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Meena, S.,Nagar, S and Meena, MK (2021) Brassinosteroids: A novel phytohormone regulating root growth in plants. Food and Scientific Reports. 2 (9) 43-51.

ABSTRACT

Plant survival largely depends on an appropriate root system for mechanical support and its function in water and nutrient uptake. The growth and development of a plant root system require coordinated regulation of endogenous stimulus as well as environmental signals. Previous studies demonstrated that plant root growth and development are inextricably linked with phytohormones. Plant growth regulators such as auxin, cytokinin, abscisic acid, brassinosteroids, ethylene, and gibberellins are involved in root growth through a range of complex interactions and exhibit either synergistic or antagonistic interactions. Brassinosteroids (BRs) are a class of polyhydroxylated steroidal phytohormone playing pivotal roles throughout the plant life cycle and are essentially required for plant growth and development, such as elongation, division and differentiation of cells, senescence, differentiation of vascular tissues, reproduction, photomorphogenesis, and responses to various biotic and abiotic stresses. A variety of BRs was identified in the roots of different plant species, such as maize and Arabidopsis. For root growth and elongation, BR ligand directly binds to the membrane steroidal receptor BRI1 (BRASSINOSTEROID INSENSITIVE 1) and triggering a signaling cascade in the cytoplasm that leads to the transcription of BR-responsive genes that drive cellular growth processes such as cell proliferation and cell differentiation. In this article, we summarize our current understanding of the BR functions in primary root development and growth.            

Keywords: Brassinolide, Reactive oxygen species, Hydrogen peroxide, Superoxide anion, Auxin, Ethylene, Cytokinin

 

Plants are a primary producers of food and they feed all the life forms such as human beings, animals, bacteria, fungus (provide substrate for decomposition), etc. Brassinosteroid is one of the recently discovered sets of naturally occurring plant steroidal hormones which have been found to play a crucial role in growth, development, and stress management (Vardhini et al., 2015). Recent studies showed that they can be synthesized in vitro (Bartwal et al., 2013) and interact with other phytohormones (cross-talk with biosynthesis, signaling, and homeostasis) regulates a wide range of physiological and developmental processes, and also plays an important role in mitigating stress in plants (Saini et al., 2015). Plant survival and existence in the environment largely depend on an appropriate root system and it is the important plant ground organs, which absorb water and nutrients to control growth and development. In higher plants, root growth is maintained by coordinating cell proliferation, differentiation, and elongation (Dello et al., 2008; Ji et al., 2015). Cell division is more dominant in the mitotically active meristem zone leads to the production of new cells, whereas their differentiation and elongation occur in the more proximal part of the root tip.

Phytohormones such as auxin, cytokinin, abscisic acid, brassinosteroids, ethylene, and gibberellins have been known to play a critical role in organ growth and development by regulating specific growth processes such as cell proliferation, differentiation, or expansion in distinct tissues (Ubeda-Tomas et al., 2008). It has been shown that several hormones are also involved in root growth through a range of complex interactions and exhibit either synergistic or antagonistic interactions (Pacifici et al., 2015). For example, ethylene regulates Arabidopsis seedling’s root growth either through modulating the auxin transport machinery or by enhancing auxin biosynthesis via upregulating the expression of ASA1 and ASB1 (Swarup et al., 2007). In addition, cytokinin was also found to control root growth through transcriptional regulation of the PIN genes and thus influencing auxin distribution (Ruzicka et al., 2009). It is very crucial for having a balance between auxin and cytokinin for proper root growth. In Arabidopsis, cell division and cell differentiation largely determine root meristem size, which is under the control of cytokinin and auxin through an ARR1/SHY2/PIN circuit (Dello et al., 2008). All these studies suggest that hormonal cross-talk plays a significant role in the regulation of root growth.

Brassinosteroid is one of the recently discovered sets of naturally occurring plant steroid hormone which has been found to play a pivotal role in various developmental processes such as elongation, division and differentiation of cells, senescence, differentiation of vascular tissues, reproduction, photomorphogenesis, and responses to various biotic and abiotic stresses (Clouse and Sasse, 1998, Divi and Krishna, 2009). It is also involved in the root growth and developmental process (Wei et al., 2016). They were originally discovered in Brassica napus pollen based on their ability to promote growth (Mitchell et al., 1970). Brassinolide, principally active component, was the first polyhydroxylated steroidal plant hormone isolated from rape pollen in crystalline form by Grove (1979) and it can be synthesized from campesterol (membrane sterol) through a series of reactions like reduction, hydroxylation, epimerization, and oxidation, etc. Till now, more than 70 brassinosteroid-related compounds have been identified and characterized so far from different plant organs including pollen grains, seeds, ad vegetative shoots (Zhao & Li, 2012).

