The interaction between glucose and cytokinin signaling in controlling <i>Arabidopsis thaliana</i> seedling root growth and development

<p>Cytokinin (CK) and glucose (GLC) control several common responses in plants. There is an extensive overlap between CK and GLC signal transduction pathways in Arabidopsis. Physiologically, both GLC and CK could regulate root length in light. CK interacts with GLC via HXK1 dependent pathway for root length control. Wild-type (WT) roots cannot elongate in the GLC free medium while CK-receptor mutant <i>ARABIDOPSIS HISTIDINE KINASE4</i> (<i>ahk4</i>) and type B ARR triple mutant <i>ARABIDOPSIS RESPONSE REGULATOR1, 10,11</i> (<i>arr1, 10,11</i>) roots could elongate even in the absence of GLC as compared with the WT. The root hair initiation was also found defective in CK signaling mutants <i>ahk4, arr1,10,11</i> and <i>arr3,4,5,6,8,9</i> on increasing GLC concentration (up to 3%); and lesser number of root hairs were visible even at 5% GLC as compared with the WT. Out of 941 BAP regulated genes, 103 (11%) genes were involved in root growth and development. Out of these 103 genes, 60 (58%) genes were also regulated by GLC. GLC could regulate 5736 genes, which include 327 (6%) genes involved in root growth and development. Out of these 327 genes, 60 (18%) genes were also regulated by BAP. Both GLC and CK signaling cannot alter root length in light in auxin signaling mutant <i>AUXIN RESPONSE3/INDOLE-3-ACETIC ACID17</i> (<i>axr3</i>/<i>iaa17</i>) suggesting that they may involve auxin signaling component as a nodal point. Therefore CK- and GLC- signaling are involved in controlling different aspects of root growth and development such as root length, with auxin signaling components working as downstream target.</p

Secondary structure pattern and sequence conservation of FLZ domain.

<p>(A) Secondary structure conservation of FLZ domain. Red color indicates alpha helix and blue color indicates beta-sheet. Confidence gradient of secondary structure formation is given on the top. (B) Sequence logo of <i>Arabidopsis</i>, <i>Medicago,</i> poplar and rice FLZ domains showing amino acid conservation.</p

Distribution of <i>FLZ</i> gene family in sequenced genomes.

<p>Distribution of <i>FLZ</i> gene family in sequenced genomes.</p

DUF581 Is Plant Specific FCS-Like Zinc Finger Involved in Protein-Protein Interaction

<div><p>Zinc fingers are a ubiquitous class of protein domain with considerable variation in structure and function. Zf-FCS is a highly diverged group of C₂-C₂ zinc finger which is present in animals, prokaryotes and viruses, but not in plants. In this study we identified that a plant specific domain of unknown function, DUF581 is a zf-FCS type zinc finger. Based on HMM-HMM comparison and signature motif similarity we named this domain as <u>F</u>CS-<u>L</u>ike <u>Z</u>inc finger (FLZ) domain. A genome wide survey identified that FLZ domain containing genes are bryophytic in origin and this gene family is expanded in spermatophytes. Expression analysis of selected <i>FLZ</i> gene family members of <i>A. thaliana</i> identified an overlapping expression pattern suggesting a possible redundancy in their function. Unlike the zf-FCS domain, the FLZ domain found to be highly conserved in sequence and structure. Using a combination of bioinformatic and protein-protein interaction tools, we identified that FLZ domain is involved in protein-protein interaction.</p></div

Schematic representation of domain organization in FLZ protein family.

<p>The FLZ proteins are scanned by InterProScan to identify conserved domains. <i>A. thaliana</i> FLZ1 is shown as a representative model for proteins which contain FLZ domain only. Proteins which possess other domains along with FLZ are also shown. The domains are abbreviated as follows, FLZ (FCS like zinc finger, PF04570), PPR (Pentatricopeptide Repeat, PF01535), Cupin (Cupin 1, PF00190), ICF (Ion channel family, PF00520), CND (Cyclic nucleotide-binding domain, PF00027) and DUF3354 (Domain of unknown function 3354, PF11834).</p

FLZ acts as the module for protein-protein interaction.

<p>(A) FLZ1 interacts with PFA-DSP3 and STH2 in Y2H. Murine p53 and SV40 large T-antigen interaction taken as positive control and p53 and lamin interaction is taken as negative control. (B) The deletion constructs of FLZ1, N-terminal (1–88 amino acids), FLZ (89–140 amino acids) and C-terminal (141–177 amino acids). (C) Y2H with deletion constructs of FLZ1 with PFA-DSP3 and STH2 showing FLZ is essential for their interaction. (D) And (E) beta-galactosidase activity of full length FLZ1 and deletion constructs interaction with PFA-DSP3 and STH2.</p

Sub-cellular localization of FLZ1 and PFA-DSP3 in onion epidermal cells.

