The CIN-TCP transcription factors promote commitment to differentiation in Arabidopsis leaf pavement cells via both auxin-dependent and independent pathways.

Cells in organ primordia undergo active proliferation at an early stage to generate sufficient number, before exiting proliferation and entering differentiation. However, how the actively proliferating cells are developmentally reprogrammed to acquire differentiation potential during organ maturation is unclear. Here, we induced a microRNA-resistant form of TCP4 at various developmental stages of Arabidopsis leaf primordium that lacked the activity of TCP4 and its homologues and followed its effect on growth kinematics. By combining this with spatio-temporal gene expression analysis, we show that TCP4 commits leaf cells within the transition zone to exit proliferation and enter differentiation. A 24-hour pulse of TCP4 activity was sufficient to impart irreversible differentiation competence to the actively dividing cells. A combination of biochemical and genetic analyses revealed that TCP4 imparts differentiation competence by promoting auxin response as well as by directly activating HAT2, a HD-ZIP II transcription factor-encoding gene that also acts downstream to auxin response. Our study offers a molecular link between the two major organ maturation factors, CIN-like TCPs and HD-ZIP II transcription factors and explains how TCP activity restricts the cell number and final size in a leaf.

Introduction TCP-mediated cell differentiation and organ maturation has been linked to the biosynthesis and response pathways of the major phytohormone, auxin [21]. Auxin controls cell proliferation and maturation in a context-dependent manner. Plants expressing a dominant, repressive form of TCP15 display up-regulation of multiple auxin biosynthesis genes YUCCA1, 4 and 6, suggesting that TCP15 is a negative regulator of auxin biosynthesis [25]. Similarly, TCP3 inhibits auxin response by inducing IAA3 (INDOLE-3-ACETIC ACID INDUCIBLE3)/ SHY2 (SHORT HYPOCOTYL2), a negative regulator of auxin signaling, and PIN1 (PIN-FORMED1), 5, 6, AUX1 (AUXIN RESISTANT1), the polar auxin transporters [21,26]. By contrast, TCP4 induces auxin response by activating YUC5, an auxin biosynthetic gene [27]. Even though the regulation of auxin response by TCP proteins has been studied in some detail, the role of auxin in cell maturation and transition zone formation has been studied only in roots [1] but not in leaves.
The role of the plant-specific homeodomain-leucine zipper (HD-ZIP) transcription factors has been implicated in leaf morphogenesis. The HD-ZIP members are classified into four subgroups (I to IV) based on sequence similarity within the DNA-binding homeodomain [28,29]. Function of the class III HD-ZIP proteins such as REVOLUTA (REV), PHABOLUSA (PHB), PHAVOLUTA (PHV), ARABIDOPSIS THALIANA HOMEOBOX 8 (ATHB8) and ATHB15 is the most well-studied among the family members and their role has been linked to the establishment of leaf/ embryo polarity and meristem maintenance [30][31][32]. Members of the class II subgroup including HOMEODOMAIN ARABIDOPSIS THALIANA1 (HAT1) and HAT2 have been implicated in growth adaptation to the environmental signals and in organ maturation in response to hormone signaling [33][34][35]. Recent reports show that the class II HD-ZIP proteins ATHB4 and HAT3 physically interact with REV and form a functional repression complex which suppresses the miR165/166 expression, thereby establishing the adaxial/abaxial polarity in leaves [36]. Interestingly, ATHB4 and HAT3 are downstream targets of REV, pointing to a complex interconnection among the HD-ZIP genes during leaf morphogenesis. In addition, some members of the class II HD-ZIP genes, specifically HAT1/2, are activated by auxin response and promote cell expansion and organ maturation [33,34,37,38]. Plants overexpressing either HAT1 or HAT2 produce smaller leaves with reduced cell number and elongated hypocotyl cells [34,37,38], which is also seen in plants with gain-of-function of class II TCP genes [22,23,27]. However, it is not clear whether and how the HD-ZIP II proteins are linked to other cell differentiation factors in promoting organ maturation.
Here, we induced a dominant form of TCP4 protein at various developmental stages of Arabidopsis leaf primordium that lacked class II TCP function and followed its effect on leaf growth kinematics. By combining this with spatio-temporal expression of endogenous TCP4, we show that the class II TCP proteins irreversibly reprogram the mitotic cells to exit division and acquire differentiation competence within the transition zone. Induction of cell maturation is mediated by auxin response as well as by direct activation of HAT2 transcription by TCP4.

Altered number and size of pavement cells in the leaves with modified TCP activity
The class II TCP genes are heterochronic regulators of leaf development and their function has been implicated in the repression of cell division [22] and the promotion of differentiation [17]. In order to examine the specific role of the five redundant, miR319-targeted, class II TCP genes-TCP2, 3, 4, 10 & 24-in cell division/ differentiation of Arabidopsis leaves, we compared the mature first leaf pair and their epidermal cells of wild type (Col-0) with those in the loss-of-function mutants tcp2;4; 10 [27] and jaw-D, where all the five TCP transcripts are down-regulated due to the over-expression of miR319a [15]. The tcp2;4;10 and jaw-D leaves measured >1.5 times larger than the Col-0 leaves (Fig 1A), as was reported previously [9,17]. The number of the abaxial epidermal cells in the mutant leaves increased~2.5-fold (Fig 1B), suggesting that the increased mutant leaf area is due to an excess of cell proliferation. To determine if class II TCP activity is sufficient to repress proliferation, we induced a miR319-resistant, dexamethasone (DEX)-inducible form of TCP4 protein (mTCP4-GR) expressed under ProTCP4 and Pro35S [27]. DEX-grown, homozygous ProTCP4:mTCP4:GR or Pro35S:mTCP4: GR plants in the Col-0 background (hereafter referred to as Col-0;GR or 35S;GR, respectively) showed smaller leaves with reduced cell number (Fig 1C and 1D and S1A-S1D Fig). A more pronounced relative reduction in the final leaf size (by~70%; Fig 1C and 1E) and cell number (by >80%; Fig 1D and 1F) were observed in the DEX-grown ProTCP4:mTCP4:GR plants in the jaw-D background (hereafter referred to as jaw-D;GR) compared to the mock treatment, demonstrating that TCP4 is a strong cell proliferation inhibitor. Similar negative effect of TCP4 on leaf size and cell number was also observed in three independent transgenic lines where three different gain-of-function forms of TCP4 were expressed constitutively under a leaf primordia-specific promoter (ProBLS:rTCP4:GFP) [17], a ubiquitous promoter (Pro35S: TCP4:3F6H) [39] or the endogenous promoter (ProTCP4:TCP4:VP16) [23] (S1F-S1I Fig).
Concomitant with the increased number, the area of mature pavement cells reduced to almost half in jaw-D and tcp2;4;10 leaves ( Fig 1G and S2A Fig). Induction of TCP4 activity, however, had little effect on the final cell area in the Col-0;GR and 35S;GR leaves ( Fig 1H and  1I and S1E Fig) and merely restored the cell size defect found in the jaw-D;GR leaves to the wild-type level (Fig 1I and S2B Fig). Pavement cell area also remained unaltered in the three other dominant lines of TCP4, ProBLS:rTCP4:GFP, Pro35S:TCP4:3F6H and ProTCP4:TCP4: VP16 (S1J and S1K Fig). These results suggest that the class II TCP proteins are required for pavement cell maturation during leaf development but are not sufficient to promote pavement cell expansion per se, even though they do so in the hypocotyl [27]. Contrary to the miR319-resistant inducible form of TCP4, constitutive expression of a miR319-susceptible form of TCP4 protein in the homozygous Pro35S:TCP4:GR plants (hereafter referred to as 35S;sGR) showed no effects on leaf size, cell number and pavement cell area (Fig 1C, 1D and 1I), demonstrating that the effects of mTCP4:GR induction described above resulted specifically from miR319 activity on the class II TCP transcripts and not from artifacts due to GR fusion or to the site of insertion [27].
The requirement of TCP genes for developmental cell maturation was also apparent in the reduced differentiation status of the pavement cells in the TCP loss-of-function leaves. The abaxial surface of mature Col-0 leaf is composed mostly of large epidermal cells with jigsaw shape (Fig 1H, S2A and S2C Fig), a characteristic marker for differentiation [17,40]. By contrast, mature tcp2;4;10 and jaw-D leaves were composed mostly of smaller cells (Fig 1J, S2A  and S2C Fig), a differentiation defect that was totally rescued by TCP4 induction (Fig 1K). However, induction of TCP4 in the Col-0 background did not alter the cell size distribution (S2D Fig).