Brassinosteroids (BRs) weas discovered as a stimulating agent for cell elongation and division in plants. Brassinosteroids are universally found throughout the plant Kingdom and play an essential role in modulating various phenomena related to the growth and differentiation at nanomolar to micromolar concentrations. They are also involved in promoting plant tropisms by modulating polar auxin transport (Xu, 2006). BRs are found in all the plant parts but their concentration varies from part to part. Young growing tissues such as pollen grain and immature seeds are the richest source of BRs than mature tissues such as shoots and leaves. The amount of BRs in growing tissues ranges from 1-100µg/kg (Fresh weight) and in mature tissues ranges from 0.01-0.1µg/kg (Fresh weight) (Clouse & Sasse, 1998).

 

Brassinosteroids perception mechanism

They are universally present in the entire plant kingdom at a very low level and regulate the several physiological processes involved in growth and development in young growing tissues. A large number of BRs was identified in the roots of different plant species, such as maize and Arabidopsis (Yokota et al., 2001; Shimada et al., 2003; Kim et al., 2005b). BR perception occurs at membrane-localized receptors but in the case of the animal systems, receptors for steroidal hormones are intracellularly located (Bartwal et al., 2013). It has been speculated that the action mechanism involved in brassinosteroids being similar to animal steroidal hormones. BRASSINOSTEROID INSENSITIVE 1 (BRI1), a leucine-rich repeat (LRR)-receptor-like kinase (RLK), which triggers an intracellular signaling cascade upon extracellular BR perception (Li & Chory, 1997). BRs are perceived by the extracellular domain of BRI1, just like a lock and key mechanism. BR-perception inactivates downstream cytosolic regulators such as GSK3/SHAGGY-LIKE kinase BRASSINOSTEROID-INSENSITIVE 2 (BIN2) by dephosphorylating a conserved tyrosine residue thereby enabling the activation of BRASSINAZOLE-RESISTANT 1 (BZR1) and BRI1-EMS-SUPPRESSOR 1 (BES1) to promote a transcriptional regulation of BR-responsive genes that drive cellular growth response genes expression such as cell wall biosynthesis and remodeling genes (Zhao and Li, 2012).

BRs are known to participate in root growth and development but mutations in BR synthesis and signaling pathways genes result in severe dwarfism, foreshortened roots (Lv et al., 2018), impaired growth, and development of organs, and limited plant fertility and hence effects the yield. It has been observed that single or double loss-of-function mutants (brl1 or brl3) lack perceptible phenotypes; however, the severe dwarfism of bri1 mutants is enhanced in bri1 brl1 brl3 triple mutants. It has been found that the expression of BR biosynthetic genes gets elicited under BR deficient conditions, while an increase in endogenous BR concentration has feedback regulation of the BR metabolic genes expression, hence in this way they can maintain the homeostasis of BR within the plant cell. It has been reported that the excessive application of bioactive BR hampered the normal growth and developmental pattern of plants (Zhu et al., 2013). Therefore, a BR homeostasis within the plant cell is important for the proper cellular function and developmental processes.

BR binding to receptor kinase BRI1 involves recruitment of the BAK1 (BRI1-ASSOCIATED RECEPTOR KINASE 1), a co-receptor kinase (Gou et al., 2012), and disassociation of the inhibitory protein i.e. BRI1 KINASE INHIBITOR 1 (BKI1)(Jaillais et al., 2011; Wang et al., 2011). BR binding promotes the transphosphorylation sequence between the kinase domains of BRI1 and BAK1 (Clouse, 2011). Phosphorylated BKI 1 also promotes the downstream cascade event by interacting with the 14-3-3 family of phosphopeptide binding proteins (Wang et al., 2011). Upon BR recognition by its plasma membrane-bound receptor BRI1 leads to phosphorylation of BRASSIONOSTEROID-SIGNALLING KINASE 1 (BSK1) and CONSTITUTIVE DIFFERENTIAL GROWTH 1 (CDG1), are plasma membrane-anchored cytoplasmic kinases (Kim et al., 2011; Tang et al., 2008). The above event promotes the binding of BSK1 and CDG1 and also phosphorylates BR1- SUPPRESSOR 1 (BSU1) phosphatase (Kim et al., 2011; Kim et al., 2009). BSU1 inactivates BRASSINOSTEROID INSENSITIVE 2 (BIN2), GSK3-LIKE KINASE by dephosphorylating a conserved tyrosine residue (Kim & Wang, 2010). Ultimately, activates the transcription factor which in turn governs the transcription of a large number of genes (Wang et al., 2013).

