Abstract:
Secondary growth or the increase in girth of plant organs is a primordial developmental process in seed plants as vascular expansion limits the transport of water and solute and provides mechanical support for the plant. Secondary growth involves the vascular cambium which is a cylindrical meristematic tissue producing inwards the xylem and outwards the phloem. It leads to thickening of plant organs and formation of secondary xylem or wood. Wood is the principal sink for excess atmospheric CO2 and fundamental source of natural renewable energy. Arabidopsis hypocotyl provides a good model to study secondary growth where two phases can be distinguished: an early phase in which xylem and phloem are produced at roughly the same rate, followed by a later phase of xylem expansion, in which xylem is produced at higher rate accompanied by fiber differentiation. Gibberellin (GA) triggers the shift between the two phases upon flowering, it moves from the shoot apex to the hypocotyl where it induces locally the degradation of DELLA proteins which are known to act as repressors of secondary growth. Although secondary growth is crucial for the plant and environment, it is surprising that little is known about the molecular mechanisms underlying it. In this study, we found that among the DELLA gene family REPRESSOR OF ga1-3 (RGA) and GA INSENSITIVE (GAI) mediate most of the GA related secondary growth, with a more pronounced role of RGA as repressor. The role of RGA and GAI seems to be conserved across ecotypes, which greatly differ in xylem occupancy. We identified novel regulators of secondary growth through evaluating DELLA known interactors as well as analyzing the transcriptome related to RGA upon transition to the xylem expansion phase. We found that AUXIN RESPONSIVE FACTOR (ARF6), ARF7 and ARF8 act as positive regulators of xylem expansion and fiber differentiation whereas type-B ARABIDOPSIS RESPONSE REGULATORS (ARR1) and ARR2, CORONATINE-INSENSITIVE 1 (COI1) and TRANSCRIPTION FACTOR MYC2 (MYC2) activate phloem proliferation with a specific role for ARR1 and ARR2 in repressing fiber differentiation in the hypocotyl. Our genetic interaction analyses indicate that DELLA sequesters the identified ARF’s and possibly BREVIPEDICELLUS/KNAT1/ (BP/KNAT1) during the early phase. During the second phase ARF6, ARF7, ARF8 and probably BP are released from DELLA repression. We also showed that BP expression does not depend on ARF6 and ARF8 in the hypocotyl, instead our data indicate a 3 possible interaction between BP and ARF’s to regulate cambium activity. We performed RNA seq experiments in order to gain more insights into the regulatory network related to the positive regulation of xylem expansion mediated by ARF6 and ARF8, we identified several candidates involved in xylem development. Apart from the DELLA mediated control of secondary growth; we also identified a specific role for Jasmonate in promoting fiber formation, in the xylem and ectopically in the phloem, without altering the Xylem/Total area (X/A) ratio. Taken together, our findings shed light on a complex hormone cross-talk between GA, Auxin, Cytockinin and Jasmonate where DELLA function as central hub regulating xylem expansion and fiber differentiation.
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