Kinematic analysis of TCP-mediated leaf growth
The results described above suggest that the class II TCP proteins restrict cell number and promote cell maturation during leaf development. To examine their role in cell proliferation/ maturation at early growth phase, we compared the kinematics of growth of the first pair of jaw-D; GR leaves under TCP4 inductive and non-inductive conditions. Col-0 leaf blade expanded slowly at an early growth stage, from 6-10 days after stratification (DAS), during which the average area of the abaxial pavement cells remained small (~500 μm 2 or less) and their number increased exponentially (Fig 2A-2C, S3A and S3B Fig). At 10 DAS, the blade acquired~10% of its final area and~75% of its final pavement cell number. Later, the blade expanded at an exponential rate up to 16-18 DAS, before reaching a plateau at~22 DAS (Fig 2A and S3A Fig). The pavement cells also expanded faster during this period, and continued to do so beyond 22 DAS (Fig 2C and S3B Fig). The number of pavement cells reached its maximum at the mid-log phase of blade expansion and no further proliferation was observed beyond 12 DAS (Fig 2B), at which stage the blade acquired approximately half of its final area. These kinematics results are in general agreement with the earlier reports on Arabidopsis leaf published by other laboratories [5,41]. The overall trend of the growth kinematics of jaw-D leaf blade was by and large similar to Col-0, with a slow (6-10 DAS), exponential (10-18 DAS) and saturating (18)(19)(20)(21)(22) phase. However, the jaw-D blades started expanding at a rate faster than that of Col-0 from the earliest stage measured (6 DAS; inset in Fig 2A), and consequently its area remained higher throughout the growth. The increased jaw-D blade area was due to excess number, and despite reduced size, of the pavement cells. The jaw-D cells accumulated at a faster rate than Col-0, but the proliferation ceased at almost similar growth stage (~12 DAS) in both the genotypes, resulting in~2.5 times more cells in the mature jaw-D leaf compared to Col-0 (Figs 1B and 2B). As in Col-0, the size of the jaw-D pavement cells remained small in the slow phase (6-10 DAS) and then increased exponentially until 16 DAS, before saturating rather abruptly ( Fig  2C). However, the average area of jaw-D pavement cells remained 1.6 ± 0.16 fold smaller than that in Col-0 throughout the growth phase of the leaf (S1 Table).
All the cellular defects of the jaw-D leaf were rescued by continuous induction of TCP4 throughout the growth phases (Fig 2A-2C and S3A Fig). The DEX-grown jaw-D;GR blade expanded at the same rate as Col-0 until 12 DAS and then at a slower rate, before ceasing to expand at 18 DAS to a final area that was smaller than the Col-0 blade (Fig 2A and S3A Fig). Upon TCP4 induction, the pavement cells accumulated in the jaw-D;GR blade for almost the TCP4 induces irreversible exit from cell proliferation same duration (till~12 DAS) as in Col-0, but at a slower rate, resulting in fewer cells at maturity compared to Col-0 (Figs 2B and 1D). While both blade area and cell number were smaller than the Col-0 values in the jaw-D;GR leaves grown under DEX, the average size of the pavement cells was rescued up to the Col-0 value at all stages of leaf growth (Figs 2C and 1I).
The growth kinematics described above shows that class II TCP activity is essential for reducing the total number, and for increasing the average size, of the pavements cells during leaf development. A growing leaf blade, however, is composed of cells of heterogeneous size and proliferation status; the smaller, dividing cells are located at the proximal end and the larger, differentiating cells towards more distal side [4]. To examine if TCP4 activity is specific to a stage of cell maturation, we determined the proportions of smaller (arbitrarily defined as <1500 μm 2 in area) and larger (>3000 μm 2 ) pavement cells in expanding Col-0 lamina (S2C Fig) and compared with jaw-D. During early growth (6)(7)(8), nearly all Col-0 pavement cells measured <1500 μm 2 , and their proportion sharply declined to~30% at 22 DAS as the blade expanded ( Fig 2D and S3C Fig). The larger cells started appearing at 10 DAS and steadily increased to~40% at 22 DAS (Fig 2E). The fraction of the smaller cells in the jaw-D;GR blade without TCP4 induction, however, declined at a slower rate and stabilized at >60% at 16 DAS ( Fig 2D and S3D Fig). Taken together, these kinematic results suggest that a major function of the class II TCP proteins is to convert the smaller pavement cells to larger cells, possibly by promoting their proliferation to differentiation competence. The differentiation-inducing activity of the TCP proteins is also apparent in the altered stomatal index (the proportion of epidermal cells assuming stomatal lineage), a measure of leaf maturation [41], in the mutant leaves. 20.5 ± 1.2% Col-0 epidermal cells assumed stomatal identity at 6 DAS, a value that steadily increased during leaf maturation and reached to a maximum value of 37.4 ± 2.6% at 22 DAS ( Fig 2F). However, only 13.8 ± 2.7% jaw-D;GR cells were converted to stomatal lineage at 6 DAS, and this value increased to a maximum of 26.7 ± 2.8% at 22 DAS. Induction of TCP4 activity in the jaw-D;GR leaves nearly restored the defect in stomatal index to wild-type level.