In the absence or low levels of BR, active BIN2 phosphorylates two homologous transcription factors, BRASSINAZOLE RESISTANT 1 (BZR1) and BZR2 {also known as BRI1-EMS-SUPPRESSOR 1 (BES1)} (Wang et al., 2002). This event results in abolish of their DNA-binding activity and retain in the cytoplasm by 14-3-3 protein (Bai et al., 2007; Gampala et al., 2007). When BR levels are high, BSUI becomes active and leads to BIN2 inactivation by proteasomal degradation (Peng et al., 2008). PP2A (PROTEIN PHOSPHATASE 2A) involves in the dephosphorylation of BZR1 and BZR2 (Tang et al., 2011). Unphosphorylated transcription factors (BZR1 and BZR2) can then move into the nucleus and binds to the target genes promoters, resulting in activation or repression of the genes (Sun et al., 2010).

 

The mechanism involved in Brassinosteroids mediated growth

In the absence of BR, the steroidal membrane receptor kinase BRI1 (BRASSINOSTEROID INSENSITIVE 1) remain under their inactive forms as it does not form a heterodimer with its co-receptor BAK1 (BRI1-ASSOCIATED RECEPTOR KINASE 1). Consequently, BIN2 (BRASSINOSTEROID-INSENSITIVE 2) which is a negative regulator of the BR signaling pathway, is constitutively express and results in the phosphorylation of BR-induced transcription factors, i.e., BZR1 (BRASSINAZOLE RESISTANT 1) and BES1 (BRI1-EMS SUPPRESSOR 1), hence inducing their interactions with 14-3-3 proteins that, in turn, promotes the cytoplasmic retention of BZR1/BES1 and also enhances the suppression of their DNA-binding activity by stimulating proteasomal degradation. On the other side, in the presence of BR, the BRI1 activation triggers its autophosphorylation and partial kinase activity and dissociates from its inhibitor BKI1, which is attached at the BRI1 kinase domain.

       

 

This leads to the formation of a heterodimer with BAK1, and transphosphorylation to complete BRI1 kinase activity. Activated BRI1 then phosphorylates the downstream elements such as BSKs (BR-SIGNALING KINASES) and CDG1 (CONSTITUTIVE DIFFERENTIAL GROWTH 1) which both in turn, phosphorylate BSU1 (BRI1 SUPPRESSOR 1, act as a phosphatase), leading to BIN2 dephosphorylation. BIN2 is subsequently restrained by KIB1 (KINK SUPPRESSED IN BZR1-1D), which prevents the association of BIN2 with BZR1/BES1 and facilitates its ubiquitination and degradation.The inactivated form of BIN2 allows transcription factors i.e., BZR1 and BES1, to enter into the nucleus and regulate the expression of BR target genes. Additionally, PP2A (PHOSPHATASE 2A) also positively regulates the brassinosteroids signaling transduction pathway in higher plants (Peres et al., 2019).

 

