Unraveling mechanisms of cell expansion linking solute transport, metabolism, plasmodesmtal gating and cell wall dynamics

Abstract

Cell expansion is a major component of plant cell development and plays a key role in organ growth, hence realization of crop productivity. Thus, unraveling mechanisms controlling plant cell expansion is essential not only for understanding fundamental plant biology but also for designing innovative approaches to increase crop yield and quality. The multi-cellular plant tissues, however, impose enormous technique challenges to assess the contribution of molecular or cellular events to the expansion of given cell types as they often deeply embedded within the tissues, thus are not readily accessible for sampling or measurement. In this context, cotton fibers, single-celled hairs developed from the seed coat epidermis represent an ideal system for studying regulation of cell expansion, owing to their rapid and synchronized elongation (up to 3∼5 cm long) and high accessibility for experimentation. Recently, we demonstrated the essential role of vacuolar invertase (VIN) in early fiber elongation. Remarkably, we discovered that VIN controls cotton fiber and Arabidopsis root elongating through osmotic dependent and independent pathways, respectively. This shows mechanistic complexity of cell expansion. Here, we evaluate the coordinated actions of multiple pathways in regulating cotton fiber elongation linking solute transport and metabolism with plasmodesmatal gating, water flow and cell wall dynamics and we outline future directions for deepening our understanding of plant cell expansion.

Cotton fibers are trichome-like single cells derived from the epidermis of the outer seed coat. The cellulosic mature cotton fiber is of great economical importance for the textile industry. The development of cotton fibers is classified into four distinct but overlapping stages: cell initiation (−1 to 0 days after anthesis [DAA]), elongation (0 – ∼20 DAA), secondary cell wall cellulose synthesis (∼16 – ∼30 DAA), and maturation (∼30 – ∼60 DAA)¹ (see Fig. 1). Cotton fiber can elongate to 3–5 cm within ∼20 DAA in the cultivated tetraploid species, rendering them one of the longest and fastest growing cell type in the plant kingdom.² Given their unicellular nature, rapid and synchronized growth pattern, high degree of accessibility for harvest and growth measurement, cotton fiber has proven to be an excellent system to study the mechanisms of cell expansion.³

Figure 1.

A schematic diagram of temporal expression pattern of turgor-related genes in cotton fiber of Gossypium hirsutum. The degree of darkness in a given bar represents level of gene expression as such that the darker the color the higher the gene expression level. Dash lines indicate the period when no expression data are available. GhSUT1, GhKT1, GhEXP1 are genes encoding sucrose and K⁺ transporter and expansin, respectively;⁵ GhSus: sucrose synthase genes;¹⁰,¹⁹ GhVIN1: vacuolar invertase gene;¹² GhPIPs and TIPs encode plasma membrane and tonoplast intrinsic proteins, respectively (Ashley J, Patrick JW, Ruan YL, unpublished data); GhPEPC 1 and 2, and GhGluc1 are genes encoding phosphoenolpyruvate carboxylase¹¹ and β1, 3-glucanase,⁹ respectively. PD: plasmodesmata.⁵,⁹

As an unidirectional cell expansion process, cotton fiber elongation is a result of a complex interplay between cell turgor and cell wall extensibility.^4,5 These two cellular processes require extensive involvement of various transport, metabolic and biosynthetic pathways, and not surprisingly, are coordinately regulated by developmental signals.⁶ Cytological studies, along with transcriptional investigations, revealed that after a slow start, the elongation rate of cotton fiber increases at ∼2–3 DAA followed with a log phase during the first ten days.^1,7 The elongation reaches the maximal rate at ∼10–15 DAA⁵ and rapidly declines at ∼16 DAA onwards when fibers switch to intensive secondary cell wall cellulose biosynthesis.^5,8 The rate and duration of fiber elongation establish the final length attained, which is a key factor determining fiber quality and yield.

