought-induced senescence by regulating papain-like cysteine protease activity and free iron levels. This strategy may allow the drought-stressed plants to extract the maximum benefit from residual N fixation and nutrient recovery resulting from the breakdown of macromolecules.