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The key transcriptional regulators that regulate root hair initiation and tip growth are unknown. While many genes have been reported that participate in root hair development, especially root hair tip growth, none that regulate root hair initiation and tip growth has been identified to date. The aim of this thesis is to characterise the role of a family of transcription factors that control these processes and to construct a regulatory network for root hair growth and initiation. Previously, we isolated two bHLH transcription factors, RHD6 and RSL1, that positively regulate root hair initiation. These two genes belong to the bHLH VIllc subfamily, which includes four other paralogues (RSL2 to RSL5) that form a sister clade to RHD6 and RSL1. To characterize the function of the sister clade genes, I examined the phenotypes of all the available knockout mutants and determined their expression patterns. The genetic data shows that the sister clade genes function downstream of RHD6 and RSLl in root hair development, with RSL4 regulating both root hair initiation and tip growth and RSL2, RSL3 and RSL5 controlling root hair tip growth. Root hair tip growth is totally abolished in plants having mutations in both RSL2 and RSL4. Consistent with the genetic data, expression of RHD6 family genes follows a temporal pattern that reflects the timing of their roles during development. RHD6 and RSLl are expressed in root hair cells around the root meristem and elongation zones to regulate the early event of root hair initiation. RSL4 is expressed in root hair cells around root hair initiation zone to direct late event of root hair initiation; then, together with RSL2 and RSL3, which are also expressed in root hair cells around root hair initiation zone, RSL4 maintains root hair tip growth; RSL5, which is regulated by the other members, is also expressed in root hair cells. As auxin and ethylene also regulate root hair initiation and tip growth, and can partially restore the root hair development in rhd6/rsll mutant, I also investigated how auxin and ethylene interact with the RHD6 family genes to regulate root hair development. I showed that auxin and ethylene can bypass RHD6 and RSLl to regulate the down stream sister clade genes during root hair development. Furthermore, RHD6 and auxin/ethylene regulate root hair tip growth through RSL2 and RSL4. And low phosphate stress also regulates root hair by modulating RSL2 and RSL4 transcription. In summary, we found a subgroup of key regulator genes in the same bHLH subfamily form a regulatory network and temporally control root hair initiation and tip growth, and the internal and external (phosphate availability) factors regulate root hair development through these genes.

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Leaf shape formation is a fascinating process involving the coordinated regulation of cell division and cell expansion, which ultimately relies on the control of cell wall expansion. Expansins comprise a large family of cell wall proteins which can non-enzymatically trigger the relaxation of the cell wall for expansion. They have been implicated in organ initiation, cell differentiation and organ growth. However, the exact mechanism of expansin action and functional role of different members of the expansin gene family is unclear. Here, I focus on the largest subgroup of expansins (EXPA) to identify which genes are expressed during different stages of leaf development in Arabidopsis. This information was used to design an inducible artificial microRNA construct to target the knock-down of multiple expansin genes expressed during early leaf development. Through a series of experiments knocking down expansin gene expression at different stages of leaf development, I show that the suppression of expansin gene expression at later stages of leaf development is sufficient to repress leaf growth and alter leaf shape. This molecular genetic approach is combined with an atomic force microscopy (AFM) approach to investigate the role of cell wall mechanics in leaf growth. I describe the use of AFM for probing the leaf primordium mechanics and outline consideration for future work.

Senescence is the last stage of leaf development. During leaf senescence essential nutrients are remobilised to the growing parts of the plant. This highly regulated process involves the expression of senescence-associated genes and a decrease in photosynthesis accompanied by chloroplast protein degradation. The following studies were performed in order to characterise the functions of the papain-like cysteine proteases and the WHIRLY1 protein in the regulation of senescence. Transgenic Arabidopsis lines expressing the rice cystatin, oryzacystatin-I (OCI) in either the chloroplasts or cytosol were characterized. These lines had a slow growth phenotype relative to the wild type. Furthermore, ectopic OCI expression in Arabidopsis increased leaf numbers and enhanced shoot and root branching at flowering. Transgenic barley lines with decreased levels of WHIRLY1 transcripts were grown under optimal and low nitrogen stress conditions. Under optimal conditions, the WHIRLY1 transgenic lines had similar CO2 assimilation rates to those of the wild type but they had significantly more chlorophyll and less sucrose. Moreover, these lines produced fewer tillers and seeds than the wild type. The WHIRLY1 transgenic plants grew better than the wild type under nitrogen deficiency, maintaining higher photosynthetic capacity and chlorophyll content. The WHIRLY1 protein influenced the expression of specific subsets of transcripts encoding chloroplast proteins including thylakoid NADH and cytochrome b6/f complexes. Additionally, the transcript profile revealed new target proteins for WHIRLY1, supporting the involvement of this protein in chloroplast signalling and stress responses. Finally, this study allowed the successful production of barley plants expressing SAG21 and wheat plants expressing the OCI gene in the cytosol, which have the potential to improve stress tolerance and increase seed yield.

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Stomata are small pores found in the leaf epidermis. Changes in stomatal aperture regulate leaf gas exchange. An increase in atmospheric [C02] causes a partial stomatal closure, reducing ~vapotranspiration and increasing leaf temperature. The aim of this study was' to use infrared thermal imaging to track changes in leaf temperature in response to high [C02], in order to isolate and characterize Arabidopsis thaliana mutants with aberrant stomatal responses. Two thermal imaging cameras were used to measure leaftemperature difference between Ler and abil-l plants $ld it was found that this was the largest (0.9-1.5 °C) at air relative humidity of 40-60%. Subsequently, a Teflon covered chamber was constructed to test the stomatal response to high [C02] of Col-O plants from 20-35 days old. Plants were imaged while subjected to 360 ppm [C02] for 40 min and then subjected to 1500 ppm [C0 2] for 40- 60 min. 20 day old Col-O plants did not show an increase in leaf temperature, whereas older plants did show an increase of between I-2°C after 40 min of higher [C02]' 20,000 Arabidopsis EMS mutants were screened comparing their leaf temperature at 360 and 1500 ppm [C02]' 68 mutants showed a higher or lower leaf temperature than Col-O at 1500 ppm [C02]' Five of the 44 mutant lines that were fertile were chosen for genetic and physiological characterization: the 'hot' mutant N2, and the 'cool' mutants F, EEA8, E26.2 and MQl. N2 and BEA8 carried recessive mutations while the others carried dominant mutations. Interestingly, the lower epidermis of the N2 mutant had 14.2 % lower SI, 85 % higher SD and 128 % higher ED and the EEA8 mutant had 41 % higher SD and 52 % higher ED compared to Col-O plants. In addition, leaf gas exchange analysis showed that MQl has higher & than Col-O when exposed to 2100 ppm [C02] in the light, as well as in the dark at 360 ppm [C02].

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