Over the last two decades, great effort has been made to identify genes and cellular mechanisms controlling fiber elongation, primarily focusing on transport of solutes into^5,9 and metabolism within fiber cytoplasm.^10,11 However, little is known how osmotically active solutes are accumulated in the vacuole driving the influx of water for cell expansion. In this context, the recent discovery on the important role of vacuolar invertase (VIN) in cotton fiber and Arabidopsis root elongation¹² represents a major advance in our understanding of mechanisms controlling plant cell expansion. Here, we provide an assessment on our current understanding on the coordinated action of multiple pathways regulating fiber elongation and discuss the underlying implications and future directions in our effort to elucidate the molecular and cellular basis of cell expansion, a key component for plant cell development and realization of crop yield.⁶

Multiple Players in Controlling of Cell Turgor during Fiber Elongation

Cell turgor is generated and maintained through influx of water down a water potential difference driven by the relatively high concentration of osmotically active solutes.¹³ In elongating fibers, soluble sugars, potassium and malate are the three major osmotic active solutes, together accounting for ∼80% of fibre sap osmolality.^2,14 Potassium and sucrose are imported from the underlying seed coat cells, either symplasmically through the plasmodesmata (PD) or apoplasmically across cell wall space and plasma membrane by their respective transporters, energized by the plasma membrane and tonoplast H⁺-ATPases, respectively.^4,5 GhKT1, encodes a cotton K⁺ transporter, is weakly expressed in fibers at 6 DAA when the PD are open. However, its mRNA abundance increases significantly at 10 DAA when the PD are closed and then rapidly decreases to undetectable level at 16 DAA when the PD are re-opened (Fig. 1).⁵ GhSUT1, a plasma membrane H⁺/sucrose symporter localized at the base regions of fibers, displays a similar temporal expression pattern with that of GhKT1⁵ (Fig. 1). The closure and re-opening of PD are probably mediated by deposition and degradation of callose catalyzed by β1,3-glucan synthase and β1,3-glucanase, respectively.⁹ Importantly, the duration of PD closure correlates positively with the fiber length at maturity, indicating a critical role PD may play in regulating fiber elongation.⁹ It is hypothesized that the closure of PD and the coordinated upregulation of GhKT1 and GhSUT1 at the mid-phase of elongation (fibers are at ∼1 cm long at this stage) may provide a cellular basis for the fibers to sustain turgor pressure to drive fiber elongation to further extent.^2,5,9 However, the precise role of PD closure and opening and the casualty between PD closure and expression of the plasma membrane GhKT1 and GhSUT1 during fiber elongation remain to be determined.

Once taken up into the fiber cells, sucrose could be degraded into UDPglucose and fructose by sucrose synthesis (GhSus) in the cytoplasm¹⁰ or hydrolyzed by acid invertase into glucose and fructose in the vacuole, thus doubling the osmotic contribution of sucrose.¹² While collective expression level of GhSus gene members appears to be strong throughout the fiber elongation period¹⁵ (Fig. 1), GhVIN1, a major vacuolar invertase in cotton fiber, shows evidently higher expression level early in fiber elongation (0–10 DAA) matching high VIN activity observed in this stage.¹² Transgenic analyses revealed that suppression of GhSus expression reduced fiber length and cellulose content¹⁰ and silencing GhVIN1 decreases fiber elongation.¹² These findings indicate that (1) GhSus play roles in both fiber elongation and cellulose biosynthesis through osmotic regulation and supplying UDP-glucose as substrate for cellulose production and hexoses for generation of ATP as an energy source for a variety of transport and metabolic processes (2) GhVIN1 appears to be more specifically required for the formation and enlargement of the central vacuole, pivotal for early fiber elongation (see below).

Unlike potassium and sucrose that are imported into fibers from phloem in the seed coat, malata is synthesized within the fiber cells by refixing CO₂ through the activity of phosphoenolpyruvate carboxylase (GhPEPCs).^1,4 GhPEPC 1 and 2 are two major PEPC cDNAs highly expressed cotton fiber.¹¹ Their transcription levels, along with PEPC activities and malata concentrations, are relatively high early in elongation, increase to maximal level around 10–12 DAA, and decline rapidly at 15 DAA onwards when elongation is dramatically slowed down¹¹ (Fig. 1).

The accumulation of osmotically active solutes, sugars, K⁺ and malate (see above) lower the water potential of the cell to ‘attract’ water influx. Transcellular water flow to the fiber cells appear to be facilitated by a group of aquaporins, mainly plasma membrane and tonoplast intrinsic proteins (PIP and TIP).^4,16,17 The observation that a number of GhPIPs and GhTIPs are abundantly expressed in elongating but not in elongated fibers (Ashley J; Patrick and Ruan YL unpublished data) indicates concerted actions between genes controlling solute accumulation and water uptake.

