Wild radish seedlings growing outdoors in late autumn
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Synthetic auxin resistance in wild radish is still an enigma

Guest author Danica Goggin finds wild radish is a problem weed in many agricultural regions. It is also very good at concealing its herbicide resistance strategies from inquisitive researchers. But with the introduction of synthetic auxin-resistant transgenic crops in North America, we need to keep chipping away at this plant’s shield of secrecy.

Wild radish running wild

Wild radish (Raphanus raphanistrum) is a prominent dicot weed in agricultural and roadside scenarios across the world. One reason for its success is the high genetic variability between populations and individuals, allowing it to adapt to local conditions and stresses. In Australian cropping systems, which are largely based on minimal disturbance of the fragile soils, a major selection pressure imposed on weeds is the intensive use of herbicides. Consequently, wild radish has developed resistance to nearly all of the most commonly-used modes of herbicide action (Owen et al. 2015).

The synthetic auxin herbicides, such as 2,4-D and dicamba, belong to an old mode of action which mimics the growth-promoting effects of the plant hormone auxin, but at an uncontrolled and amplified level. Resistance to synthetic auxins is increasing in wild radish, as farmers turn to 2,4-D to kill populations that are resistant to newer, yet over-used herbicides. Our initial foray into discovering how the plants survive this herbicide revealed an apparently clear-cut mechanism: in the two populations studied, the plants prevented 2,4-D from travelling out of their leaves and into their vulnerable growing points (Goggin et al. 2016). There was also a possible difference in how the resistant plants responded to auxin, but this seemed less important. We decided to expand our investigation to another 10 wild radish populations, to see if they had all followed the same pathway to resistance. This was our first mistake…

Wild radish seedlings growing outdoors in late autumn
Wild radish seedlings growing outdoors in late autumn, as part of a dose-response experiment using the synthetic auxin herbicides 2,4-D and dicamba.

How to study auxin resistance whilst maintaining sanity

A red-tailed black cockatoo
A red-tailed black cockatoo feeding on gumnuts in a marri (Corymbia calophylla) tree outside the lab window

We looked at the diversity of 2,4-D resistance mechanisms in a lucky total of 13 wild radish populations by measuring 2,4-D resistance, movement, fate and response in each. We also compared global gene expression in one resistant vs. one susceptible population to try and find out how the two populations differed in their short-term auxin response, and which transporter might be responsible for the reduced movement of 2,4-D. Altogether, this involved an outdoor dose-response study with 2,4-D and dicamba; measurement of root elongation in the presence of different auxins; tracking the movement and metabolic fate of radioactive 2,4-D in young plants; measuring activation of a part of the plant’s defence response (known as the MAPK pathway) in the presence and absence of 2,4-D; and a 24-sample RNA-sequencing (RNA-seq) experiment followed by targeted measurement of certain exciting genes. Luckily, the gum trees outside the lab window are occasionally visited by black cockatoos, which provide a welcome distraction for auxin-saturated researchers. (The cockies also happen to be very fond of wild radish seeds, so maybe we should be collaborating with them on non-chemical weed control methods.)

So many populations, such little agreement

The susceptible and resistant populations used in the gene expression study very obligingly showed hugely different responses to 2,4-D. The resistant population expressed genes involved in plant defence, and a gene that dials down the auxin response (Goggin et al. 2018). There was even a possible candidate for the 2,4-D transporter. However, this was where the clarity ended. There was, dismayingly, no relationship between resistance level and extent of 2,4-D movement, no consistency of response to different auxins, and no pattern to the expression levels of two of the most promising genes identified in the original population (an auxin-repressing gene and a key defence gene in the MAPK pathway). In fact, the various wild radish populations appeared to agree on only two things: there was no enhanced metabolic detoxification of the herbicide, and a higher baseline level of MAPK protein activation is very useful when it comes to resisting 2,4-D.

Variation in 2,4-D movement in plants from different 2,4-D-resistant populations
Variation in 2,4-D movement in plants from different 2,4-D-resistant populations. The visible parts of the plant are those that contain radioactively labelled 2,4-D. The resistant plant on the far right shows an identical pattern of 2,4-D movement to the susceptible plants.

Can wild radish researchers be as persistent as wild radish populations?

So far, there is no sign of a magic bullet for overcoming 2,4-D resistance in wild radish, because there is still so much we don’t understand. Although we yearned for this study to come up with an unequivocal (and highly publishable) explanation for 2,4-D resistance in wild radish, ideally “2,4-D resistance in wild radish is caused by loss of function of the auxin efflux transporter X”, the plants themselves were unwilling to cooperate. In fact, our most glaring finding was that there is too much variation between weed populations, especially in cross-pollinating species like wild radish, to conclude anything about anything. Nevertheless, the question of enhanced plant defence is worth pursuing further. Our hot-off-the-presses proteomics study has given some promising results, which open new avenues of study. Firstly, we have to hope that the proteins of the 11 populations not included in the proteomics study will show a consistency that their genes did not… and that the black cockatoos will be visiting again soon.

A highly speculative diagram
A highly speculative diagram of how the products of the up-regulated genes in a 2,4-D-resistant wild radish population might interact to confer resistance.

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