Temporal control of leaf growth by TCP4
All jaw-D;GR rosette leaves were smaller with smoother margin when grown with continuous presence of DEX (Fig 1L), indicating that TCP4 induction is active throughout the vegetative growth. However, when jaw-D;GR seedlings were grown under inductive condition for first 10 days and then transferred to non-inductive medium, the first few leaves resembled those grown under continuous DEX, but the leaves that emerged later showed progressively more jaw-D-like phenotype. Conversely, when the seedlings were grown under non-inductive condition for first 10 days and then transferred to inductive condition, the first few leaves resembled jaw-D leaves but the later leaves were smaller with smoother margin (Fig 1L). This suggests that DEX application can be used to induce TCP4 activity at any point of time during plant development.
To determine the developmental timing of TCP4 function during leaf maturation, we induced TCP4 activity in the jaw-D;GR leaves after growing them in the non-inductive medium for various number of days after stratification (4 to 15 DAS; Fig 3 and S4A Fig) and compared their blade area, cell number and cell size at maturity (29 DAS). Without TCP4 induction, the jaw-D;GR lamina grew to 32.6 ± 2.5 mm 2 that contained 17450 ± 1336 pavement cells of average area 1870 ± 170 μm 2 (Fig 3). On the other hand, the lamina under TCP4 induces irreversible exit from cell proliferation continuous TCP4 induction grew to 12.0 ± 1.7 mm 2 with much fewer cells (4130 ± 579) of the average area 2920 ± 140 μm 2 . The blade area progressively increased from 12.0 ± 1.7 mm 2 to the non-inductive value of 32.6 ± 2.5 mm 2 when TCP4 was induced at 4, 5 or 6 DAS, even though their final cell numbers were much fewer than the non-inductive value. When TCP4 was induced at 6 DAS, its blade area at maturity was close to the jaw-D value (30.4 ± 2.3 mm 2 ), even though its cell number was almost half of that in jaw-D leaf (Fig 3A-3C). This is because the defect in the pavement cell size was rescued in these leaves to the wild-type value (3130 ± 170 μm 2 ) (Fig 3D).
When TCP4 was induced at 7, 8, 9 or 10 DAS, the final jaw-D;GR blade area progressively increased to a maximum of 46 mm 2 , nearly 1.5-fold bigger than the jaw-D blade (Fig 3A and  3B and S5A and S5B Fig). When TCP4 was induced at 10 DAS, the mature first leaf had pavement cell number (15420 ± 1220) that was close to the jaw-D value, but the cells (2940 ± 240 μm 2 ) were larger than the jaw-D cells (1870 ± 170 μm 2 ), ultimately yielding a mature leaf that was bigger than even the jaw-D leaf. It may be noted here that the jaw-D leaves were at the peak of its exponential phase of proliferation at 10 DAS, when almost all pavement cells were smaller in size (<1500 mm 2 ; Fig  These results, together with those shown in Fig 2, strongly suggest that TCP4 promotes maturation in proliferating pavement cells, perhaps by committing them to differentiation, within the developmental window of the proliferative phase.

TCP4 irreversibly changes the cell fate from proliferation to differentiation
Developmental cell maturation is a unidirectional process where the fully expanded pavement cells do not revert back to proliferative fate under normal circumstances. The transcripts of the miR319-regulated class II TCP genes that induce leaf differentiation are abundant in young lamina and their level steadily declines as the lamina matures [42]. Therefore, it is possible that these TCP genes activate the proliferation!differentiation transition in the pavement cells in an irreversible manner. To test this, we grew jaw-D;GR seedlings under TCP4 inductive condition for 0.5, 1, 2, 3, 4, 5 or 6 DAS, shifted them to non-inductive condition until 29 DAS (S4B Fig) and monitored leaf area and cell number of their mature first leaves (Fig 4A-4D). Arabidopsis seeds germinated (emergence of radicle) at 1 DAS and the first leaf pair initiated at 2 DAS ( Fig 4A). When the plants were grown for 0.5 or 1 DAS in the presence of DEX, there was no effect of TCP4 induction on the final leaf area (Fig 4B and 4C), possibly because the leaf primordia were barely formed at 1 DAS. When TCP4 was induced for 2 DAS, the leaf margin defect was totally rescued ( Fig 4B) and leaf area reduced to almost half ( Fig 4C). TCP4 induction for 4 DAS yielded mature leaf as small as what was observed with continuous DEX induction ( Fig 4B and 4C), suggesting that miR319-mediated inhibition of class II TCP transcripts for the first 2 days post leaf initiation (that is, 4 DAS), when the average length of leaf primordium was 274 ± 35 μm (Figs 4A and 5B), is crucial for normal leaf growth. Consistent with the reduction of blade area, TCP4 induction for 4 DAS also reduced the pavement cell number to 4550 ± 910, similar to the value obtained with continuous TCP4 induction ( Fig 4D). Lower cell proliferation activity was also reflected in the reduction of the cell cycle marker CyclinD3;2 [43] in young leaves. Up to 1 DAS of TCP4 induction did not reduce the GUS activity to a noticeable extent in 8-day old jaw-D;ProTCP4:mTCP4:GR X Pro-CyclinD3;2:GUS seedlings ( Fig 4E). However, the GUS activity much reduced when TCP4 was induced for 2 or 4 DAS, and totally disappeared at 6 DAS of induction.
In order to determine the minimum duration of TCP4 activity that is sufficient to induce differentiation commitment in proliferating cells, we provided pulse of TCP4 function of various durations from 0 to 48 hours in 4-day old jaw-D;GR seedlings and compared their mature leaf area at 29 DAS. Leaves without TCP4 induction grew to a maximum area of 36 ± 3.8 mm 2 (Fig 4F). A 3-hour pulse of TCP4 induction reduced the area to a noticeable extent, and the area continued to reduce with longer pulses, finally stabilizing to a minimum value (23.2 ± 2.0 mm 2 ) with a 24-hour pulse. This suggests that TCP4 activity for the duration of 24 hours is sufficient to impart commitment to differentiation in a dividing cell. Consequently, a 24-hour DEX pulse at 4, 6, 8, 9, 10 and 12 DAS (S6 Fig) altered the mature leaf area of mock-grown jaw-D;GR seedlings to nearly the same extent ( Fig 4G) as in continuous DEX-induction starting from the respective DAS ( Fig 3B). When we treated 3-day old jaw-D;GR seedlings with hydroxyurea, a widely used cell cycle inhibitor that induces reversible G1/S arrest [44,45], only for a duration of 36 hours, the mature first leaf grew as big as the untreated control (Fig 4H), implying that reversible cell arrest for a short while does not adversely affect the overall cell proliferation in a growing leaf. However, similar treatment with DEX reduced the final leaf size to a great extent (Fig 4H), suggesting that TCP4 initiates irreversible cell cycle arrest in a proliferating leaf.
When TCP4 activity was induced in the 35S:GR seedlings for 0.5, 1, 2, 3, 4, 5 or 6 DAS, the final leaf area progressively reduced with increased duration of induction ( Fig 4I, 4J and S7  Fig), as observed for jaw-D;GR leaves ( Fig 4B and 4C). However, when similar induction experiment was performed on 35S;sGR seedlings, the final leaf area remained unaltered, suggesting that miR319 activity at early stage of leaf growth contributes to blade expansion.
Increased cell maturation upon TCP4 induction was also reflected in the elevated level of nuclear ploidy, a typical cell differentiation marker [46], in cells of DEX-induced leaves. Flow cytometry analysis of the nuclei isolated from 8-day old Col-0 leaves revealed a ploidy distribution of 57.5% 2C, 39.6% 4C and 2.9% 8C cells (Fig 4K). In the jaw-D;GR leaves grown in mock condition, the proportion of 2C nuclei increased modestly to 69.5% and that of 4C (28.7%) and 8C (1.7%) nuclei decreased, suggesting a less differentiation status of this cell population. Conversely, when TCP4 function was induced in these leaves, the fraction of 2C nuclei reduced to 34.8%, with a concomitant increase in the proportion of 4C (55.7%) and 8C (9.5%) nuclei. Together these results demonstrate that TCP4 makes the proliferative cells exit division cycle and enter into endoreplication.

