ADP1 Affects Plant Architecture by Regulating Local Auxin Biosynthesis

Plant architecture is one of the key factors that affect plant survival and productivity. Plant body structure is established through the iterative initiation and outgrowth of lateral organs, which are derived from the shoot apical meristem and root apical meristem, after embryogenesis. Here we report that ADP1, a putative MATE (multidrug and toxic compound extrusion) transporter, plays an essential role in regulating lateral organ outgrowth, and thus in maintaining normal architecture of Arabidopsis. Elevated expression levels of ADP1 resulted in accelerated plant growth rate, and increased the numbers of axillary branches and flowers. Our molecular and genetic evidence demonstrated that the phenotypes of plants over-expressing ADP1 were caused by reduction of local auxin levels in the meristematic regions. We further discovered that this reduction was probably due to decreased levels of auxin biosynthesis in the local meristematic regions based on the measured reduction in IAA levels and the gene expression data. Simultaneous inactivation of ADP1 and its three closest homologs led to growth retardation, relative reduction of lateral organ number and slightly elevated auxin level. Our results indicated that ADP1-mediated regulation of the local auxin level in meristematic regions is an essential determinant for plant architecture maintenance by restraining the outgrowth of lateral organs.     

Cleavage of INDOLE-3-ACETIC ACID INDUCIBLE28 mRNA by MicroRNA847 Upregulates Auxin Signaling to Modulate Cell Proliferation and Lateral Organ Growth in Arabidopsis

MicroRNAs function in a range of developmental processes. Here, we demonstrate that miR847 targets the mRNA of the auxin/indole acetic acid (Aux/IAA) repressor-encoding gene IAA28 for cleavage. The rapidly increased accumulation of miR847 in Arabidopsis thaliana coincided with reduced IAA28 mRNA levels upon auxin treatment. This induction of miR847 by auxin was abolished in auxin receptor tir1-1 and auxin-resistant axr1-3 mutants. Further analysis demonstrates that miR847 functions as a positive regulator of auxin-mediated lateral organ development by cleaving IAA28 mRNA. Importantly, the ectopic expression of miR847 increases the expression of cell cycle genes as well as the neoplastic activity of leaf cells, prolonging later-stage rosette leaf growth and producing leaves with serrated margins. Moreover, both miR847 and IAA28 mRNAs are specifically expressed in marginal meristems of rosette leaves and lateral root initiation sites. Our data indicate that auxin-dependent induction of miR847 positively regulates meristematic competence by clearing IAA28 mRNA to upregulate auxin signaling, thereby determining the duration of cell proliferation and lateral organ growth in Arabidopsis. IAA28 mRNA encodes an Aux/IAA repressor protein, which is degraded through the proteasome in response to auxin. Altered signal sensitization to IAA28 mRNA levels, together with targeted IAA28 degradation, ensures a robust signal derepression.     

Involvement of HLS1 in sugar and auxin signaling in Arabidopsis leaves

Sugar regulates a variety of genes and controls plant growth and development similarly to phytohormones. As part of a screen for Arabidopsis mutants with defects in sugar-responsive gene expression, we identified a loss-of-function mutation in the HOOKLESS1 (HLS1) gene. HLS1 was originally identified to regulate apical hook formation of dark-grown seedlings (Lehman et al., 1996, Cell 85: 183-194). In hls1, sugar-induced gene expression in excised leaf petioles was more sensitive to exogenous sucrose than that in the wild type. Exogenous IAA partially repressed sugar-induced gene expression and concomitantly activated some auxin response genes such as AUR3 encoding GH3-like protein. The repression and the induction of gene expression by auxin were attenuated and enhanced, respectively, by the hls1 mutation. These results suggest that HLS1 plays a negative role in sugar and auxin signaling. Because AUR3 GH3-like protein conjugates free IAA to amino acids (Staswick et al., 2002, Plant Cell 14: 1405-1415; Staswick et al., 2005, Plant Cell 17: 616-627), enhanced expression of GH3-like genes would result in a decrease in the free IAA level. Indeed, hls1 leaves accumulated a reduced level of free IAA, suggesting that HLS1 may be involved in negative feedback regulation of IAA homeostasis through the control of GH3-like genes. We discuss the possible mechanisms by which HLS1 is involved in auxin signaling for sugar- and auxin-responsive gene expression and in IAA homeostasis.     

