PhEXPA1, a Petunia hybrida expansin, is involved in cell wall metabolism and in plant architecture specification

Abstract

Expansins are wall-loosening proteins that induce wall stress relaxation and irreversible wall extension in a pH-dependent manner. Despite a substantial body of work has been performed on the characterization of many expansins genes in different plant species, the knowledge about their precise biological roles during plant development remains scarce. To yield insights into the expansion process in Petunia hybrida, PhEXPA1, an expansin gene preferentially expressed in petal limb, has been characterized. The constitutive overexpression of PhEXPA1 significantly increased expansin activity, cells size and organ dimensions. Moreover, 35S::PhEXPA1 transgenic plants exhibited an altered cell wall polymer composition and a precocious timing of axillary meristem development compared with wild-type plants. These findings supported a previous hypothesis that expansins are not merely structural proteins involved in plant cell wall metabolism but they also take part in many plant development processes. Here, to support this expansins dual role, we discuss about differential cell wall-related genes expressed in PhEXPA1 expression mutants and gradients of altered petunia branching pattern.

Expansins are pH-dependent wall-loosening proteins required for cell enlargement and expansions in many developmental processes.¹ Although to date their precise mechanism of action remains unclear, several lines of evidence point toward a role in dissociating the cell wall polysaccharide complex that links together wall components, thus promoting slippage between wall polymers and, eventually, expansion in cell wall.²

The characterization of Petunia hybrida PhEXPA1, an expansin preferentially expressed in petal limbs, has been reported by our group. By reporter genes assay, it was shown that PhEXPA1 is located in the cell wall of expanding tissues and its gene expression occurs during limb development and in axillary branch emergence zones. Both downregulation and overexpression of PhEXPA1 affected cell expansion and final organ size.^3,4 Moreover, both expansin expression mutants showed an altered cell wall polymer composition: while reduced PhEXPA1 levels lead to a decrease in crystalline cellulose and pectin content, its overexpression caused the reduction of pectin and hemicellulose amount in the cell wall. These results sustain the hypothesis that expansins promote cell wall creep and affect cellulose deposition during wall expansion.

Many biological functions had been ascribed to expansins.⁵ Fleming et al. proposed them as key regulators in early organ development by promoting physical stress in meristem and tissue bulging.^6-8 We have recently showed that 35S::PhEXPA1 transgenic plants exhibited an earlier axillary meristem outgrowth which resulted in an altered branching and inflorescence pattern compared with wild-type plants.³ This prompted us to hypothesize a role for PhEXPA1 in stimulating the outgrowth of petunia axillary buds, thus contributing to the shaping of the plant final habitus.

Cell Wall Related Genes are Differentially Modulated in PhEXPA1 Expression Mutants

A cDNA-amplified fragment length polymorphism transcript profiling (AFLP-TP) approach⁹ was initially used to identify Petunia hybrida genes showing a differential modulation between wild-type and 35S::antisensePhEXPA1 transgenic lines. Petal limbs harvested at four developmental stages, considering the altered petal growth rate affecting the antisense line (Fig. 1A,⁴), were used as starting material. Around 190 transcript-derived fragments (TDFs) displaying different transcription patterns between the two examined lines, have been sequenced. Among TDFs showing significant sequence homology with known genes, we found transcripts belonging to major cell-wall modifying gene families, suggesting a putative role of expanins in cell wall metabolism. To gain deeper insight about PhEXPA1 action, a β-1,3-glucanase, a β-1,4-glucanase, a pectinmethylesterase (PME) and a cellulose synthase transcript, were monitored by real-time RT-PCR in petal limbs of wild-type, 35S::antisensePhEXPA1 and in 35S::PhEXPA1 lines, harvested at the same four developmental stages used for cDNA AFLP-TP experiment (Fig. 1A).

Figure 1.

Differentially modulated genes in PhEXPA1 expression mutants. (A) Different petunia flower lengths corresponding to four developmental stages in wild-type and PhEXPA1 expression mutants. (B) Real-time RT-PCR analyses of four cell wall-related genes. Total RNA extracted from petal limbs of flower at the indicated developmental stages was pooled from different individuals of each plant line. Actin was used as control gene. Errors bars represent standard deviations (n = 3).

