SummaryNovel classes and applications of cell-penetrating peptides (CPPs) are being constantly discovered since they were first identified 2 decades ago. These short cationic peptides (nanomolecules) either by covalent binding or by noncovalent binding can traverse cell membranes and deliver a variety of molecules that are unable to overcome the permeability barrier in their own capacity. The ability of the CPPs to deliver variety of macromolecules, such as oligonucleotides, therapeutic drugs, proteins, and medical imaging agents, by forming nanoparticulate carriers in a range of cells has led them to emerge as a potential tool for both macromolecule delivery application and to gain insight into the fundamentals of mechanism of cellular uptake across the plasma membrane. This review explores the recent advances, challenges, and future prospects in the field of CPP-mediated cargo delivery in mammalian and plant cells. Studies have been conducted into the peptide chemistry and stability of CPP-macromolecular complexes. Most of the CPPs have been shown to be nontoxic and do not interfere with the functionality of the macromolecules delivered across the cell membrane. The mechanism of uptake of CPP-cargo complexes and the uptake of CPPs alone across the plasma membrane remains unresolved. As the world of CPPs is rapidly advancing in both mammalian and plant system, there is a promising future for the various applications of transduction and transfection into intact cells.

Microspore culture is contributing significantly in the field of plant breeding for crop improvement in general and cereals, in particular. In the present study, we investigated the uptake of fluorescently labeled cell-penetrating peptides (CPP; Tat, Tat(2), M-Tat, peptide vascular endothelial-cadherin, transportan) in the freshly isolated triticale microspores (mid-late uninucleate stage). We demonstrated that Tat (RKKRRQRRR) and Tat(2) (RKKRRQRRRRKKRRQRRR) are able to efficiently transduce GUS enzyme (272 kDa) in its functional form in 5 and 14% of the microspores, respectively, in a noncovalent manner. Pep-1, a synthetic CPP, was able to transduce GUS enzyme in its active form in 31% of the microspores. The effect of various endocytic and macropinocytic inhibitors on Tat(2)-mediated GUS enzyme delivery was studied and revealed a preferred micropinocytosis entry. DNase I protection assay and confocal laser microscopy was carried out to recommend a ratio of 4:1 Tat(2)-linear plasmid DNA (pActGUS) in complex preparation for microspore transfection. We further show that Tat(2) can successfully deliver GUS gene in near to 2% triticale microspores. The negative control mutated Tat (M-Tat: AKKRRQRRR) failed to transducer the GUS protein and transfect the GUS gene in microspore nucleus. The ability of CPPs to deliver macromolecules (protein as well as linear plasmid DNA) noncovalently has been demonstrated in triticale isolated microspores. It further confirms potential applications of CPPs in developing simple, time saving, cost effective plant genetic engineering technologies.

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The plant hormone auxin plays a central role in regulating many aspects of plant growth and development. This largely occurs as a consequence of changes in gene expression. The Aux/IAA genes are best characterized among the early auxin-responsive genes, which encode short-lived transcriptional repressors. In most plants examined, including Arabidopsis, soybean, and rice, the Aux/IAA genes constitute a large gene family. By screening the available databases, at least 15 expressed sequence tags (ESTs) have been identified from wheat (Triticum aestivum), which exhibit high sequence identity with Aux/IAA homologues in other species. One of these Aux/IAA genes, TaIAA1, harbouring all the four conserved domains characteristic of the Aux/IAA proteins, has been characterized in detail. The expression of TaIAA1 is light-sensitive, tissue-specific, and is induced within 15-30 min of exogenous auxin application. Also, the TaIAA1 transcript levels increase in the presence of a divalent cation, Ca(2+), and this effect is reversed by the calcium-chelating agent, EGTA. The TaIAA1 gene qualifies as the primary response gene because an increase in its transcript levels by auxin is unaffected by cycloheximide. In addition to auxin, the TaIAA1 gene is also induced by brassinosteroid, providing evidence that interplay between hormones is crucial for the regulation of plant growth and development.