MiR319-mediated spatial regulation of class II TCP transcripts establishes the transition zone
To correlate the function of the miR319-TCP module with their expression dynamics in expanding leaf blade, we localized miR319c and TCP4 promoter activity and TCP4 protein at various growth stages of transgenic leaves expressing GUS reporter. In a 19-day old ProTCP4: GUS seedling, strong GUS activity was detected throughout the blade of the 9 th rosette leaf (1.2 mm long), which was progressively restricted towards the proximal end in the more mature 8 th , 7 th and 6 th leaves (Fig 5A). Since the maturation states of younger to older leaves in an Arabidopsis rosette recapitulate the ontogeny of a leaf on a specific node [17], it can be concluded that TCP4 promoter activity progressively gets restricted towards leaf base as the blade differentiates, before disappearing completely, as observed previously for TCP4 and its orthologues [8,14,47]. GUS activity in the ProCyclinD3;2:GUS leaves [43] resembled that of ProTCP4:GUS leaves (Fig 5A), suggesting that TCP4 promoter is active in the proliferative zone of the expanding leaf blade.
GUS reporter was detected in a small domain at the base of the young 9 th leaf (0.8 mm long) of PromiR319c:GUS seedlings (Fig 5A), and expression was reduced and restricted to more proximal region in the 8 th leaf (1.9 mm long); no detectable GUS activity was found in the 7 th and the 6 th leaves. These results are in agreement with the earlier reports [48], and suggests that miR319c promoter is active during a small temporal window at the earliest stage of leaf growth. Consequently, the TCP4:GUS reporter activity in young ProTCP4:TCP4:GUS leaf at the 9 th position was not detected in the corresponding basal region where miR319c promoter is active at this stage, suggesting that miR319 activity excludes TCP4 transcript [15] and its protein product (Fig 5A) from its expression domain. To compare the relative expression domains of miR319c, TCP4 transcript and TCP4 protein in young leaf primordia where miR319 promoter is active, we studied the GUS expression pattern in 4-day old transgenic seedlings where the first leaf pair just initiated (Fig 4A). Iñ 300 μm long first leaf, TCP4 and CyclinD3;2 promoters were active throughout the leaf, whereas the miR319 promoter was detected in a small region at the very base ( Fig 5B and S8  Fig). Activity of the TCP4:GUS fusion protein was, however, detected only in the distal half of young leaf primordia (Fig 5B and 5D and S8 Fig), and was excluded from the proximal region where miR319c promoter is active. Similar expression patterns have been reported for endogenous TCP4 transcript in wild-type [15], and for a miR319-susceptible form of ProTCP4:GUS transcript in transgenic leaf primordia [47].
In young fifth leaf primordium (~300 μm long), barely detectable TCP4:GUS activity was found towards the distal extreme (Fig 5C), implying that miR319 activity is present in a broader basal domain in this leaf. In agreement with this, strong GUS activity was detected across the basal half of a PromiR319c:GUS leaf primordium of similar length (Fig 5C). At a later growth stage, PromiR319c activity was restricted more towards the base, where it reduced the TCP4:GUS signal. Since TCP4:GUS activity retracted from the distal end in these leaves owing to the lack of ProTCP4 activity in differentiated cells, the TCP4:GUS protein was detected in a band-like domain in the middle of the leaf with diffused proximal and distal boundaries (Fig 5A, 5C and 5D), which has been proposed as the transition zone where proliferating cells acquire differentiation competence [3,4,8,24]. Ectopic expression of miR319 throughout the leaf blade completely abolished the TCP4:GUS activity in the jaw-D;GR X ProTCP4:TCP4:GUS seedlings (Fig 5D and S8 Fig), highlighting the importance of class II TCP activity within the transition zone in leaf maturation.
Cells proximal to the transition zone are expected to remain proliferative whereas the distal cells would differentiate. In agreement with this, the promoter activity of YUCCA5 (YUC5), a known differentiation marker [27,49], was initiated at the distal end in a~300 μm long first leaf of 4-day old ProYUC5:GUS seedlings, and the expression extended towards more proximal direction as the leaf matured (Fig 5E). The GUS signal reduced when the class II TCP genes were down-regulated in the jaw-D;GR X ProYUC5:GUS leaves (Fig 5F), and increased when gain-of-function TCP4 activity was induced by DEX application, suggesting that the distal expression of YUC5 in young leaves is indeed mediated by TCP4 activity.