β-1,3-glucanases catalyze endo-type hydrolytic cleavage of the 1,3-β-D-glucosidic linkages in cell wall glucans. The β-1,3-glucanase transcription profile showed a positive correlation with the amount of PhEXPA1 transcript expressed in transgenic lines. Especially during the latest developmental stage, when flower expansion becomes more prominent, β-1,3-glucanase expression was lower in antisense plants and higher in overexpressors compared with wild type plants (Fig. 1B), suggesting that this class of enzymes can act downstream expansins. In addition, a member of β-1,4-glucanases family, cellulolytic proteins that hydrolyze the 1,4-β-glucosidic linkages between glucose residues, was downregulated in the latest petal developmental stages in PhEXPA1 antisense and overexpressor compared with wild-type lines. In our previous work⁴ we demonstrated that downregulation of PhEXPA1 reduces the amount of cellulose in floral cell walls. It might be inferred that β-1,4-glucanase can have a role in the deposition and/or organization of cellulose newly synthesized microfibrils, hydrolyzing not-crystalline cellulose regions. In this sense, β-1,4-glucanase would act in concert with expansins in the cell wall loosening process. Interestingly, Arabidopsis kor mutants for a β-1,4-glucanase showed a strong reduction in cell wall crystalline cellulose.¹⁰ Notably, in PhEXPA1 overexpression plants there was no effect on transgenic flowers cellulose content, possibly due to physical limitations in cell wall cellulose abundance,³ and β-1,4-glucanase was not strongly modulated except in the fourth stage when, concurrently, the β-1,3-glucanase genes show the highest expression level. Thus, at this stage, the two glucanases could complement their actions, responding to an altered cell wall polymers metabolism induced by the overexpression of PhEXPA1.

PMEs catalyze the specific demethylesterification of homogalacturonans within plant cell walls, releasing methanol and protons and creating negatively charged carboxyl groups in the process,¹¹ thus they can be included in the “cell wall loosening agents” pool promoting wall creeping and distension. The real-time analysis showed that PME expression pattern was unchanged in PhEXPA1 overexpressor line compared with wild-type, while in antisense plants the increase of PME expression level, especially at the third developmental stage, was strongly reduced. PMEs and expansins could act in a cyclic metabolic pathway in which the pH lowering due to the former enzymes action and the increasing in the cell wall polymers accessibility raised by the activity of the latter proteins exert a mutual influence, as inferred by PME expression pattern in 35S::antisense PhEXPA1 plant.

Lastly, a cellulose synthase transcript was differentially regulated in both PhEXPA1 antisense and overexpressor lines. As previously stated, PhEXPA1 amount influences the cellulose quantity in petunia limb cell wall,^3,4 so it is interesting to note here a transcriptional modulation of a cellulose synthase gene. However, since growing evidence indicates that different cellulose synthase genes are normally required to make a functional cellulose-synthesizing complex¹² and that different sets of genes are involved in the formation of the primary and secondary wall,² deeper studies are required to solve the complex relationship among cell wall polymers metabolic enzymes.

Overexpression of PhEXPA1 Affects Petunia Lateral Branching and Inflorescence Pattern

In our recent work, we provided evidence that PhEXPA1 might have a role also in plant development, particularly in plant architecture specification, affecting the timing of petunia axillary meristem outgrowth.³ The 35S::PhEXPA1 plants altered branching was here further investigated (Fig. 2). The abnormal outgrowth of axillary buds phenotype showed a gradient of intensity along the principal stem: as a result, while all transgenic plants count an extra inflorescence shoot at the 4th and the 3rd node, only the 75% and the 15% of examined plants (n = 40) did it at the 2nd and 1st node, respectively. The persistence of the altered phenotype was monitored also along the first lateral branching generated from the 4th node. Likewise the principal stem, the percentage of plants exhibiting an extra shoot decreases upward to the branching apex. Taken together, these observations corroborate our previous hypothesis that expansin activity could counteract petunia apical dominance in stimulating the development of lateral meristem. Indeed, we early showed that PhEXPA1::GUS expression is restricted to nodes and decreases toward the tips of the plant,³ on the contrary, apical dominance decreases toward the base of the plant. Whether expansin exerts this influence directly, i.e., through the control of cell expansion during the initial development of axillary meristem, or indirectly, i.e., through the determination of internode length, is still a matter of investigation. Interestingly, according to the auxin-inhibition hypothesis of apical dominance, apically-produced auxin is transported basipetally along the stem and inhibits axillary bud outgrowth in a concentration-dependent manner.¹³ However, auxin does not enter the bud, indicating that its inhibitory effects are indirect.¹⁴ As expansins expression is well-known to be regulated by auxin¹⁵ and promoter analysis revealed the presence of phytohormones-response elements, including auxin, also in PhEXPA1 promoter region, it is fascinating to suppose a putative role for expansins as auxin-secondary messangers for axillary bud activation.

Figure 2.

Altered petunia branching pattern in 35S::PhEXPA1 plants. Percentages of PhEXPA1 overexpression plants (n = 40), displaying the altered branching phenotype along the principal stem at each node (upper inner table), and along the first lateral branching at each node (lower inner table).

Zenoni S, Fasoli M, Tornielli GB, Dal Santo S, Sanson A, de Groot P, et al. Overexpression of PhEXPA1 increases cell size, modifies cell wall polymer composition and affects the timing of axillary meristem development in Petunia hybrida. New Phytol. 2011;191:662–77. doi: 10.1111/j.1469-8137.2011.03726.x.