Brassinosteroids regulates root growth by ROS signaling

Besides plant hormones, the regulation of root growth has also been tightly linked to reactive oxygen species (ROS). While ROS were earlier believed to merely represent a harmful by-product of the plant’s stress response, but nowadays they have been recognized as signaling molecules involving in various growth and development responses (Mittler et al., 2013).  ROS are an important driver of plants root growth that includes cell proliferation and cell differentiation and it has been proposed that play a critical signaling molecule during lateral root formation (Tsukagoshi et al., 2010). It has been reported that ROS produced in mitochondria localized P-loop NTpase of root tip cells were also involved in the regulation of the quiescent center cell division and maintain distal stem cell identity by regulating the ROS level inside the Arabidopsis root cell (Yu et al., 2016). In cucumber plants exposed to exogenous BR, H2O2 accumulates as a result of increased activity of NADPH oxidase (Xia et al., 2009), while in tomatoes, the same result is achieved by the up-regulation of RBOH1 (Yu et al., 2016). A BR receptor-mediated increase of the cytosolic concentration of calcium ions (Ca2+) stimulates NADPH oxidase-dependent ROS production (Ogasawara et al., 2008). It has been found that BR serves as a stimulator for root growth in A. thaliana seedling by inhibiting the synthesis of O₂ - via the peroxidase pathway rather than via the NADPH oxidase pathway (Lv et al., 2018, Tian et al., 2018). It has been reported that the det2-9 mutant is the O₂ - hyper-accumulator, which in itself likely contributed to the short root phenotype. The accumulation of the O₂ - is also partially controlled by ethylene signaling in a peroxidase-independent manner and the O₂ - accumulation can enhance ethylene signaling by increasing the expression of ACSs and ACOs. Thus, although the participation of BR in root growth and development is accepted, it’s cross-regulation with ROS signaling in root growth is largely unknown.

 

Brassinosteroids regulates root growth by ethylene biosynthesis

Ethylene inhibits unidirectional cell expansion via its well-established and complex interactions with auxin, which involve mutual elevation of their corresponding biosynthesis genes and auxin transport from the root tip to elongating cells (Ruzicka et al. 2007; Swarup et al. 2007; Robles et al. 2013). Previous studies    have    shown    that    exogenous    application    of    BRs    stimulates  ethylene production in Arabidopsis seedlings (Woeste et al. 1999), but it is remains elusive that the significance of this effect during root growth (Mussig et al. 2003). Hence, while moderate BR levels have promotory effects i.e. promotion of root growth, their high levels are inhibitory  (Mussig et al. 2003). This inhibitory effect has been recently explained by a premature cell exit from mitosis (Gonzalez-Garcia et al. 2011). Under normal growth conditions, there is a low level of BR, which inhibits the biosynthesis of ethylene by activating the transcription factors BZR1 and BES1. These transcription factors bind directly to the promoters of the ACS gene, thereby damping the level of ethylene by suppressing ACS expression.

 

Brassinosteroids regulates root growth by auxin signaling

Over the years, there are two important phytohormones i.e. BR and auxin, that have been considered as BR and auxin have been considered as master regulators in different plant development processes such as root development and stem elongation (Wei et al., 2015; Zhao et al., 2010). The developmental phenomenon in roots has been determined by the balance between cell division and differentiation in the root apical meristem (RAM). It has been observed that despite the well-known synergistic interaction in various developmental processes, BR and auxin interact antagonistically in controlling gene expression, stem cell maintenance, and cell elongation in the case of root tips.. Additionally, a finely balanced concentration between these hormones is required for optimal root growth (Chaiwanon et al., 2015). It has been observed that BR controls the size of  root apical meristem by regulating root growth in a concentration-dependent manner.

The high levels of BR induce ethylene biosynthesis either through increasing the stability of ACSs such as via ACS5 and ACS9 protein (Hansen et al., 2009) or influencing auxin signaling regulated ethylene production. The possible regulation mechanism involves the BZR1 interaction with ARF proteins which directly targets multiple auxins signaling components and genes involved in auxin metabolism such as transport and signaling, including AUX/IAA, PINs, TIR1, and ARFs, etc. (Sun et al., 2010). It has been reported that functional BR biosynthesis is partly required for auxin-dependent gene expression. This has been proved by the exogenous application  of  brassinolide  (BL) that could   induce   the   expression   of   auxin-responsive   genes   such as IAA5, IAA19, IAA17, etc., but in the BR biosynthetic mutant de-etiolated2 (det2), the expression level of the above genes is down-regulated (Nakamura et al., 2003; Kim et al., 2006). DET2 encodes a steroid 5α-reductase involved in BR biosynthesis, catalyzing the formation of campestanol with campesterol as substrates. A recent study has found that ARF3 acts as a repressor or activator depend on auxin concentration (Li et al., 2002).