TCP4-induced auxin response is required for pavement cell maturation
The expression dynamics of TCP4 described above, together with the kinematic data (Figs 2-4), suggests that TCP4 activity imparts irreversible differentiation fate to the proliferating cells within the transition zone of an expanding leaf blade. Auxin is known to play a key role in tissue maturation by coordinating the cell division with differentiation [1,50]. TCP4 function has been linked to the auxin biosynthesis and response in hypocotyl cells [27]. We observed that TCP4 activation increased GUS reporter activity in both jaw-D;GR X ProYUC5:GUS and jaw-D;GR X ProDR5:GUS rosettes (Figs 5F and 6A), demonstrating that TCP4 promotes YUC5 transcription and auxin response in developing leaves. In agreement with this observation, the levels of many SAUR and HD-ZIP II transcripts, which are known targets of auxin signaling [34, 51,52], were altered upon TCP4 induction (S9 Fig) when a previously reported jaw-D;GR microarray dataset was analyzed [27]. Fourteen SAUR transcripts were also elevated upon heightened auxin response or upon external application of auxin (Fig 6B) in the microarray datasets available in the GENEVESTIGATOR database [33,34, [51][52][53]. The SAUR and HD-ZIP II genes are known to be involved in multiple developmental events including cell proliferation and differentiation [34,37,50,51], suggesting a link between TCP4 function and auxin responsive genes in cell differentiation. RT-qPCR analysis demonstrated that the transcripts of SAUR19, 20, 21, 24, 62 and 63 increased in the jaw-D;GR seedlings grown under constitutive TCP4 activation (Fig 6C). In addition, GUS reporter activity in the jaw-D;GR X ProSAUR63: GUS seedlings increased upon dexamethasone treatment (Fig 6D), suggesting that TCP4 activates the SAUR genes in planta. SAUR activation was abolished in the jaw-D;GR seedlings when TCP4 was induced for 4 h in the presence of cycloheximide, a general protein synthesis inhibitor (Fig 6E) [54], suggesting that the promotion of SAUR by TCP4 is an indirect effect, possibly mediated by elevated auxin response. To examine whether the elevated auxin response by TCP4 is capable of inducing leaf cell maturity, we studied the effect of TCP4 induction on pavement cell area/number in short hypocotyl2-2 (shy2-2), where cells are insensitive to auxin response due to the sequestration of AUXIN RESPONSE FACTORs (ARF6 and 8) by a dominant, non-degradable form of AUX/ INDOLE-3-ACETIC ACID3 (IAA3) inhibitor protein [55]. Mature first leaves of jaw-D;GR X Col-0 heterozygous seedlings grew to 29 ± 2.6 mm 2 , consisting of 18850 ± 1670 pavement cells with average area of 1550 ± 165 μm 2 (Fig 6F, 6G and 6H). TCP4 induction in these leaves reduced the blade area to 19.0 ± 2.2 mm 2 and cell number to 5240 ± 650, while increased the average cell area to 3490 ± 180 μm 2 . The average area of mature jaw-D;GR X shy2-2 leaves (Fig 6I) under non-inductive condition was 18.0 ± 2.3 mm 2 , with the total cell number 14080 ± 1660 and average cell area 1350 ± 200 μm 2 (Fig 6F, 6G and 6H), possibly indicating a TCP-independent role for auxin response in leaf blade expansion. TCP4 induction, however, failed to reduce the jaw-D;GR X shy2-2 leaf area to any noticeable extent (Fig 6F), even though it increased the pavement cell area~2-fold, as in jaw-D;GR X Col-0 control leaves (Fig 6H). While TCP4 induction reduced the total pavement cell number by~75% in the jaw-D;GR X Col-0 leaves, it reduced the jaw-D;GR X shy2-2 cell number by~50% (Fig 6G and 6J), suggesting that at least a part of the TCP4 effect on the repression of cell number is mediated by IAA3-dependent suppression of auxin signaling.
The pavement cell population of the mature jaw-D;GR X Col-0 and jaw-D;GR X shy2-2 leaves showed similar size distribution under un-induced condition (Fig 6K), with~70% cells of smaller area (<1500 μm 2 ) and <10% cells of larger area (>3000 μm 2 ). Upon TCP4 induction, the fraction of the smaller cells decreased to~25% and that of larger cells increased tõ 50% in jaw-D;GR X Col-0, indicating a rescue of the cell size defect by TCP4 activity. The extent of rescue was, however, less (~40% smaller cells and~30% larger cells) in the jaw-D;GR X shy2-2 leaves, suggesting that TCP4 partly requires auxin response to induce cell maturity.

TCP4 activates HAT2 independent of auxin response
HAT2 and its closest homologue HAT1 have been recognized as immediate auxin responsive genes and control cell proliferation in leaves and cell elongation in hypocotyls, functions that overlap with miR319-targeted TCP genes [22,27,33,34]. HAT1 and HAT2 transcripts were consistently up-regulated in multiple independent microarray datasets performed upon external auxin application (Fig 7A) [34]. HAT2 transcript was also up-regulated at both 2 and 4 h of TCP4 induction in an earlier microarray experiment (S9 Fig) [27]. RT-qPCR analysis showed that the transcript of HAT2 alone, and not of HAT1 or the clade members, was up-regulated upon constitutive TCP4 induction in the Pro35S:mTCP4:GR seedlings (Fig 7B). HAT2 was induced within 1 h of TCP4 induction (Fig 7C), and the level was maintained for at least 4 h. In addition, HAT2 level was elevated in TCP4:VP16 seedlings, a gain-of-function line of TCP4 with enhanced transcriptional activity [23], while that was significantly reduced in the loss-offunction line jaw-D (Fig 7D). Together, these results suggest that HAT2 is an early downstream target of TCP4.
Since class II TCP proteins induce auxin biosynthesis [27,56] and HAT2 is an early auxin responsive gene, it would appear that HAT2 activation is an indirect effect of TCP4 induction. However, we found that TCP4 induction activates HAT2, but not the SAUR genes, in the jaw-D;ProTCP4:mTCP4:GR seedlings even when both auxin biosynthesis and polar auxin transport were inhibited by administering a combination of L-kynurenine and N-1-naphthylphthalamic acid (Fig 7E) [57]. Moreover, TCP4 induction in the jaw-D;GR X shy2-2 seedlings, where auxin response was genetically depleted by expressing a degradation-resistant form of IAA3 [55], also promoted HAT2 expression (Fig 7F). These results suggest that TCP4 is capable of promoting HAT2 expression independent of auxin response, and possibly by direct transcriptional activation.

HAT2 is a direct target of TCP4
Consistent with this hypothesis, TCP4 induction increased HAT2 transcript level in jaw-D; ProTCP4:mTCP4:GR and Pro35S:mTCP4:GR seedlings even when new protein synthesis was blocked by applying cycloheximide (Fig 7G and 7H). This suggests that TCP4 directly activates HAT2 possibly by binding to its genomic locus. Analysis of HAT2 genomic sequence yielded three putative TCP4 binding elements (BS1-3); two in the promoter region and the third in the coding region (Fig 7I). Electrophoretic mobility shift assay showed that recombinant MBP (maltose binding protein)-tagged TCP4 protein [20] binds to oligonucleotides corresponding to these elements with sequence specificity (Fig 7J). To test whether the TCP4 protein is recruited to the HAT2 chromatin in planta, we performed chromatin immuno-precipitation (ChIP) assay in the Pro35S:TCP4:3F6H transgenic line using anti-FLAG antibody [39]. TCP4 recruitment was observed in the HAT2 upstream regulatory regions that corresponded to the TCP4 binding motifs (Fig 7K), but no recruitment was found in the HAT2 coding regions. Consistent with this result, formaldehyde-assisted isolation of regulatory elements (FAIRE) experiment [58] suggested that the HAT2 chromatin assumed more open conformation in the regions corresponding to the putative TCP4-binding sites and not in a distant region when TCP4 was activated in the Pro35S:mTCP4:GR seedlings by dexamethasone treatment (S10A Fig).
Even though we identified one putative TCP4 binding site in the HAT1 upstream regulatory region and another in the coding region (S10B Fig), we suspected that HAT1 is not a target of TCP4 since its transcript level remained unaltered upon TCP4 induction (Fig 7B). ChIP assay also suggested that TCP4 protein is not recruited at the HAT1 locus (S10C Fig). Based on these results, and on the observation of a general overlap in the expression levels of TCP4 and HAT2 at various developmental stages (S11 Fig), we conclude that HAT2, and not HAT1, is a direct target of TCP4.