Increased leaf mesophyll porosity following transient retinoblastoma‐related protein silencing is revealed by microcomputed tomography imaging and leads to a system‐level physiological response to the altered cell division pattern

The causal relationship between cell division and growth in plants is complex. Although altered expression of cell‐cycle genes frequently leads to altered organ growth, there are many examples where manipulation of the division machinery leads to a limited outcome at the level of organ form, despite changes in constituent cell size. One possibility, which has been under‐explored, is that altered division patterns resulting from manipulation of cell‐cycle gene expression alter the physiology of the organ, and that this has an effect on growth. We performed a series of experiments on retinoblastoma‐related protein (RBR), a well characterized regulator of the cell cycle, to investigate the outcome of altered cell division on leaf physiology. Our approach involved combination of high‐resolution microCT imaging and physiological analysis with a transient gene induction system, providing a powerful approach for the study of developmental physiology. Our investigation identifies a new role for RBR in mesophyll differentiation that affects tissue porosity and the distribution of air space within the leaf. The data demonstrate the importance of RBR in early leaf development and the extent to which physiology adapts to modified cellular architecture resulting from altered cell‐cycle gene expression.     

The Arabidopsis auxin-inducible gene ARGOS controls lateral organ size

During plant development, the final size of an organ is regulated and determined by various developmental signals; however, the molecular mechanisms by which these signals are transduced and the mediators involved are largely unknown. Here, we show that ARGOS, a novel Arabidopsis gene that is highly induced by auxin, is involved in organ size control. Transgenic plants expressing sense or antisense ARGOS cDNA display enlarged or reduced aerial organs, respectively. The alteration in organ size is attributable mainly to changes in cell number and the duration of organ growth. Ectopic expression of ARGOS prolongs the expression of AINTEGUMENTA (ANT) and CycD3;1 as well as the neoplastic activity of leaf cells. Moreover, organ enlargement in plants overexpressing ARGOS can be blocked by the loss of function of ANT, implying that ARGOS functions upstream of ANT to affect the meristematic competence of organ cells. The induction of ARGOS by auxin is attenuated or abolished in auxin-resistant1 (axr1), and overexpression of ARGOS partially restores axr1 organ development. These results suggest that ARGOS may transduce auxin signals downstream of AXR1 to regulate cell proliferation and organ growth through ANT during organogenesis.     

Arabidopsis RETINOBLASTOMA-RELATED Is Required for Stem Cell Maintenance, Cell Differentiation, and Lateral Organ Production

Several genes involved in the regulation of postembryonic organ initiation and growth have been identified. However, it remains largely unclear how developmental cues connect to the cell cycle. RETINOBLASTOMA RELATED (RBR) is a plant homolog of the tumor suppressor Retinoblastoma (pRb), which is a key regulator of the cell cycle. Using inducible RNA interference (RNAi) against Arabidopsis thaliana RBR (RBRi), we reduced RBR expression levels at different stages of plant development. Conditional reduction or loss of RBR function disrupted cell division patterns, promoted context-dependent cell proliferation, and negatively influenced establishment of cell differentiation. Several lineages of toti- and pluripotent cells, including shoot apical meristem stem cells, meristemoid mother cells, and procambial cells, failed to produce appropriately differentiated cells. Meristem activity was altered, leading to a disruption of the CLAVATA-WUSCHEL feedback loop and inhibition of lateral organ formation. Release of RBR from RNAi downregulation restored meristem activity. Gene profiling analyses soon after RBRi induction revealed that a change in RBR homeostasis is perceived as a stress, even before genes regulated by RBR-E2F become deregulated. The results establish RBR as a key cell cycle regulator required for coordination of cell division, differentiation, and cell homeostasis.     

Immunodetection of retinoblastoma-related protein and its phosphorylated form in interphase and mitotic alfalfa cells

Plant retinoblastoma-related (RBR) proteins are primarily considered as key regulators of G(1)/S phase transition, with functional roles in a variety of cellular events during plant growth and organ development. Polyclonal antibody against the C-terminal region of the Arabidopsis RBR1 protein also specifically recognizes the alfalfa 115?kDa MsRBR protein, as shown by the antigen competition assay. The MsRBR protein was detected in all cell cycle phases, with a moderate increase in samples representing G(2)/M cells. Antibody against the human phospho-pRb peptide (Ser807/811) cross-reacted with the same 115?kDa MsRBR protein and with the in vitro phosphorylated MsRBR protein C-terminal fragment. Phospho-MsRBR protein was low in G(1) cells. Its amount increased upon entry into the S phase and remained high during the G(2)/M phases. Roscovitine treatment abolished the activity of alfalfa MsCDKA1;1 and MsCDKB2;1, and the phospho-MsRBR protein level was significantly decreased in the treated cells. Colchicine block increased the detected levels of both forms of MsRBR protein. Reduced levels of the MsRBR protein in cells at stationary phase or grown in hormone-free medium can be a sign of the division-dependent presence of plant RBR proteins. Immunolocalization of the phospho-MsRBR protein indicated spots of variable number and size in the labelled interphase nuclei and high signal intensity of nuclear granules in prophase. Structures similar to phospho-MsRBR proteins cannot be recognized in later mitotic phases. Based on the presented western blot and immunolocalization data, the possible involvement of RBR proteins in G(2)/M phase regulation in plant cells is discussed.