It was reported that some proteins such as Aux/IAA proteins, involved in the auxin signaling pathway are also involved in BR responses and mutation in this protein (i.e., iaa7/axr2-1 and iaa17/axr3- 3 mutants) showed aberrant BR sensitivity and aberrant BR-induced gene expression in an organ- dependent manner (Nakamura et al., 2006). Additionally, it also affects the flow of auxin by regulating the expression of auxin exporters such as PIN4 and PIN7 (Nakamura et al., 2004). During gravitropic responses in plants, BRs affects the redistribution of auxin from the root tip towards the elongation zone by enhances the polar accumulation of the auxin exporter PIN2 in the root meristem zone, result in the difference of IAA levels in both sides of roots i.e., upper and lower, hence leads to the induction of the plant gravitropism. During this process, BR activated ROP2 plays an important role in modulating the functional localization of PIN2 through the regulation of the assembly/reassembly of F-actins (Li et al., 2005). Further studies showed that decreased perception and/or concentration of BL could induce CYP79B2, the gene encoding an enzyme converting tryptophan to indole-3-acetaldoxime and thus diverting the auxin biosynthetic pathway, hence, also affect the distribution of auxin. (Kim et al., 2007).

In addition, it was found that BR as a signaling agent could regulate auxin signaling output by BIN2, which is BR negative regulator GSK3 kinase. Studies such as yeast two-hybrid screen showed that BIN2 could phosphorylate ARF2 by acting as a BIN2 interacting factor. The phosphorylation of ARF2 by the interaction of BIN2 results in the loss of its DNA binding ability and repression activity of the target genes (Vert et al., 2008). ARF2 expression is reduced by BR treatment and it acts as BZR1 target genes (Sun et al., 2010). Besides ARF2, BIN2 can phosphorylate ARF7 and ARF19 which leads to the suppression of their interaction with AUX/IAAs and thereby enhancing the transcriptional activity on their target genes such as  LATERAL ORGAN BOUNDARIES-DOMAIN16 (LBD16) and LBD29,  to regulate lateral root organogenesis (Cho et al., 2014). However, BR plays a minor role during this process and BIN2 is under the regulatory control of the TRACHEARY ELEMENT DIFFERENTIATION INHIBITORY FACTOR (TDIF)–TDIF RECEPTOR (TDR) module (Cho et al., 2014). Together, BR can regulate auxin responses by influencing different signaling components of the auxin.

                                   

 

The perception of BR by its plasma membrane-bound steroidal receptor i.e., BRASSINOSTEROID INSENSITIVE 1 (BRI1), which in turn, controls the biosynthesis of ethylene in a dose-dependent manner by activating the BR signaling cascade. Under low levels of BR, ethylene biosynthesis is inhibited by activation of BZR1 and BES1 transcription factors which suppress the transcription of ethylene biosynthetic genes such as ACSs.  BZR1/BES1 binds to the promoter of the ACC synthase (ACS) and ACC-oxidase (ACO) genes, thus inhibiting their transcription and consequently repressing the biosynthetic pathway of ethylene (Peres et al., 2019). While high levels of BR induce the biosynthesis of ethylene either through enhancing the stability of the ACSs proteins by preventing its degradation by the 26S proteasome or influencing auxin-regulated ethylene biosynthesis. At the same time, BR serves to regulate the growth of the A. thaliana seedling root by inhibiting the synthesis of O^{2 -} via the peroxidase pathway, but not the NADPH oxidase pathway (Lv et al., 2018).

 

Conclusion

Phytohormones act as the signaling molecules in controlling plant growth and developmental processes.  Plant hormones play a critical role in regulating various plant developmental processes and the flexible shaping of the plant architecture in response to variable environmental conditions. The developmental and physiological processes regulated by the hormonal signaling in plants are the typical result of combined actions of several hormonal pathways. It is known that BRs influence several biological processes, such as growth, metabolism of protein, transport and signaling in the cell, biosynthesis of the cell wall, the formation of chromatin and cytoskeleton components, stomatal closure, and environmental responses. BRs play a crucial role in the root meristem size regulation and development of lateral roots in a concentration-dependent manner. A low concentration of BRs promotes root growth by inhibiting the ethylene biosynthesis, whereas the high concentration of BRs inhibits root growth by influencing the ethylene biosynthetic pathway or auxin-regulated ethylene synthesis. It also interplays with other signals for the regulation of root growth and development at various stages either synergistically or antagonistically. Studies showed that BRs are well recognized as promoters of cell elongation. They control the root elongation process in plants either positively or negatively regulated ethylene biosynthesis in a concentration-dependent manner. Based on the current understanding and several research evidence, it has been proposed that BRs controls the growth of roots through the regulation of ethylene biosynthesis and superoxide anions accumulatio

 

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Brassinosteroids A novel phytohormone regulating root growth in plants_compressed.pdf