CIN-TCPs require HD-ZIP II transcription factors for limiting cell number and leaf size
Both TCP4 and HAT2 restrict cell number in leaves and promote cell expansion in hypocotyl [27,34]. Since TCP4 directly activates HAT2 (Fig 7), it is likely that TCP4 requires HAT2 activity in planta to promote developmental cell maturation. To test this, we crossed the jaw-D;GR line to the hat1;hat2 double mutant, since there is a high degree of functional redundancy between HAT1 and HAT2 and their single mutants cause little phenotypic alterations [59]. However, the dominant jaw-D phenotype was suppressed in the jaw-D;GR X hat1;hat2 F 1 individuals possibly due to transgene silencing, a phenomenon that commonly occurs with the viral 35S promoter [60,61]. To overcome this, we established the Col-0;GR;hat1;hat2 line and analyzed its leaf parameters upon TCP4 induction. The Col-0;GR leaves, which resemble Col-0 under non-inductive condition [27], were reduced to~38% in area upon TCP4 induction, primarily due to a corresponding reduction in the number of pavement cells (Fig 8A-8C) that were mildly but significantly larger than the un-induced cells (Fig 8D). Though the mature hat1;hat2 leaves resembled wild type leaves in shape and size (Fig 8A), they were made up of an increased number of pavement cells that were smaller in area (Fig 8B and 8D), an indication of differentiation defect frequently observed in the mutants of CIN-TCP genes (Fig 1) [17]. When TCP4 was activated in the absence of HAT1 and HAT2 gene function in the hat1;2;GR line, leaf blade area reduced only to~70% with a corresponding reduction in the pavement cell number. Further, TCP4 activation failed to sustain the increase in the pavement cell size in the absence of HAT function (Fig 8D). Taken together, these results suggest that TCP4 requires HAT2, and perhaps HAT1 as well, to limit cell proliferation and leaf size.
It has been shown that overexpression of HAT2 inhibits the leaf size and promotes the cell elongation in hypocotyl similar to that of TCP4 activity in leaf and hypocotyl, respectively [27,34,59]. Pro35S:HAT2 line shows only a mild effect in reducing leaf size and cell number in the wild-type background (60). However, this line reduced the size of the jaw-D leaf by~30% and its pavement cell number by~50% (Fig 8E-8G). Further, Pro35S:HAT2 rescued the smallsize phenotype of the jaw-D pavement cells nearly to the wild type level (Fig 8H and 8I).
Since HAT1 and HAT2 are functionally redundant partners and their overexpression yields comparable changes in leaf and hypocotyl [33-35,37], we tested whether HAT1 overexpression can also rescue jaw-D phenotype. Similar to TCP4 dominant effect (Fig 8A)  Error bars indicate SD. � indicates p <0.05. Unpaired Student's t-test was used. (J) A schematic diagram proposed to describe the mechanism of TCP4-induced commitment to differentiation of proliferating cells in a wild type leaf. The proliferative basal zone (green) remains devoid of TCP4 activity due to the action of miR319 and expresses cyclins and GRF [22,65]. The transition zone (red) above the base expresses miR319-regulated class II TCP proteins that induce cell commitment within this region via auxin response as well as HAT2 activation. TCP4 activity declines in the differentiated zone towards the tip (white), which expresses the direct targets of TCP4 such as YUC5 and miR396 [22,27]. Scale bars, 2 mm (A), 5 mm (E) and 100 μm (H).
https://doi.org/10.1371/journal.pgen.1007988.g008 TCP4 induces irreversible exit from cell proliferation HAT1 is a strong cell proliferation inhibitor in developing leaves. Moreover, HAT1 overexpression rescued the effect of class II TCP down-regulation on the leaf size and pavement cell number of the jaw-D;GR leaves nearly to the Col-0 values (S12A-S12D Fig). Intriguingly, overexpression of HAT1 resulted in a significant decrease in pavement cell area (S12E and S12F Fig), a phenotype that contrasts TCP4 overexpression. This negative effect of HAT1 on pavement cell size remained unaltered even in the jaw-D;GR background, possibly indicating an effect of HAT1 independent of TCP4.
Taken together, these genetic interaction studies show that TCP4 induces pavement cell maturation in part via direct activation of HAT2, and not HAT1. However, HAT1 (along with HAT2) is likely to be an indirect target of TCP4 as it is an auxin response gene (Fig 7A) [34] and TCP4 is known to promote auxin level through YUC5 activation (Fig 6A) [27]. Consequently, overexpression of each of these clade-members (HAT1 and HAT2) rescued jaw-D phenotype (Fig 8E-8I and S12 Fig).