<p>(A) Vector alone. (B) FLZ1. (C) PFA-DSP3. Left to right, YFP only, bright field, merged. FLZ1 is localized in both nucleus and cytoplasm while PFA-DSP3 is exclusively localized in nucleus. YFP were excited at 514 nm and emission was recorded at 530 nm.</p

Root growth, auxin transport and intracellular localization of PIN proteins in Arabidopsis plants grown in the presence and absence of light.

<p>(A) A 5-day-old seedling grown under light developed a long root, short hypocotyl and two fully-expanded cotyledons (left); by contrast, a dark-grown seedling developed a short root, long hypocotyl, two un-expanded cotyledons and an apical hook (right). Arrows marked hypocotyl-root junction. (B) Root elongation rate was 6.4±1 and 1.8±0.1 mm/day for light- and dark-grown plants, respectively (n = 10; repeated three times, <i>p</i><0.05). (C) Root diameter was 148±12 and 90±5 µm for light- and dark-grown seedlings, respectively (n = 10; repeated three times, <i>p</i><0.05). Normalized root basipetal auxin transport (D) and acropetal auxin transport (E) in dark-grown plants was 77% and 50% that of light-grown counterparts (n = 8; repeated three times, <i>p</i><0.05). (F-K) Shown were median optical sections of root tips of plants grown in light (F, H, I) and dark (G, I, K), expressing PIN2-eGFP (F, G), PIN1-eGFP (H, I) and PIN7-eGFP (J, K), and counter stained for cell walls with propidium iodide (red). Error bars represent standard deviations. Scale bars, 2 mm (A); 50 µm (F-K; left panels); 10 µm (F-K; right panels).</p

Vacuolar accumulation of PIN2-eGFP did not require de novo protein synthesis.

<p>Five-day-old light-grown PIN2-eGFP seedlings were pulse-labeled with an endocytosis marker, FM4-64, and pre-treated on growth media with or without cycloheximide (CHX; 50 µM) for 30 min. The plants were then shifted to dark and incubated for 4 hrs. In the absence of cycloheximide, PIN2-eGFP (green) accumulated in vacuolar compartments marked by FM4-64 (red) (A). Similarly, in the presence of cycloheximide, PIN2-eGFP also accumulated in vacuolar compartments in plants after shift to dark (B). Shown were root epidermis cells. Scale bar, 10 µm.</p

Time course of PIN2-eGFP vacuolar targeting.

<p>Five-day-old light-grown PIN2-eGFP seedlings were pulse-labeled with an endocytosis marker, FM4-64, and then transferred to dark (A–D) or kept in light (E–H). Both PIN2-eGFP (green) and FM4-64 (red) were restricted to the plasma membrane (PM) at T = 0 (A, E). At T = 30 min after transfer to dark, FM4-64 internalized to early endosomes and marked PIN2-eGFP-labeled endosomes (B). At T = 4 hrs after transfer to dark, PIN2-eGFP accumulated in vacuolar compartments, whose membrane was now labeled with FM4-64 (C). At T = 8 hrs after transfer to dark, strong vacuolar accumulation of PIN2-eGFP and FM4-64 labeling of vacuolar membrane were visible (D). On the other hand, FM4-64 labeled endosomes at T = 30 min (F) and vacuolar compartments at T = 4 and 8 hrs in seedling kept in the light condition (E–H). But, PIN2-eGFP remained at the PM under light (E–H). (A–H) Shown were root epidermal cells. Left, PIN2-eGFP (green); middle, FM4-64 (red); right, merged images. Scale bars, 10 µm. (I) RT-PCR analysis of steady state <i>PIN2</i> transcript levels in seedlings grown in continuous light (left) or after light-to-dark transition (right) for up to 24 hrs. The steady state transcript levels of an <i>Actin</i> gene were used as internal loading controls. (J) Real-time qRT-PCR analysis of steady state <i>PIN2</i> levels, normalized against the level of the <i>Actin</i> gene. Shown were the average <i>PIN2/Actin</i> ratios of three independent experiments. (K) Fluorescence intensities of PIN2-eGFP at the PM of root epidermis cells of light-grown plants kept in light (blue line) or shifted to dark (red line) for 0, 4, 8 and 12 hrs. Significant differences were observed between light- and dark-shifted plants at T = 4, 8 and 12 hrs (n = 168–466; Student's <i>t</i>-test, <i>p</i><0.0001).</p