Increasing seed yields by the targeted modulation of seed number in Arabidopsis thaliana and seed size in Brassica oleracea

Abstract

With the global population set to reach 9.7 billion by 2050, maximising crop yields is a focus for food security. Seed number and seed size are the main components of yield in seed-yielding crops. Whilst trade-offs can exist between seed number and size, research in Arabidopsis thaliana (hereafter Arabidopsis) suggests each component is governed by distinct loci and one trait could be improved without hindering the other. In this thesis, two mechanisms of increasing yield have been explored – altering seed number in Arabidopsis and modifying seed size in Brassica oleracea (hereafter B. oleracea). Seeds are derived from ovules in the gynoecium (the female reproductive organ) and ovules arise from ovule primordia. Previous research has shown that brassinosteroids (BR) play a role in the initiation of ovule primordia, influencing the number of ovules and seeds produced. This study has indicated a potential role for the BR-biosynthesis gene DWARF 4 (DWF4) and floral development gene AINTEGUMENTA-LIKE 7 (AIL7) in the initiation of ovule primordia. Experiments with transgenic Arabidopsis lines ectopically expressing DWF4, AIL7 and CY85A2 (another BR-biosynthesis gene) under the control of a gynoecium-specific SEEDSTICK (STK) promoter revealed an altered arrangement of ovules within the pistil and an increased density of seeds inside the silique. Transcriptome analysis of transgenic gynoecia indicated changes in expression for ovule identity genes and genes which dictate the boundaries between ovule primordia, hinting to a potential mechanism underlying this phenotype. These transgenic lines also exhibited a dwarf phenotype therefore, a promoter optimisation study was performed to investigate the expression profile of the STK promoter and explore the possibility of uncoupling changes in plant architecture from the ovule and seed phenotype. Following this, transgenic Arabidopsis plants were generated which ectopically expressed DWF4 under an optimised STK promoter. Interestingly, the optimised transgenic plants were no longer dwarfed whilst the altered ovule arrangement and increased seed density phenotypes were retained, however further experiments are required to confirm the impact on seed yield. Control of seed size in B. oleracea was also explored in this thesis. The transcription factor AUXIN RESPONSE FACTOR 2 (ARF2) is known to play a role in negatively regulating seed size in Arabidopsis by altering cell proliferation in the integument (the ovule layer that develops into the final seed coat). However, the potential for altering ARF2 in a crop species remained unexplored. This study reports that ARF2 CRISPR-edited B. oleracea lines displayed no evidence of alterations in seed size or yield when one ARF2 homolog (Bol007816) was edited, but plants did display altered traits similar to those reported in past literature (lower biomass and altered fertility). To our knowledge, this is the first example of editing ARF2 in Brassica and it is speculated the remaining two ARF2 homologs may require editing to observe a potential seed size phenotype.

Date of Award	15 Nov 2023
Original language	English
Awarding Institution	University of Bath
Supervisor	James Doughty (Supervisor), Hans-Wilhelm Nützmann (Supervisor), Smita Kurup (Supervisor) & Peter Eastmond (Supervisor)