Discussion
Constitutive down-regulation of class II TCP activity resulted in the accumulation of more cells with smaller average area while increased TCP function reduced cell number without affecting the average size (Figs 1-3) [22], thus confounding the exact effect of TCP proteins on cell proliferation and expansion. Frequency distribution of the pavement cell population based on cell area identified increased proportion of smaller cells in the jaw-D and tcp2;4;10 leaves, a defect that was rescued by TCP4 induction (Figs 1 and 2). Cell size in an organ primordium remains small as long as cells proliferate and starts to expand when they differentiate [2], suggesting that TCP4 converts the dividing cells to differentiation. This is further supported by our growth kinematics analysis that demonstrated that the effect of TCP4 on cell number is confined within the proliferation phase and TCP4 induction beyond this phase had no effects on final cell number and leaf area (Fig 3 and S5 Fig) [17], thus discounting any role for TCP4 in regulating differentiation-associated leaf cell expansion. TCP4 activity for the first two days of leaf initiation, when the primordium measured <300 μm in length, had the same effect on cell number and leaf size as observed with continuous TCP4 activity throughout the growth phase (Fig 4). Further, a 24-hour pulse of TCP4 activity in the leaf primordium reduced the final leaf area as much as the continuous TCP4 activity did (Fig 4). These results suggest that TCP4 imparts irreversible differentiation competence to the dividing cells of leaf primordium.
The mature miR319 is active at the base of young leaves (Fig 5A-5C) [48], restricting its target TCP transcripts towards more distal region of the blade [15,47]. Since the promoter activity of these TCP genes are excluded from the distal differentiated zone (Fig 5A) [62], their transcripts and protein products are restricted to a broad transition zone within a growing leaf blade that interfaces, and overlaps with, the more distal differentiation zone and the proximal proliferation zone, where they provide morphogenetic competence to the leaf blade ( Fig 8J) [8,24].
To explain the precise role of class II TCP proteins in leaf cell proliferation/ maturation, here we propose a model (Fig 8J) that incorporates the expression domain of the miR319/TCP module and the function of the TCP proteins based on the growth kinematics results. The transcripts of class II TCP genes are undetectable at the base of the leaf primordia due to the activity of mature miR319, even though their promoters are active in this zone. Lack of TCP activity helps the basal cells maintain their mitotic status [4]. Distal to this proliferation zone, TCP proteins are active in the transition zone where they commit the dividing cells to exit division and acquire differentiation competence. As the basal region expands due to cell proliferation, more dividing cells are incorporated within the transition zone where they encounter TCP proteins that commit them to exit cell division cycle. Since TCP activity imparts irreversible differentiation status to the leaf cells in the transition zone (Figs 3 and 4), the cells in the distal differentiation zone, though devoid of TCP activity, remain committed to differentiation and start expanding.
Our model explains the leaf phenotype with reduced or elevated TCP activity. Due to the absence of TCP proteins from jaw-D leaves (Fig 5D), increased proportion of cells within the transition zone remain mitotic and therefore smaller in size (Fig 2D) since fewer cells exit the division cycle (Fig 2E). This yields a lower cell area when averaged over the entire lamina ( Fig  2C). Higher proportion of mitotic cells in the jaw-D leaves ultimately results in increased cell number at maturity (Fig 2B). When TCP4 activity was induced in the transition zone of the jaw-D;ProTCP4:mTCP4:GR leaves, the proportion of cells exiting proliferation was restored to the wild-type level (Fig 2D and 2E), resulting in the correction of the defects in cell size and number. However, since jaw-D;ProTCP4:mTCP4:GR leaves express a dominant form of TCP4 that cannot be degraded by miR319 [15], the ectopic TCP4 activity at the base prematurely converts the proliferating cells to differentiated fate, thereby reducing the final cell number and leaf size at maturity (Fig 2).
Young leaf primordia with or without TCP4 induction contained nearly similar number of pavement cells (Fig 2B), but the TCP4-induced leaves had higher fraction of cells with increased ploidy compared to the un-induced leaves ( Fig 4K). Increased nuclear ploidy resulting from endoreplication cycle is used as a differentiation marker since it commits the cells at G2 phase to bypass mitosis and directly enter into the S-phase [46]. The cyclin-dependent kinase inhibitors KRP1 and KRP2 inhibit entry to mitosis and promote the onset of endoreplication in leaves [63,64]. Further, KRP1 is directly activated by TCP4 during leaf development [22], suggesting that KRP-induced endoreplication forms an important component of TCPmediated onset of cell differentiation within the transition zone.
Suppression of the GRFs that redundantly promote cell proliferation in the leaf primordia is yet another likely mechanism of TCP-mediated restriction of cell number in leaves [12,65,66]. This possibility is further supported by a recent observation that the promoter of miR396, a microRNA that degrades the transcripts of several GRF genes, is activated by TCP4 [22]. However, the effects of the miR319-TCP module on cell proliferation and differentiation are distinct from those of the miR396-GRF module in several aspects. First, TCP activation restricts the pavement cell number without a noticeable increase in size, whereas lack of GRF products results in a reduction in pavement cell number with a concomitant increase in cell size. Second, lack of TCP activity increases the cell number as well as decreases its average size, whereas overexpression of GRF proteins increase cell number without affecting the size. Third, while the class II TCP proteins are expressed only within the transition zone, the activity of miR396 promoter in the leaf primordia is initiated in the distal expansion zone and that of its target proteins is detected in the entire leaf base including the proliferation zone [12,22,66]. These differences point to a GRF-independent function of TCP proteins in promoting cell differentiation. Nevertheless, a yet unidentified link between the activities of TCP and GRF proteins within the transition zone cannot be ruled out.
The GRF-independent TCP function is likely mediated by multiple parallel pathways that possibly converge to the commitment of proliferating cells to differentiation within the transition zone. One arm of this function is via auxin response originating from TCP-mediated YUCCA activation [27,56]. An auxin minimum has been shown to promote a cellular switch from division to differentiation in root transition zone [50], whereas auxin response appears to be required for TCP-mediated differentiation commitment (Figs 6 and 8J). Auxin response activates the transcription of HAT2, a gene that restricts cell number and leaf size in Arabidopsis [33,34,37,38]. Consistent with this, hat1;hat2 leaves contain excess cells with reduced size (Fig 8B and 8D). Interestingly, the class II TCP proteins also promote division to differentiation switch in leaf pavement cells independent of auxin by directly activating HAT2 transcription (Fig 7). These parallel pathways possibly ensure a robust HAT activation by TCP proteins. The downstream events of HAT activation triggering cell differentiation are currently unclear. The possibility of the HAT transcription factors directly promoting the KRP cell cycle inhibitors to initiate endoreduplication, as shown for TCP4 [22], needs to be tested.
The TCP4 cDNA fragment of pBSKS-TCP4: GR [27] was digested with the SalI and BamHI and moved into pCAMBIA-1391Xa to generate TCP4:GUS cassette. The NotI released 2.16 kb ProTCP4 fragment of pGEMT-Easy-ProTCP4, described above, was end filled with Klenow DNA polymerase and cloned upstream of the TCP4:GUS cassette by blunt end ligation to generate ProTCP4:TCP4:GUS construct. The transgenic lines were generated in Col-0 background by Agrobacterium tumefaciens-mediated floral dip method [69].

Measurement of leaf size, epidermal cell size and cell number
For the measurement of leaf size the crinkled leaves were flattened and photographed using a Canon PowerShot S110 camera. Leaf area measurement was made in the photographs using Image J software (rsbweb.nih.gov/ij/). The first leaf from 29-day old plants were cleared with 70% alcohol and processed in choral hydrate:water:glycerol (8:2:1) solution and lactic acid for one to three weeks. In another method, leaves were collected in 70% ethanol followed by shifting them to ethanol:glacial acetic acid (7:1) and kept overnight (12-14 h) at room temperature. After clearing, the leaf tissues were treated with 1 M KOH for maximum 30 min in constant rotating followed by two times washing with double-distilled water. Image of abaxial epidermal cells were taken using differential interference contrast microscope (Olympus, USA) and analyzed with Image J software. For cell size analysis, 400-750 cells area was measured from different fields of 4-7 leaves and averaged to obtain the final cell size. The total number of cells per leaf was calculated by using the average cell size and leaf area.