GhVIN1 as a Key Player in Early Fiber Elongation Likely through Osmotic Regulation of Vacuole Enlargement

Despite the major progress in understanding cotton fiber elongation as discussed above, much less is known about the formation of the central vacuole which accounts for >90% of fiber volume during elongation. Recently, we demonstrated that high activity of vacuolar invertase, probably mediated by GhVIN1, contributes to rapid cotton fiber elongation both developmentally and genotypically.¹² In situ localization of invertase activities and GhVIN1 mRNA levels revealed much stronger VIN activity and mRNA signals in fiber initials than the adjacent non-differentiated epidermal cells at 0 DAA and no signals were detected in seed surface at −1 DAA prior to fiber elongation.¹² Most noticeably, our in situ hybridization analyses showed that GhVIN1 transcript exhibited highest level in fibers at 0–2 DAA and were maintained at high level at 5 and decreased at 10 DAA onwards. These observations, together with the finding on the reduction of fiber elongation by RNAi-mediated silencing of GhVIN1, identified GhVIN1 as a key player in early fiber elongation.

Cotton fibers start their elongation on the day of anthesis (Fig. 1) characterized by their protruding above the seed coat epidermis through the formation of a large central vacuole at 0–2 days after anthesis.^18,19 The high VIN activity at this stage would rapidly hydrolyze sucrose into two molecules of hexoses in the vacuole, hence representing a major osmotic force to drive the influx of water to expand the vacuole. Consistently, a tonoplast aquaporin, GhTIP1,2 exhibited highest expression in cotton fiber at 5 DAA as compared to later stage of elongation (Jones A, Patrick JW, Ruan YL, unpublished data), which would facilitate the influx of water across the tonoplast. It will be fascinating to explore the potential signaling pathway linking GhVIN1 and GhTIP1,2 expression and activity. It will also be important to examine the possibility that, in addition to its osmotic regulation, GhVIN1 may contribute to early fiber elongation through other mechanisms such as those involving in cell wall loosening exerted by expansin⁵ (Fig. 1) or wall-associated kinase (WAK).¹²

In conclusion, extensive studies on cotton fiber elongation have shed a light on the mechanisms underlying the complex and dynamic plant cell expansion process which may be summarized as follows: First, at 0–2 DAA, high activity of VIN rapidly doubles the osmotic contributions of sucrose, which would drive the influx of water into vacuole for its enlargement, hence the protrusion of the fibers above the seed epidermal surface.²⁰ Second, with the rapid enlargement of fiber cell, it becomes increasingly difficult to maintain cell turgor. Thus, the expression of multiple turgor-generating genes, such as GhSUT1, GhKT1, GhPEPCs, in combination with GhVIN1 and GhSus, become necessary to maintain a lower water potential in the fibers to drive continuous influx of water for the log phase of expansion. The high expression of expansin gene GhEXP1 at this stage (Fig. 1) would increase the cell wall extensibility, allowing fiber elongation to proceed readily. Third, at ∼10 DAA, coinciding with the peak expression levels of turgor-generating genes, GhSUT1 and GhKT1, fiber PD are closed which may provide a mechanism to generate and maintain a high turgor driving the maximum rate of elongation occurring at ∼12–13 DAA. Finally, by ∼16 DAA onwards, the expression of turgor-generating genes is dramatically reduced and fiber PD, are re-opened, which releases turgor. This together with the increased cell wall rigidity as indicated by diminished expression of expansin⁵ terminates the elongation process (Fig. 1).

There is no doubt that the cellular and molecular events underpinning fiber elongation outlined above provide exciting opportunities for further studies to elucidate the signaling pathways that interconnect and modulate activities of multiple players. This includes for example, (1) the identification of signaling components that may couple the VIN activity with aquaporins or cell wall properties and (2) the linkage between plasmodesmatal gating and sugar signaling mediated by sugar transport and metabolism. Moreover, the rapid elongation rate and the high accessibility of fiber cell for harvesting and experimentation allows quantitative assessment of contributions made by a given protein such as an aquaporin to cell expansion. Indeed, cotton fiber is ideal for single-cell systems biology analyses of cell expansion and cell wall cellulose biosynthesis in both qualitative and quantitative manners.