Kinematic analysis
The kinematic analysis of Col-0 and mock and DEX-grown jaw-D;GR leaf development was performed as described earlier [5,9,41,70]. Initially seedlings were grown on Murashige Skoog (MS) medium up to 10 days and then the plants were transplanted to soil. For DEX induction, the MS medium was supplemented with 12 μM DEX or ethanol control. For continuous DEX induction, seedlings were transplanted to DEX (12 μM)-or ethanol (0.04%)-treated soil and sprayed with DEX or ethanol on alternate days until experiment was completed. One of the first leaf pair from at least 9-12 plants was used for each time point of kinematic study. The leaf area, cell number and cell size were analyzed as described above. The stomatal index was analyzed as described earlier [5,41].

Gene expression and GUS reporter analyses
Total RNA samples were isolated by Trizol (Sigma, USA) method after treatment with ethanol solvent (Mock) or 12 μM dexamethasone. To check the relative expression of respective genes, quantitative RT-PCR was performed using SYBER Green qPCR kit (SensiFAST SYBR Lo-ROX Kit, Bioline, USA) according to the manufacturer's instructions. Results were analyzed using ABI Prism 7900HT SDS software (Applied Biosystems, USA) and ΔΔCT values were determined after normalization with internal control. Intensity ratios were calculated using the formula 2 -ΔΔCT . Detailed experimental protocols for dexamethasone induction and primer sequences for RT-qPCR were described earlier [27,70]. GUS assay, EMSA and FAIRE experiments were performed according to the earlier protocols [9,27]. The list of primers used in this study is provided in S2 Table. Flow cytometry 1 st pair of leaves from 8-day old seedlings of indicated genotypes were collected after removing hypocotyl and cotyledons. Ploidy levels were measured using the protocol provided earlier [71,72]. In brief, leaves were placed in ice-cold Galbraith's buffer (45 mM MgCl 2 , 30 mM sodium citrate, 20 mM 4-morpholinepropane sulfonate, and 1% Triton X-100, pH 7.0) and finely chopped using sharp razor blade followed by homogenizing in the buffer. The homogenate was filtered through Miracloth (22-25 μm) and treated with 10 μl/ml RNaseA (1mg/ml stock) for 20-30 min followed by nuclear DNA staining with 50 μl/ml of propidium iodide (1 mg/ml stock) and kept in dark at 4˚C for 30 min. Samples were run through BD FACS verse (USA) flow cytometer and analyzed using BD FACS unit software. It was considered that the left-most peak corresponds to 2C [71,72] Chromatin immunoprecipitation (ChIP) assay ChIP protocol was followed as mentioned earlier [39]. In brief, approximately 1.5-2 gm of fresh 10-day old Pro35S:TCP4:3F6H seedlings were harvested and crushed into fine powder in liquid nitrogen. The powder was homogenized in the nuclei extraction buffer 1 (0.4 M sucrose, 10 mM Tris-HCl pH 8.0, 10 mM MgCl 2 , 5 mM β-mercaptoethanol, 0.1 mM PMSF, 1 mM Na 3 VO 4 , 1 mM NaF, and Complete protease inhibitor cocktail tablets [Roche]) and proceeded for cross-linking. 37% formaldehyde was used for crosslinking by gentle rotating in 4˚C for 10 min and 2 M glycine was added to stop the reaction. After quenching the cross-linked samples were filtered through two layers of Miracloth and centrifuged for 10 min at 10 k rpm at 4˚C. The pellets containing the nuclei were resuspended and washed twice with extraction buffer 2 (0.25 M sucrose, 10 mM Tris-HCl pH 8.0, 10 mM MgCl 2 , 1% (w/v) Triton X-100, 5 mM βmercaptoethanol, 0.1 mM PMSF, 1 mM Na 3 VO 4 , 1 mM NaF, and Complete protease inhibitor cocktail tablets) and transferred to ice-cold 1.5mL Eppendorf tubes followed by centrifugation at maximum rotor speed for 10 min at 4˚C to isolate the nuclei. The isolated nuclei were lysed using Nuclei lysis buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS, 1 mM PMSF, and Complete protease inhibitor cocktail tablets) and proceeded for sonication to shear the chromatin (~500 bp to 1 kb size fragments) using Bioruptor (Condition; 30 sec ON 45 sec OFF, 45 cycles). The sheared chromatin were diluted in ChIP dilution buffer (16.7 mM Tris-HCl pH 8.0, 167 mM NaCl, 1.1% Triton X-100, 1.2 mM EDTA, 0.1 mM PMSF, 1 mM Na 3 VO 4 , 1 mM NaF, and Complete protease inhibitor cocktail tablets) to make the volume up to 1.5 mL and centrifuged at maximum speed for 5 min at 4˚C. From the collected chromatin solution, 30 μL (2%) was kept as input for the assay and rest of the solution was divided into two parts from which one was immunoprecipited using anti-FLAG antibody (Sigma; F1804; 1 mg/mL) and the other using Anti-IgG antibody (Sigma; A9044). Magnetic Dynabeads Protein G (Life technologies) were used for Immunoprecipitation for which first the beads were pretreated with the anti-FLAG antibody (Sigma; F1804; 1 mg/mL) or anti-IgG antibody for 20 min by rotating at room temperature and diluted in ChIP dilution buffer after washing twice with 1X PBST. The antibody-bound beads were incubated with chromatin solution (26 μL Dynabeads and 3 μL antibody per 600 μL of chromatin solution) for 20 min in ice followed by 2 h rotating at 4˚C. After immunoprecipitation, the immunocomplex was washed using low salt buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 2 mM EDTA) and high salt buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 0.1% SDS, 1% Triton X-100, 2 mM EDTA) and TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA). To elute the immunocomplex from the beads, 50 μL of nuclei lysis buffer was added and incubated for 20 min at 65˚C and the elution was repeated twice. Eluted immunocomplex along with the input samples were reverse cross-linked by adding 6 μL of 5 M NaCl and incubating at 65˚C overnight. Proteinase K and RNase A was treated to digest all proteins and RNA, respectively, and finally DNA were cleaned up by phenol:chloroform:isoamyl alcohol (15:24:1) followed by precipitation using sodium acetate and 100% ethanol. The final extracted DNA was dissolved in 50 μL of nuclease-free water and proceeded for qPCR using 0.8 μL DNA as template per reaction.
To quantify the relative enrichment on the respective gene element, qPCR was performed using 0.8 μL of immunoprecipited DNA and SYBER Green qPCR kit (SensiFAST SYBR Lo-ROX Kit, Bioline, USA) in a 10 μL reaction volume according to manufacturer's protocol. Relative enrichment was calculated using the formula 0.02 X 2 (Ct input-Ct IP) X 100 and normalized to IgG control. The final fold enrichment was plotted in relative to Col-0 value.  Table. List of primers used in this study. (XLSX)